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Effect of Folate and Vitamin B12 Status and Related Genetic Polymorphisms on Congenital Heart Defect Risk

Permanent Link: http://ufdc.ufl.edu/UFE0021947/00001

Material Information

Title: Effect of Folate and Vitamin B12 Status and Related Genetic Polymorphisms on Congenital Heart Defect Risk A Pilot Study
Physical Description: 1 online resource (92 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: 677, b12, chd, congenital, ct, defects, folate, heart, mthfr, polymorphisms, tt, vitamin
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: One-carbon (1-C) metabolism reactions require folate as the central substrate that provides essential one carbon moieties. One carbon groups provided by folate are vital for many reactions including those required for amino acid metabolism, purine and pyrimidine synthesis, DNA methylation, and the formation of S-adenosylmethionine (SAM). Low folate status can alter this central role in maintaining 1-C metabolism which can lead to birth defects. Birth defects are the leading cause of death in newborn infants, with congenital heart defects (CHDs) accounting for one death in every three defect-related infant deaths. Congenital heart defect risk may possibly be related to folate and vitamin B12 status and this risk may be exacerbated in the presence of polymorphisms (MTHFR 677 C?T and 1298 A?C, MTRR 66 A?G and TC 776 C?G) related to folate and vitamin B12 metabolism. To assess the roles of folate and vitamin B12 status and associated genetic polymorphisms on CHD risk, a case-control pilot study was conducted including 390 non-pregnant mothers. Blood samples were obtained from subjects and were analyzed to identify biochemical variables and genotypes. Serum folate and vitamin B12 concentrations were inversely associated (p < 0.05) with CHD risk in the case group relative to the control group in subjects who had a delivery within 3 years from the time the blood was drawn. The MTHFR 677 CT and TT genotype groups were combined and compared between cases and controls resulting in a significant (P= 0.0356) increase in risk for CHD in the case group. Future studies need to be conducted to provide a careful assessment of status at the appropriate time during gestation or within a very short time following the index pregnancy to allow a more accurate measure of status indicators that are likely to interact with polymorphisms affecting folate and vitamin B12.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Bailey, Lynn B.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021947:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021947/00001

Material Information

Title: Effect of Folate and Vitamin B12 Status and Related Genetic Polymorphisms on Congenital Heart Defect Risk A Pilot Study
Physical Description: 1 online resource (92 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: 677, b12, chd, congenital, ct, defects, folate, heart, mthfr, polymorphisms, tt, vitamin
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: One-carbon (1-C) metabolism reactions require folate as the central substrate that provides essential one carbon moieties. One carbon groups provided by folate are vital for many reactions including those required for amino acid metabolism, purine and pyrimidine synthesis, DNA methylation, and the formation of S-adenosylmethionine (SAM). Low folate status can alter this central role in maintaining 1-C metabolism which can lead to birth defects. Birth defects are the leading cause of death in newborn infants, with congenital heart defects (CHDs) accounting for one death in every three defect-related infant deaths. Congenital heart defect risk may possibly be related to folate and vitamin B12 status and this risk may be exacerbated in the presence of polymorphisms (MTHFR 677 C?T and 1298 A?C, MTRR 66 A?G and TC 776 C?G) related to folate and vitamin B12 metabolism. To assess the roles of folate and vitamin B12 status and associated genetic polymorphisms on CHD risk, a case-control pilot study was conducted including 390 non-pregnant mothers. Blood samples were obtained from subjects and were analyzed to identify biochemical variables and genotypes. Serum folate and vitamin B12 concentrations were inversely associated (p < 0.05) with CHD risk in the case group relative to the control group in subjects who had a delivery within 3 years from the time the blood was drawn. The MTHFR 677 CT and TT genotype groups were combined and compared between cases and controls resulting in a significant (P= 0.0356) increase in risk for CHD in the case group. Future studies need to be conducted to provide a careful assessment of status at the appropriate time during gestation or within a very short time following the index pregnancy to allow a more accurate measure of status indicators that are likely to interact with polymorphisms affecting folate and vitamin B12.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Bailey, Lynn B.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021947:00001


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e6b137fcc78677bea5a3985e0fb9ecdd
1103e3ede9c79a6b84d8e4a6d5acf2641c1e3464







EFFECT OF FOLATE AND VITAMIN B12 STATUS AND RELATED GENETIC
POLYMORPHISMS ON CONGENITAL HEART DEFECT RISK: A PILOT STUDY





















By

YOUNIS ALI SALMEAN


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2008


































2008 Younis Ali Salmean




























To my wife and parents









ACKNOWLEDGMENTS

I would like first to thank god for the gracious and wonderful opportunity that has been

giving to me and the wonderful people that I have met through this academic journey. I would

like to sincerely thank my supervisory committee members Lynn B. Bailey, PhD, Gail P.A.

Kauwell, PhD, RD, LDN, and Linda Young, PhD for the unique contributions that each of them

made to my research project. In particular, I would like to thank my committee chair, Dr. Lynn

Bailey for guiding and encouraging me through this challenging experience. Dr. Lynn Bailey

was a great console at times of stress, and she was always there to guide me through this stage of

my academic career. The past couple of years were very important for my intellectual

development and maturity and therefore I am grateful for everyone who has contributed to my

success.

I would like to especially thank James Huhta, MD who is acknowledged as the originator

of the study design for the pilot project, the director of the research conducted at the University

of South Florida, All Children's Hospital in St. Petersburg FL. Through the efforts of Dr. Huhta

funding was obtained for support of this project.

I would like to thank my parents and family for they always stood behind me and

believed in me. They were truly a great support, especially, to bare the hardships of being

thousands of miles away for years. I especially thank my mother for her constant encouragement

and prayers.

My dear wife was an amazing person who stood behind me all the time. She was a

source of support, comfort and encouragement. I always found her there when I needed

someone the most, and her love and care were the light that guided me in my pathway to

complete and succeed in this program.









TABLE OF CONTENTS

Page

A CK N O W LED G M EN T S ................................................................. ........... ............. .....

LIST OF TABLES ......... ........... .............................................. 8

LIST OF FIGURES .................................. .. ..... ..... ................. .9

LIST OF ABBREVIATION S ............................. ................... ........................ 10

A B S T R A C T ................................ ............................................................ 13

CHAPTER

1 IN TR OD U CTION .......................................................................... .. ... ... .. 15

H y p o th e se s ................................................................17
O v erall G o al ................................................................17
Specific Objectives ..................................................... ............ ...... .... 17

2 L IT E R A TU R E R E V IE W ......................................................................... ........................ 18

Folate and Vitamin B 12 .................. .................. ................... .......... ........ ..... 18
F o la te ................... ...................1...................8..........
C h e m istry ........................................................................................................... 1 8
B ioav ailab ility ............................................ .. ......................................... 18
Metabolism (Absorption, transport, storage and excretion)................................ 19
V itam in B 1 2 .............................................................................2 0
C h e m istry ........................................................................................................... 2 0
B ioav ailab ility ............................................ .. ......................................... 2 0
Metabolism (Absorption, transport, storage and excretion)..................................21
Biochemical Functions of Folate and Vitamin B 12 .................................... ...............22
P olym orphism s ............................................................................................... 24
Dietary Sources of Folate and Vitamin B12 Intake Recommendations............................. 26
C ongenital H heart D efects............................................................................... .. .............27
P rev alence and E tiology .......................................................................... ......................27
Effect of Low Folate Status on CHD Risk.... .................. ..........................28
Effect of Low Vitamin B12 Status on CHD Risk ...................................... ..................30
P olym orphism s and C H D R isk ............................................................ .....................3 1
MTHFR (677 C-T and 1298 A-C) polymorphisms ................. ... ........... 31
MTRR 66 A-G and TC 776 C-G polymorphisms ...........................................33
Environmental and Sociodemographic Risk Factors............................................................35
Cigarette Sm king ..................................................... ............... ...... ... ... 35
A alcohol C onsum ption ......... ................................................................ ........... .. 35
O b e sity ................... .......................................................... ................ 3 6
Febrile Illnesses ...................................... ................................ ........... 36









Use of M medications .......... ........ ...... ....... ............. .. ... .... 37
D diabetes .......................................................................................................... 37
E th n ic ity ................................................................3 7
Family History .......................................................................38
Biochemical Markers: Folate and Vitamin B12 Status Indicators ......................................38
Serum and Red Blood Cell Folate Concentrations ............... ...... ........... 38
S eru m V itam in B 12 ............................................................................................... 3 9
M eth y lm alo n ic A cid ................................................................. ...............................3 9
H om ocy steine ................................................................ ............................ 40

3 M A TER IA L S A N D M ETH O D S ..................................................................................... 47

Study Design and Methods Overview .............................................................................. 47
Sam ple Collection and Processing................................................. 48
A analytical M ethods.............................................................................49
Measurement of Serum Concentrations of Status Indicators .......................................49
Serum folate and vitamin B 12 concentrations ........................................ ...49
Red blood cell folate concentration.......................... ...... ......... 50
Homocysteine and methylmalonic acid .................................. ...............50
Genotype Identification ................................. ........................... .... ....... 51
P re g n an cy In d ex ................................................................... ...................................5 1
S u p p lem en t A n aly sis ................................................................................................. 52
Statistical M methods ................................................................52

4 R E S U L T S .............................................................................................5 4

Part I: Exploring the D ata ..................................................................54
Demographic Characteristics of the Study Sample Population ...............................54
Subjects ............................................................. 54
Demographic Characteristics ................. .................................54
S u p p lem en t U se .................................................................................. 5 5
Cigarettes and Alcohol Consumption .......................................... 55
M medical C conditions .............................................................55
H em atological Indices ........................................................................................ 55
Part II: Folate and Vitamin B 12 and Status Indicators (Objective 1)..................................55
Status Indicators and CH D Risk ......................................................... 56
Relationship of Status Indicator Variables in the Sample Population ..................56
Status Indicators in the Sub-Sample Population ..................... ...............56
Part III: Polymorphisms and CHD Risk (Objective 2) ................... .... .....................57
MTHFR 677 C- T and MTHFR 1298 A-C Polymorphisms and CHD Risk .......57
M TH FR 677 C- T Polym orphism ................................................................... ..57
M THFR 677 (CT/TT) Genotypes ...................................................................... 57
MTHFR 1298 A- C Polymorphism...................... _.. ........... ........................58
MTHFR 677 (CT/TT) Compared to CC Genotype and Status Indicators in the
sam ple population ................5.....................58
MTRR 66 and TC 776 Genotypes and CHD Risk............... ...................58



6









Part V: Relationship Between Polymorphisms Related to Folate and Vitamin B12
MTHFR 677 C-T, MTHFR 1298 A-C, MTRR 66 A-G, and TC 776 C-G and
Status Indicators and CHD Risk (Objective 3) ............. .............................................59

5 D ISC U S SIO N ..............................................................................................65

Summary of Pilot Study Design Limitation ............ ............................... ...............74
Public H health Significance of R research ................................. ...................................... 75

APPENDIX QUESTIONNAIRE ................................................................. ............... 76

L IST O F R E FE R E N C E S ......... .. ..................... ......... ............................................................79

B IO G R A PH IC A L SK E T C H ............................................ ......... ................... ...........................92









LIST OF TABLES


Table Page

2-1 Definitions of Dietary Reference Intake (DRI) recommendations...............................46

2-2 Folate intake recommendations for men and non-pregnant women >19 years. ................46

4-1 Demographic characteristics of sample population. .................................. ............... 60

4-2 Distribution and response rates for prenatal use of folic acid in the sample
p opu nation ............................................................. ................ 6 0

4-3 Distribution and response rates for cigarette and alcohol use in the sample
p o p u latio n ............................................................................. 6 1

4-4 Distribution and response rates for diabetes, seizures, and anemia during pregnancy
in the sample population. ................................ .. ... ....... ...............61

4-5 Mean comparison of hematological indices between cases and controls in the sample
p opu nation ................................................................................6 1

4-6 Comparison of serum and RBC folate, Hcy, serum B12, and MMA concentrations by
group (case/control) in the sample population........................................ ............... 62

4-7 Comparison of serum and RBC folate, Hcy, serum B12, and MMA concentrations by
group (case/control) in the sub-sample population ......................................................62

4-8 Genotypes combination distribution in the sample population for specific
polym orphism s..................................... .......................... .... ..... ........ 63

4-9 Frequency comparison of the MTHFR 677 C--T and 1298 A--C distribution in the
sample population by group type (case/control). ........................................ ............... 63

4-10 Frequency comparison of the MTHFR 677 genotype (CC, CT, TT) distribution by
group type (case/control) in the sample population...................... .......................... 63

4-11 Frequency comparison between MTHFR 677 CC genotype and the combined
genotypes (CT/TT) for the MTHFR 677 C--T polymorphism by group type
(case/control) in the sam ple population. ................................................. .....................64

4-12 Frequency comparison of the MTHFR 1298 genotype (AA, AC, CC) distribution by
group type (case/control) in the sample population...................... ............................... 64

4-13 Comparison of serum and RBC folate, Hcy, serum B12, and MMA concentrations
between the MTHFR 677 CC genotype and the combined (CT/TT) genotypes in the
sam ple p opu lation ...................................... ............................... ................. 64









LIST OF FIGURES

Figure Page

2-1 Structure of folate/folic acid ......................................................................................... 42

2-2 Cystosolic and mitochondrial pathways of THF metabolism in the body .......................43

2-3 Vitam in B 12 structure ..................................................... .......... ...... ... .... 44

2-4 One-carbon metabolism folate-dependent pathway................................ ...............45











1-C

5, 10-MTHF

5-MTHF

10-CHO-THF

Adenosyl-Cbl

AHA

AI

ANOVA

B6

B12

BMI

BWIS

Cbl

CBS

CH3-Cbl

CH3-THF

CHD

CLP

CI

CN-Cbl

Cyanobacteria

d

DHF

DFEs


LIST OF ABBREVIATIONS

one-carbon

5, 10-methylenetetrahydrofolate

5-methyltetrahydrofolate

10-formyltetrahydrofolate

adenosylcobalamin

American Heart Association

Adequate Intake

Analysis of variance

pyridoxal phosphate

vitamin B 12

body mass index

Baltimore Washington Infant Study

cobalamin

cystathionine B-synthase

methylcobalamin

methyltetrahydrofolate

congenital heart defects

cleft palate

confidence interval

cyanocobalamin

blue-green algae

day

dihydrofolate

Dietary Folate Equivalents









DNA Deoxyribonucleic acid

DRIs Dietary Reference Intakes

dTMP thymidylate

dUMP deoxyuridylate

EAR Estimated Average Requirements

FDA Food and Drug Administration

g gram

HCL hydrochloric acid

Hct hematocrit

Hcy homocysteine

Hgb hemoglobin

IF intrinsic factor

IM intramuscular injection

IOM Institute of Medicine

MCH mean corpuscular hemoglobin

MCHC mean cell hemoglobin concentration

MCV mean corpuscular volume

min minutes

[IM micromole

MMA methylmalonic acid

MTHFR methyltetrahydrofolate reductase

MTR methionine synthase

MTRR methionine synthase reductase

NCCs neural crest cells

nM nanomole









nmol/L nanomoles per liter

NO nitric oxide

NTD neural tube defect

Oh-Cbl hydroxocobalamin

PCC population-based case-control studies

pmol/L pica mol per litter

RBC red blood cell

RCT randomized control intervention trial

RDA Recommended Dietary Allowance

ROS reactive oxygen species

SAH S-adenosylhomocysteine

SAM S-adenosylmethionine

SHMT serine hydroxymethyltransferase

TC transcobalamin

TDT transmission disequilibrium test

THF tetrahydrofolate

UL Tolerable upper level









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

EFFECTS OF FOLATE AND VITAMIN B 12 RELATED GENETIC POLYMORPHISMS ON
CONGENITAL HEART DEFECT RISK: A PILOT STUDY

By

Younis Ali Salmean

May 2008

Chair: Lynn B. Bailey
Major: Food Science and Human Nutrition

One-carbon (1-C) metabolism reactions require folate as the central substrate that

provides essential one carbon moieties. One carbon groups provided by folate are vital for many

reactions including those required for amino acid metabolism, purine and pyrimidine synthesis,

DNA methylation, and the formation of S-adenosylmethionine (SAM). Low folate status can

alter this central role in maintaining 1-C metabolism which can lead to birth defects. Birth

defects are the leading cause of death in newborn infants, with congenital heart defects (CHDs)

accounting for one death in every three defect-related infant deaths. Congenital heart defect risk

may possibly be related to folate and vitamin B 12 status and this risk may be exacerbated in the

presence of polymorphisms (MTHFR 677 C-T and 1298 A-C, MTRR 66 A-G and TC 776

C--G) related to folate and vitamin B12 metabolism. To assess the roles of folate and vitamin

B12 status and associated genetic polymorphisms on CHD risk, a case-control pilot study was

conducted including 390 non-pregnant mothers. Blood samples were obtained from subjects and

were analyzed to identify biochemical variables and genotypes. Serum folate and vitamin B12

concentrations were inversely associated (p < 0.05) with CHD risk in the case group relative to

the control group in subjects who had a delivery within 3 years from the time the blood was

drawn. The MTHFR 677 CT and TT genotype groups were combined and compared between









cases and controls resulting in a significant (P= 0.0356) increase in risk for CHD in the case

group. Future studies need to be conducted to provide a careful assessment of status at the

appropriate time during gestation or within a very short time following the index pregnancy to

allow a more accurate measure of status indicators that are likely to interact with polymorphisms

affecting folate and vitamin B 12.









CHAPTER 1
INTRODUCTION

Folate and vitamin B 12 play critical roles in one-carbon (1-C) metabolism including

deoxyribonucleic acid (DNA) synthesis and methylation, and homocysteine (Hcy) remethylation.

Folate is required for the formation of 5-methyltetrahydrofolate (5-MTHF), a substrate critical

for donating methyl groups to Hcy in a co-dependent manner with vitamin B 12 serving as a

cofactor. Folate is the 1-C donor required for the formation of S-adenosylmethionine (SAM)

from methionine. While folate is involved in the formation of the methyl donor substrate,

vitamin B12 is required to facilitate the transfer of the methyl group from the 5-MTHF to Hcy.

Impaired folate or vitamin B12 status can lead to impaired generation of substrates

needed for the de novo synthesis of DNA and cell division. It will also affect formation and

maturation of red and white blood cells in the bone marrow. Abnormalities in 1-C metabolism

may result in elevated concentrations of Hcy and reduced DNA methylation, both of which

possibly can affect development of embryos. Both nutrients alone and together have been linked

to increased Hcy concentration, which is a risk factor for pregnancy complications with evidence

for an association with congenital malformations.

Folate and vitamin B12 metabolism can be adversely affected by the presence of genetic

polymorphisms, which are common mutations that may affect the structure and function of

folate/vitamin B12-dependent enzymes and transport proteins. Certain polymorphisms (677

C-T plus 1298 A->C) are responsible for the reduced activity of the methyltetrahydrofolate

reductase (MTHFR) enzyme which may lead to impaired Hcy remethylation and DNA

methylation. The MTHFR 677C->T polymorphism has been associated with an increased risk

of neural tube defects (NTDs) especially when folate status is low.









Congenital heart defect (CHD) risk may possibly be related to folate and vitamin B 12

status and this risk may be exacerbated in the presence of MTHFR polymorphisms. Methionine

synthase reductase (MTRR) is an enzyme required for the activation of methionine synthase

(MTR) for the remethylation of Hcy to methionine which may be negatively impacted by a

genetic polymorphism (MTRR 66 A--G). Investigation of the association of the MTRR 66

A--G polymorphism with CHD risk is warranted since impaired activity of MTRR may

negatively affect 1-C metabolism. The MTRR 66 A--G was previously found to be positively

associated with NTDs when vitamin B12 status was low, which provides some basis for a

possible link to CHD since the embryonic origins of NTDs and CHDs are similar.

Transcobalamin (TC) 776 C--G is a polymorphism affecting the protein responsible for

the cellular uptake of vitamin B12. It is proposed that individuals with TC 776 GG genotype may

have an impaired ability to transport vitamin B12, which may reduce cellular uptake and lead to

abnormal function including embryonic development. The critical role of TC in vitamin B12

function in 1-C metabolism necessitates the assessment of its effect on risk of CHD, however,

only a limited number of studies have investigated the role of TC 776 C--G and CHD risk and

the conclusions from these studies are not definitive due to limitations of sample size and other

factors. A comprehensive investigation of the association of folate and vitamin B12 status and

related genetic polymorphisms with CHD risk has not been conducted and is the long term goal

of future studies to follow this initial pilot study.









Hypotheses

* low folate and/or vitamin B 12 status are associated with increased risk for CHD;

* genotype status for the MTHFR 677C -* T and 1298 A C polymorphisms and the
MTRR gene 66 A -- G and the TC 776 C G polymorphisms will exacerbate the effect
of low folate and/or vitamin B 12 status on CHD risk;

* congenital heart defect risk associated with either folate and/or vitamin B12 related
polymorphisms will be exacerbated by low folate and/or vitamin B 12 status.

Overall Goal

The primary goal of this study was to assess the roles of folate and vitamin B12 status

and associated genetic polymorphisms on CHD risk in women with affected pregnancies

compared to women who delivered infants with no birth defects.

Specific Objectives

* assess the association between maternal folate and vitamin B12 status biomarkers (serum
and red blood cell (RBC) folate, vitamin B12, methylmalonic acid (MMA) and Hcy) and
infant CHD risk;

* assess the association between maternal MTHFR 677 C-T and 1298 A-C and MTRR 66
A--G and TC 776 C--G genotypes on infant CHD risk;

* determine the interaction between maternal folate and vitamin B12 related polymorphisms
and folate and vitamin B 12 status indicators on infant CHD risk.









CHAPTER 2
LITERATURE REVIEW

Folate and Vitamin B12

Folate

Chemistry

Folate is a water-soluble vitamin. The generic name for the vitamin is folate and it is

related to a family of substances containing pteridine rings joined with p-aminobenzoic acid and

one or more glutamic acid molecules (1) (Figure 2-1). The form of naturally occurring folate

depends on the side chain composition in terms of the number of glutamic acid molecules as well

as the specific one-carbon moiety attached to the vitamin. The folate molecule can vary in

structure through reduction of the pteridine moiety to dihydrofolate (DHF) and tetrahydrofolate

(THF), elongation of the glutamate chain, and substitution of the 1-C unit at the N-5, N-10, or

both positions (2,3). Folic acid, which rarely occurs in nature, is the fully oxidized

monoglutamate form of the vitamin folate, and it is used extensively in food fortification and

supplementation due to its chemical stability (1).

Bioavailability

Bioavailability may be defined as the proportion of an ingested nutrient that is used and

stored in the body. Bioavailability of food folate is considerably dependent on the individual's

ability to digest, absorb and metabolize the vitamin. The bioavailability of folate depends on the

food source and often it is incomplete (1). The bioavailability of food folate is estimated using

folic acid as the standard since folic acid exhibits nearly complete absorption (4), and is

considered to be 100% bioavailable. The bioavailability of folate from a mixed diet is

approximately 50% or less of the bioavailability of folic acid (5). When folic acid is consumed









with food, the bioavailability is reduced by 15% compared to when it is consumed alone as a

supplement, therefore folic acid in fortified foods is estimated to be -85% bioavailable (6).

Metabolism (Absorption, transport, storage and excretion)

Folic acid is a fully oxidized monoglutamate form of folate and can be absorbed readily

at the brush border membrane of the jejunum. However, food folate needs to be converted into

the monoglutamate form by the folylpoly-y-glutamate carboxypeptidase enzyme also known as

folate conjugase (7,8). The monoglutamate form of folate is transported into the enterocyte via a

pH-dependent carrier-mediated mechanism (9). At high concentrations of folate (>10 .imol/L),

ion-mediated transport becomes the means of transport into the cell (10). After the entry into the

mucosal cell, the monoglutamate form of folate is reduced and a portion is converted to

methyltetrahydrofolate (CH3-THF) before it's released into circulation (11).

Most folate in the plasma is bound to albumin in the form of 5-CH3-THF, and a smaller

amount is bound to a high affinity folate-binding protein (2). Cellular uptake of folate by cells

is mediated by membrane-associated folate-binding proteins (12). After its uptake, 5-CH3-THF

is demethylated by MTR and then is converted into a polyglutamyl form by folypolylglutamate

synthase (13). The metabolism of folate is divided into pathways occurring in either the

cytoplasm or mitochondria. Both pathways facilitate the regeneration of THF through the

reduction of various folate coenzymes. This allows THF to accept a 1-C unit from the folate

pool, which is donated by both the cystosolic and mitochondrial pathways, leading to purine

synthesis, DNA methylation, DNA synthesis, and Hcy remethylation to methionine (Figure 2-2)

While folate in the monoglutamate form is subsequently taken up by the cells, it is the

polyglutamate form that helps sequester folate inside the cell due to the polarity of the side

chains (13). Tissues do not store large amounts of folate beyond their metabolic needs. The total

body content of folate is estimated to be between 15 to 30 mg (14). A small amount of the









dietary folate is excreted in the urine (15). The small quantity of free folate in the plasma is

filtered through the glomerulus but mostly reabsorbed in the proximal renal tubules (16). The

amount of folate excreted in the urine is estimated to be similar to the amount excreted in the

feces (1).

Vitamin B12

Chemistry

Vitamin B12, also known as cobalamin (Cbl), refers to a family of substances composed

of a central cobalt atom surrounded by macrocyclic corrin tetrapyrrole which are four reduced

pyrrole rings connected by nucleotide side chains attached to the central atom (Figure 2-3).

There are various forms of the vitamin B12 molecule, including methylcobalamin (CH3-Cbl),

cyanocobalamin (CN-Cbl), adenosylcobalamin (adenosyl-Cbl) and hydroxocobalamin (OH-Cbl).

The commercially produced form of vitamin B12 is known as CN-Cbl, which is generally

used in supplements. The major form of vitamin B12 in blood is CH3-Cbl, with smaller amounts

as adenosyl-Cbl and OH-Cbl (17). Vitamin B12 in the form of OH-Cbl is widely used as an

intramuscular injection (IM). Hydroxocobalamin is retained longer in the body than CN-Cbl. In

the human body and higher animals, vitamin B12 is utilized in two major metabolic processes

where it serves as a coenzyme. The first vitamin B12 dependent reaction is the conversion of

methylmalonyl-CoA to succinyl-CoA, which uses adenosyl-Cbl as a cofactor, and the second

conversion is the remethylation of Hcy to methionine, which uses CH3-Cbl as a cofactor (18).

Cobalamin will be referred to subsequently as vitamin B12.

Bioavailability

Vitamin B 12 it is a product of microbial synthesis in all higher animals, and the sole

source of vitamin B12 is from animal-related sources. Although some edible algae and blue-

green algae (cyanobacteria), contain large amounts of vitamin B12, vitamin B 12 from these two









sources appears to be inactive in mammals (19). Bioactivity of each of the forms of vitamin B12

is different. The various forms of vitamin B12 will have different dissociation, absorption, and

protein carrier binding qualities which result in various degrees of bioavailability. As the

intrinsic factor (IF)-mediated intestinal absorption system is estimated to be saturated at about

1.5-2.0 pg per meal under normal physiologic conditions, vitamin B12 bioavailability

significantly decreases with increasing intake of vitamin B12 per meal (20). Recently reported

by Watanabe, the bioavailability of vitamin B12 in healthy humans from fish, meat, and chicken

averaged 42%, 56%-89%, and 61%-66%, respectively, while vitamin B12 in eggs appeared to

be poorly absorbed (<9%) relative to other animal food products (20).

Metabolism (Absorption, transport, storage and excretion)

Release of vitamin B12 from proteins must occur prior to absorption. Saliva contains a

vitamin B12 binding protein referred to a an R protein (18). Saliva activity starts the dissociation

of vitamin B12 from the protein to which it is bound. Further dissociation of vitamin B12 occurs

in the stomach with the action of hydrochloric acid (HCL) and pepsin. The dissociated vitamin

B12 enters the duodenum from the stomach, bound to the R-protein. A protein synthesized by

the gastric parietal cells referred to as IF binds to vitamin B12 forming the vitamin B12 intrinsic

factor complex (IF-Cbl). This happens after the stomach acid is neutralized and the R protein is

removed by digestive enzymes (21).

