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1.  Evidence from case–control and longitudinal studies supports associations of genetic variation in APOE, CETP, and IL6 with human longevity 
Age  2012;35(2):487-500.
In this study, we investigated 102 single-nucleotide polymorphisms (SNPs) covering the common genetic variation in 16 genes recurrently regarded as candidates for human longevity: APOE; ACE; CETP; HFE; IL6; IL6R; MTHFR; TGFB1; APOA4; APOC3; SIRTs 1, 3, 6; and HSPAs 1A, 1L, 14. In a case–control study of 1,089 oldest-old (ages 92–93) and 736 middle-aged Danes, the minor allele frequency (MAF) of rs769449 (APOE) was significantly decreased in the oldest-old, while the MAF of rs9923854 (CETP) was significantly enriched. These effects were supported when investigating 1,613 oldest-old (ages 95–110) and 1,104 middle-aged Germans. rs769449 was in modest linkage equilibrium (R2 = 0.55) with rs429358 of the APOE-ε4 haplotype and adjusting for rs429358 eliminated the association of rs769449, indicating that the association likely reflects the well-known effect of rs429358. Gene-based analysis confirmed the effects of variation in APOE and CETP and furthermore pointed to HSPA14 as a longevity gene. In a longitudinal study with 11 years of follow-up on survival in the oldest-old Danes, only one SNP, rs2069827 (IL6), was borderline significantly associated with survival from age 92 (P-corrected = 0.064). This advantageous effect of the minor allele was supported when investigating a Dutch longitudinal cohort (N = 563) of oldest-old (age 85+). Since rs2069827 was located in a putative transcription factor binding site, quantitative RNA expression studies were conducted. However, no difference in IL6 expression was observed between rs2069827 genotype groups. In conclusion, we here support and expand the evidence suggesting that genetic variation in APOE, CETP, and IL6, and possible HSPA14, is associated with human longevity.
Electronic supplementary material
The online version of this article (doi:10.1007/s11357-011-9373-7) contains supplementary material, which is available to authorized users.
doi:10.1007/s11357-011-9373-7
PMCID: PMC3592963  PMID: 22234866
Human longevity; Candidate gene association study; Case–control data; Longitudinal data
2.  Genome-wide linkage analysis for human longevity: Genetics of Healthy Ageing Study 
Aging cell  2013;12(2):184-193.
Summary
Clear evidence exists for heritability of human longevity, and much interest is focused on identifying genes associated with longer lives. To identify such longevity alleles, we performed the largest genome-wide linkage scan thus far reported. Linkage analyses included 2118 nonagenarian Caucasian sibling pairs that have been enrolled in fifteen study centers of eleven European countries as part of the Genetics of Healthy Ageing (GEHA) project. In the joint linkage analyses we observed four regions that show linkage with longevity; chromosome 14q11.2 (LOD=3.47), chromosome 17q12-q22 (LOD=2.95), chromosome 19p13.3-p13.11 (LOD=3.76) and chromosome 19q13.11-q13.32 (LOD=3.57). To fine map these regions linked to longevity, we performed association analysis using GWAS data in a subgroup of 1,228 unrelated nonagenarian and 1,907 geographically matched controls. Using a fixed effect meta-analysis approach, rs4420638 at the TOMM40/APOE/APOC1 gene locus showed significant association with longevity (p-value=9.6 × 10−8). By combined modeling of linkage and association we showed that association of longevity with APOEε4 and APOEε2 alleles explain the linkage at 19q13.11-q13.32 with p-value=0.02 and p-value=1.0 × 10−5, respectively. In the largest linkage scan thus far performed for human familial longevity, we confirm that the APOE locus is a longevity gene and that additional longevity loci may be identified at 14q11.2, 17q12-q22 and 19p13.3-p13.11. Since the latter linkage results are not explained by common variants, we suggest that rare variants play an important role in human familial longevity.
doi:10.1111/acel.12039
PMCID: PMC3725963  PMID: 23286790
Human familial longevity; genome-wide linkage analysis; APOE gene; association analysis; nonagenarian sibling pairs
3.  Transcriptome and genome sequencing uncovers functional variation in humans 
Nature  2013;501(7468):506-511.
Summary
Genome sequencing projects are discovering millions of genetic variants in humans, and interpretation of their functional effects is essential for understanding the genetic basis of variation in human traits. Here we report sequencing and deep analysis of mRNA and miRNA from lymphoblastoid cell lines of 462 individuals from the 1000 Genomes Project – the first uniformly processed RNA-seq data from multiple human populations with high-quality genome sequences. We discovered extremely widespread genetic variation affecting regulation of the majority of genes, with transcript structure and expression level variation being equally common but genetically largely independent. Our characterization of causal regulatory variation sheds light on cellular mechanisms of regulatory and loss-of-function variation, and allowed us to infer putative causal variants for dozens of disease-associated loci. Altogether, this study provides a deep understanding of the cellular mechanisms of transcriptome variation and of the landscape of functional variants in the human genome.
doi:10.1038/nature12531
PMCID: PMC3918453  PMID: 24037378
4.  Effects of β-Lactam Antibiotics and Fluoroquinolones on Human Gut Microbiota in Relation to Clostridium difficile Associated Diarrhea 
PLoS ONE  2014;9(2):e89417.
