Human Mutation

Research Article

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

Table 2

Inactivating post-zygotic TSC1 and TSC2 variants identified using HaloPlex custom capture NGS. Individuals fulfilling the clinical criteria for definite TSC [3] are indicated with “TSC”; those fulfilling only criteria for possible TSC are indicated with “?”; individuals for whom clinical information was not available to us are indicated with “n/a.” VAF, variant allele frequency, refers to the proportion of reads containing the corresponding variant. #For large (> 150 bp) deletions, the VAF was estimated from the allele counts for informative benign variants within the deleted region (data not shown). Cases for which multiple family members or DNA samples were tested are indicated. Evidence for effects on pre-mRNA splicing was obtained by analysis of subject RNA isolated from peripheral blood (RNA1.) and/or by in vitro exon trap assay (RNA3; see Supplementary Information Tables S7 and S8). For functional studies please refer to the TSC2 Leiden Open Variation Database (LOVD; www.lovd.nl/TSC2). ClinVar (pathogenic, unless stated otherwise), the LOVD, and gnomAD were accessed on 13/2/2023. P: pathogenic; LP: likely pathogenic; VUS: variant of uncertain clinical significance; LB: likely benign. Variants were classified according to the American College of Medical Genetics and Genomics (ACMG) criteria [15] using the ALAMUT Visual Plus (version 1.7) software package.
SubjectClinical diagnosisVariant hg19 (GRCh37); NG_005895.1, NM_000548.3(TSC2); NG_012386.1, NM_000368.4(TSC1)VAF (%)Evidence for pathogenicityLOVDACMG
2.1TSCTSC2 c.139_140del, p.(Glu47Thrfs19), chr16:2098753_2098754del35/295 (12%)Frameshift; PPVS1, PM2, PP5 (LP)
2.2n/aTSC2 c.139-2A>G, p.?, chr16:2100399A>G128/1714 (7%)Destroys acceptor site; PPM2,PP3, PP5 (VUS)
2.3TSCTSC2 c.268C>T, p.(Gln90), chr16:2103385C>T67/1000 (7%)Stopgain; PPVS1, PM2, PP5 (LP)
2.4?TSC2 c.268C>T, p.(Gln90), chr16:2103385C>T53/993 (5%)Stopgain; PPVS1, PM2, PP5 (LP)
2.5n/aTSC2 c.299del, p.(Ala100Glyfs6), chr16:2103416del21/1197 (2%)FrameshiftPPVS1, PM2, PP5 (LP)
2.6TSCTSC2 c.352dup, p.(Val118Glyfs8), chr16:2104312dup23/1133 (2%)Frameshift, PPVS1, PM2, PP5 (LP)
2.7, duoTSCTSC2 c.848+281C>T, p.? chr16:2107460C>T70/1830 (4%)Creates a cryptic donor site; (affected parent of subject 1.5 (Table 1)PPP1, PP3, PM2, PP5 (VUS)
2.8TSCTSC2 c.848+281C>T, p.? chr16:2107460C>T183/642 (29%)Creates a cryptic donor site; PPP3, PM2, PP5 (VUS)
2.9TSCTSC2 c.976-15G>A, p.?, chr16:2110656G>A16/412 (4%)Creates a cryptic acceptor site; RNA1.; PPM2, PP3, PP5 (VUS)
2.10n/aTSC2 c.990_1005del, p.(Asn331Metfs27), chr16:2110685_2110700del248/2317 (11%)FrameshiftnovelPVS1, PM2 (LP)
2.11TSCTSC2 c.1120-28_1120-10del, p.?, chr16:2111845_2111863del103/1416 (7%)Destroys acceptor sitenovelPM2, PP3, PM2 (VUS)
