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

Quantitative evaluation of the rate of myocardial interstitial fibrosis using a personal computer

金沢大学大学院医学系研究科　金沢大学大学院医学系研究科　We investigated the reliability and reproducibility of an image- analyzing system run on a personal computer for measurement of myocardial interstitial fibrosis. Measurements of myocardial interstitial fibrosis in right ventricular endomyocardial biopsies obtained from patients with hypertrophic cardiomyopathy determined by this image-analyzing system were compared with measurements determined by the point-counting method. We also investigated the correlation between measurements of interstitial fibrosis obtained by image analysis and biochemical measurements of myocardial levels of hydroxyproline in normal and cardiomyopathic hamsters. The intra- and interobserver variability were significantly lower for measurements obtained by the image-analyzing system than for measurements obtained by the point- counting system. Reproducibility was superior with the image-analyzing method. The rate of myocardial interstitial fibrosis determined by the computer image-analyzing method was positively correlated with the hydroxyproline measurement (r=0.89). Our results suggest that an image- analyzing system using a personal computer provides reproducible results with a high level of reliability

Heterogeneity of clinical manifestation of hypertrophic cardiomyopathy caused by deletion of lysine 183 in cardiac troponin I gene: Insight from two autopsy cases with an identical sarcomeric gene mutation

Hypertrophic cardiomyopathy (HCM) is associated with gene mutations that encode sarcomeric proteins. However, the relationship between genotype and histopathologic fndings is unclear. We report on two autopsy cases with advanced HCM associated with deletion of lysine 183 mutation in the cardiac troponin I gene. One case, a 74-year-old female exhibited dilated cardiomyopathy-like features. Transmural scarring was diffuse and circumferential, involving the whole left ventricle, especially the ventricular septum which was replaced with extensive fbrosis and showed marked wall thinning. The other case, a 92-year-old male revealed typical HCM fndings. Patchy scars which corresponded to replacement fbrosis were found extending from the septum to the anterior wall. These two autopsy cases indicate the clinical heterogeneity of HCM even within the same disease-causing mutation and suggest that the degree and extent of fbrosis determine differences in the clinical manifestations of HCM. This is the frst autopsy report that demonstrates identical sarcomeric gene mutations causing different clinical manifestations and histologic fndings. The fndings suggest that additional genetic or environmental factors infuence the phenotypic expressions and clinical courses of HCM caused by genetic mutation of sarcomeric proteins