Major Histocompatibility Complex class IIB polymorphism in an ancient Spanish breed.

Genes from the Major Histocompatibility Complex class II region are involved in the presentation of antigens. Therefore, they have the key role in regulating the immune response and in the resistance to infections. We investigated the Major Histocompatibility Complex class IIB genes, DRB and DQB, in Churra sheep, one of the most important indigenous breeds of Spain. These genes are among the most polymorphic in the mammalian genome. Furthermore, often different numbers of class IIB genes per haplotype exist, complicating the genotyping and sequencing of these genes. Especially the DQB region is only partially characterized in sheep and the repertoire of DRB and DQB alleles in Churra sheep, an ancient breed, is unknown. Here, we sequenced the class IIB genes for 15 rams that are the pedigree heads of a selection Nucleus herd. In total, we found 12 DRB and 25 DQB alleles. From these, 3 and 15 were new, respectively. Fourteen haplotypes carrying one or two DQB alleles could be deduced and the evolutionary relationship of these was investigated by phylogenetic trees. Based on the sequences of these most common class II alleles, a more efficient genotyping system for larger numbers of Churra sheep will be developed.

Arachnomelia syndrome in Simmental cattle is caused by a homozygous 2-bp deletion in the molybdenum cofactor synthesis step 1 gene (MOCS1).

BACKGROUND: Arachnomelia syndrome is an autosomal recessive inherited disease in cattle. Affected calves die around birth and show malformations of the skeleton mainly affecting the legs, the spinal column and the skull. A number of arachnomelia syndrome affected Simmental calves were recently detected by a surveillance system of anomalies with a peak of more than 120 recorded cases in the year 2006. The causative mutation was previously mapped to a 9 cM-region on bovine chromosome 23. We herein report the fine-mapping and identification of the gene causing arachnomelia syndrome in Simmental cattle. RESULTS: By using a dense set of markers, the arachnomelia syndrome linked region could be refined to 1.5 cM harbouring three protein coding genes. Comparative sequencing of these genes revealed a two-bp-deletion in the bovine MOCS1 gene resulting in a frame-shift and a premature termination codon. We genotyped affected calves and their ancestors and found that all affected were homozygous for the deletion whereas all carriers were heterozygous. Furthermore, cattle from the same population, but not directly related to known carriers mostly showed the wild type genotype. CONCLUSIONS: MOCS1 encodes two proteins that are involved in the first synthesis step of molybdenum cofactor. A non functional sulfite-oxydase, one of the enzymes requiring molybdenum cofactor, leads to a similar pathology in Brown Swiss cattle. In combination the perfect association of the mutation with the phenotype and the obvious disruption of protein translation provide strong evidence for the causality of the MOCS1 mutation. Our results are the first example for an oligogenic lethal inherited disease in cattle. Furthermore, they show the potential involvement of sulfite metabolism in aberrant bone development.

A robust, low- to medium-throughput prnp genotyping system in sheep.

BACKGROUND: In many countries breeding programs for resistance to scrapie in sheep are established. Therefore, the demand on genotyping capacities of the polymorphisms of the prion protein gene (prnp) relevant to presently known disease associations and EU regulations is steadily increasing. Most published typing methods are not well suited for routine typing of large sample numbers in smaller service laboratories for different reasons: they require partly manual data processing, sophisticated and sensitive protocols, high efforts regarding time and manpower, multiple step reactions or substantial hardware investments. To overcome these drawbacks, we developed a prnp typing method that is based on a 'multiplex amplification refractory mutation system' (ARMS) reaction. METHODS: In this study we combined the amplification refractory mutation system (ARMS) with standard fluorescent based fragment length analyses method to develop a prnp genotyping method (PRNP ARMS). RESULTS: By optimised primer design it was possible to type the 4 relevant single nucleotide polymorphisms (SNPs) in the prnp simultaneously in one multiplex reaction. Automated fragment length analysis enabled automated allele designation. Suitability of the PRNP ARMS for routine application was proven by typing samples with known genotypes and larger sample numbers from half-sib families. CONCLUSION: The ARMS PRNP typing method established in this study is universally suited for a broad range of typing projects with different requirements. It provides an efficient and inexpensive diagnostic mutation analysis that will improve the quality of prnp genotyping compared with other low-cost methods. It can be implemented by most molecular genetic laboratories using standard equipment.