Biochemical markers of renal function and maternal hypothyroidism in early pregnancy.

Objective: The physiological adaptations during a normal pregnancy affect renal and thyroid function and levels of associated biochemical markers. An association between cystatin C (CysC), creatinine, and thyroid function has been considered in nonpregnant individuals but not in pregnant women specifically. Methods: Cohort study within the North Denmark Region Pregnancy Cohort (2011-2015) with assessment of thyroid function and autoantibodies (ADVIA Centaur XPT, Siemens Healthineers) in serum residues from the early pregnancy. Consecutive samples (n = 1112) were selected for measurement of CysC and creatinine (Atellica CH 930, Siemens Healthineers), and results were linked to information in Danish nationwide registers for (i) establishment of pregnancy-specific reference intervals for CysC and creatinine and (ii) evaluation of the prevalence of maternal hypothyroidism in early pregnancy according to levels of CysC and creatinine. Results: The established reference intervals (2.5-97.5 percentiles) differed by week of pregnancy (week 4-8, 9-11, 12-15) and were CysC: 0.58-0.92 mg/L; 0.54-0.91 mg/L; 0.52-0.86 mg/L; creatinine: 46.9-73.0 µmol/L; 42.0-68.4 µmol/L; 38.8-66.4 µmol/L. The prevalence of maternal autoimmune hypothyroidism in early pregnancy differed by the level of CysC and creatinine (<25th percentile; 25th-75th percentile; >75th percentile) and was for CysC 1.7%, 3.8%, 7.4% and for creatinine 2.5%, 4.1%, 7.1%. Conclusions: Reference intervals for CysC and creatinine were dynamic in early pregnancy and decreased with increasing gestational age. Furthermore, higher levels of CysC and creatinine associated with a higher prevalence of maternal autoimmune hypothyroidism. Results encourage considerations on the underlying mechanisms for the association between markers of renal and thyroid function.

Parathyroid hormone reference intervals in adults using second- and third-generation assays.

Parathyroid hormone (PTH) is a routine biochemical analysis, and it varies whether a second- or third-generation assay is used. Information on the levels obtained with different assays and evidence to substantiate local assay-specific reference ranges are important to inform clinical practice. Prior to a shift from the second- to the third-generation PTH assay (Cobas 8000, Roche Diagnostics) in the Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark, a total of 59 EDTA-plasma samples were collected for method comparison (Passing-Bablok). Furthermore, 120 EDTA-plasma samples were randomly obtained from adult blood donors and used for the establishment of reference intervals using the third-generation PTH assay (Cobas 8000, Roche Diagnostics) and two second-generation assays (Atellica, Siemens Healthineers; Alinity, Abbott Laboratories). Method comparison (Cobas 8000, Roche Diagnostics) showed lower levels with the third-generation (y) as compared to the second-generation assay (x) depending on the measurement range (PTH < 10 pmol/L: y = 0.8 (95% CI: 0.7; 0.9) x + 0.3 (95% CI: 0.2; 0.5), PTH ≥ 10 pmol/L: y = 0.6 (95% CI: 0.5; 0.6) x + 3.2 (95% CI: 1.1; 5.2)). Method-specific reference intervals (2.5 and 97.5 percentiles) after the exclusion of samples (n = 31) with 25-hydroxy-vitamin D below 50 nmol/L were: 1.8-8.5 pmol/L (second-generation, Atellica, Siemens Healthineers); 2.4-10.9 pmol/L (second-generation, Alinity, Abbott Laboratories), and 1.8-7.0 pmol/L (third-generation, Cobas 8000, Roche Diagnostics). PTH levels with second- and third-generation assays are not interchangeable. Clinicians should be informed when a laboratory assay is changed, and method-specific reference ranges are needed.

Disentangling the genetics of lean mass.

