Peter Vögeli

The receptor locus for Escherichia coli F4ab/F4ac in the pig maps distal to the MUC4–LMLN region

Enterotoxigenic Escherichia coli (ETEC) with fimbriae of the F4 family are one of the major causes of diarrhea and death among neonatal and young piglets. Bacteria use the F4 fimbriae to adhere to specific receptors expressed on the surface of the enterocytes. F4 fimbriae exist in three different antigenic variants, F4ab, F4ac, and F4ad, of which F4ac is the most common. Resistance to ETEC F4ab/F4ac adhesion in pigs has been shown to be inherited as an autosomal recessive trait. In previous studies the ETEC F4ab/F4ac receptor locus (F4bcR) was mapped to the q41 region on pig chromosome 13. A polymorphism within an intron of the mucin 4 (MUC4) gene, which is one of the possible candidate genes located in this region, was shown earlier to cosegregate with the F4bcR alleles. Recently, we discovered a Large White boar from a Swiss experimental herd with a recombination between F4bcR and MUC4. A three–generation pedigree including 45 offspring was generated with the aim to use this recombination event to refine the localization of the F4bcR locus. All pigs were phenotyped using the microscopic adhesion test and genotyped for a total of 59 markers. The recombination event was mapped to a 220-kb region between a newly detected SNP in the leishmanolysin-like gene (LMLN g.15920) and SNP ALGA0072075. In this study the six SNPs ALGA0072075, ALGA0106330, MUC13-226, MUC13-813, DIA0000584, and MARC0006918 were in complete linkage disequilibrium with F4bcR. Based on this finding and earlier investigations, we suggest that the locus for F4bcR is located between the LMLN locus and microsatellite S0283.

Refined localization of the Escherichia coli F4ab/F4ac receptor locus on pig chromosome 13

Diarrhoea in newborn and weaned pigs caused by enterotoxigenic Escherichia coli (ETEC) expressing F4 fimbriae leads to considerable losses in pig production. In this study, we refined the mapping of the receptor locus for ETEC F4ab/F4ac adhesion (F4bcR) by joint analysis of Nordic and Swiss data. A total of 236 pigs from a Nordic experimental herd, 331 pigs from a Swiss experimental herd and 143 pigs from the Swiss performing station were used for linkage analysis. Genotyping data of six known microsatellite markers, two newly developed markers (MUC4gt and HSA125gt) and an intronic SNP in MUC4 (MUC4-8227) were used to create the linkage map. The region for F4bcR was refined to the interval SW207-S0075 on pig chromosome 13. The most probable position of F4bcR was in the SW207-MUC4 region. The order of six markers was supported by physical mapping on the BAC fingerprint contig from the Wellcome Trust Sanger Institute. Thus, the region for F4bcR could be reduced from 26 to 14 Mb.

Intragenic deletion in the gene encoding L-gulonolactone oxidase causes vitamin C deficiency in pigs.

The absence of L-ascorbic acid (L-AA, or AA) synthesis in scurvy-prone organisms, including humans, other primates, guinea pigs, and flying mammals, was traced to the lack of L-gulonolactone oxidase (GULO) activity. GULO is a microsomal enzyme that catalyzes the terminal step in the biosynthesis of L-AA. Clinical cases of scurvy were described in a family of Danish pigs. This trait is controlled by a single autosomal recessive allele designated od (osteogenic disorder). Here we demonstrate that the absence of GULO activity and the associated vitamin C deficiency in od/od pigs is due to the occurrence of a 4.2-kbp deletion in the GULO gene. This deletion includes 77 bp of exon VIII, 398 bp of intron 7 and 3.7 kbp of intron 8, which leads to a frame shift. The mutant protein is truncated to 356 amino acids, but only the first 236 amino acids are identical to the wild-type GULO protein. In addition, the od allele seems to be less expressed in deficient and heterozygous pigs compared with the normal allele in heterozygous and wild-type animals as determined by ribonuclease protection assay. We also developed a DNA-based test for the diagnosis of the deficient allele. However, we failed to identify the mutated allele in other pig populations.

Characterisation of five candidate genes within the ETEC F4ab/ac candidate region in pigs.

