Genome-wide association study using family-based cohorts identifies the WLS and CCDC170/ESR1 loci as associated with bone mineral density.

BACKGROUND: Osteoporosis is a common and debilitating bone disease that is characterised by a low bone mineral density (BMD), a highly heritable trait. Genome-wide association studies (GWAS) have proven to be very successful in identifying common genetic variants associated with BMD adjusted for age, gender and weight, however a large portion of the genetic variance for this trait remains unexplained. There is evidence to suggest significant genetic correlation between body size traits and BMD. It has also recently been suggested that unintended bias can be introduced as a result of adjusting a phenotype for a correlated trait. We performed a GWAS meta-analysis in two populations (total n = 6,696) using BMD data adjusted for only age and gender, in an attempt to identify genetic variants associated with BMD including those that may have potential pleiotropic effects on BMD and body size traits. RESULTS: We observed a single variant, rs2566752, associated with spine BMD at the genome-wide significance level in the meta-analysis (P = 3.36 × 10(-09)). Logistic regression analysis also revealed an association between rs2566752 and fracture rate in one of our study cohorts (P = 0.017, n = 5,654). This is an intronic variant located in the wntless Wnt ligand secretion mediator (WLS) gene (1p31.3), a known BMD locus which encodes an integral component of the Wnt ligand secretion pathway. Bioinformatics analyses of variants in moderate LD with rs2566752 produced strong evidence for a regulatory role for the variants rs72670452, rs17130567 and rs1430738. Expression quantitative trait locus (eQTL) analysis suggested that the variants rs12568456 and rs17130567 are associated with expression of the WLS gene in whole blood, cerebellum and temporal cortex brain tissue (P = 0.034-1.19 × 10(-23)). Gene-wide association testing using the VErsatile Gene-based Association Study 2 (VEGAS2) software revealed associations between the coiled-coil domain containing 170 (CCDC170) gene, located adjacent to the oestrogen receptor 1 (ESR1) gene, and BMD at the spine, femoral neck and total hip sites (P = 1.0 × 10(-06), 2.0 × 10(-06) and 2.0 × 10(-06) respectively). CONCLUSIONS: Genetic variation at the WLS and CCDC170/ESR1 loci were found to be significantly associated with BMD adjusted for only age and gender at the genome-wide level in this meta-analysis.

Genes contributing to pain sensitivity in the normal population: an exome sequencing study.

Sensitivity to pain varies considerably between individuals and is known to be heritable. Increased sensitivity to experimental pain is a risk factor for developing chronic pain, a common and debilitating but poorly understood symptom. To understand mechanisms underlying pain sensitivity and to search for rare gene variants (MAF<5%) influencing pain sensitivity, we explored the genetic variation in individuals' responses to experimental pain. Quantitative sensory testing to heat pain was performed in 2,500 volunteers from TwinsUK (TUK): exome sequencing to a depth of 70× was carried out on DNA from singletons at the high and low ends of the heat pain sensitivity distribution in two separate subsamples. Thus in TUK1, 101 pain-sensitive and 102 pain-insensitive were examined, while in TUK2 there were 114 and 96 individuals respectively. A combination of methods was used to test the association between rare variants and pain sensitivity, and the function of the genes identified was explored using network analysis. Using causal reasoning analysis on the genes with different patterns of SNVs by pain sensitivity status, we observed a significant enrichment of variants in genes of the angiotensin pathway (Bonferroni corrected p = 3.8×10(-4)). This pathway is already implicated in animal models and human studies of pain, supporting the notion that it may provide fruitful new targets in pain management. The approach of sequencing extreme exome variation in normal individuals has provided important insights into gene networks mediating pain sensitivity in humans and will be applicable to other common complex traits.

Ischemic stroke is associated with the ABO locus: the EuroCLOT study.

