Integument, mortality, and skeletal strength in extended production cycles for laying hens - effects of genotype and dietary zinc source.

1. This study on long-life layers, covering the period 20-100 weeks of age, investigated longitudinal effects on mortality, layer integument, and skeletal properties in Bovans White (BoW) and Lohmann Selected Leghorn Classic (LSL), with or without supplementation with dietary organic zinc (Zn).2. Two experiments, using 1440 layers in furnished small group cages (FC) and 1836 layers in a traditional floor housing system (Floor), were run in parallel. Each replicate consisted of five adjacent cages containing eight hens in each FC, or a pen with 102 layers in the Floor group.3. Mortality was recorded daily. Integument and keel bone condition were scored at 35, 55, 85, and 100 weeks of age on 20% of the layers. Tibial strength was recorded from 933 layers at 100 weeks. Statistical analyses were performed on replicate means, with four to five and nine replicates per combination of hybrid and diet in Floor and FC groups, respectively.4. Cumulative mortality was 9.6% and 16.3% in FC and Floor, respectively, and increased in the latter part of the production cycle, particularly in the Floor group.5. In FC, LSL had inferior feather cover, less keel bone deviation, and shorter claws than BoW. In Floor, LSL had superior feather cover, less severe vent wounds, more bumble foot, and cleaner plumage than BoW. In both production systems, claws grew longer and keel bone deviation became more severe with age.6. In FC, layers fed organic Zn had lower body weight and less keel bone deviation at 100 weeks of age.7. In conclusion, keel bone integrity, claw length, and mortality rate are potential threats to welfare in long-life layers. Feather pecking is a problem that needs addressing at an early stage in the production period. On the whole, organic Zn did not improve welfare conditions in long-life layers.

Tooth agenesis in osteogenesis imperfecta related to mutations in the collagen type I genes.

BACKGROUND: Osteogenesis imperfecta (OI) is a heterogeneous group of disorders of connective tissue, mainly caused by mutations in the collagen type I genes (COL1A1 and COL1A2). Tooth agenesis is a common feature of OI. We investigated the association between tooth agenesis and collagen type I mutations in individuals with OI. SUBJECTS AND METHODS: In this cohort study, 128 unrelated individuals with OI were included. Panoramic radiographs were analyzed regarding dentinogenesis imperfecta (DGI) and congenitally missing teeth. The collagen I genes were sequenced in all individuals, and in 25, multiplex ligation-dependent probe amplification was performed. RESULTS: Mutations in the COL1A1 and COL1A2 genes were found in 104 of 128 individuals. Tooth agenesis was diagnosed in 17% (hypodontia 11%, oligodontia 6%) and was more frequent in those with DGI (P = 0.016), and in those with OI type III, 47%, compared to those with OI types I, 12% (P = 0.003), and IV, 13% (P = 0.017). Seventy-five percent of the individuals with oligodontia (≥6 missing teeth) had qualitative mutations, but there was no association with OI type, gender, or presence of DGI. CONCLUSION: The prevalence of tooth agenesis is high (17%) in individuals with OI, and OI caused by a qualitative collagen I mutation is associated with oligodontia.

Onset of sexual maturity in female chickens is genetically linked to loci associated with fecundity and a sexual ornament.

Onset of sexual maturation is a trait of extreme importance both evolutionarily and economically. Unsurprisingly therefore, domestication has acted to reduce the time to sexual maturation in a variety of animals, including the chicken. In comparison with wild progenitor chickens [the Red Junglefowl (RJF)], domestic layer hens attain maturity approximately 20% earlier. In addition, domestic layers also possess larger combs (a sexual ornament), produce more eggs and have denser bones. A large quantitative trait loci (QTL) analysis (n=377) was performed using an F(2) intercross between a White Leghorn layer breed and a RJF population, with onset of sexual maturity measured and mapped to three separate loci. This cross has already been analysed for comb mass, egg production and bone allocation. Onset of sexual maturity significantly correlated with comb mass, whilst the genetic architecture for sexual maturity and comb mass overlapped at all three loci. For two of these loci, the QTL for sexual maturity and comb mass were statistically indistinguishable from pleiotropy, suggesting that the alleles that increase comb mass also decrease onset of sexual maturity.

