Vitamin D deficiency is associated with susceptibility to tuberculosis, and its biologically active metabolite, 1alpha,25 dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), has pleiotropic immune effects. The mechanisms by which 1alpha,25(OH)(2)D(3) protects against tuberculosis are incompletely understood. 1alpha,25(OH)(2)D(3) reduced the growth of mycobacteria in infected human PBMC cultures in a dose-dependent fashion. Coculture with agonists or antagonists of the membrane or nuclear vitamin D receptors indicated that these effects were primarily mediated by the nuclear vitamin D receptors. 1alpha,25(OH)(2)D(3) reduced transcription and secretion of protective IFN-gamma, IL-12p40, and TNF in infected PBMC and macrophages, indicating that 1alpha,25(OH)(2)D(3) does not mediate protection via these cytokines. Although NOS2A was up-regulated by 1alpha,25(OH)(2)D(3), inhibition of NO formation marginally affected the suppressive effect of 1alpha,25(OH)(2)D(3) on bacillus Calmette Guérin in infected cells. By contrast, 1alpha,25(OH)(2)D(3) strongly up-regulated the cathelicidin hCAP-18 gene, and some hCAP-18 polypeptide colocalized with CD14 in 1alpha,25(OH)(2)D(3) stimulated PBMC, although no detectable LL-37 peptide was found in supernatants from similar 1alpha,25(OH)(2)D(3)-stimulated PBMC cultures. A total of 200 mug/ml of the active peptide LL-37, in turn, reduced the growth of Mycobacterium tuberculosis in culture by 75.7%. These findings suggest that vitamin D contributes to protection against TB by "nonclassical" mechanisms that include the induction of antimicrobial peptides.

The evidence for a human genetic component in susceptibility to tuberculosis (TB) is incontrovertible. Quite apart from studies of rare disease events illustrating the importance of key genes in humans and animals, TB at the population level is also influenced by the genetics of the host. Heritability of disease concordance and immune responses to mycobacterial antigens has been clearly shown, and ranges up to 71%. Linkage studies, designed to identify major susceptibility genes in a disease, have produced a number of candidate loci but few, except for regions on chromosome 5p15, 20p and 20q, have been replicated. The region on 5p15 regulates the intensity of the response to the tuberculin skin test, and another locus on 11p14 appears to control resistance to the bacterium. In addition, numerous genes and pathways have been implicated in candidate gene association studies, with validation of polymorphisms in IFNG, NRAMP1, and NOS2A and equivocal results for IL10, CCL2, DC-SIGN, P2RX7, VDR, TLR2, TLR9 and SP110. Other more recently researched candidate genes such as TNFRSF1B remain to be validated, preferably in meta-analyses. New approaches have provided early evidence for the importance of gene-gene interactions in regulating resistance to disease, and also the prospect that applying host genetics in the field of vaccinomics could lead to a more targeted approach in designing interventions to aid the human immune system in combating mycobacteria. Genome-wide association studies and admixture mapping are approaches that remain to be applied to TB, and it is not clear, as is the case with other complex diseases, how much of the heritability of the TB susceptibility phenotype will be determined by multiple genes of small effect versus rare variants with disproportionately large effects.

The region of conserved synteny on mouse chromosome 11/human 17q11-q21 is known to carry a susceptibility gene(s) for intramacrophage pathogens. The region is rich in candidates including NOS2A, CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL5/RANTES, CCR7, STAT3 and STAT5A/5B. To examine the region in man, we studied 92 multicase tuberculosis (627 individuals) and 72 multicase leprosy (372 individuals) families from Brazil. Multipoint nonparametric analysis (ALLEGRO) using 16 microsatellites shows two peaks of linkage for leprosy at D17S250 (Z(lr) score 2.34; P=0.01) and D17S1795 (Z(lr) 2.67; P=0.004) and a single peak for tuberculosis at D17S250 (Z(lr) 2.04; P=0.02). Combined analysis shows significant linkage (peak Z(lr) 3.38) at D17S250, equivalent to an allele sharing LOD score 2.48 (P=0.0004). To determine whether one or multiple genes contribute, 49 informative single nucleotide polymorphisms were typed in candidate genes. Family-based allelic association testing that was robust to family clustering demonstrated significant associations with tuberculosis susceptibility at four loci separated by intervals (NOS2A-8.4 Mb-CCL18-32.3 kb-CCL4-6.04 Mb-STAT5B) up to several Mb. Stepwise conditional logistic regression analysis using a case/pseudo-control data set showed that the four genes contributed separate main effects, consistent with a cluster of susceptibility genes across 17q11.2.

