Genome-wide association study using extreme truncate selection identifies novel genes affecting bone mineral density and fracture risk

Osteoporotic fracture is a major cause of morbidity and mortality worldwide. Low bone mineral density (BMD) is a major predisposing factor to fracture and is known to be highly heritable. Site-, gender-, and age-specific genetic effects on BMD are thought to be significant, but have largely not been considered in the design of genome-wide association studies (GWAS) of BMD to date. We report here a GWAS using a novel study design focusing on women of a specific age (postmenopausal women, age 55-85 years), with either extreme high or low hip BMD (age- and gender-adjusted BMD z-scores of +1.5 to +4.0, n = 1055, or -4.0 to -1.5, n = 900), with replication in cohorts of women drawn from the general population (n = 20,898). The study replicates 21 of 26 known BMD-associated genes. Additionally, we report suggestive association of a further six new genetic associations in or around the genes CLCN7, GALNT3, IBSP, LTBP3, RSPO3, and SOX4, with replication in two independent datasets. A novel mouse model with a loss-of-function mutation in GALNT3 is also reported, which has high bone mass, supporting the involvement of this gene in BMD determination. In addition to identifying further genes associated with BMD, this study confirms the efficiency of extreme-truncate selection designs for quantitative trait association studies. © 2011 Duncan et al.

A novel serogenetic approach determines the community prevalence of celiac disease and informs improved diagnostic pathways

Background: Changing perspectives on the natural history of celiac disease (CD), new serology and genetic tests, and amended histological criteria for diagnosis cast doubt on past prevalence estimates for CD. We set out to establish a more accurate prevalence estimate for CD using a novel serogenetic approach.Methods: The human leukocyte antigen (HLA)-DQ genotype was determined in 356 patients with 'biopsy-confirmed' CD, and in two age-stratified, randomly selected community cohorts of 1,390 women and 1,158 men. Sera were screened for CD-specific serology.Results: Only five 'biopsy-confirmed' patients with CD did not possess the susceptibility alleles HLA-DQ2.5, DQ8, or DQ2.2, and four of these were misdiagnoses. HLA-DQ2.5, DQ8, or DQ2.2 was present in 56% of all women and men in the community cohorts. Transglutaminase (TG)-2 IgA and composite TG2/deamidated gliadin peptide (DGP) IgA/IgG were abnormal in 4.6% and 5.6%, respectively, of the community women and 6.9% and 6.9%, respectively, of the community men, but in the screen-positive group, only 71% and 75%, respectively, of women and 65% and 63%, respectively, of men possessed HLA-DQ2.5, DQ8, or DQ2.2. Medical review was possible for 41% of seropositive women and 50% of seropositive men, and led to biopsy-confirmed CD in 10 women (0.7%) and 6 men (0.5%), but based on relative risk for HLA-DQ2.5, DQ8, or DQ2.2 in all TG2 IgA or TG2/DGP IgA/IgG screen-positive subjects, CD affected 1.3% or 1.9%, respectively, of females and 1.3% or 1.2%, respectively, of men. Serogenetic data from these community cohorts indicated that testing screen positives for HLA-DQ, or carrying out HLA-DQ and further serology, could have reduced unnecessary gastroscopies due to false-positive serology by at least 40% and by over 70%, respectively.Conclusions: Screening with TG2 IgA serology and requiring biopsy confirmation caused the community prevalence of CD to be substantially underestimated. Testing for HLA-DQ genes and confirmatory serology could reduce the numbers of unnecessary gastroscopies. © 2013 Anderson et al.; licensee BioMed Central Ltd.

