Targeted Next Generation Sequencing reveals previously unidentified TSC1 and TSC2 mutations

Abstract

Background: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in TSC1 and TSC2. Conventional DNA diagnostic screens identify a TSC1 or TSC2 mutation in 75 - 90% of individuals categorised with definite TSC. The remaining individuals either have a mutation that is undetectable using conventional methods, or possibly a mutation in another as yet unidentified gene.

Methods: Here we apply a targeted Next Generation Sequencing (NGS) approach to screen the complete TSC1 and TSC2 genomic loci in 7 individuals fulfilling the clinical diagnostic criteria for definite TSC in whom no TSC1 or TSC2 mutations were identified using conventional screening methods.

Results: We identified and confirmed pathogenic mutations in 3 individuals. In the remaining individuals we identified variants of uncertain clinical significance. The identified variants included mosaic changes, changes located deep in intronic sequences and changes affecting promoter regions that would not have been identified using exon-only based analyses.

Conclusions: Targeted NGS of the TSC1 and TSC2 loci is a suitable method to increase the yield of mutations identified in the TSC patient population.

Figure 1

Coverage expressed as number of reads (forward + reverse) per base. Bases with >100 reads were considered to be adequately covered; coverage was inadequate for bases where the number of reads was <10. a. TSC1; b. TSC2.

Figure 2

Allele-specific amplification of TSC2 mosaic variants. a. Allele-specific amplification of the TSC2 c.3099C>G (p.Y1033*) mosaic variant. Specific amplification of the mutant c.3099G (upper panel) and normal c.3099C (lower panel) alleles from DNA from an unrelated healthy individual (control), DNA from individual III (III), both parents, and an individual with TSC heterozygous for the TSC2 c.3099C>G (p.Y1033*) pathogenic variant (c.3099C/G). b. Allele-specific amplification of the TSC2 c.2838-122G>A mosaic variant. Specific amplification of the mutant c.2838-122A (upper panel) and normal c.2838-122G (lower panel) alleles from DNA from an unrelated healthy individual (control), individual VI (VI) and both parents. c. RT-PCR of TSC2 mRNA from individual VI (VI) and 5 unrelated individuals (controls). An extra splice variant was amplified from RNA from individual VI (arrow), but not from the controls. d. Sequence of the additional RT-PCR product identified in individual VI (see Figure 2c, arrow). Sequences derived from exons 25 and 26 are in capitals; the premature stop codon is underlined. e. PCR amplification of the TSC2 c.226-1086del10 (intron 3; chr16 g.2102256del10) variant (rs140492671) from DNA from individuals IV and VII, the parents of individual IV and DNA from an unrelated healthy individual (control).

Figure 3

Pie charts showing the diagnostic yield in individuals with TSC. Percentages of individuals with definite TSC and a pathogenic TSC1 or TSC2 mutation (TSC mutation), an unclassified variant (UV) or no mutation identified (NMI) are indicated. a. Results of conventional molecular testing in individuals with definite TSC [12]. b. Results of targeted NGS of the TSC1 and TSC2 loci in individuals classified as TSC NMI after conventional molecular testing (this study).