Download Common RyR2 variants associate with ventricular arrhythmias and

Clinical Science (2010) 119, 215–223 (Printed in Great Britain) doi:10.1042/CS20090656
Common RyR2 variants associate with
ventricular arrhythmias and sudden cardiac
death in chronic heart failure
Yuqin RAN∗ , Jingzhou CHEN†1 , Ning LI‡, Weili ZHANG†, Li FENG‡,
Rongrong WANG‡, Rutai HUI†, Shu ZHANG∗ and Jielin PU∗ ‡1
∗
Center for Arrhythmia Diagnosis and Treatment, Fu Wai Cardiovascular Hospital, Peking Union Medical College and Chinese
Academy of Medical Sciences, Beijing 100037, China, †Sino-German Laboratory for Molecular Medicine, Fu Wai Cardiovascular
Hospital, Chinese Academy of Medical Sciences, Beijing 100037, China, and ‡Pathology and Physiology Research Center, Fu
Wai Cardiovascular Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100037, China
A
B
S
T
R
A
C
T
Ca2+ cycling plays a critical role in heart failure and lethal arrhythmias. As susceptibility to sudden
cardiac death is considered to be a heritable trait in general population, we have therefore
investigated whether potentially functional variants of genes encoding RyR2 (ryanodine receptor
2) and the L-type Ca2+ channel are related to the risk of ventricular arrhythmias and sudden
cardiac death in CHF (chronic heart failure) in a case-control study. We found that the A allele of
rs3766871 in RYR2 was associated with an increased risk of ventricular arrhythmias in patients with
CHF {odds ratio, 1.66 [95 % CI (confidence interval), 1.21–2.26]; P = 0.002}. During a median
follow-up period of 32 months in 1058 (85.0 %) patients, 296 (28.0 %) patients died from heart
failure, of whom 141 (47.6 %) had sudden cardiac death. After adjustment for age, gender and
suspected risk factors, patients carrying the A allele of rs3766871 had an increased risk of cardiac
death {HR (hazard ratio), 1.53 [95 % CI, 1.11–2.12]; P = 0.010} and sudden cardiac death [HR,
1.92 (95 % CI, 1.25–2.94); P = 0.003]. Patients carrying the A allele of rs790896 in RYR2 had a
reduced risk of sudden cardiac death [HR, 0.65 (95 % CI, 0.45–0.92); P = 0.015]. In conclusion,
the A allele of rs3766871 in RYR2 not only associates with ventricular arrhythmias, but also serves
as an independent predictor of sudden cardiac death, and the A allele of rs790896 in RYR2 is a
protective factor against sudden cardiac death in patients with CHF.
INTRODUCTION
HF (heart failure) has become an increasingly prevalent
disorder and a leading cause of hospital admissions.
For individuals over 65 years of age, the prevalence
of HF is up to 10 % [1]. Ventricular remodelling, a
hallmark of HF, plays a key role in dysfunction of
the failing heart [2]. Ischaemic heart disease and dilated
cardiomyopathy are the most common structural heart
diseases in patients with HF. Clinical HF increases the
risk of SCD (sudden cardiac death) 5-fold, regardless
of the aetiology [3]. Susceptibility to SCD has been
considered to be a heritable trait in the general population.
More than ten genes have been linked to QT interval
Key words: calcium cycling, genetic variant, heart failure, sudden cardiac death, ventricular arrhythmia.
Abbreviations: ARVC, arrhythmogenic right ventricular cardiomyopathy; BMI, body mass index; BP, blood pressure; CACNA1C,
L-type Ca2+ channel α subunit; CI, confidence interval; DR3, divergent region 3; HF, heart failure; CHF, chronic HF; HR, hazard
ratio; LD, linkage disequilibrium; LV, left ventricular; LVEF, LV ejection fraction; MHR, mean heart rate; NYHA, New York
Heart Association; OR, odds ratio; RyR2, cardiac ryanodine receptor; SCD, sudden cardiac death; NSCD, non-SCD; SNP, single
nucleotide polymorphism; UTR, untranslated region; VA, ventricular arrhythmia.
1
These authors contributed equally to the study.
Correspondence: Dr Jielin Pu (email [email protected]).
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prolongation, which is an intermediate phenotype for
SCD and a surrogate marker of SCD risk [4]. Several
genes {such as HSPB7 (heat shock 27 kDa protein family,
member 7), FRMD4B (FERM domain containing 4B) [5],
GRK5 (G-protein-coupled-receptor kinase 5) [6], RYR2
(cardiac ryanodine receptor 2) and CNTN5 (contactin
5)[7]} and some variants (such as rs10757274 in 9p21
[8], rs740363 and rs10515869 [7]) in the specific regions
of chromosomes, but not in the known genes, have
been linked to HF in genome-wide association studies.
However, the contribution of individual risk factors to
SCD in HF is still incompletely understood.
