In the current study, our aim was to assess the utility of clinical exome sequencing in the diagnosis of rare neurological disorders in India. Exome sequencing of 19 patients with intellectual disability and/ or developmental delay provided confirmed diagnosis of 10 patients, whereby, ~50% of the mutations were of de novo origin. The study also elucidated 15 rare diseases that were diagnosed in these patients that would have otherwise been difficult to diagnose with cheaper but lower-resolution orthogonal methods such as microarray and karyotyping.
Eleven patients were identified carrying autosomal dominant de novo mutations, which is a known disease mechanism in rare neurological disorders [26, 27]. Furthermore, 8 out of 11 variants have previously been reported in other studies, thereby, further strengthening the evidence for the role of these variants in causing respective diseases. It is noteworthy that out of these 8 known de novo variants, 50% were missense variants and the remaining were either splice site or nonsense mutations. This finding has direct implication on genetic counselling whereby, de novo missense mutations can be associated with incomplete penetrance, whereas, nonsense and splice site mutations are not, as shown for SCN1A gene .
Despite finding known disease associated variants in majority of the patients, our study identified 5 novel variants in 5 genes- SCN1A, SCN2A, PGAP1, AFG3L2 and BSCL2. Diseases associated with these genes include Dravet syndrome (OMIM#607208), early infantile epileptic encephalopathy type 11 (OMIM#613721), mental retardation type 42 (OMIM#615802), spastic ataxia type 5 (OMIM#614487) and hereditary spastic paraplegia (OMIM#270685), respectively. Whilst 3 of the 5 variants are classed as pathogenic or likely pathogenic according to the ACMG-AMG classification , they are to be interpreted with caution as these variants would require replication in other patient and control cohorts as well as functional follow-up to implicate them as disease causing . None of the novel variants identified in the current study had an autosomal recessive or X-linked recessive inheritance pattern. This suggests an intriguing hypothesis of a reduced probability of finding novel recessive genes compared to dominant genes in neurological diseases in the Indian population; one that is supported by the data available from studies in the European population .
Interestingly, the diagnostic yield of clinical exome in our cohort was 53%, which is in concordance with the published literature . However, this needs to be placed in contrast with the role of de novo copy number variants (CNVs) that also play role in neurological disorder pathogenesis. Genomic microarray-based studies have shown a strong correlation between the number of genes affected by a CNV and phenotypic severity [27, 31]. Indeed, microarray-based studies have shown presence of rare, autosomal dominant form of de novo CNVs in approximately 10% of patients . Whilst microarray has been the mainstay for detection of CNVs, exome sequencing based large CNV detection (>400 kb) are increasingly becoming prominent in diagnosing neurological disorders . Furthermore, it is estimated that 45-60 de novo single nucleotide variants occur per genome per generation whereas the frequency of de novo >500kb CNVs is approximately 0.01 per genome per generation [33, 34]. This difference in mutation rates together with difference in mutation detection abilities could explain an enhancement in the diagnostic yield of exome sequencing by 24-33% over microarray . Therefore, utilization of an exome sequencing technique in identifying de novo variants (both SNV and CNV) compared to microarray-based approach in identifying only de novo CNVs in neurological diseases is likely to be an attractive approach.
Whilst the current study highlights several benefits of using an exome sequencing based approach in diagnosing neurological diseases, there are some caveats which needs to be highlighted. First, the diagnostic yield of 53% in our study could be misleading if taken at face value. Indeed, overall diagnostic yield has been reported between 50-70% in diagnosing moderate to severe intellectual disability diseases , depth and quality of patient phenotyping can impact diagnostic yield . The current study carried out an in-depth patient phenotype which may have aided in interpreting genotype data and disease diagnosis. Second, 11 patients in whom de novo SNVs were identified, Sanger validation for the mode of inheritance confirmation wasn’t carried out due to the unavailability of parental samples. Without Sanger sequencing confirmation in parental samples, it is conceivable that these variants may have been inherited from one of the parents. However, since these disorders have a significant impact on patient’s fitness , it is unlikely for either of the parents to be a carrier of these mutations. Hence, despite the absence of parental samples, replication of variants from the literature together with heterozygous status in the patient’s sample suggests these variants to be likely of de novo origin. Third, the current study had a small sample size compared to the large multicenter project like Deciphering Developmental Disorders (https://www.ddduk.org). However, the study was aimed to assess the utility of clinical exome sequencing in the Indian population rather than identification of novel genes and pathways involved in neurological disorders, hence had a requirement for a small sample size.