Molecular and Functional Assessment of TSC1 and TSC2 in Individuals with Tuberous Sclerosis Complex

Abstract

Tuberous sclerosis complex (TSC) is an autosomal dominant neurodevelopmental disorder and multisystem disease caused by pathogenic DNA alterations in the TSC1 and TSC2 tumor suppressor genes. A molecular genetic diagnosis of TSC confirms the clinical diagnosis, facilitating the implementation of appropriate care and surveillance. TSC1 and TSC2 encode the core components of the TSC1/2 complex (TSC1/2), a negative regulator of the mechanistic target of rapamycin (MTOR) complex 1 (TORC1). Functional analysis of the effects of TSC1 and TSC2 variants on TORC1 activity can help establish variant pathogenicity. We searched for pathogenic alterations to TSC1 and TSC2 in DNA isolated from 116 individuals with a definite clinical diagnosis of TSC. Missense variants and in-frame deletions were functionally assessed. Pathogenic DNA alterations were identified in 106 cases (91%); 18 (17%) in TSC1 and 88 (83%) in TSC2. Of these, 35 were novel. Disruption of TSC1/2 activity was demonstrated for seven TSC2 variants. Molecular diagnostics confirms the clinical diagnosis of TSC in a large proportion of cases. Functional assessment can help establish variant pathogenicity and is a useful adjunct to DNA analysis.

Keywords: Alu repeat; TSC1; TSC2; missense variant; neurodevelopmental disorder; pathogenic alteration; splicing variant; tuberous sclerosis complex.

Figure 1

Schematic representation of TSC1 (A) and TSC2 (B) protein domains according to NP_000359.1 and NP_000539.2. (A) Location of each pathogenic variant detected in TSC1. (B) Location of each pathogenic variant detected in TSC2. Splicing variants and CNVs are not represented in this figure.

Figure 2

Schematic representation of pathogenic TSC2 splice variants. Intron canonical splice sites and splicing branchpoint are underlined in a putative RNA sequence. Exon-intron borders are represented by vertical bars.

Figure 3

TSC2 c.(975+627)_(1716+41)del intragenic deletion. (A) Sanger sequencing of the intragenic TSC2 deletion c.(975+627)_(1716+41)del identifies the breakpoints. (B) UCSC genome browser mapping of the breakpoints in TSC2 introns 10 and 16. (C) Diagram illustrating the positions of SINE/Alu and LINE/L1 repeat sequences in TSC2 introns 10 and 16, respectively, within and adjacent to each breakpoint (BP1 and BP2). Exons are shown as patterned boxes. The segments of introns 10 and 16 that display 83% similarity are indicated by lines.

Figure 4

Functional assessment of the TSC2 c.52C>G (p.L18V), c.499T>C (p.W167R), c.3132_3282del (p.1044_1094del) indicated as 1044del50, c.4493G>T (p.S1498I), c.4823_4825del (p.1608del), c.4840_4842del (p.1614del), and c.5227C>G (p.R1743G) variants. 3H9-1B1 (TSC2/TSC1 double knockout HEK 293T) cells were transfected with the indicated TSC2 variant expression constructs, together with expression constructs for myc-tagged TSC1 and S6K. WT-TSC2 as well as the pathogenic TSC2 c.1832G>A (p.R611Q) variant (R611Q) and cells with no TSC2 expression (TSC1/S6K only) were included as controls. GFP-TSC2 (GFP-TSC2 only) was employed to monitor transfection efficiency. Twenty-four hours after transfection, the cells were harvested, and the cleared cell lysates were analyzed by immunoblotting (A). The signals for TSC2, TSC1, total S6K (S6K), and T389-phosphorylated S6K (T389) were determined per variant, relative to the wild-type control (TSC2) in two independent experiments. Mean TSC2 (B), TSC1 (C), and S6K (D) signals and mean T389/S6K ratio (E) are shown for each variant. In each case, the dotted line indicates the signal/ratio for wild-type TSC2 (normalized to 1.0). Error bars represent the standard error of the mean. Statistical significance for comparisons with WT-TSC2 is indicated with an asterisk (p < 0.025; Student’s paired t-test).

Figure 5

Structural analysis of TSC2 variants affecting the TSC2 GAP domain. (A) Amino acid sequence alignment of human TSC2 (NP_000539.2) and the human RAP1GAP domain (NP_001337453.1). TSC2 Y1608 and RAP1GAP Y256 are indicated in red. (B) The TSC2:Y1608 residue is highlighted in red in the gray β-sheet part of the ribbon diagram of protein data bank structure (accession number: 1SRQ).