Loss-Of-Function DNA Sequence Variant in the CLCNKA Chloride Channel Implicates the Cardio-Renal Axis in Interindividual Heart Failure Risk Variation

Loss-of-function DNA sequence variant in the CLCNKA chloride channel implicates the cardio-renal axis in interindividual heart failure risk variation

Thomas P. Cappolaa,1, Scot J. Matkovichb, Wei Wangc, Derek van Boovenb, Mingyao Lid, Xuexia Wangd, Liming Qud, Nancy K. Sweitzere, James C. Fangf, Muredach P. Reillya, Hakon Hakonarsong, Jeanne M. Nerbonnec, and Gerald W. Dorn IIb,1

aPenn Cardiovascular Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; bCenter for Pharmacogenomics, Department of Medicine, and cDepartment of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110; dCenter for Clinical Epidemiology and Biostatistics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; eDivision of Cardiovascular Medicine, University of Wisconsin, Madison, WI 53792; fDivision of Cardiovascular Medicine, Case Western Reserve University, Cleveland, OH 44106; and gCenter for Applied Genomics, Children’s Hospital of Philadelphia, Philadelphia, PA 19104

Edited* by Jonathan G. Seidman, Harvard Medical School, Boston, MA, and approved December 20, 2010 (received for review November 22, 2010) Common heart failure has a strong undefined heritable component. The Institute for Translational Medicine and Therapeutics/ Two recent independent cardiovascular SNP array studies identified Broad Institute/Candidate-gene Association Resource (IBC) con- a common SNP at 1p36 in intron 2 of the HSPB7 gene as being asso- sortium developed a high-density SNP cardiovascular subgenome ciated with heart failure. HSPB7 resequencing identified other risk array to complement genome-wide platforms in heart disease (13). alleles but no functional gene variants. Here, we further show no Using this array, we previously identified in two US Caucasian heart effect of the HSPB7 SNP on cardiac HSPB7 mRNA levels or splicing, failure referral populations a heart failure risk locus on chromo- suggesting that the SNP marks the position of a functional variant in some 1p36 (rs1739843), within the cardiovascular heat shock pro- another gene. Accordingly, we used massively parallel platforms to tein gene HSPB7 (14). A European consortium using the IBC array resequence all coding exons of the adjacent CLCNKA gene, which recently reported that the identical variant was their strongest ge- K K CLCNKA encodes the a renal chloride channel (ClC- a). Of 51 exonic netic predictor of nonfamilial dilated cardiomyopathy (15). Thus, variants identified, one SNP (rs10927887, encoding Arg83Gly) was taken together, rs1739843 has been linked with heart failure in six common, in linkage disequilibrium with the heart failure risk SNP in independent populations on two continents, establishing it as a HSPB7, and associated with heart failure in two independent Cauca- bona fide heart failure risk variant. rs1739843 occurs in intron 2 sian referral populations (n = 2,606 and 1,168; combined P =2.25× of HSPB7 and has no predicted affect on its protein sequence −6 10 ). Individual genotyping of rs10927887 in the two study popula- or function. Accordingly, we previously resequenced the entire tions and a third independent heart failure cohort (combined HSPB7 gene to screen for nearby functional variants (16). Al- n = 5,489) revealed an additive allele effect on heart failure risk though these experiments further confirmed the rs1739843 as- that is independent of age, sex, and prior hypertension (odds ratio = sociation and revealed 11 additional heart failure-associated P × −7 1.27 per allele copy; =8.3 10 ). Functional characterization