RNA-Seq for EBJ-ACC affected and control. sheep Skin transcriptome

In this study, we demonstrate the use of a genome-wide association mapping together with RNA-seq in a reduced number of samples, as an efficient approach to detect the causal mutation for a Mendelian disease. Junctional epidermolysis bullosa is a recessive genodermatosis that manifests with neonatal mechanical fragility of the skin, blistering confined to the lamina lucida of the basement membrane and severe alteration of the hemidesmosomal junctions. In Spanish Churra sheep, junctional epidermolysis bullosa with aplasia cutis congenita (JEB-ACC) has been detected in two commercial flocks. The JEB-ACC locus was mapped to Ovis aries chromosome 11 by GWAS and subsequently fine-mapped to an 868-kb homozygous segment using the identical-by-descent method. The ITGB4, which is located within this region, was identified as the best positional and functional candidate gene. The RNA-seq variant analysis enabled us to discover a 4-bp deletion within exon 33 of the ITGB4 gene (c.4412_4415del). The c.4412_4415del mutation causes a frameshift resulting in a premature stop codon at position 1472 of the integrin 4 protein. A functional analysis of this deletion revealed decreased levels of mRNA in JEB-ACC skin samples and the absence of integrin 4 labeling in immunohistochemical assays. Genotyping of c.4412_4415del showed perfect concordance with the recessive mode of the disease phenotype. Selection against this causal mutation will now be used to solve the problem of JEB-ACC in flocks of Churra sheep. Furthermore, the identification of the ITGB4 mutation means that affected sheep can be used as a large mammal animal model for the human form of epidermolysis bullosa with aplasia cutis. Our approach evidences that RNA-seq offers cost-effective alternative to identify variants in the species in which high resolution exome-sequencing is not straightforward.

本研究展示了将全基因组关联作图（genome-wide association mapping）与少量样本的RNA测序（RNA-seq）相结合的方法，可作为检测孟德尔遗传病致病突变的高效策略。交界型大疱性表皮松解症（Junctional epidermolysis bullosa）是一种隐性遗传性皮肤病，临床表现为新生儿皮肤机械脆性增加，水疱局限于基底膜透明层（lamina lucida），且半桥粒连接（hemidesmosomal junctions）发生严重结构改变。在西班牙丘拉绵羊中，两群商业养殖羊群中已检出伴先天性皮肤发育不全的交界型大疱性表皮松解症（JEB-ACC）。全基因组关联分析（GWAS）将JEB-ACC位点定位到绵羊（Ovis aries）11号染色体，随后通过同源一致性（identical-by-descent）方法将其精细定位至一段868 kb的纯合区段。位于该区段内的ITGB4基因被确定为最佳的位置候选与功能候选基因。通过RNA-seq变异分析，我们在ITGB4基因的第33号外显子中发现了一处4 bp缺失突变（c.4412_4415del）。该c.4412_4415del突变可引发移码突变，使整合素β4（integrin β4）蛋白在第1472位氨基酸处提前出现终止密码子。针对该缺失的功能分析显示，JEB-ACC皮肤样本中的mRNA水平降低，且免疫组化检测中未观察到整合素β4的标记信号。对c.4412_4415del突变的基因分型结果显示，其与该病的隐性遗传表型完全契合。目前，针对该致病突变的选育工作将用于解决丘拉绵羊群中的JEB-ACC问题。此外，ITGB4突变的鉴定意味着患病绵羊可作为人类伴先天性皮肤发育不全的大疱性表皮松解症的大型哺乳动物动物模型。本研究方法证实，对于高分辨率外显子测序难以开展的物种而言，RNA-seq是一种兼具成本效益的变异鉴定替代方案。