Identification of a variant hotspot in MYBPC3 and of a novel CSRP3 autosomal recessive alteration in a cohort of Polish patients with hypertrophic cardiomyopathy

Abstract

Introduction: Hypertrophic cardiomyopathy (HCM) is a heart disorder caused by autosomal dominant alterations affecting both sarcomeric genes and other nonsarcomeric loci in a minority of cases. However, in some patients, the occurrence of the causal pathogenic variant or variants in homozygosity, compound heterozygosity, or double heterozygosity has also been described. Most of the HCM pathogenic variants are missense and unique, but truncating mutations of the MYBPC3 gene have been reported as founder pathogenic variants in populations from Finland, France, Japan, Iceland, Italy, and the Netherlands.

Objectives: This study aimed to assess the genetic background of HCM in a cohort of Polish patients.

Patients and methods: Twenty-nine Polish patients were analyzed by a next-generation sequencing panel including 404 cardiovascular genes.

Results: Pathogenic variants were found in 41% of the patients, with ultra-rare MYBPC3 c.2541C>G (p.Tyr847Ter) mutation standing for a variant hotspot and correlating with a lower age at HCM diagnosis. Among the nonsarcomeric genes, the CSRP3 mutation was found in a single case carrying the novel c.364C>T (p.Arg122Ter) variant in homozygosity. With this finding, the total number of known HCM cases with human CSRP3 knockout cases has reached 3.

Conclusions: This report expands the mutational spectrum and the inheritance pattern of HCM.

What’s new?

Most of the DNA mutations leading to hypertrophic cardiomyopathy (HCM) are inherited with an autosomal dominant pattern and are unique or seen in a limited number of families. However, in some ethnic groups, founder mutations have been described. We report the analysis of 404 genes in a cohort of 29 Polish patients affected by HCM. Our results showed that an ultra-rare alteration in the MYBPC3 gene, absent in databases from large-scale sequencing projects, was a mutation hotspot in the present Polish cohort and could represent a novel founder pathogenic variant. Moreover, we found that 1 patient was homozygous for a novel truncating mutation in the CSRP3 gene, thus increasing the number of known human CSRP3 knockout cases to 3. This finding also suggests that the autosomal recessive inheritance pattern could be more frequent in HCM than reported thus far.

Introduction

Hypertrophic cardiomyopathy (HCM) is a cardiac disease characterized by left ventricular hypertrophy (thickness ≥15 mm) unexplained by secondary causes, and a nondilated left ventricle with preserved or increased ejection fraction. It is an important cause of sudden cardiac death, and its prevalence has been estimated at approximately 1/500 individuals in the adult population.¹ Hypertrophic cardiomyopathy is considered a disease of the cardiac sarcomere mainly caused by pathogenic variants in over 10 loci, with MYBPC3, MYH7 and TNNT2 accounting for approximately 50% of the HCM families and encoding cardiac myosin-binding protein C, β-myosin heavy chain, and cardiac troponin T, respectively.² Other inheritable causes of the disease include pathogenic variants in genes encoding proteins involved in calcium handling and for proteins of the Z-disk.³ Most of the pathogenic variants found in HCM genes are missense and unique or private within families.² These alterations are believed to have a dominant negative effect, acting as a “poison polypeptide” on sarcomere function.⁴ The only exception is MYBPC3, in which about two-thirds of pathogenic variants are truncating and haploinsufficiency is postulated as a pathogenetic mechanism of the disease.⁵ To date, at least 12 different truncating MYBPC3 pathogenic mutations have been reported as founder variants in populations from Finland, France, Japan, Iceland, Italy, Spain, and the Netherlands.^5-8

In most adolescents and adults, HCM is inherited as an autosomal dominant trait with a clinical outcome characterized by incomplete penetrance and variable expression. The disease phenotype can indeed be modulated by environmental factors, by the genetic context (including polymorphisms of the renin–angiotensin system [RAS]),⁹ and by the occurrence of the causal pathogenic variant or variants in homozygosity, compound heterozygosity, or double heterozygosity.¹⁰ Janin et al¹¹ have recently reported 2 unrelated kindreds with homozygous truncating variants in the cysteine and glycine-rich protein 3 (CSRP3) gene encoding a member of the CSRP family of muscle LIM protein (MLP).

It is likely that HCM individuals are underdiagnosed,¹² especially in countries where the availability of genetic testing based on next-generation sequencing (NGS) is limited. Considering the prevalence of HCM, the estimated number of HCM patients in Poland is approximately 78 000. Until now, only single reports describing genetic causes of HCM in Polish patients have been reported.^13-15 Therefore, the aim of the current study was to assess the genetic background of HCM in a cohort of patients from the south-eastern part of Poland, analyzed by an NGS panel including 404 genes known to harbor alterations affecting cardiovascular system function. Thanks to this approach, we could identify a MYBPC3 truncating alteration that could represent a novel Polish founder MYBPC3 pathogenic variant. Also, we detected a new patient with the homozygous CSRP3 variant; thus, the number of known HCM cases with null homozygous alterations in this gene has increased to 3.

