Genome-wide analysis of mitochondrial DNA copy number reveals loci implicated in nucleotide metabolism, platelet activation, and megakaryocyte proliferation.

Mitochondrial DNA copy number (mtDNA-CN) measured from blood specimens is a minimally invasive marker of mitochondrial function that exhibits both inter-individual and intercellular variation. To identify genes involved in regulating mitochondrial function, we performed a genome-wide association study (GWAS) in 465,809 White individuals from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium and the UK Biobank (UKB). We identified 133 SNPs with statistically significant, independent effects associated with mtDNA-CN across 100 loci. A combination of fine-mapping, variant annotation, and co-localization analyses was used to prioritize genes within each of the 133 independent sites. Putative causal genes were enriched for known mitochondrial DNA depletion syndromes (p = 3.09 × 10-15) and the gene ontology (GO) terms for mtDNA metabolism (p = 1.43 × 10-8) and mtDNA replication (p = 1.2 × 10-7). A clustering approach leveraged pleiotropy between mtDNA-CN associated SNPs and 41 mtDNA-CN associated phenotypes to identify functional domains, revealing three distinct groups, including platelet activation, megakaryocyte proliferation, and mtDNA metabolism. Finally, using mitochondrial SNPs, we establish causal relationships between mitochondrial function and a variety of blood cell-related traits, kidney function, liver function and overall (p = 0.044) and non-cancer mortality (p = 6.56 × 10-4).

Serum fatty acid chain length associates with prevalent symptomatic end-stage osteoarthritis, independent of BMI.

Higher body mass index (BMI) is associated with osteoarthritis (OA) in both weight-bearing and non-weight-bearing joints, suggesting a link between OA and poor metabolic health beyond mechanical loading. This risk may be influenced by systemic factors accompanying BMI. Fluctuations in concentrations of metabolites may mark or even contribute to development of OA. This study explores the association of metabolites with radiographic knee/hip OA prevalence and progression. A 1H-NMR-metabolomics assay was performed on plasma samples of 1564 cases for prevalent OA and 2,125 controls collected from the Rotterdam Study, CHECK, GARP/NORREF and LUMC-arthroplasty cohorts. OA prevalence and 5 to 10 year progression was assessed by means of Kellgren-Lawrence (KL) score and the OARSI-atlas. End-stage knee/hip OA (TJA) was defined as indication for arthroplasty surgery. Controls did not have OA at baseline or follow-up. Principal component analysis of 227 metabolites demonstrated 23 factors, of which 19 remained interpretable after quality-control. Associations of factor scores with OA definitions were investigated with logistic regression. Fatty acids chain length (FALen), which was included in two factors which associated with TJA, was individually associated with both overall OA as well as TJA. Increased Fatty Acid chain Length is associated with OA.

Rare, low frequency and common coding variants in CHRNA5 and their contribution to nicotine dependence in European and African Americans.

The common nonsynonymous variant rs16969968 in the α5 nicotinic receptor subunit gene (CHRNA5) is the strongest genetic risk factor for nicotine dependence in European Americans and contributes to risk in African Americans. To comprehensively examine whether other CHRNA5 coding variation influences nicotine dependence risk, we performed targeted sequencing on 1582 nicotine-dependent cases (Fagerström Test for Nicotine Dependence score⩾4) and 1238 non-dependent controls, with independent replication of common and low frequency variants using 12 studies with exome chip data. Nicotine dependence was examined using logistic regression with individual common variants (minor allele frequency (MAF)⩾0.05), aggregate low frequency variants (0.05>MAF⩾0.005) and aggregate rare variants (MAF<0.005). Meta-analysis of primary results was performed with replication studies containing 12 174 heavy and 11 290 light smokers. Next-generation sequencing with 180 × coverage identified 24 nonsynonymous variants and 2 frameshift deletions in CHRNA5, including 9 novel variants in the 2820 subjects. Meta-analysis confirmed the risk effect of the only common variant (rs16969968, European ancestry: odds ratio (OR)=1.3, P=3.5 × 10(-11); African ancestry: OR=1.3, P=0.01) and demonstrated that three low frequency variants contributed an independent risk (aggregate term, European ancestry: OR=1.3, P=0.005; African ancestry: OR=1.4, P=0.0006). The remaining 22 rare coding variants were associated with increased risk of nicotine dependence in the European American primary sample (OR=12.9, P=0.01) and in the same risk direction in African Americans (OR=1.5, P=0.37). Our results indicate that common, low frequency and rare CHRNA5 coding variants are independently associated with nicotine dependence risk. These newly identified variants likely influence the risk for smoking-related diseases such as lung cancer.

