Novel polymorphisms in PDLIM3 and PDLIM5 gene encoding Z‐line proteins increase risk of idiopathic dilated cardiomyopathy

Abstract Idiopathic dilated cardiomyopathy (IDCM), characterized by ventricular dilation and impaired systolic function, is a primary cardiomyopathy resulting in heart failure. During heart contraction, the Z‐line is responsible for transmitting force between sarcomeres and is also a hot spot for muscle cell signalling. Mutations in Z‐line proteins have been linked to cardiomyopathies in both humans and mice. Actinin‐associated LIM protein (ALP) and enigma homolog protein (ENH), encoded by PDLIM3 and PDLIM5, are components of the muscle cytoskeleton and localize to the Z‐line. A PDLIM3 or PDLIM5 deficiency in mice leads to dilated cardiomyopathy. Since PDLIM3 and PDLIM5 are candidate IDCM susceptibility genes, the current study aims to investigate whether polymorphisms within PDLIM3 and PDLIM5 could be correlated with IDCM. We designed a case‐control study, and exons of the PDLIM3 and PDLIM5 were amplified by polymerase chain reactions in 111 IDCM patients and 137 healthy controls. We found that five synonymous polymorphisms had statistical distribution differences between IDCM patients and controls, including rs4861669, rs4862543, c.731 + 131 T > G, c.1789‐3 C > T and rs7690296, according to genotype and allele distribution. Haplotype G‐C‐C‐C and A‐T‐C‐T (rs2306705, rs10866276, rs12644280 and rs4635850 synthesized) were regarded as risk factors for IDCM patients when compared with carriers of other haplotypes (all P < .05). Furthermore, IDCM patients with two novel polymorphisms (c.731 + 131 T > G and c.1789‐3 C > T) had lower systolic blood pressure. In conclusion, these five synonymous polymorphisms might constitute a genetic background that increases the risk of the development of IDCM in the Chinese Han population.


| INTRODUC TI ON
Dilated cardiomyopathy (DCM) is a structural heart disease, characterized by ventricular dilation and impaired systolic function, in the absence of hypertension, coronary artery disease or valvular abnormalities, 1 with an estimated prevalence of up to 1:2500. 2 DCM is the third most frequent cause of heart failure and the most common condition requiring heart transplantation. 3 Accumulating evidence has shown that the pathogenesis of DCM is associated with various factors, including virus-mediated myocardial infection, autoimmune disease, toxic and metabolic damage, tachycardia, and genetic history. 2 World Health Organization/International Society and Federation of Cardiology Task Force classifies primary DCM as idiopathic dilated cardiomyopathy (IDCM) with unknown causes differently from secondary DCM, such as hypertensive and ischaemic cardiomyopathy. 4 Various genetic polymorphisms have been shown to be associated with increased risk of developing DCM. 5 In most cases, these genes are involved in the encoding of myocardial skeleton, nuclear membrane, sarcomere, mitochondrial proteins and the calcium homeostasis regulating protein. 6 The Z-line, constituents of the myocyte cytoskeleton, defines the lateral boundaries of the sarcomere, provides an anchoring site for actin, titin and nebulin filaments, and mediates the transmission of tension between sarcomeres during contraction. 7 Mutations in Z-line proteins have been proven to be associated with cardiomyopathy or myofibrillar myopathy in humans. 8 Actinin-associated LIM protein (ALP) and enigma homolog protein (ENH), encoded by PDLIM3 and PDLIM5, belong to the PDZ-LIM protein family. 9 Functionally, these proteins bind to αactinin at the Z-line through their N-terminal PDZ domains and bind to other proteins through their C-terminal LIM domains. 9 Actinin-associated LIM protein, highly expressed in skeletal and cardiac muscle, has been suggested to play a pivotal role in myocyte stability, force generation and transmission, signal transduction, and mechanical signalling, especially in growth and remodelling processes. 9 As for ENH, cardiac-specific PDLIM5 knockout mice developed DCM. 10 We hypothesized that ALP and ENH, like its homologue Cypher, had an important role in the formation and maintenance of the normal Z-line in cardiac muscle and are associated with human normal cardiac function. We hypothesized that the single nucleotide polymorphisms (SNPs) in PDLIM3 and PDLIM5 might be associated with susceptibility to IDCM. In order to confirm our hypothesis, we conducted this case-control study, tested the candidate genes and analysed their association with IDCM, in the Chinese Han population.

