J Waves for Predicting Cardiac Events in Hypertrophic Cardiomyopathy

13301甲第4597号博士（医学）金沢大学博士論文要旨Abstract 以下に掲載：Journal American College of Cardiology Clinical Electrophysiology 3(10) pp.1136-1142 2017. Elsevier. 共著者：Toyonobu Tsuda, Kenshi Hayashi, Tetsuo Konno, Kenji Sakata, Takashi Fujita, Akihiko Hodatsu, Yoji Nagata, Ryota Teramoto, Akihiro Nomura, Yoshihiro Tanaka, Hiroshi Furusho, Masayuki Takamura, 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

Microstructure and nanomechanical properties of cubic boron nitride films prepared by bias sputter deposition

Nanoindentations, using a triangular based cube-corner shaped diamond tip, were tried to study the mechanical properties of cubic boron nitride (cBN) films. Since the cBN film has a layered structure consisting of an initial sp^2-bonded BN (amorphous BN and turbostratic BN) layer and a sp^3-bonded BN (cBN) layer, which was previously studied by cross-sectional transmission electron microscopy (TEM), cBN films in different growth stages were prepared for the measurements. Hardness and elastic modulus were evaluated as a function of the penetration depth from the obtained loading-unloading curves using a method described by Oliver et al. The maximum hardness and elastic modulus of a 110-nm-thick cBN film (including an approximately 50-nm-thick initial layer) were approximately 10 and 3 times, respectively, larger than the values evaluated for a silicon wafer substrate. The dependence of the evaluated hardness and the elastic modulus on film thickness and penetration depth can be explained in terms of the layered structure of the cBN film, i. e. soft initial sp^2-bonded BN layer with low elastic modulus, and hard cBN layer with high elastic modulus

Growth process of cubic boron nitride films in bias sputter deposition

Intending to study phase evolution of cubic boron nitride (cBN) films during RF bias sputter deposition, surfaces of the cBN films in different growth stages were examined systematically. Argon content at the film surface increased during the deposition of initial sp^2 bonded layer growth, and saturated at about 1.5 at.%, and remained constant after the formation and growth of cubic phase, accompanied with an increase of compressive stress up to 5 GPa. This increase of stress is confirmed to be the result of averaging film stress over the double-layered cBN film, that is, over low-stressed initial layer and high-stressed cubic layer. Atomic force microscope (AFM) observations in tapping mode revealed self-affine fractal nature and kinetic roughening of film surface during the growth, and root mean square (rms) roughness changed from 0.3 nm of an initial layer, to 0.9 nm accompanied with peculiar surface smoothing at the initial stage of cBN formation. Moreover, the evolution of the surface roughness was clearly characterized by the change of modes in each growth process, that is, an initial layer growth stage, a transition stage, and a cubic layer growth stage