[Polychondritis: a new disease in Swiss Braunvieh?]. / Polychondritis: Eine neue Erkrankung beim Braunvieh?

Over the last 10 years Swiss Braunvieh cattle with malformations of the pinnae have repeatedly been reported. Endoscopy revealed a shortened and thickened epiglottis and malformations of the arytenoid cartilage in some of these animals. In most cases the elastic cartilage was replaced by fibrocartilage and hyaline cartilage. The direct cause and pathogenesis of the malformations could not be determined.

Clinical and molecular characterization of a re-established line of sheep exhibiting hemophilia A.

BACKGROUND: Large animal models that accurately mimic human hemophilia A (HA) are in great demand for developing and testing novel therapies to treat HA. OBJECTIVES: To re-establish a line of sheep exhibiting a spontaneous bleeding disorder closely mimicking severe human HA, fully characterize their clinical presentation, and define the molecular basis for disease. PATIENTS/METHODS: Sequential reproductive manipulations were performed with cryopreserved semen from a deceased affected ram. The resultant animals were examined for hematologic parameters, clinical symptoms, and responsiveness to human FVIII (hFVIII). The full coding region of sheep FVIII mRNA was sequenced to identify the genetic lesion. RESULTS AND CONCLUSIONS: The combined reproductive technologies yielded 36 carriers and 8 affected animals. The latter had almost non-existent levels of FVIII:C and extremely prolonged aPTT, with otherwise normal hematologic parameters. These animals exhibited bleeding from the umbilical cord, prolonged tail and nail cuticle bleeding time, and multiple episodes of severe spontaneous bleeding, including hemarthroses, muscle hematomas and hematuria, all of which responded to hFVIII. Inhibitors of hFVIII were detected in four treated animals, further establishing the preclinical value of this model. Sequencing identified a premature stop codon and frame-shift in exon 14, providing a molecular explanation for HA. Given the decades of experience using sheep to study both normal physiology and a wide array of diseases and the high homology between human and sheep FVIII, this new model will enable a better understanding of HA and facilitate the development and testing of novel treatments that can directly translate to HA patients.

Veterinary cytogenetics: past and perspective.

Cytogenetics was conceived in the late 1800s and nurtured through the early 1900s by discoveries pointing to the chromosomal basis of inheritance. The relevance of chromosomes to human health and disease was realized more than half a century later when improvements in techniques facilitated unequivocal chromosome delineation. Veterinary cytogenetics has benefited from the information generated in human cytogenetics which, in turn, owes its theoretical and technical advancement to data gathered from plants, insects and laboratory mammals. The scope of this science has moved from the structure and number of chromosomes to molecular cytogenetics for use in research or for diagnostic and prognostic purposes including comparative genomic hybridization arrays, single nucleotide polymorphism array-based karyotyping and automated systems for counting the results of standard FISH preparations. Even though the counterparts to a variety of human diseases and disorders are seen in domestic animals, clinical applications of veterinary cytogenetics will be less well exploited mainly because of the cost-driven nature of demand on diagnosis and treatment which often out-weigh emotional and sentimental attachments. An area where the potential of veterinary cytogenetics will be fully exploited is reproduction since an inherited aberration that impacts on reproductive efficiency can compromise the success achieved over the years in animal breeding. It is gratifying to note that such aberrations can now be tracked and tackled using sophisticated cytogenetic tools already commercially available for RNA expression analysis, chromatin immunoprecipitation, or comparative genomic hybridization using custom-made microarray platforms that allow the construction of microarrays that match veterinary cytogenetic needs, be it for research or for clinical applications. Judging from the technical refinements already accomplished in veterinary cytogenetics since the 1960s, it is clear that the importance of the achievements to date are bound to be matched or out-weighed by what awaits to be accomplished in the not-too-far future.

Analysis and mapping of CACNB4, CHRNA1, KCNJ3, SCN2A and SPG4, physiological candidate genes for porcine congenital progressive ataxia and spastic paresis.

