Evaluation of Cardiomyopathy-Related Target Genes by Next-Generation Sequencing Method and Investigation of the Phenotype-Genotype Relationship

Abstract

Background

Primary heart muscle diseases called cardiomyopathy (CMP) constitute an important group of subsequent heart disorders. CMPs are basically divided into four subgroups associated with the heart muscle but clinically distinguishable: hypertrophic CMP (HCM), dilated CMP (DCM), restrictive CMP (RCM), and left ventricular non-compaction CMP.

Material and Methods

The results of the patients who applied to the Genetic Diseases Evaluation Center with the preliminary diagnosis of clinical CMP were evaluated retrospectively in the current study. In the current study, 103 cases were included and evaluated for phenotype-genotype association with the CMP next-generation sequencing (NGS) panel.

Results

Fifty-eight different variants were identified in 45 patients. Sixteen out of those 58 variants were novel. Of these variants, 19 (32.75%) were likely pathogenic (LP)/pathogenic (P), and 35 (60.34%) were variants of uncertain significance.

Conclusion

The prevalence of pathogenic variants in target genes associated with CMP is important for our current country's population, and multiple gene groups associated with CMP can be screened through NGS. The contribution rate to the clinical diagnosis was 18.44% in terms of the individual population who applied to our medical genetics center and were compatible with the CMP indication.

Introduction

As the leading cause of sudden death and/or congestive heart failure, cardiomyopathy (CMP) represents a heterogeneous group of heart muscle disorders. CMP is a term used to describe the morphological and physiological characteristics of myocardial Tissue [1]. In the classification made by the American Heart Association (AHA), only heart-specific diseases caused by myocardial tissue were called “primary,” and myocardial diseases caused by its participation in systemic diseases were called “secondary” CMP [2]. This classification is in the main plan, but in addition to this main classification, the European Heart Association in 2008 created a new classification with hereditary and nonhereditary subgroups [3]. In order to reduce the confusion in the classification of CMPs in clinical practice, Westphal et al. [4] made an updated classification that includes genetic variations, viral infections, and intramyocardial inflammations. The functional classification of the most basic CMPs for clinical practice is hypertrophic CMP (HCM), dilated CMP (DCM), restrictive CMP (RCM), and left ventricular non-compaction CMP (LVNC). HCM: abnormal thickening of the myocardium makes it difficult to pump blood from the heart to the body. DCM: systemic diseases occur due to environmental factors. RCM: cardiac movements are restricted by thickening of the endocardium and myocardium. LVNC: it is the appearance of the heart muscle as a finger-like structure that occurs due to hereditary reasons.

In addition to all this classification, genetic studies are of great importance for the diagnosis and follow-up of individuals [5]. HCM (#115197) is mainly caused by heterozygous, homozygous, or compound heterozygous variation in the gene encoding cardiac myosin-binding protein C (MYBPC3). The MYBPC3 (OMIM *600958) gene is strung crosswise in sarcomere A-bands and binds myosin heavy chain (OMIM *160710/MYH6) in thick filaments and titin (OMIM *188840/TTN) in elastic filaments. Cardiac myosin-binding protein C (MYBPC3) has been more associated with DCM and HCM. The myosin beta heavy chain (OMIM *160760/MYH7) gene is predominantly expressed in fetal life [6]. MYH7 mRNA is expressed in skeletal muscle tissue. Its expression is important for slow-twitch type I muscle fibers in the soleus muscle and fast-twitch type II in the vastus medialis [7].

RCM (# 617047) is caused by heterozygous variations in the FLNC gene (OMIM *102565) on chromosome 7q32. The filamin C protein is a member of the family of actin-binding proteins involved in the remodeling of the cytoskeleton. Lim domaın-binding 3 (OMIM *605906/LDB3) is located on chromosome 10p23. Heterozygous variants on the LDB3 gene have been associated with LVNC (# 601493). In addition to these, there is another type of CMP called arrhythmogenic right ventricular dysplasia (ARVD). ARVD (# 610476) is caused by heterozygous variations in the desmocollin-2 gene (OMIM *125645/DSC2) on chromosome 18q. The DSC2 gene, a calcium-dependent glycoprotein, is expressed in the human heart, pancreas, lung, placenta, brain, skeletal muscle, liver, and kidney tissues [8].

