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The Effectiveness of Cornstarch Therapy Treatment in Individuals with Glycogen Storage Disease Type VI

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Title:
The Effectiveness of Cornstarch Therapy Treatment in Individuals with Glycogen Storage Disease Type VI
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Hernandez, Alejandra M.
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English

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Cholesterols ( jstor )
Cornstarch ( jstor )
Exons ( jstor )
Gels ( jstor )
Gene therapy ( jstor )
Glycogen ( jstor )
Glycogen storage diseases ( jstor )
Liver ( jstor )
Standard deviation ( jstor )
Triglycerides ( jstor )
Cornstarch
Glycogen storage disease
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Undergraduate Honors Thesis

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Abstract:
Sustenance of blood glucose concentrations is imperative to provide the brain with energy. Blood sugar levels are maintained through the metabolism of glycogen, a polymer of glucose. Glycogen Storage Disease (GSD) is a genetic enzymatic disorder affecting the pathway of glycogen metabolism. GSD Type VI is due to a deficiency of glycogen phosphorylase and is associated with hyperketosis, hypoglycemia, growth retardation and hepatomegaly. We intend to prove that when these patients are given cornstarch therapy similar to that titrated for other GSD types, their liver function tests, lipid panels, growth velocity, and hepatomegaly all improve significantly over time. Patients were recruited from the GSD Program at the University of Florida. Diagnosis of GSD VI was confirmed for all subjects by sequencing of the glycogen phosphorylase gene (PYGL). Following attainment of informed consent, clinical and biochemical parameters were analyzed, revealing a significant reduction in markers of metabolic control, improvement in growth and reduction in liver size. Adversely, there was a significant increase in weight. These results support the hypothesis and indicate beneficial effects of cornstarch therapy in individuals with GSD Type VI. Future studies are warranted to determine whether improved biochemical control will result in fewer complications and improved final height. ( en )
General Note:
Awarded Bachelor of Science; Graduated May 4, 2010 magna cum laude. Major: Interdisciplinary Studies, Emphasis/Concentration: Integrative Biology -Preprofessional. College/School: College of Liberal Arts and Sciences
General Note:
Awarded Bachelor of Science; Graduated May 4, 2010 cum laude. Major: Food Science and Human Nutrition, Emphasis/Concentration: Nutritional Sciences. College/School: College of Agricultural and Life Sciences
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Advisor: Dr. David Weinstein
General Note:
Legacy honors title: Only abstract available from former Honors Program sponsored database.

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University of Florida
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University of Florida
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Copyright Alejandra M. Hernandez. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.

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1 The Effectiveness of Cornstarch Therapy Treatment in Individuals with Glycogen Storage Disease Type VI by Alejandra Mandisa Hernandez Bachelor of Science i n Integrative Biology University of Florida 2010 SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIA L FULFILLMENT OF THE REQUIREMENTS FOR HIGH/HIGHEST HONORS IN BIOLOGY AT THE UNIVERISTY OF FLORIDA MARCH 2010 2010 Alejandra M. Hernandez All rights reserved. The author hereby grants to the University of Florida permission to reproduce and to distrib ut e publicly paper and electronic copies of this thesis document in whole or in part in any medium now known or hereafter created Author ______________________________________________________________ Alejandra Hernandez Department of Biology Wednesday, March 24, 2010 Ce rtified by __________________________________________________________ David A. Weinstein M.D., M.M.Sc. Associate Professor of Pediatrics College of Medicine Thesis Supervisor Accepted by _________________________________________________ _________ Bernard A. Hauser Ph.D. Associate Professor of Zoology Undergraduate Coordinator Department of Biology

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2 The Effectiveness of Cornstarch Therapy Treatment in Individuals with Glycogen Storage Disease Type VI B y Alejandra Mandisa Hernandez SUB MITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMEN TS FOR HIGH/HIGHEST HONORS IN BI OLOGY AT THE UNIVERISTY OF FLORIDA ABSTRACT Sustenance of blood glucose concentr ations is imperative to provide the brain with energy. Blood sugar levels are maintained through the production and breakdown of glycogen, a polymer of glucose and the main storage of glucose in the body. Glycogen Storage Disease (GSD) is a genetic enzymatic disorder affecting the pathway of glycogen production and break down. GSD Type VI is due to a deficiency of glycogen phosphorylase and is a ssociated with hyperketosis, hypoglycemia, growth retardation and hepatomegaly Current literature suggests merely diet ary intervention is all that is required to combat these sympt oms However, we intend to prove that when these patients are given cornstarch therapy similar to that titrated for other GSD types, their liver function tests, lipid panels, growth velocity, and hepatomegaly all improve significantly over time. Patient s were recruited from the Glycogen Storage Disease Program at t he University of Florida. Since types VI and IX GSD are phenotypically similar, the diagnosis of GSD VI was confirmed for all subjects by direct sequencing of th e glycogen phosphorylase gene ( PYGL). Following attainment of informed consent, the clinical and biochemical parameters were analyzed. Analys is of these values revealed a significant reduction in markers of metabolic control including triglyceride AST, ALT Improvement in growth was n oted, and there was a significant reduction in liver size. While biochemical and physical markers of control improved, there was also a significant increase in weight with aggressive therapy. These results support the hypothesis and indicate beneficial e ffects of cornstarch therapy treatment in individuals with GSD Type VI. Due to the rarity of GSD Type VI there are very little data available concerning this disease Future studies are warranted to determine whether improved biochemical control will re sult in fewer complications and improved final height.

