MOLECULAR-GENETIC PROFILE IN PATIENTS WITH CARDIOMYOPATHY IN BULGARIA

. Introduction : Cardiomyopathies are a clinically and genetically heterogeneous group of diseases, that are associated with significant morbidity and mortality. The aim of the present study is to clarify the molecular-genetic characteristics of cardiomyopathies in patients in Bulgaria. Material and methods : In the present study, targeted analysis of an expanded panel of 242 genes, associated with cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease with a prevalence of 1:500 to 1:200 in the general population [3,4].It is distributed in countries around the world and affects different ethnic groups, races and both sexes equally, with no significant differences in genetic mutations аndphenotypic expression of the disease depending on the demographic characteristics of patients established [5].HCM is characterized by a complex pathophysiology and is caused by pathogenic variants in at least 30 genes, encoding proteins of the cardiac sarcomere and other proteins, and in a proportion of patients no specific mutation is identified [6,7].More than 2,000 variants, associated with HCM have been reported in literature, while the frequency of occurrence of spontaneous or de novo mutations remains unclear [5,8].Inheritance of HCM is defined as autosomal dominant, but in rare cases autosomal recessive, X-linked or mitochondrial inheritance is possible [9].Pathogenic variants in the MYH7 (β-myosin heavy chain) and MYBPC3 (myosin binding protein C) genes are found with the highest frequency in patients with genetically clarified disease -in approximately 75-84% [10].Mutations in the troponin T2 (TNNT2) gene are reported in 5-15% of cases, in the troponin I gene (TNNI3) in 5%, and in the α-tropomyosin gene (TPМ1) in 3% of patients with HCM.Pathogenic mutations have also been identified in the genes MYL2, LBD3, MYL3, ACTC1 and in other genes, respectively with a lower frequency, while genocopies and phenocopies represent hereditary and non-hereditary diseases that account for 5 to 10% of HCM cases.
Arrhythmogenic cardiomyopathy (ACM) is a progressive, inherited disease of the myocardium, which is a leading cause of ventricular arrhythmia and sudden cardiac death (SCD) in young adults [17].ACM can affect primarily the right ventricle (ARVC), primarily the left ventricle (ALVC), or both ventricles of the heart.The prevalence of ACM is estimated to be 1:5000 and higher in cohorts of Italian and German origin (1:2000) [18].In approximately 50% of ACM patients, pathogenic variants are found in the desmosomal genes, including PKP2, DSP, DSG2, DSC2, and JUP, and mutations in the PKP2 gene, encoding the plakophilin-2 protein, have been reported most commonly -in 20-45% of patients [19].ACM is most often inherited in an autosomal dominant pattern, while autosomal recessive forms are rare and are mainly associated with the cardiocutaneous syndromes of Naxos and Carvajal, which are caused by homozygous mutations in the JUP and DSP genes, respectively [17,20].Pathogenic mutations in the DES, FLNC, PLN, SCN5A and TMEM43 genes have also been reported in patients with ACM [20].
Restrictive cardiomyopathy (RCM) is a rare cardiomyopathy that may result from genetic, non-genetic causes, or be associated with systemic diseases [19].Inheritance of RCM may be autosomal dominant, autosomal recessive, X-linked, or mitochondrial.The most commonly detected mutations in patients with RCM are in the sarcomeric and cytoskeletal genes, including MYH7, TNNI3, TNNT2, ACTC1, TTN, and FLNC.It is possible that relatives in a family with familial RCM exhibit different phenotypes, such as HCM in the presence of pathogenic variants in the MYH7 or TNNI3 genes, including atrioventricular block and/or skeletal myopathy [24].Data in patients with RCM indicate that genetic findings are likely to be established in up to 60% of cases [19].
Current clinical guidelines recommend genetic counseling and genetic testing as an indispensable part of the complex approach in contemporary care for patients with cardiomyopathy and their families [6,19,[25][26][27][28].Genetic testing in clinical practice is key to confirming the diagnosis in clinical uncertainty, and the identified genetic variants may facilitate cascade screening to identify close relatives at risk of developing the disease or exclude this risk in others in accordance with up-to-date clinical recommendations.
At present, to the best of our knowledge, no pooled data on the molecular-genetic characteristics of patients with cardiomyopathy in the Bulgarian population has been published.