In the ileum, a specific IF-Cbl receptor initiates the uptake of vitamin B12 complex into

the enterocytes. During uptake, the IF-Cbl complex is internalized by receptor mediated

endocytosis where vitamin B12 is then processed and subsequently bound to TC and released

into the circulation as TC-Cbl (22). Transcobalamin accounts for approximately 20% of all

vitamin B12 bound in circulation, while haptocorrin accounts for the remaining 80% (23).

However, only TC-Cbl has receptors on the cells surface which makes it essential for cellular









uptake, therefore TC-Cbl is considered the bioavailable form of the vitamin. Once TC-Cbl is

taken up by receptor-mediated endocytosis, the lysosomic activity releases vitamin B12 from the

complex into the tissue.

Vitamin B12 can be stored in the body for long periods of time. The liver is the main

storage tissue accounting for -60% of total storage while muscle tissues account for -30%.

There are approximately 2-5 mg of vitamin B 12 stored in the body predominately in the form of

adenosyl-Cbl and CH3-Cbl. As stated earlier the IF-mediated intestinal absorption system is

estimated to be saturated at about 1.5-2.0 tg per meal under normal physiologic conditions,

therefore an increase in dose will result in an increase in urinary loss. Other factors such as

bioavailability and diet content can also affect excretion of the vitamin.

Biochemical Functions of Folate and Vitamin B12

One-carbon metabolism reactions require folate as the central substrate that provides

essential one carbon moieties. The methyl group is vital for many reactions including amino

acid metabolism, purine and pyrimidine synthesis, and the formation of S-adenosylmethionine

(SAM) (1). The formation of SAM in the 1-C cycle is critical for a large number of methylation

reactions. The production of creatine, phospholipids, and neurotransmitters from SAM is

important for normal physiological function. More importantly, SAM is a methylating agent for

more than 100 compounds including proteins, RNA and DNA (24).

Folate in the polyglutamate form of THF is converted to 5, 10-methylenetetrahydrofolate

(5, 10-MTHF) by accepting a methyl group from the conversion of serine to glycine via serine

hydroxymethyltransferase (SHMT) (Figure 2-4) reaction 3. This reaction is important because it

provides the methyl group needed for the production of thymidylate (dTMP) from

deoxyuridylate (dUMP) by thimidylate synthase (1) (Figure 2-4) reaction 1. Folate in the form

of DHF is reduced back to THF by dihydrofolate reductase. Folate also is involved in de novo









synthesis of adenine and guanine from 10-formylTHF (10-CHO-THF) resulting in purine

synthesis and regeneration of THF (Figure 2-4) reactions 4, 12 and 13. These reactions

collectively underline the critical role of folate in pyrimidine and purine synthesis. Thus,

impaired folate status can be detrimental to the body since DNA synthesis required for cellular

growth may be suppressed.

Another critical role for folate in 1-C metabolism is DNA methylation. DNA methylation

depends on the reduction of 5, 10-MTHF to 5-MTHF by MTHFR. Methylenetetrahydrofolate

reductase is an important enzyme since its only function in the body is in the above step, which

is critical for the remethylation of Hcy to form methionine. Methionine serves as the dominate

precursor for SAM. The coenzyme 5-MTHF is the methyl donor, and vitamin B12 is the

cofactor needed for transferring the methyl group from 5-MTHF to Hcy to form methionine

(remethylation), a reaction that requires the MTR enzyme (Figure 2-4) reactions 5, 6 and 7. The

MTR reaction requires vitamin B 12 as a cofactor for the remethylation of Hcy to methionine.

During the MTR reaction, transfer of the methyl group from methylcobalamin-III results in the

formation of the highly reactive cobalamin-I, which may become oxidized to cobalamin-II,

resulting in MTR inactivation (25). Methionine synthase reductase is required for the reductive

methylation of cobalamin-II (with SAM providing the methyl group), which reactivates MTR

(26).

If either folate or vitamin B12 are deficient, MTR cannot remethylate Hcy to form

methionine which leads to megaloblastic changes in rapidly dividing cells, such as bone marrow

in addition to elevation of Hcy and depletion of SAM (1). The initial methyl group that was

accepted by folate is the same methyl group that is donated by SAM to over 100

transmethylation reactions (1). When SAM donates its methyl group, it is converted to S-









adenosylhomocysteine (SAH), which then is hydrolyzed back into Hcy and adenosine by SAH

hydrolase (1)( Figure 2-4) reactions 9 and 10.

Dietary protein is another source of methionine; however, the tightly regulated

remethylation of Hcy to methionine also contributes to the body's need for this essential amino

acid. When methionine is converted back to Hcy via the hydrolysis of SAH an elevation of Hcy

may occur if the MTR reaction is impaired. For this reason, Hcy conversion to methionine needs

to be optimized to maintain a balance between Hcy and the methionine pool and to prevent

hyperhomocysteinaemia. Homocysteine can also be catabolized into cystathionine through the

condensation with cysteine by pyridoxal phosphate (vitamin B6) and cystathionine B-synthase

(CBS) (18) (Figure 2-4) reaction 11. However, this transsulfuration step is dependent on SAM

activation and a surplus of methionine (1). When vitamin B12 and folate are deficient,

cystathionine levels are elevated (27).

Polymorphisms

Polymorphisms are mutations that are present in more than 1% of the population. The

relationship among the four polymorphisms occurring in genes involved in 1-C metabolism and

the risk for CHD will be discussed in a subsequent section. Methyltetrahydrofolate reductase

677 C->T is the most widely studied polymorphism in which a transition of cytosine to thymine

occurs in the gene for MTHFR at base pair 677, resulting in an alanine to valine substitution in

the enzyme (28,29). A second polymorphism of the MTHFR gene occurs at base pair 1298 due

to the substitution of cytosine for adenine. This substitution results in a coding change which

that alanine is substituted for glutamate in the protein product (30,31). Both mutations alter the

activity of the MTHFR enzyme which leads to lower concentration of 5-MTHF and less

substrate available for the remethylation of Hcy. As discussed previously, the initial step in the









remethylation pathway leading to DNA methylation starts early with the reduction of 5, 10-

MTHF to 5-MTHF via the MTHFR enzyme (Figure 2-4) reaction 5.

While these two polymorphisms MTHFR 677 C-T and 1298 A-C affect the activity of

MTHFR and folate metabolism, two other polymorphisms related to vitamin B 12 have also been

investigated for their possible role as risk factors for CHD. The first is MTRR 66 A--G in

which methionine replaces isoleucine in the enzyme protein. This mutation affects the activity of

the MTR enzyme and results in lower remethylation of Hcy into methionine (Figure 2-4)

reaction 6. The MTRR 66 A--G polymorphism was associated with an increase in plasma Hcy,

with the GG genotype having a greater effect than the AG genotype (32). Also, it was found that

MTRR 66 GG genotype was associated with an increased risk for having an NTD-affected

pregnancy when maternal vitamin B 12 concentrations were low (26).

The second polymorphism is the TC 776 C--G, which can affect the total amount of

vitamin B12 carried in the blood and the amount that the cell takes up. As mentioned earlier,

cells only have receptors for vitamin B 12 bound to TC, and therefore TC-Cbl serves as the

bioavailable form of the vitamin. Vitamin B12 bound to haptocorrin is not bioavailable because

cells lack receptors for this carrier protein.

Several base pair substitutions that may occur in the gene that encodes the TC protein

lead to decreased uptake of vitamin B12 (33). The most common polymorphism in the TC gene

is a cytosine-to-guanine transition at base pair 776 (TC 776 C--G) that results in replacement of

proline with arginine (34). This TC 776 C--G polymorphism negatively affects the serum holo-

TC concentration and studies suggest that the TC 776 C--G polymorphism may affect TC

binding affinity for vitamin B12 and the ability to transport vitamin B12 into tissues (35-38).

Miler et al. observed a reduced mean holo-TC concentration, a lower percentage of total vitamin









B 12 bound to TC, and a higher MMA concentration in individuals with the TC 776 GG genotype

than in those with the TC 776 CC genotype (36). These results indicate that the TC 776 C-*G

polymorphism may alter the cellular availability of vitamin B12 and exacerbate the effects of low

vitamin B12 status.

Dietary Sources of Folate and Vitamin B12 Intake Recommendations

Folic acid is the synthetic form of folate and is used for all enriched products in the US

since January 1998 as mandated by the Food and Drug Administration (FDA). Enriched cereal

and grain products are fortified with folic to a target level of 140 .ig/100 g (39). In addition to

cereal and grain products, folic acid fortified products are available throughout the US

marketplace in the form of ready-to-eat breakfast cereals, snacks, meal replacements and many

others. Naturally occurring dietary folate can be found mainly in orange juice, strawberries,

peanuts, legumes, asparagus and dark green leafy vegetables.

The Dietary Reference Intakes (DRIs) represent the most current recommendations for

each vitamin and mineral. It is the newest approach adopted by the Food and Nutrition Board to

provide quantitative estimates of recommended nutrient intakes for different age and gender

groups. The DRIs include the Estimated Average requirements (EAR), Recommended Dietary

Allowances (RDA), Adequate Intake (AI), and Tolerable Upper Intake Level (UL). See Table 2-

1 for definitions of each DRI (40).

The National Academy of Sciences Institute of Medicine (IOM) published the most

recent DRI recommendations in 1998 (40). The goal of these DRI recommendations was to

ensure optimum health rather than prevent clinical deficiencies. Folate recommendations based

on the DRIs are presented in Table 2-2. The DRIs also include the new RDA for folate in dietary

folate equivalents (DFEs). Dietary Folate Equivalents are units that account for differences in

the absorption of naturally occurring food folate and the more bioavailable synthetic folic acid









(41). To calculate DFEs 1.7 is multiplied times the micrograms of folic acid and added to the

micrograms of food folate. The RDA for men and non-pregnant women 19 years and older is

400 pg DFE/d. The recommendations are increased to 600 pg DFE/d during pregnancy and 500

.g DFE/d during lactation. The UL for folic acid is 1,000 [g/d. This intake level is established

solely based on the fact folic acid supplementation can mask the diagnosis of vitamin B12

deficiency; as folate itself is not associated with any toxic side-effects. It is recommended that

all women of childbearing age consume 400 [g/d of folic acid from fortified foods and/or

supplements. This is in addition to consuming food folate from varied dietary sources to reduce

the risk of an NTD-affected pregnancy (41).

Dietary sources of vitamin B 12 include animal-based products and fortified foods.

Concentrated dietary sources of vitamin B 12 include meat, dairy products, and eggs. It is not

very common to develop a vitamin B12 deficiency due to short-term dietary restriction because

liver stores of these vitamin can last for a few years. The most common reasons for developing a

vitamin B 12 deficiency are stomach and intestinal disorders that limit the release and/or

absorption of vitamin B12 (42). The RDA is 2.4 [g/d for adults, 2.6 [g/d for pregnant women,

and 2.8 [g/d during lactation (40).

Congenital Heart Defects

Prevalence and Etiology

One million children per year are born with a CHD worldwide (43). Birth defects are the

leading cause of death in new born infants (44), with CHD accounting for one death in every

three defect-related infant deaths (45). Congenital heart defects occur as an isolated

malformation due to abnormal organogenesis during embryonic development. The development

of the cardiovascular system occurs between the third and eight weeks of embryonic growth

after conception (46). During this period, both genetic and environmental factors play a critical









role in the development of the heart through proliferation and apoptosis, in which, inadequate

proliferation or excess apoptosis can lead to congenital malformations such as CHD (47,48).

The interaction between environmental and genetic factors has been the focus of many

research studies to identify the role of dietary and supplement intake in the prevention of CHD.

Factors such as low folate and vitamin B12 status have been identified as risk factors for NTD.

Neural tube defects are malformations occurring during the early embryonic stage (49-53).

Both NTDs and CHDs share the same neural crest cells (NCCs) during the embryonic stage.

Therefore, it is logical to assume a possible association between the status of both folate and

vitamin B12 and the risk of CHD based on the similarity between NTD and CHD developmental

stages.

These assumptions are supported by recent findings from investigations in which the role

of both nutrients and CHD risk was evaluated (54-57). Recently, the American Heart

Association (AHA) recommended that periconceptional use of multivitamins containing folic

acid be taken by women of reproductive age to reduce the risk of CHD (58). The AHA

recommendation was established in support of the possible protective effect of folate to reduce

the risk of CHD, although the association could not be definitively confirmed due to the small

number of studies available on which to base the recommendation (57,59).

Effect of Low Folate Status on CHD Risk

Low folate status alters 1-C metabolism resulting in impaired remethylation of Hcy to

methionine, DNA methylation, and reduction of various polyglutamate forms of folate to THF.

Consequently, this will lead to lower availability of THF which is required to form 5, 10 MTHF

necessary for cell division reactions and nucleotide synthesis. In addition, it will lead to

elevation of Hcy concentrations and alteration of the primary DNA methyl donor SAM (Figure

2-4) reactions 4,2, 6 and 8 (60).









It has long been recognized that there is an association between folate deficiency and

increased incidence of congenital abnormalities (61). The relationship between folate status and

CHD development is through impaired cardiac neural crest development. The neural crest cells

develop into many organs and tissues during embryonic development, one of which is the

cardiovascular system. Folate is required for cardiovascular development during the early stages

through the migration of NCC, differentiation, dispersal and cell cycle programming. It has also

been suggested that Hcy directly affects cardiac NCC function, however, the mechanism has

not been elucidated (61). Both impaired DNA synthesis and elevation of Hcy concentration

occur in response to low folate status. However, it is not clear whether it is the lower THF

availability or the elevated Hcy concentrations associated with low folate status or both that may

be associated with the impaired NCC development.

Periconceptional use of folic acid is well established in preventing NTDs, and emerging

evidence suggests that multivitamins containing folic acid may also protect against CHD

(57,62). An association between folate deficiency and CHD has been examined in both human

epidemiological studies and animal experimentation (60,63-67). The first studies to show an

association between B-vitamins and CHD were published in 1952 and 1954 (63,68). Congenital

heart defects were associated with the consumption of folate-deficient diets in both studies using

animal models. Developmental defects involving the heart and inhibition of myocardial

proliferation were also associated with folate deficient diets in other investigations (69,70).

Hobbs et al. reported that Hcy, SAH, and methionine concentrations are important biomarkers

predictive of CHD case or control status (71).

The strongest evidence that folate status is involved in the CHD formation and prevention

comes from a Hungarian randomized control intervention trial (RCT) in which multivitamins









containing folic acid reduced the risk of CHD up to 60% when taken during the periconceptional

period (72). These results were confirmed in two population-based case-control studies (PCC)

in the USA in which a 24% reduction in CHD risk was observed (55,57). In another study, the

Baltimore Washington Infant Study (BWIS), an inverse relationship between daily maternal

intake of folic acid and the incidence of cardiac outflow tract defects in offspring was found (56).

There are few possible links to how folate may be affecting the risk of CHD. As stated

earlier, folate is a critical substrate for DNA methylation, as well as DNA and nucleotide

synthesis, both of which are possibly associated. Research studies provide evidence that elevated

Hcy concentration (hyperhomocysteinaemia) is associated with an increased CHD risk, which

may be associated with low folate and vitamin B12 status (73). However, due to the small

number of investigations, and the inconsistency of findings between some studies, further

research assessing the effect of folic acid and the reduction of CHD risk is warranted.

Effect of Low Vitamin B12 Status on CHD Risk

The roles of folate and vitamin B 12 are interrelated with regard to DNA and nucleotide

synthesis as well DNA methylation. Folate and vitamin B12 participate in 1-C metabolism in

which both are needed for the regeneration of THF, remethylation of Hcy, and production of

SAM. As discussed earlier, folate is required to form the 5-MTHF through the action of the 5,

10 MTHFR. The methyl group is donated by 5-MTHF to form methionine via remethylation of

Hcy, and to regenerate THF. This step cannot be completed without the cofactor vitamin B 12,

which is essential for the function of the MTR enzyme. Deficiencies of vitamin B12 and/or

folic acid result in hyperhomocysteinaemia, which is associated with an increased risk of CHD

(73-76). Similar to folate, vitamin B12 deficiency is linked to an increase risk ofNTDs (77).

Several studies linked vitamin B12 status with the risk of CHD through impaired methylation of









Hey to methionine. This biochemical derangement leads to hyperhomocysteinaemia and low

DNA methylation, which might be associated with the increase risk for CHD.

It is not clear whether vitamin B12 alone or in conjunction with low folate status is a risk

factor for CHD. The association between specific polymorphisms in 1-C metabolism and

nutrient status further complicate the relationship. More comprehensive studies that account for

both folate and vitamin B12 and the polymorphisms related to their 1-C metabolism is required

to determine risk associations with CHD.

Polymorphisms and CHD Risk

MTHFR (677 C-T and 1298 A-C) polymorphisms

In this section, the association of key folate and vitamin B 12-related polymorphisms with

CHD risk will be discussed. Polymorphisms in the MTHFR, MTRR, and TC genes are

recognized to have some effect on CHD; whether this effect is dependent or independent of

folate and vitamin B12 status is the subject of on-going investigations (78-81). Although folic

acid containing supplements may have a protective effect in reducing CHD risk, the role that

folate and vitamin B 12-related polymorphisms may play in CHD risk is not clear enough to draw

definitive conclusions and more research needs to be done in this area (82).

Studies examining the roles of these polymorphisms in affecting CHD risk have produced

conflicting results. The MTHFR 677 TT genotype has been associated with reduced enzyme

activity, decreased plasma and red blood cell folate, and mildly elevated Hcy concentrations

especially when combined with the MTHFR 1298 A->C polymorphism (83-86). Data from

clinical studies indicate that there is a positive association between the MTHFR 677 C--T

polymorphism and CHD risk (79,80,87,88). In 2006, Beynum et al. reported that maternal

MTHFR 677 C--T is a risk factor for CHD in offspring (80). Beynum group's conclusion was

based on an observed increased risk of CHD in the offspring of mothers who did not use









supplements containing folic acid with either the MTHFR 677 CT or TT genotype. The risk

increased 3-fold in mothers in the CT genotype group and 6-fold in the TT genotype group

compared to women who had used supplements containing folic acid. However, there was no

risk association between the MTHFR polymorphisms and CHD in the family-based transmission

disequilibrium test (TDT). Briefly, the TDT is a family-based association test to detect the

presence of linkage between a genetic marker and a trait by measuring the over-transmission of

an allele from heterozygous parents to affected offspring.

Hobbs et al. reported that the highest estimated risk for having a CHD-affected pregnancy

was among women who were in the highest quartile for Hcy with the MTHFR 677 CC genotype

and were smokers (89). The group reported that maternal MTHFR 677 C --T polymorphism did

not have an independent impact on the estimated risk of having a CHD-affected pregnancy. The

sample size however, may have limited the power to detect an association between the MTHFR

and CHD risk. Based on a 2007 meta-analysis, Van Beynum et al. concluded that there was no

substantial evidence of increased CHD risk in individuals with either the MTHFR 677 CT or TT

genotypes (90). The group suggested that heterogeneity regarding population background, study

design and type of heart defects complicates the pooling and comparison of the studies (90).

Methyltetrahydrofolate reductase 1298 A--C polymorphism results in reduced enzyme

activity (91). Combined heterozygosity for both MTHFR 677 C-T and 1298 A-C

polymorphisms results in a lower MTHFR activity compared to hetrozygosity of either

polymorphism alone (92). The MTHFR 677 C->T polymorphism alone has a greater effect on

Hcy than the MTHFR 1298 A->C polymorphism (93). In studies concerning NTD risk, an

effect of the MTHFR 1298 A--C polymorphism was observed only when combined with the

MTHFR 677 C--T polymorphism (93). In another study concerning cleft palate (CLP), the risk









increased when the MTHFR 677 CT and TT genotypes and the MTHFR 1298 AC were

accompanied by low folate status during pregnancy (94). Due to the similarity in embryonic

origin, evaluating other congenital conditions can be critical for the understanding of the etiology

and risk factors associated with the condition.

The MTHFR 1298 and 677 AC and CT genotypes combined are more associated with

elevation of Hcy when folate and vitamin B12 concentrations are low. The role of nutrient-gene

interaction and gene-gene interaction in the development of CHD has not been fully determined.

More research and well-defined phenotypic subcategory analyses as well as status assessment of

both folate and vitamin B12 are needed to definitively determine whether the MTHFR 677 C-T

and/or MTHFR 1298 A--C polymorphisms of the mothers are risk factors for the development

of CHD.

MTRR 66 A-G and TC 776 C-G polymorphisms

Both MTRR and TC are essential proteins required for vitamin B12 function and in the

remethylation of Hcy and DNA methylation. Unlike the MTHFR polymorphisms, MTRR and

TC are two separate genes that directly involve vitamin B 12 but not folate. The MTRR enzyme,

officially known as 5-methyltetrahydrofolate homocysteine reductase, is required to activate the

MTR through the reduction of vitamin B12. The later enzyme is important for the conversion of

Hcy to methionine by accepting the methyl group from vitamin B 12 and transferring it to Hcy, a

reaction in which Hcy is remethylated to methionine. The importance of this enzyme in 1-C

metabolism is based on its critical role in keeping Hcy and methionine concentrations in balance,

which will result in normal DNA methylation and nucleotide synthesis.

The MTRR 66 A--G polymorphism is associated with an increased risk of NTD (26,95).

In a case-control study to investigate the influence of the MTRR 66 A--G polymorphism on

CHD risk and the possible interaction between the variant and MMA concentrations, Van









Beynum et al. reported that the MTRR 66 GG genotype in combination with a high MMA

concentration was associated with a 3-fold increase in CHD risk (81). The increase risk was

dependent on the elevation of MMA, which indicates that compromised vitamin B12 status may

possibly be a risk factor for CHD risk.

A C--G substitution at base pair 776 in the gene that encodes transcobalamin (i.e. TC

776 C->G) may affect TC binding affinity for vitamin B12 which will lead to reduced cellular

uptake (35,96,97). Von Castel-Dunwoody found that the presence of the TC 776 CG genotype

negatively affected vitamin B12 metabolism and increased Hcy concentration in 359 non

pregnant young women (38). These researchers found that the TC 776 GG genotype group had a

significantly lower vitamin B 12 concentration and slightly higher Hcy concentration when

compared to CC and CG among CHD cases. However, folate, vitamin B 12, and Hcy

concentrations were not different in the case group with any of the MTRR genotypes. The group

suggested that a larger sample size might provide a more definitive answer to whether these two

polymorphisms are associated with CHD risk.

The limited number of studies, the complexity of gene-gene interactions, and nutrient-

gene interactions limit the ability to conclude whether the MTRR and/or TC polymorphisms

alone or in combination with low vitamin B12 has an effect on CHD risk. Further research

investigating both MTRR 66 and TC 776 with the MTHFR 677 and 1298 polymorphisms and

folate and vitamin B12 status is needed. This will help expand the understanding of the

relationship among folate, vitamin B12 and polymorphisms occurring in genes that codes for key

enzymes in folate and vitamin B12 metabolism and how these factors influence CHD risk.









Environmental and Sociodemographic Risk Factors

Environmental and sociodemographic factors such as maternal cigarette smoking, alcohol

consumption, obesity, febrile illnesses, and use of medications, diabetes, ethnicity, and family

history are important to consider when assessing the risk of CHD. There is relatively little

research on the potential adverse effects of these non-inherited factors on the development of the

fetal hear. However, a growing body of epidemiological studies investigating the critical

involvement of such modifiable factors on the development of the fetal heart and risk for

congenital malformations is emerging. It is estimated that up to 30% of congenital heart defect

cases are attributable to identifiable and potentially modifiable factors (98).

Cigarette Smoking

Cigarette smoking during pregnancy has been documented over the years to be associated

with various negative effects including severe impairment of embryonic development and infant

mortality. A meta-analysis of studies published between 1971 and 1999 found no association

with smoking for all types of heart defects combined and mixed outcomes for the analysis of

specific groups or phenotypes (99). Some recent studies found an association between maternal

smoking and various types of heart defects (100,101). Hobbs et al reported an association

between smoking and CHD risk especially when combined with more elevated concentrations of

Hcy (89). While these studies suggest an association, a study by Kallen et al., as well as a study

by Correa-Villasenor et al. failed to corroborate these results (99,102). The difference in

methods, classification, control of confounding factors, and sample size could be the cause of the

inconclusive results.

Alcohol Consumption

It has been suggested that ethanol may produce fetal tissue edema and affect the turgor of

the primitive cardiac loop (58). There are a large number of studies in which a wide range of









teratogenic effects of alcohol consumption during pregnancy were investigated. In a case-control

study in Spain, the risk of congenital anomalies with different daily intakes of alcohol doses was

investigated and an increase risk of CHD was only found with the highest level of maternal

consumption of alcohol per day (ie, > 92 g/d) (103). In the study by Correa-Villasenor et al,

alcohol consumption in early pregnancy was associated in a dose dependent manner (102).

There is no conclusive evidence that moderate alcohol consumption is associated with increased

risk of CHD; however, the studies provide more evidence associating heavy consumption with

CHD risk (102,103).

Obesity

A number of studies have examined the association between maternal pre-pregnancy

obesity and CHD. In two studies, no statistically significant increase in risk for any heart defect

in relation to maternal obesity was observed (104,105). Walker et al reported a positive

association between maternal obesity, defined as a body mass index (BMI) of> 26 kg/m2, and

defects of the great vessels (106). In a recent study, a 6.5-fold increased risk in aggregate

cardiac defects was detected among black obese mothers (107). Careful assessment of an

obesity effect on CHD risk when conducting observational studies is required to minimize the

confounding complications by other factors such as diabetes.

Febrile Illnesses

There is strong evidence that febrile illnesses are associated with increased risk of

congenital anomalies. Studies suggest that maternal febrile illnesses during the first trimester of

pregnancy is associated with increased risk for certain heart defects (108-110). Mothers

reporting any febrile illnesses during the first trimester had a 2-fold increase risk of having a

child with heart defects (110).









Use of Medications

In a recently published paper by the AHA, research findings related to the use of different

medications and the risk of congenital defects were reviewed (58). There was no conclusive

evidence that the drugs that were investigated were associated with an increased risk of CHD.

Maternal therapeutic drugs such as oral contraceptives, penicillin, ampicillin, and corticosteroids

as well as maternal non-therapeutic caffeine were not associated with any variation of congenital

anomalies. The exception was ibuprofen which was reported to be associated with some specific

variations of congenital defects (98).

Diabetes

Studies have clearly documented a link between maternal pre-gestational diabetes and a

range of congenital defects. This association is less consistent with gestational diabetes (111-

114). The large body of evidence reviewed by the AHA recently suggested that diabetes is a

well known prenatal maternal risk factor for CHD (58). As illustrated by the study ofFerencz et

al., specific types of CHD associated with maternal pre-gestational diabetes include laterality

and looping defects, transposition of the great vessels, hypoplastic left heart syndrome

cardiomyopathy, and nonchromosomal atrioventricular septal defects (115).

Ethnicity

Ethnicity among different groups has been associated with various types of congenital

defects. Increased risk prevalence of specific CHD has been reported among white infants

compared to black infants (116,117). However, in a population-based study of variations in

prevalence of birth defects comparing Hispanic and black mothers to non-Hispanic white

mothers in California between 1987 and 1997, no differences in prevalence were detected

(117,118).









Family History

The evidence for an association between family history and CHD is not strong.

However, family history is critical in studies evaluating the risk of CHD. Family history may be

a risk factor for CHD in association with other confounding factors. Although there is no direct

evidence that CHD risk is associated with family history, careful assessment is required when

evaluating related data in future studies.

Biochemical Markers: Folate and Vitamin B12 Status Indicators

Serum and Red Blood Cell Folate Concentrations

Serum folate concentration is a good indicator of current and recent folate intake. It is a

sensitive measure that reflects the short-term folate status (119). Serum folate concentration

decreases within one to three weeks after lower folate intake is maintained (86,120). Lower

serum folate concentration is detected before any change occurs to RBC folate concentration.