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3 TABLE OF CONTENTS Page # 0 3 .. 0 4 Introduc tion 6 Methods .. 1 0 Confirmation of GSD VI by Sequencing of the PYGL Gene 10 Assessment of Clinical Response to Cornstarch Therapy 13 Statistical Analysis 13 Results 1 4 Genetic Confirmation of GSD VI 1 4 Clinical Response to Cornstarch Therapy 14 Discussion 16 18 19 ... ... 21 Appendix A: .. 21 Appendix B: Graphs Illustrating C120 ... 26 Appendix C: Graphs Illustrating D067 .. 31

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4 LIST OF FIGURES Page # 07 08 21 21 22 22 23 23 24 24 25 26 26 27 27 28 28 29 29 30 31 31 32 32 33 33 34 34

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5 LIST OF TABLES Page # .. 0 8 .. 11 .. 11 .. 14 .. 15 15 .. 15

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6 INTRODUCTION The body requires an imm ense amount of energy on a daily basis. Much of the energy needed is provided by glucose, a basic carbohydrate monomer. When blood glucose levels are high, and more energy than needed is available, the body stores this monomer for f uture use (Berg, Tymoczko, & and Stryer, 2002) Adversely, when blood glucose levels are low, and thus not providing the body with the sufficient energy needed to function, the body converts the stored glucose back to glucose for use by the body. The polymer storage form of glucose is termed glycogen and is found in the muslces, kidney, and liver (Berg, Tymoczko, & and Stryer, 2002) Muscle lacks glucose 6 phosphatase, however, and therefore cannot contribute to maintenanc e of glucose concentrations during fasting (Champe, Harvey, & Ferrier, 2008, p. 121) The process of breaking down and storing glucose is a complex process and controlled by numerous enzymes. Glycogen Storage Disease (GSD) is a collection of inherited metabolic disorders that affect the breakdown and storage of glycogen. There are numerous types of GSD, each pertaining to a deficiency of a specific enzyme in glycogen synthesis or breakdow n (The Association for Glycogen Storage Disease, 2006) Figure 1 explores the pathways of glucose storage and glycogen breakdown, specifying the enzymes required at each point in the pathway and the G SD types associated with each enzyme in the case of def iciency or mutation.

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7 Lysosome \ IN LIVER In Brain & Muscle In Blood Lysosome In Brain & Muscles In Blood Glycogen Glucose 1,4 Glucosidase ( GSD II ) Branching Enzyme ( GSD IV ) Glycogen Synthase Debranching Enzyme ( GSD III ) Glucose 1 Phosphate Gluc ose 6 Phosphate ( GSD I ) Glucose 6 Phosphate Transporter ( GSD IB ) Phosophoglucomutase Glucose 6 Phosphate Glucose Fructose 6 Phosphate Phosofructokinase ( GSD VII ) Liver Phosphorylase ( GSD VI ) Muscle Phosphorylase ( GSD V ) Glycogen Fructose 1,6 Phosphate UDPG 2 ( GSD VIII and IX, X linked ) Figure 1 : Glycogen Storage and Breakdown Pathways

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8 Figure 2 : Glycogen Storage and Breakdown Pathway Tabl e 1 provides a list of the known Glycogen Storage Disease Types that effect the liver and the ir resp ective enzymes Table 1 : Glycogen Storage Disease Types and Their Corresponding Affected Enzyme (Kishnan i, Koeberl, & Chen, 2009) Disease Type Enzyme Affected Type 0 Glycogen Synthase Type I a Glucose 6 phosphatase Type Ib Glucose 6 phosphate t ransporter Type IIIa Glycogen d ebranch ing enzyme (liver and muscle) Type IIIb Glycogen debranching enzyme (l iver only) Type VI Glycogen p hosphorylase Type IX Hepatic phosphorylase k inase Type XI Glucose Transporter 2 (GLUT 2) Glycogen Storage Disease Type VI is a result of mutations in the PYGL gene which encodes for the hepatic enzyme glycogen phosphorylase (Dagli & Weinstein, 2009) A deficiency in this enzyme results in impaired mobilization of g lucose from glycogen (Ierardi Curto, 2008) GSD VI generally manifests in childhood as poor growth, developmental dealy, and hepatomegaly (Dagli & Weinstein, 2009) A ffected patients tend to show signs of hyp erketosis, hypoglycemia, hyperlipidemia, and hepatic transaminase elevation (Ierardi Curto, 2008)

