Marcel Bruinenberg

Obesity is heritable and predisposes to many diseases. To understand the genetic basis of obesity better, here we conduct a genome-wide association study and Metabochip meta-analysis of body mass index (BMI), a measure commonly used to define obesity and assess adiposity, in up to 339,224 individuals. This analysis identifies 97 BMI-associated loci (P < 5 × 10(-8)), 56 of which are novel. Five loci demonstrate clear evidence of several independent association signals, and many loci have significant effects on other metabolic phenotypes. The 97 loci account for ∼2.7% of BMI variation, and genome-wide estimates suggest that common variation accounts for >20% of BMI variation. Pathway analyses provide strong support for a role of the central nervous system in obesity susceptibility and implicate new genes and pathways, including those related to synaptic function, glutamate signalling, insulin secretion/action, energy metabolism, lipid biology and adipogenesis.

Using genome-wide data from 253,288 individuals, we identified 697 variants at genome-wide significance that together explained one-fifth of the heritability for adult height. By testing different numbers of variants in independent studies, we show that the most strongly associated ∼2,000, ∼3,700 and ∼9,500 SNPs explained ∼21%, ∼24% and ∼29% of phenotypic variance. Furthermore, all common variants together captured 60% of heritability. The 697 variants clustered in 423 loci were enriched for genes, pathways and tissue types known to be involved in growth and together implicated genes and pathways not highlighted in earlier efforts, such as signaling by fibroblast growth factors, WNT/β-catenin and chondroitin sulfate-related genes. We identified several genes and pathways not previously connected with human skeletal growth, including mTOR, osteoglycin and binding of hyaluronic acid. Our results indicate a genetic architecture for human height that is characterized by a very large but finite number (thousands) of causal variants.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10−8). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms. Genome-wide association meta-analyses of waist-to-hip ratio adjusted for body mass index in more than 224,000 individuals identify 49 loci, 33 of which are new and many showing significant sexual dimorphism with a stronger effect in women; pathway analyses implicate adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution. In the first of a pair of Articles in this issue from the GIANT Consortium, genome-wide association meta-analyses of waist and hip circumference-related traits in more than 200,000 individuals have been used to identify 49 loci — 33 of them new — associated with waist-to-hip ratio adjusted for body mass index and an additional 19 loci associated with related waist and hip circumference measures. A subset of these loci shows significant sexual dimorphism, with many showing a stronger effect in women. Analyses implicate adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms and offer potential targets for interventions in the risks associated with abdominal fat accumulation.

Through genome-wide association meta-analyses of up to 133,010 individuals of European ancestry without diabetes, including individuals newly genotyped using the Metabochip, we have increased the number of confirmed loci influencing glycemic traits to 53, of which 33 also increase type 2 diabetes risk (q < 0.05). Loci influencing fasting insulin concentration showed association with lipid levels and fat distribution, suggesting impact on insulin resistance. Gene-based analyses identified further biologically plausible loci, suggesting that additional loci beyond those reaching genome-wide significance are likely to represent real associations. This conclusion is supported by an excess of directionally consistent and nominally significant signals between discovery and follow-up studies. Functional analysis of these newly discovered loci will further improve our understanding of glycemic control.

Our genome-wide association study of celiac disease previously identified risk variants in the IL2-IL21 region. To identify additional risk variants, we genotyped 1,020 of the most strongly associated non-HLA markers in an additional 1,643 cases and 3,406 controls. Through joint analysis including the genome-wide association study data (767 cases, 1,422 controls), we identified seven previously unknown risk regions (P < 5 x 10(-7)). Six regions harbor genes controlling immune responses, including CCR3, IL12A, IL18RAP, RGS1, SH2B3 (nsSNP rs3184504) and TAGAP. Whole-blood IL18RAP mRNA expression correlated with IL18RAP genotype. Type 1 diabetes and celiac disease share HLA-DQ, IL2-IL21, CCR3 and SH2B3 risk regions. Thus, this extensive genome-wide association follow-up study has identified additional celiac disease risk variants in relevant biological pathways.

Approaches exploiting trait distribution extremes may be used to identify loci associated with common traits, but it is unknown whether these loci are generalizable to the broader population. In a genome-wide search for loci associated with the upper versus the lower 5th percentiles of body mass index, height and waist-to-hip ratio, as well as clinical classes of obesity, including up to 263,407 individuals of European ancestry, we identified 4 new loci (IGFBP4, H6PD, RSRC1 and PPP2R2A) influencing height detected in the distribution tails and 7 new loci (HNF4G, RPTOR, GNAT2, MRPS33P4, ADCY9, HS6ST3 and ZZZ3) for clinical classes of obesity. Further, we find a large overlap in genetic structure and the distribution of variants between traits based on extremes and the general population and little etiological heterogeneity between obesity subgroups.

To identify genetic factors contributing to type 2 diabetes (T2D), we performed large-scale meta-analyses by using a custom ∼50,000 SNP genotyping array (the ITMAT-Broad-CARe array) with ∼2000 candidate genes in 39 multiethnic population-based studies, case-control studies, and clinical trials totaling 17,418 cases and 70,298 controls. First, meta-analysis of 25 studies comprising 14,073 cases and 57,489 controls of European descent confirmed eight established T2D loci at genome-wide significance. In silico follow-up analysis of putative association signals found in independent genome-wide association studies (including 8,130 cases and 38,987 controls) performed by the DIAGRAM consortium identified a T2D locus at genome-wide significance (GATAD2A/CILP2/PBX4; p = 5.7 × 10−9) and two loci exceeding study-wide significance (SREBF1, and TH/INS; p < 2.4 × 10−6). Second, meta-analyses of 1,986 cases and 7,695 controls from eight African-American studies identified study-wide-significant (p = 2.4 × 10−7) variants in HMGA2 and replicated variants in TCF7L2 (p = 5.1 × 10−15). Third, conditional analysis revealed multiple known and novel independent signals within five T2D-associated genes in samples of European ancestry and within HMGA2 in African-American samples. Fourth, a multiethnic meta-analysis of all 39 studies identified T2D-associated variants in BCL2 (p = 2.1 × 10−8). Finally, a composite genetic score of SNPs from new and established T2D signals was significantly associated with increased risk of diabetes in African-American, Hispanic, and Asian populations. In summary, large-scale meta-analysis involving a dense gene-centric approach has uncovered additional loci and variants that contribute to T2D risk and suggests substantial overlap of T2D association signals across multiple ethnic groups. To identify genetic factors contributing to type 2 diabetes (T2D), we performed large-scale meta-analyses by using a custom ∼50,000 SNP genotyping array (the ITMAT-Broad-CARe array) with ∼2000 candidate genes in 39 multiethnic population-based studies, case-control studies, and clinical trials totaling 17,418 cases and 70,298 controls. First, meta-analysis of 25 studies comprising 14,073 cases and 57,489 controls of European descent confirmed eight established T2D loci at genome-wide significance. In silico follow-up analysis of putative association signals found in independent genome-wide association studies (including 8,130 cases and 38,987 controls) performed by the DIAGRAM consortium identified a T2D locus at genome-wide significance (GATAD2A/CILP2/PBX4; p = 5.7 × 10−9) and two loci exceeding study-wide significance (SREBF1, and TH/INS; p < 2.4 × 10−6). Second, meta-analyses of 1,986 cases and 7,695 controls from eight African-American studies identified study-wide-significant (p = 2.4 × 10−7) variants in HMGA2 and replicated variants in TCF7L2 (p = 5.1 × 10−15). Third, conditional analysis revealed multiple known and novel independent signals within five T2D-associated genes in samples of European ancestry and within HMGA2 in African-American samples. Fourth, a multiethnic meta-analysis of all 39 studies identified T2D-associated variants in BCL2 (p = 2.1 × 10−8). Finally, a composite genetic score of SNPs from new and established T2D signals was significantly associated with increased risk of diabetes in African-American, Hispanic, and Asian populations. In summary, large-scale meta-analysis involving a dense gene-centric approach has uncovered additional loci and variants that contribute to T2D risk and suggests substantial overlap of T2D association signals across multiple ethnic groups.

Cannabis is the most widely produced and consumed illicit psychoactive substance worldwide. Occasional cannabis use can progress to frequent use, abuse and dependence with all known adverse physical, psychological and social consequences. Individual differences in cannabis initiation are heritable (40-48%). The International Cannabis Consortium was established with the aim to identify genetic risk variants of cannabis use. We conducted a meta-analysis of genome-wide association data of 13 cohorts (N=32 330) and four replication samples (N=5627). In addition, we performed a gene-based test of association, estimated single-nucleotide polymorphism (SNP)-based heritability and explored the genetic correlation between lifetime cannabis use and cigarette use using LD score regression. No individual SNPs reached genome-wide significance. Nonetheless, gene-based tests identified four genes significantly associated with lifetime cannabis use: NCAM1, CADM2, SCOC and KCNT2. Previous studies reported associations of NCAM1 with cigarette smoking and other substance use, and those of CADM2 with body mass index, processing speed and autism disorders, which are phenotypes previously reported to be associated with cannabis use. Furthermore, we showed that, combined across the genome, all common SNPs explained 13-20% (P<0.001) of the liability of lifetime cannabis use. Finally, there was a strong genetic correlation (rg=0.83; P=1.85 × 10(-8)) between lifetime cannabis use and lifetime cigarette smoking implying that the SNP effect sizes of the two traits are highly correlated. This is the largest meta-analysis of cannabis GWA studies to date, revealing important new insights into the genetic pathways of lifetime cannabis use. Future functional studies should explore the impact of the identified genes on the biological mechanisms of cannabis use.

Although physical activity and sedentary behavior are moderately heritable, little is known about the mechanisms that influence these traits. Combining data for up to 703,901 individuals from 51 studies in a multi-ancestry meta-analysis of genome-wide association studies yields 99 loci that associate with self-reported moderate-to-vigorous intensity physical activity during leisure time (MVPA), leisure screen time (LST) and/or sedentary behavior at work. Loci associated with LST are enriched for genes whose expression in skeletal muscle is altered by resistance training. A missense variant in ACTN3 makes the alpha-actinin-3 filaments more flexible, resulting in lower maximal force in isolated type IIA muscle fibers, and possibly protection from exercise-induced muscle damage. Finally, Mendelian randomization analyses show that beneficial effects of lower LST and higher MVPA on several risk factors and diseases are mediated or confounded by body mass index (BMI). Our results provide insights into physical activity mechanisms and its role in disease prevention.

Infectious uveitis entities are usually rapidly progressive blinding diseases that can be prevented by prompt administration of specific antimicrobial therapy. With the aim of improving early diagnosis in patients with infectious uveitis, intraocular fluid samples from patients with sight-threatening posterior uveitis were investigated to determine the causative agent.Thirty-eight patients with acquired immunodeficiency syndrome (AIDS) and retinitis, eight immunosuppressed patients with retinitis, 16 immunocompetent patients with acute retinal necrosis, and 22 immunocompetent patients with toxoplasmic retinochoroiditis were analyzed by polymerase chain reaction for the presence of herpesviruses and Toxoplasma gondii DNA and for local antibody production against these microorganisms.In patients with AIDS and retinitis, polymerase chain reaction was positive for cytomegalovirus DNA in 21 (91%) of the 23 ocular fluid samples obtained during active cytomegalovirus retinitis, whereas local antibody production analysis was negative in all cases. In acute retinal necrosis, varicella-zoster virus or herpes simplex virus could be established as the inciting agent in 81% of the cases, using the combination of both techniques. Polymerase chain reaction was positive in all samples obtained within two weeks after the onset of disease. Toxoplasma gondii DNA was detected in 4 of 13 samples (31%) from immuno-competent patients with active toxoplasmic retinochoroiditis; in each case, local antibody production was also detected. In contrast, no local antibody production was observed in two of three samples from transplant recipients that were positive for T. gondii DNA. All the control samples tested were negative for the above-mentioned tests.In patients with AIDS, polymerase chain reaction analysis is preferable above local antibody production in detecting the inciting agent of retinitis. In other cases, the combination of both techniques can make a valuable contribution to the diagnosis.

Rationale: A disintegrin and metalloprotease 33 (ADAM33) has been identified as a susceptibility gene for asthma and single nucleotide polymorphisms (SNPs) in this gene have been associated with excessive decline of lung function in individuals with asthma. Objectives: To assess whether SNPs in ADAM33 are associated with accelerated lung function loss in the general population and with chronic obstructive pulmonary disease (COPD). Methods: DNA was collected from subjects of the Vlagtwedde–Vlaardingen cohort participating in the last survey in 1989–1990 after a follow-up of 25 years. Information was collected every 3 years, including lung function measurements. We defined COPD as GOLD stage 2 or higher at the last survey. A total of 1,390 subjects from the cohort was genotyped for the following SNPs in ADAM33: F+1, Q-1, S_1, S_2, T_1, T_2, V_4, and ST+5. Differences in prevalence of SNPs were analyzed with χ2 tests. Linear mixed effects models were used to analyze FEV1 decline according to genotype. Measurements and Main Results: In the whole population, mean adjusted decline was 18.7 and 12.7 ml/year in females and males, respectively. Individuals homozygous for minor alleles of SNPs S_2 and Q-1 and heterozygous for SNP S_1 had a significantly accelerated decline in FEV1 of, respectively, 4.9, 9.6, and 3.6 ml/year compared with wild type. We found a significantly higher prevalence of SNPs F+1, S_1, S_2, and T_2 in subjects with COPD. Conclusions: We demonstrated that SNPs in ADAM33 are associated with accelerated lung function decline in the general population. These SNPs are also risk factors for COPD.

Our previous coeliac disease genome-wide association study (GWAS) implicated risk variants in the human leucocyte antigen (HLA) region and eight novel risk regions. To identify more coeliac disease loci, we selected 458 single nucleotide polymorphisms (SNPs) that showed more modest association in the GWAS for genotyping and analysis in four independent cohorts.458 SNPs were assayed in 1682 cases and 3258 controls from three populations (UK, Irish and Dutch). We combined the results with the original GWAS cohort (767 UK cases and 1422 controls); six SNPs showed association with p<1 x 10(-04) and were then genotyped in an independent Italian coeliac cohort (538 cases and 593 controls).We identified two novel coeliac disease risk regions: 6q23.3 (OLIG3-TNFAIP3) and 2p16.1 (REL), both of which reached genome-wide significance in the combined analysis of all 2987 cases and 5273 controls (rs2327832 p = 1.3 x 10(-08), and rs842647 p = 5.2 x 10(-07)). We investigated the expression of these genes in the RNA isolated from biopsies and from whole blood RNA. We did not observe any changes in gene expression, nor in the correlation of genotype with gene expression.Both TNFAIP3 (A20, at the protein level) and REL are key mediators in the nuclear factor kappa B (NF-kappaB) inflammatory signalling pathway. For the first time, a role for primary heritable variation in this important biological pathway predisposing to coeliac disease has been identified. Currently, the HLA risk factors and the 10 established non-HLA risk factors explain approximately 40% of the heritability of coeliac disease.

Obesity promotes inflammation in adipose tissue (AT) and this is implicated in pathophysiological complications such as insulin resistance, type 2 diabetes and cardiovascular disease. Although based on the classical hypothesis, necrotic AT adipocytes (ATA) in obese state activate AT macrophages (ATM) that then lead to a sustained chronic inflammation in AT, the link between human adipocytes and the source of inflammation in AT has not been in-depth and systematically studied. So we decided as a new hypothesis to investigate human primary adipocytes alone to see whether they are able to prime inflammation in AT.Using mRNA expression, human preadipocytes and adipocytes express the cytokines/chemokines and their receptors, MHC II molecule genes and 14 acute phase reactants including C-reactive protein. Using multiplex ELISA revealed the expression of 50 cytokine/chemokine proteins by human adipocytes. Upon lipopolysaccharide stimulation, most of these adipocyte-associated cytokines/chemokines and immune cell modulating receptors were up-regulated and a few down-regulated such as (ICAM-1, VCAM-1, MCP-1, IP-10, IL-6, IL-8, TNF-α and TNF-β highly up-regulated and IL-2, IL-7, IL-10, IL-13 and VEGF down-regulated. In migration assay, human adipocyte-derived chemokines attracted significantly more CD4+ T cells than controls and the number of migrated CD4+ cells was doubled after treating the adipocytes with LPS. Neutralizing MCP-1 effect produced by adipocytes reduced CD4+ migration by approximately 30%.Human adipocytes express many cytokines/chemokines that are biologically functional. They are able to induce inflammation and activate CD4+ cells independent of macrophages. This suggests that the primary event in the sequence leading to chronic inflammation in AT is metabolic dysfunction in adipocytes, followed by production of immunological mediators by these adipocytes, which is then exacerbated by activated ATM, activation and recruitment of immune cells. This study provides novel knowledge about the prime of inflammation in human obese adipose tissue, opening a new avenue of investigations towards obesity-associated type 2 diabetes.

Rationale: Asthma is a chronic inflammatory airway disease that affects more than 300 million individuals worldwide.Asthma is caused by interaction of genetic and environmental factors.Bronchial hyperresponsiveness (BHR) is a hallmark of asthma and results from increased sensitivity of the airways to physical or chemical stimulants.BHR and asthma are linked to chromosome 5q31-q33.Objectives: To identify a gene for BHR on chromosome 5q31-q33.Methods: In 200 Dutch families with asthma, linkage analysis and fine mapping were performed, and the Protocadherin 1 gene (PCDH1) was identified.PCDH1 was resequenced in 96 subjects from ethnically diverse populations to identify novel sequence variants.Subsequent replication studies were undertaken in seven populations from The Netherlands, the United Kingdom, and the United States, including two general population samples, two family samples, and three case-control samples.PCDH1 mRNA and protein expression was investigated using polymerase chain reaction, Western blotting, and immunohistochemistry. Measurements and Main Results: In seven out of eight populations (n 5 6,168) from The Netherlands, United Kingdom, and United States, PCHD1 gene variants were significantly associated with BHR (P values, 0.005-0.05)This association was present in both families with asthma and general populations.PCDH1 mRNA and protein were expressed in airway epithelial cells and in macrophages.Conclusions: PCDH1 is a novel gene for BHR in adults and children.The identification of PCDH1 as a BHR susceptibility gene may suggest that a structural defect in the integrity of the airway epithelium, the first line of defense against inhaled substances, contributes to the development of BHR.

