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. 2012 Oct 1;1:44–46. doi: 10.1016/j.atg.2012.06.001

Mutation analysis of the candidate genes SCN1B-4B, FHL1, and LMNA in patients with arrhythmogenic right ventricular cardiomyopathy

Lena Refsgaard a,b,c,1, Morten Salling Olesen a,b,c,⁎,1, Daniel Vega Møller e, Michael Christiansen e, Stig Haunsø a,b,c,d, Jesper Hastrup Svendsen a,b,c,d, Alex Hørby Christensen a,b,c
PMCID: PMC5121199  PMID: 27896052

Abstract

Introduction

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetically determined heart disease characterized by fibrofatty infiltrations in the myocardium, right and/or left ventricular involvement, and ventricular tachyarrhythmias. Although ten genes have been associated with ARVC, only about 40% of the patients have an identifiable disease-causing mutation. In the present study we aimed at investigating the involvement of the genes SCN1B-SCN4B, FHL1, and LMNA in the pathogenesis of ARVC.

Methods

Sixty-five unrelated patients (55 fulfilling ARVC criteria and 10 borderline cases) were screened for variants in SCN1B-4B, FHL1, and LMNA by direct sequencing and LightScanner melting curve analysis.

Results

A total of 28 sequence variants were identified: seven in SCN1B, three in SCN2B, two in SCN3B, two in SCN4B, four in FHL1, and ten in LMNA. Three of the variants were novel. One of the variants was non-synonymous. No disease-causing mutations were identified.

Conclusions

In our limited sized cohort the six studied candidate genes were not associated with ARVC.

Keywords: ARVC, Genetics, Nav beta-subunits, Sodium current, Lamin A/C, FHL1

Highlights

► Arrhythmogenic right ventricular cardiomyopathy (ARVC). ► Tachyarrhythmias. ► DNA sequencing the genes SCN1B-4B, FHL1, and LMNA. ► No disease-causing mutations were identified.

1. Introduction

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inheritable cardiomyopathy pathologically characterized by fibrofatty infiltrations in the myocardium leading to thinning of the wall and aneurysms. Clinical presentation is mainly due to ventricular arrhythmias and may include palpitations, syncope, and sudden cardiac death (Basso et al., 2009). Diagnosis is based on Task Force criteria (Marcus et al., 2010), which encompass functional and anatomical characteristics of the right ventricle, electrical abnormalities, family history, and genetic information. The original Task Force criteria were published in 1994, and in 2010 revised criteria were published (Marcus et al., 2010).

To this date ARVC has been associated with mutations in five desmosomal (DSC2, DSG2, DSP, JUP, and PKP2) and five non-desmosomal genes (TGFβ3, RYR2, TMEM43, DES, and TTN) (Basso et al., 2009, Taylor et al., 2011, van Tintelen et al., 2009). Although ten genes have been associated with ARVC, these only account for approximately 40% of the patients with identifiable mutations (Christensen et al., 2010, Bhuiyan et al., 2009) underlining the genetic heterogeneity of the disease.

In the present study we aimed at investigating the involvement of six candidate genes which have not previously been investigated in the pathogenesis of ARVC: SCN1B-SCN4B (Sodium channel, voltage gated, type I-IV, beta), encoding β-subunits for the cardiac sodium channel, FHL1 (Four and a half LIM domains 1), encoding a member of the four-and-a-half LIM only protein family, and LMNA (Lamin A/C), encoding a nuclear envelope protein. All are highly expressed in the heart and are known to interact with ARVC-associated genes. They have also been associated with heritable arrhythmia syndromes and/or cardiomyopathy (Brackenbury and Isom, 2008, Cowling et al., 2011, Lu et al., 2011).

2. Methods

2.1. Study subjects

The study comprised 65 unrelated patients recruited from Rigshospitalet, Copenhagen University Hospital, Denmark. All patients were of Northern European descent. The clinical characteristics of the study population have previously been published (Christensen et al., 2010). All patients have previously been screened for mutations in the known ARVC-related genes (DSC2, DSG2, DSP, JUP, PKP2, TGFb3, and TMEM43) and mutations were identified in 36% of patients fulfilling the 1994 Task Force criteria (Christensen et al., 2010, Christensen et al., 2011). Since compound inheritance has been described (Bhuiyan et al., 2009) we chose not to exclude patients with known mutations from further screening. The study was approved by the local ethics committee, followed the Helsinki II declaration, and all subjects gave written informed consent before inclusion.

