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Additional File 3. Supplementary Figures.

Additional File 1. Purity plots of T cell suspensions.

Multiple sclerosis is a complex neurological disease, with ∼20% of risk heritability attributable to common genetic variants, including >230 identified by genome-wide association studies. Multiple strands of evidence suggest that much of the remaining heritability is also due to additive effects of common variants rather than epistasis between these variants or mutations exclusive to individual families. Here, we show in 68,379 cases and controls that up to 5% of this heritability is explained by low-frequency variation in gene coding sequence. We identify four novel genes driving MS risk independently of common-variant signals, highlighting key pathogenic roles for regulatory T cell homeostasis and regulation, IFNγ biology, and NFκB signaling. As low-frequency variants do not show substantial linkage disequilibrium with other variants, and as coding variants are more interpretable and experimentally tractable than non-coding variation, our discoveries constitute a rich resource for dissecting the pathobiology of MS.

Background: Several environmental exposures, including infection with Epstein-Barr virus, low levels of vitamin D and smoking are established risk factors for multiple sclerosis (MS). Also, high hygienic standard and infection with parasites have been proposed to influence MS risk. The aim of this study was to investigate the influence of various environmental exposures on MS risk in a Norwegian cohort, focusing on factors during childhood related to the hygiene hypothesis. Methods: A questionnaire concerning environmental exposures, lifestyle, demographics and comorbidity was administrated to 756 Norwegian MS patients and 1090 healthy controls. Logistic regression was used to calculate odds ratio (OR) with 95% confidence interval (CI) for the risk of MS associated with the variables infectious mononucleosis, severe infection during childhood, vaccination and animals in the household during childhood. Age, gender, HLA-DRB1*15:01, smoking and infectious mononucleosis were included as covariates. General environmental exposures, including tobacco use, were also evaluated. Results: Infectious mononucleosis was confirmed to be significantly associated with increased MS risk, also after adjusting for the covariates (OR?=?1.79, 95% CI: 1.12-2.87, p?=?0.016). The controls more often reported growing up with a cat and/or a dog in the household, and this was significant for ownership of cat also after adjusting for the covariates (OR?=?0.56, 95% CI: 0.40-0.78, p?=?0.001). More patients than controls reported smoking and fewer patients reported snuff use. Conclusions: In this Norwegian MS case-control study of environmental exposures, we replicate that infectious mononucleosis and smoking are associated with increased MS risk. Our data also indicate a protective effect on MS of exposure to cats during childhood, in accordance with the hypothesis that risk of autoimmune diseases like MS may increase with high hygienic standard.

Background The procoagulant state in cancer increases the thrombotic risk, but also supports tumor progression. To investigate the molecular mechanisms controlling cancer and hemostasis, we conducted a case-control study of genotypic and phenotypic variables of the tissue factor (TF) pathway of coagulation in breast cancer. Methods 366 breast cancer patients and 307 controls were genotyped for SNPs (n = 41) in the F2, F3 (TF), F5, F7, F10, TFPI and EPCR genes, and assayed for plasma coagulation markers (thrombin generation, activated protein C (APC) resistance, D-dimer, antithrombin, protein C, protein S, and TF pathway inhibitor (TFPI)). Associations with breast cancer were evaluated using logistic regression to obtain odds ratios (ORs) and 95% confidence intervals (CIs), or the chi-square test. Results Four SNPs in F5 (rs12120605, rs6427202, rs9332542 and rs6427199), one in F10 (rs3093261), and one in EPCR (rs2069948) were associated with breast cancer. EPCR rs2069948 was associated with estrogen receptor (ER) and progesterone receptor (PR) positivity, while the SNPs in F5 appeared to follow hormone receptor negative and triple negative patients. The prothrombotic polymorphisms factor V Leiden (rs6025) and prothrombin G20210A (rs1799963) were not associated with breast cancer. High APC resistance was associated with breast cancer in both factor V Leiden non-carriers (OR 6.5, 95% CI 4.1-10.4) and carriers (OR 38.3, 95% CI 6.2-236.6). The thrombin parameters short lag times (OR 5.8, 95% CI 3.7-9.2), short times to peak thrombin (OR 7.1, 95% CI 4.4-11.3), and high thrombin peak (OR 6.1, 95% CI 3.9-9.5) predicted presence of breast cancer, and high D-dimer also associated with breast cancer (OR 2.0, 95% CI 1.3-3.3). Among the coagulation inhibitors, low levels of antithrombin associated with breast cancer (OR 5.7, 95% CI 3.6-9.0). The increased coagulability was not explained by the breast cancer associated SNPs, and was unaffected by ER, PR and triple negative status. Conclusions A procoagulant phenotype was found in the breast cancer patients. Novel associations with SNPs in F5, F10 and EPCR to breast cancer susceptibility were demonstrated, and the SNPs in F5 were confined to hormone receptor negative and triple negative patients. The study supports the importance of developing new therapeutic strategies targeting coagulation processes in cancer. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-845) contains supplementary material, which is available to authorized users.