OBJECTIVE: End-stage coagulation and the structure/function of fibrin are implicated in the pathogenesis of ischemic stroke. We explored whether genetic variants associated with end-stage coagulation in healthy volunteers account for the genetic predisposition to ischemic stroke and examined their influence on stroke subtype. METHODS: Common genetic variants identified through genome-wide association studies of coagulation factors and fibrin structure/function in healthy twins (n = 2,100, Stage 1) were examined in ischemic stroke (n = 4,200 cases) using 2 independent samples of European ancestry (Stage 2). A third clinical collection having stroke subtyping (total 8,900 cases, 55,000 controls) was used for replication (Stage 3). RESULTS: Stage 1 identified 524 single nucleotide polymorphisms (SNPs) from 23 linkage disequilibrium blocks having significant association (p < 5 × 10(-8)) with 1 or more coagulation/fibrin phenotypes. The most striking associations included SNP rs5985 with factor XIII activity (p = 2.6 × 10(-186)), rs10665 with FVII (p = 2.4 × 10(-47)), and rs505922 in the ABO gene with both von Willebrand factor (p = 4.7 × 10(-57)) and factor VIII (p = 1.2 × 10(-36)). In Stage 2, the 23 independent SNPs were examined in stroke cases/noncases using MOnica Risk, Genetics, Archiving and Monograph (MORGAM) and Wellcome Trust Case Control Consortium 2 collections. SNP rs505922 was nominally associated with ischemic stroke (odds ratio = 0.94, 95% confidence interval = 0.88-0.99, p = 0.023). Independent replication in Meta-Stroke confirmed the rs505922 association with stroke, beta (standard error, SE) = 0.066 (0.02), p = 0.001, a finding specific to large-vessel and cardioembolic stroke (p = 0.001 and p = < 0.001, respectively) but not seen with small-vessel stroke (p = 0.811). INTERPRETATION: ABO gene variants are associated with large-vessel and cardioembolic stroke but not small-vessel disease. This work sheds light on the different pathogenic mechanisms underpinning stroke subtype.

The architecture of gene regulatory variation across multiple human tissues: the MuTHER study.

While there have been studies exploring regulatory variation in one or more tissues, the complexity of tissue-specificity in multiple primary tissues is not yet well understood. We explore in depth the role of cis-regulatory variation in three human tissues: lymphoblastoid cell lines (LCL), skin, and fat. The samples (156 LCL, 160 skin, 166 fat) were derived simultaneously from a subset of well-phenotyped healthy female twins of the MuTHER resource. We discover an abundance of cis-eQTLs in each tissue similar to previous estimates (858 or 4.7% of genes). In addition, we apply factor analysis (FA) to remove effects of latent variables, thus more than doubling the number of our discoveries (1,822 eQTL genes). The unique study design (Matched Co-Twin Analysis--MCTA) permits immediate replication of eQTLs using co-twins (93%-98%) and validation of the considerable gain in eQTL discovery after FA correction. We highlight the challenges of comparing eQTLs between tissues. After verifying previous significance threshold-based estimates of tissue-specificity, we show their limitations given their dependency on statistical power. We propose that continuous estimates of the proportion of tissue-shared signals and direct comparison of the magnitude of effect on the fold change in expression are essential properties that jointly provide a biologically realistic view of tissue-specificity. Under this framework we demonstrate that 30% of eQTLs are shared among the three tissues studied, while another 29% appear exclusively tissue-specific. However, even among the shared eQTLs, a substantial proportion (10%-20%) have significant differences in the magnitude of fold change between genotypic classes across tissues. Our results underline the need to account for the complexity of eQTL tissue-specificity in an effort to assess consequences of such variants for complex traits.

A locus on chromosome 1p36 is associated with thyrotropin and thyroid function as identified by genome-wide association study.

Thyroid hormones are key regulators of cellular growth, development, and metabolism, and thyroid disorders are a common cause of ill health in the community. Circulating concentrations of thyrotropin (TSH), thyroxine (T4) and triiodothyronine (T3) have a strong heritable component and are thought to be under polygenic control, but the genes responsible are mostly unknown. In order to identify genetic loci associated with these metabolic phenotypes, we performed a genome-wide association study of 2,120,505 SNPs in 2014 female twins from the TwinsUK study and found a significant association between rs10917469 on chromosome 1p36.13 and serum TSH (p = 3.2 × 10(-8)). The association of rs10917469 with serum TSH was replicated (p = 2.0 × 10(-4)) in an independent community-based sample of 1154 participants in the Busselton Health Study. This SNP is located near CAPZB, which might be a regulator of TSH secretion and thus of pituitary-thyroid axis function. Twenty-nine percent of white individuals carry the variant, and the difference in mean TSH concentrations between wild-type individuals and those homozygous for the minor G allele was 0.5 mU/l, which is likely to be clinically relevant. We also provide evidence of suggestive association (p < 5.0 × 10(-6)) of other SNPs with serum TSH, free T4, and free T3 concentrations, and these SNPs might be good targets for further studies. These results advance understanding of the genetic basis of pituitary-thyroid axis function and metabolic regulation.

