Spectrum of Mutations in Hypertrophic Cardiomyopathy Genes Among Tunisian Patients

Abstract

Background: Hypertrophic cardiomyopathy (HCM) is a common cardiac genetic disorder associated with heart failure and sudden death. Mutations in the cardiac sarcomere genes are found in approximately half of HCM patients and are more common among cases with a family history of the disease. Data about the mutational spectrum of the sarcomeric genes in HCM patients from Northern Africa are limited. The population of Tunisia is particularly interesting due to its Berber genetic background. As founder mutations have been reported in other disorders. Methods: We performed semiconductor chip (Ion Torrent PGM) next generation sequencing of the nine main sarcomeric genes (MYH7, MYBPC3, TNNT2, TNNI3, ACTC1, TNNC1, MYL2, MYL3, TPM1) as well as the recently identified as an HCM gene, FLNC, in 45 Tunisian HCM patients. Results: We found sarcomere gene polymorphisms in 12 patients (27%), with MYBPC3 and MYH7 representing 83% (10/12) of the mutations. One patient was homozygous for a new MYL3 mutation and two were double MYBPC3 + MYH7 mutation carriers. Screening of the FLNC gene identified three new mutations, which points to FLNC mutations as an important cause of HCM among Tunisians. Conclusion: The mutational background of HCM in Tunisia is heterogeneous. Unlike other Mendelian disorders, there were no highly prevalent mutations that could explain most of the cases. Our study also suggested that FLNC mutations may play a role on the risk for HCM among Tunisians.

Introduction

Hypertrophic cardiomyopathy (HCM) is the most common genetic myocardial disease and is the most common cause of sudden cardiac death (SCD) among young patients. In more than half of the patients, the disease has been associated with mutations in nine sarcomere protein genes with MYH7 (cardiac beta-myosin heavy chain) and MYBPC3 (cardiac myosin-binding protein C) accounting for most of the mutations (Richard et al., 2003). The frequency of mutations in each HCM gene may differ between the studies, a likely consequence of the different population genetic backgrounds (Mohiddin et al., 2004; Curila et al., 2009; Otsuka et al., 2012). Most of the HCM genetic studies have been based on populations from Europe and Asia, but data from African cohorts are limited (Moolman-Smook et al., 1999; El-Saiedi et al., 2014; Chiou et al., 2015). The identification of mutations in HCM patients is important not only to establish the diagnosis but also for the genetic counseling in family members who could be mutation carriers and be at risk of developing the disease (Christiaans et al., 2009).

Tunisia is a North African country of 11 million inhabitants, located at the crossroad between Europe and Africa. The Amazigh (also known as Berbers) settled in the region in the seventh century BC. Since then, Tunisia has witnessed successive invasions by different ethnic groups, which have genetically shaped the population and influenced the spectrum and frequency of genetic diseases (Chaabani et al., 1988; Romdhane and Abdelhak, 2011). Despite the changes that have taken place during the last decades, familial and geographical endogamy still exists in Tunisia at high frequencies, especially in rural areas. The health implications of consanguinity in Tunisian families include an increased risk of autosomal recessive diseases and particular phenotypic expressions. In agreement with the homogeneous genetic background, several studies reported a high incidence of particular mutations linked to genetic diseases among Berbers, such as the LRRK2 mutation (p.G2019S) among familial dominant Parkinson's disease (Ishihara et al., 2007). In reference to HCM, a recent study examining 10 sarcomeric genes based on only 11 patients has found a putative mutation in five cases. Despite these preliminary results, this study pointed to the heterogeneous genetic background, with no founder mutations present at a high rate among Tunisians (Jaafar et al., 2015).

The aim of this study is to determine the mutational spectrum of the main sarcomeric genes in a cohort of 45 HCM patients in Tunisia. In addition, these patients were sequenced for the FLNC gene, which was recently linked to HCM among Caucasians.

Methods

Patients' characteristics

A total of 45 HCM nonrelated index cases (29 male and 16 female) were recruited through the Habib Thameur Hospital (n = 20) and Abderrahmen Mami Hospital (n = 25) in Tunis. Most of the patients were from North West Tunisia (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/gtmb). All the patients were diagnosed during the years 2014 and 2015 by qualified cardiologists from the two hospitals. HCM was diagnosed based on clinical symptoms and left ventricular septum (LVS) ≥15 mm in the absence of any other conditions that could explain the cardiac hypertrophy. Patients with at least one relative diagnosed with HCM were defined as familial cases. The evaluation of each patient was made based upon the medical history, the electrocardiogram, and the echocardiography. This research was approved by the Ethics Committees of Hospital Universitario Central Asturias (HUCA), Habib Thameur Hospital, and Abderrahmen Mami Hospital. None of patients that were considered as eligible for the genetic study refused to participate, and all subsequently signed an informed consent to participate in the study.

