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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>MOCS1 </it>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.</p> <p>Conclusions</p> <p><it>MOCS1 </it>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 <it>MOCS1 </it>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.</p

Identification of the amino acids in the major histocompatibility complex class II region of scottish blackface sheep that are associated with resistance to nematode infection

Lambs with the Major Histocompatibility Complex DRB1*1101 allele have been shown to produce fewer nematode eggs following natural and deliberate infection. These sheep also possess fewer adult Teladorsagia circumcincta than sheep with alternative alleles at the DRB1 locus. However, it is unclear if this allele is responsible for the reduced egg counts or merely acts as a marker for a linked gene. This study defined the MHC haplotypes in a population of naturally infected Scottish Blackface sheep by PCR amplification and sequencing, and examined the associations between MHC haplotypes and faecal egg counts by generalised linear mixed modelling. The DRB1*1101 allele occurred predominately on one haplotype and a comparison of haplotypes indicated that the causal mutation or mutations occurred in or around this locus. Additional comparisons with another resistant haplotype indicated that mutations in or around the DQB2*GU191460 allele were also responsible for resistance to nematode infections. Further analyses identified six amino acid substitutions in the antigen binding site of DRB1*1101 that were significantly associated with reductions in the numbers of adult T. circumcincta

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

The genetic architecture of the MHC class II region in British Texel sheep

Understanding the structure of the major histocompatibility complex, especially the number and frequency of alleles, loci and haplotypes, is crucial for efficient investigation of the way in which the MHC influences susceptibility to disease. Nematode infection is one of the most important diseases suffered by sheep, and the class II region has been repeatedly associated with differences in susceptibility and resistance to infection. Texel sheep are widely used in many different countries and are relatively resistant to infection. This study determined the number and frequency of MHC class II genes in a small flock of Texel sheep. There were 18 alleles at DRB1, 9 alleles at DQA1, 13 alleles at DQB1, 8 alleles at DQA2 and 16 alleles at DQB2. Several haplotypes had no detectable gene products at DQA1, DQB1 or DQB2, and these were defined as null alleles. Despite the large numbers of alleles, there were only 21 distinct haplotypes in the population. The relatively small number of observed haplotypes will simplify finding disease associations because common haplotypes provide more statistical power but complicate the discrimination of causative mutations from linked marker loci

A novel RNAseq–assisted method for MHC class I genotyping in a non-model species applied to a lethal vaccination-induced alloimmune disease

BACKGROUND: MHC class I genotyping is essential for a wide range of biomedical, immunological and biodiversity applications. Whereas in human a comprehensive MHC class I allele catalogue is available, respective data in non-model species is scarce in spite of decades of research. RESULTS: Taking advantage of the new high-throughput RNA sequencing technology (RNAseq), we developed a novel RNAseq-assisted method (RAMHCIT) for MHC class I typing at nucleotide level. RAMHCIT is performed on white blood cells, which highly express MHC class I molecules enabling reliable discovery of new alleles and discrimination of closely related alleles due to the high coverage of alleles with reads. RAMHCIT is more comprehensive than previous methods, because no targeted PCR pre-amplification of MHC loci is necessary, which avoids preselection of alleles as usually encountered, when amplification with MHC class I primers is performed prior to sequencing. In addition to allele identification, RAMHCIT also enables quantification of MHC class I expression at allele level, which was remarkably consistent across individuals. CONCLUSIONS: Successful application of RAMHCIT is demonstrated on a data set from cattle with different phenotype regarding a lethal, vaccination-induced alloimmune disease (bovine neonatal pancytopenia), for which MHC class I alleles had been postulated as causal agents. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-016-2688-0) contains supplementary material, which is available to authorized users

SPE and HPLC of metabolites of melatonin

MHC class I and SNP haplotype tracking in a F2 full-sib family. Detection of paternally and maternally inherited MHC class I haplotypes. Bluish strands: paternal chromatids; reddish strands: maternal chromatids; rs-number: Reference SNP cluster ID; green boxes: SNP alleles; yellow boxes: classical MHC class I alleles; grey boxes: non-classical MHC class I alleles; alleles deduced from genotypes of progeny are indicated in italic. (PDF 286 kb

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

Additional file 4: Table S2. of A novel RNAseq–assisted method for MHC class I genotyping in a non-model species applied to a lethal vaccination-induced alloimmune disease

Allele specific primers used for experimental MHC class I allele confirmation by Sanger sequencing. The table lists the oligonucleotide primers used for amplification and sequencing of MHC class I sequences. (DOCX 16 kb

Syndrome of arachnomelia in Simmental cattle

<p>Abstract</p> <p>Background</p> <p>The syndrome of arachnomelia is an inherited malformation mainly of limbs, back and head in cattle. At present the arachnomelia syndrome has been well known mainly in Brown Swiss cattle. Nevertheless, the arachnomelia syndrome had been observed in the Hessian Simmental population during the decade 1964–1974. Recently, stillborn Simmental calves were observed having a morphology similar to the arachnomelia syndrome. The goal of this work was the characterization of the morphology and genealogy of the syndrome in Simmental to establish the basis for an effective management of the disease.</p> <p>Results</p> <p>The first pathologically confirmed arachnomelia syndrome-cases in the current Simmental population appeared in the year 2005. By 2007, an additional 140 calves with the arachnomelia syndrome were identified. The major pathological findings were malformed bones affecting the head, long bones of the legs and the vertebral column. It could be shown that, with the exception of two cases that were considered as phenocopies, all of the paternal and about two-third of the maternal pedigrees of the affected calves could be traced back to one common founder. Together with the data from experimental matings, the pedigree data support an autosomal recessive mutation being the etiology of the arachnomelia syndrome. The frequency of the mutation in the current population was estimated to be 3.32%.</p> <p>Conclusion</p> <p>We describe the repeated occurrence of the arachnomelia syndrome in Simmental calves. It resembles completely the same defect occurring in the Brown Swiss breed. The mutation became relatively widespread amongst the current population. Therefore, a control system has to be established and it is highly desirable to map the disease and develop a genetic test system.</p