Molecular basis of a new ovine model for human 3M syndrome-2.

BACKGROUND: Brachygnathia, cardiomegaly and renal hypoplasia syndrome (BCRHS, OMIA 001595-9940 ) is a previously reported recessively inherited disorder in Australian Poll Merino/Merino sheep. Affected lambs are stillborn with various congenital defects as reflected in the name of the disease, as well as short stature, a short and broad cranium, a small thoracic cavity, thin ribs and brachysternum. The BCRHS phenotype shows similarity to certain human short stature syndromes, in particular the human 3M syndrome-2. Here we report the identification of a likely disease-causing variant and propose an ovine model for human 3M syndrome-2. RESULTS: Eight positional candidate genes were identified among the 39 genes in the approximately 1 Mb interval to which the disease was mapped previously. Obscurin like cytoskeletal adaptor 1 (OBSL1) was selected as a strong positional candidate gene based on gene function and the resulting phenotypes observed in humans with mutations in this gene. Whole genome sequencing of an affected lamb (BCRHS3) identified a likely causal variant ENSOARG00000020239:g.220472248delC within OBSL1. Sanger sequencing of seven affected, six obligate carrier, two phenotypically unaffected animals from the original flock and one unrelated control animal validated the variant. A genotyping assay was developed to genotype 583 animals from the original flock, giving an estimated allele frequency of 5%. CONCLUSIONS: The identification of a likely disease-causing variant resulting in a frameshift (p.(Val573Trpfs*119)) in the OBSL1 protein has enabled improved breeding management of the implicated flock. The opportunity for an ovine model for human 3M syndrome and ensuing therapeutic research is promising given the availability of carrier ram semen for BCRHS.

Suspected hereditary cervicothoracic vertebral subluxation with cervical myopathy in Poll Merino sheep.

BACKGROUND: Cervicothoracic vertebral subluxation in sheep presents as a postural and locomotor disorder, and has been described in several breeds in Australia and overseas. Cervical myopathy may also be present in these cases. CASE REPORT: A New South Wales sheep producer reported a postural and locomotor disorder with a low prevalence in his Poll Merino stud flock, affecting neonate, weaner and adult sheep. Animals with postural abnormalities, variable degrees of ataxia and proprioceptive deficits involving both fore and hind limbs were described. Abnormalities of the cervicothoracic vertebral column were identified grossly during necropsy, with misalignment and consequent narrowing of the posterior cervical spinal canal. Lesions ranging from pallor (cellular degeneration) to white streaky lesions with pinpoint haemorrhage (necrosis) were identified in the cervicothoracic paravertebral musculature of affected animals. Boney abnormalities were further characterised by imaging studies. Pedigree analysis of the very extensive breeding and disease incident records available for this flock suggested that the disease was inherited. A similar case recognised in a separate New South Wales Poll Merino flock is also described. CONCLUSION: This report describes an entity of cervicothoracic vertebral subluxation in two Poll Merino sheep flocks, with cervical myopathy also identified in one, with preliminary evidence in the primary case that there is likely to be a hereditary basis. The two cases outlined in this report resemble the findings of several historical investigations into ovine flock postural disorders in Australia and beyond.

An ABCA12 missense variant in a Shorthorn calf with ichthyosis fetalis.

Two clinical forms of ichthyosis in cattle have been reported, ichthyosis fetalis and congenital ichthyosis. Ichthyosis poses animal welfare and economic issues and the more severe form, ichthyosis fetalis, is lethal. A Shorthorn calf with ichthyosis fetalis was investigated and a likely causal missense variant on chromosome 2 in the ABCA12 gene (NM_001191294.2:c.6776T>C) was identified by whole genome sequencing. Mutations in the ABCA12 gene are known to cause ichthyosis fetalis in cattle and Harlequin ichthyosis in humans. Sanger sequencing of the affected calf and the dam confirmed the variant was homozygous in the affected calf and heterozygous in the dam. Further genotyping of 130 Shorthorn animals from the same property revealed an estimated allele frequency of 3.8%. The presented findings enable genetic testing for breeding and diagnostics.

Development and validation of an inexpensive and efficient method for the extraction of Theileria orientalis DNA from blood.

Theileria orientalis is an emerging bovine pathogen in Australasia. PCR-based detection methods for this parasite are sensitive but relatively expensive, partly due to costs associated with DNA extraction. An inexpensive and efficient technique was developed for the extraction of T. orientalis DNA from blood based on hypotonic erythrocyte lysis and detergent-proteinase K treatment (DPK method). The DPK method compares favourably to a commercial extraction kit when paired with a T. orientalis multiplex qPCR.

Inferring the recent ancestry of myostatin alleles affecting muscle mass in cattle.

Double muscling is an inherited condition in cattle characterised by large increases in muscle mass. Mutations in the myostatin (MSTN) gene, responsible for double muscling, were targeted in this study to estimate the time since the most recent common ancestor (TMRCA) for Q204X (p.Gln204*), E226X (p.Glu226*), 821del11 (c.821del11), E291X (p.Glu291*), C313Y (p.Cys313Tyr) and the more phenotypically moderate F94L (p.Phe94Leu) mutation. Genetic variability was examined in eight regions upstream and downstream of the MSTN locus. The molecular distance of the homozygous region associated with each MSTN allele was used to estimate the TMRCA. Long homozygous segments were associated with the MSTN alleles (mostly > 2 Mb), compared to short segments (130 kb) for cattle wild type at the double muscling and F94L sites. Estimates of time indicated that each MSTN allele had a recent common ancestor (<400 years ago). The results from this study, and the increasing frequency of these MSTN alleles in some cattle breeds, demonstrate recent positive selection.

Replication of staphylococcal multiresistance plasmids.

Based on structural and functional properties, three groups of large staphylococcal multiresistance plasmids have been recognized, viz., the pSK1 family, pSK41-like conjugative plasmids, and beta-lactamase-heavy-metal resistance plasmids. Here we describe an analysis of the replication functions of a representative of each of these plasmid groups. The replication initiation genes from the Staphylococcus aureus plasmids pSK1, pSK41, and pI9789::Tn552 were found to be related to each other and to the Staphylococcus xylosus plasmid pSX267 and are also related to rep genes of several plasmids from other gram-positive genera. Nucleotide sequence similarity between pSK1 and pI9789::Tn552 extended beyond their rep genes, encompassing upstream divergently transcribed genes, orf245 and orf256, respectively. Our analyses revealed that genes encoding proteins related to the deduced orf245 product are variously represented, in several types of organization, on plasmids possessing six seemingly evolutionarily distinct types of replication initiation genes and including both theta-mode and rolling-circle replicons. Construction of minireplicons and subsequent functional analysis demonstrated that orf245 is required for the segregational stability of the pSK1 replicon. In contrast, no gene equivalent to orf245 is evident on the conjugative plasmid pSK41, and a minireplicon encoding only the pSK41 rep gene was found to exhibit a segregational stability approaching that of the parent plasmid. Significantly, the results described establish that many of the large multiresistance plasmids that have been identified in clinical staphylococci, which were formerly presumed to be unrelated, actually utilize an evolutionarily related theta-mode replication system.