2.2. Genetic screening

Genomic DNA was isolated from blood samples using the QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany). The coding sequences and splice sites of SCN1B (NM_001037.3, NM_199037.2), SCN2B (NM_004588.4), SCN3B (NM_018400.3), SCN4B (NM_174934.2), FHL1 (NM_001449, NM_001159700, NM_001159704, NM_001167819, NM_001159702, NM_001159703), and LMNA (NM_005572, NM_0170707.2), corresponding to a total of 38 exons, were amplified with intronic primers and bidirectionally sequenced using Big Dye chain-termination chemistry (DNA analyzer 3730, Applied Biosystems, CA, USA). Some amplicons were prescreened with a LightScanner high-resolution melting curve analysis system (Idaho Technology, UT, USA). All fragments with an abnormal melting profile were sequenced. Primers and PCR conditions are available on request.

3. Results

3.1. Study cohort

A total of 65 (38 men) patients were included in the study. 55 of the patients fulfilled 1994 Task Force criteria for ARVC: ≥ two major criteria (n = 15), one major and ≥ two minor criteria (n = 36), and four minor criteria (n = 4). Ten patients had some ARVC characteristics but did not fulfill the criteria: one major and one minor criterion (n = 5), three minor criteria (n = 4), and two minor criteria (n = 1).

3.2. Genetic screening

The variant analysis identified a total of 28 sequence variants: Seven in SCN1B, three in SCN2B, two in SCN3B, two in SCN4B, four in FHL1, and ten in LMNA. The identified variants are listed in Table 1. Three of the variants were novel. All novel variants were positioned in flanking introns and did not affect a canonical splice site. One non-synonymous variant was identified (FHL1 p.D275N). No disease-causing mutations were identified.

Table 1.

Identified sequence variants.

Gene Variant Exon/intron Amino acid Minor allele MAFa dbSNP
SCN1B c.40+15G>T Intron T 0.238 rs72556351
c.448+181T>C Intron C 0.423 rs55742440
c.448+296C>A Intron A 0.169 rs67701503
c.448+301G>C Intron C 0.169 rs67486287
c.860T>C Exon p.I167I C 0.046 rs16969930
c.591-25T>G Intron G 0.008 rs28365107
c.591-14C>A Intron A 0.008 rs28365109
SCN2B c.70+32G>A Intron A 0.023 –
c.449-12C>A Intron A 0.454 rs8192613
c.648+38C>T Intron T 0.092 rs8192614
SCN3B c.438C>T Exon p.T146T T 0.054 rs1275085
c.445+37T>G Intron G 0.008 –
SCN4B c.174C>T Exon p.C58C T 0.046 rs45539032
c.639C>T Exon p.N213N T 0.023 rs72544155
FHL1 c.688+37G>A Intron A 0.008 –
c.689-8C>T Intron T 0.546 rs2076705
c.689-13C>T Intron C 0.031 rs11798700
c.823G>A Exon p.D275N A 0.008 –
LMNA c.612G>A Exon p.L204L A 0.015 rs12117552
c.369+56G>T Intron T 0.031 rs11264442
c.639+73C>T Intron T 0.031 rs11264443
c.640-52C>T Intron T 0.008 rs41314033
c.861T>C Exon p.A287A C 0.054 rs538089
c.937-83G>T Intron T 0.054 rs80356810
c.1157+16G>A Intron A 0.054 rs534807
c.1338T>C Exon p.D446D C 0.054 rs505058
c.1489-41C>T Intron T 0.054 rs553016
c.1698C>T Exon p.H566H T 0.231 rs4641
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a

Minor allele frequency in the 130 patient alleles tested.

4. Discussion

This study is the first comprehensive attempt to associate ARVC with genetic variation in the accessory subunits of the cardiac Nav1,5 channel, FHL1, and LMNA. Screening of these genes, representing 38 exons, revealed no disease-causing mutations and these genes are therefore unlikely to be common causes of ARVC.