Patients and methods

Patients

Twenty-nine unrelated patients were selected from those attending an outpatient service dedicated to the diagnosis and management of HCM at the Institute of Cardiology, John Paul II Hospital (Kraków, Poland). Patients underwent clinical history taking, physical examination, electrocardiography (ECG), echocardiography, cardiopulmonary exercise test coupled with ambulatory ECG monitoring, and cardiac magnetic resonance imaging. Diagnostic criteria for HCM were defined in adults by a maximal left ventricular wall thickness of 15 mm or higher on echocardiography, or of 13 mm or higher in relatives, in the absence of loading conditions.¹⁶ Family history of sudden cardiac death, syncope episodes, and the presence of nonsustained ventricular tachycardia were defined as described by O’Mahony et al.¹⁷ Electrocardiographic abnormalities that were considered of clinical significance included abnormal Q waves (0.04 s or 25% depth of the R wave), left ventricular hypertrophy (voltage criteria), and marked repolarization changes (eg, T-wave inversion in at least 2 leads). Familial HCM cases were defined if at least 1 additional affected family member with HCM or 1 case of sudden cardiac death was present in the pedigree. When available, the relatives of index cases were recruited for genetic testing. All patients gave informed consent for the DNA analyses, and the study was approved by local ethics committees in accordance with the principles of the Declaration of Helsinki.

Genetic analyses

Genomic DNA from peripheral blood was tested by NGS with the Ion AmpliSeq™ Cardiovascular Research Panel (ThermoFisher, Carlsbad, California, United States), including the 16 “core HCM genes” defined by the American College of Medical Genetics and Genomics (ACMG),¹⁸ as well as other 388 genes known to harbor alterations affecting cardiovascular functioning (Supplementary material, Table S1). The Ion Chef System (ThermoFisher) was employed for the automated library and template preparation, as well as for chip loading. Sequencing reactions were carried out on the Ion S5 XL System (ThermoFisher). Sequencing reads were aligned on the GRCh37/hg19 reference sequence by the Torrent Suite Software v.5.4.0 (ThermoFisher). For every patient, the panel coverage as well as the mean and median read depth reached for each of 404 genes is given in Supplementary material, Table S1. The mapped reads were analyzed to determine the presence of DNA point variants by Variant Caller v5.4.0.46 plugin using “germline – low stringency” parameters (ThermoFisher). Variants’ calls were scored and prioritized by the TGex software (LifeMap Sciences, Alameda, California, United States; http://tgex.genecards.org/), which ranks variants according to their association to the phenotype (ie, HCM). Between the variants scored by TGex and matching with the HCM phenotype, only the ones meeting all the following parameters were filtered: 1) nonsynonymous exonic or ±10-bp intronic variants; 2) minor allele frequency (MAF) in the Genome Aggregation Database (GnomAD) of less than 0.01; 3) high quality of the call (ie, Q&R score = Coverage ≥20 × and GQ ≥50); and 4) at least 20% of reads showing the alternative allele (% Alt >20%). The resulting variants were confirmed by Sanger sequencing (polymerase chain reaction [PCR] primers are listed in Supplementary material, Table S2) and classified into 5 categories presented in the 2015 guidelines of the ACMG / Association for Molecular Pathology (AMP),¹⁹ modelled by a Bayesian framework as previously described.²⁰ This approach allowed a better categorization of the DNA variants into 7 classes: 1) pathogenic; 2) likely pathogenic; 3) variants of unknown significance (VUS)–favoring pathogenic; 4) VUS; 5) VUS-favoring benign; 6) likely benign; and 7) benign. Data for this classification were obtained from the CardioVai (www.cardioclassifier.org), Cardio Classifier (www.cardiovai.engenome.com), Intervar (www.wintervar.wglab.org), and Varsome (www.varsome.com) systems, as well as from the ClinVar database (www.ncbi.nlm.nih.gov/clinvar). Regarding MYH7, the ACMG/AMP 2015 classification was adapted following the ClinGen’s Inherited Cardiomyopathy Expert Panel.²¹ Variants not yet reported in literature were referred to as “novel.”

The presence of copy number alterations encompassing the CSRP3 c.364C>T variant was investigated by a SYBR Green–based quantitative PCR on an ABI7900 HT Fast Real Time PCR System (ThermoFisher), with the following primer pair mapping in the CSRP3 gene: FW-5’-TGGGAATTCTGGTTTGCTTTG-3’ and Rv-5’-GAGGCATGTAAGATCCAGTGGTT-3’. The experiment included patient 58 as well as 4 unaffected control individuals. The reference gene, TERT, was simultaneously quantified in a separate tube for each specimen.