Clostridium difficile infections are an emerging health problem in the modern hospital environment. Severe alterations of the gut microbiome with loss of resistance to colonization against C. difficile are thought to be the major trigger, but there is no clear concept of how C. difficile infection evolves and which microbiological factors are involved. We sequenced 16S rRNA amplicons generated from DNA and RNA/cDNA of fecal samples from three groups of individuals by FLX technology: (i) healthy controls (no antibiotic therapy); (ii) individuals receiving antibiotic therapy (Ampicillin/Sulbactam, cephalosporins, and fluoroquinolones with subsequent development of C. difficile infection or (iii) individuals receiving antibiotic therapy without C. difficile infection. We compared the effects of the three different antibiotic classes on the intestinal microbiome and the effects of alterations of the gut microbiome on C. difficile infection at the DNA (total microbiota) and rRNA (potentially active) levels. A comparison of antibiotic classes showed significant differences at DNA level, but not at RNA level. Among individuals that developed or did not develop a C. difficile infection under antibiotics we found no significant differences. We identified single species that were up- or down regulated in individuals receiving antibiotics who developed the infection compared to non-infected individuals. We found no significant differences in the global composition of the transcriptionally active gut microbiome associated with C. difficile infections. We suggest that up- and down regulation of specific bacterial species may be involved in colonization resistance against C. difficile providing a potential therapeutic approach through specific manipulation of the intestinal microbiome.
doi:10.1371/journal.pone.0089417
PMCID: PMC3938479  PMID: 24586762
5.  Ablation of Gly96/Immediate Early Gene-X1 (gly96/iex-1) Aggravates DSS-Induced Colitis in Mice: Role for gly96/iex-1 in the Regulation of NF-κB 
Inflammatory bowel diseases  2010;16(2):320-331.
Background
Inflammatory bowel diseases (IBDs) result from environmental and genetic factors and are characterized by an imbalanced immune response in the gut and deregulated activation of the transcription factor NF-κB. Addressing the potential role of gly96/iex-1 in the regulation of NF-κB in IBD, we used the dextran sodium sulfate (DSS) colitis model in mice in which the gly96/iex-1 gene had been deleted.
Methods
C57BL/6 mice of gly96/iex-1–/– or gly96/iex-1+/+ genotype were treated continuously with 4% DSS (5 days) and repeatedly with 2% DSS (28 days) for inducing acute and chronic colitis, respectively. In addition to clinical and histological exploration, colon organ culture and bone marrow-derived cells (BMCs) were analyzed for chemo/cytokine expression and NF-κB activation.
Results
Compared to wildtype littermates, gly96/iex-1–/– mice exhibited an aggravated phenotype of both acute and chronic colitis, along with a greater loss of body weight and colon length. Colonic endoscopy revealed a higher degree of hyperemia, edema, and bleeding in gly96/iex-1–/– mice, and immunohistochemistry detected massive mucosal infiltration of leukocytes and marked histological changes. The expression of proinflammatory chemoand cytokines was higher in the colon of DSS-treated gly96/iex-1–/– mice, and the NF-κB activation was enhanced particularly in the distal colon. In cultured BMCs from gly96/iex-1–/– mice, Pam3Cys4 treatment induced expression of proinflammatory medi ators to a higher degree than in gly96/iex-1+/+ BMCs, along with greater NF-κB activation.
Conclusions
Based on the observation that genetic ablation of gly96/iex-1 triggers intestinal inflammation in mice, we demonstrate for the first time that gly96/iex-1 exerts strong antiinflammatory activity via its NF-κB-counterregulatory effect.
doi:10.1002/ibd.21066
PMCID: PMC3927407  PMID: 19714745
intestinal inflammation; signal transduction; cytokines; feedback regulation
6.  Genetic investigation of FOXO3A requires special attention due to sequence homology with FOXO3B 
Our study demonstrates that the genetic investigation of forkhead box O3A gene (FOXO3A), a validated human longevity gene, is greatly hampered by the fact that its exonic regions have 99% sequence homology with the FOXO3B pseudogene. If unaccounted for, this high degree of homology can cause serious genotyping or sequencing errors. Here, we present an experimental set-up that allows reliable data generation for the highly homologous regions and that can be used for the evaluation of assay specificity. Using this design, we exemplarily showed FOXO3A-specific results for two single-nucleotide polymorphisms (SNPs) (rs4945816 and rs4946936) that are significantly associated with longevity in our centenarian-control sample (Peach=0.0008). Because both SNPs are located in the 3′ untranslated region of FOXO3A, they could be of functional relevance for the longevity phenotype. Our experimental set-up can be used for reliable and reproducible data generation for further sequencing and genotyping studies of FOXO3A with the aim of discovering new SNPs of functional relevance.
doi:10.1038/ejhg.2012.83
PMCID: PMC3548564  PMID: 22588664
centenarians; human longevity; aging; FOXO3A; FOXO3B
7.  Investigation of Complement Component C4 Copy Number Variation in Human Longevity 
PLoS ONE  2014;9(1):e86188.