Defining inadequate serum folate status using the microbiological assay is based on the lower

limit of the normal range <13.6 nmol/L (121). The radiobinding assay is also used as an

alternative method to assess folate status, however, this method has been shown to yield lower

blood folate values relative to the microbiological assay (122). The lower limit for the

radiobinding assay is <7 nmol/L.

Red blood cell folate concentration is considered a better long-term indicator of folate

status than serum folate concentration (120). While serum folate concentration reflects the early

and short-term changes in folate status, RBC folate concentration reflects tissue storage. Folate

uptake into the erythrocytes only occurs during early stages of erythropoiesis which takes place

exclusively in the bone marrow. Folate therefore cannot permeate the membrane of mature RBC

during its 120-day life span, thus RBC folate concentration is a good reflection of folate status

four months prior to the time of blood sampling. Inadequate status defined using the lower limit









of normal of RBC folate concentrations using radiobinding assay is <317 nmol/L (120). Using

the microbiological assay, deficient status is defined as a concentration of 362.6 nmol/L. The

lower limit of normal or acceptable is 453.2 nmol/L (123).

Serum Vitamin B12

In the general US population, the mean serum vitamin B12 concentration for healthy

individuals over four years of age is 381 pmol/L (124). In the clinical setting, serum vitamin

B12 is the primary method for assessing vitamin B12 status; however other measures are

necessary to avoid false diagnosis of impaired status (18). This is due to the nature by which

vitamin B12 is metabolized as well as the way it is stored in the body. Vitamin B12 can be

stored in some tissues such as the liver and kidney for longer periods of times, while some other

tissues can be deficient. This can lead to inaccurate estimates as some individuals can have low

normal serum vitamin B12 concentrations even though their body stores are deficient in vitamin

B12. A plasma concentration >221 pmol/L is considered normal, concentrations between 148

and 221 pmol/L are considered marginally deficient or "low normal", and a concentration <148

pmol/L is considered deficient.

Methylmalonic Acid

It is preferable to rely on status indicators that reflect vitamin B12 function. Specifically

an elevation in MMA concentration is highly specific for a vitamin B12 deficiency.

Methylmalonic acid is a four carbon molecule related to valine, isoleucine, and propionic acid

catabolism (17). Vitamin B12 is required for the conversion of methylmalonyl-CoA into

succinyl-CoA, a reaction sensitive to vitamin B12 status in which it prevents elevation of MMA

concentration. When vitamin B12 is low, the conversion is impaired leading to accumulation of

methylmalonyl-CoA, which eventually is converted into MMA. For that reason, MMA is a

better predictor of B12 status than serum B12 (17).









Elevated MMA concentrations can be detected in the early stages of vitamin B 12

deficiency before a decrease in serum vitamin B12 can be measured (17). Van Beynum et al

reported that maternal subjects with the MTRR 66 GG genotype in combination with high

MMA levels above 80th percentile had a 3-fold increased risk for all types of CHD in offspring

(81). Therefore, using MMA in conjunction with serum B12 to fully assess the risk of vitamin

B12 and polymorphisms related to its function in 1-C metabolism is preferable. Normal serum

MMA concentration is < 271 nmol/L, with reported references ranges for serum MMA

concentration of 50 to 400 nmol/L (125,126).

Homocysteine

Homocysteine is a dimer containing a sulfur group. Plasma Hcy concentration is

inversely related to the concentration of plasma folate. This was evident from the implementation

of folic acid fortification of cereal grains in 1998 by the US government with the aim of reducing

the incidence of NTDs in pregnancy (127). This fortification strategy was successful in that it

resulted in an improved folate status and lower plasma Hcy concentration (128). The mean

Hcy concentration was significantly lower in the 1999-2000 post-fortification period compared

to (1994-1998) pre-fortification period. In the 1994-1998 period, Hcy concentration was 9.5

pmol/L while in the 1999-2000 period it was 7.9 imol/L (129).

Plasma Hcy concentration is maintained within narrow ranges through enzymes that

metabolize methionine and Hcy. The cutoff for plasma Hcy concentration is >12 \ mol/L.

Values in this higher range have been associated with increased risks for adverse health effects

(130). Kepusta et al. reported an association between mild maternal hyperhomocysteinemia and

CHD risk, which was confirmed by Hobbs et al. (71,131). Bailey et al reported that vitamin B12

status < 221 pmol/L has a negative effect on Hcy concentration independent of the genotype (93).

Homocysteine evokes oxidative stress through production of reactive oxygen species (ROS),









binds to nitric oxide (NO), or leads to accumulation of its precursor SAH which is a potent

inhibitor of biological transmethylation (132). As discussed earlier, impaired folate and vitamin

B12 status and polymorphisms related to both nutrients and the elevation of Hcy concentration

were common factors for the risk of CHD in several investigations.










OH H 0 H COOH
N CH2-N / --C -N-C-CH2-CH2-COOH
IX H
H2 N K' Folic (Pteroyl-L-Glutamic) Acid


P olyglutamyl Ttrahydrofolates


Substituent (R) Position
-CH3 (methyl) 5
-CHO (fonnyl) 5or10
-CH=NH (form imino) 5
--CH (methulenel 5 and 10


-CH- (methenyl)


Figure 2-1.


5 and 10


Structure of folate/folic acid. [Reprinted with permission from Baiely, L. 2006.
Bailey, L. & Gregory, J. (2006) Folate. In: Present knowledge in nutrition, 9th ed.
(Bowman, B. & Russell, R., eds.), pp. 278-301. ILSI Press, Washington, D.C.]
Folic acid consists of a para-aminobenzoic acid molecule linked on one side by a
methylene bridge to a pteridine ring, and joined by peptide linkage to a glutamic
acid molecule on the other side. Naturally occurring food folates exist in various
chemical forms, containing a side-chain composed of two to ten additional
glutamate residues (n) joined to the first glutamic acid. The pteridine ring of the
folate/folic acid structure can be reduced to form dihydrofolic acid and
tetrahydrofolic acid (THF). Folate coenzymes are formed by substitution of one
carbon units at the N5, N10, or both positions (R) to the polyglutamyl form of
THF.










Cytosol
C2O+THF
cf3t


Mitochondrial Matrix


Cystosolic and mitochondrial pathways of THF metabolism in the body.
[Reprinted with permission from Baiely, L. 2006. Bailey, L. & Gregory, J. (2006)
Folate. In: Present knowledge in nutrition, 9th ed. (Bowman, B. & Russell, R.,
eds.), pp. 278-301. ILSI Press, Washington, D.C.]


Figure 2-2.









0i" N2


H-N




H2 W


Vitamin B 12 structure.


Figure 2-3.










I Nucleotide Biosynthesis


I Methylation Reactions I


/O
10-CHO-THF



Purine Synthesis + THF


upregulated
Cystathi~nine


Transulfuration


I
Cysteine


Glutathione


Figure 2-4.


One-carbon metabolism folate-dependent pathway. [Reprinted with permission
from Baiely, L. 2006. Bailey, L. & Gregory, J. (2006) Folate. In: Present
knowledge in nutrition, 9th ed. (Bowman, B. & Russell, R., eds.), pp. 278-301.
ILSI Press, Washington, D]










Table 2-1. Definitions of Dietary Reference Intake (DRI) recommendations.


DRI recommendation
Estimated Average Requirement (EAR)



Recommended Dietary
Allowance (RDA)




Adequate Intake (AI)





Tolerable Upper Intake
Level (UL)


Definition
A daily nutrient intake value that is estimated to meet
the requirement of half the healthy individuals in a
group.

The average daily dietary intake level that is sufficient
to meet the nutrient requirement of nearly all (97 to
98%) healthy individuals in a particular life stage and
gender group.

Individuals should aim for this intake level.
A recommended daily intake value based on observed
or experimentally determined approximations of
nutrient intake by a group (or groups) of healthy
people that are assumed to be adequate-used when an
RDA cannot be determined.

The highest level of daily nutrient intake that is likely
to pose no risk of adverse health effects to almost all
individuals in the general population. As intake
increases above the UL, the risk for adverse health
effects increase.


Table 2-2. Folate intake recommendations for men and non-pregnant women >19 years.


DRI recommendation
Estimated Average Requirement (EAR)
Recommended Dietary Allowance (RDA)
Adequate Intake (AI)
Tolerable Upper Intake Level (UL)


320 gg DEF/d
400 gg DEF/d
Not Applicable
1,000 pg synthetic folic
acid/d









CHAPTER 3
MATERIALS AND METHODS

Study Design and Methods Overview

This study was designed by clinical investigators at the University of South Florida and

All Children's Hospital in St. Petersburg, Florida as an initial pilot study to provide data for

future grant proposals. The long-term goal of future studies would be to conduct large-scale

well-designed investigations to evaluate the association between folate and vitamin B12 status

and genetic polymorphisms and the etiology of CHDs. Following the initiation of the study, Drs.

Bailey and Kauwell were invited to join the collaborative research team to analyze the folate and

vitamin B12 status indicators, to do the data analysis, interpret the findings, and to provide

advice regarding how the study design and data collection should be revised in future studies.

This section provides a description of the design and methods used in the pilot study and the

discussion section includes a critique and explanation of specific aspects of this pilot study that

need revision for future investigations.

The study protocol was approved by the Institutional Review Board at the University of

South Florida and All Children's Hospital where the study was conducted and the University of

Florida, which received blood samples for analysis. Three hundred ninety (n= 390) non-

pregnant volunteer subjects were enrolled in the pilot study. The subjects recruited were mothers

of children and juvenile patients from the pediatric cardiology practice at All Children Hospital,

St. Petersburg, FL, as well as staff of All Children's Hospital or the University of South Florida

at St. Petersburg, FL. Inclusion criteria (a) "women who had previously delivered an infant"; (b)

"previous pregnancy resulted in any variation of structural CHD-related complications (cases)";

(c) "previous pregnancy with no related structural CHD complications (controls)"; and (d)" not

pregnant at time of data collection." Mothers were primarily recruited and informed of the study









at the time that they brought their children to All Children's Hospital for a clinical appointment.

In addition, staff members were recruited who met the inclusion criteria. After determining

eligibility, subjects were provided information regarding the study and were asked to sign an

informed consent form. After obtaining the informed consent, blood samples were collected for

the determination of polymorphisms in the MTHFR, MTRR and TC genes (MTHFR 677 C-T,

MTHFR 1298 A-C, MTRR 66 A-G, and TC 776 C-G) and concentrations of serum folate,

RBC folate, vitamin B 12, Hcy, MMA, and hematological parameters. The categorization of the

control (n=186) and the case group (n=204) was based on the diagnosis of the presence of CHD

using prenatal and postnatal ultrasonography. Subjects were instructed to complete a self-

administered questionnaire (Appendix A) designed to collect demographical data including

information about their health status, supplement use, alcohol consumption, cigarette use, and

prescription medication use during their index pregnancy.

Sample Collection and Processing

Blood samples were collected from each participant by a phlebotomist in vacutainer tubes

(Vacutainer Blood Collection Set; Becton Dickinson, Vacutainer Systems; Franklin Lakes,

NJ). A total of 30 mL of blood was collected for analysis of the following indices "RBC folate,

plasma vitamin B12, serum MMA, serum Hcy, serum folate, and hematocrit." The vacutainer

tubes were immediately shipped on dry ice to the University of Florida for processing and frozen

storage prior to analysis.

Blood for serum folate samples was collected in 8.3 ml serum separator gel clot activator

tubes (Vacutainer, Becton Dickinson, Rutherford, NJ) and kept at room temperature for 30 to

60 minutes to allow time for clotting. Serum was obtained by centrifuging the tubes at 650 x g

for 15 minutes at 210C (International Equipment Compant; Model HN-S II Centrifuge, Needham









Heights, MA). Supernatant sera were mixed with sodium ascorbate (1 mg/ml), aliquoted into

200 [tl samples, and stored at -30C until analysis.

Whole blood was collected in 7 ml tubes containing K3 ethylenediaminetraacetic acid to

prevent clotting (Vacutainer, Becton Dickinson, Rutherford, NJ). Blood for plasma

homocysteine was kept on ice prior to processing. A small aliquot of whole blood held at room

temperature was diluted 20-fold in 1 mg/ml ascorbic acid and aliquoted into 200 [l samples and

frozen for measurement of RBC folate concentration. The iced blood was centrifuged at 2000 x

g at 4C for 30 minutes. The plasma from these samples was frozen and used to measure the

plasma homocysteine concentration.

Following removal of the plasma, the samples were used to extract DNA to be used to

determine genotype for polymorphisms (MTHFR, TC, and MTRR). Aliquots were frozen at

30C for subsequent analysis of serum and RBC folate concentrations.

Analytical Methods

Measurement of Serum Concentrations of Status Indicators

Serum folate and vitamin B12 concentrations

The serum folate concentrations of all subjects were determined using the MP

Biomedicals, Inc. SimulTRAC-S Radioassay Kit (Orangebury, New York). The radiobinding

assay is conducted by adding dithiothreitol solution to the folate tracer (125I, borate buffer with

human serum albumin, dextran, potassium cyanide, dye and preservative). This mixture is added

to serum folate samples and heated in a water bath at 100C for 15 minutes. Once cooled, the

folate binder is added to the mixture, which is protected from exposure to light, and incubated for

one hour. During incubation, endogenous folate and 125I compete for binding sites to the folate

binder. Samples are centrifuged and bound folates and microbeads precipitated. The bound

folate (labeled and unlabeled) accumulates in a pellet, while unbound folate is in the supernatant,









which is gently discarded. The radioactivity of the pellet is measured using a scintillation

gamma counter. Sample serum folate concentration was calculated using a standard curve on

which the radioactivity was inversely related to serum folate concentration.

Plasma B12 was determined by RIA using a commercially available kit (Quantaphase II,

Bio-Rad). Specifically, samples were incubated with a 57Co labeled B12 tracer in a 100C water

bath to convert all forms of B 12 to cyanocobalamin. Samples were brought to room temperature

after boiling for 20 min, and then mixed with purified porcine IF bound to polymer beads and

incubated for one hour. During incubation labeled and unlabeled B 12 compete for binding to IF

at rates that match their relative concentrations. Finally, samples were centrifuged, and

supernatant containing unbound B 12 was removed. Sample radioactivity was measured by

gamma counter and B12 concentration was calculated using a standard curve on which the

radioactivity was inversely related to B 12 concentration. Folate concentrations were inversely

related to the measured radioactivity. Serum folate and vitamin B12 concentrations >12 nmol/L

and >148 nmol/L, respectively, represented the lower limit of normal values for this study.

Red blood cell folate concentration

The red blood cell folate concentrations of blood specimens were determined using the

Lactobacillus casei microbiological assay in a 96-well microplate system adapted from Tamura

(133) and Home and Patterson (134). The intra- and interassay CV for the microbiological assay

were 8.7 and 7.1%.

Homocysteine and methylmalonic acid

Samples were shipped to the (Metabolite Labortories, Inc. Denver, Colorado) for analysis

to determine serum Hcy and MMA concentrations. Samples were analyzed using gas

chromatography mass spectrometry (135,136).









Genotype Identification

Genotypes of potential subjects were determined using Dynamic Allele Specific

Hybridization (DASH) performed by DynaMetrix. Samples were sent to DynaMetrix Limited,

University of Leicester (United Kingdom, where primers and probes were designed for the

genotype polymorphisms and analyses were performed using the DASHTM method with

DynaScore Software v. 0.7 (http://www.dynametrix-ltd.com).

Briefly, a short PCR product was created spanning the polymorphic position. One PCR

primer was 5'-labeled with biotin for attachment of the amplified targets to streptavidin-coated

96-well microtiter plates. Following denaturation and a wash to remove the unbound strand, an

allele-specific probe was hybridized to the bound target DNA strand at low temperature in the

presence of the double-strand specific intercalating dye Sybr Green. Finally, the temperature

was steadily increased while recording the probe-target duplex melting temperature, as

monitored by diminution of Sybr Green fluorescence with a quantitative PCR analysis device.

Pregnancy Index

Pregnancy index is a term that indicates the elapsed time from the delivery of the affected

infant for the case mothers or for the normal infant for the control mothers, and the time when

the blood was drawn. The pregnancy index was calculated using the date the blood was drawn

(transformed into numerical counts) minus the date of the delivery (transformed into numerical

counts). The product was then divided by 365 which is the number of days in a year to obtain

the number of years. The pregnancy index was then divided into three categories: < 3 years, 3-5

year, and > 5 years.

Selection of sub-sample population: Since the primary focus of the study was to determine

if folate and vitamin B 12 status during pregnancy was associated with risk for CHD, it was

important to focus on women who had delivered their infants in a relatively short time from the









time the blood sample was drawn for nutrient and metabolite concentration determinations. For

this reason, a subgroup of the study participants, those with a pregnancy index of less than three

years were carefully evaluated. Since status indicators were more likely to reflect status during

gestation.

Supplement Analysis

The results from the questionnaire regarding supplement use were modified to fit our

model due to the low response rate. We combined all responses to either (Yes or No) categories

instead of the actual categories (yes, no, infrequently, sometimes, or routinely). The following

responses "yes", "sometimes", or "routinely" were counted as "yes" responses. A" no" or

"infrequently" response was considered a "no" responses. Data on other supplements and herbal

use were not assessed due to the high non-response rate.

Statistical Methods

The statistical analysis was performed using SAS, version 9.1, SAS Institute Inc. Cary,

NC, USA. An initial analysis was conducted to determine basic statistics of the demographic

and background variables and hematological profile: ethnicity, maternal age, pregnancy index,

supplement use, cigarette and alcohol consumption, and hematological indices. One-way

analysis of variance (ANOVA) test was used to determine if the continuous demographic and

background variables means differed among the two groups (case and control). A Chi-square

test was used to determine whether the proportion of responses in each of the categories differed

between categorical variables. Status indicators were log-transformed so that the assumptions of

normality are met, and the reported values were back-log transformed. Linear regression

analysis was performed to check correlation between status indicators. An ANOVA test was

used to compare the means of the transformed status indicators for both folate and vitamin B 12

(Hcy, MMA, vitamin B12, serum folate, and RBC folate) and determine whether the status









indicators differed between cases and controls with regard to CHD risk. A Chi-square test was

used to determine whether there was a relationship between subject status and genotype groups

concerning the MTHFR, MTRR, and TC polymorphisms. Logistic regression was used to

evaluate a complete model of the subject status (case/control) versus the genotype classification

and status indicators. All first order interactions were included in the full model.









CHAPTER 4
RESULTS

Part I: Exploring the Data

Demographic Characteristics of the Study Sample Population

Subjects

Three hundred ninety (n= 390) non-pregnant subjects were enrolled in the pilot study.

Subjects were primarily mothers of children and juvenile patients from the pediatric cardiology

practice at All Children Hospital, St Petersburg, Florida. Subjects were both non-pregnant

mothers of children with any variation of structural heart defects referred to as the case group

(n=204) and non-pregnant mothers of non-CHD affected offspring referred to as controls

(n=186).

Demographic Characteristics

Demographic characteristics of the sample population are presented in Table 4-1. No

difference (p > 0.05) between case and control based on ethnicity, maternal age at birth, or

pregnancy index was detected. Ethnicity was reported as African-American, Hispanic, white-

American, or other, and there was no difference (p > 0.05) in the ethnic distribution between the

case and control groups. When the subjects were categorized into different age groups (<18, 18-

30, 30-45, and >45 years of age), no differences (p > 0.05) between the case and control groups

were detected. Maternal age was not different (p > 0.05) between cases (28.5 0.65 years) and

controls (27.7 0.74 years). Subjects were then categorized into three categories according to

pregnancy index (< 3 years, 3-5, > 5 years). No significant difference (p > 0.05) was detected

between cases and controls based on pregnancy index category.









Supplement Use

Low response rate for the supplement question in the questionnaire was recorded. Based

on the response of one-third of the subjects in either the case or control group, use of either

prenatal and/or folic acid supplements was determined not to differ (p > 0.05) between case and

control groups as presented in (Table 4-2).

Cigarettes and Alcohol Consumption

Limited data were collected about cigarette and alcohol consumption among participants.

Due to the limited data and response rate in the "yes" category (Table 4-3), a statistical analysis

test to evaluate an association between smoking cigarettes and alcohol use and increased CHD

risk was not conducted.

Medical Conditions

Data were collected for a small percentage of subjects related to diabetes, seizures, and

anemia which developed during pregnancy (Table 4-4). No data on other medical conditions

were available. Data were insufficient to conduct a statistically valid comparison.

Hematological Indices

Hematological indices are presented in (Table 4-5). No differences (p > 0.05) in any of

the hematological indices (Hgb, MCV, MCH, MCHC, and Hct) were found between case and

control groups.

Part II: Folate and Vitamin B12 and Status Indicators (Objective 1)

The first hypothesis was that low folate and/or vitamin B12 status are associated with

increased risk for CHD. To test this hypothesis, association between maternal folate and vitamin

B12 status biomarkers (serum and RBC folate, vitamin B12, Hcy, and MMA concentrations) on

infant CHD risk was observed.









Status Indicators and CHD Risk

The association between maternal folate and vitamin B12 status and risk of having a child

with CHD was evaluated. The specific status indicators for folate status that were compared

included serum and RBC folate and Hcy concentrations and the vitamin B12 status indicators,

that included serum vitamin B12, MMA and Hcy concentrations. There were no significant

differences between any of the status indicators in the case versus control group (Table 4-6).

One entry in the MMA concentrations reported for a subject was omitted from the MMA dataset

because it was 10-fold higher than the maximum value for any of the remaining subjects, a value

that is physiologically implausible.

Relationship of Status Indicator Variables in the Sample Population

The means for vitamin B 12, RBC folate, and serum folate concentrations were inversely

correlated with Hcy concentration (p < 0.0001), (p < 0.002) and (p < 0.0001), respectively.

Mean concentrations for MMA and Hcy were positively correlated (p < 0.0001).

Status Indicators in the Sub-Sample Population

Subjects who delivered their babies in less than 3 years from the time the blood sample

was drawn were selected as for this sub-sample. A total of 62 subjects had a pregnancy index of

< 3 years, 35 of whom were cases and 27 were controls (Table 4-1). When folate and vitamin

B12 status indicators were compared between the cases and controls in the sub-sample

population groups (Table 4-7), a relationship (p < 0.05) between serum folate-concentration and

CHD risk was detected. The mean serum vitamin B12 concentration between case and controls

was associated with CHD risk in the case group (p = 0.0016) lower. No differences (p > 0.05)

between case and control groups for the other status indicators (Hcy, MMA, RBC folate

concentration).









Part III: Polymorphisms and CHD Risk (Objective 2)

The second hypothesis is that polymorphisms affecting genes encoding MTHFR, MTRR,

and TC genes (i.e. MTHFR 677C-T and 1298 A-C, MTRR gene 66 A-G and TC 776 C ->

G) will exacerbate the effect of low folate and/or vitamin B12 status on CHD risk. To test this

hypothesis, the association between maternal MTHFR 677 C--T and 1298 A--C, MTRR 66

A--G, and TC 776 C--G polymorphisms on infant CHD risk was associated. The distribution

for all possible genotypes is presented in (Table 4-8).

MTHFR 677 C-T and MTHFR 1298 A-C Polymorphisms and CHD Risk

The relationship between both MTHFR 677 C--T and 1298 A--C polymorphism and the

risk of CHD independent of the other two polymorphisms TC 776 C--G and MTRR 66 A-G

was investigated using the Chi-square test. No association (p > 0.05) was detected between the

MTHFR genotype groups and CHD (Table 4-9). More cases (n=39) have the double

heterozygote (AC-CT) MTHFR polymorphism compared to the control group (n=24). When the

double heterozygous AC-CT MTHFR genotype groups were compared between the case and

control group, there was a trend (P= 0609) for a greater number of case mothers to have the AC-

CT MTHFR genotype compared with all of the remaining genotypes (Table 4-9).

MTHFR 677 C-T Polymorphism

Comparison of the three genotypes (CC, CT, TT) between cases and controls resulted in

no significant association (P=0.1073) (Table 4-10). Although not significant, the somewhat

higher number of cases with the CT and TT genotypes compared to controls suggests a trend.

MTHFR 677 (CT/TT) Genotypes

Based on the higher number of cases observed having the CT and TT genotypes for the

MTHFR gene, the MTHFR 677 CT and TT genotype groups were combined independently of









the MTHFR 1298 A--C polymorphism. This combination resulted in an association (P=

0.0356) between CHD and the MTHFR 677 C-T polymorphism (Table 4-11).

MTHFR 1298 A-C Polymorphism

Similar to the MTHFR 677 C--T comparison between cases and controls based on the

MTHFR 1298 A--C genotypes were carried out. No association (p >0.05) between the MTHFR

1298 genotypes and CHD (independent of the MTHFR 677) was detected (Table 4-12).

MTHFR 677 (CT/TT) Compared to CC Genotype and Status Indicators in the sample
population

Based on the findings from (Table 4-11), the data were further explored by comparing the

status indicators between all subjects with either the CT or TT genotype for the MTHFR 677

C--T polymorphism to subjects with the CC genotype. There was no difference (p > 0.05)

among status indicators between the two groups based on this method of comparison using the

entire sample (Table 4-13).

MTRR 66 and TC 776 Genotypes and CHD Risk

The relationship of both MTRR 66 A-- G polymorphism and the TC 776 C--G

polymorphism and CHD risk was assessed and was not found to differ (p > 0.05). Number of

subjects with the MTRR 66 GG genotype in the case group is lower than the control group (39,

48, respectively). To further characterize this relationship, the AA and AG genotype groups

were compared to the double homozygous of the gene MTRR 66 GG genotype and a trend (p =

0.0917) for an association was detected.

For the TC 776 genotype groups, no association (p>0.05) for any of the genotype groups

and CHD risk was detected. Although not significant, the number of individuals with CG and

GG genotypes tended to be higher in case compared to control groups (104, 92) and (47, 33),









respectively. The TC 776 CG and GG genotype groups were combined and case and control

groups were compared, but no difference was detected (p > 0.05).

Part V: Relationship Between Polymorphisms Related to Folate and Vitamin B12 MTHFR
677 C-T, MTHFR 1298 A-C, MTRR 66 A-G, and TC 776 C-G and Status Indicators
and CHD Risk (Objective 3)

The third hypothesis is that CHD risk associated with either folate and/or vitamin B 12

related polymorphisms will be exacerbated by low folate and/or vitamin B12 status. Therefore,

the third objective was to determine the interaction between maternal folate and vitamin B12

related polymorphisms and folate and vitamin B 12 status indicators and infant CHD risk.

Status indicators and genotype relationship and CHD risk: The relationships among

probable risk for CHD and status indicators for both folate and vitamin B12 and polymorphisms

affecting the MTHFR, MTRR, and TC genes were assessed. The first model used was a

stepwise procedure to evaluate all genotype groups and their first-order interactions. Only the

MTHFR CT/TT combinations were associated with an increased risk of CHD (p < 0.05). No

other polymorphism interactions were identified in the model. In the second model, a stepwise

procedure was used to evaluate all status indicators and their first order interactions. At the (a =

0.05) level of significance, no significant associations between all status indicators and the risk

of CHD were detected. In the third model, both genotypes and status indicators were included.

A step wise procedure was performed, and no association (p > 0.05) was ascertained for all status

indicators and all genotypes which were removed from the model except for the MTHFR CT/TT

genotypes group which was significant (p < 0.05).









Table 4-1. Demographic characteristics of sample population.


a One-way ANOVA was used for statistical comparisons between groups.
b Chi-square test was used for statistical comparisons between groups.
SMean standard deviation (SD). NA: not applicable


Table 4-2. Distribution and response rates for prenatal use of folic acid in the sample population.
Supplement Case Control Total (n) P-valuea
Prenatal 0.5579
Yes 117 98 215
No 8 9 17
NAb 104
Folic Acid 0.2410
Yes 26 16 42
No 114 105 219
NAb 129
aChi-square test was used for statistical comparisons between groups. Data were missing (NA,
not available).