In CMPs, variations in more than 40 genes that encode important components of cardiomyocytes such as sarcomeric filaments, calcium metabolizing proteins, and mitochondrial enzyme that reveal the phenotype of the disease have been identified [9–11]. However, the definition of genotype-phenotype correlations for CMP is rather difficult due to the overlap between phenotype and gene groups [12]. In this context, the prevalence of pathogenic variants in CMP-related target genes is important for the current population. Multiple gene groups associated with CMP can be screened through next-generation sequencing (NGS). Gene numbers may vary in panel-based NGS studies. The aim of our study was to evaluate retrospectively the contribution of NGS and multigene panel study to clinical diagnosis and the genotype-phenotype relationship in individuals with clinical pre-diagnosis of CMP.

Materials and Methods

Study Group

One hundred and three individuals who applied to our Genetic Diseases Evaluation Center with a clinical diagnosis of CMP between February 2017 and January 2022 were included in the study. Two-dimensional/doppler echocardiography (2D echocardiography) was applied to all cases that were included in the current study. This study was conducted by the Ethics Committee of our University inaccordance with the principles of the Declaration of Helsinki with the approval code numbered 2022/70.Informed consent forms were obtained from all individuals included in the study and from their families.

DNA Isolation and Characterization

Two mL of peripheral blood samples of cases were collected into EDTA-containing tubes. Genomic DNA was isolated from peripheral blood with the EZ1 Advanced XL device using the EZ1 DNA Blood Kit (Qiagen, Hilden, Germany). The first concentration measurements after isolation were made with the Qubit 4 fluorometer (Thermo Fisher Scientific-Waltham/MA) device. The optimum range for samples to be included in the NGS panel study should be 10–40 ng/ul.

CMP-Associated NGS Panel Study

The cardiomyopathy panel (Qiagen, Hilden, Germany-CDHS-14644Z-5714) was used for the NGS panel study. The gene information in the cardiomyopathy panel (Qiagen, Hilden, Germany-CDHS-14644Z-5714) is as in Table 1. The libraries were prepared by barcoding the panel study samples. The samples prepared in the library were pooled by taking the primary lengths into appropriate pools. The panel study pools were loaded on the Nextseq 550 (Illumina, San Diego, CA, USA) sequence device. We performed a family segregation study for variant confirmation of a case (sample ID A1) with the pathogenic variant.

Table 1.

Targeted genes involved in cardiomyopathy panel (90 genes)

ABCC9	ACTC1	ACTN2	ADRB1	ADRB2	ADRB3	AGL	ANK2	ANKRD1	BAG3	BRAF	CALR3
CAV3	CBL	CRYAB	CSRP3	CTF1	DES	DMD	DSC2	DSG2	DSP	DTNA	EMD
EYA4	FHL1	FHL2	FKTN	FLNC	FXN	GAA	GLA	HRAS	ILK	JPH2	JUP
KRAS	LAMA4	LAMP2	LDB3	LMNA	MAP2K1	MAP2K2	MYBPC3	MYH6	MYH7	MYL2	MYL3
MYLK2	MYOT	MYOZ2	MYPN	NEBL	NEXN	NRAS	PDLIM3	PKP2	PKP4	PLEC	PLN
PNN	PRKAG2	PSEN1	PSEN2	PTPN11	RAF1	RBM20	RPSA	RYR2	SCN5A	SDHA	SGCD
SHOC2	SLC25A4	SOS1	SPRED1	SYNE1	SYNE2	TAZ	TCAP	TGFB3	TMEM43	TMPO	TNNC1
TNNI3	TNNT2	TPM1	TTN	TTR	VCL

NGS Data Analysis

Variant screening and analysis QIAGEN Clinical Insight (QCI) software IGV_2.3.6 (Integrative Genomics Viewer) program was used for the visual evaluation of the data. 2015 American College Medical Genetics and Genomics (ACMG-2015) guidelines followed for the classification of all variants [13, 14]. ClinVar, dbSNP, HGMD-Professional, GnomAD, Exac, HapMap, 1000 Genomes, Varsome, and Franklin databases were also looked at for variant classification.