RyR2, a Ca2+ -releasing channel protein located
in the sarcoplasmic reticulum membrane, plays a
crucial role in cardiac excitation–contraction coupling.
Diastolic Ca2+ leak through RyR2 is one of the most
serious pathophysiological problems in failing hearts.
Spontaneous Ca2+ release through RyR2 during diastole
not only decreases sarcoplasmic reticulum Ca2+ content,
but also induces delayed after-depolarization, which leads
to lethal arrhythmias [9].
In the myocardium, the opening of voltage-dependent
L-type Ca2+ channels following membrane depolarization results in the entry of a modest amount of Ca2+ that
induces Ca2+ release from the sarcoplasmic reticulum.
A large increase in intracellular Ca2+ concentration
ultimately initiates contraction of the heart. L-type Ca2+
channel function is altered in failing hearts [10], and
mutations in the L-type Ca2+ channel gene cause severe
arrhythmic disorders [11].
We therefore tested the hypothesis that potentially
functional genetic variants of the genes encoding the
RyR2 and the L-type Ca2+ channel may influence
the prognosis of CHF (chronic HF) in a case-controlled
study in a Chinese Han population. We prospectively
followed the CHF population to assess the predictive
value of these genetic variants in predicting SCD risk.
MATERIALS AND METHODS
Study population
From July 2005 to January 2008, consecutive patients
with CHF referred to Fu Wai Cardiovascular Hospital
were recruited. Inclusion criteria included: CHF caused
by ischaemic heart disease arising from coronary
artery disease or CHF caused by idiopathic dilated
cardiomyopathy; symptomatic HF with NYHA (New
York Heart Association) functional class II–IV despite
optimized medical therapy; and LV (left ventricular)
systolic dysfunction with an LVEF (LV ejection fraction)
50 % in ischaemic heart disease and 45 % in dilated
cardiomyopathy. HF severity was defined as mild (40 %<
LVEF 50 %), moderate (30 %< LVEF 40 %) or
severe (LVEF 30 %). Ischaemic heart disease was
defined as 70 % luminal stenosis of at least one major
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coronary artery diagnosed by coronary angiography and
a history of myocardial infarction more than 3 months
before enrolment. Idiopathic dilated cardiomyopathy
was diagnosed according to the guidelines for the study
of familial dilated cardiomyopathies [12]. Patients were
excluded if they had malignancies, other severe systemic
disease, pregnancy, unwillingness to participate in the
present study, or any missing clinical data.
Controls were selected from inpatients with coronary
artery heart disease, but without acute coronary syndrome, unstable angina pectoris, myocardial infarction,
HF or VAs (ventricular arrhythmias; 41.2 % of the
control group), and from community-based inhabitants
who underwent an annual health examination and were
free of structural heart disease and any type of cardiac
arrhythmias (58.8 % of the control group). The gender
and ages were matched with the cases, and the exclusion
criteria were the same as for cases.
The investigation conformed with the principles
outlined in the Declaration of Helsinki and was approved
by the Ethics Committee of Fu Wai Cardiovascular
Hospital (Beijing, China). All subjects who participated
in the study provided written informed consent and
reported themselves as being of Chinese Han nationality.
Clinical data and blood sample collection
Clinical data were collected after the participants were
hospitalized or during their annual physical examination.
All subjects underwent transthoracic echocardiography
to evaluate valves, atrial and ventricular chamber
dimensions, thickness of the ventricular walls and
interventricular septum, right and left ventricular
function, wall motion abnormalities and morphological
abnormalities. When possible, these evaluations included
cardiac magnetic resonance imaging, especially in patients
with dilated cardiomyopathy. At baseline, a 12-lead
ECG and 24-h Holter recording were obtained to
look for arrhythmias. In some patients, arrhythmias
were recorded while they were being monitored using
inpatient telemetry. VAs included sustained or nonsustained ventricular tachycardia, premature ventricular
beats (more than 3000 beats/24 h) and ventricular
fibrillation. In addition, a history of hypertension,
diabetes mellitus, hyperlipidaemia or atrial fibrillation,
as well as treatment strategies for these conditions, such
as drug, invasive and surgical therapies, were recorded.
Renal and liver function, and full blood counts were
routinely examined. All examinations were performed in
the central laboratory of our hospital. Blood samples for
genetic testing were collected during hospitalization or
the annual physical examination.
End point assessment
The study population was followed up for a median
period of 32 months until October 2009. A standard HF
questionnaire was completed either by telephone contact
Genetics and sudden cardiac death
or by mailing to family members of the patients, as well
as by consulting the records of the most recent hospitalization. The end points included cardiac death (heart
transplantation regarded as cardiac death) and SCD. SCD
was defined operationally as a witnessed natural death
attributable to cardiac causes heralded by an abrupt loss
of consciousness within 1 h of onset of acute symptoms,
or as an unwitnessed unexpected death of someone seen
in a stable medical condition <24 h previously with no
evidence of a non-cardiac cause [13,14].