Structure and vascular tissue expression of duplicated TERMINAL EAR1-like paralogues in poplar

TERMINAL EAR1-like (TEL) genes encode putative RNA-binding proteins only found in land plants. Previous studies suggested that they may regulate tissue and organ initiation in Poaceae. Two TEL genes were identified in both Populus trichocarpa and the hybrid aspen Populus tremula × P. alba, named, respectively, PoptrTEL1-2 and PtaTEL1-2. The analysis of the organisation around the PoptrTEL genes in the P. trichocarpa genome and the estimation of the synonymous substitution rate for PtaTEL1-2 genes indicate that the paralogous link between these two Populus TEL genes probably results from the Salicoid large-scale gene-duplication event. Phylogenetic analyses confirmed their orthology link with the other TEL genes. The expression pattern of both PtaTEL genes appeared to be restricted to the mother cells of the plant body: leaf founder cells, leaf primordia, axillary buds and root differentiating tissues, as well as to mother cells of vascular tissues. Most interestingly, PtaTEL1-2 transcripts were found in differentiating cells of secondary xylem and phloem, but probably not in the cambium itself. Taken together, these results indicate specific expression of the TEL genes in differentiating cells controlling tissue and organ development in Populus (and other Angiosperm species).

Common variants near ATM are associated with glycemic response to metformin in type 2 diabetes

Metformin is the most commonly used pharmacological therapy for type 2 diabetes. We report a genome-wide association study for glycemic response to metformin in 1,024 Scottish individuals with type 2 diabetes with replication in two cohorts including 1,783 Scottish individuals and 1,113 individuals from the UK Prospective Diabetes Study. In a combined meta-analysis, we identified a SNP, rs11212617, associated with treatment success (n = 3,920, P = 2.9 P×-9, odds ratio = 1.35, 95% CI 1.22-1.49) at a locus containing ATM, the ataxia telangiectasia mutated gene. In a rat hepatoma cell line, inhibition of ATM with KU-55933 attenuated the phosphorylation and activation of AMP-activated protein kinase in response to metformin. We conclude that ATM, a gene known to be involved in DNA repair and cell cycle control, plays a role in the effect of metformin upstream of AMP-activated protein kinase, and variation in this gene alters glycemic response to metformin. © 2011 Nature America, Inc. All rights reserved.

Interaction between ERAP1 and HLA-B27 in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27 in disease susceptibility

Ankylosing spondylitis is a common form of inflammatory arthritis predominantly affecting the spine and pelvis that occurs in approximately 5 out of 1,000 adults of European descent. Here we report the identification of three variants in the RUNX3, LTBR-TNFRSF1A and IL12B regions convincingly associated with ankylosing spondylitis (P < 5 × 10-8 in the combined discovery and replication datasets) and a further four loci at PTGER4, TBKBP1, ANTXR2 and CARD9 that show strong association across all our datasets (P < 5 × 10-6 overall, with support in each of the three datasets studied). We also show that polymorphisms of ERAP1, which encodes an endoplasmic reticulum aminopeptidase involved in peptide trimming before HLA class I presentation, only affect ankylosing spondylitis risk in HLA-B27-positive individuals. These findings provide strong evidence that HLA-B27 operates in ankylosing spondylitis through a mechanism involving aberrant processing of antigenic peptides.

Elucidating the chromosome 9 association with AS; CARD9 is a candidate gene

Ankylosing spondylitis (AS) is polygenic with contributions from the immunologically relevant genes HLA-B27, ERAP1 and IL23R. A recent genome-wide association screen (GWAS) identified associations (P0.005) with the non-synonymous single-nucleotide polymorphisms (nsSNPs), rs4077515 and rs3812571, in caspase recruitment domain-containing protein 9 (CARD9) and small nuclear RNA-activating complex polypeptide 4 (SNAPC4) on chromosome 9q that had previously been linked to AS. We replicated these associations in a study of 730 AS patients compared with 2879 historic disease controls (rs4077515 P0.0004, odds ratio (OR)1.2, 95% confidence interval (CI)1.1-1.4; rs3812571 P0.0003, OR1.2, 95% CI1.1-1.4). Meta-analysis revealed strong associations of both SNPs with AS, rs4077515 P0.000005, OR1.2, 95% CI1.1-1.3 and rs3812571 P0.000006, OR1.2, 95% CI1.1-1.3. We then typed 1604 AS cases and 1020 controls for 13 tagging SNPs; 6 showed at least nominal association, 5 of which were in CARD9. We imputed genotypes for 13 additional SNPs but none was more strongly associated with AS than the tagging SNPs. Finally, interrogation of an mRNA expression database revealed that the SNPs most strongly associated with AS (or in strong linkage disequilibrium) were those most associated with CARD9 expression. CARD9 is a plausible candidate for AS given its central role in the innate immune response.