ATP5H/KCTD2 locus is associated with Alzheimer's disease risk.

To identify loci associated with Alzheimer disease, we conducted a three-stage analysis using existing genome-wide association studies (GWAS) and genotyping in a new sample. In Stage I, all suggestive single-nucleotide polymorphisms (at P<0.001) in a previously reported GWAS of seven independent studies (8082 Alzheimer's disease (AD) cases; 12 040 controls) were selected, and in Stage II these were examined in an in silico analysis within the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium GWAS (1367 cases and 12904 controls). Six novel signals reaching P<5 × 10(-6) were genotyped in an independent Stage III sample (the Fundació ACE data set) of 2200 sporadic AD patients and 2301 controls. We identified a novel association with AD in the adenosine triphosphate (ATP) synthase, H+ transporting, mitochondrial F0 (ATP5H)/Potassium channel tetramerization domain-containing protein 2 (KCTD2) locus, which reached genome-wide significance in the combined discovery and genotyping sample (rs11870474, odds ratio (OR)=1.58, P=2.6 × 10(-7) in discovery and OR=1.43, P=0.004 in Fundació ACE data set; combined OR=1.53, P=4.7 × 10(-9)). This ATP5H/KCTD2 locus has an important function in mitochondrial energy production and neuronal hyperpolarization during cellular stress conditions, such as hypoxia or glucose deprivation.

A K(ATP) channel gene effect on sleep duration: from genome-wide association studies to function in Drosophila.

Humans sleep approximately a third of their lifetime. The observation that individuals with either long or short sleep duration show associations with metabolic syndrome and psychiatric disorders suggests that the length of sleep is adaptive. Although sleep duration can be influenced by photoperiod (season) and phase of entrainment (chronotype), human familial sleep disorders indicate that there is a strong genetic modulation of sleep. Therefore, we conducted high-density genome-wide association studies for sleep duration in seven European populations (N=4251). We identified an intronic variant (rs11046205; P=3.99 × 10(-8)) in the ABCC9 gene that explains ≈5% of the variation in sleep duration. An influence of season and chronotype on sleep duration was solely observed in the replication sample (N=5949). Meta-analysis of the associations found in a subgroup of the replication sample, chosen for season of entry and chronotype, together with the discovery results showed genome-wide significance. RNA interference knockdown experiments of the conserved ABCC9 homologue in Drosophila neurons renders flies sleepless during the first 3 h of the night. ABCC9 encodes an ATP-sensitive potassium channel subunit (SUR2), serving as a sensor of intracellular energy metabolism.

No modification of the beneficial effect of NSAIDs on colorectal cancer by CYP2C9 genotype.

BACKGROUND: CYP2C9 enzymes are involved in non-steroidal anti-inflammatory drug (NSAID) metabolism. Therefore, we investigated whether CYP2C9*2 and *3 variant alleles, encoding for enzymes with lower activity, increased the protective effect of NSAIDs on colorectal cancer. METHODS: Individual and combined associations of NSAIDs and CYP2C9*2 and *3 variant alleles with colorectal cancer were studied in 7757 Caucasian individuals of The Rotterdam Study, a population-based prospective cohort since 1990. Additive and multiplicative effect modification models were used to examine drug-gene interactions. RESULTS: There were 212 incident cases of colorectal cancer during follow-up. A reduced risk of colorectal cancer was observed in individuals who used NSAIDs for more than a year (HR 0.45; 95% CI 0.28 to 0.71), and in carriers of an CYP2C9 variant allele associated with lower enzymatic activity (HR 0.67; 95% CI 0.47 to 0.96). The combination of both determinants was associated with a further risk reduction but without synergy. CONCLUSION: Both NSAID use and CYP2C9*2 and/ or *3 carriage are associated with a reduced risk of colorectal cancer. However, no interaction between the determinants was found, which might indicate independent pathophysiological mechanisms.