Results

One hundred and three cases with CMP, HCM, DCM, and familial CMP history were included in the study. The mean age of the 40 female individuals was 38.3 ± 19.16 (standard deviation) years, and the mean age of 63 male individuals was 34.6 ± 18.43 (standard deviation) years. Diagnostic information and percentages of cases with variants are as in Figure 1.

Fig. 1.

Distribution of the clinical diagnosis across the studied population in the current study.

The number of variants detected in a total of cases was 45 (43.68%). The total number of different variants detected in 45 cases was 58; of these, 19 (32.75%) were likely pathogenic (LP)/pathogenic (P) and 35 (60.34%) variants were of uncertain significance (VUS). Table 2 contains the LP/P variant information of the cases, and Table 3 contains the variant information of the cases with VUS and others. A total of 4 LP/P variants and 12 VUS were not registered in dbSNP. The novel variants we have identified have not yet been presented in any publication.

Table 2.

Classification of known and novel pathogenic and LP variants in individuals

Sample ID	Age	Gender	Gene	Diagnosis	Nucleotide HGVS	Protein HGVS	Zygosity	Mutation type	dbSNP	ACMG 2015	Variant classification	ClinVar	GnomAD frequency
A1	17	F	TNNI3	Restrictive cardiomyopathy, pulmonary hypertension	NM_000363.4:c.532_534del	(p.Lys178del)	HT	In frame-deletion	rs397516351	Pathogenic	PS2/PM4	NA	NA
											PM5
A2	5	M	MYBPC3	Hypertrophic CMP	NM_000256.3:c.1484G>A	(p.Arg495Gln)	HT	Missense	rs200411226	Pathogenic	PM2/PM5	Pathogenic/LP	0.000043
A3	22	M	MYBPC3	Brugada syndrome? CMP	NM_000256.3:c.1468G>A	(p.Gly490Arg)	HT	Missense	rs200625851	Pathogenic	PM2/PM5	VUS	0.000242
A4	41	M	EMD	Emery Dreifuss?_CMP	NM_000117.3:c.123C>G	(p.Tyr41Ter)	HMZ	Nonsense	Novel	Pathogenic	PM2/PVS1	LP	NA
A5	25	M	RYR2	Brugada Syndrome? CMP	NM_001035.3: c.10555–2A>C		HT	Intronic	rs1251118085	Pathogenic	PM2/PVS1	NA	0.000004
A6	62	M	MYBPC3	CMP	NM_000256.3:c.772G>A	(p.Glu258Lys)	HT	Missense	rs397516074	Pathogenic	PM2/PP3	Pathogenic	0.000043
A7	6	F	BRAF	Hypertrophic CMP	NM_004333.5:c.1406G>A	(p.Gly469Glu)	HT	Missense	rs121913355	Pathogenic	PM2/PM5	Pathogenic	0.000004
A8	69	F	MYH7	Hypertrophic obstructive CMP	NM_000257.4: c.1988G>A	(p.Arg663His)	HT	Missense	rs371898076	Pathogenic	PM2/PM5	Pathogenic	0.000050
A9	23	M	TTN	Familial CMP	NM_133378.4: c.78601_78602dup	(p.Ala26202ValfsTer6)	HT	Frameshift	Novel	Pathogenic	PM2/PVS1	NA	NA
A10	28	F	MYBPC3	Hypertrophic CMP	NM_000256.3:c.772G>A	(p.Glu258Lys)	HT	Missense	rs397516074	Pathogenic	PM2/PP3	Pathogenic	0.000043
A11	40	M	TPM1	Hypertrophic CMP	NM_001018005.1:c.842T>C	(p.Met281Thr)	HT	Missense	rs199476321	LP	PM2/PP2	Conflicting Interpretations of pathogenicity	0.000014
A12	23	F	MYBPC3	Familial CMP	NM_000256.3:c.1457+1G>T		HT	Intronic	Novel	Pathogenic	PM2/PVS1	NA	NA
A13	72	F	MYBPC3	Hypertrophic CMP	NM_000256.3:c.3697C>T	(p.Gln1233Ter)	HT	Nonsense	rs397516037	Pathogenic	PM2/PVS1	Pathogenic	0.000008
A14	51	F	MYBPC3	Hypertrophic CMP	NM_000256.3:c.2441_2443del	(p.Lys814del)	HT	In frame-deletion	rs727504288	LP	PM2/PM4	Conflicting Interpretations of pathogenicity	0.000029
A15	2	M	MYH7	Hypertrophic CMP	NM_000257.4:c.3158G>A	p.Arg1053Gln (p.Leu1492ArgfsTer21)	HT	Missense	rs587782962	Pathogenic	PM2/PM5	Pathogenic	0.000078
					NM_000257.4:c.4475del		HT	Frameshift	Novel	VUS	PM2	NA	NA
A16	62	M	MYH7	Hypertrophic CMP	NM_000257.4:c.5135G>A	(p.Arg1712Gln)	HT	Missense	rs193922390	Pathogenic	PM2/PM5	Pathogenic	0.000021
A17	34	M	CAV3	Hypertrophic CMP	NM_001234.5:c.233C>T	(p.Thr78Met)	HT	Missense	rs72546668	LP	PM1/PM2/PP2/PP3	Conflicting Interpretations of pathogenicity	0.003165
A18	69	F	ABCC9	Hypertrophic CMP	NM_005691.4:c.4572_4573insT	(p.Val1525CysfsTer4)	HT	Frameshift	rs761784169	Pathogenic	PM2/PVS1	Conflicting Interpretations of pathogenicity	0.000431