The cause of porcine congenital progressive ataxia and spastic paresis (CPA) is unknown. This severe neuropathy manifests shortly after birth and is lethal. The disease is inherited as a single autosomal recessive allele, designated cpa. In a previous study, we demonstrated close linkage of cpa to microsatellite SW902 on porcine chromosome 3 (SSC3), which corresponds syntenically to human chromosome 2. This latter chromosome contains ion channel genes (Ca(2+), K(+) and Na(+)), a cholinergic receptor gene and the spastin (SPG4) gene, which cause human epilepsy and ataxia when mutated. We mapped porcine CACNB4, KCNJ3, SCN2A and CHRNA1 to SSC15 and SPG4 to SSC3 with the INRA-Minnesota porcine radiation hybrid panel (IMpRH) and we sequenced the entire open reading frames of CACNB4 and SPG4 without finding any differences between healthy and affected piglets. An anti-epileptic drug treatment with ethosuximide did not change the severity of the disease, and pigs with CPA did not exhibit the corticospinal tract axonal degeneration found in humans suffering from hereditary spastic paraplegia, which is associated with mutations in SPG4. For all these reasons, the hypothesis that CACNB4, CHRNA1, KCNJ3, SCN2A or SPG4 are identical with the CPA gene was rejected.

Radiation hybrid mapping of 18 positional and physiological candidate genes for arthrogryposis multiplex congenita on porcine chromosome 5.

We report the chromosomal assignment of 18 porcine genes to human homologues using the INRA-Minnesota swine radiation hybrid panel (IMpRH). These genes (CACNA1C, COL2A1, CPNE8, C3F, C12ORF4, DDX11, GDF11, HOXC8, KCNA1, MDS028, TMEM106C, NR4A1, PHB2, PRICKLE1, Q6ZUQ4, SCN8A, TUBA8 and USP18) are located on porcine chromosome 5 (SSC5) and represent positional and functional candidates for arthrogryposis multiplex congenita (AMC), which maps to SSC5. CPNE8, PRICKLE1, Q6ZUQ4 and TUBA8 were mapped to the interval for pig AMC between microsatellites SW152 and SW904. Three SNPs in TUBA8 co-segregated with the AMC phenotype in 230 pigs of our research population without recombination and could be used as a genetic marker test for AMC. In addition, we provide evidence that a small chromosomal region of HSA22q11.2 evolutionarily corresponds to SSC5q12-q22 (and contains the human homologues of porcine SW152, Q6ZUQ4, TUBA8 and USP18), while the regions flanking HSA22q11.2 on SSC5 correspond to HSA12p13 and HSA12q12. We identified seven distinct chromosomal blocks, further supporting extensive rearrangements between genes on HSA12 and HSA22 in the AMC region on SSC5.

Application of bovine microsatellite markers for genetic diversity analysis of Swiss yak (Poephagus grunniens).

In order to assess the applicability of bovine microsatellite markers for population genetic studies in Swiss yak, 131 bovine microsatellite markers were tested on a panel of 10 animals. Efficient amplification was observed for 124 markers (94.6%) with a total of 476 alleles, of which 117 markers (94.3%) were polymorphic. The number of alleles per locus among the polymorphic markers ranged from two to nine. Seven loci (ILSTS005, BMS424B, BMS1825, BMS672, BM1314, ETH123 and BM6017) failed to amplify yak genomic DNA. Two cattle Y-chromosome specific microsatellite markers (INRA126 and BM861) amplified genomic DNA from both male and female yaks. However, two additional markers on cattle Y-chromosome (INRA124 and INRA189) amplified DNA from only males. Of the polymorphic markers, 24 microsatellites proposed by CaDBase for within- and cross-species comparisons and two additional highly polymorphic markers (MHCII and TGLA73) were used to investigate the genetic variability and the population structure of a Swiss yak herd that included 51 additional animals. The polymorphic information content ranged from 0.355 to 0.752, while observed heterozygosity (HO) ranged from 0.348 to 0.823. Furthermore, a set of 13 markers, organized into three multiplex polymerase chain reactions, was evaluated for routine parentage testing. This set provided an exclusion probability in a family of four yaks (both parents and two offspring) of 0.995. These microsatellites serve as useful tools for genetic characterization of the yak, which continues to be an important domestic livestock species.

Inheritance of the F4ab, F4ac and F4ad E. coli receptors in swine and examination of four candidate genes for F4acR.

