Original Article Common Variant Rs7597774 in ADD2 Is Associated with Dilated Cardiomyopathy in Chinese Han Population

Int J Clin Exp Med 2015;8(1):1188-1196 www.ijcem.com /ISSN:1940-5901/IJCEM0003312

Original Article Common variant rs7597774 in ADD2 is associated with dilated cardiomyopathy in Chinese Han population

Fei F Chen1*, Yun L Xia1*, Cheng Q Xu2, Si S Li2, Yuan Y Zhao2, Xiao J Wang2, Shan S Chen2, Lian J Gao1, Yang Zhong3, Xin Tu2, Qing Wang2,4, Yan Z Yang1

1Department of Cardiology, First Affiliated Hospital of Dalian Medical University, Dalian, China; 2College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technol- ogy, Wuhan, China; 3Department of Cardiology, Fifth People’s Hospital of Dalian, Dalian, China; 4Department of Molecular Cardiology, Center for Cardiovascular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA. *Equal contributors. Received October 21, 2014; Accepted January 7, 2015; Epub January 15, 2015; Published January 30, 2015

Abstract: Two polymorphisms, rs7597774 and rs1739843 in ADD2 and HSPB7 respectively, were found to be associated with dilated cardiomyopathy (DCM) in European cohorts but the results were not validated in the Chinese Han population. We aimed to test the association of the two variants with DCM in a cohort of Chinese Han population. DCM (399) and control (1384) individuals were identified from the GeneID database in China, and DNA was isolated from peripheral blood lymphocytes for genotyping. Alleles were amplified by PCR, and amplicons harboring polymorphisms rs1739843 and rs7597774 were directly genotyped using high-resolution melting analysis. Sta- tistical analysis was subsequently performed to evaluate the association of the variants with DCM. Allelic analysis demonstrated that rs7597774 was significantly related to DCM (P = 0.0157), and an increased risk of DCM was -adj specifically associated with the minor allele A (OR = 1.582). High-grade cardiac dysfunction (NYHA III/IV) was a clinical parameter significantly associated with the rs7597774 genotypes AA + AC relative to genotype CC (P = 0.021). Furthermore, DCM patients with the rs7597774 genotype AA tended to undergo more invasive medical interven- tions than those with the genotype CC (P = 0.008). No association was detected between rs1739843 and DCM under any allelic (P = 0.407, OR = 0.920) or genotypic model. In the Chinese Han population, rs7597774 but not -adj rs1739843 was found to be associated with DCM. This study is the first to demonstrate that underlying genotypes of rs7597774 may assist in assessing the heart functional status of DCM patients and also in the prediction of the benefit of particular therapies for these patients.

Keywords: ADD2, dilated cardiomyopathy, HSPB7, NYHA heart functional classification, polymorphisms, therapeutic regimens

Introduction fied in monogenic forms of DCM, with most of them encoding cytoskeletal, sarcomeric, and Dilated cardiomyopathy (DCM) is a heteroge- regulatory proteins, or ion channels [6]. neous heart disease with variable aetiological and clinical features. The disease is character- A recent genome wide association study (GWAS) ized by ventricular chamber enlargement and revealed that two polymorphisms, ADD2 systolic dysfunction that ultimately result in rs7597774 (adducin 2) and HSPB7 rs1739843 sudden cardiac death or progressive heart fail- (heat-shock 27 kDa protein family, member 7), ure with the eventual consequence of cardiac were associated with DCM in European popula- transplantation [1-4]. The pathophysiology of tions [7]. In a second GWAS, however, HSPB7 DCM is multifactorial with a possible implica- rs1739843 was not found to be associated tion of environmental factors and the existence with sporadic DCM in various western popula- of a strong genetic component as demonstrat- tions [8]. Furthermore, no relationship between ed by a high rate of familial aggregation [5]. To the HSPB7 single nucleotide polymorphisms date, approximately 33 genes have been identi- (SNPs) and genetic susceptibility to idiopathic Variant rs7597774 in ADD2 and dilated cardiomyopathy

