The bovine fibroblast growth factor receptor 3 (FGFR3) gene is not the locus responsible for bovine chondrodysplastic dwarfism in Japanese brown cattle

Fibroblast growth factor receptor 3 (FGFR3) is one of the four distinct membrane-spanning tyrosine kinase receptors for fibroblast growth factors. The FGFR3 is a negative regulator of endochondral ossification and mutations in the FGFR3 gene have been found in patients of human hereditary diseases with chondrodysplastic phenotypes. Recently, we mapped the locus responsible for hereditary chondrodysplastic dwarfism in Japanese brown cattle to the distal region of bovine chromosome 6 close to the FGFR3 gene, suggesting that FGFR3 was a positional candidate gene for this disorder. In the present study, we isolated complementary DNA (cDNA) clones containing the entire coding region of the bovine FGFR3 gene. Comparison of the nucleotide sequence between affected and normal animals revealed no disease-specific differences in the deduced amino acid sequences. We further refined the localization of FGFR3 by radiation hybrid mapping, which is distinct from that of the disease locus. Therefore we conclude that bovine chondrodysplastic dwarfism in Japanese brown cattle is not caused by mutation in the FGFR3 gene.     

Localization of the locus responsible for Chediak-Higashi syndrome in cattle to bovine chromosome 28

Chediak-Higashi syndrome in Japanese black cattle is a hereditary disease with prolonged bleeding time and partial albinism. In the present study, we mapped the locus responsible for the disease (CHS) by linkage analysis using microsatellite genotypes of paternal half-sib pedigrees obtained from commercial herds. Analysis revealed significant linkage between the CHS locus and marker loci on the proximal end of bovine chromosome 28. The CHS locus was mapped on the region incorporating the microsatellite markers BMC6020, BM2892, and RM016 with recombination fraction 0 and lod score 4.9-11.2. We also assigned the bovine CHS1/LYST, the homologue of the gene responsible for human Chediak-Higashi syndrome, to bovine chromosome 28 using a bovine/murine somatic cell hybrid panel. These findings suggest that a mutation in the CHS1/LYST gene is likely to be responsible for Chediak-Higashi syndrome in Japanese black cattle.     

Homozygosity mapping of the locus responsible for renal tubular dysplasia of cattle on bovine Chromosome 1

Renal tubular dysplasia is a hereditary disease of Japanese black cattle showing renal failure and growth retardation with an autosomal recessive trait. In the present study, we mapped the locus responsible for the disease (RTD) by linkage analysis with an inbred paternal half-sib pedigree obtained from commercial herds. By analyzing segregation of microsatellite markers in the half-sibs, significant linkage was observed between the RTD locus and markers on bovine Chromosome (Chr) 1 with the highest lod score of 11.4. Homozygosity mapping with the inbred pedigree further defined the localization of the RTD locus in a 4-cM region between microsatellite markers BMS4003 and INRA119. Mapping of the RTD locus on bovine Chr 1 will facilitate cloning and characterization of the gene responsible for this disease. Received: 24 September 1999 / Accepted: 14 December 1999     

Defective splicing of thyroglobulin gene transcripts in the congenital goitre of the Afrikander cattle

The structure of thyroglobulin mRNA was analyzed in an inbred herd of Afrikander cattle with hereditary goitre. Northern transfer of RNA from affected animals revealed both a shorter (approximately 7100 bases) and a normal-sized (approximately 8200 bases) thyroglobulin mRNA when hybridized to bovine thyroglobulin cDNA clones. S1 nuclease mapping experiments established that 1100 bases are deleted in the 5' region of the smaller mRNA. Electron microscopy of RNA from animals with goitre hybridized to a bovine genomic DNA clone showed that the region deleted corresponds to exon 9 of the thyroglobulin gene. Southern blot analysis of the exon 9 region revealed differences between affected and control animals with the enzymes PstI and TaqI. Although they could reflect a linkage disequilibrium between the mutation and restriction fragment length polymorphism, it is noteworthy that these differences map in the region of the exon 9/intron 9 junction. Our results show that a genetic lesion in the thyroglobulin gene causes aberrant splicing of the pre-mRNA, and suggest that the responsible mutation is at the exon 9/intron 9 junction.     