2.12TSCTSC2 c.1210C>T, p.(Gln404), chr16:2111962C>T169/1509 (11%)Stopgain; PPVS1,PM2, PP5 (LP)
2.13TSCTSC2 c.1221C>A, p.(Tyr407), chr16:2111973C>A66/1929 (3%)Nonsense; PPVS1, PM2, PP5 (LP)
2.14, trioTSCTSC2 c.1258-1G>A, p.?, chr16:2112497G>A596/7538 (8%)Disrupts acceptor site; : no assertion providedPPP3, PM2, PP5 (VUS)
2.15, trioTSCTSC2 c.1361+1G>A, p.?, chr16:2112602G>A46/1439 (3%)Destroys donor site; PPP3, PM2. PP5 (VUS)
2.16TSCTSC2 c.1372C>T p.(Arg458), chr16:2112983C>T21/505 (4%)Nonsense: PPVS1, PM2, PP5 (LP)
2.17n/aTSC2 c.1492G>T p.(Glu498), chr16:2114321G>T226/990 (23%)Nonsense; novelPVS1, PM2, PP5 (LP)
2.18TSCTSC2 c.1636del, p.(Glu546Lysfs15), chr16:2115556del22/910 (2%)FrameshiftP/LPPVS1, PS3, PM2, PP3, PP5 (LP)
2.19TSCTSC2 c.1831C>T, p.(Arg611Trp), chr16:2120571C>T24/542 (4%)Missense; functional study (LOVD); P/LPPS3, PM2, PP3, PP5 (LP)
2.20TSCTSC2 c.1831C>T, p.(Arg611Trp), chr16:2120571C>T60/2193 (3%)Missense; functional study (LOVD); P/LPPS3, PM2, PP3, PP5 (LP)
2.21TSCTSC2 c.1832G>A, p.(Arg611Gln), chr16:2120572G>A19/1305 (1%)Missense; functional study (LOVD); PPS3, PM2, PP3, PP5 (LP)
2.22, trioTSCTSC2 c.1852del p.(Leu618Cysfs80), chr16:2121523del102/545 (19%)FrameshiftPPVS1, PM2, PP5 (LP)
2.23, 2 DNAsTSCTSC2 c.2108G>A, p.(Trp703), chr16:2122252G>Ablood 1: 119/1459 (8%)
blood 2: 49/690 (7%)		Stopgain; PPVS1, PM2, PP5 (LP)
2.24TSCTSC2 c.2119_2120ins[2098_2119;GTCT], p.(Lys707Argfs3), chr16:2122263insGAGTCTGACTGGAAGGTGCTGAGTCT19/1056 (2%)FrameshiftnovelPVS1, PM2 (LP)
2.25TSCTSC2 c.2251C>T p.(Arg751), chr16:2122880C>T54/1761 (3%)Stopgain; PPVS1, PM2, PP5 (LP)
2.26, trioTSCTSC2 c.2590C>T, p.(Gln864), chr16:2125844C>T83/2359 (4%)Stopgain; ; confirmed in DNA from an angiofibroma by DD PCRPPVS1, PM2, PP5 (LP)
2.27?TSC2 c.2590_2593dup, p.(Tyr865fs19), chr16:2125844_2125847dup10/1698 (0.6%)Frameshift; variant confirmed in DNA isolated from a facial angiofibroma: 41/888 (4%) (R. Oegema, personal communication)novelPVS1, PM2 (LP)
2.28TSCTSC2 c.2687G>A, p.(Trp896), chr16:2126116G>A30/1796 (2%)Stopgain; PPVS1, PM2, PP5 (LP)
2.29?TSC2 c.2713C>T, p.(Arg905Trp), chr16:2126142C>T8/706 (1%)Missense; functional study (LOVD); PPS3, PM2, PP3, PP5 (LP)
2.30, trioTSCTSC2 c.2742G>A, p.(Lys914=), chr16:2126171G>A206/1536 (13%)Destroys donor site; : LPP/LP/VUSPM2, PP5, PP3, BP4 (VUS)
2.31TSCTSC2 c.2838-122G>A, r.2837ins120fs, p.( Ser949Argins4), chr16:2127477G>A133/368 (36%)Creates cryptic acceptor site; RNA [11], RNA3.; LPPM2, PS3, PP3, PP5 (VUS)
2.32TSCTSC2 c.2838-122G>A, r.2837ins120fs, p.( Ser949Argins4), chr16:2127477G>A172/551 (31%)Creates cryptic acceptor site; RNA [11], RNA3.; LPPM2, PS3, PP3, PP5 (VUS)
2.33, duoTSCTSC2 c.2838-122G>A, r.2837ins120fs, p.(Ser949Argins4), chr16:2127477G>A1426/10593 (15%)Creates cryptic acceptor site; RNA [11], RNA3.; LPPM2, PS3, PP3, PP5 (VUS)