Gene variant selection and genotyping
RYR2 is located on chromosome 1q42.1-q43 and
CACNA1C (encoding the L-type Ca2+ channel α subunit) is located on chromosome 12p13.3. A total of 5784
SNPs (single nucleotide polymorphisms) in RYR2 and
3337 SNPs in CACNA1C have been identified in public
databases (dbSNP; http://www. ncbi.nlm.nih.gov/SNP/)
up until October 2009. After ruling out the rare allele
variants (minor allele frequency <0.05), according to
the genotyping data in the CHB (Chinese Han in
Beijing, China) population in the International HapMap
Project (http://hapmap.cshl.org/), we calculated the LDs
(linkage disequilibriums; Supplementary Figures S1 and
S2 at http://www.clinsci.org/cs/119/cs1190215add.htm)
between the variants in each gene using the standard
definition (r2 0.8) of LDs. However, both genes are
large and consisted of many SNP LDs. More than
300 tagSNPs in RYR2 and more than 200 tagSNPs in
CACNA1C were identified, and most of them were
located in the introns of genes. Therefore we used the
candidate approach to select the variants and undertook a
search of variants in the potentially functional regions of
RYR2 and CACNA1C, including the promoter region,
coding regions and 3 -UTR (untranslated region). In the
CHB population, we found two potentially functional
variants, rs3766871 (G1886S) and rs790896 (G > A),
located in exon 37 and the 3 -UTR of RYR2, and one
potentially functional variant, rs723672 (T > C), located
in the promoter region of CACNA1C. Milting et al.
[15] reported that the variants rs41315858 (G1885E) and
rs3766871 together were a risk factor for ARVC (arrhythmogenic right ventricular cardiomyopathy), so we
performed a direct sequencing to determine the frequency
of the variant G1885E of RYR2 (Supplementary Figure S3
at http://www.clinsci.org/cs/119/cs1190215add.htm) in
50 healthy Han Chinese subjects. We found the
frequency of rs41315858 in Han Chinese is 6 % (3
out of 50). rs41315858 and rs3766871 locate in the
DR3 (divergent region 3) domain which is important
for ligand binding of RyR2. The amino acid change
of G1886S and G1885E/G1886S has been shown to
result in the functional change of RyR2 [15–17]. The
biological functions of rs790896 and rs723672 are unclear.
Therefore we selected these four variants for genotyping
in our study population. Genomic DNA from white
blood cells was extracted using standard methods [18]
and kept at −70 ◦ C. Participants were genotyped for
these four variants using ligase detection reactions [19]
in a blinded manner (e.g. sample status was covered
during genotyping). A fluorescently labelled primer was
employed to detect the genotype of the (GT)n repeat using
Megabase 1000 capillary electrophoresis (Amersham
Biosciences). The reproducibility of genotyping was
confirmed by direct sequencing in 114 (5 %) random
samples, and the reproducibility was 100 %. Finally,
20 (1.61 %) cases and 17 (1.65 %) controls were excluded
because of a failure of genotyping.
Statistical analysis
Continuous values are expressed as means +
− S.D., and
categorical variables are shown as numbers (percentage).
LDs of SNPs were analysed by Haploview 4.0
(http://hapmap.cshl.org/). χ 2 tests with one degree of
freedom were used for the Hardy–Weinberg equilibrium
test for each variant and categorical variable comparison
to determine statistical significance between groups.
The association between the variants and case/control
status was assessed by unconditional multivariate logistic
regression analysis using dominant genetic models
(Mm+mm compared with MM), in which M denotes the
major allele and m denotes the minor allele. Multivariate
analyses were adjusted for age, gender and suspected risk
factors in each analysis. Person-months of the followup period started from the initial date of diagnosis of
CHF in the patient population and from the date of
enrolment for controls until October 2009. We performed
survival analysis in the CHF population. Patients lost to
follow-up in the CHF population [186 (14.95 %)] were
not included in the survival analysis. HRs (hazard ratios)
for time to cardiac death and SCD from baseline were
estimated using Cox proportional hazards models. In
addition to considering the influence of rs3766871 and
rs790896, we analysed Kaplan–Meier curves for time to
probability of survival using the log rank test according
to presence or absence of the minor allele. Analyses were
performed with SPSS 11.0. A two-tailed P < 0.05 was
considered significant. Bonferroni correction was used
for correction of multiple comparisons.