Abstract Large consortia have revealed hundreds of genetic loci associated with anthropometric traits, one trait at a time. We examined whether genetic variants affect body shape as a composite phenotype that is represented by a combination of anthropometric traits. We developed an approach that calculates averaged PCs (AvPCs) representing body shape derived from six anthropometric traits (body mass index, height, weight, waist and hip circumference, waist-to-hip ratio). The first four AvPCs explain &gt;99% of the variability, are heritable, and associate with cardiometabolic outcomes. We performed genome-wide association analyses for each body shape composite phenotype across 65 studies and meta-analysed summary statistics. We identify six novel loci: LEMD2 and CD47 for AvPC1, RPS6KA5 / C14orf159 and GANAB for AvPC3, and ARL15 and ANP32 for AvPC4. Our findings highlight the value of using multiple traits to define complex phenotypes for discovery, which are not captured by single-trait analyses, and may shed light onto new pathways.

Genome wide association studies have been hugely successful in identifying disease risk variants, yet most variants do not lead to coding changes and how variants influence biological function is usually unknown.We correlated gene expression and genetic variation in untouched primary leucocytes (n = 110) from individuals with celiac disease - a common condition with multiple risk variants identified. We compared our observations with an EBV-transformed HapMap B cell line dataset (n = 90), and performed a meta-analysis to increase power to detect non-tissue specific effects.In celiac peripheral blood, 2,315 SNP variants influenced gene expression at 765 different transcripts (< 250 kb from SNP, at FDR = 0.05, cis expression quantitative trait loci, eQTLs). 135 of the detected SNP-probe effects (reflecting 51 unique probes) were also detected in a HapMap B cell line published dataset, all with effects in the same allelic direction. Overall gene expression differences within the two datasets predominantly explain the limited overlap in observed cis-eQTLs. Celiac associated risk variants from two regions, containing genes IL18RAP and CCR3, showed significant cis genotype-expression correlations in the peripheral blood but not in the B cell line datasets. We identified 14 genes where a SNP affected the expression of different probes within the same gene, but in opposite allelic directions. By incorporating genetic variation in co-expression analyses, functional relationships between genes can be more significantly detected.In conclusion, the complex nature of genotypic effects in human populations makes the use of a relevant tissue, large datasets, and analysis of different exons essential to enable the identification of the function for many genetic risk variants in common diseases.

Arginase probably plays an important role in asthma development, severity and progression. Polymorphisms in arginase 1 and arginase 2 genes have been associated with childhood asthma and FEV1 reversibility to beta2 agonists.We investigated the association between arginase 1 and arginase 2 polymorphisms and adult asthma, asthma severity and treatment response in a longitudinal cohort of 200 asthma patients.Patients were studied during 1962-1975 and reexamined during 1990-1999, together with their families. Longitudinal data on lung function and treatment were extracted from medical records. Associations between haplotype-tagging polymorphisms in arginase 1 (n=3) and arginase 2 (n=8) and asthma, asthma severity, acute response to bronchodilators and chronic response to inhaled corticosteroids were analyzed.Two polymorphisms in arginase 2 (rs17249437 and rs3742879) were associated with asthma and with more severe airway obstruction. Increased airway hyperresponsiveness and lower beta2 agonist reversibility, but not anticholinergic reversibility, were associated with both arginase 1 and arginase 2. Inhaled corticosteroids slowed down the annual FEV1 decline, which was significantly less effective in homozygote carriers of the C-allele of the arginase 1 polymorphism, rs2781667.We show that previously reported associations between arginase polymorphisms and childhood asthma are also present in adult asthma and the previously found associations with lower reversibility are specific for beta2 agonists. Furthermore, we identified associations of arginase 1 and arginase 2 genes with asthma severity, as reflected by a lower lung function, more severe airway hyperresponsiveness, and less long-term response to inhaled corticosteroids. Studies on the functionality of the polymorphisms are warranted to further unravel the complex mechanisms underlying these observations.

Despite the recent explosive rise in number of genetic markers for complex disease traits identified in genome-wide association studies, there is still a large gap between the known heritability of these traits and the part explained by these markers. To gauge whether this 'heritability gap' is closing, we first identified genome-wide significant SNPs from the literature and performed replication analyses for 32 highly relevant traits from five broad disease areas in 13 436 subjects of the Lifelines Cohort. Next, we calculated the variance explained by multi-SNP genetic risk scores (GRSs) for each trait, and compared it to their broad- and narrow-sense heritabilities captured by all common SNPs. The majority of all previously-associated SNPs (median=75%) were significantly associated with their respective traits. All GRSs were significant, with unweighted GRSs generally explaining less phenotypic variance than weighted GRSs, for which the explained variance was highest for height (15.5%) and varied between 0.02 and 6.7% for the other traits. Broad-sense common-SNP heritability estimates were significant for all traits, with the additive effect of common SNPs explaining 48.9% of the variance for height and between 5.6 and 39.2% for the other traits. Dominance effects were uniformly small (0-1.5%) and not significant. On average, the variance explained by the weighted GRSs accounted for only 10.7% of the common-SNP heritability of the 32 traits. These results indicate that GRSs may not yet be ready for accurate personalized prediction of complex disease traits limiting widespread adoption in clinical practice.

Meta-analysis of genome-wide association studies (GWASs) has led to the discoveries of many common variants associated with complex human diseases. There is a growing recognition that identifying "causal" rare variants also requires large-scale meta-analysis. The fact that association tests with rare variants are performed at the gene level rather than at the variant level poses unprecedented challenges in the meta-analysis. First, different studies may adopt different gene-level tests, so the results are not compatible. Second, gene-level tests require multivariate statistics (i.e., components of the test statistic and their covariance matrix), which are difficult to obtain. To overcome these challenges, we propose to perform gene-level tests for rare variants by combining the results of single-variant analysis (i.e., p values of association tests and effect estimates) from participating studies. This simple strategy is possible because of an insight that multivariate statistics can be recovered from single-variant statistics, together with the correlation matrix of the single-variant test statistics, which can be estimated from one of the participating studies or from a publicly available database. We show both theoretically and numerically that the proposed meta-analysis approach provides accurate control of the type I error and is as powerful as joint analysis of individual participant data. This approach accommodates any disease phenotype and any study design and produces all commonly used gene-level tests. An application to the GWAS summary results of the Genetic Investigation of ANthropometric Traits (GIANT) consortium reveals rare and low-frequency variants associated with human height. The relevant software is freely available.

