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BackgroundHirschsprung disease (HSCR) is a heterogeneous genetic disease characterized by the absence of ganglion cells in the intestinal tract. The REarranged during Transfection (RET) is the most responsible gene for its pathogenesis. RET’s somatic mosaicisms have been reported for HSCR; however, they are still under-recognized. Therefore, we determined the frequency of somatic mutation of RET rs2435357 in HSCR patients at our institution.MethodsWe performed RET rs2435357 genotyping from 73 HSCR formalin-fixed and paraffin-embedded (FFPE) rectal and 60 non-HSCR controls using the PCR-RFLP method. Subsequently, we compared those frequencies of genotypes for RET rs2435357 with our previous genotyping data from 93 HSCR blood specimens.ResultsThe frequencies of genotypes for RET rs2435357 in HSCR paraffin-embedded rectal were CC 0, CT 11 (15%), and TT 62 (85%), whereas their frequencies in HSCR blood samples were CC 4 (4.3%), CT 22 (23.7%), and TT 67 (72%). Those frequencies differences almost reached a significant level (p = 0.06). Moreover, the frequency of RET rs2435357 risk allele (T) was significantly higher in HSCR patients (135/146, 92.5%) than controls (46/120, 38.3%) (p = 3.4 × 10–22), with an odds ratio of 19.74 (95% confidence interval = 9.65–40.41).ConclusionOur study suggests somatic mosaicism in HSCR patients. These findings further imply the complexity of the pathogenesis of HSCR. Moreover, our study confirms the RET rs2435357 as a significant genetic risk factor for HSCR patients.

Contains fulltext : 220495.pdf (Publisher’s version ) (Open Access) Background: The semaphorin 3D (SEMA3D) gene has been implicated in the pathogenesis of Hirschsprung disease (HSCR), a complex genetic disorder characterized by the loss of ganglion cells in varying lengths of gastrointestinal tract. We wished to investigate the role of SEMA3D variants, both rare and common variants, as well as its mRNA expression in Indonesian HSCR patients. Methods: Sanger sequencing was performed in 54 HSCR patients to find a pathogenic variant in SEMA3D. Next, we determined SEMA3D expression in 18 HSCR patients and 13 anorectal malformation colons as controls by quantitative real-time polymerase chain reaction (qPCR). Results: No rare variant was found in the SEMA3D gene, except one common variant in exon 17, p.Lys701Gln (rs7800072). The risk allele (C) frequency at rs7800072 among HSCR patients (23%) was similar to those reported for the 1,000 Genomes (27%) and ExAC (28%) East Asian ancestry controls (p = 0.49 and 0.41, respectively). A significant difference in SEMA3D expression was observed between groups (p = 0.04). Furthermore, qPCR revealed that SEMA3D expression was strongly up-regulated (5.5-fold) in the ganglionic colon of HSCR patients compared to control colon (ΔC(T) 10.8 ± 2.1 vs. 13.3 ± 3.9; p = 0.025). Conclusions: We report the first study of aberrant SEMA3D expressions in HSCR patients and suggest further understanding into the contribution of aberrant SEMA3D expression in the development of HSCR. In addition, this study is the first comprehensive analysis of SEMA3D variants in the Asian ancestry.