RESULTS
Clinical characteristics
The clinical characteristics of CHF patients and controls
are shown in Supplementary Table S1 (at http://www.
clinsci.org/cs/119/cs1190215add.htm). In brief, a total of
1244 CHF patients (1017 males; 81.8 %) with a mean
age of 56.6 +
− 13.4 years were included. The underlying
aetiologies of CHF were ischaemic heart disease in
733 (58.9 %) patients with a mean age of 61.4 +
− 10.5 years
and idiopathic dilated cardiomyopathy in 511 (41.1 %)
patients with a mean age of 49.8 +
− 14.3 years. The controls
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Table 1 Association of the rs3766871 genotype with VAs in patients with CHF
*Determined using the χ 2 test; †obtained with multivariate logistic regression analysis by adjusting for age, gender, BMI, BP, MHR, and a history of hypertension,
diabetes mellitus and hyperlipidaemia. The MM genotype was the reference. M, major allele; m, minor allele.
Genotype (n)
Dominant model (Mm+mm) compared with MM
Genetic variant
MM
Mm
mm
rs41315858 (G1885E)
Controls (n = 1032)
CHF (n = 1244)
With VA (n = 676)
Without VA (n = 568)
rs3766871 (G1886S)
Controls (n = 1032)
CHF (n = 1244)
With VA (n = 676)
Without VA (n = 568)
rs790896 (G > A)
Controls (n = 1032)
CHF (n = 1244)
With VA (n = 676)
Without VA (n = 568)
rs723672 (T > C)
Controls (n = 1032)
CHF (n = 1244)
With VA (n = 676)
Without VA (n = 568)
GG
984 (95.3 %)
1176 (94.5 %)
637 (94.2 %)
539 (94.9 %)
GG
935 (90.6 %)
1078 (86.7 %)
576 (85.2 %)
502 (88.4 %)
GG
608 (58.9 %)
731 (58.8 %)
402 (59.3 %)
329 (59.3 %)
TT
645 (62.5 %)
739 (59.4 %)
406 (60.1 %)
333 (58.5 %)
GA
48 (4.7 %)
68 (5.5 %)
39 (5.8 %)
29 (5.1 %)
GA
96 (9.3 %)
164 (13.2 %)
98 (14.5 %)
66 (11.6 %)
GA
374 (36.2 %)
459 (36.9 %)
255 (37.6 %)
204 (36.0 %)
TC
331 (32.1 %)
431 (34.6 %)
224 (33.2 %)
207 (36.4 %)
AA
0
0
0
0
AA
1 (0.1 %)
2 (0.2 %)
2 (0.3 %)
0
AA
50 (4.8 %)
54 (4.3 %)
21 (3.1 %)
33 (5.8 %)
CC
56 (5.4 %)
74 (5.9 %)
45 (6.7 %)
29 (5.1 %)
consisted of 1032 subjects, in whom there were 867 males
(84.0 %) and the mean age was 58.2 +
− 11.7 years. Possible
risk factors for CHF, including a history of hypertension,
diabetes mellitus, hyperlipidaemia and atrial fibrillation,
were more prevalent in the CHF group than in controls.
The variant G1886S of RYR2 is associated
with VAs in patients with CHF
The distribution of the genotypes of the four variants are
shown in Tables 1 and 2. All genotypes were in Hardy–
Weinberg equilibrium in each group. In variant rs3766871
(G1886S), AA was a rare genotype (3 out of 2274).
rs41315858 (G1885E) of RYR2 is a minor allele variant
in the Han Chinese population (4.7 % in the controls and
5.5 % in the CHF population) and only a heterozygous
genotype was found. We did not find the G1885E/1886S
genotype of RYR2 in our study population. The genotype
frequencies of the A allele homozygote and heterozygote
were significantly higher in patients with CHF than
in controls {13.4 compared with 9.4 % respectively;
crude OR (odds ratio), 1.48 [95 % CI (confidence
interval), 1.13–1.93]; P = 0.004}. Moreover, the A allele
of rs3766871 was found significantly more often in
CHF patients with VAs than in controls [14.8 compared
with 9.4 % respectively; crude OR, 1.66 (95 % CI, 1.23–
2.24); P = 0.001]. After adjustment with multivariate
logistic regression analysis for age, gender, BMI (body
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Crude OR (95 % CI)*
P value
Adjusted OR (95 % CI)†
Adjusted P value
1
1.19 (0.81–1.73)
1.26 (0.81–1.94)
1.10 (0.69–1.77)
1
0.379
0.305
0.685
1.23 (0.80–1.74)
1.23 (0.79–1.92)
1.11 (0.69–1.81)
0.411
0.359
0.665
1
1.48 (1.13–1.93)
1.66 (1.23–2.24)
1.27 (0.91–1.77)
1
0.004
0.001
0.158
1.49 (1.13–1.97)
1.66 (1.21–2.26)
1.30 (0.92–1.83)
0.005
0.002
0.137
1
1.01 (0.85–1.19)
0.99 (0.81–1.20)
1.03 (0.84–1.27)
1
0.937
0.906
0.785
1.05 (0.88–1.25)
1.02 (0.83–1.26)
1.08 (0.87–1.34)
0.613
0.826
0.507
1
1.14 (0.96–1.35)
1.11 (0.91–1.35)
1.18 (0.91–1.35)
1
0.128
0.319
0.117
1.13 (0.94–1.35)
1.11 (0.90–1.36)
1.16 (0.93–1.44)
0.183
0.343
0.189
mass index), BP (blood pressure), MHR (mean heart
rate), and a history of hypertension, diabetes mellitus,
hyperlipidaemia and atrial fibrillation, the association
remained significant [adjusted OR, 1.66 (95 % CI, 1.21–
2.26); P = 0.002], supporting the hypothesis that the A
allele of rs3766871 is a risk factor for CHF with VAs.
rs790896 was not associated with CHF or VAs in our
present study population.