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9 The symptoms that GSD Type VI patients experience are similar to those of individuals with GSD Typ e IX (hepatic glycogen phosphorylase kinase deficiency) (Dagli & Weinstein, 2009) T he two types are clinically indistinguishable since impairment of phosphorylase kinase results in abnormal glycogen phosphorylase activity (Dagli & Weinstein, 2009) Hence proper diagnosis is best done by extrac ting DNA gathe red from patients, reviewing the gene re sponsible for each disease type and looking for mutations within that gene The gene responsible for GSD Type VI termed PYGL, consists of 20 exons; two mutations within this gene (termed a heterozygous mutation) or a single homozygous mutation would indicate having GSD Type VI (Dagli & Weinstein, 2009) Despite the symptoms associated with Type VI, t he enzymatic deficiency seen in in this type of Glycogen Storage Disease is not as sev ere as other types, because people with GSD VI can utilize protein and fat for energy (Weinstein & Woldsdorf, 2003, p. 100) (Refer to Figure 2 ). In addition, ketones produced from fatty acid oxidation can be used during peri ods of hypoglycemia as an alternative fuel limiting brain damage and seizure activity (Stafstrom, 2006, p. 91) Because the se symptoms are usually mild, current treatment recommendations for GSD Type VI merely suggest diet alteratio n rather than the more aggressive treatment methods used to treat other types of GSD including cornstarch therapy (Ierardi Curto, 2008) While the brain is protected in GSD as a result of ketone formation, accumu lation of ketones from counterregulation can have untoward effects. High concentrations of ketones can result in ketoacidosis and recurrent vomiting (American Diabetes Association) Chronically elevated ketones can impair bo ne mineralization due to osteoblastic dysfunction possibly causing osteopenia and osteoporosis (Arnett, Brandao Burch, Orriss, & Utting, 2005 p. 167 ; Deutschmann, Kotanko Skrabal, Weger, & Weger, 1999 pp. 325 326 ) Impaired growth also occurs in the set ting of systemic acidosis resulting in short stature (McSherry, 1978, p. 349) Cornstarch is a complex polymer of glucose that is digested by pancreatic amylase in the small intestine. Due to the complex structure of this starch, cornstarch i s digested over the course of about 4 6 hours thereby maintaining blood glucose concentrations during periods of fasting (Weinstein & Woldsdorf, 2003, p. 98) In other forms of GSD, cornstarch has been used to prevent hypoglycemia and counterregulation, and it is hypothesized that treatment with cornstarch in GSD VI will prevent hypoglycemia, lower hyperlipidemia, decrease liver size, and improve growth (Weinstein & Wo lfsdord, 2002)

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10 METHODS Patients enrolled in the Glycogen Storage Disease Program at the University of Florida showing symptoms characteristic of GSD Type VI were recruited for these investigations. All studies were approved by the institutional review board and the General Clinical Research Center scientific advisory committee of the University of Florida. Informed consent and assent of minors were obtained for each participant. Confirmation of GSD VI by Sequencing of the PYGL Gene: Due to the simila rities GSD Type VI and GSD Type IX, genetic testing was performed to confirm each patient with GSD Type VI. DNA was extracted buccal cells obtained in a saliva sample or from a direct buccal swab. The freshly obtained saliva was transferred fro m the fiel d container to a sterile 15 mL tube. Orangene Corporation Purifier was added to the tube (1/25th the volume of spit was the volume of Orange Purifier given; for example, 80 L was added for 2 mL of spit sample). The sample was then placed on ice for 10 mi minutes. After centrifugation, the clear supernatant was transferred with a clean pipette to a new 15 mL centrifuge tube; the remaining pellet was discarded. Ethanol was added in the same volume as the supernatant present. This sample was mixed by inversion 10 times and then sat on the bench top for 10 minutes. Next, the sample was centrifuged for 10 minutes at 3000 rpm and the supernatant was removed by rotating and pouring off the t ube. The tube was flipped and tilted at an angle and the remaining ethanol evaporated for 5 10 minutes. After the ethanol h ad evaporated, 1 mL of DNA rehydrating solution was added. The sample was then placed in the Vortex at 4 rpm for 30 seconds and subse quently placed in the heating block for 1 hour at 50 degrees. Over the hour in the heating block, the sample was rem oved for vortexing after 30 minutes and 50 minutes. The heated sample was then cooled to room temperature over a minimum period of 1 hour. The sample was then prepared to be placed in the Peltier Thermal Cycler (PTC) to undergo Polymer ase Chain Reaction (PCR) The following components were added to a 250 L PCR tube in the following order: 35 L double deoxidized water, 5 L PCR buffer, 1 L each of forward and reverse primer (both specific to the GSD Type and exon as outlined in Table 2 1 L dNTP, 5 L Template DNA and .5 L of TAQ Polymerase. Immediately upon adding TAQ Polymerase, the sample was loaded into the PTC and the machine was set to r un at specific conditions as outlined in Table 3

