Group Title: BMC Medical Genetics
Title: Protective effect of KCNH2 single nucleotide polymorphism K897T in LQTS families and identification of novel KCNQ1 and KCNH2 mutations
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Title: Protective effect of KCNH2 single nucleotide polymorphism K897T in LQTS families and identification of novel KCNQ1 and KCNH2 mutations
Physical Description: Book
Language: English
Creator: Zhang, Xianqin
Chen, Shenghan
Zhang, Li
Liu, Mugen
Redfearn, Sharon
Bryant, Randall
Oberti, Carlos
Vincent, G. M.
Wang, Qing
Publisher: BMC Medical Genetics
Publication Date: 2008
 Notes
Abstract: BACKGROUND:KCNQ1 and KCNH2 are the two most common potassium channel genes causing long QT syndrome (LQTS), an inherited cardiac arrhythmia featured by QT prolongation and increased risks of developing torsade de pointes and sudden death. To investigate the disease expressivity, this study aimed to identify mutations and common variants that can modify LQTS phenotype.METHODS:In this study, a cohort of 112 LQTS families were investigated. Among them two large LQTS families linkage analysis with markers spanning known LQTS genes was carried out to identify the specific gene for mutational analysis. All exons and exon-intron boundaries of KCNH2 and KCNQ1 were sequenced for mutational analysis.RESULTS:LQTS-associated mutations were identified in eight of 112 families. Two novel mutations, L187P in KCNQ1 and 2020insAG in KCNH2, were identified. Furthermore, in another LQTS family we found that KCNH2 mutation A490T co-segregated with a common SNP K897T in KCNH2. KCNH2 SNP K897T was reported to exert a modifying effect on QTc, but it remains controversial whether it confers a risk or protective effect. Notably, we have found that SNP K897T interacts with mutation A490T in cis orientation. Seven carriers for A490T and the minor allele T of SNP K897T showed shorter QTc and fewer symptoms than carriers with A490T or A490P (P < 0.0001).CONCLUSION:Our family-based approach provides support that KCNH2 SNP K897T confers a protective effect on LQTS patients. Our study is the first to investigate the effect of SNP K897T on another KCNH2 mutation located in cis orientation. Together, our results expand the mutational and clinical spectrum of LQTS and provide insights into the factors that determine QT prolongation associated with increased risk of ventricular tachycardia and sudden death.
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BMC Medical Genetics BiolledCentral


Research article


Protective effect of KCNH2 single nucleotide polymorphism K897T
in LQTS families and identification of novel KCNQ I and KCNH2
mutations
Xianqin Zhangt1,2, Shenghan Chent2, Li Zhang3, Mugen Liu2,
Sharon Redfearn4, Randall M Bryant4, Carlos Oberti2, G Michael Vincent3
and Qing K Wang*1,2


Address: 'Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human
Genome Research, Huazhong University of Science and Technology, Wuhan, Hubei 430074, PR China, 2Department of Molecular Cardiology,
Lerner Research Institute, Cleveland Clinic, and Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western
Reserve University, Cleveland, Ohio 44195, USA, 3Department of Internal Medicine, LDS Hospital and University of Utah School of Medicine,
Salt Lake City, Utah 84103, USA and 4Department of Pediatric Cardiology, University of Florida Health Science Center, Jacksonville, FL, USA
Email: Xianqin Zhang zhangx3@ccf.org; Shenghan Chen chens@ccf.org; Li Zhang Li.Zhang@intermountainmail.org;
Mugen Liu lium@mail.hust.edu.cn; Sharon Redfearn sharon.redfearn@jax.ufl.edu; Randall M Bryant randy.bryant@jax.ufl.edu;
Carlos Oberti Obertic@ccf.org; G Michael Vincent G.Michael.Vincent@intermountainmail.org; Qing K Wang* wangq2@ccf.org
* Corresponding author tEqual contributors