RYR2 variants have an impact on SCD in
patients with CHF
During a median follow-up period of 32 months
(range, 0.1–52 months) in 1058 (85.0 %) patients with
CHF, 296 (28.0 %) patients died from CHF, of whom
141 (47.6 %) had SCD. No end point event occurred in
the controls. Unexpectedly, 168 (15.0 %) patients in cases
and 114 (11.0 %) subjects in controls were lost to followup because of changes in phone numbers or emigration.
There were no differences in the clinical data between the
follow-up and lost-to-follow-up groups in the cases. We
analysed the end points with the four variants using a
survival Cox regression analysis in the CHF population
(Table 3). In this prospective model, after adjustment for
age, gender and suspected risk factors for SCD (including
BMI, BP, MHR, a history of hypertension, diabetes
mellitus, hyperlipidaemia or atrial fibrillation, NYHA
class, LVEF, causes of HF and taking β-blockers), patients
Genetics and sudden cardiac death
Table 2 No association between potentially functional variants of RYR2 , CACNA1C and HF severity in a CHF cohort
*Obtained with multivariate logistic regression analysis by adjusting for age and gender; †obtained with multivariate logistic regression analysis by adjusting for age,
gender, BMI, BP, MHR, a history of hypertension, diabetes mellitus, hyperlipidaemia and atrial fibrillation, NYHA class and cause of heart failure. The MM genotype was
the reference. The severity of HF was defined as: mild, 40 % < LVEF 50 %; moderate, 30 % < LVEF 40 %; severe, LVEF 30 %. M, major allele; m, minor
allele.
Genotype (n)
Dominant model (Mm+mm) compared with MM
Genetic variant
MM
Mm
mm
rs41315858 (G1885E)
Mild (n = 405)
Moderate (n = 441)
Severe (n = 398)
rs3766871 (G1886S)
Mild (n = 405)
Moderate (n = 441)
Severe (n = 398)
rs790896 (G > A)
Mild (n = 405)
Moderate (n = 441)
Severe (n = 398)
rs723672 (T > C)
Mild (n = 405)
Moderate (n = 441)
Severe (n = 398)
GG
383 (94.6 %)
422 (95.7 %)
371 (93.2 %)
GG
349(85.5 %)
380 (86.2 %)
349 (88.4 %)
GG
226 (55.8 %)
268 (60.8 %)
237 (59.5 %)
TT
239 (58.6 %)
263 (59.5 %)
237 (60.2 %)
GA
22 (5.4 %)
19 (4.3 %)
27 (6.8 %)
GA
59 (14.5 %)
59 (13.4 %)
46 (11.6 %)
GA
160 (39.5 %)
158 (35.8 %)
141 (35.4 %)
TC
146 (35.8 %)
150 (33.9 %)
135 (34.3 %)
AA
0
0
0
AA
0
2 (0.5)
0
AA
19 (4.7)
15 (3.4)
20 (5.0 %)
CC
23 (5.6 %)
29 (6.6 %)
22 (5.6 %)
carrying the A allele of rs3766871 had an increased risk of
cardiac death [HR, 1.53 (95 % CI, 1.11–2.12); P = 0.010]
and SCD [HR, 1.92 (95 % CI, 1.25–2.94); P = 0.003].
Patients carrying the A allele of rs790896 had a reduced
risk of SCD [HR, 0.65 (95 % CI, 0.45–0.92); P = 0.015].
These results suggest that the A allele of rs3766871 is a risk
factor for SCD, but the A allele of rs790896 is a protective
factor against SCD. When we created Kaplan–Meier
curves using the log-rank test for time to probability of
survival according to the presence or absence of the A
allele of rs3766871 and the presence or absence of the A
allele of rs790896, the association remained significant
(P = 0.004 for rs3766871, and P = 0.020 for rs790896;
Figures 1 and 2 respectively).
For rs723672 (T > C) of CACNA1C, no significance
was found in any of the analyses.