DCM was found in a small Chinese Han cohort Clinical characteristics [9]. An association between ADD2 rs7597774 and DCM, however, has not yet been examined The clinical data included age, gender, hyper- in a population of non-European ancestry. tension, type 2 diabetes mellitus (T2DM), and left ventricular ejection fraction (LVEF), left ven- To further investigate the role of rs7597774 tricular end-diastolic diameter (LVEDD), and left and rs1739843 in DCM, allelic and genotypic atrial diameter (LAD) of echocardiographic association analyses were performed on DNA parameters in both groups. New York Heart samples from an independent case-control Association (NYHA) heart functional classifica- DCM cohort with a total of 1783 Chinese Han tion, therapeutic regimens, and serum brain subjects from central and northeastern China. natriuretic peptide (BNP) concentrations were Specific clinical features of DCM patients were also obtained from medical records for DCM also included in the analyses in order to poten- patients. Hypertension was defined as systolic tially reveal associations with the functional blood pressure ≥ 140 mmHg or diastolic blood consequences of the disease and to provide a pressure ≥ 90 mmHg. The criteria set by the molecular basis for the selection of treatment American Diabetes Association was applied for options. a diagnosis of T2DM [11]. Serum BNP levels were determined using a high-sensitivity Materials and methods enzyme-linked immunosorbent assay kit (R & D Systems; Minneapolis, MN, USA). Ethics statement SNP genotyping All protocols performed in this study were approved by the Ethics Committee of First Genomic DNA was isolated from peripheral Affiliated Hospital of Dalian Medical University blood lymphocytes with the TIANamp Blood and Huazhong University of Science and Tech- DNA Kit (TiangenBiotect; Beijing, China) accord- nology. Written informed consent was obtained ing to the manufacturer’s protocols. PCR was from all subjects participating in the study. The performed in a 25 μL final volume containing study conformed to the principles outlined in 1.5 mM Mg2+, 0.2 mM dNTPs, 0.5 μM each the Declaration of Helsinki. primer (rs1739843: forward, 5’-ACC CGC ATC Study subjects CGC CCC CCT ATA G-3’, reverse, 5’-GGG GGT GGG GCT TGA GGG TG-3’; rs7597774: forward, All subjects were selected from GeneID, which 5’-AGC CCT GTC CAG CCC TGA G-3’, reverse, is an ongoing Chinese population database. 5’-TTG GGC ACT GAG GCA CCT G-3’), 25 ng of DNA samples and clinical information are col- human genomic DNA template, 5 μM SYTO9 lected from individuals with cardiovascular and green fluorescent intercalating agent, and 0.15 cerebrovascular diseases national wide in an U of Taq DNA polymerase. PCR was performed effort to identify susceptibility loci related to on an ABI 9700 System (Life Technologies; these diseases [10]. Grand Island, NY, USA) with the following ther- mal profile: 95°C for 5 min; 40 cycles of 95°C The study subjects were from central and north- for 10 s, corresponding annealing temperature eastern China and were ethnic Chinese Han by (57.4°C for rs1739843, 60.3°C for rs7597774) self-description. The criteria for a diagnosis of for 10 s, and 72°C for 15 s; and a final cycle of DCM were in accordance with the guidelines 72°C for 10 min. PCR amplicons were directly established by the America Heart Association genotyped using high-resolution melting analy- in 2006. Patients with known causes such as sis (HRM) on the Rotor-Gene 6000 System acute viral myocarditis, coronary artery dis- (Corbett Life Science, Australia) under standard ease, valvular disease, congenital heart defects protocols with minor modifications [12]. During and a positive family history of DCM were each run of HRM, three positive control DNA excluded. The controls were individuals without samples with known genotypes (TT, CT, and CC DCM as determined by echocardiography or for rs1739843; and AA, AC, and CC for medical history at the time of enrollment, and rs7597774), as well as a negative control with- who underwent annual physical exams with/ out DNA template were included. To validate without any diseases. the accuracy of HRM genotyping data, ten sam-

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Table 1. Baseline clinical characteristics of study groups responding 95% confidential intervals (95% CI) were also Characteristics DCM Group Control Group P-value calculated. Genotypic asso- Number 399 1384 N/A ciation analysis under three Age (M ± SD, y) 55.97 ± 15.194 57.3 ± 16.074 0.67 genetic models (dominant, Gender (Male) 62.66% 56.79% 0.038 recessive, and additive) was Hypertension (1/0) 143/256 616/768 0.002 performed using 2 × 3 con- T2DM (1/0) 53/344 237/1149 0.076 tingency tables assessed by LVEDD (M ± SD, mm) Pearson’s chi-squared (χ2) Male 65.00 ± 9.329 44.49 ± 4.196 < 0.001 test. Multiple logistic regres- Female 60.70 ± 7.523 42.04 ± 4.481 < 0.001 sion analysis was used to LVEF (M ± SD, %) 35.88 ± 11.98 59.62 ± 6.358 < 0.001 adjust covariates such as BNP levels (M ± SD, pg/ml) 1022.55 ± 910.86 N/A - sex, age, hypertension, T2D- LAD (M ± SD, mm) M. When the case-control Male 44.08 ± 6.529 33.00 ± 5.144 < 0.001 samples were divided into Female 40.69 ± 9.160 31.84 ± 6.597 < 0.001 several subgroups, basic sta- M ± SD, mean ± standard deviation; T2DM, type 2 diabetes mellitus; LVEDD, left tistical methods were applied ventricular end-diastolic diameter; LAD, left atrial diameter; BNP, brain natriuretic as described. peptide; N/A, data not available. For the association between age, LVEF, LVEDD, LAD, BNP ples for each genotype of both SNPs were ran- levels and rs7597774, One-Way analysis of domly selected for Sanger sequencing. Primers variance (ANOVA) was used. For the association for sequencing the SNPs were the following: between rs7597774 and NYHA heart functional rs1739843: forward, 5’-TCC CCA CCT ACC CGC classification or therapeutic regimens, a non- ATC C-3’ and reverse, 5’-GCC CCC TCA CTG CCT parametric test, Ridit analysis and the Kruskal- CTC TT-3’ for; rs7597774: forward, 5’-CGG CCT Wallis H test, were used, respectively. Two- GTG TCT CTG CGT TT-3’ and reverse 5’-TTG GGC tailed P < 0.05 was accepted as statistically ACT GAG GCA CCT G-3’. All sequencing results significant. were consistent with the genotypes as determined by HRM analysis. Results