Genetic factors have been estimated to account for about 25% of the variation in an adult's life span. The complement component C4 with the isotypes C4A and C4B is an effector protein of the immune system, and differences in the overall C4 copy number or gene size (long C4L; short C4S) may influence the strength of the immune response and disease susceptibilities. Previously, an association between C4B copy number and life span was reported for Hungarians and Icelanders, where the C4B*Q0 genotype, which is defined by C4B gene deficiency, showed a decrease in frequency with age. Additionally, one of the studies indicated that a low C4B copy number might be a genetic trait that is manifested only in the presence of the environmental risk factor “smoking”. These observations prompted us to investigate the role of the C4 alleles in our large German longevity sample (∼700 cases; 94–110 years and ∼900 younger controls). No significant differences in the number of C4A, C4B and C4S were detected. Besides, the C4B*Q0 carrier state did not decrease with age, irrespective of smoking as an interacting variable. However, for C4L*Q0 a significantly different carrier frequency was observed in the cases compared with controls (cases: 5.08%; controls: 9.12%; p = 0.003). In a replication sample of 714 German cases (91–108 years) and 890 controls this result was not replicated (p = 0.14) although a similar trend of decreased C4L*Q0 carrier frequency in cases was visible (cases: 7.84%; controls: 10.00%).
doi:10.1371/journal.pone.0086188
PMCID: PMC3899116  PMID: 24465950
8.  Copy number variation in patients with cervical artery dissection 
European Journal of Human Genetics  2012;20(12):1295-1299.
Cervical artery dissection (CeAD) occurs in healthy young individuals and often entails ischemic stroke. Skin biopsies from most CeAD-patients show minor connective tissue alterations. We search for rare genetic deletions and duplication that may predispose to CeAD. Forty-nine non-traumatic CeAD-patients with electron microscopic (EM) alterations of their dermal connective tissue (EM+ patients) and 21 patients with normal connective tissue in skin biopsies (EM− patients) were analyzed. Affymetrix 6.0 microarrays (Affymetrix) from all patients were screened for copy number variants (CNVs). CNVs absent from 403 control subjects and from 2402 published disease-free individuals were considered as CeAD-associated. The genetic content of undentified CNVs was analyzed by means of the Gene Ontology (GO) Term Mapper to detect associations with biological processes. In 49 EM+ patients we identified 13 CeAD-associated CNVs harboring 83 protein-coding genes. In 21 EM− patients we found five CeAD-associated CNVs containing only nine genes (comparison of CNV gene density between the groups: Mann–Whitney P=0.039). Patients' CNVs were enriched for genes involved in extracellular matrix organization (COL5A2, COL3A1, SNTA1, P=0.035), collagen fibril organization COL5A2, COL3A1, (P=0.0001) and possibly for genes involved in transforming growth factor beta (TGF)-beta receptor signaling pathway (COL3A1, DUPS22, P=0.068). We conclude that rare genetic variants may contribute to the pathogenesis of CeAD, in particular in patients with a microscopic connective tissue phenotype.
doi:10.1038/ejhg.2012.82
PMCID: PMC3499738  PMID: 22617347
carotid and vertebral artery dissection; copy number variants (CNVs); connective tissue morphology; cerebral ischemic stroke
9.  Diagnosing Fatty Liver Disease: A Comparative Evaluation of Metabolic Markers, Phenotypes, Genotypes and Established Biomarkers 
PLoS ONE  2013;8(10):e76813.
Background
To date, liver biopsy is the only means of reliable diagnosis for fatty liver disease (FLD). Owing to the inevitable biopsy-associated health risks, however, the development of valid noninvasive diagnostic tools for FLD is well warranted.
Aim
We evaluated a particular metabolic profile with regard to its ability to diagnose FLD and compared its performance to that of established phenotypes, conventional biomarkers and disease-associated genotypes.
Methods
The study population comprised 115 patients with ultrasound-diagnosed FLD and 115 sex- and age-matched controls for whom the serum concentration was measured of 138 different metabolites, including acylcarnitines, amino acids, biogenic amines, hexose, phosphatidylcholines (PCs), lyso-PCs and sphingomyelins. Established phenotypes, biomarkers, disease-associated genotypes and metabolite data were included in diagnostic models for FLD using logistic regression and partial least-squares discriminant analysis. The discriminative power of the ensuing models was compared with respect to area under curve (AUC), integrated discrimination improvement (IDI) and by way of cross-validation (CV).
Results
Use of metabolic markers for predicting FLD showed the best performance among all considered types of markers, yielding an AUC of 0.8993. Additional information on phenotypes, conventional biomarkers or genotypes did not significantly improve this performance. Phospholipids and branched-chain amino acids were most informative for predicting FLD.
Conclusion
We show that the inclusion of metabolite data may substantially increase the power to diagnose FLD over that of models based solely upon phenotypes and conventional biomarkers.
doi:10.1371/journal.pone.0076813
PMCID: PMC3793954  PMID: 24130792
10.  Metabolic Signature of Electrosurgical Liver Dissection 
PLoS ONE  2013;8(9):e72022.
Background and Aims
High frequency electrosurgery has a key role in the broadening application of liver surgery. Its molecular signature, i.e. the metabolites evolving from electrocauterization which may inhibit hepatic wound healing, have not been systematically studied.
Methods
Human liver samples were thus obtained during surgery before and after electrosurgical dissection and subjected to a two-stage metabolomic screening experiment (discovery sample: N = 18, replication sample: N = 20) using gas chromatography/mass spectrometry.
Results
In a set of 208 chemically defined metabolites, electrosurgical dissection lead to a distinct metabolic signature resulting in a separation in the first two dimensions of a principal components analysis. Six metabolites including glycolic acid, azelaic acid, 2-n-pentylfuran, dihydroactinidiolide, 2-butenal and n-pentanal were consistently increased after electrosurgery meeting the discovery (p<2.0×10−4) and the replication thresholds (p<3.5×10−3). Azelaic acid, a lipid peroxidation product from the fragmentation of abundant sn-2 linoleoyl residues, was most abundant and increased 8.1-fold after electrosurgical liver dissection (preplication = 1.6×10−4). The corresponding phospholipid hexadecyl azelaoyl glycerophosphocholine inhibited wound healing and tissue remodelling in scratch- and proliferation assays of hepatic stellate cells and cholangiocytes, and caused apoptosis dose-dependently in vitro, which may explain in part the tissue damage due to electrosurgery.
