Newborn DNA-methylation, childhood lung function, and the risks of asthma and COPD across the life course.

RATIONALE: We aimed to identify differentially methylated regions (DMRs) in cord blood DNA associated with childhood lung function, asthma and chronic obstructive pulmonary disease (COPD) across the life course. METHODS: We meta-analysed epigenome-wide data of 1688 children from five cohorts to identify cord blood DMRs and their annotated genes, in relation to forced expiratory volume in 1 s (FEV1), FEV1/forced vital capacity (FVC) ratio and forced expiratory flow at 75% of FVC at ages 7-13 years. Identified DMRs were explored for associations with childhood asthma, adult lung function and COPD, gene expression and involvement in biological processes. RESULTS: We identified 59 DMRs associated with childhood lung function, of which 18 were associated with childhood asthma and nine with COPD in adulthood. Genes annotated to the top 10 identified DMRs were HOXA5, PAOX, LINC00602, ABCA7, PER3, CLCA1, VENTX, NUDT12, PTPRN2 and TCL1A. Differential gene expression in blood was observed for 32 DMRs in childhood and 18 in adulthood. Genes related with 16 identified DMRs were associated with respiratory developmental or pathogenic pathways. INTERPRETATION: Our findings suggest that the epigenetic status of the newborn affects respiratory health and disease across the life course.

Multi-ethnic SULT1A1 copy number profiling with multiplex ligation-dependent probe amplification.

AIM: To develop a SULT1A1 multiplex ligation-dependent probe amplification assay and to investigate multi-ethnic copy number variant frequencies. METHODS: A novel multiplex ligation-dependent probe amplification assay was developed and tested on 472 African-American, Asian, Caucasian, Hispanic and Ashkenazi Jewish individuals. RESULTS: The frequencies of atypical total copy number (i.e., greater or less than two) were 38.7% for Hispanics, 38.9% for Ashkenazi Jewish, 43.2% for Caucasians, 53.6% for Asians and 64.1% for African-Americans. Heterozygous SULT1A1 deletion carriers (slow sulfators) were most common among Caucasians (8.4%), whereas African-Americans had the highest frequencies of three or more copies (rapid sulfators; 60.9%). CONCLUSION: Different ethnic and racial populations have varying degrees of SULT1A1-mediated sulfation activity, which warrants further research and that may have utility for drug response prediction among SULT1A1-metabolized medications.

Genetic Determinants of Circulating Estrogen Levels and Evidence of a Causal Effect of Estradiol on Bone Density in Men.

Context: Serum estradiol (E2) and estrone (E1) levels exhibit substantial heritability. Objective: To investigate the genetic regulation of serum E2 and E1 in men. Design, Setting, and Participants: Genome-wide association study in 11,097 men of European origin from nine epidemiological cohorts. Main Outcome Measures: Genetic determinants of serum E2 and E1 levels. Results: Variants in/near CYP19A1 demonstrated the strongest evidence for association with E2, resolving to three independent signals. Two additional independent signals were found on the X chromosome; FAMily with sequence similarity 9, member B (FAM9B), rs5934505 (P = 3.4 × 10-8) and Xq27.3, rs5951794 (P = 3.1 × 10-10). E1 signals were found in CYP19A1 (rs2899472, P = 5.5 × 10-23), in Tripartite motif containing 4 (TRIM4; rs17277546, P = 5.8 × 10-14), and CYP11B1/B2 (rs10093796, P = 1.2 × 10-8). E2 signals in CYP19A1 and FAM9B were associated with bone mineral density (BMD). Mendelian randomization analysis suggested a causal effect of serum E2 on BMD in men. A 1 pg/mL genetically increased E2 was associated with a 0.048 standard deviation increase in lumbar spine BMD (P = 2.8 × 10-12). In men and women combined, CYP19A1 alleles associated with higher E2 levels were associated with lower degrees of insulin resistance. Conclusions: Our findings confirm that CYP19A1 is an important genetic regulator of E2 and E1 levels and strengthen the causal importance of E2 for bone health in men. We also report two independent loci on the X-chromosome for E2, and one locus each in TRIM4 and CYP11B1/B2, for E1.