The channels carrying the cardiac sodium current (Nav1.5) is composed of an α-subunit, encoded by SCN5A, and two of the four β-subunits (Brackenbury and Isom, 2008). The β subunits (β1–β4), encoded by SCN1B-4B, are multifunctional and members of the immunoglobulin superfamily. Some of their functions may be independent of the α-subunit (Brackenbury and Isom, 2008) and several studies have demonstrated that β1 and β2 both play important roles in homophilic and heterophilic cell adhesion and function as cell adhesion molecules (Brackenbury and Isom, 2008, Chioni et al., 2009). A recent study demonstrated a physical and functional interaction between Nav1.5 and the ARVC-associated gene PKP2 at the intercalated disc and an altered sodium current after knockdown of PKP2 (Sato et al., 2009). In addition, Deo et al. (2011) showed that these alterations of the sodium current complex lead to an increased susceptibility to arrhythmias due to reentrant activity, even without anatomical obstacles. Furthermore, it has been demonstrated that 16% of the ARVC patients exhibit inducible coved-type ST elevations in right precordial leads (Peters, 2008), an ECG finding usually characteristic of Brugada Syndrome, and that mutations affecting Nav1.5 are prevalent in Brugada syndrome patients with structural heart abnormalities resembling ARVC (Frustaci et al., 2005, Frigo et al., 2007). Both SCN1B and SCN3B have recently been associated with Brugada Syndrome (Olesen et al., 2012). These findings thus suggest that the sodium current complex could play a role in the pathogenesis of ARVC. A variety of other diseases have also been associated with mutations in SCN1B-4B, including cardiac arrhythmia, cancer, and neurological diseases (Brackenbury and Isom, 2008, Chioni et al., 2009, Olesen et al., 2011).

The FHL1 gene, located on the X chromosome, is characterized by a half LIM domain in the N-terminus and four following complete LIM domains. The protein is localized both in the cytosol and the nucleus and has several functions including transcription regulation, cell signaling, and sarcomere assembly (Cowling et al., 2011). FHL1 interacts with TTN recently associated with ARVC4 (Taylor et al., 2011, Cowling et al., 2011). A wide variety of human muscle diseases including non-compaction, hypertrophic and dilated cardiomyopathies, have been associated with FHL1 mutations (Cowling et al., 2011). In our study the non-synonymous variant FHL1 p.D275N was present in one patient. The variant affects an unconserved residue located in the C-terminus of the FHL1 peptide. The variant has previously been described in control populations (Schoser et al., 2009) and was present in 31 out of 1669 alleles of American/European descent in the NHLBI GO Exome Sequencing Project (Exome Variant Server). We therefore consider it to be a polymorphism.

The LMNA gene, encoding the intermediate filament proteins lamin A and C, plays a role in maintaining the structural integrity of the inner nuclear membrane, in gene expression, and in organization of chromatin (Lu et al., 2011). The lamins interact with several proteins, including TMEM43, associated with ARVC5 (Basso et al., 2009, Bengtsson and Otto, 2008). The ARVC-genes TMEM43 and JUP are believed to cause dysregulation of the adipogenic pathway regulated by PPAR-γ, resulting in adipogenesis, fibrogenesis and myocyte apoptosis (Merner et al., 2008), and may explain the fibrofatty replacement in the myocardium characteristic of ARVC. Also LMNA have been suggested to affect PPAR-γ. (Capanni et al., 2005). Mutations in LMNA have been associated with numerous extracardiac disorders, conduction defects, ventricular arrhythmia, and dilated cardiomyopathy (Lu et al., 2011). Dilated cardiomyopathy may mimic ARVC and a predominantly left-sided form of ARVC is becoming increasingly recognized (Sen-Chowdhry et al., 2008) making LMNA a plausible candidate gene for ARVC.

This study has the following limitations: We only analyzed the coding regions of SCN1B-4B, FHL1, and LMNA, and the possibility of variants occurring in other regions of the genes cannot be excluded. Our study cohort has a limited size and all patients were of Northern European descent, thus we cannot exclude the possibility of different results in cohorts of different ethnicity. The diagnosis of our cohort is based on the 1994 Task Force criteria. We were not able to apply the revised criteria published in 2010 to our cohort, due to lack of necessary clinical data, e.g. all myocardial biopsies are obtained from the septum.

In conclusion, we found no disease-causing mutations in ARVC patients within the SCN1B-4B, FHL1, and LMNA genes. The present study is to our knowledge the first attempt to associate genetic variation in these genes with ARVC. Our results indicate that these six genes are not common causes of ARVC.

Disclosures

None.

Acknowledgments

This work was supported by The Danish Heart Foundation [grant no. 07-10-R60-A1815-B573-22398], The Danish National Research Foundation Centre for Cardiac Arrhythmia, The John and Birthe Meyer Foundation, The Research Foundation of the Heart Centre Rigshospitalet, Lægernes Forsikringsforening af 1891, The Arvid Nilsson Foundation, Director Ib Henriksens Foundation, King Christian X's Foundation, National Association for the Control of Circulatory Diseases, The scholarship of Stockbroker Henry Hansen and wife Karla Hansen born Westergaard, The Director Kurt Bønnelycke and wife Mrs. Grethe Bønnelyckes Foundation, The Aase and Ejnar Danielsens Foundation, and University of Copenhagen.

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