DNA kahjustuste reparatsioon ja genoomi terviklikkuse tagamine pseudomonaadides

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Efektiivne DNA replikatsioon ja DNA kahjustuste eemaldamine on erakordse tähtsusega elusrakkude genoomi terviklikkuse säilitamisel. Kõrgelt koordineeritud DNA reparatsiooni mehhanismid võimaldavad rakkudel ellu jääda ka ulatuslike rakuväliselt indutseeritud DNA kahjustuste korral, kuid on ilmselgelt olulised ka spontaanselt tekkivate DNA kahjustuste kõrvaldamisel. Nukleotiidi väljalõike reparatsioon (NER) on üks põhilisi DNA reparatsioonisüsteeme, kus UvrA, UvrB, UvrC ja UvrD valkude toimel eemaldatakse DNA kaksikahelast 12-13 nukleotiidi pikkune üksikahelaline DNA kahjustust sisaldav lõik ning seejärel sünteesib DNA polümeraas I (Pol I) selle asemele uue DNA ahela. Pol I põhifunktsiooniks rakus on DNA replikatsioonil RNA praimerite eemaldamine ja nende asemele uute DNA lõikude süntees. DNA kahjustuste korral osaleb Pol I ka reparatsioonilisel DNA sünteesil. Seega on nii NER kui ka Pol I olulised geneetilise informatsiooni säilitamiseks rakkude paljunemisel. Doktoritöös uuriti NER, Pol I ning samuti ka DNA-d kahjustatavate ühendite rakkudest aktiivse väljaviimise osalust genoomi terviklikkuse tagamisel pseudomonaadides, mullabakteris Pseudomonas putida ning oportunistlikus inimese patogeenis P. aeruginosa. Uuringute tulemusena selgus, et NER valkude puudumisel suureneb oluliselt homoloogilise rekombinatsiooni (HR) sagedus P. putida kromosoomis ning väheneb rakkude eluvõime. Samas tekivad NER-defektsuse korral kiiresti kompensatoorsed mutatsioonid, mis taastavad bakterite normaalse kasvu ja vähendavad HR-i sagedust. Sellist efekti NER valkude puudumisele ei ole teistes organismides varem kirjeldatud. Teiseks selgus uurimistööst, et Pol I on seni arvatust olulisem endogeenselt tekkivate reaktiivsete hapniku radikaalide tolereerimisel. Samuti ilmnes, et Pol I puudumisel osalevad DNA sünteesil spetsialiseeritud DNA polümeraasid Pol II, Pol IV ja DnaE2. Lisaks selgus, et P. aeruginosa PAO1 liini laboratoorsete tüvede puhul toimub rakke kahjustavate kemikaalide rakkudest välja viimine erineva efektiivsusega. Selline varieeruvus mõjutab bakterite tundlikkust DNA-d kahjustatavatele ühenditele ja antibiootikumidele ning raskendab oluliselt erinevate töörühmade poolt saadud tulemuste võrdlust.Every time a living cell divides, it is confronted with a challenge to replicate its genome with high fidelity and maintain its integrity despite numerous endogenous and exogenous DNA damage. The primary strategy to avoid replication blocks is to excise the damage from the DNA strand and fill in the missing nucleotides using the intact strand as a template. In nucleotide excision repair (NER), one of the major DNA repair pathways, DNA damage is recognized and excised through a coordinated action of UvrA, UvrB, UvrC and UvrD proteins. As a next step DNA polymerase I (Pol I) fills in the resulting 12-13 nucleotide-long gap. Pol I is also involved in DNA repair synthesis in the base excision repair pathway, although the major function of Pol I is the replacement of RNA primers on the lagging strand with DNA stretches during chromosome replication. This thesis aimed to investigate the roles of NER, DNA polymerase I and the active efflux of the damaging agents in combating DNA damage in pseudomonads, a soil bacterium Pseudomonas putida and an opportunistic human pathogen P. aeruginosa. This thesis identifies that in contrast to all studied bacteria, NER deficiency results in deleterious effects on morphology, growth and viability of P. putida and enhances homologous recombination between chromosomal loci both in growing and starving cells. Furthermore, this study reveals the involvement of P. putida specialized DNA polymerases Pol II, Pol IV and DnaE2 in DNA synthesis in the absence of Pol I. This work also shows that the need of Pol I for the growth of bacteria on the rich medium is caused by the inability of the Pol I-deficient cells to cope with oxidative DNA damage more than has been thought before. Finally, this study demonstrates that the levels of active efflux extrusion of noxious compounds, one of the strategies to prevent DNA damage, vary in laboratory strains of P. aeruginosa contributing to selected resistance to various chemicals and antibiotics

GWAS of peptic ulcer disease implicates Helicobacter pylori infection, other gastrointestinal disorders and depression.

