Int J Clin Exp Pathol 2014;7(4):1359-1368 www.ijcep.com /ISSN:1936-2625/IJCEP1401084 Original Article WNT3A gene expression is associated with isolated Hirschsprung disease polymorphism and disease status Dong Chen1, Jie Mi1, Xiaomei Liu2, Juan Zhang3, Weilin Wang1, Hong Gao2 1Department of Pediatric Surgery, Shengjing Hospital of China Medical University, Shenyang 110004, China; 2Key Laboratory of Pediatric Congenital Malformations, Ministry of Public Health, Shengjing Hospital of China Medical University, Shenyang 110004, China; 3Jinzhou Women and Children’s Hospital, Jinzhou, Liaoning, 121000, China Received January 22, 2014; Accepted March 11, 2014; Epub March 15, 2014; Published April 1, 2014 Abstract: WNT3A has been regarded as an activator of the canonical Wnt signaling pathway. It has been found Wnt signaling pathway is closely related with embrionic development and Hirschsprung disease (HSCR). A com- mon haplotype consisting of minor SNPs alleles located in the WNT3A gene has been described as a risk factor for various genetic disorders. However, whether WNT3A contributes to the onset of HSCR has not been identified. The present study aims to detect the interactions of genetic variations in the WNT3A gene and examine the biological expression levels with Hirschsprung disease (HSCR) in the Chinese people. We analyzed WNT3A gene (rs61743220, rs192966556 and rs145882986) variants in the whole blood samples from HSCR patients and normal children (control groups). WNT3A expression was also examined by quantitative real-time PCR (qRT-PCR), western blotting and immunostaining. Consequently, when rs192966556 and rs145882986 alleles of the WNT3A gene lack the SNPs, they are especially associated with a greater risk of HSCR (OR [95% confidence interval] = 1.791, p = 0.001; OR [95% confidence interval] = 1.556, p = 0.003, respectively). The mRNA and protein expressions of WNT3A were higher in the aganglionic colon segment tissues than in the normal ganglionic segments tissues. Immunostaining indicates that the staining of WNT3A was much stronger (brown) in the aganglionic colon segment tissues than that in the normal ganglionic colon segment tissues (colorless or light yellow) in the mucous layer and muscular layer. Although preliminary, these results suggest that WNT3A may play an important role in the pathogenesis of HSCR. Keywords: Hirschsprung disease, WNT3A, polymorphism, gene and protein, expression Introduction sporadic HSCR cases carry RET mutations. Besides RET and EDNRB, other genes have Hirschsprung disease (HSCR) is a congenital been identified in sporadic affected individuals, disorder of the enteric nervous system (ENS) such as endothelin 3 (EDN3) [5], glial derived and is characterized by the absence of intesti- neurotrophic factor (GDNF) [6], sex determining nal ganglion cells in myenteric and submucosal region Y-box 10 (SOX10) [7], paired-like homeo- plexuses. Its incidence is approximately 1/ box 2B (PHOX2B) [8] and ZFHX1B (SIP1) [9]. 5,000 human live births, and has a male pre- Thus, HSCR has become a model of a complex ponderance of 4:1 [1]. At present, the cause of polygenic disorder in which the interaction of HSCR still remains unclear, but there is a com- different genes need to be elucidated. mon understanding that HSCR is a complex dis- ease influenced by multiple genetic and envi- Wnt signaling is one of the most critical signal- ronmental factors. Recent studies have shown ing pathways in many aspects of development that the genetic etiology of the neurocristopa- [10, 11]. Wnt signaling is subdivided into the thy is complex and many genes may be involved canonical β-catenin dependent and the non- in the development of HSCR [2-4]. It is well canonical β-catenin independent branch. known that RET and EDNRB are primary genes Canonical Wnt signaling is characterized by the in the etiology of HSCR. The RET mutations are stabilization and subsequent nuclear transport associated with the development of HSCR in of β-catenin resulting in the activation of tran- 50% of cases in familial series, but only 3% of scriptional responses. Non-canonical Wnt sig- Polymorphism and expression of WNT3A in HSCR naling is more diverse and includes several dif- 3.5 years old including 156 males and 44 ferent signaling modes that regulate cell females (average age, 1.5 ± 0.3 years), and behavior. Genes of the Wnt family encode were recruited as the HSCR groups. An addi- structurally related, cystein-rich glycoproteins tional 200 healthy children that matched with involved in cell-cell signalling for a wide variety the HSCR group in age and gender were used of developmental processes [12-15]. Wnt can as control groups (average age, 2 ± 0.5 years). act either on the cell that secrets them (auto- The control groups had no history of constipa- crine feedback) or on neighboring cells by para- tion. Tissue samples (aganglionic and normal crine signaling. Wnt signaling pathway plays ganglionic colon segment tissues) were