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11 Table 2 : PYGL Primers Used for the Polymerase Chain Reaction Primer Exons Primer Exons 1F* Exon 1 11F Exon 11 1R* Exon 1 11R Exon 11 2F Exon 2 12F Exon 12 2R Exon 2 12R Exon 12 3F Exon 3 13F Exon 13 3R Exon 3 13R Exon 13 4F Exon 4 14F Exon 14 4R Exon 4 14R Exon 14 5F Exon 5 15F Exons 15 & 16 5R Exon 5 15R Exons 15 & 16 6F Exon 6 17F Exon 17 6R Exon 6 17R Exon 17 7F Exon 7 18F Exon 18 7R Exon 7 18R Exon 18 8/9F Exons 8 & 9 19F Exon 19 8/9R Exons 8 & 9 19R Exon 19 10F Exon 10 20F Exon 20 10R Exon 10 20R Exon 20 Table 3 : PYGL Polymerase Chain Reaction Heating Conditions Exon (s) Heat 1 66C (or 63C) 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 19 56C 13 58C 5, 14, 18, 20 60C for further denaturing, then lowered to 50 renaturation. This 1.5 minute process of denaturing, annealing and renaturing occurs 30 times allow ing for the DNA to be greatly amplified. After 30 cycles

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12 sample was removed from the machine. In o rder to assess the success of DNA ampli fi cation and purification of the product, the PCR product then underwent gel electrophoresis, a process that separates DNA based upon size. Prior to preparing the samples for gel electrophoresis, a 2% Agarose gel was cre ated. T he gel box was prepared by inserting a gel comb and two gel plates and set aside until the gel mixture was ready. To make the gel, 2 g of Agarose was weighed and placed in a 250 mL flask. 100 mL 1X TBE buffer was added to the flask. This mixture was microwaved until the liquid ge latinized. Then, 0.4 L of ethidium b romide was added to the flask and swiveled to mix the Ethidium Bromide with the gelatinous mixture. The mixture was then poured into the gel box and sat for thirty minutes to harden fo r use. While the gel was hardening, the samples (that had recently underwent PCR) were prepared for gel electrophoresis. Tubes were obtained and labeled, one for each sample. 5 L of PCR product was inserted into its respective tube. To each tube, 1 L of gel loading dye was added and the contents of the tube were mixed gently with the pipette tip. This was repeated until all PCR samples were prepared for gel electrophoresis. After all samples had been prepared and the g el had hardened, the gel box was fil led nearly to the top with 1X TBE. The first well was loaded with 2 L DNA ladder (DNA ladder is used as a comparison to the injected DNA sample, providing an expected progression of electrophoresis and indicates the size of the replicated DNA ). Then, 5 L of each PCR product was loaded in the wells. All of this was noted in the lab notebook, especially the order i n which the samples were added. Electrophoresis was performed with a 110 voltage current. Once the samples had reached the middle of the gel, t he electrophoresis machine was turned off. The gel was removed from the gel box and transferred to a UV camera machine. Here, a photo was taken of the gel. The UV light allows for fluorescence of the ethidium bromide that binds to the DNA allowing the DNA to be visualized. A copy of this photo was placed in the lab notebook Once it was determined that a successful PCR reaction had occurred, the PCR products were purified. This process starts by labeling fresh tubes specific for purification (spin columns ); each sample is designated one tube. 450 L PBI Buffer was added to each PCR product in its original container and this new mixture was mixed using a pipette. The mixed PCR product plus PBI Buffer was then transferred to the spin columns. Next, the spin columns were centrifuged at 8000 rpm for .5 minutes; the flow thru created from this centrifugation was discarded and the bottom tube of each spin column was replaced. 750 L PE

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13 Buffer was then added to the spin columns and they were centrifuged twice for .5 min at 8000 rpm; after the first centrifugation, the flow thru was discarded and bottom tube replaced, however, after the second, the bottom tube was discarded with the flow thru liquid. The upper part of the spin columns were then placed into 1.5 mL Ep itubes and 50 L EB Buffer was added. Making sure that the caps were properly aligned, the spin columns were centrifuged for .5 min at 8000 rpm. After the final centrifugation, the spin columns were discarded and the remaining liquid in the Epitubes was pu rified DNA product. 10 L of and reverse primers were sent to sequencing. Sequencing also required a great deal of paperwork and the photo taken earlier for proof of DNA. T he s results were printed and compared to that of the expected sequences of each exon. Differences in the sample from t he known DNA indicated for compared with databases of known benign polymorphis ms. Two errors within the entire gene or a single homozygous mutation were deemed supportive of the diagnosis of GSD Type VI. Assessment of Clinical Response to Cornstarch Therapy: After permission was obtained from the IRB and Compliance office, review of clinical information from the patient medical records was performed. Physical parameters assessed included height, weight, height standard deviation score, weight standard deviation score, and liver size. Laboratory markers of biochemical control asse ssed included the following: aspartate aminotransferase (AST), alanine transaminase (ALT), cholesterol, triglyceride, and creatine kinase (CK). In order to assess the impact of treatment, data were obtained in a l ongitudinal way The final value was used for statistical analysis, but all values are included in the Figures included in the Appendix. Statistical Analysis: The clinical information obtained from patient medical records was exposed to statistical analysis. The mean and standard deviation for AS T, AL T, cholesterol, triglyceride, CK height standard deviation score, weight standard deviation score and liver size were determined using a 2007 Excel Spreadsheet Program Additionally, two tailed t s core values were determined for each value to evalua te the significance of our findings.

