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Background Several CD14 gene–environment interactions in relation to the development of allergic diseases have been reported, but the underlying biological mechanisms are unclear. We recently showed that CD14 methylation increased during childhood, parallelling a decreased impact of CD14 polymorphisms on soluble CD14 levels. Here, we aim to explore whether environmental stimuli during childhood affects CD14 methylation, thereby providing a biological mechanism through which environment may modulate genetic effect. Methods CD14 methylation levels were quantified in 157 children from the prospective Environment and Childhood Asthma birth cohort at ages 2 and 10. Associations between CD14 methylation levels and house dust levels of endotoxin, β(1,3)-glucans (at 2 yr only), allergens (dog, cat, and house dust mite), pet keeping and tobacco smoke exposure (TSE; questionnaire data) at 2 and 10 yr were explored. Results Children in homes without pets had larger increases in CD14 methylation through childhood (2–10 yr) compared with children with pets (2.1% increase (p = 0.003) vs. 0.4% decrease (n.s.), global p = 0.04). At 10 yr of age, lower CD14 methylation values were found in children with pets compared with children without pets at both 2 and 10 yr (5.4% vs. 7.5% [p = 0.02]). A similar trend was detected for TSE; children not exposed show larger increases in CD14 methylation, most pronounced in school-age girls exposed vs. not exposed to tobacco (5.5% vs. 7.5% methylation, p = 0.037). Conclusion Pet keeping and TSE appears to limit increase in CD14 methylation from 2 to 10 yr of age. This may partly explain the diverging CD14 allele associations with allergic diseases detected in different environments.

Background: The CHRNA 3 and 5 genes on chromosome 15 encode the alpha subunits of the nicotinic acetylcholine receptor, mediating airway cholinergic activity. Polymorphisms are associated with cigarette smoking, chronic obstructive pulmonary disease, and lung cancer. Aims: To determine possible associations between CHRNA 3/5 SNP rs8034191 and asthma or lung function in children in one local and one replicate multinational population, and assess if tobacco smoke modified the associations. Materials and methods: The rs8034191 SNP genotyped in 551 children from the environment and childhood asthma (ECA) birth cohort study in Oslo, Norway, and in 516 families from six European centers [the Genetics of Asthma International Network (GAIN) study] was tested for genotypic or allelic associations to current or history of asthma, allergic sensitization (= one positive skin prick tests), bronchial hyperresponsiveness (BHR), and lung function (FEV1% of predicted and FEV1/FVC ratio over/ below the 5th percentile). Results: Although the TT and CT genotypes at SNP rs 8034191 were overall significantly associated with BHR (OR = 3.9, 95% CI 1.510.0, p = 0.005), stratified analyses according to exposure to maternal smoking in-utero or indoor smoking at 10 yrs of age showed significant association (OR = 4.4, 95% CI 1.512.6, p = 0.006 and OR 5.6, 95% CI 1.718.5, p = 0.004, respectively) only in the non-exposed and not in exposed children. The SNPBHR association was replicated in the non-tobacco-smoke-exposed subjects in one of the GAIN centers (BHR associated with the T allele (p = 0.034)), but not in the collated GAIN populations. Asthma, allergic sensitization, and lung function were not associated with the rs8034191 alleles. Conclusion: An interaction between tobacco smoke exposure and a CHRNA3/5 polymorphism was found for BHR in children, but CHRNA3/5 was not associated with asthma or lung function.

Obesity has a strong genetic etiology involving numerous identified metabolic pathways and others not yet examined. We investigated the association between severe obesity and genetic variation in selected candidate genes, including three drug-related genes: cannabinoid receptor 1 (CNR1), N-acyl phosphatidylethanolamine phospholipase D (NAPEPLD), and gastric lipase (LIPF); and three genes related to inflammation: nicotinamide phosphoribosyltransferase, six-transmembrane epithelial antigen of the prostate 4 (STEAP4) and interleukin 18 (IL-18). Subjects were 1,632 individuals with severe obesity (BMI ≥ 35 kg/m²) and 3,379 controls (BMI 20-24.9 kg/m²) that took part in a Norwegian population based cohort study. Tagging single-nucleotide polymorphisms (SNPs) of the coding region of these genes were analyzed. SNP-haplotypes for each gene were constructed in order to analyze allelic, genotypic, and haplotypic association to obesity. A single SNPs rs17605251 in NAPEPLD was nominally associated with BMI ≥ 35 kg/m² (P = 0.035). A common haplotype in NAPEPLD was associated with BMI ≥ 35 kg/m² after correction for multiple testing. The allele frequency was 56.8% in cases and 60.3% in controls, giving an odds ratio (OR) of 0.87 (95% confidence interval (CI) 0.79, 0.95; P = 0.0016). Homozygosity for this haplotype was protective against obesity (OR 0.79 (CI 0.70-0.91); P = 0.00059). The SNP rs7913071 in LIPF was associated with obesity, but the association lost statistical significance after correction for multiple testing. The CNR1, IL-18, STEAP4, and nicotinamide phosphoribosyltransferase genes were not associated with obesity. In conclusion a common haplotype in NAPEPLD, an enzyme involved in endocannabinoid synthesis, was protective against obesity.