MYBPC3 haplotype analysis

To test whether the carriers of the same MYBPC3 variant share a common haplotype, a linkage analysis around the MYBPC3 region on chromosome 11 was performed with 8 microsatellite markers upstream the gene (ie, D11S4109, D11SA1, D11S1784, D11S4165, D11S1395, and D11S1765), 6 single nucleotide polymorphisms within the gene (ie, rs1052373, rs11570078, rs2856650, rs3729989, rs11570051, and rs11570050), and 6 microsatellite markers downstream MYBPC3 (ie, D11S905, D11S1763, D11S986, D11S4174, D11S4137, D11S1385, D11S1344, and D11S1252). These polymorphic markers cover about 19.8 Mb around MYBPC3.

Statistical assessment of genotype–phenotype correlations

All statistical analyses were performed using the SPSS software package version 20.0 (IBM SPSS Statistics, Milan, Italy). For each patient, the clinical and molecular data were tabulated (Figure 1). Phenotype data were presented as continuous variables obtained from clinical data and instrumental measurements, and they were summarized using means and standard deviations. The mean values of age at diagnosis, maximal wall thickness, and left ventricular mass were compared using the t test for independent data between the subgroups of patients with different genotypes. Also, associations between sex, the genotype of 2 polymorphisms in the genes of the RAS, and clinical features were tested. The penetrance of HCM, according to age at diagnosis, was analyzed by the Kaplan–Meier method, and differences between cumulative hazard were evaluated with the log-rank test. Due to the relatively low number of patients, no adjustments were planned for multiple testing. Therefore, the analysis is exploratory and the results should be considered as hypothesis generating.

**Figure 1**. Clinical and molecular data of the 29 Polish cases affected by hypertrophic cardiomyopathy. The x-axis shows the 29 patients. The y-axis shows the clinical parameters (top), “core HCM genes” (middle), and other genes found mutated (bottom). The last 2 rows show the genotype of the rs5186 and rs699 polymorphisms, mapping respectively in the *AGTR1* and *AGT* genes of the renin–angiotensin system. The color code employed in the Figure is shown at the bottom.
a Patient on drug treatment
Abbreviations: +, present; AF, atrial fibrillation; AMI, acute myocardial infarction; EF, ejection fraction; F, female; LAFB, left anterior fascicular block; LBBB, left bundle branch block; M, male; MRI, magnetic resonance imaging; NE, not evaluated; NSTEMI, non–ST-segment elevation myocardial infarction; NYHA, New York Heart Association; PSVT, paroxysmal supraventricular tachycardia; RAS, renin–angiotensin system; RBBB, right bundle branch block; SA, sinus arrest

Results and discussion

In total, 12 of the 29 patients (41%) were found to carry at least 1 pathogenetic, likely pathogenetic, or VUS-favoring pathogenic alteration of the “core HCM genes,” as classified by the 2015 ACMG/AMP guidelines and ClinVar database (Table 1 and Figure 1). Most of the changes were identified in the MYBPC3, MYH7 and TNNT2 genes. The genotype of the rs5186 and rs699 polymorphisms, mapping respectively in the angiotensin receptor type 1 (AGTR1) and angiotensinogen (AGT) genes, belonging to the RAS, was also recorded (Figure 1). Indeed, some studies have shown that rs5186 and rs699 may influence the clinical phenotype of HCM,²² since the RAS regulates cardiac function, blood pressure, and electrolyte homeostasis.²³ However, in the present cohort, no significant correlation with the disease expression was found.