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14 RESULTS Genetic Confirmation of GSD VI: Genetic analysis was performed on 24 of patients with ketotic forms of glycogen storage disease. Of these GSD type VI was confirmed in 6 subjects, but longitudinal data wer e not available for two of these subjects. The genetic abnormalities from the four available subjects are depicted in Table 4 : Table 4 : Mutation Analysis Results Patient Mutation A118 exon 2: R94Stop exon 20: I806L B 119 exon 20: Y821H homozygous C 120 exon 2: R94Stop exon 16: D634H D0 67 exon 15: G607D and G608S E150 exon 1: T 71P exon 16: D634H Of these mutations, all but the D634H mutation are novel mutations, but are predicted based upon the changes to be pat hologic. It is noteworthy that exons 15, 16, and 20 where 5 of the 6 newly found mutations are located are known to frequent mutations in people with GSD type VI. Clinical Response to Cornstarch Therap y : edical files was obtained. Because patient B119 was not complying with clinical recommendations of cornstarch therapy, his data was removed from the statistical analysis. Thus, t he clinical information of A118, C120 and D067 w as subjected to statistical an alysis and the mean and standard deviation of pre treatment and post treatment values of AST, ALT, CK, triglycerides and cholesterol were determined. These values are displayed in Table 5 and Table 6 To determine the significance of our results, the pre treatment and post treatment values were also subjected to a two tailed t score test. These v alues are displayed in Table 7. Clinical data obtained indicating individual clinical response to cornstarch therapy overtime can be seen in Appendix A, Appendix B and Appendix C.

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15 Table 5 : Normal, Pre Treatment and Post Treatment Lipid Panels and Liver Functioning Tests Normal Pre Treatment Mean and Standard Deviation Post Treatment Mean and Standard Deviation AST 15 46 U /L 85.33 17.67 36.00 11.00 ALT 8 36 U /L 86.00 33.42 29.67 5.69 CK 30 170 U/L 115.33 44.38 102 25.12 Triglycerides <150 mg/dL 365.33 146.66 139.33 24.17 Cholesterol <170 mg/dL 187.67 64.51 187.67 47.35 Table 6 : Pre trea tment and Post treatment Clinical Evaluation Values Clinical Evaluation Pre Treatment Mean and Standard Deviation Post Treatment Mean and Standard Deviation Height (cm) 89.07 5.45 114.73 6.35 Height SDS 1.19 1.07 0.26 1.55 Weight (kg) 14.10 1.15 24.67 4.55 Weight SDS 0.12 1.35 1.18 2.27 Liver Size (cm) 8.45 5.30 3.35 0.21 Table 7 : T Score Values Evaluating Significance of Pre treatment and Post Treatment Improvement Clinical Evaluation T Score Value AST 0.058 ALT 0.076 CK 0.419 Triglycerides 0.091 Cholesterol 1.000 Height SDS 0.187 Weight SDS 0.380 Liver Size 0.416

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16 DISCUSSION The results gathered strongly support the hypothesis; p roviding GSD Type VI pa tients with cornstarch t herapy significantl y improve s the values of AST, ALT triglyceride, height, height SDS and liver size over time. L iver function test values were greatly lowered over the time of treatment The mean AST and ALT values of patients A11 8, C120 and D067 decreased from 85.33 U/L and 86.00 U/L to 36.00 U/L and 29.67 U/L respectively. Looking specifically at A118 AST and ALT values dropped from 65 U/L and 49 U/L to 36 U/L and 25 U/L respec tively, over a four year period; these results can be viewed in Figure 3. In patient C 120, A ST and ALT values decreased from 94 U/L and 114 U/L to 47 U/L and 36 U/L respectively over a three year period, as shown in Figure 12. In patient D067, AST and ALT values decreased from 97 U/L and 95 U/L to 25 U/L and 28 U/L respectively over a six yea r period; these re sults can be viewed in Figure 21 These dramatic decreases in AST and ALT values indicate impr ovement in liver function and decrease in liver inflammation. Lipid pane l tests exhibited varied responses to cornstarch therapy. Triglycerides were perhaps the most affected lab value, gr eatly decreasing in all patients; the mean pre treatment and post treatment values are 365.33 mg/dL and 139.33 mg/dL, respectively. Specifically, A mg/dL o ver a four year period with cornstarch therapy as indicated by Figure 4 In patient C120, triglycerides decreased from 256 mg/dL to 136 mg/dL, over a four year period, as indicated by Figure 13 D0 67 showed an overwhelming decreas e as well, from 532 mg/dL to 165 mg/dL, over a six year p eriod, as indicated by Figure 22 Cholesterol levels in al l patients were less responsive to cornstarch therapy. A118 actually saw a slight increase in cholesterol b etween their initial blood draw at diagnosis and their most recent lab data, as indicated by Figure 5 C12 0 and D067 saw slight improvement of cholesterol levels pre and post treatment; their values decreased from 119 mg/dL and 247 mg/dL to 143 mg/dL and 229 mg/dL, respectively as illustrated in Figures 14 and 2 3 The cholesterol levels of these patients m ay be explained by familial genetics or dietary effects Food intake particularly of dietary fat, can create a temporarily high cholesterol reading. Thus, cholesterol values may not be the best indicators of lo ngitudinal lipid status. Creatine kinase levels exhibited varied responses to cornstarch therapy. A118 and C 1 2 0 both experienced a decrease in CK, however D067 experienced an increase. Creatine kinase is an enzyme in the muscle that aids in making energy r eadably available to the muscle cells Since GSD Type VI is an enzymatic de ficiency of liver phosphorylase, treatment geared towards alleviating a liver enzymatic deficiency may not affect muscle cells and the activity of their respective enzymes

