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DNA methylation patterns in the genome both reflect and help to mediate transcriptional regulatory processes. The digital nature of DNA methylation, present or absent on each allele, makes this assay capable of quantifying events in subpopulations of cells, whereas genome-wide chromatin studies lack the same quantitative capacity. Testing DNA methylation throughout the genome is possible using whole-genome bisulfite sequencing (WGBS), but the high costs associated with the assay have made it impractical for studies involving more than limited numbers of samples. We have optimized a new transposase-based library preparation assay for the Illumina HiSeq X platform suitable for limited amounts of DNA and providing a major cost reduction for WGBS. By incorporating methylated cytosines during fragment end repair, we reveal an end-repair artifact affecting 1%-2% of reads that we can remove analytically. We show that the use of a high (G + C) content spike-in performs better than PhiX in terms of bisulfite sequencing quality. As expected, the loci with transposase-accessible chromatin are DNA hypomethylated and enriched in flanking regions by post-translational modifications of histones usually associated with positive effects on gene expression. Using these transposase-accessible loci to represent the cis -regulatory loci in the genome, we compared the representation of these loci between WGBS and other genome-wide DNA methylation assays, showing WGBS to outperform substantially all of the alternatives. We conclude that it is now technologically and financially feasible to perform WGBS in larger numbers of samples with greater accuracy than previously possible., (© 2018 Suzuki et al.; Published by Cold Spring Harbor Laboratory Press.)

In ∼30% of patients with EGFR -mutant lung adenocarcinomas whose disease progresses on EGFR inhibitors, the basis for acquired resistance remains unclear. We have integrated transposon mutagenesis screening in an EGFR -mutant cell line and clinical genomic sequencing in cases of acquired resistance to identify mechanisms of resistance to EGFR inhibitors. The most prominent candidate genes identified by insertions in or near the genes during the screen were MET , a gene whose amplification is known to mediate resistance to EGFR inhibitors, and the gene encoding the Src family kinase YES1. Cell clones with transposon insertions that activated expression of YES1 exhibited resistance to all three generations of EGFR inhibitors and sensitivity to pharmacologic and siRNA-mediated inhibition of YES1 Analysis of clinical genomic sequencing data from cases of acquired resistance to EGFR inhibitors revealed amplification of YES1 in five cases, four of which lacked any other known mechanisms of resistance. Preinhibitor samples, available for two of the five patients, lacked YES1 amplification. None of 136 postinhibitor samples had detectable amplification of other Src family kinases ( SRC and FYN ). YES1 amplification was also found in 2 of 17 samples from ALK fusion-positive lung cancer patients who had progressed on ALK TKIs. Taken together, our findings identify acquired amplification of YES1 as a recurrent and targetable mechanism of resistance to EGFR inhibition in EGFR -mutant lung cancers and demonstrate the utility of transposon mutagenesis in discovering clinically relevant mechanisms of drug resistance., Competing Interests: Conflict of interest statement: H.A.Y. has served on the advisory boards for AstraZeneca and Boehringer Ingelheim. Y.Y.J. has received consulting fees from Bristol–Myers Squibb and honoraria from Pfizer, Genentech, and Boehringer Ingelheim. J.E.C. has received consulting fees from AstraZeneca, Genentech, Bristol–Myers Squibb, and Merck. M.G.K. has served as a consultant for AstraZeneca. C.M.L. has served on the Advisory Board for Cepheid Oncology and has received consulting fees from Pfizer, Novartis, AstraZeneca, Genoptix, Sequenom, Ariad, Takeda, and Foundation Medicine. G.J.R. has received consulting fees from Roche, and Memorial Sloan Kettering Cancer Center (MSKCC) has received support from Pfizer and Roche to fund G.J.R.’s clinical research. K.P. has received research funding from AstraZeneca, Roche, Kolltan, and Symphogen; honoraria for consulting or advisory roles from AstraZeneca, Merck, Novartis, and Tocagen; and royalties from intellectual property licensed by MSKCC to Molecular MD. M.L. has received advisory board compensation from Boehringer Ingelheim, AstraZeneca, Bristol-Myers Squibb, Takeda, and Bayer, and research support from LOXO Oncology., (Copyright © 2018 the Author(s). Published by PNAS.)

Rationale: Albuterol, a bronchodilator medication, is the first-line therapy for asthma worldwide. There are significant racial/ethnic differences in albuterol drug response., Objectives: To identify genetic variants important for bronchodilator drug response (BDR) in racially diverse children., Methods: We performed the first whole-genome sequencing pharmacogenetics study from 1,441 children with asthma from the tails of the BDR distribution to identify genetic association with BDR., Measurements and Main Results: We identified population-specific and shared genetic variants associated with BDR, including genome-wide significant (P < 3.53 × 10 -7 ) and suggestive (P < 7.06 × 10 -6 ) loci near genes previously associated with lung capacity (DNAH5), immunity (NFKB1 and PLCB1), and β-adrenergic signaling (ADAMTS3 and COX18). Functional analyses of the BDR-associated SNP in NFKB1 revealed potential regulatory function in bronchial smooth muscle cells. The SNP is also an expression quantitative trait locus for a neighboring gene, SLC39A8. The lack of other asthma study populations with BDR and whole-genome sequencing data on minority children makes it impossible to perform replication of our rare variant associations. Minority underrepresentation also poses significant challenges to identify age-matched and population-matched cohorts of sufficient sample size for replication of our common variant findings., Conclusions: The lack of minority data, despite a collaboration of eight universities and 13 individual laboratories, highlights the urgent need for a dedicated national effort to prioritize diversity in research. Our study expands the understanding of pharmacogenetic analyses in racially/ethnically diverse populations and advances the foundation for precision medicine in at-risk and understudied minority populations.

