Two new Loci for body-weight regulation identified in a joint analysis of genome-wide association studies for early-onset extreme obesity in French and german study groups.

Meta-analyses of population-based genome-wide association studies (GWAS) in adults have recently led to the detection of new genetic loci for obesity. Here we aimed to discover additional obesity loci in extremely obese children and adolescents. We also investigated if these results generalize by estimating the effects of these obesity loci in adults and in population-based samples including both children and adults. We jointly analysed two GWAS of 2,258 individuals and followed-up the best, according to lowest p-values, 44 single nucleotide polymorphisms (SNP) from 21 genomic regions in 3,141 individuals. After this DISCOVERY step, we explored if the findings derived from the extremely obese children and adolescents (10 SNPs from 5 genomic regions) generalized to (i) the population level and (ii) to adults by genotyping another 31,182 individuals (GENERALIZATION step). Apart from previously identified FTO, MC4R, and TMEM18, we detected two new loci for obesity: one in SDCCAG8 (serologically defined colon cancer antigen 8 gene; p = 1.85x10(-8) in the DISCOVERY step) and one between TNKS (tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase gene) and MSRA (methionine sulfoxide reductase A gene; p = 4.84x10(-7)), the latter finding being limited to children and adolescents as demonstrated in the GENERALIZATION step. The odds ratios for early-onset obesity were estimated at approximately 1.10 per risk allele for both loci. Interestingly, the TNKS/MSRA locus has recently been found to be associated with adult waist circumference. In summary, we have completed a meta-analysis of two GWAS which both focus on extremely obese children and adolescents and replicated our findings in a large followed-up data set. We observed that genetic variants in or near FTO, MC4R, TMEM18, SDCCAG8, and TNKS/MSRA were robustly associated with early-onset obesity. We conclude that the currently known major common variants related to obesity overlap to a substantial degree between children and adults.

Coronary artery calcification and its relationship to validated genetic variants for diabetes mellitus assessed in the Heinz Nixdorf recall cohort.

OBJECTIVE: To examine the association between genomewide association study-based diabetes mellitus-related single-nucleotide polymorphisms (SNPs) and coronary artery calcification (CAC), a valid risk factor for coronary heart disease, in a large, unselected, population-based cohort. METHODS AND RESULTS: We genotyped 11 validated genomewide association study-based diabetes SNPs in 4459 participants of the Heinz Nixdorf Recall Study. We applied generalized linear regression models to explore the impact of the diabetes SNPs on CAC and to jointly model the effect of the SNPs and CAC on diabetes status. We observed a significant association between cyclin-dependent kinase inhibitor 2A/2B (CDKN2A/2B) variant rs564398 and quantitative CAC (P=1.81 x 10(-5) and adjusted P=4.02 x 10(-4); odds ratio for the presence of CAC, 1.12 [95% CI, 1.02 to 1.25]). Moreover, we observed no strong impact of CAC on diabetes risk in the presence of the other genetic variants. CONCLUSIONS: We show that a genetic variant near CDKN2A/2B that has been reported to be strongly associated with diabetes is strongly associated with CAC. In contrast, variants near insulin-like growth factor-binding protein 2 (IGFBP2), CDK5 regulatory subunit associated protein 1-like 1 (CDKAL1), solute carreir family 30 (zinc transporter), member 8 (SLC30A8), hematopoietically-expressed homeobox (HHEX), and transcription factor 7-like2 (TCF7L2) were clearly associated with diabetes; no evidence for an association to CAC was observable. This differential association pattern underlines the potential of endophenotypes, such as CAC, to extend the scope of disease outcome associations.

Relationship between MTNR1B (melatonin receptor 1B gene) polymorphism rs10830963 and glucose levels in overweight children and adolescents.

AIMS: The G-allele of the single nucleotide polymorphism (SNP) rs10830963 in MTNR1B (melatonin receptor 1B gene) is associated with type 2 diabetes mellitus and glucose levels in adults. The aim of this study was to analyze whether there is an allele-dosage effect on glucose metabolism in overweight children and to explore if changes in glucose metabolism in a lifestyle intervention do also depend on genotype. METHODS: We genotyped rs10830963 in 1118 overweight children and adolescents [mean age 10.7 yr, mean body mass index (BMI) 27.8 kg/m2]; 340 of these individuals completed a 1-yr lifestyle intervention (mean age 10.7 yr, mean BMI 27.9 kg/m2). The degree of overweight [BMI-SDS (standard deviation score)], fasting insulin, glucose, homeostasis model assessment for insulin resistance (HOMA-IR), and quantitative insulin sensitivity check index (QUICKI) were measured before and after intervention. RESULTS: We showed a significant relationship between rs10830963 and basal glucose levels [ß:1.101, 95% confidence interval (CI) 0.316-1.886 mg/dL per risk allele; p = 0.006] by linear regression adjusted for age, age(2), and sex. There was no effect of the allele on insulin or indices of insulin resistance or sensitivity. After the 1-yr lifestyle intervention, we observed a significant reduction of BMI-SDS as well as an improvement of HOMA-IR and QUICKI, but no evidence for an association between rs10830963 genotype and changes of glucose levels. CONCLUSIONS: The G-allele of rs10830693 in the MTNR1B gene was significantly related to glucose levels, while an impact of this genetic variant on the changes in glucose metabolism in children participating in a lifestyle intervention was not observable.

