Gerrit van der Steege

Novel association in chromosome 4q27 region with rheumatoid arthritis and confirmation of type 1 diabetes point to a general risk locus for autoimmune diseases.

Recently, association of celiac disease with common single-nucleotide polymorphism (SNP) variants in an extensive linkage-disequilibrium block of 480 kb containing the KIAA1109, Tenr, IL2, and IL21 genes has been demonstrated in three independent populations (rs6822844P combined=1.3 x 10(-14)). The KIAA1109/Tenr/IL2/IL21 block corresponds to the Idd3 locus in the nonobese diabetic mouse model of type 1 diabetes (T1D). This block was recently found to be associated with T1D in a genomewide association study, although this finding lacks unequivocal confirmation. We therefore aimed to investigate whether the KIAA1109/Tenr/IL2/IL21 region is involved in susceptibility to multiple autoimmune diseases. We tested SNP rs6822844 for association with disease in 350 T1D-affected and 1,047 rheumatoid arthritis (RA)-affected Dutch patients and in 929 controls. We replicated the association with T1D (P=.0006; OR 0.64 [95% CI 0.50-0.83]), and revealed a similar novel association with RA (P=.0002; OR 0.72 [95% CI 0.61-0.86]). Our results replicate and extend the association found in the KIAA1109/Tenr/IL2/IL21 gene region with autoimmune diseases, implying that this locus is a general risk factor for multiple autoimmune diseases.

Association between Toll-like receptor 4 and inflammatory bowel disease.

Background: The human Toll‐like receptor 4 (TLR4) participates in the innate response. Recently, the TLR4 variant Asp299Gly has been described to affect the response of this receptor to lipopolysaccharide. As such, there is a potentially important role of TLR4 in the pathogenesis of inflammatory bowel disease (IBD). We studied the involvement of TLR4 in IBD in a large population of Dutch patients with IBD and in family‐based controls. Methods: In 781 IBD cases and 315 controls, genotyping was performed for Asp299Gly and Thr399Ile variants and for 4 microsatellite markers flanking TLR4. Association analysis and the were applied. In addition, interaction of TLR4 with the caspase recruitment domain containing protein 15 gene (CARD15) was studied in patients with Crohns disease (CD). Results: The haplotype sharing statistic showed association at microsatellite marker D9S1864 with IBD (P = 0.0019), and in particular with CD (P = 0.0025) and at TLR406 with ulcerative colitis (UC; P = 0.027). No association was found for Asp299Gly and Thr399Ile. However, the frequencies of both variant allele carriers were higher among CD cases with a disease onset ≥40 years than among controls. No evidence for interaction between TLR4 and CARD15 was found. Conclusions: Haplotype analysis shows that TLR4 is associated with both CD and UC. The Asp299Gly and Thr399Ile variants do not show an association with CD, UC, or IBD as a group, indicating that these polymorphisms are likely not the causal ones. We propose that the 2 polymorphisms are in linkage with (the) disease susceptibility variant(s) located elsewhere on TLR4.

Genetic susceptibility has a more important role in pediatric‐onset Crohn's disease than in adult‐onset Crohn's disease

