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

Figure 2

Examples of HaloPlex discovery and validation data for TSC2 variants identified in the TSC ' no mutation identified' (NMI) cohort. (a–c) Subject 1.15, germline TSC2 c.2967-460G>A variant. (a) Effect of the TSC2 c.2967-460G>A variant on pre-mRNA splicing as predicted using the ALAMUT Visual Plus software package (version 1.7). Green blocks indicate a possible 3 acceptor site, and blue blocks indicate a non-canonical 5 donor site. (b) RT-PCR was performed on RNA isolated from subject 1.15, 2 control individuals (c), and a sample lacking RNA (-) using primers specific for TSC2 exons 26 and 27. An abnormal product only was amplified from RNA from subject 1.15, most likely due to preferential amplification of the abnormal transcript. Exon 26 is skipped in the majority of wild-type TSC2 transcripts in blood and the wild-type NM_000548.3(TSC2) transcript that includes exon 26 is often present at very low levels (data not shown). Size markers are indicated; bp, base pairs. (c) The Sanger sequencing of the RT-PCR products revealed the insertion of intronic sequence TSC2 r.2966ins2967-458_2967-263, p.(Ser989Argfs82) in subject 1.15, but not in controls. Sequence corresponding to TSC2 exons 26 and 27 is indicated in blue and yellow, respectively. (d–g) Subject 2.24, post-zygotic TSC2 c.2119_2120ins[2098_2119;GTCT] variant. (d) Screenshot of the HaloPlex variant discovery data in the IGV. Reads are shown as grey bars; the insertion is shown in purple in multiple reads. The TSC2 locus reference sequence is indicated; nucleotides corresponding to TSC2 exon 20 are boxed. (e) Screenshot of the Nextera XT variant validation data in the IGV. Reads are shown as grey bars; the insertion is shown in purple in multiple reads. The TSC2 locus reference sequence is shown in (d). (f) Allele-specific (AS)-PCR to show the presence of the TSC2 c.2119_2120ins[2098_2119;GTCT] variant in genomic DNA from subject 2.24, and the absence of the variant from control samples with (c) or without (-) genomic DNA. Size markers are indicated; bp, base pairs. The AS primers are shown, with the variant-specific primer (AG>GT-f) and nucleotides indicated in red. (g) Schematic of the TSC2 c.2119_2120ins[2098_2119;GTCT] variant. Nucleotides corresponding to the WT-f and AG>GT-f primers are underlined, the insertion is shown in red with the duplicated sequence shaded in blue. Sequences corresponding to TSC2 exon 20 are boxed. (h–j) Subject 2.6, post-zygotic TSC2 c.352dup variant, and subject 2.29, post-zygotic TSC2 c.2713C>T variant. (h) Screenshot of the HaloPlex variant discovery data in the IGV for subject 2.6. Reads are shown as grey bars; a G insertion is shown in purple in multiple reads. The TSC2 locus reference sequence is indicated; nucleotides corresponding to TSC2 exon 5 are boxed. (i) Screenshot of RNASeq variant validation data in the IGV. Reads are shown as grey bars; the G insertion is shown in purple in multiple reads. The TSC2 locus reference sequence is shown as in (h); RNA for RNASeq analysis was prepared from cultured skin fibroblasts; +CHX indicates that the fibroblasts were treated with cycloheximide. (j) Screenshot of the HaloPlex variant discovery data in the IGV for subject 2.29. Reads are shown as grey bars; a C>T transition is shown in red in multiple reads. The TSC2 locus reference sequence is indicated; nucleotides corresponding to TSC2 exon 24 are boxed. (k) AS-PCR to confirm the presence of the TSC2 c.352dup and TSC2 c.2713C>T variants in genomic DNA from subjects 2.6 and 2.29 respectively, but not in control genomic DNA samples (c) or in the absence of DNA (-). Size markers are indicated; bp, base pairs. The AS primers are shown, with the variant-specific primer and nucleotides indicated in red.

(a) TSC2 c.2967-460G>A splice site prediction analysis

(b) Subject 1.15: RT-PCR

(c) Subject 1.15: TSC2 c.2967-460G>A, r.2966ins2967-458_2967-263, p.(Ser989Argfs82) Sanger sequence analysis

(d) Subject 2.24, HaloPlex NGS

(e) Subject 2.24, Nextera NGS

(f) Subject 2.24, AS-PCR

(g) Subject 2.24, TSC2 c.2119_2120ins[2098_2119;GTCT], p.(Lys707Serfs3)

(h) Subject 2.6, HaloPlex NGS

(i) Subject 2.6, RNASeq

(j) Subject 2.29, HaloPlex NGS

(k) Subjects 2.6 and 2.29, AS-PCR