Susceptibility to enterotoxigenic Escherichia coli with fimbriae F4ac is dominantly inherited in the pig. A three-generation pedigree was created to refine the position of F4acR on chromosome 13 comprising 202 pigs: eight parents, 18 F1 and 176 F2 pigs. The 17-point analysis indicates that F4acR lies between Sw207 and S0283. Recombinant offspring specify that the most probable order is Sw207-S0075-F4acR-Sw225-S0283. We observed six phenotypes for the three fimbrial variants F4ab, F4ac and F4ad. The two missing phenotypes F4abR-/F4acR+/F4adR+ and F4abR-/F4acR+/F4adR- indicate that pigs susceptible to F4ac are always susceptible to F4ab. Furthermore, a weak and a strong adhesion of F4ab and F4ad bacteria was observed. The weak receptor F4abR (F4abRw) was present only in pigs devoid of the receptor F4acR (F4abR+/F4acR-). In contrast, in pigs with the phenotype F4abR+/F4acR+, F4ab bacteria adhered to the majority of enterocytes. F4abRw constitutes a frequently observed phenotype whose inheritance is still unclear. Strong adhesion of F4ab and F4ac bacteria is most likely influenced by the same receptor that we name F4bcR. The number of F4ad bacteria that adhered to enterocytes was very variable in the adhesion test. Moreover, expression of F4adR was independent of age. Our segregation analyses indicated a dominant inheritance of F4adR, although the number of susceptible pigs was smaller than expected. We examined four genes as candidates for the F4acR locus: the transferrin receptor gene (TFRC) and three genes members of the glucosyl/galactosyltransferase family (B3GnT5, B3GALT3 and B4GALT4). Comparison of sequences from resistant and homozygous susceptible F4ac pigs did not reveal any causative single nucleotide polymorphism in the four genes. Two silent mutations at the positions 295 (C/T) and 313 (T/C) in B3GALT3 were found. Using the somatic cell hybrid panel, B3GnT5 and B3GALT3 were assigned to the chromosomal region SSC13q23-q41. No mutations were found in the cDNA sequences of these genes associated with the F4acR genotypes.

Identification and characterization of novel peroxisome proliferator-activated receptor-gamma (PPAR-gamma) transcriptional variants in pig and human.

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the steroid/thyroid/retinoid receptor superfamily, and is primarily expressed in fat tissue. To date, two major PPAR-gamma isoforms have been identified in pig, PPAR-gamma1 and PPAR-gamma2. Porcine PPAR-gamma1a consists of two leader exons, designated A1 and A2, followed by six exons containing the open reading frame. Here, we report the isolation and characterization of three novel PPAR-gamma1 transcripts. PPAR-gamma1b is derived from exon A1, with exon A2 spliced out. PPAR-gamma1c and PPAR-gamma1d are derived from the new exon, A', containing exon A2 (gamma1c) or without exon A2 (gamma1d). Based on PCR analysis of PAC clones that included sequences from the 5'-untranslated region of the PPAR-gamma gene, the new A' exon is located between the known exons A1 and A2. We also isolated the human homologue to exon A', as well as the two new PPAR-gamma1c and -gamma1d splice variants, from human adipose tissue. Studies of the expression of porcine PPAR-gamma by real time reverse transcription-polymerase chain reaction analysis show that transcripts derived from exon A1 were not expressed at significantly different levels in visceral fat (lamina subserosa) or subcutaneous fat (back fat, inner and outer layer). In contrast, exon A'-derived transcripts were expressed at progressively higher levels in the inner and outer layers of subcutaneous fat than in visceral fat. The same expression pattern was also observed for PPAR-gamma2. We hypothesize that there are three promoters, which differentially regulate PPAR-gamma1 and PPAR-gamma2 gene expression, depending on the specific localization of the fat tissue.

Phylogenetic analysis and species allocation of individual equids using microsatellite data.

The taxonomic status of all equid species is not completely unravelled. This is of practical relevance for conservation initiatives of endangered, fragmented equid populations, such as the Asiatic wild asses (in particular Equus hemionus onager and E. hemionus kulan). In this study, a marker panel consisting of 31 microsatellite loci was used to assess species demarcation and phylogeny, as well as allocation of individuals (n = 120) to specific populations of origin (n = 11). Phylogenetic analysis revealed coalescence times comparable with those previously published from fossil records and mtDNA data. Using Bayesian approaches, it was possible to distinguish between the studied equids, although individual assignment levels varied. The observed results support the maintenance of separate captive conservation herds for E. hemionus onager and E. hemionus kulan. The first molecular genetic results for E. hemionus luteus remained contradictory, as they unexpectedly indicated a closer genetic relationship between E. hemionus luteus and E. kiang holderi compared with the other hemiones.