Purpose: This study aimed to identify the genetic cause of autosomal dominant pericentral retinal dystrophy (adPRD) in a large Norwegian family with 35 affected members. Methods: The family was characterized by clinical ophthalmological examination along with fundus photography, dark adaptometry and electroretinography. We performed a genome-wide linkage analysis followed by sequencing of a candidate gene to identify the mutation causing the disease. Results: The ophthalmological examinations revealed an atypical form of retinitis pigmentosa (RP), which we prefer to call adPRD. Compared with classical RP, this phenotype has a favourable prognosis. Linkage analysis showed a linkage peak covering the most recently reported adRP gene TOPORS. This gene was sequenced in 19 family members and a novel missense mutation, c.1205a>c, resulting in an amino acid substitution p.Q402P, was detected in all affected members. The mutation showed complete co-segregation with the disease in this family, with a LOD score of 7.3. It is located in a highly conserved region and alignment with the appropriate DNA sequence from other species shows complete conservation of this amino acid. The mutation was not detected in 207 healthy, unrelated controls of Norwegian origin. Conclusions: We present a novel mutation in the TOPORS gene co-segregating with a distinct phenotype of adPRD in a large Norwegian family.

Background: The HLA (human leukocyte antigen) class II genes DQB1 and DRB1 and the Tumor Necrosis Factor α gene (TNFA) within the HLA complex (chromosome 6p21) have been associated with asthma and allergy. Due to the strong linkage disequilibrium characterizing this complex and the multiple asthma/allergy expressions, we aimed to determine which of these genes were primarily involved in specific asthma/allergy traits. Methods: The DRB1–DQB1 alleles and TNFA-308 polymorphism were genotyped in 959 children from the Environment and Childhood Asthma study and analyzed for possible associations with allergic and non-allergic asthma (with/without at least one positive skin prick test for allergens) and specific allergic sensitization, as well as bronchial hyperresponsiveness and total IgE, using both allele and extended haplotype analyses. Results: Different genes within the HLA complex were associated with separate asthma and allergy traits. Nonallergic asthma was associated with both the TNFA-308A allele [Odds ratio (OR) 1.7 (1.3–2.3)] and DRB1*03 allele [OR 1.6(1–2.6)], but extended DRB1*03-TNFA-308 haplotype analysis suggested that the DRB1–DQB1 association was secondary to linkage disequilibrium with the TNFA-308 polymorphism. Allergies were associated with HLA class II alleles only; birch sensitization with DQB1*0603-DRB1*13 [OR 2.3 (1.4–4.0)] and mugwort sensitization with DQB1*0609-DRB1*13 [OR 7.1 (1.9–27.0)] and DQB1*0501-DRB1*01 [OR 2.0 (1.0–4.0)]. Conclusions: Our data suggests that asthma is not associated with DRB1 or DQB1 but rather TNFA or a gene(s) in linkage disequilibrium, while sensitization to specific allergens is associated with particular alleles at the DQ and/or DR loci. A novel association between DQB1*0603-DRB1*13 and birch allergy is identified.

The direct involvement of the human leukocyte antigen class II DR-DQ genes in type 1 diabetes (T1D) is well established, and these genes display a complex hierarchy of risk effects at the genotype and haplotype levels. We investigated, using data from 38 studies, whether the DR-DQ haplotypes and genotypes show the same relative predispositional effects across populations and ethnic groups. Significant differences in risk within a population were considered, as well as comparisons across populations using the patient/control (P/C) ratio. Within a population, the ratio of the P/C ratios for two different genotypes or haplotypes is a function only of the absolute penetrance values, allowing ranking of risk effects. Categories of consistent predisposing, intermediate ('neutral'), and protective haplotypes were identified and found to correlate with disease prevalence and the marked ethnic differences in DRB1-DQB1 frequencies. Specific effects were identified, for example for predisposing haplotypes, there was a statistically significant and consistent hierarchy for DR4 DQB1*0302s: DRB1*0405 =*0401 =*0402 > *0404 > *0403, with DRB1*0301 DQB1*0200 (DR3) being significantly less predisposing than DRB1*0402 and more than DRB1*0404. The predisposing DRB1*0401 DQB1*0302 haplotype was relatively increased compared with the protective haplotype DRB1*0401 DQB1*0301 in heterozygotes with DR3 compared with heterozygotes with DRB1*0101 DQB1*0501 (DR1). Our results show that meta-analyses and use of the P/C ratio and rankings thereof can be valuable in determining T1D risk factors at the haplotype and amino acid residue levels.