We compared coding region variants of 53 cognitively healthy centenarians and 45 patients with Alzheimer's disease (AD), all of Ashkenazi Jewish (AJ) ancestry. Despite the small sample size, the known AD risk variant APOE4 reached genome-wide significance, indicating the advantage of utilizing 'super-controls'. We restricted our subsequent analysis to rare variants observed at most once in the 1000 Genomes database and having a minor allele frequency below 2% in our AJ sample. We compared the burden of predicted protein altering variants between cases and controls as normalized by the level of rare synonymous variants. We observed an increased burden among AD subjects for predicted loss-of-function (LoFs) variants defined as stop-gain, frame shift, initiation codon (INIT) and splice site mutations (n = 930, OR = 1.3, P = 1.5×E-5). There was no enrichment across all rare protein altering variants defined as missense plus LoFs, in frame indels and stop-loss variants (n = 13 014, OR = 0.97, P = 0.47). Among LoFs, the strongest burden was observed for INIT (OR = 2.16, P = 0.0097) and premature stop variants predicted to cause non-sense-mediated decay in the majority of transcripts (NMD) (OR = 1.98, P = 0.02). Notably, this increased burden of NMD, INIT and splice variants was more pronounced in a set of 1397 innate immune genes (OR = 4.55, P = 0.0043). Further comparison to additional exomes indicates that the difference in LoF burden originated both from the AD and centenarian sample. In summary, we observed an overall increased burden of rare LoFs in AD subjects as compared to centenarians, and this enrichment is more pronounced for innate immune genes., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Species sensitivity evaluation represents an approach to avoid chronic toxicity testing of aquatic vertebrates in accordance with the animal welfare concept of the EU chemicals regulation. In this study a data set of chemicals is analysed for relative species sensitivity between Daphnia and fish in chronic testing to evaluate under what condition chronic fish tests can be waived without underestimating the environmental hazard. Chronic fish toxicity is covered in 84% of the evaluated substances by the chronic invertebrate test and an assessment factor of 50. Thus, animal testing can be avoided in environmental hazard assessment for many chemicals. Moreover, it is shown that species sensitivity in chronic testing is associated with species sensitivity in acute testing. The more sensitive species in chronic testing is predicted with a high probability if a species is >5x more sensitive in acute testing. If substances are comparable or more toxic to Daphnia in acute testing than to fish chronic fish toxicity is covered by the chronic Daphnia test and an assessment factor of 50 in about 95% of the evaluated cases. To provide decision support for the regulation of chemicals a categorization scheme on relative sensitivity comparison is presented., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Unlabelled: Respiratory paramyxoviruses, including the highly prevalent human parainfluenza viruses, cause the majority of childhood croup, bronchiolitis, and pneumonia, yet there are currently no vaccines or effective treatments. Paramyxovirus research has relied on the study of laboratory-adapted strains of virus in immortalized cultured cell lines. We show that findings made in such systems about the receptor interaction and viral fusion requirements for entry and fitness-mediated by the receptor binding protein and the fusion protein-can be drastically different from the requirements for infection in vivo. Here we carried out whole-genome sequencing and genomic analysis of circulating human parainfluenza virus field strains to define functional and structural properties of proteins of circulating strains and to identify the genetic basis for properties that confer fitness in the field. The analysis of clinical strains suggests that the receptor binding-fusion molecule pairs of circulating viruses maintain a balance of properties that result in an inverse correlation between fusion in cultured cells and growth in vivo. Future analysis of entry mechanisms and inhibitory strategies for paramyxoviruses will benefit from considering the properties of viruses that are fit to infect humans, since a focus on viruses that have adapted to laboratory work provides a distinctly different picture of the requirements for the entry step of infection., Importance: Mechanistic information about viral infection-information that impacts antiviral and vaccine development-is generally derived from viral strains grown under laboratory conditions in immortalized cells. This study uses whole-genome sequencing of clinical strains of human parainfluenza virus 3-a globally important respiratory paramyxovirus-in cell systems that mimic the natural human host and in animal models. By examining the differences between clinical isolates and laboratory-adapted strains, the sequence differences are correlated to mechanistic differences in viral entry. For this ubiquitous and pathogenic respiratory virus to infect the human lung, modulation of the processes of receptor engagement and fusion activation occur in a manner quite different from that carried out by the entry glycoprotein-expressing pair of laboratory strains. These marked contrasts in the viral properties necessary for infection in cultured immortalized cells and in natural host tissues and animals will influence future basic and clinical studies., (Copyright © 2016 Palermo et al.)