Genome-wide association study reveals a complex genetic architecture underpinning-induced CYP3A4 enzyme activity.

Atypical cytochrome P450 3A4 (CYP3A4) enzyme activity-induced and inhibited-is thought to be the driver of numerous poor or adverse therapeutic responses to up to 50 % of all commonly prescribed drugs. We carried out a genome-wide association study to identify common genetic variants associated with variation in induced CYP3A4 activity. A total of 310 twins were included in this study. Each participant had already completed a 14 days course of St John's Wort to induce CYP3A4, which was quantified through the metabolic ratio of exogenous 3-hydroxyquinine to quinine. We failed to detect any genome-wide significant associations (P < 1 × 10(-8)) with variation in induced CYP3A4 activity although several genomic regions were highlighted which may play minor roles. We report the first GWAS of variation in induced CYP3A4 activity and our preliminary results indicate a complex genetic architecture underpinning induced CYP3A4 enzyme activity.

Genetic epidemiology of induced CYP3A4 activity.

AIM: The cytochrome P450 3A4 (CYP3A4) enzyme is implicated in the metabolism of more than 50% of all prescribed medications and its activity - including induced or inhibited activity - is deemed to be a crucial determinant of interindividual variability in drug disposition, poor therapeutic efficacy, and adverse response to medication. METHODS: We used the classical twin model in conjunction with an induction experiment to uncover the relative contribution of genetic and environmental factors to interindividual variation in induced CYP3A4 activity. A total of 367 healthy twins participated in the study. Each volunteer was administered a potent inducer of CYP3A4 (St John's Wort) for 14 days and the activity of CYP3A4 was quantified through the metabolism of the exogenously administered probe drug quinine sulfate. RESULTS: Baseline and induced CYP3A4 activity were highly variable with a seven-fold and 11-fold difference among our population, respectively. Alcohol consumption, BMI, and smoking were significantly associated with induced CYP3A4 activity, collectively explaining 20% of the variation (P<1×10(-4)). The narrow-sense heritability of induced CYP3A4 activity was estimated at 66%, whereas the remainder of the variation was attributed to unique environmental factors. CONCLUSION: To our knowledge, this is the first genetic epidemiological study of induced CYP3A4 activity. Our results motivate further research to identify common and rarer genetic variants that underpin the heritable component of variation in induced CYP3A4 activity.

Identification of quantitative trait loci for fibrin clot phenotypes: the EuroCLOT study.

OBJECTIVE: Fibrin makes up the structural basis of an occlusive arterial thrombus, and variability in fibrin phenotype relates to cardiovascular risk. The aims of the current study from the EU consortium EuroCLOT were to (1) determine the heritability of fibrin phenotypes and (2) identify QTLs associated with fibrin phenotypes. METHODS AND RESULTS: 447 dizygotic (DZ) and 460 monozygotic (MZ) pairs of healthy UK white female twins and 199 DZ twin pairs from Denmark were studied. D-dimer, an indicator of fibrin turnover, was measured by ELISA and measures of clot formation, morphology, and lysis were determined by turbidimetric assays. Heritability estimates and genome-wide linkage analysis were performed. Estimates of heritability for d-dimer and turbidometric variables were in the range 17% to 46%, with highest levels for maximal absorbance which provides an estimate of clot density. Genome-wide linkage analysis revealed 6 significant regions with LOD >3 on 5 chromosomes (5, 6, 9, 16, and 17). CONCLUSIONS: The results indicate a significant genetic contribution to variability in fibrin phenotypes and highlight regions in the human genome which warrant further investigation in relation to ischemic cardiovascular disorders and their therapy.

The association between physical activity in leisure time and leukocyte telomere length.