Statistical analysis Baseline characteristics

Statistical power analysis of study populations Clinical characteristics of 399 DCM cases and was conducted with the program PS (Power 1384 non-DCM controls were examined to and Sample size Calculations, version 3.0.43). determine their distribution across the cohort. Hardy-Weinberg linkage equilibrium was tested Clinical characteristics of DCM and control in the control group with PLINK, version 1.07 groups are compared in Table 1. Age and T2DM [13]. were two clinical features that were distributed similarly between the two groups, but a greater Pearson’s chi-squared (χ2) and unpaired stu- number of males were in the DCM (62.66%) dent’s t tests were performed with SPSS ver- than the control group (56.79%). Measurements sion 22.0 software (IBM Inc.; Armonk, NY, USA) of the heart were more indicative of declining for categorical traits (gender, hypertension, function in the DCM cases. LVEDD and LAD T2DM, NYHA heart functional classification, were greater in DCM patients than controls, and therapeutic regimens) and continuous regardless of gender (P < 0.001), whereas LVEF traits (age, LVEF, LVEDD, LAD, and BNP levels), was significantly lower in cases than controls respectively. For allelic association analysis, 2 (35.88 ± 11.98 versus 59.62 ± 6.358, P < × 2 contingency tables assessed by Pearson’s 0.001). Serum BNP concentrations were chi-squared (χ2) test were used to compare dif- 1022.55 ± 910.86 pg/ml in DCM patients. ferences in the frequencies of the rs1739843 Hypertension, however, was surprisingly less and rs7597774 minor alleles between the case frequent among cases than controls (P = 0.002, and control groups. Odds ratios (ORs) and cor- P < 0.001, respectively).

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Table 2. Allelic analysis of rs7597774 association with DCM

rs7597774 (Case/Control) Frequency of A Allele (Case/Control) P-value OR (95% CI) P-adj Exp (B) (95% CI) Total (399/1384) 0.269/0.202 0.016 1.457 (1.215-1.748) 0.016 1.582 (1.296-1.930) Male (250/786) 0.276/0.196 2.21 × 10-4 1.564 (1.24-1.974) 0.053 1.581 (1.234-2.025) Female (149/598) 0.258/0.210 0.072 1.312 (0.977-1.761) 0.006 1.609 (1.150-2.253) Hypertension (143/616) 0.234/0.221 0.637 1.080 (0.796-1.465) 0.556 1.105 (0.792-1.543) T2DM (53/237) 0.349/0.217 5.62 × 10-3 1.931 (1.225-3.045) 0.018 1.837 (1.110-3.041)

OR, odds ratio; P-adj, adjusted P value; Exp (B), adjusted OR; CI, confidence interval; T2DM, type 2 diabetes mellitus.

Table 3. Genotypic analysis of rs1739843/rs7597774 with DCM under Overall, the minor three genetic models allele A of rs7597774 showed a significant Models P-value OR (95% CI) P-adj Exp (B) (95% CI) allelic association wi- rs1739843 th an increased risk Addictive 0.785 N/A 0.744 1.032 (0.856-1.244) of DCM (Total P = Recessive 0.511 1.157 (0.763-1.754) 0.601 1.124 (0.725-1.742) 0.0156, OR 1.457; Dominant 0.776 1.036 (0.829-1.296) 0.890 1.017 (0.802-1.290) Table 2). The associ- rs7597774 ation remained sig- Addictive 2.79 × 10-4 N/A 1.7 × 10-5 1.506 (1.249-1.815) nificant with an OR of -3 -4 Recessive 1.12 × 10 1.978 (1.304-3.001) 3.27 × 10 2.208 (1.433-3.402) 1.582 (P-adj = 0.01- Dominant 1.01 × 10-3 1.465 (1.169-1.836) 3.19 × 10-4 1.557 (1.224-1.982) 57) after adjusting