Background: Acute-to-chronic extrapolation is an important approach to predict acceptable no-effect levels from acute data which has some uncertainties, but is valuable for risk assessment of chemical substances. With regard to the still limited and heterogenic data of chronic fish tests, conclusions on aquatic hazard estimation need to be checked and the question arises whether the chronic toxicity to fish can be adequately derived from acute data. A comprehensive dataset including ecotoxicological studies of 203 substances was used to investigate acute-to-chronic ratios (ACR) for both fish and Daphnia . To address potential uncertainty parameters of the approach, the influence of the octanol-water partition coefficient Kow, the mode of action, and the acute toxicity levels on the ACR was evaluated., Results: For industrial chemicals, median ACRs of 12.0 for fish and 8.8 for Daphnia and 90th percentiles of 68.0 and 50.2, respectively, were determined. The ACR for the most sensitive aquatic trophic level (ACRaqu) is derived by comparing the lowest acute and chronic effect value of Daphnia and fish. The median ACRaqu was 9.9, and the 90th percentile was determined to 58.5. The influence of the Kow on the ACR value was analysed and a correlation could not be confirmed. Non-polar narcosis was associated with a lower ACR, whereas polar narcosis was associated with an increased ACR., Conclusions: The result suggests that an acute-to-chronic extrapolation factor of 100 is protective for more than 90 % of the chemicals. Polar narcosis may represent a predictor for an increased ACR and an increased uncertainty of the approach. The result further suggests that a high Kow is probably not associated with increased ACRs and does not necessarily represent a determinant for chronic toxicity testing within this context.

To identify previously reported disease mutations that are compatible with extraordinary longevity, we screened the coding regions of the genomes of 44 Ashkenazi Jewish centenarians. Individual genome sequences were generated with 30× coverage on the Illumina HiSeq 2000 and single-nucleotide variants were called with the genome analysis toolkit (GATK). We identified 130 coding variants that were annotated as "pathogenic" or "likely pathogenic" based on the ClinVar database and that are infrequent in the general population. These variants were previously reported to cause a wide range of degenerative, neoplastic, and cardiac diseases with autosomal dominant, autosomal recessive, and X-linked inheritance. Several of these variants are located in genes that harbor actionable incidental findings, according to the recommendations of the American College of Medical Genetics. In addition, we found risk variants for late-onset neurodegenerative diseases, such as the APOE ε4 allele that was even present in a homozygous state in one centenarian who did not develop Alzheimer's disease. Our data demonstrate that the incidental finding of certain reported disease variants in an individual genome may not preclude an extraordinarily long life. When the observed variants are encountered in the context of clinical sequencing, it is thus important to exercise caution in justifying clinical decisions.

Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare liver tumor affecting adolescents and young adults with no history of primary liver disease or cirrhosis. We identified a chimeric transcript that is expressed in FL-HCC but not in adjacent normal liver and that arises as the result of a ~400-kilobase deletion on chromosome 19. The chimeric RNA is predicted to code for a protein containing the amino-terminal domain of DNAJB1, a homolog of the molecular chaperone DNAJ, fused in frame with PRKACA, the catalytic domain of protein kinase A. Immunoprecipitation and Western blot analyses confirmed that the chimeric protein is expressed in tumor tissue, and a cell culture assay indicated that it retains kinase activity. Evidence supporting the presence of the DNAJB1-PRKACA chimeric transcript in 100% of the FL-HCCs examined (15/15) suggests that this genetic alteration contributes to tumor pathogenesis.

Objectives: To determine whether heterogeneity in interleukin-6 (IL-6), IL-6 receptor and other components of the IL-6 signalling pathway/network, at the gene, transcript and protein levels, correlate with disease activity in patients with rheumatoid arthritis (RA) and with clinical response to tocilizumab., Design: Biomarker samples and clinical data for five phase 3 trials of tocilizumab were analysed using serum (3751 samples), genotype (927 samples) and transcript (217 samples) analyses. Linear regression was then used to assess the association between these markers and either baseline disease activity or treatment response., Results: Higher baseline serum IL-6 levels were significantly associated (p<0.0001) with higher baseline DAS28, erythrocyte sedimentation rate, C reactive protein and Health Assessment Questionnaire in patients whose responses to disease-modifying antirheumatic drugs (DMARD-IR) and to antitumour necrosis factor (aTNF-IR) were inadequate and patients who were naive/responders to methotrexate (MTX). Higher baseline serum IL-6 levels were also significantly associated with better clinical response to tocilizumab (versus placebo) measured by cDAS28 in the pooled DMARD-IR (p<0.0001) and MTX-naive populations (p=0.04). However, the association with treatment response was weak. A threefold difference in baseline IL-6 level corresponded to only a 0.17-unit difference in DAS28 at week 16. IL-6 pathway single nucleotide polymorphisms and RNA levels also were not strongly associated with treatment response., Conclusions: Our analyses illustrate that the biological activity of a disease-associated molecular pathway may impact the benefit of a therapy targeting that pathway. However, the variation in pathway activity, as measured in blood, may not be a strong predictor. These data suggest that the major contribution to variability in clinical responsiveness to therapeutics in RA remains unknown.