Estrogen receptor alpha polymorphisms and postmenopausal breast cancer risk.

BACKGROUND: The estrogen receptor alpha (ESR1) is a mediator of estrogen response in the breast. The most studied variants in this gene are the PvuII and XbaI polymorphisms, which have been associated to lower sensitivity to estrogen. We evaluated whether these polymorphisms were associated with breast cancer risk by means of an association study in a population of Caucasian postmenopausal women from the Rotterdam study and a meta-analysis of published data. METHODS: The PvuII and XbaI polymorphisms were genotyped in 3,893 women participants of the Rotterdam Study. Baseline information was obtained through a questionnaire. We conducted logistic regression analyses to assess the risk of breast cancer by each of the ESR1 genotypes. Meta-analyses of all publications on these relations were done by retrieving literature from Pubmed and by further checking the reference lists of the articles obtained. RESULTS: There were 38 women with previously diagnosed breast cancer. During follow-up, 152 were additionally diagnosed. The logistic regression analyses showed no difference in risk for postmenopausal breast cancer in carriers of the PvuII or XbaI genotypes neither in overall, incident or prevalent cases. No further evidence of a role of these variants was found in the meta-analysis. CONCLUSIONS: Our results suggest that the ESR1 polymorphisms do not play a role in breast cancer risk in Caucasian postmenopausal women.

IGF-1 CA repeat variant and breast cancer risk in postmenopausal women.

IGF-I is an important growth factor for the mammary gland. We evaluated the relationship of the IGF-I CA(n) polymorphism with breast cancer risk in Caucasian postmenopausal women and performed a meta-analysis of published data. The IGF-I CA(n) polymorphism was genotyped in 4091 from the Rotterdam Study. A disease-free survival analysis was performed along with a meta-analysis of all available data on IGF-I CA(n) polymorphism and breast cancer risk. During follow-up 159 women were diagnosed with breast cancer. The disease-free survival analysis adjusted for age at entry, age at menopause, body mass index and waist hip ratio yielded a HR=0.97 (95% CI=0.59-1.58) for CA(19) non-carriers against carriers. The meta-analysis using the random-effects model gave a pooled OR of 1.26 (95% CI=0.95-1.82) for IGF-I CA(19) non-carriers versus CA(19) homozygous carriers. According to these results, the IGF-I CA(19) promoter polymorphism is not likely to predict the risk of breast cancer.

Plasma amino acids and neopterin in healthy persons with Down's syndrome.

In persons with Down's syndrome (DS) immunological abnormalities as well as hypothyroidism and Alzheimer type dementia are frequently observed. In addition, the activity of the enzyme cystathionine beta-synthase (CBS) is over-expressed which results in an altered homocysteine metabolism. In the present study, 48 older healthy DS persons without signs of dementia, psychiatric or somatic comorbidity and free of medication were analyzed for plasma levels of amino acids, neopterin and monoaminergic metabolites. Data were compared with those obtained from age and sex matched healthy controls. It was found that the spectrum of amino acids showed widespread differences in that levels of nearly all essential amino acids were lower in DS patients as compared to healthy controls. In addition, a significantly lower methionine and higher taurine concentration were observed which is in accordance with a disturbed homocysteine metabolism. With respect to the monoamine metabolites, the concentration of 5-hydroxyindoleacetic acid was not altered whereas that of homovanillic acid was significantly increased. Finally, the concentration of the immune activation marker neopterin was increased in persons with DS. It is concluded that healthy DS persons of older age show extensive biochemical abnormalities suggesting a compromised homocysteine metabolism, an activated cell-mediated immune response and an enhanced turnover of dopamine.

TGF-beta1 polymorphisms and arterial stiffness; the Rotterdam Study.