OR, odds ratio; P-adj, adjusted P value; Exp (B), adjusted OR; CI, confidence interval. for covariates. In the subgroup analyses of males and T2DM, Prior statistical power was estimated for the a significant association between the A allele of proposed study on the 1783 participants. A rs7597774 and DCM was found with all param- type I error of 0.05 and a minor allele frequency eters (P = 2.205 × 10-4 and 5.624 × 10-3, (MAF) of 0.256 for rs1739843 and 0.167 for respectively). After adjusting covariates, a cor- rs7597774 in the Chinese Han population relation was also found between the A allele

(NCBI data) generated odds ratios (OR) of 0.67 and DCM in the female subgroup (P-adj = 0.006). for rs1739843 and 1.44 for rs7597774 in DCM No relationship between the A allele and DCM [7]. The statistical power was calculated to be in the hypertension subgroup was found before 98.5% and 94.9% for rs1739843 and or after adjustment. These results demonstrat- rs7597774, respectively. The SNPs were also ed that the A allele of rs7597774 was associ- tested and found to be in Hardy-Weinberg equi- ated with DCM in the cohort, but this associa- librium (HWE) in the cohort (P > 0.05). tion was not specific to any subgroup based on individual clinical parameters. Allelic association between rs1739843/ rs7597774 and DCM Genotypic association between rs1739843/ rs7597774 and DCM Analyses were performed to illuminate whether either of the SNPs was related to DCM suscep- Genotypic association of the different alleles tibility, and more specifically to individual from the two SNPs with DCM was analyzed alleles. Analysis was also performed to identify under the three common genetic models, addi- allelic associations specific to clinical subgroup tive, dominant and recessive (Table 3). For (s). Comparisons of the minor allele frequen- rs1739843, no association with DCM was cies of rs1739843 among the subgroups are revealed under the three models, even after summarized in Supplementary Table 1. None of adjusting for covariates. However, for the subgroups were associated with the minor rs7597774, a significant genotypic association allele T of rs1739843 (Total P = 0.6159, OR was found between the A allele and an 1.05). After adjusting for age, gender, hyperten- increased risk of DCM under the three models sion, T2DM, rs1739843 still did not correlate both before and after adjusting for covariates in with DCM (Total P-adj = 0.407, OR 0.920). the study groups (additive model: P = 2.79 ×

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Table 4. Distribution of rs7597774 genotype relative to NYHA heart functional grade of DCM patients rs7597774 genotype NYHA Heart Functional Grade P Total AA AC CC -value I 2 (4.88%) 7 (4.49%) 14 (6.93%) 24 (6.02%) II 7 (17.07%) 30 (19.23%) 65 (32.18%) 102 (25.56%) III 21 (51.22%) 77 (49.36%) 81 (40.10%) P = 0.021 179 (44.86%) IV 11 (26.83%) 42 (26.92%) 42 (20.79%) 94 (23.56%) Total 41 156 202 399 NYHA, New York Heart Association.

(P = 0.021, Table 4 and Figure 1). In NYHA I/II, the proportion of genotype AA (2/41 (4.8- 8%)/7/41 (17.07%)) was lower than genotype CC (14/202 (6.93%)/65/202 (32.18%)). In contrast, the proportion of genotype AA in NYHA III/IV (21/41 (51.22%)/11/41 (26. 83%)) was strikingly greater than genotype CC (81/202 (40.10%)/42/202 (20.79%)). The results further demonstrated that the A allele of rs7597774 was associated with an increased risk of DCM, and that patients with risk allele A displayed more severe Figure 1. Patients with rs7597774 risk allele A display more severe symp- symptoms than patients with toms than patients with the genotype CC. Ridit analysis was performed to es- CC. tablish the nature of relationship between rs7597774 genotypes and NYHA heart functional grade in DCM patients. Groups 1, 2, and 3 represent ridit Assessment of association values for genotypes AA, AC, and CC. The ridit value is represented as a 95% between rs7597774 and confidence interval (CI) of the mean (M). The average ridit value of groups 1 and 2 is significantly greater than group 3. therapeutic regimens of DCM patients