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17 Growth w as an additional area of significant improvement in GSD Type VI individuals who received cornstarch therapy treatment. The mean height standard deviation for A118, C120 and D067 pre and post treatment improved from 1.19 and 0.26, respectively. The resul ts per individual are incredibly suppor tive, as indicated in Figures 7, 8, 16, 17, 25 and 26 A 118, for example, had a height SDS of 0.73 when first height SDS improved to 1.14, as indicated by Figu re 8. Additionally, C 120 improved from a height standard deviation score of 0.42 to 0.02 As indicated by Figure 17 C120 returned to negative height standard deviation scores after a year of cornstarch therapy and improved height SDS; these results can b e explained by the eds due to increased adolescent growth When suit his growth needs, his height standard deviation scores positively improved. results impress t hat t hough cornstarch therapy improves and normalizes blood glucose levels proper monitoring and altering of uncooked cornstarch therapy is required to maintain blood sugar s throughout periods of substantial growth. Without proper monitoring, improvement s in height SDS are less drastic, as a result of inadequate blood sugars. The weight standard deviation scores of the followed patients receiving cornstarch therapy did not show the same significant improvement as seen in height standard deviation scores. For patient A118 and D067 weight SDS values pre and post treatment increased from 0.87 and 1.44 to 3.72 and 0.67 respectively. Patients C120 showed slight improvement, from 0.92 to 0.50, respectively. Though biochemical control improves with cornstar ch therapy, this treatment method does so by adding calories to the daily diet; these extra calories may explain the noted increase in weight SDS in some participants. Additional factors such as genetics and environment may also explain our weight SDS resu lts. Liver size was also noted to decrease with cornsta rch therapy treatment. D067 exhibited the most compelling liver size data C120 12.2 cm in 2007 to 3.2 cm in 2009 A118 also illustrated a decrease in hepatomegaly liver size decreased from 4.7 cm in 2008 to 3.5 cm in 2009 having a 1.2 cm decrease. A118 s hepatomegaly seemingly decreased more than that of D067, however this is explicable by comparing the drastically different diagnostic liver sizes of A118 and D067; such values indicate that each patient significantly improved their liver size in relation to their individual liver size upon initial diagnosis. There are a variety of clinical assessments that could not be obtained from the patient medical files of the participants of this study. Diagnostic procedures such as liver pathology and bone density exam s were not performed, thus these values could not be included. It is important to note, however, that including these values in future studies could provide even greater insight on the effect cornstarch therapy has on the physical well being of Glycogen Storage Disease Type VI patients.