Aims: We examined the risk association between single nucleotide polymorphisms (SNPs) in eleven candidate genes with type 2 diabetes (T2D). T2D-associated polymorphisms were also examined for prediction of incident CHD., Methods: 113 tagging SNPs were genotyped in stage 1 (467 T2D cases, 290 controls), and 15 SNPs were analyzed in the final cohort (1462 T2D cases, 600 controls). Three T2D-associated SNPs were further tested for prediction of CHD within a subset of 1417 T2D cases free of CHD at enrolment., Results: In the case-control analysis, PPARG rs1801282 (Pro12Ala) (OR=1.48 (1.02-2.16)), ADIPOQ rs1063539 (OR=1.17 (1.01-1.35)), and HNF4A rs1884614 (OR=1.16 (1.00-1.32) were associated with T2D (P(allelic)<0.05). Joint analysis of rs1801282-C, rs1063539-G, and rs1884614-T risk alleles showed an additive dosage effect (P for trend=0.001). Moreover, carriers with two PPARG rs1801282-C risk alleles were associated with an increased risk of incident CHD (HR=4.38 (1.03-18.57), P=0.045) in T2D patients in the prospective analysis., Conclusions: Genetic variants of PPARG, ADIPOQ and HNF4A were individually and jointly associated with T2D in Hong Kong Chinese. The PPARG Pro12 risk allele contributed to increased risk for both T2D and CHD., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Background & Aims: Although interleukin 28B (interferon, lambda 3) (IL28B) genotype affects the response of patients with chronic hepatitis C to peginterferon and ribavirin, little is known regarding its effect on response to direct-acting antivirals in interferon-free combinations. We analyzed the effects of IL28B genotype on the viral kinetic (VK) response to an interferon-free combination of the nucleoside polymerase inhibitor mericitabine (RG7128) and the hepatitis C virus (HCV) protease inhibitor danoprevir., Methods: We performed a double-blind, dose-escalation study of patients with chronic HCV genotype 1 infection who were interferon treatment naive or had not responded to previous therapy with peginterferon and ribavirin. Patients were sequentially assigned to 1 of 7 cohorts then randomly assigned to groups that received up to 13 days of treatment with mericitabine (500 or 1000 mg, twice daily) plus danoprevir (100 or 200 mg, every 8 hours, or 600 or 900 mg, twice daily) or placebo. Eighty-three of 87 patients were genotyped for the IL28B single-nucleotide polymorphism rs12979860. VKs were analyzed only in patients who received 13 days of treatment, at optimal doses, using a biphasic model to describe first- and second-phase slopes of viral decay during therapy., Results: At day 14 (the end of interferon-free treatment), the mean reduction in the serum level of HCV RNA was slightly greater in patients with the CC polymorphism (5.01 log(10) IU/mL) than those without (4.59 log(10) IU/mL). Modeling revealed that patients with the CC polymorphism had slightly better early VKs, most apparent in the β-phase of viral decay. A mixed effect on the α-phase was observed, which was reduced in magnitude but prolonged in patients with CC, who also had better on-treatment response to peginterferon and ribavirin during follow up., Conclusions: IL28B genotype appears to affect early VKs in patients with chronic hepatitis C receiving interferon-free treatment., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

A high percentage of patients with BRAF(V600E) mutant melanomas respond to the selective RAF inhibitor vemurafenib (RG7204, PLX4032) but resistance eventually emerges. To better understand the mechanisms of resistance, we used chronic selection to establish BRAF(V600E) melanoma clones with acquired resistance to vemurafenib. These clones retained the V600E mutation and no second-site mutations were identified in the BRAF coding sequence. Further characterization showed that vemurafenib was not able to inhibit extracellular signal-regulated kinase phosphorylation, suggesting pathway reactivation. Importantly, resistance also correlated with increased levels of RAS-GTP, and sequencing of RAS genes revealed a rare activating mutation in KRAS, resulting in a K117N change in the KRAS protein. Elevated levels of CRAF and phosphorylated AKT were also observed. In addition, combination treatment with vemurafenib and either a MAP/ERK kinase (MEK) inhibitor or an AKT inhibitor synergistically inhibited proliferation of resistant cells. These findings suggest that resistance to BRAF(V600E) inhibition could occur through several mechanisms, including elevated RAS-GTP levels and increased levels of AKT phosphorylation. Together, our data implicate reactivation of the RAS/RAF pathway by upstream signaling activation as a key mechanism of acquired resistance to vemurafenib, in support of clinical studies in which combination therapy with other targeted agents are being strategized to combat resistance.

Objective: Tocilizumab, a monoclonal antibody to interleukin-6 receptor, was recently approved for the treatment of moderate-to-severe rheumatoid arthritis. Two patients during clinical development met laboratory, but not clinical, criteria for Hy's law with bilirubin elevations suspected as a result of genetic variation in uridine diphosphoglucose glucuronosyltransferase (UGT1A1) typical of Gilbert syndrome., Methods: Genotyping of the two cases potentially meeting with Hy's law was performed using commercially available procedures. UGT1A1 single nucleotide polymorphism data were extracted from a genome-wide array database for 1187 patients from tocilizumab trials, and associations of UGT1A1 genotypes with bilirubin elevations were analyzed using logistic regression for associations with baseline and change from baseline in bilirubin levels as continuous variables., Results: Bilirubin elevations were not associated with clinical adverse events. Both patients potentially meeting Hy's law carry homozygous UGT1A1*28 alleles and UGT1A1*60 alleles. UGT1A1*28 and three additional single nucleotide polymorphisms showed odds ratios greater than 25 for associations with elevated bilirubin. The presence of rs6742078 accounted for 32% of the total variance in bilirubin (P=2.2×10)., Conclusion: Bilirubin increases occurring with tocilizumab appear to be related to anti-inflammatory effects extending to the liver. Thus, in the absence of other signs of hepatic dysfunction, bilirubin elevations after treatment with tocilizumab have a high probability of association with UGT1A1 polymorphism, which should alleviate concerns of serious hepatotoxicity. Our results underscore the value of genotyping in the clinical trial setting to avoid misinterpretations that could lead to terminating development of a promising new agent.