**Table 1**. Genetic alterations identified in 12 of the 29 Polish patients affected by hypertrophic cardiomyopathy (for references 51–67, see Supplementary material)
Patient	Gene	Location	Ref	Alt	RefSeq	Nucleotide	AA	Coding impact	Zygosity	dbSNP	GnomAD-Exomes allele frequency, %		ClinVar	ACMG / AMP 2015		ClinVar conditions	Comment
Patient	Gene	Location	Ref	Alt	RefSeq	Nucleotide	AA	Coding impact	Zygosity	dbSNP	European Non-Finnish	Total	ClinVar	Classification	Activeted rules	ClinVar conditions	Comment
Patient	Gene	Location	Ref	Alt	RefSeq	Nucleotide	AA	Coding impact	Zygosity	dbSNP	GnomAD-Exomes allele frequency, %		ClinVar	ACMG / AMP 2015		ClinVar conditions	Comment
Patient	Gene	Location	Ref	Alt	RefSeq	Nucleotide	AA	Coding impact	Zygosity	dbSNP	Pt 9	MYBPC3	ClinVar	11:47369975	C	ClinVar conditions	Comment	T	NM_000256.3	c.772G>A	Glu258Lys	Missense	Het	rs397516074	0.003	0.001	P, LP	P	PP2, PP3, PP5, PM1, PM2, PS4	HCM	Reported in unrelated patients with HCM (Niimura et al³⁴; Richard et al³⁵; Nanni et al³⁶; Van Driest et al³⁷; Song et al³⁸; Murphy et al³⁹)
Pt 10	MYBPC3	11:47359003	G	C	NM_000256.3a	c.2541C>Ga	Tyr847Tera	Nonsense	Het	rs397515974	0	0	P, LP	P	PP3, PP5, PM1, PM2, PS1, PVS1	HCM	Reported in individuals with HCM (Van Driest et al³⁷; Berge et al⁴⁰; Kapplinger et al⁴¹; Viswanathan et al⁴²; Zhao et al⁴³)
Pt 18	MYBPC3	11:47369403	C	T	NM_000256.3	c.821+5G>A	–	Intronic	Het	rs397516077	0	0	P, LP	P	PP3, PP5, PM1, PM2, PS1, PVS1	HCM	Reported in association with HCM (Carrier et al⁴⁴; Millat et al⁴⁵)
Pt 22	MYBPC3	11:47359003	G	C	NM_000256.3a	c.2541C>Ga	Tyr847Tera	Nonsense	Het	rs397515974	0	0	P, LP	P	PP3, PP5, PM1, PM2, PS1, PVS1	HCM	Reported in individuals with HCM (Van Driest et al³⁷; Berge et al⁴⁰; Kapplinger et al⁴¹; Viswanathan et al⁴²; Zhao et al⁴³)
Pt 22	MYH7	14:23891518	T	C	NM_000257.3	c.3116A>G	Glu1039Gly	Missense	Het	rs199573700	0.004	0.01	VUS	VUS-3B	PP2, PP3, PM2	HCM	Novel but present in ClinVar
Pt 57	MYH7	14:23886875	A	C	NM_000257.3	c.4190T>G	Leu1397Arg	Missense	Het	–	0	0	–	P	PP3, PP5, PM1, PM2, PS1, PVS1	–	Novel
Pt 58	CSRP3	11:19207813	G	A	NM_003476.4	c.364C>T	Arg122Ter	Nonsense	Hom	rs902082118	0	0.001	LP	P	PP2, PP3, PM2, PVS1	Not assessed	Novel
Pt 64	MYBPC3	11:47364270	G	A	NM_000256.3	c.1483C>T	Arg495Trp	Missense	Het	rs397515905	0	0	P/VUS	LP	PP2, PP3, PM1, PM2, PM5	HCM	Reported in at least 4 individuals with HCM (Garcia-Castro et al⁴⁶; Rodriguez-Garcia⁴⁷; Coto⁴⁸; Martin⁴⁹)
Pt 74	MYBPC3	11:47359003	G	C	NM_000256.3a	c.2541C>Ga	Tyr847Tera	Nonsense	Het	rs397515974	0	0	P, LP	P	PP3, PP5, PM1, PM2, PS1, PVS1	HCM	Reported in individuals with HCM (Van Driest et al³⁷; Berge et al⁴⁰; Kapplinger et al⁴¹; Viswanathan et al⁴²; Zhao et al⁴³)
Pt 161	MYH7	14:23901922	C	T	NM_000257.3	c.428G>A	Arg143Gln	Missense	Het	rs397516209	0.0009	0.0004	LP	P	PP1, PP3, PM2, PM5, PS4	HCM	Reported in association with HCM (Kimura et al⁵⁰; Van Driest et al³⁷; Song et al³⁸; Coto et al⁴⁸; Marsiglia et al⁵¹)
Pt 3566	MYBPC3	11:47359003	G	C	NM_000256.3a	c.2541C>Ga	Tyr847Tera	Nonsense	Het	rs397515974	0	0	P	P	PP3, PP5, PM1, PM2, PS1, PVS1	HCM	Reported in individuals with HCM (Van Driest et al³⁷; Berge et al⁴⁰; Kapplinger et al⁴¹; Viswanathan et al⁴²; Zhao et al⁴³)
Pt 3937	TNNT2	1:201334426	G	A	NM_000364.3	c.304C>T	Arg102Trp	Missense	Het	rs397516456	0.001	0.0004	P	LP	PP3, PP5, PM1, PM2	HCM	Reported in association with HCM (Koga et al⁵²; Moolman et al⁵³; Moolman-Smook et al⁵⁴; Palm et al⁵⁵; Van Driest et al⁵⁶; Revera et al⁵⁷; Ho et al⁵⁸; Ripoll-Vera et al⁵⁹). Some authors suggested that it is associated with a higher risk of sudden death (Moolman et al⁵⁵; Moolman-Smook et al⁵⁴; Ripoll-Vera et al⁵⁹)
a The MYBPC3 variant hotspot Abbreviations: AA, amino acid; ACMG / AMP, American College of Medical Genetics and Genomics and the Association for Molecular Pathology; Alt, alternative; HCM, hypertrophic cardiomyopathy; Het, heterozygous; Hom, homozygous; LP, likely pathogenic; P, pathogenic; Ref, reference; Pt, patient; VUS-3B, variant of unknown significance–favoring pathogenic

