Determining the pathogenicity of patient-derived TSC2 mutations by functional characterization and clinical evidence

Abstract

Tuberous sclerosis complex (TSC) is a genetic condition characterized by the growth of benign tumours in multiple organs, including the brain and kidneys, alongside intellectual disability and seizures. Identification of a causative mutation in TSC1 or TSC2 is important for accurate genetic counselling in affected families, but it is not always clear from genetic data whether a sequence variant is pathogenic or not. In vitro functional analysis could provide support for determining whether an unclassified TSC1 or TSC2 variant is disease-causing. We have performed a detailed functional analysis of four patient-derived TSC2 mutations, E92V, R505Q, H597R and L1624P. One mutant, E92V, functioned similarly to wild-type TSC2, whereas H597R and L1624P had abnormal function in all assays, consistent with available clinical and segregation information. One TSC2 mutation, R505Q, was identified in a patient with intellectual disability, seizures and autistic spectrum disorder but who did not fulfil the diagnostic criteria for TSC. The R505Q mutation was also found in two relatives, one with mild learning difficulties and one without apparent phenotypic abnormality. R505Q TSC2 exhibited partially disrupted function in our assays. These data highlight the difficulties of assessing pathogenicity of a mutation and suggest that multiple lines of evidence, both genetic and functional, are required to assess the pathogenicity of some mutations.

Functional Assessment ofTSC2 Variants Identified in Individuals with Tuberous Sclerosis Complex

“…Therefore, we also classified this variant as possibly pathogenic. The R505Q variant was originally identified in a family that did not fulfill the diagnostic criteria for TSC and was shown to have reduced activity relative to wild‐type TSC2 [Dunlop et al, ]. The variant has also been identified once in a control population of 10 758 individuals not selected according to their TSC status (http://evs.gs.washington.edu/EVS/).…”

“…In TSC‐associated lesions, loss or inactivation of the TSC1–TSC2 complex results in TORC1 activation and the constitutive phosphorylation of the downstream TORC1 targets including p70 S6 kinase (S6K), ribosomal protein S6 and 4E‐BP1 [Huang and Manning, ]. To determine whether unclassified TSC1 and TSC2 variants are disease‐causing, the effect of the changes on TSC1–TSC2 complex formation, on the activation of RHEB GTPase activity by the complex, and on the phosphorylation status of S6K, S6, and 4E‐BP1 can be determined [Dunlop et al, ; Hodges et al, ; Hoogeveen‐Westerveld et al, ; Inoki et al, ; Rendtorff et al, ; Qin et al, ].…”

Genetics, genomics, and genotype–phenotype correlations of TSC: Insights for clinical practice

“…When a novel variant in TSC1 or TSC2 is identified, the laboratory should follow the genetic criteria (Northrup and Krueger, 2013) and the American College of Medical Genetics and Genomics guidelines (Richards et al, 2015) to determine if the variant is studies may provide support in this process (Dunlop et al, 2011;Hoogeveen-Westerveld et al, 2011.…”

Measurement of epithelial thickness within the oral cavity using optical coherence tomography

“…TSC1 is important for maintaining the stability, activity, and correct intracellular localization of the TSC1‐TSC2 complex [Benvenuto et al, 2000; Cai et al, 2006; Chong‐Kopera et al, 2006]. To determine whether unclassified TSC1 and TSC2 variants are disease causing, the effect of the changes on TSC1‐TSC2 complex formation, on the activation of RHEB GTPase activity by the complex, and on the phosphorylation status of S6K, S6, and 4E‐BP1 can be determined [Dunlop et al, 2011; Nellist et al, 2005; Nellist et al, 2009].…”