Published: 23 September 2008 Received: 16 March 2008
BMC Medical Genetics 2008, 9:87 doi:10.1 186/1471-2350-9-87 Accepted: 23 September 2008
This article is available from: http://www.biomedcentral.com/1471-2350/9/87
2008 Zhang et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.ore/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: KCNQI and KCNH2 are the two most common potassium channel genes causing long QT
syndrome (LQTS), an inherited cardiac arrhythmia featured by QT prolongation and increased risks of
developing torsade de points and sudden death. To investigate the disease expressivity, this study aimed
to identify mutations and common variants that can modify LQTS phenotype.
Methods: In this study, a cohort of I 12 LQTS families were investigated. Among them two large LQTS
families linkage analysis with markers spanning known LQTS genes was carried out to identify the specific
gene for mutational analysis. All exons and exon-intron boundaries of KCNH2 and KCNQI were sequenced
for mutational analysis.
Results: LQTS-associated mutations were identified in eight of I 12 families. Two novel mutations, L 187P
in KCNQI and 2020insAG in KCNH2, were identified. Furthermore, in another LQTS family we found that
KCNH2 mutation A490T co-segregated with a common SNP K897T in KCNH2. KCNH2 SNP K897T was
reported to exert a modifying effect on QTc, but it remains controversial whether it confers a risk or
protective effect. Notably, we have found that SNP K897T interacts with mutation A490T in cis
orientation. Seven carriers for A490T and the minor allele T of SNP K897T showed shorter QTc and
fewer symptoms than carriers with A490T or A490P (P < 0.0001).
Conclusion: Our family-based approach provides support that KCNH2 SNP K897T confers a protective
effect on LQTS patients. Our study is the first to investigate the effect of SNP K897T on another KCNH2
mutation located in cis orientation. Together, our results expand the mutational and clinical spectrum of
LQTS and provide insights into the factors that determine QT prolongation associated with increased risk
of ventricular tachycardia and sudden death.



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Background
Long QT syndrome (LQTS) is an inheritable cardiac ion
channelopathy characterized by prolonged QT intervals
and characteristic T wave morphology changes on electro-
cardiograms (ECG). Patients with LQTS have increased
risks of developing life-threatening polymorphic ventricu-
lar tachycardia/fibrillation (VT/VF) when subjected to
physical or emotional stress, or upon exposure to QT pro-
longing drugs. VT/VF can result in cardiac events such as
syncope, cardiac arrest and sudden death [1]. LQTS-
related cardiac events often occur in the young, otherwise
healthy individuals, and sudden death may present as the
first symptom in many cases.

Multiple genes have been found to cause LQTS and these
include KCNQ1 (LQT1), KCNH2 (LQT2), SCN5A
(LQT3), ANK2 (LQT4), KCNE1 (LQT5), KCNE2 (LQT6),
KCNJ2 (LQT7), CACNAIC (LQT8), CAV3 (LQT9) and
SCNB4 (LQT10). LQT4-10 are rare and there is a contro-
versy as whether some of the subtypes are truly LQTS.
LQT1 and LQT2 remain the mainstay as the most com-
mon LQTS genotypes. Previously, we reported a study of
mutational analysis of KCNQ1 in 102 families and
patients with a family history of lethal cardiac events,
including LQTS [2]. The present study was carried out as a
continued investigation in the same cohort of patients
plus 10 new LQTS families/patients enrolled since the ear-
lier report, and focused on mutational analysis of the
KCNQ1 gene in the new families/patients and the LQT2
gene, KCNH2 in the whole cohort. Multiple mutations
were identified. Most importantly, we provide the first
family-based genetic evidence to show that two variants in
KCNH2, mutation A490T and a common single nucle-
otide polymorphism (SNP) K897T, interact with each
other in cis orientation to reduce the risk of LQTS (shorter
QTc, less severe symptoms).

Methods
Study subjects
Patients with a clinical diagnosis of LQTS or idiopathic
VT/VF and their first degree blood related family members
were enrolled from medical clinics in North America. A
total 154 families/patients were involved in this study.
This study was approved by the Cleveland Clinic Founda-
tion Institutional Review Board on Human Subject
Research and informed consent was obtained from the
study participants or their guardians.

Clinical examinations
Diagnosis of LQTS was based on the QT interval and T
wave morphology from 12-lead ECG and medical history
of syncope, cardiac arrest and sudden death as described
previously [3-7]. All the 12-lead ECG tracings available to
the study were recorded at 25 mm/sec as standard in
North America. QT interval was manually measured in


leads II or V5 or the lead with longest QT interval and QTc
was calculated using Bazett's formula for heart rate correc-
tion. A LQTS clinical diagnosis was considered if QTc >
0.47 s in males or QTc > 0.48 s in females [8]. Borderline
QT prolongation (0.45-0.47 s) associated with symptoms
were also considered as affected. Asymptomatic family
members with QTc of <0.44 s and with a normal T wave
morphology were phenotyped as non-affected. Other
individuals were classified as having uncertain phenotype
with regard to LQTS.