Background: Genetic susceptibility may play a more important role in the etiology of early‐onset inflammatory bowel disease (IBD) than in late‐onset IBD, and therefore pediatric‐onset IBD patients can be expected to have a higher frequency of gene mutations. We aimed to determine genotypes and phenotypes of patients with pediatric‐onset IBD, to compare them with those of patients with adult‐onset IBD and with controls, and to identify genotype–phenotype associations. Methods: Polymorphisms R702W, G908R, and 3020insC of CARD15 (caspase activating recruitment domain 15); Asp299Gly and Thr399Ile of TLR4; ‐207G→C, 1672C→T (L503F), rs3792876, rs274551, rs272893, and rs273900 of SLC22A4/5; and 113G→A as well as rs2289311, rs1270912, and rs2165047 of DLG5 (Drosophila discs large homologue 5) were assessed in 103 pediatric‐onset and 696 adult‐onset IBD patients. Phenotypic classification was based on disease localization and behavior. Results: Homozygosity for 3020insC in CARD15 was significantly higher in patients with pediatric‐onset Crohns disease (CD) than in patients with adult‐onset CD (4.2% versus 0.6%, 95% confidence interval [CI] 1.2–42.0). Homozygosity for single‐nucleotide polymorphism (SNP) rs3792876 in SLC22A4/5 was significantly higher in patients with pediatric‐onset CD than in patients with adult‐onset CD (6.1% versus 1.1%, P = 0.02). Polymorphism 3020insC in CARD15 was associated with ileal involvement (1.9% versus 13.3%, CI 1.0–53.8) and a positive family history (6.1% versus 20%, CI 1.2–9.0). DLG5 SNP rs2165047 was significantly associated with perianal disease (50% versus 21.2%, CI 1.4–4). Conclusions: Polymorphisms 3020insC in CARD15 and SNP rs3792876 in SLC22A4/5 occurred statistically significantly more often in patients with pediatric‐onset CD than in patients with adult‐onset CD. Polymorphisms 3020insC in CARD15 and SNP rs2165047 in DLG5 were associated with specific phenotypes in this pediatric‐onset CD cohort.

Serum chemokine levels in Hodgkin lymphoma patients: highly increased levels of CCL17 and CCL22

Hodgkin lymphoma (HL) is characterized by a minority of neoplastic Hodgkin‐Reed Sternberg (HRS) cells surrounded by a non‐neoplastic reactive infiltrate. As immunological mechanisms appear to be crucial in classical HL pathogenesis, altered serum chemokine levels might be related to disease activity. Serum levels of nine chemokines were examined in 163 untreated HL patients and 334 controls. We investigated single nucleotide polymorphisms (SNPs) for association with serum CCL17 (thymus and activation‐regulated chemokine, TARC) levels and HL susceptibility. Serum CCL17 and CCL22 (macrophage‐derived chemokine, MDC) levels were significantly increased in 82% and 57% of the HL patients. Nodular sclerosis cases showed increased serum CCL17 and CCL22 levels (Pu2003<u20030·001) and serum levels were correlated with Ann Arbor stage. Of nine patients with pre‐ and post‐treatment serum samples, the majority showed decreased CCL17 and CCL22 levels after treatment. HRS cells expressed CCL17 and CCL22 in 77% and 75% of 74 cases. Three SNPs showed a trend of increased serum CCL17 levels with minor alleles in controls, but were not associated with HL susceptibility. CCL17 and CCL22 were the only chemokines with increased serum levels in the vast majority of HL patients, which provides further insight into the molecular mechanism(s) leading to infiltrations of reactive lymphocytes in HL.

Variation in Bleomycin Hydrolase Gene Is Associated With Reduced Survival After Chemotherapy for Testicular Germ Cell Cancer

PURPOSEnResponse to chemotherapy may be determined by gene polymorphisms involved in metabolism of cytotoxic drugs. A plausible candidate is the gene for bleomycin hydrolase (BLMH), an enzyme that inactivates bleomycin, an essential component of chemotherapy regimens for disseminated testicular germ-cell cancer (TC). We investigated whether the single nucleotide polymorphism (SNP) A1450G of the BLMH gene (rs1050565) is associated with survival.nnnPATIENTS AND METHODSnData were collected on survival and BLMH genotype of 304 patients with TC treated with bleomycin-containing chemotherapy at the University Medical Center Groningen, the Netherlands, between 1977 and 2003. Survival according to genotype was analyzed using Kaplan-Meier curves with log-rank testing and Cox regression analysis with adjustment for confounders.nnnRESULTSnBLMH gene SNP A1450G has a significant effect on TC-related survival (log-rank P = .001). The homozygous variant (G/G) genotype (n = 31) is associated with decreased TC related survival compared with the heterozygous variant (A/G; n = 133) and the wild-type (A/A; n = 140). With Cox regression the G/G genotype proves to be an unfavorable prognostic factor, in addition to the commonly used International Germ Cell Consensus Classification prognosis group, with a hazard ratio of 4.97 (95% CI, 2.17 to 11.39) for TC-related death. Furthermore, the G/G genotype shows a higher prevalence of early relapses.nnnCONCLUSIONnThe homozygous variant G/G of BLMH gene SNP A1450G is associated with reduced survival and higher prevalence of early relapses in TC patients treated with bleomycin-containing chemotherapy. This association is hypothesis generating and may eventually be of value for risk classification and selection for alternative treatment strategies in patients with disseminated TC.