DISCUSSION
The results of our present study demonstrate that the
A allele of rs3766871 (G1886S) in RYR2 is not only
associated with VAs, but also serves as a predictor of
SCD in patients with CHF. In addition, it was not related
to other risk factors, such as a history of hypertension,
diabetes mellitus, hyperlipidaemia and atrial fibrillation,
age, gender and cause of CHF, suggesting that the
contribution of the A allele of rs3766871 to VAs and SCD
was independent of other risk factors. After adjustment
Crude OR* (95 % CI)
P value
Adjusted OR† (95 % CI)
Adjusted P value
1
0.80 (0.42–1.51)
1.28 (0.71–2.34)
1
0.488
0.412
0.70 (0.35–1.39)
1.12 (0.53–2.37)
0.303
0.774
1
0.92 (0.62–1.35)
0.76 (0.50–1.15)
1
0.657
0.195
0.95 (0.61–1.47)
0.77 (0.45–1.32)
0.818
0.342
1
0.83 (0.63–1.09)
0.86 (0.66–1.18)
1
0.176
0.403
0.75 (0.55–1.03)
0.76 (0.52–1.10)
0.071
0.141
1
0.97 (0.73–1.27)
0.94 (0.70–1.26)
1
0.802
0.672
0.98 (0.72–1.35)
1.04 (0.76–1.44)
0.920
0.933
Figure 1 Kaplan–Meier curves for the probability of
survival from SCD in patients with CHF according to the
presence or absence of the A allele of rs3766871 in RYR2
Each censored case due to NSCD (non-SCD) is marked with a dot. The Table at
the bottom of the curves indicates the number of patients with CHF at risk for
every 6 months (the last period was 4 months) of follow-up. Patients carrying
the A allele of rs3766871 were more susceptible to SCD compared with G allele
carriers. Log rank test, P = 0.004.
of these co-factors, patients carrying the A allele of the
G1886S variant of RYR2 had an approx. 2-fold greater
HR for SCD compared with those not carrying the A
allele. It appears that the G1886S variant may alter Ca2+
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Table 3 HRs (95 % CI) of SCD in a prospective CHF cohort
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*Model I, Cox regression models by adjusting for age and gender; †model II, Cox regression models by adjusting for age, gender and suspected risk factors of SCD, including BMI, BP, MHR, a history of hypertension, diabetes mellitus, hyperlipidaemia
and atrial fibrillation, NYHA class, LVEF, causes of heart failure and taking β-blockers.
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Cardiac death
SCD
HR (95 % CI) for cardiac death
NSCD
HR (95 % CI) for SCD
HR (95 % CI) for NSCD
Genetic variant
Survival (n)
n
Model I*
Model II†
n
Model I*
Model II†
n
Model I*
Model II†
rs41315858 (G1885E)
GG (n = 1004)
726 (95.3 %)
278 (93.9 %)
18 (6.1 %)
1
1.19 (0.58–2.42)
(P = 0.640)
1
1.16 (0.56–2.39)
(P = 0.696)
145 (93.5 %)
36 (4.6 %)
1
1.23 (0.76–2.00)
(P = 0.405)
133 (94.3 %)
GA+AA (n = 54)
rs3766871 (G1886S)
GG (n = 930)
1
1.26 (0.78–2.03)
(P = 0.343)
1
1.33 (0.70–2.52)
(P = 0.389)
1
1.29 (0.67–2.49)
(P = 0.446)
678 (89.0 %)
252 (85.1 %)
GA+AA (n = 128)
rs790896 (G > A)
GG (n = 623)
84 (11.0 %)
44 (14.9 %)
1
1.42 (1.03–1.96)
(P = 0.032)
1
1.53 (1.11–2.12)
(P = 0.010)
1
1.81 (1.18–2.77)
(P = 0.006)
1
1.92 (1.25–2.94)
(P = 0.003)
1
1.08 (0.66–1.77)
(P = 0.748)
1
1.19 (0.73–1.95)
(P = 0.488)
440 (57.7 %)
183 (61.8 %)
1
0.83 (0.66–1.05)
(P = 0.123)
1
0.83 (0.66–1.06)
(P = 0.129)
1
0.65 (0.46–0.93)
(P = 0.017)
1
0.65 (0.45–0.92)
(P = 0.015)
1
1.03 (0.75–1.41)
(P = 0.868)
1
1.06 (0.76–1.46)
(P = 0.742)
1
1.14 (0.90–1.43)
(P = 0.271)
1
1.19 (0.95–1.50)
(P = 0.140)
1
1.12 (0.80–1.56)
(P = 0.502)
1
1.18 (0.84–1.65)
(P = 0.338)
1
1.15 (0.84–1.58)
(P = 0.379)
1
1.21 (0.88–1.66)
(P = 0.245)
GG+GA (n = 435)
rs723672 (T > C)
TT (n = 629)
322 (43.2 %)
113 (38.2 %)
464 (60.9 %)
165 (55.7 %)
TC+CC (n = 429)
298 (39.1 %)
131 (44.3 %)
8 (5.4 %)
115 (81.6 %)
26 (18.4 %)
95 (67.4 %)
46 (32.6 %)
79 (56.0 %)
62 (44.0 %)
10 (6.5 %)
137 (88.4 %)
18 (11.6 %)
88(56.8 %)
67 (43.2 %)
86 (55.5 %)
69 (44.5 %)
Genetics and sudden cardiac death
Figure 2 Kaplan–Meier curves for the probability of
survival from SCD in patients with CHF according to the
presence or absence of the A allele of rs790896 in RYR2
Each censored case due to NSCD is marked with a dot. The Table at the bottom
of the curves indicates the number of patients with CHF at risk for every 6
months (the last period was 4 months) of follow-up. Patients carrying the A allele
of rs790896 were more resistant to SCD compared with G allele carriers. Log
rank test, P = 0.020.
