The de novo paradigm. De novo mutations play a significant role in many neurodevelopmental disorders including autism, intellectual disability and schizophrenia. In addition, several smaller studies have indicated a role for de novo mutations in severe epilepsies. However, unless known genes for human epilepsies are involved, findings from large-scale genetic studies are difficult to interpret. De novo mutations are also seen in unaffected individuals and only very few genes are observed more than once. Now, a publication in Nature by the Epi4K and EPGP collaborators uses a novel framework to tell pathogenic mutations from genomic noise. Their study provides very strong evidence for a predominant role of de novo mutations in Infantile Spasms and Lennox-Gastaut Syndrome.
Exomes. The current publication on Infantile Spasms (IS) and Lennox-Gastaut Syndrome (LGS) presents the data of 264 proband-parent trios that were analyzed using trio exome sequencing. The authors found 329 de novo mutations (1.25 mutations per trio on average) including de novo mutations in known epilepsy genes such as SCN1A, STXBP1, SCN2A, CDKL5, KCNT1 or DCX. For some of these genes, particularly SCN1A and SCN2A, this study expands the phenotypic spectrum and suggests that they are also relevant in IS/LGS, albeit to a much lesser extent than in the classical phenotypes such as Dravet Syndrome (SCN1A). However, these mutations in known genes for epileptic encephalopathies explain less than 10% of cases, depending on how strictly the definition of a known causative gene is applied. The majority of de novo mutations were in genes that were not previously associated with epilepsy.
Double hits? Deducing a possible pathogenic role for novel genes can occur through different ways. First, as the most straightforward case, a gene can be assumed to be causative if de novo mutations are observed in several patients. Using this “double hit” paradigm, the authors identify GABRB3 as a novel gene in IS/LGS that was found in four patients. However, the interpretation of genes found to be mutated in two patients was complicated, as the classic “double hit scenario” has at least two downsides. First, since mutations in a given gene are rare, large patient cohorts would be needed to find a second or third hit with certainty. This is beyond the capacity of most groups and consortia. Secondly, there is a bias towards large genes, as they have more coding regions to accumulate random mutations. For example, de novo mutations in Titin (TTN) have been recurrently found in various neurodevelopmental phenotypes. However, as Titin is the largest gene of the human genome, these “double hits” are usually ignored. Also, some benign mutation hotspots might be considered real findings, such as the MUC genes.
Mutation intolerance. The Epi4K/EPGP researchers have addressed this conflict using a novel framework that considers intolerance to mutations. Basically, the authors assess the normal variation in a given gene and develop a score on how mutation-resistant a gene is based on the finding in controls. Using this framework, mutations in TTN and MUCs would not be surprising. The authors found that there is a clear excess of mutations in intolerant genes and that most known EE genes belong to this group. Identified de novo mutations in CACNA1A, CHD2, FLNA, GABRA1, GRIN1, GRIN2B, HDAC4, HNRNPU, IQSEC2, MTOR, and NEDD4L were considered pathogenic based on this concept. In addition, the authors found a recurrent mutation in ALG13, i.e. the same mutation in two independent patients.
Summary. Overall, including known and novel genes, the authors identified likely disease-causing mutations in 39/264 patients (15%). This clearly indicates that de novo mutations play a strong role in epileptic encephalopathies. The estimate of 15% is a conservative estimate, as most “single hits” that did not fulfill the criteria of being mutation-resistant are not considered causative, including some double hits. However, with increasing sample sizes, the fraction of cases explained is likely to rise.
Bench to bedside. The Epi4K/EPGP study is a major milestone in epilepsy genetics. For the first time, we can claim that we have a reasonable chance of identifying causative genetic variants in a significant subset of patients. However, the current study also suggests a considerable genetic heterogeneity, suggesting a limitation for candidate or panel studies. Trio exome sequencing is likely to become a diagnostic gold standard for epileptic encephalopathies and future efforts will likely focus on lowering prices and providing better access to this technology.