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case_images: A novel homozygous TTC7A missense mutation was identified in the patient with familial MIA. (A) Pedigree chart of the proband's family. Consanguinity is indicated with a double horizontal line. Males are depicted as squares, females as circles, decreased individuals with a diagonal line. Abortion is depicted as a triangle with a diagonal line. Black filled symbols represent individuals who are clinically affected, while grey filled symbols indicate individuals who do not receive medical testing. The carrier is indicated by a dot in squares or circles. The proband who underwent WES is indicated by an arrow. (B) H&E staining of the patient's ileum of the atretic areas shows mucosal erosion, epithelial depolarization, small vascular hyperplasia and dense immune-cell infiltration within the lamina propria. Glands located in the lamina propria are dilated and twisted. (C) Sanger sequencing chromatogram depicts the c. 206 T>C missense mutation of TTC7A.
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case_images: The proband was a boy born to consanguineous parents of Chinese origin, and he was the fifth child of the couple ( Figure 1A ). The mother had a history of induced abortion (the fifth pregnancy,  Figure 1A ). Glands located in the lamina propria were dilated and twisted, with diffused submucosal lymphoproliferative changes ( Figure 1B ). TTC7A gene was inherited from his father, and the other from the mother ( Figure 1C ). This homozygous missense mutation was predicted as pathogenic by MutationTaster software. T cells was markedly reduced (6.01% CD3+ cells in the patient versus 68.9% and 60.4% in two healthy controls,  Supplementary Figure 1A ). There was a significantly reduced absolute number of T (both CD4 and CD8) cells (CD3, 182 cells/mul, reference 2179-4424 cells/mul; CD4, 47 cells/mul, reference 1461-3018 cells/mul; CD8, 123 cells/mul, reference 556-1687 cells/mul,  Table 2 ). Likewise, the proportion of CD19+ B cells in lymphocytes was markedly reduced (0.82% CD19+ cells in the patient versus 18.0% and 22.9% in two healthy controls,  Supplementary Figure 1B ). Our study found that the frequency of NK cells in this patient was comparable with that of age- and sex-matched healthy controls (5.10% CD3-CD56+ cells in the patient versus 5.41% and 5.67% in two healthy controls,  Supplementary Figure 1C ).
case_images: 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 3A 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.
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case_images: Phylogenetic analysis indicated that the amino acid sequences surrounding the proline 69 residue were highly conserved across different species by UGENE software ( Figure 2A ). As shown in  Figure 2B , this site-mutation would cause a conversion from leucine 69 into proline in exon 2 (p. In silico analysis indicated that the hydrogen bond present between Gly65 and Leu69 in the wild-type TTC7A was disrupted by the Leu69Pro mutation ( Figure 2C ).
case_images: Increased frequencies of DNT, transitional B cells and reduced TREC, KREC levels. (A) The absolute lymphocyte counts during treatment. (B) Reduced TREC levels of the patient as determined by multiple fluorescence qPCR. ***p < 0.001, ****p < 0.0001. (C) Representative dot plots showed markedly elevated frequencies of DNT compared with age- and sex-matched healthy control (HC), as measured by flow cytometry. (D) Reduced KREC levels of the patient as determined by multiple fluorescence qPCR. ***p < 0.001, ****p < 0.0001. (E) Representative dot plots showed significantly increased frequencies of CD24brightCD38bright transitional B cells, as measured by flow cytometry.
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case_images: Moreover, the dropped naive T cells were consistent with a subdued thymic output as the recent thymic emigrants evaluated by our TRECs determination indicated an obvious reduction (29.4 copies/mul in the patient versus 454.8, 153.9, 123.4 copies/mul in three age- and sex-matched healthy controls,  Figure 3B ). Besides, the patient presented with a markedly elevated double negative T (DNT, CD3+CD4-CD8-, 6.04% versus 15.9%,  Figure 3C ) cells, which suggested that an impeded or skewed T-cell differentiation does exist. The BCR recombination activity assessed by KREC levels was shown to decrease significantly (54.3 copies/mul in the patient versus 423.8, 145.6, 181.7 copies/mul in three age- and sex-matched healthy controls,  Figure 3D ). The residue B cells in circulation were predominantly transitional B cells (CD24brightCD38bright) and virtually no CD24+CD38dim naive B cells or CD24+CD38- memory B cells were presented (naive B cells, 11.4% versus 60.5%; memory B cells, 1.49% versus 14.2%; transitional B cells, 77.2% versus 13.6%  Figure 3E ).
case_images: 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). beta-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 x50, magnification for right panel is x100.
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case_images: Indeed, we found that patient PBMCs expressed very low level of TTC7A compared with age- and sex-matched healthy controls by western blot ( Figures 4A, B ). We then tested the TTC7A expression in mRNA level using colon tissue of the patient, qPCR analysis indicated that the expression level of TTC7A was lower in the patient compared with age- and sex-matched HD patients as control ( Figure 4C ). TTC7A expression in patient ileum tissue compared with that from meconium ileus patient as control ( Figure 4D ). These results confirmed that this homozygous missense mutation in TTC7A gene led to a reduced expression in lymphocytes and intestinal tissues.
case_images: GO and KEGG pathway analysis indicated the downregulation of genes implicated in lymphocyte development. GO terms (A) and KEGG pathways (B) enriched by the downregulated genes in the patient PBMCs compared with those of healthy controls. Enriched pathways with an adjusted p value of less than 0.05 were considered significantly enriched.
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case_images: The downregulated genes significantly enriched in GO terms and KEGG pathways were clustered into the function of T- and B-cell proliferation, differentiation and activation, as well as NK-cell mediated cytotoxicity ( Figures 5A, B ). The downregulation of genes implicated in lymphocyte functions might explain the TlowB-NKlow immune-phenotype we observed in this patient.
case_images: Downregulation of genes implicated in the PI4K-FAM126A-EFR3A pathway of colon tissue. (A-E, G, H) qPCR analysis of patient-derived resected narrow colon tissue showed reduced PI4KA
  (A), PI4KB
  (B), FAM126A
  (C), FAM126B
  (D), EFR3A
  (E), ROCK1
  (G), and ROCK2
  (H) expression compared with age- and sex-matched patients with Hirschsprung's disease (HD) as control. (F) qPCR analysis of patient-derived resected narrow colon tissue showed comparable EFR3B expression compared with HD. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant.
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case_images: We next examined the transcription level of PI4KA and PI4KB in colon tissue and found that PI4KA and PI4KB expression were significantly reduced in patient colon tissue compared with that of the same tissue location in HD controls ( Figures 6A, B ). Through qPCR analysis, we found that patient expressed much lower levels of FAM126A and FAM126B mRNA compared with that of HD controls ( Figures 6C, D ). Interestingly, we observed a reduced expression of EFR3A mRNA in patient colon tissue but not EFR3B ( Figures 6E, F ). RhoA signaling pathway, and crucial to normal ROCK function. Reduced levels of ROCK1 and ROCK2 expression were found in the patient colon tissue as shown in  Figures 6G, H .