Single nucleotide polymorphisms of the serotonin transporter gene in migraine--an association study.

We report a case-control study with 251 unrelated migraine patients and 192 unrelated healthy controls to evaluate an association between the polymorphisms of the 5-HT transporter (5-HTT) gene rs2066713 and rs1979572 and different migraine phenotypes. We found a genetic association for the A allele of rs1979572 for migraine with aura (MA) especially in women as well as a significant lower prevalence for MA for carrier of the A allele of rs2066713 in women. These findings support previous results suggesting that the 5-HTT gene is involved in the polygenic etiology of MA. These data further suggest that women are more likely to be clinically affected by mutations in the 5-HTT gene than men.

New resampling method for evaluating stability of clusters.

BACKGROUND: Hierarchical clustering is a widely applied tool in the analysis of microarray gene expression data. The assessment of cluster stability is a major challenge in clustering procedures. Statistical methods are required to distinguish between real and random clusters. Several methods for assessing cluster stability have been published, including resampling methods such as the bootstrap. We propose a new resampling method based on continuous weights to assess the stability of clusters in hierarchical clustering. While in bootstrapping approximately one third of the original items is lost, continuous weights avoid zero elements and instead allow non integer diagonal elements, which leads to retention of the full dimensionality of space, i.e. each variable of the original data set is represented in the resampling sample. RESULTS: Comparison of continuous weights and bootstrapping using real datasets and simulation studies reveals the advantage of continuous weights especially when the dataset has only few observations, few differentially expressed genes and the fold change of differentially expressed genes is low. CONCLUSION: We recommend the use of continuous weights in small as well as in large datasets, because according to our results they produce at least the same results as conventional bootstrapping and in some cases they surpass it.

Increased cause-specific mortality associated with 2003 heat wave in Essen, Germany.

During the 2003 heat wave an increase in mortality was observed in several European countries. Evidence suggests that the heat wave effect on mortality varies based upon underlying disease. In this study we examined the effects of the 2003 heat wave on all-cause and cause-specific mortality (neoplasms, cardiovascular and respiratory diseases) in a large west German city. Daily weather data for Essen was obtained from the German meteorological service. Death certificates for all deaths in Essen from 2002 to 2003 were coded according to the World Health Organization (WHO) guidelines. Mean numbers of daily deaths during and after the heat wave were compared with the average mortality in summer months (reference period). Poisson generalized additive models, adjusted for weekday and season, were fitted for overall and cause-specific mortality for the entire study period. During the 2003 heat wave (August 6-12), daily mortality increased by 15% (neoplasms), 30% (cardiovascular), and 61% (respiratory), with a decrease in the week after the heat wave of 17% for neoplasms and a sustained rise for respiratory mortality (77%). Regression analysis showed an association between heat and overall mortality in 2003 and greatest associations for respiratory mortality. Even the comparatively short heat wave in Essen in the year 2003 was associated with a rise in overall and cause-specific mortality. Different mechanisms appear to influence cause-specific mortality, with strongest associations for respiratory mortality. Harvesting might play a role in mortality due to neoplasms.

Prevalence of pulmonary hypertension associated with severe acute respiratory distress syndrome: predictive value of computed tomography.

OBJECTIVE: To evaluate the prevalence of pulmonary hypertension associated with severe acute respiratory distress syndrome (ARDS) and to asses the value of pulmonary artery trunk diameter (PAT) to predict pulmonary hypertension. DESIGN: Prospective study SETTING: University teaching hospital and ARDS referral center. PATIENTS: 103 patients with ARDS, who received both right heart catheterization and chest computed tomography. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: 95 patients (92.2%) with ARDS had pulmonary artery hypertension, 16 of them (16.8%) mild, 72 (75.8%) moderate, and 7 (7.4%) severe, as assessed by right heart catheterization. Of the patients with moderate and severe pulmonary hypertension, 43 had a pulmonary artery trunk diameter >or=29 mm yielding a sensitivity of 0.54 and a specificity of 0.63. Pulmonary artery trunk diameter correlated significantly but weakly with mean pulmonary artery pressure (r=0.34, p=0.0004). The positive predictive value was 0.83, and the negative predictive value was 0.28. The diagnosis of pulmonary hypertension by PAT diameter measurements was incorrect in 43.7% of patients with ARDS. CONCLUSIONS: Pulmonary artery hypertension has a high prevalence in patients with severe ARDS. Measurement of PAT diameter on admission CT scan is an unreliable tool for identification of ARDS patients with pulmonary hypertension.

