Editors: Donna E. Hansel, MD, PhD, chief, Division of Anatomic Pathology, and professor, Department of Pathology, University of California, San Diego; John A. Thorson, MD, PhD, associate professor of pathology, director of the Clinical Genomics Laboratory, Center for Advanced Laboratory Medicine, UCSD; Sarah S. Murray, PhD, associate professor, Department of Pathology, and director of genomic technologies, Center for Advanced Laboratory Medicine, UCSD; and James Solomon, MD, PhD, resident, Department of Pathology, UCSD.
A gene panel to examine mosaic somatic mutations in cerebral malformations
Somatic mutations are widely recognized in cancer, often affecting prognosis and determining candidacy for use of molecular targeted treatments. These somatic mutations may lead to a mosaic population of cells. Recent advances in technology involving deep next-generation sequencing have allowed for detection and quantification of these mosaic variants. In addition to cancer, somatic mutations often occur randomly during normal mitosis in many tissues, and those that occur during embryonic development are generating interest. Postzygotic somatic mutations are thought to play a role in the pathogenesis of many neurodevelopmental disorders and brain malformations. It is also hypothesized that these mutations may play a role in epilepsy, autism, and other neurologic or psychiatric conditions. The authors examined 158 patients with MRI-diagnosed brain malformations, including double-cortex syndrome, polymicrogyria with megalencephaly, periventricular nodular heterotopia, and pachygyria. They obtained blood samples from the patients and, using high-coverage next-generation sequencing, screened mutations in a panel of genes implicated in the patients’ specific disorders. Known causative genes and candidate genes were examined to a mean depth of at least 200×. After identification, the mutations were verified with traditional Sanger sequencing and analyzed in silico to verify pathogenicity. If the frequency of mutant alleles significantly deviated from 50 percent, which would represent a heterozygous germline mutation, then the genes were subcloned and screened to confirm the variant and quantify the mosaicism using a Sanger sequencing assay. Using this approach, causative mutations were discovered in 27 of 158 patients (17 percent), and eight of those mutations were mosaic. Many of the mosaic somatic mutations were in the DCX and LIS1 genes associated with double-cortex syndrome. Of those eight mosaic mutations, five were undetectable with traditional Sanger sequencing, which has a detection threshold for mosaic variants of approximately 15 to 20 percent alternate-allele frequency. Using the high-coverage sequencing gene panel approach described, mosaic somatic mutations in as few as five percent of alleles (or 10 percent of cells) can be detected. One caveat is that this approach assumes the same level of mosaicism in all tissues, yet many somatic mutations may not be present in blood cells or may be concentrated within affected tissue. The authors concluded that until cost-effective, whole-exome sequencing at a sufficient read-depth is developed, this targeted gene panel high-coverage approach will serve as an accurate method of examining somatic mutations that may be present at a low allelic frequency. It is likely that many gene panels will be developed to examine other disorders with known mosaicism or high rates of somatic mutations.
Jamuar SS, Lam ATN, Kircher M, et al. Somatic mutations in cerebral cortical malformations. N Engl J Med. 2014;371(8):733–743.
Correspondence: Christopher A. Walsh at christopher.walsh@childrens.harvard.edu