Demographic Case Control Total Missing P-value
Variable (n) (n) (n)
Ethnicity (%)b 0.2177
African American 10 9 19 NA
Hispanic 17 21 38 NA
White American 115 88 203 NA
Other 5 10 15 NA
Total (n) 147 128 275 115

Maternal Agea"c 28.5 + 0.65 27.7 + 0.74 0.4166

Maternal Age Groupb 0.4224
Less than 18 3 4 7 NA
18-30 61 45 106 NA
20-45 41 32 73 NA
45 > 0 1 1 NA
Total (n) 105 82 187 203

Pregnancy Index (years)b 0.2852
<3 35 27 62 NA
3-5 13 6 19 NA
>5 150 150 300 NA
Total (n) 198 183 381 9









Table 4-3. Distribution and response rates for cigarette and alcohol use in the sample population.
Factor Case Control Total (n)
Cigarettes
Yes 2 1 3
No 23 27 50
NAa 337
Alcohol


Yes 1
No 24
NAa
aData were missing (NA, not available).


1
51
338


Table 4-4. Distribution and response rates for diabetes, seizures, and anemia during pregnancy
in the sample population.
Condition Yesa Nob Missing' Total
(n)d


Diabetes
Seizures


336
337


390
390
390


Anemia 12 41 337
aNumber of subjects who answered with yes for the condition.
bNumber of subjects who answered with no for the condition.
cNumber of subjects who did not specify an answer for the condition.
dTotal number of subjects enrolled in the study.


Table 4-5. Mean comparison of hematological indices between cases and controls in the sample
population.a'b
Index Case Control Total (n) P-value
Hgb (g/dl) 13.3 (13.1, 13.4) 13.3 (13.2, 13.5) 380 0.5643

MCV (fl) 87.9 (87.1, 88.7) 88.4 (87.6, 89.1) 380 0.4311

MCH (pg) 29.2 (28.9, 29.5) 29.4 (29.1, 29.7) 380 0.3674

MCHC (g/dl) 33.2 (33.0, 33.2) 33.3 (33.1, 33.4) 380 0.5003

Hct (%) 39.9 (39.5, 40.3) 40.7 (39.7, 40.5) 388 0.6325
aMean (5%, 95% CI). bOne-way ANOVA was used for statistical comparisons between groups.









Table 4-6. Comparison of serum and RBC folate, Hcy, serum B12, and MMA concentrations by
group (case/control) in the sample population.a


Variable


Case


Serum folate (nmol/L) 21.1 (19.3, 22.9)


RBC folate (nmol/L)


Hcy (iM)


1447 (1328.2, 1565.8)


6.6 (5.9, 7.3)


Serum B12 (pmol/L)


Control


21.1 (19.2, 23.0)

1447.3 (1324.5,
1570.1)


7.3 (6.5, 8.1)


367.7 (335.95, 399.45) 395.5 (362.3, 428.7) 390


MMA (nM) 230.6 (213.5, 247.7) 222.5 (204.3, 240.7) 367 0.5255
aMean (5%, 95% CI). One-way ANOVA was used for statistical comparisons between groups.
bp-values were based on normalized (log transformed) data. The results have been back-
transformed to original scale.


Table 4-7. Comparison of serum and RBC folate, Hcy, serum B12, and MMA concentrations by
group (case/control) in the sub-sample population b, c
Variable Case Control P-value
(n = 35) (n =27)


Serum folate (nmol/L) 17.7 (14.8, 21.1) 24.2 (19.8, 29.8) 0.0258d

RBC folate (nmol/L) 1254.6 (1044.1, 1507.6) 1418.3 (1123.0, 1791.1) 0.4246

Hcy (FM) 5.9 (5.4, 6.5) 5.4 (4.8, 6.0) 0.2060

Vitamin B12 (pmol/L) 307.3 (253.1, 372.9) 503.5 (403.8, 627.8) 0.0016d

MMA (nM) 208.8 (185.2, 235.5) 189.8 (165.1, 218.1) 0.3113
aSub-sample population is a category for all subjects who had pregnancy index of < 3 years.
bMean (5%, 95% CI). One-way ANOVA was used for statistical comparisons between groups.
cP-values were based on normalized (log transformed) data. The results have been back-
transformed to original scale. dSignificantly lower than controls.


Total
(n)
388


P-value

0.9384

0.8233


0.2555

0.2366









Table 4-8. Genotypes combination distribution in the sample population for specific
polymorphisms.a


TC 776/MTRR 66
CC/AA
CC/AG
CC/GG
CG/AA
CG/AG
CG/GG
GG/AA
GG/AG
GG/GG


CC/AA
8
7


None


CT/AA
10
10


MTHFR (677/1298)
TT/AA CT/AC
6 3


CC/AC
7


CC/CC
5


"Numbers indicate the occurrence of each genotype combination in the sample population
according to their genetic makeup in the genes selected in this study (MTHFR, MTRR and TC).

Table 4-9. Frequency comparison of the MTHFR 677 C--T and 1298 A--C distribution in the


sample population by group type (case/control).
MTHFR (677/1298) Case Control
(n = 204) (n = 186)
CC-AA 25 33


Total
(n = 387)
58


P-valuea

0.3223


CT-AA 56 49 105
TT-AA 28 20 48
CC-AC 42 46 88
CT-AC 39 24 63 0.0609b
CC-CC 14 14 28
aChi-square test was used for statistical comparisons between genotype groups.
bChi-square test was used to compare CT-AC genotypes between case and control.

Table 4-10. Frequency comparison of the MTHFR 677 genotype (CC, CT, TT) distribution by
group type (case/control) in the sample population.
MTHFR 677 Case Control Total P-valuea
(n = 202) (n = 185) (n = 387)


0.1073


168
48


CC 79 92
CT 95 78
TT 28 20
aChi-square test was used for statistical comparisons between groups.









Table 4-11. Frequency comparison between MTHFR 677 CC genotype and the combined
genotypes (CT/TT) for the MTHFR 677 C--T polymorphism by group type
(case/control) in the sample population.
MTHFR 677 Case Control Total P-valuea
(n = 202) (n = 185) (n = 390)


CC 79 92 171 0.0356
CT/TTb 123 93 216
Missing -- 3
aChi-square test was used for statistical comparisons between groups. Significantly higher
frequency of CT/TT in cases compared to controls. bSubjects with either one of these genotypes
were combined in one category.


Table 4-12. Frequency comparison of the MTHFR 1298 genotype (AA, AC, CC) distribution by
group type (case/control) in the sample population.
MTHFR 1298 Case Control Total P-valuea
(n = 204) (n = 186) (n = 390)


AA 109 101 210
AC 81 70 151
CC 14 15 29
aChi-square test was used for multiple statistical comparisons between groups.


0.8562


Table 4-13. Comparison of serum and RBC folate, Hcy, serum B12, and MMA concentrations
between the MTHFR 677 CC genotype and the combined (CT/TT) genotypes in the
sample population.a b
MTHFR 677 Variable Total (n) P-value


Hcy (iM)
7.2 (6.4, 8.0)
6.7 (6.0, 7.4)


MMA (nM)
232.7 (214.1, 251.3)
222.4 (205.5, 239.3)

Serum B12 (pmol/L)
364.0 (329.3, 379.1)
394.0 (363.2, 424.8)

Serum Folate (nmol/L)
20.7 (18.7, 22.7)
21.1 (19.3, 22.9)


365 0.4781


362



386



384


0.4230



0.2113



0.6889


RBC Folate (nmol/L) 211 0.8526
CC 1466.6 (1347.0, 1585.6)
CT/TT 1450.3 (1326.6, 1574.0)
aMean (5%, 95% CI). One-way ANOVA was used for statistical comparisons between groups.
bSubjects carrying either one of these two genotypes (CT/TT) were grouped together.


CC
CT/TT


CC
CT/TT


CC
CT/TT


CC
CT/TT









CHAPTER 5
DISCUSSION

The primary goal of this retrospective observational pilot study was to assess the roles of

folate and vitamin B 12 status and associated genetic polymorphisms on CHD risk and to provide

a framework for future studies. The first objective of the retrospective pilot study was to assess

the association between maternal folate and vitamin B12 status biomarkers on risk for having an

infant with a CHD. An association between means of folate and vitamin B12 status indicators

and CHD risk was not detected in this pilot study. A key issue to consider in evaluating this

finding is whether the status at the time of the blood sampling for this study was similar to that

when the fetal heart was developing. Since the average period of time that had elapsed from the

time of delivery for both cases and controls was 11.8 years, it is likely that there may have been

significant changes in folate and/or vitamin B 12 status during this period of time due to changes

in the diet or changes in the use of supplements. To address this issue, a small subset of women

whose pregnancies occurred within three years of the time of the blood draw was evaluated. The

case women in this subgroup were found to have significantly lower serum folate and B12

concentrations compared to that of control women. The sub-sample population used in this

study, subjects with pregnancy index < 3 years proved more relevant to the study objectives

related to the association of status indicators and CHD risk.

A more definitive answer related to this issue would be provided from a prospective

study in which status indices were determined during the pregnancy in both case and controls. In

a retrospective study conducted by Hobbs et al., folate and vitamin B12 status were evaluated

using a shorter pregnancy index range. The median time between the end of pregnancy and the

blood draw in the study was 24 months for controls and 14.9 months for cases, thus providing a

narrower window of time for changes in folate and vitamin B12 status to occur (89,137).









Although, these investigators did not detect differences in folate and vitamin B12 status between

case and control mothers, there were significant differences in related biomarkers including

Hcy, SAH, methionine and SAM, which suggests that the metabolism of these two nutrients may

be abnormal in case mothers (137). However, in the study by Hobbs et al., case subjects had a

pregnancy index median of 14.9 months while control subjects had a pregnancy index median of

24 months which was addressed as a limitation in a study by Verkleij-Hagoort et al. (73). In the

study by Verkleij-Hagoort et al., a range of -17 months was used as a pregnancy index for both

case and control subjects. Both studies, however, suggest a shorter pregnancy index to be more

relevant to measure status indicators (73).

When evaluating the association between folic acid and vitamin B12 status and CHD

risk, it is important to link potential differences in the use of supplements during gestation to

birth outcomes so that conclusions can be drawn for public health recommendations. In a

previous study conducted by Shaw et al., women who took multivitamins containing folic acid

during critical periods of heart development were found to have a reduced risk of CHD (57).

The strongest evidence that folate status is involved in CHD formation and prevention comes

from a Hungarian RCT in which multivitamins containing folic acid reduced the risk of CHD up

to 60% when taken during the periconceptional period (72). These results were confirmed in two

population-based case-control studies in the US in which a 24% reduction in CHD risk was

observed (55,57). In addition to the reduction in CHD risk associated with folic acid supplement

use seen the these two studies, investigators from the Baltimore Washington Infant Study

reported an inverse relationship between daily maternal intake of folic acid and the incidence of

cardiac outflow tract defects in offspring (56).









In the present pilot study, information obtained from subjects regarding prenatal

supplement use was limited in that information was missing from approximately one-third of the

women which limited the power to detect a reduction in risk associated with folic acid and

vitamin use during gestation. To address this issue in future investigations, detailed information

regarding supplement use including specific brand name to obtain the exact nutrient composition

and timing of the use of the supplement during pregnancy should be obtained. The ideal study

design for an observational, non-intervention protocol is a prospective study in which

information regarding supplement use prior to and during pregnancy is obtained and the outcome

of pregnancy subsequently monitored. Another important source of folic acid for which

information should be obtained in future studies are foods that are fortified with folic acid

including ready-to-eat breakfast cereals that may provide daily folic acid doses that are

comparable to supplements. No information was obtained regarding consumption of folic acid

fortified products in the current pilot study and this limitation of the study design is shared by

other previously conducted studies (138-141). Future investigations should incorporate the use

of a detailed dietary intake questionnaire to allow for a quantitative estimate of daily folic acid

intake during the index pregnancy.

The second objective of this pilot study was to assess the association between the

MTHFR 677 C-T, 1298 A-C, MTRR 66 A-G and TC 776 C-G polymorphisms on

maternal CHD risk. Detailed statistical analyses involving multiple comparisons were conducted

to determine if there were any associations between specific genotypes associated with these

polymorphisms and CHD risk. Initially, MTHFR 677 C--T genotypes proportions were

compared to one another between cases and controls (CC, CT, and TT). There was a pattern for

the T allele at the MTHFR 677 position in the case group but was not significant which warrants









further exploring. When the MTHFR 677 (CT/TT) genotype groups were combined in one

group, a significantly higher number of cases with either of these genotypes were found

compared to controls. Findings that are consistent with this observational study include those of

Wenstrom et al. who reported that CHD risk was associated with the combined MTHFR 677

(CT/TT) genotypes compared to controls (79). Van Beynum et al. found that the MTHFR 677

(CT/TT) genotypes of the mother, when combined with no use of periconceptional folic acid

supplements, increased the risk for CHDs in offspring (142).

Hobbs et al. reported that there was a higher risk for CHDs associated with the MTHFR

677 CC compared to the TT genotype, which is in contrast to the findings in the present pilot

study and those of other investigators (89). A possible explanation for this inconsistency may be

due to differences in folate status of the groups since a higher status is known to alleviate the

metabolic abnormalities such as elevations in plasma homocysteine associated with this

polymorphism (84). The results from Hobbs et al. have not been confirmed by other

investigators and may be related to other risk factors that may not have been controlled for in

that study. An elevation in plasma Hcy concentration is an established risk factor for CHD and

may be associated with the MTHFR 677C --T polymorphism in women who delivered infants

affected by CHD (79).

The findings in the present pilot study are consistent with the study by Junker et al. in

which mothers carrying the MTHFR 677 TT genotype were found to be at significantly

increased risk for the development of structural congenital heart malformations compared to

mothers with the CC genotype (87). However, a meta-analysis by Verkleij-Hagoort et al. did not

confirm that MTHFR 677 C--T polymorphism is independently associated with CHD risk (143).

In addition, negative results were reported by other investigators concerning the association









between MTHFR 677 C--T polymorphism and CHD risk (71,88). The inconsistency of these

findings regarding the MTHFR 677 C--T polymorphism might be due to the heterogeneity

regarding population background, sample size, study design and the type of heart defects that

complicates the pooling and comparison of the studies.

Only a few studies have investigated the combined effect of the MTHFR (677 C--T and

1298 A-C) polymorphism on CHD risk. The results of this study indicate that the MTHFR

1298 A--C polymorphism was not associated with an increased risk of CHD unless it was

combined with the MTHFR 677 CT genotype group. Association between CHD and MTHFR

1298 AC was only observed when the MTHFR 1298 AC genotype group was combined with the

MTHFR 677 CT genotype group. The frequency of MTHFR 1298 AC genotype was only higher

in cases when it was combined with the MTHFR 677 CT genotype. The meta-analysis by

Verkleij-Hagoort et al. concluded that the MTHFR polymorphisms 677 C-T and 1298 A-C in

mothers are not independently associated with CHDs and further studies are needed (143).

To address the third objective, which was to determine the interaction between folate and

vitamin B 12 related polymorphisms and status indicators on maternal CHD risk, comparisons

between all genotype groups for the polymorphisms and status indicators were compared and no

differences were detected. Other investigations that have evaluated these associations include

Van Beynum et al. who reported that the maternal MTRR 66 GG genotype in combination with

high MMA concentration (above the 80th percentile) was associated with a 3-fold increased risk

for all types of CHD in the offspring (81). Hobbs et al. did not find any of the biochemical

indicators to be associated with increased CHD risk in conjunction with MTHFR C-T

polymorphism except for Hcy, which was defined as an independent risk factor (89). Their

results indicated that maternal MTHFR 677 C--T polymorphism did not have an independent









impact on the estimated risk of having a CHD-affected pregnancy. Their data also indicated that

CHD risk in the group with the combined CT and TT genotypes increased only with elevated

Hcy and smoking and that the CC genotype had a greater increase in risk compared to the CT

and TT genotypes.

Vaughn et al. reported that MTRR 66 AA, AG and GG genotypes are associated with

increased plasma Hcy concentration when combined with the MTHFR 677 TT genotype

compared to other combinations of the MTHFR 677 C->T and 1298 A->G polymorphisms (93).

Since the MTRR 66 GG genotype has previously been associated with elevated Hcy and

elevation in Hcy has been shown to be associated with CHD in several studies, there is a

plausible link between the presence of this polymorphism and CHD risk although an association

was not observed in the present pilot study (93,144). Further analysis of the relationship

between this polymorphism and the risk of CHD in this pilot study suggested a slightly lower

incidence of MTRR 66 GG genotype in the cases (n=39) compared to the controls (n=45). When

the genotype frequency distribution was evaluated, more individuals with the MTRR 66 AA and

AG genotypes were observed in cases group and more controls with the GG genotype (p= 0.09)

unlike what was observed and reported by Van Beynum et al. (81).

These findings suggest that MTRR 66 GG genotype may have a protective effect

reducing CHD risk. A potential explanation for this observation is that the MTRR 66 GG

genotype may impair the utilization of the methyl donor from the 5-MTHF resulting in less

folate entering this irreversible pathway for making 5-MTHF, which would shunt this coenzyme

toward the nucleotide and DNA synthesis part of the cycle. It is possible however, that these

results are coincidental and that more controls by chance have the MTRR 66 GG genotype.









It is also important to consider that due to study design weaknesses it is not possible to

thoroughly assess the interaction between genotype and folate/vitamin B 12 status. Further

studies need to be conducted to provide a careful assessment of status at the appropriate time

during gestation or within a very short time following the index pregnancy to allow a more

accurate measure of status indicators that are likely to interact with polymorphisms affecting

folate and vitamin B 12 status.

The primary purpose of this pilot study was to establish an infrastructure for future well-

designed studies to determine the association between maternal folate and vitamin B12 status

and related environmental and genetic factors on CHD risk. It is critical to identify the study

limitations so that improvements can be made in the design of future large-scale studies to

evaluate similar objectives.

One study limitation relates to pregnancy index, which is an important consideration

when considering biochemical profile because status indicators can change over time in response

to dietary and supplement intake. This retrospective study was designed to include women who

had previous pregnancies that could be characterized as either case or control. No

comprehensive exclusion criterions were used to select the subjects. As a result, subjects were

recruited regardless of the period of time between the index pregnancy and the time the blood

sample was drawn for analysis of folate, vitamin B12 and homocysteine concentrations. To

determine the effect of folate and/or vitamin B 12 status on risk of CHD during the index

pregnancy, maternal blood samples must be collected in a timely manner following delivery of

the affected infant (case) or normal infant (control) since changes in blood indices may change

significantly following delivery of the infant due to factors including alterations in diet,

supplement use, and drug use. For the majority of study participants, the index pregnancy









occurred more than five years prior to the time that the blood sample was drawn. Previous

studies designed to assess the association between status indicators and CHD risk used

pregnancy indexes of -17 and 24 months from the time of delivery to the time of blood

collection and ascertainment of information related to supplement use and environmental

exposure (73,137).

Another limitation of not excluding subjects whose index pregnancies were > 3 years is

the inability for participants to recall significant details associated with the pregnancy. In this

study, the index pregnancy occurred > 5 years (average 11.8 years) from the time that the blood

sample was drawn and questionnaire information was obtained. Since the data collected by

questionnaire were highly dependent on memory recall, the long period of time between the

index pregnancy and time of study introduced probable error and bias in the subjects' responses.

For example, some of the questions included in the questionnaire requested at least one of the

following: supplement brand, frequency of use, and dosages, questions that require very exact

recall, which becomes increasingly difficult over time. For this reason the data obtained in this

study may have been biased, inaccurate, and incomplete due to issues related to poor recall

Selection of the type of questions to ask is a critical part of any study. The questionnaire

used in the study (Appendix A) was not developed to address the objectives of the study. The

primary problems associated with the questionnaire include the fact that the questions were

insufficient to obtain essential information required to address the study objectives and the fact

that the data collection was incomplete. For example, questions related to medical conditions

prior to pregnancy, drugs use, and supplement use had low response rates. In addition, some

questions were inaccurately answered (e.g. a question was answered with a "yes" while the

response requested was the supplement dose). The fact that the questionnaire was self-









conducted could have led to the low and inaccurate response rate. To obtain detailed and more

accurate and detailed information, the answers to study questions should be obtained by trained

personnel in an interview situation. Reviewing medical files is another way to ensure

completeness and accuracy of answers, which is critical to obtaining valid results.

The collection of demographic and background information was a weakness of this study

as the data collected for each subject were very limited. For example, no data were collected on

marital status, educational level, employment status, weight and height (BMI), family and/or

offspring history of congenital malformations, and number of prior pregnancies. The lack of

these data limits the ability to describe the sample population and to either control for or

determine the association of these factors with risk for CHD. Even though the questionnaire

included questions about employment and father's ethnicity, data were not collected. Reviewing

medical records and having trained personal ask the questions could have improved the response

rate.

The data on supplement use both during the index pregnancy and at the time of blood

collection were poorly asked and collected. Questions on supplement use are very critical in this

type of study since supplements may have had a significant effect on folate and vitamin B12

status indicators. Large amounts of data regarding the prenatal and folic acid intake from

supplements were missing. One factor that may have reduced the amount of data obtained

related to prenatal supplement use was the length of time that had elapsed from the index

pregnancy to the time of the study reducing the ability of the subjects to recall this information.

The supplement questions requested information related to specific brands, the exact time of use

during pregnancy, whether the subject switched the type of supplement used or not, and the

name of the later brand. In addition, the categories provided as an answer to specify the









frequency of supplement use were very confusing (yes, no, infrequently, sometimes and

routinely). This could be improved by limiting the number of categorical answers to yes, no, and

sometimes and/or providing numerical values for each category for example: routinely can be

described as (2-3 times a week).

Another weakness of this study was the fact that no data were obtained related to

consumption of folate or vitamin B 12 and folic acid or B 12-fortified foods since dietary intake

may have significantly impacted status. A primary objective of this study was to determine

whether there was a relationship between low concentrations of blood folate and/or vitamin B 12

and an increased risk for CHD, which makes it important to assess the intake of these nutrients

from both supplements and dietary sources. Medical conditions are critical when assessing

biochemical status and risk for congenital malformations since medical conditions and/or

medications used to treat their conditions associations might explain and/or indicate unusual

concentrations ofbiomarkers in the blood. However, in this study no data were collected on

medical conditions prior to pregnancy, and only limited data were available during pregnancy

Cigarette and alcohol consumption are risk factors for various malformations (145),

however, there were very low response rates regarding the use of substances. This is a limitation

in the study because assessing risk of CHD requires comprehensive analysis of all possible

elements, especially the ones with known risk associations.

Summary of Pilot Study Design Limitation

This pilot study presented an opportunity to identify key study design issues that can be

addressed in future studies. Limitations included (1)" restricted exclusion criteria"; (2)" large

pregnancy index range"; (3)" low response rate"; (4)" large number of questions that depended

heavily on memory recall"; (5)" critical responses were missing"; (6)" self-administrated test";

(7)" inconclusive questionnaire"; and (8)" small sample size especially after adjusting for









pregnancy index." Improvement for future studies should include an exclusion criterion for

pregnancy index that limits the number of years between delivery and sample collection. A more

detailed and comprehensive questionnaire that would include dietary intake, and drug and

supplement use related questions should be administered to participants, which would minimize

the number of unknown variables that may affect the outcomes of the study, thus enabling

researchers to draw better conclusions related to the effects of both folate and vitamin B12 status

on CHD risk. Trained personnel to supervise the administration of the questionnaire and

collection of data would minimize errors and increase response rate in future studies. Last but

not least, sample size must be considered especially in observational studies due to their nature

and design complexity. In this study, the initial sample size was similar to other studies, but after

correcting for pregnancy index, it became very small (73,138).

Public Health Significance of Research

The primary goal of this retrospective observational pilot study was to provide a

framework for future studies in the area of folate and vitamin B 12 and related genetic

polymorphisms and CHD risk. Congenital heart defect-related studies that involve modifiable

factors are critical to further minimize the limits surrounding the intervention efforts to reduce

the occurrence of CHD. This initial pilot study provides data for future studies and preliminary

findings to support future grant requests for funding large scale well-designed studies to

investigate the association between folate and vitamin B 12 in the etiology of CHDs. Future

studies should correct for the limitations inherent in this pilot study to allow for more definitive

results and conclusions to be drawn. Future studies are needed to advance our understanding of

CHD etiology and the risk factors associated with it. Effective intervention studies designed to

reduce the risk of CHD will depend on the outcome of future well-designed studies.









APPENDIX
QUESTIONNAIRE

Folic acid study: mother's questionnaire

(All answers will be kept strictly confidential)

Name:

Date of Birth:

Height: Weight:

Child's name: Date of Birth:

Reason child is seen by pediatric cardiologist:



Child's birth weight: Full Term or Preterm

How many weeks:

Gestational age when born:

Any malformations or syndromes for this child? Y N if so, what?



Your ethnicity: African-American Asian Hispanic Native-American White

Other:

Father's occupation:

Please answer the following questions about your pregnancy with this child:

Any significant health problems prior to your pregnancy? Y N if so, what?



Did you take supplements prior to your pregnancy? Y N

Which ones?

How long did you take them prior to your pregnancy?









Did you take a prenatal vitamin? Y N Infrequently Sometimes Routinely

How far along into the pregnancy were you when you started taking the prenatal vitamins?



Brand name:

Did you take the same ones throughout the whole pregnancy? Y N

Did you switch to a different pre-natal vitamin during the pregnancy and which one:



Did you take additional folic acid? Y N Infrequently Sometimes Routinely

What was the dose?

Did you take other nutritional supplement or herbs? Y N

Which ones?

Did you develop diabetes during the pregnancy? Y N Was insulin used? Y N

Any seizures before or during your pregnancy? Y N if so, what were you treated with:

Did you develop anemia during your pregnancy? Y N if so, what were you treated with:

Please check if you took any of the following medications during your pregnancy:

Aspirin

Ibuprofen (Advil, Motrin)

Anti-depressants:

Anti-seizure medication:

Diabetes medicine:

Other:

How many cigarettes did you average per day during pregnancy? packs/day

How much alcohol did you drink during pregnancy?









How much coffee/tea/chocolate/caffeinated soft drinks did you drink during your

pregnancy?

Did you drink or eat products containing Aspartame (Nutrasweet, no sugar added or diet

on the label)? Y N Infrequently Sometimes Routinely

Did you use any other artificial sweeteners? Y N Type:

Were you on a special diet during your pregnancy? Y N Type:

Family History:

Any family history of a heart defect from birth? Y N if so, what?



How many pregnancies have you had?

Number of miscarriages?

How many other children do you have? Ages:

Congenital heart defects in your other children?

Any other malformations or syndromes for your other children? Y N if so, what?



Any health problems for your other children?

Are you planning to have more children? Y N Undecided

May we contact you in the future? Y N

Do you want to be contacted with your lab results? Y N

Phone no:

Thank you for your assistance!










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BIOGRAPHICAL SKETCH

Younis was born in Kuwait. After completing high school in Kuwait, he went to the US to

earn his Bachelor of Science in food science and human nutrition from California State

University Fresno in 2005. After graduating in 2005, Younis went back to Kuwait and worked

for the Kuwait health system. Younis worked as a weight and disease management consultant.

He was also granted a part time job at Kuwait University to work as a teaching assistant in the

Nutrition Department before he was awarded a scholarship to pursue graduate studies. In 2006,

Younis was accepted into the University of Florida's nutritional sciences' Master of Science

degree program. Younis plans to pursue his academic career and earn his PhD in nutritional

sciences to later become a faculty member in the Nutrition Department at Kuwait University.





