Candidate gene approach

To identify candidate genes, we searched for related eye disorders in man which show similarities in the phenotype compared to BCSE in cattle. The genes or loci were mapped on the bovine genome (Btau_4.0).

In human, some of the misinnervation syndromes show striking similarities in pathology and clinical features to BCSE in cattle. Progressive external ophthalmoplegia (PEO), Duane retraction syndrome (DURS), and congenital fibrosis of extraocular (CFEOM) belong to this group of diseases in human.

The candidate genes POLG (van Goethem et al. 2001), ANT1 (Kaukonen et al.

2000) and C10orf2 (Spelbrink et al. 2001) responsible for PEO in human were excluded in a previous study as responsible for BCSE in German Brown cattle (Hauke 2003).

The loci responsible for DURS1 and DURS2 were mapped to BTA14 between 30.2 and 30.7 Mb (DURS1) and BTA2 between 14.7 and 21.3 Mb (DURS2), respectively.

Therefore, these loci can also be eliminated as candidates for BCSE. There are different forms of CFEOM which are characterised by a dysinnervation of the oculomotor and/or trochlear nerve innervated ocular muscles. The CFEOM phenotypes causing genes or loci include KIF21A (CFEOM1) on HSA12q12, (Engle et al. 1994, 1995), ARIX (CFEOM2) on HSA11q.13.3-q13.4 (Wang et al. 1998) and CFEOM3 on HSA16q24.2-24.3 (Doherty et al. 1999). The bovine syntenic regions are located on BTA5 at 45.3 Mb (KIF21A), on BTA15 at 51.34 Mb (ARIX) and on BTA18 from 11.5 to 14.0 Mb (CFEOM3). KIF21A which is located 9.7 Mb distally of the QTL was screened for polymorphisms in the coding sequence and excluded as involved in the pathogenesis of BCSE because none of the SNPs detected within the gene was associated with BCSE (Fink et al. 2009). The loci/genes responsible for CFEOM2 and CFEOM3 were not more analysed due to the mapping results on the bovine genome.

None of the loci for related eye disorders in man could be mapped to our linked QTL regions for BCSE. Therefore further molecular genetic studies were necessary to identify a causal gene responsible for BCSE.

5.3 Association analysis

In the next step we performed an association study with SNPs located within both BCSE regions on BTA5 and BTA18 (Fink et al. 2008, 2009). We used published SNP markers to achieve a high density of markers in both QTL regions. Most of the SNPs in the different bovine databases are located within intergenic regions. Therefore the detection of the causal mutation for BCSE could not be expected. But an intergenic with BCSE associated marker could be in very close linkage with a SNP located in an adjacent gene.

In addition, we screened candidate genes for polymorphisms, which were chosen due to their expression profile and known function in human, and location in the two BCSE regions. The identification of intragenic SNP markers includes the possibility of finding mutations that cause the phenotypic effects.

On BTA5, we performed a casecontrol analysis of 213 SNPs and screened the candidate genes PLXNC1 and SOCS2 for polymorphisms. Subsequently, we permutated three to five SNPs and calculated the proportion of phenotypic variance and haplotype association of different marker combinations. Within the coding sequence of SOCS2 no polymorphism was found whereas within the ORF of PLXNC1 three SNPs were detected. A SNP within exon1 (DN825458:c.168G>T) was significantly associated with BCSE (χ² from 9.87 to 11.23 with error probabilities from 0.0009 to 0.003). However, the SNP did not result in an amino acid exchange, but may exert influence on the tertiary structure of the bovine PLXNC1 protein. We found a second SNP (ARS-BFGL-NGS-12640) which is located 220 kb proximally to PLXNC1 reaching also significant results in genotype, allele and trend test statistics (χ² from 7.51 to 11.76 with error probabilities from 0.002 to 0.008). Both SNPs were not in LD. Therefore, both SNPs can be used as single markers for BCSE.

Due to our haplotype association, a refinement of the BCSE region on BTA5 was possible. The significantly associated haplotype included the SNPs Hapmap42731-BTA-92931, ARS-BFGL-NGS-12640, BTA-73209-no-rs and ARS-BFGL-NGS-49972 and spanned an 8.39 Mb interval from 26.49 Mb to 32.85 Mb. 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. Excluding the proximally located Hapmap42731-BTA-92931SNP the haplotype of the three SNPs ARS-BFGL-NGS-12640, BTA-73209-no-rs and ARS-BFGL-NGS-49972 showed a lower error probability. Therefore, it is most likely that the smaller 6.36 Mb interval from 26.49 Mb to 32.85 Mb harbours a causal gene for BCSE on BTA5.

On BTA18, we developed a total of 29 intragenic SNPs within the coding sequence and exon flanking intronic sequences of six candidate genes (CPT1C, SYT3, SYT5;

TNNT1, RDH13 and TFPT) and 25 PCR-products within genes distributed equally over the complete QTL. Within the genes, CPT1C and NALP7, two intronic SNPs were associated with BCSE in association tests for single markers. Within the coding sequence of CPT1C, SYT3 and SYT5 no polymorphisms were detected. Within the ORF of the candidate genes TNNT1, RDH13 and TFPT no significant results from χ²

tests for distribution of genotypes and alleles was estimated for the detected SNPs.