To the Editor: Several genome-wide screens, including our own, have shown strong linkage with asthma, lung function, and atopy on chromosome 2q.1Koppelman G.H. Stine O.C. Xu J. Howard T.D. Zheng S.L. Kauffman H.F. et al.Genome-wide search for atopy susceptibility genes in Dutch families with asthma.J Allergy Clin Immunol. 2002; 109: 498-506Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar The IL-1 receptorlike-1 (IL1RL1) gene, also known as ST2, is a promising candidate gene for asthma and atopy. IL1RL1 is located on 2q12 and resides in a cluster of IL1 receptor genes (IL-1 receptor 2 [IL1R2], IL1R1, IL1RL2, IL1RL1, IL-18 receptor 1 [IL18R1], IL-18 receptor accessory protein [IL18RAP]).2Dale M. Nicklin M.J. Interleukin-1 receptor cluster: gene organization of IL1R2, IL1R1, IL1RL2 (IL-1Rrp2), IL1RL1 (T1/ST2), and IL18R1 (IL-1Rrp) on human chromosome 2q.Genomics. 1999; 57: 177-179Crossref PubMed Scopus (90) Google Scholar IL1RL1, a member of the Toll–IL-1 receptor superfamily, is a receptor located on mast cells, TH2 cells, regulatory T cells, and macrophages and is also present in serum in a soluble form. IL1RL1 binds IL-33 and exerts its role through Toll-like receptor pathways. Various forms of IL1RL1 can either stimulate or inhibit TH2 responses.3Brint E.K. Xu D. Liu H. Dunne A. McKenzie A.N. O'Neill L.A. et al.ST2 is an inhibitor of interleukin 1 receptor and Toll-like receptor 4 signaling and maintains endotoxin tolerance.Nat Immunol. 2004; 5: 373-379Crossref PubMed Scopus (433) Google Scholar, 4Mangan N.E. Dasvarma A. McKenzie A.N. Fallon P.G. T1/ST2 expression on Th2 cells negatively regulates allergic pulmonary inflammation.Eur J Immunol. 2007; 37: 1302-1312Crossref PubMed Scopus (55) Google Scholar, 5Schmitz J. Owyang A. Oldham E. Song Y. Murphy E. McClanahan T.K. et al.IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines.Immunity. 2005; 23: 479-490Abstract Full Text Full Text PDF PubMed Scopus (2798) Google Scholar, 6Hayakawa H. Hayakawa M. Kume A. Tominaga S. Soluble ST2 blocks interleukin-33 signaling in allergic airway inflammation.J Biol Chem. 2007; 282: 26369-26380Crossref PubMed Scopus (428) Google Scholar Interestingly, single nucleotide polymorphisms (SNPs) located in the IL1RL1 gene are associated with atopic dermatitis.7Shimizu M. Matsuda A. Yanagisawa K. Hirota T. Akahoshi M. Inomata N. et al.Functional SNPs in the distal promoter of the ST2 gene are associated with atopic dermatitis.Hum Mol Genet. 2005; 14: 2919-2927Crossref PubMed Scopus (153) Google Scholar The adjacently located family members IL18R1 and IL18RAP may also be important in the development of asthma and atopy. The gene products of both IL18R1 and IL18RAP form the α-chain and β-chain of the IL-18 receptor (IL-18R).8Torigoe K. Ushio S. Okura T. Kobayashi S. Taniai M. Kunikata T. et al.Purification and characterization of the human interleukin-18 receptor.J Biol Chem. 1997; 272: 25737-25742Crossref PubMed Scopus (433) Google Scholar IL18R is a key regulator of TH1 cells. Binding of IL-18 to IL-18R stimulates TH1 cytokine release, but also TH2-type cytokines, depending on its cytokine milieu.9Nakanishi K. Yoshimoto T. Tsutsui H. Okamura H. Interleukin-18 is a unique cytokine that stimulates both Th1 and Th2 responses depending on its cytokine milieu.Cytokine Growth Factor Rev. 2001; 12: 53-72Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar A recent publication showed strong associations with SNPs located in IL18R1 and asthma and atopic phenotypes.10Zhu G. Whyte M.K. Vestbo J. Carlsen K. Carlsen K.H. Lenney W. et al.Interleukin 18 receptor 1 gene polymorphisms are associated with asthma.Eur J Hum Genet. 2008; (Apr 2 [epub ahead of print])Google Scholar Our aim was to investigate whether SNPs in the IL1RL1 gene are associated with asthma, atopy, and allergic rhinitis. Given the strong linkage disequilibrium (LD) in this region, we also investigated the adjacently located family members IL18R1 and IL18RAP. We analyzed 200 well-characterized Dutch asthma families (n = 1259) with an asthma proband and used 407 independent asthma trios and 226 rhinitis trios to replicate our results. Furthermore, we performed a combined analysis of asthma families and trios to increase power. The medical ethics committee of the University Medical Center Groningen approved all studies, and all participants signed written informed (parental) consent. Families were assessed as described previously.11Panhuysen C.I. Bleecker E.R. Koeter G.H. Meyers D.A. Postma D.S. Characterization of obstructive airway disease in family members of probands with asthma: an algorithm for the diagnosis of asthma.Am J Respir Crit Care Med. 1998; 157: 1734-1742Crossref PubMed Scopus (63) Google Scholar The trios were characterized using the standardized protocol similar to that used in the family study. Bronchial hyperresponsiveness (BHR) to histamine was measured as described previously.11Panhuysen C.I. Bleecker E.R. Koeter G.H. Meyers D.A. Postma D.S. Characterization of obstructive airway disease in family members of probands with asthma: an algorithm for the diagnosis of asthma.Am J Respir Crit Care Med. 1998; 157: 1734-1742Crossref PubMed Scopus (63) Google Scholar BHR was defined as a PC20 ≤32 mg/mL (30 seconds inhalation). Intracutaneous skin testing was performed with 16 (for the families) and 12 (for the trios) common aeroallergens. Asthma was defined by a previously published algorithm.11Panhuysen C.I. Bleecker E.R. Koeter G.H. Meyers D.A. Postma D.S. Characterization of obstructive airway disease in family members of probands with asthma: an algorithm for the diagnosis of asthma.Am J Respir Crit Care Med. 1998; 157: 1734-1742Crossref PubMed Scopus (63) Google Scholar Twenty-one SNPs located in IL1RL1, IL18R1, and IL18RAP were genotyped. SNPs were selected from the Celera database (http://www.celeradiscoverysystem.com; Celera, Alameda, Calif), and primers were ordered through assay by design service (Applied Biosystems, Foster City, Calif). SNP selection was based on minor allele frequency >10% and location. Whenever possible, the SNPs were located in coding regions with ≈5 kb distance between each other. Eleven selected SNPs are present in the HapMap database (release 22, April 2007),12The International HapMap Consortium. The International HapMap Project.Nature. 2003; 426: 789-796Crossref PubMed Scopus (4941) Google Scholar of which 7 were haplotype tagging SNPs capturing ≈75% of SNPs located in the IL1RL1, IL18R1, and IL18RAP gene cluster. PCR was performed by using an ABI Prism 9700 HT real-time thermal cycler (Applied Biosystems). In-house software was used to test for LD (D′ and r2) between the SNPs. Family-Based Association Tests (version 2.0.2) analyses were used for investigation of preferential transmission of alleles (www.biostat.harvard.edu).13Laird N.M. Horvath S. Xu X. Implementing a unified approach to family-based tests of association.Genet Epidemiol. 2000; 19: S36-S42Crossref PubMed Scopus (744) Google Scholar Sliding window analysis with 3 consecutive SNPs was used with Haplotype-Based Association Tests.12The International HapMap Consortium. The International HapMap Project.Nature. 2003; 426: 789-796Crossref PubMed Scopus (4941) Google Scholar Results were considered significant if P < .05. Family and trio characteristics are shown in this article's Table E1 in the Online Repository at www.jacionline.org. All SNPs were in Hardy-Weinberg equilibrium (P > .01). Minor allele frequencies within the asthma families, asthma trios, and rhinitis trios were similar (see this article's Table E2 in the Online Repository at www.jacionline.org). Genotyping of 2 of 21 SNPs failed because of design problems. Strong LD between the SNPs was present in all 3 populations (eg, asthma families; Fig 1). Asthma families: Eight of 19 SNPs located in the IL1RL1, IL18R1, and IL18RAP genes were strongly associated with BHR, severity of BHR, and total serum IgE (Table I and Table E3, Table E4, Table E6 in the Online Repository at www.jacionline.org). SNPs located in IL18R1 and IL18RAP were also associated with serum eosinophils (see this article's Table E7 in the Online Repository at www.jacionline.org). Haplotype analysis using sliding windows of 3 consecutive SNPs in all 3 genes showed even stronger associations (asthma, P = .003-.05; BHR, P = .005-.05; severity of BHR, P = .0003-.05; IgE, P = .03-.05; and serum eosinophils, P = .03-.05).Table IFamily-Based Association Test of IL1RL1, IL18R1, and IL18RAP with BHR, asthma, and total IgE in the separate and combined analysis of Dutch asthma families and asthma triosAsthma familiesAsthma triosAsthma families and trios combinedGeners No.Allele∗Allele 1, major allele; 2, minor allele.z (P values)z (P values)Informative familiesz (P values)BHRIL1RL1rs142010121.311 (.190)1.474 (.141)2111.927 (.054)rs192162212.067 (.039)1.432 (.152)2122.463 (.014)rs186124611.969 (.049)1.292 (.196)1752.314 (.021)IL1RL1 /IL18R1rs1299936421.660 (.097)1.777 (.076)1982.402 (.016)IL18R1rs155862712.291 (.022)0.526 (.599)1801.973 (.049)rs227029712.208 (.027)0.590 (.555)1851.975 (.048)rs103513021.496 (.142)1.273 (.203)1741.981 (.048)IL18RAPrs142010612.418 (.016)0.813 (.416)1842.277 (.023)Intergenicrs146879112.376 (.018)0.221 (.825)1881.816 (.069)HaplotypeIL1RL1rs1921622, rs1861246, rs102067531112.803 (.005)1.844 (.065)1923.311 (.0009)IL1RL1 /IL18R1rs10206753, rs12999364, rs14200991212.125 (.033)1.800 (.072)1792.770 (.006)Asthma†Asthma based on algorithm.AlleleIL1RL1rs192162211.508 (.132)1.451 (.147)1932.074 (.038)rs186124611.201 (.230)1.552 (.121)1631.952 (.050)IL1RL1 /IL18R1rs1299936421.702 (.089)1.622 (.105)1852.312 (.021)IL18R1rs103513021.547 (.122)1.205 (.228)1591.996 (.046)HaplotypeIL1RL1rs1921622, rs1861246, rs102067531113.020 (.003)1.766 (.077)1703.354 (.0008)IL18R1rs1420099, rs1558627, rs22702971112.749 (.006)1.694 (.090)1803.086 (.002)Total IgEAlleleIL1RL1rs10540962−0.971 (.331)1.998 (.046)1270.246 (.806)rs192162211.729 (.084)1.335 (.182)2762.207 (.027)rs186124612.069 (.039)1.070 (.285)2302.320 (.020)IL18R1rs155862712.554 (.011)0.229 (.819)2382.203 (.028)rs227029712.142 (.032)0.364 (.716)2391.930 (.054)IL18RAPrs142010612.659 (.008)0.554 (.579)2342.516 (.012)Intergenicrs146879112.118 (.034)0.257 (.797)2431.833 (.067)HaplotypeIL1RL1rs1921622, rs1861246, rs102067531111.988 (.046)1.888 (.059)2452.719 (.007)Significant SNP results and consistent haplotype results are shown. z Scores and (uncorrected) P values are shown for the asthma families and asthma trios. Numbers of informative families, z scores, and P values are shown for the combined analysis.∗ Allele 1, major allele; 2, minor allele.† Asthma based on algorithm. Open table in a new tab Significant SNP results and consistent haplotype results are shown. z Scores and (uncorrected) P values are shown for the asthma families and asthma trios. Numbers of informative families, z scores, and P values are shown for the combined analysis. Asthma trios: Significant associations were observed with total serum IgE, serum eosinophils, and skin test positivity and 2 SNPs located in the IL1RL1 and IL18R1 gene (Table I and Table E6, Table E7, Table E8). Haplotype analysis showed the associations to be present with SNPs located in the IL1RL1 and IL18R1 gene with total IgE (P = .03-.05), serum eosinophils (P = .04-.05), and skin test positivity (P = .02-.05). Three haplotype combinations, [rs1921622, rs1861246, and rs10206753], [rs10206753, rs12999364, and rs1420099], and [rs1420099, rs1558627, and rs2270297], strictly replicated results in the asthma families with similar (borderline) associations with BHR, asthma, and total IgE (Table I) and serum eosinophils (P = .03-.06). Asthma families and asthma trios combined: Combination of asthma families and trios resulted in similar or more significant results (Table I and Table E3, Table E4, Table E5, Table E6). Interestingly, significant associations between asthma and SNPs located in IL1RL1 and IL18R1 were found that were not present when analyzing the both cohorts separately (Tables I and E5). Also, haplotype analysis showed a more significant association with asthma (P = .0008-.05), BHR (P = .0009-.05), BHR severity (P = .0006-.05), total IgE (P = .007-.05), and serum eosinophils (P = .005-.05) in all 3 genes, compared with the analysis of both cohorts separately. Rhinitis trios: No significant associations were found with SNPs located in the IL1RL1 and IL18R1 gene and atopic phenotypes in the rhinitis trios (data not shown). This is the first study that provides suggestive evidence for associations of SNPs in the IL1RL1 gene and adjacently located family members IL18R1 and IL18RAP with asthma and atopy in 2 independent Dutch asthma populations, but not with rhinitis. This study thus shows the importance of analyzing SNPs in adjacently located genes because it is not decisive whether the associations are caused by the IL1RL1, IL18R1, and/or IL18RAP gene as a result of strong LD in the region. Tabled 1Clinical characteristics of Dutch families from probands with asthmaProbandsSpousesChildrenGrandchildrenNo. (% female)200 (38)201∗One proband married twice; both spouses participated in the study. (62)556 (54)194 (51)Age (y), median (range)52.5 (37.8-75.3)51.5 (33.1-76.5)24.5 (6.8-53.4)12.7 (4.6-41.8)Asthma (%)9110.631.439.6BHR (%)10025.645.359.8Total IgE (IU/mL), geometric mean (range)92.9 (1.0-2880)26.2 (0.5-1940)63.5 (0.5-3360)63.8 (0.5-3710)Positive skin tests (%)82.831.053.332.8Blood eosinophils 10E7, mean (range)15.8 (0-126.5)9.4 (0-63.8)15.8 (0-294.8)20.5 (0-177)Clinical characteristics of probands of asthma and rhinitis triosAsthma probandsRhinitis probandsNo. (% female)407 (63.1)226 (56.2)Age (y), median (range)34.3 (18.1-64.7)32.5 (18-46.7)Asthma (%)86.915.3BHR (%)9133Total IgE (IU/mL), geometric mean (range)108.5 (0.5-12400)115.7 (5-6915)Positive skin tests (%)82.995.5Blood eosinophils 10E7, mean (range)16.5 (1.0-94)16.3 (1.0-78)∗ One proband married twice; both spouses participated in the study. Open table in a new tab Tabled 1SNPs selected for IL1RL1, IL18R1 and IL18RAP in Dutch asthma family study, asthma trios and rhinitis triosMinor allele frequencyGeneRs no.FunctionAllele∗Major allele first.Asthma familiesAsthma triosRhinitis triosIL1RL1rs1041973MissenseC/A0.150.180.19IL1RL1rs873022IntronG/T0.330.290.29IL1RL1rs1420101Intron/silentG/A0.380.350.36IL1RL1rs129053′UTR/intronG/A0.320.290.29IL1RL1rs1054096IntronT/C0.080.090.11IL1RL1rs1921622IntronA/G0.460.460.46IL1RL1rs1861246IntronC/T0.220.240.23IL1RL1rs10206753MissenseT/C0.380.390.38IL1RL1 /IL18R1rs129993643′UTR/promoterC/T0.360.380.39IL18R1rs1420099IntronC/G0.380.380.38IL18R1rs1558627IntronA/G0.220.250.23IL18R1rs2270297IntronC/T0.230.25NGIL18R1rs1035130MissenseA/G0.330.290.29IL18R1rs1420096IntronC/T0.470.46NGIL18R1rs37321273′UTRG/C0.130.13NGIL18RAPrs1420106PromoterG/A0.220.25NGIL18RAPrs887971IntronT/C0.320.33NGIL18RAPrs2058659IntronG/A0.480.45NGIntergenicrs1468791IntergenicG/A0.220.25NGNG, Not genotyped; UTR, untranslated region.∗ Major allele first. Open table in a new tab NG, Not genotyped; UTR, untranslated region. Tabled 1Family-Based Association Test of IL1RL1, IL18R1, and IL18RAP with BHR in the separate and combined analysis of Dutch asthma families and asthma triosBHRFamiliesTriosAsthma families and trios combinedGeneRs no.Allelez (P values)z (P values)Informative familiesz (P values)IL1RL1rs104197310.028 (.978)0.266 (.790)1540.212 (.831)rs87302221.204 (.229)0.587 (.557)1971.297 (.194)rs142010121.311 (.190)1.474 (.141)2111.927 (.054)rs1290521.513 (.130)0.899 (.368)1901.741 (.082)rs105409620.186 (.852)1.395 (.163)911.016 (.310)rs192162212.067 (.039)1.432 (.152)2122.463 (.014)rs186124611.969 (.049)1.292 (.196)1752.314 (.021)rs1020675310.337 (.736)1.237 (.216)2141.044 (.297)IL1RL1 /IL18R1rs1299936421.660 (.097)1.777 (.076)1982.402 (.016)IL18R1rs142009910.335 (.738)1.504 (.133)2141.210 (.226)rs155862712.291 (.022)0.526 (.599)1801.973 (.049)rs227029712.208 (.027)0.590 (.555)1851.975 (.048)rs103513021.496 (.142)1.273 (.203)1741.981 (.048)rs14200961−0.302 (.763)0.469 (.639)2270.072 (.942)rs37321271−0.043 (.966)0.749 (.454)1260.596 (.551)IL18RAPrs142010612.418 (.016)0.813 (.416)1842.277 (.023)rs88797120.795 (.427)0.840 (.401)2051.157 (.247)rs20586591−0.217 (.828)0.668 (.504)2330.281 (.777)Intergenicrs146879112.376 (.018)0.221 (.825)1881.816 (.069)z Scores and (uncorrected) P values are shown for the asthma families and asthma trios. Numbers of informative families, z scores, and P values are shown for the combined analysis. Open table in a new tab z Scores and (uncorrected) P values are shown for the asthma families and asthma trios. Numbers of informative families, z scores, and P values are shown for the combined analysis. Tabled 1Family-Based Association Test of IL1RL1, IL18R1, and IL18RAP with BHR severity in the separate and combined analysis of Dutch asthma families and asthma triosBHR severityFamiliesTriosAsthma families and trios combinedGeneRs no.Allelez (P values)z (P values)Informative familiesz (P values)IL1RL1rs104197320.703 (.482)−0.184 (.854)1600.446 (.656)rs87302221.637 (.101)0.106 (.916)1981.413 (.158)rs142010122.145 (.032)−0.028 (.978)2131.79 (.073)rs1290521.742 (.082)0.228 (.819)1921.578 (.114)rs105409621.693 (.090)0.032 (.974)1011.512 (.131)rs192162212.637 (.008)−0.246 (.806)2141.979 (.048)rs186124612.235 (.025)1.113 (.266)1762.420 (.016)rs1020675311.045 (.296)0.241 (.810)2081.032 (.301)IL1RL1 /IL18R1rs1299936423.150 (.002)0.142 (.887)2032.640 (.008)IL18R1rs142009911.004 (.315)−0.106 (.916)2100.827 (.409)rs155862712.631 (.009)−0.199 (.841)1801.881 (.060)rs227029712.442 (.015)0.083 (.934)1831.925 (.054)rs103513021.836 (.066)0.376 (.707)1761.747 (.081)rs142009620.272 (.785)−0.149 (.881)2210.152 (.880)rs373212720.926 (.355)0.227 (.821)1270.851 (.395)IL18RAPrs142010612.633 (.008)−0.006 (.995)1812.000 (.045)rs88797121.494 (.135)−0.334 (.739)2041.010 (.312)rs205865920.196 (.844)−0.028 (.978)2260.151 (.880)Intergenicrs146879112.673 (.008)−0.224 (.823)1841.887 (.059)z Scores and (uncorrected) P values are shown for the asthma families and asthma trios. Numbers of informative families, z scores, and P values are shown for the combined analysis. Open table in a new tab z Scores and (uncorrected) P values are shown for the asthma families and asthma trios. Numbers of informative families, z scores, and P values are shown for the combined analysis. Tabled 1Family-Based Association Test of IL1RL1, IL18R1, and IL18RAP with asthma in the separate and combined analysis of Dutch asthma families and asthma triosAsthma∗Asthma based on algorithm.15FamiliesTriosAsthma families and trios combinedGeneRs no.Allelez (P values)z (P values)Informative familiesz (P values)IL1RL1rs104197320.020 (.984)0.092 (.926)1390.083 (.934)rs87302221.382 (.167)0.300 (.764)1781.201 (.230)rs142010121.816 (.069)1.155 (.248)1962.063 (.039)rs1290521.460 (.144)0.688 (.491)1711.540 (.124)rs105409620.000 (1.000)1.612 (.107)791.212 (.225)rs192162211.508 (.132)1.451 (.147)1932.074 (.038)rs186124611.201 (.230)1.552 (.121)1631.952 (.050)rs1020675311.397 (.162)0.827 (.408)1901.575 (.115)IL1RL1 /IL18R1rs1299936421.702 (.089)1.622 (.105)1852.312 (.021)IL18R1rs142009911.479 (.139)1.109 (.268)1901.823 (.068)rs155862711.387 (.166)0.846 (.397)1641.527 (.127)rs227029711.309 (.191)0.905 (.366)1711.536 (.125)rs103513021.547 (.122)1.205 (.228)1591.996 (.046)rs142009610.381 (.703)0.000 (1.000)2010.266 (.790)rs373212711.175 (.240)0.385 (.700)1221.019 (.308)IL18RAPrs142010611.427 (.154)1.134 (.257)1701.796 (.073)rs88797121.592 (.111)0.641 (.521)1851.556 (.120)rs205865910.892 (.372)0.206 (.837)2030.762 (.446)Intergenicrs146879111.180 (.238)0.526 (.599)1731.169 (.243)z Scores and (uncorrected) P values are shown for the asthma families and asthma trios. Numbers of informative families, z scores, and P values are shown for the combined analysis.∗ Asthma based on algorithm.15 Open table in a new tab z Scores and (uncorrected) P values are shown for the asthma families and asthma trios. Numbers of informative families, z scores, and P values are shown for the combined analysis. Tabled 1Family-Based Association Test of IL1RL1, IL18R1, and IL18RAP with total IgE in the separate and combined analysis of Dutch asthma families and asthma triosTotal IgEFamiliesTriosAsthma families and trios combinedGeneRs no.Allelez (P values)z (P values)Informative familiesz (P values)IL1RL1rs104197320.343 (.732)0.513 (.879)1970.364 (.716)rs87302220.872 (.383)0.396 (.692)2650.952 (.341)rs142010120.840 (.401)1.385 (.166)2761.469 (.142)rs1290521.091 (.275)0.891 (.373)2551.406 (.160)rs10540962−0.971 (.331)1.998 (.046)1270.246 (.806)rs192162211.729 (.084)1.335 (.182)2762.207 (.027)rs186124612.069 (.039)1.070 (.285)2302.320 (.020)rs102067531−0.124 (.901)1.351 (.179)2740.638 (.524)IL1RL1 /IL18R1rs1299936420.100 (.920)1.800 (.072)2661.212 (.226)IL18R1rs14200991−0.181 (.856)1.743 (.081)2740.824 (.410)rs155862712.554 (.011)0.229 (.819)2382.203 (.028)rs227029712.142 (.032)0.364 (.716)2391.930 (.054)rs103513020.582 (.561)1.131 (.258)2401.109 (.267)rs142009621.062 (.288)−0.441 (.659)2890.598 (.550)rs373212710.432 (.666)0.760 (.447)1580.872 (.383)IL18RAPrs142010612.659 (.008)0.554 (.579)2342.516 (.012)rs8879712−0.018 (.986)0.583 (.560)2740.324 (.746)rs205865920.568 (.570)−0.675 (.499)2960.057 (.954)Intergenicrs146879112.118 (.034)0.257 (.797)2431.833 (.067)z Scores and (uncorrected) P values are shown for the asthma families and asthma trios. Numbers of informative families, z scores, and P values are shown for the combined analysis. Open table in a new tab z Scores and (uncorrected) P values are shown for the asthma families and asthma trios. Numbers of informative families, z scores, and P values are shown for the combined analysis. Tabled 1Family-Based Association Test of IL1RL1, IL18R1, and IL18RAP with serum eosinophils in the separate and combined analysis of Dutch asthma families and asthma triosSerum eosinophilsFamiliesTriosAsthma families and trios combinedGeneRs no.Allelez (P values)z (P values)Informative familiesz (P values)IL1RL1rs104197320.010 (.992)0.298 (.766)1900.183 (.855)rs87302220.720 (.471)0.461 (.645)2540.853 (.394)rs142010120.784 (.433)1.379 (.168)2671.369 (.171)rs1290520.904 (.366)0.834 (.404)2491.203 (.229)rs10540962−0.330 (.742)1.791 (.073)1250.566 (.572)rs192162211.512 (.130)1.438 (.150)2682.040 (.041)rs186124611.861 (.063)1.538 (.124)2272.374 (.018)rs1020675310.012 (.990)1.226 (.220)2660.657 (.511)IL1RL1 /IL18R1rs1299936420.147 (.883)2.012 (.044)2571.281 (.200)IL18R1rs14200991−0.074 (.941)1.705 (.088)2650.844 (.399)rs155862712.411 (.016)0.640 (.522)2352.323 (.020)rs227029712.086 (.037)0.773 (.440)2342.127 (.033)rs103513020.444 (.657)0.979 (.328)2330.886 (.376)rs142009621.007 (.314)−0.155 (.877)2790.754 (.451)rs373212710.661 (.509)0.318 (.750)1520.717 (.473)IL18RAPrs142010612.879 (.004)1.081 (.280)2302.968 (.003)rs8879712−0.105 (.916)0.891 (.373)2650.397 (.691)rs205865920.841 (.400)−0.343 (.731)2860.513 (.608)Intergenicrs146879112.123 (.034)0.684 (.494)2392.093 (.036)z Scores and (uncorrected) P values are shown for the asthma families and asthma trios. Numbers of informative families, z scores, and P values are shown for the combined analysis. Open table in a new tab z Scores and (uncorrected) P values are shown for the asthma families and asthma trios. Numbers of informative families, z scores, and P values are shown for the combined analysis. Tabled 1Family-Based Association Test of IL1RL1, IL18R1, and IL18RAP with positive skin test result in the separate and combined analysis of Dutch asthma families and asthma triosPositive skin test resultFamiliesTriosAsthma families and trios combinedGeneRs no.Allele∗Allele 1, major allele; 2, minor allele.z (P values)z (P values)Informative familiesz (P values)IL1RL1rs104197320.573 (.567)−0.191 (.849)1370.249 (.080)rs87302210.307 (.759)−0.234 (.815)1920.096 (.932)rs14201012−0.176 (.860)1.199 (.230)2020.584 (.560)rs1290520.063 (.950)0.318 (.750)1840.244 (.807)rs10540962−0.863 (.388)2.151 (.031)880.796 (.426)rs192162210.000 (1.000)1.474 (.141)2050.968 (.333)rs186124611.020 (.308)0.811 (.417)1691.273 (.203)rs102067531−0.338 (.735)1.631 (.103)2030.751 (.452)IL1RL1 /IL18R1rs129993642−1.121 (.262)1.818 (.069)1970.321 (.748)IL18R1rs14200991−0.283 (.777)1.613 (.107)2050.798 (.424)rs155862711.256 (.209)0.160 (.873)1801.030 (.303)rs227029710.787 (.431)0.157 (.875)1820.676 (.499)rs10351302−0.512 (.609)0.828 (.408)1720.102 (.919)rs142009621.211 (.226)−0.489 (.625)2170.578 (.563)rs37321271−0.388 (.698)1.294 (.196)1200.699 (.485)IL18RAPrs142010611.693 (.090)0.552 (.581)1741.626 (.104)rs88797110.660 (.509)−0.455 (.649)1940.211 (.833)rs205865920.668 (.504)−0.626 (.532)2180.077 (.939)Intergenicrs146879111.065 (.287)−0.078 (.938)1830.724 (.469)z Scores and (uncorrected) P values are shown for the asthma families and asthma trios. Numbers of informative families, z scores, and P values are shown for the combined analysis.∗ Allele 1, major allele; 2, minor allele. Open table in a new tab z Scores and (uncorrected) P values are shown for the asthma families and asthma trios. Numbers of informative families, z scores, and P values are shown for the combined analysis.