-4 -5 10 , P-adj = 1.7 × 10 ; recessive model: P = To further characterize the clinical significance -3 -4 1.12 × 10 , P-adj = 3.27 × 10 ; dominant model: of the rs7597774 variant, the data for the -3 -4 P = 1.01 × 10 , P-adj = 3.19 × 10 ). method of treatment were analyzed for genotypic association under a dominant model for rs7597774 is significantly correlated with allele A. The therapeutic regimens of the 399 NYHA heart functional classification DCM patients were recorded into two groups: classified drug therapy and catheterization/sur- To determine whether the any clinical parame- gical intervention. The latter included dual ter could be a manifestation of the underlying chamber pacing (DDD), implantable cardiovert- rs7597774 genotype, the clinical data and the er defibrillator (ICD), cardiac resynchronization genotypes were examined for associations. The therapy (CRT), cardiac resynchronization thera- distribution of rs7597774 genotypes in DCM py defibrillator (CRTD), and cardiac transplanta- patients was similar when age, LVEDD, LAD, tion (CTX). Only 31/399 DCM patients (7.77%) BNP levels, and LVEF were examined (Supp- underwent invasive therapy (Table 5). The dis- lementary Table 2). However, a significant cor- tribution of rs7597774 genotypes (AA, AC and relation between rs7597774 genotypes and CC) and therapeutic regimens in the invasive NYHA heart functional classification was found therapy group were examined by nonparamet-

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Table 5. Genotype distribution of rs7597774 relative to invasive association remained statis- procedure performed tically significant in three rs7597774 genotypes validation groups (Germany, Therapeutic Regimens P Total AA AC CC -value France 1, and France 2) [7]. DDD 1 (20%) 3 (23.08%) 6 (46.15%) 10 This finding revealed HSPB7 as a risk factor for idiopathic ICD 1 (20%) 5 (38.46%) 2 (15.38%) 8 DCM at the first diagnosis. CRT 0 (0%) 0 (0%) 5 (38.46%) 5 0.008 An association of 12 SNP CRTD 2 (40%) 2 (15.38%) 0 (0%) 4 HSPB7 polymorphisms, inclu- CTX 1 (20%) 3 (23.08%) 0 (0%) 4 ding rs1739843, was made Total 5 13 13 31 with heart failure, but the DDD, dual chamber pacing; ICD, implantable cardioverter defibrillator; CRT, cardiac diagnosis of DCM was not resynchronization therapy; CRTD, cardiac resynchronization therapy defibrillator; included in the analysis in CTX, cardiac transplantation. this third study [15]. Other groups did not find an asso- ric analysis in the Kruskal-Wallis H test. Analysis ciation of rs1739843 with sporadic DCM in of the data from the 31 patients demonstrated various western populations [8]. Similar results that a greater proportion of genotype AA (3/5 were demonstrated in a small Chinese Han (60%)) than genotype CC (5/13 (38.46%)) population [9]. The sample size of our study was patients underwent more invasive interven- larger than this previous study on Chinese Han, tions, including CRT, CRTD and CTX. Corre- and the statistical power was greater at 98.5% spondingly, a smaller proportion of genotype AA for rs1739843. Our result that no association (2/5 (40%)) than genotype CC (8/13 (61.54%)) of rs1739843 with DCM exists in Chinese Han patients received intervention with DDD and cohorts is in all probability real. The most likely ICD (P = 0.008). These results indicated that reason for the different results is differences in genotypes of rs7597774 might be used to ethnic origin (European/Caucasian versus guide the selection for treatment of individual Chinese Han). First, the minor allele frequency DCM patients. However, the study sample was of rs1739843 was different among the cohorts small, and the results require further testing in (0.39 in Europeans; 0.256 in Chinese Han). a larger cohort. Second, in the German population, the T allele Discussion of rs1739843 was found to be a protective allele to DCM [7], and these results were cor- The hope for molecular characterization of roborated in the study where rs1739843 was heart dysfunction is that it will contribute to the associated with heart failure [14]. development of therapies for prevention and HSPB7 encodes the small heat shock protein treatment of the disease. Here, relationships cvHsp and is known to be expressed in cardio- between SNPs rs1739843 and rs7597774 and DCM were examined in a cohort of Chinese Han vascular and insulin-sensitive tissues [16]. In where the statistical power was 98.5% and general, the expression and activation of heat 94.9%, respectively. No significant association shock proteins is influenced by elevated tem- was found between rs1739843 and DCM peratures as well as ischemia, hypoxia, and regardless of the genetic model applied, before acute cellular stress [17, 18]. However, none of or after adjusting for covariates. A significant the DCM-associated SNPs identified through association between rs7597774 and increased systematic sequencing actuallyaffects the cod- risk of DCM was however revealed before and ing sequence of HSPB7 [14]. HSPB7 exons after adjusting for covariates. have also been sequenced in 168 independent index cases diagnosed with familial DCM, but Several studies have applied a 50K bead chip no coding variant was identified [8]. Therefore, to reveal associations between SNPs and heart the biological function of the polymorphisms of dysfunction. In one study, rs1739843 was HSPB7 in DCM/heart failure risk remains found to be associated with heart failure [14]. A unclear. second set of studies also revealed a significant association between rs1739843 and idio- A statistically significant association between pathic DCM in German populations, and the ADD2 rs7597774 and idiopathic DCM was pre-

