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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 (JEB) has been detected in two commercial flocks. The JEB 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 β4 protein. A functional analysis of this deletion revealed decreased levels of mRNA in JEB skin samples and the absence of integrin β4 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 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. [ABSTRACT FROM AUTHOR]

In this study, we used the Illumina OvineSNP50 BeadChip to conduct a genome-wide association (GWA) analysis for milk production traits in dairy sheep by analyzing a commercial population of Spanish Churra sheep. The studied population consisted of a total of 1,681 Churra ewes belonging to 16 half-sib families with available records for milk yield (MY), milk protein and fat yields (PY and FY) and milk protein and fat contents (PP and FP). The most significant association identified reached experiment-wise significance for PP and FP and was located on chromosome 3 (OAR3). These results confirm the population-level segregation of a previously reported QTL affecting PP and suggest that this QTL has a significant pleiotropic effect on FP. Further associations were detected at the chromosome-wise significance level on 14 other chromosomal regions. The marker on OAR3 showing the highest significant association was located at the third intron of the alpha-lactalbumin (LALBA) gene, which is a functional and positional candidate underlying this association. Sequencing this gene in the 16 Churra rams of the studied resource population identified additional polymorphisms. One out of the 31 polymorphisms identified was located within the coding gene sequence (LALBA_g.242T.C) and was predicted to cause an amino acid change in the protein (Val27Ala). Different approaches, including GWA analysis, a combined linkage and linkage disequilibrium study and a concordance test with the QTL segregating status of the sires, were utilized to assess the role of this mutation as a putative QTN for the genetic effects detected on OAR3. Our results strongly support the polymorphism LALBA_g.242T.C as the most likely causal mutation of the studied OAR3 QTL affecting PP and FP, although we cannot rule out the possibility that this SNP is in perfect linkage disequilibrium with the true causal polymorphism. [ABSTRACT FROM AUTHOR]

Extending genome wide association analysis by the inclusion of gene expression data may assist in the dissection of complex traits. We examined piebald, a pigmentation phenotype in both human and Merino sheep, by analysing multiple data types using a systems approach. First, a case control analysis of 49,034 ovine SNP was performed which confirmed a multigenic basis for the condition. We combined these results with gene expression data from five tissue types analysed with a skin-specific microarray. Promoter sequence analysis of differentially expressed genes allowed us to reverse-engineer a regulatory network. Likewise, by testing two-loci models derived from all pair-wise comparisons across piebald-associated SNP, we generated an epistatic network. At the intersection of both networks, we identified thirteen genes with insulin-like growth factor binding protein 7 (IGFBP7), platelet-derived growth factor alpha (PDGFRA) and the tetraspanin platelet activator CD9 at the kernel of the intersection. Further, we report a number of differentially expressed genes in regions containing highly associated SNP including ATRN, DOCK7, FGFR1OP, GLI3, SILV and TBX15. The application of network theory facilitated co-analysis of genetic variation with gene expression, recapitulated aspects of the known molecular biology of skin pigmentation and provided insights into the transcription regulation and epistatic interactions involved in piebald Merino sheep. [ABSTRACT FROM AUTHOR]