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

Clinical Impact of Dual Time Point 18F-Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography Fusion Imaging in Pancreatic Cancer

We examined the value of preoperative dual time point (DTP) 18F-fluorodeoxyglucose positron emission tomography/computed tomography fusion imaging (FDG PET/CT) as a predictor of early recurrence or the outcomes in patients with pancreatic cancer. Standardized uptake values (SUVs) in DTP FDG PET/CT were performed as preoperative staging. SUVmax1 and SUVmax2 were obtained in 60 min and 120 min, respectively. ΔSUVmax% was defined as (SUVmax2 − SUVmax1)/SUVmax1 × 100. The optimal cut-off values for SUVmax parameters were selected based on tumor relapse within 1 year of surgery. Optimal cut-off values for SUVmax1 and ΔSUVmax% were 7.18 and 24.25, respectively. The combination of SUVmax1 and ΔSUVmax% showed higher specificity and sensitivity, and higher positive and negative predictive values for tumor relapse within 1 year than SUVmax1 alone. Relapse-free survival (RFS) was significantly worse in the subgroups of high SUVmax1 and high ΔSUVmax% (median 7.0 months) than in the other subgroups (p < 0.0001). The multivariate Cox analysis of RFS identified high SUVmax1 and high ΔSUVmax% as independent prognostic factors (p = 0.0060). DTP FDG PET/CT may effectively predict relapse in patients with pancreatic cancer. The combination of SUVmax1 and ΔSUVmax% identified early recurrent patient groups more precisely than SUVmax1 alone

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

Comparative Complete Genome Sequence Analysis of the Amino Acid Replacements Responsible for the Thermostability of Corynebacterium efficiens

Corynebacterium efficiens is the closest relative of Corynebacterium glutamicum, a species widely used for the industrial production of amino acids. C. efficiens but not C. glutamicum can grow above 40°C. We sequenced the complete C. efficiens genome to investigate the basis of its thermostability by comparing its genome with that of C. glutamicum. The difference in GC content between the species was reflected in codon usage and nucleotide substitutions. Our comparative genomic study clearly showed that there was tremendous bias in amino acid substitutions in all orthologous ORFs. Analysis of the direction of the amino acid substitutions suggested that three substitutions are important for the stability of the C. efficiens proteins: from lysine to arginine, serine to alanine, and serine to threonine. Our results strongly suggest that the accumulation of these three types of amino acid substitutions correlates with the acquisition of thermostability and is responsible for the greater GC content of C. efficiens