Genetic Screening and Double Mutation in Japanese Patients With Hypertrophic Cardiomyopathy

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1 Circulation Journal Official Journal of the Japanese Circulation Society ORIGIAL ARTICLE Molecular Cardiology Genetic Screening and Double Mutation in Japanese Patients With Hypertrophic Cardiomyopathy Toru Kubo; Hiroaki Kitaoka; Makoto Okawa; Yuichi Baba; Takayoshi Hirota; Kayo Hayato; aohito Yamasaki; Yoshihisa Matsumura; Haruna Otsuka; Takuro Arimura; Akinori Kimura; Yoshinori L. Doi Background: Hypertrophic cardiomyopathy (HCM) is a primary myocardial disorder with an autosomal-dominant pattern of inheritance mainly caused by single heterozygous mutations in sarcomere genes. Although multiple gene mutations have recently been reported in Western countries, clinical implications of multiple mutations in Japanese subjects are not clear. Methods and Results: A comprehensive genetic analysis of 5 sarcomere genes (cardiac β-myosin heavy chain gene [MYH7 ], cardiac myosin-binding protein C gene [MYBPC3 ], cardiac troponin T gene [TT2 ], α-tropomyosin gene [TPM1 ] and cardiac troponin I gene [TI3 ]) was performed in 93 unrelated patients and 14 mutations were identified in 28 patients. Twenty-six patients had single heterozygosity (20 in MYBPC3, 4 in MYH7, 1 in TT2, 1 in TI3 ), whereas 2 proband patients with familial HCM had double heterozygosity: 1 with P106fs in MYBPC3 and R869C in MYH7 and 1 with R945fs in MYBPC3 and E1049D in MYH7. From the results of the family survey and the previous literature on HCM mutations, P106fs, R945fs and R869C seemed to be pathological mutations and E1049D might be a rare polymorphism. The proband patient with P106fs and R869C double mutation was diagnosed as having HCM at an earlier age (28 years of age) than her relatives with single mutation, and had greater wall thickness with left ventricular outflow obstruction. Conclusions: One double mutation was identified in a Japanese cohort of HCM patients. Further studies are needed to clarify the clinical significance of multiple mutations including phenotypic severity. (Circ J 2011; 75: ) Key Words: Double mutation; Genetic screening; Hypertrophic cardiomyopathy; Sarcomere protein genes Hypertrophic cardiomyopathy (HCM) is a primary myocardial disorder with heterogeneous morphologic, functional, and clinical features. 1 5 Recent molecular genetic studies have shown that HCM is mainly caused by single heterozygous mutations in genes for sarcomere proteins. 6 8 There is a wide allelic heterogeneity, given that numerous mutations have been identified. Although the mechanisms by which disease-causing mutations result in the characteristic pathological and morphologic features of HCM are unclear, preliminary genetic studies on HCM have suggested that particular gene abnormalities are associated with specific clinical phenotypes such as degree of hypertrophy, risk of sudden death, onset time of the disease, and disease penetrance in families. 6 8 Recently, multiple gene mutations have been reported in Western countries, which may further contribute to the disease heterogeneity There has been no report, however, on multiple mutations in Japanese HCM patients, and the clinical implications of double mutations are unclear. Here, we present the results of genetic analysis including the identification of a double mutation in the present cohort of HCM patients. Methods Subjects The subjects were 93 patients with familial or sporadic HCM. Twenty-seven patients had a family history of HCM and the other 66 subjects were not confirmed to have relatives with HCM. The diagnosis of HCM was based on echocardiography showing unexplained left ventricular hypertrophy (LVH), that is, maximum left ventricular wall thickness (MLVWT) 15 mm. Relatives of the proband patients were contacted by the probands themselves and visited the clinic of their own free will. Following the identification of mutations, pedigree analysis, including both clinical evaluation and genotyping, was performed. Informed consent was given by all subjects or their Received January 11, 2011; revised manuscript received June 4, 2011; accepted June 17, 2011; released online July 29, 2011 Time for primary review: 12 days Department of Medicine and Geriatrics, Kochi Medical School, ankoku (T.K., H.K., M.O., Y.B., T.H., K.H.,.Y., Y.M., Y.L.D.); Department of Molecular Pathogenesis, Medical Research Institute, Tokyo Medical and Dental University, Tokyo (H.O., T.A., A.K.), Japan Mailing address: Hiroaki Kitaoka, MD, Department of Medicine and Geriatrics, Kochi Medical School, Oko-cho, ankoku , Japan. kitaokah@kochi-u.ac.jp ISS doi: /circj.CJ All rights are reserved to the Japanese Circulation Society. For permissions, please cj@j-circ.or.jp