M, male; F, female; HGVS, human genome variation society; dbSNP, single nucleotide polymorphism database; LP, likely pathogenic; VUS, variants of uncertain significance; NA, not available; HT, heterozygous; HMZ, hemizygous.

Table 3.

Classification of uncertain significance and other variants in individuals

Sample ID	Age	Gender	Gene	Diagnosis	Nucleotide HGVS	Protein HGVS	Zygosity	Mutation type	dbSNP	ACMG 2015	ClinVar
B1	44	M	TTN	CMP	NM_001267550.2:c.72766A>G	(p.Asn24256His)	HT	Missense	rs187868672	LB	Conflicting interpretations of pathogenicity
B2	10	M	TTN	CMP	NM_133378.4:c.875C>T	(p.Pro292Leu)	HT	Missense	rs768278225	VUS	NA
B3	14	M	TTN	CMP	NM_133378.4:c.88976C>G	(p.Ala29659Gly)	HT	Missense	Novel	VUS	NA
					NM_133378.4:c.3895A>G	(p.Asn1299Asp)	HT	Missense	Novel	VUS	NA
B4	19	M	SCN5A	CMP	NM_198056.3:c.3133G>A	(p.Val1045Met)	HT	Missense	rs527480102	VUS	VUS
B5	36	F	MYL3	DCM	NM_000258.3:c.170C>A	(p.Ala57Asp)	HT	Missense	rs139794062	B	Conflicting interpretations of pathogenicity
			ACTN2		NM_001103.4:c.2125G>C	(p.Asp709His)	HT	Missense	Novel	VUS	NA
B6	5	F	MYH7	DCM	NM_000257.3:c.4187G>A	(p.Arg1396Gln)	HT	Missense	rs370069461	VUS	VUS
B7	9	M	RYR2	HCM	NM_001035.3:c.6677G>A	(p.Ser2226Asn)	HT	Missense	rs1558104099	VUS	VUS
			SDHA		NM_004168.4:c.1861C>T	(p.His621Tyr)	HT	Missense	rs1554002475	VUS	NA
B8	60	F	DSC2	HCM	NM_024422.6:c.907G>A	(p.Val303Met)	HM	Missense	rs145560678	VUS	C
			SYNE1		NM_033071.4:c.512A>C	(p.Lys171Thr)	HT	Missense	rs1280830331	VUS	NA
			MYH1		NM_005963.4:c.1425C>A	(p.Ser475Arg)	HT	Missense	Novel	VUS	NA
B9	29	M	TTN	HCM	NM_133379.4:c.15428C>A	(p.Thr5143Lys)	HT	Missense	Novel	VUS	NA
					NM_133379.4:c.55735G>A	(p.Ala18579Thr)	HT	Missense	rs72646853	VUS	VUS
B10	15	M	DSC2	DCM	NM_004949.4:c.2138C>T	(p.Thr713Met)	HT	Missense	rs180863872	VUS	VUS
B11	71	F	SGCD	HCM	NM_000337.5:c.839C>T	(p.Ser280Phe)	HT	Missense	rs397516337	VUS	NA
			SYNE1		NM_033071.3:c.1985A>G	(p.Gln662Arg)	HT	Missense	rs9397509	Benign	LB
			TTN		NM_133378.4:c.73453T>A	(p.Tyr24485Asn)	HT	Missense	rs776943572	VUS	Conflicting interpretations of pathogenicity
B12	39	F	MYH6	CMP	NM_002471.3:c.2717G>A	(p.Arg906His)	HT	Missense	rs527636904	VUS	Conflicting interpretations of pathogenicity