POTATO GRANULE-BOUND STARCH SYNTHASE PROMOTER-CONTROLLED GUS EXPRESSION - REGULATION OF EXPRESSION AFTER TRANSIENT AND STABLE TRANSFORMATION

Chimaeric genes of promoter sequences from the potato gene encoding granule-bound starch synthase (GBSS) and the β-glucuronidase (GUS) reporter gene were used to study GBSS expression and regulation. Analysis of stable transformants revealed that a GBSS promoter sequence of 0.4 kb was sufficient to result in tissue-dependent GUS expression: levels in stably transformed microtubers exceeded levels in corresponding leaves by orders of magnitude. GBSS-GUS constructs could be transiently expressed in leaf protoplasts from wild-type and amylose-free potato lines, etuberosumSolanum brevidens, Nicotiana tabacum andArabidopsis thaliana. Transient expression levels in potato leaf protoplasts were clearly lower than in corresponding suspension cell protoplasts. This lower expression in leaf protoplasts could not be elevated by increasing DNA concentrations during transfection. Light incubation of electroporated suspension cell protoplasts reduced transient GBSS-GUS expression, whereas incubation of transfected protoplasts in media with different sucrose concentrations did not affect transient expression levels. However, electroporated protoplasts, isolated from suspensions, which had been grown on media with increasing amounts of sucrose showed a sucrose concentration-dependent transient expression profile. This indicates that studying GBSS regulation by transient expression experiments needs pre-treatment of the protoplast source. Sequence data of the GBSS promoter were compared to those of two other potato alleles.

Possible Association Between Telomere Length and Renal Dysfunction in Patients With Chronic Heart Failure

Renal function impairment relates to poor outcome in patients with chronic heart failure (HF). Differences in biological aging could affect the susceptibility to develop renal dysfunction in chronic HF. In the present study, we explored the association of leukocyte telomere length with renal function in patients with chronic HF. We studied 610 patients with HF, aged 40 to 80 years, NYHA class II-IV, with left ventricular ejection fraction of 0.40 or less. Glomerular filtration rate was estimated by the Modification of Diet in Renal Diseases (MDRD) formula, and telomere length of leukocytes was determined by a validated quantitative polymerase chain reaction-based method. Age-and gender-adjusted telomere length ratio decreased steadily with decreasing quartile of the MDRD formula (mean 0.80, 95% confidence interval [CI] 0.73 to 0.88; mean 0.74, 95% CI 0.68 to 0.81; 0.70 mean, 95% CI 0.63 to 0.76; mean 0.67, 95% CI 0.61 to 0.73; p <0.01). Telomere length of leukocytes correlated positively with the MDRD formula (correlation coefficient 0.141; p <0.001). These findings remained significant after adjustment for baseline differences and sensitivity analysis based on propensity score one-to-one matching. In conclusion, shorter leukocyte telomere length is associated with decreased renal function as estimated by the MDRD formula in patients with HF. Further studies will be needed to determine whether shorter leukocyte telomere length is the cause or consequence in this population and whether it plays a role in the prognosis of renal dysfunction in HF.