Background: Lean body mass (LM) plays an important role in mobility and metabolic function. We previously identified five loci associated with LM adjusted for fat mass in kilograms. Such an adjustment may reduce the power to identify genetic signals having an association with both lean mass and fat mass. Objectives: To determine the impact of different fat mass adjustments on genetic architecture of LM and identify additional LM loci. Methods: We performed genome-wide association analyses for whole-body LM (20 cohorts of European ancestry with n = 38,292) measured using dual-energy X-ray absorptiometry) or bioelectrical impedance analysis, adjusted for sex, age, age2, and height with or without fat mass adjustments (Model 1 no fat adjustment; Model 2 adjustment for fat mass as a percentage of body mass; Model 3 adjustment for fat mass in kilograms). Results: Seven single-nucleotide polymorphisms (SNPs) in separate loci, including one novel LM locus (TNRC6B), were successfully replicated in an additional 47,227 individuals from 29 cohorts. Based on the strengths of the associations in Model 1 vs Model 3, we divided the LM loci into those with an effect on both lean mass and fat mass in the same direction and refer to those as "sumo wrestler" loci (FTO and MC4R). In contrast, loci with an impact specifically on LM were termed "body builder" loci (VCAN and ADAMTSL3). Using existing available genome-wide association study databases, LM increasing alleles of SNPs in sumo wrestler loci were associated with an adverse metabolic profile, whereas LM increasing alleles of SNPs in "body builder" loci were associated with metabolic protection. Conclusions: In conclusion, we identified one novel LM locus (TNRC6B). Our results suggest that a genetically determined increase in lean mass might exert either harmful or protective effects on metabolic traits, depending on its relation to fat mass.

Identification of a novel locus on chromosome 2q13, which predisposes to clinical vertebral fractures independently of bone density.

OBJECTIVES: To identify genetic determinants of susceptibility to clinical vertebral fractures, which is an important complication of osteoporosis. METHODS: Here we conduct a genome-wide association study in 1553 postmenopausal women with clinical vertebral fractures and 4340 controls, with a two-stage replication involving 1028 cases and 3762 controls. Potentially causal variants were identified using expression quantitative trait loci (eQTL) data from transiliac bone biopsies and bioinformatic studies. RESULTS: A locus tagged by rs10190845 was identified on chromosome 2q13, which was significantly associated with clinical vertebral fracture (P=1.04×10-9) with a large effect size (OR 1.74, 95% CI 1.06 to 2.6). Bioinformatic analysis of this locus identified several potentially functional SNPs that are associated with expression of the positional candidate genes TTL (tubulin tyrosine ligase) and SLC20A1 (solute carrier family 20 member 1). Three other suggestive loci were identified on chromosomes 1p31, 11q12 and 15q11. All these loci were novel and had not previously been associated with bone mineral density or clinical fractures. CONCLUSION: We have identified a novel genetic variant that is associated with clinical vertebral fractures by mechanisms that are independent of BMD. Further studies are now in progress to validate this association and evaluate the underlying mechanism.

Genome-wide association study for radiographic vertebral fractures: a potential role for the 16q24 BMD locus.

Vertebral fracture risk is a heritable complex trait. The aim of this study was to identify genetic susceptibility factors for osteoporotic vertebral fractures applying a genome-wide association study (GWAS) approach. The GWAS discovery was based on the Rotterdam Study, a population-based study of elderly Dutch individuals aged > 55 years; and comprising 329 cases and 2666 controls with radiographic scoring (McCloskey­Kanis) and genetic data. Replication of one top-associated SNP was pursued by de-novo genotyping of 15 independent studies across Europe, the United States, and Australia and one Asian study. Radiographic vertebral fracture assessment was performed using McCloskey­Kanis or Genant semi-quantitative definitions. SNPs were analyzed in relation to vertebral fracture using logistic regression models corrected for age and sex. Fixed effects inverse variance and Han­Eskin alternative random effects meta-analyses were applied. Genome-wide significance was set at p < 5 × 10− 8. In the discovery, a SNP (rs11645938) on chromosome 16q24 was associated with the risk for vertebral fractures at p = 4.6 × 10− 8. However, the association was not significant across 5720 cases and 21,791 controls from 14 studies. Fixed-effects meta-analysis summary estimate was 1.06 (95% CI: 0.98­1.14; p = 0.17), displaying high degree of heterogeneity (I2 = 57%; Qhet p = 0.0006). Under Han­Eskin alternative random effects model the summary effect was significant (p = 0.0005). The SNP maps to a region previously found associated with lumbar spine bone mineral density (LS-BMD) in two large meta-analyses from the GEFOS consortium. A false positive association in the GWAS discovery cannot be excluded, yet, the low-powered setting of the discovery and replication settings (appropriate to identify risk effect size > 1.25) may still be consistent with an effect size < 1.10, more of the type expected in complex traits. Larger effort in studies with standardized phenotype definitions is needed to confirm or reject the involvement of this locus on the risk for vertebral fractures.