Background: Disordered cellular calcium regulation has been implicated in the pathophysiology of diabetes through mechanisms that remain unresolved. The non-selective calcium channel, transient receptor potential cation channel, subfamily M, member 2 (TRPM2), has been recently reported to play a role in insulin secretion by pancreatic beta-cells. We hypothesized that gene variation of TRPM2 may play a role in the pathophysiology of type 2 diabetes mellitus (T2DM)., Methods: Using a case-control study from a community-based population sample of the Boston metropolitan area (all whites: 455 controls and 467 cases), we assessed the relationship of 9 TRPM2 tag-SNPs with (i) diabetes-related intermediate phenotypes and (ii) the presence of T2DM., Result: All SNPs examined were in Hardy-Weinberg equilibrium. Overall allele, genotype, and haplotype distributions were similar between cases and controls. Three TRPM2 variants (rs2838553, rs2838554 and rs4818917) were associated with homeostasis model assessment of beta-cell function (HOMA-%B), but not with HOMA-insulin resistance (HOMA-IR), fasting glucose levels nor hemoglobin A1c levels. Marker-by-marker logistic regression analysis, adjusted for potential risk factors, showed no evidence for an association of any of the tag-SNPs tested with T2DM. Further investigation using an entropy blocker-defined haplotype-based approach showed similar null findings., Conclusions: The present investigation found no evidence for an association of the variants tested with T2DM, although HOMA-%B was negatively associated with three TRPM2 variants (rs2838553, rs2838554 and rs4818917). More importantly, our present findings require replication/confirmation in future large-scale, prospective investigations., (Copyright 2010 Elsevier B.V. All rights reserved.)

Background: Previous studies identified melatonin receptor 1B (MTNR1B), islet-specific glucose 6 phosphatase catalytic subunit-related protein (G6PC2), glucokinase (GCK) and glucokinase regulatory protein (GCKR) as candidate genes for type 2 diabetes (T2D) acting through elevated fasting plasma glucose (FPG). We examined the associations of the reported common variants of these genes with T2D and glucose homeostasis in three independent Chinese cohorts., Methodology/principal Findings: Five single nucleotide polymorphisms (SNPs), MTNR1B rs10830963, G6PC2 rs16856187 and rs478333, GCK rs1799884 and GCKR rs780094, were genotyped in 1644 controls (583 adults and 1061 adolescents) and 1342 T2D patients. The G-allele of MTNR1B rs10830963 and the C-alleles of both G6PC2 rs16856187 and rs478333 were associated with higher FPG (0.0034

Recent data have implicated leukocyte telomere length shortening as a potential risk predictor for type 2 diabetes mellitus (T2DM) and its associated phenotypes. However, to date, epidemiologic data are scarce. Using a case-control study from a community-based population sample of the Boston metropolitan area (all whites: 424 controls and 432 cases), we examined the relationship of mean leukocyte telomere repeat copy number to single gene copy number (TSR) and T2DM. Associations of log(e)-transformed TSR with age, race, sex, body mass index (BMI), current smoking status, fasting insulin levels, fasting glucose levels, and hemoglobin A1c (HbA1c) were examined by multivariable linear regression analysis. A logistic regression analysis was performed to evaluate the association of log(e)-transformed TSR with T2DM with or without adjustment for potential confounders. The log(e)-transformed TSR was significantly shorter in the white cases than the white controls (P=0.003). In a multivariable linear regression analysis, an inverse association of log(e)-transformed TSR with BMI was observed (P=0.04). Furthermore, in a multivariable logistic regression analysis, decreased log(e)-transformed TSR was significantly associated with T2DM (adjusted odds ratio=1.748; 95% confidence interval [CI]=1.015-3.012; P=0.044). In summary, the current investigation has shown an association of mean leukocyte telomere length shortening with T2DM in white subjects. If corroborated in other studies, our findings suggest the potential importance of telomere biology in T2DM.

Investigation of the benzopyranones from the roots of PELARGONIUM SIDOIDES led to the identification of 15 benzopyranones, among them the novel compounds 12 and 14. Furthermore, benzopyranones 4 and 15 were detected in PELARGONIUM SIDOIDES for the first time. Structure determinations were performed by one- and two-dimensional NMR spectroscopy. An HPLC system for the discrimination of these benzopyranones has been developed., (Copyright Georg Thieme Verlag KG Stuttgart New York.)

Background: Recent data have implicated telomere length shortening as a potential risk predictor for cardiovascular disease. However, to date, prospective epidemiological data are scarce., Methods: Using leukocyte DNA samples collected at baseline in a prospective cohort of 14,916 initially healthy American men, we examined the relationship of mean telomere repeat copy number to single gene copy number (T/S ratio), using a re-modified quantitative polymerase chain reaction protocol, among 337 white males who subsequently developed an incident myocardial infarction (MI), and among an equal number of age- and smoking-matched white males who remained free of reported vascular disease during follow-up (controls)., Results: The mean follow-up time since randomization was 3.85 y. The T/S ratio was inversely correlated with age in the controls (R=-0.114; p=0.036). The log(e)-transformed T/S ratios were significantly smaller in the MI cases (3.41+/-0.63) than the MI controls (3.52+/-0.78) (p=0.01). In a multi-variable adjusted analysis, decreased T/S ratio was significantly associated with risk of MI (odds ratio=1.621; 95%CI=1.140-2.304; p=0.007)., Conclusions: The present investigation has shown an association of telomere length shortening with increased risk of incident myocardial infarction, further suggesting the importance of telomere biology in atherogenesis.

