ABSTRACT & COMMENTARY
By Eric Mallack, MD, MBE and Barry Kosofsky, MD, PhD
Dr. Mallack is Fellow, Department of Pediatrics, Division of Pediatric Neurology, Weill Cornell Medical College. Dr. Kosofsky is Chief, Division of Pediatric Neurology, Goldsmith Professor of Pediatrics, Neurology and Neuroscience, and Radiology, Weill Cornell Medical College.
Drs. Mallack and Kosofsky report no financial relationships relevant to this field of study.
Synopsis: Targeted high-coverage sequencing for causal somatic mutations in patients with cortical malformations is more sensitive than traditional Sanger and whole-exome sequencing.
Source: Jamuar SS, et al. Somatic mutations in cerebral cortical malformations. N Eng J Med 2014;371:733-743.
The role of somatic mutations in cancer has been well established. However, the analogous role of somatic mutations in normally dividing cells, and its relation to non-cancerous disease states, is less well described. This study evaluates the improved diagnostic sensitivity of targeted high-coverage DNA sequencing using "Next-Gen," a method used commonly in detecting somatic mutations in tumor samples, vs traditional Sanger sequencing in detecting cerebral cortical malformations. The authors analyzed DNA acquired from blood samples derived from human subjects with the double-cortex syndrome (subcortical band heterotopia), polymicrogyria with megalencephaly, periventricular nodular heterotopia, or pachygyria, and used this novel, high throughput, targeted DNA diagnostic approach to estimate the prevalence of mosaicism in each condition.
A total of 158 affected individuals were studied, all diagnosed with cortical malformations radiographically by MRI. Two targeted high-coverage gene panels were designed to detect a combination of 68 genes known to be the most frequent genetically identifiable causes of cerebral cortical malformations. To detect mosaicism, libraries of sequence data were generated from DNA isolated from peripheral blood samples and analyzed using this high-coverage sequencing method. The inclusion criteria for pathogenic mutations were defined as mutations that are known to cause the disorder, are absent from controls, alter the sequence of the encoded protein, and alter the function of the encoded protein (as predicted by SIFT or Poly-Phen-2 software algorithms).
The authors report that by using this sophisticated Next-Gen DNA diagnostic approach specifically designed to identify somatic mutations in a set of genes known to affect structural brain development, study of peripheral blood samples revealed the following:
- 17% (n = 27) of the 158 subjects were found to have causative mutations.
- 30% (n = 8) of the 27 were found to be somatic, non-germline, mosaic mutations by targeted high coverage sequencing: six occurred among 30 patients with Double-Cortex Syndrome who had a somatic mutation in either the DCX or LIS1 gene; one of eight patients with periventricular nodular heterotopia had a somatic mutation in the FLNA gene; one of eight patients with pachygyria had a somatic mutation in the TUBB2B gene.
- As compared to Next-Gen sequencing, five of those eight mutations were misread by traditional Sanger sequencing as either undetected or classified as germline anomalies.
- Standard (Sanger) DNA sequencing methods routinely identify a corresponding mutation only when it is present in at least 10% of circulating blood cells.
This study brings to light the practical exigencies that exist in our era of increasing options for and sophistication of genetic testing for developmental brain disorders and other neurologic diseases. It nicely illustrates the limitations of our current genetic testing methods. The authors have demonstrated that the traditional Sanger method of DNA sequencing has a lower sensitivity for correctly detecting and classifying somatic mutations vs targeted high-coverage sequencing, as shown by testing for DCX and LIS1 in the Double-Cortex Syndrome. Even more striking, with the promise of whole-exome sequencing as being a "diagnostic promised land," Next-Gen sequencing was able to detect a somatic mutation in a patient with pachygyria, whereas whole-exome sequencing returned a false negative result for the TUBB2B gene. This is consistent with other limitations of whole-exome sequencing, such as an inability to test for triplicate repeat disorders, requiring additional specific gene panels to be utilized for such testing.
The article also advances an understanding of the clinical severity secondary to somatic mosaicism in cerebral cortical malformations vs that of germline mutations. The DCX mutation (R186C) in the germline of one subject produced the expected phenotype of thick-band heterotopia in both anterior and posterior portions of the brain. The same mosaic mutation in a different subject in whom approximately only 10% of brain cells expressed that same mutation, produced a milder phenotype of predominantly anterior, thin-band heterotopia.
The study does make a critical assumption; the degree of mosaicism found in peripheral cells for a mutation is quantitatively reflective of that which exists in the brain. The reality of the matter is that in this study, brain tissue was not directly tested, so the corresponding incidence and expression of the somatic mutations reported from the blood in the brain is unknown. However, these same authors have additionally pioneered approaches to enable such single brain cell DNA diagnostics.1
In terms of shedding light on the full clinical picture of these disorders, the study showed the ability of targeted Next-Gen DNA sequencing to implicate new genes that may cause certain cerebral cortical malformations, in this case pachygyria. The approach reports de novo mutations in two previously unknown genes: DYNC1H1 (which matched the genotype of two participants in a parallel study) and a novel mutation in KIF5C. It also implicated known genes KIF7, KIF1A, and KIF2A, thought previously to not play a role in disease. This may be the most important point of this research, as the multiple advanced genetic diagnostic modalities utilized in this study only identified 27 mutations (17%) in the 158 subjects diagnosed with a brain malformation. These investigators have confirmed that targeted high-coverage sequencing is an example of a novel approach to genetic testing that provides enhanced diagnostic sensitivity as well as the potential for discovering new disease-causing mutations, which can increasingly be applied to many developmental brain and neurologic disorders.
- Cai X, et al. Single-cell, genome-wide sequencing identifies clonal somatic copy-number variation in the human brain. Cell Rep 2014;8:1280-1289..