The identified mutational spectrum included 10 distinct substitutions: 6 missense, 3 nonsense, and 1 intronic change. Most of the missense variants were sarcomeric, and, among them, 3 mapped in MYH7, 2 in MYBPC3, and 1 in TNNT2. On the other hand, 2 of the nonsense alterations were found in MYBPC3 and 1 in CSRP3. The GnomAD allele frequency of all the sarcomeric alterations was smaller than 0.01%, that is, the MAF threshold suggested for HCM pathogenic variants.²⁴

A single variant was found in more than 1 patient (Supplementary material, Figure S1A). The MYBPC3 c.2541C>G variant, resulting in the premature insertion of a stop codon in exon 24 (p.Tyr847Ter), was indeed identified in 4 unrelated cases, despite the fact that it is absent in the GnomAD. Carriers of the MYBPC3 c.2541C>G showed a lower age at HCM diagnosis compared with carriers of other DNA variants, with a 50% probability of HCM diagnosis at 38 years for MYBPC3 c.2541C>G carriers, in comparison with the age of 49 years for the carriers of other DNA variants (P = 0.04; Figure 2). The MYBPC3 c.2541C>G mutation was the only identified variant in cases 10, 74, and 3566, while it was associated with another sarcomeric alteration (ie, the MYH7 c.3116A>G,p.Glu1039Gly) in patient 22. The allele frequency of the MYH7 c.3116A>G was very close to the 0.01% threshold, and we infer that this variant could be a modifier allele or a low penetrance variant contributing to the severity of HCM expression in patient 22. Among the 4 carriers of the MYBPC3 c.2541C>G, patient 22 indeed manifested the earliest age at diagnosis, the need for septal myectomy at the age of 41 years, and the presence of a long QTc interval (Figure 1). The segregation of MYBPC3 c.2541C>G and of MYH7 c.3116A>G was tested in 4 relatives of case 22: a 50-year-old sister affected by HCM and 3 children aged 9, 12, and 16 years, who are currently asymptomatic. All of them were found to carry MYBPC3 c.2541C>G in heterozygosity, while they had the wild-type MYH7 c.3116A variant (Supplementary material, Figure S1A).

**Figure 2**. Penetrance of hypertrophic cardiomyopathy, according to age at diagnosis, in *MYBPC3* c.2541C>G carriers (blue) and in carriers of other DNA variants (green). Plot represents 1 minus the probability of not having a diagnosis of hypertrophic cardiomyopathy (HCM) at each age. This probability is estimated by the Kaplan–Meier method. Patient 22 was not included in this analysis due to being a double-variant carrier.

The MYBPC3 c.2541C>G variant was also detected in heterozygosity in a 19-year-old daughter of patient 74 (Supplementary material, Figure S1A). On clinical evaluation, she did not display any chronic cardiac or noncardiac disease. Her echocardiogram showed normal left ventricular (left ventricular end-diastolic diameter, 43 mm) and atrial size (left atrial appendage, 17 cm²; right atrial appendage, 13.5 cm²), as well as normal thickness of intraventricular septum (end-diastolic diameter, 10 mm). Also, left ventricular systolic function was normal (70%), and she did not show any valvular abnormalities, signs of systolic anterior motion, or left ventricular outflow obstruction. Therefore, at present, the children of case 22 and the daughter of case 74 are asymptomatic carriers of c.2541C>G and possibly would show later onset of the disease due to an age-related penetrance.

To investigate whether carriers of the MYBPC3 c.2541C>G variant could share a common ancestor, we performed a markers analysis. A haplotype, spanning about 5 Mb, was found to segregate with the c.2541G variant allele in the family of patient 22. The alleles of the c.2541G-haplotype were also present in all the MYBPC3 c.2541C>G carriers (Table 2), even if we could not reconstruct their phase. This finding supports the hypothesis that the MYBPC3 c.2541C>G variant could have a founder role in the Polish population. In support of this hypothesis, the MYBPC3 c.2541C>G variant has been recently found in another Polish patient with HCM, not related to any of the cases reported herein.²⁵