Genome-wide association study identifies eight loci associated with blood pressure

Elevated blood pressure is a common, heritable cause of cardiovascular disease worldwide. To date, identification of common genetic variants influencing blood pressure has proven challenging. We tested 2.5 million genotyped and imputed SNPs for association with systolic and diastolic blood pressure in 34,433 subjects of European ancestry from the Global BPgen consortium and followed up findings with direct genotyping (N 71,225 European ancestry, N 12,889 Indian Asian ancestry) and in silico comparison (CHARGE consortium, N = 29,136). We identified association between systolic or diastolic blood pressure and common variants in eight regions near the CYP17A1 (P = 7 × 10 24), CYP1A2 (P = 1 × 10 23), FGF5 (P = 1 × 10 21), SH2B3 (P = 3 × 10 18), MTHFR (P = 2 × 10 13), c10orf107 (P = 1 × 10 9), ZNF652 (P = 5 × 10 9) and PLCD3 (P = 1 × 10 8) genes. All variants associated with continuous blood pressure were associated with dichotomous hypertension. These associations between common variants and blood pressure and hypertension offer mechanistic insights into the regulation of blood pressure and may point to novel targets for interventions to prevent cardiovascular disease.

Fine mapping of the MHC Class III region demonstrates association of AIF1 and rheumatoid arthritis

Objective. The heritability of RA has been estimated to be ∼55%, of which the MHC contributes about one-third. HLA-DRB1 alleles are strongly associated with RA, but it is likely that significant non-DRB1 MHC genetic susceptibility factors are involved. Previously, we identified two three-marker haplotypes in a 106-kb region in the MHC class III region immediately centromeric to TNF, which are strongly associated with RA on HLA-DRB1*0404 haplotypes. In the present study, we aimed to refine these associations further using a combination of genotyping and gene expression studies. Methods. Thirty-nine nucleotide polymorphisms (SNPs) were genotyped in 95 DRB1*0404 carrying unrelated RA cases, 125 DRB1*0404 - carrying healthy controls and 87 parent-case trio RA families in which the affected child carried HLA-DRB1*04. Quantitative RT-PCR was used to assess the expression of the positional candidate MHC class III genes APOM, BAT2, BAT3, BAT4, BAT5, AIF1, C6orf47, CSNK2β and LY6G5C, and the housekeeper genes, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and β2-microglobulin (B2M) in 31 RA cases and 21 ethnically, age- and sex-matched healthy controls. Synovial membrane specimens from RA, PsA and OA cases were stained by an indirect immunoperoxidase technique using a mouse-anti-human AIF1 monoclonal antibody. Results. Association was observed between RA and single markers or two marker haplotypes involving AIF1, BAT3 and CSNK. AIF1 was also significantly overexpressed in RA mononuclear cells (1.5- to 1.9-fold difference, P = 0.02 vs HPRT, P = 0.002 vs B2M). AIF1 protein was clearly expressed by synovial macrophages in all the inflammatory synovial samples in contrast to the non-inflammatory OA samples. Conclusions. The results of the genotyping and expression studies presented here suggest a role for AIF1 in both the aetiology and pathogenesis of RA.

