| dc.description.abstracteng | Musculoskeletal anomalies are a heterogeneous group of fairly common, often with severe clinical symptoms, but mostly fatal disorders in human and animals. By far, complex vertebral malformation (CVM) and Brachyspina are the most important lethal osteodysplasias described in Holstein Friesian (HF) cattle. First aim of this thesis was to elucidate the genetic cause of a lethal case of osteogenesis imperfecta (OI) in a newborn HF calf. The molecular mechanism underlying OI in humans has been clearly explored, with a series of candidate genes reported. However, none of these hitherto known genes harboured any causative variant in the affected OI calf. To identify genomic regions associated with OI, a genome wide association study was performed. A total of 6 significantly associated SNP loci were detected on 1, 5, 6, 17, 18, and 24 (p < 0.05). In addition, a whole genome re-sequencing of the affected calf and its parents as well as gene prioritization using CANDID was performed. From these analyses 16 candidate genes with functional variants remained. Validation was performed in 2612 randomly chosen healthy German HF using high-resolution melting curve analysis. Finally, a haplotype of 4 functional variants in 3 candidate genes (ABCA13: ENSBTAT00000061018.2:c.12553+1A>G, p.Gln4393Arg; QRFPR: p.Arg412Ter; IFITM5: p.Ala30Ser) was determined explaining the development of OI in the calf. This was in agreement with the results of the GWAS indicating a so far unknown oligogenic origin of OI.
Bovine interdigital hyperplasia (IH) is a typical claw disorder affecting cattle welfare and causing serious economic losses. IH shows the highest estimated heritabilities in comparison to other feet/claw disorders. Second aim of this thesis was to elucidate the molecular genetic cause of IH in HF. To investigate the pathogenesis, the IH status was inspected during hoof trimming of first-lactation dairy cows. To study the clinical course, the cows were examined seven times every three months. From herds with highest IH prevalence, cows were selected to perform a genome-wide association study. Two significantly associated chromosomal positions were revealed with both located in the tyrosine-protein kinase transmembrane recepetor ROR2 gene. Due to its previously reported function, ROR2 was a reasonable candidate for IH. A comparative sequencing between cases and controls was conducted and 2 missense mutations were identified. Large-scale genotyping showed that one variant ROR2 p.Trp9Arg was highly significantly associated with IH (p < 0.0001). Quantification of mRNA and protein levels proved significant reduction of ROR2 expression due to this variant.
Ribosome biogenesis is the process of generating ribosomes which serve as the site of biological protein synthesis in all living cells. Impaired or decreased ribosome biosynthesis is associated with a group of diseases called ribosomopathies. Due to the lack of functional ribosomes, these diseases broadly fall under the category of cellular hypo-proliferation phenotypes. Before supportive care was developed, such hypo-proliferation phenotypes were severe even lethal in the past. Molecular dissection of ribosomopathies in humans has revealed
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a list of causal genes, however, ribosomopathies have not been reported in cattle so far. Third aim of this thesis was to address the question whether lethal variants in the causal genes of ribosomopathies exist in cattle. A 2bp deletion has been deposited into bovine genomic databases in bovine uL5 gene on chromosome 2, which would result in a frameshift and a premature stop codon. The deletion causes a truncation of bovine uL5. A probe for this variant had been included as expert-selected marker in the custom add-on part of the Illumina BovineLD BeadChip and can therefore be monitored during routine genotyping. To determine the frequency of the variant, we genotyped 370,527 cattle, including 18 different dairy and beef cattle breeds. 299,218 homozygous wild type and 71,249 heterozygous cattle were called from the bead chip. Cattle harboring the homozygous mutant genotype were not detected. According to Hardy-Weinberg equilibrium around 4,241 homozygous individuals carrying the deletion should have been present in the cohort. Hence, the data apparently indicated that the homozygous 2 bp deletion in uL5 was a lethal variant interfering with ribosome biogenesis and resulting in embryonic death. However, using Sanger sequencing we could not detect the putative deleterious variant in randomly selected heterozygous cattle identified by the BeadChip genotyping. An alignment of the BeadChip probe to the bovine genome showed a perfect match to a processed uL5 pseudogene on bovine chromosome 18 mimicking the assumed exonic deletion. Subsequently, an examination by Sanger sequencing showed that the previously detected heterozygous cattle were homozygous for the 2 bp deletion within the pseudogene. The BeadChip genotyping results were clearly confounded by the erroneous detection of this variant. Therefore, special care should be taken when designing probes and/or primers for a specific assay in high-throughput genotyping platforms to avoid severe misinterpretations led by pseudogenes. We not yet identify a lethal variant, however, our strategy can be used to farther explore potentially recessive lethal variants causing ribosomopathies in cattle. | de |