**Table 2.** Haplotype analysis in cases carrying the *MYBPC3* c.2541C>G variant
Marker	Genomic position start (Hg19)	c.2541G-haplotype^a	Pt 10	Pt 74	Daughter of Pt 74	Pt 3566
D11S1252	11:46446790	153*	153*-153	153*-153	153*-153	153*-161
MYBPC3 start	11:47353396
rs1052373	11:47354787	A*	A*-G	A*-G	A*-G	A*-G
c.2541C>G	11:47359003	G*	G*-C	G*-C	G*-C	G*-C
rs11570078	11:47365014	G*	G*-G	G*-A	G*-G	G*-G
rs2856650	11:47365199	C*	C*-T	C*-C	C*-T	C*-T
rs3729989	11:47370041	A*	A*-A	A*-G	A*-A	A*-A
rs11570051	11:47371442	T*	T*-C	T*-C	T*-C	T*-C
rs11570050	11:47371485	C*	C*-del	C*-C	C*-del	C*-del
MYBPC3 end	11:47374253
D11S4109	11:47601406	153*	153*-167	153*-171	153*-165	153*-167
D11SA1	11:47741121	257*	257*-251	257*-245	257*-253	257*-251
D11S1784	11:48022707	143*	143*-141	143*-149	143*-141	143*-145
D11S4165	11:50137951	217*	217*-217	217*-217	217*-217	217*-217
D11S1395	11:51382783	223*	223*-223	223*-231	223*-227	223*-227
a The c.2541G-haplotype was found by segregation analysis performed in patient 22 and her relatives. Abbreviations: see Table 1

Case 58 was found to carry the truncating CSRP3 c.364C>T (p.Arg122Ter) variant in homozygosity (Supplementary material, Figure S1B), as confirmed by SYBR Green–based quantitative PCR on ABI7900 HT Fast Real Time PCR System (ThermoFisher) (Supplementary material, Figure S1C). Patient 58 was a 56-year-old woman diagnosed with HCM at the age of 54 due to early fatigue and chest pain. She mainly complained of nonspecific and episodic chest pain (discomfort) on exertion. Echocardiography showed asymmetric interventricular septal hypertrophy (18 mm), posterior wall of 12 mm, ejection fraction of 70%, normal right ventricle, and maximal (provoked) left ventricular outflow tract gradient of 18 mm Hg without the systolic anterior motion of the mitral valve. Also, severe diastolic dysfunction with no signs of pulmonary hypertension was observed. The 24-hour electrocardiogram highlighted 5 runs of nonsustained ventricular tachycardia (the longest one of 11 beats). Family history was negative for HCM, but the CSRP3 c.364C>T variant was found to segregate in heterozygosity in a 29-year-old son (Supplementary material, Figure S1B), who currently does not suffer from any chronic cardiac or noncardiac diseases. He has no symptoms and is physically active. On echocardiography, he has normal left ventricular (left ventricular end-diastolic diameter, 46 mm) and atrial sizes (left atrial appendage, 17.5 cm²; right atrial appendage, 17 cm²). However, his intraventricular septum was mildly thickened (end-diastolic diameter, 13 mm), with normal systolic function (ejection fraction, 67%), no valvular abnormalities, and no sign of systolic anterior motion and left ventricular outflow tract obstruction.

The CSRP3 c.364C>T (p.Arg122Ter) mutation is not a common variant since it is absent in GnomAD-Genomes and GnomAD-Exomes-European databases. In total, only 3 CSRP3 c.364C>T alleles are listed in GnomAD-Exomes (ie, 2 in African and 1 in South Asian populations), but never in a homozygous state. It has not been described in the literature before, but it is listed as likely pathogenic in the ClinVar database because of the following evidence: 1) it is a rare variant; 2) it is predicted to cause the loss of protein function either by protein truncation or nonsense-mediated mRNA decay; 3) CSRP3-null mice develop cardiomyopathy and heart failure due to a disrupted cardiomyocyte architecture²⁶;and 4) myocardial biopsies of a HCM patient with a heterozygous CSRP3 missense variant showed myocyte disarray and a reduced level of MLP, suggesting that cardiomyopathy may stem from CSRP3 haploinsufficiency.²⁷ To date, 24 carriers of a CSRP3 alteration were reported in the literature or included in the ClinVar database: 18 had HCM and 6 were affected by dilated cardiomyopathy. Among the HCM patients, 9 carried a heterozygous CSRP3 variant (including 7 amino acid substitutions and 2 truncating variants), 2 cases were heterozygous but also carried a second variant in another gene, while 2 patients harbored a truncating alteration in homozygosity (Table 3).¹¹ Therefore, patient 58 described herein is the third CSRP3 human knockout case reported so far. Our finding strengthens the assumption that at least several CSRP3 variants lead to HCM with an autosomal recessive inheritance¹¹ rather than with an autosomal dominant transmission as recorded in the Online Mendelian Inheritance in Man (OMIM) database (OMIM: *600824). The mouse model knock-in for CSRP3 pathogenic variants also corroborates the hypothesis of autosomal recessive inheritance of the cardiac disease and recalls the findings displayed by patient 58 and his son.²⁸ Indeed, the heterozygous mice for CSRP3 pathogenic variants do not display an overt cardiac phenotype (except for an increase in anterior wall thickness), while the homozygous mutated mouse shows a clear cardiomyopathy phenotype.²⁸