A variant in LIN28B is associated with 2D:4D finger-length ratio, a putative retrospective biomarker of prenatal testosterone exposure

The ratio of the lengths of an individual's second to fourth digit (2D:4D) is commonly used as a noninvasive retrospective biomarker for prenatal androgen exposure. In order to identify the genetic determinants of 2D:4D, we applied a genome-wide association approach to 1507 11-year-old children from the Avon Longitudinal Study of Parents and Children (ALSPAC) in whom 2D:4D ratio had been measured, as well as a sample of 1382 12- to 16-year-olds from the Brisbane Adolescent Twin Study. A meta-analysis of the two scans identified a single variant in the LIN28B gene that was strongly associated with 2D:4D (rs314277: p = 4.1 x 10(-8)) and was subsequently independently replicated in an additional 3659 children from the ALSPAC cohort (p = 1.53 x 10(-6)). The minor allele of the rs314277 variant has previously been linked to increased height and delayed age at menarche, but in our study it was associated with increased 2D:4D in the direction opposite to that of previous reports on the correlation between 2D:4D and age at menarche. Our findings call into question the validity of 2D:4D as a simplistic retrospective biomarker for prenatal testosterone exposure.

Dominant negative ATM mutations in breast cancer families

BACKGROUND: The ATM gene encoding a putative protein kinase is mutated in ataxia-telangiectasia (A-T), an autosomal recessive disorder with a predisposition for cancer. Studies of A-T families suggest that female heterozygotes have an increased risk of breast cancer compared with noncarriers. However, neither linkage analyses nor mutation studies have provided supporting evidence for a role of ATM in breast cancer predisposition. Nevertheless, two recurrent ATM mutations, T7271G and IVS10-6T-->G, reportedly increase the risk of breast cancer. We examined these two ATM mutations in a population-based, case-control series of breast cancer families and multiple-case breast cancer families. METHODS: Five hundred twenty-five or 262 case patients with breast cancer and 381 or 68 control subjects, respectively, were genotyped for the T7271G and IVS10-6T-->G ATM mutations, as were index patients from 76 non-BRCA1/2 multiple-case breast cancer families. Linkage and penetrance were analyzed. ATM protein expression and kinase activity were analyzed in lymphoblastoid cell lines from mutation carriers. All statistical tests were two-sided. RESULTS: In case and control subjects unselected for family history of breast cancer, one case patient had the T7271G mutation, and none had the IVS10-6T-->G mutation. In three multiple-case families, one of these two mutations segregated with breast cancer. The estimated average penetrance of the mutations was 60% (95% confidence interval [CI] = 32% to 90%) to age 70 years, equivalent to a 15.7-fold (95% CI = 6.4-fold to 38.0-fold) increased relative risk compared with that of the general population. Expression and activity analyses of ATM in heterozygous cell lines indicated that both mutations are dominant negative. CONCLUSION: At least two ATM mutations are associated with a sufficiently high risk of breast cancer to be found in multiple-case breast cancer families. Full mutation analysis of the ATM gene in such families could help clarify the role of ATM in breast cancer susceptibility.

G-protein beta3 subunit gene (GNB3) variant in causation of essential hypertension

-Essential hypertensives display enhanced signal transduction through pertussis toxin-sensitive G proteins. The T allele of a C825T variant in exon 10 of the G protein beta3 subunit gene (GNB3) induces formation of a splice variant (Gbeta3-s) with enhanced activity. The T allele of GNB3 was shown recently to be associated with hypertension in unselected German patients (frequency=0.31 versus 0.25 in control). To confirm and extend this finding in a different setting, we performed an association study in Australian white hypertensives. This involved an extensively examined cohort of 110 hypertensives, each of whom were the offspring of 2 hypertensive parents, and 189 normotensives whose parents were both normotensive beyond age 50 years. Genotyping was performed by polymerase chain reaction and digestion with BseDI, which either cut (C allele) or did not cut (T allele) the 268-bp polymerase chain reaction product. T allele frequency in the hypertensive group was 0.43 compared with 0.25 in the normotensive group (chi2=22; P=0.00002; odds ratio=2.3; 95% CI=1.7 to 3.3). The T allele tracked with higher pretreatment blood pressure: diastolic=105+/-7, 109+/-16, and 128+/-28 mm Hg (mean+/-SD) for CC, CT, and TT, respectively (P=0.001 by 1-way ANOVA). Blood pressures were higher in female hypertensives with a T allele (P=0.006 for systolic and 0.0003 for diastolic by ANOVA) than they were in male hypertensives. In conclusion, the present study of a group with strong family history supports a role for a genetically determined, physiologically active splice variant of the G protein beta3 subunit gene in the causation of essential hypertension.

Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans: 1. Molecular modeling studies

Platelet endothelial cell adhesion molecule 1 (PECAM-1) has many functions, including its roles in leukocyte extravasation as part of the inflammatory response and in the maintenance of vascular integrity through its contribution to endothelial cell−cell adhesion. PECAM-1 has been shown to mediate cell−cell adhesion through homophilic binding events that involve interactions between domain 1 of PECAM-1 molecules on adjacent cells. However, various heterophilic ligands of PECAM-1 have also been proposed. The possible interaction of PECAM-1 with glycosaminoglycans (GAGs) is the focus of this study. The three-dimensional structure of the extracellular immunoglobulin (Ig) domains of PECAM-1 were constructed using homology modeling and threading methods. Potential heparin/heparan sulfate-binding sites were predicted on the basis of their amino acid consensus sequences and a comparison with known structures of sulfate-binding proteins. Heparin and other GAG fragments have been docked to investigate the structural determinants of their protein-binding specificity and selectivity. The modeling has predicted two regions in PECAM-1 that appear to bind heparin oligosaccharides. A high-affinity binding site was located in Ig domains 2 and 3, and evidence for a low-affinity site in Ig domains 5 and 6 was obtained. These GAG-binding regions were distinct from regions involved in PECAM-1 homophilic interactions.

Multistage genome-wide association meta-analyses identified two new loci for bone mineral density

Aiming to identify novel genetic variants and to confirm previously identified genetic variants associated with bone mineral density (BMD), we conducted a three-stage genome-wide association (GWA) meta-analysis in 27 061 study subjects. Stage 1 meta-analyzed seven GWA samples and 11 140 subjects for BMDs at the lumbar spine, hip and femoral neck, followed by a Stage 2 in silico replication of 33 SNPs in 9258 subjects, and by a Stage 3 de novo validation of three SNPs in 6663 subjects. Combining evidence from all the stages, we have identified two novel loci that have not been reported previously at the genome-wide significance (GWS; 5.0 × 10-8) level: 14q24.2 (rs227425, P-value 3.98 × 10-13, SMOC1) in the combined sample of males and females and 21q22.13 (rs170183, P-value 4.15 × 10-9, CLDN14) in the female-specific sample. The two newly identified SNPs were also significant in the GEnetic Factors for OSteoporosis consortium (GEFOS, n 5 32 960) summary results. We have also independently confirmed 13 previously reported loci at the GWS level: 1p36.12 (ZBTB40), 1p31.3 (GPR177), 4p16.3 (FGFRL1), 4q22.1 (MEPE), 5q14.3 (MEF2C), 6q25.1 (C6orf97, ESR1), 7q21.3 (FLJ42280, SHFM1), 7q31.31 (FAM3C, WNT16), 8q24.12 (TNFRSF11B), 11p15.3 (SOX6), 11q13.4 (LRP5), 13q14.11 (AKAP11) and 16q24 (FOXL1). Gene expression analysis in osteogenic cells implied potential functional association of the two candidate genes (SMOC1 and CLDN14) in bone metabolism. Our findings independently confirm previously identified biological pathways underlying bone metabolism and contribute to the discovery of novel pathways, thus providing valuable insights into the intervention and treatment of osteoporosis. © The Author 2013. Published by Oxford University Press.