Understanding The Molecular Mechanism Of External Ear Innate Defect By Using Pig As A Model

Microtia is a complex genetic disease causing various types of outer earmalformition with high morbidity in humans. It is also accompanied by hearingimpairment and seriously affects the patient’s appearance and social interactions. Thegenetic basis of this innate defect is complicated and remains poorly understood. Pig isan ideal model for researches on human genetic diseases. The size and type of the outerear is always described as one of its morphological characteristics. So, we performed astudy on the molecular mechanism of external ear innate defect using pig as a model. Wehave observed some individuals with auricle malformation at different levels resemblinghuman microtia in an Erhualian×Shaziling F2pig inbred population. The segregationpattern in the F2pedigree of the inbred population revealed that the disorder is anautosomal recessive monogenic trait.To map the disease locus, we genotyped47individuals in the pedigree usingillumina porcine60k chips and performed GWAS on these individuals. The strongestsignals were detected in a cluster of50SNPs on SSC18. All11affected pigs shared a5.0-Mb homozygous region on this chromosome. Recombination breakpoint analysisrefined the critical region to a2.0-Mb segment harboring18annotated genes including acluster of11HOXA family genes.We further performed deep resequencing for a2.6Mb-region covering the target2.0-Mb segment and identified14candidate causal mutations. By using a broad panel ofsamples including643individuals with normal ear phenotype and all103individuals inthe Erhualian×Shaziling inbreeding pedigree and concordance analysis, we show that adeletion and insertion mutation c.451G>TC in the CDS region of HOXA1gene is thetrancting underlying the genetic disorder. The mutant protein lacks the homeobox domainthat is essential for the biological function of HOXA1.To elucidate the effect of the mutation on downstream genes of HOXA1, weconducted a RNA-Seq experiment using two RNA pools each from two normal andaffected embryos at day14.25of pregnancy. We identified a total of337differentialexpressed genes (DEGs) between the case and control samples, of which several areimportant for external ear development. The reliability of RNA-Seq results wasconfirmed by qPCR analysis of10randomly selected DEGs. By analysis of GO,Pathway and gene network, DEGs are found to be enriched in biological processesincluding drug and lipid metabolism, celluar assembly, cancer, organism deformity andnerver system development and fumction, which is in accordance with the role ofHOXA1gene. To identify causal mutations in146isolated human Microtia patients, we thenperformed exome sequencing of HOXA1, HOXC4, PDX1, NKX2-8, EVC2, FGF1,FGFR3and CTCF genes prioritied from the337DEGs on the basis of threebioinformatic analyses. We finally identified four candidate causal mutations including amissense mutation p.1094D>N of EVC2, a deletion mutation p.65-67HHHdel and twomutations g.2613G>A and g.2944delinsGT in3’UTR of HOXA1gene. These fourcandidates are novel variants, conservative across mammals and probably damaging withfunctional impacts, highlighting the importance of these genes in the development ofexternal ears in both pigs and humans.In conclusion, our findings provide novel insights into the molecular mechanism forhuman microtia and its associated syndromes.