Arterial stiffness is a risk factor for cardiovascular disease. Transforming growth factor beta1 is a pleiotropic cytokine, with many functions, including influence on the vascular wall (e.g., on angiogenesis, endothelial cells and the extracellular matrix). We investigated five functional polymorphisms in the transforming growth factor beta1 gene (-800 G/A, -509 C/T, codon 10 Leu/Pro, codon 25 Arg/Pro and codon 263 Thr/Ile) in relation to arterial stiffness in a population-based study. A total of 3863 participants of the Rotterdam Study, a prospective population-based study, were included in the current study. The relations of the genotypes and haplotypes with arterial stiffness (pulse wave velocity (PWV), distensibility coefficient (DC) and pulse pressure (PP)) were studied using analyses of variance and linear regression. The analyses were adjusted for age, sex, mean arterial pressure, heart rate, conventional cardiovascular risk factors and measures of atherosclerosis. There were no associations between PWV and -800 G/A (P=0.56), -509 C/T (P=0.29), codon 10 (P=0.98) and, codon 25 (P=0.28). These polymorphisms were not associated with the DC or with PP. The haplotype-based analyses yielded similar results. The results of this study show that the TGF-beta1 -800 G/A, -509 C/T, codon 10 Leu/Pro and codon 25 Arg/Pro polymorphisms are not associated with arterial stiffness.

Differential roles of Angiotensinogen and Angiotensin Receptor type 1 polymorphisms in breast cancer risk.

While angiotensinogen (AGT) seems to have anti proliferative properties, angiotensin II (ATII) is a potent growth factor and it mediates its actions through the angiotensin type 1 receptor (AGTR1). In the AGT gene, the M235T polymorphism has been associated with the variation in angiotensinogen levels and in the AGTR1 gene; the C573T variant is associated with different pathologies. We aimed to evaluate the relationship of these two variants and the risk of breast cancer. These polymorphisms were genotyped in 3787 women participating the Rotterdam Study. We performed a logistic regression and a disease free survival analysis by genotype. The logistic regression yielded an odds ratio of 1.4 (95% CI: 1.1-1.9) for the MM genotype carriers versus the T allele carriers. The breast cancer free survival by AGT genotype was significantly reduced in MM genotype carriers compared to non-carriers (hazard ratio (HR) = 1.5; 95% CI: 1.1-2.2). We did not find any association of the AGTR1 polymorphism and breast cancer risk or disease free survival. Our results suggest that AGT plays a role in breast cancer risk in postmenopausal women, whereas the role of AGTR1 needs further studying.

Transforming-growth factor beta1 Leu10Pro polymorphism and breast cancer morbidity.

TGF-beta1 has a dual role in carcinogenesis. In this gene, a leucine to proline substitution in codon 10 leads to higher circulating levels of TGF-beta1. This variant has been studied in relationship to the risk for breast cancer yielding contradicting results. We aim to unravel the relationship of this polymorphism and the risk of breast cancer. Women participating in the Rotterdam Study including 143 patients with incident breast cancer were genotyped for this polymorphism. We carried out a logistic regression and a survival analysis using age as the time variable. The logistic regression analysis showed an increased risk of breast cancer for Proline carriers (OR=1.4; 95% confidence interval (CI)=1.1-2.0) versus non-carriers. The survival analysis showed that carriers of the same allele had an increased risk of breast cancer (HR=1.4, 95% CI=1.1-2.0) against non-carriers. Our data suggest that the TGF-beta1 Leu10Pro polymorphism might play a role in breast cancer risk.

Heritability of serum iron, ferritin and transferrin saturation in a genetically isolated population, the Erasmus Rucphen Family (ERF) Study.

BACKGROUND: Iron has been implicated in the pathogenesis of various disorders. Mutations in the HFE gene are associated with an increase in serum iron parameters. The aim of this study was to estimate the heritability in serum iron parameters explained by HFE. METHODS: Ninety families (980 subjects) were included in the present analysis. Heritability estimation was conducted using the variance component method. The likelihood ratio test was used to compare models. Phenotypic and genetic correlations between serum iron parameters were calculated. RESULTS: The heritability (h(2) +/- SE) estimates were 0.23 +/- 0.07 (p < 0.0001) for iron, 0.29 +/- 0.09 (p < 0.0001) for ferritin and 0.28 +/- 0.07 (p < 0.0001) for transferrin saturation while adjusting for age, age(2) and sex. The HFE genotypes explained between 2 to 6% of the sex and age-adjusted variance in serum iron, ferritin and transferrin saturation. There was a high genetic correlation between serum iron parameters, suggesting pleiotropy between these traits. CONCLUSION: A substantial proportion of the variance of iron, ferritin and transferrin saturation can be explained by additive genetic effects, independent of sex and age. The HFE genotypes explained a considerable proportion of serum iron parameters and may be an important factor in the complex iron network.