For the purpose of LQTS diagnosis, we used a stationary
bike ergometerr) exercise protocol as described previ-
ously[9]. Superior to treadmill tests, ergometer tests elim-
inates the upper body movement to reduce ECG artifacts.
An accurate QT measurement therefore can be achieved
for exercise ECG recordings. 24-h Holter ECG monitoring
was performed using Lifecard CF Digital Holter Recorder
(Renolds Medical). LQTS phenotyping including ECG
measurement was performed by G.M.V. and R.B. prior to
the genetic testing.

For the bike test, we recorded standard 12-lead ECG in a
paper speed of 25 mm/sec with voltage calibrated as 1 mV
= 10 mm (or 10 mm/mV) for the baseline in supine and
sitting positions, and exercise (3, 6, 9, 12, 15 minutes)
and recovery (1, 3, 5 and 8 minutes) stages. QT interval
was measured in the lead with the longest QT interval
(mostly in lead II or V5) as described previously[9].

Isolation of human genomic DNA
Each study participant donated 5-20 ml of blood sam-
ples, which were used for isolation of genomic DNA using
the DNA Isolation Kit for Mammalian Blood (Roche
Diagnostic Co.)

Linkage analysis
For the two large families with LQTS, linkage analysis with
markers spanning known LQTS genes was carried out to
identify the specific gene for mutational analysis. Poly-
morphic microsatellite markers linked to LQTS genes
were selected from the ABI PRISM Linkage Mapping Set-
MD 10 panel for linkage analysis, including D11 S4046 for
KCNQ1, D7S798 for KCNH2, D3S1277 for SCN5A,
D4S402 for ANKB and D21S266 for KCNE1 and KCNE2.
Markers were genotyped using an ABI 3100 Genetic Ana-
lyzer with the standard protocols provided by Applied
Biosystems (Foster City, CA). Genotypes were analyzed
using the GeneMapper 2 Software program (Applied Bio-
systems, Foster City, CA).

Mutational and single-strand conformational
polymorphism (SSCP) analyses
Mutational analysis was carried out using direct DNA
sequence analysis. All exons and exon-intron boundaries


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of KCNQ1 and KCNH2 were amplified by polymerase
chain reaction (PCR) using primers previously published
[10-12]. PCR was performed in 10 tl of standard PCR
buffer containing 1.5 mM MgCl2, 0.2 mM of each dNTP,
0.5 gM of each primer, 0.1 unit of Taq DNA polymerase,
and 25 ng of DNA. The amplification program was one
cycle of denaturation at 94 C for 5 min, 35 cycles of 94 C
for 30 s, 620 C or other annealing temperatures for each
specific pair of primers for 30 s, and 72 C for 1 min, and
one 7 min extension step at 72 C. The PCR products were
purified using the QIAquick Gel Extraction Kit (Qiagen
Inc., Valencia, CA), and sequenced with both forward and
reverse primers. DNA sequencing analysis was performed
using the BigDye Terminator Cycle Sequencing v3.1 kit
(Applied Biosystems, Foster City, CA).

SSCP analysis was used to determine whether a mutation
co-segregated with the disease in the family and whether
the mutation was present in normal controls. SSCP anal-
ysis was carried out as described previously [5,6].

Statistical analysis
Statistical differences between groups were analyzed by
Student's t tests and were considered significant when P <
0.01.

Results
We investigated 112 families with a family history of
lethal cardiac events, including LQTS, for mutations and
common variants in KCNQ1 and KCNH2, the two most
common potassium channel genes associated with LQTS.
LQTS-associated mutations were identified in eight fami-
lies. No mutation was detected in the rest of 104 families.
We carried out mutational analysis in 112 families with a
family history of lethal cardiac events, including LQTS.
LQTS-associated mutations were identified in eight fami-
lies. No mutation was detected in the rest of 104 families.
The results are described below.