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18 Exploring this study, a few limitations can be noted such as the sample size and the limited data available from p atients. Due to the rarity of Glycogen Storage Disease Type VI, this study included a very small number of participants More partic ipants w o u l d have provided more data and could have improved the t score values. Further mutation analysis of individuals w ith all types of Glycogen Storage Disease could provide a larger pool of data that could be beneficial for future studies. Data for this study were l imited by how frequent patients visited for physical exams and blood draws. Consistent physical exams and b lood draws could provide improved statistical analysis. S tressing compliance through education of the patient on the benefits of achieving good metabolic control through regular visits and monitoring would lead to more data. A future study that gathered mo re confirmed GSD Type VI individuals could be of immense benefit to those that have this rare disease. A myriad of reasons have provided limited data on GSD Type VI including the prior dependence on liver biopsy for diagnosis, the relatively mild presentat ion which could lead to a misdiagnosis, the recent institution and widespread acceptance of DNA testing and prior misdiagnosis of Type VI and Type IX. Because there is very limited data available concerning Type VI, more comprehensive longitudinal studies analyzing the effect of cornstarch therapy on physical wel l being could provide h i g h l y u s e f u l i n f o r m a t i o n t o G S D T y p e V I p a t i e n t s CONCLUSION T hese investigations support our hypothesis, treatment with cornstarch in GSD T y p e VI will lower hyperlipidemia, decrease liver size, and improve growth. We have shown that cornstarch therapy treatment is indeed beneficial to individuals with Glycogen Storage Disease Type VI as it has improved AST, ALT, and triglycerid e levels in all patients treated. Additionally, height standard deviation scores greatly improved, along with a reduction in liver size. Though cholesterol levels appear to be less responsive to cornstarch therapy treatment, these results can be explained by the affect familial genetics and dietary intake have on cholesterol levels. S i m i l a r l y though creatine kinase values do not significantly improve, CK i s not a direct indicator of liver activity, the focus of GSD Type VI treatment. Furthermore, a dditional s tudies are warranted to understand the full implication of treatment, specifically evaluating the effect cornstarch therapy has weight status. Creation of a worldwide database of patients with G S D Type VI would be beneficial a l l o w i n g t h e e f f e c t o f c o r n s t a r c h t h e r a p y o n T y p e V I t o b e b e s t e x p l o r e d

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19 REFERENCES 1. American Diabetes Association (n.d.). Ketoacidosis (DKA). Retrieved March 28, 2010, from Living with Diabetes: www.diabetes.org/living with diabetes 2. Arnett, T., Brandao Burch, A., Orriss, I., & Utting, J. (2005). Acidosis Inhibits Bone Formation by Osteoblasts In Vitro By Preventing Mineralization. Calcified Tissue International 167 174. 3. Berg, J. M., Tymoczko, J. L., & and Stryer, L. (2002). Biology. New York: W. H. Freeman and Co. 4. Champe, P. C., Harvey, R. A., & Ferrier, D. R. (2008). Biochemistry. Baltimore: Lippincott Williams & Wilkins. 5. Dagli, A. I., & Weinstein, D. A. (2009, April 23). Glycogen Storage Disease Type VI. Retrieved March 23, 2010, from NCBI Gene Reviews: www.ncbi.nlm.nih.gov 6. Deutschmann, H., Kotanko, P., Skrabal, F., Weger, M., & Weger, W. (1999). Incomplete R enal Tubular Acidosis in 'Primary' Osteoporosis. Osteoporosis International 325 329. 7. Ierardi Curto, M. P. (2008, August 4). Glycogen Storage Disease Type VI. Retrieved March 21, 2010, from eMedicine: http://emedicine.medscape.com/ 8. Kishnani, P. S., Koeb erl, D., & Chen, Y. T. (2009, March). Chapter 71: Glycogen Storage Disease. Retrieved March 21, 2010, from The Online Metabolic & Molecular Bases of Inherited Diseases: http://www.ommbid.com/ 9. McSherry. (1978). Acidosis and Growth in Nonuremic Renal Diseas e. Kidney International 349 354. 10. Stafstrom, C. E. (2006). Ketones Keep Neurons Alive. Epilepsy Curr e n t 91 92. 11. The Association for Glycogen Storage Disease. (2006, October). "What is Glycogen Stoage Disease?". Retrieved March 21, 2010, from The Association for Glycogen Storage Disease: http://www.agsdus.org/html/whatisglycogenstoragedisease.html

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20 12. Weinstein, D. A., & Wolfsdord, J. I. (2002). Effect of Continuous Glucose Therapy with Uncooked Cornstarch on the Long term Clinical Course of Type 1a Glycogen Storage Disease. European Journal of Pediatrics 35 39. 13. Weinstein, D. A., & Woldsdorf, J. I. (2003). Glycogen Storage Diseases. Reviews in Endocrine & Metabolic Disorders 95 102.

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21 APPENDICES Appendix A. Graphs Clinical Response to Cornstarch Therapy Figure 3 : AST and ALT Values for A 118 from 2005 2009 Figure 4 : Triglyceride Values for A 118 from 2005 20 09 0 20 40 60 80 100 120 1 2 3 4 5 6 7 8 9 10 11 12 U/L Collection Date A118 AST and ALT Values from 2005 2009 ALT AST 0 50 100 150 200 250 300 350 1 2 3 4 5 6 7 8 9 10 11 Triglycerides mg/dL Collection Date A118 Triglyceride Values from 2005 2009 TRI

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22 Figure 5 : Cholesterol Values for A 118 from 2007 2009 Figure 6 : CK Values for A118 from 2007 2009 0 50 100 150 200 250 1 2 3 4 5 6 7 8 9 Cholesterol mg/dL Collection Number A118 Cholesterol Values from 2007 2009 Cholesterol 0 50 100 150 200 1 2 3 4 5 6 CK (U/L) Collection Date A118 CK Values from 2006 2009 CK (U/L)