MicroRNAs (miRNAs) are small single-stranded non-coding RNAs with an average length of ~ 22 nucleotides. The main function of miRNA is post-transcriptional regulation of gene expression by targeting specific mRNAs. The value of miRNA regulation and expression has become an interesting focus in diagnostics. Extracellular miRNAs have been found in a variety of body fluids, making miRNAs suitable as noninvasive biomarkers. For the understanding of miRNAs biological roles and for identifying potential biomarkers, reliable technologies are required for detection and quantitation. Reverse transcription of miRNA to cDNA template followed by quantitative PCR (RT-qPCR) are often the method of choice, however different RT-qPCR technologies have been observed to give varying results. In this thesis, a systematic evaluation has been conducted for three kits and protocols to perform RT-qPCR for detection and quantitation of miRNA. The three different methods evaluated was miRCURY RT-qPCR setup (Qiagen), TaqMan Advanced RT-qPCR setup (Thermo Fisher Scientific) with and without preamplification, and TaqMan MicroRNA RT- qPCR setup (Thermo Fisher Scientific) with and without uracil-N glycosylase (UNG) included in the master mix. All setups included assays for six miRNAs represented by synthetic material. The miRNAs were analyzed once with added synthetical noise and once with added biological noise. The objective of the evaluation was to find a preferred RT-qPCR setup that later can be used for verifying miRNA-findings from RNA sequencing. It was indicated that miRCURY RT- qPCR setup gave best result for verifying lowly expressed miRNAs. This was based on least deviation in the reaction efficiency, low indication of false positives and unspecific amplification and a wide range of input of total RNA. This study indicated that TaqMan Advanced RT-qPCR setup without the preamplification reaction, should not be used for detection and quantitation of lowly expressed miRNA. This was based on the setup’s poor reaction efficiency, observed detection range and precision. TaqMan MicroRNA RT-qPCR setup indicated being advantageous when few miRNA-targets and replicates are required due to the lowest price per reaction for one target and the feasibility. By including UNG in the master mix the deviation of the reaction efficiency decreased. MicroRNA (miRNA) er korte, ikke-kodene, enkeltrådige RNA med en gjennomsnittlig størrelse på ~ 22 nukleotider. Hovedfunksjonen til miRNA er regulering av genuttrykk etter transkripsjonen. Dette gjøres ved at miRNA bindes til komplementære sekvenser på målmRNA. Genregulering av miRNA har fått økende fokus i sykdomssammenheng. MicroRNA har blitt funnet i ulike kroppsvæsker, blant annet i plasma, og dette gjør miRNA egnet som ikke-invasive biomarkører. For å identifisere potensielle biomarkører er det nødvendig med nøyaktige metoder til deteksjon og kvantifisering. Reversert transkripsjon av miRNA til cDNA, etterfulgt av kvantitativ PCR (RT-qPCR) er ofte den foretrukne metoden, men ulike RT-qPCR fremgangsmåter har vist varierende resultat. I denne studien ble det utført en systematisk evaluering av tre ulike fremgangsmåter for å utføre RT-qPCR til deteksjon og kvantifisering av miRNA. De tre ulike fremgangsmåtene som ble vurdert var miRCURY RT-qPCR oppsett (Qiagen), TaqMan Advanced RT-qPCR oppsett (Thermo Fisher Scientific) med og uten pre-amplifisering og TaqMan MicroRNA RTqPCR oppsett (Thermo Fisher Scientific) med og uten uracil-N glycosylase (UNG) inkludet i reaksjonsmiksen. Vurderingen ble utført med assay for seks miRNA representert av syntetisk materiale. MiRNAene ble analysert både med inkludert syntetisk og biologisk bakgrunnsstøy i to ulike oppsett. M-BIOTEK