Objective: Recent studies in European populations have reported a reciprocal association of glucokinase regulatory protein (GCKR) gene with triglyceride versus fasting plasma glucose (FPG) levels and type 2 diabetes risk. GCKR is a rate-limiting factor of glucokinase (GCK), which functions as a key glycolytic enzyme for maintaining glucose homeostasis. We examined the associations of two common genetic polymorphisms of GCKR and GCK with metabolic traits in healthy Chinese adults and adolescents., Research Design and Methods: Two single nucleotide polymorphisms (SNPs), rs780094 at GCKR and rs1799884 at GCK, were genotyped in 600 healthy adults and 986 healthy adolescents. The associations of these SNPs with metabolic traits were assessed by linear regression adjusted for age, sex, and/or BMI. We also tested for the epistasis between these two SNPs and performed a meta-analysis among European and Asian populations., Results: The T-allele of GCKR rs780094 was associated with increased triglycerides (P = 5.4 x 10(-7)), while the A-allele of GCK rs1799884 was associated with higher FPG (P = 3.1 x 10(-7)). A novel interaction effect between the two SNPs on FPG was also observed (P = 0.0025). Meta-analyses strongly supported the additive effects of the two SNPs on FPG and triglycerides, respectively. CONCLUSIONS;- In support of the intimate relationship between glucose and lipid metabolisms, GCKR and GCK genetic polymorphisms interact to increase FPG in healthy adults and adolescents. These risk alleles may contribute to increased diabetes risk in subjects who harbor other genetic or environmental/lifestyle risk factors.

Decabromodiphenyl ether (decaBDE) is a widely used brominated flame retardant, considered to be of low toxicity. However, previous toxicity studies applied exposure methods with low bioavailability of this compound, and the actual hazard of decaBDE for humans, which are environmentally exposed to decaBDE, may thus be underestimated in current risk assessments. The present 28 days oral toxicity study in Wistar rats was designed to facilitate detection of endocrine and immune modulating effects of decaBDE using an exposure protocol with improved bioavailability. A technical preparation of high purity decaBDE was thus tested by daily exposure through gavage with an emulsion of soy phospholipon/lutrol as a carrier. Most sensitive effect in males were increased weight of seminal vesicle/coagulation gland with BMDL of 0.2mg/kg bw/day and increased expression of hepatic CYP1A and CYP2B (BMDLs 0.5-0.7 mg/kg bw/day). In females the most sensitive effect was decreased activity of P450c17 (CYP17), which is a key enzyme in the androgen synthesis pathway, in adrenals (BMDL 0.18 mg/kg bw/day). These results suggest that decaBDE may represent an as yet unreported hazard for reproductive health.

Extracts of PELARGONIUM SIDOIDES, commonly known as EPs 7630, are produced by extraction of milled roots with 11 % (w/w) ethanol in water. This solvent leads to a spectrum of constituents which differs significantly from extracts obtained by extraction with non-polar solvents. EPs 7630 is composed of six main groups of constituents, namely unsubstituted and substituted oligomeric prodelphinidins, monomeric and oligomeric carbohydrates, minerals, peptides, purine derivatives and highly substituted benzopyranones. The oligoprodelphinidins, frequently supposed to be compounds with unspecific tanning interactions, show, in contrast to other polyphenols, an amazing variety of substructures and connectivities which results in an uncommon diversity even at a low degree of polymerization. Three distinct purine derivatives, second messengers and probably intermediates of DNA synthesis, were identified and characterized by phytochemical means. The main benzopyranones of EPs 7630 are highly oxygenated at the phenyl moiety (three to four oxygens) and in addition sulfated at distinct positions. A disulfate of 6,7,8-trihydoxybenzopyranone has been identified for the first time in plants. Taken together, these constituents amount to about 70 to 80 % of the total weight of EPs 7630, the active ingredient of the phytopharmaceutical Umckaloabo (Iso Arzneimittel Ettlingen).

Objective: C-reactive protein (CRP) gene variation, in particular an rs2794521 variant was recently associated with type 2 diabetes mellitus (T2DM) in Pima Indians., Research Design and Methods: The present investigation was conducted to replicate this previous association, and to further examine the potential association of a set of common CRP gene variants with the prevalence of T2DM in a case-control investigation. A total of 629 T2DM cases (476 Whites, and 153 Blacks), and 579 controls (481 Whites, and 98 Blacks) were examined. Seven CRP variants were evaluated: rs3093059, rs2794521, rs3091244, rs1417938, rs1800947, rs1130864, and rs1205., Results: Using a marker-by-marker logistic regression analysis, adjusting for age, smoking, gender, and body mass index, we found an association of rs3093059 (recessive: OR, 7.01; 95% CI, 1.16-42.22; p=0.03) with T2DM in the white study population, and an association, albeit not statistically significant, of rs2794521 with T2DM in the Black study population. Moreover, further analysis using a haplotype-based analysis showed no evidence for an association of the haplotypes tested with T2DM., Conclusion: Further studies are needed to examine the possible involvement of C-reactive protein gene variation in the pathogenesis of type 2 diabetes mellitus.