Identification of two co-segregating variants, K897T and
A490T of KCNH2 associated with LQTS in a mid-size
family
Family QW2648 is a LQTS family with 11 family mem-
bers (Fig. 1A). Two family members, I: land 11:2, had QTc
of 0.47 s or higher (Fig. 1A). Linkage analysis for family
QW2648 showed linkage to D7S798 near the LQT2 locus
(Fig. 1A). Direct DNA sequence analysis of the DNA from
the proband in the family identified two variants in
KCNH2: a heterozygous G->A transition at nucleotide
1468, which results in a substitution of amino acid resi-
due alanine by threonine (A490T) (Fig. 1B) and a hetero-
zygous A->C transition at nucleotide 2690, which results
in a substitution of amino acid residue lysine by threonine
(K897T) (Fig. IC). In the family, A490T co-segregated
with K897T, suggesting that the K897T and A490T vari-


ants are in the cis orientation on the same chromosome
(Fig. 1A). The normal family members did not carry either
of the two variants.

The clinical characteristics of seven mutation carriers in
family QW2648 are shown in Table 1. The maximum QTc
ranged from 0.43 s to 0.48 s (0.46 0.01) and the mini-
mum QTc was from 0.41 s to 0.47 s (0.43 0.01). Exercise
ECG testing was performed for three carriers (III-3, III-4,
and III-5), and showed that QTc was significantly pro-
longed compared to QTc before testing (P = 0.002).
Bradycardia, defined as a resting heart rate of under 60
beats per minute, was seen in five of seven carriers with an
average heart rate of 46 beats/min by resting ECG (Table
1). Holter monitoring in 111:4 and 111:5 showed 27 and 32
runs of bradycardia, respectively and the slowest rate was
34 beats/min. Syncopal episodes were reported in carriers
I-1 (once while on a high protein diet), II-3 (several at the
age of 11), and III-3 (once at the age of 17), but not in
other carriers. There was no family history of cardiac arrest
or of sudden cardiac death.

Identification of a novel KCNQ I mutation, L 187P, in a
large family
We identified and characterized a 7-generation LQTS fam-
ily with over 300 family members (QW1648, Fig. 2A).
Five affected members in the family had a history of syn-
cope. No aborted cardiac arrest was reported. One LQTS
related sudden death had occurred before LQTS diagnosis.

DNA samples for forty-two family members from this
large Utah family were available for genetic analysis. Link-
age analysis with five markers for known LQTS genes
revealed that the large Utah family was linked to
D11S4046 at LQT1 on chromosome 11pl5.5. The LOD
score reached 6.11 at a recombination fraction of 0, which
strongly suggests that the disease-causing gene in the fam-
ily is KCNQ1. DNA sequence analysis of the DNA from
the proband revealed a heterozygous T->C transition at
nucleotide 560 of KCNQ1, which results in a substitution
of amino acid residue leucine by proline (L187P) (Fig.
2B). The L187P mutation is located between domains S2
and S3 of KCNQ1.

To verify that the L187P mutation causes LQTS, SSCP
analysis was carried out for the 42 family members with
DNA samples. As shown in Fig. IC, all affected members
in the family carried the heterozygous mutation as dem-
onstrated by the presence of two bands, whereas the nor-
mal family members showed one band, the wild type
allele (Fig. 2C). The mutant SSCP band was not identified
in 200 normal control individuals. These results suggest
that the L187P mutation of KCNQ1 is a pathogenic muta-
tion that causes LQTS in the family.



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QW264


04-0.4 0-44
S 113 -- -

LO KOMANIT 4-
0.47 045 OJ39
II2I 111.31 1114 1 III 5.