Microvillus inclusion disease (MVID) is a rare autosomal recessive enteropathy characterized by intractable diarrhea and malabsorption. Recently, various MYO5B gene mutations have been identified in patients with MVID. Interestingly, several patients with MVID showed only a MYO5B mutation in 1 allele (heterozygous) or no mutations in the MYO5B gene, illustrating the need to further functionally characterize the cell biological effects of the MYO5B mutations.The genomic DNA of 9 patients diagnosed as having MVID was screened for MYO5B mutations, and quantitative polymerase chain reaction and immunohistochemistry on the material of 2 patients was performed to investigate resultant cellular consequences.We demonstrate for the first time that MYO5B mutations can be correlated with altered myosin Vb messenger RNA expression and with an aberrant subcellular distribution of the myosin Vb protein. Moreover, we demonstrate that the typical and myosin Vb-controlled accumulation of Rab11a- and FIP5-positive recycling endosomes in the apical cytoplasm of the cells is abolished in MVID enterocytes, which is indicative of altered myosin Vb function. Moreover, we report 8 novel MYO5B mutations in 9 patients of various ethnic backgrounds with MVID, including compound heterozygous mutations.Our functional analysis indicates that MYO5B mutations can be correlated with an aberrant subcellular distribution of the myosin Vb protein, and apical recycling endosomes, which, together with the additional compound heterozygous mutations, significantly strengthen the link between MYO5B and MVID.

Asthma and chronic obstructive pulmonary disease (COPD) are thought to share a genetic background ("Dutch hypothesis"). We investigated whether asthma and COPD have common underlying genetic factors, performing genome-wide association studies for both asthma and COPD and combining the results in meta-analyses. Three loci showed potential involvement in both diseases: chr2p24.3, chr5q23.1 and chr13q14.2, containing DDX1, COMMD10 (both participating in the nuclear factor (NF) κβ pathway) and GNG5P5, respectively. Single nucleotide polymorphisms (SNPs) rs9534578 in GNG5P5 reached genome-wide significance after first replication phase (p=9.96×10(-9)). The second replication phase, in seven independent cohorts, provided no significant replication. Expression quantitative trait loci (eQTL) analysis in blood cells and lung tissue on the top 20 associated SNPs identified two SNPs in COMMD10 that influenced gene expression. Inflammatory processes differ in asthma and COPD and are mediated by NF-κβ, which could be driven by the same underlying genes, COMMD10 and DDX1. None of the SNPs reached genome-wide significance. Our eQTL studies support a functional role for two COMMD10 SNPs, since they influence gene expression in both blood cells and lung tissue. Our findings suggest that there is either no common genetic component in asthma and COPD or, alternatively, different environmental factors, e.g. lifestyle and occupation in different countries and continents, which may have obscured the genetic common contribution.

Excessive accumulation of body fat, in particular in the visceral fat depot, is a major risk factor to develop a variety of diseases such as type 2 diabetes. The mechanisms underlying the increased risk of obese individuals to develop co-morbid diseases are largely unclear.We aimed to identify genes expressed in subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) that are related to blood parameters involved in obesity co-morbidity, such as plasma lipid and glucose levels, and to compare gene expression between the fat depots.Whole-transcriptome SAT and VAT gene expression levels were determined in 75 individuals with a BMI >35 kg/m2. Modules of co-expressed genes likely to be functionally related were identified and correlated with BMI, plasma levels of glucose, insulin, HbA1c, triglycerides, non-esterified fatty acids, ALAT, ASAT, C-reactive protein, and LDL- and HDL cholesterol.Of the approximately 70 modules identified in SAT and VAT, three SAT modules were inversely associated with plasma HDL-cholesterol levels, and a fourth module was inversely associated with both plasma glucose and plasma triglyceride levels (p < 5.33 x 10(-5)). These modules were markedly enriched in immune and metabolic genes. In VAT, one module was associated with both BMI and insulin, and another with plasma glucose (p < 4.64 x 10(-5)). This module was also enriched in inflammatory genes and showed a marked overlap in gene content with the SAT modules related to HDL. Several genes differentially expressed in SAT and VAT were identified.In obese subjects, groups of co-expressed genes were identified that correlated with lipid and glucose metabolism parameters; they were enriched with immune genes. A number of genes were identified of which the expression in SAT correlated with plasma HDL cholesterol, while their expression in VAT correlated with plasma glucose. This underlines both the singular importance of these genes for lipid and glucose metabolism and the specific roles of these two fat depots in this respect.

Decorin, an extracellular matrix (ECM) proteoglycan, and TGF-beta1 are both involved in lung ECM turnover. Decorin and TGF-beta1 expression are decreased respectively increased in COPD lung tissue. Interestingly, they act as each other's feedback regulator. We investigated whether single nucleotide polymorphisms (SNPs) in decorin and TGF-beta1 underlie accelerated decline in FEV1 and development of COPD in the general population.We genotyped 1390 subjects from the Vlagtwedde/Vlaardingen cohort. Lung function was measured every 3 years for a period of 25 years. We tested whether five SNPs in decorin (3'UTR and four intron SNPs) and three SNPs in TGF-beta1 (3'UTR rs6957, C-509T rs1800469 and Leu10Pro rs1982073), and their haplotypes, were associated with COPD (last survey GOLD stage = II). Linear mixed effects models were used to analyze genotype associations with FEV1 decline.We found a significantly higher prevalence of carriers of the minor allele of the TGF-beta1 rs6957 SNP (p = 0.001) in subjects with COPD. Additionally, we found a significantly lower prevalence of the haplotype with the major allele of rs6957 and minor alleles for rs1800469 and rs1982073 SNPs in TGF-beta1 in subjects with COPD (p = 0.030), indicating that this association is due to the rs6957 SNP. TGF-beta1 SNPs were not associated with FEV1 decline. SNPs in decorin, and haplotypes constructed of both TGF-beta1 and decorin SNPs were not associated with development of COPD or with FEV1 decline.Our study shows for the first time that SNPs in decorin on its own or in interaction with SNPs in TGF-beta1 do not underlie the disturbed balance in expression between these genes in COPD. TGF-beta1 SNPs are associated with COPD, yet not with accelerated FEV1 decline in the general population.

partly overlapping individuals were genotyped, phenotyped and their data analyzed in genetic association studies, reflecting a huge communal effort by the substance use/addiction genetics community. These genome-wide association study (GWAS) efforts considered different stages of substance use: lifetime use (ever versus never use) was analyzed for cannabis and smoking, quantity of use (in users) was analyzed for coffee, alcohol, and smoking and age of initiation and cessation were analyzed for smoking. There are other GWA efforts and publications in the realm of addiction (see ref. 5), but here we limit ourselves to the largest meta-analyses per substance in order to maximize power. The GWA meta-analyses of substance-related traits identified many substance-specific genetic variants of moderate to small effect, which provided insight in the genetic etiology of substance use and its comorbidities. There are substantial phenotypic correlations among use of different substances, and both twin and polygenic risk prediction studies have shown that these phenotypic correlations are partly due to common genetic influences. 6, 7 Here we estimate genetic correlations (rg) between substance use-related variables based on the GWA summary statistics. These estimates of rg are based on all polygenic effects captured by single nucleotide polymorphisms. We used the recently developed linkage disequilibrium (LD) score regression method to estimate the proportion of covariance between traits that is due to single nucleotide polymorphisms, based on the expected relationship between LD and strength of association under a polygenic model. 8,9 The genetic correlation matrix revealed important information about common versus substance-specific genetic effects as well as specific patterns of cross-substance comorbidity (Figure 1). The substantial negative correlation between smoking cessation and smoking initiation reveals that the genes that predispose to initiation are negative predictors of success at cessation. Likewise, the genes that predispose individuals to smoke more cigarettes per day are negative predictors of successful cessation. Age at first cigarette is only associated with smoking initiation, not with cigarettes per day or smoking cessation. Interestingly, high genetic correlations are also observed across substance, between cannabis initiation and smoking initiation (rg=0.83, se=0.148), but also between quantity of nicotine consumption (cigarettes per day) and quantity of coffee consumed (cups per day) (rg=0.44, se=0.151), between coffee consumed and nicotine consumption (rg=0.38, se=0.16), and between alcohol consumption (alcohol per week) and cigarettes per day (rg=0.44, se=0.17). Most significant cross-substance correlations reflect genetic correlations within stage. However, both coffee per day and cigarettes per day are negatively associated with successful smoking cessation, indicating that frequent use, irrespective of substance, is genetically related to more problematic use of a different substance. The pattern of correlations observed implies a genetic model for substance use where both substance-specific and stagespecific genetic effects play a role. GWA meta-analyses of smoking, alcohol, cannabis and coffee use have shed light on the specific genetic effects for each substance. Here we show substance- and stage-specific GWAS results can be leveraged to elucidate the genetic architecture of substance use vulnerability in general. The next generation of large well-powered substance use GWA studies should systematically target all stages of use, for a broad spectrum of substances (e.g., cocaine and sugar rich foods) or addictive behavior (e.g., gambling, gaming and compulsive Internet use). Such an effort can aid in distinguishing between genes that are substance specific from genes that contribute to a specific stage of use, irrespective of substance or addictive behavior.

There is ongoing debate on the association between eosinophil count and diseases, as previous studies were inconsistent. We studied the relationship of eosinophil count with 22 complex metabolic, cardiac, and pulmonary traits and diseases. From the population-based LifeLines Cohort Study (N = 167,729), 13,301 individuals were included. We focused on relationship of eosinophil count with three classes of metabolic (7 traits, 2 diseases), cardiac (6 traits, 2 diseases), and pulmonary (2 traits, 2 diseases) outcomes. Regression analyses were applied in overall, women and men, while adjusted for age, sex, BMI and smoking. A p-value of <0.00076 was considered statistically significant. 58.2% of population were women (mean±SD 51.3±11.1 years old). In overall, one-SD higher of ln-eosinophil count was associated with a 0.04 (±SE ±0.002;p = 6.0×10−6) SD higher levels in ln-BMI, 0.06 (±0.007;p = 3.1×10−12) SD in ln-TG, 0.04 (±0.003;p = 7.0×10−6) SD in TC, 0.04 (±0.004;p = 6.3×10−7) SD in LDL, 0.04 (±0.006;p = 6.0×10−6) SD in HbA1c; and with a 0.05 (±0.004;p = 1.7×10−8) SD lower levels in HDL, 0.05 (±0.007;p = 3.4×10−23) SD in FEV1, and 0.09 (±0.001;p = 6.6×10−28) SD in FEV1/FVC. A higher ln-eosinophil count was associated with 1.18 (95%CI 1.09–1.28;p = 2.0×10−5) odds ratio of obesity, 1.29 (1.19–1.39;p = 1.1×10−10) of metabolic syndrome, 1.40 (1.25–1.56;p = 2.7×10−9) of COPD and 1.81 (1.61–2.03;p = 1.0×10−23) of asthma. Similar results were found in women. We found no association between ln-eosinophil count either with blood pressure indices in overall, women and men; or with BMI, LDL, HbA1c and obesity in men. In a large population based cohort, we confirmed eosinophil count as a potential factor implicated in metabolic and pulmonary outcomes.

To investigate whether routine testing for Epstein-Barr virus (EBV) is necessary in the examination of a patient with uveitis.Intraocular EBV DNA was determined in 183 ocular fluid samples taken from patients with AIDS and uveitis, HIV negative immunocompromised uveitis, acute retinal necrosis, toxoplasma chorioretinitis, intraocular lymphoma, anterior uveitis, and miscellaneous uveitis of unknown cause. In 82 samples from this group of patients paired serum/ocular fluid analysis was performed to detect local antibody production against EBV. Controls (n = 46) included ocular fluid samples taken during surgery for diabetic retinopathy, macular pucker, or cataract.Serum antibody titres to EBV capsid antigen proved to be significantly increased in HIV negative immunocompromised patients with uveitis (p < 0.01) compared with controls. Local antibody production revealed only three positive cases out of 82 patients tested, two results were borderline positive and one patient had uveitis caused by VZV. EBV DNA was detected in three out of 46 control ocular fluid samples. In the different uveitis groups EBV DNA was noted, but was not significantly higher than in the controls, except in six out of 11 HIV negative immunocompromised patients (p = 0.0008). In four out of these six cases another infectious agent (VZV, HSV, CMV, or Toxoplasma gondii) had previously been identified as the cause of the uveitis.When comparing various groups of uveitis patients, EBV DNA was found more often in HIV negative immunocompromised patients with uveitis. Testing for EBV does not have to be included in the routine management of patients with uveitis, since indications for an important role of this virus were not found in the pathogenesis of intraocular inflammation.

Purpose To describe a case of acute retinal necrosis with concurrent encephalitis and determine the causative virus. The patient had a history of presumed acute retinal necrosis in the left eye at the age of 8 years and recurrent genital herpes. Methods Diagnostic anterior chamber puncture of the eye and lumbar puncture for laboratory analysis. Results Polymerase chain reaction identified herpes simplex virus type 2 in the eye, and local antibody production to herpes simplex virus was demonstrated in the aqueous of this eye and in the cerebrospinal fluid. Conclusion Herpes simplex virus type 2 may cause bilateral acute retinal necrosis with long delay of fellow eye involvement and concurrent encephalitis. To describe a case of acute retinal necrosis with concurrent encephalitis and determine the causative virus. The patient had a history of presumed acute retinal necrosis in the left eye at the age of 8 years and recurrent genital herpes. Diagnostic anterior chamber puncture of the eye and lumbar puncture for laboratory analysis. Polymerase chain reaction identified herpes simplex virus type 2 in the eye, and local antibody production to herpes simplex virus was demonstrated in the aqueous of this eye and in the cerebrospinal fluid. Herpes simplex virus type 2 may cause bilateral acute retinal necrosis with long delay of fellow eye involvement and concurrent encephalitis.

BackgroundAsthma and allergic phenotypes are complex genetic diseases with known linkage to chromosome 5q. This region has many candidate genes, including serine protease inhibitor Kazal type 5 (SPINK5), which has been associated with asthma and atopic dermatitis in family-based studies of children with atopic dermatitis.ObjectiveWe sought to investigate whether single nucleotide polymorphisms in SPINK5 are associated with asthma, atopic phenotypes, and atopic dermatitis.MethodsWe investigated whether single nucleotide polymorphisms in SPINK5 (ie, −785 A/G, Asn368Ser, and Lys420Glu) are associated with asthma, atopic phenotypes, and atopic dermatitis in 200 families ascertained by a proband with asthma (nonaffected spouses served as a matched control population) and an independent set of 252 trios with asthma.ResultsWe found no association with asthma, atopic phenotypes, and atopic dermatitis after correction for multiple testing.ConclusionThe negative results in this study suggest that SPINK5 is not associated with asthma or atopic phenotypes in individuals ascertained by a proband with asthma. This is consistent with the finding that SPINK5 is not expressed in the lung. Because our patients were ascertained for asthma, a role of SPINK5 in atopic dermatitis cannot be excluded. Asthma and allergic phenotypes are complex genetic diseases with known linkage to chromosome 5q. This region has many candidate genes, including serine protease inhibitor Kazal type 5 (SPINK5), which has been associated with asthma and atopic dermatitis in family-based studies of children with atopic dermatitis. We sought to investigate whether single nucleotide polymorphisms in SPINK5 are associated with asthma, atopic phenotypes, and atopic dermatitis. We investigated whether single nucleotide polymorphisms in SPINK5 (ie, −785 A/G, Asn368Ser, and Lys420Glu) are associated with asthma, atopic phenotypes, and atopic dermatitis in 200 families ascertained by a proband with asthma (nonaffected spouses served as a matched control population) and an independent set of 252 trios with asthma. We found no association with asthma, atopic phenotypes, and atopic dermatitis after correction for multiple testing. The negative results in this study suggest that SPINK5 is not associated with asthma or atopic phenotypes in individuals ascertained by a proband with asthma. This is consistent with the finding that SPINK5 is not expressed in the lung. Because our patients were ascertained for asthma, a role of SPINK5 in atopic dermatitis cannot be excluded.

