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Somatic mutations in cerebral cortical malformations.

ABSTRACT: BACKGROUND:Although there is increasing recognition of the role of somatic mutations in genetic disorders, the prevalence of somatic mutations in neurodevelopmental disease and the optimal techniques to detect somatic mosaicism have not been systematically evaluated. METHODS:Using a customized panel of known and candidate genes associated with brain malformations, we applied targeted high-coverage sequencing (depth, ?200×) to leukocyte-derived DNA samples from 158 persons with brain malformations, including the double-cortex syndrome (subcortical band heterotopia, 30 persons), polymicrogyria with megalencephaly (20), periventricular nodular heterotopia (61), and pachygyria (47). We validated candidate mutations with the use of Sanger sequencing and, for variants present at unequal read depths, subcloning followed by colony sequencing. RESULTS:Validated, causal mutations were found in 27 persons (17%; range, 10 to 30% for each phenotype). Mutations were somatic in 8 of the 27 (30%), predominantly in persons with the double-cortex syndrome (in whom we found mutations in DCX and LIS1), persons with periventricular nodular heterotopia (FLNA), and persons with pachygyria (TUBB2B). Of the somatic mutations we detected, 5 (63%) were undetectable with the use of traditional Sanger sequencing but were validated through subcloning and subsequent sequencing of the subcloned DNA. We found potentially causal mutations in the candidate genes DYNC1H1, KIF5C, and other kinesin genes in persons with pachygyria. CONCLUSIONS:Targeted sequencing was found to be useful for detecting somatic mutations in patients with brain malformations. High-coverage sequencing panels provide an important complement to whole-exome and whole-genome sequencing in the evaluation of somatic mutations in neuropsychiatric disease. (Funded by the National Institute of Neurological Disorders and Stroke and others.).

SUBMITTER: Jamuar SS

PROVIDER: S-EPMC4274952 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Somatic mutations in cerebral cortical malformations.

Jamuar Saumya S SS Lam Anh-Thu N AT Kircher Martin M D'Gama Alissa M AM Wang Jian J Barry Brenda J BJ Zhang Xiaochang X Hill Robert Sean RS Partlow Jennifer N JN Rozzo Aldo A Servattalab Sarah S Mehta Bhaven K BK Topcu Meral M Amrom Dina D Andermann Eva E Dan Bernard B Parrini Elena E Guerrini Renzo R Scheffer Ingrid E IE Berkovic Samuel F SF Leventer Richard J RJ Shen Yiping Y Wu Bai Lin BL Barkovich A James AJ Sahin Mustafa M Chang Bernard S BS Bamshad Michael M Nickerson Deborah A DA Shendure Jay J Poduri Annapurna A Yu Timothy W TW Walsh Christopher A CA

The New England journal of medicine 20140801 8

<h4>Background</h4>Although there is increasing recognition of the role of somatic mutations in genetic disorders, the prevalence of somatic mutations in neurodevelopmental disease and the optimal techniques to detect somatic mosaicism have not been systematically evaluated.<h4>Methods</h4>Using a customized panel of known and candidate genes associated with brain malformations, we applied targeted high-coverage sequencing (depth, ≥200×) to leukocyte-derived DNA samples from 158 persons with bra ...[more]

PMID: 25140959

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Project description:BackgroundSporadic arteriovenous malformations of the brain, which are morphologically abnormal connections between arteries and veins in the brain vasculature, are a leading cause of hemorrhagic stroke in young adults and children. The genetic cause of this rare focal disorder is unknown.MethodsWe analyzed tissue and blood samples from patients with arteriovenous malformations of the brain to detect somatic mutations. We performed exome DNA sequencing of tissue samples of arteriovenous malformations of the brain from 26 patients in the main study group and of paired blood samples from 17 of those patients. To confirm our findings, we performed droplet digital polymerase-chain-reaction (PCR) analysis of tissue samples from 39 patients in the main study group (21 with matching blood samples) and from 33 patients in an independent validation group. We interrogated the downstream signaling pathways, changes in gene expression, and cellular phenotype that were induced by activating KRAS mutations, which we had discovered in tissue samples.ResultsWe detected somatic activating KRAS mutations in tissue samples from 45 of the 72 patients and in none of the 21 paired blood samples. In endothelial cell-enriched cultures derived from arteriovenous malformations of the brain, we detected KRAS mutations and observed that expression of mutant KRAS (KRASG12V) in endothelial cells in vitro induced increased ERK (extracellular signal-regulated kinase) activity, increased expression of genes related to angiogenesis and Notch signaling, and enhanced migratory behavior. These processes were reversed by inhibition of MAPK (mitogen-activated protein kinase)-ERK signaling.ConclusionsWe identified activating KRAS mutations in the majority of tissue samples of arteriovenous malformations of the brain that we analyzed. We propose that these malformations develop as a result of KRAS-induced activation of the MAPK-ERK signaling pathway in brain endothelial cells. (Funded by the Swiss Cancer League and others.).

Project description:Cerebral cavernous malformations (CCM) are hamartomatous vascular malformations characterized by abnormally enlarged capillary cavities without intervening brain parenchyma. They cause seizures and focal neurological deficits due to cerebral hemorrhages. CCM loci have already been assigned to chromosomes 7q (CCM1), 7p (CCM2), and 3q (CCM3) and have been identified in 40%, 20%, and 40%, respectively, of families with CCM. Loss-of-function mutations have been identified in CCM1/KRIT1, the sole CCM gene identified to date. We report here the identification of MGC4607 as the CCM2 gene. We first reduced the size of the CCM2 interval from 22 cM to 7.5 cM by genetic linkage analysis. We then hypothesized that large deletions might be involved in the disorder, as already reported in other hamartomatous conditions, such as tuberous sclerosis or neurofibromatosis. We performed a high-density microsatellite genotyping of this 7.5-cM interval to search for putative null alleles in 30 unrelated families, and we identified, in 2 unrelated families, null alleles that were the result of deletions within a 350-kb interval flanked by markers D7S478 and D7S621. Additional microsatellite and single-nucleotide polymorphism genotyping showed that these two distinct deletions overlapped and that both of the two deleted the first exon of MGC4607, a known gene of unknown function. In both families, one of the two MGC4607 transcripts was not detected. We then identified eight additional point mutations within MGC4607 in eight of the remaining families. One of them led to the alteration of the initiation codon and five of them to a premature termination codon, including one nonsense, one frameshift, and three splice-site mutations. All these mutations cosegregated with the disease in the families and were not observed in 192 control chromosomes. MGC4607 is so far unrelated to any known gene family. Its implication in CCMs strongly suggests that it is a new player in vascular morphogenesis.

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Somatic mutations in cerebral cortical malformations.

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Somatic mutations in cerebral cortical malformations.

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