A 28-day subacute oral toxicity study was performed in Wistar rats with a purified preparation of the commercial pentabromodiphenyl ether (pentaBDE), DE-71. The applied OECD407 protocol was enhanced for endocrine and immune parameters, and to enable benchmark dose analysis. A vehicle control group and 7 dose groups were included, which received 0.27, 0.82, 2.47, 7.4, 22.2, 66.7 or 200 mg pentaBDE/kg bw/d (mkd). The liver appeared to be a key target organ, showing a marked increase of weight and centrilobular hepatocellular hypertrophy, probably due to the observed induction of P450 enzymes, notably CYP1A and CYP2B. A marked decrease of circulating total thyroxine (TT4) and an increase of plasma cholesterol were probably secondary to the liver effects. Furthermore, dose-dependently decreased weight of epididymis, seminal vesicles, and prostate, as well as sperm head deformities in males, and induction of CYP17 activity in adrenals in females were observed, all possibly related to anti-androgenic activity. Finally, we observed a substantial increase of large unstained cells in the blood and a decrease of apolar retinoids in the liver. All these effects had benchmark doses at the lower confidence bound (BMDL) in the low- or mid-dose range, but particular sensitive, potentially adverse effects were TT4 decrease (BMDLs 1.1 in males and 1.8 mkd in females), and decrease of hepatic apolar retinoids (BMDLs 0.5 mkd in males and 2.3 mkd in females). These results contribute to refinement of the hazard identification of pentaBDE and improved risk assessment of human exposure to this industrial chemical and environmental pollutant.

Despite many recent advances in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, there is still a great need for inexpensive and flexible methods with a reasonable throughput. Here we report substantial modifications and improvements to an existing homogenous allele-specific PCR-based SNP genotyping method, making it an attractive new option for researchers engaging in candidate gene studies or following up on genome-wide scans. In this advanced version of the melting temperature (Tm)-shift SNP genotyping method, we attach two GC-rich tails of different lengths to allele-specific PCR primers, such that SNP alleles in genomic DNA samples can be discriminated by the Tms of the PCR products. We have validated 306 SNP assays using this method and achieved a success rate in assay development of greater than 83% under uniform PCR conditions. We have developed a standalone software application to automatically assign genotypes directly from melting curve data. To demonstrate the accuracy of this method, we typed 592 individuals for 6 SNPs and showed a high call rate (>98%) and high accuracy (>99.9%). With this method, 6-10,000 samples can be genotyped per day using a single 384-well real-time thermal cycler with 2-4 standard 384-well PCR instruments.

We have evaluated the use of allele-specific PCR (AS PCR) on DNA pools as a tool for screening inherited genetic variants that may be associated with risk of adult acute myeloid leukemia (AML). Two DNA pools were constructed, one of 444 AML cases, and another of 823 matched controls. The pools were validated using individual genotyping data for GSTP1 and LTalpha variants. Allele frequencies for variants in GSTP1 and LTalpha were estimated using quantitative AS PCR, and when compared to individual genotyping data, a high degree of concordance was seen. AS primer pairs were designed for nine candidate genetic variants in DNA repair and cell cycle/apoptotic regulatory genes, including Cyclin D1 [codon 870 splice site variant (A>G)]; BRCA1, P871L; ERCC2, K751Q; FAS -1377 (G>A); hMLH1 -93 (G>A) and V219I; p21, S31R; and the XRCC1 R194W and R399Q variants. For six of these assays, there was at least 95% concordance between AS PCR genotyping and an alternative approach carried out on individual samples. Furthermore, these six AS PCR assays all accurately estimated allele frequencies in the pools that had been calculated using individual genotyping data. A significant disease association was seen with AML for the -1377 variant in FAS (odds ratio 1.76, 95% confidence interval 1.26-2.44). These data suggest that quantitative AS PCR can be used as an efficient screening technique for disease associations of genetic variants in DNA pools made from case-control studies.

The ideal technology for screening single-nucleotide polymorphisms requires high throughput with minimal cost per sample, minimal usage of valuable DNA resources, and maximal flexibility for assessment of new polymorphisms. We demonstrate here the feasibility of kinetic allele-specific PCR with DNA pooling (S. Germer et al., Genome Res., 10: 258-266, 2000) in a population study that satisfies all of the mentioned criteria and offers a powerful new tool for detecting meaningful polymorphic differences in candidate gene association studies and genome-wide linkage dysequilibrium scans. Three individuals prepared pooled DNA samples from 269 individuals separated into three racial/ethnic groups: Caucasians (n = 56), African-Americans (n = 86), and Hispanics (n = 127). We used kinetic allele-specific PCR to determine the allele frequencies of the common paraoxonase 1 polymorphism, PON1 Q191R, in these pools. Paraoxonase 1 is a critical enzyme for inactivating neurotoxic intermediates in the metabolism of organophosphates. In a blinded test of the technology, these nine pooled DNA samples were sent to Roche for genotyping by kinetic allele-specific PCR. The allele frequencies found were 0.266 +/- 0.011, 0.386 +/- 0.011, and 0.617 +/- 0.010, respectively, which were comparable to the frequencies of 0.269, 0.403, and 0.622 determined by PCR-restriction fragment length polymorphism analysis. These same samples were genotyped on two kinetic PCR platforms from different manufacturers, using three different DNA polymerases. The results were comparable between both platforms and among all three polymerases. The results demonstrate a powerful new technology for determining frequencies of single-nucleotide polymorphisms in an epidemiological study.

