A. T. Bowling

A missense mutation in the endothelin-B receptor gene is associated with Lethal White Foal Syndrome: An equine version of Hirschsprung Disease

Abstract. Lethal White Foal Syndrome is a disease associated with horse breeds that register white coat spotting patterns. Breedings between particular spotted horses, generally described as frame overo, produce some foals that, in contrast to their parents, are all white or nearly all white and die shortly after birth of severe intestinal blockage. These foals have aganglionosis characterized by a lack of submucosal and myenteric ganglia from the distal small intestine to the large intestine, similar to human Hirschsprung Disease. Some sporadic and familial cases of Hirschsprung Disease are due to mutations in the endothelin B receptor gene (EDNRB). In this study, we investigate the role of EDNRB in Lethal White Foal Syndrome. A cDNA for the wild-type horse endothelin-B receptor gene was cloned and sequenced. In three unrelated lethal white foals, the EDNRB gene contained a 2-bp nucleotide change leading to a missense mutation (I118K) in the first transmembrane domain of the receptor, a highly conserved region of this protein among different species. Seven additional unrelated lethal white foal samples were found to be homozygous for this mutation. No other homozygotes were identified in 138 samples analyzed, suggesting that homozygosity was restricted to lethal white foals. All (40/40) horses with the frame overo pattern (a distinct coat color pattern that is a subset of overo horses) that were tested were heterozygous for this allele, defining a heterozygous coat color phenotype for this mutation. Horses with tobiano markings included some carriers, indicating that tobiano is epistatic to frame overo. In addition, horses were identified that were carriers but had no recognized overo coat pattern phenotype, demonstrating the variable penetrance of the mutation. The test for this mutant allele can be utilized in all breeds where heterozygous animals may be unknowingly bred to each other including the Paint Horse, Pinto horse, Quarter Horse, Miniature Horse, and Thoroughbred.

Synteny and Regional Marker Order Sssignment of 26 type I and Microsatellite Markers to the Horse X- and Y-Chromosomes

The hypothesis that the conservation of sex-chromosome-linked genes among placental mammals could be extended to the horse genome was tested using the UCDavis horse–mouse somatic cell hybrid (SCH) panel. By exploiting the fluorescence in-situ hybridization (FISH) technique to localize an anchor locus, X-inactivation-specific transcript (XIST) on the horse X chromosome, together with the fragmentation and translocation of the X- and Y-chromosome fragments in a somatic cell hybrid panel, we regionally assigned 13 type I and 13 type II (microsatellite) markers to the horse X- and Y-chromosomes. The synteny groups that correspond to horse X- and Y-chromosomes were identified by synteny mapping of sex-specific loci zinc finger protein X-linked (ZFX), zinc finger protein Y-linked (ZFY) and sex-determining region Y (SRY) on the SCH panel. A non-pseudoautosomal gene in the human steroid sulfatase (STS) was identified in both X- and Y-chromosome- containing clones. The regional order of the X-linked type I markers examined in this study, from Xp- to Xq-distal, was [STS- X, the voltage-gated chloride channel 4 (CLCN4)], [ZFX, delta- aminolevulinate synthase 2 (ALAS2)], XIST, coagulation factor IX (F9) and [biglycan (BGN), equine F18, glucose-6-phosphate dehydrogenase (G6PD)] (precise marker order could not be determined for genes within the same brackets). The order of the Y-linked type I markers was STS-Y, SRYand ZFY. These orders are the same arrangements as reported for the human X- and Y-chromosomes, supporting the conservation of genomic organization between the human and the horse sex chromosomes. Regional ordering of X- linked type I and microsatellite markers provides the first integration of type I and type II markers in the horse X chromosome.

Comparative mapping of 18 equine type I genes assigned by somatic cell hybrid analysis.