Erratum: Large meta-analysis of genome-wide association studies identifies five loci for lean body mass.

A correction to this article has been published and is linked from the HTML version of this article.

Homocysteine levels associate with subtle changes in leukocyte DNA methylation: an epigenome-wide analysis.

AIM: Homocysteine (Hcy) is a sensitive marker of one-carbon metabolism. Higher Hcy levels have been associated with global DNA hypomethylation. We investigated the association between plasma Hcy and epigenome-wide DNA methylation in leukocytes. METHODS: Methylation was measured using Illumina 450 k arrays in 2035 individuals from six cohorts. Hcy-associated differentially methylated positions and regions were identified using meta-analysis. RESULTS: Three differentially methylated positions cg21607669 (SLC27A1), cg26382848 (AJUBA) and cg10701000 (KCNMA1) at chromosome 19, 14 and 10, respectively, were significantly associated with Hcy. In addition, we identified 68 Hcy-associated differentially methylated regions, the most significant of which was a 1.8-kb spanning domain (TNXB/ATF6B) at chromosome 6. CONCLUSION: We identified novel epigenetic loci associated with Hcy levels, of which specific role needs to be further validated.

Genetically defined elevated homocysteine levels do not result in widespread changes of DNA methylation in leukocytes.

BACKGROUND: DNA methylation is affected by the activities of the key enzymes and intermediate metabolites of the one-carbon pathway, one of which involves homocysteine. We investigated the effect of the well-known genetic variant associated with mildly elevated homocysteine: MTHFR 677C>T independently and in combination with other homocysteine-associated variants, on genome-wide leukocyte DNA-methylation. METHODS: Methylation levels were assessed using Illumina 450k arrays on 9,894 individuals of European ancestry from 12 cohort studies. Linear-mixed-models were used to study the association of additive MTHFR 677C>T and genetic-risk score (GRS) based on 18 homocysteine-associated SNPs, with genome-wide methylation. RESULTS: Meta-analysis revealed that the MTHFR 677C>T variant was associated with 35 CpG sites in cis, and the GRS showed association with 113 CpG sites near the homocysteine-associated variants. Genome-wide analysis revealed that the MTHFR 677C>T variant was associated with 1 trans-CpG (nearest gene ZNF184), while the GRS model showed association with 5 significant trans-CpGs annotated to nearest genes PTF1A, MRPL55, CTDSP2, CRYM and FKBP5. CONCLUSIONS: Our results do not show widespread changes in DNA-methylation across the genome, and therefore do not support the hypothesis that mildly elevated homocysteine is associated with widespread methylation changes in leukocytes.

Large meta-analysis of genome-wide association studies identifies five loci for lean body mass.

Lean body mass, consisting mostly of skeletal muscle, is important for healthy aging. We performed a genome-wide association study for whole body (20 cohorts of European ancestry with n = 38,292) and appendicular (arms and legs) lean body mass (n = 28,330) measured using dual energy X-ray absorptiometry or bioelectrical impedance analysis, adjusted for sex, age, height, and fat mass. Twenty-one single-nucleotide polymorphisms were significantly associated with lean body mass either genome wide (p < 5 × 10-8) or suggestively genome wide (p < 2.3 × 10-6). Replication in 63,475 (47,227 of European ancestry) individuals from 33 cohorts for whole body lean body mass and in 45,090 (42,360 of European ancestry) subjects from 25 cohorts for appendicular lean body mass was successful for five single-nucleotide polymorphisms in/near HSD17B11, VCAN, ADAMTSL3, IRS1, and FTO for total lean body mass and for three single-nucleotide polymorphisms in/near VCAN, ADAMTSL3, and IRS1 for appendicular lean body mass. Our findings provide new insight into the genetics of lean body mass.Lean body mass is a highly heritable trait and is associated with various health conditions. Here, Kiel and colleagues perform a meta-analysis of genome-wide association studies for whole body lean body mass and find five novel genetic loci to be significantly associated.

Genomic analyses identify hundreds of variants associated with age at menarche and support a role for puberty timing in cancer risk.