OBJECTIVE

Cytokines and other inflammatory mediators are involved in the pathogenesis of otitis media. We hypothesized that polymorphisms in inflammatory response genes contribute to the increased susceptibility to acute otitis media in otitis-prone children.

METHODS

DNA samples from 348 children with>> or = 2 acute otitis media episodes, who were participating in a randomized, controlled vaccination trial, and 463 healthy adult controls were included. Polymorphisms in TNFA, IL1B, IL4, IL6, IL10, IL8, NOS2A, C1INH, PARP, TLR2, and TLR4 were genotyped. Genotype distributions in children with recurrent acute otitis media were compared with those in controls. Within the patient group, the number of acute otitis media episodes before vaccination and the clinical and immunologic response to pneumococcal conjugate vaccinations were analyzed.

RESULTS

The IL6-174 G/G genotype was overrepresented in children with acute otitis media when compared with controls. In the patient group, TNFA promoter genotypes -238 G/G and -376 G/G and the TLR4 299 A/A genotype were associated with an otitis-prone condition. Furthermore, lower specific anticapsular antibody production after complete vaccination was observed in patients with the TNFA-238 G/G genotype or TNFA-863 A allele carriage. Finally, the IL10-1082 A/A genotype contributed to protection from the recurrence of acute otitis media after pneumococcal vaccination.

CONCLUSIONS

Variation in innate immunoresponse genes such as TNFA-863A, TNFA-376G, TNFA-238G, IL10-1082 A, and IL6-174G alleles in the promoter sequences may result in altered cytokine production that leads to altered inflammatory responses and, hence, contributes to an otitis-prone condition.

The historical impression that tuberculosis was an inherited disorder has come full circle and substantial evidence now exists of the human genetic contribution to susceptibility to tuberculosis. This evidence has come from several whole-genome linkage scans, and numerous case-control association studies where the candidate genes were derived from the genome screens, animal models and hypotheses pertaining to the disease pathways. Although many of the associated genes have not been validated in all studies, the list of those that have been is growing, and includes NRAMP1, IFNG, NOS2A, MBL, VDR and some TLR. Certain of these genes have consistently been associated with tuberculosis in diverse populations. The future investigation of susceptibility to tuberculosis is almost certain to include genome-wide association studies, admixture mapping and the search for rare variants and epigenetic mechanisms. The genetic identification of more vulnerable individuals is expected to inform personalized treatment and perhaps vaccination strategies.

BACKGROUND

Genetic variation in arginase (ARG) and nitric oxide synthase (NOS) has been associated with exhaled nitric oxide (FeNO) levels in children. Little is known about whether epigenetic variation in these genes modulates FeNO.

OBJECTIVE

To evaluate whether DNA methylation in ARG and NOS genes is associated with FeNO.

METHODS

A subset of 940 participants in the Children's Health Study were selected for this study. Children were eligible if they had FeNO measurements and buccal cells collected on the same day. CpG loci located in the promoter regions of NOS1, NOS2A, NOS3, ARG1, and ARG2 genes were analyzed. Multiple loci in each gene were evaluated individually and averaged together. DNA methylation was measured using a bisulfite-polymerase chain reaction pyrosequencing assay. Linear regression models were used to investigate the association between DNA methylation and FeNO and whether associations differed by asthma status.

RESULTS

DNA methylation in ARG2 was significantly associated with FeNO. A 1% increase in average DNA methylation of ARG2 was associated with a 2.3% decrease in FeNO (95% confidence interval, -4 to -0.6). This association was significantly larger in children with asthma (%diff = -8.7%) than in children with no asthma (%diff = -1.6%; p(int) = 0.01). Differences in FeNO by asthma status were also observed for ARG1 (%diff(asthma) = -4.4%; %diff(non-asthma) = 0.3%; p(int) = 0.02). DNA methylation in NOS genes was not associated with FeNO.

CONCLUSIONS

DNA methylation in ARG1 and ARG2 is associated with FeNO in children with asthma and suggests a possible role for epigenetic regulation of nitric oxide production.

