Hypermathylation Of SHH May Contribute To The Development Of Congenital Anorectal Malformation

Congenital anorectal malformation (ARM) is the most common digestive tract malformation, and is one of the congenital diseases in routine surveillance by the World Health Organization. The incidence is2.81/10000in China. It is well known that surgical correction of ARM is highly risk and very likely subject to some postoperative complications, especially in high and intermediateARM patients. Because of the diversity of risk factors and complexity of pathological changes, it is still unclear on its etiology and embryogenesis. Now, it is mostly accepted that it is closely related to the abnormal inner conditions of uterus caused by virus infection, chemicals, environmental and nutritional factors in pregnancy, especially in the early stage which is the key period for anorectal development.To better understand and clarify the mechanism of ARM, we performed a genome-wide DNA methylation study in distal rectum and observed the methylation and expression of development-related gene SHH in order to provide the theory basis for the treatment and prevention of ARM. Part One Genome-wide DNA methylation patterns analysis in distal rectum from children with ARMPurposeThe pathogenesis of congenital anorectal malformation (ARM) is still unknown. Recent evidence strongly supports an epigenetic contribution to the pathogenesis of ARM. To better understand and clarify the mechanism of ARM, we performed a genome-wide DNA methylation study in distal rectum from infants with ARM.Methods Distal rectum was collected from patients and controls. Whole genomic DNA was then prepared using a DNEasy kit and bisulfite-treated using a DNA Methylation Kit. Genome-wide DNA methylation was measured using the Illumina Infinium HumanMethylation450BeadChip assay, which interrogates more than450,000cytosine-guanine (CpG) dinucleotide sites, selected predominantly from the promoter regions of the entire genome. Two probes were designed to each CpG site:a ’methylated’ and an ’unmethylated’ query probe. Meanwhile, bioinformatic analysis including Normalization, Background, Average and None was applied to classify the differentially methylated sites in ARM. Absolute methylation values (β-values) were extracted with the GenomeStudioTM software and then subjected to detailed analysis.Results We identified32731CG loci with differential methylation in ARM between cases and controls. Among them,15141are hypomathylated and17590are hypermathylated. According to the different functions of genes, we found some genes belonging to the "development and differentiation" category were hypermathylated. Of interest, the SHHgene which played an important role in the development of anal, rectum and ENS was also hypermethylated in ARM patients.Conclusions Our data indicate the presence of hypomethylated and hypermethylated loci in ARM distal rectum. We observed that CpG sites in some development and differentiation genes of ARM were hypermethylated, and ARM-related SHH gene was also hypermathylated. This work provides novel insights that could help to better understand the pathogenesis of ARM Part Two Abnormal DNA methylation and mRNA expression of SHH gene in distal rectum from ARMPurposeRecent study shows abnormal DNA methylation patterns may contribute to development of ARM. In this study, we have examined DNA methylation patterns in ARM, and investigated genomic and gene specific DNA methylation levels in distal rectum of patients with ARM, especiallySHH gene.Methods We quantified global methylcytosine levels in distal rectum from20patients with ARM and6controls without digestive tract malformation. mRNA levels ofSHHgene was measured by real-time reverse transcriptase-PCR. Methylation of an SHHregulatory element domain was determined by bisulfite sequencing PCR.ResultsCompared with controls, promoter region of SHH in intermediate-high and low group was hypermathylated (p=0.0036, p=0.0087respectively) and SHH mRNA levels were significantly lower (p=0.0065and p=0.0156, respectively). Compared with low group, promoter region of SHH in intermediate and high group was hypermathylated (p=0.0095) and SHH mRNA levels were significantly lower (p=0.0252). The SHH mRNA levels was negatively correlated with DNA methylation (r=-0.89, p<0.05).ConclusionsThese data shows that the levels of SHH mRNA are negatively correlated with DNA methylation in ARM, which suggest that DNA methylation changes may contribute to the development of ARM.