A genome-wide copy number association study of osteoporotic fractures points to the 6p25.1 locus.

BACKGROUND: Osteoporosis is a systemic skeletal disease characterised by reduced bone mineral density and increased susceptibility to fracture; these traits are highly heritable. Both common and rare copy number variants (CNVs) potentially affect the function of genes and may influence disease risk. AIM: To identify CNVs associated with osteoporotic bone fracture risk. METHOD: We performed a genome-wide CNV association study in 5178 individuals from a prospective cohort in the Netherlands, including 809 osteoporotic fracture cases, and performed in silico lookups and de novo genotyping to replicate in several independent studies. RESULTS: A rare (population prevalence 0.14%, 95% CI 0.03% to 0.24%) 210 kb deletion located on chromosome 6p25.1 was associated with the risk of fracture (OR 32.58, 95% CI 3.95 to 1488.89; p = 8.69 × 10(-5)). We performed an in silico meta-analysis in four studies with CNV microarray data and the association with fracture risk was replicated (OR 3.11, 95% CI 1.01 to 8.22; p = 0.02). The prevalence of this deletion showed geographic diversity, being absent in additional samples from Australia, Canada, Poland, Iceland, Denmark, and Sweden, but present in the Netherlands (0.34%), Spain (0.33%), USA (0.23%), England (0.15%), Scotland (0.10%), and Ireland (0.06%), with insufficient evidence for association with fracture risk. CONCLUSIONS: These results suggest that deletions in the 6p25.1 locus may predispose to higher risk of fracture in a subset of populations of European origin; larger and geographically restricted studies will be needed to confirm this regional association. This is a first step towards the evaluation of the role of rare CNVs in osteoporosis.

Assessment of gene-by-sex interaction effect on bone mineral density.

Sexual dimorphism in various bone phenotypes, including bone mineral density (BMD), is widely observed; however, the extent to which genes explain these sex differences is unclear. To identify variants with different effects by sex, we examined gene-by-sex autosomal interactions genome-wide, and performed expression quantitative trait loci (eQTL) analysis and bioinformatics network analysis. We conducted an autosomal genome-wide meta-analysis of gene-by-sex interaction on lumbar spine (LS) and femoral neck (FN) BMD in 25,353 individuals from 8 cohorts. In a second stage, we followed up the 12 top single-nucleotide polymorphisms (SNPs; p < 1 × 10(-5) ) in an additional set of 24,763 individuals. Gene-by-sex interaction and sex-specific effects were examined in these 12 SNPs. We detected one novel genome-wide significant interaction associated with LS-BMD at the Chr3p26.1-p25.1 locus, near the GRM7 gene (male effect = 0.02 and p = 3.0 × 10(-5) ; female effect = -0.007 and p = 3.3 × 10(-2) ), and 11 suggestive loci associated with either FN- or LS-BMD in discovery cohorts. However, there was no evidence for genome-wide significant (p < 5 × 10(-8) ) gene-by-sex interaction in the joint analysis of discovery and replication cohorts. Despite the large collaborative effort, no genome-wide significant evidence for gene-by-sex interaction was found to influence BMD variation in this screen of autosomal markers. If they exist, gene-by-sex interactions for BMD probably have weak effects, accounting for less than 0.08% of the variation in these traits per implicated SNP. © 2012 American Society for Bone and Mineral Research.

Genome-wide meta-analysis identifies 56 bone mineral density loci and reveals 14 loci associated with risk of fracture.