BACKGROUND: Physical inactivity is an important risk factor for many aging-related diseases. Leukocyte telomere dynamics (telomere length and age-dependent attrition rate) are ostensibly a biological indicator of human aging. We therefore tested the hypothesis that physical activity level in leisure time (over the past 12 months) is associated with leukocyte telomere length (LTL) in normal healthy volunteers. METHODS: We studied 2401 white twin volunteers, comprising 2152 women and 249 men, with questionnaires on physical activity level, smoking status, and socioeconomic status. Leukocyte telomere length was derived from the mean terminal restriction fragment length and adjusted for age and other potential confounders. RESULTS: Leukocyte telomere length was positively associated with increasing physical activity level in leisure time (P< .001); this association remained significant after adjustment for age, sex, body mass index, smoking, socioeconomic status, and physical activity at work. The LTLs of the most active subjects were 200 nucleotides longer than those of the least active subjects (7.1 and 6.9 kilobases, respectively; P= .006). This finding was confirmed in a small group of twin pairs discordant for physical activity level (on average, the LTL of more active twins was 88 nucleotides longer than that of less active twins; P= .03). CONCLUSIONS: A sedentary lifestyle (in addition to smoking, high body mass index, and low socioeconomic status) has an effect on LTL and may accelerate the aging process. This provides a powerful message that could be used by clinicians to promote the potentially antiaging effect of regular exercise.

Genetic loci linked to pituitary-thyroid axis set points: a genome-wide scan of a large twin cohort.

OBJECTIVE: Previous studies have shown that circulating concentrations of TSH, free T4, and free T3 are genetically regulated, but the genes responsible remain largely unknown. The aim of this study was to identify genetic loci associated with these parameters. DESIGN: We performed a multipoint, nonparametric genome-wide linkage scan of 613 female dizygotic twin pairs. All subjects were euthyroid (TSH 0.4-4.0 mU/liter) with negative thyroid peroxidase antibodies and no history of thyroid disease. The genome scan comprised 737 microsatellite markers supplemented with dinucleotide markers. Data were analyzed using residualized thyroid hormone data after adjustment for age, smoking, and body mass index. RESULTS: Multipoint linkage analysis gave linkage peaks for free T4 on chromosome 14q13 and 18q21 [logarithm of odds (LOD) 2.4-3.2]; TSH on chromosomes 2q36, 4q32, and 9q34 (LOD 2.1-3.2); and free T3 on chromosomes 7q36, 8q22, and 18q21 (LOD 2.0-2.3). CONCLUSIONS: This study has identified eight genomic locations with linkage of LOD of 2.0 or greater. These results should enable targeted positional candidate and positional cloning studies to advance our understanding of genetic control of the pituitary-thyroid axis.

Higher serum vitamin D concentrations are associated with longer leukocyte telomere length in women.

BACKGROUND: Vitamin D is a potent inhibitor of the proinflammatory response and thereby diminishes turnover of leukocytes. Leukocyte telomere length (LTL) is a predictor of aging-related disease and decreases with each cell cycle and increased inflammation. OBJECTIVE: The objective of the study was to examine whether vitamin D concentrations would attenuate the rate of telomere attrition in leukocytes, such that higher vitamin D concentrations would be associated with longer LTL. DESIGN: Serum vitamin D concentrations were measured in 2160 women aged 18-79 y (mean age: 49.4) from a large population-based cohort of twins. LTL was measured by using the Southern blot method. RESULTS: Age was negatively correlated with LTL (r = -0.40, P < 0.0001). Serum vitamin D concentrations were positively associated with LTL (r = 0.07, P = 0.0010), and this relation persisted after adjustment for age (r = 0.09, P < 0.0001) and other covariates (age, season of vitamin D measurement, menopausal status, use of hormone replacement therapy, and physical activity; P for trend across tertiles = 0.003). The difference in LTL between the highest and lowest tertiles of vitamin D was 107 base pairs (P = 0.0009), which is equivalent to 5.0 y of telomeric aging. This difference was further accentuated by increased concentrations of C-reactive protein, which is a measure of systemic inflammation. CONCLUSION: Our findings suggest that higher vitamin D concentrations, which are easily modifiable through nutritional supplementation, are associated with longer LTL, which underscores the potentially beneficial effects of this hormone on aging and age-related diseases.