					NM_002471.3:c.3427C>T	(p.Arg1143Trp)	HT	Missense	rs755209382	VUS	VUS
B13	19	M	MYH7	Familial CMP	NM_000257.4:c.481G>A	(p.Ala161Thr)	HM	Missense	rs552302426	VUS	NA
B14	39	F	DMD	Systolic heart failure	NM_004006.2:c.941G>T	(p.Arg314Leu)	HT	Missense	rs751164104	VUS	VUS
B15	38	M	PLEC	Left ventricular hypertrophy, arrhythmia in family	NM_000445.4:c.8701G>A	(p.Asp2901Asn)	HT	Missense	Novel rs397517701	VUS	NA
			TTN		NM_133378.4:c.68030G>A	(p.Arg22677Lys)	HT	Missense		VUS	Conflicting interpretations of pathogenicity
B16	41	F	JPH2	CMP	NM_020433.4:c.1714C>T	(p.Arg572Cys)	HT	Missense	rs377366285	VUS	VUS
B17	67	M	ACTN2	HCM	NM_001103.3:c.1961A>C	(p.Gln654Pro)	HT	Missense	Novel	VUS	NA
B18	3	M	SYNE2	CMP	NM_182914.2: c.12001_12 002delinsCA	(p.Trp4001Gln)	HT	Missense	rs373323825	VUS	NA
B19	58	M	FLNC	HCM	NM_001458.4:c.5161G>A	(p.Gly1721Arg)	HT	Missense	rs759786433	VUS	VUS
B20	34	F	DMD	Familial CMP	NM_004006.3:c.3850G>A	(p.Glu1284Lys)	HT	Missense	rs1557359192	VUS	VUS
			RYR2		NM_001035.3:c.9454C>T	(p.Arg3152Cys)	HT		rs181105904	VUS	VUS
B21	62	F	EYA4	HCM	NM_001301012.1:c.1305A>C	(p.Lys435Asn)	HT	Missense	Novel	VUS	NA
B22	49	F	RYR2	HCM	NM_001035.3:c.10345A>G	(p.Arg3449Gly)	HT	Missense	Novel	VUS	NA
B23	22	M	ADRB3	Syncope	NM_000025.3:c.286A>C	(p.Thr96Pro)	HT	Missense	Novel rs397517523	VUS	NA
			TTN		NM_001256850.1:c.26792C>A	(p.Thr8931Asn)	HT	Missense		VUS	VUS
B24	36	M	MYH7	HCM	NM_000257.4:c.3627C>A	(p.Asn1209Lys)	HT	Missense	Novel	VUS	NA
B25	49	F	DSC2	DCM, systolic heart failure	NM_004949.5:c.76A>G	(p.Ile26Val)	HT	Missense	Novel	VUS	NA
B26	61	F	DSG2	HCM	NM_001943.5:c.2001+3C>G		HT	Splice	rs746471051	VUS	Conflicting interpretations of pathogenicity

M, Male; F, Female; HGVS, human genome variation society; dbSNP, single nucleotide polymorphism database; LB, likely benign; VUS, variants of uncertain significance; NA, not available; HT, heterozygous; HM, homozygous.

MYBPC3 and MYH7 genes are the most frequent LP/P variants detected. The number of VUS and other variants in TTN, RYR2, and MYH7 genes is higher than in other genes in the cardiomyopathy panel (Qiagen, Hilden, Germany-CDHS-14644Z-5714).