Bone mineral density (BMD) is the most widely used predictor of fracture risk. We performed the largest meta-analysis to date on lumbar spine and femoral neck BMD, including 17 genome-wide association studies and 32,961 individuals of European and east Asian ancestry. We tested the top BMD-associated markers for replication in 50,933 independent subjects and for association with risk of low-trauma fracture in 31,016 individuals with a history of fracture (cases) and 102,444 controls. We identified 56 loci (32 new) associated with BMD at genome-wide significance (P < 5 × 10(-8)). Several of these factors cluster within the RANK-RANKL-OPG, mesenchymal stem cell differentiation, endochondral ossification and Wnt signaling pathways. However, we also discovered loci that were localized to genes not known to have a role in bone biology. Fourteen BMD-associated loci were also associated with fracture risk (P < 5 × 10(-4), Bonferroni corrected), of which six reached P < 5 × 10(-8), including at 18p11.21 (FAM210A), 7q21.3 (SLC25A13), 11q13.2 (LRP5), 4q22.1 (MEPE), 2p16.2 (SPTBN1) and 10q21.1 (DKK1). These findings shed light on the genetic architecture and pathophysiological mechanisms underlying BMD variation and fracture susceptibility.

Single-nucleotide polymorphisms in the P2X7 receptor gene are associated with post-menopausal bone loss and vertebral fractures.

The purinergic P2X7 receptor has a major role in the regulation of osteoblast and osteoclast activity and changes in receptor function may therefore affect bone mass in vivo. The aim of this study was to determine the association of non-synonymous single-nucleotide polymorphisms in the P2RX7 gene to bone mass and fracture incidence in post-menopausal women. A total of 1694 women (aged 45-58) participating in the Danish Osteoporosis Prevention Study were genotyped for 12 functional P2X7 receptor variants. Bone mineral density was determined at baseline and after 10 years. In addition, vertebral fracture incidence was documented at 10 years. We found that the rate of bone loss was clearly associated with the Arg307Gln amino acid substitution such that individuals heterozygous for this polymorphism had a 40% increased rate of bone loss. Furthermore, individuals carrying the Ile568Asn variant allele had increased bone loss. In contrast, the Gln460Arg polymorphism was associated with protection against bone loss. The Ala348Thr polymorphism was associated with a lower vertebral fracture incidence 10 years after menopause. Finally, we developed a risk model, which integrated P2RX7 genotypes. Using this model, we found a clear association between the low-risk (high-P2X7 function) alleles and low rate of bone loss. Conversely, high-risk (reduced P2X7 function) alleles were associated with a high rate of bone loss. In conclusion, an association was demonstrated between variants that reduce P2X7 receptor function and increased rate of bone loss. These data support that the P2X7 receptor is important in regulation of bone mass.

The expression of IL-6 by osteoblasts is increased in healthy elderly individuals: stimulated proliferation and differentiation are unaffected by age.

Increasing age is associated with reduced bone mineral content and increased risk of fractures. This is caused by a relative insufficiency of osteoblasts compared with osteoclasts. We therefore wanted to examine the potential differences in proliferation, differentiation, and expression of cytokines between human osteoblasts (hOBs) obtained from young and elderly individuals. Cultures of hOBs were obtained from 11 elderly (73-85 years) and 15 young (21-27 years) healthy individuals. The cells were stimulated with hGH, IGF-I, hGH + IGF-I, and TGF-ß1. Proliferation was evaluated by thymidine incorporation, and differentiation was evaluated by alkaline phosphatase, OPG, and PINP production. Expression of IL-6, TGF-ß1, OPG, and RANKL was investigated using real-time PCR and three carefully selected housekeeping genes. Combined stimulation with hGH and IGF-I increased proliferation without differences between hOBs obtained from young and elderly individuals. hOBs from young individuals responded to stimulation with vitamin D with a more pronounced increase in alkaline phosphatase: 107 ± 17% vs. 43 ± 5%, P < 0.01. Stimulation with TGF-ß1 decreased OPG production by hOBs from elderly individuals but not from young individuals, P < 0.05. hOBs from elderly individuals expressed significantly higher amounts of IL-6 mRNA (P < 0.05) and less OPG and TGF-ß1 mRNA (P = 0.08 and P = 0.08, respectively) compared with hOBs from young individuals. In conclusion, hOBs from elderly individuals express more IL-6 mRNA and less OPG and TGF-ß1 mRNA than hOBs from young individuals. This could partly explain the reduced bone mass and increased fracture risk seen in the elderly. hOBs from young and elderly individuals responded similarly to short-term stimulation of proliferation and differentiation.