Vitamin D receptor genotypes in primary hyperparathyroidism.

Vitamin D and parathyroid hormone (PTH) constitute the main regulators of systemic calcium homeostasis. As well as its calcaemic effects, active vitamin D3(1,25(OH)2D3) has a direct regulatory role on parathyroid cells. Active vitamin D3 acts via its receptor (VDR), and binding of the ligand-receptor complex to specific promoter regions of the PTH gene inhibits transcription. Active vitamin D3 constitutes a principal regulator of parathyroid cell growth, and polymorphism in the VDR gene has recently been related to bone mineral density and suggested as predisposing to osteoporosis. Impaired effects of active vitamin D3 may contribute to the relatively enhanced secretion and cell proliferation seen in hyperparathyroidism (HPT). Indeed, VDR dysfunction, of essentially unknown character, has been demonstrated in the pathological parathyroid tissue of primary HPT as well as HPT secondary to uraemia. Consistent with the essential role of active vitamin D3 in parathyroid regulation, the VDR gene polymorphism was studied in 90 postmenopausal women with primary hyperparathyroidism. The VDR genotype bb was found in 60.0% of HPT patients and in 33.3% of the postmenopausal female controls (P < 0.001). As the b allele has been linked to decreased transcriptional activity or messenger RNA stability, reduced VDR expression may impede regulatory actions of vitamin D and may contribute to parathyroid tumorigenesis in these patients.

The genetic architecture of domestication in the chicken: effects of pleiotropy and linkage.

The extent of pleiotropy and epistasis in quantitative traits remains equivocal. In the case of pleiotropy, multiple quantitative trait loci are often taken to be pleiotropic if their confidence intervals overlap, without formal statistical tests being used to ascertain if these overlapping loci are statistically significantly pleiotropic. Additionally, the degree to which the genetic correlations between phenotypic traits are reflected in these pleiotropic quantitative trait loci is often variable, especially in the case of antagonistic pleiotropy. Similarly, the extent of epistasis in various morphological, behavioural and life-history traits is also debated, with a general problem being the sample sizes required to detect such effects. Domestication involves a large number of trade-offs, which are reflected in numerous behavioural, morphological and life-history traits which have evolved as a consequence of adaptation to selective pressures exerted by humans and captivity. The comparison between wild and domestic animals allows the genetic analysis of the traits that differ between these population types, as well as being a general model of evolution. Using a large F(2) intercross between wild and domesticated chickens, in combination with a dense SNP and microsatellite marker map, both pleiotropy and epistasis were analysed. The majority of traits were found to segregate in 11 tight 'blocks' and reflected the trade-offs associated with domestication. These blocks were shown to have a pleiotropic 'core' surrounded by more loosely linked loci. In contrast, epistatic interactions were almost entirely absent, with only six pairs identified over all traits analysed. These results give insights both into the extent of such blocks in evolution and the development of domestication itself.

Variations in the vitamin D receptor gene are not associated with measures of muscle strength, physical performance, or falls in elderly men. Data from MrOS Sweden.

The vitamin D receptor (VDR) has been proposed as a candidate gene for several musculoskeletal phenotypes. However, previous results on the associations between genetic variants of the VDR with muscle strength and falls have been contradictory. The MrOS Sweden survey, a prospective population-based cohort study of 3014 elderly men (mean age 75 years, range 69-81) offered the opportunity to further investigate these associations. At baseline, data were collected on muscle strength and also the prevalence of falls during the previous 12 months. Genetic association analysis was performed for 7 Single Nucleotide Polymorphisms (SNPs), covering the genetic region surrounding the VDR gene in 2924 men with available samples of DNA. Genetic variations in the VDR were not associated with five different measurements of muscle strength or physical performance (hand grip strength right and left, 6 m walking test (easy and narrow) and timed-stands test). However, one of the 7 SNPs of the gene for the VDR receptor, rs7136534, was associated with prevalence of falls (33.6% of the AA, 14.6% of the AG and 16.5% of the GG allele). In conclusion, VDR genetic variants are not related to muscle strength or physical performance in elderly Swedish men. The role of the rs7136534 SNP for the occurrence of falls is not clear.