Linkage mapping of the locus responsible for forelimb-girdle muscular anomaly of Japanese black cattle on bovine chromosome 26

Forelimb-girdle muscular anomaly is an autosomal recessive disorder of Japanese black cattle characterized by tremor, astasia and abnormal shape of the shoulders. Pathological examination of affected animals reveals hypoplasia of forelimb-girdle muscles with reduced diameter of muscle fibres. To identify the gene responsible for this disorder, we performed linkage mapping of the disorder locus using an inbred pedigree including a great-grand sire, a grand sire, a sire and 26 affected calves obtained from a herd of Japanese black cattle. Two hundred and fifty-eight microsatellite markers distributed across the genome were genotyped across the pedigree. Four markers on the middle region of bovine chromosome 26 showed significant linkage with the disorder locus. Haplotype analysis using additional markers in this region refined the critical region of the disorder locus to a 3.5-Mb interval on BTA26 between BM4505 and MOK2602 . Comparative mapping data revealed several potential candidate genes for the disorder, including NRAP , PDZD8 and HSPA12A , which are associated with muscular function.     

Genome-wide scan identifies loci associated with classical BSE occurrence

Classical bovine spongiform encephalopathy (BSE) is an acquired prion disease that is invariably fatal in cattle and has been implicated as a significant human health risk. Sequence variations in the coding region of the prion gene (PRNP) have been associated with acquired transmissible spongiform encephalopathy (TSE) susceptibility in mammals; however, this is not the case in cattle. It has been hypothesized that genes, in addition to the prion gene, contribute to genetic susceptibility of acquired TSEs. Accordingly, genetic studies of classical BSE in cattle identified loci other than PRNP that are associated with disease incidence. The objective of this study was to utilize a genome-wide association study to test for genetic loci associated with classical BSE. The samples include 143 BSE affected (case) and 173 unaffected half sib (control) animals collected in the mid 1990s in Southern England. The data analysis identifies loci on two different chromosomes associated with BSE disease occurrence. Most notable is a single nucleotide polymorphism on chromosome 1 at 29.15 Mb that is associated with BSE disease (p = 3.09E-05). Additionally, a locus on chromosome 14, within a cluster of SNPs showed a trend toward significance (p = 5.24E-05). It is worth noting that in a human vCJD study markers on human chromosome 8, a region with shared synteny to the region identified on cattle chromosome 14, were associated with disease. Further, our candidate genes appear to have plausible biological relevance with the known etiology of TSE disease. One of the candidate genes is hypothetical gene LOC521010, similar to FK506 binding protein 2 located on chromosome 1 at 29.32 Mb. This gene encodes a protein that is a member of the immunophilin protein family and is involved in basic cellular processes including protein folding. The chromosomal regions identified in this study and candidate genes within these regions merit further investigation.     

An integrated radiation hybrid map of bovine chromosome 18 that refines a critical region associated with multiple ocular defects in cattle

Congenital multiple ocular defects (MOD) of Japanese black cattle is a hereditary ocular disorder with an autosomal recessive mode of inheritance showing developmental defects of the lens, retina and iris, persistent embryonic eye vascularization and microphthalmia. The MOD locus has been mapped by linkage analysis to a 6.6-cM interval on the proximal end of bovine chromosome 18, which corresponds to human chromosome 16q and mouse chromosome 8. To refine the MOD region in cattle, we constructed an integrated radiation hybrid (RH) map of the proximal region of bovine chromosome 18, which consisted of 17 genes and 10 microsatellite markers, using the SUNbRH7000 panel. Strong conservation of gene order was found among the corresponding chromosomal regions in cattle, human and mouse. The MOD-critical region was fine mapped to a 59.5-cR region that corresponds to a 6.3-Mb segment of human chromosome 16 and a 4.8-Mb segment of mouse chromosome 8. Several positional candidate genes, including FOXC2 and USP10, were identified in this region.     

Comparative mapping of genes from human chromosome 12 by genetic linkage mapping in cattle.

Loci from human chromosome 12 were mapped in cattle to compare the gene order between species. Polymorphisms were detected in cattle in six loci that had been mapped with high precision in humans. Four of these loci, LALBA, SLC2A3, SYT1, and TPI1, mapped to bovine chromosome 5, and one, PLA2G1B, mapped to bovine chromosome 17. The sixth locus, SLC2A3L, due to a fragment produced by the SLC2A3 primers, maps to the telomeric region of BTA18. The differences in gene order between human chromosome 12 and cattle chromosome 5, when these loci are added to others already mapped in cattle, show evidence of significant rearrangement in gene order requiring several evolutionary events. There is also evidence in cattle chromosome 5 of the interspersal of material conserved on human chromosome 22 into the material conserved on human chromosome 12, consistent with ZOOFISH analyses. This analysis indicates that the larger block near the centromere is conserved on the long arm of human chromosome 12 and the smaller block near the telomere is conserved as part of the short arm of human chromosome 12. The level of variation detected in the amplified cattle DNA was approximately 1 variant per 464 nucleotides of haploid DNA using single-strand conformation polymorphism analysis. This corresponds to a per individual level of 1 variant per 1, 961 nucleotides of haploid DNA. This confirms lower genetic variability in cattle compared to humans but indicates the potential for millions of single nucleotide polymorphisms in cattle.     