Conclusion
Hepatic electrosurgery generates a metabolic signature with characteristic lipid peroxidation products. Among these, azelaic acid shows a dose-dependent toxicity in liver cells and inhibits wound healing. These observations potentially pave the way for pharmacological intervention prior liver surgery to modify the metabolic response and prevent postoperative complications.
doi:10.1371/journal.pone.0072022
PMCID: PMC3772850  PMID: 24058442
11.  Deep Resequencing of GWAS Loci Identifies Rare Variants in CARD9, IL23R and RNF186 That Are Associated with Ulcerative Colitis 
PLoS Genetics  2013;9(9):e1003723.
Genome-wide association studies and follow-up meta-analyses in Crohn's disease (CD) and ulcerative colitis (UC) have recently identified 163 disease-associated loci that meet genome-wide significance for these two inflammatory bowel diseases (IBD). These discoveries have already had a tremendous impact on our understanding of the genetic architecture of these diseases and have directed functional studies that have revealed some of the biological functions that are important to IBD (e.g. autophagy). Nonetheless, these loci can only explain a small proportion of disease variance (∼14% in CD and 7.5% in UC), suggesting that not only are additional loci to be found but that the known loci may contain high effect rare risk variants that have gone undetected by GWAS. To test this, we have used a targeted sequencing approach in 200 UC cases and 150 healthy controls (HC), all of French Canadian descent, to study 55 genes in regions associated with UC. We performed follow-up genotyping of 42 rare non-synonymous variants in independent case-control cohorts (totaling 14,435 UC cases and 20,204 HC). Our results confirmed significant association to rare non-synonymous coding variants in both IL23R and CARD9, previously identified from sequencing of CD loci, as well as identified a novel association in RNF186. With the exception of CARD9 (OR = 0.39), the rare non-synonymous variants identified were of moderate effect (OR = 1.49 for RNF186 and OR = 0.79 for IL23R). RNF186 encodes a protein with a RING domain having predicted E3 ubiquitin-protein ligase activity and two transmembrane domains. Importantly, the disease-coding variant is located in the ubiquitin ligase domain. Finally, our results suggest that rare variants in genes identified by genome-wide association in UC are unlikely to contribute significantly to the overall variance for the disease. Rather, these are expected to help focus functional studies of the corresponding disease loci.
Author Summary
Genetic studies of common diseases have seen tremendous progress in the last half-decade primarily due to recent technologies that enable a systematic examination of genetic markers across the entire genome in large numbers of patients and healthy controls. The studies, while identifying genomic regions that influence a person's risk for developing disease, often do not pinpoint the actual gene or gene variants that account for this risk (called a causal gene/variant). A prime example of this can be seen with the 163 genetic risk factors that have recently been associated with the chronic inflammatory bowel diseases known as Crohn's disease and ulcerative colitis. For less than a handful of these 163 is the causative change in the genetic code known. The current study used an approach to directly look at the genetic code for a subset of these and identified a causative change in the genetic code for eight risk factors for ulcerative colitis. This finding is particularly important because it directs biological studies to understand the mechanisms that lead to this chronic life-long inflammatory disease.
doi:10.1371/journal.pgen.1003723
PMCID: PMC3772057  PMID: 24068945
12.  Polymorphisms in the glial glutamate transporter SLC1A2 are associated with essential tremor 
Neurology  2012;79(3):243-248.
Objective:
Sporadic, genetically complex essential tremor (ET) is one of the most common movement disorders and may lead to severe impairment of the quality of life. Despite high heritability, the genetic determinants of ET are largely unknown. We performed the second genome-wide association study (GWAS) for ET to elucidate genetic risk factors of ET.
Methods:
Using the Affymetrix Genome-Wide SNP Array 6.0 (1000K) we conducted a two-stage GWAS in a total of 990 subjects and 1,537 control subjects from Europe to identify genetic variants associated with ET.
Results:
We discovered association of an intronic variant of the main glial glutamate transporter (SLC1A2) gene with ET in the first-stage sample (rs3794087, p = 6.95 × 10−5, odds ratio [OR] = 1.46). We verified the association of rs3794087 with ET in a second-stage sample (p = 1.25 × 10−3, OR = 1.38). In the subgroup analysis of patients classified as definite ET, rs3794087 obtained genome-wide significance (p = 3.44 × 10−10, OR = 1.59) in the combined first- and second-stage sample. Genetic fine mapping using nonsynonymous single nucleotide polymorphisms (SNPs) and SNPs in high linkage disequilibrium with rs3794087 did not reveal any SNP with a stronger association with ET than rs3794087.
Conclusions:
We identified SLC1A2 encoding the major glial high-affinity glutamate reuptake transporter in the brain as a potential ET susceptibility gene. Acute and chronic glutamatergic overexcitation is implied in the pathogenesis of ET. SLC1A2 is therefore a good functional candidate gene for ET.
doi:10.1212/WNL.0b013e31825fdeed
PMCID: PMC3398434  PMID: 22764253
13.  Identification of fifteen new psoriasis susceptibility loci highlights the role of innate immunity 
Nature genetics  2012;44(12):1341-1348.