The clinical findings of the cases in which we found the LP/P variant are HCM and familial CMP. In addition, in the case with a familial history NM_133378.4: c.78601_78602dup variant in TTN, mild pericardiac effusion was detected in the 2D echocardiography. Asymmetric septal hypotrophy was detected in the 2D echocardiography scan of the case with HCM indication, for which we detected the NM_000256.3:c.772G>A variant in MYBPC3. Our case with the NM_000363.4:c.532_534del variant in TNNI3 has an indication for RCM, and it was observed that the left atrium was wide and the pulmonary trunk diameter increased in cardiac MRI.

On the other hand, the case in which we detected novel NM_000117.3:c.123C>G pathogenic variant in EMD carries CMP and Emery Dreifuss indications. It was determined that a lead image was observed in the right heart cavity in the 2D echocardiography of this case. In the case who presented with Brugada syndrome and suspected CMP, NM_001035.3:c.10555-2A>C pathogenic variant in RYR2 was detected, and a trace amount of insufficiency was found in the tricuspid valve in 2D echocardiography. DCM and familial CMP are the indications of the majority of our cases in which we found a VUS. In the 2D echocardiography profile of individuals with NM_000257.3:c.4187G>A variant in MYH7 variant has an indication for DCM, the cardiac contraction was found to be decreased. 2D echocardiography and other screening test information of the cases in which we detected the LP/P variant are given in Table 4.

Table 4.

2D echocardiography and other screening test information of cases with LP/P variants

Sample ID	Age	Gender	Clinical findings	2D echocardiography results	Cardiac magnetic resonance imaging (MRI) results
1	17	F	RCM, pulmonary hypertension	Mild mitral regurgitation	Thickening of the pulmonary trunk diameter
2	5	M	HCM	Mild mitral regurgitation	No finding was detected in functional and morphological MRI
3	22	M	Brugada syndrome? CMP	Left ventricular hypertophy	No finding was detected in functional and morphological MRI
4	41	M	Emery Dreifuss? CMP	Tricuspid regurgitation	Less interventricular septum contraction
5	25	M	Brugada syndrome? CMP	Tricuspid regurgitation	No finding was detected in functional and morphological MRI
6	62	M	CMP	Left ventricular hypertrophy	No finding was detected in functional and morphological MRI
7	6	F	HCM	Asymmetric septal hypertrophy	Increased end diastolic septum wall
				Left ventricle diastolic dysfunction
				Mild mitral regurgitation
				Mild tricuspid regurgitation
8	69	F	Hypertrophic obstructive CMP	Mild mitral regurgitation	No MRI findings in favor of HCM
				Moderate pulmonary regurgitation
9	23	M	Familial CMP	Mild pericardiac effusion	Mild global hypokinesia in the left ventricle
10	28	F	HCM	Asymmetric septal hypertrophy	No MRI findings in favor of HCM
11	40	M	HCM	Left ventricle diastolic dysfunction	No MRI findings in favor of HCM
				Asymmetric septal hypertrophy
12	23	F	Familial CMP	Left ventricle hypertrophy	No finding was detected in functional and morphological MRI
13	72	F	HCM	Mild Mitral regurgitation	No MRI findings in favor of HCM
				Mild Aortic regurgitation
14	51	F	HCM	Asymmetric septal hypertrophy	No MRI findings in favor of HCM
				Mild mitral regurgitation
				Left ventricle hypertrophy
15	2	M	HCM	Asymmetric septal hypertrophy	Increased left ventricular wall thickness
					Increased interventricular septum thickness
16	62	M	HCM	Left ventricle hypertrophy	No MRI findings in favor of HCM
				Enlargement in right heart and left atrium
				Moderate mitral regurgitation
				Moderate tricuspite regurgitation
				Pulmonary hypertension
17	34	M	HCM	Moderate mitralregurgitation	No MRI findings in favor of HCM
				Left ventricle hypertrophy
				Dynamic left ventricle outflow obstruction
				Left verticuler diastolic disfunction
18	69	F	HCM	Mild mitral regurgitation	No MRI findings in favor of HCM

Discussion

CMPs are important in our country as well as in the world. Due to the complexity of terminology and different application methods, it is very difficult to know the statistical status of CMPs in the world and in Turkey. According to the World Health Organization (WHO) data, HCM and DCM are more common than RCM. Basically, it can be thought of as a classification according to the functional and morphological features that are most useful for clinical practice for CMP patients. In addition, the emergence of different CMPs caused by genetic variation in unrelated and related individuals is important for solving some clinical problems involving the evolution of one disease phenotype to another over time. The contribution rate to the clinical diagnosis was 18.44% in terms of the individual population who applied to our genetic center and were compatible with the CMP indication. Since the number of LP/P variants we detected compatible with the indication of 103 cases was 19, our contribution rate to the diagnosis is 18.44%.