Genetic factors are recognized to contribute to peptic ulcer disease (PUD) and other gastrointestinal diseases, such as gastro-oesophageal reflux disease (GORD), irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Here, genome-wide association study (GWAS) analyses based on 456,327 UK Biobank (UKB) individuals identify 8 independent and significant loci for PUD at, or near, genes MUC1, MUC6, FUT2, PSCA, ABO, CDX2, GAST and CCKBR. There are previously established roles in susceptibility to Helicobacter pylori infection, response to counteract infection-related damage, gastric acid secretion or gastrointestinal motility for these genes. Only two associations have been previously reported for duodenal ulcer, here replicated trans-ancestrally. The results highlight the role of host genetic susceptibility to infection. Post-GWAS analyses for PUD, GORD, IBS and IBD add insights into relationships between these gastrointestinal diseases and their relationships with depression, a commonly comorbid disorder

Estimation and implications of the genetic architecture of fasting and non-fasting blood glucose

This upload includes the sample code that was used in the paper "Estimation and implications of the genetic architecture of fasting and non-fasting blood glucose", which has been accepted for publication in Nature Communications. Abstract The genetic regulation of post-prandial glucose levels is poorly understood. Here, we characterise the genetic architecture of blood glucose variably measured within 0 and 24 hours of fasting in 368,000 European ancestry participants of the UK Biobank. We found a near-linear increase in the heritability of non-fasting glucose levels over time, which plateaus to its fasting state value after 5 hours post meal (h2=11%; standard error: 1%). The genetic correlation between different fasting times is > 0.77, suggesting that the genetic control of glucose is largely constant across fasting durations. Accounting for heritability differences between fasting times leads to a ~16% improvement in the discovery of genetic variants associated with glucose. Newly detected variants improve the prediction of fasting glucose and type 2 diabetes in independent samples. Finally, we meta-analysed summary statistics from genome-wide association studies of random and fasting glucose (N=518,615) and identified 156 independent SNPs explaining 3% of fasting glucose variance. Altogether, our study demonstrates the utility of random glucose measures to improve discovery of genetic variants associated with glucose homeostasis, even in fasting conditions

GWAS of peptic ulcer disease implicates Helicobacter pylori infection, other gastrointestinal disorders and depression

Abstract: Genetic factors are recognized to contribute to peptic ulcer disease (PUD) and other gastrointestinal diseases, such as gastro-oesophageal reflux disease (GORD), irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Here, genome-wide association study (GWAS) analyses based on 456,327 UK Biobank (UKB) individuals identify 8 independent and significant loci for PUD at, or near, genes MUC1, MUC6, FUT2, PSCA, ABO, CDX2, GAST and CCKBR. There are previously established roles in susceptibility to Helicobacter pylori infection, response to counteract infection-related damage, gastric acid secretion or gastrointestinal motility for these genes. Only two associations have been previously reported for duodenal ulcer, here replicated trans-ancestrally. The results highlight the role of host genetic susceptibility to infection. Post-GWAS analyses for PUD, GORD, IBS and IBD add insights into relationships between these gastrointestinal diseases and their relationships with depression, a commonly comorbid disorder

Linear Mixed Models Minimise False Positive Rate and Enhance Precision of Mass Univariate Vertex-Wise Analyse of Grey-Matter

International audienceWe evaluated the statistical power, family wise error rate (FWER) and precision of several competing methods that perform mass-univariate vertex-wise analyses of grey-matter (thickness and surface area). In particular, we compared several generalised linear models (GLMs, current state of the art) to linear mixed models (LMMs) that have proven superior in genomics. We used phenotypes simulated from real vertex-wise data and a large sample size (N=8,662) which may soon become the norm in neuroimaging. No method ensured a FWER<5% (at a vertex or cluster level) after applying Bonferroni correction for multiple testing. LMMs should be preferred to GLMs as they minimise the false positive rate and yield smaller clusters of associations. Associations on real phenotypes must be interpreted with caution, and replication may be warranted to conclude about an association

Randomized, placebo controlled trial of experimental hookworm infection for improving gluten tolerance in Celiac disease

INTRODUCTION: Celiac disease is an autoimmune disorder where intestinal immunopathology arises after gluten consumption. Previous studies suggested that hookworm infection restores gluten tolerance; however, these studies were small (n = 12) and not placebo controlled. METHODS: We undertook a randomized, placebo-controlled trial of hookworm infection in 54 people with celiac disease. The 94-week study involved treatment with either 20 or 40 Necator americanus third-stage larvae (L3-20 or L3-40) or placebo, followed by escalating gluten consumption (50 mg/d for 12 weeks, 1 g intermittent twice weekly for 12 weeks, 2 g/d sustained for 6 weeks, liberal diet for 1 year). RESULTS: Successful study completion rates at week 42 (primary outcome) were similar in each group (placebo: 57%, L3-20: 37%, and L3-40: 44%; P = 0.61), however gluten-related adverse events were significantly reduced in hookworm-treated participants: Median (range) adverse events/participant were as follows: placebo, 4 (1–9); L3-20, 1 (0–9); and L3-40, 0 (0–3) (P = 0.019). Duodenal villous height:crypt depth deteriorated similarly compared with their enrolment values in each group (mean change [95% confidence interval]: placebo, −0.6 [−1.3 to 0.2]; L3-20, −0.5 [−0.8 to 0.2]; and L3-40, −1.1 [−1.8 to 0.4]; P = 0.12). A retrospective analysis revealed that 9 of the 40 L3-treated participants failed to establish hookworm infections. Although week 42 completion rates were similar in hookworm-positive vs hookworm-negative participants (48% vs 44%, P = 0.43), quality of life symptom scores were lower in hookworm-positive participants after intermittent gluten challenge (mean [95% confidence interval]: 38.9 [33.9–44] vs 45.9 [39.2–52.6]). DISCUSSION: Hookworm infection does not restore tolerance to sustained moderate consumption of gluten (2 g/d) but was associated with improved symptom scores after intermittent consumption of lower, intermittent gluten doses