of SNPs in tight linkage disequilibrium, the additional HSPB7 SNPs K recombinant wild-type Arg83 and variant Gly83 ClC- a chloride chan- were also intronic or synonymous. As such, the causative gene ≈ nel currents revealed 50% loss-of-function of the variant channel. variant(s) remain unknown, suggesting either that rs1739843 fi fi These ndings identify a common, functionally signi cant genetic marks an expression quantitative trait locus (eQTL) that modifies CLCNKA risk factor for Caucasian heart failure. The variant risk allele, HSPB7 expression (17) or that it marks the position of a geneti- HSPB7 telegraphed by linked variants in the adjacent gene, uncovers cally linked functional variant located outside of HSPB7. a previously overlooked genetic mechanism affecting the cardio- Here we demonstrate that there is no association between renal axis. rs1739843 genotype and HSPB7 mRNA expression levels or splicing in human myocardium, making an eQTL mechanism cardiomyopathy | genetic association unlikely. For this reason, we resequenced the coding exons of the neighboring gene, CLCNKA, which is within the same linkage- he lifetime risk of developing heart failure is estimated at one disequilibrium block as HSPB7.Of51CLCNKA sequence var- Tin five (1, 2). Although rare familial cardiomyopathies can iants identified, rs10927887 (encoding Arg83Gly) is positively lead to heart failure that is almost entirely attributable to genetic associated with heart failure risk in three independent Caucasian factors, common heart failure has a smaller, poorly defined her- heart failure populations. Functional analysis shows the Gly83 itable component. Framingham Heart Study data show that pa- variant channel to exhibit markedly abnormal chloride currents, rental heart failure confers a 70% greater disease risk than in suggesting that this common loss of function variant in CLCNKA individuals without a family history (3). Cardiac hypertrophy that at 1p36 confers heart failure risk in Caucasians and accounts for predisposes to heart failure is also heritable, suggesting that un- derlying genetic variation contributes to interindividual variability in heart failure risk (4). A fraction of common heart failure may Author contributions: T.P.C., J.M.N., and G.W.D. designed research; T.P.C., S.J.M., W.W., be due to unrecognized monogenic cardiomyopathy (5), but most D.v.B., M.L., X.W., L.Q., N.K.S., J.C.F., M.P.R., H.H., J.M.N., and G.W.D. performed research; T.P.C., S.J.M., W.W., D.v.B., M.L., N.K.S., J.C.F., M.P.R., H.H., J.M.N., and G.W.D. contributed cases are explained by multiple interacting environmental and as- new reagents/analytic tools; T.P.C., S.J.M., W.W., D.v.B., M.L., X.W., L.Q., M.P.R., H.H., J.M.N., yet unidentified genetic susceptibility factors. Previous efforts to and G.W.D. analyzed data; and T.P.C., S.J.M., H.H., and G.W.D. wrote the paper. identify genetic risk variants uncovered a rare combination of The authors declare no conflict of interest. adrenergic receptor polymorphisms that increases heart failure *This Direct Submission article had a prearranged editor. susceptibility 10-fold among African Americans (6), although this Freely available online through the PNAS open access option. particular combination of risk alleles has not yet been replicated 1To whom correspondence may be addressed. E-mail: [email protected] (7, 8). A few population-based cohort studies have implicated or [email protected]. – genomic regions in heart failure risk (9 11) or mortality (12), but This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. specific causative risk alleles are still unknown. 1073/pnas.1017494108/-/DCSupplemental.