Szabadosova et al. in 2016 noted that the first gene mutation (MHC *170260/major histocompatibility complex) that causes heart disease was reported in 1993 [15, 16]. After the heart diseases started to be defined, a variant was detected in the MYH7 gene as a result of the linkage analyses performed in a French Canadian family with clinical findings of HCM in 1990 [17].

Variations in genes that are effective in the emergence of CMP types differ. The genes for which we detected LP/P variants were MYBPC3, MYH7, TNNI3, EMD, RYR2, BRAF, TTN, TPM1, CAV3, and ABCC9. And the genes we detected novel variants were TTN, EMD, MYBPC3, and MYH7.

Variations in MYBPC3, MYH7, and TTN genes have clinical importance for HCM and DCM [18]. But in our study, the LP/P variant was detected in CAV3 (OMIM *601253/Caveolin 3) and ABCC9 (OMIM *601439/Atp-binding cassette, subfamily c, member 9) genes in 2 cases with HCM findings. CAV3 is the main protein component found in the extensions of the plasma membrane and plays a role in muscle development [19]. It has also been reported to be involved in the energy metabolism pathway and may be important for CMP in this respect [20]. ABCC9 is the gene associated with DCM in animal model studies [21] and is involved in ATP-sensitive potassium channels in the heart and skeletal muscle [22, 23].

We detected a TNNI3 (OMIM *191044/troponın I) pathogenic variant in our case, who presented with RCM findings. Although variations in the TNNI3 gene are generally associated with RCM findings, they are also found in individuals with findings such as DCM. Murphy et al. [24] (2004) found homozygous and missense variants in the TNNI3 gene in a study of 235 individuals with DCM findings. Since TNNI3 is one of the three subunits that make up the striated muscle filament complex, it has been associated with various heart diseases [25, 26]. Variations in the TNNI3 gene have been associated with various CMP subtypes. In a study, they stated that the TNNI3 gene is important in cardiac function and that the resulting variation may be related to RCM [27]. In addition, there are studies showing that variations in the TNNI3 gene may be related to different CMP profiles [28, 29].

In our study, we have identified a case with a pathogenic novel variant in the EMD (OMIM *300384/emerin) gene whose clinical finding was Emery Dreifuss. This inherited syndrome, which is a type of muscular dystrophy, may cause muscle weakness and life-threatening progressive CMP in the later years of the individual [30]. EMD gene communicates directly with the nucleoplasm in the nuclear membrane of the heart and skeletal muscle cells and maintains stabilization during contraction and relaxation [31]. With this function, it is important for CMP.

The genes with variants of uncertain clinical significance were TTN, RYR2, MYH7, SCN5A, MYL3, ACTN2, SDHA, DSC2, SYNE1, MYH1, SGCD, MYH6, PLEC, SYNE2, FLNC, EYA4, and DSG2. And the genes we have detected novel variants were TTN, ACTN2, MYH1, and PLEC. We reinvestigate variants every 6 months for other cases in which we have detected VUS.

On the other hand, we have detected a novel variant of uncertain significance in the EYA4 (OMIM *603550/Eya Transcriptional Coactivator and Phosphatase 4) gene in a case presenting with clinical findings of HCM. The EYA4 gene, which is defined as the transcription factor, has been mostly associated with DCM in studies [32]. Schonberger et al. [33] in 2005 found a large deletion in the EYA4 gene at position 6q23-q24 in a case with hearing loss and DCM finding.

The RYR2 gene is one of the main sources of calcium required for the contraction of the heart [34]. According to studies, defects in this gene are mostly associated with ventricular arrhythmia Benkusky et al. ARVD and ventricular tachycardia [35–37]. In our study, we detected 3 different VUS in the RYR2 gene, and related individuals had HCM clinical findings.

