Polymorphisms within Toll-like receptors are associated with systemic lupus erythematosus in a cohort of Danish females.

OBJECTIVES: Toll-like receptors (TLRs) are pattern-associated receptors in innate immunity that may be involved in the recognition of self-antigens and the production of pathogenic autoantibodies. This study was undertaken to examine whether polymorphisms of TLR genes are associated with SLE and to determine the expression of various TLRs in peripheral blood mononuclear cells (PBMCs) of patients with SLE. METHODS: The TLR polymorphisms in a cohort of 143 Danish lupus patients and 432 healthy Danish blood donors were analysed. Groups were age matched. Genotyping for the TLR single-nucleotide polymorphisms (SNPs) was performed using Sequenom Multiplex technology. In addition, the mRNA expression of TLRs in PBMCs from 56 SLE patients and 56 healthy controls was studies by quantitative real-time PCR. RESULTS: We found a genetic association with SLE and three SNPs located within the TLR3, TLR8 and TLR9 genes (rs3775291, P = 0.006; rs37648, P = 0.013; rs352143, P < 0.02). Furthermore, the relative TLR7, TLR8, IFN-α and LY6E mRNA expression levels were significantly higher in SLE patients than in healthy controls (P < 0.0001, P < 0.0001, P = 0.0004 and P < 0.0001, respectively). CONCLUSION: These results obtained from a female lupus population of Danish ancestry suggest that variations in TLR3, TLR8 and TLR9 genes are implicated in the pathogenesis of the disease. If these polymorphisms are associated with innate immune dysfunction they may add to the growing field of theoretically well founded new therapeutic targets.

Expression of HERV-Fc1, a human endogenous retrovirus, is increased in patients with active multiple sclerosis.

Multiple sclerosis (MS) is considered to be an autoimmune disease with an unknown cause and with immune system dysregulation. Among environmental factors, viruses are most often connected with the etiology of MS. Human endogenous retroviruses (HERVs) constitute 5 to 8% of human genomic DNA and have been detected as transcripts and proteins in the central nervous system (CNS) and peripheral blood, frequently in the context of neuroinflammation. HERV-Fc1, which belongs to the HERV-H/F family, has received our attention largely because of the genetic association with MS. We studied the expression of a capsid (Gag) protein of HERV-H/F origin by flow cytometry in peripheral blood mononuclear cells (PBMCs) from healthy controls and from MS patients with nonactive or active disease. There was a significant increase in HERV-H/F Gag expression in CD4(+) (P < 0.001) and CD8(+) (P < 0.001) T lymphocytes and in monocytes (P = 0.0356) in PBMCs from MS patients with active disease. Furthermore, we have undertaken the first rigorous SYBR green-based absolute quantitative PCR (Q-PCR) evaluation approach to quantify extracellular HERV-Fc1 RNA viral loads in plasma from MS patients and healthy controls. We found a 4-fold increase in extracellular HERV-Fc1 RNA titers in patients with active MS compared with healthy controls (P < 0.001). These findings strengthen the link between HERV-Fc1 and the pathology of MS. The cause and biological consequences of these differential expression levels will be the subject of further investigation. HERV-Fc1 biology could be a compelling area for understanding the pathology of MS and possibly other autoimmune disorders.

The importance of a sub-region on chromosome 19q13.3 for prognosis of multiple myeloma patients after high-dose treatment and stem cell support: a linkage disequilibrium mapping in RAI and CD3EAP.

The gene RAI was originally described as an inhibitor of RelA/p65 subunit of nuclear factor κB (NF-κB). Here, we analyse the association between genetic variation in the genes RAI and CD3EAP and outcome of 348 myeloma patients treated with high-dose treatment (HDT), 146 patients treated with interferon-α (INF-α) as maintenance treatment, 177 patients treated with thalidomide, and 74 patients treated with bortezomib at relapse and address if the effects of polymorphisms in CD3EAP and RAI are modified by a functional polymorphism in NFКB1. By linkage disequilibrium mapping, we found that variant alleles of several polymorphisms in a sub-region of 19q13.3 spanning the regions RAI-intron1-1 to RAI intron1-3 and the region exon1 to exon3­6 in CD3EAP were associated with prolonged time-to-treatment failure (TTF; p = 0.003) and overall survival (OS; p = 0.02). Haplotype analyses revealed that none of the haplotypes were more strongly associated to TTF or OS than the two strongly linked SNPs, RAI-intron1-1 (rs4572514) and CD3EAP G-21A (rs967591). The association of RAI-intron1-1 and CD3EAP G-21A with TTF was independent of NFKB1-94 ins/del, but homozygous ins-allele carriers which were also variant allele carriers of RAI-intron1-1 or CD3EAP G-21A had the longest OS. Among patients treated with INF-α or thalidomide, no effect was seen in relation to genotype. Our results indicate that polymorphism in RAI and CD3EAP are associated with outcome of myeloma patients treated with HDT. Combination analyses with the functional polymorphism in NFKB1 suggest that a possibly functional effect of RAI or CD3EAP could be related to NF-κB availability.