2456–2461 | PNAS | February 8, 2011 | vol. 108 | no. 6 www.pnas.org/cgi/doi/10.1073/pnas.1017494108 Downloaded by guest on September 29, 2021 the previously described association between some HSPB7 SNPs variants marked heart failure risk alleles within the broader ge- and heart failure. netic region. Accordingly, we resequenced all 19 coding exons and intron–exon boundaries of the adjacent CLCNKA gene in Results 1,742 DNA samples from the Cincinnati Heart Failure Study rs1739843 Is Not Associated with Altered Myocardial HSPB7 Expression (18, 19). (Detailed clinical characteristics of the study pop- or Splicing. The rs1739843 intronic HSPB7 SNP has been associated ulations are in Table S1.) Polymorphism screening by pooled with heart failure in multiple independent cohorts. Neither it nor resequencing identified 51 nonprivate exonic CLCNKA SNPs other linked polymorphisms alter protein coding. To examine (nine in dbSNP and 42 previously unreported; 11 synonymous whether this SNP might instead confer HSPB7 dysfunction by al- and 40 nonsynonymous; Table S2). Only seven of these SNPs tering transcription, we measured HSPB7 mRNA levels from human (five nonsynonymous) are common, defined as having an allele left-ventricular heart specimens of 111 Caucasian heart failure sub- frequency of ≥0.05. Thus, the CLCNKA gene exhibits limited jects genotyped for rs1739843. Using microarrays, we found no dif- polymorphic variability. ference in HSPB7 expression across rs1739843 genotypes, and we To determine whether CLCNKA polymorphisms were associ- verified this finding using RT–quantitative PCR (RT-qPCR) (Fig. ated with heart failure, allele frequencies for the 51 SNPs were 1A). HSPB7 exon expression revealed identical splicing patterns compared between 1,117 Caucasian heart failure cases and 625 regardless of rs1739843 genotype (Fig. 1B). Thus, the association unaffected controls. Nine SNPs (seven nonsynonymous) had between rs1739843 and heart failure risk is unlikely to be caused by significant associations with heart failure (P < 0.001, corre- an eQTL mechanism or by alterations in HSPB7 splicing. sponding to a Bonferroni-adjusted P < 0.05). In all but one in- stance allele frequencies for these SNPs were <0.05, whereas that A Common Nonsynonymous CLCNKA SNP Is Associated with Systolic for the marker HSPB7 SNP was ≈0.5 (14, 16). However, the allele Heart Failure. HapMap data reveal the HSPB7 locus on 1p36 to be frequency of rs10927887 was ≈0.50 in controls and ≈0.57 in heart − an area of high linkage disequilibrium (Fig. 2A). Because the failure cases (P = 7.09 × 10 5; Table S2), which is similar to the HSPB7 heart failure-associated SNPs do not alter protein coding marker HSPB7 SNP. This CLCNKA variant encodes a change HSPB7 or cardiac gene expression, we hypothesized that the risk from Arg to Gly at amino acid 83 (exon 3) of the renal ClC-Ka chloride channel (Fig. 2B). Because the association between rs10927887 and heart failure was identified during comprehensive screening of all CLCNKA coding exons, we performed a technical replication of the findings by targeted resequencing of just CLCNKA exon 3 at greater se- MEDICAL SCIENCES quencing depth (90-fold vs. 19-fold) in the same study population. Allele frequencies in controls and heart failure cases were similar to the screening study (≈0.50 in controls and ≈0.56 in heart failure − cases; P =8.7× 10 4; Table 1, primary cohort). Sequence data from Caucasians in the 1000Genomes project show that rs10927887 is in strong linkage disequilibrium (R2 = 0.87) with the previously identified heart failure-associated intronic HSPB7 SNP, rs1739843 (14, 16). On the basis of these findings, we considered that rs1739843 on the IBC array tagged the position of the nonsynonymous CLCNKA variant encoded by rs10927887. To validate the association between rs10927887 and heart failure, we resequenced CLCNKA exon 3 in an independent cohort of 857 Caucasian heart failure cases and 311 unaffected Caucasian controls from the Penn Heart Failure Study (20). Again, rs10927887 was overrepresented in heart failure (≈0.56 − vs. ≈0.48 in controls; P = 4.4 × 10 4; Table 1, secondary cohort). Combined analysis of cases (n = 1,974) and controls (n = 936) from both study cohorts gives a P value for association of 2.25 × − 10 6 (Table 1, combined analysis). Our study design compares carefully selected nonaffected controls with systolic heart failure of any etiology (i.e., all-cause heart failure). To determine whether the rs10927887 association was stronger with ischemic or nonischemic heart failure, we performed subgroup analyses in the primary and secondary cohorts. The association was present for heart failure of either etiology in both − groups, with combined analyses giving P values of 9.88 × 10 5 for − ischemic heart failure and 2.82 × 10 6 for nonischemic heart failure.

