Compound heterozygosity deteriorates phenotypes of hypertrophic cardiomyopathy with founder MYBPC3 mutation: evidence from patients and zebrafish models

13301甲第4178号博士（医学）金沢大学博士論文要旨Abstract 要約Outline 以下に掲載：American Journal of Physiology Heart and Circulatory Physiology 307(11) pp.H1594-H1604 2014. American Physiological Society. 共著者：Akihiko Hodatsu, Tetsuo Konno, Kenshi Hayashi, Akira Funada, Takashi Fujita, Yoji Nagata,Noboru Fujino, Masa-aki Kawashiri, Masakazu Yamagish

Impact of functional studies on exome sequence variant interpretation in early-onset cardiac conduction system diseases

Aims The genetic cause of cardiac conduction system disease (CCSD) has not been fully elucidated. Whole-exome sequencing (WES) can detect various genetic variants; however, the identification of pathogenic variants remains a challenge. We aimed to identify pathogenic or likely pathogenic variants in CCSD patients by using WES and 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines as well as evaluating the usefulness of functional studies for determining them. Methods and Results We performed WES of 23 probands diagnosed with early-onset (<65 years) CCSD and analyzed 117 genes linked to arrhythmogenic diseases or cardiomyopathies. We focused on rare variants (minor allele frequency < 0.1%) that were absent from population databases. Five probands had protein truncating variants in EMD and LMNA which were classified as “pathogenic” by 2015 ACMG standards and guidelines. To evaluate the functional changes brought about by these variants, we generated a knock-out zebrafish with CRISPR-mediated insertions or deletions of the EMD or LMNA homologs in zebrafish. The mean heart rate and conduction velocities in the CRISPR/Cas9-injected embryos and F2 generation embryos with homozygous deletions were significantly decreased. Twenty-one variants of uncertain significance were identified in 11 probands. Cellular electrophysiological study and in vivo zebrafish cardiac assay showed that 2 variants in KCNH2 and SCN5A, 4 variants in SCN10A, and 1 variant in MYH6 damaged each gene, which resulted in the change of the clinical significance of them from “Uncertain significance” to “Likely pathogenic” in 6 probands. Conclusions Of 23 CCSD probands, we successfully identified pathogenic or likely pathogenic variants in 11 probands (48%). Functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants in patients with CCSD. SCN10A may be one of the major genes responsible for CCSD. Translational Perspective Whole-exome sequencing (WES) may be helpful in determining the causes of cardiac conduction system disease (CCSD), however, the identification of pathogenic variants remains a challenge. We performed WES of 23 probands diagnosed with early-onset CCSD, and identified 12 pathogenic or likely pathogenic variants in 11 of these probands (48%) according to the 2015 ACMG standards and guidelines. In this context, functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants, and SCN10A may be one of the major development factors in CCSD

High sensitivity of late gadolinium enhancement for predicting microscopic myocardial scarring in biopsied specimens in hypertrophic cardiomyopathy

金沢大学医薬保健研究域医学系Background: Myocardial scarring can be assessed by cardiac magnetic resonance imaging with late gadolinium enhancement and by endomyocardial biopsy. However, accuracy of late gadolinium enhancement for predicting microscopic myocardial scarring in biopsied specimens remains unknown in hypertrophic cardiomyopathy. We investigated whether late gadolinium enhancement in the whole heart reflects microscopic myocardial scarring in the small biopsied specimens in hypertrophic cardiomyopathy. Methods and Results: Twenty-one consecutive patients with hypertrophic cardiomyopathy who were examined both by cardiac magnetic resonance imaging and by endomyocardial biopsy were retrospectively studied. The right interventricular septum was the target site for endomyocardial biopsy in all patients. Late gadolinium enhancement in the ventricular septum had an excellent sensitivity (100%) with a low specificity (40%) for predicting microscopic myocardial scarring in biopsied specimens. The sensitivity of late gadolinium enhancement in the whole heart remained 100% with a specificity of 27% for predicting microscopic myocardial scarring in biopsied specimens. Quantitative assessments of fibrosis revealed that the extent of late gadolinium enhancement in the whole heart was the only independent variable related to the microscopic collagen fraction in biopsied specimens (β = 0.59, 95% confident interval: 0.15-1.0, p = 0.012). Conclusions: Although there was a compromise in the specificity, the sensitivity of late gadolinium enhancement was excellent for prediction of microscopic myocardial scarring in hypertrophic cardiomyopathy. Moreover, the severity of late gadolinium enhancement was independently associated with the quantitative collagen fraction in biopsied specimens in hypertrophic cardiomyopathy. These findings indicate that late gadolinium enhancement can reflect both the presence and the extent of microscopic myocardial scarring in the small biopsied specimens in hypertrophic cardiomyopathy. © 2014 Konno et al