Genetic and physical mapping of the locus for autosomal dominant renal Fanconi syndrome, on chromosome 15q15.3

Autosomal dominant renal Fanconi syndrome is a genetic model for the study of proximal renal tubular transport pathology. We were able to map the locus for this disease to human chromosome 15q15.3 by genotyping a central Wisconsin pedigree with 10 affected individuals. After a whole-genome scan with highly polymorphic simple sequence repeat markers, a maximum LOD score of 3.01 was calculated for marker D15S659 on chromosome 15q15.3. Linkage and haplotype analysis for an additional 24 markers flanking D15S659 narrowed the interval to approximately 3 cM, with the two highest single-point LOD scores observed being 4.44 and 4.68 (for D15S182 and D15S537, respectively). Subsequently, a complete bacterial artificial chromosome contig was constructed, from the High Throughput Genomic Sequence Database, for the region bounded by D15S182 and D15S143. The identification of the gene and gene product altered in autosomal dominant renal Fanconi syndrome will allow the study of the physiology of proximal renal tubular transport.     

Arachnomelia in Brown Swiss cattle maps to chromosome 5

Arachnomelia in Brown Swiss cattle is a monogenic autosomal recessive inherited congenital disorder of the skeletal system giving affected calves a spidery look (OMIA ID 000059). Over a period of 20 years 15 cases were sampled in the Swiss and Italian Brown cattle population. Pedigree data revealed that all affected individuals trace back to a single acknowledged carrier founder sire. A genome scan using 240 microsatellites spanning the 29 bovine autosomes showed homozygosity at three adjacent microsatellite markers on bovine Chr 5 in all cases. Linkage analysis confirmed the localization of the arachnomelia mutation in the region of the marker ETH10. Fine-mapping and haplotype analysis using a total of 34 markers in this region refined the critical region of the arachnomelia locus to a 7.19-Mb interval on bovine Chr 5. The disease-associated IBD haplotype was shared by 36 proven carrier animals and allows marker-assisted selection. As the corresponding human and mouse chromosome segments do not contain any clear functional candidate genes for this disorder, the mutation causing arachnomelia in the Brown Swiss cattle might help to identify an unknown gene in bone development. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Replication-Defective Bovine Adenovirus Type 3 as an Expression Vector

Although recombinant human adenovirus (HAV)-based vectors offer several advantages for somatic gene therapy and vaccination over other viral vectors, it would be desirable to develop alternative vectors with prolonged expression and decreased toxicity. Toward this objective, a replication-defective bovine adenovirus type 3 (BAV-3) was developed as an expression vector. Bovine cell lines designated VIDO R2 (HAV-5 E1A/B-transformed fetal bovine retina cell [FBRC] line) and 6.93.9 (Madin-Darby bovine kidney [MDBK] cell line expressing E1 proteins) were developed and found to complement the E1A deletion in BAV-3. Replication-defective BAV-3 with a 1.7-kb deletion removing most of the E1A and E3 regions was constructed. This virus could be grown in VIDO R2 or 6.93.9 cells but not in FBRC or MDBK cells. The results demonstrated that the E1 region of HAV-5 has the capacity to transform bovine retina cells and that the E1A region of HAV-5 can complement that of BAV-3. A replication-defective BAV-3 vector expressing bovine herpesvirus type 1 glycoprotein D from the E1A region was made. A similar replication-defective vector expressing the hemagglutinin-esterase gene of bovine coronavirus from the E3 region was isolated. Although these viruses grew less efficiently than the replication-competent recombinant BAV-3 (E3 deleted), they are suitable for detailed studies with animals to evaluate the safety, duration of foreign gene expression, and ability to induce immune responses. In addition, a replication-competent recombinant BAV-3 expressing green fluorescent protein was constructed and used to evaluate the host range of BAV-3 under cell culture conditions. The development of bovine E1A-complementing cell lines and the generation of replication-defective BAV-3 vectors is a major technical advancement for defining the use of BAV-3 as vector for vaccination against diseases of cattle and somatic gene therapy in humans.     

