Exome sequencing identifies a novel TRPV4 mutation in a CMT2C family.

The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters. Guida Landouré contributed to patient characterization, completed some sequencing, mutagenesis and cell death assays, and drafted the manuscript. Barrington G. Burnett designed experiments and completed mutagenesis and cell death assays. Charlotte J. Sumner directed the study, examined patients, performed cell death assays and completed the manuscript. Authors report no disclosure. Bryan J. Traynor is a member of the editorial board of Neurology. Introduction Mutations in over 50 genes have been associated with Charcot-Marie-Tooth (CMT) disease. As new genes continue to be discovered, the task of sequencing each of them individually with traditional techniques becomes increasingly burdensome. In addition, these approaches have disadvantages that may reduce their sensitivity. Exome sequencing uses targeted sequence capture of exons and next-generation sequencing to evaluate the sequence of all exons simultaneously. Here, we report a family with the CMT2C phenotype in whom exome sequencing revealed a novel mutation in the transient receptor potential vanilloid 4 (TRPV4) gene missed by previous Sanger sequencing. Subjects were evaluated after giving written consent to an NINDS Institutional Review Board-approved protocol. DNA extracted from peripheral blood was sequenced on a Genome Analyzer IIx platform (Illumina, CA). Sequence variants were confirmed by Sanger sequencing using newly-designed primers. Cellular studies Calcium imaging and cell death assays were performed using transiently-transfected HEK293 cells as previously described. 1 Statistical significance was determined using two-tailed unpaired t-tests. Results Exome sequencing reveals a novel mutation in a family with CMT2C Neurology #2011/409300 We evaluated nine family members (figure, A), three of whom exhibited features consistent with the CMT2C phenotype, 1,2 including progressive distal limb muscle weakness and atrophy, hoarseness of voice, and stridor on exertion (Supplemental Table). Nerve conduction studies confirmed an axonal neuropathy with phrenic nerve involvement; laryngoscopy showed reductions in vocal fold opening and closing. Two individuals had scoliosis, but skeletal dysplasia was not evident on bone radiographs. One individual had bilateral sensorineural hearing loss. Previous Sanger sequencing failed to identify TRPV4 mutations in this family (figure, B), designated Family 3. 1 In the present study, we performed exome sequencing in one affected individual and, surprisingly, identified a novel sequence variant (c.557G>A) in TRPV4. No significant variants were identified in other CMT-causing genes. A review of the Sanger sequencing primers used to amplify this region of TRPV4 revealed that the forward and reverse primers each …