1193 Int J Clin Exp Med 2015;8(1):1188-1196 Variant rs7597774 in ADD2 and dilated cardiomyopathy viously found in German populations, but the genotype of rs7597774 might assist in assess- results were not validated in other samples/ ing the cardiac function during heart failure as cohorts [7]. Our study was the first to confirm a well as to be able to identify the DCM patients statistically significant association between who will benefit most from particular rs7597774 and DCM, with an allelic or geno- therapies. typic model, before or after adjusting covariates. Interestingly, the minor allele C of The results require further investigation both rs7597774 in European ancestry conferred a for validation and more importantly to under- risk of DCM, whereas in Chinese Han samples, stand the biological function of the SNPs in it was the minor allele A of rs7597774 that was DCM. First, although an association between found to increase the risk of DCM. The minor ADD2 rs7597774 and DCM is reported for the allele and MAF of rs7597774 were, however, first time in the Chinese Han population, this different in the two races (European C allele, result as well as the association with treat- MAF 0.325; Chinese Han A allele, MAF 0.167). ment, must be validated in larger Chinese Han cohorts. Such studies can ultimately assess Adducin 2 (Add2) is a heterodimeric cytoskele- the utility of rs7597774 in predicting therapy tal protein. ADD2 spans 108 kb including 17 for DCM patients. Second, although the analy- exons that are alternatively spliced to code for sis here revealed only rs7597774 from ADD2 to at least five known protein isoforms [19]. Add2 be associated with DCM, these results never- has been proposed to regulate renal tubular theless implicate a genetic component to the transport of Na+ reabsorption, which in turn disease, and thus highlight the importance of regulates body sodium, fluid volumes, and the examining more variants, especially low-pene- development of hypertension [20, 21]. A series trance variants from other suspected DCM- of parallel studies indicated that an altered susceptibility genes, such as BAG3 [8]. Third, function in Add2 might cause hypertension because DCM is a multifactorial disease, the through enhanced constitutive tubular sodium interaction of multiple variants from different reabsorption. A variant of ADD2 was also found chromosomes should be analyzed in order to to be associated with blood pressure in rats gain better insight into the genetic component [20]. Furthermore, eight SNPs in ADD2 were of DCM. In this study, two polymorphisms that found to be significantly associated with sys- are located on different chromosomes were tolic blood pressure in untreated hypertensive assessed. However as rs1739843 was not patients, and SNPs were also identified that found to be associated with DCM, the interac- were associated with gene-by-drug interactions tion between the two variants could not be cal- on systolic blood pressure in drug-treated culated. Finally, the nature of functional rela- hypertensive patients [22]. The mechanism tionship of the ADD2 polymorphisms in the underlying the association between the poly- etiology of DCM remains to be elucidated. morphisms of ADD2 and DCM risk, however, still remains unclear. ADD2 rs7597774 but not HSPB7 rs1739843 was found to be associated with the risk of Based on NYHA functional classification, the DCM in the Chinese Han population. Including results of the study demonstrated that geno- the genotype of rs7597774 in the clinical data type AA DCM patients had more severe heart collected for DCM patients may assist in failure symptoms than genotypes AC + CC. assessing heart function and predicting those Furthermore, DCM patients harboring genotype who will benefit most from particular thera- AA underwent more complicated medical inter- pies. ventions, although the selection of therapies is related to many additional factors such as Acknowledgements symptoms (syncope, dyspnea), LVEF < 40%, malignant arrhythmia, NYHA grade > II, cardiac This work was supported by grants from the arrest, history of myocardial infarction or family Specialized Research Fund for the Doctoral history of sudden death, QRS duration > 120 Program of Higher Education (No. 2009210- ms, and even economic issues. Although many 5110003), Dalian Science and technology proj- factors are involved in the selection of the right ect (No. 2011503391), and the National Basic therapeutic strategy for individual patients, our Research Program of China (No. 2013CB- ultimate goal was to determine whether the 531100).

