Date Published: January 13, 2010
Publisher: Public Library of Science
Author(s): Doreen Becker, Jens Tetens, Adrian Brunner, Daniela Bürstel, Martin Ganter, James Kijas, Cord Drögemüller, Alfred Lewin. http://doi.org/10.1371/journal.pone.0008689
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.
Partial Text: Human microphthalmia, characterized by small eyes and other ocular abnormalities in newborns, is highly variable with the most severe cases anophthalmic , . Anophthalmia and microphthalmia cause congenital blindness and affect up to 30 per 100,000 people worldwide . Both anophthalmia and microphthalmia may occur in isolation or as part of a syndrome, as in one-third of cases . Morphological studies showed that impaired lens formation seems to be the major cause of anophthalmia and microphthalmia, although the precise pathogenesis of these phenotypes remains unknown . Lens development is a critical embryonic period in vertebrate eye development during which many inductive signals are exchanged between the optic vesicle and surface ectoderm , . This stage is characterized by formation of the lens placode, a thickening of the surface ectoderm that comes into contact with the optic vesicle , . Coordinated invagination of the lens placode and the optic vesicle results in the formation of the lens vesicle and a double-layered optic cup and provides the first indication of the final shape of the eye , . Genetic studies have identified some of the critical determinants of eye formation. A set of putative transcription factors required for the earliest step of eye development were identified in Drosophila. The involvement of homologous proteins in vertebrate lens development was subsequently elucidated by the characterization of mutations that cause congenital human or murine ocular disorders and their comparison to mutations in model organisms . Analyzing inherited isolated microphthalmia/anophthalmia in humans revealed a total of eight genes (SOX2, PAX6, OTX2, RAX, CHX10, FOXE3, PITX3, CRYBA4) carrying causative mutations –. For some human non-syndromic microphthalmia cases the underlying mutation has not yet been found . The role of the eight genes during lens development was confirmed by studying spontaneous mouse mutants and genetically engineered mice with more or less similar ocular phenotypes as in human . Besides CRYBA4, encoding a lens specific structural protein, seven of these genes encode transcription factors which are required for appropriate lens formation during eye development .
Historically, the development of genomic tools for the sheep genome has lagged behind those of other major livestock species such as the cattle and chicken. This has limited the ability to identify genes controlling specific traits of interest . The development of low density microsatellite based linkage maps  have lead to the mapping of Mendelian diseases – and subsequent discovery of mutations underlying at least three genetic diseases in sheep –, however many others remain uncharacterized . The recent development of a set of SNP markers distributed across the sheep genome has changed the prerequisites for such gene mapping projects. For the first time, we demonstrate the use of a genome-wide ovine SNP array for efficient positional cloning of a Mendelian trait in sheep. The result illustrates the power of genome-wide association analysis in domestic animals for the genetic dissection of trait loci .