Genome-wide pharmacogenetic investigation of a hepatic adverse event without clinical signs of immunopathology suggests an underlying immune pathogenesis.

One of the major goals of pharmacogenetics is to elucidate mechanisms and identify patients at increased risk of adverse events (AEs). To date, however, there have been only a few successful examples of this type of approach. In this paper, we describe a retrospective case-control pharmacogenetic study of an AE of unknown mechanism, characterized by elevated levels of serum alanine aminotransferase (ALAT) during long-term treatment with the oral direct thrombin inhibitor ximelagatran. The study was based on 74 cases and 130 treated controls and included both a genome-wide tag single nucleotide polymorphism and large-scale candidate gene analysis. A strong genetic association between elevated ALAT and the MHC alleles DRB1(*)07 and DQA1(*)02 was discovered and replicated, suggesting a possible immune pathogenesis. Consistent with this hypothesis, immunological studies suggest that ximelagatran may have the ability to act as a contact sensitizer, and hence be able to stimulate an adaptive immune response.

Novel estrogen receptor alpha promoter polymorphism increases ventricular hypertrophic response to hypertension.

Given the strong genetic contribution to blood pressure and left ventricular hypertrophy (LVH), and the influence of estrogen on these parameters, we hypothesized that polymorphisms in the estrogen receptor alpha (ERalpha) promoter may influence LVH. Three novel polymorphisms were identified upstream of the ERalpha alternatively spliced exon 1E, within sequence which demonstrated significant promoter activity in vitro. Demonstration of ERalpha E isoform expression in human ventricle by RT-PCR supported a possible functional role for the 1E novel polymorphisms in estrogen signaling in the heart. Indeed, G>A (-721 E) was significantly associated with LVH after controlling for systolic blood pressure and sex in a healthy population (n=74), contributing to 23% of interventricular septum (IVS) width variance (p<0.001) and 9.4% of left ventricular mass index (LVMI) variance (p=0.035). In a separate hypertensive cohort, male carriers of the A allele (n=8) had a 17% increase in IVS (95% CI: 6-28%) and a 19% increase in LVMI (3-34%) compared to GG homozygotes (n=84). We conclude that a novel polymorphism in the promoter of a cardiac mRNA splice isoform of ERalpha is associated with LVH.

Decreased fracture rate, pharmacogenetics and BMD response in 79 Swedish children with osteogenesis imperfecta types I, III and IV treated with Pamidronate.

BACKGROUND: Osteogenesis imperfecta (OI) is an inherited heterogeneous bone fragility disorder, usually caused by collagen I mutations. It is well established that bisphosphonate treatment increases lumbar spine (LS) bone mineral density (BMD), as well as improves vertebral geometry in severe OI; however, fracture reduction has been difficult to prove, pharmacogenetic studies are scarce, and it is not known at which age, or severity of disease, treatment should be initiated. MATERIALS AND METHODS: COL1A1 and COL1A2 were analyzed in 79 children with OI (type I n=33, type III n=25 and type IV n=21) treated with Pamidronate. Data on LS BMD, height, and radiologically confirmed non-vertebral and vertebral fractures were collected prior to, and at several time points during treatment. RESULTS: An increase in LS BMD Z-score was observed for all types of OI, and a negative correlation to Δ LS BMD was observed for both age and LS BMD Z-score at treatment initiation. Supine height Z-scores were not affected by Pamidronate treatment, The fracture rate was reduced for all OI types at all time points during treatment (overall p<0.0003, <0.0001 and 0.0003 for all OI types I, III and IV respectively). The reduced fracture rate was maintained for types I and IV, while an additional decrease was observed over time for type III. The fracture rate was reduced also in individuals with continued low BMD after >4yrs Pamidronate. Twice as many boys as girls with OI type I were treated with Pamidronate, and the fracture rate the year prior treatment was 2.2 times higher for boys (p=0.0236). Greater Δ LS BMD, but smaller Δ fracture numbers were observed on Pamidronate for helical glycine mutations in COL1A1 vs. COL1A2. Vertebral compression fractures did not progress in any individual during treatment; however, they did not improve in 9%, and these individuals were all >11years of age at treatment initiation (p<0.0001). CONCLUSION: Pamidronate treatment in children with all types of OI increased LS BMD, decreased fracture rate, and improved vertebral compression fractures. Fracture reduction was prompt and maintained during treatment, irrespective of age at treatment initiation and collagen I mutation type.