Background: Eosinophil cationic protein (ECP) is a potent cytotoxic secretory protein with bactericidal and antiviral properties. ECP is released by activated eosinophils and regarded as a marker of eosinophilic inflammation. High levels of ECP have been reported in cases of active asthma and other allergic diseases. This study aimed to assess whether three single-nucleotide polymorphisms (SNPs) in the ECP gene (RNASE3) on chromosome 14 q24–q31 or their haplotypes are associated with asthma, allergy, or related phenotypes. Methods: The three SNPs −38CA, +371CG and +499CG in RNASE3 and their haplotypes were analyzed for associations with asthma, serum-ECP (s-ECP) levels, allergic sensitization (positive skin-prick test to common allergens), bronchial hyperresponsiveness (BHR) assessed by methacholine inhalation, and serum-IgE (s-IgE) levels in 177 families from Norway and the Netherlands identified through siblings with asthma. Results: Transmission disequilibrium test (TDT) demonstrated significant associations between the A-G-G haplotype and asthma as well as the specific phenotypes allergic asthma (but not non-allergic asthma), high s-ECP, high s-IgE and BHR, while the C-G-G haplotype was associated with reduced occurrence of these traits. In addition, the −38A allele was associated with high s-ECP levels and allergic asthma. Conclusion: The present study suggests that the A-G-G haplotype in the RNASE3 gene influences the development of asthma, in particular, an allergic form of asthma. Furthermore, as the −38CA SNP lies in close vicinity of known intron-regulatory sites, results of SNP analysis suggest that the detected association is possibly linked to a genetic transcriptional control of s-ECP levels.

Alleles at the HLA-DQB1, -DQA1 and -DRB1 loci are major determinants for susceptibility to develop type 1 diabetes (T1D). Increasing evidence supports that also other genes in, or near, the HLA complex contribute to the HLA-encoded risk. Alleles at the DPB1 locus have been suggested to directly influence the risk conferred by DQB1, DQA1 and DRB1 alleles, but the results are conflicting. We therefore genotyped 217 families from Norway, Denmark, Sweden and southern France to address the role of DPB1 alleles in T1D. After taking into account linkage disequilibrium (LD) with DQB1, DQA1 and DRB1 alleles, we found evidence that some DPB1 alleles are associated with modulating the risk of developing T1D. However, we show that the strong LD in the HLA complex, and the presence of extended haplotypes complicate the interpretation of the results. On DQ2-DR3 haplotypes, both allele 3 at microsatellite D6S2223 located 5.3-Mb telomeric of DPB1 and the extended DQ2-DR3-B18 haplotype display much stronger association than DPB1 alleles. When we exclude these effects, most of the apparent association of DPB1 alleles on DQ2-DR3 haplotypes disappear. Taken together, although we cannot completely rule out an effect of some DPB1 alleles, we propose that the statistically significant, albeit weak, DPB1 associations found are most likely the result of LD with another unidentified disease-susceptibility gene(s) in this region.

Objective Juvenile idiopathic arthritis (JIA) is associated with particular alleles at 3 different HLA loci: HLA–A, HLA–DR/DQ, and HLA–DP. These associations are independent of each other (i.e., they cannot be explained by the known linkage disequilibrium between alleles at these loci). The purpose of this study was to look for additional JIA susceptibility genes in the HLA complex. Methods One hundred two Norwegian JIA patients and 270 healthy individuals, all carrying the DQ4;DR8 haplotype, were investigated by scanning ∼10 megabases of DNA covering the HLA complex with microsatellite polymorphisms. An expectation-maximization algorithm was used to estimate haplotype frequencies, and the distribution of microsatellite alleles on the high-risk DQ4;DR8 haplotype was compared between patients and controls, to exclude effects secondary to linkage disequilibrium with these susceptibility genes. Results Allele 5 at the microsatellite locus D6S265 (D6S265* 5), 100 kb centromeric of HLA–A, showed a strong positive association with the disease (odds ratio 4.7, corrected P < 10−6). Haplotype analysis demonstrated that the D6S265*5 association was not caused by linkage disequilibrium to the gene encoding HLA–A*02, which has previously been reported to be associated with JIA. Instead, our data suggested that a gene in linkage disequilibrium with D6S265*5, but distinct from HLA–A*02, is involved in the predisposition to JIA. Conclusion We found that D6S265*5 could be a marker for an additional susceptibility gene in JIA which is distinct from A*02, adding to the risk conferred by DQ4;DR8.