Linkage and potential association of obesity-related phenotypes with two genes on chromosome 12q24 in a female dizygous twin cohort.

Obesity is a multifactorial disorder with a complex phenotype. It is a significant risk factor for diabetes and hypertension. We assessed obesity-related traits in a large cohort of twins and performed a genome-wide linkage scan and positional candidate analysis to identify genes that play a role in regulating fat mass and distribution in women. Dizygous female twin pairs from 1,094 pedigrees were studied (mean age 47.0+/-11.5 years (range 18-79 years)). Nonparametric multipoint linkage analyses showed linkage for central fat mass to 12q24 (141 cM) with LOD 2.2 and body mass index to 8q11 (67 cM) with LOD 1.3, supporting previously established linkage data. Novel areas of suggestive linkage were for total fat percentage at 6q12 (LOD 2.4) and for total lean mass at 2q37 (LOD 2.4). Data from follow-up fine mapping in an expanded cohort of 1243 twin pairs reinforced the linkage for central fat mass to 12q24 (LOD 2.6; 143 cM) and narrowed the -1 LOD support interval to 22 cM. In all, 45 single-nucleotide polymorphisms (SNPs) from 26 positional candidate genes within the 12q24 interval were then tested for association in a cohort of 1102 twins. Single-point Monks-Kaplan analysis provided evidence of association between central fat mass and SNPs in two genes - PLA2G1B (P = 0.0067) and P2RX4 (P = 0.017). These data provide replication and refinement of the 12q24 obesity locus and suggest that genes involved in phospholipase and purinoreceptor pathways may regulate fat accumulation and distribution.

Genome-wide association study with 1000 genomes imputation identifies signals for nine sex hormone-related phenotypes.

Genetic factors contribute strongly to sex hormone levels, yet knowledge of the regulatory mechanisms remains incomplete. Genome-wide association studies (GWAS) have identified only a small number of loci associated with sex hormone levels, with several reproductive hormones yet to be assessed. The aim of the study was to identify novel genetic variants contributing to the regulation of sex hormones. We performed GWAS using genotypes imputed from the 1000 Genomes reference panel. The study used genotype and phenotype data from a UK twin register. We included 2913 individuals (up to 294 males) from the Twins UK study, excluding individuals receiving hormone treatment. Phenotypes were standardised for age, sex, BMI, stage of menstrual cycle and menopausal status. We tested 7,879,351 autosomal SNPs for association with levels of dehydroepiandrosterone sulphate (DHEAS), oestradiol, free androgen index (FAI), follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, progesterone, sex hormone-binding globulin and testosterone. Eight independent genetic variants reached genome-wide significance (P<5 × 10(-8)), with minor allele frequencies of 1.3-23.9%. Novel signals included variants for progesterone (P=7.68 × 10(-12)), oestradiol (P=1.63 × 10(-8)) and FAI (P=1.50 × 10(-8)). A genetic variant near the FSHB gene was identified which influenced both FSH (P=1.74 × 10(-8)) and LH (P=3.94 × 10(-9)) levels. A separate locus on chromosome 7 was associated with both DHEAS (P=1.82 × 10(-14)) and progesterone (P=6.09 × 10(-14)). This study highlights loci that are relevant to reproductive function and suggests overlap in the genetic basis of hormone regulation.

Whole-genome sequence-based analysis of thyroid function.

Normal thyroid function is essential for health, but its genetic architecture remains poorly understood. Here, for the heritable thyroid traits thyrotropin (TSH) and free thyroxine (FT4), we analyse whole-genome sequence data from the UK10K project (N=2,287). Using additional whole-genome sequence and deeply imputed data sets, we report meta-analysis results for common variants (MAF≥1%) associated with TSH and FT4 (N=16,335). For TSH, we identify a novel variant in SYN2 (MAF=23.5%, P=6.15 × 10(-9)) and a new independent variant in PDE8B (MAF=10.4%, P=5.94 × 10(-14)). For FT4, we report a low-frequency variant near B4GALT6/SLC25A52 (MAF=3.2%, P=1.27 × 10(-9)) tagging a rare TTR variant (MAF=0.4%, P=2.14 × 10(-11)). All common variants explain ≥20% of the variance in TSH and FT4. Analysis of rare variants (MAF<1%) using sequence kernel association testing reveals a novel association with FT4 in NRG1. Our results demonstrate that increased coverage in whole-genome sequence association studies identifies novel variants associated with thyroid function.