Feather pecking in chickens is genetically related to behavioural and developmental traits.

Feather pecking (FP) is a detrimental behaviour in chickens, which is performed by only some individuals in a flock. FP was studied in 54 red junglefowl (ancestor of domestic chickens), 36 White Leghorn laying hens, and 762 birds from an F(2)-intercross between these two lines. From all F(2)-birds, growth and feed consumption were measured. Age at sexual maturity and egg production in females, and corticosterone levels in males were also measured. From 333 F(2)-birds of both sexes, and 20 parental birds, body composition with respect to bone mineral content, muscle and fat was obtained by post-mortem examinations using Dual X-Ray Absorptiometry (DXA). In femurs of the same birds, the bone density and structure were analysed using DXA and Peripheral Quantitative Computerized Tomography (pQCT), and a biomechanical analysis of bone strength was performed. Furthermore, plumage condition was determined in all birds as a measure of being exposed to feather pecking. Using 105 DNA-markers in all F(2)-birds, a genome-wide scan for Quantitative Trait Loci (QTL), associated with the behaviour in the F(2)-generation was performed. FP was at least as frequent in the red junglefowl as in the White Leghorn strain studied here, and significantly more common among females both in the parental strains and in the F(2)-generation. In the F(2)-birds, FP was phenotypically linked to early sexual maturation, fast growth, weak bones, and, in males, also high fat accumulation, indicating that feather peckers have a different resource allocation pattern. Behaviourally, F(2) feather peckers were more active in an open field test, in a novel food/novel object test, and in a restraint test, indicating that feather pecking might be genetically linked to a proactive coping strategy. Only one suggestive QTL with a low explanatory value was found on chromosome 3, showing that many genes, each with a small effect, are probably involved in the causation of feather pecking. There were significant effects of sire and dam on the risk of being a victim of feather pecking, and victims grew faster pre- and post-hatching, had lower corticosterone levels and were less active in a restraint test. Hence, a wide array of behavioural and developmental traits were genetically linked to FP.

The vitamin D receptor (VDR) start codon polymorphism in primary hyperparathyroidism and parathyroid VDR messenger ribonucleic acid levels.

Vitamin D regulates parathyroid cell proliferation and secretion of PTH. Increased prevalence of the polymorphic vitamin D receptor (VDR) alleles b, a, and T has been reported in sporadic primary hyperparathyroidism (PHPT), suggesting that these genetic variants may predispose to the disease. Recently, another polymorphism in the VDR gene was related to bone mineral density, and this VDR-FokI polymorphism causes different lengths of the VDR, implying possible functional consequences. The VDR-FokI polymorphism was studied in 182 postmenopausal women with sporadic PHPT and in matched controls. No significant differences in distribution of the VDR-FokI genotypes could be detected between the groups, although there was a tendency toward overrepresentation of the F allele in the PHPT patients (P = 0.05). There were no significant associations with age, serum calcium, serum PTH, bone mineral density, or parathyroid tumor weight. The VDR genotypes were unrelated to VDR and PTH messenger ribonucleic acid levels in the parathyroid adenomas of 42 PHPT patients. In 23 PHPT patients, the Ca2+-PTH set-points were determined in vivo and were unrelated to the VDR alleles. We suggest that the VDR-FokI polymorphism has at most a minor pathogenic importance in the development of PHPT.

A deletion polymorphism in the RIZ gene, a female sex steroid hormone receptor coactivator, exhibits decreased response to estrogen in vitro and associates with low bone mineral density in young Swedish women.

