Exome sequencing allows for rapid gene identification in a Charcot-Marie-Tooth family

Abstract

Objective: Charcot-Marie-Tooth (CMT) disease comprises a large number of genetically distinct forms of inherited peripheral neuropathies. The relative uniform phenotypes in many patients with CMT make it difficult to decide which of the over 35 known CMT genes are affected in a given patient. Genetic testing decision trees are therefore broadly based on a small number of major subtypes (eg, CMT1, CMT2) and the observed mutation frequency for CMT genes. Since conventional genetic testing is expensive many rare genes are not being tested for at all.

Methods: Whole-exome sequencing has recently been introduced as a novel and alternative approach. This method is capable of resequencing a nearly complete set of coding exons in an individual. We performed whole-exome sequencing in an undiagnosed family with CMT.

Results: Within over 24,000 variants detected in 2 exomes of a CMT family, we identified a nonsynonymous GJB1 (Cx32) mutation. This variant had been reported previously as pathogenic in X-linked CMT families. Sanger sequencing confirmed complete cosegregation in the family. Affected individuals had a marked early involvement of the upper distal extremities and displayed a mild reduction of nerve conduction velocities.

Interpretation: We have shown for the first time in a genetically highly heterogeneous dominant disease that exome sequencing is a valuable method for comprehensive medical diagnosis. Further improvements of exon capture design, next-generation sequencing accuracy, and a constant price decline will soon lead to the adoption of genomic approaches in gene testing of Mendelian disease.

Conflict of interest statement

Potential conflict of interest

C.S., E.P., G.B., G.M., J.H., J.V., M.S., C.Z., W.H., and Y.E. have received a research grant from the NIH.

Copyright © 2011 American Neurological Association.

Figures

FIGURE 1

Pedigree of the studied family. Filled black symbols are phenotypically affected individuals. Each individual has an identifier and the mutational state is indicated. Question marks refer to unclear phenotypes. While (individual 2000) was not genetically tested and phenotypic information is unclear, it can be assumed that she was the carrier of the mutation, based on the inheritance pattern of CMT1X.

FIGURE 2

Identification of a nonsynonymous mutation in GJB1. (A) Next-generation sequencing output at the position of the identified mutation in GJB1 (black box). A coverage depth of 36 individual sequencing reads covered this particular site, with 22 of them being forward-aligned reads (green) and 14 being reverse-aligned reads (red). Out of 36 reads 12 (33%), showed the mutation in a female individual. (B) Sanger sequencing traces confirmed the change c.283G>A, V95M. As expected, male individuals showed a homozygous change and female participants carried a heterozygous change.

FIGURE 3

Typical sequence coverage (log10 scale) of peripheral neuropathy–related genes. Exome sequencing results in a range of sequence coverage depth. Some areas of targeted CMT genes (x-axis) were insufficiently covered in the current version of the whole-exome kits (<5 reads, dotted line). This will likely improve in future versions of exome-enrichment kits and with increasing sequence read length from ~75 bases to up to 125 bases on the Illumina Genome Analyzer platform.