BACKGROUND

Asthma pathogenesis and susceptibility involves a complex interplay between genetic and environmental factors. Their interaction modulates the airway inflammation and remodelling processes that are present even in mild asthma and governs the appearance and severity of symptoms of airway hyperresponsiveness. While asthma is felt to develop as the result of interaction among many different genes and signalling pathways, only a few genes have been linked to an increased risk of developing this condition.

RESULTS

We report the results of expression microarray studies using tissue obtained from bronchial biopsies of healthy controls and of subjects with allergic asthma, both before and following inhaled corticotherapy. We identified 79 genes that show significant differences in expression (following Bonferroni cutoff using p < 6.6 x 10(-6) to correct for multiple testing) in asthmatics compared to controls at significance levels. These included 21 genes previously implicated in asthma, such as NOS2A and GPX3, as well as new potential candidates, such as ALOX15, CTSC and CX3CR1. The expression levels of one third of these transcripts were partially or completely corrected following inhaled corticosteroid therapy.

CONCLUSIONS

The study shows that bronchial biopsies obtained from healthy and asthmatic subjects display distinct expression profiles. These differences provide a global view of physiopathologic processes active in the asthmatic lung and may provide invaluable help to clarify the natural history of asthma.

Evidence supporting the contribution of oxidative stress to key pathways in cancer, such as inflammation and DNA damage, continues to mount. We investigated variations within genes mediating oxidative stress to determine whether they alter risk for non-Hodgkin lymphoma (NHL). Thirteen single nucleotide polymorphisms (SNPs) from 10 oxidative stress genes (AKR1A1, AKR1C1, CYBA, GPX, MPO, NOS2A, NOS3, OGG1, PPARG and SOD2) were genotyped in 1172 NHL cases and 982 population-based controls from a USA multicenter case-control study. For NHL and five subtypes (diffuse large B-cell, follicular, marginal zone, small lymphocytic and T-cell), SNP associations were calculated. Odds ratios (OR) and 95% confidence intervals (CI) were adjusted for sex, age (<45, 45-64, 65+ years), race (white, black, other) and study site. Overall, the oxidative stress pathway was associated significantly with the B-cell NHL subtype, diffuse large B-cell lymphoma (DLBCL) (global P-value=0.003). Specifically, for nitric oxide synthase (NOS2A Ser608Leu, rs2297518) Leu/Leu homozygotes, there was a 2-fold risk increase for NHL (OR=2.2, 95% CI=1.1-4.4) (referent=Ser/Ser and Ser/Leu). This risk increase was consistent by cell lineage (B- and T-cell NHL) and pronounced for the two most common subtypes, diffuse large B-cell (OR=3.4, 95% CI=1.5-7.8) and follicular lymphoma (OR=2.6, 95% CI=1.0-6.8). In an analysis of manganese superoxide dismutase (SOD2 Val16Ala, rs1799725) Ala/Ala homozygotes, we observed moderately increased risks for B-cell lymphomas (OR=1.3, 95% CI=1.0-1.6; referent=Val/Val and Val/Ala) that was consistent across the B-cell subtypes. Genetic variations that result in an increased generation of reactive oxygen species appear to increase risk for NHL and its major subtypes, particularly DLBCL. Independent replication of our findings are warranted and further evaluation of oxidative stress in the context of inflammation, DNA repair and the induction of the NF-kappaB pathway may further reveal important clues for lymphomagenesis.

Nitric oxide synthase (NOS) genes (NOS1, NOS2A, and NOS3) may create excess nitric oxide that contributes to neurodegeneration in Parkinson's disease (PD). NOS genes might also interact with one another or with environmental factors in PD. Coding and tagging single nucleotide polymorphisms (SNPs) (27 NOS1, 18 NOS2A, and five NOS3 SNPs) were genotyped in families with PD (1,065 cases and 1,180 relative and other controls) and were tested for allelic associations with PD using the association in the presence of linkage test and the pedigree disequilibrium test (PDT), allelic associations with age-at-onset (AAO) using the quantitative transmission disequilibrium test, and interactions using the multifactor dimensionality reduction-PDT. Gene-environment interactions involving cigarette smoking, caffeine, nonsteroidal anti-inflammatory drugs, and pesticides were examined using generalized estimating equations in participants with environmental data available. Significant associations with PD were detected for the NOS1 SNPs rs3782218, rs11068447, rs7295972, rs2293052, rs12829185, rs1047735, rs3741475, and rs2682826 (range of p = 0.00083-0.046) and the NOS2A SNPs rs2072324, rs944725, rs12944039, rs2248814, rs2297516, rs1060826, and rs2255929 (range of p = 0.0000040-0.047) in earlier-onset families with sporadic PD, and some SNPs were also associated with earlier AAO. There was no compelling statistical evidence for gene-gene interactions. However, of the significantly associated SNPs, interactions were found between pesticides and the NOS1 SNPs rs12829185, rs1047735, and rs2682826 (range of p = 0.012-0.034) and between smoking and the NOS2A SNPs rs2248814 (p = 0.021) and rs1060826 (p = 0.013). These data implicate NOS1 and NOS2A as genetic risk factors for PD and demonstrate that their interactions with established environmental factors may modulate the environmental effects.

