A Novel Nonsense Mutation in the DMP1 Gene Identified by a Genome-Wide Association Study Is Responsible for Inherited Rickets in Corriedale Sheep

Abstract

Inherited rickets of Corriedale sheep is characterized by decreased growth rate, thoracic lordosis and angular limb deformities. Previous outcross and backcross studies implicate inheritance as a simple autosomal recessive disorder. A genome wide association study was conducted using the Illumina OvineSNP50 BeadChip on 20 related sheep comprising 17 affected and 3 carriers. A homozygous region of 125 consecutive single-nucleotide polymorphism (SNP) loci was identified in all affected sheep, covering a region of 6 Mb on ovine chromosome 6. Among 35 candidate genes in this region, the dentin matrix protein 1 gene (DMP1) was sequenced to reveal a nonsense mutation 250C/T on exon 6. This mutation introduced a stop codon (R145X) and could truncate C-terminal amino acids. Genotyping by PCR-RFLP for this mutation showed all 17 affected sheep were “T T” genotypes; the 3 carriers were “C T”; 24 phenotypically normal related sheep were either “C T” or “C C”; and 46 unrelated normal control sheep from other breeds were all “C C”. The other SNPs in DMP1 were not concordant with the disease and can all be ruled out as candidates. Previous research has shown that mutations in the DMP1 gene are responsible for autosomal recessive hypophosphatemic rickets in humans. Dmp1_knockout mice exhibit rickets phenotypes. We believe the R145X mutation to be responsible for the inherited rickets found in Corriedale sheep. A simple diagnostic test can be designed to identify carriers with the defective “T” allele. Affected sheep could be used as animal models for this form of human rickets, and for further investigation of the role of DMP1 in phosphate homeostasis.

Approach to Investigating Congenital Skeletal Abnormalities in Livestock

“…68 Sequencing of this gene in our sheep demonstrated a nonsense mutation in exon 6 of the DMP1 gene, which led to a premature stop codon and truncation of the protein. 114 The chances of successfully detecting a mutation using this technique is increased if the inheritance of the disease is known and if DNA from family members, including unaffected siblings, dams, and sires is available. Accurate recognition of phenotype is also important, as misdiagnosis of affected animals will obviously affect the analysis.…”

A missense mutation in AGTPBP1 was identified in sheep with a lower motor neuron disease

“…To define the molecular genetic basis for the LMN disease in these Romney sheep, a search for putative homozygous-by-descent (also named IBD) regions was initiated by performing a genome-wide scan using an in-house UNIX script as described in our previous publication (Zhao et al, 2011). The SNP genotypes were ordered according to their genomic location as provided in the Illumina manifest, and used to identify regions of consecutive homozygous SNP loci that were common to all affected animals.…”

“…Among the many genes in the identified homozygous regions, AGTPBP1 was considered to be the best candidate gene because of its role in neural functions as reported by several publications (Harris et al, 2000;FernandezGonzalez, et al, 2002). PCR was performed using a 10-ml cocktail mixture and a standard program as described previously (Zhao et al, 2011), with an annealing temperature of 58 or 60 1C. Negative controls were performed to check for the presence of contaminants in all reactions.…”

Genetic testing for phenotype-causing variants in sheep and goats

“…This enabled the identification of genes which contain phenotype-affecting variants in sheep by a powerful genome wide association analysis approach. The SNP chip was already successfully employed to narrow down the involved chromosomal region to a size which allowed the identification of the genes, and then of the causative variants for the following phenotypes: microphthalmia in Texel sheep [12], epidermolysis bullosa in German Black Headed Mutton [13], and inherited rickets in Corriedale sheep [14]. A genome wide association study using the ovine SNP50 beadchip gave also strong evidence that the relaxin/ insulin-like family peptide receptor 2 gene (RXFP2) is involved in the control of the horned/polled status of Soay sheep.…”