2 Genetic Screening and Double Mutation in HCM 2655 Table 1. Identified Mutations in Japanese HCM Patients o. Gene Mutation Mutation type o. probands Mutation publication status 1 MYBPC3 P106fs Frameshift mutation 1 ovel 2 MYBPC3 S297X onsense mutation 2 Previously published 15 3 MYBPC3 H379P Missense mutation 1 ovel 4 MYBPC3 V593fs Frameshift mutation 15 Previously published 16 5 MYBPC3 G805S Missense mutation 1 ovel 6 MYBPC3 R945fs Frameshift mutation 2 Previously published 17,18 7 MYH7 R243C Missense mutation 1 ovel 8 MYH7 562K Missense mutation 1 ovel 9 MYH7 R663C Missense mutation 1 Previously published MYH7 R869C Missense mutation 1 Previously published MYH7 E1049D Missense mutation 1 ovel 12 MYH7 T1929M Missense mutation 1 Previously published TT2 D46V Missense mutation 1 ovel 14 TI3 R162W Missense mutation 1 Previously published 21 HCM, hypertrophic cardiomyopathy; MYBPC3, cardiac myosin-binding protein C gene; MYH7, cardiac β-myosin heavy chain gene. a not investigated mutation absent, clinically unaffected mutation present, clinically affected mutation present, clinically unaffected deceased, not investigated sudden death, not investigated proband patient b H020 I II + / - + / / / / + + / / / - - / - - / - III c I II III + / / - - / - - / - + / / - + / / - - / - - / - + / + 2 H053 I II Genotypes P106fs in MYBPC3/ R869C in MYH7 H085 + / - 1 Genotypes R945fs in MYBPC3/ E1049D in MYH7 Figure. (a) Genotype and phenotype symbols. (b) Pedigree of family H020. +/+, presence of heterozygous P106fs in MYBPC3 and presence of heterozygous R869C in MYH7; +/, presence of heterozygous P106fs in MYBPC3 and absence of heterozygous R869C in MYH7; /+, absence of heterozygous P106fs in MYBPC3 and presence of heterozygous R869C in MYH7; /, absence of heterozygous P106fs in MYBPC3 and absence of heterozygous R869C in MYH7. (c) Pedigree of families H053 and H085. +/+, presence of heterozygous R945fs in MYBPC3 and presence of heterozygous E1049D in MYH7; +/, presence of heterozygous R945fs in MYBPC3 and absence of heterozygous E1049D in MYH7. MYBPC3, cardiac myosin-binding protein C gene; MYH7, cardiac β-myosin heavy chain gene.

3 2656 KUBO T et al. Table 2. Clinical Characteristics of Patients With a P106fs Mutation in MYBPC3 and/or R869C in MYH7 at Initial Evaluation (H020 Family) Patient no. Mutation Gender Age at diagnosis (years) Rhythm LVH phenotype MLVWT LVEDD FS (%) LVOTO H020-II-1 P106fs F 69 SR H020-II-2 P106fs M 67 SR H020-II-3 P106fs M 65 AF H020-II-4 P106fs M 63 SR H020-II-5 R869C M 75 Pacemaker H020-II-6 P106fs F 62 SR H020-II-7 P106fs M 60 SR H020-II-8 P106fs M 56 SR H020-III-1 P106fs and F 22 SR R869C H020-III-2 P106fs F 40 SR H020-III-3 P106fs F 37 SR Age at last evaluation; these patients were phenotype negative (no LVH). LVH, left ventricular hypertrophy; MLVWT, maximum left ventricular wall thickness; LVEDD, left ventricular end-diastolic diameter; FS, fractional shortening; LVOTO, left ventricular outflow tract obstructio; SR, sinus rhythm; AF, atrial fibrillation. Other abbreviations see in Table 1. parents in accordance with the guidelines of the Ethics Committee on Medical Research of Kochi Medical School. Clinical Evaluation Evaluation of proband patients and relatives included history, clinical examination, 12-lead electrocardiography, M-mode, 2-D and Doppler echocardiography, and ambulatory 24-h Holter electrocardiogram analysis. The severity and distribution of LVH were assessed in the parasternal short-axis plane at the mitral valve and papillary muscle levels. MLVWT was defined as the greatest thickness in any single segment. Left ventricular end-diastolic diameter (LVEDD) and end-systolic diameter (LVESD) were measured from M-mode and 2-D images obtained from parasternal long-axis views, and fractional shortening (%FS = (LVEDD LVESD) / LVEDD 100) was calculated. LV outflow tract gradient was calculated from continuous-wave Doppler using the simplified Bernoulli equation. Significant basal LV outflow tract obstruction was defined as pressure gradient 30 mmhg at rest on echocardiography. Genetic Analysis Peripheral blood samples were taken at the time of clinical evaluation, and they were frozen and stored at 20ºC. DA was extracted using a DA purification kit from Qiagen (no ; Hilden, Germany). In vitro amplification of genomic DA was