Alphabet Soup: Recurrent De Novo Mutations in Novel Genes Causing Developmental and Epileptic Encephalopathies

Commentary

Developmental and epileptic encephalopathies (DEE) is a term that refers to the co-occurrence of epilepsy and intellectual disability where the underlying mechanism of either or both disorders may play a role (1). These disorders have a wide spectrum of etiologies ranging from acquired lesions, including strokes and malformations of cortical development, to genetic causes, including single gene disorders through chromosomal abnormalities (1). Determining the cause for DEE in a given patient can be challenging given the heterogeneous nature of potential pathogenic etiologies. In 2003, the American Academy of Neurology and Child Neurology Society published practice guidelines for the diagnostic workup of children with global developmental delay. The guideline recommends routine brain imaging, an EEG in children with features of epilepsy, and basic genetic testing including cytogenetic tests and Fragile X molecular testing (2). Further genetic testing assessing subtelomeric chromosomal rearrangements was recommended in moderate to severe cases of global developmental delay (2). However, newer genetic tests, including targeted gene panels and whole exome sequencing, were not clinically available routinely at the time of publication. Deciding what genetic test to perform for a patient with DEE can be challenging and should be based on clinical features and performed in a stepwise approach (3, 4).

More recently large cohorts of trios comprised of unaffected parents and children with sporadic DEE (e.g., Epi4K Consortium, Epilepsy Phenotype/Genotype Project, EuroEPINOMICS-RES Consortium) have undergone whole exome sequencing resulting in identification of de novo mutations (DNMs) in a minority of cases, although many cases remain unresolved or unclear (5, 6). If whole exome sequencing reveals a variant of unknown significance (VUS), it can be difficult to determine if that DNM is pathogenic or a benign variant, especially for a single patient. One strategy to address this challenge employs a statistical approach to look for gene enrichment in specific types of variants (e.g., missense, frameshift, nonsense, or splice site) that may validate the pathogenicity of DNM (7).

The central principle of the current study by Hamdan and colleagues was to identify new genes causing sporadic DEE in rare cases. To accomplish this, the authors focused on recurrent DNMs, including point mutations, small insertions or deletions (indels), and copy-number variations (CNVs) affecting both coding sequences and splice-site regions in novel genes from the Canadian Epilepsy Network (CENet), a cohort of 197 DEE patients and their unaffected parents. First, whole genome sequencing was performed followed by Sanger sequencing confirmation to identify high-quality DNMs affecting canonical splice sites and coding regions and CNVs with similar mutation rates to those of previous studies. Approximately 8% of the single nucleotide DNMs were canonical splice-site variants and nonsense variants predicted to cause a loss of function, a higher percentage than in control populations suggesting that some of these variants were likely pathogenic.

Next, several candidate genes were picked for further investigation because at least two individuals had DNMs that were predicted to be damaging. Additional variants were found by targeted sequencing of these genes in 595 unsolved DEE cases, as well as through searching the DECIPHER database and using the GeneMatcher tool (8, 9). These methods identified DNMs in NTRK2, GABRB2, CLTC, DHDDS, and NUS1 as causative or possibly causative of DEE. Finally, meta-analyses including variants identified in other studies of DEE or intellectual disability trios were used to determine if there was enrichment of DNMs in the candidate genes. This method provided additional support for the involvement of RAB11A, GABBR2, and SNAP25 in the pathogenesis of DEE. For each of these genes, the variants were mapped to putative functional domains of the protein or put into the context of global protein function with a resultant hypothesis about how these variants could cause DEE. On the whole, this combination of approaches provided a molecular/genetic diagnosis in 63/197 (32%) DEE patients in the CENet cohort.

Overall, this study highlighted the importance of combining relevant clinical phenotype, in this case both epilepsy and developmental encephalopathy, and evaluating recurrence of a DNM in a particular gene to assess whether a variant is benign or pathogenic. This approach can improve diagnostic yield as seen with the 1/3 of solved genetic diagnoses in the CENet cohort in this study. In most cases, there were multiple DNMs throughout different regions of the gene with variable phenotypes, but for two of the genes there were multiple recurrences of DNMs affecting the same highly conserved amino acid residue. Hamdan and colleagues provided evidence of a high rate of recurrence of DNMs in eight genes elucidating their role as pathogenic in rare cases of DEE. To evaluate recurrence of DNMs in candidate genes, the authors employed several methods in addition to whole genome sequencing. The GeneMatcher tool and DECIPHER database are increasingly an important way to harness the power of rare individual variants within a broadly and globally accessible platform (8, 9). Furthermore, the meta-analysis and statistical gene enrichment strategies will be valuable for validating candidate genes in further large DEE cohort sequencing studies.

Interestingly, many of the genes identified in the current study by Hamdan et al. encode proteins that interact with proteins already implicated in genetic forms of epilepsy. Thus, many of the fundamental biological processes that can go awry in epilepsy and developmental delay may have already been identified. For example, several GABA receptor family members are causative of epilepsy and this study discovered an additional two GABA receptor subunits that can cause DEE. Further novel gene discovery in DEE will likely implicate other genes in similar essential pathways that may be amenable to modulation by novel therapeutics. As more detailed genetic testing becomes widely available and more affordable for routine clinical practice, practitioners will need updated guidelines for determining what genetic test is appropriate for a given case. For VUS on whole exome sequencing or whole genome sequencing, awareness of the continually rising number of genes involved in the key biological pathways producing epilepsy and developmental delay will help treating clinicians and genetic counselors inform families about the relevance and impact of these variants for the individual patient with sporadic DEE.

Supplementary Material