ゼブラフィッシュを用いた肥大型心筋症の発症、進展機序の解明

金沢大学医薬保健研究域医学系肥大型心筋症例で見いだされた遺伝子変異を再現するため、ゼブラフィッシュ胚にMYBPC3に対するアンチセンスモルフォリノ及びゼブラフィッシュMYBPC3 mRNAをインジェクションし、心臓表現型を解析した。野生型mRNAのみ、野生型mRNAおよびA745D mRNA、A745DおよびR815Q mRNAをインジェクションし、それぞれ野生型、単独変異モデル、複合ヘテロ接合体モデルを作成した。モルフォリノ単独群に比し、野生型モデルおよび単独変異モデルは心室径、心室内径短縮率、心房面積、心拍数に有意な変化を認めなかったが、複合ヘテロ接合体モデルでは有意に心室の拡大、心拍数の増加を認めた。MYBPC3 antisense morpholino oligonucleotide (MO) and MYBPC3 mRNA were injected in one cell stage zebrafish embryos to assess the functional consequences of the MYBPC3 mutations found in human hypertrophic cardiomyopathy patients. Wild type MYBPC3 mRNA, wild type and A745D mutant mRNA, or double mutant mRNA (A745D and R815Q mutants) were co-injected. They corresponds wild type, single mutation (heterozygote) and compound heterozygote, respectively. Ventricular dimensionand heart rate were similar between the MYBPC3 MO group and the MYBPC3 MO with WT mRNA group or the MYBPC3 MO with Ala745Asp mRNA and WT mRNA group, whereas ventricular dimension and heart rate were significantly increased in the MYBPC3 MO with Ala745Asp mRNA and Arg815Gln mRNA group compared with the MYBPC3 MO group.研究課題/領域番号:25860589, 研究期間(年度):2013-04-01 – 2016-03-3

Huge right ventricular mass lesion associated with genital malignant tumor: a case report

Abstract Background Primary heart tumors are rare, whereas metastatic heart tumors occur more frequently. Case presentation We report a case of a 75-year-old Japanese woman who had metastatic heart tumors of the right ventricle. Although she initially received antibiotic therapy following a diagnosis of pneumonia and pleuritis, her symptoms worsened, and she developed dyspnea and bilateral lower limb edema. Echocardiography showed a huge mass lesion occupying the entire right ventricle. Because the patient’s tumor markers were elevated, we used computed tomography to search for the primary lesion, which was located in the vagina or the uterus. Histology demonstrated the presence of basaloid squamous cell carcinoma in the vaginal tissue. Chemotherapy with paclitaxel and carboplatin was initiated. Conclusions These data suggest that the tumor in the right ventricle metastasized from the genital organs

High sensitivity of late gadolinium enhancement for predicting microscopic myocardial scarring in biopsied specimens in hypertrophic cardiomyopathy.

BACKGROUND: Myocardial scarring can be assessed by cardiac magnetic resonance imaging with late gadolinium enhancement and by endomyocardial biopsy. However, accuracy of late gadolinium enhancement for predicting microscopic myocardial scarring in biopsied specimens remains unknown in hypertrophic cardiomyopathy. We investigated whether late gadolinium enhancement in the whole heart reflects microscopic myocardial scarring in the small biopsied specimens in hypertrophic cardiomyopathy. METHODS AND RESULTS: Twenty-one consecutive patients with hypertrophic cardiomyopathy who were examined both by cardiac magnetic resonance imaging and by endomyocardial biopsy were retrospectively studied. The right interventricular septum was the target site for endomyocardial biopsy in all patients. Late gadolinium enhancement in the ventricular septum had an excellent sensitivity (100%) with a low specificity (40%) for predicting microscopic myocardial scarring in biopsied specimens. The sensitivity of late gadolinium enhancement in the whole heart remained 100% with a specificity of 27% for predicting microscopic myocardial scarring in biopsied specimens. Quantitative assessments of fibrosis revealed that the extent of late gadolinium enhancement in the whole heart was the only independent variable related to the microscopic collagen fraction in biopsied specimens (β  =  0.59, 95% confident interval: 0.15 - 1.0, p  =  0.012). CONCLUSIONS: Although there was a compromise in the specificity, the sensitivity of late gadolinium enhancement was excellent for prediction of microscopic myocardial scarring in hypertrophic cardiomyopathy. Moreover, the severity of late gadolinium enhancement was independently associated with the quantitative collagen fraction in biopsied specimens in hypertrophic cardiomyopathy. These findings indicate that late gadolinium enhancement can reflect both the presence and the extent of microscopic myocardial scarring in the small biopsied specimens in hypertrophic cardiomyopathy