**Table 3.** *CSRP3* variants to date reported in literature or listed in the ClinVar database (for references 51–67, see Supplementary material)
CSRP3 variant	Zygosity	Reference(s)	Condition(s)
c.46A>T (p.Thr16Ser)	Not reported	ClinVar	DCM
c.50insGCAGATTTCTT (p.Tyr18GlnfsX194)	Het	van Rijsingen et al⁶⁰	HCM
c.96G>A (p.Lys32=)	Not reported	ClinVar	DCM
c.122_123dupGG (p.Lys42Glyfs)	Het	Bos et al⁶¹	HCM
c.131T>C (p.Leu44Pro)	Het	Geier et al²⁷; Geier et al⁶²; ClinVar	Familial HCM 12, DCM 1M, not specified, cardiovascular phenotype
c.131T>C (p.Leu44Pro)	Het, in association with MYBPC3 p.Gly1041fs	Bos et al⁶¹	HCM
c.136A>C (p.Ser46Arg)	Het, in association with TNNI3 p.Arg162Gln	Bos et al⁶¹; ClinVar	Familial HCM 12, DCM 1M, cardiomyopathy, not specified
c.160_164delTCGGAinsAGGGG (p.Ser54_Glu55delinsArgGly)	Het	Geier et al²⁷; ClinVar	Familial HCM 12
c.172T>G (p.Cys58Gly)	Het	Geier et al⁶²; ClinVar	Familial HCM 12
c.190C>T (p.Arg64Cys)	Het	Bos et al⁶¹	HCM
c.197A>G (p.Tyr66Cys)	Het	Bos et a⁶¹	HCM
c.206A>G (p.Lys69Arg)	Not reported	ClinVar	Familial HCM 12, DCM 1M, not specified, cardiovascular phenotype
c.214G>A (p.Gly72Arg)	Het	Hershberger et al⁶³	DCM
c.233G>T (p.Gly78Val)	Not reported	ClinVar	Cardiovascular phenotype, DCM
c.272A>T (p.Gln91Leu)	Het	Bos et al⁶¹	HCM
c.299G>A (p.Arg100His)	Het	Andersen et al⁶⁴	HCM
c.336G>A (p.Ala112=)	Not reported	ClinVar	Not specified, HCM, cardiovascular phenotype, DCM, DCM, dominant
c.354G>A (p.Glu118=)	Not reported	ClinVar	DCM
c.364C>T (p.Arg122Ter)	Hom	This report	HCM
c.365G>A (p.Arg122Gln)	Not reported	ClinVar	Familial HCM 12, DCM 1M, cardiomyopathy
c.369T>A (p.Cys123Ter)	Hom	Janin et al¹¹	HCM
c.420G>C (p.Trp140Cys)	Not reported	ClinVar	DCM
c.449G>A (p.Cys150Tyr)	Not reported	ClinVar	Familial HCM 12, DCM 1M, not specified, cardiovascular phenotype
c.483dup (p.Lys162GlnfsX52)	Hom	Janin et al¹¹	Hypertrophic cardiomyopathy
c.536C>T (p.Thr179Met)	Not reported	ClinVar	Familial HCM 12, DCM 1M, not specified
Abbreviations: DCM, dilated cardiomyopathy; others, see Table 1

The “non–core HCM genes” with mutations in the present HCM cohort were GYG1, GUSB, PMM2 and SCO2. Cases 64, 161, 211, and 218 were indeed identified as heterozygous carriers of autosomal recessive alterations associated with PMM2, SCO2, GUSB, and GYG1 deficiency, respectively (Table 4). The GYG1, GUSB, PMM2, and SCO2 variants were prioritized by the TGex software since they were related to the cardiomyopathy phenotype: GYG1 c.304G>C, found in patient 218, has previously been described in homozygosity in cases affected by severe cardiomyopathy without skeletal muscle weakness²⁹; the GUSB and PMM2 genes are responsible for genetic disorders associated with HCM³⁰; the SCO2 c.418G>A variant has been previously reported by the name of “c.1541G>A” in homozygosity or in compound heterozygosity in individuals with SCO2-related clinical features including HCM.^31,32 Therefore, these results can be considered “secondary findings” for which we cannot exclude a possible hypomorphic impact over the patients’ phenotype. Also, the SCO2 p.Glu140Lys variant was previously associated with autosomal dominant high-grade myopia.³³ In the present patient, presenting low myopia of –2.00 diopters in both eyes, the SCO2 c.418G>A variant exhibited reduced penetrance.