BACKGROUND

Angiogenesis is required for development and progression of prostate cancer. Potentially functional single nucleotide polymorphisms (SNP) in genes important in prostate angiogenesis (VEGF, HIF1A, and NOS3) have previously been associated with risk or severity of prostate cancer.

METHODS

Prostate cancer cases (n = 1,425) and controls (n = 1,453) were selected from the Cancer Prevention Study II Nutrition Cohort. We examined associations between 58 SNPs in nine angiogenesis-related candidate genes (EGF, LTA, HIF1A, HIF1AN, MMP2, MMP9, NOS2A, NOS3, VEGF) and risk of overall and advanced prostate cancer. Unconditional logistic regression was used to estimate odds ratios, adjusted for matching factors.

RESULTS

Our results did not replicate previously observed associations with SNPs in VEGF, HIF1A, or NOS3, nor did we observe associations with SNPs in EGF, LTA, HIF1AN, MMP9, or NOS2A. In the MMP2 gene, three intronic SNPs, all in linkage disequilibrium, were associated with overall and advanced prostate cancer (for overall prostate cancer, P(trend) = 0.01 for rs1477017, P(trend) = 0.01 for rs17301608, P(trend) = 0.02 for rs11639960). However, two of these SNPs (rs17301608 and rs11639960) were examined and were not associated with prostate cancer in a recent genome-wide association study using prostate cancer cases and controls from the Prostate, Lung, Colorectal, and Ovary study cohort. Furthermore, when we pooled our results for these two SNPs with those from the Prostate, Lung, Colorectal, and Ovary cohort; neither SNP was associated with prostate cancer.

CONCLUSIONS

None of the SNPs examined seem likely to be importantly associated with risk of overall or advanced prostate cancer.

Trachoma is a poorly understood immunofibrogenic disease process, initiated by Chlamydia trachomatis. Differences in conjunctival gene expression profiles between Ethiopians with trachomatous trichiasis (with [TTI] or without [TT] inflammation) and controls (C) were investigated to identify relevant host responses. Tarsal conjunctival swab samples were collected for RNA isolation and C. trachomatis PCR. Transcriptome-wide microarray experiments were conducted on 42 samples (TTI, n = 13; TT, n = 15; C, n =14). Specific results were confirmed by using multiplex quantitative reverse transcription-PCR for 16 mRNA targets in an independent collection of case-control samples: 386 case-control pairs (TTI, n = 244; TT, n = 142; C, n = 386). The gene expression profiles of cases were consistent with squamous metaplasia (keratins, SPRR), proinflammatory cytokine production (IL1β, CXCL5, and S100A7), and tissue remodeling (MMP7, MMP9, MMP12, and HAS3). There was no difference in the level of IFNγ between cases and controls. However, cases had increased INDO, NOS2A, and IL13RA2 and reduced IL13. C. trachomatis was detected in 1/772. Cases show evidence of ongoing inflammation and tissue remodeling, which were more marked where clinical inflammation was also present. Significantly, these processes appear to be active in the absence of current C. trachomatis infection. There was limited evidence of a T(H)1 response (INDO and NOS2A) and no association between a T(H)2 response and cases. The epithelium appears to be actively involved in late cicatricial stages of trachoma through the production of proinflammatory factors (IL1β, CXCL5, and S100A7). Longitudinal studies are needed to investigate which etiological factors and pathways are associated with progressive scarring and whether simply controlling chlamydial infection will halt progression in people with established cicatricial disease.

