Targeted Genomic Sequencing of TSC1 and TSC2 Reveals Causal Variants in Individuals for Whom Previous Genetic Testing for Tuberous Sclerosis Complex Was Normal

Abstract

Tuberous sclerosis complex (TSC) is caused by inactivating variants in TSC1 and TSC2. Somatic mosaicism, as well as the size and complexity of the TSC1 and TSC2 loci, makes variant identification challenging. Indeed, in some individuals with a clinical diagnosis of TSC, diagnostic testing fails to identify an inactivating variant. To improve TSC1 and TSC2 variant detection, we screened the TSC1 and TSC2 genomic regions using targeted HaloPlex custom capture and next-generation sequencing (NGS) in genomic DNA isolated from peripheral blood of individuals with definite, possible or suspected TSC in whom no disease-associated variant had been identified by previous diagnostic genetic testing. We obtained >95% target region coverage at a read depth of 20 and >50% coverage at a read depth of 300 and identified inactivating TSC1 or TSC2 variants in 83/155 individuals (54%); 65/113 (58%) with clinically definite TSC and 18/42 (43%) with possible or suspected TSC. These included 19 individuals with deep intronic variants and 54 likely cases of mosaicism (variant allele frequency 1-28%; median 7%). In 13 cases (8%), we identified a variant of uncertain significance (VUS). Targeted genomic NGS of TSC1 and TSC2 increases the yield of inactivating variants found in individuals with suspected TSC.

The Genetics of Tuberous Sclerosis Complex and Related mTORopathies: Current Understanding and Future Directions

“…Newer tools, such as SpliceAI, are proving to be more sensitive for detecting deep intronic variants [ 73 ]. Many recent studies have also shown the utility in re-phenotyping and re-genotyping cases with NMI, where deep intronic variants have improved the diagnostic yield, including a recent study on TSC1/TSC2 [ 74 , 75 , 76 ]. Most deep intronic variants will require functional studies for their pathogenicity to be evaluated as per guidelines from the American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) [ 71 , 77 , 78 ].…”

“…In neurological tissue, intronic pathogenic variants that cause aberrant exonization have been associated with milder phenotypes (sometimes with only one clinical feature present in a systemic disease) hypothesized to be explained by significantly abrogated, but not eliminated, canonical transcript expression [ 76 , 102 , 103 ]. In a recent cohort study reevaluating NMI TSC patients with a definite or possible clinical TSC diagnosis, 19/155 individuals presented with deep intronic variants; of these intronic carriers, several of them did not have a definite clinical diagnosis nor did they display as many systemic features as did the rest of the cohort [ 74 ].…”