Therefore, these 6 candidate genes are unlikely to be causal for BCSE in German Brown cattle.

In addition, 136 published SNPs located within the QTL on BTA18 were tested for association with BCSE. The Hapmap42211-BTA-43910 SNP located at 57.78 Mb reached significant results in single marker association tests (χ² from 4.45 to 4.84 with error probabilities from 0.028 to 0.034).

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.

We were able to identify two well defined QTLs for BCSE in German Brown cattle on BTA5 and BTA18 using linkage analysis and subsequent association study.

Haplotype analysis was a valuable tool to determine and refine the BCSE intervals.

Especially the QTL on BTA5 was narrowed down to a 6.36 Mb interval by permutation of different marker haplotypes.

Due to the work of this thesis we found associated haplotypes of specific marker combinations on both chromosomes which can be used to calculate the risk of a German Brown diary cattle to develop BCSE in life. In addition, we found three single markers DN825458:c.168G>T within exon 1 of PLXNC1 and two intergenic markers Hapmap42211-BTA-43910 on BTA18 and ARS-BFGL-NGS-12640 on BTA5 which were associated with BCSE in a large sample of affected cattles and controls. These SNPs could also be used as markers for BCSE in cattle. The proportion of phenotypic variance explained by the genotypes of these three SNPs was at 9.23%

(p<0.0001).

The interaction of both BCSE loci is not yet clear and further analyses are necessary to unravel this mystery. Further candidate genes should be sequenced in both BCSE regions on BTA5 and BTA18 to identify more BCSE associated markers and genes, which may be involved in the pathogenesis of this eye disorder.

This work made a fundamental contribution to the development of a gene test to identify genetic carrier for BCSE and prevent them from breeding. We found haplotypes highly associated with BCSE. Therefore, the detection and selection of animals which will develop BCSE with a high risk in their life is now possible. Further on we identified haplotypes suggesting a low risk for a cow to develop BCSE. Thus breeding with these animals will reduce the risk of an offspring to develop BCSE.

Nevertheless there is a lot work to do. We were not able to identify the disease causing mutation. Most of the candidate genes were ruled out as causative for BCSE and the function of associated gene PLXNC1 in BCSE remains unknown.

However, the DNA of both BCSE regions should be sequenced completely using next generation sequencing technology. With this technology it is possible to detect copy number repeats, duplications and polymorphisms within regulatory elements of a gene which could be causal for BCSE.

5.4 References

Distl O. Analysis of pedigrees in dairy cattle segregating for bilateral strabismus with exophthalmus. Anim Genet 1993; 110: 393-400.

Doherty EJ, Macy ME, Wang SM, Dykeman CP, Melanson MT, Engle EC. CFEOM3:

a new extraocular congenital fibrosis syndrome that maps to 16q24.2-q24.3.

Invest Ophthalmol Vis Sci 1999; 40:1687-94.

Engle EC, Kunkel LM, Specht LA, Beggs AH. Mapping a gene for congenital fibrosis of the extraocular muscles to the centromeric region of chromosome 12. Nat Genet 1994; 7:69-73

Engle EC, Marondel I, Houtman WA, de Vries B, Loewenstein A, Lazar M, Ward DC, Kucherlapati R, Beggs AH. Congenital fibrosis of the extraocular muscles (autosomal dominant congenital external ophthalmoplegia): genetic homogeneity, linkage refinement, and physical mapping on chromosome 12.

Fink S, Mömke S, Wöhlke A, Distl O. Genes on bovine chromosome 18 associated with bilateral convergent strabismus with exophthalmos in German Brown cattle. Mol Vis 2008; 14:1737-51.

Fink S, Mömke S, Distl O. Association study in two potential BCSE regions for German Brown cattle located on BTA5 and BTA18. Mol Vis 2009; accepted

POLG is associated with progressive external ophthalmoplegia characterized by mtDNA deletions. Nat Genet 2001; 28:211-2.

Hauke G. Candidate gene analysis for bilateral convergent strabismus with exophthalmus in German Brown cattle. Diss. med. vet., Tierärztliche Hochschule Hannover 2000.

Kaukonen J, mtDNA maintenance.

Mömke S, Fink S, Wöhlke A, Drögemüller C, Distl O. Linkage of bilateral convergent strabismus with exophthalmus (BCSE) to BTA5 and BTA18 in German Brown cattle. Anim Genet 2008; 39:544-9.

Spelbrink JN, Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria.

Nat Genet 2001; 28:223-31.

Vogt C, Distl O. Untersuchungen zum bilateralen Strabismus convergens mit Exophthalmus beim Deutschen Braunvieh. Tierärztl Prax 2002; 30:148–52.

Wang SM, Zwaan J, Mullaney PB, Jabak MH, Al-Awad A, Beggs AH, Engle EC.

Congenital fibrosis of the extraocular muscles type 2, an inherited exotropic strabismus fixus, maps to distal 11q13. Am J Hum Genet 1998; 63:517-25.

CHAPTER 6

Summary

6 Summary

Steffen Martin Fink (2009)

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 χ²-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.

CHAPTER 7

Erweiterte Zusammenfassung