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Significance of Neurexin and Neuroligin Polymorphisms in Regulating Risk of Hirschsprung’S Disease Yanhong Li, Hui Liu, Yubin Dong

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Significance of Neurexin and Neuroligin Polymorphisms in Regulating Risk of Hirschsprung’S Disease Yanhong Li, Hui Liu, Yubin Dong Original research J Investig Med: first published as 10.1136/jim-2017-000623 on 4 April 2018. Downloaded from Significance of neurexin and neuroligin polymorphisms in regulating risk of Hirschsprung’s disease Yanhong Li, Hui Liu, Yubin Dong ► Additional material is ABSTRact published online only. To By performing a basic case–control study among a Significance of this study view please visit the journal Chinese population, the aims of this study were to online (http:// dx. doi. org/ 10. What is already known about this subject? 1136/ jim- 2017- 000623). explore if single nucleotide polymorphisms (SNPs) within neurexin and neuroligin were associated with ► The morbidity of Hirschsprung’s disease Department of Pediatrics, (HD) was ranked high at second of all Zhoukou Central Hospital, susceptibility to Hirschsprung’s disease (HD). Eleven Zhoukou, Henan Province, SNPs within neurexin and neuroligin were selected in disorders relevant to newborn congenital China this basic case–control study, and this study recruited digestive tract malformation. 210 children with HD and 187 healthy children. The ► About 20% of patients with HD were Correspondence to t-test and Χ2 test were used to find the difference accompanied with family heredity. Dr Yanhong Li, Department between case and control in their clinical variables. ► The development of HD may be related of Pediatrics, Zhoukou with migration of neural crest cells in the Central Hospital, Henan OR and 95% CI were used to assess the association Province 466000,China; between HD susceptibility and neurexin/neuroligin embryonic period. xushuai_ yy@ 163. com polymorphisms/haplotypes. Several SNPs were What are the new findings? significantly associated with altered risk of HD in the The C allele of NRXN1 rs1421589 acted as a Accepted 29 January 2018 Chinese Han population, including rs1421589 within ► protective element for HD risk. NRXN1, rs11795613 and rs4844285 within NLGN3, The mutant alleles of rs11795613 (A>G) as well as rs5961397, rs7157669 and rs724373 ► and rs4844285 (G>A) within NLGN3 were within NLGX4X (all P<0.05). Further studies presented both associated with significantly lower that the effects of rs1421589 within NRXN1, susceptibility to HD than their wide alleles. rs4844285 and rs11795613 within NLGN3, as well as The C allele of NLGX4X rs5961397 could rs5961397 within NLGX4X on HD phenotypes were ► reduce the risk of HD in comparison to the also statistically significant (all P<0.05). Conclusively, T allele. http://jim.bmj.com/ the polymorphisms and haplotypes situated within The subjects carrying mutant alleles of neurexin and neuroligin were markedly associated ► rs7157669 and rs724373 were more prone with the onset of HD, implying that mutations of to suffer from HD than carriers of the neurexin and neuroligin might serve as the treatment corresponding wide alleles. target for HD for the Chinese children. How might these results change the focus of research or clinical practice? on September 29, 2021 by guest. Protected copyright. ► The results strongly evidenced the INTRODUCTION hypothesis that the aberrant neuroligin and Hirschsprung’s disease (HD), a developmental neurexin expressions caused by genetic disorder, was featured by congenital malforma- mutations participated in the connections tions in the gastrointestinal tract, and it could between intestinal neurons, leading to be commonly found among cases of pediatric developmental immaturity of enteric surgery. The morbidity of HD (ie, 1/2000– nervous system and thus the presence of 1/5000) was ranked high at second of all disor- such diseases as HD. ders relevant to newborn congenital digestive tract malformation, and males possessed three- fold more susceptibility to HD than females.1 The pathological changes of HD lied in the Up to now, the familial incidence of HD was dysfunctional enteric nervous system (ENS), demonstrated to account for around 3.6%– which was particularly manifested as shortage of 7.8% of the overall incidence of HD, and about intestinal ganglion cells within myenteric nerve 20% of patients with HD were accompanied plexus or intestinal submucosa of distal spastic with family heredity.4 Thus, the consensus To cite: Li Y, Liu H, colon and disorderly arranged nerve fibers. The has been reached that one dominating etio- Dong Y. J Investig Med Epub ahead of print: above symptoms would render intestinal canal logic factor of HD was genetic variation of [please include Day Month to take the spastic shape, making the fecal sedi- ENS. For instance, single nucleotide polymor- Year]. doi:10.1136/jim- ment difficult to empty the dung and facilitating phisms (SNPs) within RET (ie, rs2435357 and 2017-000623 compensatory dilation of proximal intestine.2 3 rs2506030) and SEMA3 (ie, rs12707682) could Li Y, et al. J Investig Med 2018;0:1–8. doi:10.1136/jim-2017-000623 1 