BackgroundEnterotoxigenic Escherichia coli (ETEC) that express the F4ab and F4ac fimbriae is a major contributor to diarrhoea outbreaks in the pig breeding industry, infecting both newborn and weaned piglets. Some pigs are resistant to this infection, and susceptibility is inherited as a simple dominant Mendelian trait. Indentifying the genetics behind this trait will greatly benefit pig welfare as well as the pig breeding industry by providing an opportunity to select against genetically susceptible animals, thereby reducing the number of diarrhoea outbreaks. The trait has recently been mapped by haplotype sharing to a 2.5 Mb region on pig chromosome 13, a region containing 18 annotated genes.FindingsThe coding regions of five candidate genes for susceptibility to ETEC F4ab/ac infection (TFRC, ACK1, MUC20, MUC4 and KIAA0226), all located in the 2.5 Mb region, were investigated for the presence of possible causative mutations. A total of 34 polymorphisms were identified in either coding regions or their flanking introns. The genotyping data for two of those were found to perfectly match the genotypes at the ETEC F4ab/ac locus, a G to C polymorphism in intron 11 of TFRC and a C to T silent polymorphism in exon 22 of KIAA0226. Transcriptional profiles of the five genes were investigated in a porcine tissue panel including various intestinal tissues. All five genes were expressed in intestinal tissues at different levels but none of the genes were found differentially expressed between ETEC F4ab/ac resistant and ETEC F4ab/ac susceptible animals in any of the tested tissues.ConclusionsNone of the identified polymorphisms are obvious causative mutations for ETEC F4ab/ac susceptibility, as they have no impact on the level of the overall mRNA expression nor predicted to influence the composition of the amino acids composition. However, we cannot exclude that the five tested genes are bona fide candidate genes for susceptibility to ETEC F4ab/ac infection since the identified polymorphism might affect the translational apparatus, alternative splice forms may exist and post translational mechanisms might contribute to disease susceptibility.

Congenital progressive ataxia and spastic paresis, a hereditary disease in swine, maps to Chromosome 3 by linkage analysis

The congenital progressive ataxia (CPA) and spastic paresis in pigs, recently identified in Switzerland, is a disease with unknown etiology. A disease similar to CPA was described earlier by Rimaila-Pärnänen (1982). The affected animals show a neuropathic disorder, which occurs in piglets of both sexes within the first week after birth. The disease manifests itself within 1 or 2 days as a severe neuropathy, characterized by spastic gait, incoordination, and rapidly progressive ataxia in the hind limbs. Finally, the piglets remain lying down and are no longer able to support themselves. Histological examination of the central nervous system revealed no deficiency of stainable myelin nor any significant morphological changes. The disease was first observed in two litters of pigs (Table 1, matings 1 and 2), derived from two dams and one sire, all of Large White origin. The dams were cousins and not related to the sire, referring to the last two generations. Of the 23 offspring, seven (30.4%) were found to be affected and 16 (69.6%) were normal. The observed ratio of approximately 3:1 suggested that the disease may be controlled by a recessive allele. Therefore, affected animals were considered homozygous for the recessive allele ( cpa/cpa), and normal animals either heterozygous for the recessive allele ( CPA/cpa), or homozygous ( CPA/CPA). The present studies were conducted to: (1) confirm the autosomal recessive inheritance of CPA; and (2) map the CPA phenotype to the porcine genome. Of the 16 phenotypically normal animals, two males were mated to five females, and each female produced two litters (Table 1, mating 3–12). Of the 107 descendants, 17 animals (15.9%) showed ataxia and paresis syndromes. As their condition progressively worsened, the piglets were euthanized. Their average life span was (mean ± SD: 8.7 ± 8.3; n 4 17) days. In addition to the above offspring, nine piglets were produced from an unrelated family (Table 1, mating 13), of which three (33.3%) showed characteristics of the disease. To map the CPA phenotype, two to three highly informative microsatellite markers for each chromosome spread at intervals of about 40 cM were selected (Rohrer et al. 1996). According to conditions described in the original references (Rohrer et al. 1996), polymerase chain reaction (PCR) was carried out in a reaction volume of 25ml containing 100hg porcine genomic DNA extracted from blood, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 200 mM of each deoxynucleotide, 0.4 mM of forward and reverse primers, and 1.25 U of Taq DNA Polymerase (Pharmacia Biotech, Uppsala, Switzerland). PCR reactions (25 ml) were incubated for 30 cycles of 95°C for 30 s, 56°–62°C for 30 s, and 72°C for 30 s. With PrismTM Genescan-500 Rox marker (Applied Biosystems, Perkin-Elmer Corp., Foster City, Calif.), formamide diluted samples were analyzed on a 373A ABI Sequencer (Applied Biosystems Inc.). Linkage analysis was carried out with the CRIMAP program, version 2.4 (Green et al. 1990). In the inbreeding scheme among the phenotypically normal individuals, eight of the 12 litters produced atactic offspring (Table 1, matings No. 1, 2, 3, 6, 7, 8, 10, 12). Of the 95 descendants, 24 (25.3%) were found to be affected, while the remaining 71 (74.7%) were unaffected. The affected animals were classified as homozygous recessive ( cpa/cpa), and the unaffected as heterozygous (CPA/cpa) or homozygous ( CPA/CPA). Their parents were either heterozygous ( CPA/cpa), or homozygous ( CPA/CPA). Thex-test, calculated from the segregation data, showed that the observed ratios of thecpavs CPAalleles did not deviate significantly from the expected 1:3 ratio ( x 4 0.01; 0.9 <P < 0.95; 1 df). The genome scan revealed that the Sw902 allele (1894 size in bp), located on pig ( Sus scrofa ) Chromosome 3 (SSC3), co-segregated 100% with the recessive allele involved in the disease, while theSw902, Sw902 or Sw902 alleles cosegregated 100% with the normal allele (Table 1). Therefore, six additional markers in close proximity to marker Sw902were selected for further genotyping to generate a multipoint map covering the CPA region. Pairwise lod scores and recombination fractions forCPAand the seven marker loci are presented in Table 2. High lod scores of 16.9 and 11.6 were obtained for linkage of CPA with markersSw902and Sw1066respectively, the two markers exceeding a lod score of 47. Recombination was estimated to be 0.05 betweenSw1066andCPA,while no recombination occurred betweenSw902andCPA. It was computationally not feasible to perform a multipoint linkage analysis considering all eight loci jointly with n!/2 possible locus orders. Thus, the order Sw2618–Sw902–ACTG2–S0216 was fixed according to the genetic map of Rohrer et al. (1996), and the loci Sw460andSw1066were inserted sequentially with the CRIMAP “build” option. The most likely orderSw2618–Sw902– Sw1066–Sw460–ACTG2–S0216 fitted the data best, in accordance with Rohrer et al. (1996). The likelihood of six other loci orders did not differ by more than a factor of 1000, and they were, therefore, not considered significantly different. Similar results were obtained when other loci were assumed to be in a fixed order, and subsequently two additional loci were inserted. The marker order described by Rohrer et al. (1996), that is, Sw2618–S0094– Sw902–Sw1066–Sw460–ACTG2–S0216, was never rejected by our data. Therefore, this order was used in subsequent analyses. As expected, the estimated genetic distances and recombination rates are not completely in accordance with the data of Rohrer et al. (1996) (Fig. 1), probably owing to the different family material and limited number of meioses. The two orders ofCPA in adjacent intervals toSw902fit the Correspondence to: P. Vögeli Mammalian Genome 10, 1036–1038 (1999).

