Bioscience Reports (2019) 39 BSR20182290 https://doi.org/10.1042/BSR20182290 Research Article Associations of SLC6A20 genetic polymorphisms with Hirschsprung’s disease in a Southern Chinese population * * Xiaoli Xie , Qiuming He , Lihua Huang, Le Li, Yuxiao Yao, Huimin Xia, Jinglu Zhao, Wei Zhong and Downloaded from http://portlandpress.com/bioscirep/article-pdf/39/8/BSR20182290/863686/bsr-2018-2290.pdf by guest on 29 September 2021 Yan Zhang Department of Pediatric Surgery, Guangzhou Institute of Pediatrics, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou 510623, Guangdong, China Correspondence: Wei Zhong ([email protected]) or Yan Zhang ([email protected]) Hirschsprung’s disease (HSCR) is a neurodevelopmental disorder characterized by the ab- sence of nerves in intestine with strong genetic components. SLC6A20 was found to be associated with HSCR in Korean population waiting for replication in an independent co- hort. In the present study, ten single nucleotide polymorphisms (SNPs) in the SLC6A20 were selected from Southern Chinese with 1470 HSCR cases and 1473 ethnically matched healthy controls. Our results indicated that SNP rs7640009 was associated with HSCR and SLC6A20 has a gene–dose effect in the extent of the aganglionic segment during enteric nervous system (ENS) development. It is the first time to reveal the relationship between SNP rs2191026 and HSCR-associated enterocolitis (HAEC) susceptibility. Introduction Hirschsprung’s disease (HSCR) is a congenital disease characterized by the absence of intrinsic ganglion cells in the submucosal and myenteric plexuses of the intestinal tract involving a variable portion. It is common that HSCR leads to the mechanical intestinal obstruction in infants and children. At present, surgery is a unique way to cure HSCR in spite of its fraught complications such as constipation, diarrhea, incontinenceandsoon.Ifleftuntreated,HSCRcanleadtodeath.SimilartoWesternEurope[1]andNorth America [2], the incidence in Asians is approximately 1 in 5000 neonates [3,4]. HSCR was classified by the length of intestine lacking nerve cells, known as short-segment HSCR (SHSCR) when the aganglionic segment does not extend beyond the upper sigmoid, long-segment HSCR (LHSCR) when the agangliono- sis extends proximally to the sigmoid, or total colonic aganglionosis (TCA) when the aganglionosis ex- tends throughout the entire colon [5]. To the most extreme, total intestinal aganglionosis (TIA), whose aganglionosis extends to almost total intestines in sporadic rare cases, can be deadly [6]. While all cases present a functional intestinal obstruction, but the mode of presentations often differs. The diagnosis of HSCR is mostly made in the newborn period, only a few patients’ diagnoses are delayed into infancy *These authors contributed stage or adulthood. The typical clinical features are as follows: delayed passage of meconium (24 h after equally to this work. birth), constipation in the neonatal period, abdominal distension and vomiting during the first few days Received: 07 December 2018 of life and neonatal enterocolitis. Persistent constipation, chronic abdominal distension, loss of appetite Revised: 06 May 2019 and failure to thrive are common symptoms in older children [5]. Timely surgical treatment to remove Accepted: 24 July 2019 the affected bowel segment can save most patients’ lives, but obvious postoperative complications, such as fecal incontinence, constipation, repeated enterocolitis, nutrition disorder and so on [7], could bring Accepted Manuscript Online: 29 July 2019 the patients and their families severe economic burden and great psychological pressure [8], which might Version of Record published: affect their quality of life in adulthood [9,10]. Even more, severe complications like HSCR-related short 13 August 2019 bowel syndrome could also kill the patients after surgery [11]. © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution 1 License 4.0 (CC BY). Bioscience Reports (2019) 39 BSR20182290 https://doi.org/10.1042/BSR20182290 It is widely believed that genetic factors playing an important role though the etiology of HSCR is still unknown. In the past decades, several genes have been identified to be associated with HSCR, including RET [12], EDNRB [13], GDNF [14] and SOX10 [15], which however failed to explain the disease cause of large sporadic cases. In 2014, SLC6A20 on chromosome 3p21.3 was suggested as associated with HSCR using 124 patients and 450 controls [16]. These single nucleotide polymorphisms (SNPs) were subsequently replicated, and 13 SNPs were significantly