Genetic pleiotropy may contribute to the clustering of obesity and metabolic conditions. We assessed whether genetic variants that are robustly associated with BMI and waist-to-hip ratio (WHR) also influence metabolic and cardiovascular traits, independently of obesity-related traits, in meta-analyses of up to 37,874 individuals from six European population-based studies.We examined associations of 32 BMI and 14 WHR loci, individually and combined in two genetic predisposition scores (GPSs), with glycaemic traits, blood lipids and BP, with and without adjusting for BMI and/or WHR.We observed significant associations of BMI-increasing alleles at five BMI loci with lower levels of 2 h glucose (RBJ [also known as DNAJC27], QPTCL: effect sizes -0.068 and -0.107 SD, respectively), HDL-cholesterol (SLC39A8: -0.065 SD, MTCH2: -0.039 SD), and diastolic BP (SLC39A8: -0.069 SD), and higher and lower levels of LDL- and total cholesterol (QPTCL: 0.041 and 0.042 SDs, respectively, FLJ35779 [also known as POC5]: -0.042 and -0.041 SDs, respectively) (all p < 2.4 × 10(-4)), independent of BMI. The WHR-increasing alleles at two WHR loci were significantly associated with higher proinsulin (GRB14: 0.069 SD) and lower fasting glucose levels (CPEB4: -0.049 SD), independent of BMI and WHR. A higher GPS-BMI was associated with lower systolic BP (-0.005 SD), diastolic BP (-0.006 SD) and 2 h glucose (-0.013 SD), while a higher GPS-WHR was associated with lower HDL-cholesterol (-0.015 SD) and higher triacylglycerol levels (0.014 SD) (all p < 2.9 × 10(-3)), independent of BMI and/or WHR.These pleiotropic effects of obesity-susceptibility loci provide novel insights into mechanisms that link obesity with metabolic abnormalities.

BRCA1 and BRCA2 germline mutations account for <5% of breast cancer cases. Less penetrant breast cancer susceptibility genes are likely to exist. Earlier studies have suggested involvement of the HLA region. The HLA region was genotyped with 24 microsatellite markers and markers for two single nucleotide polymorphisms (SNPs) in TNF α and TNF β, in germline DNA from 956 breast cancer patients and 1271 family-based controls. Association analyses and the haplotype sharing statistic (HSS) were used to search for differences in haplotype sharing between patients and controls. Based on criteria known to influence genetic breast cancer risk, patients were divided into groups of high, moderate and low risk. The HSS revealed a significant difference in mean haplotype sharing between patients and controls for four consecutive markers (D6S2671, TNFa, D6S2672 and MICA), the highest being at D6S2671 ( P =0.017). Subgroup analyses showed that moderate-risk patients were responsible for this difference, with the strongest association for D6S2672 ( P =0.0009). A single haplotype was more frequent and longer in moderate-risk patients than in controls. The results were confirmed with association analyses. Individuals homozygous for haplotype 110–184 (D6S2672-MICA) were observed in 9.0% of moderate-risk patients and 1.5% of controls [odds ratio (OR)=7.14], while heterozygotes were at a lower risk (OR=1.41), suggesting a recessive effect. No association was observed between the two SNPs in TNF α (−308) and TNF β (intron 1) and breast cancer risk. The results reveal a potential role of the HLA class III subregion in susceptibility to breast cancer in patients at moderate familial risk.

The psoriasis susceptibility locus 1 (PSORS1) mutation is assumed to reside within a region around human leukocyte antigen-C spanning 250 kb, termed risk haplotype (RH) 1/2. By re-analyzing a published data set with a previously developed method, the haplotype sharing statistic, we confirm localization of PSORS1 to the RH1 region and refine its location to marker M6S168. We replicate this result in an independent patient sample. The target region harbors fragments of a human endogenous retrovirus K (HERV-K) endogenous retrovirus. Two single-nucleotide polymorphisms with alleles differing between high- and low-risk haplotypes are located within the HERV-K dUTPase. One of these encodes a predicted non-conserved Glu-Arg exchange. The HERV-K dUTPase is expressed in peripheral blood and in normal as well as lesional psoriatic skin. Our results indicate that an endogenous retroviral dUTPase constitutes a candidate gene for the PSORS1 mutation.