Summary
To gain further insight into the genetic architecture of psoriasis, we conducted a meta-analysis of three genome-wide association studies (GWAS) and two independent datasets genotyped on the Immunochip, involving 10,588 cases and 22,806 controls in total. We identified 15 new disease susceptibility regions, increasing the number of psoriasis-associated loci to 36 for Caucasians. Conditional analyses identified five independent signals within previously known loci. The newly identified shared disease regions encompassed a number of genes whose products regulate T-cell function (e.g. RUNX3, TAGAP and STAT3). The new psoriasis-specific regions were notable for candidate genes whose products are involved in innate host defense, encoding proteins with roles in interferon-mediated antiviral responses (DDX58), macrophage activation (ZC3H12C), and NF-κB signaling (CARD14 and CARM1). These results portend a better understanding of shared and distinctive genetic determinants of immune-mediated inflammatory disorders and emphasize the importance of the skin in innate and acquired host defense.
doi:10.1038/ng.2467
PMCID: PMC3510312  PMID: 23143594
14.  Genetic characterization of northeastern Italian population isolates in the context of broader European genetic diversity 
Population genetic studies on European populations have highlighted Italy as one of genetically most diverse regions. This is possibly due to the country's complex demographic history and large variability in terrain throughout the territory. This is the reason why Italy is enriched for population isolates, Sardinia being the best-known example. As the population isolates have a great potential in disease-causing genetic variants identification, we aimed to genetically characterize a region from northeastern Italy, which is known for isolated communities. Total of 1310 samples, collected from six geographically isolated villages, were genotyped at >145 000 single-nucleotide polymorphism positions. Newly genotyped data were analyzed jointly with the available genome-wide data sets of individuals of European descent, including several population isolates. Despite the linguistic differences and geographical isolation the village populations still show the greatest genetic similarity to other Italian samples. The genetic isolation and small effective population size of the village populations is manifested by higher levels of genomic homozygosity and elevated linkage disequilibrium. These estimates become even more striking when the detected substructure is taken into account. The observed level of genetic isolation in Friuli-Venezia Giulia region is more extreme according to several measures of isolation compared with Sardinians, French Basques and northern Finns, thus proving the status of an isolate.
doi:10.1038/ejhg.2012.229
PMCID: PMC3658181  PMID: 23249956
population genetics; isolated population; genetic distance
15.  Human Longevity and Variation in GH/IGF-1/Insulin Signaling, DNA Damage Signaling and Repair and Pro/antioxidant Pathway Genes: Cross Sectional and Longitudinal Studies 
Experimental Gerontology  2012;47(5):379-387.
Here we explore association with human longevity of common genetic variation in three major candidate pathways: GH/IGF-1/insulin signaling, DNA damage signaling and repair and pro/antioxidants by investigating 1273 tagging SNPs in 148 genes composing these pathways. In a case-control study of 1089 oldest-old (age 92–93) and 736 middle-aged Danes we found 1 pro/antioxidant SNP (rs1002149 (GSR)), 5 GH/IGF-1/INS SNPs (rs1207362 (KL), rs2267723 (GHRHR), rs3842755 (INS), rs572169 (GHSR), rs9456497 (IGF2R)) and 5 DNA repair SNPs (rs11571461 (RAD52), rs13251813 (WRN), rs1805329 (RAD23B), rs2953983 (POLB), rs3211994 (NTLH1)) to be associated with longevity after correction for multiple testing.
In a longitudinal study with 11 years of follow-up on survival in the oldest-old Danes we found 2 pro/antioxidant SNPs (rs10047589 (TNXRD1), rs207444 (XDH)), 1 GH/IGF-1/INS SNP (rs26802 (GHRL)) and 3 DNA repair SNPs (rs13320360 (MLH1), rs2509049 (H2AFX) and rs705649 (XRCC5)) to be associated with mortality in late life after correction for multiple testing.
When examining the 11 SNPs from the case-control study in the longitudinal data, rs3842755 (INS), rs13251813 (WRN) and rs3211994 (NTHL1) demonstrated the same directions of effect (p<0.05), while rs9456497 (IGF2R) and rs1157146 (RAD52) showed non-significant tendencies, indicative of effects also in late life survival. In addition, rs207444 (XDH) presented the same direction of effect when inspecting the 6 SNPs from the longitudinal study in the case-control data, hence, suggesting an effect also in survival from middle age to old age.
No formal replications were observed when investigating the 11 SNPs from the case-control study in 1613 oldest-old (age 95–110) and 1104 middle-aged Germans, although rs11571461 (RAD52) did show a supportive non-significant tendency (OR = 1.162, 95% CI = 0.927–1.457). The same was true for rs10047589 (TNXRD1) (HR = 0.758, 95%CI = 0.543–1.058) when examining the 6 SNPs from the longitudinal study in a Dutch longitudinal cohort of oldest-old (age 85+, N = 563).
In conclusion, the present candidate gene based association study, the largest to date applying a pathway approach, points to potential new longevity loci, but does also underline the difficulties of replicating association findings in independent study populations and thus the difficulties in identifying universal longevity polymorphisms.
doi:10.1016/j.exger.2012.02.010
PMCID: PMC3334439  PMID: 22406557
human longevity; association study; case-control data; longitudinal data
16.  A Randomized Controlled Trial of the Efficacy and Safety of CCX282-B, an Orally-Administered Blocker of Chemokine Receptor CCR9, for Patients with Crohn’s Disease 
PLoS ONE  2013;8(3):e60094.
