A Non-Coding Genomic Duplication at the HMX1 Locus Is Associated with Crop Ears in Highland Cattle

Abstract

Highland cattle with congenital crop ears have notches of variable size on the tips of both ears. In some cases, cartilage deformation can be seen and occasionally the external ears are shortened. We collected 40 cases and 80 controls across Switzerland. Pedigree data analysis confirmed a monogenic autosomal dominant mode of inheritance with variable expressivity. All affected animals could be traced back to a single common ancestor. A genome-wide association study was performed and the causative mutation was mapped to a 4 Mb interval on bovine chromosome 6. The H6 family homeobox 1 (HMX1) gene was selected as a positional and functional candidate gene. By whole genome re-sequencing of an affected Highland cattle, we detected 6 non-synonymous coding sequence variants and two variants in an ultra-conserved element at the HMX1 locus with respect to the reference genome. Of these 8 variants, only a non-coding 76 bp genomic duplication (g.106720058_106720133dup) located in the conserved region was perfectly associated with crop ears. The identified copy number variation probably results in HMX1 misregulation and possible gain-of-function. Our findings confirm the role of HMX1 during the development of the external ear. As it is sometimes difficult to phenotypically diagnose Highland cattle with slight ear notches, genetic testing can now be used to improve selection against this undesired trait.

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“…The ‘crop ear’ trait that is common in the Highland cattle breed represents a moderately to severely truncated (or cropped) ear deformity, which may vary according to gene dosage and genetic background (Scheider et al, 1994). In contrast to the rat dumbo phenotype, the bovine crop ear trait exhibits partially dominant inheritance and is due to a 76bp duplication within the most highly conserved part of the Hmx1 distal CNE (Koch et al, 2013). …”

A distal 594bp ECR specifies Hmx1 expression in pinna and lateral facial morphogenesis and is regulated by Hox-Pbx-Meis

“…However, Hmx1-related phenotypes have also been described in humans (OAS) and cattle (crop ear). In contrast to the loss-of-function mutations found in mice and humans, the mutations at the Hmx1 locus in rats and cattle implicate a putative regulatory region located ∼80 kb 3′ of the last exon (Quina et al, 2012a;Koch et al, 2013). At this site, we previously identified a highly conserved ∼600 bp segment (dmECR) that we hypothesized could represent a cis-regulatory enhancer of Hmx1 expression .…”

“…These sites are found in the most conserved portion of the dmECR, shared not only among mammalian species but also in chick, frogs and zebrafish, albeit with one or two nucleotide differences in these latter species. The likely importance of these binding sites in mammals is emphasized by the crop ear cattle allele, which is a 76 bp duplication (Koch et al, 2013) involving this central core containing the Hox-Pbx-Meis binding motifs. Consistent with the presence of predicted binding sites, genome-wide occupancy of Hoxa2 and Meis in vivo shows that both Hoxa2 and Meis bind to the dmECR in vivo.…”

“…This condition has been termed oculo-auricular syndrome (OAS) in humans (Schorderet et al, 2008;Gillespie et al, 2015), 'dumbo' and/or 'misplaced ears' in mice, 'dumbo' in rats (Munroe et al, 2009;Kuramoto et al, 2010;Quina et al, 2012a), and 'crop ear' in cattle (Koch et al, 2013).…”

“…This deleted sequence contains a ∼600 bp evolutionarily conserved region (ECR). Remarkably, the recessive crop ear allele in cattle also involves this ECR, specifically a 76 bp duplication of the central core (Koch et al, 2013).…”