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1 EFFECT OF FOLATE AND VITAMIN B 12 STATUS AND RELATED GENETIC POLYMORPHISMS ON CONGENITAL HEAR T DEFECT RISK: A PILOT STUDY By YOUNIS ALI SALMEAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Younis Ali Salmean

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3 To my wife and parents

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4 ACKNOWLEDGMENTS I would like first to thank god for the gracious and wonderful opportunity that has been giving to m e and the wonderful people that I have met through this academic journey. I would like to sincerely thank my supervisory comm ittee members Lynn B. Bailey, PhD, Gail P.A. Kauwell, PhD, RD, LDN, and Linda Young, PhD for the unique contributions that each of them made to my research project. In particular, I would like to thank my committee chair, Dr. Lynn Bailey for guiding and encouraging me through th is challenging experi ence. Dr. Lynn Bailey was a great console at times of stress, and she was always there to guide me through this stage of my academic career. The past couple of years were very important for my intellectual development and maturity and therefore I am gr ateful for everyone who has contributed to my success. I would like to especial ly thank James Huhta, MD who is acknowledged as the originator of the study design for the pilot proj ect, the director of the resear ch conducted at the University of South Florida, All Childrens Hospital in St. Petersburg FL. Through the efforts of Dr. Huhta funding was obtained for suppor t of this project. I would like to thank my parents and fam ily for they always stood behind me and believed in me. They were truly a great support especially, to bare the hardships of being thousands of miles away for years. I especially thank my mother for her constant encouragement and prayers. My dear wife was an amazing person w ho stood behind me all the time. She was a source of support, comfort and encouragement. I always f ound her there when I needed someone the most, and her love and care were th e light that guided me in my pathway to complete and succeed in this program.

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5 TABLE OF CONTENTS Page ACKNOWLEDGMENTS ............................................................................................................... 4 LIST OF TABLES ...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 LIST OF ABBREVIATIONS ........................................................................................................ 10 ABSTRACT ...................................................................................................................... .............13 CHAP TER 1 INTRODUCTION .................................................................................................................. 15 Hypotheses .................................................................................................................... ..17 Overall Goal ....................................................................................................................17 Specific Objectives ..........................................................................................................17 2 LITERATURE REVIEW .......................................................................................................18 Folate and Vitamin B12 ........................................................................................................ ..18 Folate ...............................................................................................................................18 Chemistry .................................................................................................................18 Bioavailability ..........................................................................................................18 Metabolism (Absorption, transpor t, storage and excretion) .....................................19 Vitamin B12 ................................................................................................................... .20 Chemistry .................................................................................................................20 Bioavailability ..........................................................................................................20 Metabolism (Absorption, transpor t, storage and excretion) .....................................21 Biochemical Functions of Folate and Vitam in B12 ............................................................... 22 Polymorphisms ................................................................................................................. ......24 Dietary Sources of Folate and Vitamin B12 Intake Recommendations .................................26 Congenital Heart Defects ...................................................................................................... ..27 Prevalence and Etiology ..................................................................................................27 Effect of Low Folate Status on CHD Risk ...................................................................... 28 Effect of Low Vitamin B12 Status on CHD Risk ........................................................... 30 Polymorphisms and CHD Risk ....................................................................................... 31 MTHFR (677 CT and 1298 A C) polym orphisms ............................................ 31 MTRR 66 A G and TC 776 CG polym orphisms .............................................. 33 Environmental and Sociodem ographic Risk Factors ..............................................................35 Cigarette Smoking ........................................................................................................... 35 Alcohol Consumption ...................................................................................................... 35 Obesity ....................................................................................................................... ......36 Febrile Illnesses ...............................................................................................................36

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6 Use of Medications ..........................................................................................................37 Diabetes ...................................................................................................................... .....37 Ethnicity ..................................................................................................................... .....37 Family History .................................................................................................................38 Biochemical Markers: Folate and Vitam in B12 Status Indicators ......................................... 38 Serum and Red Blood Cell Folate Concentrations ..........................................................38 Serum Vitamin B12 ......................................................................................................... 39 Methylmalonic Acid ........................................................................................................ 39 Homocysteine .................................................................................................................. 40 3 MATERIALS AND METHODS ...........................................................................................47 Study Design and Methods Overview ....................................................................................47 Sample Collection and Processing .......................................................................................... 48 Analytical Methods ............................................................................................................ .....49 Measurement of Serum Concentrat ions of Status Indicators ..........................................49 Serum folate and vitamin B12 concentrations ......................................................... 49 Red blood cell folate concentration ..........................................................................50 Homocysteine and methylmalonic acid ................................................................... 50 Genotype Identification ...................................................................................................51 Pregnancy Index ..............................................................................................................51 Supplement Analysis ....................................................................................................... 52 Statistical Methods ..........................................................................................................52 4 RESULTS ....................................................................................................................... ........54 Part I: Exploring the Data .......................................................................................................54 Demographic Characteristics of the Study Sam ple Population ............................... 54 Subjects .................................................................................................................... 54 Demographic Characteristics ...................................................................................54 Supplement Use ........................................................................................................ 55 Cigarettes and Alcohol Consumption ...................................................................... 55 Medical Conditions .................................................................................................. 55 Hematological Indices ..............................................................................................55 Part II: Folate and Vitamin B12 and Status Indicators (Objective 1) ..................................... 55 Status Indicators and CHD Risk ............................................................................... 56 Relationship of Status Indicator Va riables in the Sa mple Population ..................... 56 Status Indicators in th e Sub-Sam ple Population ...................................................... 56 Part III: Polymorphisms and CHD Risk (Objective 2) ........................................................... 57 MTHFR 677 CT and MTHFR 1298 A C Polym orphisms and CHD Risk ....... 57 MTHFR 677 CT Polymorphism ..........................................................................57 MTHFR 677 (CT/TT) Genotypes ............................................................................ 57 MTHFR 1298 AC Polym orphism ........................................................................ 58 MTHFR 677 (CT/TT) Compared to CC Genotype and Stat u s Indicators in the sample population .................................................................................................58 MTRR 66 and TC 776 Genotypes and CHD Risk ................................................... 58

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7 Part V: Relationship Between Polymorphism s Related to Folate and Vitamin B12 MTHFR 677 CT, MTHFR 1298 AC, MTRR 66 A G, and TC 776 C G and Status Indicators and CHD Risk (Objective 3) ................................................................... 59 5 DISCUSSION .................................................................................................................... .....65 Summary of Pilot Study Design Lim itation ....................................................................74 Public Health Significance of Research ..........................................................................75 APPENDIX QUESTIONNAIRE............................................................................................... 76 LIST OF REFERENCES ...............................................................................................................79 BIOGRAPHICAL SKETCH .........................................................................................................92

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8 LIST OF TABLES Table Page 2-1 Definitions of Dietary Refere nce Intake (DRI) recommendations. ................................... 462-2 Folate intake recommendations for men and nonpregnant women 19 years. ................464-1 Demographic characteristics of sample population. .......................................................... 604-2 Distribution and response rates for pren atal use of folic acid in the sample population. ................................................................................................................... ......604-3 Distribution and response rates for ci garette and alcohol use in the sample population. ................................................................................................................... ......614-4 Distribution and response rates for diabet es, seizures, and anemia during pregnancy in the sample population. ...................................................................................................614-5 Mean comparison of hematological indices between cases and controls in the sample population .................................................................................................................... ......614-6 Comparison of serum and RBC folate, Hc y, serum B12, and MMA concentrations by group (case/control) in the sample population ................................................................... 624-7 Comparison of serum and RBC folate, Hc y, serum B12, and MMA concentrations by group (case/control) in th e sub-sample population ............................................................ 624-8 Genotypes combination distribution in the sample population for specific polymorphisms ................................................................................................................. ..634-9 Frequency comparison of the MTHFR 677 C T and 1298 A C distribution in the sample population by group type (case/control). ...............................................................634-10 Frequency comparison of the MTHFR 677 genotype (CC, CT, TT) distribution by group type (case/control) in the sample population. .......................................................... 634-11 Frequency comparison between MTHF R 677 CC genotype and the combined genotypes (CT/TT) for the MTHFR 677 C T polymorphism by group type (case/control) in the sample population. ............................................................................ 644-12 Frequency comparison of the MTHFR 1298 genotype (AA, AC, CC) distribution by group type (case/control) in the sample population. .......................................................... 644-13 Comparison of serum and RBC folate, Hcy, serum B12, and MMA concentrations between the MTHFR 677 CC genotype and th e combined (CT/TT) genotypes in the sample population ..............................................................................................................64

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9 LIST OF FIGURES Figure Page 2-1 Structure of folate/folic acid ..............................................................................................42 2-2 Cystosolic and mitochondrial path ways of THF m etabolism in the body ......................... 43 2-3 Vitamin B12 structure.. ......................................................................................................44 2-4 One-carbon metabolism folate-dependent pathway ...........................................................45

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10 LIST OF ABBREVIATIONS 1-C one-carbon 5, 10-MTHF 5, 10-methylenetetrahydrofolate 5-MTHF 5-methyltetrahydrofolate 10-CHO-THF 10-formyltetrahydrofolate Adenosyl-Cbl adenosylcobalamin AHA American Heart Association AI Adequate Intake ANOVA Analysis of variance B6 pyridoxal phosphate B12 vitamin B12 BMI body mass index BWIS Baltimore Washington Infant Study Cbl cobalamin CBS cystathionine B-synthase CH3-Cbl methylcobalamin CH3-THF methyltetrahydrofolate CHD congenital heart defects CLP cleft palate CI confidence interval CN-Cbl cyanocobalamin Cyanobacteria blue-green algae d day DHF dihydrofolate DFEs Dietary Folate Equivalents

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11 DNA Deoxyribonucleic acid DRIs Dietary Reference Intakes dTMP thymidylate dUMP deoxyuridylate EAR Estimated Average Requirements FDA Food and Drug Administration g gram HCL hydrochloric acid Hct hematocrit Hcy homocysteine Hgb hemoglobin IF intrinsic factor IM intramuscular injection IOM Institute of Medicine MCH mean corpuscular hemoglobin MCHC mean cell hemoglobin concentration MCV mean corpuscular volume min minutes M micromole MMA methylmalonic acid MTHFR methyltetrahydrofolate reductase MTR methionine synthase MTRR methionine synthase reductase NCCs neural crest cells nM nanomole

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12 nmol/L nanomoles per liter NO nitric oxide NTD neural tube defect Oh-Cbl hydroxocobalamin PCC population-based cas e-control studies pmol/L pica mol per litter RBC red blood cell RCT randomized control intervention trial RDA Recommended Dietary Allowance ROS reactive oxygen species SAH S-adenosylhomocysteine SAM S-adenosylmethionine SHMT serine hydroxymethyltransferase TC transcobalamin TDT transmission disequilibrium test THF tetrahydrofolate UL Tolerable upper level

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13 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF FOLATE AND VITAMIN B12 RELATED GENETIC POLYMORPHISMS ON CONGENITAL HEART DEFECT RISK: A PILOT STUDY By Younis Ali Salmean May 2008 Chair: Lynn B. Bailey Major: Food Science and Human Nutrition One-carbon (1-C) metabolism reactions require folate as the central substrate that provides essential one carbon moieties. One car bon groups provided by fola te are vital for many reactions including those required for amino acid meta bolism, purine and pyrimidine synthesis, DNA methylation, and the formation of S-adenosyl methionine (SAM). Low folate status can alter this central role in maintaining 1-C meta bolism which can lead to birth defects. Birth defects are the leading cause of death in newborn infants, with congenital heart defects (CHDs) accounting for one death in every three defect-related infant deaths. Congen ital heart defect risk may possibly be related to folate and vitamin B12 status and this risk may be exacerbated in the presence of polymorphisms (MTHFR 677 C T and 1298 A C, MTRR 66 A G and TC 776 C G) related to folate and vitamin B12 metabolism. To assess the roles of folate and vitamin B12 status and associated genetic polymorphisms on CHD risk, a casecontrol pilot study was conducted including 390 non-pregnant mothers. Bl ood samples were obtained from subjects and were analyzed to identify biochemical variables and genotypes. Serum folate and vitamin B12 concentrations were inversely associated (p < 0.05) with CHD risk in the case group relative to the control group in subjects who had a deliver y within 3 years from the time the blood was drawn. The MTHFR 677 CT and TT genotype gr oups were combined and compared between

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14 cases and controls resulting in a significant (P= 0.0356) increase in risk for CHD in the case group. Future studies need to be conducted to provide a carefu l assessment of status at the appropriate time during gestation or within a very short time following the index pregnancy to allow a more accurate measure of status indicators that are likely to inte ract with polymorphisms affecting folate and vitamin B12.

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15 CHAPTER 1 INTRODUCTION Folate and vitam in B12 play critical roles in one-carbon (1-C ) metabolism including deoxyribonucleic acid (DNA) synthe sis and methylation, and homocysteine (Hcy) remethylation. Folate is required for the formation of 5-met hyltetrahydrofolate (5-MTHF), a substrate critical for donating methyl groups to Hcy in a co-dep endent manner with vitamin B12 serving as a cofactor. Folate is the 1-C donor required fo r the formation of S-adenosylmethionine (SAM) from methionine. While folate is involved in the formation of the methyl donor substrate, vitamin B12 is required to facilitate the transf er of the methyl group from the 5-MTHF to Hcy. Impaired folate or vitamin B12 status can lead to impaired gene ration of substrates needed for the de novo synthesis of DNA and cell division. It will also affect formation and maturation of red and white blood cells in the bone marrow. Abnormalities in 1-C metabolism may result in elevated concentrations of Hcy and reduced DNA methylation, both of which possibly can affect development of embryos. Both nutrients alone and toge ther have been linked to increased Hcy concentration, which is a risk f actor for pregnancy complications with evidence for an association with congenital malformations. Folate and vitamin B12 metabolism can be adversely affected by the presence of genetic polymorphisms, which are common mutations that may affect the structure and function of folate/vitamin B12-dependent enzymes and tran sport proteins. Certain polymorphisms (677 C T plus 1298 A C) are responsible for the reduced ac tivity of the methyltetrahydrofolate reductase (MTHFR) enzyme which may lead to impaired Hcy remethylation and DNA methylation. The MTHFR 677C T polymorphism has been associat ed with an increased risk of neural tube defects (NTDs) especia lly when folate status is low.

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16 Congenital heart defect (CHD) risk may possi bly be related to folate and vitamin B12 status and this risk may be ex acerbated in the presence of MTHFR polymorphisms. Methionine synthase reductase (MTRR) is an enzyme requi red for the activation of methionine synthase (MTR) for the remethylation of Hcy to methi onine which may be negatively impacted by a genetic polymorphism (MTRR 66 A G). Investigation of the association of the MTRR 66 A G polymorphism with CHD risk is warrant ed since impaired activity of MTRR may negatively affect 1-C meta bolism. The MTRR 66 A G was previously found to be positively associated with NTDs when vitamin B12 status was low, which provides some basis for a possible link to CHD since the embryonic origins of NTDs and CHDs are similar. Transcobalamin (TC) 776 C G is a polymorphism affecting the protein responsible for the cellular uptake of vitamin B12. It is proposed that individuals with TC 776 GG genotype may have an impaired ability to tr ansport vitamin B12, which may redu ce cellular uptake and lead to abnormal function including embryoni c development. The critical role of TC in vitamin B12 function in 1-C metabolism necessitates the assessme nt of its effect on risk of CHD, however, only a limited number of studies have investigated the role of TC 776 C G and CHD risk and the conclusions from these studies are not definiti ve due to limitations of sample size and other factors. A comprehensive investigation of the as sociation of folate and vitamin B12 status and related genetic polymorphisms with CHD risk ha s not been conducted and is the long term goal of future studies to follow this initial pilot study.

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17 Hypotheses low folate and/or vitam in B12 status are associated with increased risk for CHD; genotype status for the MTHFR 677C T and 1298 A C polymorphisms and the MTRR gene 66 A G and the TC 776 C G polymorphisms will exacerbate the effect of low folate and/or vita min B12 status on CHD risk; congenital heart defect risk associated with either folate and/or vitamin B12 related polymorphisms will be exacerbated by low folate and/or vitamin B12 status. Overall Goal The prim ary goal of this study was to assess th e roles of folate and vitamin B12 status and associated genetic polymorphisms on CHD risk in women with affected pregnancies compared to women who delivered in fants with no birth defects. Specific Objectives assess the as sociation between maternal folate and vitamin B12 status biomarkers (serum and red blood cell (RBC) folate, vitamin B 12, methylmalonic acid (MMA) and Hcy) and infant CHD risk; assess the association betw een maternal MTHFR 677 C T and 1298 A C and MTRR 66 A G and TC 776 CG genotypes on infant CHD risk; determine the interaction between maternal folate and vitamin B12 related polymorphisms and folate and vitamin B12 status indicators on infant CHD risk.

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18 CHAPTER 2 LITERATURE REVIEW Folate and Vitamin B12 Folate Chemistry Folate is a w ater-soluble vitamin. The generic name for the vitamin is folate and it is related to a family of substances containing pt eridine rings joined with p-aminobenzoic acid and one or more glutamic acid molecules (1) (Figure 2-1). The form of naturally occurring folate depends on the side chain compositi on in terms of the number of glutamic acid molecules as well as the specific one-carbon moiety attached to the vitamin. The folate molecule can vary in structure through reduction of the pteridine moiety to dihydrofolate (DHF) and tetrahydrofolate (THF), elongation of the glutamate chain, and su bstitution of the 1-C unit at the N-5, N-10, or both positions (2,3). Folic acid, which rarely occurs in nature, is the fully oxidized monoglutamate form of the vitamin folate, and it is used extensively in food fortification and supplementation due to its ch emical stability (1). Bioavailability Bioavailability m ay be defined as the proportion of an ingested nutrien t that is used and stored in the body. Bioavailability of food folate is cons iderably dependent on the individuals ability to digest, absorb and metabolize the vita min. The bioavailability of folate depends on the food source and often it is incomplete (1). The bioavailability of food folate is estimated using folic acid as the standard since folic acid exhi bits nearly complete absorption (4), and is considered to be 100% bioavailable. The bioa vailability of folate from a mixed diet is approximately 50% or less of the bi oavailability of folic acid (5). When folic acid is consumed

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19 with food, the bioavailability is reduced by ~ 15% compared to when it is consumed alone as a supplement, therefore folic acid in fortified foods is estimated to be ~85% bioavailable (6). Metabolism (Absorption, transport, storage and excretion) Folic acid is a fully oxidized m onoglutamate fo rm of folate and can be absorbed readily at the brush border membrane of the jejunum. Howe ver, food folate needs to be converted into the monoglutamate form by the folylpoly-glutamate carboxypeptidase enzyme also known as folate conjugase (7,8). The monoglutamate form of folate is transported into the entero cyte via a pH-dependent carrier-mediated mechanism (9). At high concentrations of folate (>10 mol/L), ion-mediated transport becomes the means of transport into the cell (10). After the entry into the mucosal cell, the monoglutamate form of folate is reduced and a portion is converted to methyltetrahydrofolate (CH3-THF) before its released into circulation (11). Most folate in the plasma is bound to albumin in the form of 5-CH3-THF, and a smaller amount is bound to a high affinity folate-binding prot ein (2). Cellular uptake of folate by cells is mediated by membrane-associated folate-bi nding proteins (12). After its uptake, 5-CH3-THF is demethylated by MTR and then is converted into a polyglutamyl fo rm by folypolylglutamate synthase (13). The metabolism of folate is divided into pathways occurring in either the cytoplasm or mitochondria. Both pathways facilitate the regenera tion of THF through the reduction of various folate coenzymes. This allows THF to accept a 1-C unit from the folate pool, which is donated by both the cystosolic an d mitochondrial pathways, leading to purine synthesis, DNA methylation, DNA synt hesis, and Hcy remethylation to methionine (Figure 2-2) While folate in the monoglutamate form is subsequently taken up by the cells, it is the polyglutamate form that helps sequester folate in side the cell due to the polarity of the side chains (13). Tissues do not store large amounts of folate beyond their metabolic needs. The total body content of folate is estimated to be betw een 15 to 30 mg (14). A small amount of the

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20 dietary folate is excreted in the urine (15). Th e small quantity of free folate in the plasma is filtered through the glomerulus but mostly reabso rbed in the proximal renal tubules (16). The amount of folate excreted in the urine is estimated to be simila r to the amount excreted in the feces (1). Vitamin B12 Chemistry Vitam in B12, also known as cobalamin (Cbl), refers to a family of substances composed of a central cobalt atom surrounded by macrocyclic corrin tetrapyrrole which are four reduced pyrrole rings connected by nucleotide side chains attached to th e central atom (Figure 2-3). There are various forms of the vitamin B 12 molecule, including methylcobalamin (CH3-Cbl), cyanocobalamin (CN-Cbl), adenosylcobalamin (adenosyl-Cbl) and hydroxocobalamin (OH-Cbl). The commercially produced form of vitamin B 12 is known as CN-Cbl, which is generally used in supplements. The major form of vitamin B12 in blood is CH3-Cbl, with smaller amounts as adenosyl-Cbl and OH-Cbl (17) Vitamin B12 in the form of OH-Cbl is widely used as an intramuscular injection (IM). Hydroxocobalamin is re tained longer in the body than CN-Cbl. In the human body and higher animals, vitamin B12 is utilized in two major metabolic processes where it serves as a coenzyme. The first vita min B12 dependent reactio n is the conversion of methylmalonyl-CoA to succinyl-CoA, which uses adenosyl-Cbl as a cofactor, and the second conversion is the remethylation of Hcy to methionine, which uses CH3-Cbl as a cofactor (18). Cobalamin will be referred to subsequently as vitamin B12. Bioavailability Vitam in B12 it is a product of microbial synt hesis in all higher animals, and the sole source of vitamin B12 is from animal-related s ources. Although some ed ible algae and bluegreen algae (cyanobacteria), cont ain large amounts of vitamin B 12, vitamin B12 from these two

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21 sources appears to be inactive in mammals (19). Bioactivity of each of the forms of vitamin B12 is different. The various forms of vitamin B 12 will have different dissociation, absorption, and protein carrier binding qualities which result in various degrees of bioavailability. As the intrinsic factor (IF)-medi ated intestinal ab sorption system is estimated to be saturated at about 1.5.0 g per meal under normal physiologic conditions, vitamin B12 bioavailability significantly decreases with increasing intake of vitamin B12 per meal (20). Recently reported by Watanabe, the bioavailability of vitamin B12 in healthy humans from fish, meat, and chicken averaged 42%, 56%%, and 61%%, respectivel y, while vitamin B12 in eggs appeared to be poorly absorbed (<9%) relative to othe r animal food products (20). Metabolism (Absorption, transport, storage and excretion) Release of vitam in B12 from proteins must occur prior to ab sorption. Saliva contains a vitamin B12 binding protein referred to a an R prot ein (18). Saliva activity starts the dissociation of vitamin B12 from the protein to which it is bound. Further dissociation of vitamin B12 occurs in the stomach with the action of hydrochloric acid (HCL) and pe psin. The dissociated vitamin B12 enters the duodenum from the stomach, bound to the R-protein. A protein synthesized by the gastric parietal cells referred to as IF binds to vitamin B12 forming the vitamin B12 intrinsic factor complex (IF-Cbl). This happens after the stomach acid is neutralized and the R protein is removed by digestive enzymes (21). In the ileum, a specific IF-Cbl receptor initia tes the uptake of vitamin B12 complex into the enterocytes. During uptake, the IF-Cbl co mplex is internalized by receptor mediated endocytosis where vitamin B12 is then processed and subsequently bound to TC and released into the circulation as TC-Cbl (22). Transcobalamin accounts for approximately 20% of all vitamin B12 bound in circulation, while haptocorrin accounts for the remaining 80% (23). However, only TC-Cbl has receptors on the cell s surface which makes it essential for cellular

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22 uptake, therefore TC-Cbl is c onsidered the bioavailable form of the vitamin. Once TC-Cbl is taken up by receptor-mediated endocytosis, the lyso somic activity releases vitamin B12 from the complex into the tissue. Vitamin B12 can be stored in the body for long periods of time. The liver is the main storage tissue accounting for ~60% of total storage while muscle tissues account for ~30%. There are approximately 2-5 mg of vitamin B12 stored in the body predominately in the form of adenosyl-Cbl and CH3-Cbl. As stated earlier the IF-media ted intestinal absorption system is estimated to be saturated at about 1.5.0 g per meal under normal physiologic conditions, therefore an increase in dose will result in an increase in urinary loss. Other factors such as bioavailability and diet content can al so affect excretion of the vitamin. Biochemical Functions of Folate an d Vitamin B12 One-carbon metabolism reactions require folate as the central substrate that provides essential one carbon moieties. The me thyl group is vital for many reactions including amino acid metabolism, purine and pyrimidine synthesis, and the formation of S-adenosylmethionine (SAM) (1). The formation of SAM in the 1-C cycl e is critical for a large number of methylation reactions. The production of creatine, phospholipids, and neurotransmitters from SAM is important for normal physiological function. More importantly, SAM is a methylating agent for more than100 compounds includi ng proteins, RNA and DNA (24). Folate in the polyglutamate form of THF is converted to 5, 10-methylenetetrahydrofolate (5, 10-MTHF) by accepting a methyl group from the c onversion of serine to glycine via serine hydroxymethyltransferase (SHMT) (Figure 2-4) reaction 3. This reaction is important because it provides the methyl group needed for the production of thymi dylate (dTMP) from deoxyuridylate (dUMP) by thimidylate synthase (1) (F igure 2-4) reaction 1. Folate in the form of DHF is reduced back to THF by dihydrofolate reductase. Folate also is involved in de novo

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23 synthesis of adenine and guanine from 10-formylTHF (10-CHO-THF) resulting in purine synthesis and regeneration of THF ( Figure 2-4) reactions 4, 12 and 13. These reactions collectively underline the critical role of fo late in pyrimidine and purine synthesis. Thus, impaired folate status can be detrimental to the body since DNA synthesis required for cellular growth may be suppressed. Another critical role for folate in 1-C metabolism is DNA methylation. DNA methylation depends on the reduction of 5, 10-MTHF to 5-MT HF by MTHFR. Methylenetetrahydrofolate reductase is an important enzyme since its only function in the body is in the above step, which is critical for the remethylation of Hcy to form methionine. Methionine serves as the dominate precursor for SAM. The coenzyme 5-MTHF is the methyl donor, and vitamin B12 is the cofactor needed for transferri ng the methyl group from 5-MTHF to Hcy to form methionine (remethylation), a reaction that requires the MTR enzyme (Figure 2-4) reactions 5, 6 and 7. The MTR reaction requires vitamin B12 as a cof actor for the remethylation of Hcy to methionine. During the MTR reaction, transfer of the methyl group from met hylcobalamin-III results in the formation of the highly reactive cobalamin-I, which may become oxidized to cobalamin-II, resulting in MTR inactivation (25) Methionine synthase reductas e is required for the reductive methylation of cobalamin-II (with SAM providing the methyl group), which reactivates MTR (26). If either folate or vitamin B12 are deficient, MTR cannot remethylate Hcy to form methionine which leads to megaloblastic change s in rapidly dividing cells such as bone marrow in addition to elevation of Hcy and depletion of SAM (1). The initial methyl group that was accepted by folate is the same methyl group that is donated by SAM to over 100 transmethylation reactions (1). When SAM donates its methyl group, it is converted to S-

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24 adenosylhomocysteine (SAH), which then is hy drolyzed back into Hcy and adenosine by SAH hydrolase (1)( Figure 2-4) reactions 9 and 10. Dietary protein is another source of methionine; howev er, the tightly regulated remethylation of Hcy to methionine also contribu tes to the bodys need fo r this essential amino acid. When methionine is converted back to Hcy via the hydrolysis of SAH an elevation of Hcy may occur if the MTR reaction is impaired. For this reason, Hcy conversion to methionine needs to be optimized to maintain a balance between Hcy and the methionine pool and to prevent hyperhomocysteinaemia. Homocysteine can also be catabolized into cystathionine through the condensation with cysteine by pyridoxal phosphate (vitamin B6) and cystathionine B-synthase (CBS) (18) (Figure 2-4) reaction 11. However, this transsulfuration step is dependent on SAM activation and a surplus of meth ionine (1). When vitamin B12 and folate are deficient, cystathionine levels are elevated (27). Polymorphisms Polym orphisms are mutations that are presen t in more than 1% of the population. The relationship among the four polymorphisms occurri ng in genes involved in 1-C metabolism and the risk for CHD will be discussed in a subseque nt section. Methyltetrahydrofolate reductase 677 C T is the most widely studied polymorphism in which a transition of cytosine to thymine occurs in the gene for MTHFR at base pair 677, resulting in an alanine to valine substitution in the enzyme (28,29). A second polymorphism of the MTHFR gene occurs at base pair 1298 due to the substitution of cytosine for adenine. This substitution results in a coding change which that alanine is subs tituted for glutamate in the protein product (30,31). Both mutations alter the activity of the MTHFR enzyme which leads to lower concentration of 5-MTHF and less substrate available for the remethylation of Hcy. As discussed previously, the initial step in the

