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

<div><p>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 (<i>HMX1</i>) 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 <i>HMX1</i> 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 <i>HMX1</i> misregulation and possible gain-of-function. Our findings confirm the role of <i>HMX1</i> 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.</p></div

Comparative map of the HMX1 gene region on BTA 6.

<p>(<b>A</b>) Physical map position of HMX1 and flanking transcripts (UMD3 assembly). The ultra-conserved enhancer region is located 148 kb downstream of the coding region of HMX1. (<b>B</b>) Comparison and sequence conservation of the ultra-conserved enhancer region across different species (screenshot of the human UCSC genome browser <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077841#pone.0077841-Hompage2" target="_blank">[26]</a>). (<b>C</b>) Multi-species genomic DNA sequence alignment showing the sequence conservation of the 76 bp duplication. The identifier of transcription factor matrices for potential transcription factor binding sites are shown above and were determined using Transcription factor Affinity Prediction (TRAP) software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077841#pone.0077841-ThomasChollier1" target="_blank">[27]</a>.</p

Agarose gel electrophoresis of diagnostic PCR for crop ear (CE) genotyping (L: Ladder 100 bp; CE-0: wild type control, CE-1: heterozygous affected, CE-2: homozygous mutant severely affected.

<p>Agarose gel electrophoresis of diagnostic PCR for crop ear (CE) genotyping (L: Ladder 100 bp; CE-0: wild type control, CE-1: heterozygous affected, CE-2: homozygous mutant severely affected.</p

Results of the genome-wide association study.

<p>(<b>A</b>) Manhattan-plot showing the negative decadic logarithms of the raw P values obtained from the genotypic association test (χ² test with 2 degrees of freedom). The dashed line indicates the Bonferroni-adjusted threshold for genome-wide significance (0.01 divided by 519,828 markers). (<b>B</b>) Detailed view of the significantly associated region of BTA 6. The position of the <i>HMX1</i> gene is highlighted in red. (<b>C</b>) QQ-plot reflecting the strong association signal on BTA6. The data points referring to SNPs located in the distal region of BTA6 shown in B are highlighted in blue. The genomic inflation factor λ is considerably smaller when excluding BTA 6 from the analysis (λ_-BTA6) as compared to the genome-wide analysis including the chromosome (λ_+BTA6).</p

<i>HMX1</i> sequence variants.

<p>Coding variants in the <i>HMX1</i> gene and structural non-coding variants in the <i>HMX1</i> associated highly conserved region. Note that the 76 bp duplication is perfectly associated with crop ears (bold face).</p

Mutation detection by whole genome re-sequencing.

<p>(<b>A</b>) Screenshot of the short read mapping against the reference sequence, note the red labeled reads indicating the genomic duplication. (<b>B</b>) Sanger sequencing confirmed the presence of the duplication; black box: 76 bp element in the reference sequence; red box: additional 76 bp duplication, note the enlarged region showing the heterozygous sequence using reverse PCR primer for sequencing.</p

<i>COL7A1</i> genotype frequencies.

*<p>Angus (n = 18), Aubrac (n = 1), Ayshire (n = 1), Belgian blue (n = 3), Blonde d'Aquitaine (n = 2), Brown Swiss (n = 35), Charolais (n = 17), Chianina (n = 19), Dutch belted (n = 18), Eringer (n = 16), Evolenard (n = 10), Gelbvieh (n = 1), Galloway (n = 20), Hereford (n = 3), Scotish Highland (n = 4), Holstein (n = 35), Jersey (n = 3), Limousin (n = 17), Montbéliarde (n = 4), Nelore (n = 3), Ongola (n = 1), Piedmontese (n = 2), Pinzgauer (n = 10), Pustertaler Sprinzen (n = 10), Romagnola (n = 18), Salers (n = 1), Simmentaler (n = 38), Tyrolean Grey (n = 18), Domestic Yak (n = 1).</p

Clinical features of a DEB affected Rotes Höhenvieh calf.

<p>Typical signs are the extensive epidermal loss with ulcerations at the fetlocks (A) and muzzle (B) and dysungulation (C).</p

Pedigree of Rotes Höhenvieh cattle with DEB.

<p>DNA samples were available only from red labeled cattle. The genotypes for the <i>COL7A1</i> c.4756C>T exon 49 SNP are given below the symbols. The arrow indicates the cow, which is supposed to be the founder animal. We genotyped all available male offspring of the two carrier bulls Hannibal and Oska and detected 51% and 61% carriers, respectively.</p

Genome-wide homozygosity mapping of the DEB mutation.

<p>After genotyping approximately 777,000 uniformly distributed SNP markers homozygous blocks >0.1 Mb were identified across 3 DEB affected cattle. Only on BTA 22 a very large homozygous block was identified.</p