signalling in the failing heart and result in life-threatening
arrhythmias when other risk factors co-exist.
RYR2 is one of the largest human genes (105
exons), encoding a membrane protein of 2300 kDa.
Acquired or genetic defects in RyR2 and associated
molecules can cause functional and/or structural changes
in the heart and lead to HF and SCD [20,21]. Indeed,
mutations in RYR2 have been linked to catecholaminergic
polymorphic ventricular tachycardia [22] and ARVC
[23]. The G1885E and G1886S variants of RYR2 are
located at the DR3 domain, which is involved in
excitation–contraction coupling as well as in the binding
of ligands, such as Ca2+ , Mg2+ and FKBP12.6 (FK506binding protein 12.6) [16]. Koop et al. [17] have found
that a serine residue replacing Gly1886 of rs3766871
caused a significant increase in the intracellular Ca2+
oscillation activity compared with cells expressing wildtype RyR2 channels. Milting et al. [15] reported that the
G1886S variant may create a PKC (protein kinase C)
phosphorylation site in human RyR2. This suggests that
G1886S amino-acid exchange in the DR3 region of RyR2
may modify channel gating, leading to diastolic Ca2+ leak
that can provoke delayed after-depolarizations and fatal
cardiac arrhythmias [24,25], especially in the failing heart.
However, we did not find the G1885E/G1886S genotype
in our present study population. This may be explained by
the fact that the combination of two amino acid exchanges
of G1885E/G1886S causes a modified channel gating
with a highly increased channel activity at diastolic free
Ca2+ concentrations and results in severe dysfunction of
RyR2 [15]. Therefore both amino acid replacements
of G1885E/G1886S genotype may play an important role
in the development of ARVC, but not in CHF.
By contrast, we found that the A allele of rs790896
(G > A) variant in the 3 -UTR of RYR2 was linked to
a reduced risk of SCD in patients with CHF. There
was no significance in genotype distribution between
CHF patients with or without VAs and control subjects.
The biological function of rs790896 variant remains
unknown. In fact, the 3 -UTR of the gene is usually
the target binding domain of modulating factors, such
as microRNA, which negatively modulate transcription
by binding to mRNA [26]. An in-depth exploration of
the potential function of the rs790896 variant may be of
interest for future investigations.
When analysing subgroups in the HF population, the
genotype distributions of all four variants in the moderate
and severe groups were not significantly different from
those of the mild group. This reflects the fact that
none of the four variants were not associated with the
severity of HF. One study has reported that the LEPR
(leptin receptor) Gln223Arg variant displayed an
independent prediction role for LVEF in patients
with HF caused by ischaemic heart disease or dilated
cardiomyopathy in a Czech population [27]; however, no
literature is available regarding the relationship between
genetic variants of Ca2+ -cycling-related genes and the
classification of HF.
In recent years, several prospective studies have
reported genetic variants related to HF and SCD. One
study based on a large cohort of African-Americans and
Caucasians found that the GG genotype of rs10757274
on chromosome 9p21 not only increased the risk of
coronary artery disease, but also increased the risk of HF
among Caucasians [8]. The authors also found that the G
allele at rs10757274 conferred a significantly elevated ageadjusted OR for SCD in individuals of European ancestry
[28]. A separate study found that homozygosity for
the common allele at Q27E of the β 2 -adrenoceptor was
associated with an increased risk of SCD [29]. Arvanitis
et al. [30] found that the Ser96Ala variant in histidinerich Ca2+ -binding protein was associated with lifethreatening VAs in idiopathic dilated cardiomyopathy.
The presence of multiple genetic risk factors for SCD
suggests that common variants of genes often act as
genetic markers, but are unlikely to be sufficient to cause
SCD on their own [31]. In our present Cox regression
models, a history of VAs, lower LVEF and higher NYHA
class were the significant clinical risk factors for SCD.
This is consistent with the literature [14]. Our present
results imply that genetic variants of RYR2 may modulate
the risk of SCD in patients with CHF.