CCX282-B, also called vercirnon, is a specific, orally-administered chemokine receptor CCR9 antagonist that regulates migration and activation of inflammatory cells in the intestine. This randomized, placebo-controlled trial was conducted to evaluate the safety and efficacy of CCX282-B in 436 patients with Crohn’s disease. Crohn’s Disease Activity Index (CDAI) scores were 250–450 and C-reactive protein >7.5 mg/L at study entry. In addition to stable concomitant Crohn’s medication (85% of subjects), subjects received placebo or CCX282-B (250 mg once daily, 250 mg twice daily, or 500 mg once daily) for 12 weeks. They then received 250 mg CCX282-B twice daily, open-label, through week 16. Subjects who had a clinical response (a ≥70 point drop in CDAI) at week 16 were randomly assigned to groups given placebo or CCX282-B (250 mg, twice daily) for 36 weeks. Primary endpoints were clinical response at Week 8 and sustained clinical response at Week 52. During the 12-week Induction period, the clinical response was highest in the group given 500 mg CCX282-B once daily. Response rates at week 8 were 49% in the placebo group, 52% in the group given CCX282-B 250 mg once daily (odds ratio [OR] = 1.12; p = .667 vs placebo), 48% in the group given CCX282-B 250 mg twice daily (OR = 0.95; p = .833), and 60% in the group given CCX282-B 500 mg once daily (OR = 1.53; p = .111). At week 12, response rates were 47%, 56% (OR = 1.44; p = .168), 49% (OR = 1.07; p = .792), and 61% (OR = 1.74; p = .039), respectively. At the end of the Maintenance period (week 52), 47% of subjects on CCX282-B were in remission, compared to 31% on placebo (OR = 2.01; p = .012); 46% showed sustained clinical responses, compared to 42% on placebo (OR = 1.14; p = .629). CCX282-B was well tolerated. Encouraging results from this clinical trial led to initiation of Phase 3 clinical trials in Crohn’s disease.
Trial Registration
ClinicalTrials.gov NCT00306215.
doi:10.1371/journal.pone.0060094
PMCID: PMC3603920  PMID: 23527300
17.  Smoking Cessation Induces Profound Changes in the Composition of the Intestinal Microbiota in Humans 
PLoS ONE  2013;8(3):e59260.
Background
The human intestinal microbiota is a crucial factor in the pathogenesis of various diseases, such as metabolic syndrome or inflammatory bowel disease (IBD). Yet, knowledge about the role of environmental factors such as smoking (which is known to influence theses aforementioned disease states) on the complex microbial composition is sparse. We aimed to investigate the role of smoking cessation on intestinal microbial composition in 10 healthy smoking subjects undergoing controlled smoking cessation.
Methods
During the observational period of 9 weeks repetitive stool samples were collected. Based on abundance of 16S rRNA genes bacterial composition was analysed and compared to 10 control subjects (5 continuing smokers and 5 non-smokers) by means of Terminal Restriction Fragment Length Polymorphism analysis and high-throughput sequencing.
Results
Profound shifts in the microbial composition after smoking cessation were observed with an increase of Firmicutes and Actinobacteria and a lower proportion of Bacteroidetes and Proteobacteria on the phylum level. In addition, after smoking cessation there was an increase in microbial diversity.
Conclusions
These results indicate that smoking is an environmental factor modulating the composition of human gut microbiota. The observed changes after smoking cessation revealed to be similar to the previously reported differences in obese compared to lean humans and mice respectively, suggesting a potential pathogenetic link between weight gain and smoking cessation. In addition they give rise to a potential association of smoking status and the course of IBD.
doi:10.1371/journal.pone.0059260
PMCID: PMC3597605  PMID: 23516617
18.  A Genome-Wide Analysis of Populations from European Russia Reveals a New Pole of Genetic Diversity in Northern Europe 
PLoS ONE  2013;8(3):e58552.
Several studies examined the fine-scale structure of human genetic variation in Europe. However, the European sets analyzed represent mainly northern, western, central, and southern Europe. Here, we report an analysis of approximately 166,000 single nucleotide polymorphisms in populations from eastern (northeastern) Europe: four Russian populations from European Russia, and three populations from the northernmost Finno-Ugric ethnicities (Veps and two contrast groups of Komi people). These were compared with several reference European samples, including Finns, Estonians, Latvians, Poles, Czechs, Germans, and Italians. The results obtained demonstrated genetic heterogeneity of populations living in the region studied. Russians from the central part of European Russia (Tver, Murom, and Kursk) exhibited similarities with populations from central–eastern Europe, and were distant from Russian sample from the northern Russia (Mezen district, Archangelsk region). Komi samples, especially Izhemski Komi, were significantly different from all other populations studied. These can be considered as a second pole of genetic diversity in northern Europe (in addition to the pole, occupied by Finns), as they had a distinct ancestry component. Russians from Mezen and the Finnic-speaking Veps were positioned between the two poles, but differed from each other in the proportions of Komi and Finnic ancestries. In general, our data provides a more complete genetic map of Europe accounting for the diversity in its most eastern (northeastern) populations.