*039 4 -
038 043 0.45 0.44


+ KCNH2 K897T SNP

-KCNH2 A490T MUTATION


G+A



1,
"_ .l^
-^~~~ -'-L


A+C


KCNH2 A490T

WLD TYE
GGC COC ATC GCC GTC CAC TAC TC AAG
0 R I A V H Y F K

MUTANT
GGC OGC ATC ACC GTC CAC TAC Tr AAG
O R I T V H Y F K





XNM KS7T
WHLD TYPE
,G~G OCC ACG GAC AAG GAC ACG GAG CAG CCA
tR T D K D T E Q P


0G0 COC ACO GAC ACO GAC ACO GAG CAO CCA
R R T D T D T E Q P


Figure I
Identification and co-segregation of KCNH2 mutation A490T and SNP K897T in family QW2648 and with
LQTS. (A). Pedigree structure of family QW2648. All the affected individuals carry double variants, K897T and A490T of
KCNH2 on the same chromosome (in cis orientation). (B, C). DNA sequence analysis for the proband in family QW2648. A
heterozygous G->A transition at nucleotide 1468 of KCNH2, which results in a substitution of alanine by threonine (A490T)
(B). DNA sequence for KCNH2 SNP K897T is shown in (C). (D) Comparison of mean QTc for carriers with KCNH2 mutation
A490T, A490P, and both A490T and SNP K897T.








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0-4
O44


014



04


0(1.2
OAiJ_________o_____








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Table I: Clinical characteristics of mutation carriers with KCNH2 A490T/K897T in family QW2648


Age (yr) Average HR
(beats/min)


67
66
61
56
46
55
61
58.86
22.25


Low HR
(beats/min)


N/A
N/A
43
53
44
44
47
46.20
1.55


Bradycardia
(runs/24 h x beats/
min)

N/A
N/A
N/A
N/A
N/A
27 x 34
32 x 34


High QTc (s) Low QTc (s) QTc exercise (s) Syncope (times)


0.48
0.47
0.47
0.45
0.43
0.45
0.44
0.456
0.007


0.43
0.47
0.42
0.44
0.43
0.42
0.41
0.431
0.007


N/A
N/A
N/A
N/A
0.49
0.48
0.50
0.49
0.006


I
0
multiple
0
0
0
0
0


Detailed clinical analysis revealed that the penetrance of
the L187P mutation was not 100%. In the family, 31 of 42
family members genetically characterized here were iden-
tified as mutation carriers (age 33 21 years, 13 females
and 18 males) with mean QTc of 0.468 0.022 sec and 11
of 42 members as non-carriers (age 30 22 years, 4
females and 7 males) with mean QTc of 0.426 + 0.019 sec
(Fig. 3). Typical LQT1 T wave patterns were present in
84% of affected members. Only 16% were symptomatic.
Among mutation carriers, 58% (18/31) had normal to
borderline prolonged QTc (<0.46 sec) on their initial
ECGs, which overlapped with non-carriers (Fig. 3). A bicy-
cle exercise test was performed for 24 mutation carriers
and 4 non-carriers. Mean QTc was significantly prolonged
during exercise in mutation carriers, whereas it remained
in the normal range in non-carriers (Fig. 3). All mutation
carriers with a normal or borderline prolonged QT inter-
val reached QTc of 0.48 sec or greater during exercise tests.

Identification of a novel 2-bp insertion mutation,
2020insAG in KCNH2
One novel mutation, a 2-bp insertion 2020insAG, was
identified in KCNH2 in family QW258 with LQTS (Fig.
4). The mutation was neither present in three normal fam-
ily members nor 200 controls. The proband in the family
was a 75-year-old female with a prolonged QTc of 0.596-
0.687 s and left bundle branch block (QRS = 0.122 s). The
patient developed a long run of polymorphic VT at the age
of 75 years, which was cardioverted. She also developed
two syncopal episodes at the age of 67 and 75 years, and
a transient ischemic attack at the age of 63. Telemetry Hol-
ter ECG monitoring identified frequent multiform ven-
tricular ectopic beats with occasional supraventricular
ectopy, no bradycardia, and 173 runs of polymorphic VT.
Echocardiography showed normal left and right ventri-
cles, ejection fraction of 40% and abnormal left ventricu-
lar relaxation (stage I diastolic dysfunction). EEG,
neurological and hearing tests, and troponin T and creat-
ine kinase-MB levels (0.01 ng/ml and 4.8 ng/ml, respec-
tively) were normal. Cardiac catheterization showed mild


luminal irregularities without obstructing lesions, and
mild global systolic left ventricular dysfunction consistent
with hypertensive cardiomyopathy.