Increased mRNA expression levels of ERCC1, OGG1 and RAI in colorectal adenomas and carcinomas.

BACKGROUND: The majority of colorectal cancer (CRC) cases develop through the adenoma-carcinoma pathway. If an increase in DNA repair expression is detected in both early adenomas and carcinomas it may indicate that low repair capacity in the normal mucosa is a risk factor for adenoma formation. METHODS: We have examined mRNA expression of two DNA repair genes, ERCC1 and OGG1 as well as the putative apoptosis controlling gene RAI, in normal tissues and lesions from 36 cases with adenomas (mild/moderat n = 21 and severe n = 15, dysplasia) and 9 with carcinomas. RESULTS: Comparing expression levels of ERCC1, OGG1 and RAI between normal tissue and all lesions combined yielded higher expression levels in lesions, 3.3-fold higher (P = 0.005), 5.6-fold higher (P < 3.10-5) and 7.7-fold higher (P = 0.0005), respectively. The levels of ERCC1, OGG1 and RAI expressions when comparing lesions, did not differ between adenomas and CRC cases, P = 0.836, P = 0.341 and P = 0.909, respectively. When comparing expression levels in normal tissue, the levels for OGG1 and RAI from CRC cases were significantly lower compared to the cases with adenomas, P = 0.012 and P = 0.011, respectively. CONCLUSION: Our results suggest that increased expression of defense genes is an early event in the progression of colorectal adenomas to carcinomas.

Polymorphisms of the XRCC1, XRCC3 and XPD genes and risk of colorectal adenoma and carcinoma, in a Norwegian cohort: a case control study.

BACKGROUND: Genetic polymorphisms in DNA repair genes may influence individual variation in DNA repair capacity, which may be associated with risk of developing cancer. For colorectal cancer the importance of mutations in mismatch repair genes has been extensively documented. Less is known about other DNA repair pathways in colorectal carcinogenesis. In this study we have focused on the XRCC1, XRCC3 and XPD genes, involved in base excision repair, homologous recombinational repair and nucleotide excision repair, respectively. METHODS: We used a case-control study design (157 carcinomas, 983 adenomas and 399 controls) to test the association between five polymorphisms in these DNA repair genes (XRCC1 Arg194Trp, Arg280His, Arg399Gln, XRCC3 Thr241Met and XPD Lys751Gln), and risk of colorectal adenomas and carcinomas in a Norwegian cohort. Odds ratio (OR) and 95% confidence interval (95% CI) were estimated by binary logistic regression model adjusting for age, gender, cigarette smoking and alcohol consumption. RESULTS: The XRCC1 280His allele was associated with an increased risk of adenomas (OR 2.30, 95% CI 1.19-4.46). The XRCC1 399Gln allele was associated with a reduction of risk of high-risk adenomas (OR 0.62, 95% CI 0.41-0.96). Carriers of the variant XPD 751Gln allele had an increased risk of low-risk adenomas (OR 1.40, 95% CI 1.03-1.89), while no association was found with risk of carcinomas. CONCLUSION: Our results suggest an increased risk for advanced colorectal neoplasia in individuals with the XRCC1 Arg280His polymorphism and a reduced risk associated with the XRCC1 Arg399Gln polymorphism. Interestingly, individuals with the XPD Lys751Gln polymorphism had an increased risk of low-risk adenomas. This may suggest a role in regression of adenomas.

GPX Pro198Leu and OGG1 Ser326Cys polymorphisms and risk of development of colorectal adenomas and colorectal cancer.

Little is known about genetic risk factors for colorectal cancer. We assessed the association between polymorphisms in two genes involved in DNA repair of oxidative stress, GPX and OGG1, and risk of colorectal carcinoma or adenomas. We studied 166 cases with adenocarcinoma, 974 with adenomas and 397 controls recruited from the Norwegian cohort NORCCAP. No associations were found between the polymorphism GPX Pro198Leu and risk of colorectal adenomas or carcinomas. Carriers of the variant allele OGG1 Ser326Cys polymorphism had a lowered risk of colorectal cancer, OR=0.56 (95% confidence interval 0.33-0.95), while no association were found with risk of adenomas. This indicates that a low repair capacity of oxidative DNA damage may not be a risk factor for development of colorectal adenomas or carcinoma.

Effectiveness of anti-tumour necrosis factor-α therapy in Danish patients with inflammatory bowel diseases.