doi:10.1371/journal.pone.0058552
PMCID: PMC3591355  PMID: 23505534
19.  NOD2-mediated dysbiosis predisposes mice to transmissible colitis and colorectal cancer 
Instability in the composition of gut bacterial communities (dysbiosis) has been linked to common human intestinal disorders, such as Crohn’s disease and colorectal cancer. Here, we show that dysbiosis caused by Nod2 deficiency gives rise to a reversible, communicable risk of colitis and colitis-associated carcinogenesis in mice. Loss of either Nod2 or RIP2 resulted in a proinflammatory microenvironment that enhanced epithelial dysplasia following chemically induced injury. The condition could be improved by treatment with antibiotics or an anti–interleukin-6 receptor–neutralizing antibody. Genotype-dependent disease risk was communicable via maternally transmitted microbiota in both Nod2-deficient and WT hosts. Furthermore, reciprocal microbiota transplantation reduced disease risk in Nod2-deficient mice and led to long-term changes in intestinal microbial communities. Conversely, disease risk was enhanced in WT hosts that were recolonized with dysbiotic fecal microbiota from Nod2-deficient mice. Thus, we demonstrated that licensing of dysbiotic microbiota is a critical component of disease risk. Our results demonstrate that NOD2 has an unexpected role in shaping a protective assembly of gut bacterial communities and suggest that manipulation of dysbiosis is a potential therapeutic approach in the treatment of human intestinal disorders.
doi:10.1172/JCI62236
PMCID: PMC3561825  PMID: 23281400
20.  HEPATITIS B VIRUS-INDUCED ALTERATIONS IN HEPATOCYTE CD1D LIPID ANTIGENS ACTIVATE NATURAL KILLER T CELLS AND CONTRIBUTE TO PROTECTIVE IMMUNITY 
Nature medicine  2012;18(7):1060-1068.
In most adult patients, hepatitis B is a self-limiting disease leading to life-long protective immunity, which is the consequence of a robust adaptive immune response occurring weeks after HBV infection. Intriguingly, HBV-specific T cells can be detected shortly after infection but the mechanisms underlying this early immune priming and its consequences for subsequent control of viral replication are poorly understood. Using primary human and murine hepatocytes and mouse models of transgenic and adenoviral HBV expression, we show that HBV-expressing hepatocytes produce endoplasmic reticulum (ER)-associated endogenous antigenic lipids including lysophospholipids that are generated by HBV-induced secretory phospholipases and lead to activation of natural killer T (NKT) cells. The absence of NKT cells, CD1d or a defect in ER-associated transfer of lipids onto CD1d results in diminished HBV-specific T and B cell responses and delayed viral control. NKT cells may therefore contribute to control of HBV infection through sensing of HBV-induced modified self-lipids.
doi:10.1038/nm.2811
PMCID: PMC3478098  PMID: 22706385
21.  Leveraging Ethnic Group Incidence Variation to Investigate Genetic Susceptibility to Glioma: A Novel Candidate SNP Approach 
Frontiers in Genetics  2012;3:203.
Objectives: Using a novel candidate SNP approach, we aimed to identify a possible genetic basis for the higher glioma incidence in Whites relative to East Asians and African-Americans. Methods:  We hypothesized that genetic regions containing SNPs with extreme differences in allele frequencies across ethnicities are most likely to harbor susceptibility variants. We used International HapMap Project data to identify 3,961 candidate SNPs with the largest allele frequency differences in Whites compared to East Asians and Africans and tested these SNPs for association with glioma risk in a set of White cases and controls. Top SNPs identified in the discovery dataset were tested for association with glioma in five independent replication datasets. Results: No SNP achieved statistical significance in either the discovery or replication datasets after accounting for multiple testing or conducting meta-analysis. However, the most strongly associated SNP, rs879471, was found to be in linkage disequilibrium with a previously identified risk SNP, rs6010620, in RTEL1. We estimate rs6010620 to account for a glioma incidence rate ratio of 1.34 for Whites relative to East Asians. Conclusion: We explored genetic susceptibility to glioma using a novel candidate SNP method which may be applicable to other diseases with appropriate epidemiologic patterns.
doi:10.3389/fgene.2012.00203
PMCID: PMC3469791  PMID: 23091480
glioma; candidate SNP association study; ancestry informative markers; admixture; race; ethnicity; brain cancer
22.  Genome-wide meta-analysis of Psoriatic Arthritis Identifies Susceptibility Locus at REL 
Psoriatic arthritis (PsA) is a chronic inflammatory musculoskeletal disease affecting up to 30% of psoriasis vulgaris (PsV) cases and approximately 0.25% to 1% of the general population. To identify common susceptibility loci, we performed a meta-analysis of three imputed genome-wide association studies (GWAS) on psoriasis, stratified for PsA. A total of 1,160,703 SNPs were analyzed in the discovery set consisting of 535 PsA cases and 3,432 controls from Germany, the United States and Canada. We followed up two SNPs in 1,931 PsA cases and 6,785 controls comprising six independent replication panels from Germany, Estonia, the United States and Canada. In the combined analysis, a genome-wide significant association was detected at 2p16 near the REL locus encoding c-Rel (rs13017599, P=1.18×10−8, OR=1.27, 95% CI=1.18–1.35). The rs13017599 polymorphism is known to associate with rheumatoid arthritis (RA), and another SNP near REL (rs702873) was recently implicated in PsV susceptibility. However, conditional analysis indicated that rs13017599, rather than rs702873, accounts for the PsA association at REL. We hypothesize that c-Rel, as a member of the Rel/NF-κB family, is associated with PsA in the context of disease pathways that involve other identified PsA and PsV susceptibility genes including TNIP1, TNFAIP3 and NFκBIA.
doi:10.1038/jid.2011.415
PMCID: PMC3305829  PMID: 22170493
23.  Limited Evidence for Parent-of-Origin Effects in Inflammatory Bowel Disease Associated Loci 
PLoS ONE  2012;7(9):e45287.