Abstract. Polymerase chain reaction primers designed from horse cDNA sequences and from consensus sequences highly conserved in mammalian species were used to amplify markers for synteny mapping 18 equine type I genes. These markers were used to screen a horse–mouse somatic cell hybrid panel (UCDavis SCH). Fourteen primer sets amplified horse-specific fragments, while restriction enzyme digests of PCR products were used to distinguish the fragments amplified from horse and mouse with four primer sets. Synteny assignments were made based on correlation values between each marker tested and other markers in the UCDavis SCH panel database. The 18 horse genes were assigned to previously established synteny groups. Synteny mapping of two genes previously mapped in the horse by FISH was used to anchor two UCD synteny groups to horse chromosomes. Previous chromosome assignments of three equine loci by FISH were confirmed. Comparative mapping analysis based on published human–horse Zoo-FISH data and the synteny mapping of 14 horse genes confirmed the physical assignment of 12 synteny groups to the respective horse chromosomes and was used to infer the physical location of one synteny group.

Mapping of 13 horse genes by fluorescence in-situ hybridization (FISH) and somatic cell hybrid analysis

We report fluorescence in-situ hybridization (FISH) and somatic cell hybrid mapping data for 13 different horse genes (ANP, CD2, CLU, CRISP3, CYP17, FGG, IL1RN, IL10, MMP13, PRM1, PTGS2, TNFA and TP53). Primers for PCR amplification of intronic or untranslated regions were designed from horse-specific DNA or mRNA sequences in GenBank. Two different horse bacterial artificial chromosome (BAC) libraries were screened with PCR for clones containing these 13 Type I loci, nine of which were found in the libraries. BAC clones were used as probes in dual colour FISH to confirm their precise chromosomal origin. The remaining four genes were mapped in a somatic cell hybrid panel. All chromosomal assignments except one were in agreement with human–horse ZOO-FISH data and revealed new and more detailed information on the equine comparative map. CLU was mapped by synteny to ECA2 while human–horse ZOO-FISH data predicted that CLU would be located on ECA9. The assignment of IL1RN permitted analysis of gene order conservation between HSA2 and ECA15, which identified that an event of inversion had occurred during the evolution of these two homologous chromosomes.

Equine Synteny Mapping of Comparative Anchor Tagged Sequences (CATS) From Human Chromosome 5

Abstract. Comparative anchor tagged sequences (CATS) from human Chromosome 5 (HSA5) were used as PCR primers to produce molecular markers for synteny mapping in the horse. Primer sets for 21 genes yielded eight horse-specific markers, which were mapped with the UC Davis horse–mouse somatic cell hybrid panel into two synteny groups: UCD14 and UCD21. These data, in conjunction with earlier human chromosome painting studies of the horse karyotype and synteny mapping of horse microsatellite markers physically mapped by FISH, confirm the assignment of UCD21 to ECA21 and suggest that UCD14 is located on ECA14. In addition, our results can be used to substantiate previously published data which indicate that ECA21 contains material orthologous to HSA5p and HSA5q, and to propose an approximate region for an evolutionary chromosomal rearrangement event.

HETEROGENEITY AND LINKAGE OF EQUINE C4 AND STEROID 21-HYDROXYLASE GENES

The fourth component of complement (C4) is polymorphic in most species studied, and is encoded by a gene or genes within the MHC. In man and mouse there are two closely linked C4 and steroid 21‐hydroxylase (21‐OH) genes. Therefore we have used Southern blotting to determine whether equine C4 and 21‐OH genes are linked. C4 restriction fragment length polymorphism (RFLP) was found with the enzymes EcoRI and BamHI. Comparison of the sizes of EcoRI‐digested fragments of genomic DNA hybridizing with C4 and 21‐OH probes revealed that equine C4 and 21‐OH genes are separated by no more than 13 kb. Further, there is no evidence of C4 and 21‐OH gene duplication in the horse. Segregation of ELA and different polymorphic forms of equine C4 suggest that C4 and 21‐OH genes are within the MHC. It is likely that equine MHC supratypes will provide improved markers of disease susceptibility.