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25 remethylation pathway leading to DNA methylati on starts early with the reduction of 5, 10MTHF to 5-MTHF via the MTHFR enzy me (Figure 2-4) reaction 5. While these two polymorphisms MTHFR 677 C T and 1298 A C affect the activity of MTHFR and folate metabolism, two other polymorphisms related to vitamin B12 have also been investigated for their possible role as risk factors for CHD. The first is MTRR 66 A G in which methionine replaces isoleucine in the enzyme protei n. This mutation affects the activity of the MTR enzyme and results in lower remethyl ation of Hcy into methionine (Figure 2-4) reaction 6. The MTRR 66 A G polymorphism was associated with an increase in plasma Hcy, with the GG genotype having a greater effect than the AG genotype (32). Also, it was found that MTRR 66 GG genotype was associated with an increased risk for having an NTD-affected pregnancy when maternal vitamin B12 c oncentrations were low (26). The second polymorphism is the TC 776 C G, which can affect the total amount of vitamin B12 carried in the blood a nd the amount that the cell takes up. As mentioned earlier, cells only have receptors for vitamin B12 bound to TC, and therefore TC-Cbl serves as the bioavailable form of the vitamin. Vitamin B12 bound to haptocorrin is not bioavailable because cells lack receptors for this carrier protein. Several base pair substitutions that may occur in the gene that encodes the TC protein lead to decreased uptake of vita min B12 (33). The most common polymorphism in the TC gene is a cytosine-to-guanine transition at base pair 776 (TC 776 C G) that results in replacement of proline with arginine (34). This TC 776 C G polymorphism negatively affects the serum holoTC concentration and studie s suggest that the TC 776 C G polymorphism may affect TC binding affinity for vitamin B12 and the ability to transport vitami n B12 into tissues (35-38). Miler et al. observed a reduced mean holo-TC concentration, a lower percentage of total vitamin

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26 B12 bound to TC, and a higher MMA concentration in individuals with the TC 776 GG genotype than in those with the TC 776 CC genotype (36). These results indicate that the TC 776 C G polymorphism may alter the cellular availability of vitamin B12 and exacerbate the effects of low vitamin B12 status. Dietary Sources of Folate and Vitamin B12 Intake Recommendations Folic ac id is the synthetic form of folate a nd is used for all enriched products in the US since January 1998 as mandated by the Food and Drug Administration (FDA). Enriched cereal and grain products are fortified with folic to a ta rget level of 140 g/100 g (39). In addition to cereal and grain products, folic acid fortified products ar e available throughout the US marketplace in the form of r eady-to-eat breakfast cereals, snacks, meal replacements and many others. Naturally occurring dietary folate can be found mainly in orange juice, strawberries, peanuts, legumes, asparagus and dark green leafy vegetables. The Dietary Reference Intakes (DRIs) repr esent the most current recommendations for each vitamin and mineral. It is the newest a pproach adopted by the Food and Nutrition Board to provide quantitative estimates of recommended nutrient intakes for different age and gender groups. The DRIs include the Estimated Average requirements (EAR), Recommended Dietary Allowances (RDA), Adequate Intake (AI), and To lerable Upper Intake Level (UL). See Table 21 for definitions of each DRI (40). The National Academy of Sciences Institute of Medicine (IOM) published the most recent DRI recommendations in 1998 (40). The goal of these DRI recommendations was to ensure optimum health rather than prevent clinic al deficiencies. Folate recommendations based on the DRIs are presented in Table 2-2. The DRIs also include the new RDA for folate in dietary folate equivalents (DFEs). Diet ary Folate Equivalents are units that account for differences in the absorption of naturally occurr ing food folate and the more bioa vailable synthetic folic acid

PAGE 27

27 (41). To calculate DFEs 1.7 is multiplied times the micrograms of folic acid and added to the micrograms of food folate. The RDA for men and non-pregnant women 19 years and older is 400 g DFE/d. The recommendations are increa sed to 600 g DFE/d during pregnancy and 500 g DFE/d during lactation. The UL for folic acid is 1,000 g/d. This intake level is established solely based on the fact folic acid supplement ation can mask the dia gnosis of vitamin B12 deficiency; as folate itself is not associated with any toxic side-effects. It is recommended that all women of childbearing age consume 400 g/d of folic acid from fortified foods and/or supplements. This is in addition to consuming food folate from varied dietary sources to reduce the risk of an NTD-affected pregnancy (41). Dietary sources of vitamin B12 include animal-based products and fortified foods. Concentrated dietary sources of vitamin B12 incl ude meat, dairy products, and eggs. It is not very common to develop a vitamin B12 deficiency due to short-term dietary restriction because liver stores of these vitamin can last for a few years. The most common reasons for developing a vitamin B12 deficiency are stomach and intestinal disorders that limit the release and/or absorption of vitamin B12 (42). The RDA is 2.4 g/d for adults, 2.6 g/d for pregnant women, and 2.8 g/d during lactation (40). Congenital Heart Defects Prevalence and Etiology One m illion children per year are born with a CHD worldwide (43). Birth defects are the leading cause of death in new born infants (44), with CHD accounting for one death in every three defect-related infant deaths (45). C ongenital heart defects occur as an isolated malformation due to abnormal organogenesis du ring embryonic development. The development of the cardiovascular system occurs between th e third and eight weeks of embryonic growth after conception (46). During this period, both genetic and environm ental factors pl ay a critical

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28 role in the development of the heart through pr oliferation and apoptosis in which, inadequate proliferation or excess apoptosis can lead to congenital malforma tions such as CHD (47,48). The interaction between environmental and ge netic factors has been the focus of many research studies to identify the role of dietary and supplement inta ke in the prevention of CHD. Factors such as low folate and vitamin B12 status have been identified as risk factors for NTD. Neural tube defects are malformations occu rring during the early embryonic stage (49-53). Both NTDs and CHDs share the same neural crest cells (NCCs) during the embryonic stage. Therefore, it is logical to assume a possible association between the status of both folate and vitamin B12 and the risk of CHD based on the similarity between NTD and CHD developmental stages. These assumptions are supported by recent findings from investigations in which the role of both nutrients and CHD risk was evaluated (54-57). Recently, the American Heart Association (AHA) recommended th at periconceptional use of multivitamins containing folic acid be taken by women of re productive age to reduce the risk of CHD (58). The AHA recommendation was established in support of the possibl e protective effect of folate to reduce the risk of CHD, although the association could not be definitively confirmed due to the small number of studies available on which to base the recommendation (57,59). Effect of Low Folate Status on CHD Risk Low f olate status alters 1-C metabolism resu lting in impaired remethylation of Hcy to methionine, DNA methylation, and re duction of various polyglutamate forms of folate to THF. Consequently, this will lead to lower availabil ity of THF which is requ ired to form 5, 10 MTHF necessary for cell division reactions and nucleotid e synthesis. In addition, it will lead to elevation of Hcy concentrations and alterati on of the primary DNA methyl donor SAM (Figure 2-4) reactions 4,2, 6 and 8 (60).

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29 It has long been recognized th at there is an association be tween folate deficiency and increased incidence of congenita l abnormalities (61). The relations hip between folate status and CHD development is through impaired cardiac neural crest development. The neural crest cells develop into many organs and tissues during embryonic development, one of which is the cardiovascular system. Folate is required for ca rdiovascular development during the early stages through the migration of NCC, di fferentiation, dispersal and cell cycle programming. It has also been suggested that Hcy directly affects cardiac NCC function, however, the mechanism has not been elucidated (61). Both impaired DNA synthesis and elevation of Hcy concentration occur in response to low folate status. However, it is not clear whethe r it is the lower THF availability or the elevated Hcy concentrations associated with lo w folate status or both that may be associated with the impaired NCC development. Periconceptional use of folic acid is well established in preventing NTDs, and emerging evidence suggests that multivitamins containi ng folic acid may also protect against CHD (57,62). An association between folate deficien cy and CHD has been examined in both human epidemiological studies and animal experimentat ion (60,63-67). The first studies to show an association between B-vitamins and CHD were published in 1952 and 1954 (63,68). Congenital heart defects were associated with the consumpti on of folate-deficient di ets in both studies using animal models. Developmental defects invol ving the heart and inhibition of myocardial proliferation were also associated with folate deficient diets in other investigations (69,70). Hobbs et al. reported that Hcy, SAH, and methi onine concentrations ar e important biomarkers predictive of CHD case or control status (71). The strongest evidence that folate status is involved in the CHD formation and prevention comes from a Hungarian randomized control in tervention trial (RCT) in which multivitamins

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30 containing folic acid reduced th e risk of CHD up to 60% when taken during the periconceptional period (72). These results were confirmed in two population-based case-co ntrol studies (PCC) in the USA in which a 24% reduction in CHD risk was observed (55,57). In another study, the Baltimore Washington Infant Study (BWIS), an inverse relationship between daily maternal intake of folic acid and the incidence of cardiac ou tflow tract defects in offspring was found (56). There are few possible links to how folate may be affecting the risk of CHD. As stated earlier, folate is a critical substrate fo r DNA methylation, as well as DNA and nucleotide synthesis, both of which are possibly associated. Research studies provide evidence that elevated Hcy concentration (hyperhomocysteinaemia) is a ssociated with an increased CHD risk, which may be associated with low folate and vitamin B12 status (73). However, due to the small number of investigations, and the inconsiste ncy of findings between some studies, further research assessing the effect of folic acid and the reduction of CHD risk is warranted. Effect of Low Vitamin B 12 Status on CHD Ri sk The roles of folate and vitamin B12 are inte rrelated with regard to DNA and nucleotide synthesis as well DNA methylation. Folate and vitamin B12 participate in 1-C metabolism in which both are needed for the regeneration of THF, remethylation of Hcy, and production of SAM. As discussed earlier, folate is require d to form the 5-MTHF th rough the action of the 5, 10 MTHFR. The methyl group is donated by 5-MTHF to form methionine via remethylation of Hcy, and to regenerate THF. This step cannot be completed without th e cofactor vitamin B12, which is essential for the function of the MTR en zyme. Deficiencies of vitamin B12 and/or folic acid result in hyperhomocysteinaemia, which is associated with an increased risk of CHD (73-76). Similar to folate, vitamin B12 deficiency is linked to an increase risk of NTDs (77). Several studies linked vitamin B12 status with the risk of CHD through impaired methylation of

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31 Hcy to methionine. This biochemical derangement leads to hyperhomocysteinaemia and low DNA methylation, which might be associated with the increase risk for CHD. It is not clear whether vitamin B12 alone or in conjunction with low fola te status is a risk factor for CHD. The association between specific polymorphisms in 1-C metabolism and nutrient status further complicate the relationship. More comprehe nsive studies that account for both folate and vitamin B12 and the polymorphism s related to their 1-C metabolism is required to determine risk associations with CHD. Polymorphisms and CHD Risk MTHFR (677 CT and 1298 A C) polymorphisms In this section, the associ ation of key folate and vitami n B12-related polymorphisms with CHD risk will be discussed. Polymorphisms in the MTHFR, MTRR, and TC genes are recognized to have some effect on CHD; whether this effect is depende nt or independent of folate and vitamin B12 status is the subject of on-going investigations (78-81). Although folic acid containing supplements may have a protective effect in reducing CHD risk, the role that folate and vitamin B12-related polymorphisms may play in CHD risk is not clear enough to draw definitive conclusions and more research n eeds to be done in this area (82). Studies examining the roles of these polymorphi sms in affecting CHD risk have produced conflicting results. The MTHFR 677 TT genotype has been associated with reduced enzyme activity, decreased plasma and red blood cell folate, and mildly elevated Hcy concentrations especially when combined with the MTHFR 1298 A C polymorphism (83-86). Data from clinical studies indicate that there is a positive association between the MTHFR 677 C T polymorphism and CHD risk (79,80,87,88). In 20 06, Beynum et al. reported that maternal MTHFR 677 CT is a risk factor for CHD in offs pring (80). Beynum groups conclusion was based on an observed increased risk of CHD in the offspring of mothers who did not use

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32 supplements containing folic acid with either the MTHFR 677 CT or TT genotype. The risk increased 3-fold in mothers in the CT genot ype group and 6-fold in the TT genotype group compared to women who had used supplements containing folic acid. However, there was no risk association between the MTHFR polymorphism s and CHD in the family-based transmission disequilibrium test (TDT). Briefly, the TDT is a family-based association test to detect the presence of linkage between a genetic marker and a trait by measuring the over-transmission of an allele from heterozygous pare nts to affected offspring. Hobbs et al. reported that the highest estimat ed risk for having a CHD-affected pregnancy was among women who were in the highest quar tile for Hcy with the MTHFR 677 CC genotype and were smokers (89). The group reported that maternal MTHFR 677 C T polymorphism did not have an independent impact on the estimated risk of having a CHD-affected pregnancy. The sample size however, may have limited the power to detect an association between the MTHFR and CHD risk. Based on a 2007 meta-ana lysis, Van Beynum et al. concluded that there was no substantial evidence of increased CHD risk in individuals with either the MTHFR 677 CT or TT genotypes (90). The group suggested that he terogeneity regarding population background, study design and type of heart defects complicates the pooling and compar ison of the studies (90). Methyltetrahydrofolate reductase 1298 A C polymorphism results in reduced enzyme activity (91). Combined hete rozygosity for both MTHFR 677 C T and 1298 A C polymorphisms results in a lower MTHFR activ ity compared to hetrozygosity of either polymorphism alone (92). The MTHFR 677 C T polymorphism alone has a greater effect on Hcy than the MTHFR 1298 A C polymorphism (93). In st udies concerning NTD risk, an effect of the MTHFR 1298 A C polymorphism was observed only when combined with the MTHFR 677 CT polymorphism (93). In another study co ncerning cleft palate (CLP), the risk

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33 increased when the MTHFR 677 CT and TT genotypes and the MTHFR 1298 AC were accompanied by low folate status during pregnanc y (94). Due to the similarity in embryonic origin, evaluating other congenital conditions can be critical for the understanding of the etiology and risk factors associated with the condition. The MTHFR 1298 and 677 AC and CT genotypes combined are more associated with elevation of Hcy when folate and vitamin B12 con centrations are low. Th e role of nutrient-gene interaction and gene-gene interaction in the deve lopment of CHD has not been fully determined. More research and well-defined phenotypic subcategory analys es as well as status assessment of both folate and vitamin B12 are needed to definitively determine whether the MTHFR 677 C T and/or MTHFR 1298 A C polymorphisms of the mothers are risk factors for the development of CHD. MTRR 66 A G and TC 776 C G polymorphisms Both MTRR and TC are essential proteins required for vitam in B12 function and in the remethylation of Hcy and DNA methylation. Unlike the MTHFR polymorphisms, MTRR and TC are two separate genes that directly involve vitamin B12 but not folate. The MTRR enzyme, officially known as 5-methyltetrahydrofolate ho mocysteine reductase, is required to activate the MTR through the reduction of vitamin B12. The la ter enzyme is important for the conversion of Hcy to methionine by accepting th e methyl group from vitamin B12 and transferring it to Hcy, a reaction in which Hcy is remethylated to methionine. The importance of this enzyme in 1-C metabolism is based on its critical role in keepin g Hcy and methionine conc entrations in balance, which will result in normal DNA methylation and nucleotide synthesis. The MTRR 66 A G polymorphism is associated with an increased risk of NTD (26,95). In a case-control study to investig ate the influence of the MTRR 66 A G polymorphism on CHD risk and the possible interaction between the variant and MMA concentrations, Van

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34 Beynum et al. reported that the MTRR 66 GG genotype in combination with a high MMA concentration was associated with a 3-fold increase in CHD risk (81). The increase risk was dependent on the elevation of MMA, which indica tes that compromised vitamin B12 status may possibly be a risk fact or for CHD risk. A CG substitution at base pair 776 in the gene that encodes transcobalamin (i.e. TC 776 C G) may affect TC binding affinity for vitami n B12 which will lead to reduced cellular uptake (35,96,97). Von Castel-Dunwoody found that the presence of th e TC 776 CG genotype negatively affected vitamin B12 metabolism and increased Hcy concentration in 359 non pregnant young women (38). Th ese researchers found that the TC 776 GG genotype group had a significantly lower vitamin B12 concentration and slightly hi gher Hcy concentration when compared to CC and CG among CHD cases. However, folate, vitamin B12, and Hcy concentrations were not different in the case group with any of the MTRR genotypes. The group suggested that a larger sample size might provide a more definitive answer to whether these two polymorphisms are associated with CHD risk. The limited number of studies, the complexity of gene-gene interactions, and nutrientgene interactions limit the ability to conc lude whether the MTRR and/or TC polymorphisms alone or in combination with low vitamin B12 has an effect on CHD risk. Further research investigating both MTRR 66 and TC 776 with the MTHFR 677 and 1298 polymorphisms and folate and vitamin B12 status is needed. This will help expand the understanding of the relationship among folate, vitamin B12 and polymor phisms occurring in genes that codes for key enzymes in folate and vitamin B12 metabolis m and how these factors influence CHD risk.

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35 Environmental and Sociodemographic Risk Factors Environm ental and sociodemographic factors su ch as maternal cigarette smoking, alcohol consumption, obesity, febrile illnesses, and use of medications, diabetes ethnicity, and family history are important to consider when assessing the risk of CHD. There is relatively little research on the potential adverse effects of these non-inherited f actors on the development of the fetal hear. However, a growing body of epidem iological studies invest igating the critical involvement of such modifiable factors on the development of the feta l heart and risk for congenital malformations is emergi ng. It is estimated that up to 30% of congenital heart defect cases are attributable to identifiable a nd potentially modifiable factors (98). Cigarette Smoking Cigarette sm oking during pregnancy has been do cumented over the years to be associated with various negative effects in cluding severe impairment of embryonic development and infant mortality. A meta-analysis of studies pub lished between 1971 and 1999 found no association with smoking for all types of heart defects comb ined and mixed outcomes for the analysis of specific groups or phenotypes (99). Some recen t studies found an associ ation between maternal smoking and various types of heart defects (100,101). Hobbs et al reported an association between smoking and CHD risk especi ally when combined with more elevated concentrations of Hcy (89). While these studies s uggest an association, a study by Kallen et al., as well as a study by Correa-Villasenor et al. failed to corrobor ate these results (99,102). The difference in methods, classification, control of confounding factors, and sample size could be the cause of the inconclusive results. Alcohol Consumption It has been suggested that ethanol m ay produce fetal tissue edema and a ffect the turgor of the primitive cardiac loop (58). There are a large number of studies in which a wide range of

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36 teratogenic effects of alcohol c onsumption during pregnancy were investigated. In a case-control study in Spain, the risk of congenital anomalies with different daily intakes of alcohol doses was investigated and an increase risk of CHD wa s only found with the highest level of maternal consumption of alcohol per day (ie, > 92 g/d) (103). In the study by Correa-Villasenor et al, alcohol consumption in early pregnancy was a ssociated in a dose dependent manner (102). There is no conclusive evidence that moderate al cohol consumption is associated with increased risk of CHD; however, the studies provide more evidence associating heavy consumption with CHD risk (102,103). Obesity A num ber of studies have examined the a ssociation between maternal pre-pregnancy obesity and CHD. In two studies, no statistically significant increas e in risk for any heart defect in relation to maternal obesity was observed (104,105). Walker et al reported a positive association between maternal obesity, defi ned as a body mass index (BMI) of > 26 kg/m2, and defects of the great vessels (106) In a recent study, a 6.5-fold increased risk in aggregate cardiac defects was detected among black obese mothers (107). Careful assessment of an obesity effect on CHD risk when conducting observ ational studies is required to minimize the confounding complications by other factors such as diabetes. Febrile Illnesses There is s trong evidence that febrile illnesse s are associated with increased risk of congenital anomalies. Studies suggest that matern al febrile illnesses during the first trimester of pregnancy is associated with increased risk for certain heart defect s (108-110). Mothers reporting any febrile illnesses during the first trimester had a 2-fold increase risk of having a child with heart defects (110).

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37 Use of Medications In a recently published paper by the AHA, research findings related to the use of diffe rent medications and the risk of congenital defects were reviewed (58). There was no conclusive evidence that the drugs that were investigated were associated with an increased risk of CHD. Maternal therapeutic drugs such as oral contrace ptives, penicillin, ampici llin, and corticosteroids as well as maternal non-therapeutic caffeine were not associated with any variation of congenital anomalies. The exception was ibuprofen which was reported to be associated with some specific variations of congenital defects (98). Diabetes Studies have clearly docum ented a link between maternal pre-gestational diabetes and a range of congenital defects. This association is less consistent with ge stational diabetes (111114). The large body of evidence reviewed by th e AHA recently suggested that diabetes is a well known prenatal maternal risk factor for CHD ( 58). As illustrated by the study of Ferencz et al., specific types of CHD associ ated with maternal pre-gestati onal diabetes include laterality and looping defects, transposition of the great vessels, hypoplastic left heart syndrome cardiomyopathy, and nonchromosomal atri oventricular septal defects (115). Ethnicity Ethnicity among differ ent groups has been asso ciated with various types of congenital defects. Increased risk prevalence of speci fic CHD has been reported among white infants compared to black infants (116,117). However, in a population-based study of variations in prevalence of birth defects comparing Hispan ic and black mothers to non-Hispanic white mothers in California between 1987 and 1997, no differences in prevalence were detected (117,118).

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38 Family History The evidence for an association betw een family history and CHD is not strong. However, family history is critical in studies evaluating the risk of CHD. Family history may be a risk factor for CHD in association with other conf ounding factors. Although there is no direct evidence that CHD risk is associated with fam ily history, careful asse ssment is required when evaluating related data in future studies. Biochemical Markers: Folate and Vitamin B12 Status Indicators Serum and Red Blood Cell Folate Concentrations Serum folate concentration is a good indicator of current and recent folate intake. It is a sensitive measure that reflects the short-term fola te status (119). Serum folate concentration decreases within one to three weeks after lower folate intake is maintained (86,120). Lower serum folate concentration is detected before any change occurs to RBC folate concentration. Defining inadequate serum folate status using the microbiological assay is based on the lower limit of the normal range 13.6 nmol/L (121). The radiobindi ng assay is also used as an alternative method to assess fola te status, however, this method has been shown to yield lower blood folate values relative to the microbi ological assay (122). The lower limit for the radiobinding assay is <7 nmol/L. Red blood cell folate concentration is consider ed a better long-term indicator of folate status than serum folate concentr ation (120). While serum folate concentration reflects the early and short-term changes in folate status, RBC folate concentration reflects ti ssue storage. Folate uptake into the erythrocytes only occurs during early stages of erythropoiesis which takes place exclusively in the bone marrow. Folate therefore cannot permeate the membrane of mature RBC during its 120-day life span, thus RBC folate con centration is a good reflec tion of folate status four months prior to the time of blood sampling. Inadequate st atus defined using the lower limit

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39 of normal of RBC folate concentrat ions using radiobinding assay is 317 nmol/L (120). Using the microbiological assay, deficien t status is defined as a con centration of 362.6 nmol/L. The lower limit of normal or acceptable is 453.2 nmol/L (123). Serum Vitamin B12 In the general US population, the mean serum vitamin B12 concentration for healthy individuals over four years of ag e is 381 pmol/L (124). In the clinical setting, serum vitamin B12 is the primary method for assessing vita min B12 status; however other measures are necessary to avoid false diagnosis of impaired status (18). This is due to the nature by which vitamin B12 is metabolized as well as the way it is stored in the body. Vitamin B12 can be stored in some tissues such as the liver and ki dney for longer periods of times, while some other tissues can be deficient. This can lead to inac curate estimates as some individuals can have low normal serum vitamin B12 concentrations even though their body stores are deficient in vitamin B12. A plasma concentration >221 pmol/L is considered normal, concentrations between 148 and 221 pmol/L are considered marginally deficient or low normal, and a concentration <148 pmol/L is considered deficient. Methylmalonic Acid It is preferable to rely on st atus indicators that reflect vi tam in B12 function. Specifically an elevation in MMA concentr ation is highly specific for a vitamin B12 deficiency. Methylmalonic acid is a four car bon molecule related to valine, isoleucine, and propionic acid catabolism (17). Vitamin B12 is required fo r the conversion of methylmalonyl-CoA into succinyl-CoA, a reaction sensitive to vitamin B12 status in which it prevents elevation of MMA concentration. When vitamin B12 is low, the conversion is impaired leading to accumulation of methylmalonyl-CoA, which eventually is convert ed into MMA. For that reason, MMA is a better predictor of B12 status than serum B12 (17).

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40 Elevated MMA concentrations can be detect ed in the early stages of vitamin B12 deficiency before a decrease in serum vitamin B12 can be measured ( 17). Van Beynum et al reported that maternal subjects with the MTRR 66 GG genotype in combination with high MMA levels above 80th percentile had a 3-fold increased risk for all types of CHD in offspring (81). Therefore, using MMA in conjunction with serum B12 to fully assess the risk of vitamin B12 and polymorphisms related to its function in 1-C metabolism is preferable. Normal serum MMA concentration is 271 nmol/L, with reported refe rences ranges for serum MMA concentration of 50 to 400 nmol/L (125,126). Homocysteine Hom ocysteine is a dimer containing a sulf ur group. Plasma Hcy concentration is inversely related to the concentration of plasma folate. This was evident from the implementation of folic acid fortification of cer eal grains in 1998 by the US government with the aim of reducing the incidence of NTDs in pregnancy (127). This fortifi cation strategy was succes sful in that it resulted in an improved folate status and lower plasma Hcy concentration (128). The mean Hcy concentration was significantly lower in the 1999-2000 post-fortification period compared to (1994-1998) pre-fortification period. In the 1994-1998 period, Hcy concentration was 9.5 mol/L while in the 1999-2000 period it was 7.9 mol/L (129). Plasma Hcy concentration is maintained within narrow ranges through enzymes that metabolize methionine and Hcy. The cutoff for plasma Hcy concentration is 12 mol/L. Values in this higher range have been associated with increased risks for adverse health effects (130). Kepusta et al. reported an association between mild maternal hyperhomocysteinemia and CHD risk, which was confirmed by Hobbs et al. ( 71,131). Bailey et al reported that vitamin B12 status < 22l pmol/L has a negative effect on Hcy concentration inde pendent of the genotype (93). Homocysteine evokes oxidative stress through production of reactive ox ygen species (ROS),

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41 binds to nitric oxide (NO), or leads to accumulation of its precursor SAH which is a potent inhibitor of biological transmethyl ation (132). As discussed earlier impaired folate and vitamin B12 status and polymorphisms related to both nutr ients and the elevation of Hcy concentration were common factors for the risk of CHD in several investigations.

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42 Figure 2-1. Structure of folate /folic acid. [Reprinted with permission from Baiely, L. 2006. Bailey, L. & Gregory, J. (2006) Folate. In : Present knowledge in nutrition, 9th ed. (Bowman, B. & Russell, R., eds.), pp. 278-301. ILSI Press, Washington, D.C.] Folic acid consists of a para-aminobenzoic acid molecule linked on one side by a methylene bridge to a pteridine ring, and joined by peptide linkage to a glutamic acid molecule on the other side. Naturally occurring food folates exist in various chemical forms, containing a side-chain composed of two to ten additional glutamate residues (n) joined to the firs t glutamic acid. The pteridine ring of the folate/folic acid structure can be reduced to form dihydrofolic acid and tetrahydrofolic acid (THF). Folate coenzymes are formed by substitution of one carbon units at the N5, N10, or both positio ns (R) to the polyglutamyl form of THF.

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43 Figure 2-2. Cystosolic and mitochondrial pathways of THF metabolism in the body. [Reprinted with permission from Baiely, L. 2006. Bailey, L. & Gregory, J. (2006) Folate. In: Present knowledge in nutrition, 9th ed. (Bowman, B. & Russell, R., eds.), pp. 278-301. ILSI Pr ess, Washington, D.C.]