Large-scale analysis of association between LRP5 and LRP6 variants and osteoporosis.

CONTEXT: Mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene cause rare syndromes characterized by altered bone mineral density (BMD). More common LRP5 variants may affect osteoporosis risk in the general population. OBJECTIVE: To generate large-scale evidence on whether 2 common variants of LRP5 (Val667Met, Ala1330Val) and 1 variant of LRP6 (Ile1062Val) are associated with BMD and fracture risk. DESIGN AND SETTING: Prospective, multicenter, collaborative study of individual-level data on 37,534 individuals from 18 participating teams in Europe and North America. Data were collected between September 2004 and January 2007; analysis of the collected data was performed between February and May 2007. Bone mineral density was assessed by dual-energy x-ray absorptiometry. Fractures were identified via questionnaire, medical records, or radiographic documentation; incident fracture data were available for some cohorts, ascertained via routine surveillance methods, including radiographic examination for vertebral fractures. MAIN OUTCOME MEASURES: Bone mineral density of the lumbar spine and femoral neck; prevalence of all fractures and vertebral fractures. RESULTS: The Met667 allele of LRP5 was associated with reduced lumbar spine BMD (n = 25,052 [number of participants with available data]; 20-mg/cm2 lower BMD per Met667 allele copy; P = 3.3 x 10(-8)), as was the Val1330 allele (n = 24,812; 14-mg/cm2 lower BMD per Val1330 copy; P = 2.6 x 10(-9)). Similar effects were observed for femoral neck BMD, with a decrease of 11 mg/cm2 (P = 3.8 x 10(-5)) and 8 mg/cm2 (P = 5.0 x 10(-6)) for the Met667 and Val1330 alleles, respectively (n = 25 193). Findings were consistent across studies for both LRP5 alleles. Both alleles were associated with vertebral fractures (odds ratio [OR], 1.26; 95% confidence interval [CI], 1.08-1.47 for Met667 [2001 fractures among 20 488 individuals] and OR, 1.12; 95% CI, 1.01-1.24 for Val1330 [1988 fractures among 20,096 individuals]). Risk of all fractures was also increased with Met667 (OR, 1.14; 95% CI, 1.05-1.24 per allele [7876 fractures among 31,435 individuals)]) and Val1330 (OR, 1.06; 95% CI, 1.01-1.12 per allele [7802 fractures among 31 199 individuals]). Effects were similar when adjustments were made for age, weight, height, menopausal status, and use of hormone therapy. Fracture risks were partly attenuated by adjustment for BMD. Haplotype analysis indicated that Met667 and Val1330 variants both independently affected BMD. The LRP6 Ile1062Val polymorphism was not associated with any osteoporosis phenotype. All aforementioned associations except that between Val1330 and all fractures and vertebral fractures remained significant after multiple-comparison adjustments. CONCLUSIONS: Common LRP5 variants are consistently associated with BMD and fracture risk across different white populations. The magnitude of the effect is modest. LRP5 may be the first gene to reach a genome-wide significance level (a conservative level of significance [herein, unadjusted P < 10(-7)] that accounts for the many possible comparisons in the human genome) for a phenotype related to osteoporosis.

Large-scale analysis of association between polymorphisms in the transforming growth factor beta 1 gene (TGFB1) and osteoporosis: the GENOMOS study.