Identification of five other mutations in KCNH2
DNA sequence analysis of the rest of families and patients
identified five previously-known mutations in the
KCNH2 gene. These mutations include A561T, D609N,
A614V, N629S and R366X http://www.ssi.dk/graphics/
html/lqtsdb/herg.htm. Patients with mutations A561T,
D609N, A614V, N629S all had a typical diagnosis of
LQTS. Interestingly, the LQTS patient with mutation
R366X also presented with bradycardia at the age of 30
years, torsade de points, recurrent syncopal episodes when
awakening, startling and sitting, and first degree AV block.

Discussion
In the present study, we carried out mutational analysis of
KCNQ1 and KCNH2, the two most common potassium
channel genes associated with LQTS in 112 families and
patients with a family history of lethal cardiac events,
including LQTS, Mutations were identified in 8 of 112
families. Interestingly, genotype-phenotype correlation
studies in family QW2648 showed that two variants in
KCNH2, mutation A490T and SNP K897T, interact with
each other in cis to reduce the risk of LQTS (shorter QTc,
less severe symptoms). As shown in Fig. 1, in LQTS family
QW2648, two variants in the KCNH2 gene, one common
SNP K897T and one mutation A490T, were identified.
Because both variants co-segregated or in linkage disequi-
librium in the family, they are located in the cis orienta-
tion on the same chromosome. A490T was previously
reported as a de novo mutation in a Japanese female who
showed markedly prolonged QTc of 0.61 s, bradycardia,
torsades de points and recurrent syncope. Functional stud-
ies revealed that the mutant channel showed reduced cur-
rent density by 39% compared to the wild type channel
[13]. In a sharp contrast, none of the seven mutation car-
riers with A490T and K897T showed a QTc of> 0.48 s. The
difference in QTc between A490T alone and combination



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1:1
11:2
11:3
111:1
111:3
111:4
111:5
Mean
SEM*


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NornnaI Letcne (CTC)


Patient Proi~ne (CCC)
Letiane (CTC)


K( NQImutati~on L187P
TC -r (-Cc
L P


Figure 2
Identification of a novel mutation, LI87P, in KCNQ I
in LQTS family QWI 648. (A). A partial pedigree struc-
ture for the large Utah family is shown. The family had over
300 members. For ease of illustration only a portion of the
pedigree is shown. Affected males and females are indicated
by filled squares and circles, respectively. Normal individuals
are shown as empty symbols. Individuals with uncertain
LQTS diagnosis are shown with gray symbols. Deceased indi-
viduals are shown using slashes. The identification numbers
for family members and QTc are shown below symbols. (B).
DNA sequence analysis revealed a heterozygous T->C tran-
sition at nucleotide 560 of KCNQI, which results in a substi-
tution of amino acid residue leucine by a proline residue
(LI 87P). The sequence for a normal family member is shown
on the top and the sequence for an affected family member is
shown below. (C). SSCP analysis showed co-segregation of
the mutation L I87P with the LQTS phenotype in the family.
All phenotypically affected members had two bands, and the
non-affected individuals had only one band.


of both A490T and K897T was notable (Fig. 3D). These
results suggest that the T allele of SNP K897T confers a
protective effect against QTc prolongation. On the other
hand, SNP K897T did not appear to exert any effect on
bradycardia because both A490T and A490T/K897T carri-
ers exhibited clear evidence of the bradycardia phenotype.
A mutation that changes A490 to P490 was recently
reported in a large family with nine LQTS patients by Pel-
legrino et al [14]. The penetrance of mutation A490P was
100% in the family and the phenotype of LQTS in the
family appears to be very severe. QTc ranged from 0.48 s


s I P <001


044
04B 056

I .hn QT E-1. QT-
n =31 n =11 L87PCarriers(n 24)









Before exercise QTc = 0.39 s






S. F.. 1 t .. T t .



15 min exercise QTc= 0.48 s

Figure 3
(A) Reduced penetrance of LQTS phenotype and
effect of exercise on QTc. Mean QTc for carriers with
mutation LI 87P and I I non-carriers (left) and mean QTc for
mutation carriers before (resting state) and during exercise
(right). (B, C) Example of reduced penetrance of LQTS phe-
notype at baseline. Initial ECG (B) and ECG after I5-min
exercise (C) are shown for a 29 year old, male, asympto-
matic mutation L 87P carrier.



to 0.57 s (mean QTc = 0.510 +/- 0.036), and five of the
nine patients showed QTc of >0.50 s. Recurrent syncope,
torsade de points, ventricular fibrillation, cardiac arrest,
and family history of sudden death were documented in
the family. The mean QTc between carriers with mutation
A490P in comparison and those with both A490T and
K897T was statistically significant (P = 0.0008, Fig. 3D).
Our study is the first family-based genetic investigation to
show that two variants in KCNH2, mutation A490T and
SNP K897T, interact with each other in cis orientation and
resulted in a mild LQTS phenotype.

