Fig 2: New THOC2 variants (purple). These include the first Arg77Cys recurrent (boxed) and Del-Exon-37-38 variants (red bold). Previously published THOC2 variants (black) and structural features are also shown (Kumar et al., 2015, 2018).

Fig 3: THOC2 variant protein analysis in patient-derived cell lines. (A) THOC2 protein in affected son with Del-Ex37-38, unaffected carrier mother and control fibroblasts. Total protein lysates were western blotted with an anti-THOC2-I antibody that binds to a region between amino acids 1,400–1,450 coded by Ex32-34 mRNA (upper panel) and anti-THOC2-II antibody that binds to a region between amino acids 1543–1593 coded by Ex37-38 mRNA (lower panel). Samples were probed for ß-Tubulin as a loading control. (B) THOC2 protein in p.Arg77Cys, p.Tyr881Cys, affected son with Del-Ex37-38, unaffected carrier mother and control LCLs. Total protein lysates were western blotted with anti-THOC2-I, anti-THOC2-II, anti-THOC1, anti-THOC5 and anti-ß-Tubulin (loading control) antibodies. Del-Ex37-38 affected fibroblasts (Lane 1 in panel A) and LCLs (Lane 3 in panel B) showing presence of a C-terminally deleted smaller but higher levels of the THOC2 protein when western blotted with anti-THOC2-I antibody (upper panels) that is absent in the affected fibroblast lysates probed with anti-THOC2-II antibody (lower panels). However, normal full-length THOC2 protein is present in the carrier mother and controls western blotted with both the anti-THOC2-I and anti-THOC2-II antibodies (Lane 2 in upper and lower panels in A and Lane 4 in upper and lower panels in B). The p.Arg77Cys and p.Tyr881Cys THOC2 levels are reduced. (C) THOC2 protein levels in p.Leu438Pro (reported to have reduced protein stability; Kumar et al., 2015), p.Arg77Cys, p.Tyr881Cys, Del-Ex37-38 affected son, his unaffected carrier mother and control LCLs. Total protein lysates were western blotted with anti-THOC2-II, anti-THOC5 and anti-ß-Tubulin (loading control) antibodies. (D) The p.Asn666Asp levels are moderately reduced in the affected fibroblasts. p.Asn666Asp affected son, his unaffected carrier mother and control fibroblast total lysates were western blotted with the antibodies as shown. p.Leu438Pro fibroblasts previously reported having reduced THOC2 protein stability were included as controls (Kumar et al., 2015). Vertical lines on western blot images indicate the site of deleted sample lanes.

Fig 4: THOC2 localization is unaltered in variant fibroblasts. THOC2-II antibody detects THOC2 protein in fibroblasts of the Del-Ex37-38 unaffected carrier mother (A) but not affected son (not shown, as we detected no fluorescence signal). However, the THOC2-I antibody detects THOC2 protein in fibroblasts of unaffected carrier mother (B) and the affected son with Del-Ex37-38 (C). Localization p.Asn666Asp THOC2 protein in fibroblasts of an affected son (D), his unaffected carrier mother (E) and control (F).

Fig 5: Del-Ex37-38 results in the loss of 34 C-terminal amino acids in the affected individual. (A) Part of the THOC2 gene showing Ex34 to Ex39 and positions of primers. Ex37-38 deleted mRNA translates a 1575 amino acid protein, lacking 34 C-terminal amino acids but adding 16 amino acids encoded from the 3' UTR region of the deleted mRNAs. (B) Sanger sequencing chromatograms of DNA amplified from LCL and fibroblast cDNAs of the affected individual and his heterozygous unaffected carrier mother using primers P276/P392 located within Ex34 and Ex39. Wild type (1593 amino acid) and C-terminal deleted THOC2 protein (1575 amino acids) translated from Del-Ex37-38 mRNA is also shown. (C) Ex37-38 coding sequence is deleted in both fibroblast and LCL THOC2 mRNAs of the affected son but not in the unaffected carrier mother. Total fibroblast and LCL RNAs were reverse transcribed and PCR amplified using primers P276/P392. PCR products (999 bp from the carrier mother and 876 bp from the affected son) were gel purified and Sanger sequenced using P276 and P392 primers.