INTRODUCTION: The TGFB1 gene which encodes transforming growth factor beta 1, is a strong candidate for susceptibility to osteoporosis and several studies have reported associations between bone mineral density (BMD), osteoporotic fractures and polymorphisms of TGFB1, although these studies have yielded conflicting results. METHODS: We investigated associations between TGFB1 polymorphisms and BMD and fracture in the GENOMOS study: a prospective multicenter study involving 10 European research studies including a total of 28,924 participants. Genotyping was conducted for known TGFB1 polymorphisms at the following sites: G-1639-A (G-800-A, rs1800468), C-1348-T (C-509-T, rs1800469), T29-C (Leu10Pro, rs1982073), G74-C (Arg25Pro, rs1800471) and C788-T (Thr263Ile, rs1800472). These polymorphisms were genotyped prospectively and methodology was standardized across research centers. Genotypes and haplotypes were related to BMD at the lumbar sine and femoral neck and fractures. RESULTS: There were no significant differences in either women or men at either skeletal site for any of the examined polymorphisms with the possible exception of a weak association with reduced BMD (-12 mg/cm2) in men with the T-1348 allele (p<0.05). None of the haplotypes was associated with BMD and none of the polymorphisms or haplotypes significantly affected overall risk of fractures, however, the odds ratio for incident vertebral fracture in carriers of the rare T788 allele was 1.64 (95% CI: 1.09-2.64), p<0.05. CONCLUSIONS: This study indicates that polymorphic variation in the TGFB1 gene does not play a major role in regulating BMD or susceptibility to fractures. The weak associations we observed between the C-1348-T and lumbar spine BMD in men and between C788-T and risk of incident vertebral fractures are of interest but could be chance findings and will need replication in future studies.

The association between common vitamin D receptor gene variations and osteoporosis: a participant-level meta-analysis.

BACKGROUND: Polymorphisms of the vitamin D receptor (VDR) gene have been implicated in the genetic regulation of bone mineral density (BMD). However, the clinical impact of these variants remains unclear. OBJECTIVE: To evaluate the relation between VDR polymorphisms, BMD, and fractures. DESIGN: Prospective multicenter large-scale association study. SETTING: The Genetic Markers for Osteoporosis consortium, involving 9 European research teams. PARTICIPANTS: 26,242 participants (18,405 women). MEASUREMENTS: Cdx2 promoter, FokI, BsmI, ApaI, and TaqI polymorphisms; BMD at the femoral neck and the lumbar spine by dual x-ray absorptiometry; and fractures. RESULTS: Comparisons of BMD at the lumbar spine and femoral neck showed nonsignificant differences less than 0.011 g/cm2 for any genotype with or without adjustments. A total of 6067 participants reported a history of fracture, and 2088 had vertebral fractures. For all VDR alleles, odds ratios for fractures were very close to 1.00 (range, 0.98 to 1.02) and collectively the 95% CIs ranged from 0.94 (lowest) to 1.07 (highest). For vertebral fractures, we observed a 9% (95% CI, 0% to 18%; P = 0.039) risk reduction for the Cdx2 A-allele (13% risk reduction in a dominant model). LIMITATIONS: The authors analyzed only selected VDR polymorphisms. Heterogeneity was detected in some analyses and may reflect some differences in collection of fracture data across cohorts. Not all fractures were related to osteoporosis. CONCLUSIONS: The FokI, BsmI, ApaI, and TaqI VDR polymorphisms are not associated with BMD or with fractures, but the Cdx2 polymorphism may be associated with risk for vertebral fractures.

The structure of bovine complement component 3 reveals the basis for thioester function.

The third component of complement (C3) is a 190 kDa glycoprotein essential for eliciting the complement response. The protein consists of two polypeptide chains (alpha and beta) held together with a single disulfide bridge. The beta-chain is composed of six MG domains, one of which is shared with the alpha-chain. The disulfide bridge connecting the chains is positioned in the shared MG domain. The alpha-chain consists of the anaphylatoxin domain, three MG domains, a CUB domain, an alpha(6)/alpha(6)-barrel domain and the C-terminal C345c domain. An internal thioester in the alpha-chain of C3 (present in C4 but not in C5) is cleaved during complement activation. This mediates covalent attachment of the activated C3b to immune complexes and invading microorganisms, thereby opsonizing the target. We present the structure of bovine C3 determined at 3 Angstroms resolution. The structure shows that the ester is buried deeply between the thioester domain and the properdin binding domain, in agreement with the human structure. This domain interface is broken upon activation, allowing nucleophile access. The structure of bovine C3 clearly demonstrates that the main chain around the thioester undergoes a helical transition upon activation. This rearrangement is proposed to be the basis for the high level of reactivity of the thioester group. A strictly conserved glutamate residue is suggested to function catalytically in thioester proteins. Structure-based design of inhibitors of C3 activation may target a conserved pocket between the alpha-chain and the beta-chain of C3, which appears essential for conformational changes in C3.