performed using polymerase chain reaction (PCR). Oligonucleotide primers were used to amplify protein-encoding exons of the 5 most common sarcomere protein genes: cardiac β-myosin heavy chain gene (MYH7), cardiac myosinbinding protein C gene (MYBPC3), cardiac troponin T gene (TT2), α-tropomyosin gene (TPM1) and cardiac troponin I gene (TI3). Information on primer sequences and PCR conditions is available upon request. Direct sequencing was performed in all samples and the sequencing data were analyzed on an ABI PRISM 3100-Avant Genetic Analyzer in accordance with the manual of the manufacturer. In patients in whom mutation was identified, confirmation was obtained by re-analysis with direct sequencing from a second blood sample. Results Fourteen putative HCM-susceptibility mutations were identified in 28 patients: P106fs, S297X, H379P, V593fs, G805S, R945fs in MYBPC3, R243C, 562K, R663C, R869C, E1049D, T1929M in MYH7, D46V in TT2, and R162 W in TI3 (Table 1) These were not found in at least 200 chromosomes from healthy Japanese individuals. Eighteen (67%) of 27 proband patients with familial HCM had mutations. Even though 66 patients did not have confirmation of familial HCM, 10 (15%) of them had mutations. In 28 patients with mutations, 26 had single heterozygosity (20 in MYBPC3, 4 in MYH7, 1 in TT2, 1 in TI3) and the other 2 had double heterozygosity of MYBPC3 and MYH7. To identify if these genetic variants were pathological mutations, we performed the family survey and referred to the previous literature on HCM mutations. Pathological implication of H379P, G805S in MYBPC3 and R243C, 562K, E1049D in MYH7 was unclear, although some of these variants may be identified as disease-causing mutations in the future. One double-mutation case involved P106fs in MYBPC3 and R869C in MYH7 (Figure). The proband patient (H020-III-1; Figure) was identified as having inherited double mutations, P106fs in MYBPC3 from her mother and R869C in MYH7 from her father. Many of her mother s siblings had P106fs in MYBPC3. Her father, who had R869C in MYH, had pacemaker implantation for complete atrioventricular block but did not have LV hypertrophy. Other relatives of her father could not be evaluated. Table 2 lists the clinical characteristics of this family. Patient H020-III-1 with double mutations was diagnosed as having HCM at an earlier age (28 years old) than her relatives with single mutations. In contrast, her cousins (H020-III-2 and H020-III-3) with 1 mutation in MYBPC3 had no hypertrophy at the ages of 40 and 37 years. The double-mutation patient had greater wall thickness with left ventricular obstruction on echocardiography, although other relatives carrying the single MYBPC3 mutation did not have obstruction. Figure also shows a family pedigree of another proband patient with double heterozygosity (H053 family). Two siblings (H053-III-1 and H053- III-2) had R945fs frameshift mutation in MYBPC3 and E1049D in MYH7. Although we did not have an opportunity to evaluate

4 Genetic Screening and Double Mutation in HCM 2657 Table 3. Clinical Characteristics of Patients With an R945fs Mutation in MYBPC3 and/or E1049D in MYH7 at Initial Evaluation (H053 Family and H085 Family) Patient no. Mutation Gender H053-III-1 H053-III-2 R945fs and E1049D R945fs and E1049D Age at diagnosis (years) Rhythm LVH phenotype MLVWT LVEDD FS (%) LVOTO F 55 SR M 48 SR H085-II-1 R945fs F 63 SR Abbreviations see in Tables 1,2. Table 4. Mutations in MYH7 Exons Coding the -Terminal Region of the Rod Amino acid position Conservation of cardiac β-myosin heavy chain amino acid residues Human E K S E A R R K E L E V R M D L E R A K R K Family H020 E K S E A C R K E L E V R M D L E R A K R K Family H053 E K S E A R R K E L E V R M D L D R A K R K Rat E K S E A R R K E L E V R M D L E R A K R K Mouse E K S E A R R K E L E V R M D L E R A K R K Hamster E K S E A R R K E L E V R M D L E R A K R K Rabbit E K S E A R R K E L E V R M D L E R A K R K Pig E K S E A R R K E L E V R M D L E R A K R K Abbreviations see in Table 1. the genotype and phenotype in the relatives, 3 relatives died suddenly. Another patient (H085-II-1) had the same MYBPC3 mutation (R945fs), while there was no other patient with E1049D in MYH7. From the results of the family survey and the previous literature on HCM mutations, we finally concluded that E1049D was a variant of uncertain effect. Clinical characteristics of the patients with R945fs mutation are listed in Table 3. One of the double-heterozygosity patients (H053- III-2) was diagnosed as having HCM at a younger age than the patient with a single R945fs mutation and died at the age of 62 years due to heart failure. In contrast, his sister with the same double heterozygosity (H053-III-1) has been clinically stable. Discussion HCM is a primary myocardial disorder marked by phenotypic and genotypic heterogeneity. 