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44 Figure 2-3. Vitamin B12 structure.

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45 Figure 2-4. One-carbon metabolism folate-depen dent pathway. [Reprinted with permission from Baiely, L. 2006. Bailey, L. & Gr egory, J. (2006) Folate. In: Present knowledge in nutrition, 9th ed. (Bowman, B. & Russell, R., eds.), pp. 278-301. ILSI Press, Washington, D]

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46 Table 2-1. Definitions of Dietary Reference Intake (DRI) recommendations. Table 2-2. Folate intake recommendations for men and non-pregnant women 19 years. DRI recommendation Estimated Average Requirement (EAR) 320 g DEF/d Recommended Dietary Allowance (RDA) 400 g DEF/d Adequate Intake (AI) Not Applicable Tolerable Upper Intake Level (UL) 1,000 g synthetic folic acid/d DRI recommendation Definition Estimated Average Requirement (EAR) A daily nutri ent intake value that is estimated to meet the requirement of half th e healthy individuals in a group. Recommended Dietary Allowance (RDA) The average daily dietary intake level that is sufficient to meet the nutrient requirement of nearly all (97 to 98%) healthy individuals in a particular life stage and gender group. Individuals should aim fo r this intake level. Adequate Intake (AI) A recommended da ily intake value based on observed or experimentally determined approximations of nutrient intake by a grou p (or groups) of healthy people that are assumed to be adequateused when an RDA cannot be determined. Tolerable Upper Intake Level (UL) The highest level of daily nutri ent intake that is likely to pose no risk of adverse h ealth effects to almost all individuals in the genera l population. As intake increases above the UL, the risk for adverse health effects increase.

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47 CHAPTER 3 MATERIALS AND METHODS Study Design and Methods Overview This study was designed by clinical investigator s at the University of South Florida and All Childrens Hospital in St. Petersburg, Florida as an initial pilot study to provide data for future grant proposals. The long-term goal of future studies would be to conduct large-scale well-designed investigations to evaluate the asso ciation between folate and vitam in B12 status and genetic polymorphisms and the etiology of C HDs. Following the initiation of the study, Drs. Bailey and Kauwell were invited to join the collabor ative research team to analyze the folate and vitamin B12 status indicators, to do the data an alysis, interpret the fi ndings, and to provide advice regarding how the study design and data colle ction should be revised in future studies. This section provides a description of the desi gn and methods used in the pilot study and the discussion section includes a critiq ue and explanation of specific aspects of this pilot study that need revision for future investigations. The study protocol was approved by the Institutio nal Review Board at the University of South Florida and All Childrens Hospital where the study was conducted and the University of Florida, which received blood samples for an alysis. Three hundred ninety (n= 390) nonpregnant volunteer subjects were enrolled in the pilot study. The subjects recruited were mothers of children and juvenile patients from the pediat ric cardiology practice at All Children Hospital, St. Petersburg, FL, as well as staff of All Childrens Hospital or the University of South Florida at St. Petersburg, FL. Inclusion cr iteria (a) women who had previ ously delivered an infant; (b) previous pregnancy resulted in a ny variation of struct ural CHD-related complications (cases); (c) previous pregnancy with no re lated structural CHD complicati ons (controls); and (d) not pregnant at time of data collec tion. Mothers were primarily r ecruited and inform ed of the study

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48 at the time that they brought their children to All Childrens Hospital for a clinical appointment. In addition, staff members were recruited who me t the inclusion criteria. After determining eligibility, subjects were provide d information regarding the study and were asked to sign an informed consent form. After obtaining the info rmed consent, blood samples were collected for the determination of polymorphisms in the MTHFR, MTRR and TC genes (MTHFR 677 C T, MTHFR 1298 AC, MTRR 66 A G, and TC 776 C G) and concentrations of serum folate, RBC folate, vitamin B12, Hcy, MMA, and hematologi cal parameters. The categorization of the control (n=186) and the case group (n=204) was based on the diagnos is of the presence of CHD using prenatal and postnatal ul trasonography. Subjects were instructed to complete a selfadministered questionnaire (Appendix A) designed to collect dem ographical data including information about their health status, supplemen t use, alcohol consumption, cigarette use, and prescription medication use dur ing their index pregnancy. Sample Collection and Processing Blood sam ples were collected from each participant by a phlebotomist in vacutainer tubes (Vacutainer Blood Collection Set; Becton Dickinson, Vacutainer Systems; Franklin Lakes, NJ). A total of 30 mL of blood wa s collected for analysis of th e following indices RBC folate, plasma vitamin B12, serum MMA, serum Hcy, serum folate, and hematocrit. The vacutainer tubes were immediately shi pped on dry ice to the Univ ersity of Florida for processing and frozen storage prior to analysis. Blood for serum folate samples was collected in 8.3 ml serum separator gel clot activator tubes (Vacutainer, Becton Dickinson, Rutherford, NJ) and kept at room temperature for 30 to 60 minutes to allow time for clotting. Serum was obtained by centrifuging the tubes at 650 x g for 15 minutes at 21oC (International Equipment Compant; Model HN-S II Centrifuge, Needham

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49 Heights, MA). Supernatant sera were mixed w ith sodium ascorbate (1 mg/ml), aliquoted into 200 l samples, and stored at -30oC until analysis. Whole blood was collected in 7 ml tubes cont aining K3 ethylenediaminetraacetic acid to prevent clotting (Vacutainer, Becton Dick inson, Rutherford, NJ). Blood for plasma homocysteine was kept on ice pr ior to processing. A small ali quot of whole blood held at room temperature was diluted 20-fold in 1 mg /ml ascorbic acid and aliquoted into 200 l samples and frozen for measurement of RBC folate concentr ation. The iced blood was centrifuged at 2000 x g at 4oC for 30 minutes. The plasma from these sa mples was frozen and used to measure the plasma homocysteine concentration. Following removal of the plasma, the samples were used to extract DNA to be used to determine genotype for polymorphisms (MTHFR, TC, and MTRR). Aliquots were frozen at 30oC for subsequent analysis of seru m and RBC folate concentrations. Analytical Methods Measurement of Serum Concentrations of Status Indicators Serum folate and vitamin B12 concentrations The serum folate concentrations of all subjects were determined using the MP Biomedicals, Inc. SimulTRAC-S Radioassay K it (Orangebury, New York). The radiobinding assay is conducted by adding dithiothre itol solution to the folate tracer (125I, borate buffer with human serum albumin, dextran, potassium cyanide, dye and preservative). This mixture is added to serum folate samples and heated in a water bath at 100oC for 15 minutes. Once cooled, the folate binder is added to the mixture, which is protected from exposure to light, and incubated for one hour. During incubation, endogenous folate and 125I compete for binding sites to the folate binder. Samples are centrifuged and bound fola tes and microbeads precipitated. The bound folate (labeled and unlabeled) accumulates in a pe llet, while unbound folate is in the supernatant,

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50 which is gently discarded. The radioactivity of the pellet is measured usin g a scintillation gamma counter. Sample serum folate concentrat ion was calculated using a standard curve on which the radioactivity was inversely re lated to serum folate concentration. Plasma B12 was determined by RIA using a commercially available kit (Quantaphase II, Bio-Rad). Specifically, samples were incuba ted with a 57Co labeled B12 tracer in a 100oC water bath to convert all forms of B12 to cyanocobalamin. Samples were brought to room temperature after boiling for 20 min, and then mixed with purified porcine IF bound to polymer beads and incubated for one hour. During incubation labeled and unlabeled B12 compete for binding to IF at rates that match their relative concentra tions. Finally, samples were centrifuged, and supernatant containing unbound B12 was remove d. Sample radioactivity was measured by gamma counter and B12 concentration was calcu lated using a standard curve on which the radioactivity was inversely relate d to B12 concentration. Folate concentrations were inversely related to the measured radioac tivity. Serum folate and vitami n B12 concentrations >12 nmol/L and >148 nmol/L, respectively, represented the lower limit of normal values for this study. Red blood cell folate concentration The red blood cell folate concentrations of blood specim ens were determined using the Lactobacillus casei microbiological assay in a 96-well micr oplate system adapted from Tamura (133) and Horne and Patterson (134). The intraand interassay CV for th e microbiological assay were 8.7 and 7.1%. Homocysteine and methylmalonic acid Sa mples were shipped to the (Metabolite Labor tories, Inc. Denver, Colorado) for analysis to determine serum Hcy and MMA concentratio ns. Samples were analyzed using gas chromatography mass spectrometry (135,136).

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51 Genotype Identification Genotypes of potential subjects were dete rm ined using Dynamic Allele Specific Hybridization (DASH) performed by DynaMetrix. Samples were sent to DynaMetrix Limited, University of Leicester ( United Kingdom, where primers a nd probes were designed for the genotype polymorphisms and analyses were performed using the DASH method with DynaScore Software v. 0.7 ( http://www.dynametrix-ltd.com ). Briefly, a short PCR product was created sp anning the polym orphic position. One PCR primer was 5'-labeled with biotin for attachment of the amplified targets to streptavidin-coated 96-well microtiter plates. Fo llowing denaturation and a wash to remove the unbound strand, an allele-specific probe was hybridized to the bound target DNA strand at low temperature in the presence of the double-strand specific intercala ting dye Sybr Green. Finally, the temperature was steadily increased while recording the probe-target duplex melting temperature, as monitored by diminution of Sybr Green fluorescen ce with a quantitative P CR analysis device. Pregnancy Index Pregnancy index is a term that indicates the elap sed time from the delivery of the affected infant for the case mothers or for the normal infa nt for the control mothers, and the time when the blood was drawn. The pregnancy index was calculated using the date the blood was drawn (transformed into numerical count s) minus the date of the delivery (transformed into numerical counts). The product was then divided by 365 which is the number of days in a year to obtain the number of years. The pregnancy index was then divided into three categories: < 3 years, 3-5 year, and > 5 years. Selection of sub-sample population: Since the primary focus of the study was to determine if folate and vitamin B12 status during pregna ncy was associated with risk for CHD, it was important to focus on women who ha d delivered their infants in a relatively short time from the

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52 time the blood sample was drawn for nutrient and metabolite concentration determinations. For this reason, a subgroup of the study participants, those with a pre gnancy index of less than three years were carefully evaluated. Since status indicators were more likely to reflect status during gestation. Supplement Analysis The results from the questionnaire regarding supplement use were modified to fit our model due to the low response rate. We combined all responses to either (Yes or No) categories instead of the actual categories (yes, no, infrequently, sometimes, or routinely). The following responses yes, sometimes, or routinely we re counted as yes responses. A no or infrequently response was consid ered a no responses. Data on other supplements and herbal use were not assessed due to th e high non-response rate. Statistical Methods The statistical analysis was performed using SAS, version 9.1, SAS Institute Inc. Cary, NC, USA. An initial analysis was conducted to determ ine basic statistics of the demographic and background variables and hematological prof ile: ethnicity, maternal age, pregnancy index, supplement use, cigarette and alcohol consum ption, and hematological indices. One-way analysis of variance (ANOVA) test was used to determine if the continuous demographic and background variables means differed among the two groups (case and control). A Chi-square test was used to determine whether the proportion of responses in each of the categories differed between categorical variables. Status indicators were log-transforme d so that the assumptions of normality are met, and the reported values we re back-log transformed. Linear regression analysis was performed to check correlation be tween status indicators. An ANOVA test was used to compare the means of the transformed st atus indicators for both folate and vitamin B12 (Hcy, MMA, vitamin B12, serum folate, and RBC folate) and determine whether the status

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53 indicators differed between cases and controls with regard to CHD risk. A Chi-square test was used to determine whether there was a relations hip between subject stat us and genotype groups concerning the MTHFR, MTRR, and TC polymorphi sms. Logistic regression was used to evaluate a complete model of th e subject status (case/control) versus the genotype classification and status indicators. All first order intera ctions were included in the full model.

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54 CHAPTER 4 RESULTS Part I: Exploring the Data Demographic Characteristics of the Study Sample Population Subjects Three hundred ninety (n= 390) non-pregnant subjects were enro lled in the pilot study. Subjects were prim arily mothers of children and j uvenile patients from the pediatric cardiology practice at All Children Hospital, St Petersbu rg, Florida. Subjects were both non-pregnant mothers of children with any vari ation of structural heart defect s referred to as the case group (n=204) and non-pregnant mothers of non-CHD a ffected offspring referred to as controls (n=186). Demographic Characteristics De mographic characteristics of the sample population are presented in Table 4-1. No difference (p > 0.05) between case and control ba sed on ethnicity, maternal age at birth, or pregnancy index was detected. Ethnicity was reported as African-American, Hispanic, whiteAmerican, or other, and there wa s no difference (p > 0.05) in the ethnic distribution between the case and control groups. When the subjects were categorized into diffe rent age groups (<18, 1830, 30-45, and 45 years of age), no differences (p > 0.05) between the case and control groups were detected. Maternal age was not different (p > 0.05) betw een cases (28.5 0.65 years) and controls (27.7 0.74 years). Subjects were then categorized into three categories according to pregnancy index (< 3 years, 3-5, > 5 years). No significant difference (p > 0.05) was detected between cases and controls based on pregnancy index category.

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55 Supplement Use Low response rate for the supplem ent question in the questionnaire was recorded. Based on the response of one-third of the subjects in either the case or control group, use of either prenatal and/or folic acid supplem ents was determined not to differ (p > 0.05) between case and control groups as presen ted in (Table 4-2). Cigarettes and Alcohol Consumption Lim ited data were collected about cigarette and alcohol consumption among participants. Due to the limited data and response rate in the y es category (Table 4-3), a statistical analysis test to evaluate an associa tion between smoking cigarettes and alcohol use and increased CHD risk was not conducted. Medical Conditions Data were c ollected for a small percentage of subjects related to di abetes, seizures, and anemia which developed during pregnancy (Table 4-4). No data on other medical conditions were available Data were insufficient to conduc t a statistically valid comparison. Hematological Indices He matological indices are presented in (Table 4-5). No differences (p > 0.05) in any of the hematological indices (Hgb, MCV, MCH, MCHC, and Hct) were found between case and control groups. Part II: Folate and Vitamin B12 and Status Indicators (Objective 1) The f irst hypothesis was that low folate and/or vitamin B12 status are associated with increased risk for CHD. To test this hypothesis, association betw een maternal folate and vitamin B12 status biomarkers (serum and RBC folate, vitamin B12, Hcy, and MMA concentrations) on infant CHD risk was observed.

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56 Status Indicators and CHD Risk The association between m aternal folate and v itamin B12 status and risk of having a child with CHD was evaluated. The specific status i ndicators for folate status that were compared included serum and RBC folate an d Hcy concentrations and the vi tamin B12 status indicators, that included serum vitamin B12, MMA and Hcy concentrations. There were no significant differences between any of the st atus indicators in the case vers us control group (Table 4-6). One entry in the MMA concentrations reported for a subject was omitted from the MMA dataset because it was 10-fold higher than the maximum va lue for any of the remaining subjects, a value that is physiologically implausible. Relationship of Status Indicator V ariables in the Sample Population The means for vitamin B12, RBC folate, and seru m folate concentrations were inversely correlated with Hcy concentr ation (p < 0.0001), (p < 0.002) a nd (p < 0.0001), respectively. Mean concentrations for MMA and Hcy were positively correlated (p < 0.0001). Status Indicators in the Sub-Sample Population Subjects who delivered their babies in less than 3 years from the time the blood sample was drawn were selected as for this sub-sample. A total of 62 subjects had a pregnancy index of < 3 years, 35 of whom were cases and 27 were controls (Table 4-1). When folate and vitamin B12 status indicators were compared between the cases and controls in the sub-sample population groups (Table 4-7), a relationship (p < 0.05) between serum folate-concentration and CHD risk was detected. The mean serum vitami n B12 concentration betw een case and controls was associated with CHD risk in the case group (p = 0.0016) lower. No differences (p > 0.05) between case and control groups for the other status indicat ors (Hcy, MMA, RBC folate concentration).

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57 Part III: Polymorphisms and CHD Risk (Objective 2) The second hypothesis is that polymorphism s affecting genes encoding MTHFR, MTRR, and TC genes (i.e. MTH FR 677C T and 1298 A C, MTRR gene 66 A G and TC 776 C G) will exacerbate the effect of low folate and/or vitamin B12 status on CHD risk. To test this hypothesis, the association be tween maternal MTHFR 677 C T and 1298 A C, MTRR 66 A G, and TC 776 C G polymorphisms on infant CHD risk was associated. The distribution for all possible genotypes is presented in (Table 4-8). MTHFR 677 CT and MTHFR 1298 A C P olymorphisms and CHD Risk The relationship between both MTHFR 677 C T and 1298 A C polymorphism and the risk of CHD independent of the other two polymorphisms TC 776 C G and MTRR 66 A G was investigated using the Chi-s quare test. No association (p > 0.05) was detected between the MTHFR genotype groups and CHD (Table 49). More cases (n=39) have the double heterozygote (AC-CT) MTHFR polymorphism compared to the control group (n=24). When the double heterozygous AC-CT MTHFR genotype groups were compared between the case and control group, there was a trend (P= 0609) for a greater number of case mothers to have the ACCT MTHFR genotype compared with all of the remaining genotypes (Table 4-9). MTHFR 677 CT Pol ymorphism Comparison of the three genotypes (CC, CT, TT ) between cases and controls resulted in no significant association (P=0.1073) (Table 4-10 ). Although not significant, the somewhat higher number of cases with the CT and TT genot ypes compared to controls suggests a trend. MTHFR 677 (CT/TT) Genotypes Based on the higher number of cases observed having the C T and TT genotypes for the MTHFR gene, the MTHFR 677 CT and TT genotype groups were combined independently of

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58 the MTHFR 1298 A C polymorphism. This combination resulted in an association (P= 0.0356) between CHD and the MTHFR 677 C T polymorphism (Table 4-11). MTHFR 1298 A C Po lymorphism Similar to the MTHFR 677 C T comparison between cases and controls based on the MTHFR 1298 AC genotypes were carried out. No asso ciation (p >0.05) between the MTHFR 1298 genotypes and CHD (independent of the MTHFR 677) was detected (Table 4-12). MTHFR 677 (CT/TT) Compared to CC Genotype and Status Indicators in the sample population Based on the findings from (Table 4-11), the data were further explored by comparing the status indicators between all subjects with either the CT or TT genotype for the MTHFR 677 C T polymorphism to subjects with the CC genot ype. There was no difference (p > 0.05) among status indicators between the two groups based on this method of comparison using the entire sample (Table 4-13). MTRR 66 and TC 776 Genotypes and CHD Risk The relationship of both MTRR 66 A G polymorphism and the TC 776 C G polymorphism and CHD risk was assessed and was not found to di ffer (p > 0.05). Number of subjects with the MTRR 66 GG genotype in the case group is lower than the control group (39, 48, respectively). To further characterize this relationship, the AA and AG genotype groups were compared to the double homozygous of the gene MTRR 66 GG genotype and a trend (p = 0.0917) for an association was detected. For the TC 776 genotype groups, no associati on (p>0.05) for any of the genotype groups and CHD risk was detected. Although not significant, the number of individuals with CG and GG genotypes tended to be higher in case compared to control groups (104, 92) and (47, 33),

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59 respectively. The TC 776 CG and GG genotype groups were combined and case and control groups were compared, but no diffe rence was detected (p > 0.05). Part V: Relationship Between Polymorphisms Related to Folate and Vitamin B1 2 MTHFR 677 C T, MTHFR 1298 A C, MTRR 66 AG, and TC 776 C G and Status Indicators and CHD Risk (Objective 3) The third hypothesis is that C HD risk associated with either folate and/or vitamin B12 related polymorphisms will be exacerbated by low fo late and/or vitamin B12 status. Therefore, the third objective was to determine the interac tion between maternal fo late and vitamin B12 related polymorphisms and folate and vitamin B 12 status indicators an d infant CHD risk. Status indicators and genotype relationship and CHD risk: The relationships among probable risk for CHD and status indicators for both folate an d vitamin B12 and polymorphisms affecting the MTHFR, MTRR, and TC genes we re assessed. The first model used was a stepwise procedure to evaluate all genotype groups and their firs t-order interactions. Only the MTHFR CT/TT combinations were associated with an increased risk of CHD (p < 0.05). No other polymorphism interactions were identified in the model. In the second model, a stepwise procedure was used to evaluate all status indicato rs and their first order interactions. At the ( = 0.05) level of significance, no significant associati ons between all status indicators and the risk of CHD were detected. In the third model, both genotypes and status in dicators were included. A step wise procedure was performed, and no associ ation (p > 0.05) was ascertained for all status indicators and all genotypes wh ich were removed from the model except for the MTHFR CT/TT genotypes group which was si gnificant (p < 0.05).

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60 Table 4-1. Demographic characte ristics of sample population. a One-way ANOVA was used for statis tical comparisons between groups. b Chi-square test was used for stat istical comparisons between groups. c Mean standard deviation (SD). NA: not applicable Table 4-2. Distribution and response rates for prenatal use of folic acid in the sample population. Supplement Case Control Total (n) P-valuea Prenatal 0.5579 Yes 117 98 215 No 8 9 17 NA b 104 Folic Acid 0.2410 Yes 26 16 42 No 114 105 219 NA b 129 aChi-square test was used for stat istical comparisons between groups. bData were missing (NA, not available). Demographic Variable Case (n) Control (n) Total (n) Missing P-value Ethnicity (%) b 0.2177 African American 10 9 19 NA Hispanic 17 21 38 NA White American 115 88 203 NA Other 5 10 15 NA Total (n) 147 128 275 115 Maternal Agea, c 28.5 0.65 27.7 0.74 0.4166 Maternal Age Groupb 0.4224 Less than 18 3 4 7 NA 18-30 61 45 106 NA 20-45 41 32 73 NA 45 > 0 1 1 NA Total (n) 105 82 187 203 Pregnancy Index (years)b 0.2852 < 3 35 27 62 NA 3 5 13 6 19 NA >5 150 150 300 NA Total (n) 198 183 381 9

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61 Table 4-3. Distribution and response rates for cigarette and alcohol use in the sample population. Factor Case Control Total (n) Cigarettes Yes 2 1 3 No 23 27 50 NAa 337 Alcohol Yes 1 0 1 No 24 27 51 NAa 338 aData were missing ( NA, not available). Table 4-4. Distribution and res ponse rates for diabetes, seizures and anemia during pregnancy in the sample population. Condition Yesa No b Missingc Total (n)d Diabetes 6 48 336 390 Seizures 1 52 337 390 Anemia 12 41 337 390 aNumber of subjects who answer ed with yes for the condition. bNumber of subjects who answer ed with no for the condition. cNumber of subjects who did not sp ecify an answer for the condition. dTotal number of subjects enrolled in the study. Table 4-5. Mean comparison of hematological indices between cases and controls in the sample population.a,b Index Case Control Total (n) P-value Hgb (g/dl) 13.3 (13.1, 13.4) 13.3 (13.2, 13.5) 380 0.5643 MCV (fl) 87.9 (87.1, 88.7) 88.4 (87.6, 89.1) 380 0.4311 MCH (pg) 29.2 (28.9, 29.5) 29.4 (29.1, 29.7) 380 0.3674 MCHC (g/dl) 33.2 (33.0, 33.2) 33.3 (33.1, 33.4) 380 0.5003 Hct (%) 39.9 (39.5, 40.3) 40.7 (39.7, 40.5) 388 0.6325 aMean (5%, 95% CI). bOne-way ANOVA was used for statisti cal comparisons between groups.

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62 Table 4-6. Comparison of serum and RBC folate Hcy, serum B12, and MMA concentrations by group (case/control) in the sample population.a Variable Case Control Total (n) P-value Serum folate (nmol/L) 21.1 (19.3, 22.9) 21.1 (19.2, 23.0) 388 0.9384 RBC folate (nmol/L) 1447 (1328.2, 1565.8) 1447.3 (1324.5, 1570.1) 211 0.8233 Hcy (M) 6.6 (5.9, 7.3) 7.3 (6.5, 8.1) 367 0.2555 Serum B12 (pmol/L) 367.7 (335.95, 399.45) 395.5 (362.3, 428.7) 390 0.2366 MMA (nM) 230.6 (213.5, 247.7) 222.5 (204.3, 240.7) 367 0.5255 aMean (5%, 95% CI). One-way ANOVA was used for statisti cal comparisons between groups. bP-values were based on normalized (log transformed) data. The results have been backtransformed to original scale. Table 4-7. Comparison of serum and RBC folate Hcy, serum B12, and MMA concentrations by group (case/control) in the sub-sample population.a, b, c Variable Case (n = 35) Control ( n = 27) P-value Serum folate (nmol/L) 17.7 (14.8, 21.1) 24.2 (19.8, 29.8) 0.0258 d RBC folate (nmol/L) 1254.6 (1044.1, 1507.6) 1418.3 (1123.0, 1791.1) 0.4246 Hcy (M) 5.9 (5.4, 6.5) 5.4 (4.8, 6.0) 0.2060 Vitamin B12 (pmol/L) 307.3 (253.1, 372.9) 503.5 (403.8, 627.8) 0.0016 d MMA (nM) 208.8 (185.2, 235.5) 189.8 (165.1, 218.1) 0.3113 aSub-sample population is a category for all subj ects who had pregnancy index of < 3 years. bMean (5%, 95% CI). One-way ANOVA was used for statis tical comparisons between groups cP-values were based on normalized (log transformed) data. The results have been backtransformed to original scale. dSignificantly lower than controls.

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63 Table 4-8. Genotypes combina tion distribution in the samp le population for specific polymorphisms.a MTHFR (677/1298) TC 776/MTRR 66 CC/AA CT/AA TT/AA CT/AC CC/AC CC/CC CC/AA 8 10 6 3 7 5 CC/AG 7 10 8 9 3 2 CC/GG 2 7 1 5 7 2 CG/AA 8 16 8 10 8 5 CG/AG 14 28 14 11 28 3 CG/GG 10 5 1 12 12 2 GG/AA 4 8 1 5 5 3 GG/AG 3 8 5 3 7 4 GG/GG None 9 4 5 3 1 aNumbers indicate the occurrence of each genotype combination in the sample population according to their genetic makeup in the genes selected in this study (MTHFR, MTRR and TC). Table 4-9. Frequency comp arison of the MTHFR 677 C T and 1298 A C distribution in the sample population by group type (case/control). MTHFR (677/1298) Case (n = 204) Control (n = 186) Total (n = 387) P-valuea CC-AA 25 33 58 0.3223 CT-AA 56 49 105 TT-AA 28 20 48 CC-AC 42 46 88 CT-AC 39 24 63 0.0609 b CC-CC 14 14 28 aChi-square test was used for statisti cal comparisons between genotype groups. bChi-square test was used to compare CT-AC genotypes between case and control. Table 4-10. Frequency comparison of the MTHFR 677 genotype (CC, CT, TT) distribution by group type (case/control) in the sample population. MTHFR 677 Case (n = 202) Control (n = 185) Total (n = 387) P-valuea CC 79 92 171 0.1073 CT 95 78 168 TT 28 20 48 aChi-square test was used for stat istical comparisons between groups.