Porcine arthrogryposis multiplex congenita (AMC): new diagnostic test and narrowed candidate region.

In the Swiss Large White pig population a genetically caused arthrogryposis multiplex congenita (AMC) variant was identified. The disease is autosomal recessively inherited and is a fatal defect. Affected piglets are of normal size, but show malformed and permanently contracted joints in their legs. Often the spinal cord is curved and the lower jaw is shortened. Originally, AMC was mapped to a 5 Mb region on pig chromosome 5 (SSC5) between microsatellite markers SW152 and SW904. In order to detect unaffected carriers a diagnostic test using markers within the candidate region was developed. However, two independent recombination events occurred in a diseased and in a healthy piglet. Therefore, we selected 24 consecutive markers (3 microsatellites, 19 SNPs and 2 indels) in the candidate region, and determined the haplotypes in the two pedigrees with the recombinations. The parents and five offspring were investigated. In consequence, we were able to narrow down the candidate region and map AMC between SNPs ALGA0032767 and DRGA0006010 on SSC5 which span around 2.32 Mb. The candidate region shares homology to human chromosome 12. However, we are still lacking good candidate genes. A PCR-RFLP was developed and is used as an improved genetic test for AMC.

Elimination of INPP4A and SLC5A7 as candidate genes for congenital progressive ataxia and spastic paresis in pigs

The gene causing congenital progressive ataxia and spastic paresis (CPA) in Large White piglets remains unknown. This lethal neuropathy manifests shortly after birth, and is inherited as a single autosomal recessive allele cosegregating with the microsatellite SW9021 on SSC3, which approximately corresponds to position 90–110 Mb on HSA2.2 INPP4A (inositol polyphosphate-4-phosphatase, type 1) and SLC5A7 (solute carrier family 5, choline transporter, member 7) are attractive positional and functional candidates as they map within this region and are also involved in diseases with similar phenotypes. A 1-bp deletion in INPP4A causes the weeble (wbl) mutation in mice, a disorder characterized by severe locomotor instability and ataxia.3 SLC5A7 encodes a transmembrane transporter in neurons; a missense mutation in a gene of the same family in cattle (SLC35A3) has been shown to cause complex vertebral malformations and locomotor instability.4