1194 Int J Clin Exp Med 2015;8(1):1188-1196 Variant rs7597774 in ADD2 and dilated cardiomyopathy

Disclosure of conflict of interest ter T, Germain M, Dubourg O, Tavazzi L, Au- mont MC, DeGroote P, Fauchier L, Trochu JN, None. Gibelin P, Aupetit JF, Stark K, Erdmann J, Het- zer R, Roberts AM, Barton PJ, Regitz-Zagrosek Address correspondence to: Dr. Yan Z Yang, De- V, Aslam U, Duboscq-Bidot L, Meyborg M, partment of Cardiology, First Affiliated Hospital of Maisch B, Madeira H, Waldenström A, Galve E, Dalian Medical University, 193, Lianhe Road, Cleland JG, Dorent R, Roizes G, Zeller T, Blan- kenberg S, Goodall AH, Cook S, Tregouet DA, Shahekou District, Dalian 116000, China. E-mail: Tiret L, Isnard R, Komajda M, Charron P, Cam- [email protected] bien F. A genome-wide association study identifies two loci associated with heart failure due References to dilated cardiomyopathy. Eur Heart J 2011; 32: 1065-1076. [1] Maekawa Y, Ouzounian M, Opavsky MA, Liu PP. [9] Li X, Luo R, Mo X, Jiang R, Kong H, Hua W, Wu Connecting the missing link between dilated X. Polymorphism of ZBTB17 gene is associated cardiomyopathy and viral myocarditis: Virus, with idiopathic dilated cardiomyopathy: a case cytoskeleton, and innate immunity. Circulation control study in a Han Chinese population. Eur 2007; 115: 5-8. J Med Res 2013; 18: 10-17. [2] Cihakova D, Rose NR. Pathogenesis of myocar- [10] Shi L, Li C, Wang C, X, Xia Y, Wu G, Wang F, Xu ditis and dilated cardiomyopathy. Adv Immunol C, Wang P, Li X, Wang D, Xiong X, Bai Y, Liu M, 2008; 99: 95-114. Liu J, Ren X, Gao L, Wang B, Zeng Q, Yang B, [3] Morimoto S. Sarcomeric proteins and inherited Ma X, Yang Y, Tu X, Wang QK. Assessment of cardiomyopathies. Cardiovasc Res 2008; 77: association of rs2200733 on chromosome 659-666. 4q25 with atrial fibrillation and ischemic stroke [4] Maron BJ, Towbin JA, Thiene G, Antzelevitch C, in a Chinese Han population. Hum Genet Corrado D, Arnett D, Moss AJ, Seidman CE, 2009; 26: 843-849. Young JB. Contemporary definitions and clas- [11] American Diabetes Association. Standards of sification of the cardiomyopathies: an Ameri- medical care in diabetes--2008. Diabetes Care can Heart Association Scientific Statement 2008; 31: S12-S54. from the Council on Clinical Cardiology, Heart [12] Wittwer CT. High-resolution DNA melting analy- Failure and Transplantation Committee; Quali- sis: advancements and limitations. Hum Mutat ty of Care and Outcomes Research and Func- 2009; 30: 857-859. tional Genomics and Translational Biology In- [13] Purcell S, Neale B, Todd-Brown K, Thomas L, terdisciplinary Working Groups; and Council on Ferreira MA, Bender D, Maller J, Sklar P, de Epidemiology and Prevention. Circulation Bakker PI, Daly MJ, Sham PC. PLINK: a tool set 2006; 113: 1807-1816. for whole-genome association and population- [5] Jefferies JL, Towbin JA. Dilated cardiomyopa- based linkage analyses. Am J Hum Genet thy. Lancet 2010; 375: 752-762. 2007; 81: 559-575. [6] Hershberger RE, Norton N, Morales A, Li D, [14] Cappola TP, Li M, He J, Ky B, Gilmore J, Qu L, Siegfried JD, Gonzalez-Quintana J. Coding se- Keating B, Reilly M, Kim CE, Glessner J, Frack- quence rare variants identified in MYBPC3, elton E, Hakonarson H, Syed F, Hindes A, Mat- MYH6, TPM1, TNNC1, and TNNI3 from 312 pa- kovich SJ, Cresci S, Dorn GW. Common vari- tients with familial or idiopathic dilated cardio- ants in HSPB7 and FRMD4B associated with myopathy. Circ Cardiovasc Genet 2010; 3: advanced heart failure. Circ Cardiovasc Genet 155-161. 2010; 3: 147-154. [7] Stark K, Esslinger UB, Reinhard W, Petrov G, [15] Matkovich SJ, Van Booven DJ, Hindes A, Kang Winkler T, Komajda M, Isnard R, Charron P, Vil- MY, Druley TE, Vallania FL, Mitra RD, Reilly MP, lard E, Cambien F, Tiret L, Aumont MC, Du- Cappola TP, Dorn GW. Cardiac signaling genes bourg O, Trochu JN, Fauchier L, Degroote P, exhibit unexpected sequence diversity in spo- Richter A, Maisch B, Wichter T, Zollbrecht C, radic cardiomyopathy, revealing HSPB7 poly- Grassl M, Schunkert H, Linsel-Nitschke P, Erd- morphisms associated with disease. J Clin In- mann J, Baumert J, Illig T, Klopp N, Wichmann vest 2010; 120: 280-289. HE, Meisinger C, Koenig W, Lichtner P, Meit- [16] Krief S, Faivre JF, Robert P, Le Douarin B, Bru- inger T, Schillert A, König IR, Hetzer R, Heid IM, ment-Larignon N, Lefrère I, Bouzyk MM, Ander- Regitz-Zagrosek V, Hengstenberg C. Genetic son KM, Greller LD, Tobin FL, Souchet M, Bril A. Association Study Identifies HSPB7 as a Risk Identification and characterization of cvHsp. A Gene for Idiopathic Dilated Cardiomyopathy. novel human small stress protein selectively PloS Genet 2010; 6: e1001167-e1001176. expressed in cardiovascular and insulin-sensi- [8] Villard E, Perret C, Gary F, Proust C, Dilanian G, tive tissues. J Biol Chem 1999; 274: 36592- Hengstenberg C, Ruppert V, Arbustini E, Wich- 36600.