rather important roles in the process of the obtained from 50 HSCR patients (41 males and embryonic development. It has been shown 9 females) with pathologically confirmed HSCR that the Wnt signaling pathway plays important pre- or post-operatively at Shengjing Hospital role in intestinal cell proliferation [16]. of China Medical University. Age ranged from 0.5 to 4.5 years old with an average of 1.5 WNT3A, a canonical Wnt ligand, has been years. None of these cases received any preop- regarded as an activator of the canonical Wnt erative treatment or had a history of HSCR’s signaling pathway. WNT3A has been reported enterocolitis or active enterocolitis at the time to induce the accumulation of β-catenin and of surgery. Tissue samples were collected activation of the canonical Wnt signaling path- immediately after operation and were stored at way [17]. Mice that are homozygous for a hypo- -80°C until use. morphic wnt3a allele display vertebral defects, a short tail due to loss of caudal vertebrae, defi- Genomic DNA extraction cient cloacal development and incomplete uro- rectal septation [18]. Moreover, studies involv- Venous blood (200 µl) was obtained from the ing human pluripotent stem cells have shown study participants using EDTA as an anticoagu- that WNT3A is required for hindgut specifica- lant. Genomic DNA of peripheral blood white tion [19]. Thus, WNT3A is a pivotal component blood cells (WBCs) was extracted according to of the mesoderm gene that regulates network the QIAamp® DNA Blood Mini Kit Handbook. with ramifications for embryonic development. For the present study, the absorbance value at The objective of this study was to investigate 260/280 nm (A260/A280) ranged from 1.6 to 2.0, whether genetic variations in the WNT3A gene which met the requirements for further are associated with HSCR and examine the bio- experiments. logical expression levels of WNT3A in the Chinese people. The single nucleotide polymor- Polymerase chain reaction (PCR), DNA se- phisms (SNPs) in WNT3A gene (rs61743220, rs quence analysis and statistical analysis 192966556 and rs145882986) were selected and analyzed in a group of patients with HSCR We have chosen rs61743220, rs192966556 and matched control samples. In this study, we and rs145882986 for WNT3A gene as our poly- further detected differential expressions of morphism analysis loci based on the values of WNT3A by qRT-PCR, western blot and MAF. Specific primers to WNT3A gene were immunostaining. designed using DNASTAR primer design pro- gram and synthesized by Invitrogen Co. Materials and methods (Shanghai) (Table 1). The primers have no homology with other genes as determined by Patients and specimens BLAST analysis on homology. This study was approved by Ethics Committee Genomic DNA from peripheral blood was of China Medical University (Ethical Number: obtained with QIAamp Blood kits (Takara, 2013PS07K). Blood samples of 200 HSCR Dalian, China) using standard methods [20]. patients were collected from the Department of Basically, 150 ng of genomic DNA was ampli- Pediatric Surgery, Shengjing Hospital of China fied in a 50 μl reaction that contained 20 pmol Medical University. Patients with familial consti- each primer and 2U Taq DNA polymerase pation and a history of other congenital gastro- (TaKaRa Biotechnology Co.). Conditions for PCR intestinal tract malformations were excluded amplification in rs61743220, rs192966556 from the study. The patients ranged from 0.5 to and rs145882986 of the WNT3A gene frag- 1360 Int J Clin Exp Pathol 2014;7(4):1359-1368 Polymorphism and expression of WNT3A in HSCR Table 1. Primer sequences and conditions for polymorphism analysis value was recorded. The average CT value was the Location & Annealing SNP locus Sequence (5’-3’) Amplicon temperature extreme CT value of the sample. The expression dif- rs61743220 F: ATA CAC CAC CCA ACC TCA CG 306 bp 57°C ference of the gene was cal- R: AGA CAC CAT CCC ACC AAA CT ‘A/G’ culated by the 2-ΔΔct method rs192966556 F: AGA GCA AAG GGT CTG TAG C 237 bp 55°C [21]. R: CAC TCC TGG ATG CCA AT ‘C/T’ rs145882986 F: CAG GCA AGG GCT GGA AGT 316 bp 59°C Western blot analysis R: GCT GTG GTG TAG CTG GAT GG ‘C/T’ Approximately 50 mg speci- men was minced to small ments are shown in Table 1. A 50 µl reaction pieces using surgical blades and sonicated in system included 10 × PCR buffer solutions, 1 protein lysis buffer. Protein concentrations mmol/L of MgCl2, 0.1 mmol/L of dNTP, 10 were measured by the Bradford method, and pmol/L of each of the upstream and down- specimens were adjusted to the same protein stream primers, 100 ng of DNA template, and concentration, aliquoted and stored at -80°C. 1U of Taq DNA polymerase. PCR condition was: Equal amounts of total proteins from tissues denaturing at 95°C for 3 min, then 35 cycles of were separated on SDS-polyacrylamide gels denaturing at 95°C for 30 sec, annealing at and then electro-transfer to PVDF membranes 55~59°C for 30 sec and elongation at 72°C for (Millipore, USA).