To the Editor:Worldwide, millions of patients are affected by asthma, an inflammatory airway disease. Corticosteroids suppress virtually every step of the inflammatory cascade and constitute the cornerstone of asthma treatment. Their beneficial effects on a group level are well accepted, but the individual response to inhaled corticosteroids (ICSs) varies widely. The latter might be due to an underlying genetic background, as suggested by Tantisira and colleagues.1Tantisira K.G. Lake S. Silverman E.S. Palmer L.J. Lazarus R. Silverman E.K. et al.Corticosteroid pharmacogenetics: association of sequence variants in CRHR1 with improved lung function in asthmatics treated with inhaled corticosteroids.Hum Mol Genet. 2004; 13: 1353-1359Crossref PubMed Scopus (277) Google Scholar, 2Weiss S.T. Lake S.L. Silverman E.S. Silverman E.K. Richter B. Drazen J.M. et al.Asthma steroid pharmacogenetics. A study strategy to identify replicated treatment responses.Proc Am Thorac Soc. 2004; 1: 364-367Crossref PubMed Scopus (35) Google Scholar They have shown that 3 haplotype-tagging single nucleotide polymorphisms (SNPs) in the corticotropin-releasing hormone receptor 1 gene (CRHR1) are associated with lung function improvement after 8 weeks' treatment with ICSs in patients with asthma.1Tantisira K.G. Lake S. Silverman E.S. Palmer L.J. Lazarus R. Silverman E.K. et al.Corticosteroid pharmacogenetics: association of sequence variants in CRHR1 with improved lung function in asthmatics treated with inhaled corticosteroids.Hum Mol Genet. 2004; 13: 1353-1359Crossref PubMed Scopus (277) Google Scholar, 2Weiss S.T. Lake S.L. Silverman E.S. Silverman E.K. Richter B. Drazen J.M. et al.Asthma steroid pharmacogenetics. A study strategy to identify replicated treatment responses.Proc Am Thorac Soc. 2004; 1: 364-367Crossref PubMed Scopus (35) Google Scholar If this association would last over many years, it might open avenues for pharmacogenetic long-term management of asthma.We assessed, in an asthma cohort followed for 22 years, whether the effects of ICS treatment on the immediate improvement in lung function and long-term decrease in lung function were associated with the 3 previously described CRHR1 SNPs.We evaluated 281 patients given diagnoses of symptomatic asthma who were initially studied in 1962 through 1975 and re-examined in 1991 through 1999.3Xu J. Postma D.S. Howard T.D. Koppelman G.H. Zheng S.L. Stine O.C. et al.Major genes regulating total serum immunoglobulin E levels in families with asthma.Am J Hum Genet. 2000; 67: 1163-1173Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar All were younger than 45 years and had airway hyperresponsiveness to histamine.3Xu J. Postma D.S. Howard T.D. Koppelman G.H. Zheng S.L. Stine O.C. et al.Major genes regulating total serum immunoglobulin E levels in families with asthma.Am J Hum Genet. 2000; 67: 1163-1173Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar After initial testing, participants had routine check-ups for asthma at least once a year. Lung function tests were performed with the same type of water-sealed spirometer throughout follow-up (Lode Spirograph D53; Lode Instruments, Groningen, The Netherlands). Data on lung function and corticosteroid use during check-ups were extracted from medical records, excluding lung function data during asthma exacerbations and pregnancies. The Medical Ethics Committee of the University Hospital Groningen approved this study. All participants provided written informed consent.We genotyped the 3 CRHR1 SNPs described by Tantisira et al1Tantisira K.G. Lake S. Silverman E.S. Palmer L.J. Lazarus R. Silverman E.K. et al.Corticosteroid pharmacogenetics: association of sequence variants in CRHR1 with improved lung function in asthmatics treated with inhaled corticosteroids.Hum Mol Genet. 2004; 13: 1353-1359Crossref PubMed Scopus (277) Google Scholar: rs1876828, rs242939, and rs242941 (TaqMan Universal PCR Master Mix and ABI prism 7900 HT, Applied Biosystems TaqMan SNP Genotyping Assays, Nieuwekerk aan de IJessel, The Netherlands).Linear mixed-effects models were used to investigate the effect of CRHR1 SNPs on annual decrease in FEV1. The age of 30 years was the starting point for analyses of the decrease in FEV1. Subjects had to have more than 2 FEV1 measurements over more than 2 years to be included in the analyses. Annual FEV1 decrease was estimated for the different genotypes by including genotype, time, and their interaction in the analyses. Individual variation in these decreases was accounted for by estimating random effects for this variable. Only subjects who started ICSs during follow-up were analyzed. To minimize potential bias by subjects stopping ICSs or using ICSs intermittently, we included only data on lung function in the first period with continuous ICS use. Interactions between SNPs and the use of ICSs were included in the model to investigate whether the effects of ICSs on the change in level of FEV1 (immediate effect), rate of decline in FEV1 (long-term effect), or both differed between the genotypes. Explanatory variables were sex, height, the first available FEV1 measurement after age 30 years (centered at 2.8 L), pack-years smoking, and oral corticosteroid and ICS use over time and their interaction with time. Oral steroid use was accounted for only on the FEV1 level (as a time-varying variable) because oral steroids were often used intermittently, which makes it impossible to estimate their effect on FEV1 decrease.Of the 281 individuals, 68 were excluded from analyses because they had insufficient data on FEV1 or missing lung function records, incomplete or unknown data on smoking history, or intermittent ICS use. Of the remaining 213 patients, 164 had data on CRHR1 genotyping (Table I), and the median number of FEV1 measurements per individual was 25 (interquartile range [IQR], 8-43). There existed no demographic differences between subjects with different CRHR1 genotypes.Table IPopulation characteristicsMale/female sex (n)96/72No. of corticosteroid users (n [%])98 (60)Age of onset of symptoms (y)6 (3-21)Mean daily dose of ICSs (μg/d)∗Calculated for individuals who ever used ICSs only. The dose of ICSs was calculated to an equivalent daily dose of beclomethasone, with 500 μg of fluticasone, 1000 μg of budesonide, and 500 μg of budesonide administered through a Turbuhaler being equipotent to 1000 μg of beclomethasone.749 (426-1152)Duration of use of ICSs (y)13.0 (7-19)Visit 1962-1975Visit 1990-1999Age (y)27 (20-35)53 (46-60)FEV1 % predicted after BD (%)89 (70-99)82 (66-100)Reversibility (% predicted)23 (16-31)12 (7-17)AHR ≤16.0 mg/mL (%)†Percentage of all subjects with an airway hyperresponsiveness of less than 16 mg/mL histamine. All subjects had airway hyperresponsiveness (≤32 mg/mL histamine).8779≥1 Positive skin test result (%)9180Blood eosinophils (× 107/L)35 (22-54)12 (7-21)Smoking Pack-years smoking0.3 (0.0-6.3)4.5 (0.0-15.6) Nonsmoker/exsmoker/current smoker (%)51/10/4042/34/25Data are presented as median values (IQR) unless stated otherwise.BD, Bronchodilator; AHR, airway hyperresponsiveness to histamine.∗ Calculated for individuals who ever used ICSs only. The dose of ICSs was calculated to an equivalent daily dose of beclomethasone, with 500 μg of fluticasone, 1000 μg of budesonide, and 500 μg of budesonide administered through a Turbuhaler being equipotent to 1000 μg of beclomethasone.† Percentage of all subjects with an airway hyperresponsiveness of less than 16 mg/mL histamine. All subjects had airway hyperresponsiveness (≤32 mg/mL histamine). Open table in a new tab Within the period of analysis on the course of FEV1, 98 of the 164 included subjects started using ICSs. The median time period without and with ICSs was 11.2 (IQR, 7-22) and 13.0 (IQR, 7-19) years, respectively. There was no significant association of the CRHR1 polymorphisms with FEV1 decrease, neither in all subjects before the start of ICS use (results not shown) nor during ICS use. The effect of ICS use was not significantly different between CRHR1 genotypes with respect to both the immediate effect on FEV1 level (effect within 3-6 months) and the long-term effect of ICSs on the decrease in FEV1 (Fig 1). Estimates and 95% CIs of the differences between genotypes in the effect of ICSs on the change in FEV1 decrease are presented in Table II.Table IIThe effect of ICS use per genotype on the mean annual change in lung function (long-term effect) compared with no ICS use and differences in these effects between genotypes in human CRHR1 polymorphismsMean annual FEV1 change (mL/y), 95% CICRHR1 polymorphismsChange in annual FEV1 during ICS use compared with no ICS useDifferences in these effects between genotypes∗Wild-type as the reference.rs1876828CC27.2(4.9 to 49.4)CT34.1(4.0 to 64.1)7.0(−25.5 to 39.6)TT31.4(−32.8 to 95.5)4.4(−60.8 to 69.5)rs242939AA37.1(17.0 to 57.2)GA4.0(−37.6 to 45.7)−33.0(−77.3 to 11,3)GG−9.3(−86.2 to 67.5)−46.4(−124.2 to 31.4)rs242941CC33.9(5.9 to 61.9)CA26.4(3.3 to 49.6)−7.4(−40.0 to 25.1)AA25.9(−14.4 to 66.1)−8.1(−50.1 to 33.8)∗ Wild-type as the reference. Open table in a new tab The rs242939 SNP showed the largest difference in effect of ICS use on the change in FEV1 decrease. Pooling the heterozygous and homozygous subjects for this SNP is justified given the absence of change in FEV1 decrease by ICS use in patients having at least 1 mutant allele. The estimated difference in this change in FEV1 decrease compared with the wild-type was −34.1 mL/y (95% CI, −73.1 to 4.9 mL/y), which is almost significant (P = .087).Corticotropin-releasing hormone binds to CRHR1, which exerts indirect anti-inflammatory effects through ACTH-induced cortisol production and direct proinflammatory effects through, for example, mast cell degranulation.4Agelaki S. Tsatsanis C. Gravanis A. Margioris A.N. Corticotropin-releasing hormone augments proinflammatory cytokine production from macrophages in vitro and in lipopolysaccharide-induced endotoxin shock in mice.Infect Immun. 2002; 70: 6068-6074Crossref PubMed Scopus (133) Google Scholar, 5Theoharides T.C. Singh L.K. Boucher W. Pang X. Letourneau R. Webster E. et al.Corticotropin-releasing hormone induces skin mast cell degranulation and increased vascular permeability, a possible explanation for its proinflammatory effects.Endocrinology. 1998; 139: 403-413Crossref PubMed Google Scholar SNPs in this gene might thus affect airway inflammation and treatment effects of ICSs in asthma. Three variants in CRHR1 have been previously shown to be associated with improvement in FEV1 after 8 weeks' ICS treatment in childhood and adult asthma.1Tantisira K.G. Lake S. Silverman E.S. Palmer L.J. Lazarus R. Silverman E.K. et al.Corticosteroid pharmacogenetics: association of sequence variants in CRHR1 with improved lung function in asthmatics treated with inhaled corticosteroids.Hum Mol Genet. 2004; 13: 1353-1359Crossref PubMed Scopus (277) Google Scholar, 2Weiss S.T. Lake S.L. Silverman E.S. Silverman E.K. Richter B. Drazen J.M. et al.Asthma steroid pharmacogenetics. A study strategy to identify replicated treatment responses.Proc Am Thorac Soc. 2004; 1: 364-367Crossref PubMed Scopus (35) Google Scholar We investigated adult asthmatic subjects who were followed for more than 20 years, a population in which we previously have shown that ICS treatment could reduce long-term lung function decrease in a dose-dependent way.6Dijkstra A. Vonk J.M. Jongepier H. Koppelman G.H. Schouten J.P. ten Hacken N.H.T. et al.Lung function decline in asthma: association with inhaled corticosteroids, smoking and sex.Thorax. 2006; 61: 105-110Crossref PubMed Scopus (160) Google Scholar We found no association of the 3 CRHR1 variants with long-term lung function decrease (both with and without the use of ICSs). Importantly, there was also no significant association with the immediate effects of ICS treatment on lung function in the same asthmatic patients. These findings suggest that variability of long-term improvement and preservation of lung function with corticosteroid treatment cannot simply be predicted by genotyping CRHR1 variants in asthmatic patients.Our study seems to contradict the work of Tantisira and colleagues.1Tantisira K.G. Lake S. Silverman E.S. Palmer L.J. Lazarus R. Silverman E.K. et al.Corticosteroid pharmacogenetics: association of sequence variants in CRHR1 with improved lung function in asthmatics treated with inhaled corticosteroids.Hum Mol Genet. 2004; 13: 1353-1359Crossref PubMed Scopus (277) Google Scholar, 2Weiss S.T. Lake S.L. Silverman E.S. Silverman E.K. Richter B. Drazen J.M. et al.Asthma steroid pharmacogenetics. A study strategy to identify replicated treatment responses.Proc Am Thorac Soc. 2004; 1: 364-367Crossref PubMed Scopus (35) Google Scholar However, Tantisira and colleagues showed significant effects of CRHR1 variants on the short-term increase in FEV1 level with ICS use and demonstrated the greatest effects in their pediatric cohort. We did not find this effect in our adult asthmatic subjects and extend these observations by investigating long-term effects of ICS use on FEV1 decrease, which can only be investigated in adults.We are aware that our study is observational in nature and that there is a possibility that the lack of significance of CRHR1 variants on corticosteroid response can be spurious. However, we believe the outcome to be of relevance given the large number of FEV1 measurements per individual and hence accurate analyses of longitudinal FEV1 decrease. Moreover, our previous analyses on a disintegrin and metalloprotease gene (ADAM33) variants in association with FEV1 decrease in the same asthmatic patients contained even fewer subjects with a homozygous ADAM33 genotype, and these findings were replicated in the general population.7Jongepier H. Boezen H.M. Dijkstra A. Howard T.D. Vonk J.M. Koppelman G.H. et al.Polymorphisms of the ADAM33 gene are associated with accelerated lung function decline in asthma.Clin Exp Allergy. 2004; 34: 757-760Crossref PubMed Scopus (184) Google Scholar, 8van Diemen C.C. Postma D.S. Vonk J.M. Bruinenberg M. Schouten J.P. Boezen H.M. A disintegrin and metalloprotease 33 polymorphisms and lung function decline in the general population.Am J Respir Crit Care Med. 2005; 172: 329-333Crossref PubMed Scopus (180) Google Scholar Therefore we believe that the lack of significance might not be due to inadequate power.We studied the same 3 SNPs as Tantisira et al1Tantisira K.G. Lake S. Silverman E.S. Palmer L.J. Lazarus R. Silverman E.K. et al.Corticosteroid pharmacogenetics: association of sequence variants in CRHR1 with improved lung function in asthmatics treated with inhaled corticosteroids.Hum Mol Genet. 2004; 13: 1353-1359Crossref PubMed Scopus (277) Google Scholar that were positively associated with lung function improvement in interaction with ICS treatment. Because the SNPs cover about 10% of the total variation in CRHR1, we cannot rule out that other SNPs in CRHR1 or genes in the vicinity are associated with ICS effects on lung function improvement in our patients. However, the magnitude of pharmacogenetic effects of scarce variants in genes, such as the CRHR1 variants, are likely small or less common and therefore would not have sufficient magnitude to affect our clinical approach to individual asthma therapy.9Wechsler M.E. Managing asthma in the 21st century: role of pharmacogenetics.Pediatr Ann. 2006; 35 (664-9): 660-662PubMed Google ScholarIt would be of interest to analyze the effect of homozygosity of the GAT haplotype described by Tantisira and colleagues.1Tantisira K.G. Lake S. Silverman E.S. Palmer L.J. Lazarus R. Silverman E.K. et al.Corticosteroid pharmacogenetics: association of sequence variants in CRHR1 with improved lung function in asthmatics treated with inhaled corticosteroids.Hum Mol Genet. 2004; 13: 1353-1359Crossref PubMed Scopus (277) Google Scholar, 2Weiss S.T. Lake S.L. Silverman E.S. Silverman E.K. Richter B. Drazen J.M. et al.Asthma steroid pharmacogenetics. A study strategy to identify replicated treatment responses.Proc Am Thorac Soc. 2004; 1: 364-367Crossref PubMed Scopus (35) Google Scholar In our study only 9 subjects had 2 GAT copies, and these subjects showed the largest reduction in FEV1 decrease with ICS use (44.5 mL/y less decline than without ICS use (95% CI, −7.3 to 96.3 mL/y). Subjects with 1 or 0 GAT copies had smaller and nonsignificant effects.Our data suggest that CRHR1 polymorphisms are not associated with immediate or long-term improvement in FEV1 by ICSs or with prevention of accelerated FEV1 decrease in adult asthma. To the Editor: Worldwide, millions of patients are affected by asthma, an inflammatory airway disease. Corticosteroids suppress virtually every step of the inflammatory cascade and constitute the cornerstone of asthma treatment. Their beneficial effects on a group level are well accepted, but the individual response to inhaled corticosteroids (ICSs) varies widely. The latter might be due to an underlying genetic background, as suggested by Tantisira and colleagues.1Tantisira K.G. Lake S. Silverman E.S. Palmer L.J. Lazarus R. Silverman E.K. et al.Corticosteroid pharmacogenetics: association of sequence variants in CRHR1 with improved lung function in asthmatics treated with inhaled corticosteroids.Hum Mol Genet. 2004; 13: 1353-1359Crossref PubMed Scopus (277) Google Scholar, 2Weiss S.T. Lake S.L. Silverman E.S. Silverman E.K. Richter B. Drazen J.M. et al.Asthma steroid pharmacogenetics. A study strategy to identify replicated treatment responses.Proc Am Thorac Soc. 2004; 1: 364-367Crossref PubMed Scopus (35) Google Scholar They have shown that 3 haplotype-tagging single nucleotide polymorphisms (SNPs) in the corticotropin-releasing hormone receptor 1 gene (CRHR1) are associated with lung function improvement after 8 weeks' treatment with ICSs in patients with asthma.1Tantisira K.G. Lake S. Silverman E.S. Palmer L.J. Lazarus R. Silverman E.K. et al.Corticosteroid pharmacogenetics: association of sequence variants in CRHR1 with improved lung function in asthmatics treated with inhaled corticosteroids.Hum Mol Genet. 2004; 13: 1353-1359Crossref PubMed Scopus (277) Google Scholar, 2Weiss S.T. Lake S.L. Silverman E.S. Silverman E.K. Richter B. Drazen J.M. et al.Asthma steroid pharmacogenetics. A study strategy to identify replicated treatment responses.Proc Am Thorac Soc. 2004; 1: 364-367Crossref PubMed Scopus (35) Google Scholar If this association would last over many years, it might open avenues for pharmacogenetic long-term management of asthma. We assessed, in an asthma cohort followed for 22 years, whether the effects of ICS treatment on the immediate improvement in lung function and long-term decrease in lung function were associated with the 3 previously described CRHR1 SNPs. We evaluated 281 patients given diagnoses of symptomatic asthma who were initially studied in 1962 through 1975 and re-examined in 1991 through 1999.3Xu J. Postma D.S. Howard T.D. Koppelman G.H. Zheng S.L. Stine O.C. et al.Major genes regulating total serum immunoglobulin E levels in families with asthma.Am J Hum Genet. 2000; 67: 1163-1173Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar All were younger than 45 years and had airway hyperresponsiveness to histamine.3Xu J. Postma D.S. Howard T.D. Koppelman G.H. Zheng S.L. Stine O.C. et al.Major genes regulating total serum immunoglobulin E levels in families with asthma.Am J Hum Genet. 2000; 67: 1163-1173Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar After initial testing, participants had routine check-ups for asthma at least once a year. Lung function tests were performed with the same type of water-sealed spirometer throughout follow-up (Lode Spirograph D53; Lode Instruments, Groningen, The Netherlands). Data on lung function and corticosteroid use during check-ups were extracted from medical records, excluding lung function data during asthma exacerbations and pregnancies. The Medical Ethics Committee of the University Hospital Groningen approved this study. All participants provided written informed consent. We genotyped the 3 CRHR1 SNPs described by Tantisira et al1Tantisira K.G. Lake S. Silverman E.S. Palmer L.J. Lazarus R. Silverman E.K. et al.Corticosteroid pharmacogenetics: association of sequence variants in CRHR1 with improved lung function in asthmatics treated with inhaled corticosteroids.Hum Mol Genet. 2004; 13: 1353-1359Crossref PubMed Scopus (277) Google Scholar: rs1876828, rs242939, and rs242941 (TaqMan Universal PCR Master Mix and ABI prism 7900 HT, Applied Biosystems TaqMan SNP Genotyping Assays, Nieuwekerk aan de IJessel, The Netherlands). Linear mixed-effects models were used to investigate the effect of CRHR1 SNPs on annual decrease in FEV1. The age of 30 years was the starting point for analyses of the decrease in FEV1. Subjects had to have more than 2 FEV1 measurements over more than 2 years to be included in the analyses. Annual FEV1 decrease was estimated for the different genotypes by including genotype, time, and their interaction in the analyses. Individual variation in these decreases was accounted for by estimating random effects for this variable. Only subjects who started ICSs during follow-up were analyzed. To minimize potential bias by subjects stopping ICSs or using ICSs intermittently, we included only data on lung function in the first period with continuous ICS use. Interactions between SNPs and the use of ICSs were included in the model to investigate whether the effects of ICSs on the change in level of FEV1 (immediate effect), rate of decline in FEV1 (long-term effect), or both differed between the genotypes. Explanatory variables were sex, height, the first available FEV1 measurement after age 30 years (centered at 2.8 L), pack-years smoking, and oral corticosteroid and ICS use over time and their interaction with time. Oral steroid use was accounted for only on the FEV1 level (as a time-varying variable) because oral steroids were often used intermittently, which makes it impossible to estimate their effect on FEV1 decrease. Of the 281 individuals, 68 were excluded from analyses because they had insufficient data on FEV1 or missing lung function records, incomplete or unknown data on smoking history, or intermittent ICS use. Of the remaining 213 patients, 164 had data on CRHR1 genotyping (Table I), and the median number of FEV1 measurements per individual was 25 (interquartile range [IQR], 8-43). There existed no demographic differences between subjects with different CRHR1 genotypes. Data are presented as median values (IQR) unless stated otherwise. BD, Bronchodilator; AHR, airway hyperresponsiveness to histamine. Within the period of analysis on the course of FEV1, 98 of the 164 included subjects started using ICSs. The median time period without and with ICSs was 11.2 (IQR, 7-22) and 13.0 (IQR, 7-19) years, respectively. There was no significant association of the CRHR1 polymorphisms with FEV1 decrease, neither in all subjects before the start of ICS use (results not shown) nor during ICS use. The effect of ICS use was not significantly different between CRHR1 genotypes with respect to both the immediate effect on FEV1 level (effect within 3-6 months) and the long-term effect of ICSs on the decrease in FEV1 (Fig 1). Estimates and 95% CIs of the differences between genotypes in the effect of ICSs on the change in FEV1 decrease are presented in Table II. The rs242939 SNP showed the largest difference in effect of ICS use on the change in FEV1 decrease. Pooling the heterozygous and homozygous subjects for this SNP is justified given the absence of change in FEV1 decrease by ICS use in patients having at least 1 mutant allele. The estimated difference in this change in FEV1 decrease compared with the wild-type was −34.1 mL/y (95% CI, −73.1 to 4.9 mL/y), which is almost significant (P = .087). Corticotropin-releasing hormone binds to CRHR1, which exerts indirect anti-inflammatory effects through ACTH-induced cortisol production and direct proinflammatory effects through, for example, mast cell degranulation.4Agelaki S. Tsatsanis C. Gravanis A. Margioris A.N. Corticotropin-releasing hormone augments proinflammatory cytokine production from macrophages in vitro and in lipopolysaccharide-induced endotoxin shock in mice.Infect Immun. 2002; 70: 6068-6074Crossref PubMed Scopus (133) Google Scholar, 5Theoharides T.C. Singh L.K. Boucher W. Pang X. Letourneau R. Webster E. et al.Corticotropin-releasing hormone induces skin mast cell degranulation and increased vascular permeability, a possible explanation for its proinflammatory effects.Endocrinology. 1998; 139: 403-413Crossref PubMed Google Scholar SNPs in this gene might thus affect airway inflammation and treatment effects of ICSs in asthma. Three variants in CRHR1 have been previously shown to be associated with improvement in FEV1 after 8 weeks' ICS treatment in childhood and adult asthma.1Tantisira K.G. Lake S. Silverman E.S. Palmer L.J. Lazarus R. Silverman E.K. et al.Corticosteroid pharmacogenetics: association of sequence variants in CRHR1 with improved lung function in asthmatics treated with inhaled corticosteroids.Hum Mol Genet. 2004; 13: 1353-1359Crossref PubMed Scopus (277) Google Scholar, 2Weiss S.T. Lake S.L. Silverman E.S. Silverman E.K. Richter B. Drazen J.M. et al.Asthma steroid pharmacogenetics. A study strategy to identify replicated treatment responses.Proc Am Thorac Soc. 2004; 1: 364-367Crossref PubMed Scopus (35) Google Scholar We investigated adult asthmatic subjects who were followed for more than 20 years, a population in which we previously have shown that ICS treatment could reduce long-term lung function decrease in a dose-dependent way.6Dijkstra A. Vonk J.M. Jongepier H. Koppelman G.H. Schouten J.P. ten Hacken N.H.T. et al.Lung function decline in asthma: association with inhaled corticosteroids, smoking and sex.Thorax. 2006; 61: 105-110Crossref PubMed Scopus (160) Google Scholar We found no association of the 3 CRHR1 variants with long-term lung function decrease (both with and without the use of ICSs). Importantly, there was also no significant association with the immediate effects of ICS treatment on lung function in the same asthmatic patients. These findings suggest that variability of long-term improvement and preservation of lung function with corticosteroid treatment cannot simply be predicted by genotyping CRHR1 variants in asthmatic patients. Our study seems to contradict the work of Tantisira and colleagues.1Tantisira K.G. Lake S. Silverman E.S. Palmer L.J. Lazarus R. Silverman E.K. et al.Corticosteroid pharmacogenetics: association of sequence variants in CRHR1 with improved lung function in asthmatics treated with inhaled corticosteroids.Hum Mol Genet. 2004; 13: 1353-1359Crossref PubMed Scopus (277) Google Scholar, 2Weiss S.T. Lake S.L. Silverman E.S. Silverman E.K. Richter B. Drazen J.M. et al.Asthma steroid pharmacogenetics. A study strategy to identify replicated treatment responses.Proc Am Thorac Soc. 2004; 1: 364-367Crossref PubMed Scopus (35) Google Scholar However, Tantisira and colleagues showed significant effects of CRHR1 variants on the short-term increase in FEV1 level with ICS use and demonstrated the greatest effects in their pediatric cohort. We did not find this effect in our adult asthmatic subjects and extend these observations by investigating long-term effects of ICS use on FEV1 decrease, which can only be investigated in adults. We are aware that our study is observational in nature and that there is a possibility that the lack of significance of CRHR1 variants on corticosteroid response can be spurious. However, we believe the outcome to be of relevance given the large number of FEV1 measurements per individual and hence accurate analyses of longitudinal FEV1 decrease. Moreover, our previous analyses on a disintegrin and metalloprotease gene (ADAM33) variants in association with FEV1 decrease in the same asthmatic patients contained even fewer subjects with a homozygous ADAM33 genotype, and these findings were replicated in the general population.7Jongepier H. Boezen H.M. Dijkstra A. Howard T.D. Vonk J.M. Koppelman G.H. et al.Polymorphisms of the ADAM33 gene are associated with accelerated lung function decline in asthma.Clin Exp Allergy. 2004; 34: 757-760Crossref PubMed Scopus (184) Google Scholar, 8van Diemen C.C. Postma D.S. Vonk J.M. Bruinenberg M. Schouten J.P. Boezen H.M. A disintegrin and metalloprotease 33 polymorphisms and lung function decline in the general population.Am J Respir Crit Care Med. 2005; 172: 329-333Crossref PubMed Scopus (180) Google Scholar Therefore we believe that the lack of significance might not be due to inadequate power. We studied the same 3 SNPs as Tantisira et al1Tantisira K.G. Lake S. Silverman E.S. Palmer L.J. Lazarus R. Silverman E.K. et al.Corticosteroid pharmacogenetics: association of sequence variants in CRHR1 with improved lung function in asthmatics treated with inhaled corticosteroids.Hum Mol Genet. 2004; 13: 1353-1359Crossref PubMed Scopus (277) Google Scholar that were positively associated with lung function improvement in interaction with ICS treatment. Because the SNPs cover about 10% of the total variation in CRHR1, we cannot rule out that other SNPs in CRHR1 or genes in the vicinity are associated with ICS effects on lung function improvement in our patients. However, the magnitude of pharmacogenetic effects of scarce variants in genes, such as the CRHR1 variants, are likely small or less common and therefore would not have sufficient magnitude to affect our clinical approach to individual asthma therapy.9Wechsler M.E. Managing asthma in the 21st century: role of pharmacogenetics.Pediatr Ann. 2006; 35 (664-9): 660-662PubMed Google Scholar It would be of interest to analyze the effect of homozygosity of the GAT haplotype described by Tantisira and colleagues.1Tantisira K.G. Lake S. Silverman E.S. Palmer L.J. Lazarus R. Silverman E.K. et al.Corticosteroid pharmacogenetics: association of sequence variants in CRHR1 with improved lung function in asthmatics treated with inhaled corticosteroids.Hum Mol Genet. 2004; 13: 1353-1359Crossref PubMed Scopus (277) Google Scholar, 2Weiss S.T. Lake S.L. Silverman E.S. Silverman E.K. Richter B. Drazen J.M. et al.Asthma steroid pharmacogenetics. A study strategy to identify replicated treatment responses.Proc Am Thorac Soc. 2004; 1: 364-367Crossref PubMed Scopus (35) Google Scholar In our study only 9 subjects had 2 GAT copies, and these subjects showed the largest reduction in FEV1 decrease with ICS use (44.5 mL/y less decline than without ICS use (95% CI, −7.3 to 96.3 mL/y). Subjects with 1 or 0 GAT copies had smaller and nonsignificant effects. Our data suggest that CRHR1 polymorphisms are not associated with immediate or long-term improvement in FEV1 by ICSs or with prevention of accelerated FEV1 decrease in adult asthma.

Asthma is characterised by chronic airway inflammation and remodelling, which can be (partially) suppressed by inhaled corticosteroids (ICSs). Plasminogen activator inhibitor-1, encoded by the SERPINE1 gene, is the key inhibitor of the plasminogen activator system, which affects tissue repair and remodelling. We studied associations between a functional SERPINE1 -675 4G/5G promoter polymorphism and asthma development, severity and response to ICSs. Longitudinal cohorts of 281 asthmatics and their nonasthmatic spouses, and the general population (n=1,390) were studied. No significant associations were found with asthma development and immunoglobulin (Ig)E levels, or with forced expiratory volume in 1 s (FEV₁) in nonasthmatic controls. Asthmatic subjects carrying the SERPINE1 5G allele had higher IgE and lower lung function levels at follow-up, lower maximally attained lung function levels, and faster lung function decline compared with individuals with the 4G/4G genotype. ICS treatment showed an immediate improvement in FEV₁ in asthmatics carrying the 5G allele. However, these asthmatics still had the fastest rate of FEV₁ decline after initiating ICS treatment. Finally, the 5G allele was associated with a lower prevalence of complete asthma remission at follow-up. These findings suggest that SERPINE1 is not an asthma susceptibility gene, but rather affects the severity, progression and long-term ICS response in asthma.