The timing of puberty is a highly polygenic childhood trait that is epidemiologically associated with various adult diseases. Using 1000 Genomes Project-imputed genotype data in up to ∼370,000 women, we identify 389 independent signals (P < 5 × 10-8) for age at menarche, a milestone in female pubertal development. In Icelandic data, these signals explain ∼7.4% of the population variance in age at menarche, corresponding to ∼25% of the estimated heritability. We implicate ∼250 genes via coding variation or associated expression, demonstrating significant enrichment in neural tissues. Rare variants near the imprinted genes MKRN3 and DLK1 were identified, exhibiting large effects when paternally inherited. Mendelian randomization analyses suggest causal inverse associations, independent of body mass index (BMI), between puberty timing and risks for breast and endometrial cancers in women and prostate cancer in men. In aggregate, our findings highlight the complexity of the genetic regulation of puberty timing and support causal links with cancer susceptibility.

Genetic variation and bone mineral density in long-term adult survivors of childhood cancer.

PURPOSE: Despite similarities in upfront treatment of childhood cancer, not every adult survivor of childhood cancer (CCS) has an impaired bone mineral density (BMD). No data are available on the role of genetic variation on impairment of BMD in CCS. METHODS: This cross-sectional single-center cohort study included 334 adult CCSs (median follow-up time after cessation of treatment: 15 years; median age at follow-up: 26 years). Total body BMD (BMDTB ) and lumbar spine BMD (BMDLS ) were measured by dual x-ray absorptiometry. We selected 12 candidate single-nucleotide polymorphisms (SNPs) in 11 genes (COL1A1, TNFSF11, TNFRSF11, TNRFSA11B, VDR, ESR1, WLS, LRP5, MTHFR, MTRR, IL-6). RESULTS: Multivariate analyses revealed that lower BMD was associated with lower weight and height at follow-up, male sex, and previously administered radiotherapy. Survivors with the homozygous minor allele (GG) genotype of rs2504063 (ESR1: estrogen receptor type 1) had a lower BMDTB values (-1.16 vs. -0.82; P = 0.01) than those with the AG/AA genotype; however, BMDLS was not different. Carriers of two minor alleles (GG) of rs599083 (LRP5: low-density lipoprotein receptor) revealed lower BMDTB (-1.20 vs. -0.78; P = 0.02) and lower BMDLS (-0.95 vs. -0.46; P = 0.01) values than those with the TT/TG genotype. CONCLUSION: CCSs who are carriers of candidate SNPs in the ESR1 or LRP5 genes seem to have an impaired bone mass at an early adult age. Information on genetic variation, in addition to patient- and treatment-related factors, may be helpful in identifying survivors who are at risk for low bone density after childhood cancer treatment.

Lower sex hormone levels are associated with more chronic musculoskeletal pain in community-dwelling elderly women.

Chronic pain is more prevalent in women than in men, with increasing differences between sexes in advanced age. This could be caused by differences in sex hormone levels. We therefore studied the relationship between sex hormones and the prevalence and incidence of chronic pain. The association between sex hormone levels and chronic pain was examined in 9717 participants aged 45 years and older from the Rotterdam Study, a population-based study. Chronic pain was defined as pain in the lower back, hands, knees and/or hips for at least 3 months. Sex hormone levels included estrogen, testosterone, androstenedione, and 17-hydroxyprogesterone. Relationships between hormones and prevalent and new onset chronic pain were analyzed using linear and logistic regression, stratified by gender. Women with androstenedione or estradiol levels in the lowest tertile had more chronic pain (odds ratio, 1.20; 95% CI, 1.03-1.39 and odds ratio, 1.27; 95% CI, 1.10-1.48, respectively). Mean estradiol levels were lower among men with chronic pain (mean difference -3.88 pmol/L; P = 0.005). Lowest tertile 17-hydroxyprogesterone in women was associated with 38% more new onset pain. All these associations were independent from age, body mass index, health and lifestyle factors, and osteoarthritis. Lower sex hormone levels are associated with chronic musculoskeletal pain, independent from lifestyle and health-related factors, in community-dwelling elderly women. These results suggest that sex hormones play a role in chronic pain and should be taken into account when a patient presents with chronic pain. Therefore, sex hormones may be a potential treatment target for these patients.