To identify low-penetrance susceptibility alleles for chronic lymphocytic leukemia (CLL), we performed a case-control study genotyping 768 single-nucleotide polymorphisms (SNP) in 692 cases of CLL and 738 controls. We investigated nonsynonymous SNPs, SNPs with potential functional effect, and tag SNPs in regulatory gene regions in a total of 172 genes involved in cancer biology. After adjustment for multiple testing, we found a strong association between CLL risk and six genetic variants: CCNH (rs2266690, V270A), APAF1 (rs17028658, 3'region), IL16 (rs4505265, first intron), CASP8 (rs1045485, D302H), NOS2A (rs2779251, promoter), and CCR7 (rs3136687, intron 1). We found association with CLL susceptibility and 22 haplotypes in APAF1, IL6, TNFRSF13B, IL16, CASP3, CCR7, LTA/TNF, BAX, BCL2, CXCL12, CASP10/CASP8, CASP1, CCL2, BAK1, and IL1A candidate genes. Finally, we evaluated using public data sets the potential functional effect on gene expression levels of the CLL associated genetic variants detected in regulatory regions. Minor alleles for APAF1 and IL16 were associated with lower mRNA levels; no expression differences were observed for CCR7, whereas NOS2A could not be assessed. This study suggests that common genetic variation in apoptosis- and immunoregulation-related genes is associated with the CLL risk.

Tuberculosis (TB) has substantial mortality worldwide with 5-10% of those exposed progressing to active TB disease. Studies in mice and humans indicate that the inducible nitric oxide synthase (iNOS) molecule plays an important role in immune response to TB. A mixed case-control association study of individuals with TB, relatives, or close contact controls was performed in 726 individuals (279 case and 166 control African-Americans; 198 case and 123 control Caucasians). Thirty-nine single nucleotide polymorphisms (SNPs) were selected from the NOS2A gene for single SNP, haplotype, and multilocus interaction analyses with other typed candidate genes using generalized estimating equations. In African-Americans, ten NOS2A SNPs were associated with TB. The strongest associations were observed at rs2274894 (odds ratio (OR) = 1.84, 95% confidence interval (CI) [1.23-2.77], p = 0.003) and rs7215373 (OR = 1.67, 95% CI [1.17-2.37], p = 0.004), both of which passed a false discovery rate correction for multiple comparisons (q* = 0.20). The strongest gene-gene interactions were observed between NOS2A rs2248814 and IFNGR1 rs1327474 (p = 0.0004) and NOS2A rs944722 and IFNGR1 rs1327474 (p = 0.0006). Three other SNPs in NOS2A interacted with TLR4 rs5030729 and five other NOS2A SNPs interacted with IFNGR1 rs1327474. No significant associations were observed in Caucasians. These results suggest that NOS2A variants may contribute to TB susceptibility, particularly in individuals of African descent, and may act synergistically with SNPs in TLR4 and IFNGR1.

Aging-related erectile dysfunction is characterized by a loss of smooth muscle cells (SMCs) and fibrosis in the corpora cavernosa, and functionally by corporal veno-occlusive dysfunction (CVOD). Phosphodiesterase 5 (PDE5A) inhibitors, in part via upregulating inducible nitric oxide synthase (NOS2A), have antifibrotic properties in penile tissues. We aimed to determine whether in the aged rat the chronic long-term treatment with sildenafil ameliorates corporal SMC loss and fibrosis, stimulates NOS2A induction, and corrects the associated CVOD. Aged male rats (20 mo old) received sildenafil in their drinking water (20 mg/kg per day) or plain water for 45 days, and untreated young rats (5 mo old) served as controls (n = 8 per group). CVOD was assessed by dynamic infusion cavernosometry (DIC). Collagen:SMC (Masson trichrome) and collagen III:I (picrosirius red) ratios, SMC content (alpha-smooth muscle actin [ACTA2]), cell proliferation (proliferating nuclear antigen [PCNA]), apoptotic death (TUNEL), and NOS2A induction were measured by histochemistry and immunohistochemistry followed by quantitative image analysis. Collagen content was determined by hydroxyproline assay, and transforming growth factor beta-1 (TGFB1); xanthine oxidoreductase (XDH); ACTA2; NOS2A; and the Rho kinase inhibitor protein tyrosine phosphatase, nonreceptor type 11 (PTPN11), and activator, VAV, were measured by quantitative Western blot. In the aged rats treated with sildenafil, the erectile response by DIC was normalized, and the corporal SMC:collagen ratio and SMC number were increased. In addition, sildenafil reduced the corporal collagen content without affecting the collagen III:I ratio, increased the PCNA:apoptosis ratio, and stimulated NOS2A induction, although there was no effect on XDH, TGFB1, PTPN11, or VAV levels. These data show that long-term PDE5A treatment corrected CVOD in the aged rat and partially reversed the aging-related fibrosis and loss of SMC in the corpora cavernosa without affecting TGFB1 or PTPN11 levels, which are markers of oxidative stress. It may be speculated that similar effects may be achieved with this paradigm in men.