Comparative chromosomal studies of E. caballus (ECA) and E. przewalskii (EPR) in a female F1 hybrid.

Previous research revealed that the karyotypes of Equus przewalskii (2n = 66) and Equus caballus (2n = 64) differ by one pair of metacentric chromosomes, present in ECA but not in EPR, and two pairs of acrocentric chromosomes found only in the EPR karyotype. The formation of a trivalent during meiosis in a male F1 hybrid and the homologies in G-banding patterns suggest that ECA 5 corresponds to two acrocentric EPR chromosomes resulting from a Robertsonian fusion or fission event. Chromosomal investigations of a female interspecies F1 hybrid including banded karyograms and fluorescence in situ hybridization (FISH) studies focusing on the p and q arm of ECA 5 were conducted. Q- and G-banding patterns of E. caballus, E. przewalskii and the hybrid revealed interspecies homology between all chromosome pairs except for ECA 5, EPR 23 and EPR 24, which were unique for that particular species. Furthermore, they indicated homology between ECA 5p and EPR 23 as well as between ECA 5q and EPR 24. FISH revealed hybridization of the BACs laminin beta 3 (LAM B3) and laminin gamma 2 (LAM C2) to ECA 5p and EPR 23. However, nuclear factor I (NFIA) and immunoglobulin lambda (IGL@), primarily assigned to ECA 5q, mapped to ECA 7 and EPR 6 respectively. Thus the karyotypes of E. caballus and E. przewalskii differ solely by one Robertsonian translocation (ECA 5 =EPR 23 + EPR 24).

Bovine spinal muscular atrophy: AFG3L2 is not a positional candidate gene.

Bovine spinal muscular atrophy (BSMA) is a neurodegenerative disorder, which is widespread in Brown Swiss cattle. Main symptoms of the disease are muscular atrophy and recumbency. Affected calves die within few days or weeks. BSMA seems to be inherited as a recessive trait and the disease allele appears to have a common origin. In this study, a pedigree with 30 affected BSMA calves was used to genetically localize the BSMA locus. Linkage analysis was performed between microsatellite markers of seven chromosomes, where the homologous genes of human neurodegenerative disorders are located according to comparative mapping data, and the disease genotype. BSMA was mapped to chromosome 24 confirming the recently published localization (Medugorac et al. 2003). The candidate gene AFG3L2 was physically mapped to chromosome 24q24 using fluorescence in situ hybridization. Due to their different localizations AFG3L2 is not a positional candidate for BSMA. An informative marker localized on the telomeric side of the BSMA locus would be beneficial for marker-assisted selection as well as searching for the causative gene. However, finding a marker distal to BSMA locus is difficult because of its position at the end of the chromosome.

Application of bovine microsatellite markers on Saola (Pseudoryx nghetinhensis).

The aim of the present study was to assess the applicability of bovine microsatellite markers on Saola (Pseudoryx nghetinhensis). A total of 127 microsatellite markers were tested on a male and a young female Saola. An efficient amplification was observed for 123 markers (96.8%), 73 markers (59.3%) were polymorphic. Four loci (BM2304, BMS1928, BMS779 and ILSTS006) on cattle chromosomes 1, 4, 7 and 8, respectively, failed to amplify in Saola. Two cattle Y-chromosome-specific microsatellite markers (INRA126 and BM861) were successfully amplified from both sexes in Saola. However, two additional markers (INRA124 and INRA189) on Y-chromosome failed to amplify in the female animal. These results show that most of the bovine microsatellite markers are applicable in Saola and therefore they can be used to study the phylogenetic relationships and the genetic diversity of the Saola population.

Cytogenetic comparison of saola (Pseudoryx nghetinhensis) and cattle (Bos taurus) using G- and Q-banding and FISH.

In order to cytogenetically describe the new bovid species saola (Pseudoryx nghetinhensis), comparative G- and Q-banding of saola and cattle (Bos taurus) chromosomes as well as FISH-mapping of 32 type-I markers (29 Texas markers and three additional markers) on saola chromosomes were performed. Saola was shown to have a diploid number of 2n = 50 chromosomes possessing five biarmed autosomal pairs and an acrocentric X chromosome. Homology of saola and cattle chromosomes was indicated by banding patterns and by marker hybridization suggesting that all five biarmed pairs in saola originate from centric fusions involving ten cattle autosomes. However, small intrachromosomal rearrangements cannot be excluded. In this study the first preliminary homology map of these two species is presented.