Interestingly, no bradycardia was reported for mutations
carriers with A490P, suggesting that the effect of mutation
A490T on bradycardia may be mutation-specific. The
molecular mechanism for mutation-specific development
of bradycardia is unknown, but it is an interesting subject
that warrants future investigation.


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KCNH2 2020insAG


KCNH2 Forward Sequence


C, C :%' C TG TGTC TG C G G GTG C G G TG T; T
io'A 160 170
lion A


KCNH2 Reverse Sequence

CT CCTG TG TG T[ N
150 160 I


TN i 'NN


18 N
180


WILD TYPE
ACA GCC CGC TAC CAC ACA CAG ATG CTG CGG
T A R Y H T Q M L R


MUTANT
ACA GCC
T A


KCNH2 2020insAG

CGC TAC C ACA CAC AGA TGC TGC
R Y G T H R C C


Figure 4
Identification of a novel mutation in KCNH2, 2020insAG, in family QW258. Top, pedigree structure; Middle, DNA
sequence for the patient using the forward primer; Bottom, DNA sequence for the patient using the reverse primer.




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The modifying effect of KCNH2 SNP K897T has been
studied previously, but inconsistency exists among differ-
ent studies, mostly population-based studies. In this
study, we investigated the effect of SNP K897T on QTc
prolongation in multiple LQTS patients in a moderate-
size family. This family-based approach has the advantage
that the influence by environmental factors is more lim-
ited than a population-based approach. As described
above, our results indicated that the rare T allele of SNP
K897T has a protective effect against QTc prolongation
(Fig. 3D). This is consistent with the results from a recent
population-based study in 2,515 men and women
involved in the Framingham Heart Study. In September,
2007, Newton-Cheh et al. showed that the common K
allele of SNP K897T was associated with a 1.6 ms increase
of QTc per allele copy [15], although the effect was very
mild. Similarly, in 2005 in a population of 3,966 involved
in the German KORA S4 study, Pfeufer et al. found that
the rare T allele was associated with 1.9 ms and 3.5 ms
decreases of QTc in heterozygotes and homozygotes,
respectively [16]. In 2005, Gouas et al. studied a French
population with 200 subjects with short QTc (100 men
with QTc of 0.333-0.363 s and 100 women with QTc of
0.336-0.365 s) and 198 subjects with longer QTc (99 men
with QTc of 0.394-0.433 s and 99 women with QTc of
0.400-0.432 s) [17]. The rare T allele was significantly
more frequent in the group with short QTc than the group
with long QTc (odds ratio = 0.64, P = 0.006), suggesting
that the T allele was associated with shorter QTc. In 2003,
Bezzina et al. found that TT homozygotes had shorter QTc
than heterozygotes and KK homozygotes in a Caucasian
population of 1,030 from Netherlands [181. The associa-
tion of SNP K897T with shorter QTc was not without con-
troversy. In 2002, Pietila et al studied 267 women and 259
men from Finland and found that the rare T allele (KT +
TT) was significantly associated with longer maximum
QTc than the K allele (KK) in females only (0.441 s vs.
0.477 s, P = 0.005) [19]. In males, maximum QTc was
longer in the KK group than the KT + TT group (0.465 s vs.
0.447 s), though the difference was not statistically signif-
icant (the small sample size may account for the finding
of non-significance). In an Italian family Crotti et al. stud-
ied a small LQT2 family and reported a KCNH2 mutation
(All1116V) and the T allele of SNP K897T on the non-
mutant allele (i.e. in trans orientation) in one severe,
symptomatic LQTS patient, whereas 3 relatives with
Al 116V alone were asymptomatic and showed mild QTc
prolongation [20]. Thus, the T allele was associated with
increased QTc in the Italian family, which is contrary to
the results from our study. The cis-localization between
the mutation A490T and SNP K897T in our family vs.
trans-localization in the study by Crotti et al. may be one
of the potential causes for the discrepancy. Overall, our
results are more consistent with the finding that the minor
allele T of SNP K897T plays a protective role on QTc
lengthening.


