Molecular genetic analysis of bilateral convergent strabismus with exophthalmus in German Brown cattle

The objective of the present thesis was to localize genomic regions and genes that are responsible for bilateral convergent strabismus with exophthalmus (BCSE) in German Brown cattle and mutation analysis of potential candidate genes. By non-parametric linkage analysis with a set of 164 microsatellites and 13 SNPs in a total sample of 159 animals spread across 10 families, we identified two putative gene loci involved in the development of BCSE in German Brown cattle. These putative BCSE regions were located on BTA5 between 17.29 and 47.0 cM and on BTA18 between 72.01 and 78.84 cM. The corresponding regions on the current Bos taurus genome assembly 4.0 were determined between 14.38 and 35.53 Mb on BTA5 and between 55.23 and 63.0 Mb on the telomeric end of BTA18 using BLAST analysis. We compared the gene order within both candidate regions on BTA5 and BTA18 with the gene assignment on HSA12 and HSA19, respectively, to improve the gene annotation on the bovine genome assembly 4.0 and to detect potential candidate genes for BCSE. On the one hand we screened PCR products of 17 genes located within the BCSE region on BTA18 for new SNP markers and developed 9 SNPs within 9 different genes. On the other hand we have chosen 349 published SNPs within both putative BCSE regions derived from the Bovine HapMap Consortium, the bovine genome reference assembly Btau3.1 and the Illumina’s Genome Analyzer. In addition we performed cDNA analysis of three candidate genes on BTA5 (PLXNC1, SOCS2 and KIF21A) and six candidate genes on BTA18 (CPT1C, SYT3, SYT5, TNNT1, RDH13 and TFPT). Within the five candidate genes PLXNC1, KIF21A, TNNT1, RDH13 and TFPT we identified 15 exonic and 10 intronic SNPs. The genes SOCS2, CPT1C, SYT3, and SYT5 did not harbour any polymorphism within their coding sequences. For all the SNPs we performed a case-control association study based on c2-tests for genotypes, alleles and trend of the alleles. Subsequently we tested the combination of different markers for haplotype association. On BTA5, we found a significant association to BCSE in allele, genotype and trend test statistics for the intergenic SNP ARS-BFGL-NGS-12640 at 26.49 Mb and the SNP DN825458:c.168G>T located within exon 1 of PLXNC1 at 26.71 Mb. This SNP did not affect the amino acid structure of the protein. Both SNPs were located in close vicinity, but they were not in LD. By haplotype analysis we found a significant association to BCSE for the combination of the SNPs Hapmap42731-BTA-92931, ARS-BFGL-NGS-12640, BTA-73209-no-rs and ARS-BFGL-NGS-49972 in the sample of 340 German Brown cows. We showed that the haplotype A-G-G-A-G indicate a high risk of an animal to develop BCSE later in life whereas the haplotypes G-A-A-C-G, A-G-G-C-C and A-A-A-C-G were related with low risk to BCSE. Exclusion of the proximal located SNP Hapmap42731-BTA-92931 in the calculation, showed a lower error probability for the haplotype of the three SNPs ARS-BFGL-NGS-12640, BTA-73209-no-rs and ARS-BFGL-NGS-49972. Thus, the most likely interval which harbours the causal gene for BCSE spanned 6.36 Mb on BTA5. On BTA18, the intergenic Hapmap42211-BTA-43910 SNP located at 57.78 Mb reached significant results in single marker association tests. By permutation of haplotypes composed of three to five SNPs we found a haplotype which was significantly associated with BCSE. This haplotype included SNPs of CPT1C, SYT5, RDH13, and NAPL7. The A-G-C-C-G haplotype indicated a high probability of a German Brown dairy cattle to contract BCSE later in life. On the other hand, animals which show the haplotypes A-G-A-T-T or G-G-C-C-T will develop BCSE with a low risk. In conclusion, the haplotype association refined the BCSE-region to a 6.82 Mb interval.