Heart Failure Risk Conferred by rs10927887 Increases with Gene Dose. For a more detailed assessment of the association between rs10927887 and heart failure risk, we individually genotyped Cau- casian heart failure cases and controls from the primary Cincinnati Fig. 1. No association between rs1739843 genotype and HSPB7 mRNA ex- Heart Failure Study cohort, an expanded secondary Penn Heart pression or splicing in human myocardium. Data are from n = 111 left- Failure Study cohort, and from a third independent heart failure ventricular free-wall specimens from genetically inferred Caucasians with cohort (total n = 5,489 subjects; Table 2). In age- and gender- heart failure. (A) HSPB7 RT-qPCR showed no differences in mRNA abundance CLCNKA across genotypes (P = 0.3; diamonds show median with interquartile range). adjusted models, the Gly83 allele was associated with all- (B) Exon arrays revealed a typical pattern of splicing observed in GenBank cause heart failure in all three cohorts, with an overall 1.26-fold −6 that was identical across rs1739843 genotypes (P = 0.8; diamonds show increase in risk per allele copy (P =3.8× 10 ; Table 2). Further median expression of each exon probeset, spaced along Chr1). adjusting for age, gender, and hypertension did not alter the associ-

Cappola et al. PNAS | February 8, 2011 | vol. 108 | no. 6 | 2457 Downloaded by guest on September 29, 2021 Fig. 2. Genetic characteristics of CLCNKA.(A) Structure (Upper) and linkage disequilibrium (Lower) at 1p36 for HapMap Caucasian Europeans (CEU). (B) Representative rs10927887 genotypes by Sanger sequencing and associated changes in amino acid coding. (C) Transmembrane structural diagram showing position of heart failure risk variant Arg83Gly (R83G) relative to Bartter’s syndrome Cys80 mutation (W80C) and hypertension-associated Ala447 variant (T447A). Analogous position of common ClC-Kb variant T481S is also shown.

ation between Gly83 and heart failure risk (odds ratio 1.27, P =8.3× (Fig. 3B) having nearly linear current–voltage relations and − − 10 7; Table 2). Analysis of multiple race-informative genetic markers a reversal potential (in symmetrical Cl )of−0.2 ± 0.9 mV (Fig. showed no evidence for population stratification (λ = 1.010 and 3C). By comparison, currents evoked by ClC-Ka Gly83 had di- 1.055). These findings indicate that risk of heart failure is increased minished amplitudes at all test potentials (P < 0.001; Fig. 3C). by ≈27% and 54% in Gly83 heterozygotes and homozygotes, Immunoblot analysis revealed similar expression levels of Arg83 respectively, independent of age, sex, or hypertension. and Gly83 ClC-Ka, suggesting that reduced currents are an intrinsic property of the Gly83 ClC-Ka variant (Fig. 3D). The rs10927887 Confers Loss of Function of Renal Chloride Channel CLC- mean ± SEM (n = 10) reversal potential of the ClC-KaArg83– K a. rs10927887 encodes a substitution of Gly for normal Arg at encoded currents shifted to 12.2 ± 0.7 mV and 23.2 ± 1.8 mV − amino acid position 83 of the ClC-Ka renal chloride channel. We when the extracellular Cl concentration was reduced to 50 mM examined the functional implications of this gene variant by and 20 mM, respectively (Fig. 3E). In contrast, the mean ± SEM comparative analysis of recombinantly expressed Arg83 and (n = 13) reversal potential of the ClC-KaGly83–encoded cur- − Gly83 ClC-Ka channels in tsA201cells (21). Because barttin is rents (n = 13) was less affected by changes in extracellular Cl , necessary for ClC-K functional expression (22, 23), inward and shifting to 4.0 ± 2.7 mV and 10.0 ± 3.7 mV in 50 mM and 20 mM − outward currents in cells expressing ClC-Ka Arg83 or Gly83 extracellular Cl , respectively (Fig. 3F). These values are sig- alone or barttin alone were no different from nontransfected or nificantly lower than those determined for the ClC-KaArg83– YFP-expressing cells (Fig. 3A). However, coexpression of ClC-Ka encoded currents at both 50 mM (P = 0.017) and 20 mM − Arg83 with barttin evoked large inward and outward currents (P = 0.008) extracellular Cl . Overall these findings indicate that

Table 1. Association and subgroup analysis of CLCNKA rs10927887 G allele with nonfamilial heart failure G allele frequency

Cohort Controls Cases P

Primary cohort (n = 625 controls, 1,117 cases) 0.4979 0.5565 8.7 × 10−4 Ischemic cardiomyopathy (n = 691) 0.5440 0.0172 − Nonischemic cardiomyopathy (n = 426) 0.5767 4.3 × 10 4 Secondary cohort: (n = 311 controls, 857 cases) 0.4777 0.5600 4.4 × 10−4 − Ischemic cardiomyopathy (n = 422) 0.5613 1.5 × 10 3 − Nonischemic cardiomyopathy (n = 435) 0.5587 2.3 × 10 3 Combined analysis (n = 936 controls, 1,974 cases) 0.4912 0.5580 2.25 × 10−6 Ischemic cardiomyopathy (n = 1113) 0.5526 9.88 × 10−5 − Nonischemic cardiomyopathy (n = 861) 0.5697 2.82 × 10 6

2458 | www.pnas.org/cgi/doi/10.1073/pnas.1017494108 Cappola et al. Downloaded by guest on September 29, 2021 Table 2. Odds of advanced heart failure associated with each copy of the of CLCNKA risk allele in three study cohorts Age-, sex-, and Age- and sex-adjusted HTN-adjusted