The transcriptional landscape of age in human peripheral blood.

Disease incidences increase with age, but the molecular characteristics of ageing that lead to increased disease susceptibility remain inadequately understood. Here we perform a whole-blood gene expression meta-analysis in 14,983 individuals of European ancestry (including replication) and identify 1,497 genes that are differentially expressed with chronological age. The age-associated genes do not harbor more age-associated CpG-methylation sites than other genes, but are instead enriched for the presence of potentially functional CpG-methylation sites in enhancer and insulator regions that associate with both chronological age and gene expression levels. We further used the gene expression profiles to calculate the 'transcriptomic age' of an individual, and show that differences between transcriptomic age and chronological age are associated with biological features linked to ageing, such as blood pressure, cholesterol levels, fasting glucose, and body mass index. The transcriptomic prediction model adds biological relevance and complements existing epigenetic prediction models, and can be used by others to calculate transcriptomic age in external cohorts.

Large-scale genomic analyses link reproductive aging to hypothalamic signaling, breast cancer susceptibility and BRCA1-mediated DNA repair.

Menopause timing has a substantial impact on infertility and risk of disease, including breast cancer, but the underlying mechanisms are poorly understood. We report a dual strategy in ∼70,000 women to identify common and low-frequency protein-coding variation associated with age at natural menopause (ANM). We identified 44 regions with common variants, including two regions harboring additional rare missense alleles of large effect. We found enrichment of signals in or near genes involved in delayed puberty, highlighting the first molecular links between the onset and end of reproductive lifespan. Pathway analyses identified major association with DNA damage response (DDR) genes, including the first common coding variant in BRCA1 associated with any complex trait. Mendelian randomization analyses supported a causal effect of later ANM on breast cancer risk (∼6% increase in risk per year; P = 3 × 10(-14)), likely mediated by prolonged sex hormone exposure rather than DDR mechanisms.

Causal mechanisms and balancing selection inferred from genetic associations with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common reproductive disorder in women, yet there is little consensus regarding its aetiology. Here we perform a genome-wide association study of PCOS in up to 5,184 self-reported cases of White European ancestry and 82,759 controls, with follow-up in a further ∼2,000 clinically validated cases and ∼100,000 controls. We identify six signals for PCOS at genome-wide statistical significance (P<5 × 10(-8)), in/near genes ERBB4/HER4, YAP1, THADA, FSHB, RAD50 and KRR1. Variants in/near three of the four epidermal growth factor receptor genes (ERBB2/HER2, ERBB3/HER3 and ERBB4/HER4) are associated with PCOS at or near genome-wide significance. Mendelian randomization analyses indicate causal roles in PCOS aetiology for higher BMI (P=2.5 × 10(-9)), higher insulin resistance (P=6 × 10(-4)) and lower serum sex hormone binding globulin concentrations (P=5 × 10(-4)). Furthermore, genetic susceptibility to later menopause is associated with higher PCOS risk (P=1.6 × 10(-8)) and PCOS-susceptibility alleles are associated with higher serum anti-Müllerian hormone concentrations in girls (P=8.9 × 10(-5)). This large-scale study implicates an aetiological role of the epidermal growth factor receptors, infers causal mechanisms relevant to clinical management and prevention, and suggests balancing selection mechanisms involved in PCOS risk.

Trans-ancestry genome-wide association study identifies 12 genetic loci influencing blood pressure and implicates a role for DNA methylation.

We carried out a trans-ancestry genome-wide association and replication study of blood pressure phenotypes among up to 320,251 individuals of East Asian, European and South Asian ancestry. We find genetic variants at 12 new loci to be associated with blood pressure (P = 3.9 × 10(-11) to 5.0 × 10(-21)). The sentinel blood pressure SNPs are enriched for association with DNA methylation at multiple nearby CpG sites, suggesting that, at some of the loci identified, DNA methylation may lie on the regulatory pathway linking sequence variation to blood pressure. The sentinel SNPs at the 12 new loci point to genes involved in vascular smooth muscle (IGFBP3, KCNK3, PDE3A and PRDM6) and renal (ARHGAP24, OSR1, SLC22A7 and TBX2) function. The new and known genetic variants predict increased left ventricular mass, circulating levels of NT-proBNP, and cardiovascular and all-cause mortality (P = 0.04 to 8.6 × 10(-6)). Our results provide new evidence for the role of DNA methylation in blood pressure regulation.

