Fig 2: In silico analysis of the mutant TTC7A and amino acid conservation of p. L69P. (A) Protein alignment indicated that p. L69P amino acids were highly conserved across different species. Residue L69 is shown in red arrow. (B) The schematic structure of TTC7A shows the location of the variant identified in this study. Illustration of TTC7A protein with TPR domains in red and the identified mutation is highlighted by an arrow. (C) The hydrogen bond presenting between Gly65 and Leu69 in the wild-type TTC7A is disrupted by the L69P mutation. The residue 69 together with certain nearby residues within 3Å is illustrated in the wild-type and the mutant by PyMOL. Computed hydrogen bonds are shown as yellow dashed lines. Residues Leu69/Pro69 are highlighted in blue.

Fig 3: Diminished TTC7A expression in lymphocytes and intestinal tissues. (A) Western blot of patient-derived PBMCs showed a severely impairment of TTC7A protein expression compared with age- and sex-matched healthy controls (HC). ß-actin was used as an internal loading control in the western blot. The result was a representative of three independent experiments. (B) The protein band intensity was quantified and analyzed by ImageJ software *p < 0.05, **p < 0.01. (C) The qPCR results demonstrated that patient’s colon tissue had a significant reduction in the amount of TTC7A expression compared with those from age- and sex-matched Hirschsprung's disease (HD) patients as control. TTC7A expression was normalized to 18S rRNA, and results were represented as relative expression normalized to the control. **p < 0.01, ***p < 0.001. (D) In the patient’s ileum of the atretic areas, immunohistochemistry demonstrated that TTC7A protein expression was diminished compared with the ileum tissue of age- and sex-matched meconium ileus patient (control). Magnification for left panel is ×50, magnification for right panel is ×100.