Abstract
Somatic Mutations in Cerebral Cortical Malformations
Jamuar SS, Lam AT, Kircher M, D'Gama AM, Wang J, Barry BJ, Zhang X, Hill RS, Partlow JN, Rozzo A, Servattalab S, Mehta BK, Topcu M, Amrom D, Andermann E, Dan B, Parrini E, Guerrini R, Scheffer IE, Berkovic SF, Leventer RJ, Shen Y, Wu BL, Barkovich AJ, Sahin M, Chang BS, Bamshad M, Nickerson DA, Shendure J, Poduri A, Yu TW, Walsh CA N Engl J Med. 2014;371:733–743.
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.)
Commentary
Cortical malformations, including double-cortex syndrome, polymicrogyria, periventricular nodular heterotopia, pachygyria, and others, are a common cause of refractory epilepsy (1, 2). These epilepsies are thought to arise from disruptions in cortical development, such as failed cell migration, altered proliferation, or stalled cellular maturation. Cortical malformations have a number of known genetic causes, including mutations in DCX, Lis1, FLNA, Tsc1/2, and others. Many times, however, clear genetic linkages cannot be made. Recent revolutions in genomic technologies are now addressing this issue. Next-generation (NextGen) sequencing allows massively parallel sequencing of genes of interest, or even the entire genome, in a high throughput manner (3). The amount of genetic information that can now be accessed in a relatively cost-effective manner is something that would have been impossible even a decade ago. Implementation of this approach has allowed significant advances in understanding the genetics of many diseases, including the epilepsies.
The Epi4K project is an example of a powerful application of NextGen sequencing. DNA sequences isolated from leukocytes of more than 200 patients were analyzed, leading to the identification of novel genes linked to epileptic encephalopathies (4). Here, we highlight recent work by Jamuar and colleagues using NextGen sequencing in a different way to investigate the role of somatic mutations in patients with cortical malformations. Somatic mutations are postzygotic events that result in groups of cells with different genetic sequences. If a somatic mutation induces a change in DNA sequence in a single cell early in development, all the cells that subsequently arise from that cell will contain that DNA alteration, whereas all the remaining cells in the body will not. This is a simple example of how a somatic mutation can lead to diversity in the genome of an individual. In reality, the genomic diversity within an individual is much more complex. Recent studies of postmortem brain show an abundance of single-neuron genomic diversity (or mosaicism), including megabase scale deletions and duplications (5). Using NextGen sequencing, Jamuar and colleagues asked whether somatic mutation plays a role in the pathology of cortical malformations.
Patients with cortical malformations were identified and enrolled in the study. Leukocytes were isolated as a source of DNA, and a panel of genes was investigated. These genes included known and potential candidate genes thought to be associated with cortical malformations. Exons, intronexon boundaries, and target gene flanking sequences were sequenced. To examine somatic mutations, allele frequencies between 5 and 40 percent were investigated. In other words, variants in the DNA sequence that occurred in 5 to 40 percent of the DNA reads generated by massively parallel sequencing were of interest. Some amount of allele frequency variation is inherent in high throughput genetic sequencing as the chemistry involved is not error free. In general, a control sequence can have up to 1% variation in allele frequency. Therefore, picking a lower cut off of 5% ensured that variants identified were well above the threshold for artifact-based variation. Inherited germline variants occur with a 50% allele frequency (i.e., one normal allele from the mother and one variant allele form the father). By choosing variants with an allele frequency <40%, germline variants were eliminated from further analysis.
Using this approach, the study identified mutations that were likely disease causing in 17% of the patients. Of those, 30% were mosaic mutations present in some, but not all, of the leukocytes examined. Some of these causative mosaic mutations were present in as few as 10% of the cells examined, which suggests that mutation in a relatively small number of cells can lead to disease. Furthermore, 63% of mosaic mutations identified using NextGen sequencing were not found using traditional Sanger sequencing and required further subcloning for conformation. This highlights the strength of NextGen sequencing in the identification of rare variants and the exciting findings, both scientifically and clinically, that can be made when cutting-edge genetic approaches are used.
Of the mosaic mutations identified, all were in genes previously associated with genetic epilepsies, including LIS1, TUBA1A, FLNA, AKT3, and PIK3CA. There was a great deal of consistency in the genes identified and the subtype of cortical malformation in a given patient; for example, DCX mutations were found in patients with double-cortex syndrome but not in patients with pachygyria. Novel, potentially pathogenic, germline mutations were found in DYNC1H1, KIF5C, KIF7, KIF1A, and KIF26A. Of particular interest, NextGen sequencing identified a number of variants that fell below the 15 to 20 percent allele variation cutoff associated with traditional sequencing technologies. Furthermore, targeted sequencing provides better read coverage and, therefore. an increased ability to detect low-abundance variants compared with whole exome sequencing. Even though whole exome sequencing is a NextGen approach, read depth (the number of DNA reads at a given location in the genome) is decreased at the expense of genome coverage. Here the authors focused on relatively few genes of interest and had relatively high read depth, which is essential to power the statistical analysis required to identify mosaic variants that can be missed by exome sequencing.
These findings are of great interest to the field of epilepsy because they help to dispel the long-held belief that all cells in the body (and brain) have an identical genome. In diseases of cortical malformation, there is often a focal region of the brain where structural pathology occurs. Perhaps the focal nature of malformations results from mosaic variation in neurons. Can a genetic disruption of a critical precursor cell create a group of dysfunctional daughter cells responsible for a malformation? This question has yet to be addressed, but it seems within the realm of possibility. Can smaller cortical malformations (e.g., focal cortical dysplasia) result from mosaicism in an even smaller number of cells? The advent of single-cell genomics may allow us to address this question in the future. It is critical to consider the appropriateness of using a blood-based DNA assay to perform diagnostic or mechanistic studies of a neurologic disease. There are known examples of mutations that exist in the brain but are undetectable in the blood (6). Also, we do not know whether the scale of mosaicism in the brain and blood are equivalent. All in all, this study demonstrates that somatic mutations and genetic mosaicism can play a causative role in epilepsies associated with cortical malformations. Recent work by others also highlights the importance of somatic mutations in autism (7) and other neurodevelopmental disorders (8, 9). With time, this type of work will spur innovation in epilepsy diagnosis and treatment (10).