Next generation sequencing

All the patients were sequenced for the MYH7, MYBPC3, TNNT2, TNNI3, ACTC1, TNNC1, MYL2, MYL3, TPM1, and FLNC genes using NGS with AmpliSeq and the Ion Torrent Personal Machine Sequencer (PGM) semiconductor chip technology (Life-Technologies). The two-tubes AmpliSeq was designated online (IonAmpliSeq™ Designer v1.2; www.ampliseq.com) to cover the coding exons and at least five intron flanking nucleotides of the 10 genes. We compared several primer design options and ordered the one that gave the maximum target sequence coverage. Primer pairs to amplify fragments that covered 98.22% of the target sequence (a total of ∼14 Kb) were provided by the manufacturer.

The basic NGS procedure was previously reported (Gómez et al., 2014a, 2014b). Briefly, the DNA from each patient was obtained and adjusted to a final concentration of 10 ng/μL. Each DNA was amplified with the Ion AmpliSeq™ Library Kit 2.0 in conjunction with Ion AmpliSeq Custom Primer Pool protocols according to the manufacturer procedures (Thermo Fisher Scientific). We performed an emulsion PCR using the Ion PGM template Hi-Q OT2 Kit in the Ion One-Touch instrument 2 (Thermo Fisher Scientific), and template-positive spheres were recovered using Dynabeads MyOne Streptavidin C1 beads. The sequencing was performed in the PGM using the Ion Hi-Q sequencing Kit and in 318-v2 semiconductor chips. We used 500-flow runs, which support a template read length of ∼200 bp. The raw PGM data were processed with the Torrent Suite v5 software (Life Technologies). For reads assembling and variant identification, we used the Variant Caller (VC) v5 and Ion Reporter v5 software. We used the Integrative Genome Viewer (IGV, Broad Institute) for the analysis of depth coverage, sequence quality, and variants identification with the somatic sample default algorithm (Gómez et al., 2014a).

Variants classification and Sanger sequencing validation

Nucleotide changes that result in nonsense or frame-shift amino acid changes or changes in the splice site consensus sequence (that would affect introns in the pre-mRNA) were classified as mutations. Missense changes were classified as likely pathogenic (putative mutations) if they have not been reported in the Exome Sequencing Project database (ESP; http://evs.gs.washington.edu/EVS) and were predicted to be pathogenic with bioinformatics' tools Poly-phen2 (Adzhubei et al., 2010), SIFT (Kumar et al., 2009), and Mutation Taster (Schwarz et al., 2014), and meta-predictors, Condel (Gonzalez-Perez and Lopez-Bigas, 2011), and combined annotation-dependent depletion (Kircher et al., 2014). All the putative mutations identified by NGS were confirmed through Sanger sequencing using BigDye chemistry in ABI3130 equipment (Life Technologies).

MYBPC3 haplotype characterization

A total of 10 SNPs in the coding exons of MYBPC3 were genotyped through Sanger sequencing of PCR fragments in the three Tunisian and in three Spanish MYBPC3 c.772G>A (p.E258K) carriers to characterize the haplotypes and a possible founder effect for this mutation (Table 3).

Results

Our study involved a total of 45 HCM patients (64% male and 36% female) with a mean onset age of 50 years (range 14-74 years). The mean LVS size was 21 mm (range 15-32 mm). A total of 11 patients (24%) had a family history of HCM, and 15 cases (33%) had relatives with SCD history. We performed NGS on the nine most common HCM genes, including MYH7, MYBPC3, TNNT2, TNNI3, ACTC1, TNNC1, MYL2, MYL3, and FLNC. We identified a total of 12 putative sarcomere mutations (Table 1). Five patients had a single mutation in the MYH7 gene and three had mutations in MYBPC3 (Table 2). Three patients were heterozygous for MYBPC3 p.E258K (c.772G>A). This MYBPC3 mutation was also identified among the Spanish HCM patients studied in the HUCA reference laboratory (3 mutation carriers among 350 index cases). To determine whether this mutation has a common origin (being thus in the same haplotype in all the patients), we genotyped several MYBPC3 SNPs in the Tunisian and Spanish patients. We found two different haplotypes defined by an AG polymorphism at codon 216 (rs201098973, c.646 GA, p.A216T): the p.258K mutation was linked to the c.646 A in the Spanish patients (demonstrated by family segregation in two of the patients) and to the c.646 G in the three Tunisian cases (who were GG homozygotes) (Table 3). This suggested that the p.E258K change originated independently in the two populations, although a common origin with a subsequent acquisition of the rare c.646 A allele in the Spanish haplotype cannot be ruled out.

