Obscurin Mutations Cause Haploinsufficiency and are Common in Patients with Familial Dilated Cardiomyopathy (FDCM)

Abstract

Whole exon sequence data from 28 FDCM patient cardiac muscle samples was screened for potentially disease-causing mutations in 58 genes previously implicated in HCM or DCM. We identified OBSCN gene mutations in 5 samples; one sample had two OBSCN mutations, one also had a DSP mutation and another also had a SCN5A mutation Also identified were 6 truncating mutations in TTN, 3 mutations in MYH7, 2 in DSP and one each in TNNC1, TNNI3, MYOM1, VCL, GLA, PLB, PKP2 and LAMA4.The mean level of obscurin mRNA was significantly greater and more variable in donor samples than the FDCM samples (1.69±0.53, n=58 compared with 0.57±0.10,n= 68, p=0.0025). The mRNA content of FDCM samples was not significantly different with and without OBSCN mutations.The obscurin protein band was estimated to be <1% of the abundance of titin; it was identified and quantified with antibodies. The apparent mass was 960± 60 kDa. The OBSCN mutation samples had levels of expression, significantly different from FDCM samples without obscurin mutations, donor hearts or myectomies. Four OBSCN mutant samples exhibited low levels of obscurin protein (48, 61, 51,and 70% of the level in control FDCM samples). One OBSCN mutant sample exhibited 34% more expression.Immunofluorescence microscopy using obscurin, myomesin and a-actinin-specific antibodies showed that obscurin was located at the level of the M-line and preferentially labelled the sides of the myofibrils. There was no apparent differences between wild-type and mutant samples.Mutations in the obscurin gene should be considered as a significant cause of FDCM, alone or in concert with another mutation. Disease-related OBSCN mutations cause demonstrably abnormal expression in myofibrils that could account for the development of a DCM phenotype.