Interleukin 6 G-174 C polymorphism and breast cancer risk.

Interleukin-6 (IL-6) is a growth factor involved in many processes including carcinogenesis. The C allele of the G-174 C promoter single nucleotide polymorphism (SNP) in the IL-6 gene decreases levels of IL-6 expression and it has been studied in the context of breast cancer progression yielding contradicting results. Furthermore a recent study found that carriers of the C allele were at an increased risk for this disease. We aim to evaluate the association between this variant and breast cancer risk in Caucasian postmenopausal women. Women participating in the Rotterdam Study (N=3822), including 171 patients with breast cancer were genotyped for this polymorphism. In order to assess the relationship between this SNP and breast cancer we carried out a logistic regression in relation to the incidence of breast cancer. The C allele frequency was 41.3% and the genotypes followed Hardy-Weinberg distribution (p=0.3). The logistic regression analysis showed a slight increase of risk for C allele carriers (odds ratio=1.24, 95% CI: 0.8-1.9), compared to non-carriers of this allele. This increased risk was not statistically significant. Our data suggest that the IL-6 G-174 C polymorphism does not seem to play a role in breast cancer risk, although its role as a prognostic factor remains to be studied.

An insulin-like growth factor-I gene polymorphism modifies the risk of microalbuminuria in subjects with an abnormal glucose tolerance.

OBJECTIVE: Microalbuminuria (MA) is related to cardiovascular disease both in diabetic patients and non-diabetic subjects. DESIGN: We investigated whether a polymorphism near the promoter region of the IGF-I gene was related to the development of MA. METHODS: For this study, 1069 participants of the Rotterdam study were selected (440 participants with an abnormal glucose tolerance (AGT), 220 participants with type 2 diabetes and 254 subjects with pre-diabetes, and 595 subjects with a normal glucose tolerance (NGT). RESULTS: 787 subjects were carriers of the wild type IGF-I genotype (73.6%) and 282 subjects were variant carriers (26.4%) of this IGF-I gene polymorphism. Compared to subjects with NGT the risk for microalbuminuria was higher (Odds Ratio (OR): 3.1 (95% CI: 1.2-7.7); P = 0.02) in variant carriers with AGT than in carriers of the wild type of this IGF-I gene polymorphism (OR: 2.2 (95% CI: 1.2-4.0); P = 0.009). Compared with wild type carriers with AGT, the relative risk for MA was unadjusted and non-significantly increased in variant carriers with AGT (1.6; 95% CI: 0.8-2.9). However, after adjustment for possible confounding factors (age, gender, mean blood pressure, fasting insulin, fasting glucose and smoking) this risk became significant (OR: RR 2.1; 95% CI:1.1-4.4; P = 0.04). CONCLUSIONS: In subjects with AGT, a higher risk for MA was observed in variant carriers than in carriers of the wild type genotype of this IGF-I gene polymorphism. Since MA is primarily associated with cardiovascular disease in subjects with AGT, our study suggests that variant carriers have a higher risk for cardiovascular disease than carriers of the wild type when they develop an AGT.

Stable incidence of childhood and adult glioma in The Netherlands, 1989-2003.

Time trends in the incidence of glioma may reflect changes in the prevalence of environmental risk factors for glioma. We therefore investigated trends in the incidence of childhood and adult glioma in The Netherlands from 1989 to 2003. We used population-based incidence data from the Netherlands Cancer Registry. We calculated European standardised incidence rates for glioma, and stratified for age, gender and glioma subgroups. Changes in the incidence were estimated by calculating the Estimated Annual Percentage Change. Similar to other countries, the overall incidence of glioma was fairly stable in The Netherlands during the period 1989 to 2003, for both children and adults. In adult astrocytic glioma, a significantly increasing incidence of high-grade astrocytoma was balanced by simultaneous decreases of low-grade astrocytoma, astrocytoma with unknown malignancy grade and glioma of uncertain histology. Most of these time trends can be explained by improving detection and diagnostic precision. Stable incidence rates of adult and childhood glioma suggest that no major changes in environmental risk factors have occurred, which influenced the incidence of glioma in the studied period.