Low bone mineral density (BMD) is a major risk factor for osteoporotic fracture, and the trait is under genetic control by a large number of genes. It is recognized that estrogen plays an important role in the maintenance of bone mass by binding to estrogen receptor alpha (ERalpha). RIZ1 has previously been shown to be a specific ERalpha coactivator and strongly enhances its function both in vivo and in vitro. We performed in vitro studies comparing the abilities of RIZ1 P704 polymorphic variants (homozygous presence, P704+; absence, P704-; heterozygosity P704(+/-) of a proline at position 704) to coactivate the ERalpha and also examined the polymorphism associated to BMD of 343 Swedish women, aged 20-39 yr. The expression vector containing P704- RIZ1 showed an impaired response in coactivating ERalpha in a ligand- and dose-dependent manner compared with P704+ RIZ (P < 0.0001). The genotype frequencies were 19% (P704+), 32% (P704-), and 49% (P704(+/-)) and were in Hardy-Weinberg equilibrium. BMD at the heel was higher in the P704+ genotype group than in the P704(+/-) group (P = 0.02), which was evident also after corrections for fat and lean mass (P = 0.03). We conclude that RIZ1 may be a new candidate gene for involvement in the variation seen in BMD.

Estrogen receptor gene polymorphism in postmenopausal primary hyperparathyroidism.

BACKGROUND: Estrogen and its receptor play a key role in calcium homeostasis, with implications in both the development and treatment of primary hyperparathyroidism. METHODS: Leukocyte DNA was isolated from 101 postmenopausal female patients with primary hyperparathyroidism and age-matched female control subjects (n = 101). Polymorphic estrogen receptor alleles denoted P/p and X/x were detected by polymerase chain reaction and restriction digestion with Pvu II and Xba 1 RESULTS: Allelic prevalences were similar in the patients and control subjects. In contrast to normocalcemic control subjects, patients with primary hyperthyroidism homozygous for alleles P and X displayed less elevated serum calcium concentrations (mean, 2.63 and 2.66 mmol/L) versus those with Pp and pp (2.72 mmol/L; p < 0.01) and Xx and xx (2.71; p < 0.05), respectively, as well as trends for higher intact serum parathyroid hormone values (p = 0.09). The PP genotype was accompanied by lower bone mineral density in the lumbar spine (p < 0.05). CONCLUSIONS: Estrogen receptor gene polymorphisms are presumably not directly involved in the parathyroid tumorigenesis. However, they seem to interact with the extent of biochemical derangements of primary hyperthyroidism by possible influences on both the peripheral action of parathyroid hormone and calcium regulation of its secretion.

Inhibitory effects of 9-cis and all-trans retinoic acid on 1,25(OH)2 vitamin D3-induced bone resorption.

The effects of retinoic acid (RA), and calcitriol are mediated by specific nuclear receptors (RARs and VDR, respectively). Induction of RAR and VDR responsive elements in target genes requires a cofactor, the retinoid-X-receptor (RXR), with its ligand 9-cis RA. We have previously demonstrated the expression of RARs and RXRs in osteoblasts, and herein investigated the effects of the retinoids all-trans RA and 9-cis RA alone and combined with calcitriol on bone resorption in vitro, measured by 45Ca-release from prelabeled neonatal mouse calvarial bones. All-trans RA and 9-cis RA were powerful stimulators of bone resorption and essentially equipotent. At threshold concentrations (1 nM) both 9-cis RA and at-RA markedly inhibited the resorption induced by calcitriol (1 pM). The findings are compatible with a physiological role for retinoids in bone metabolism.

Cloning of a novel retinoic acid-inducible mRNA from human osteoblast-like cells.

Retinoids have long been known to influence skeletal development and bone remodeling. Cells of the osteoblastic lineage play a key role in these processes. In this study we have used the differential display PCR technique to identify retinoic acid (RA)-induced mRNAs in human osteoblast-like cells. We report the cloning and sequencing of one such mRNA, AT-RA 6, which was specifically induced by all-trans RA both in normal human osteoblast-like cells and in MG-63 osteosarcoma cells. Maximal expression was found after 60 min, suggesting that this may be an early response gene. Expression was found in all tissues examined. No homology to known mRNA sequences was detected.

Reverse transcription-polymerase chain reaction assay demonstrates that the 9-cis retinoic acid receptor alpha is expressed in human osteoblasts.