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64 Table 4-11. Frequency compar ison between MTHFR 677 CC genotype and the combined genotypes (CT/TT) for the MTHFR 677 C T polymorphism by group type (case/control) in the sample population. MTHFR 677 Case (n = 202) Control (n = 185) Total (n = 390) P-valuea CC 79 92 171 0.0356 CT/ TT b 123 93 216 Missing 3 aChi-square test was used for statistical co mparisons between groups. Significantly higher frequency of CT/TT in cases compared to controls. bSubjects with either one of these genotypes were combined in one category. Table 4-12. Frequency comparison of the MTHF R 1298 genotype (AA, AC, CC) distribution by group type (case/control) in the sample population. MTHFR 1298 Case (n = 204) Control (n = 186) Total (n = 390) P-valuea AA 109 101 210 0.8562 AC 81 70 151 CC 14 15 29 aChi-square test was used for multiple statistical comparisons between groups. Table 4-13. Comparison of serum and RBC folate Hcy, serum B12, and MMA concentrations between the MTHFR 677 CC genotype and th e combined (CT/TT) genotypes in the sample population.a, b MTHFR 677 Variable Total (n) P-value Hcy (M) 365 0.4781 CC 7.2 (6.4, 8.0) CT/TT 6.7 (6.0, 7.4) MMA (nM) 362 0.4230 CC 232.7 (214.1, 251.3) CT/TT 222.4 (205.5, 239.3) Serum B12 (pmol/L) 386 0.2113 CC 364.0 (329.3, 379.1) CT/TT 394.0 (363.2, 424.8) Serum Folate (nmol/L) 384 0.6889 CC 20.7 (18.7, 22.7) CT/TT 21.1 (19.3, 22.9) RBC Folate (nmol/L) 211 0.8526 CC 1466.6 (1347.0, 1585.6) CT/TT 1450.3 (1326.6, 1574.0) aMean (5%, 95% CI). One-way ANOVA was used for statisti cal comparisons between groups. bSubjects carrying either one of these two genotypes (CT/TT) were grouped together.

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65 CHAPTER 5 DISCUSSION The prim ary goal of this retrospective observational pilot study was to assess the roles of folate and vitamin B12 status and associated genetic polymorphisms on CHD risk and to provide a framework for future studies. The first objectiv e of the retrospective pilot study was to assess the association between maternal folate and vita min B12 status biomarkers on risk for having an infant with a CHD. An association between mean s of folate and vitamin B12 status indicators and CHD risk was not detected in this pilot st udy. A key issue to consider in evaluating this finding is whether the status at the time of the blood sampling for this study was similar to that when the fetal heart was developi ng. Since the average period of time that had elapsed from the time of delivery for both cases and controls was 11.8 years, it is likely that there may have been significant changes in folate and/or vitamin B12 status during this period of time due to changes in the diet or changes in the use of supplements. To address this issue, a small subset of women whose pregnancies occurred within three years of the time of the blood draw was evaluated. The case women in this subgroup were found to have significantly lower serum folate and B12 concentrations compared to that of control women. The sub-sample population used in this study, subjects with pregnancy index < 3 years proved more relevant to the study objectives related to the association of stat us indicators and CHD risk. A more definitive answer related to this issue would be provided from a prospective study in which status indices were determined during the pregnancy in both case and controls. In a retrospective study conducted by Hobbs et al., fo late and vitamin B12 st atus were evaluated using a shorter pregnancy index range. The me dian time between the end of pregnancy and the blood draw in the study was 24 months for contro ls and 14.9 months for cases, thus providing a narrower window of time for changes in folate and vitamin B12 status to occur (89,137).

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66 Although, these investigators did not detect differences in folate and vitamin B12 status between case and control mothers, there were significan t differences in related biomarkers including Hcy, SAH, methionine and SAM, which suggests th at the metabolism of th ese two nutrients may be abnormal in case mothers (137). However, in the study by Hobbs et al., case subjects had a pregnancy index median of 14.9 mont hs while control subjects had a pregnancy index median of 24 months which was addressed as a limitation in a study by Verkleij-Hagoort et al. (73). In the study by Verkleij-Hagoort et al., a range of ~17 months was used as a pregnancy index for both case and control subjects. Both studies, however, suggest a shorter pregnancy index to be more relevant to measure stat us indicators (73). When evaluating the association between fo lic acid and vitamin B12 status and CHD risk, it is important to link pot ential differences in the use of supplements during gestation to birth outcomes so that conclusions can be drawn for public health recommendations. In a previous study conducted by Shaw et al., wome n who took multivitamins containing folic acid during critical periods of heart development were found to have a reduced risk of CHD (57). The strongest evidence that folate status is involved in CHD formation and prevention comes from a Hungarian RCT in which multivitamins c ontaining folic acid redu ced the risk of CHD up to 60% when taken during the periconceptional peri od (72). These results were confirmed in two population-based case-control studies in the US in which a 24% reduction in CHD risk was observed (55,57). In addition to the reduction in CHD risk associated with folic acid supplement use seen the these two studies investigators from the Ba ltimore Washington Infant Study reported an inverse relationship between daily maternal intake of folic acid and the incidence of cardiac outflow tract defects in offspring (56).

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67 In the present pilot study, information obt ained from subjects regarding prenatal supplement use was limited in that information wa s missing from approximately one-third of the women which limited the power to detect a reduc tion in risk associated with folic acid and vitamin use during gestation. To address this issu e in future investigatio ns, detailed information regarding supplement use includi ng specific brand name to obtain the exact nutrient composition and timing of the use of the supplement during pregnancy should be obtained. The ideal study design for an observational, non-intervention protocol is a prospective study in which information regarding supplement use prior to an d during pregnancy is obtained and the outcome of pregnancy subsequently monitored. Another important source of folic acid for which information should be obtained in future studie s are foods that are fortified with folic acid including ready-to-eat breakfast cereals that may provide daily folic acid doses that are comparable to supplements. No information was obtained regarding consumption of folic acid fortified products in the current pilot study and this limitation of the study design is shared by other previously conducted studies (138-141). Future i nvestigations should incorporate the use of a detailed dietary in take questionnaire to allow for a quant itative estimate of daily folic acid intake during the index pregnancy. The second objective of this pilot study was to assess th e association between the MTHFR 677 CT, 1298 A C, MTRR 66 A G and TC 776 CG polymorphisms on maternal CHD risk. Detailed statistical anal yses involving multiple comparisons were conducted to determine if there were any associations between specific genotypes associated with these polymorphisms and CHD ris k. Initially, MTHFR 677 C T genotypes proportions were compared to one another between cases and controls (CC, CT, and TT). There was a pattern for the T allele at the MTHFR 677 position in the ca se group but was not significant which warrants

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68 further exploring. When the MTHFR 677 (CT/TT) genotype groups were combined in one group, a significantly higher number of cases with either of these genotypes were found compared to controls. Findings that are consiste nt with this observati onal study include those of Wenstrom et al. who reported that CHD risk was associated with the combined MTHFR 677 (CT/TT) genotypes compared to co ntrols (79). Van Beynum et al. found that the MTHFR 677 (CT/TT) genotypes of the mother, when combined with no use of periconceptional folic acid supplements, increased the risk for CHDs in offspring (142). Hobbs et al. reported that th ere was a higher risk for CHDs associated with the MTHFR 677 CC compared to the TT genotype, which is in contrast to the findings in the present pilot study and those of other investigat ors (89). A possible explanation for this inconsistency may be due to differences in folate st atus of the groups since a higher st atus is known to alleviate the metabolic abnormalities such as elevations in plasma homocysteine associated with this polymorphism (84). The results from Hobbs et al. have not been confirmed by other investigators and may be related to other risk f actors that may not have been controlled for in that study. An elevation in plasma Hcy concentr ation is an established risk factor for CHD and may be associated with the MTHFR 677C T polymorphism in women who delivered infants affected by CHD (79). The findings in the present pilot study are cons istent with the st udy by Junker et al. in which mothers carrying the MTHFR 677 TT genot ype were found to be at significantly increased risk for the development of structur al congenital heart malformations compared to mothers with the CC genotype (87). However, a meta-analysis by Verkleij-Hagoort et al. did not confirm that MTHFR 677 C T polymorphism is independently asso ciated with CHD risk (143). In addition, negative results were reported by ot her investigators con cerning the association

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69 between MTHFR 677 C T polymorphism and CHD risk (71,88). The inconsistency of these findings regarding the MTHFR 677 CT polymorphism might be due to the heterogeneity regarding population background, sample size, study design and the type of heart defects that complicates the pooling and comparison of the studies. Only a few studies have investigated th e combined effect of the MTHFR (677 C T and 1298 A C) polymorphism on CHD risk. The results of this study indicate that the MTHFR 1298 A C polymorphism was not associated with an increased risk of CHD unless it was combined with the MTHFR 677 CT genotype gr oup. Association between CHD and MTHFR 1298 AC was only observed when the MTHFR 1298 AC genotype group was combined with the MTHFR 677 CT genotype group. The frequenc y of MTHFR 1298 AC genotype was only higher in cases when it was combined with the MTHFR 677 CT genotype. The meta-analysis by Verkleij-Hagoort et al. concluded that the MTHFR polymorphisms 677 C T and 1298 A C in mothers are not independently associated with CHDs and further studies are needed (143). To address the third objective, which was to determine the interaction between folate and vitamin B12 related polymorphisms and status indicators on maternal CHD risk, comparisons between all genotype groups for the polymorphisms and status indicators were compared and no differences were detected. Othe r investigations that have evaluated these associations include Van Beynum et al. who reported that the mate rnal MTRR 66 GG genotype in combination with high MMA concentration (above the 80th percentile) was associated with a 3-fold increased risk for all types of CHD in the offspring (81). H obbs et al. did not find any of the biochemical indicators to be associated with increased CHD risk in conjunction with MTHFR C T polymorphism except for Hcy, which was defined as an independent risk factor (89). Their results indicated that maternal MTHFR 677 C T polymorphism did not have an independent

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70 impact on the estimated risk of having a CHD-aff ected pregnancy. Their data also indicated that CHD risk in the group with the combined CT and TT genotypes increased only with elevated Hcy and smoking and that the CC genotype had a gr eater increase in risk compared to the CT and TT genotypes. Vaughn et al. reported that MTRR 66 AA, AG and GG genotypes are associated with increased plasma Hcy concentration when combined with the MTHFR 677 TT genotype compared to other combinations of the MTHFR 677 C T and 1298 A G polymorphisms (93). Since the MTRR 66 GG genotype has previously been associated with elevated Hcy and elevation in Hcy has been shown to be associated with CHD in several studies, there is a plausible link between the presence of this pol ymorphism and CHD risk although an association was not observed in the presen t pilot study (93,144). Further analysis of the relationship between this polymorphism and the risk of CHD in this pilot study sugge sted a slightly lower incidence of MTRR 66 GG genotype in the cases (n=39) compared to the c ontrols (n=45). When the genotype frequency distribut ion was evaluated, more indivi duals with the MTRR 66 AA and AG genotypes were observed in cases group and more controls with the GG genotype (p= 0.09) unlike what was observed and repor ted by Van Beynum et al. (81). These findings suggest that MTRR 66 GG ge notype may have a protective effect reducing CHD risk. A potential explanation for this observation is that the MTRR 66 GG genotype may impair the utiliza tion of the methyl donor from the 5-MTHF resulting in less folate entering this irreversible pathway for making 5-MTHF, which would shunt this coenzyme toward the nucleotide and DNA synthesis part of the cycle. It is possible however, that these results are coincidental and that more controls by chance have the MTRR 66 GG genotype.

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71 It is also important to cons ider that due to study design w eaknesses it is not possible to thoroughly assess the interaction between genotype and folate/vitamin B12 status. Further studies need to be conducted to provide a careful assessment of status at the appropriate time during gestation or within a very short time following the index pregnancy to allow a more accurate measure of status indicators that are likely to interact with polymorphisms affecting folate and vitamin B12 status. The primary purpose of this pilot study was to establish an infrastructure for future welldesigned studies to determine the association between maternal folate and vitamin B12 status and related environmental and gene tic factors on CHD risk. It is critical to identify the study limitations so that improvements can be made in the design of future large-scale studies to evaluate similar objectives. One study limitation relates to pregnancy i ndex, which is an important consideration when considering biochemical profile because stat us indicators can change over time in response to dietary and supplement intake. This retros pective study was designed to include women who had previous pregnancies that could be char acterized as either case or control. No comprehensive exclusion criterions were used to se lect the subjects. As a result, subjects were recruited regardless of the period of time be tween the index pregnancy and the time the blood sample was drawn for analysis of folate, vita min B12 and homocysteine concentrations. To determine the effect of folate and/or vitami n B12 status on risk of CHD during the index pregnancy, maternal blood samples must be co llected in a timely manner following delivery of the affected infant (case) or normal infant (cont rol) since changes in blood indices may change significantly following delivery of the infant due to factors including alterations in diet, supplement use, and drug use. For the major ity of study participants, the index pregnancy

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72 occurred more than five years prior to the ti me that the blood sample was drawn. Previous studies designed to assess the association be tween status indicator s and CHD risk used pregnancy indexes of ~17 and 24 months from the time of delivery to the time of blood collection and ascertainme nt of information related to supplement use and environmental exposure (73,137). Another limitation of not excl uding subjects whose index pre gnancies were > 3 years is the inability for participants to recall significant de tails associated with th e pregnancy. In this study, the index pregnancy occurred > 5 years (average 11.8 years) from the time that the blood sample was drawn and questionnaire informati on was obtained. Since the data collected by questionnaire were highly de pendent on memory recall, the long period of time between the index pregnancy and time of study introduced probable error and bias in the subjects responses For example, some of the questions included in the questionnaire requested at least one of the following: supplement brand, frequency of use, a nd dosages, questions that require very exact recall, which becomes increasingly difficult over time. For this reason the data obtained in this study may have been biased, inaccurate, and incomp lete due to issues rela ted to poor recall Selection of the type of questions to ask is a critical part of any study. The questionnaire used in the study (Appendix A) was not develope d to address the objectives of the study. The primary problems associated with the questionnaire include the f act that the questions were insufficient to obtain essential information requi red to address the study objectives and the fact that the data collection was incomplete. For ex ample, questions relate d to medical conditions prior to pregnancy, drugs use, and supplement use had low response rates. In addition, some questions were inaccurately answered (e.g. a que stion was answered with a yes while the response requested was the supplement dose). The fact that the questionnaire was self-

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73 conducted could have led to the low and inaccurate response rate. To obtain detailed and more accurate and detailed information, the answers to study questions should be obtained by trained personnel in an interview situ ation. Reviewing medical file s is another way to ensure completeness and accuracy of answers, which is critical to obtaining valid results. The collection of demographic and background information was a weakness of this study as the data collected for each subject were very limited. For example, no data were collected on marital status, educational level, employment stat us, weight and height (BMI), family and/or offspring history of congenital malformations, and number of prior pregnancies. The lack of these data limits the ability to describe the sample population and to either control for or determine the association of these factors with risk for C HD. Even though the questionnaire included questions about employment and fathers ethnicity, data were not collected. Reviewing medical records and having trained personal ask the questions could have improved the response rate. The data on supplement use both during the i ndex pregnancy and at the time of blood collection were poorly asked and collected. Questi ons on supplement use are very critical in this type of study since supplements may have had a significant effect on fo late and vitamin B12 status indicators. Large amount s of data regarding the prenat al and folic acid intake from supplements were missing. One factor that ma y have reduced the amount of data obtained related to prenatal supplement use was the le ngth of time that had elapsed from the index pregnancy to the time of the study reducing the ability of the subjec ts to recall this information. The supplement questions requested information rela ted to specific brands, the exact time of use during pregnancy, whether the subject switched th e type of supplement used or not, and the name of the later brand. In addition, the categories provided as an answer to specify the

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74 frequency of supplement use were very c onfusing (yes, no, infrequently, sometimes and routinely). This could be improved by limiting the number of categorical answers to yes, no, and sometimes and/or providing numerical values fo r each category for example: routinely can be described as (2-3 times a week). Another weakness of this study was the fact that no data were obtained related to consumption of folate or vitamin B12 and folic ac id or B12-fortified foods since dietary intake may have significantly impacted status. A prim ary objective of this st udy was to determine whether there was a relationship between low con centrations of blood folate and/or vitamin B12 and an increased risk for CHD, which makes it im portant to assess the inta ke of these nutrients from both supplements and dietary sources. Me dical conditions are cr itical when assessing biochemical status and risk for congenital ma lformations since medical conditions and/or medications used to treat their conditions asso ciations might explain and/or indicate unusual concentrations of biomarkers in the blood. Ho wever, in this study no data were collected on medical conditions prior to pregnancy, and only limited data were available during pregnancy Cigarette and alcohol consumption are risk factors for various malformations (145), however, there were very low response rates rega rding the use of substan ces. This is a limitation in the study because assessing risk of CHD requi res comprehensive analysis of all possible elements, especially the ones w ith known risk associations. Summary of Pilot Study Design Limitation This pilot study presented an opportunity to identify key study design issues that can be addressed in future studies. Lim itations included (1) restricted exclusion criteria; (2) large pregnancy index range; (3) low response rate; (4) large numb er of questions that depended heavily on memory recall; (5) cr itical responses were missing; (6 ) self-administrated test; (7) inconclusive questionnaire; and (8) sm all sample size especia lly after adjusting for

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75 pregnancy index. Improvement for future stud ies should include an exclusion criterion for pregnancy index that limits the number of years between delivery and sample collection. A more detailed and comprehensive questionnaire that would include dietar y intake, and drug and supplement use related questions should be admini stered to participants, which would minimize the number of unknown variables that may affect the outcomes of the study, thus enabling researchers to draw better conclu sions related to the effects of bot h folate and vitamin B12 status on CHD risk. Trained personnel to supervise the administration of the questionnaire and collection of data would minimize errors and increase response rate in future studies. Last but not least, sample size must be considered especi ally in observational stud ies due to their nature and design complexity. In this st udy, the initial sample size was si milar to other studies, but after correcting for pregnancy index, it became very small (73,138). Public Health Significance of Research The prim ary goal of this retrospective observational pilot study was to provide a framework for future studies in the area of folate and vitamin B12 and related genetic polymorphisms and CHD risk. Conge nital heart defect-related studi es that involve modifiable factors are critical to further minimize the li mits surrounding the intervention efforts to reduce the occurrence of CHD. This initial pilot study provides data for future studies and preliminary findings to support future grant requests for funding large scale well -designed studies to investigate the association between folate and vi tamin B12 in the etiology of CHDs. Future studies should correct for the limitations inherent in this pilot study to allow for more definitive results and conclusions to be drawn. Future st udies are needed to advance our understanding of CHD etiology and the risk factors associated with it. Effective intervention studies designed to reduce the risk of CHD will depend on the out come of future well-designed studies.

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76 APPENDIX QUESTIONNAIRE Folic acid study: mother s questionnaire (All answers will be kept strictly confidential) Name: ______________________________________________________________ Date of Birth: ________________________________________________________ Height: __________________ Weight: ___________________________________ Childs name: _____________ Date of Birth: ____________________________ Reason child is seen by pe diatric cardiologist: ______________________________ ____________________________________________________________________ Childs birth weight: __________ Full Term or Preterm How many weeks: ____________________________________________________ Gestational age when born: _____________________________________________ Any malformations or syndromes for th is child? Y N if so, what? ____________________________________________________________ Your ethnicity: African-American Asian Hispanic Native-American White Other: _____________________________________________________________ Fathers occupation: __________________________________________________ Please answer the following questions about your pregnancy with this child: Any significant health problems prior to your pregnancy? Y N if so, what? ___________________________________________________________________ Did you take supplements prior to your pregnancy? Y N Which ones? ________________________________________________________ How long did you take them prior to your pregnancy? _______________________

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77 Did you take a prenatal vitami n? Y N Infrequently Sometimes Routinely How far along into the pregnancy were you when you started taking the prenatal vitamins? _____________________________________________________________ Brand name: ______________________________________________________ Did you take the same ones throughout the whole pregnancy? Y N ___________ Did you switch to a different pre-natal vitamin during the pregnancy and which one: _________________________________________________________________ Did you take additional folic aci d? Y N Infrequently Sometimes Routinely What was the dose? _________________________________________________ Did you take other nutritional supplement or herbs? Y N Which ones? _______________________________________________________ Did you develop diabetes during the pregnancy? Y N Was insulin used? Y N Any seizures before or during your pregnancy? Y N if so, what were you treated with: Did you develop anemia during your pregnancy? Y N if so what were you treated with: Please check if you took any of the fo llowing medications during your pregnancy: __Aspirin __Ibuprofen (Advil, Motrin) __Anti-depressants: _________________________________ __Anti-seizure medication: ___________________________ __Diabetes medicine: ________________________________ __Other: __________________________________________ How many cigarettes did you average per day during pregnancy? ____ packs/day How much alcohol did you drink during pregnancy? ___________________________

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78 How much coffee/tea/chocolate/caffeinated soft drinks did you drink during your pregnancy? _________________________________________________________________ Did you drink or eat products containing Aspa rtame (Nutrasweet, no sugar added or diet on the label)? Y N Infrequently Sometimes Routinely Did you use any other artificial sw eeteners? Y N Type: _______________________ Were you on a special diet during your pregnancy? Y N Type: __________________ Family History: Any family history of a heart defect from birth? Y N if so, what? ______________________________________________________________________ How many pregnancies have you had?_______________________________________ Number of miscarriages? _________________________________________________ How many other children do you have? ____________ Ages: ___________________ Congenital heart defects in your other children? ________________________________ Any other malformations or syndromes for your other children? Y N if so, what? ______________________________________________________________________ Any health problems for your other children? _________________________________ Are you planning to have more children? Y N Undecided May we contact you in the future? Y N Do you want to be contacted with your lab results? Y N Phone no: _____________________________________________________________ Thank you for your assistance!

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79 LIST OF REFERENCES 1. Bailey, L. & Gregory, J. (2006) Folate. In: Present knowledge in nutrition, 9th ed. (Bowm an, B. & Russell, R., eds.), pp. 278-301. ILSI Press, Washington, D.C. 2. Stokstad, E. L. R. (1990) Historical perspect ive on key advances in the biochemistry and physiology of folates. In: Folic acid metabolism in health and disease (Picciano, M. F., Stokstad, E. L. R., Gregory, J. F. & eds., eds.), pp. 1-15. Wiley-Liss, New York. 3. Carmel, R. & Jacobson, D. (2001) Homocyst eine in Health and Disease. Cambridge University Press, Cambridge, UK. 4. Gregory, J. F., 3rd (1997) Bi oavailability of folate. Eur J Clin Nutr 51 Suppl 1: S54-59. 5. Sauberlich, H. E., Kretsch, M. J., Skala, J. H ., Johnson, H. L. & Taylor, P. C. (1987) Folate requirement and metabolism in nonpregnant women. Am J Clin Nutr 46: 1016-1028. 6. Pfeiffer, C. M., Rogers, L. M., Bailey, L. B. & Gregory, J. F., 3rd (1997) Absorption of folate from fortified cereal-grain products and of suppl emental folate consumed with or without food determined by using a dual-label stable-iso tope protocol. Am J Clin Nutr 66: 1388-1397. 7. Persad, V. L., Van den Hof, M. C., Dube, J. M. & Zimmer, P. (2002) Incidence of open neural tube defects in Nova Scotia after folic acid fortificati on. Can Med Assoc J 167: 241-245. 8. Lopez-Camelo, J. S., Orioli, I. M., da Graca Du tra, M., Nazer-Herrera, J., Rivera, N., Ojeda, M. E., Canessa, A., Wettig, E., Fontannaz, A. M. et al. (2005) Reduction of birth prevalence rates of neural tube defects af ter folic acid fortification in Chile. Am J Med Genet A 135: 120125. 9. Botto, L. D. & Correa, A. (2003) Decreasing the burden of congenital heart anomalies: an epidemiologic evaluation of risk factors and su rvival. Progress in Pediatric Cardiology 18: 111121. 10. Botto, L. D., Olney, R. S. & Erickson, J. D. (2004) Vitamin supplements and the risk for congenital anomalies other than neural tube defects. Am J Med Genet C Semin Med Genet. 125C: 12-21. 11. Bailey, L. B. & Berry, R. J. (2005) Folic acid supplementation and the occurrence of congenital heart defects, orofacial clefts, multiple births, and miscarriage. Am J Clin Nutr 81: 1213S-1217. 12. Henderson, G. B. (1990) Folate-binding proteins. Annu Rev Nutr 10: 319-335. 13. Shane, B. (1989) Folylpolyglutamate synthe sis and role in the regulation of one-carbon metabolism. Vitam Horm 45: 263-335.

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80 14. Hoppner, K. & Lampi, B. (1980) Folate levels in human liver from autopsies in Canada. Am J Clin Nutr 33: 862-864. 15. Scott, J. M. (1986) Catabolism of folates. In: Folates and pterins (Bla kley, R. L., Whitehead, V. M. & eds., eds.), pp. 307-327. John Wiley & Sons, New York. 16. Whitehead, V. M. (1986) Pharmacokinetics and physiological disposition of folate and its derivatives. In: Folates and pter ins (Blakley, R. L., Whitehead, V. M. & eds., eds.), pp. 177-205. John Wiley & Sons, New York. 17. Klee, G. G. (2000) Cobalamin and Folate Ev aluation: Measurement of Methylmalonic Acid and Homocysteine vs Vitamin B12 and Folate. Clin Chem 46: 1277-1283. 18. Stabler, S. P. (2001) Vitamin B12. In: Pres ent Knowledge in Nutrition (Bowman, B. A., Russell, R. M. & eds., eds.), pp. 230-240. ILSI Press, Washington, D.C. 19. Watanabe F, T. S., Kittaka-Katsura H, Eb ara S, Miyamoto E (2002) Characterization and bioavailability of vitamin B12-compounds from ed ible algae. J Nutr Sci Vitaminol (Tokyo) 48: 325-331. 20. Watanabe, F. (2007) Vitamin B12 Sources an d Bioavailability. Experimental Biology and Medicine 232: 1266-1274. 21. Festen, H. P. (1991) Intrinsic factor secr etion and cobalamin absorption. Physiology and pathophysiology in the gastroin testinal tract. Scand J Gast roenterol Suppl 188: 1-7. 22. Quadros, E. V., Regec, A. L., Khan, K. M., Quadros, E. & Rothenberg, S. P. (1999) Transcobalamin II synthesized in the intestinal villi facilitates transfer of cobalamin to the portal blood. Am J Physiol 277: G161-166. 23. Allen, R. H. (1976) The plasma transport of vitamin B12. Br J Haematol 33: 161-171. 24. Hase, Y. (1998) [Disorders of transsulfuratio n; disorders of sulfur-aminoacids metabolism]. Ryoikibetsu Shokogun Shirizu: 205-218. 25. Leclerc, D., Wilson, A., Dumas, R., Gafuik, C., Song, D., Watkins, D., Heng, H. H., Rommens, J. M., Scherer, S. W. et al. (1998) Cloning and mapping of a cDNA for methionine synthase reductase, a fl avoprotein defective in patients with homocystinuria. Proc Natl Acad Sci U S A 95: 3059-3064. 26. Wilson, A., Platt, R., Wu, Q., Leclerc, D., Ch ristensen, B., Yang, H., Gravel, R. A. & Rozen, R. (1999) A common variant in methionine synt hase reductase combined with low cobalamin (vitamin B12) increases risk for sp ina bifida. Mol Genet Metab 67: 317-323. 27. Stabler, S., Lindenbaum, J., Savage, D. & Allen, R. (1993) Elevation of serum cystathionine levels in patients with cobalamin and folate deficiency. Blood 81: 3404-3413.

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91 144. Barbosa, P. R., Stabler, S. P., Machado, A. L ., Braga, R. C., Hirata, R. D., Hirata, M. H., Sampaio-Neto, L. F., Allen, R. H. & Guerra-S hinohara, E. M. (2007) Association between decreased vitamin levels and MTHFR, MTR an d MTRR gene polymorphisms as determinants for elevated total homocysteine concentrati ons in pregnant women. Eur J Clin Nutr. 145. Shaw, G. M., Nelson, V., Carmichael, S. L., Lammer, E. J., Finnell, R. H. & Rosenquist, T. H. (2002) Maternal periconceptional vitamins: inte ractions with selected factors and congenital anomalies? Epidemiology 13: 625-630.

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92 BIOGRAPHICAL SKETCH Younis was born in Kuwait. After completing high school in Kuwait, he went to the US to earn his Bachelor of Science in food scienc e and hum an nutrition from California State University Fresno in 2005. Afte r graduating in 2005, Younis went back to Kuwait and worked for the Kuwait health system. Younis worked as a weight and disease management consultant. He was also granted a part time job at Kuwait University to work as a teaching assistant in the Nutrition Department before he was awarded a scholarship to pursue graduate studies. In 2006, Younis was accepted into the University of Flor idas nutritional sciences Master of Science degree program. Younis plans to pursue his academic career and earn his PhD in nutritional sciences to later become a faculty member in the Nutrition Department at Kuwait University.