We did not find any evidence that the common
variant rs723672 (T > C) in the promoter region of
CACNA1C was associated with the risk of CHF or SCD
in our study population. As the L-type Ca2+ channel is
essential for the development of the embryo, mutations of
CACNA1C are usually fatal and may be inherited in such
condition as Timothy syndrome (long QT syndrome
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Y. Ran and others
type 8) [11]. Genetic variants of CACNA1C have not yet
been reported in population-based studies, and further
studies need to be performed in other ethnic/race-based
populations.
However, the present study is subject to several
potential limitations. First, it is difficult to find another
independent cohort with CHF and VAs with the same
inclusion criteria and follow-up of at least 2 years. In
addition, a lack of replication in a second population
limits the confirmation of our findings. Secondly, the rate
of patients with CHF lost to follow-up was relatively
high.
In conclusion, our present findings show that the A
allele of rs3766871 (G1886S) variant in RYR2 not only
associates with VAs in patients with CHF, but also serves
as an independent predictor of SCD, and the A allele of
rs790896 (G > A) variant located at the 3 -UTR of RYR2
is a protective factor against SCD in patients with CHF.
Our observations support the hypothesis that genetic
variants in RYR2 may influence the progression of HF
and modify the risk of SCD in patients with CHF.
FUNDING
This work was supported by the 973 National
Basic Research Program of China [grant numbers
2007CB512000; 2007CB512008 (to J.P.)].
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Received 22 December 2009/15 April 2010; accepted 21 April 2010
Published as Immediate Publication 21 April 2010, doi:10.1042/CS20090656
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Clinical Science (2010) 119, 215–223 (Printed in Great Britain) doi:10.1042/CS20090656
SUPPLEMENTARY ONLINE DATA
Common RyR2 variants associate with
ventricular arrhythmias and sudden cardiac
death in chronic heart failure
Yuqin RAN∗ , Jingzhou CHEN†1 , Ning LI‡, Weili ZHANG†, Li FENG‡, Rongrong
WANG‡, Rutai HUI†, Shu ZHANG∗ and Jielin PU∗ ‡1
∗
Center for Arrhythmia Diagnosis and Treatment, Fu Wai Cardiovascular Hospital, Peking Union Medical College and Chinese
Academy of Medical Sciences, Beijing 100037, China, †Sino-German Laboratory for Molecular Medicine, Fu Wai Cardiovascular
Hospital, Chinese Academy of Medical Sciences, Beijing 100037, China, and ‡Pathology and Physiology Research Center, Fu
Wai Cardiovascular Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100037, China
Figure S1 Map of LDs of RYR2 in the Chinese Han population
Rare allele variants (minor allele frequency <0.05) were not included. The definition of LDs is r 2 0.8.
1
These authors contributed equally to the study.
Correspondence: Dr Jielin Pu (email [email protected]).
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Y. Ran and others
Figure S2 Map of LDs of CACNA1C in the Chinese Han population
Rare allele variants (minor allele frequency < 0.05) were not included. The definition of LDs is r 2 0.8.
Figure S3 Direct sequencing of PCR-amplified genomic DNA
from the Chinese population revealing the rs41315858
(G > A) variant in (a) wild-type and (b) variant sequences
The arrow indicates the variant nucleotide position.
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Genetics and sudden cardiac death
Table S1 Clinical characteristics of patients with CHF and
control subjects
Values are means +
− S.D. or numbers (%). AF, atrial fibrillation; DBP, diastiolic
BP; LVD, LV diameter; SBP, systolic BP.
Characteristic
Controls (n = 1032)
Patients with CHF
(n = 1244)
P value
Age (years)
Male gender (n)
LVEF (%)
LVD (mm)
NYHA Class (n)
Not applicable
II
III
IV
Hypertension (n)
Hyperlipidaemia (n)
Diabetes mellitus (n)
AF (n)
VA (n)
MHR (beats/min)
SBP (mmHg)
DBP (mmHg)
BMI (kg/m2 )
58.2 +
− 1.7
867 (84.0%)
62.8 +
− 5.3
49.9 +
− 3.9
56.6 +
− 13.4
1017 (81.8%)
35.9 +
− 9.0
63.7 +
− 9.6
0.003
0.164
<0.001
<0.001
1032 (100%)
0
0
0
255 (24.7%)
140 (13.6%)
119 (11.5%)
44 (4.3%)
0
72.6 +
− 10.1
115.9 +
− 11.1
74.4 +
− 9.2
24.5 +
− 3.2
0
410 (33.0%)
521 (41.9%)
313 (25.2%)
483 (38.3%)
238 (19.1%)
255 (20.5%)
388 (31.2%)
676 (54.3%)
78.8 +
− 16.1
114.0 +
− 18.0
72.1 +
− 12.2
24.4 +
− 3.6
<0.001
<0.001
<0.001
<0.001
<0.001
0.003
<0.001
0.323
Received 22 December 2009/15 April 2010; accepted 21 April 2010
Published as Immediate Publication 21 April 2010, doi:10.1042/CS20090656
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