Study cohort OR (95% CI) P OR (95% CI) P

Primary cohort (603 controls, 1.21 (1.04–1.39) 0.011 1.28 (1.05–1.55) 0.012 1,113 cases) − − Secondary cohort (1,887 controls, 1.26 (1.11–1.43) 1.9 × 10 4 1.26 (1.11–1.44) 2.0 × 10 4 755 cases) Tertiary cohort (316 controls, 1.31 (1.08–1.58) 0.0026 1.29 (1.06–1.57) 0.0048 785 cases) Metaanalysis (2,806 controls, 1.26 (1.14–1.38) 3.8 × 10−6 1.27(1.16–1.40) 8.3 × 10−7 2,653 cases)

HTN, hypertension; OR, odds ratio per copy of the risk allele (G, encoding Gly) at rs10927887 in additive genetic models; CI, conﬁdence interval. P values are two-sided for primary cohort and metaanalysis and one- sided for secondary and tertiary cohorts.

the heart failure-associated Gly83 variant substantially impairs focused on Caucasian disease. However, HapMap data indicate K ClC- a function. that the ClC-Ka Gly83 allele is ≈40% more prevalent in African- derived than European-derived individuals (≈70% vs. ≈50%, Discussion respectively). African Americans are known to have an increased Here, we performed exomic sequencing within the heart failure burden of heart failure compared with Caucasians [prevalence of risk locus on chromosome 1p36 and identified rs10927887 3% vs. 2%, respectively (36)] and a greater risk for heart failure K encoding ClC- a Gly83 as a common risk allele for heart failure progression with mild or moderate ventricular systolic dysfunc- among Caucasians. These findings also provide genetic evidence tion (37). It is interesting to consider that hyperreninemic MEDICAL SCIENCES supporting clinical observations that individual differences in hyperaldosteronism induced by the Gly83 ClC-Ka polymorphism salt-sensitivity can predispose to cardiovascular complications may be one of the genetic factors that contribute to dispropor- (24, 25). As expected for a common polymorphism, the increase tionate heart failure risk among African Americans. Our studies in heart failure risk conferred by the variant allele is modest are limited to Caucasians, and large-scale genetic association (≈27% per copy of the risk allele). However, approximately one studies in African American populations will be necessary to fourth of all Caucasians are homozygous for the rs10927887 address this hypothesis. variant allele, with an expected 54% increase in heart failure risk, Our findings suggest that the association with heart failure of the indicating a potentially broad impact at the population level. seminal 1p36 SNP in intron 2 of HSPB7 is at least in part due to the In analyzing the functional consequences of the CLCNKA heart linked ClC-Ka Gly-83 polymorphism. Although the functional data failure risk polymorphism, we determined that the Gly83 renal ClC- are striking and the reported associations of unexplained cardio- K a chloride channel variant reduces chloride currents. Intrinsic renal myopathy with Bartter’s syndrome are suggestive, these clinical mechanisms have been largely overlooked in assessing genetic risk data do not establish either causality of the risk allele or define the for heart failure, and this aspect of our work reinforces a strength of – – responsible mechanism. Such efforts will require structure genetic studies directed by array-based SNP genotype phenotype function studies to define how Gly83 alters the biophysical prop- associations, their mechanistic agnosticism. Although it seems erties of ClC-K and development of novel experimental systems to counterintuitive that a genetic variant that diminishes the ability of a assess the effects of ClC-Ka Gly83 on cardio-renal physiology. kidney cells to retain NaCl would increase risk for heart failure, CLCNKA K Furthermore, if the proposed concepts linking to heart nature has provided a possible explanation: the Gly83 renal ClC- a failure are correct, it is likely that other gene variants with similar variant is located near, and has a similar loss of function profile to, effects on the cardio-renal axis can also modify heart failure risk. a previously described Cys80 ClC-K mutation that in combination a We consider that the possibilities for such risk variants include rare with disruption of the related CLCNKB gene caused a Bartter’s-like or private nonsynonymous loss-of-function variants in CLCNKA syndrome in one affected individual (26). Bartter’s syndrome is (26, 38), variants of the related CLCNKB gene that confer similar a multifactorial inherited salt-wasting disorder produced by loss-of- channel dysfunction, or variants of other genes that affect renal salt function mutations in NCCT, NKCC2, ROMK, ClC-Kb, BSND, or – barttin (i.e., factors determining renal NaCl reabsorption) (27). The handling and therefore the renin angiotensin set point. Thus, on hallmark abnormality in all forms of Bartter’s syndrome is hyper- the basis of the current genetic and functional data, we postulate reninemia independent of volume status (28, 29). Hyperreninemia that multiple genetic events inducing a common cardio-renal physiological response can modify heart failure risk. Indeed, likewise mediates heart failure in the cardiorenal syndrome (30) and K was an independent risk factor for heart failure in the Heart Out- multiple or combinatorial interactions are likely. Because ClC- a comes Prevention Evaluation Study (31). Thus, the critical physio- Gly83 is both common and functional, its relative importance as a heart failure risk modifier gene seems clear. logic abnormality conferred by loss of ClC-Ka function with the Gly83 variant may be increased renin, predisposing affected subjects to Methods heart failure when a second “hit,” such as infarction or mechanical overload, damages the heart. Indeed, unexplained cardiomyopathies Study Subjects. Human study protocols were approved by the institutional ’ – review boards of the University of Cincinnati, Cincinnati, OH; the University of are a rare but recognized complication of Bartter s syndrome (32 Pennsylvania, Philadelphia, PA; Case Western Reserve University, Cleveland, 34), and pharmacological blockade of renin–angiotensin–aldoste- fi OH; or the University of Wisconsin, Madison, WI. All subjects provided written rone system components is rst-line therapy for heart failure (35). informed consent. Flash-frozen left ventricular myocardial samples from heart Because the seminal findings localizing heart failure risk to failure patients were obtained at time of cardiac surgery as previously de- chromosome 1p36 derived from a microarray study of Caucasian scribed (39, 40). Subjects with advanced systolic heart failure were recruited heart failure (14), chromosome walking in the present study also into one of two National Heart, Lung, and Blood Institute-funded longitu-