Background
Genome-wide association studies of two main forms of inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), have identified 99 susceptibility loci, but these explain only ∼23% of the genetic risk. Part of the ‘hidden heritability’ could be in transmissible genetic effects in which mRNA expression in the offspring depends on the parental origin of the allele (genomic imprinting), since children whose mothers have CD are more often affected than children with affected fathers. We analyzed parent-of-origin (POO) effects in Dutch and Indian cohorts of IBD patients.
Methods
We selected 28 genetic loci associated with both CD and UC, and tested them for POO effects in 181 Dutch IBD case-parent trios. Three susceptibility variants in NOD2 were tested in 111 CD trios and a significant finding was re-evaluated in 598 German trios. The UC-associated gene, BTNL2, reportedly imprinted, was tested in 70 Dutch UC trios. Finally, we used 62 independent Indian UC trios to test POO effects of five established Indian UC risk loci.
Results
We identified POO effects for NOD2 (L1007fs; OR = 21.0, P-value = 0.013) for CD; these results could not be replicated in an independent cohort (OR = 0.97, P-value = 0.95). A POO effect in IBD was observed for IL12B (OR = 3.2, P-value = 0.019) and PRDM1 (OR = 5.6, P-value = 0.04). In the Indian trios the IL10 locus showed a POO effect (OR = 0.2, P-value = 0.03).
Conclusions
Little is known about the effect of genomic imprinting in complex diseases such as IBD. We present limited evidence for POO effects for the tested IBD loci. POO effects explain part of the hidden heritability for complex genetic diseases but need to be investigated further.
doi:10.1371/journal.pone.0045287
PMCID: PMC3459955  PMID: 23028907
24.  Accurate variant detection across non-amplified and whole genome amplified DNA using targeted next generation sequencing 
BMC Genomics  2012;13:500.
Background
Many hypothesis-driven genetic studies require the ability to comprehensively and efficiently target specific regions of the genome to detect sequence variations. Often, sample availability is limited requiring the use of whole genome amplification (WGA). We evaluated a high-throughput microdroplet-based PCR approach in combination with next generation sequencing (NGS) to target 384 discrete exons from 373 genes involved in cancer. In our evaluation, we compared the performance of six non-amplified gDNA samples from two HapMap family trios. Three of these samples were also preamplified by WGA and evaluated. We tested sample pooling or multiplexing strategies at different stages of the tested targeted NGS (T-NGS) workflow.
Results
The results demonstrated comparable sequence performance between non-amplified and preamplified samples and between different indexing strategies [sequence specificity of 66.0% ± 3.4%, uniformity (coverage at 0.2× of the mean) of 85.6% ± 0.6%]. The average genotype concordance maintained across all the samples was 99.5% ± 0.4%, regardless of sample type or pooling strategy. We did not detect any errors in the Mendelian patterns of inheritance of genotypes between the parents and offspring within each trio. We also demonstrated the ability to detect minor allele frequencies within the pooled samples that conform to predicted models.
Conclusion
Our described PCR-based sample multiplex approach and the ability to use WGA material for NGS may enable researchers to perform deep resequencing studies and explore variants at very low frequencies and cost.
doi:10.1186/1471-2164-13-500
PMCID: PMC3534403  PMID: 22994565
High-throughput targeted next-generation resequencing; Microdroplet-based multiplex PCR; Sample pooling or multiplexing; Whole-genome amplified DNA samples; Cost reduction
25.  Gene Expression and Physiological Changes of Different Populations of the Long-Lived Bivalve Arctica islandica under Low Oxygen Conditions 
PLoS ONE  2012;7(9):e44621.
The bivalve Arctica islandica is extremely long lived (>400 years) and can tolerate long periods of hypoxia and anoxia. European populations differ in maximum life spans (MLSP) from 40 years in the Baltic to >400 years around Iceland. Characteristic behavior of A. islandica involves phases of metabolic rate depression (MRD) during which the animals burry into the sediment for several days. During these phases the shell water oxygen concentrations reaches hypoxic to anoxic levels, which possibly support the long life span of some populations. We investigated gene regulation in A. islandica from a long-lived (MLSP 150 years) German Bight population and the short-lived Baltic Sea population, experimentally exposed to different oxygen levels. A new A. islandica transcriptome enabled the identification of genes important during hypoxia/anoxia events and, more generally, gene mining for putative stress response and (anti-) aging genes. Expression changes of a) antioxidant defense: Catalase, Glutathione peroxidase, manganese and copper-zinc Superoxide dismutase; b) oxygen sensing and general stress response: Hypoxia inducible factor alpha, Prolyl hydroxylase and Heat-shock protein 70; and c) anaerobic capacity: Malate dehydrogenase and Octopine dehydrogenase, related transcripts were investigated. Exposed to low oxygen, German Bight individuals suppressed transcription of all investigated genes, whereas Baltic Sea bivalves enhanced gene transcription under anoxic incubation (0 kPa) and, further, decreased these transcription levels again during 6 h of re-oxygenation. Hypoxic and anoxic exposure and subsequent re-oxygenation in Baltic Sea animals did not lead to increased protein oxidation or induction of apoptosis, emphasizing considerable hypoxia/re-oxygenation tolerance in this species. The data suggest that the energy saving effect of MRD may not be an attribute of Baltic Sea A. islandica chronically exposed to high environmental variability (oxygenation, temperature, salinity). Contrary, higher physiological flexibility and stress hardening may predispose these animals to perform a pronounced stress response at the expense of life span.
doi:10.1371/journal.pone.0044621
PMCID: PMC3446923  PMID: 23028566

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