Treatment of severe acute respiratory distress syndrome: role of extracorporeal gas exchange.

OBJECTIVE: To evaluate the effect of extracorporeal gas exchange (ECMO) on mortality of patients referred with severe acute respiratory distress syndrome (ARDS). DESIGN AND SETTING: Prospective observational study in a university hospital ICU. PATIENTS: 150 patients with severe ARDS. INTERVENTIONS: Multimodal treatment with and without ECMO. MEASUREMENTS AND MAIN RESULTS: We treated 118 patients (78.7%) conservatively and 32 patients with ECMO. Patients in the ECMO group presented with significantly more severe disease (lung injury score 3.8+/-0.3 vs. 3.3+/-0.4; SOFA score 52+/-14 vs. 43+/-12; and SAPS score 14+/-3.3 vs. 10+/-3.5). Mortality in ECMO-treated patients tended to be higher than that with conservative treatment (46.9% vs. 28.8%, p=0.059). Multivariate logistic regression analyses with backward selection excluded ECMO as predictor of mortality (p=0.79). Independent predictors of mortality were age (odds ratio 1.044, 95% confidence interval 1.014-1.075, p=0.004), mean pulmonary artery pressure (1.082, 1.026-1.141, p=0.036), sequential organ failure assessment score (1.148, 1.018-1.294, p=0.024), and days of mechanical ventilation prior to referral (1.064, 1.008-1.123, p=0.025). CONCLUSION: ECMO treatment does not predict mortality in patients with most severe ARDS.

Two-part permutation tests for DNA methylation and microarray data.

BACKGROUND: One important application of microarray experiments is to identify differentially expressed genes. Often, small and negative expression levels were clipped-off to be equal to an arbitrarily chosen cutoff value before a statistical test is carried out. Then, there are two types of data: truncated values and original observations. The truncated values are not just another point on the continuum of possible values and, therefore, it is appropriate to combine two statistical tests in a two-part model rather than using standard statistical methods. A similar situation occurs when DNA methylation data are investigated. In that case, there are null values (undetectable methylation) and observed positive values. For these data, we propose a two-part permutation test. RESULTS: The proposed permutation test leads to smaller p-values in comparison to the original two-part test. We found this for both DNA methylation data and microarray data. With a simulation study we confirmed this result and could show that the two-part permutation test is, on average, more powerful. The new test also reduces, without any loss of power, to a standard test when there are no null or truncated values. CONCLUSION: The two-part permutation test can be used in routine analyses since it reduces to a standard test when there are positive values only. Further advantages of the new test are that it opens the possibility to use other test statistics to construct the two-part test and that it avoids the use of any asymptotic distribution. The latter advantage is particularly important for the analysis of microarrays since sample sizes are usually small.

SDCCAG8 obesity alleles and reduced weight loss after a lifestyle intervention in overweight children and adolescents.

Genome-wide association analyses (GWAS) contributed to the detection of a number of single-nucleotide polymorphisms (SNPs) associated with obesity. However, little is known about the impact of the obesity-risk alleles on weight loss-related phenotypes after lifestyle interventions. A recent meta-analysis of GWAS reported five genomic loci near or in the genes FTO, MC4R, TMEM18, SDCCAG8, TNKS/MSRA that were associated with obesity in children and adolescents. Here, we analyzed the effect of the 10 SNPs representative of the five loci on measures of weight loss and cardiometabolic risk after a 1-year lifestyle intervention in 401 children and adolescents (mean age 10.74 years; 55.4% female; mean BMI 27.42 kg/m(2), mean BMI-standard deviation score (SDS) 2.37). For confirmation of one locus genotyping of three intronic SNPs in SDCCAG8 was performed in 626 obese adults who completed the 10-week hypoenergetic diet program. Intronic variants of SDCCAG8, which are associated with early onset obesity, are associated with reduced weight loss after a 1-year lifestyle intervention in overweight children and adolescents even after adjusting for age, sex, baseline measurement, or multiple testing (all P < 10(-6)). However, our results could not be confirmed in 626 obese adults undertaking a hypoenergetic diet intervention.