The 9-cis retinoic acid receptor (RXR) alpha, a co-regulator of the thyroid hormone and vitamin D receptors, has previously been shown to be expressed predominantly in metabolic organs such as the liver and the kidney. In this study we have used a reverse transcription polymerase chain reaction (RT-PCR) to examine the expression of retinoic acid (RA) nuclear receptors in primary human osteoblasts and in SaOS-2, a human osteosarcoma-derived cell line with osteoblastic characteristics. Our results demonstrate that human osteoblasts express RXR alpha, as well as the all-trans RA receptors (RAR) alpha, beta, and gamma. These data further establish bone as a major target for retinoids and suggest that RA can regulate vitamin D and thyroid hormone actions in osteoblasts.

A poly adenosine repeat in the human vitamin D receptor gene is associated with bone mineral density in young Swedish women.

Peak bone mass (PBM) and subsequent bone loss are important risk factors for development of osteoporosis later in life, and twin studies have reported strong genetic influence on PBM. The genetic factor influencing PBM is polygenetic, and many genes most likely exert relatively small effects on bone mass. The poly adenosine (A) microsatellite in the 3' untranslated region (UTR) of the VDR gene has been associated with both prostate and breast cancer risk but little is known about the effect of bone mineral density (BMD). In this report the poly A microsatellite and the linked BsmI SNP have been investigated in a population-based cohort of 343 Swedish women, aged 20-39. BMD was measured by dual x-ray absorptiometry at the spine, proximal femur, total body and heel and by quantitative ultrasound at the heel. Correlations were found between VDR genotypes and BMD at lumbar spine L2-L4, (ss versus LL, P = 0.03 and BB versus bb, P = 0.02, respectively), with a similar pattern concerning total hip (ss versus LL, P = 0.12 and BB versus bb, P = 0.16 respectively). After corrections for age, height, fat and lean mass, the VDR BsmI genotype was still associated to BMD at the lumbar spine (BB versus bb, P = 0.03). The polymorphisms were in linkage disequilibrium (Chi-square = 566, P < 0.0001). In conclusion, genetic variation in the VDR is associated with BMD in premenopausal women, and further studies are needed to evaluate a possible functional role of the VDR 3'UTR poly A repeat, a region that has shown to be of important for mRNA stability.

Cellular retinoic acid-binding protein type II is expressed in adult human osteoblasts and in adult liver.

In this study we have used a reverse transcription polymerase chain reaction (RT-PCR) to demonstrate that adult primary human osteoblasts and SaOS-2, a human osteosarcoma-derived cell line with osteoblastic properties, express cellular retinol-binding protein I (CRBP I), cellular retinoic acid-binding protein II (CRABP II), and very low levels of CRABP I. We also show that CRABP II is expressed in the adult liver, which does not express CRABP I. The results suggest that CRABP II is the important isoform in the adult bone as well as in the adult liver. Since the 9-cis retinoic acid receptor (RXR) alpha previously has been shown to be expressed predominantly in the liver, CRABP II might be involved in the transport of 9-cis retinoic acid to its nuclear receptor.

Vitamin D receptor alleles b, a, and T: risk factors for sporadic primary hyperparathyroidism (HPT) but not HPT of uremia or MEN 1.

Polymorphisms in the vitamin D receptor (VDR) gene have been hypothezised to interfere with VDR expression. VDR alleles (Bb, Aa and Tt) were examined in 254 Caucasian patients with sporadic primary hyperparathyroidism (spHPT, n = 206), HPT of multiple endocrine neoplasia type 1 (MEN-1; n = 17), and HPT of uremia (n = 31). In comparison to age- and sex-matched controls, the b, a and T alleles were overrepresented in 100 menopausal females with spHPT (p = 0.006-0.0004), equivalent to an odds ratio of 2.6-3.4 for spHPT in homozygotes for the b, a and, T alleles. The association between VDR genotypes and spHPT was restricted to female patients and those with parathyroid adenoma (p = 0.0006-0.0001), whereas HPT of MEN 1 and uremia seemed unrelated to the VDR polymorphisms (p = 0.26-0.96). The results suggest that the VDR alleles b, a, and T are novel risk factors in the essentially uncharacterized pathogenesis of spHPT.