Cappola et al. PNAS | February 8, 2011 | vol. 108 | no. 6 | 2459 Downloaded by guest on September 29, 2021 − Fig. 3. Functional characterization of ClC-Ka Arg and Gly-83. (A) Whole-cell Cl currents, evoked in response to voltage steps ranging from −165 mV to +85 mV (in 10-mV increments) from a holding potential of 0 mV recorded from tsA201 cells expressing YFP, barttin, ClC-KaArg83 (WT), or ClCKaGly83 (mutant); the voltage clamp paradigm is illustrated adjacent to the current records. (B) Representative whole-cell currents recorded from YFP + barttin + ClC-KaArg83 (WT) and from YFP + barttin + ClC-KaGly83 (mutant)-expressing cells. (C) Mean ± SEM peak inward and outward currents determined from records similar to those illustrated in B plotted as a function of test potential; n = numbers of cells. (D) Representative anti-FLAG immunoblots of tsA201 cells transiently expressing FLAG-tagged ClC-KaArg83 or ClC-KaGly83 alone and in the presence of barttin. Anti-transferrin receptor is loading control. (E and F) Reversal − potentials of ClC-Ka Arg83 (E) and Gly83 (F) currents as a function of extracellular Cl concentration.

dinal studies of heart failure genomics (P50 HL77101 and R01 HL88577) from initial CLCNKA whole exon screening of the primary Cincinnati Heart Failure patients presenting to the heart failure referral program at the University of Study cohort and 91-fold for targeted exon 3 resequencing of the primary Cincinnati (16, 19) or the University of Pennsylvania (20), according to pre- (90.4-fold) and secondary Penn Heart Failure Study cohorts (92.7-fold). In- specified criteria. The same infrastructure was used to recruit nonaffected dividual genotypes for the Cincinnati Heart Failure Study cohort were de- controls. Additional cases and controls were analyzed from Case Western termined by dye terminator sequencing of exon 3 using an ABI3730xl Reserve University, University of Wisconsin at Madison, and the PennCATH capillary sequencer. Each sequence pherogram was blindly and inde- study (41) to constitute a tertiary study cohort. pendently examined by two researchers (S.J.M. and G.W.D.). Success rate was 98.9% and conforming to predictions of Hardy-Weinberg equilibrium Assessing Myocardial HSPB7 Expression and Splicing by rs1739843 Genotype. (P = 0.44 controls, P = 0.75 heart failure). Because rs10927887 genotypes are DNA and RNA were isolated directly from left-ventricular myocardium using available as part of the 1000Genomes project (42), existing IBC geno- Gentra and miRNeasy kits. Genotypes at rs1739843 were obtained using the types (14) were imputed with 1000Genomes data using the software pack- IBC array, and multidimensional scaling of all IBC genotypes was used to age MACH (43) (http://www.sph.umich.edu/csg/abecasis/mach/) to obtain identify a subgroup of 111 genetically inferred Caucasians. Gene expression rs10927887 genotypes for the Penn Heart Failure Study, Case Western Re- and splicing were assessed using Affymetrix ST1.0 arrays. Expression was serve, University of Wisconsin, and PennCATH (41). Imputed genotypes further assessed using RT-qPCR with ABI Taqman assays on demand for HSPB7 showed good quality with imputation R2 > 0.92; frequency of the G allele (Hs00205296_m1) with RPLP0 (Hs99999902_m) as a housekeeping gene. All was similar to 1000Genomes CEU (0.53). samples were run in duplicate, and expression was quantified as the average Δ change in cycle threshold (- CT). Expression was compared across rs1739843 Functional