Common variants near MC4R: exploring gender effects in overweight and obese children and adolescents participating in a lifestyle intervention.

OBJECTIVE: Association with obesity and increased insulin levels have been reported for two variants (rs17782313 and rs12970134) located downstream of the melanocortin-4 receptor gene (MC4R). METHODS: We investigated whether these variants have sex-specific effects on overweight, obesity and 14 related phenotypes in 889 overweight and obese children and adolescents. We also explored the impact of the variants on weight change in 367 of the 889 cases who participated in an intervention program. Prior to these analyses we showed that both variants were associated with overweight/obesity in the analyzed 889 cases versus 442 normal-weight and lean controls (case-control study). RESULTS: In explorative analyses we observed higher diastolic blood pressure levels in males (rs17782313: ß = 2.52 mm Hg per risk allele; p = 0.003) but reduced blood pressure level in females for the same risk allele (ß = -1.72 mm Hg; p = 0.039). We also detected a greater BMI standard deviation score (BMI-SDS) reduction in females with the risk allele at rs17782313 (ß = 0.086 per risk allele; p = 0.021). Additionally, we observed evidence for an association of the same risk allele with insulin levels (ß = 0.029 log (µU/ml); p = 0.044) with no sex-specific effect. For the remaining 11 phenotypes, we observed no evidence for a (sex-specific) association. CONCLUSIONS: In sum, our data support the associations of variants rs17782313 and rs12970134 near MC4R with early onset obesity and increased insulin levels. Exploratory evidence for sex-specific effects of the risk alleles were observed for diastolic blood pressure and BMI-SDS reduction.

Gene dosage effects in chronic lymphocytic leukemia.

To understand the influence of chromosomal alterations on gene expression in a genome-wide view, chromosomal imbalances detected by single nucleotide polymorphism (SNP) chips were compared with global gene expression in 16 cases of chronic lymphocytic leukemia (CLL). A strong concordance between chromosomal gain or loss and increased or reduced expression of genes in the affected regions was found, respectively. Regions of uniparental disomy (UPD) were rare and had usually no consistent influence on gene expression, but in one instance, a large UPD was associated with a downregulation of most genes in the affected chromosome. The frequently deleted miRNAs, MIRN15A and MIRN16-1, did not show a reduced expression in cases with monoallelic deletions. The BCL2 protein, considered to be downregulated by these miRNAs, was upregulated not only in CLL with biallelic deletion of MIRN15A and MIRN16-1, but also in cases with monoallelic deletion. This suggests a complex regulation of BCL2 levels in CLL cells. Taken together, in CLL, a global gene dosage effect exists for chromosomal gains and deletions and in some instances for UPDs. We did not confirm a consistent correlation between MIRN15A and MIRN16-1 expression levels and BCL2 protein levels, indicating a complex regulation of BCL2 expression.

Gene expression profiles in murine hematopoietic stem cells revisited: analysis of cDNA libraries reveals high levels of translational and metabolic activities.

Gene expression studies from hematopoietic stem cell (HSC) populations purified to variable degrees have defined a set of stemness genes. Unexpectedly, results also hinted toward a HSC chromatin poised in a wide-open state. With the aim of providing a robust tool for further studies into the molecular biology of HSCs, the studies herein describe the construction and comparative molecular analysis of lambda-phage cDNA libraries from highly purified HSCs that retained their long-term repopulating activities (long-term HSCs [LT-HSCs]) and from short-term repopulating HSCs that were largely depleted of these activities. Microarray analysis of the libraries confirmed the previous results but also revealed an unforeseen preferential expression of translation- and metabolism-associated genes in the LT-HSCs. Therefore, these data indicate that HSCs are quiescent only in regard of proliferative activities but are in a state of readiness to provide the metabolic and translational activities required after induction of proliferation and exit from the HSC pool.

Combining DNA expression with positional information to detect functional silencing of chromosomal regions.

MOTIVATION: Loss of chromosomal material is often observed in cancer cells. In this situation the expression of genes is related to their position on the genome. Epigenetic phenomena may also silence several genes in the same region of a chromosome. While cytogenetic or other molecular genetic methods spot changes of DNA copy number, they cannot detect other causes of silencing. RESULTS: We propose a method that utilizes the link from expression information gained from high-density DNA microarrays to the gene locus according to current databases. Statistical methods adequate to spot conspicuous runs of non-expressed genes are introduced and compared to one another by merit of their power and robustness against false positives. AVAILABILITY: Code for the formulae can be obtained (R code) via http://www.panix.com/~derwisch/hannes/longrun