Methylenetetrahydrofolate reductase (MTHFR) and susceptibility for (pre)neoplastic cervical disease

Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme regulating the metabolism of folate and methionine. The potential influence of MTHFR activity on DNA methylation and on the availability of uridylates and thymidylates for DNA synthesis and repair presents MTHFR as a candidate for being a cancer-predisposing gene. In the present study, we have examined a large study population to determine whether the C677T polymorphism at the MTHFR locus affects susceptibility for cervical cancer or its precursor, cervical intraepithelial neoplasia (CIN). In addition, we have investigated whether this polymorphism is causal, and not merely associated, by typing microsatellite markers in the region surrounding the MTHFR gene. A total of 311 CIN and 695 cervical cancer patients and 115 family-based and 586 unrelated controls was analysed. Association analysis showed a decreased cervical cancer risk for individuals heterozygous or homozygous for the T-allele, both for squamous cell carcinoma (heterozygous odds ration [OR] 0.66 [0.51–0.86]; homozygous OR 0.76 [0.49–1.16]) and adenocarcinoma (heterozygous OR 0.71 [0.49–1.03]; homozygous OR 0.34 [0.14–0.81]). No difference was found for high grade CIN (heterozygous OR 1.03 [0.76–1.40]; homozygous OR 0.91 [0.54–1.55]). A microsatellite haplotype containing the C allele was associated with an increased risk for cervical cancer and CIN (both among squamous cell carcinomas, adenocarcinomas and CIN II–III; OR=2.61 [1.59–4.27]). Our study thus lends further support to the hypothesis that the MTHFR C677T polymorphism is involved in susceptibility cervical cancer but also illustrates that, despite the large sample size analysed, still larger studies are needed to establish fully the nature of this association.

Absence of association between the multidrug resistance (MDR1) gene and inflammatory bowel disease

Objective. The multidrug resistance (MDR1) gene encodes for P-glycoprotein, a drug efflux pump. Mice deficient for the MDR1a gene spontaneously develop colitis. In humans, a polymorphism in exon 26 (C3435T) is associated with reduced expression levels and function of MDR1. Currently there are controversial data on the association between MDR1 and inflammatory bowel disease (IBD). The purpose of this study was to examine the involvement of this gene in IBD in a large population of Dutch patients with IBD and family-based controls. Material and methods. A total of 781 IBD cases and 315 controls were investigated. CD phenotypes were determined according to the Vienna Classification. Individuals were genotyped for six single nucleotide polymorphisms (SNPs) close to and in the MDR1 locus. This included the C3435T variant and six microsatellite markers close to and in the MDR1 locus. Single locus association analysis, haplotype association analysis and haplotype sharing statistic (HSS) were used to search for differences between patients and controls. Results. No association was observed for any of the SNPs with IBD as a group, or for ulcerative colitis, Crohns disease and Crohns disease phenotypes, either by single locus or haplotype association analysis or by HSS. Conclusions. No association was observed between the MDR1 gene and IBD. This suggests that it is unlikely that MDR1 plays a role in IBD susceptibility.

The human leukocyte antigen class I region is associated with EBV-positive Hodgkin's lymphoma : HLA-A and HLA Complex Group 9 are putative candidate genes

Various studies have indicated that the human leukocyte antigen (HLA) region is associated with Hodgkins lymphoma. We recently showed a specific association of the HLA class I region with EBV-positive Hodgkins lymphoma cases. One haplotype of two consecutive microsatellite markers (D6S265 and D6S510) was overrepresented in the patient group, whereas another haplotype was underrepresented. Here, we did fine mapping of this region of ∼400 kb as a next step to find the causative single-nucleotide polymorphism(s) (SNP). To select candidate SNPs for screening the total study population, several known SNPs were determined by sequencing two individuals homozygous for either of the above-mentioned associated haplotypes. Seven SNPs displayed different alleles in these two individuals and were therefore analyzed in the total study population, including 238 Hodgkins lymphoma patients and 365 family-based controls. All seven SNPs showed significant association with the EBV-positive patient group. Two of these SNPs were analyzed in a Scottish Hodgkins lymphoma population and revealed significant associations as well. The associated SNPs are located nearby two putative candidate genes: HLA-A and HLA complex group 9. HLA-A represents the most interesting target because of its consistent expression in EBV-positive Hodgkins lymphoma cases and its ability to present EBV-derived peptides to cytotoxic T cells. (Cancer Epidemiol Biomarkers Prev 2006;15(11):2280–4)