Hematopoietic stem cells (HSCs) are rare multipotent cells that contribute to all blood lineages. During inflammatory stress, hematopoietic stem and progenitor cells (HSPCs) can be stimulated to proliferate and differentiate into the required immune cell lineages. Manipulating signaling pathways that alter HSPC capacity holds great promise in the treatment of hematological malignancies. To date, signaling pathways that influence HSPC capacity, in response to hematopoietic stress, remain largely unknown. Using a zebrafish model of demand-driven granulopoiesis to explore the HSPC response to infection, we present data supporting a model where the zebrafish ortholog of the cytokine-inducible form of nitric oxide synthase (iNOS/NOS2) Nos2a acts downstream of the transcription factor C/ebpβ to control expansion of HSPCs following infection. These results provide new insights into the reactive capacity of HSPCs and how the blood system is "fine-tuned" in response to inflammatory stress.

OBJECTIVE

There is evidence to suggest that vasospasm and vascular dysregulation play a role in the etiology of glaucoma. This effect may be particularly relevant in patients with glaucoma who have a history of migraine or vasospastic tendencies. This study was conducted to investigate the role of genes with products that regulate blood flow to ocular tissues. The candidate genes were the two isoforms of nitric oxide synthase (NOS), NOS3 and -2A, and endothelin (ET)-l. The frequency of the T786C mutation in NOS3 was also examined.

METHODS

DNA was obtained from 58 patients with primary open-angle glaucoma (POAG), 76 with normal tension glaucoma (NTG), and 38 control subjects. Polymerase chain reactions (PCR) were used to compare the frequency of the alleles between the subjects with glaucoma and the control subjects and the subjects with glaucoma with vasospasm or migraine. The PCR product was sequenced to identify the frequency of the T786C mutation.

RESULTS

The distribution of the NOS3 repeat alleles in subjects with glaucoma and control subjects just failed to reach statistical significance (P = 0.06). The distribution in subjects with NTG or POAG did not differ significantly. A significant difference was found (P < 0.001) in the distribution of allele frequencies of the NOS3 marker in subjects who had glaucoma with migraine versus control subjects. There were no differences in the distribution of the NOS2A or ET-1 markers between the subjects with glaucoma and the control subjects.

CONCLUSIONS

This study provides evidence of an association between the NOS3 gene and subjects with glaucoma who have a history of migraine. Unlike in other studies, no evidence was found of an association between ET-1 and glaucoma.

Oxidative damage caused by reactive oxygen species (ROS) and other free radicals is involved in a number of pathological conditions including cancer. In a population-based case-control study of non-Hodgkin lymphoma (NHL) (n = 518 cases, 597 controls) among women in Connecticut, we analyzed one or more single nucleotide polymorphisms (SNPs) in ten candidate genes (AKR1A1, AKR1C1, AKR1C3, CYBA, GPX1, MPO, NOS2A, NOS3, OGG1, and SOD2) that mediate oxidative stress directly or indirectly in the NADPH oxidase-dependent respiratory burst. Odds ratios (OR) and 95% confidence intervals (CI) were adjusted for age and race. Polymorphisms in AKR1A1 and CYBA were significantly associated with increased risk of NHL. There was a 1.7-fold (95% CI = 1.2-2.4, P = 0.0047) increased risk of NHL for individuals who were variant homozygous for the AKR1A1 (IVS5 + 282T>> C) SNP. The effect was most pronounced for risk of diffuse large B-cell lymphoma, but risk estimates were non-significantly elevated for other common B-cell histologies and T-cell lymphomas as well. In addition, individuals variant homozygous for the CYBA (Ex4 + 11C>> T) SNP had a 1.6-fold (95% CI = 1.1-2.4, P = 0.019) increased risk of NHL that was particularly pronounced for T-cell lymphoma (OR = 3.5, 95% CI = 1.3-9.6, P = 0.013), but was also associated with non-significant increased risks for each of the common B-cell histologies. These results suggest that SNPs in genes related to the oxidative stress pathway may be associated with increased risk of NHL.

