A genome-screen of a large twin cohort reveals linkage for quantitative ultrasound of the calcaneus to 2q33-37 and 4q12-21.

UNLABELLED: A genome-wide screen was performed on a large cohort of dizygous twin pairs to identify regions of the genome that contain QTL for QUS of bone. Suggestive linkage of QUS parameters to 2q33-37 and 4q12-21 highlighted these regions as potentially important for studies of genes that regulate bone. INTRODUCTION: The genetics of osteoporotic fracture is only partly explained by bone mineral density (BMD). Quantitative ultrasound (QUS) of the calcaneus can also be used for independent clinical assessment of osteoporotic fracture risk. Two specific indices are derived from this assessment: broadband ultrasound attenuation (BUA) and velocity of sound (VOS). Both parameters provide information on fracture risk; however, BUA has been studied more extensively and may be favored because it is thought to have a stronger predictive value for osteoporotic fracture and incorporates aspects of trabecular structure and bone quality as well as BMD. Studies of QUS in twins have shown that both derived parameters are under substantial genetic control, independent of BMD. MATERIALS AND METHODS: To identify regions of the genome that contain quantitative trait loci (QTL) for QUS of bone, we performed a genome-wide screen on a large cohort of dizygous twin pairs. Unselected female dizygous twins from 1067 pedigrees from the St Thomas' UK Adult Twin Registry were genome scanned (737 highly polymorphic microsatellite markers). Multipoint linkage analyses provided maximum evidence of linkage for BUA (LOD 2.1-5.1) to 2q33-37. Linkage for VOS (LOD 2.2-3.4) was maximal at 4q12-21. Potential evidence of linkage in the cohort indicated five other possible locations of QTL (LOD > 2.0) relevant to bone density or structure on chromosomes 1, 2, 13, 14, and X. RESULTS AND CONCLUSIONS: This study has identified eight genomic locations with linkage of LOD > 2.0. This data should be of value in assisting researchers to localize genes that regulate bone mass and microstructure. These results should complement genome screens of BMD and bone structure and serve to enable further targeted positional candidate and positional cloning studies to advance our understanding of genetic control of bone quality and risk of fracture.

Comparison of genome screens for two independent cohorts provides replication of suggestive linkage of bone mineral density to 3p21 and 1p36.

Low bone mineral density (BMD) is a major risk factor for osteoporotic fracture. Studies of BMD in families and twins have shown that this trait is under strong genetic control. To identify regions of the genome that contain quantitative trait loci (QTL) for BMD, we performed independent genomewide screens, using two complementary study designs. We analyzed unselected nonidentical twin pairs (1,094 pedigrees) and highly selected, extremely discordant or concordant (EDAC) sib pairs (254 pedigrees). Nonparametric multipoint linkage (NPL) analyses were undertaken for lumbar spine and total-hip BMD in both cohorts and for whole-body BMD in the unselected twin pairs. The maximum evidence of linkage in the unselected twins (spine BMD, LOD 2.7) and the EDAC pedigrees (spine BMD, LOD 2.1) was observed at chromosome 3p21 (76 cM and 69 cM, respectively). These combined data indicate the presence, in this region, of a gene that regulates BMD. Furthermore, evidence of linkage in the twin cohort (whole-body BMD; LOD 2.4) at chromosome 1p36 (17 cM) supports previous findings of suggestive linkage to BMD in the region. Weaker evidence of linkage (LOD 1.0-2.3) in either cohort, but not both, indicates the locality of additional QTLs. These studies validate the use, in linkage analysis, of large cohorts of unselected twins phenotyped for multiple traits, and they highlight the importance of conducting genome scans in replicate populations as a prelude to positional cloning and gene discovery.

Mapping of serum amylase-1 and quantitative trait loci for milk production traits to cattle chromosome 4.

The present study was undertaken to confirm and refine the mapping of a quantitative trait locus in cattle for milk fat percentage that had earlier been reported to be linked to the serum amylase-1 locus, AM1. Five half-sib families from the previous study and 7 new ones were genotyped for nine microsatellite markers spanning chromosome 4. AM1 was mapped between the microsatellite markers BMS648 and BR6303. In a granddaughter design, interval mapping based on multiple-marker regression was utilized for an analysis of five milk production traits: milk yield, fat percentage and yield, and protein percentage and yield. In the families reported on previously, significant effects for fat and protein percentages were detected. In the new families, an effect on milk and fat yields was found. The most likely positions of the quantitative trait locus in both groups of families were in the same area of chromosome 4 in the vicinity of the obese locus. Direct effects of the obese locus were tested for using polymorphism in two closely linked microsatellites located 2.5 and 3.6 top downstream of the coding sequence. No firm evidence was found for an association between the obese locus and the tested traits.

