Characterization of the equine ITGAX gene and its association with recurrent airway obstruction in European Warmblood horses

Abstract

Supporting informationAdditional supporting information may be found in the online version of this article. Table S1 PCR primers and amplification conditions for porcine ID1, ID2, ID3 and ID4. Table S2 Selected polymorphisms used for genotyping and linkage mapping. Table S3 Frequencies of SNP and indel alleles in ID2, ID3 and ID4 in several breeds of pigs. Figure S1 Comparison of deduced amino acid sequences of porcine ID1, ID2, ID3 and ID4.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Source/description: Recurrent airway obstruction (RAO) in horses is clinically characterized by reversible bronchoconstriction, increased mucus production and neutrophilic airway inflammation following exposure of susceptible horses to hay and stable dust. 1,2 Equine RAO is a naturally occurring respiratory disease and is considered an animal model of human asthma. 3 Recently we identified two QTL for RAO on ECA 13 and 15 in two Warmblood half-sib families. 4 Integrins are heterodimeric membrane-bound glycoproteins. 5 In humans, haplotype blocks close to the integrin alpha X gene (ITGAX) were potentially associated with higher allergy risk. 6 The equine ITGAX gene is located in the RAO QTL region on ECA 13 and was thus chosen as a positional and functional candidate gene.Gene characterization: In the EquCab 2.0 assembly, the ITGAX sequence is interrupted by a gap containing the putative exons 12 and 13. We PCR-amplified and sequenced the gap region. We annotated the 30 exons by comparison to an equine mRNA sequence (NM_001114177) and submitted a complete genomic sequence of the~20 kb equine ITGAX gene to the EMBL database (FN561630). SNP identification:We amplified each of the 30 ITGAX exons with flanking regions from four half-sib offspring of the Warmblood family, in which the QTL on ECA 13 was detected. We selected two RAO-affected and two non-affected animals. We identified a total of 22 polymorphisms, including two missense mutations (Table S1).Association with RAO: The two missense mutations are located close together in exon 11 and 13. Therefore, we amplified and sequenced a PCR product containing exons 11, 12 and 13 from 340 European Warmblood horses that were unrelated at the grandparental level. The animals consisted of 177 RAO cases (HOARSI 3 or 4) and 163 controls (HOARSI 1). 7 We did not detect an allelic association to any of the five SNPs, which were present on the PCR product (chi square-test, P > 0.05).We then analyzed specific haplotypes at the ITGAX gene. We obtained the genotype information for the markers BIEC2-214642, BIEC2-214653, BIEC2-214689, spanning over 88 kb in the region of the ITGAX, from Illumina equine SNP50 data, and estimated the haplotypes using the HAPLOVIEW software. 8 In the QTL family, the ha...

Replication and fine‐mapping of a QTL for recurrent airway obstruction in European Warmblood horses

“…No associations with RAO were found in the ITGAX gene on ECA 13 encoding integrin alpha X, a part of the complement receptor that plays a role in immune‐complex mediated hypersensitivity (Shakhsi‐Niaei et al . ). Two other studies have shown that non‐coding regulatory variants in the IL4R gene, which is also located in the wider QTL region on ECA13, are associated with RAO phenotype in this half‐sib family (Jost et al .…”