[Molecular genetic studies of the "Einsiedler" horse population]. / Molekulargenetische Untersuchungen beim Einsiedler-Pferd.

In this study it was investigated whether the "Einsiedler" warmblood horse, a historically old horse population from central Switzerland (Abbey of Einsiedeln), is distinguishable from micellaneous horse breeds, using molecular genetic techniques. The breeding history of Einsiedler horses is characterised by systematic line breeding through the dams. Therefore, two Einsiedler dam lines (N = 28), going back to the middle of the 19th century according to pedigree entries, were the focus of the survey. Random samples of diverse warmblood horse populations, but also samples from more distinct types of horse breeds, served as comparison populations (N = 52). Variation in the mitochondrial genome appeared to be only partially informative to demarcate the studied horses, as horses of distinct breeds may share identical mtDNA sequence fragments. Both dam lines revealed haplotypes commonly found in Iberian horse breeds. This is to take as an indication on the genetic origin of Einsiedler horses. Furthermore, the Klima dam line held a homologous mtDNA sequence fragment with E. ferus przewalskii. Therefore, this seems to be a phylogenetically old haplotype. The analysis of microsatellite loci revealed that horses from the two Einsiedler dam lines were in fact distinguishable from more distinct types of horses, but not from closely related European warmblood horse breeds and English thoroughbred.

Characterization of G-banded chromosomes of a female saola (Pseudoryx nghetinhensis,2n = 50) and X chromosome i dentification by means of fluorescent in situ hybridization.

The saola (Pseudoryx nghetinhensis) is a newly discovered large mammal species, belongs to the subfamily Bovinae and is listed as being endangered. Due to the limitation of the material available, no cytogenetic studies have been carried out on this species. In the present study, preliminary cytogenetic analysis was undertaken on cultured female fibroblast cells to characterize the karyotype organization of saola. An examination of 120 Giemsa stained metaphases showed the diploid chromosome number of 2n = 50, including five bi-armed chromosome pairs. The distribution of constitutive heterochromatin in saola was studied. However, the variability in the size of C-bands was not significant on all the homologous chromosomes. The X chromosome pair, corresponding to the largest telocentric chromosomes, was identified by fluorescent in situ hybridization (FISH) using a bacterial artificial chromosome clone (BAC 0577G05, which maps to BTAXq25-->q33). In comparison to the standard karyotype of cattle (ISCNDB 2000), a G-banded ideogram of saola (about 390 band level) was presented. This work, therefore, provided a basic insight into the karyotype organization of this endangered species and will be particularly useful to improve the understanding of differences of genomes between related species.

Bayesian inference on major loci in related multigeneration selection lines of laying hens.

A mixed inheritance model, postulating a polygenic effect and differences between the 3 genotypes of a biallelic locus, was fitted separately to the data of 2 multigeneration selection lines and a control evolving from a common base population. Inferences about the model were drawn from the application of the Gibbs sampler. Body weight at 20 and 40 wk (BW20, BW40) and average egg weight to 40 wk (EW40) were included in the analyses. Significance of differences between posterior means of parameters was established by comparing their 95% highest probability density regions. Significant (P < 0.05) additive and dominance effects due to the genotypes at the major locus were found for all traits. The allele causing a lower trait value was the (partial) dominant one in all traits, leading to a negative dominance effect. The additive variance due to the major locus was also significant, i.e., greater than zero (P < 0.05) in all traits, whereas the dominance variance was only important for EW40. With the exception of the residual variances of one selection and the control line, no (P > 0.05) differences of posterior means of any parameter could be observed between lines. No significant genotypic or polygenic selection response was found for BW40. On the contrary, both genetic responses were found significant for EW40 in the selected lines, but not in the control. No differences (P > 0.05) between lines could be observed for the derived frequencies of the allele causing the higher trait value and the frequencies of one homozygote and the heterozygote genotypes at the major locus. The detection of a major locus with relatively modest effect confirmed that this type of analysis with data from selected lines was indeed powerful.