2.34, trioTSCTSC2 c.2838-122G>A, r.2837ins120fs, p.( Ser949Argins4), chr16:2127477G>A1130/4097 (15%)Creates cryptic acceptor site; RNA [11], RNA3.; LPPM2, PS3, PP3, PP5 (VUS)
2.35?TSC2 c.2838-122G>A, r.2837ins120fs, p.( Ser949Argins4), chr16:2127477G>A123/625 (20%)Creates cryptic acceptor site; RNA [11], RNA3.; LPPM2, PS3, PP3, PP5 (VUS)
2.36TSCTSC2 c.3094C>T, p.(Arg1032), chr16:2129160C>T12/636 (2%)Stopgain; PPVS1, PM2, PP5 (LP)
2.37TSCTSC2 c.3412C>T p.(Arg1138), chr16:2130180C>T15/1444 (1%)Stopgain; PPVS1, PM2, PP5 (LP)
2.38TSCTSC2 c.3412C>T p.(Arg1138), chr16:2130180C>T33/201 (16%)Stopgain; PPVS1, PM2, PP5 (LP)
2.39, trioTSCTSC2 c.3520del, p.(Arg1174Glyfs17), chr16:2130288del105/3175 (3%)Frameshift; PPVS1, PM2, PP5 (LP)
2.40TSCTSC2 c.3696dup, p.(Asn1233), chr16:2131681dup72/2640 (3%)Stopgain; PPVS1, PM2, PP5 (LP)
2.41, trioTSCTSC2 c.4351dup p.(Arg1451Profs73), chr16:2134574dup64/880 (7%)Frameshift; PPVS1, PM2, PP5 (LP)
2.42?TSC2 c.4488_4491C[6], p.(Ser1498Profs79), chr16:2134713_2134714dup132/1217 (11%)FrameshiftnovelPVS1, PM2 (LP)
2.43TSCTSC2 c.4490C>T, p.(Pro1497Leu), chr16:2134713C>T7/55 (13%)Missense; functional study (see Supplementary Information Figure S2)PPVS1, PM2, PP5 (LP)
2.44TSCTSC2 c.4537G>T, p.(Glu1513), chr16:2134995G>T69/521 (13%)StopgainPPVS1, PM2, PP5 (LP)
2.45, trioTSCTSC2 c.4959C>A, p.(Ser1653=), chr16:2136842C>A270/1100 (25%)Creates cryptic donor site; RNA3.; 1 × ClinVar: VUSLBPM2, BP4, PS3, PVS1 (LP)
2.46, 2 DNAsTSCTSC2 c.5024C>T p.(Pro1675Leu), chr16:2137898C>Tblood: 106/592 (18%)
cultured normal skin fibroblasts: 133/556 (24%)		Missense; functional study (LOVD); 4 × ClinVarPPM2, PS3, PP5 (LP)
2.47, trioTSCTSC2 c.5069-2A>G, p.?, chr16:2138047A>G17/551 (3%)Disrupts acceptor site; PPM2, PP3, PP5 (VUS)
2.48, trioTSCTSC2 c.5183_5184insGCCG, p.(Ser1728Argfs48), chr16:2138250_2138251insGCCG295/1487 (20%)FrameshiftnovelPVS1, PM2 (LP)
2.49, trioTSCTSC2 c.5227C>T, p.(Arg1743Trp), chr16:2138294C>T247/2031 (12%)Missense; functional study (LOVD); PPM1, PM2, PM5, PS3, PP3, PP5 (LP)
2.50, trioTSCTSC2 c.5228G>A, p.(Arg1743Gln), chr16:2138295G>A271/4114 (7%)Missense; functional study (LOVD); : LPPPM1, PM2, PM5, PS3, PP3, PP5 (LP)
2.51TSCTSC2 c.5228G>A, p.(Arg1743Gln), chr16:2138295G>A12/71 (17%)Missense; functional study (LOVD); : LPPPM1, PM2, PM5, PS3, PP3, PP5 (LP)
2.52, duoTSCTSC2 c.(?_-106)_(?_1362-50)del, p.?, chr16: (?_2097990)_(?_2112923)del(~15%)#Large deletion (~24 kb); confirmed by SNP array, MLPAnovelPVS1, PM2 (LP)
2.53TSCTSC2 c.(?_-106)_(102_?)del, p.?, chr16: (?_2097990)_(2138713_?)del(~10%)#Large deletion (~52 kb); confirmed by MLPAPPVS1, PM2, PP5 (LP)
2.54TSCTSC1 c.587C>T p.(Pro196Leu), chr9:135797282G>A137/803 (17%)Missense; functional study (see Supplementary Information Figure S2)LPPS3, PM2, PP5 (VUS)