Assessment of age-related changes in heritability and IGF-1 gene effect on circulating IGF-1 levels.

It is well established that insulin-like growth factor 1 (IGF-1) circulating levels correlate with age and that heritability and influence of IGF-1 gene variation on IGF-1 levels also well-known. However, the influence of age on the genetic factors determining IGF-1 levels is not clear. In this study, we compared heritability estimates between younger (<52 years) and older (>52 years) twins and tested: (a) whether single nucleotide polymorphisms (SNPs) lying within 100 kbp of the IGF-1 gene are also associated with IGF-1 variation and (b) whether associated SNPs show interaction with age on IGF-1 levels. To achieve these aims, we measured plasma levels of IGF-1 and genotyped 18 SNPs with minor allele frequency >0.1 in a large sample, 4,471 UK female twins. Heritability explained 42 % of IGF-1 variation adjusted for age and in unadjusted sample was independent of age. Ten SNPs in four haploblocks showed significant association with IGF-1 levels, with p = 0.01-0.0005. The most distal SNP was located up to 90 kbp from the IGF-1 gene. When their age-dependent effects were examined, one SNP, rs855203, showed significant (p = 0.0009) age-dependent interaction effect on IGF-1 levels variation. This is the first study to test the age × genotype interaction in IGF-1 levels. The genomic region marked by rs855203 may consequently be of significance for further molecular and pharmacogenetic research, in particular in advanced age.

Elevated plasma fractalkine levels are associated with higher levels of IL-6, Apo-B, LDL-C and insulin, but not with body composition in a large female twin sample.

OBJECTIVE: Plasma fractalkine (FRACT) is involved in the development of numerous inflammatory conditions including atherosclerosis. It is associated with type 2 diabetes mellitus and adipose inflammation. However, whether FRACT is associated with major risk factors for cardiovascular disease, in particular obesity, metabolic syndrome and blood lipids, is virtually unknown. METHODS: The study included a large community-based sample of 3306 middle-aged women drawn from the general UK population. Blood samples were analyzed for circulating levels of FRACT, leptin, insulin, glucose, LDL-C, HDL-C, Apo-A, ApoB and IL-6. Obesity was assessed by fat body mass (FBM) using dual-energy x-ray absorptiometry and by body mass index (BMI). RESULTS: We found no association between FRACT and body composition, in particular adiposity. Obese and non obese subjects with metabolic syndrome tended to have higher levels of FRACT compared with non-obese subjects without metabolic syndrome but this did not reach statistical significance. Most importantly we report significant correlations between FRACT and circulating IL-6, Apo-B, LDL-C and insulin. The associations with IL-6 and Apo-B were particularly significant (P-value<0.001), and survived correction for multiple testing and adjustment for age and other covariates. CONCLUSION: Higher FRACT levels correlated with elevated levels of IL-6, Apo-B, LDL-C and insulin, all known risk factors for several clinical related diseases suggesting a potential role of FRACT in inflammation and tissue injury. Variations of FRACT levels are not influenced by body composition and are not correlated with leptin indicating that fat mass alone is not responsible for elevation of FRACT seen in obese individuals.

Contribution of putative genetic factors and candidate gene variants to inter-individual variation of circulating fractalkine (CX3CL1) levels in a large UK twins' sample.

OBJECTIVE: Soluble fractalkine (sFRACT) is involved in the pathogenesis of several clinical diseases. Our major objective was to determine to what extent its variation is governed by genetic factors and whether this genetic variation could be attributable to SNPs in five candidate genes: CX3CL1, CX3CR1, ADAM10, ADAM17 and AREG. METHODS: Plasma levels of sFRACT and 38 SNPs, with minor allele frequency >0.1 were examined in a large twin sample drawn from the general UK population. The discovery sample included 3306 middle-aged females: 1172 MZ twins and 2134 DZ twins. A replication sample of 1675 twins was used to validate the major association results obtained in genetic association analysis in the discovery sample. We implemented variance component analysis to estimate contribution of putative genetic, (including above SNPs) and environmental factors to sFRACT variation. RESULTS: sFRACT was found not to vary with either age or BMI. Putative genetic factors (heritability) explained 43.6±3% of the total variation of plasma sFRACT levels. However, we found no evidence of association between sFRACT and any of the examined SNPs, despite having >85% power to detect an association of just 1% of the variance explained. The results in the discovery and replication samples were in good agreement suggesting these findings are real. CONCLUSION: Our results suggest involvement of genetic factors to inter-individual variation of sFRACT levels in a general human population. However, further studies are required to determine genetic polymorphisms affecting sFRACT variation.