Table 1.

Summary of the Sarcomeric Gene Putative Mutations Identified in the NGS of 45 Tunisian HCM Patients

Gene	Exon	Variant	Effect	Spanish HCM	SIFT	Polyphen-2	Mutation Taster	ESP carriers (Africans)	Exac carriers (Africans)	CADD
MYBPC3	5	c.530G>A	R177H	No	Delet	Benign	Dis causing	43 (42)	92 (84)	23.5
MYBPC3	6	c.772G>A	E258K	N = 5	Delet	Prob Dam	Dis causing	No	3 (0)	28.8
MYBPC3	29	c.3296G>A	G1099E	No	Delet	Prob Dam	Dis causing	No	No	28.7
MYH7	5	c.353C>T	S118 L	No	Delet	Prob Dam	Dis causing	No	No	28.1
MYH7	20	c.2213G>A	S738 N	No	Delet	Prob Dam	Dis causing	No	No	21.5
MYH7	23	c.2792A>C	E931A	No	Delet	Prob Dam	Dis causing	No	No	28.8
MYH7	24	c.3016G>A	A1006T	No	Delet	Prob Dam	Dis causing	No	No	8.1
MYH7	34	c.4909G>A	A1637T	No	Tolerated	Benign	Dis causing	6 (5)	7 (5)	19.8
MYH7	38	c.5639G>A	R1880H	No	Delet	Prob Dam	Dis causing	No	No	33
MYH7	39	c.5779A>T	I1927F	N = 2	Tolerated	Benign	Dis causing	No	4 (0)	14.1
MYL3	3	c.170C>A	A57D	No	Delet	Prob Dam	Dis causing	No	14 (0)	29.8
TNNC1	1	c.23C>T	A8 V	No	Tolerated	Benign	Dis causing	No	No	23.6
FLNC	15	c.2375G>T	S792I	No	Delet	Prob Dam	Dis causing	No	No	32
FLNC	27	c.4651G>A	A1551T	No	Delet	Benign	Dis causing	No	No	15.3
FLNC	36	c.5996G>A	R1999Q	No	Delet	Benign	Dis causing	No	No	27.5

ESP carriers = number of carriers in ∼6000 exomes (http://evs.gs.washington.edu/EVS).

Spanish HCM = number of carriers in 400 HCM patients from Spain (recruited through HUCA).

CADD, combined annotation-dependent depletion; Delet, deleterious; Dis, disease; HCM, hypertrophic cardiomyopathy; Prob Dam, probably damaging.

Table 2.

Main Characteristics of the HCM Patients with Sarcomeric Mutations

Patients ID	Sex	Gene	Mutation	Onset age	Current age	Septum size	Family history HCM	Family history SCD	Symptoms
N29	F	MYH7	c.2792A>C (p.E931A)	60	61	21	No	No	Chest pain, palpitation
N22	M	MYH7	c.5779A>T (p.I1927F)	36	37	15	No	No	Palpitation
N17	M	MYH7	c.3016G>A (p.A1006T)	74	74	17	No	No	Palpitation
N16	F	MYH7	c.4909G>A (A1637T)	72	76	18	No	Yes	Chest pain
N40	F	MYH7	c.2213G>A (p.S738 N)	56	D	20	No	Yes	Chest pain, dyspnea
N2	M	MYBPC3	c.530G>A (p.R177H)	56	58	15	No	No	Chest pain
N50	M	MYBPC3	c.772G>A (p.E258K)	49	51	15	Yes	No	Chest pain, palpitation
N4	F	MYBPC3	c.772G>A (p.E258K)	70	73	17	No	No	Chest pain, dyspnea
N39	M	MYBPC3 + MYH7	c.3296G>A (p.G1099E); c.353C>T (p.S118 L)	30	40	26	No	No	Chest pain, dyspnea
N3	F	MYBPC3 + MYH7	c.772G>A (p.E258K); c.5639G>A (p.R1880H)	28	30	25	Yes	Yes	Chest pain
N44	F	MYL3	c.170C>A (p.A57D)	45	50	28	No	Yes	Chest pain, dyspnea
N46	M	TNNC1	c.23C>T (p.A8 V)	53	62	21	Yes	No	Chest pain, dyspnea
N20	M	FLNC	c.4651G>A (p.A1551T)	31	36	15	No	No	Chest pain
N31	F	FLNC	c.2375G>T (S792I)	60	63	16	No	No	Palpitation
N49	M	FLNC	c.2375G>T (S792I)	48	52	16	No	No	Chest pain
N11	F	FLNC	c.5996G>A (p.R1999Q)	74	79	22	No	Yes	Chest pain
Mutation, mean values				52 Years	55 Years	20 mm	n = 3	n = 5
No mutation, mean values				48 Years	67 Years	21 mm	n = 8	n = 9