Activation by melanocortins of the melanocortin 1 receptor (MC1R), expressed in epidermal melanocytes, stimulates melanogenesis. Human MC1R gene loss-of-function mutations are associated with fair skin, poor tanning and increased skin cancer risk. We identified two natural alleles: Ile40Thr, probably associated with skin types I-II, and Val122Met. Val122Met bound [(125)I][Nle(4), D-Phe(7)]-alpha-melanocyte stimulating hormone with lower affinity than the wild-type. Dose-response curves of cAMP accumulation were right-shifted for both forms. The Val122Met form failed to achieve maximal cAMP responses comparable to the wild-type or Ile40Thr receptors. Thus, the Ile40Thr and Val122Met variants are partial loss-of-function natural mutations of MC1R.

Experimental murine genetic models of complex human disease show great potential for understanding human disease pathogenesis. To reduce the time required for analysis of such models from many months down to milliseconds, a computational method for predicting chromosomal regions regulating phenotypic traits and a murine database of single nucleotide polymorphisms were developed. After entry of phenotypic information obtained from inbred mouse strains, the phenotypic and genotypic information is analyzed in silico to predict the chromosomal regions regulating the phenotypic trait.

The prevalence and severity of allergic asthma continue to rise, lending urgency to the search for environmental triggers and genetic substrates. Using microarray analysis of pulmonary gene expression and single nucleotide polymorphism-based genotyping, combined with quantitative trait locus analysis, we identified the gene encoding complement factor 5 (C5) as a susceptibility locus for allergen-induced airway hyperresponsiveness in a murine model of asthma. A deletion in the coding sequence of C5 leads to C5-deficiency and susceptibility. Interleukin 12 (IL-12) is able to prevent or reverse experimental allergic asthma. Blockade of the C5a receptor rendered human monocytes unable to produce IL-12, mimicking blunted IL-12 production by macrophages from C5-deficient mice and providing a mechanism for the regulation of susceptibility to asthma by C5. The role of complement in modulating susceptibility to asthma highlights the importance of immunoregulatory events at the interface of innate and adaptive immunity in disease pathogenesis.

It would be easier and cheaper, and there would be less risk of cerebral oedema, if bacterial meningitis could be adequately treated without the intravenous administration of fluid. Fifty children with bacterial meningitis were treated with intramuscular injections of benzyl penicillin, probenecid given orally and chloramphenicol palmitate suspension given orally, and the outcome was evaluated prospectively. Seven (14%) of the 50 children died. In a control group of 50 children with bacterial meningitis treated with the intravenous administration of benzyl penicillin and chloramphenicol sodium succinate, the outcome was determined retrospectively. Twelve (24%) of the 50 children died. The difference in mortality rate was 10% +/- 15.7% (+/- 2 SE), which is not significant. Except in the rare case of a child with shock or persistent vomiting, bacterial meningitis can be effectively treated with six-hourly intramuscular injections of penicillin, and probenecid and chloramphenicol given orally.

To determine the aetiology of pneumonia in 83 children admitted to Goroka Hospital, Papua New Guinea, lung aspirates and blood were cultured for bacteria. Haemophilus infuenzae, Streptococcus pneumoniae, or both, were isolated from 43 (52%) of the children, other bacteria from 8 (10%), and no bacteria from 32 (39%). Of the 32 strains of H influenzae tested, 18 (56%) were non-serotypable, 8 (25%) were serotypes other than type b, and only 6 (19%) were type b. Viruses were isolated from lung or nasopharyngeal aspirates from 18 (29%) of the 62 children for whom viral cultures were done. It seems that, although viruses may initiate infection, death from pneumonia in children in developing countries is often due to H influenzae, S pneumoniae, or both. Antibiotic therapy would prevent many of these deaths. There is an urgent need for vaccines, effective in children less than 6 months old, that protect against all strains of H influenzae, and S pneumoniae.

In the five year period 1973-77, 278 children in Goroka Hospital paediatric ward were found to have a white cell count (W.C.C.) over 30,000/mm3. Of those with a W.C.C. over 40,000/mm3 in 1977 where the outcome was known, 13 of the 17 (76%) died. It is suggested that "leukaemoid reaction" in Highlands children be defined as a total W.C. over 40,000/mm3 that is not due to leukaemia. A leukaemoid reaction is a useful, but grave, prognostic sign. Such children comprise a quarter of all deaths in the Goroka paediatric ward.

Rotavirus infections were detected by electron microscopy examinations in 54 of 66 children (82%) with acute gastroenteritis which necessitated admission to hospital during April to July, 1979, in the Highlands of Papua New Guinea. Longitudinal epidemiological studies may confirm rotavirus infections to be more important aetiolgical agents of childhood gastroenteritis in this region than in many other countries studied to date.

We have reviewed the clinical presentation of pneumonia to the Goroka paediatric ward. In comparison to survivors, children dying from pneumonia more often (p less than 0.05) had malnutrition (weight-for-age under 80%), anaemia (haemoglobin under 9g%), and a marked leucocytosis (total white cell count over 30,000 cells per c.m.m.). Children dying from pneumonia had been ill for longer and had been given more antibiotics prior to admission. There was no significant difference between children dying from pneumonia and survivors in age distribution, pulse rate, incidence of cardiac failure or duration of stay in hospital. 70% of the children dying from pneumonia at Goroka Hospital are infants under 12 months of age. Pneumococcal vaccine gives a poor antibody response in infants, and overseas studies using lung aspiration suggest that Haemophilus influenzae and Staphylococcus aureus might be causative organisms as well as Streptococcus pneumoniae. A study to determine the aetiology of pneumonia in Highlands children is required to enable a rational choice of routine antibiotic therapy and to plan further research on vaccination against pneumonia.