Copy number variation of the APC gene is associated with regulation of bone mineral density.

INTRODUCTION: Genetic studies of osteoporosis have commonly examined SNPs in candidate genes or whole genome analyses, but insertions and deletions of DNA, collectively called copy number variations (CNVs), also comprise a large amount of the genetic variability between individuals. Previously, SNPs in the APC gene have been strongly associated with femoral neck and lumbar spine volumetric bone mineral density in older men. In addition, familial adenomatous polyposis patients carrying heterozygous mutations in the APC gene have been shown to have significantly higher mean bone mineral density than age- and sex-matched controls suggesting the importance of this gene in regulating bone mineral density. We examined CNV within the APC gene region to test for association with bone mineral density. METHODS: DNA was extracted from venous blood, genotyped using the Human Hap610 arrays and CNV determined from the fluorescence intensity data in 2070 Caucasian men and women aged 47.0 ± 13.0 (mean ± SD) years, to assess the effects of the CNV on bone mineral density at the forearm, spine and total hip sites. RESULTS: Data for covariate adjusted bone mineral density from subjects grouped by APC CNV genotype showed significant difference (P=0.02-0.002). Subjects with a single copy loss of APC had a 7.95%, 13.10% and 13.36% increase in bone mineral density at the forearm, spine and total hip sites respectively, compared to subjects with two copies of the APC gene. CONCLUSIONS: These data support previous findings of APC regulating bone mineral density and demonstrate that a novel CNV of the APC gene is significantly associated with bone mineral density in Caucasian men and women.

Mapping cis- and trans-regulatory effects across multiple tissues in twins.

Sequence-based variation in gene expression is a key driver of disease risk. Common variants regulating expression in cis have been mapped in many expression quantitative trait locus (eQTL) studies, typically in single tissues from unrelated individuals. Here, we present a comprehensive analysis of gene expression across multiple tissues conducted in a large set of mono- and dizygotic twins that allows systematic dissection of genetic (cis and trans) and non-genetic effects on gene expression. Using identity-by-descent estimates, we show that at least 40% of the total heritable cis effect on expression cannot be accounted for by common cis variants, a finding that reveals the contribution of low-frequency and rare regulatory variants with respect to both transcriptional regulation and complex trait susceptibility. We show that a substantial proportion of gene expression heritability is trans to the structural gene, and we identify several replicating trans variants that act predominantly in a tissue-restricted manner and may regulate the transcription of many genes.

The basis of differential responses to folic acid supplementation.

BACKGROUND/AIMS: Elevated levels of total homocysteine (tHcy) are associated with an increased risk of many common diseases. Supplementation with folic acid has been shown to significantly reduce tHcy levels. We used the classical twin model to partition the variability in changes in plasma tHcy levels through folic acid supplementation into genetic, environmental, and confounding epidemiological factors. METHODS: We carried out an intervention study of folic acid using 101 healthy, female, identical and non-identical twins aged 50-80 years. Each twin was administered folic acid (0.8 mg/day) for 6 weeks. Total plasma folate, cobalamin and tHcy were measured at both baseline and after dosing. We calculated the heritability and tested for associations between the MTHFR C677T functional variant and response to folic acid supplementation. RESULTS: Supplementation with folic acid led to a significant reduction in tHcy levels. The mean tHcy changed from 12.14 to 10.42 µmol/l after supplementation (p < 10(-5)). Moreover, the change in tHcy levels was highly heritable (64%), not associated with the C677T functional variant at MTHFR and not confounded by age, BMI or diet. CONCLUSIONS: Our results highlight the need to identify genetic factors associated with biomarkers of response to folate supplementation.