Materials and methods
The study was approved by the Ethics Committee of the Medical University of Sofia, Protocol No 17/16.12.2022.Written informed consent was obtained from the patients included in the study or their guardians, as well as from all relatives tested.
For the purpose of this study, DNA from venous blood of 20 Bulgarian patients, diagnosed with cardiomyopathy, was isolated using the salting-out method.Assessment of the quality of the extracted DNA was carried out by direct spectrophotometry.
Whole exome sequencing (WES) was conducted in the partner laboratory "Admera Health, LLC", USA.The analysis of the whole exome sequencing data was performed with the specialized software GensearchNGS, PhenoSystems SA, using an expanded panel of 242 genes, associated with cardiomyopathy, and an additional target panel of 20 genes, associated with hereditary amyloidosis, in two of the patients.
The target regions of the human genome, where the genetic variants were found during the whole exome sequencing, were multiplied by polymerase chain reaction.The amplified fragments were sequenced using Sanger's method with BigDye® Terminator v.3.1 sequencing kit (Applied Biosystems, Foster City, CA, USA).Electrophoretic separation of sequence products was performed via an ABI Prism 3130 Sequence Genetic Analyzer sequencer.The obtained data were processed automatically by the program ABI3130 Data Collection Software and received in the form of an electrophoregram with Sequencing Analysis software v.5.1.1.
The interpretation of the detected genetic variants was performed according to the classification criteria of the guidelines of the American College of Medical Genetics and Genomics/Association of Molecular Pathology (ACMG/AMP), taking into account the patient's clinical manifestations and the results of the segregation analyses conducted in the affected families [29].
Only in the patients with RCM, no genetic findings related to the manifested clinical symptoms were identified (10%).Pathogenic/likely pathogenic variants were found in ~58% of the patients with HCM and in ~83% of the patients with DCM.The estimated frequency of likely pathogenic/pathogenic variants in the study was ~32% of all variants found, with the frequency in patients with HCM and DCM being 28% and ~38%, respectively.
The results about the frequency of pathogenic/likely pathogenic variants and variants with uncertain significance, only of the pathogenic/likely pathogenic variants in the studied patient group, and also the results of the analyses in the subgroups of patients with HCM and DCM are presented in Figure 1.The results about the frequency of the genetic variants (all pathogenic/likely pathogenic variants and variants with uncertain significance) found in the Bulgarian patients with cardiomyopathy show that the most commonly affected genes were the sarcomeric genes (TTN, MYBPC3, TNNI3, MYH7, MYH6) -in half of the cases, ion channel genes (SCN5A, KCNA5, KCNH2, CACNA1C), cytoskeletal genes (LAMA4, ILK, FLNC), Z-disc genes (TCAP, MYPN), genes for mitochondrial proteins (TAZ, NDUFB11), as well as genes for cell-adhesion protein (CDH2), desmosome structures (DSP), cell membrane protein (ANK2) and for specific receptor molecules (LDLR, IL31RA).

