title: Hypermethylation of SHH in the pathogenesis of congenital anorectal malformations.
abstract: OBJECTIVE
  This study sought to examine promoter methylation and expression of the identified sonic hedgehog (SHH) gene in terminal rectal tissues of children with congenital anorectal malformations (ARMs).
  
  
  METHODS
  Tissue samples from the terminal rectum of pediatric patients with ARMs (five cases each of high and intermediate malformation - two cases of rectovesical fistula, two cases of rectourethral prostatic fistula, one case of cloaca with >3cm common channel, four cases of rectourethral bulbar fistula and one case of imperforate anus without fistula, respectively, and ten cases of low malformation - five cases of perineal fistula and five cases of vestibular fistula, respectively), and patients with non-gastrointestinal tract malformation (six cases, anal fistula) were collected and divided into three groups: high-intermediate ARM (ARMhi-int), low ARM (ARMlo), and control (Cont.). Real-time RT-PCR was used to detect mRNA expression levels of the verified differentially methylated gene SHH, and bisulfite genomic sequencing was performed to evaluate DNA methylation in the SHH promoter region.
  
  
  RESULTS
  The average methylation levels of the SHH promoter were significantly higher in ARMhi-int (0.850±0.030, P=0.0036) and ARMlo (0.540±0.053, P=0.0087) groups than in Cont. group (0.280±0.032). SHH mRNA expression levels were lower in ARMhi-int (0.340±0.015, P=0.0065) and ARMlo (0.530±0.042, P=0.0156) groups than in Cont. group (0.870±0.046). The average methylation levels of the SHH promoter were higher in ARMhi-int group than in ARMlo group (0.850±0.030 vs. 0.540±0.053, P=0.0095), while SHH expression was significantly reduced in ARMhi-int group compared to ARMlo group (0.340±0.15 vs. 0.530±0.042, P=0.0252). The methylation levels of the SHH promoter in ARMhi-int group were negatively correlated with SHH gene expression (r=-0.89, P<0.01).
  
  
  CONCLUSIONS
  The SHH gene, which plays a major role in the development of the anorectum and enteric nervous system, is hypermethylated at its promoter, and this is correlated with low levels of SHH gene expression. This epigenetic modification may therefore be responsible for the observed changes in SHH expression, which could in turn underlie the pathogenesis of congenital ARMs.
authors.affiliation: Department of Pediatric Surgery, Children's Hospital of Fudan University, Shanghai 201102, P.R. China. Electronic address: yanleihuang@163.com.
authors.forename: Yanlei
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Department of Pediatric Surgery, Children's Hospital of Fudan University, Shanghai 201102, P.R. China.
authors.forename: Pei
authors.initials: P
authors.lastname: Zhang
authors.affiliation: Department of Pediatric Surgery, Children's Hospital of Fudan University, Shanghai 201102, P.R. China. Electronic address: szheng@shmu.edu.cn.
authors.forename: Shan
authors.initials: S
authors.lastname: Zheng
authors.affiliation: Department of Pediatric Surgery, Children's Hospital of Fudan University, Shanghai 201102, P.R. China.
authors.forename: Rui
authors.initials: R
authors.lastname: Dong
mesh_terms: D001003:Anal Canal / Q000002:abnormalities* / Q000378:metabolism*; D019175:DNA Methylation; D005260:Female; D018507:Gene Expression Regulation, Developmental; D053823:Hedgehog Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007223:Infant; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012007:Rectum / Q000002:abnormalities* / Q000378:metabolism*
chemical_list: D053823:Hedgehog Proteins; D012333:RNA, Messenger; C509239:SHH protein, human
keywords: Congenital anorectal malformations; DNA methylation; SHH