Type 1 diabetes is a complex disease where numerous genes are involved in the pathogenesis. Genes that account for approximately 50% of the familial clustering of the disease are located within or in the vicinity of the HLA complex on chromosome 6. Some DRB1, DQA1 and DQB1 genes are known to be involved, in addition to as yet unidentified HLA-linked genes. The DR4-DQ8 and DR3-DQ2 haplotypes are known to confer high risk for developing the disease, particularly when occurring together. Approximately 10% of patients, however, do not carry any of these high-risk HLA class II haplotypes. We have performed genotyping of DRB1, DQA1 and DQB1 alleles in non-DR3-DQ2/non-DR4-DQ8 patients and controls from Sweden and Norway to test if any HLA associations were observed in these patients. Our results clearly demonstrate several statistically significant differences in the frequency of HLA haplotypes between patients and controls. Case-control analysis including the relative predispositional effect test, and transmission disequilibrium test (TDT) analysis in Norwegian type 1 diabetes families revealed that the DQA1*03-DQB1*0301, DQA1*0401-DQB1*0402, DQA1*0101-DQB1*0501, DQA1*03-DQB1*0303 and DQA1*0102-DQB1*0604 haplotypes may also confer risk. Our analyses also supported independent risks of certain DRB1 alleles. The study clearly demonstrates that HLA associations in type 1 diabetes extends far beyond the well-known associations with the DR4-DQ8 and DR3-DQ2 haplotypes. Our data suggest that there is a hierarchy of HLA class II haplotypes conferring risk to develop type 1 diabetes.

We have recently shown that an as yet unidentified gene within or in the vicinity of the HLA complex, in linkage disequilibrium with microsatellite D6S2223, modifies the risk to develop type 1 diabetes independently of HLA-DR and -DQ genes. This microsatellite is located 2.5 Mb telomeric to HLA-F and particular alleles at this microsatellite modifies the risk encoded by the high-risk DRB1*03-DQA1*0501-DQB1*0201 (hereafter called DR3) haplotype. Coeliac disease and type 1 diabetes share some susceptibility HLA class II haplotypes, in Scandinavia particularly the DR3 haplotype. We therefore investigated whether the marker D6S2223 might also be associated with coeliac disease. In order to keep the contributions from the DRB1-DQA1-DQB1 genes constant (i.e., eliminate the effects of linkage disequilibrium to disease associated DR and/or DQ alleles), we only used cases and controls being homozygous for DR3. We found the frequency of allele 3 at D6S2223 to be reduced among patients with coeliac disease compared to controls, to a similar extent as seen in type 1 diabetes, which could not be explained by a different distribution of HLA-B alleles (as ascertained by typing for the MIB microsatellite). This negatively associated allele 3 at D6S2223 occurred in a homozygous combination at a significantly lower frequency among patients than controls. Thus, allele 3 at D6S2223 on DR3 haplotypes is associated with reduced susceptibility for development of both type 1 diabetes and coeliac disease. This suggests that a gene(s) in the vicinity of D6S2223 is involved in the pathogenesis of both of these immune-mediated diseases.

The TAP1 and TAP2 genes, located in the HLA class II region, encode subunits of a peptide transporter. Both genes display limited genetic variability; four different nucleotide substitutions have been found in the TAP2 gene. Here studies on linkage disequilibrium between TAP2 variants and HLA class II alleles are reported, in an attempt to evaluate whether TAP2 variants are associated with insulin-dependent diabetes mellitus (IDDM). As reported previously, a significant decrease of homozygosity for TAP2 alleles encoding alanine at residue 665 (665 Ala) and glutamine at 687 (687 Gln) paralleled by an increase in homozygosity for TAP2 alleles encoding threonine at residue 665 (665 Thr) and a stop codon at 687 (687 Stop), was found in both Finnish and Norwegian IDDM patients compared to random controls. However, a strong linkage disequilibrium between these TAP2 polymorphisms and given HLA-DR and -DQ genes was observed among healthy controls. The frequent 665 Thr and 687 Stop variants were in linkage disequilibrium both with the DR4-DQ8 and the DR3-DQ2 haplotypes, haplotypes which are strongly associated with IDDM. In contrast, the DR1-DQ5 and DR13-DQ6 (e.g. DQB1*0603) haplotypes, which are decreased among IDDM patients, were associated with the 665 Ala and 687 Gln variants. Thus, when DR- and DQ-matched patients and controls were compared, associations of the investigated TAP2 variants and IDDM were no longer detectable. These data, therefore, indicate that the associations previously found between certain TAP2 variants and IDDM are secondary to a primary association between this disease and particular DQ alpha beta heterodimers.