Space-time clustering of glioma cannot be attributed to specific histological subgroups.

We previously showed that infectious exposures may be involved in the aetiology of adult glioma, by analysing for space-time clustering using population-based data from the South of the Netherlands. Here we extended these analyses and describe in detail the space-time clustering patterns in glioma subgroups, gender and age-categories. Knox tests for space-time interactions between cases were applied with fixed thresholds of close in space, <5 km, and close in time, <1 year apart. We used the spatial coordinates of the addresses at diagnosis in the analyses. Tests were repeated replacing geographical distance with distance to the Nth nearest neighbour. N was chosen such that the mean distance was 5 km. Data were also analysed by a second order procedure based on K-functions. There was only statistically significant space-time clustering for oligodendroglioma. Clustering was present for adults aged 30-54 years and was more pronounced among males. Given the low prior probability of an infectious aetiology for this specific subgroup, these results should probably be interpreted as false-positive. We conclude that space-time clustering of glioma cannot be attributed to a specific glioma subgroup. The observed clustering in our previous study is therefore probably an overall effect within and between glioma subgroups.

The association between antihypertensive drugs and glioma.

We pursued an association between hypertension and gliomas by investigating whether antihypertensive drugs (AHD) are associated with an increased glioma risk by a population-based nested case-control study using the PHARMO database; this links dispensing records of prescription drugs to hospital discharge data on an individual basis. Pathological data were derived from the Dutch nationwide registry of histo- and cytopathology. A total of 306 glioma cases incident between 1997 and 2003 were matched to 1108 controls for year of birth, sex, geographical region and duration of follow-up. Exposure was defined as cumulative duration of AHD use and, in an alternative analysis, as cumulative dose. We estimated the magnitude of the association with conditional logistic regression analysis. Cumulative use of any AHD for more than 6 months was associated with an increased risk of glioma (OR 1.45; 95% CI 1.03-2.04). After stratification for different groups of AHD, no significantly increased risk of glioma was found for any class of AHD. After excluding a latency period of 3 years before the date of diagnosis, no association was found. In conclusion, the use of AHD seems to be associated with an increased risk of glioma, but this is probably not causal.

Space-time clustering patterns of gliomas in The Netherlands suggest an infectious aetiology.

To test the hypothesis that infectious exposures may be involved in glioma aetiology, we have analysed space-time clustering and seasonal variation using population-based data from the South of The Netherlands between 1983 and 2001. Knox tests for space-time interactions between cases were applied, with spatial coordinates of the addresses at time of diagnosis, and with distance to the Nth nearest neighbour. Data were also analysed by a second order procedure based on K-functions. Tests for heterogeneity and Edwards' test for sinusoidal variation were applied to examine seasonal variation of incidence. There was statistically significant space-time clustering in the Eastern, but not in the Western part of the region. Clustering was only present in adults, particularly in less densely populated areas. There was no evidence for seasonal variation. The results support a role for infectious exposures in glioma aetiology that may act preferentially in certain geographical areas.

[Gliomas: the role of environmental risk factors and genetic predisposition]. / Gliomen: de rol van omgevingsfactoren en genetische predispositie.

Exposure to ionising radiation is the only established risk factor for glioma. Although gliomas are looked upon as a non-heritable disease, physicians regularly see patients with affected relatives in practice. A few monogenetic tumour syndromes explain < 5% of all gliomas: neurofibromatosis type 1 and 2, Li-Fraumeni syndrome, tuberous sclerosis, Turcot syndrome, Gorlin syndrome, and the melanomaastrocytoma syndrome. Aggregation ofgliomas in families without these tumour syndromes also occurs. The overall familial pattern is generally atypical for hereditary cancers. Relatives are estimated to have a 2- to 9-fold increased risk. The occurrence ofgliomas in families can probably best be explained by a multifactorial model: environmental risk factors with a genetically determined susceptibility for these risk factors. The identity of the genetic variants leading to this predisposition remains to be determined. When there are indications of a hereditary tumour syndrome, additional diagnostic investigation is indicated.