Based On A Molecular Genetics Study Of Hypertrophic Cardiomyopathy

Background The MYH7 gene encodes the myosin heavy chain,MYH7 gene has been found to be the main disease-causing gene associated with hypertrophic cardiomyopathy（HCM）.In our previous study,we had identified a new mutation of the MYH7 gene,p.E370Q,in one HCM family.In addition,this MYH7disease-causing mutation was associated with variable clinical presentation and incomplete penetrance in the family.Materials and methods First,in order to determine the pathogenic consequence of this mutation in MYH7 gene,we used the zebrafish model for studying larva cardiac structure and function in vivo.We constructed mammalian expression plasmids for WT and mutant EGFP-tagged MYH7 with p.E370Q,and each PCR product was microinjected into zebrafish embryos,and the heart rate（HR）,shortening fraction（FS）,and heart rate variability（HRV）were calculated;The effects of mutations on cardiac hypertrophic growth were analyzed by tissue sections,in situ hybridization and RT-PCR;The signaling changes caused by the mutations were assessed by RNA-seq analysis.Second,the next generation sequencing technology（NGS）was performed to search for potential disease-modifying genes in the family.The effects of TTN variant on protein structure and stability were analyzed by circular dichroism,fluorescence spectroscopy and western blot analysis;GST-pulldown technique was used to analyze the effect of TTN variation on the binding of titin to putative interacting protein.Results We found that the mutant zebrafish embryos had abnormal heart function,slow heart rate（HR）,decreased fractional shortening（FS）,and increased heart rate variability（HRV）at 72hpf;Histological examination revealed that the ventricular wall thickness increased and the area of ventricular was significantly larger in mutant group than in WT group.RT-PCR analysis demonstrated that the expression of NPPA and VMHCL were up-regulated.RNA-seq analysis also revealed abnormalities in a series of signaling pathways related to cardiac function in mutant zebrafish embryos.We found that TTN Y5193C mutation may be the potential modified gene through whole exon sequencing.The variant altered tertiary structure of titin and displayed a slightly redshift of the wavelength of titin protein’s maximum absorption value;Under the stress condition of H₂O₂treatment,the protein of TTN Y5193C is more prone to degradation;In addition,TTN mutation Y5193C affected the interaction between titin andαB-crystallin.