SCD, sudden cardiac death.

Table 3.

Genotypes for the MYBPC3 SNPs Used to Determine the Haplotypes Linked to the p.E258K Mutation in the Tunisian Patients (N3, N4, N50) and Three Spanish Patients (SP1-3)

SNP	Amino acid	N3	N4	N50	SP1	SP2	SP3
Rs3729986, c.472 GA	p.V158M	AG	GG	GG	GG	GG	GG
Rs11570050^*, c.506-12delC	Intron 5	Del/Del	Del/Del	Del/Del	Del/Del	Del/Ins	Del/Del
Rs11570051, c.537 CT	p.R179H	CC	CC	CC	CC	CC	CC
Rs11570052, c.565 GA	p.V189I	AG	GG	GG	GG	GG	GG
Rs201098973, c.646 GA	p.A216T	GG	GG	GG	A G	A G	A G
Rs3729989, c.706 AG	p.S236G	AA	AA	AA	AA	AA	AA
Mutation	p.E258K	AG	AG	AG	AG	AG	AG
Rs11570058, c.786 TC	p.T262T	CC	CC	CC	CC	CC	CC
Rs11570076, c.1144CT	p.R382 W	CC	CC	CC	CC	CC	CC
Rs3729952, c.2498 CT	p.A833V	CC	CC	CC	CC	CC	CC
Rs3729953. C.2457 CT	p.V849V	CC	CC	CC	CC	CC	CC
Rs11570097, c.2601 CT	p.I867I	CC	CC	CC	CC	CC	CC
Rs554694434, c.2854 CG	p.R952A	GG	GG	GG	GG	GG	GG

For the c.646 GA SNP, underlined is the A allele that segregated with the disease among the Spanish patients.

Intron 5 variant, delC (CCCC = Ins, CCC = Del).

In reference to the main clinical findings among the Tunisian HCM patients, there was no significant difference in the mean septum size (19 mm vs. 21 mm) and the mean onset age (53 years vs. 50 years) between patients with and without mutations. Furthermore, there were no significant differences in frequency of family history of HCM between the two groups (25% vs. 29%) (Table 2). Of the genotype-positive patients, two had double mutations in MYH7 + MYBPC3. The patient carrying both the MYBPC3 (p.E258K) and the MYH7 (p.R1880H) was 28 years old with a septal size of 25, larger than those carrying the MYBPC3 (p.E258K) variant only (Table 2).

One patient was homozygous for the MYL3 (p.A57D; c.170C>A) (Supplementary Fig. S2). She is a 45-year-old female with dyspnea as the presenting symptom and a septum of 28 mm. Two sisters died from sudden cardiac cause at the age of 40 and 39, probably HCM but there is no confirmation. The mother was healthy (no HCM symptoms), but she was not available for echocardiography study. The father died from an unknown cardiac cause. These data suggest a recessive inheritance for the Asp57 MYL3 mutation. Unfortunately, this patient was the only one available for the genetic study, therefore, we would be unable to determine the presence of the MYL3 mutation in other family members (Supplementary Fig. S2).

We also sequenced the FLNC gene, recently linked to HCM by some authors. Four patients were carrying three putative FLNC mutations (Supplementary Fig. S3), including two cases with a new variant p.S792I (c.2375G>T) (Fig. 1). None of these FLNC variants was found in the Spanish cohort of 350 HCM patients (unpublished data). These three amino acid changes were predicted to be pathogenic with at least two bioinformatics programs (Table 2; Supplementary Fig. S4).

FIG. 1.

Schematic representation of the filamin C protein with the position of the eight mutations identified in the Spanish patients (up) as well as the three putative mutations found in the Tunisian patients (down).