| Genotyping
Genomic DNA of each individual was extracted from 200 µL EDTA-  Table S1. PCR amplification was carried out to determine the exonic sequences of PDLIM3 and PDLIM5, and the amplification products were purified. All gene exon sequences underwent sequencing in both the positive and negative directions followed by analysis using the DNAMAN software. The results were compared with the standard template sequences using NCBI BLAST and the CHROMAS software, to identify the gene mutation loci (sequencing was accomplished by China Hangzhou Tsingke Biological Testing Company).

| Statistical analysis
Statistical analyses were performed with SPSS 19.0.0 (SPSS Inc).
Continuous variables were reported as mean ± SD; categorical variables were summarized in terms of number and percentages. The chi-square test was utilized to compare the difference in gender between IDCM patients and healthy controls, while the non-parametric test was performed to determine any distinctions in age.
Deviation from Hardy-Weinberg equilibrium (HWE) for each SNP in the control group was assessed with a chi-square goodness of fit test. Genotypic association tests in a case-control pattern, assuming dominant, recessive, additive, over dominant and codominant genetic models, were performed using the Pearson chi-square test.
If expected frequencies in the cells of 2 × 2 tables were <5, Fisher's exact test was used. Odds ratio (OR) and respective 95% confidence intervals (CIs) were determined to assess the influence of any difference between genotypes, alleles and haplotypes. Correlation between variables and two new SNPs was determined using a nonparametric test. Linkage disequilibrium (LD) among the SNPs and haplotype analysis were used by SHESIS software (Available online: http://analy sis.bio-x.cn/myAna lysis.php). All reported P values were 2-tailed, and statistical significance was set at P < .05.

| Characteristics of participants
There were 248 unrelated participants, including 111 IDCM cases (77 men and 34 women, median age = 57.0 years) and 137 controls (90 men and 47 women, median age = 53.0 years). The baseline clinical characteristics of all participants are summarized in Table 1. In the IDCM group, the LVEF was 31.5 ± 8.7% and the left ventricular end-diastolic dimensions (LVEDD) were 6.8 ± 0.9 cm on average. Of those, 45 underwent ICD implantation.

| Identification of polymorphisms
Eight exons for PDLIM3-a corresponding to PDLIM3-b-d, and 16 exons for PDLIM5-a corresponding to isoforms PDLIM5-b-I, were screened.
This screening strategy was based on high and specific expression of these isoforms in cardiac muscle, as well as mapping of the PDZ and LIM domains, within these isoforms. In the IDCM group, we found two new SNPs, the c.731 + 131 T > G and c.1789-3 C > T ( Figure S1).
Moreover, these two new SNPs were not present in the 137 controls.

| Association analysis of genotype and haplotypes of SNPs with the IDCM susceptibility
Because some participants' genes were failed to be sequenced, the total number of genotypes for some SNPs was less than 248. The frequency of each genotype in controls was consistent with the HWE hypothesis (Table 2). Table 3 shows the alleles and genotype frequencies of 11 SNPs, and the corresponding statistical analysis results. As shown in Table 3, significant differences in genotypes and allele frequencies were found at rs4861669, rs4862543, rs7690296 and two new SNPs, indicating that these polymorphisms may play an important role in the pathogenesis of IDCM.
Our results showed that the minor allele of the two novel SNPs  We estimated LD among the six SNPs in PDLIM3 and five SNPs in PDLIM5 by using SHESIS software. As displayed in Figure 1, the SNP pairs rs2306705/rs10866276/rs12644280/rs4635850 in PDLIM3 exhibited strong linkage disequilibrium (D′ > 0.75).
Subsequently, a total of 16 haplotypes were obtained after randomly combining the four SNPs in PDLIM3 based on SHESIS software; however, five haplotypes were finally excluded for their low frequency in the studied population (each lower than 3%). Partial haplotype frequencies of the PDLIM3 gene, in patients and controls, are summarized in Table 4. It was finally indicated, by haplotype analyses, that haplotypes G-C-C-C and A-T-C-T (rs2306705,

| Two new SNPs within PDLIM5 were significantly associated with systolic blood pressure in patients with IDCM
Subgroup analysis was performed to evaluate the potential relation-  Table 5). However, no significant relationship was found between LVEDD and LVEF and the genotype of the two new SNPs, in IDCM subjects.