asso- ciated with HSCR using 187 patients and 283 unaffected controls [17]. The haplotype analysis also indicated their significant associations with HSCR susceptibility. Limited by the sample size and incomplete clinical records in pre- vious studies, the functional mechanisms of the variants related to the identified HSCR were not clarified. In addition, it is still necessary to further validate the associations between these SNPs derived from one single causal variant or the congregation of multiple variants that is still awaited for further validation in independent cohorts. In order to further investigate the association between SLC6A20 and HSCR susceptibility, a case–control study was conducted to verify the effects of selected SNPs from an independent South Chinese population with 1470 cases and Downloaded from http://portlandpress.com/bioscirep/article-pdf/39/8/BSR20182290/863686/bsr-2018-2290.pdf by guest on 29 September 2021 1473 controls. We failed to further verify the association between nine previously confirmed SNPs and HSCR except SNP rs7640009. However, six of the other SNPs were found to contribute to the risk of severe subtypes of HSCR in the following detailed analysis stratified by clinical subphenotypes. Furthermore, the relationships between selected SNPs and HSCR-associated enterocolitis (HAEC) susceptibility were also examined. Materials and methods Study population From 2005 to 2015, 1470 HSCR patients from Guangzhou Women and Children’ Medical Center were selected in our study, including 1034 SHSCR, 295 LHSCR, 82 TCA and 3 TIA patients. Inclusion: (1.) sporadic HSCR with histological examination of biopsy specimens with the absence of the enteric ganglia; (2.) age <18 years; (3.) claimed as south Chinese. Exclusion: diagnosis of familial HSCR or syndromic HSCR. The blood samples of 1473 individuals matched geographically and ethnically with no history of HSCR or neurologically associated disorders were collected as controls. Written informed consent was obtained from all the subjects and the legal guardians of every child. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Ethics Committee on Human Research of the Faculty of Guangzhou Women and Children’ Medical Center. SNP genotyping and quality control Nine SNPs (rs2191027, rs6770261, rs2191026, rs10461016, rs4299518, rs764177, rs2108917, rs17279465 and rs78962450 ) of SLC6A20 involved in the present study were selected from to the replication study with 187 Ko- rean HSCR cases [17]. Rs4299518 in SLC6A20 was chosen from the GWAS of HSCR by Kim et al. [16]. Ten SNPs in SLC6A20 weregenotypedbyMassARRAYiPLEXGoldsystem(Sequenom)onallthesamples.Theassociatedstudies searched by NCBI based on the criteria as follows: (1.) SNP with high probability to be regulatory variants were kept for further pursuing (Regulome DB score higher than 2. http://regulomedb.org/). (2.) Removed one of the two SNPs with Linkage disequilibrium (LD) (r2) larger than 0.8 were kept one. (3.) SNP with minor allele frequency larger than 5% in the Chinese population (Han Chinese in Beijing (CHB)) was kept (https://www.ncbi.nlm.nih.gov/snp/?term=). We carried out quality control of the ten SNPs using the following two criteria: (1.) SNPs with >10% missing data were removed (one SNP). (2.)Three SNPs were removed according to the genotyping allele intensity plots for clus- tering quality and violation of Hardy–Weinberg equilibrium (HWE). SNPs were removed if HWE, P<1.0E-04, was calculated by control subjects. After quality control, all ten SNPs were kept for further analysis consisted of 1470 cases and 1473 controls). Association analysis and subphenotype analysis TheSNPswereanalyzedforassociationswiththediseaseusingacomparisonoftheminorallelefrequencyinpatients and controls (basic allelic test) as well as other tests using PLINK1.9 (genotype test of 3 × 2contingencytables, Cochran–Armitage trend test, a test of dominant and recessive models). Association of the SNPs with disease risk was also corrected by logistic regression using age and sex as covariates and the associations found in the present study remain significant. Association with subphenotype was analyzed by comparing cases with a certain subphenotype with controls, cases without the subphenotype with controls. Independence testing LD patterns and values were obtained using HaploView. SNPTEST v2.5b was used to perform the logistic regression tests in the present study. Tests of independent contributions toward disease associations for SNPs in a single locus 2 © 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Bioscience Reports (2019)