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23 Figure 7 : A 118 Height from 2005 2009 Figure 8 : A 118 Height Standard Deviation Score from 2005 2009 0 20 40 60 80 100 120 140 1 2 3 4 5 6 7 8 Height (cm) Collection Date A118 Height (cm) from 2005 to 2009 Height (cm) 1 0.5 0 0.5 1 1.5 1 2 3 4 5 6 7 8 Height SDS Collection Date A118 Height SDS from 2005 2009 Height Standard Deviation Score

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24 Figure 9 : A118 Weight from 2005 2009 Figure 10 : A118 Weight SDS from 2005 2009 0 5 10 15 20 25 30 1 2 3 4 5 6 7 8 Weight (kg) Collection Date A118 Weight from 2005 2009 Weight (kg) 0 0.5 1 1.5 2 2.5 3 3.5 4 1 2 3 4 5 6 7 8 Weight SDS Collection Date A118 Weight SDS from 2005 2009 Weight SDS

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25 Figure 11 : A118 Liver Si ze from 2008 2009 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 1 2 3 Liver Size (cm) Collection Date A118 Liver Size (cm) from 2008 2009 Liver Size (cm)

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26 Figure 12 : C120 AST and ALT Values from 2006 2010 Figure 13 : C120 Triglyceride V alues from 2006 2010 0 50 100 150 200 250 1 2 3 4 5 6 7 8 9 10 11 U/L Collection Date C120 AST and ALT Values from 2006 2010 ALT AST 0 50 100 150 200 250 300 1 2 3 4 5 6 7 8 9 10 Triglycerides (mg/dL) Collection Date C120 Triglyceride Values from 2006 2010 Triglycerides

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27 Figure 14 : C120 Cholesterol Values from 2006 2009 Figure 15 : C120 CK Values from 2007 2009 0 20 40 60 80 100 120 140 160 180 1 2 3 4 5 6 7 8 9 Cholesterol (mg/dL) Collection Date C120 Cholesterol Values from 2006 2010 Cholesterol 0 50 100 150 200 250 1 2 3 4 5 6 7 8 CK (U/L) Collection Date C120 CK Values from 2007 2009 CK

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28 Figure 16 : CD120 Height from 2007 2010 Figure 17 : C120 Height SDS from 2007 2010 0 20 40 60 80 100 120 1 2 3 4 5 6 Height (cm) Collection Date CD120 Height from 2007 2010 Height 0.5 0.4 0.3 0.2 0.1 0 0.1 0.2 0.3 0.4 1 2 3 4 5 6 Height SDS Collection Date C120 Height SDS from 2007 2010 Height SDS

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29 Figure 18 : C120 Weight from 2007 2010 Figure 19 : C120 Weight SDS from 2007 2010 0 5 10 15 20 25 1 2 3 4 5 6 Weight (kg) Collection Date C120 Weight from 2007 2010 Weight 0 0.2 0.4 0.6 0.8 1 1.2 1 2 3 4 5 6 Weight SDS Collection Date C120 Weight SDS from 2007 2010 Weight SDS

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30 Figure 20 : C120 Liver Size from 2007 2009 0 2 4 6 8 10 12 14 1 2 3 4 5 6 Liver Size (cm) Collection Date C120 Liver Size (cm) from 2007 2009 Liver Size (cm)

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31 Figure 21 : D067 AST and ALT Values from 2003 2009 Figure 22 : D 067 Triglyceride Values from 2003 2009 0 50 100 150 200 250 1 2 3 4 5 6 7 U/L Collection Date D067 AST and ALT Values from 2003 2009 ALT AST 0 100 200 300 400 500 600 1 2 3 4 5 6 7 8 Triglycerides (mg/dL) Collection Date D067 Triglyceride Values from 2003 2009 Triglycerides

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32 Figure 23 : D067 Cholesterol Values from 2003 2009 Figure 24 : D067 CK Values from 2006 2009 0 50 100 150 200 250 300 1 2 3 4 5 6 7 8 Cholesterol (mg/dL) Collection Date D067 Cholesterol Values from 2003 2009 Cholesterol 0 10 20 30 40 50 60 70 80 90 100 1 2 3 4 5 CK (U/L) Collection Date D067 CK Values from 2006 2009 CK (U/L)

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33 Figure 25 : D067 Height from 2006 2009 Figure 26 : D067 Height SDS from 2006 2009 104 105 106 107 108 109 110 111 112 113 1 2 Height (cm) Collection Date D067 Height (cm) from 2006 2009 Height (cm) 3 2.5 2 1.5 1 0.5 0 1 2 Height SDS Collection Date D067 Height SDS from 2006 2009 Height SDS

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34 Figure 27 : D067 Weight from 2006 2009 Figure 28 : D067 Weight SDS from 2006 2009 0 5 10 15 20 25 30 1 2 Weight (kg) Collection Date D067 Weight from 2006 2009 Weight (kg) 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 1 2 Weight SDS Collection Date D067 Weight SDS from 2006 2009 Weight SDS