1 5 More than 200 different gene mutations have been reported for HCM, and the disease is usually caused by single heterozygous mutations in the gene encoding for sarcomere proteins (which is an autosomal-dominant pattern of inheritance). 6 8 In the present study, mutational analysis of a rural cohort (Kochi Prefecture in Japan) of unrelated patients derived from a single institution and comprehensive screening of the 5 most common disease genes in HCM were carried out. One remarkable characteristic of the present cohort was that an identical mutation (V593fs in MYBPC3) was found in 15 of 94 unrelated patients, and this mutation was likely to have arisen from a common ancestor (founder effect) according to the haplotype analysis. 16 Such a high frequency of identical disease-causing mutations in an HCM cohort is uncommon. This unique situation might have been caused by both the geographic features of Kochi Prefecture and the nature of this mutation. First, Kochi Prefecture is located in the southwestern part of Japan and is far from a central urban area. Kochi Prefecture is often isolated by bad weather conditions because it is surrounded by mountains on 3 sides and faces the Pacific Ocean. Second, the onset of HCM caused by this mutation (V593fs in MYBPC3) tended to be late in life, although in 2 exceptional cases the patients were diagnosed as having the disease as teenagers. 16 This mutation may therefore have been easily passed on to the next generation. Although there have been several studies showing multiple mutations in HCM patients in Western countries, to the best of our knowledge, this is the first report of a double mutation in Japanese HCM patients We investigated the clinical findings of patients carrying the double heterozygosity (P106fs and R945fs in MYBPC3 and R869C and E1049D in MYH7) and their relatives to clarify whether the mutations were disease causing or not. P106fs and R945fs in MYBPC3 mutations were predicted to result in a truncation of the protein, including loss of C-terminal myosin and titin binding sites. These 2 mutations were thought to be disease causing based on the presence of the mutations in all affected individuals (P106fs and R945fs) and the appearance of the same mutation in 2 unrelated patients (R945fs in H053 and H085 families). As for the 2 variants (R869C and E1049D) in MYH7, Table 4 lists amino acid residues among different species in cardiac β-myosin heavy chain. These 2 were located in the evolutionarily conserved regions. It has been reported that 4 individuals in 1 HCM family had R869C in MYH7, but there is no report for the mutation E1049D. 20 These observations suggest that R869C in MYH7 seems to be a pathological mutation. In contrast, the pathological implication of E1049D was not clear due to the lack of information about the clinical phenotypes of carriers with single E1049D variant. We found a double mutation in 1 Japanese HCM patient, which was heterozygous in 2 different genes. In previous reports, other types of multiple mutations have been described The reported multiple mutations include compound mutations, which are different mutations in the same gene on each chromosome, and homozygous mutations, which are the same

5 2658 KUBO T et al. mutations on both chromosomes. Recent studies in which comprehensive sequencing of sarcomere genes was carried out have found multiple mutations in 2 5% of subjects in whole cohorts and in 5 17% of subjects carrying the diseasecausing mutations, and MYBPC3 and MYH7 mutations were the most frequent in multiple mutations as well as in single mutations The present data are in accordance with the findings for HCM in Western countries. Furthermore, in the present study, clinical features of a patient with double pathological mutation (H020-III-1) were more severe in terms of onset of disease and clinical presentation than those of the single mutations. Previous studies on multiple mutations in HCM have found similar clinical phenotypes such as diagnosis at an early age, more significant hypertrophy, and requiring more advanced treatment A double dose mutation effect may be related to the severe phenotypes in these patients. The double-mutant murine model of HCM (Gly203Ser cardiac troponin I mutation and Arg403Gln α-myosin heavy chain mutation) has been reported to generate a severe cardiac phenotype characterized by heart failure and early death. 