Analysis of ClC-Ka Arg83 and Gly83. tsA201 cells were transiently genotypes using the Kruskal-Wallis test. transfected using Lipofectamine with a plasmid (pBK-CMV-YFP) encoding enhanced YFP, pcDNA3.1-barttin, pCMV-Sport6-CLCNKA WT, or pCMV- Resequencing and Genotyping. In the primary and secondary cohorts, pooled Sport6-CLCNKAR83G, individually or in combination as described. The in- DNA library preparation and Illumina Genome Analyzer II resequencing of clusion of the plasmid encoding YFP allowed transfected cells to be identified CLCNKA exons and exon–intron boundaries were performed as previously before electrophysiological recordings. Approximately 15 h after trans- described (16). Exon-spanning primer pairs (Table S3) were developed for all fection, cells were washed and allowed to recover for 20–24 h before current 19 CLCNKA coding exons. Average sequence coverage depth was 19-fold for recordings were obtained. Whole cell recordings were obtained at room

2460 | www.pnas.org/cgi/doi/10.1073/pnas.1017494108 Cappola et al. Downloaded by guest on September 29, 2021 temperature (22–25 °C). Data were collected using a Dagan Model 3900A Statistical Analysis. For pooled resequencing, allele frequencies between patch-clamp amplifier; experimental parameters were controlled with Dell study groups were compared using Fisher’s exact test. The P value threshold personal computer through a Digidata 1322 interface using pCLAMP9 (Axon for significance in the primary heart failure case–control analysis was <0.001 Instruments). ClC-Ka currents were evoked during 400-ms voltage steps to using a Bonferroni correction for multiple testing (n = 51 exonic SNPs at an α test potentials between −165 and +85 mV in 10-mV increments. Data were level of 0.05). Only one common SNP met criteria for association and was compiled and analyzed using CLAMPFIT (Axon) and Excel (Microsoft) and evaluated in the replication study. After comparison of all-cause heart fail- presented as means ± SEM. ure with controls, cases were stratified into ischemic and nonischemic Western blot analyses were performed on 20-μg/lane protein lysates groups. For individual CLCNKA genotypes, SNP association was tested using prepared from tsA201 cells transfected as above, except with FLAG epitope- logistic regression with the genotype score or the imputation dosage score tagged ClC-Ka. After blocking, membranes were incubated with the mouse included as a covariate, adjusting for age, gender, and hypertension. Met- monoclonal anti-FLAG antibody (Sigma) at 4 °C overnight. To ensure equal aanalysis was carried out using the software package METAL (http://www. protein loading of lanes, membranes were also probed with a mouse sph.umich.edu/csg/abecasis/Metal/). Comparison of whole-cell chloride cur- monoclonal anti-transferrin receptor (Invitrogen) antibody. After washing, rents used Student t test, with P < 0.05. membranes were incubated with a rabbit anti-mouse horseradish peroxi- dase-conjugated secondary antibody (Bethyl Laboratories) followed by ACKNOWLEDGMENTS. This work was supported by National Institutes of SuperSignal West Dura Extended Duration substrate (Pierce). Signals were Health/National Heart, Lung, and Blood Institute Cardiac Translational detected using a Molecular Imager Chemidoc XRS system running Quantity Implementation Program Grants RC2 HL102222, HL088577, HL034161, and One software version 4.6 (Bio-Rad). ULI RR024992.

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