Type 1 interferon can trigger flares of psoriasis. Hypersensitivity to type 1 interferon signaling causes a psoriasis-like skin disease in mice deficient for the transcription factor interferon regulatory factor 2 (IRF2). The human IRF2 gene is located at a previously identified candidate psoriasis susceptibility locus on chromosome 4q (PSORS3 at D4S1535). Therefore, we tested association of psoriasis with IRF2. We generated a sample consisting of 157 families with a total of 521 individuals. Five novel microsatellite markers were developed and typed, and complemented with three known markers to yield a set of eight markers spaced within 600 kb around the IRF2 gene, three of which are located in the gene. We detected association of IRF2 with type 1 psoriasis at two markers in the IRF2 gene. Haplotype sharing analysis confirmed association of IRF2 with type 1 psoriasis (p=0.0017; p_corr=0.03). The 921G/A SNP in exon 9 was found to obliterate a predicted exon splice enhancer in an allele-specific manner. There was a suggestive increase of homozygosity for the splicing-deficient allele in type 1 psoriasis patients. Our data identify IRF2 as a potential susceptibility gene for psoriasis.

The CHEK2 1100delC mutation was recently identified as a low-penetrance breast cancer susceptibility allele. The mutation occurred more frequently in families with clustering of breast and colorectal cancers (CRCs) than in families with clustering of breast cancer only. Hence, the 1100delC mutation could also be a low-penetrance CRC susceptibility allele. To test this hypothesis, we examined the mutation in 629 unselected CRC cases, 230 controls, and 105 selected CRCs diagnosed in patients before age 50. The mutation was observed in 1.6% of unselected patients and in 0.3% of controls (Not significant (NS)). After stratifying unselected patients according to defined genetic risk (on the basis of age at diagnosis and family history of colorectal and endometrial cancer), the highest frequency was observed in high-risk patients (12.5%), followed by moderate-risk patients (3.3%), and was lowest in low-risk patients (1.0%, P(trend) 0.014). In selected patients, 1.6% carried the mutation (NS). Subgroup analyses for tumor localization, gender, and age at diagnosis did not reveal an association with the 1100delC genotype. In addition, a pooled analysis, combining data of one published study in unselected CRC cases and our study, also did not reveal an association. In conclusion, the frequency of the 1100delC genotype was neither significantly increased in unselected CRC patients nor in selected CRC patients diagnosed before age 50. However, after stratifying unselected CRC patients according to defined genetic risk, a significant trend of increasing frequency was observed. Together, the results are consistent with a low-penetrance effect (OR 1.5-2.0) of the CHEK2 1100delC on CRC risk. Large case-control studies are required to clarify the exact role of the CHEK2 1100delC mutation in CRC.

The renin-angiotensin system (RAS) is thought to play a major role in the pathophysiology of de-novo restenotic lesions and in-stent restenosis after percutaneous coronary intervention (PCI). Heme oxygenase-1 (HO-1), is thought to beneficially influence these processes. We examined the effect of pharmacologic as well as genetic RAS interactions on restenosis in a large population of consecutive patients undergoing PCI, and evaluated possible gene-gene interactions in both systems.The GENDER project is a multicenter prospective follow-up study, including 3146 patients after successful PCI. Genotyping in these patients was performed for the ACE gene insertion/deletion, the angiotensinogen 235Met/Thr, T174M and A(-6)G, the angiotensin-II type 1 receptor (AT1R) 1166A/C and T810A, the angiotensin-II type 2 receptor (AT2R) 1675G/A and 3123A polymorphisms and the length polymorphism in the HO-1 promoter region.A total of 3104 patients were followed for 10 months. In 2975 patients at least one of the nine genotypes could be determined. The AT1R 1166 CC genotype showed a significant association with TVR; the other polymorphisms did not. RAS-inhibitory drugs were not associated with the incidence of TVR, nor did they interact with any of the investigated polymorphisms. Patients with the ACE I/I polymorphism showed a trend towards a better outcome if they had a short number of repeats in the HO-1 promoter. This relationship was inversely present in carriers of the ACE D/D polymorphism.We could only establish a role for the AT1R 1166A/C polymorphism in restenosis after PCI. However, significant gene-gene interaction was suggested for the ACE gene and the HO-1 promotor. The RAS and HO-1 relation in restenosis merits further investigation.

To cite this article: Niedoszytko M, Bruinenberg M, van Doormaal JJ, de Monchy JGR, Nedoszytko B, Koppelman GH, Nawijn MC, Wijmenga C, Jassem E, Oude Elberink JNG. Gene expression analysis predicts insect venom anaphylaxis in indolent systemic mastocytosis. Allergy 2011; 66: 648–657. Background: Anaphylaxis to insect venom (Hymenoptera) is most severe in patients with mastocytosis and may even lead to death. However, not all patients with mastocytosis suffer from anaphylaxis. The aim of the study was to analyze differences in gene expression between patients with indolent systemic mastocytosis (ISM) and a history of insect venom anaphylaxis (IVA) compared to those patients without a history of anaphylaxis, and to determine the predictive use of gene expression profiling. Methods: Whole-genome gene expression analysis was performed in peripheral blood cells. Results: Twenty-two adults with ISM were included: 12 with a history of IVA and 10 without a history of anaphylaxis of any kind. Significant differences in single gene expression corrected for multiple testing were found for 104 transcripts (P < 0.05). Gene ontology analysis revealed that the differentially expressed genes were involved in pathways responsible for the development of cancer and focal and cell adhesion suggesting that the expression of genes related to the differentiation state of cells is higher in patients with a history of anaphylaxis. Based on the gene expression profiles, a naïve Bayes prediction model was built identifying patients with IVA. Conclusions: In ISM, gene expression profiles are different between patients with a history of IVA and those without. These findings might reflect a more pronounced mast cells dysfunction in patients without a history of anaphylaxis. Gene expression profiling might be a useful tool to predict the risk of anaphylaxis on insect venom in patients with ISM. Prospective studies are needed to substantiate any conclusions.

Insulin resistance (IR) is accompanied by chronic low grade systemic inflammation, obesity, and deregulation of total body energy homeostasis. We induced inflammation in adipose and liver tissues in vitro in order to mimic inflammation in vivo with the aim to identify tissue-specific processes implicated in IR and to find biomarkers indicative for tissue-specific IR. Human adipose and liver tissues were cultured in the absence or presence of LPS and DNA Microarray Technology was applied for their transcriptome analysis. Gene Ontology (GO), gene functional analysis, and prediction of genes encoding for secretome were performed using publicly available bioinformatics tools (DAVID, STRING, SecretomeP). The transcriptome data were validated by proteomics analysis of the inflamed adipose tissue secretome. LPS treatment significantly affected 667 and 483 genes in adipose and liver tissues respectively. The GO analysis revealed that during inflammation adipose tissue, compared to liver tissue, had more significantly upregulated genes, GO terms, and functional clusters related to inflammation and angiogenesis. The secretome prediction led to identification of 399 and 236 genes in adipose and liver tissue respectively. The secretomes of both tissues shared 66 genes and the remaining genes were the differential candidate biomarkers indicative for inflamed adipose or liver tissue. The transcriptome data of the inflamed adipose tissue secretome showed excellent correlation with the proteomics data. The higher number of altered proinflammatory genes, GO processes, and genes encoding for secretome during inflammation in adipose tissue compared to liver tissue, suggests that adipose tissue is the major organ contributing to the development of systemic inflammation observed in IR. The identified tissue-specific functional clusters and biomarkers might be used in a strategy for the development of tissue-targeted treatment of insulin resistance in patients.

[This corrects the article DOI: 10.1371/journal.pgen.1005378.].

To cite this article: Niedoszytko M, Oude Elberink JNG, Bruinenberg M, Nedoszytko B, de Monchy JGR, te Meerman GJ, Weersma RK, Mulder AB, Jassem E, van Doormaal JJ. Gene expression profile, pathways, and transcriptional system regulation in indolent systemic mastocytosis. Allergy 2011; 66: 229–237. Background: Mastocytosis is an uncommon disease resulting from proliferation of abnormal mast cells infiltrating skin, bone marrow, liver, and other tissues. The aim of this study was to find differences in gene expression in peripheral blood cells of patients with indolent systemic mastocytosis compared to healthy controls. The second aim was to define a specific gene expression profile in patients with mastocytosis. Methods: Twenty-two patients with indolent systemic mastocytosis and 43 healthy controls were studied. Whole genome gene expression analysis was performed on RNA samples isolated from the peripheral blood. For amplification and labelling of the RNA, the Illumina TotalPrep 96 RNA Amplification Kit was used. Human HT-12_V3_expression arrays were processed. Data analysis was performed using GeneSpring, Genecodis, and Transcriptional System Regulators. Results: Comparison of gene expression between patients and controls revealed a significant difference (P < 0.05 corrected for multiple testing) and the fold change difference >2 in gene expression in 2303 of the 48.794 analysed transcripts. Functional annotation indicated that the main pathways in which the differently expressed genes were involved are ubiquitin-mediated proteolysis, MAPK signalling pathway, pathways in cancer, and Jak-STAT signalling. The expression distributions for both groups did not overlap at all, indicating that many genes are highly differentially expressed in both groups. Conclusion: We were able to find abnormalities in gene expression in peripheral blood cells of patients with indolent systemic mastocytosis and to construct a gene expression profile which may be useful in clinical practice to predict the presence of mastocytosis and in further research of novel drugs.

BackgroundVenom immunotherapy (VIT) enables longtime prevention of insect venom allergy in the majority of patients. However, in some, the risk of a resystemic reaction increases after completion of treatment. No reliable factors predicting individual lack of efficacy of VIT are currently available.ObjectiveTo determine the use of gene expression profiles to predict the long-term effect of VIT.MethodsWhole genome gene expression analysis was performed on RNA samples from 46 patients treated with VIT divided into 3 groups: (1) patients who achieved and maintained long-term protection after VIT, (2) patients in whom insect venom allergy relapsed, and (3) patients still in the maintenance phase of VIT.ResultsAmong the 48.071 transcripts analyzed, 1401 showed a >2 fold difference in gene expression (P < .05); 658 genes (47%) were upregulated and 743 (53%) downregulated. Forty-three transcripts still show significant differences in expression after correction for multiple testing; 12 of 43 genes (28%) were upregulated and 31 of 43 genes (72%) downregulated. A naive Bayes prediction model demonstrated a gene expression pattern characteristic of effective VIT that was present in all patients with successful VIT but absent in all subjects with failure of VIT. The same gene expression profile was present in 88% of patients in the maintenance phase of VIT.ConclusionGene expression profiling might be a useful tool to assess the long-term effectiveness of VIT. The analysis of differently expressed genes confirms the involvement of immunologic pathways described previously but also indicates novel factors that might be relevant for allergen tolerance. Venom immunotherapy (VIT) enables longtime prevention of insect venom allergy in the majority of patients. However, in some, the risk of a resystemic reaction increases after completion of treatment. No reliable factors predicting individual lack of efficacy of VIT are currently available. To determine the use of gene expression profiles to predict the long-term effect of VIT. Whole genome gene expression analysis was performed on RNA samples from 46 patients treated with VIT divided into 3 groups: (1) patients who achieved and maintained long-term protection after VIT, (2) patients in whom insect venom allergy relapsed, and (3) patients still in the maintenance phase of VIT. Among the 48.071 transcripts analyzed, 1401 showed a >2 fold difference in gene expression (P < .05); 658 genes (47%) were upregulated and 743 (53%) downregulated. Forty-three transcripts still show significant differences in expression after correction for multiple testing; 12 of 43 genes (28%) were upregulated and 31 of 43 genes (72%) downregulated. A naive Bayes prediction model demonstrated a gene expression pattern characteristic of effective VIT that was present in all patients with successful VIT but absent in all subjects with failure of VIT. The same gene expression profile was present in 88% of patients in the maintenance phase of VIT. Gene expression profiling might be a useful tool to assess the long-term effectiveness of VIT. The analysis of differently expressed genes confirms the involvement of immunologic pathways described previously but also indicates novel factors that might be relevant for allergen tolerance.

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous disease characterized by recurrent respiratory tract infections, sinusitis, bronchiectasis and male infertility. The pulmonary phenotype in PCD is caused by the impaired motility of cilia in the respiratory epithelium, due to ultrastructural defects of these organelles. We hypothesized that defects of multi-protein ciliary complexes should be reflected by gene expression changes in the respiratory epithelium. We have previously found that large group of genes functionally related to cilia share highly correlated expression pattern in PCD bronchial tissue. Here we performed an explorative analysis of differential gene expression in the bronchial tissue from six PCD patients and nine non-PCD controls, using Illumina HumanRef-12 Whole Genome BeadChips. We observed 1323 genes with at least 2-fold difference in the mean expression level between the two groups (t-test p-value <0.05). Annotation analysis showed that the genes down-regulated in PCD biopsies (602) were significantly enriched for terms related to cilia, whereas the up-regulated genes (721) were significantly enriched for terms related to cell cycle and mitosis. We assembled a list of human genes predicted to encode ciliary proteins, components of outer dynein arms, inner dynein arms, radial spokes, and intraflagellar transport proteins. A significant down-regulation of the expression of genes from all the four groups was observed in PCD, compared to non-PCD biopsies. Our data suggest that a coordinated down-regulation of the ciliome genes plays an important role in the molecular pathomechanism of PCD.

Insect venom immunotherapy (VIT) is the only causative treatment of insect venom allergy (IVA). The immunological mechanism(s) responsible for long-term protection achieved by VIT are largely unknown. A better understanding is relevant for improving the diagnosis, prediction of anaphylaxis, and monitoring and simplifying treatment of IVA.To find genes that are differentially expressed during the maintenance phase of VIT and after stopping, to get clues about the pathways involved in the long-term protective effect of immunotherapy.Whole genome gene expression analysis was performed on RNA samples from 50 patients treated with VIT and 43 healthy controls. Patients were divided into three groups: (1) before the start of VIT; (2) on maintenance phase of VIT for at least 3 years still receiving injections; and (3) after VIT.Of all 48,804 probes present in the array, 48,773 transcripts had sufficient data for further analysis. The list of genes that were differentially expressed (at least log2 FC > 2; P < .05 corrected for multiple testing) during the maintenance phase of VIT as well as after successful VIT contains 89 entities. The function of these genes affects cell signaling, cell differentiation, and ion transport.This study shows that a group of genes is differentially expressed both during and after VIT in comparison with gene expression in patients before VIT. Although the results of this study should be confirmed prospectively, the relevance of these findings is supported by the fact that they are related to putative mechanisms of immunotherapy.

Blood eosinophil count is associated with a variety of common complex outcomes in epidemiological observation. The aim of this study was to explore the causal association between determined blood eosinophil count and 20 common complex outcomes (10 metabolic, 6 cardiac, and 4 pulmonary). Through Mendelian randomization, we investigated genetic evidence for the genetically determined eosinophil in association with each outcomes using individual-level LifeLines cohort data ( n = 13,301), where a weighted eosinophil genetic risk score comprising five eosinophil associated variants was created. We further examined the associations of the genetically determined eosinophil with those outcomes using summary statistics obtained from genome-wide association study consortia (6 consortia and 14 outcomes). Blood eosinophil count, by a 1- SD genetically increased, was not statistically associated with common complex outcomes in the LifeLines. Using the summary statistics, we showed that a higher genetically determined eosinophil count had a significant association with lower odds of obesity (odds ratio ( OR ) 0.81, 95% confidence interval (CI) [0.74, 0.89]) but not with the other traits and diseases. To conclude, an elevated eosinophil count is unlikely to be causally associated to higher risk of metabolic, cardiac, and pulmonary outcomes. Further studies with a stronger genetic risk score for eosinophil count may support these results.

To isolate tissue-specific gene products that contribute to corneal integrity.A cDNA library was constructed and differentially hybridized. Cornea-specific clones were purified and further characterized.In this study cornea-specific gene products were isolated by differential cDNA hybridization. In addition to known cornea-specific gene products, a transcript was isolated coding for a protein homologous to the angiopoietins, a recently described family of (anti)angiogenic factors. Subsequently, the full cDNA was sequenced, and the identified open reading frame was named cornea-derived transcript 6 (CDT6). Similar to the angiopoietins, CDT6 contains a hydrophobic NH2-terminal sequence, a coiled-coil domain, and a COOH-terminal fibrinogenlike domain. Expression of CDT6 could be detected only in the cornea and not in several other adult human tissues. Within the cornea, expression of CDT6 is confined to the stromal layer.The human cornea shows high-level expression of a gene product homologous to the (anti)angiogenic factors, the angiopoietins. This homology, together with stromal-specific expression, suggests that this factor may contribute to the avascularity of the human cornea.

CHEK2 is low-penetrance breast cancer susceptibility gene. The 1100delC mutation may interact with variants/mutations in other breast cancer susceptibility loci. We identified a risk haplotype in the HLA class III region in breast cancer patients [de Jong MM, Nolte IM, de Vries EGE, et al. The HLA class III subregion is responsible for an increased breast cancer risk. Hum Mol Genet 2003, 12, 2311–2319] and tested whether it interacted with 1100delC mutation. The CHEK2 1100delC mutation was analysed in the same series of patients and controls as in the HLA breast cancer study. In 962 unselected breast cancer patients, the 1100delC mutation was observed in 2.9% and in 367 controls in 1.4% (NS). The highest 1100delC frequency occurred in high-risk (4.4%), followed by moderate-risk (3.8%), and lowest in low genetic risk patients (2.4%, Ptrend 0.029). In HLA risk haplotype carriers no increased breast cancer risk was observed in the presence of 1100delC mutation. Patients more often had one than both genetic risk factors. The 1100delC mutation and the HLA risk haplotype confer increased breast cancer risks, but an interactive effect on breast cancer between both factors is unlikely. In contrast, the effect of 1100delC mutation on breast cancer risk was limited to individuals without HLA risk haplotype, suggesting a mutual excluding effect between these risk factors.