| D ISCUSS I ON
IDCM is estimated to be of genetic origin in up to 50% of all cases. 13 To date, mutations in many of Z-line-associated proteins have been proven to be linked to cardiomyopathy in both humans and transgenic mouse models, 8,14 suggesting that Z-line proteins play a pivotal role in the pathogenesis of cardiomyopathy. One such protein is Cypher, a PDZ and LIM domain-containing protein, that was first cloned in 1999. 15 Cypher-deficient mice display premature lethality with severe DCM and disorganized Z-lines. 16   factors. 9 ALP and ENH are essential for proper heart development and contractility in hearts, as a PDLIM3 or PDLIM5 deficiency in mice results in DCM. 9,10,17 The important roles of ALP and ENH in heart function make them two promising, candidate genes for cardiomyopathy. The association of polymorphisms of PDLIM3 and PDLIM5 with susceptibility to IDCM, however, remains unexplored.
The present analysis of the data, from 111 IDCM patients and 137 controls, identified five SNPs in the PDLIM3 and PDLIM5 genes associated with IDCM. These two genes are considered in more detail below.
ALP is highly expressed in striated muscle (Xia, 1997 was reported in a patient with DCM. 13 Meanwhile, in mice, ablation of PDLIM3 results in primarily right ventricular dysmorphogenesis, a decrease in trabeculation and mild DCM. 17 Moreover, Elisabeth and coworkers found that tight regulation of ALP is essential for a proper balance between maintenance of the extracellular matrix and the formation of fibrotic scars. 20 Cardiac fibrosis is a prominent feature of several cardiomyopathies, which reduces cardiac contractility and electric conductivity. 21 In addition, many disease-related polymorphisms in non-coding regions can affect the expression level of coding proteins by disrupting the transcription factor recognition sequence in related cell types. 22 Within the PDLIM3 gene, we found two SNPs (rs4861669 and rs4862543) associated with IDCM. Allele A of rs4861669 was closely related, with higher risk of IDCM (allele A: OR = 1.71, 95% CI = 1.08-2.69). By using the chi-square test, we found that a significantly increased risk of IDCM was associated with the AA/GA genotypes, compared with GG genotype, in the dominant model. A significantly increased risk of IDCM was also found to be associated with the AA genotype of rs4861669 in the additive model, compared with GG genotypes, which shows homozygote advantage. The same held true for rs4862543.
The PDLIM5 gene is located at 4q22.3. PDLIM5 encodes several splice variants, whose expression is tissue specific and temporally regulated. 9 Alternative splicing plays an important role in heart development and in the development of cardiopathies. 23 The splice variants of PDLIM5 can be divided into two groups, long isoform (ENH1), containing the three LIM domains, and short isoforms, lacking the three LIM domains. 9 Maturana and coworkers showed that ENH1 forms a complex with protein kinase D1 and the alpha1C subunit of cardiac L-type voltage-gated calcium channels in rat neonatal cardiomyocytes to regulate the activity of the channel. 24  to explore the role of these two novel SNPs in the pathophysiologic mechanisms of DCM. Also, the haplotype consisting of four highly linked SNPs implied that G-C-C-C and A-T-C-T carriers were more inclined to suffer from DCM than carriers of other haplotypes.
There are still some limitations to our research. The main limitations are the relatively small sample size of the study population and the lack of replication of this significant association in a second, independent cohort of IDCM patients. This is also a single-centre study; thus, we cannot exclude the presence of selection bias in patient enrolment.
Our study was limited to the genetic level, and functional studies are therefore required to explore the molecular mechanisms by which SNPs would affect the gene expression of PDLIM3 and PDLIM5.

| CON CLUS ION
Our study demonstrates, for the first time, that polymorphisms in PDLIM3 (rs4861669, rs4862543) and PDLIM5 (rs1056772) were significantly associated with IDCM in Chinese Han patients. We also identified 2two novel SNPs (c.731 + 131 T > G and c.1789-3 C > T) in the intron of the PDLIM5 gene that may increase the risk of IDCM and affect SBP level in IDCM patients. This study is an important step towards establishing a link between genetic polymorphisms in Z-line protein genes, PDLIM3 and PDLIM5, and IDCM.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest associated with the manuscript.

AUTH O R S ' CO NTR I B UTI O N S
DFW, JF and JLL executed the experiments. DFW, HQC and XGG designed the study. JF, ZCP and XY performed data analysis. DFW and XGG wrote the manuscript while JF and JLL assisted with manuscript editing. The final version was approved by all authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author on request.