Clinical And Pathogenic Mechanism Of Myocardial Noncompaction And Hypertrophic Cardiomyopath

Pathogenic mechanism of TTNp.R2021X mutation in noncompaction of cardiomyopathyBackground Noncompaction of cardiomyopathy（NCCM）is a relatively rare cardiomyopathy characterized by excessively prominent trabeculations and associated deep recesses that communicate with the ventricular cavity.The predominant mode of inheritance is autosomal dominant.TTN gene encode titin,TTN mutation is the most prevalent genetic cause of dilated cardiomyopathy.Recent studies have shown the importance of TTN mutations in NCCM.However,the molecular mechanisms underlying the pathogenesis of TTN gene mutation in NCCM remains poorly understood.Objective In the present study,we identified the presence of a novel TTN p.R2021X mutation in a pedigree with noncompaction of cardiomyopathy.We further investigated the role and potential mechanism of the novel mutation in vitro using CRISPR/CAS9 technology.Methods The gene sequencing with linkage analysis was performed in a 3-generation family affected by autosomal dominant NCCM.CRISPR/CAS9 technology was used to establish novel truncating mutation in TTN in a rat cardiomyoblast H9c2 cell line in vitro,in which functional studies were carried out and characterized in comparison to its wild-type counterpart.（1）we performed real-time RT-PCR analysis to quantify cardiac TTN expression after mutation of TTN R2021X.（2）The mitochondrial respiratory activity represented by the cellular oxygen consumption rate and ATP levels were measured in WT and mutant cells.（3）Autophagy-related ultrastructural differences between WT and mutant cells were evaluated under electron microscopy.（4）Levels of phosphorylated mTOR protein,ribosomal protein S6,and eukaryotic translation initiation factor 4E-binding protein 1 were tested using western blot in mutant cells and WT cells.Results Sanger sequencing confirmed cosegregation of the heterozygous mutation p.R2021X mutation in TTN with disease in all affected individuals of the family.Single-base alteration at position 6061（c.6061C>T）resulted in a premature stop of titin protein.This mutation has not been identified in the ExAC database and in silico prediction（MutationTaster）reported a deleterious effect.Quantitative reverse transcription-polymerase chain reaction and cellular immunofluorescence supported a haploinsufficient disease mechanism in titin truncation mutation cardiomyocytes.Further functional studies suggested mitochondrial abnormities in the presence of mutation,including decreased oxygen consumption rate,reduced ATP production,impaired activity of electron translation chain,and abnormal mitochondrial structure on electron microscopy.Impaired autophagy under electron microscopy accompanied with activation of the Akt-mTORC1 signaling pathway was observed in TTN p.R2021X truncation mutation cardiomyocytes.Conclusions We identified a novel TTN p.R2021X mutation in a pedigree of noncompaction of cardiomyopathy.This finding expands the spectrum of titin’s roles in cardiomyopathies.Further functional studies suggest mitochondrial dysfunction and impaired autophagy activity in mutant cells.These findings provide novel insight into treatment of noncompaction of cardiomyopathy in future clinical practice:improving cardiac metabolism,activating autophagy,inhibiting mTORCl signaling pathway can be potential target of therapy.A novel mutation in OBSCN gene in a pedigree of noncompaction of cardiomyopathyBackground Noncompaction of cardiomyopathy（NCCM）is a rare type of genetic cardiomyopathy,and the predominant mode of inheritance is autosomal dominant.The phenotypic expression of patients are highly variable,and clinical features can range from asymptomatic to symptomatic,with a relatively stable course over several years or an evolution toward severe complications including congestive heart failure,ventricular arrhythmia and sudden cardiac death,atrial arrhythmias,and systemic embolic events.Recent studies have shown potential genotype-phenotype correlations in patients with NCCM.Therefore,it is of great importance to perform gene sequencing in index patients of NCCM and casade screening in their families.In this way,we can make diagnosis in early time and identify high-risk patients in clinical practice.Objective The purpose of the present study is to identify potential pathogenic mutation leading to disease in a Chinese NCCM family.Methods In outpatient clinic,a patient was diagnosed with NCCM by echocardiography.Then we performed clinical assessment on available family members.The proband,the proband’s daughter and mother were selected for whole genome sequencing（WGS）.By performing WGS,filtering against variants seen in normal population cohorts,and using linkage information derived from single nucleotide polymorphism（SNP）arrays,we aim to identify potential pathogenic mutation in the family.Sanger sequencing were performed in all of the surviving