22 HCM genetic testing is now in a transitional period from the research stage to the clinical arena, and the occurrence of multiple mutations in some HCM patients has led to complicated situations in genetic testing and counseling. At present, it is difficult to distinguish patients with single heterozygous mutation from patients with multiple mutations in terms of their clinical phenotypes, even though patients with multiple mutations tend to have more severe symptoms and clinical presentations. Therefore, the consequence for genetic testing is that the mutation screening should not be stopped after identification of 1 mutation. The large number of genes that need to be examined in each patient continues to limit the efficacy of a gene-based diagnosis. Given the large genetic heterogeneity of the disease, development of new strategies for genetic testing is required. For genetic counseling, an explanation of the inheritance of the disease and the assessment of risk for children become more complicated. For example, in general, each child of HCM patients with a single mutation has a 50% chance of inheriting the disease. In contrast, if a patient has double mutations such as those found in the present study, the risk that a child will receive at least 1 mutation becomes 75%. Furthermore, family members who do not carry the causative single mutation are not completely relieved from the anxiety of developing the disease later because of the possible presence of a second mutation. For the translation of genetic research into practical clinical applications and routine clinical strategy, it is important to establish a worldwide genetic database of this disease in order to clarify the actual disease-causing mutations (not all genetic variations, even non-synonymous mutations, cause HCM), the genotype phenotype correlations in each mutation, and the prevalence and distribution of multiple mutations in HCMcausing genes. There were several limitations to the present study. First, the effects of R869C and E1049D in MYH7 in these families are not clear because we did not have enough family survey data of the variant carriers (single R869C and single E1049D) to confirm that these variants in MYH7 were disease causing. R869C has been reported to be associated with HCM although the phenotype of the carrier of R869C (H020-II-5) in the present study was different from the reported phenotype. 20 R869C may be a low-penetrance mutation. In contrast, E1049D might be a rare polymorphism because we failed to confirm it as a pathological mutation and there have been no studies on this variant in HCM gene abnormalities. Second, we performed a comprehensive genetic analysis of only 5 sarcomere genes (MYBPC3, MYH7, TT2, TPM1, TI3). Other genes such as ventricular myosin essential light chain gene and cardiac α-actin gene might be associated with multiple mutations. The 2 major genes in HCM patients, however, appear to be the MYBPC3 and MYH7. Mutations of these 5 genes account for >80% of genotyped HCM cases and almost all multiple mutations previously reported were combinations of these 5 gene mutations. Third, there was only 1 patient with double pathological mutation in the present study. Further analysis with larger numbers of patients is required to clarify the effects of multiple mutations in clinical phenotypes. In conclusion, 1 double mutation was identified in a Japanese cohort of HCM patients, which indicates the importance of continuance of screening even after a single mutation has been identified. Further studies are needed to clarify the clinical significance of multiple mutations including phenotypic severity in Japanese HCM patients. Disclosure one of the authors has a conflict of interest. References 1. Spirito P, Seidman CE, McKenna WJ, Maron BJ. The management of hypertrophic cardiomyopathy. Engl J Med 1997; 336: Maron BJ, McKenna WJ, Danielson GK, Kappenberger LJ, Kuhn HJ, Seidman CE, et al. American College of Cardiology/European Society of Cardiology clinical expert consensus document on hypertrophic cardiomyopathy: A report of the American College of Cardiology Foundation Task Force on Clinical Expert Consensus Documents and the European Society of Cardiology Committee for Practice Guidelines. J Am Coll Cardiol 2003; 42: Elliott P, McKenna WJ. Hypertrophic cardiomyopathy. 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