Large-scale evidence for the effect of the COLIA1 Sp1 polymorphism on osteoporosis outcomes: the GENOMOS study.

BACKGROUND: Osteoporosis and fracture risk are considered to be under genetic control. Extensive work is being performed to identify the exact genetic variants that determine this risk. Previous work has suggested that a G/T polymorphism affecting an Sp1 binding site in the COLIA1 gene is a genetic marker for low bone mineral density (BMD) and osteoporotic fracture, but there have been no very-large-scale studies of COLIA1 alleles in relation to these phenotypes. METHODS AND FINDINGS: Here we evaluated the role of COLIA1 Sp1 alleles as a predictor of BMD and fracture in a multicenter study involving 20,786 individuals from several European countries. At the femoral neck, the average (95% confidence interval [CI]) BMD values were 25 mg/cm2 (CI, 16 to 34 mg/cm2) lower in TT homozygotes than the other genotype groups (p < 0.001), and a similar difference was observed at the lumbar spine; 21 mg/cm2 (CI, 1 to 42 mg/cm2), (p = 0.039). These associations were unaltered after adjustment for potential confounding factors. There was no association with fracture overall (odds ratio [OR] = 1.01 [CI, 0.95 to 1.08]) in either unadjusted or adjusted analyses, but there was a non-significant trend for association with vertebral fracture and a nominally significant association with incident vertebral fractures in females (OR = 1.33 [CI, 1.00 to 1.77]) that was independent of BMD, and unaltered in adjusted analyses. CONCLUSIONS: Allowing for the inevitable heterogeneity between participating teams, this study-which to our knowledge is the largest ever performed in the field of osteoporosis genetics for a single gene-demonstrates that the COLIA1 Sp1 polymorphism is associated with reduced BMD and could predispose to incident vertebral fractures in women, independent of BMD. The associations we observed were modest however, demonstrating the importance of conducting studies that are adequately powered to detect and quantify the effects of common genetic variants on complex diseases.

Localization of carbohydrate attachment sites and disulfide bridges in limulus alpha 2-macroglobulin. Evidence for two forms differing primarily in their bait region sequences.

The primary structure determination of the dimeric invertebrate alpha(2)-macroglobulin (alpha(2)M) from Limulus polyphemus has been completed by determining its sites of glycosylation and disulfide bridge pattern. Of seven potential glycosylation sites for N-linked glycosylation, six (Asn(275), Asn(307), Asn(866), Asn(896), Asn(1089), and Asn(1145)) carry common glucosamine-based carbohydrates groups, whereas one (Asn(80)) carries a carbohydrate chain containing both glucosamine and galactosamine. Nine disulfide bridges, which are homologues with bridges in human alpha(2)M, have been identified (Cys(228)-Cys(269), Cys(456)-Cys(580), Cys(612)-Cys(799), Cys(657)-Cys(707), Cys(849)-Cys(876), Cys(874)-Cys(910), Cys(946)-Cys(1328), Cys(1104)-Cys(1155), and Cys(1362)-Cys(1475)). In addition to these bridges, Limulus alpha(2)M contains three unique bridges that connect Cys(361) and Cys(382), Cys(1370) and Cys(1374), respectively, and Cys(719) in one subunit with the same residue in the other subunit of the dimer. The latter bridge forms the only interchain disulfide bridge in Limulus alpha(2)M. The location of this bridge within the bait region is discussed and compared with other alpha-macroglobulins. Several peptides identified in the course of determining the disulfide bridge pattern provided evidence for the existence of two forms of Limulus alpha(2)M. The two forms have a high degree of sequence identity, but they differ extensively in large parts of their bait regions suggesting that they have different inhibitory spectra. The two forms (Limulus alpha(2)M-1 and -2) are most likely present in an approximately 2:1 ratio in the hemolymph of each animal, and they can be partially separated on a Mono Q column at pH 7.4 by applying a shallow gradient of NaCl.