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[17] Lindquist S, Craig EA. The heat-shock proteins. [21] Hughes CA, Bennett V. Adducin: A physical Annu Rev Genet 1988; 22: 631-677. model with implications for function in assem- [18] Anckar J, Sistonen L. Heat shock factor 1 as a bly of spectrin-actin complexes. J Biol Chem coordinator of stress and developmental path- 1995; 270: 18990-18996. ways. Adv Exp Med Biol 2007; 594: 78-88. [22] Kardia SL, Sun YV, Hamon SC, Barkley RA, [19] Gilligan DM, Lozovatsky L, Silberfein A. Organi- Boerwinkle E, Turner ST. Interactions between zation of the human beta-adducin gene the adducin 2 gene and antihypertensive drug (ADD2). Genomics 1997; 43: 141-148. therapies in determining blood pressure in [20] Bianchi G, Tripodi G, Casari G, Salardi S, Bar- people with hypertension. BMC Med Genet ber BR, Garcia R, Leoni P, Torielli L, Cusi D, Fer- 2007; 8: 61-71. randi M, Pinna LA, Baralle FE, Ferrari P. Two point mutations within the adducin genes are involved in blood pressure variation. Proc Natl Acad Sci U S A 1994; 91: 3999-4003.

1196 Int J Clin Exp Med 2015;8(1):1188-1196 Variant rs7597774 in ADD2 and dilated cardiomyopathy

Supplementary Table 1. Allelic analysis of rs1739843 association with DCM Frequency of T Allele rs1739843 (Case/Control) P OR (95% CI) P Exp (B) (95% CI) (Case/Control) -value -adj Total (399/1384) 0.2707/0.2612 0.6159 1.05 (0.8788-1.254) 0.407 0.920 (0.756-1.120) Male (250/786) 0.284/0.2589 0.2695 1.135 (0.9068-1.422) 0.984 0.998 (0.784-1.270) Female (149/598) 0.2483/0.2642 0.6064 0.92 (0.6866-1.233) 0.148 0.777 (0.553-1.094) Hypertension (143/616) 0.2587/0.2549 0.8807 1.02 (0.7605-1.369) 0.607 0.918 (0.662-1.273) T2DM (53/237) 0.2736/0.23 0.3771 1.261 (0.7823-2.033) 0.843 0.948 (0.559-1.607) OR, odds ratio; P-adj, adjusted P values; Exp (B), adjusted OR; CI, confidence interval; T2DM, type 2 diabetes mellitus.

Supplementary Table 2. Genotype distribution of rs7597774 in DCM patients based on clinical parameters

Subgroups AA AC CC P-value Age (M ± SD, y) 61.56 ± 10.96 55.60 ± 14.64 55.16 ± 16.06 0.064 LVEDD (M ± SD, mm) Male 64.77 ± 10.94 63.80 ± 9.49 66.54 ± 8.50 0.276 Female 61.86 ± 5.58 61.09 ± 9.41 59.04 ± 6.03 0.536 LVEF (M ± SD, %) 33.60 ± 9.18 34.54 ± 11.17 37.31 ± 12.90 0.104 BNP (M ± SD, pg/ml) 581.91 ± 452.16 979.90 ± 966.44 1505.83 ± 1313.97 0.113 LAD (M ± SD, mm) Male 43.77 ± 5.76 43.52 ± 6.65 44.86 ± 6.69 0.399 Female 43.57 ± 4.35 42.97 ± 6.18 44.48 ± 5.17 0.498 LVEDD, left ventricular end-diastolic diameter; M ± SD, mean ± standard deviation; LVEF, left ventricular ejection fraction; BNP, brain natriuretic peptide; LAD, left atrial diameter; NYHA, New York Heart Association.