Тип ЛК хипертрофия
На фиг. 3 са представени резултатите от проведените сегрегационни анализи при 6 от засегнатите семейства в настоящото проучване.Pathogenic/likely pathogenic variants were found with the highest frequency in the MYBPC3 gene (50%), followed by the findings in the titin gene, the β-myosin heavy chain, troponin I, filamin C, tafazzin and the NDUFB11 protein (8% each) (Figure 2).The results of the analysis of the frequency of genetic findings in the subgroup of patients with HCM showed that 55% of the identified pathogenic/likely pathogenic variants and variants with uncertain significance, and 100% of the identified pathogenic/likely pathogenic variants affected the sarcomeric genes.In the subgroup of patients with DCM, pathogenic/likely pathogenic variants and variants with uncertain significance were most commonly found in the TTN gene (31%) and SCN5A gene (15%).Pathogenic/likely pathogenic variants in the DCM patients were found in genes encoding proteins involved in the sarcomeric and cytoskeletal structure, as well as mitochondrial function.
The results of the segregation analyses conducted in 6 of the affected families in the present study are showed in Figure 3.
The presence of two or three pathogenic mutations in one or more genes in patients with HCM was rarely reported -in 5% and 0.8% of cases, respectively, while the data from a study of European patients with sporadic or familial DCM, shows that a large proportion of patients were double heterozygotes in one (7%) or in different genes (38%), and that 12.8% of patients carried 3 or more variants [35][36][37].None of the patients in the study was found to have more than 1 pathogenic/likely pathogenic genetic variant, which may be due to the small number of patients studied.In accordance with literature data, the variants found in Bulgarian patients with cardiomyopathy represent rare variants that are found in single patients or families [9,31,38].
Approximately 90% of the pathogenic mutations that cause HCM, according to literature data, are missense mutations [9].Insertions/deletions and frameshift mutations have been reported in the MYBPC3 gene, as well as rare cases of deletions in the MYH7 and TNNT2 genes.In a study, conducted by Norton et al., it was reported that ~90% of variants found in patients with familial or sporadic DCM were single nucleotide substitutions (93% missense, 5% nonsense, and 2% splice site variants), and ~10% of them were insertions/deletions with a size of 1 to 4 bases leading to a frameshift in the coding sequences in 86% of cases [38].The distribution by variant type in the present study shows that a respectively 2,5 and 4-fold higher frequency of truncating variants was found compared to the frequency reports in the literature, both in patients with HCM (~26%), and in patients with DCM (~39%).Despite this high rate, missense mutations remain predominant, being found twice as often in both patient subgroups.While data from a large study of 4,756 HCM patients showed no statistically significant difference in disease phenotype, severity, and adverse events according to the MYBPC3 gene mutation type, clarification of mutation type may have important clinical implications in certain patients with DCM in terms of assessing the risk of arrhythmias [39].Truncating mutations in the filamin C gene with a high penetrance have been reported in families with arrhythmogenic DCM and a high incidence of SCD [40,41].In a large study of 1150 patients with cardiomyopathy (700 HCM, 300 DCM, 50 RCM and 100 LVNC), truncating mutations in the FLNC gene were found only in DCM patients, whereas missense or in-frame insertions/deletions were associated with the other phenotypes.Carriers of truncating mutations had a При пациентите с ХКМП в нашата група, с най-висока честота се откриват патогенни/вероятно патогенни варианти в MYBPC3 гена (71%), а ДКМП се характеризира с разнообразен генетичен профил, като данните показват, че TTN генът се засяга в 20% от случаите, което най-общо е в съответствие с докладваните литературни данни [10,11].Разликите в честотите в сравнение с литературните източници за останалите установени патогенни/вероятно патогенни находки може да се обясни с относително малкия брой изследвани пациенти в двете подгрупи.
Pathogenic/likely pathogenic variants in the MYBPC3 gene (71%) were found in our HCM patients with the highest frequency, and DCM is characterized by a diverse genetic profile, with the data showing that the TTN gene being affected in 20% of cases, which is generally in agreement with reported literature data [10,11].The differences in frequencies compared to literature sources for other detected pathogenic/likely pathogenic findings may be explained by the relatively small number of patients studied in both of our subgroups.
Interestingly, the results of family segregation analysis in patient #10, diagnosed with HCM confirmed the presence of the variant c.2791_2793del in the MYH7 gene in a heterozygous state in his mother with LVNC.
It is important to note that in the subgroup of DCM patients, genetic findings in NDUFB11 and TAZ genes, encoding proteins with a specific role in mitochondrial metabolism, were associated with severe clinical presentation in pediatric patients in the first days of the postnatal period.In this regard, genetic testing and elucidating of the genetic diagnosis in these patients may be of key importance in determining the course and prognosis of the disease, clinical management and therapeutic strategy, as well as the risk of inheritance in the family.
In conclusion, the current study provides the first pooled data regarding the molecular-genetic profile of cardiomyopathy patients in Bulgaria.The results obtained from whole exome sequencing and the performed molecular-genetic analysis in 20 Bulgarian patients with cardiomyopathy show the presence of genetic findings in 90% of them.Pathogenic/likely pathogenic variants were found in more than half of patients with HCM with no family history of SCD or cardiomyopathy, in ~67% of patients with HCM with family history or with sporadic DCM, as well as in 100% of DCM patients with a positive family history, and genetic findings related to the manifested clinical symptoms were not identified in the patients with RCM in the study.Clarification of the type of mutation may have important clinical implications in certain patients with DCM in terms of assessing the risk of arrhythmias.Pathogenic/likely pathogenic variants in the myosin-binding protein C gene were found with the highest frequency in patients with HCM, while DCM is associated with genetic findings in different genes.The genetic variants, found in the NDUFB11 and TAZ genes, were associated with severe clinical presentation in the early days of the postnatal period.Results of segregation ниране по Sanger са докладвани в 6 от засегнатите семейства.Основно ограничение на изследването представлява малкия брой включени и анализирани пациенти.Данните от проведеното проучване са в подкрепа на приложението на генетични изследвания и медико-генетично консултиране при пациентите и засегнатите семейства с кардиомиопатия в България.analyses performed by direct Sanger sequencing were reported in 6 of the affected families.A major limitation of the study is the small number of included and analyzed patients.The data from the study supports the importance of conducted genetic testing and medical-genetic counseling in patients and affected families with cardiomyopathy in Bulgaria.

Фиг. 1 .Fig. 1 .Фиг. 2 .Fig. 2 .
Fig. 1.Frequency of pathogenic/ likely pathogenic variants and variants of uncertain significance, as well as pathogenic/likely pathogenic variants in the studied patients.The results regarding the frequency of pathogenic/likely pathogenic variants and variants of uncertain significance (Panel A.1, Panel B.1 and Panel C.1) and pathogenic/likely pathogenic variants (Panel A.2, Panel B.2 and Panel C.2) in all studied patients are presented in Panel A, in the subgroup of patients with hypertrophic cardiomyopathy in Panel B, and in the subgroup of patients with dilated cardiomyopathy in Panel C

3 .Fig. 3 .
Fig. 3. Results of the segregation analyses performed in the studied patients.The results of the segregation analyses performed in patients № 10, 11, 12, 15, 17 and 18, studied within the framework of the research, are presented sequentially

Таble 6. Genetic variants, found in the studied patients.
Genetic variants that are underlined in the table are newly discovered in the study (not reported in ClinVar or in the literature)