Prenatal parental tobacco smoking, gene specific DNA methylation, and newborns size: the Generation R study.

BACKGROUND: Deleterious effects of prenatal tobacco smoking on fetal growth and newborn weight are well-established. One of the proposed mechanisms underlying this relationship is alterations in epigenetic programming. We selected 506 newborns from a population-based prospective birth cohort in the Netherlands. Prenatal parental tobacco smoking was assessed using self-reporting questionnaires. Information on birth outcomes was obtained from medical records. The deoxyribonucleic acid (DNA) methylation of the growth genes IGF2DMR and H19 was measured in newborn umbilical cord white blood cells. Associations were assessed between parental tobacco smoking and DNA methylation using linear mixed models and adjusted for potential confounders. RESULTS: The DNA methylation levels of IGF2DMR and H19 in the non-smoking group were median (90 % range), 54.0 % (44.6-62.0), and 30.0 % (25.5-34.0), in the first trimester only smoking group 52.2 % (44.5-61.1) and 30.8 % (27.1-34.1), and in the continued smoking group 51.6 % (43.9-61.3) and 30.2 % (23.7-34.8), respectively. Continued prenatal maternal smoking was inversely associated with IGF2DMR methylation (ß = -1.03, 95 % CI -1.76; -0.30) in a dose-dependent manner (P-trend = 0.030). This association seemed to be slightly more profound among newborn girls (ß = -1.38, 95 % CI -2.63; -0.14) than boys (ß = -0.72, 95 % CI -1.68; 0.24). H19 methylation was also inversely associated continued smoking <5 cigarettes/day (ß = -0.96, 95 % CI -1.78; -0.14). Moreover, the association between maternal smoking and newborns small for gestational age seems to be partially explained by IGF2DMR methylation (ß = -0.095, 95 % CI -0.249; -0.018). Among non-smoking mothers, paternal tobacco smoking was not associated with IGF2DMR or H19 methylation. CONCLUSIONS: Maternal smoking is inversely associated with IGF2DMR methylation in newborns, which can be one of the underlying mechanisms through which smoking affects fetal growth.

Rare coding variants and X-linked loci associated with age at menarche.

More than 100 loci have been identified for age at menarche by genome-wide association studies; however, collectively these explain only ∼3% of the trait variance. Here we test two overlooked sources of variation in 192,974 European ancestry women: low-frequency protein-coding variants and X-chromosome variants. Five missense/nonsense variants (in ALMS1/LAMB2/TNRC6A/TACR3/PRKAG1) are associated with age at menarche (minor allele frequencies 0.08-4.6%; effect sizes 0.08-1.25 years per allele; P<5 × 10(-8)). In addition, we identify common X-chromosome loci at IGSF1 (rs762080, P=9.4 × 10(-13)) and FAAH2 (rs5914101, P=4.9 × 10(-10)). Highlighted genes implicate cellular energy homeostasis, post-transcriptional gene silencing and fatty-acid amide signalling. A frequently reported mutation in TACR3 for idiopathic hypogonatrophic hypogonadism (p.W275X) is associated with 1.25-year-later menarche (P=2.8 × 10(-11)), illustrating the utility of population studies to estimate the penetrance of reportedly pathogenic mutations. Collectively, these novel variants explain ∼0.5% variance, indicating that these overlooked sources of variation do not substantially explain the 'missing heritability' of this complex trait.

Tobacco smoking is associated with methylation of genes related to coronary artery disease.

