Microphthalmia in Texel sheep is associated with a missense mutation in the paired-like homeodomain 3 (PITX3) gene

Abstract

Microphthalmia in sheep is an autosomal recessive inherited congenital anomaly found within the Texel breed. It is characterized by extremely small or absent eyes and affected lambs are absolutely blind. For the first time, we use a genome-wide ovine SNP array for positional cloning of a Mendelian trait in sheep. Genotyping 23 cases and 23 controls using Illumina's OvineSNP50 BeadChip allowed us to localize the causative mutation for microphthalmia to a 2.4 Mb interval on sheep chromosome 22 by association and homozygosity mapping. The PITX3 gene is located within this interval and encodes a homeodomain-containing transcription factor involved in vertebrate lens formation. An abnormal development of the lens vesicle was shown to be the primary event in ovine microphthalmia. Therefore, we considered PITX3 a positional and functional candidate gene. An ovine BAC clone was sequenced, and after full-length cDNA cloning the PITX3 gene was annotated. Here we show that the ovine microphthalmia phenotype is perfectly associated with a missense mutation (c.338G>C, p.R113P) in the evolutionary conserved homeodomain of PITX3. Selection against this candidate causative mutation can now be used to eliminate microphthalmia from Texel sheep in production systems. Furthermore, the identification of a naturally occurring PITX3 mutation offers the opportunity to use the Texel as a genetically characterized large animal model for human microphthalmia.

Figure 1. Microphthalmia phenotype in Texel sheep.

(A) Normal newborn lamb. (B) Microphthalmia affected lamb.

Figure 2. Genome-wide association mapping of microphthalmia.

(A) Case-control whole genome association analysis finds significant association to SNPs on chromosome 22. (B) Single SNP association statistic across OAR 22. (C) Homozygosity mapping of the microphthalmia mutation. The analysis of SNP genotypes from affected sheep indicated that they had extended overlapping homozygous regions on OAR 22 (indicated as black blocks). Thus – assuming that it resides on the common haplotype block – the causative mutation is located within a 2.4 Mb interval on OAR 22 (indicated as red box). All 23 affected sheep had homozygous identical by state intervals with shared alleles between 24.5 Mb and 26.9 Mb. (D) Gene content of the corresponding human chromosome 10 segment.

Figure 3. PITX3 mutation analysis.

(A) Ovine PITX3 gene structure. (B) Electropherograms of the PITX3 c. 338G>C mutation. Representative sequence traces of PCR products amplified from genomic DNA of three sheep with the different genotypes are shown. (C) Multispecies alignment of the evolutionary conserved homeodomain of the PITX3 protein sequence. The p.R113P mutation in PITX3is indicated by an arrow. It affects an arginine residue, which is perfectly conserved from human to zebrafish across all investigated species. The sequences for the alignment were taken from the following accessions: (sheep, NP_005020 (human), XP_589431 (cattle), XP_001499185 (horse), EDL41981 (mouse), NP_062120 (rat), XP_421631 (chicken), NP_001082023 (Xenopus laevis), NP_991238 (Danio rerio).