There are some limitations for this study. First, the com-
parison of the mean QTc among carriers with A490T,
A490P, and A490T/K897T was made in individuals of dif-
ferent geographical origins under different clinical set-
tings. The accuracy of the analysis might be compromised
as the ethnic and environmental influences have not been
accounted for. Furthermore, the studied family was small.
Second, no modifying variants in SCN5A, KCNE1, and
KCNE2 were identified in the carriers with A490T/K897T
in family QW2648, however, we cannot exclude the pres-
ence of variants in other ion channels that may influence
QTc and other QTc-modifiers such as NOSAP1 variants
[21]. Third, an in vitro functional study to examine the
electrophysiological effects of the mutations or combina-
tions of mutations may provide an insight into the molec-
ular mechanism underlying the pathogenesis of LQTS.

In this study, we also identified two new mutations asso-
ciated with LQTS, L187P in KCNQ1 and a KCNH2 muta-
tion, the 2-bp insertion 2020insAG. The L187P mutation
is located in the cytoplasmic loop between transmem-
brane domains S2 and S3 of the KCNQ1 channel. Two
other mutations in the same area, G189R and R190Q,
were loss-of-function mutations when expressed in Xeno-
pus oocytes [22] Thus, the L187P mutation may also act by
a loss-of-function mechanism. However, the mild pheno-
type of this large LQT1 family with 58% presented with a
normal to borderline QT prolongation and only 16% of
gene careers had a history of cardiac events, indicating that
the functional outcome of L187P may be more compli-
cated than a simple loss-of-function mechanism. The 2-bp
insertion 2020insAG occurs at codon 674 and results in
frameshift that leads to premature termination of transla-
tion. The mutant protein contains amino acid residues 1
to 673 of KCNH2 and addition of a short peptide,
YQTHRCCGCGSSSASTRSPIPCASASRSTSSTPGPTPTASX.
Alternatively, the frameshift creates an internal stop
codon that leads to degradation of KCNH2 mutant mRNA
by non-sense-mediated mRNA decay (NMD). A recent
investigation that the NMD effect resulted in a mild phe-
notype in a large LQT2 family [23]. However, since >30%
of KCNH2 mutations are nonsense mutations that may
lead to NMD, a mutation that can trigger NMD effect may
not necessarily lead to a mild LQTS phenotype. The inves-
tigation of modifier effects has opened a new chapter for
us to better understand why some LQTS patients die sud-
denly and others do not. Genotype-phenotype correlation
and further molecular electrophysiology investigation
may help the risk stratification.

Conclusion
In summary, our study identified two novel mutations
causing LQTS, the L187P mutation in KCNQ1 and the
2020insAG mutation in KCNH2. Most interestingly, we
showed that the rare T allele of SNP K897T conferred a
protective effect on LQTS patients with KCNH2 mutation


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A490T located in cis orientation. Our study is the first to
investigate the effect of SNP K897T on another KCNH2
mutation located in cis orientation. Together, our results
expand the mutational and clinical spectrum of LQTS and
provide insights into the factors that determine QT pro-
longation associated with increased risk of VT and sudden
death.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
XZ and SC carried out genetic studies including linkage
and DNA sequence analysis in the cohort of LQTS families
and patients and controls, ML carried out linkage analysis
and SSCP. LZ, SR, RB, CO, GMV performed the clinical
characterization of the patients and family pedigree anal-
ysis. QKW supervised the study, obtained the funding,
and critically revised and approved the manuscript. All the
authors read and approved the final manuscript.

Acknowledgements
We are grateful to the family members for their enthusiastic participation
in this study. This work was supported by a China National 973 Program
grant, No. 2007CB512000 and No. 2007CB512002 and an NIH/NHLBI
grant, RO I HL66251 (to Q.K.W.), and Deseret Foundation Grant DF 400
(G.M.V.) and DF464 (L.Z.). Q.K.W. is an Established Investigator of the
American Heart Association (0440157N).

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