Short Communication. Physical mapping of genetic markers to chromosome 30 using a trisomic horse and evidence for maternal origin of the extra chromosome

A clinical case of autosomal trisomy provided evidence to assign horse microsatellites VHL20 and LEX025 to a synteny group on chromosome 30. Informative genetic markers at these loci identi®ed the aged dam as the source of the extra chromosome. With the application of molecular technologies, the horse gene map is showing substantial progress although still lagging in information content behind genome maps for other livestock species, notably cattle and pigs. Correspondence of substantial blocks of chromosomal homologies between horse and human has been demonstrated using Zoo-FISH (Raudsepp et al. 1996). The consensus reference for physical location of horse genetic markers, including the standard karyotype, idiogram and landmark descriptions, is provided by the International System for Cytogenetic Nomenclature of the Domestic Horse (ISCNH) (ISCNH, in preparation). Physical mapping using in situ hybridization has placed six genes (ELA [MHC], CRC [RYR], GPI, PGD, C3 and HBA) (for references see Bowling 1996) and 20 microsatellites (Sakagami et al. 1995, Tozaki et al. 1995, Breen et al. 1997) on 12 of the 31 autosomes. We report the ®rst use of autosomal trisomy to assign genetic markers to a horse chromosome. In 1990 Bowling and Millon reported a chromosome 30 trisomy for a yearling female Arabian horse clinically characterized by small size, angular limb deformities and gaits compromised by joint stiffness (Bowling & Millon 1990). Based on the prominent association of increased maternal age with human cases of autosomal trisomy, it was proposed that the origin of the extra chromosome might be the 24-year-old dam rather than the 4-year-old sire. If genetic typing of the trisomic case provided evidence of three alleles at any locus, then that trait could be assumed to be physically located on chromosome 30. Typing of the sire, dam and offspring might substantiate the hypothesis of maternal origin for the extra chromosome. The available blood group and protein marker assays at 21 loci did not provide evidence of three alleles for any system, either because of lack of tested markers on the chromosome involved or because of lack of informative polymorphisms. The recent de®nition of horse microsatellite loci has increased the potential battery of genetic markers that can be tested for assignment to chromsome 30 and to identify the maternal or paternal source of the extra chromosome. Initially, we typed this family for a selected panel of 11 dinucleotide microsatellite loci useful for parentage testing (Bowling et al. 1997). Root bulbs from mane hair of the trisomic female and her sire were used as a DNA source. Tissue preparation, PCR ampli®cation and fragment length analysis were performed according to the protocol of Eggleston-Stott and colleagues (1996). The dam was dead and the only available biological material was on 8-year-old Giemsastained slides of alcohol±acetic acid ®xed blood cells used in the original karyotyping study. The procedures for microsatellite typing of the dam were the same as for the offspring and sire except that DNA was obtained from material scraped off an archived slide with a scalpel. Washed scrapings were incubated with 30 ìl of 200 mM NaOH for 10 min at 958C, then the pH of the alkaline extract was lowered before PCR ampli®cation with addition of 30 ìl of a solution of 200 mM HCl and 100 mM Tris-HCl, pH 8.5. In the initial typing of the offspring, one locus (VHL20) (Van Haeringen et al. 1994) presented an atypical fragment pro®le of three major peaks, consistent with location on the trisomic chromosome (chromosome 30). With a mouse/horse somatic cell hybrid panel (data not shown) we have evidence that VHL20 was potentially syntenic with LEX025 (Coogle et al. 1996); therefore, we also tested the three animals for that marker. Fragment pro®les for these two loci of the trisomic horse and her parents are presented (Figure 1). We report our microsatellite results in the standardized alphabetical nomenclature system used for horses by members of the International Society for Animal Genetics (ISAG). For VHL20, a dinucleotide repeat microsatellite, the M marker designation has been assigned to the 97-bp fragment. The trisomic horse showed a pro®le of three major peaks, corresponding to a genetic marker type of LMN. Her sire was typed as IM and the dam as LN. From this information it is clear that the sire Chromosome Research 1997, 5, 429±431