Copyright 2018 by American Federation for Medical Research (AFMR). Original research J Investig Med: first published as 10.1136/jim-2017-000623 on 4 April 2018. Downloaded from impart tremendous risk of HD development and disor- colon and terminal ileum (<30 cm far from ileocecal valve). ders of phenotypic conditions among a Chinese popula- Besides, 187 normal children receiving the physical exam- tion.5 A Chinese genome-wide association study disclosed ination in our hospital were included in the control group, that NRG3 mattered a lot to the onset of HD,6 yet later a and they all had no malformations relevant to digestive case–control study did not uncover the association of three tract and neural crest. Parents of all subjects have signed NRG3 SNPs with HD within a Chinese crowd.7 None- informed consents. theless, it was documented that rs78356888 of NRG1, as well as rs2506030 and rs2435357 of RET, was signifi- cantly correlated with susceptibility to HD.7 Since the study Selection of SNPs results about the association of SNPs with HD risk were The investigated SNPs were partly selected with aid of quite limited and were sometimes in dispute, finding novel the bioinformation database (http://www. ncbi.nlm. nih. gov/ SNP/). Specifically, with the SNP browser software, target SNPs or conduction of replicated studies were critical 2 to hindering the HD onset. tag SNPs were chosen if their r was more than 0.8 and Of note, the achievements related with the development their minor allelic frequency was at least 5%, in accordance with the pairwise r2 method. Other SNPs were deemed as of central nervous system were vital to the exploration 15 16 with respect to development of ENS, for that ENS and eligible based on related documentations. Finally, SNPs central nervous system were both originating from embry- within NRXN1 (ie, rs1363032 and rs1424589), NRXN3 onic neural crest.8 Since production of synapse symbol- (ie, rs11624704, rs7157669 and rs724373), NLGN1 ized complete development of central nervous system, the (rs13074723 and rs1488547), NLGN3 (rs11795613 and sensory and motor functions of ENS should also be guar- rs4844285) and NLGX4X (rs6529901 and rs5961379) anteed by the appropriate synaptic actions. It has been were chosen and arranged for the following studies. verified that neurexin family and neuroligin family were located within presynaptic membranes or across the subsyn- Genotyping 9 10 aptic membranes. Furthermore, neurexin might interact Peripheral blood (volume: 2 mL) was extracted from HD with neuroligin to mediate synaptic junctions within the and healthy children and 3.8% sodium citrate was used 11–13 central nervous system via affecting scaffolding protein. for anticoagulation. The Qiagen DNA extraction kit was Hence, it was hypothesized that mutants of neurexin and utilized to extract the DNA, and specific procedures were neuroligin SNPs were highly potential factors accounting carried out strictly in accordance with the manufacturers’ for ENS-relevant disorders, such as HD. protocols. The primers were designed based on the primer Accordingly, the current study was aimed to explore 5 software and were synthesized with assistance of Shanghai whether SNPs within neurexin and neuroligin were associ- Invitrogen Biotechnology Co (Supplementary table 1). All ated with susceptibility to HD. the primers were all purified via polyacrylamide gel electro- phoresis and were then recycled using C18 column. Then PCR-restriction fragment length polymor- METHODS http://jim.bmj.com/ phism (RFLP) was implemented to genotype the SNPs. Subjects The PCR reaction system (50 µL) mainly included double Totally 210 children with HD were retrospectively distilled water (32.5 µL), 10× buffer (5.0 µL), 25 mmol/L recruited from Zhoukou Central Hospital from March magnesium chloride (4.0 µL), 10 mmol/L deoxyribonu- 2011 to February 2016. They all experienced the transanal cleotide triphosphate (4.0 µL), 20 μmol/L sense primer pull-through operation for the first time. According to the (1.0 µL), 20 μmol/L antisense primer (1.0 µL), 5 U/µL Taq diagnostic criteria for HD declared by the fourth interna- 14 DNA polymerase and 100 ng DNA template (2.0 µL). on September 29, 2021 by guest. Protected copyright. tional conference on HD and related neural crest diseases, Moreover, the PCR reaction was proceeded according to the children with HD were preoperatively managed with the following process: (1) predenaturation at 95°C for barium enema examination, rectum mucosa biopsy, H&E 5 min; (2) 35 cycles of 95°C for 40 s, 60°C for 30 s and staining and acetylcholinesterase staining. Consecutive 72°C for 30 s and (3) extension at 72°C for 10 min and (4) sections were performed for each sample, and HD was conservation at 4°C. confirmed when hardly any neurons were observed and obvious hyperplasia of nerve plexus were found within submucosa. Since there existed a cell area featured by phys- Operation iological hyporesponsiveness and aganglionosis beyond the The operation methods were selected in line with the age normal rectal line, biopsies for HD diagnosis were obtained of children and the length of pathological intestinal tract.
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