family members.Results Combined whole genome sequencing with linkage analysis identified a novel missense mutation in the giant protein obscurin（OBSCN NM₀01098623,c.C19063T）,as the only plausible disease-causing variant that segregates with disease among the 4 surviving individuals,with interrogation of the entire genome excluding other potential causes.This c.C19063T missense mutation resulted in p.R6355W in the encoded OBSCN protein.It affected a highly conserved residue in the C terminus of the obscurin-B-like isoform between the PH and STKc domains,which was predicted to affect the function of the protein by different bioinformatics tools.Conclusions Here we present clinical and genetic evidence implicating the novel R6355W missense mutation in obscurin as the cause of familial NCCM.This expands the spectrum of obscurin’s roles in cardiomyopathies.This study also provides new insights into the precision treatment of patients with NCCM.Clinical characteristics of patients of hypertrophic cardiomyopathy with biventricular outflow tract obstructionBackground Hypertrophic cardiomyopathy（HCM）is the most common genetic heart disease.It is the leading cause of sudden cardiac death（SCD）in young individuals and can lead to heart failure symptoms and death at any age.Mild-to-moderate right ventricular hypertrophy in conjunction with left ventricular hypertrophy is commonly observed,while with a maximal right ventricular wall thickness of≥10 mm,even with biventricular outflow tract obstruction（BVOTO）is relatively infrequent.Limited data are available on on clinical outcomes of patients with HCM and BVOTO.Objective In this study,we aimed to investigate the clinical features,treatment therapies,and clinical outcomes of patients with HCM and BVOTO.Method The clinical data of HCM patients with BVOTO confirmed by echocardiography or magnetic resonance imaging in Fuwai Hospital from January 2006 to December 2016 were collected.The clinical characteristics of HCM patients with BVOTO were summarized and analyzed.They were compared with the data of HCM patients with single ventricular outflow tract obstruction（LVOTO）,a retrospective analysis of clinical characteristics,treatment and prognosis for BVOTO patients were carried out.Result The results show that 151 patients are diagnosed as hypertrophic obstructive cardiomyopathy（HOCM）in Fuwai Hospital from January 2006 to December 2016.Among them,15 patients（4.5%）with BVOTO,Comparing with LVOTO group,a younger diagnosis age[（29.4±18.5）years vs（39.9± 13.1）years,P<0.01],a higher NYHA class（class Ⅲ or Ⅳ）（7cases,46.7%vs 16 cases,12.1%,P<0.01）and a higher frequence of malignant arrthymia(26.7%vs 13.3%,P<0.05 are identified in BVOTO group.BVOTO group has a higher risk of sudden cardiac death.By surgical myectomy,most patients exhibited significant improvements in NYHA functional class with a substantial reduction in maximal septal thickness,LA diameter,and residual left ventricular outflow tract（LVOT）and right ventricular outflow tract（RVOT）gradients.Conclusion We demonstrated that BVOTO is an uncommon phenotype in HCM.Patients with BVOTO tend to present with severe symptoms that require complex surgical procedures.These patients’ clinical symptoms and echocardiographic parameters of clearly improved after surgical treatment.Therefore,evaluation of the right side of the heart in patients with HCM should receive more attention by clinicians.Detection of pathogenic mutations in familial hypertrophic cardiomyopathy by targeted resequencingObjective To identify the disease-causing mutation in Chinese familial hypertrophic cardiomyopathy（HCM）,and analyze the genotype-phenotype correlationMethod A Chinese family with HCM was investigated.Targeted resequencing was performed in the proband based on a custom AmpliSeq panel which includes 64 candidate pathogenic genes for cardiomyopathies.Suspicious causative mutations were validated by Sanger sequencing,and cascade genetic screening and 100 unrelated normal controls screening were then performed.The genotype-phenotype relationship in the family was analyzed.Result A frameshift mutation,c.1377delC,was identified in the MYBPC3 gene in 4 alive family members,including the proband.This mutation was not detected in 100 unrelated normal controls.Two mutation carriers were diagnosed with HCM,with the involvement of ventricular septum.And the other two asymptomatic mutation carriers were found with no abnormalities in electrocardiogram and echocardiogram.In the current family,sudden cardiac death occurred in 3 members,a history of syncope was found in 4 members,and the onset age<40 years was observed in 3 members,while 2 members developed symptoms in their 40s.ConclusionMYBPC3 is one of the most frequently mutated genes in HCM in China.Targeted resequencing is a useful method to screen disease-causing mutations in HCM,and genetic testing is of great clinical importance in the differential diagnosis and family screening of HCM.