Dissection Of The Genertic Mechanism Underlying Congential Anal Atresia In Large White Pigs

Congenital anal atresia is a complex innate defect of the rectum development in humans,which includes simple anal atresia and complicated imperforate anus with other abnormal syndrome.It is generally considered that anal atrsia is caused by failing to rupture of the cloacal membrane due to dysplasia of the posterior intestine.The most common phenotype is the absence of anus.Congenital anal atresia brings great mental stress and heavy economic burden to patients and their families.The pathogenic factors are complicated and genetic factors play the dominant role in this disease.To date,the genetic mechanism of this innate defect remains poorly understood.Pig is an ideal model for researches on human genetic diseases,as it is highly similar to human in anatomy,physiological metabolism and disease characteristics.We herein performed a study to identify causal genes and mutations for congenital anal atresia using a F₂ and F₃inbreeding population of Large White pigs,aiming to dissect the molecular mechanism of this genetic defect in Large White pigs.According to the segregation pattern（affected:unaffected≈1/3）in the F₃inbreeding population,which was derived from a backcross between an affected F₂ and a F₁ normal individual,we speculated that the disorder was controlled by two recessive genes on autosomes.Next,we performed genome-wide association（GWAS）,genome-wide linkage analysis,SNP-QTL concordance analysis and identical-by-descent（IBD）analysis using60K SNP chip data of 137 individuals and imputed whole-genome SNP makers of 31individuals in this population.We identified two candidate intervals that were located on Sus Scrofa chromosome 1（SSC1）（127.5-147.7 Mb）and SSC15（25.0-67.9 Mb）.The two confidence intervals were refined into SSC1:127.5-129.2 Mb or 145.8-147.5 Mb and SSC15:29.1-33.9 Mb by recombination breakpoint analysis.To identify the causal genes and mutations,we further performed whole-genome deep resequencing on 12 individuals（4 affected individuals,2 heterozygotes and 6 normal individuals）.We identified 15 candidate causal mutations according to an accordance analysis.By sequencing 32 affected individuals and 188 unaffected animals,we then pinpointed two disease-causing candidate mutations:one（T>A）was located at 2 Kb downstream of the CAPN3 gene on SSC1 and the other（G>A）resided in the intron 1splice region of the GLI2 gene on SSC15.Intriguingly,CAPN3 is closely related to the apoptosis pathway and GLI2 plays an important role in the SHH signaling pathway,which are involved in the development of anus.To understand the transcriptom difference between anal atresia affected individuals and normal individuals,we conducted RNA sequencing using the anus skin and rectum terminal tissues of 4 affected and 4 full-sib normal individuals.We identified a total of 247and 131 differential expression genes（DEGs）in the two tissues,respectively.The reliability of RNA-Seq results were confirmed by quantitative Real-time PCR analysis of 4DEGs and 4 genes without differential expression.GO（gene ontology）and KEGG（Kyoto Encyclopedia of Genes and Genomes）analysis showed that these DEGs enriched in several biological processes including ion transmembrane transport,cell-cell signaling,regulation of organ development,insulin secretion,digestion,development of skin,cell differentiation and apoptosis.We had a close examination on two candidate causative genes:CAPN3 and GLI2.CAPN3 expression was significantly up-regulated in the anus skin and rectum terminal of affected individuals compared with normal individuals,while GLI2 expression was significantly down-regulated in the two tissues of affected individuals.GLI2 is a main transmitter of the SHH signaling pathway.The expression of CAPN3 inhibits the apoptosis process required for the formation of the anus by the rupture of the cloaca membrane.Therefore,we assumed that GLI2 G>A splice mutation generates an abnormal transcript which likely leads to markedly decreased expression of GLI2 via nonsense transcript mediated decay and consequenty inhibit or block the SHH signal transmission.For the CAPN3 T>A mutation,it enhances the expression level of CAPN3,which may led to the failure to rupture the cloaca membrane.The two causative mutations collectively contribute to congenital anus atresia in Large White pigs.In summary,we identified the strong candidate causal genes（CAPN3 and GLI2）and mutations（CAPN3 T>A and GLI2 G>A）for congential anus atresia in Large White pigs via a series of genetic analyse,both a significantly increased expression of CAPN3 and a remarkably deduced expression of GLI2 contribute to the pathogenesis of congenital anal atresia in Large White pigs.Our findings not only provide diagnostic markers for this genetic disease that allow us to efficiently eliminate the disease-causing alleles in Large White pigs,but also provide insight into the pathogenic mechanism of imperforate anus in humans.