Designing, optimization, and validation of whole blood direct T-ARMS PCR for precise and rapid genotyping of complex vertebral malformation in cattle

Background DNA testing in the cattle industry undergoes multiple hurdles. Successful genotyping involves the transportation of samples from the field to the laboratory in a chilled environment followed by DNA extraction, and finally, a specific genotyping protocol is followed. Various researches are focused on overcoming these issues. Microcards offer blood transportation at ambient temperature. Direct PCR methods can save the time of DNA extraction but available only for simplex PCR. Tetra Primer-Amplification Refractory Mutation System based Polymerase Chain Reaction (T-ARMS PCR) can make DNA testing faster in a low-cost setting. The present study was aimed to design, optimize, and validate a T-ARMS PCR for faster DNA testing of SNP responsible for Complex Vertebral Malformation (CVM)-an important genetic disease of the cattle industry. Further, a direct T-ARMS PCR from whole blood was developed to avoid the DNA extraction steps. Lastly, using the optimized protocol, genotyping of blood spotted on Microcard eliminates the need for cold chain maintenance in the transportation of samples. Results The present study demonstrated a novel T-ARMS PCR-based genotyping of the SNP rs438228855, which is responsible for CVM. Here, wild genotypes were recognized by 389 bp and 199 bp bands in agarose gel, while the carrier genotype showed an additional 241 bp band. The developed protocol was validated using PCR-Primer Introduced Restriction Analysis (PCR-PIRA) and sequencing. The present study further established a direct T-ARMS PCR for this SNP from whole blood. Different conditions such as heparin and EDTA treated blood, the need for pre-treatment, and two different DNA Polymerases for the direct PCR were optimized. Finally, our optimized protocol successfully genotyped the whole blood samples dried on Insta™DNA cards. Conclusions The present study reported the usefulness of primer modified T-ARMS PCR for detecting CVM for the first time. To the best of our knowledge, direct PCR in T-ARMS PCR has never been reported. Lastly, the use of microcards in the developed protocol can make the assay useful in the DNA testing of field samples.

Variants Affecting the C-Terminal of CSF1R Cause Congenital Vertebral Malformation Through a Gain-of-Function Mechanism

CSF1R encodes the colony-stimulating factor 1 receptor which regulates the proliferation, differentiation, and biological activity of monocyte/macrophage lineages. Pathogenic variants in CSF1R could lead to autosomal dominant adult-onset leukoencephalopathy with axonal spheroids and pigmented glia or autosomal recessive skeletal dysplasia. In this study, we identified three heterozygous deleterious rare variants in CSF1R from a congenital vertebral malformation (CVM) cohort. All of the three variants are located within the carboxy-terminal region of CSF1R protein and could lead to an increased stability of the protein. Therefore, we established a zebrafish model overexpressing CSF1R. The zebrafish model exhibits CVM phenotypes such as hemivertebral and vertebral fusion. Furthermore, overexpression of the mutated CSF1R mRNA depleted of the carboxy-terminus led to a higher proportion of zebrafish with vertebral malformations than wild-type CSF1R mRNA did (p = 0.03452), implicating a gain-of-function effect of the C-terminal variant. In conclusion, variants affecting the C-terminal of CSF1R could cause CVM though a potential gain-of-function mechanism.

Congenital thoracic kyphosis caused by hemivertebra in a pug puppy

Congenital spine deformation caused by vertebral malformation is reported in different brachycephalic dog breeds, but also in other large dog breeds. The most severe clinical signs are due to spinal cord compression. Depending on localisation of the lesion, clinical signs can manifest differently. A five-month-old pug was brought for autopsy to the Department of Forensic Veterinary Medicine, Faculty of Veterinary Medicine, University of Belgrade. The first clinical signs, including general weakness and difficulty to jump appeared when he was three and a half months old. They became more severe until the dog was four months old. Radiography and myelography showed severe kyphosis and spinal cord compression in the T8-T9 region. After euthanasia, an autopsy was performed. Pathomorphological examination showed severe deformation of the spine, kyphosis and a wedge-shaped T8 vertebra. Although, literature data indicates that clinical signs can be latent until five or six months of age, this case shows that even in young puppies, three to four months old, serious clinical signs can be present. This is of great clinical importance, not just for veterinarians, but also for brachycephalic dog owners and breeders.