INTRODUCTION: The objective of this study was to evaluate the outcome of anti-tumour necrosis factor-α (anti-TNF) treatment in a large cohort of patients with inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC) in clinical practice and to establish a cohort for future studies of genetic markers associated with treatment response. METHODS: A national, clinically based cohort of previously naïve anti-TNF treated patients from 18 medical departments was established. The patients were screened for tuberculosis prior to treatment initiation. By combining the unique personal identification number of Danish citizens (the CPR number) from blood samples with data from the National Patient Registry, patients with International Classification of Diseases, Version 10 (ICD-10) codes K50-K63 were identified. Treatment efficacy reflected the maximum response within 22 weeks. RESULTS: Among 492 patients with CD and 267 patients with UC, 74%/13%/14% and 65%/12%/24% were responders, partial responders and non-responders to anti-TNF therapy, respectively. More patients with UC than with CD were non-responders (odds ratio (OR) = 1.96, 95% confidence interval (CI): 1.34-2.87, p = 0.001). Young age was associated with a beneficial response (p = 0.03), whereas smoking ≥ 10 cigarettes/day was associated with non-response among patients with CD (OR = 2.33, 95% CI: 1.13-4.81, p = 0.03). CONCLUSION: In this clinically based cohort of Danish patients with IBD treated with anti-TNF, high response rates were found. Heavy smoking was associated with non-response, whereas young age at treatment initiation was associated with a beneficial response among patients with CD. Thus, the results obtained in this cohort recruited from clinical practice were similar to those previously obtained in clinical trials. FUNDING: The work was funded by Health Research Fund of Central Denmark Region, Colitis-Crohn Foreningen and the University of Aarhus (PhD grant). TRIAL REGISTRATION: Clinicaltrials NCT02322008.

(Some) cellular mechanisms influencing the transcription of human endogenous retrovirus, HERV-Fc1.

DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. DNA methylation is considered an important mechanism for silencing of retroelements in the mammalian genome. However, the methylation of human endogenous retroviruses (HERVs) is not well investigated. The aim of this study was to investigate the transcriptional potential of HERV-Fc1 proviral 5'LTR in more detail, and examined the specific influence of CpG methylation on this LTR in number of cell lines. Specifically, the role of demethylating chemicals e.g. 5-aza-2' deoxycytidine and Trichostatin-A, in inducing or reactivating expression of HERV-Fc1 specific sequences and the mechanisms were investigated. In our present study, 5-aza-dC is shown to be a powerful inducer of HERV-Fc1, and at the same time it strongly inhibits methylation of DNA. Treatment with this demethylating agent 5-aza-dC, results in significantly increased levels of HERV-Fc1 expression in cells previously not expressing HERV-Fc1, or with a very low expression level. The extent of expression of HERV-Fc1 RNAs precisely correlates with the apparent extent of demethylation of the related DNA sequences. In conclusion, the results suggest that inhibition of DNA methylation/histone deacetylase can interfere with gene silencing mechanisms affecting HERV-Fc1 expression in human cells.

High-quality and -quantity DNA extraction from frozen archival blood clots for genotyping of single-nucleotide polymorphisms.

BACKGROUND: The recovery of biological samples for genetic epidemiological studies can be cumbersome. Blood clots are routinely collected for serological examinations. However, the extraction of DNA from blood clots can be difficult and often results in low yields. AIM: The aim was to compare the efficiency of commercial purification kits for extracting DNA from long-term frozen clotted blood. METHODS: Serum tubes with clotted blood were stored at -20°C for 1 to 2.5 years before DNA extraction. DNA was extracted from 10 blood clot samples using PureGene (Qiagen) with and without glycogen, the QIAamp DNA Micro kit (Qiagen), and the Nucleospin 96 Blood kit (Macherey-Nagel). Furthermore, blood clots from 1055 inflammatory bowel disease patients were purified using the Maxwell 16 Blood purification kit (Promega). The DNA was extracted according to the manufacturers` instructions and real-time PCR and the A(260)/A(280) ratio were used to evaluate the quality of the extracted DNA. RESULTS: The highest DNA yield was obtained by the Maxwell 16 Blood purification kit (Promega) with a median of 4.90 µg (range 0.8-25 µg) pr 300 µL total blood. PureGene with glycogen (Qiagen) had the second highest yield with a median of 0.65 µg (range 0.5-2.6 µg) pr 300 µL total blood. CONCLUSION: The yield obtained by the different commercial kits varied considerably. Our work demonstrates that high-quality and -quantity DNA can be extracted with the Maxwell 16 Blood purification kit (Promega) from cryopreserved blood clots, even after prolonged storage. The recovered DNA served as a reliable PCR template for single-nucleotide polymorphism assays.

Twelve single nucleotide polymorphisms on chromosome 19q13.2-13.3: linkage disequilibria and associations with basal cell carcinoma in Danish psoriatic patients.

The genetic susceptibility to basal cell carcinoma (BCC) among Danish psoriatic patients was investigated in association studies with 12 single nucleotide polymorphisms on chromosome 19q13.2-3. The results show a significant association between BCC and the A-allele of a polymorphism in ERCCI exon4 (Odds ratio 12;95% Confidence Interval 1.17-124; p(chi2, two-side) = 0.019) and to a lesser extent with XPD exon6 (p = 0.06). This is in accordance with recent studies of a different group of BCC cases (Rockenbauer et al. (in press) Carcinogenesis; Yin et al. (manuscript submitted for publication). Cancer Epidemiol. Biomarkers Prev), which places two highly influential markers between these two genes. The analysis also confirmed that considerable linkage disequilibrium exists between SNPs both within genes and between genes in this region. The combined studies suggest that genetic variation in nucleotide excision repair is of importance for the development of BCC.