Discussion

MYBPC3 and MYH7 were the most common mutated genes, accounting for 27% of the total HCM patients and 83% of the putative mutations in the main sarcomeric genes. This was in agreement with what was reported in the literature (Richard et al., 2003; Gómez et al., 2014a). In addition, we found two double MYH7 + MYBPC3 mutations carriers. Indeed, patients with two sarcomere mutations have more severe form of the disease with a higher risk for SCD compared to single mutation carriers. These two patients had onset ages below the mean value and a higher septum size.

One patient was homozygous for the MYL3 (p.A57D) mutation. We could not confirm this mutation in the parents, who were consanguineous and probably heterozygous carriers. The mother was asymptomatic, a fact that pointed to the recessive effect of this mutation. MYL3 mutations are a rare cause of HCM and have been mainly associated with adult onset (Andersen et al., 2001). However, infantile cases with asymptomatic parents have also been reported (Olson et al., 2002; Jay et al., 2013). There is a marked phenotypic heterogeneity associated with MYL3 mutations in the families: the penetrance in individuals (>18 years) was high, and most of the mutations have been associated with SCD in the young. At least one study reported a recessive inheritance pattern for the MYL3 p.Glu143Lys mutation in children with severe hypertrophy and SCD while the parents were heterozygous and asymptomatic (Olson et al., 2002).

The 45 Tunisian HCM patients were also sequenced with NGS for the filamin C (FLNC) gene. FLNC was recently identified as an HCM gene by using an exome sequencing approach in a group of patients/families negative for mutations in the known HCM genes (Valdés-Mas et al., 2014; Gómez et al., 2016). The study of heart tissue from some of these patients showed marked sarcomere abnormalities in cardiac muscle. The FLNC putative mutations result in the formation of large filamin C aggregates. FLNC encodes for a protein involved in the muscular function of the heart by interacting with the components of sarcolemma and Z-disc (Fujita et al., 2012). The FLNC p.A1551T was predicted as nonpathogenic with Polyphen-2 based on interspecies amino acid conservation, but this was a new variant and was close to p.A1539T found in our Spanish patients, and functional studies showed that this amino acid change could result in enhanced expression of actin (Valdés-Mas et al., 2014).

While FLNC mutations had been associated with myofibrillar myopathy, none of the HCM patients showed symptoms of myopathy clinically or histologically, which suggest that FLNC mutations are not restricted to skeletal muscle disorders but can also be a cause of cardiac muscle abnormalities (Finsterer and Stöllberger, 2000; Kley et al., 2007; Fürst et al., 2013). None of the Tunisian patients had symptoms of myopathy. We found a total of three rare FLNC variants in four patients that represent ∼10% of the cases. This frequency was close to the reported one by Valdes-Mas et al. (2014) among Spanish HCM patients. The FLNC mutations were found in patients with mild hypertrophy (15-16 mm) and without a family history of HCM or SCD; this was compatible with a benign disease course. Although these FLNC variants fulfilled some of the criteria to be considered as likely pathogenic, we are well aware that their pathogenicity is unclear in the absence of a clear familial segregation or functional studies.

Finally, several inherited disorders are characterized by a strong founder effect among Tunisians, with only a few mutations accounting for most of the cases (Nagara et al., 2014). For example, the LRRK2 p.G2019S mutation is common among families with dominant Parkinson's disease (Ishihara et al., 2007; Hulihan et al., 2008). In addition, a high frequency of familial hypercholesterolemia has been reported in the Tunisian population, which is related to the high frequency of consanguinity (Slimane et al., 1993; Jelassi et al., 2010). In contrast with these findings, the mutational spectrum of HCM seems to be heterogeneous since there were no recurrent mutations. Our study has some limitations, mainly the difficulty to conclude the pathological effect of some of the variants based solely on the bioinformatic analysis. In addition, it was based on a limited sample size, although we consider that the recruited cohort was representative of Tunisian HCM patients and highly prevalent (founder) mutations in this population would have been identified.

In conclusion, we report the mutational spectrum of the main sarcomeric genes in Tunisian HCM patients. As reported in other ethnic groups, the MYBPC3 and MYH7 genes were the most frequently mutated, and there were no recurrent mutations despite the genetic structure of the Tunisian population. We also provided evidence for the involvement of FLNC as an HCM gene among Tunisians.

Footnotes

Authors' Contributions

All the authors contributed to this work by recruiting the patients or performing the genetic studies. N.J. and E.C. wrote the article, and all the authors have seen the article and approved the submission.

Author Disclosure Statement

No competing financial interests exist.

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