Polygenic prediction of educational attainment within and between families from genome-wide association analyses in 3 million individuals

We conduct a genome-wide association study (GWAS) of educational attainment (EA) in a sample of ~3 million individuals and identify 3,952 approximately uncorrelated genome-wide-significant single-nucleotide polymorphisms (SNPs). A genome-wide polygenic predictor, or polygenic index (PGI), explains 12–16% of EA variance and contributes to risk prediction for ten diseases. Direct effects (i.e., controlling for parental PGIs) explain roughly half the PGI’s magnitude of association with EA and other phenotypes. The correlation between mate-pair PGIs is far too large to be consistent with phenotypic assortment alone, implying additional assortment on PGI-associated factors. In an additional GWAS of dominance deviations from the additive model, we identify no genome-wide-significant SNPs, and a separate X-chromosome additive GWAS identifies 57

Genome-wide association analyses identify 143 risk variants and putative regulatory mechanisms for type 2 diabetes

Type 2 diabetes (T2D) is a very common disease in humans. Here we conduct a meta-analysis of genome-wide association studies (GWAS) with ~16 million genetic variants in 62,892 T2D cases and 596,424 controls of European ancestry. We identify 139 common and 4 rare variants associated with T2D, 42 of which (39 common and 3 rare variants) are independent of the known variants. Integration of the gene expression data from blood (n = 14,115 and 2765) with the GWAS results identifies 33 putative functional genes for T2D, 3 of which were targeted by approved drugs. A further integration of DNA methylation (n = 1980) and epigenomic annotation data highlight 3 genes (CAMK1D, TP53INP1, and ATP5G1) with plausible regulatory mechanisms, whereby a genetic variant exerts an effect on T2D through epigenetic regulation of gene expression. Our study uncovers additional loci, proposes putative genetic regulatory mechanisms for T2D, and provides evidence of purifying selection for T2D-associated variants.</p

Risk prediction of late-onset Alzheimer’s disease implies an oligogenic architecture

© 2020, The Author(s). Genetic association studies have identified 44 common genome-wide significant risk loci for late-onset Alzheimer’s disease (LOAD). However, LOAD genetic architecture and prediction are unclear. Here we estimate the optimal P-threshold (Poptimal) of a genetic risk score (GRS) for prediction of LOAD in three independent datasets comprising 676 cases and 35,675 family history proxy cases. We show that the discriminative ability of GRS in LOAD prediction is maximised when selecting a small number of SNPs. Both simulation results and direct estimation indicate that the number of causal common SNPs for LOAD may be less than 100, suggesting LOAD is more oligogenic than polygenic. The best GRS explains approximately 75% of SNP-heritability, and individuals in the top decile of GRS have ten-fold increased odds when compared to those in the bottom decile. In addition, 14 variants are identified that contribute to both LOAD risk and age at onset of LOAD

Mapping genomic loci implicates genes and synaptic biology in schizophrenia

Schizophrenia has a heritability of 60-80%1, much of which is attributable to common risk alleles. Here, in a two-stage genome-wide association study of up to 76,755 individuals with schizophrenia and 243,649 control individuals, we report common variant associations at 287 distinct genomic loci. Associations were concentrated in genes that are expressed in excitatory and inhibitory neurons of the central nervous system, but not in other tissues or cell types. Using fine-mapping and functional genomic data, we identify 120 genes (106 protein-coding) that are likely to underpin associations at some of these loci, including 16 genes with credible causal non-synonymous or untranslated region variation. We also implicate fundamental processes related to neuronal function, including synaptic organization, differentiation and transmission. Fine-mapped candidates were enriched for genes associated with rare disruptive coding variants in people with schizophrenia, including the glutamate receptor subunit GRIN2A and transcription factor SP4, and were also enriched for genes implicated by such variants in neurodevelopmental disorders. We identify biological processes relevant to schizophrenia pathophysiology; show convergence of common and rare variant associations in schizophrenia and neurodevelopmental disorders; and provide a resource of prioritized genes and variants to advance mechanistic studies.</p