Molecular Diagnostic Approaches to Metabolic Myopathies by Target Gene Capture and Next Generation Sequencing (P07.215)

Abstract

Objective: To develop an effective molecular approach by target gene capture and next generation sequencing for the diagnosis of metabolic myopathies. Background Metabolic myopathy is a group of clinically and genetically heterogeneous diseases due to defects in cellular energy metabolism, which includes three major categories: glycogen storage diseases, fatty acid oxidation, and mitochondrial respiratory chain disorders. Clinical presentation, abnormal metabolites, and biochemical enzymatic analyses may help with the diagnosis of the disease, however, the ultimate definitive diagnosis is the identification of causative molecular defects in the genes responsible for the disease. Design/Methods: A group of 24 nuclear genes commonly found to be associated with metabolic myopathies were enriched by hybridization of genome DNA with target gene specific probes (NimbleGen) followed by next generation sequencing (NGS). In addition, one single pair of primer was used to amplify the whole 16.6 kb mitochondrial genome (mtDNA) for massively parallel sequencing by NGS. Results: The sequencing was performed using Illumina9s HiSeq 2000. Twelve individually barcoded samples were pooled and analyzed. QC samples are indexed accordingly and spiked in with each sample. The coverage depth for the nuclear genes was 500X and for the mitochondrial genome was 20,000X. This deep coverage allows accurate base calls and quantitative analysis of mtDNA mutation heteroplasmy. Our results demonstrated that mutations in POLG, OPA1, LCHAD, CPTII, PHKs , and PYGM genes were all correctly identified. In addition, heteroplasmy of mtDNA point mutations and large deletion with breakpoints were also accurately determined. Conclusions: Capture of target genes in combination with long range PCR of mtDNA followed by next generation massively parallel sequencing allows cost effective analyses of a group of genes commonly involved in metabolic myopathies. This approach also provides quantitative information of mtDNA point mutations and large deletions. Disclosure: Dr. Zhang has nothing to disclose. Dr. Cui has nothing to disclose. Dr. Wang has nothing to disclose. Dr. Wong has nothing to disclose.