Biobank sample storage is critical in population health and epidemiology studies. Biobanks bridge two very different worlds: they connect to the participants and patients at an individual level, but they also aggregate information and represent the cutting edge of scientific discovery. In this brief report, we describe how the LifeLines study in the Netherlands manages its resources for communication and services, and how it can serve as a model for the Human Heredity and Health in Africa Initiative (H3Africa Initiative).

To investigate possible differences in cytomegalovirus (CMV) strain distribution between the eye and blood in AIDS patients with CMV retinitis.CMV DNA sequences from aqueous humour and peripheral blood leukocytes (PBL), obtained from 13 AIDS patients with CMV retinitis, were compared. DNA was isolated and the CMV IE-1 sequence (part of the immediate early-1 gene) and the a-sequence (located in the a-region) were amplified by polymerase chain reaction (PCR). The PCR products of the a-sequence were analysed by Southern blotting for amplified fragment-length polymorphisms. The level of divergence between the a-sequences of aqueous humour- and PBL-derived CMV was studied in two patients by cloning these sequences followed by sequence analysis.CMV DNA could be detected in all aqueous humour samples and in 10 out of 13 paired blood samples. In the 10 patients, with CMV DNA detectable in both aqueous humour and PBL, seven cases showed differences between the amplified products of both compartments. Sequence analysis in two patients revealed that the aqueous humour and PBL of the same patient can harbour both identical, similar and highly divergent CMV a-sequences.These results indicate that despite the haematogenous spread of CMV, the eye, being a relatively shielded organ, may contain CMV strains different from those found in the blood.

To evaluate the measurement of intraocular antibody production and detection of DNA by the polymerase chain reaction (PCR) for diagnosis of the causative microorganism in patients with AIDS and necrotizing retinitis.Paired serum and aqueous humour samples obtained from 28 patients with AIDS and necrotizing retinitis, seen between January 1987 and March 1992, were analysed for intraocular antibody production against cytomegalovirus (CMV), varicella zoster virus, herpes simplex virus, Epstein-Barr virus, and Toxoplasma gondii. Specific antibody titres in the inflamed eye and in the circulation were related to total immunoglobulin G content in the aqueous humour and serum. In addition, PCR analysis was performed in 15 samples. Results were compared to the final diagnosis, which was based on the subsequent clinical course. Results were also related to parameters describing the immune state of the patients: CD4 count, time between diagnosis of an AIDS-defining illness and retinitis, and time of survival following the diagnosis of retinitis.In 11 (39%) out of 28 patients we found local intraocular antibody production which correlated with the final diagnosis (one out of two cases with acute retinal necrosis, three out of five cases with toxoplasma retinitis, and eight out of 21 patients with CMV retinitis). In all 13 patients with CMV retinitis PCR analysis detected CMV DNA. In one patient with the clinical diagnosis of Toxoplasma retinitis, Toxoplasma DNA could be determined, whereas in the same sample CMV DNA was also found. In yet another patient with Toxoplasma retinitis only CMV DNA could be detected. A relationship between results of local antibody determination with either CD4 counts, or the time interval between AIDS-defining illness and retinitis, or survival time after diagnosis of retinitis could not be established. CD4 counts were higher than 50 x 10(6)/l in eight out of 19 patients with CMV retinitis. No complications of paracentesis were seen.Detection of intraocular antibody production and PCR analysis are quick and safe procedures and helpful tools for diagnosis of the involved pathogen in AIDS patients with a necrotizing retinitis. Negative results of local antibody production, even in the presence of detectable viral DNA, could not be related to the parameters of a more deteriorated immune status of these patients.

The association with histocompatibility antigens (HLA), in particular Class II genes (DQB1, DRB1), has recently been suggested to be one of the genetic factors involved in testicular germ cell tumor (TGCT) development. The current study, which uses genotyping of microsatellite markers, was designed to replicate previous associations.In 151 patients, along with controls comprising parents or spouses, the HLA region (particularly Class II) on chromosome 6p21 was genotyped for a set of 15 closely linked microsatellite markers.In both patients and controls, strong linkage disequilibrium was observed in the genotyped region, indicating that similar haplotypes are likely to be identical by descent. However, association analysis and the transmission disequilibrium test did not show significant results. Haplotype sharing statistics, a haplotype method that derives extra information from phase and single marker tests, did not show differences in haplotype sharing between patients and controls.The current genotyping study did not confirm the previously reported association between HLA Class II genes and TGCT. As the HLA alleles for which associations were reported are also prevalent in the Dutch populations, these associations are likely to be nonexistent or much weaker than previously reported.

Obesity is heritable and predisposes to many diseases. To understand the genetic basis of obesity better, here we conduct a genome-wide association study and Metabochip meta-analysis of body mass index (BMI), a measure commonly used to define obesity and assess adiposity, in up to 339,224 individuals. This analysis identifies 97 BMI-associated loci (P 20% of BMI variation. Pathway analyses provide strong support for a role of the central nervous system in obesity susceptibility and implicate new genes and pathways, including those related to synaptic function, glutamate signalling, insulin secretion/action, energy metabolism, lipid biology and adipogenesis.

To determine the frequency of cytomegalovirus glycoprotein B (gB) genotypes in clinical samples of ocular fluids of patients with acquired immune deficiency syndrome (AIDS) who have cytomegalovirus retinitis and to compare these with the cytomegalovirus gB genotype in paired peripheral blood leukocytes.Glycoprotein B genotypes of cytomegalovirus genomic DNA were determined in 29 ocular and 9 paired blood samples of 27 patients, by polymerase chain reaction amplification followed by restriction fragment length polymorphism analysis.In the 29 ocular samples, 30 gB genotypes were determined: Glycoprotein B1 was found in 8 samples (27%), gB2 in 9 samples (30%), gB3 in 6 samples (20%), and gB4 in 3 samples (10%). In one sample, a mixed genotype was observed. In addition to these previously characterized gB genotypes, a new gB variant was observed in the ocular fluid of four patients. Partial sequence analysis revealed that this new gB genotype is closely related to gB3, and it was therefore named gB3'. In the blood samples, only gB1, gB2, and gB3 genotypes were observed. In the nine paired samples of ocular fluid and blood, four showed a difference in gB genotype between these compartments.The distribution of cytomegalovirus glycoprotein B genotypes gB1-gB4 in ocular fluids of patients with AIDS who have cytomegalovirus retinitis was determined in this study. The predominance of gB2, as described by others, was not confirmed. The glycoprotein B genotype in the eye can be different from the genotype found in the blood of the same patient. A new gB variant, gB3', was found in the ocular samples of 4 of 27 patients, but not in the blood samples tested.

PURPOSE: Recently, we found a certain haplotype in the human leukocyte antigen Class III subregion to be associated with breast cancer. Epidemiologic studies have shown that breast cancer and colorectal cancer have several risk factors in common. In view of these studies and because polymorphisms located in the human leukocyte antigen III region have been found to be associated with colorectal cancer, we wondered whether the same region also is involved in colorectal cancer susceptibility. METHODS: The human leukocyte antigen region was genotyped with 14 microsatellite markers in germline DNA from 643 colorectal cancer patients and 841 family-based controls. Association analyses and the Haplotype Sharing Statistic were used to search for differences between patients and controls. Subgroup analyses were performed for gender, age at diagnosis, and localization of the tumor. RESULTS: The Haplotype Sharing Statistic analysis revealed neither a difference in mean haplotype sharing between all patients and controls, nor in any of the subgroups. The single allele, genotype, and two-locus association analyses for all patients and for the different subgroups did not show an association with colorectal cancer for the 14 microsatellite markers. Also, no association was observed between the tumor necrosis factor-beta polymorphism and colorectal cancer. CONCLUSIONS: No association was observed between commonly occurring haplotypes and alleles in the human leukocyte antigen region and colorectal cancer risk.

The American Journal of Human Genetics, 90, 410–425; March 2012 The originally published online version of this paper omitted two authors, Peter Sever and Neil Poulter, who have now been added. Middle initials have also been added for Deepak L. Bhatt and Folkert W. Asselbergs. In addition, the ASCOT and INVEST portions of the Supplemental Acknowledgments have been updated. The authors regret the errors. Large-Scale Gene-Centric Meta-Analysis across 39 Studies Identifies Type 2 Diabetes LociSaxena et al.The American Journal of Human GeneticsFebruary 9, 2012In BriefTo identify genetic factors contributing to type 2 diabetes (T2D), we performed large-scale meta-analyses by using a custom ∼50,000 SNP genotyping array (the ITMAT-Broad-CARe array) with ∼2000 candidate genes in 39 multiethnic population-based studies, case-control studies, and clinical trials totaling 17,418 cases and 70,298 controls. First, meta-analysis of 25 studies comprising 14,073 cases and 57,489 controls of European descent confirmed eight established T2D loci at genome-wide significance. Full-Text PDF Open Archive

To the Editor: In the September 2004 issue of the Journal, Böttcher et al1Fageras Bottcher M. Hmani-Aifa M. Lindstrom A. Jenmalm M.C. Mai X.M. Nilsson L. et al.A TLR4 polymorphism is associated with asthma and reduced lipopolysaccharide-induced interleukin-12(p70) responses in Swedish children.J Allergy Clin Immunol. 2004; 114: 561-567Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar presented an interesting study reporting an association between a polymorphism in the Toll-like receptor 4 (TLR4) gene and asthma. They explained this association by altered cytokine production, presumably caused by this variation. In their study of Swedish children, TLR4 Asp299Gly (AG) heterozygosity was associated with atopic asthma and with lower LPS-induced IL-12 and IL-10 levels compared with TLR4 Asp299Gly (AA) homozygosity. In addition, CD14/-159 TT homozygote individuals showed higher LPS-induced IL-12 levels than individuals carrying CD14/-159 CC and CT. Finally, LPS-induced IL-10 and IL-12 levels were lower in individuals with atopic asthma. Thus, the TLR4 Asp299Gly (AG) polymorphism is associated with atopic asthma and asthma by itself with lower cytokine production. Consequently, the polymorphism is also associated with lower cytokine production. This raises the question whether the observation on cytokine production in the Swedish study is confounded by disease status. We can illustrate this with an example. We have investigated 40 adult individuals of a Dutch family study in whom the association of the CD14/-159 polymorphism and atopy severity previously was shown.2Koppelman G.H. Reijmerink N.E. Colin Stine O. Howard T.D. Whittaker P.A. Meyers D.A. et al.Association of a promoter polymorphism of the CD14 gene and atopy.Am J Respir Crit Care Med. 2001; 163: 965-969Crossref PubMed Scopus (233) Google Scholar We assessed the association of the CD14/-159 genotype and both membrane-bound CD14 expression and LPS-induced (1 ng/mL) IL-10 and IL-12(p70) cytokine production in vitro of PBMCs in the presence of IFN-γ. In an attempt to disentangle atopic status and CD14/-159 genotype, we investigated these associations in 4 groups: 10 CD14/-159 CC atopic individuals, 9 TT atopic individuals, 10 CC nonatopic individuals, and 11 TT nonatopic individuals. We found that CC atopic individuals had a significantly lower percentage CD14+ monocytes compared with TT nonatopic individuals (median, 62%; range, 49-82%; vs median, 73%; range, 58-80%). However, the difference was even larger comparing all atopic individuals with all nonatopic individuals (median, 61%; range, 45-82%; vs median, 73%; range, 57-86%; Fig 1). Furthermore, we also found higher levels of LPS-induced IL-10 production in atopic individuals (median, 1976 pg/mL; range, 1307-2966 pg/mL) compared with nonatopic individuals (median, 1548 pg/mL; range, 629-2937 pg/mL; P = .04) irrespective of the genotype. We could not confirm an association of the CD14/-159 genotype and LPS-induced IL-12 production. In conclusion, atopy is an important confounder of the effects of the CD14/-159 genotype on membrane-bound CD14 in our study. This could also apply to the study of Böttcher et al.1Fageras Bottcher M. Hmani-Aifa M. Lindstrom A. Jenmalm M.C. Mai X.M. Nilsson L. et al.A TLR4 polymorphism is associated with asthma and reduced lipopolysaccharide-induced interleukin-12(p70) responses in Swedish children.J Allergy Clin Immunol. 2004; 114: 561-567Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar It would be of interest if the authors present their data on the effects of the TLR4 Asp299Gly (AG) and CD14/-159 genotype on cytokines stratified by atopic asthma. Of importance, our data suggest taking the disease status into account when analyzing functional effects of single nucleotide polymorphisms in a human population.

ADAM33 (A Disintegrin and Metalloprotease 33) has been identified as a susceptibility gene for asthma and single nucleotide polymorphisms (SNPs) in this gene have been associated with excess decline of lung function in asthmatics.To assess whether SNPs in ADAM33 are associated with accelerated lung function loss in the general population and with chronic obstructive pulmonary disease (COPD).We have collected DNA from subjects of the Vlagtwedde/Vlaardingen cohort participating in the last survey in 1989/1990 after a follow up of 25 years.Information was collected every 3 years, including lung function measurements.We defined COPD as GOLD stage 2 or higher at the last survey.1390 subjects from the cohort were genotyped for the following SNPs in ADAM33: F+1, Q-1, S_1, S_2, T_1, T_2, V_4, ST+5.Differences in prevalence of SNPs were analyzed with chi-square tests.Linear mixed effects models were used to analyze FEV 1 decline according to genotype.In the whole population mean adjusted decline was 18.7 and 12.7 ml?y -1 in females and males respectively.Individuals homozygous for minor alleles of SNPs S_2 and Q-1 and heterozygous for SNP S_1 had a significantly accelerated decline in FEV 1 of respectively 4.9, 9.6 and 3.6 ml?y -1 compared with wild type.We found a significantly higher prevalence of SNPs F+1, S_1, S_2 and T_2 in subjects with COPD.We demonstrated that SNPs in ADAM33 are associated with accelerated lung function decline in the general population.These SNPs are also risk factors for COPD.

Insulin resistance (IR) is accompanied by chronic low grade systemic inflammation and deregulation of total body energy homeostasis. We induced inflammation in human adipose and liver tissue in vitro in order to mimic inflammation in vivo with the aim to identify tissue-specific processes and biomarkers implicated in IR Human adipose and liver tissues were cultured with or without LPS and DNA Microarray Technology was applied for their transcriptome analysis. Gene Ontology (GO), gene functional analysis, and prediction of genes encoding for the secretome were performed using DAVID, STRING, and SecretomeP as bioinformatics tools .The transcriptome data were validated by proteomics analysis of the inflamed adipose tissue secretome using CILAIR technology. LPS significantly affected 667 and 484 genes in adipose and liver tissues respectively. During inflammation adipose tissue, compared to liver tissue, had more significantly upregulated genes, GO terms, and functional clusters related to inflammation and angiogenesis. The secretome prediction led to identification of 399 and 236 genes in adipose and liver tissue respectively. The secretomes of both tissues shared 66 genes.The adipose tissue specific biomarkers were represented by fractalkine, tumor necrosis factor, pentraxin-related protein or interstitial collagenase (matrix metallopeptidase 1) and the liver specific biomarkers were for example chemokine (C-X-C motif) ligand 9, chemokine (C-X-C motif) ligand 3, or follistatin-like 3 (secreted glycoprotein). The transcriptome data of the inflamed adipose tissue secretome showed excellent correlation with the proteomics data. The higher number of altered proinflammatory genes, GO processes, and genes encoding for secretome during inflammation in adipose tissue compared to liver suggests that adipose tissue is the major organ in the development of systemic IR. Our study led to the identification of differential pathways and biomarkers suggesting tissue specific changes, which could be applied for tissue specific detection and treatment of IR.

Background and aims. In one small study, the DCC Arg201Gly polymorphism has been observed more frequently in colorectal cancer cases compared with controls. We wondered whether these results could be replicated in a much larger study. Methodology. The DCC Arg201Gly polymorphism was genotyped in 625 unselected Caucasian colorectal cancer patients and 220 controls. Association analysis was used to search for a difference between patients and controls. Subgroup analyses were performed for site of tumour, gender, age at diagnosis, family history of colorectal cancer and modified Dukes classification. Results. The association analyses revealed no difference in Arg201Gly genotype frequency between patients and controls, neither overall nor for different subgroups according to site of tumour, gender, age at diagnosis, family history of colorectal cancer and modified Dukes classification. Conclusion. No association was observed between the Arg201Gly polymorphism of DCC and colorectal cancer risk.

4579 Background: Testicular Germ Cell Tumours (TGCT) was found to be linked to the X chromosome. Families with at least one bilateral case appeared to be linked to a 2.7 Mb region on Xq27 (Rapley et al. Nat Genet. 2000 Feb;24(2):197–200). We tried to confirm this finding in a comprehensive association study with markers from this region. Methods: In 288 testicular cancer patients and 169 unaffected first-degree male family members, 12 microsatellite markers covering the candidate region were genotyped and matched the quality criteria to study association with Xq27 both by single locus and haplotype analysis (haplotype sharing statistics (HSS). Results: None of these 12 markers analyzed, or combinations, showed association with TGCT. Subgroup analyses of cases with bilateral TGCT (n=11), familiar TGCT (n=28) and cryptorchism (n=44) did also not reveal evidence for association with Xq27. Conclusions: We could not replicate the previously observed linkage of TGCT to Xq27 by association analysis. Our sample had 80% power to detect a relative risk of 2.0 for a risk allele with a frequency larger than 10%. The subgroups of bilateral cases, familiar cases and cases with cryptorchism did not show significant results either. No significant financial relationships to disclose.