2D Doppler echocardiography is crucial for initial screening in individuals with heart disease [38]. Some studies emphasized that 2D echocardiography is not a diagnostic tool on its own in terms of CMP patients, and it may be useful to perform genetic tests for diagnosis [39, 40]. However, it is not an adequate method for diagnosing CMP cases by itself. In some CMP cases, which can be quite heterogeneous, additional techniques such as cardiac MRI can be applied. In our study, we have a case that supports this situation NM_000363.4:c.532_534del variant in TNNI3 was identified in our case with RCM; besides 2D echocardiography, cardiac MRI was also performed. A cardiac MRI revealed an increased diameter of the pulmonary trunk, larger than normal left atrium, and hypokinesia in the anterior wall of the left ventricle and septum. We performed a family segregation study to confirm the NM_000363.4:c.532_534del variant in TNNI3 in this case. As a result of the segregation analysis, we determined that the variant was de novo.

Due to the heterogeneous classification of CMPs and the diversity of clinical findings, different approaches can be applied according to the patient’s condition. In terms of CMP, the current condition of the patient and the early diagnosis of the disease are very important in terms of target-oriented treatment and the creation of treatment strategies. It is crucial to classify the detected variants correctly (Table 5).

Table 5.

Classification criteria of pathogenic variants according to ACMG-2015 criteria

Very strong	Strong	Moderate	Supporting
PVS1: Predicted null variant in a gene where LOF is a known mechanism of disease	PS4: prevalence in affected statistically increased over controls	PM1: mutational hot spot or well-studied functional domain without benign variation	PP1: cosegregation with disease in multiple affected family members
	PS1: same amino acid change as an established pathogenic variant	PM6: de novo without paternity and maternity confirmed	PP3: multiple lines of computational evidence support a deleterious effect on the gene/gene product
	PS3: well-established functional studies show a deleterious effect	PM2: absent in population databases	PP2: missenses common
	PS2: de novo paternity and maternity confirmed	PM5: novel missense change at an amino acid residue where a different pathogenic missense change has been seen before	PP4: patient’s phenotype or highly specific for gene
		PM4: protein length changing variant
		PM3: for recessive disorders, detected in trans with a pathogenic variant

In this context, NGS technologies are important for clinically important CMPs. Understanding the functionality of the disease is very important for clinically heterogeneous diseases such as CMP. Until a certain time, the complexity underlying genetic diagnosis of hereditary heart diseases has triggered this condition. The clinical overlap of variants detected in certain gene groups with CMPs has only recently become clear. One of the biggest contributions to the clarification of this situation is the active use of the NGS method. Because with this method, panels created with targeted gene groups for CMPs are studied.

A new era of diagnosis for CMPs has begun, thanks to significant advances in genetic studies and genotyping. Therefore, the prevalence of pathogenic variants in CMP-related target genes is important for our current population. Multiple gene groups associated with CMP’s can be screened through NGS, making significant contributions to the discovery of new locus for CMPs. The clinical significance was found to be 18.44% in terms of CMP patients who applied to our genetic center.

Statement of Ethics

Written informed consent forms were obtained from the individuals/legal guardians for publication of this research and any accompanying images. The article was approved by the Ethics Committee of Trakya University with the number of 2022/70.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

None.

Author Contributions

Concept and design, data collection, and/or processing: Hazal Sezginer Guler, Drenushe Zhuri, and Sinem Yalcintepe; resource: Hazal Sezginer Guler, Drenushe Zhuri, Sinem Yalcintepe, Selma Demir, Murat Deveci, Servet Altay, Hanefi Yekta Gurlertop, and Hakan Gurkan; materials: Hazal Sezginer Guler, Drenushe Zhuri, Engin Atli, Emine İkbal Atli, and Selma Demir; analysis and interpretation: Hazal Sezginer Guler, Sinem Yalcintepe, Murat Dveci, Hanefi Yekta Gurlertop, Servet Altay, and Hakan Gurkan; literature review: Hazal Sezginer Guler, Drenushe Zhuri, Engin Atli, and Emine İkbal Atli; writing manuscript: Hazal Sezginer Guler, Sinem Yalcintepe, Selma Demir, and Hakan Gurkan; critical reviews: Sinem Yalcintepe and Hakan Gurkan.

Funding Statement

None.

Data Availability Statement

The data supporting the findings of this study are available with the corresponding author upon request.