BACKGROUND: Tobacco smoking, a risk factor for coronary artery disease (CAD), is known to modify DNA methylation. We hypothesized that tobacco smoking modifies methylation of the genes identified for CAD by genome-wide association study (GWAS). RESULTS: We selected genomic regions based on 150 single-nucleotide polymorphisms (SNPs) identified in the largest GWAS on CAD. We investigated the association between current smoking and the CpG sites within and near these CAD-related genes. Methylation was measured with the Illumina Human Methylation 450K array in whole blood of 724 Caucasian subjects from the Rotterdam Study, a Dutch population based cohort study. A total of 3669 CpG sites within 169 CAD-related genes were studied for association with current compared to never smoking. Fifteen CpG sites were significantly associated after correction for multiple testing (Bonferroni-corrected p value <1.4 × 10(-5)). These sites were located in the genes TERT, SARS, GNGT2, SMG6, SKI, TOM1L2, SIPA1, MRAS, CDKN1A, LRRC2, FES and RPH3A. In 12 sites, current smoking was associated with a 1.2 to 2.4 % lower methylation compared to never smoking; and in three sites, it was associated with a 1.2 to 1.8 % higher methylation. The effect estimates were lower in 10 of the 15 CpG sites when comparing current to former smoking. One CpG site, cg05603985 (SKI), was found to be associated with expression of nearby CAD-related gene PRKCZ. CONCLUSIONS: Our study suggests an effect of tobacco smoking on DNA methylation of CAD-related genes and thus provides novel insights in the pathways that link tobacco smoking to risk of CAD.

Genome-wide association study in an admixed case series reveals IL12A as a new candidate in Behçet disease.

INTRODUCTION: The etiology of Behçet's disease (BD) is unknown, but widely considered an excessive T-cell mediated inflammatory response in a genetically susceptible host. Recent genome-wide association studies (GWAS) have shown limited number of novel loci-associations. The rarity and unequal distribution of the disease prevalence amongst different ethnic backgrounds have hampered the use of GWAS in cohorts of mixed ethnicity and sufficient sample size. However, novel statistical approaches have now enabled GWAS in admixed cohorts. METHODS: We ran a GWAS on 336 BD cases and 5,843 controls. The cases consisted of Western Europeans, Middle Eastern and Turkish individuals. Participants from the Generation R study, a multiethnic birth cohort in Rotterdam, The Netherlands were used as controls. All samples were genotyped and data was combined. Linear regression models were corrected for population stratification using Genomic Principal Components and Linear Mixed Modelling. Meta-analysis was performed on selected results previously published. RESULTS: We identified SNPs associated at genome-wide significant level mapping to the 6p21.33 (HLA) region. In addition to this known signal two potential novel associations on chromosomes 6 and 18 were identified, yet with low minor allele frequencies. Extended meta-analysis reveal a GWS association with the IL12A variant rs17810546 on chromosome 3. DISCUSSION: We demonstrate that new statistical techniques enable GWAS analyses in a limited sized cohort of mixed ethnicity. After implementation, we confirmed the central role of the HLA region in the disease and identified new regions of interest. Moreover, we validated the association of a variant in the IL2A gene by meta-analysis with previous work. These findings enhance our knowledge of genetic associations and BD, and provide further justification for pursuing collective initiatives in genetic studies given the low prevalence of this and other rare diseases.

Homocysteine and DNA methylation: a review of animal and human literature.

Homocysteine (Hcy) is a sulfur-containing non-protein forming amino acid, which is synthesized from methionine as an important intermediate in the one-carbon pathway. High concentrations of Hcy in a condition called hyperhomocysteinemia (HHcy) are an independent risk factor for several disorders including cardiovascular diseases and osteoporotic fractures. Since Hcy is produced as a byproduct of the methyltransferase reaction, alteration in DNA methylation is studied as one of the underlying mechanisms of HHcy-associated disorders. In animal models, elevated Hcy concentrations are induced either by diet (high methionine, low B-vitamins, or both), gene knockouts (Mthfr, Cbs, Mtrr or Mtr) or combination of both to investigate their effects on DNA methylation or its markers. In humans, most of the literature involves case-control studies concerning patients. The focus of this review is to study existing literature on HHcy and its role in relation to DNA methylation. Apart from this, a few studies investigated the effect of Hcy-lowering trials on restoring DNA methylation patterns, by giving a folic acid or B-vitamin supplemented diet. These studies which were conducted in animal models as well as humans were included in this review.