Localization of the ß-subunit of follicle stimulating hormone in cattle and sheep by in situ hybridization

The locus for the beta-subunit of the follicle stimulating hormone gene (FSHB) has been determined in both cattle and sheep by in situ hybridization of a bovine and an ovine cDNA probe, respectively, to metaphase chromosomes. Our results show that the FSHB locus is on cattle chromosome 15 in the region of bands q24-qter and in sheep on the cytogenetically homologous chromosome 15, also in the region q24-qter. The mapping of the FSHB gene in cattle together with the location of other genes (CAT, HBB and PTH) previously found to be syntenic in cattle and on human chromosome 11p, defines an evolutionarily conserved synteny. The localization of the FSHB gene to a cytogenetically homologous region in cattle and sheep is consistent with the hypothesis of extensively conserved chromosome structure in these two species.     

Genetic mapping of the belt pattern in Brown Swiss cattle to BTA3

The white belt pattern of Brown Swiss cattle is characterized by a lack of melanocytes in a stretch of skin around the midsection. This pattern is of variable width and sometimes the belt does not fully circle the body. To identify the gene responsible for this colour variation, we performed linkage mapping of the belted locus using six segregating half-sib families including 104 informative meioses for the belted character. The pedigree confirmed a monogenic autosomal dominant inheritance of the belted phenotype in Brown Swiss cattle. We performed a genome scan using 186 microsatellite markers in a subset of 88 animals of the six families. Linkage with the belt phenotype was detected at the telomeric region of BTA3. Fine-mapping and haplotype analysis using 19 additional markers in this region refined the critical region of the belted locus to a 922-kb interval on BTA3. As the corresponding human and mouse chromosome segments contain no obvious candidate gene for this coat colour trait, the mutation causing the belt pattern in the Brown Swiss cattle might help to identify an unknown gene influencing skin pigmentation.     

Characterization of bovine TGM1 and exclusion as candidate gene for ichthyosis in Chianina

Ichthyosis is a heterogeneous group of keratinization disorders reported both in human and animals. Two rare, inherited forms have been reported in cattle, both characterized by autosomal recessive transmission. Because mutations of transglutaminase 1 (TGM1) gene are associated with autosomal recessive ichthyosis in people, this gene was investigated as a candidate for the diseases in cattle. Three different polymorphisms were identified in 5' end region of cattle TGM1. Marker homozygosity was not found among affected calves. Linkage analysis excluded (logarithmic odds [LOD] score -2.0) TGM1 as the cause for ichthyosis phenotype in the analyzed Chianina cases.     

SNP-based association mapping of Arachnomelia in Fleckvieh cattle

Bovine arachnomelia is an inherited congenital disorder with malformation mainly of the limbs, the vertebral column and the skull, following a monogenic autosomal recessive heredity. Despite almost identical pathological findings, arachnomelia has previously been mapped to bovine chromosome 23 and 5 in Fleckvieh and Braunvieh respectively. Therefore, this disorder may be an example of locus heterogeneity in cattle. This study aimed to refine the candidate region to allow positional cloning and sequence analyses of candidate genes in Fleckvieh cattle. For that purpose, a case-control association mapping design was set up with a case group of 16 pre-selected affected individuals and a control group of 50 unrelated animals. The subset of affected animals was selected from a total of 129 pathologically confirmed cases due to the occurrence of recombination(s) within a 14.5 cM candidate interval previously mapped to chromosome 23. Six linked microsatellites currently used for indirect gene testing in Fleckvieh were analysed for this purpose. In all selected cases, a genome-wide scan using 44 473 informative SNPs revealed shared segments of homozygosity at 15 adjacent SNPs on chromosome 23. Additional haplotype analysis of 37 carrier bulls confirmed the localization of the arachnomelia locus to a region of 927 kb (13.622-14.549 Mb) containing molybdenum cofactor biosynthesis protein 1 gene, the most likely candidate gene for arachnomelia in Fleckvieh. The number of recombinant haplotypes observed in cases was more than doubled compared with the number of expected recombinations. This remarkably increased mapping resolution and thus illustrates the benefit of pre-selection in association studies.     

Assignment of the growth hormone gene locus to 19q26-qter in cattle and to 11q25-qter in sheep by in situ hybridization

The growth hormone gene locus (GH) of cattle and sheep was mapped to a chromosomal region in each species by using in situ hybridization. The probe employed was an 830-bp cDNA sequence from the ovine growth hormone gene. Based on QFQ chromosome preparations, our results show that the GH locus is on cattle chromosome 19 in the region of bands q26-qter and in sheep on chromosome region 11q25-qter. The GH assignments together with previous localizations of type I cytokeratin genes (KRTA) and one homeobox (HOX2) gene in cattle and one type I cytokeratin gene (KRTA) in sheep identify a strongly conserved chromosomal segment on human chromosome 17, bovine chromosome 19, and sheep chromosome 11.