DNA methylation is an important epigenetic mark with a regulatory effect on the expression of genes. It has also been shown that individuals change their DNA methylation pattern over time. In mammals, methylation only occurs in cytosines, and almost exclusively in the CpG context. Differential methylation has been shown to be important for the development and differentiation of pluripotent stem cells. Rheumatoid arthritis (RA) patients have also been shown as having T-cells with reduced methylation compared to healthy controls. RA is a severe disease associated with great pain and disability and affects about 0.5-1% of the population. In this thesis, the process of establishing a protocol for multiplexed reduced representation bisulfite sequencing (mRRBS) in order to create a methylome profile of RA patients is described. The extracted DNA was cleaved by a restriction enzyme, MspI, which enriched for areas of the genome containing CpG islands. This DNA was then treated with sodium bisulfite which converted all unmethylated cytosines to uracils, while leaving the methylated cytosines intact. Through PCR amplification, and subsequent sequencing, all unmethylated cytosines was read by the sequencer as thymines, while methylated cytosines was read as normal. Four different DNA extraction methods were tested, as well as two DNA cleanup protocols. It was an aim in this study to extract RNA and protein, in addition to DNA, from the same sample, to enable further studies. The best results were achieved through the use of the QIAamp DNA micro kit, but comparable results were achieved by extracting the DNA with the Norgen DNA/RNA/Protein purification plus kit followed by a cleanup with the QIAamp DNA micro kit. In this thesis, it has been demonstrated that the established mRRBS protocol could generate methylome profiles of T cells from RA patients with a bisulfite conversion ratio >99.9%, and sequence alignment of at least 60%. An exploratory analysis of CD4+ T cells showed that the methylation level largely followed the expected pattern of high degree of methylation for genes with low expression, and vice versa, but also supported the notion that other regulatory factors were involved in addition to methylation. DNA metylering er en viktig epigenetisk faktor med en regulatorisk effekt over ekspresjonen av gener. Det har også blitt påvist at metyleringsmønsteret i DNAet til et individ vil forandre seg over tid. I pattedyr forekommer DNA metylering kun på cytosiner, og nesten alltid i en CpG kontekst. Differensiell metylering har blitt vist å være viktig under utvikling og differensiering av pluripotente stamceller. I pasienter med revmatoid artritt (RA) har det blitt påvist at T-celler har redusert metylering i forhold til hos friske kontroller. RA er en alvorlig sykdom assosiert med sterke smerter og funksjonshemming. Anslagsvis 0,5-1% av befolkningen er rammet av denne sykdommen. Arbeidet med å etablere en protokoll for multiplekset begrenset representativ bisulfittsekvensering (mRRBS), for å kunne produsere metylomprofiler for pasienter med RA er beskrevet i denne oppgaven. Det ekstraherte DNAet ble kløyvd med restriksjonsenzymet MspI. Dette restriksjonsenzymet anriket for genomiske områder som inneholdt CpG øyer. DNAet ble så behandlet med natrium bisulfitt som konverterte alle umetylerte cytosiner til uraciler, mens metylerte cytosiner sto uberørt. Gjennom PCR amplifikasjon etterfulgt av sekvensering, ble alle de umetylerte cytosinene lest som tyminer av sekvensatoren og metylerte cytosiner lest som normalt. Fire forskjellige DNA ekstraksjonsmetoder ble testet i tillegg til to opprensingsprotokoller. Det var i denne studien et mål å få ekstrahert RNA og protein i tillegg til DNA fra det samme prøvematerialet, slik at videre studier kunne gjennomføres på et senere tidspunkt. Det beste resultatet ble oppnådd ved bruk av QIAamp DNA micro kit, men sammenlignbare resultater ble oppnådd fra DNA ekstraksjon med Norgen DNA/RNA/Protein purification plus kit etterfulgt av en opprensing med QIAamp DNA micro kit. Denne oppgaven har demonstrert at den etablerte mRRBS protokollen kunne generere metylomprofiler av T-celler fra RA pasienter med en oppnådd bisulfittkonverteringsrate på >99,9% og sekvenssammenstilling på minimum 60%. En undersøkende analyse av CD4+ T celler viste at metyleringsnivået stort sett fulgte det forventede mønsteret med høy metylering i lavt utrykte gener, og motsatt, men støttet også teorien om at andre regulerende faktorer er involvert i tillegg til metylering. M-MB