**Table 4**. Secondary molecular findings (for references 51–67, see Supplementary material)
Patient	Gene	Location	Ref	Alt	RefSeq	Nucleotide	AA	Coding impact	Zygosity	dbSNP	GnomAD-Exomes allele frequency (%)		ClinVar	ACMG / AMP 2015		ClinVar conditions	Comment
Patient	Gene	Location	Ref	Alt	RefSeq	Nucleotide	AA	Coding impact	Zygosity	dbSNP	European Non-Finnish	Total	ClinVar	Classification	Activated rules	ClinVar conditions	Comment
Pt 64	PMM2	16:8941632	G	A	NM_000303.2	c.691G>A	Val231Met	Missense	Het	rs80338707	0.011	0.007	P	VUS-3B	PP3, PP5, PM1	Carbohydrate-deficient glycoprotein syndrome type Ia	Reported in several individuals affected with PMM2-CDG (Barone et al⁶⁵)
Pt 161	SCO2	22:50962423	C	T	NM_005138.2	c.418G>A	Glu140Lys	Missense	Het	rs74315511	0.017	0.008	P	VUS-3B	PP3, PP5, PM1	Cardioencephalomyo- pathy, fatal infantile, due to cytochrome c oxidase deficiency 1a Myopia 6	Previously reported as c.1541G>A in homozygosity or compound heterozygosity with a second SCO2 variant in individuals with SCO2-related clinical features including HCM (Papadopoulou et al³²; Jaksch et al³¹) Reported as a heterozygous mutation in 1 individual with autosomal dominant high-grade myopia (Tran-Viet et al³³)
Pt 211	GUSB	7:65444841	C	T	NM_000181.3	c.454G>A	Asp152Asn	Missense	Het	rs149606212	0.189	0.113	VUS	LP	PP3, PM1, PM2	MPS type VIIa	Identified in homozygosity in a child with MPS type VIIa (Vervoort et al⁶⁶). Transfection studies showed that the D152N substitution resulted in decreased enzyme activity. Vervoort et al⁶⁶ referred to D152N as a “pseudodeficiency allele” that leads to greatly reduced levels of beta-glucuronidase activity without apparent deleterious consequences.
Pt 218	GYG1	3:148714249	G	C	NM_004130.3	c.304G>C	Asp102His	Missense	Het	rs143137713	0.189	0.102	P/LP	VUS-3B	PP3, PP5, PM1	Glycogen storage disease XVa	Reported in homozygosity or compound heterozygosity in individuals with glycogenin-1 deficiency (Malfatti et al⁶⁷; Hedberg-Oldfors et al²⁹)
a Autosomal recessive Abbreviations: MPS, mucopolysaccharidosis; others, see Table 1

In conclusion, this report expands the mutational spectrum and the inheritance pattern of HCM. The ultra-rare MYBPC3 c.2541C>G (p.Tyr847Ter) alteration, found in 9 cases (of which 4 were index patients), while absent in databases from large-scale sequencing projects, acts as a variant hotspot in the present Polish cohort and correlates with a younger age at HCM diagnosis. These findings, if confirmed in a wider population of the same ethnic origin, will increase the number of truncating founder MYBPC3 alterations.

The identification of the novel homozygous null CSRP3 variant leading to HCM suggests that the autosomal recessive inheritance pattern could be more frequent in HCM than reported so far. In heterozygosity, the null CSRP3 allele seems to correlate only with a mild thickening of the intraventricular septum.

Supplementary material.pdf

Correspondence to

Irene Bottillo, PhD, Medical Genetics, Department of Molecular Medicine, Sapienza University, San Camillo-Forlanini Hospital, Circonvallazione Gianicolense, 87-00 52 Rome, Italy, phone: +39 06 58704622, email: i.bottillo@gmail.com

Received

November 26, 2019.

Revision accepted

January 2, 2020.

Published online

January 9, 2020.

Note

For references 51–67, see Supplementary material at www.mp.pl/paim.

Acknowledgments

This work was funded by the department of Molecular Medicine, Sapienza University of Rome.

Contribution statement

ML contributed to investigation, data curation, and methodology. EW contributed to data curation and methodology. MK, LT-P, PR, and PPD contributed to patients’ clinical assessment. LL and FB contributed to manuscript review and editing. DG contributed to statistical analyses. AU contributed to project administration and validation. PG contributed to project administration, funding acquisition, resources, and validation. IB contributed to conceptualization, data curation, and writing of the original draft.

Conflict of interest

None declared.

How to cite

Lipari M, Wypasek E, Karpiński M, et al. Identification of a variant hotspot in MYBPC3 and of a novel CSRP3 autosomal recessive alteration in a cohort of Polish patients with hypertrophic cardiomyopathy. Pol Arch Intern Med. 2020; 130: 88-99. doi:10.20452/pamw.15130

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