Tissue chambers--a useful model for in vivo studies of cytokine production in the pig.

An in vivo tissue chamber model was developed to enable studies of local cytokine production and cellular events during inflammatory and immune reactions in the pig. Tissue chambers made of sialistic rubber tubing were surgically implanted in the subcutaneous tissue- and samples of tissue chamber fluid (TCF) and inflammatory cells were collected by aspiration with a syringe. To evaluate the model for local cytokine production, two cytokine inducers, polyribinosinic-polyribocytidylic acid (poly I:C) and fixed Aujeszky's disease virus infected PK15 cells (ADV-PK15), were injected into the tissue chambers and samples of TCF were collected 0, 4, 8, 12, 24 and 48 h post injection. Poly I:C injections induced local production of interferon-alpha (IFN-alpha) as well as tumor necrosis factor (TNF) in the TCF but kinetic differences in the production of the cytokines were noted. Poly I:C also induced an increase in cell numbers in the TCF, mainly due to increased neutrophil numbers. Injections of ADV-PK15 induced local IFN-alpha production in the TCF as long as the pigs were serologically negative to ADV. Immunofluorescence and in situ hybridization techniques could be applied for characterization of TCF cells. Moreover, cells recovered from the tissue chambers were viable and could be used in functional in vitro tests. Taken together, this tissue chamber model could prove very useful in in vivo studies of inflammatory/immune responses and cytokine production in the pig.

Genotyping bovine milk proteins using allele discrimination by primer length and automated DNA sizing technology.

A method for genotyping kappa-casein (A, B, E), beta-casein (A1, A2, A3, A5, B) and beta-lactoglobulin (A, B) simultaneously by the use of allele discrimination by primer length combined with automated detection of fragments with a sequencing instrument is described. Seven different mutations within the milk protein genes were analysed in order to distinguish between the alleles examined. The samples were amplified in two separate multiplex polymerase chain reactions (PCRs), which were then pooled and separated according to size in a single lane on the gel. By using stringent PCR conditions, we have been able to achieve allele-specific amplifications and minimize amplification of mis-matched primer for all seven mutations.

Interferon-alpha production and tissue localization of interferon-alpha/beta producing cells after intradermal administration of Aujeszky's disease virus-infected cells in pigs.

Intradermal administration of glutaraldehyde-fixed Aujeszky's disease virus (ADV) infected autologous or allogeneic cells resulted in the induction of an interferon (IFN)-alpha response in pigs. Using a sensitive dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), IFN-alpha was detected in blood at 8 and 24 h after injection of ADV-infected cells. In parallel, by means of in situ hybridization, IFN-alpha/beta mRNA containing cells were demonstrated in regional lymph nodes. Occasional IFN-alpha/beta mRNA positive cells were also seen in injected dermal areas, but not in contralateral lymph nodes, spleen, bone marrow, blood or liver. The ability of leucocytes in whole blood cultures to produce IFN-alpha upon stimulation by ADV was markedly diminished 3-7 days after intradermal injection of ADV-infected cells. In contrast, cultures of purified peripheral blood mononuclear cells (PBMC) had intact IFN-alpha responses. Further, serum from ADV-injected pigs inhibited the in vitro ADV-induced IFN-alpha responses in PBMC from control pigs, most likely due to the demonstrated presence of anti-ADV antibodies. We suggest that the IFN-alpha/beta producing cells in lymph nodes may participate in the development of antiviral immunity and could be equivalent to Natural IFN-alpha/beta producing (NIP) cells.

Molecular cloning of a gene encoding porcine interferon-beta.

A gene encoding the porcine interferon-beta (poIFN-beta) was cloned from a genomic library. The sequence of a potential intronless coding region as well as 1,265 bp of the 5'- and 277 bp of the 3'-flanking regions is presented. The gene is predicted to encode a mature protein of 165 amino acids and a signal peptide of 21 amino acids. This probable poIFN-beta shows high homology (63%) with human (hu) IFN-beta at the amino acid level, but less with porcine (po) IFN-alpha 1 (32%). It contains three cysteines and three potential N-glycosylation sites. A region of the 5' flank (-116 to -159) of the gene is homologous to the IFN gene regulatory element (IRE) of the huIFN-beta gene which mediates virus inducibility. Southern blot analysis indicates that the poIFN-beta gene is present as a single copy in the porcine genome. Its expression in porcine peripheral blood leukocytes stimulated in vitro by pseudorabies virus (PRV) was demonstrated at the RNA level both by Northern blot analysis and by in situ hybridization. The latter approach in addition detected only about one IFN-beta mRNA-containing cell per 2,000 PRV-stimulated porcine leukocytes, a frequency in the same range as that for leukocytes containing IFN-alpha mRNA.