Chromosome 17 is known to contain TB susceptibility genes. Polymorphisms in two of these genes, namely NOS2A and CCL2, have been associated with TB in various populations. To investigate a possible association of gene variants with TB in the South African Coloured population we genotyped SNPs from NOS2A and CCL2 in over 800 TB cases and controls. We found a significant association between TB and two haplotypes, containing the functional rs9282799 and rs8078340 SNPs, in the NOS2A promoter. The T allele of rs8078340, found in the haplotype over-represented in cases (p=0.015, p(c)=0.038, OR=1.4, 95% CI [1.1-1.8]), was previously shown to decrease the quantity of DNA-protein complex bound as well as the duration of binding and may decrease nitric oxide (NO) production. The C allele of rs8078340 was present in the haplotype more frequent in controls (p=0.011, p(c)=0.029, OR=1.4, 95% CI [1.1-1.8]). In the single-point analysis of NOS2A, rs2779249 (previously associated with TB in Brazilians) and the functional rs8078340 were nominally associated with disease. No association was found between any of the other SNPs or haplotypes studied and TB. This study presents evidence that haplotypes in the NOS2A promoter influence susceptibility to TB and confirms the importance of NO production in the disease.

Preterm delivery (PTD) is the leading cause of infant mortality and morbidity worldwide. The etiology of PTD is largely unknown but is believed to be complex, encompassing multiple genetic and environmental determinants. To date, reports of genetic studies on PTD are sparse. We conducted a large-scale case-control study exploring the associations of 426 single-nucleotide polymorphisms with PTD in 300 mothers with PTD and 458 mothers with term deliveries at the Boston Medical Center. Twenty-five candidate genes were included in the final haplotype analysis, and a significant association of F5 gene haplotype with PTD was revealed and remained significant after Bonferroni correction for multiple testing (P=0.025). We applied different statistical algorithms (both Gibbs sampling and expectation-maximization) in reconstructing haplotype phases and different tests (both likelihood ratio test and permutation test) in association analyses, and all yielded similar results. We also performed exploratory ethnicity-specific analyses, which confirmed the consistent findings of the F5 gene across the ethnic groups. Moreover, IL1R2 (P=0.002 in Blacks), NOS2A (P<0.001 in Whites) and OPRM1 (P=0.004 in Hispanics) gene haplotypes were associated with PTD in specific ethnic groups but not at global significance level. In summary, our results underscore the potentially important role of F5 gene variants in the pathogenesis of PTD, and demonstrate the utility of high-throughput genotyping and a haplotype-based approach in dissecting genetic basis of complex traits.

Sheep have a varying ability to resist infection with gastrointestinal nematodes. This ability is due in part to genetic differences that exist between individuals. In order to define these differences we have used real-time PCR to quantify gene expression responses in the gut mucosal surface of genetically resistant and susceptible sheep, following a nematode challenge. Expression profiles were determined in response to two different nematode species, Haemonchus contortus and Trichostrongylus colubriformis, and in divergent sheep originating from two different genetic backgrounds. Results show that the response generated differs between resistant and susceptible animals and is further impacted by the origin of the sheep and nematode species used for challenge. However, some conserved features of a response mounted by a resistant or a susceptible animal were identified. Genes found to be more abundantly expressed in resistant animals include markers of an early inflammatory response, several Toll-like receptors (TLR2, 4, 9) and free radical producing genes (DUOX1 and NOS2A). Conversely, genes differentiating susceptible animals indicate a prolonged response and development of a chronic inflammatory state, characterised by elevated expression of members of the NF-kappabeta signalling pathway (IKBKB and NFKBIA) together with delayed expression of regulatory markers such as IL2RA (CD25), IL10 and TGFbeta2. While multiple nematode response pathways were identified, the identification of conserved aspects of the response which associate with resistance provides evidence that alternative nematode control strategies, such as breeding for resistant animals, may be feasible.

Accumulating evidence shows that the severity and rapidity of onset of diabetic retinopathy are influenced by genetic factors. Expression of the nitric oxide synthases is altered in the retinal vasculature in the early stages of diabetic retinopathy. We analyzed the allele distribution of a polymorphic pentanucleotide repeat within the 5' upstream promoter region of the NOS2A gene in samples of diabetic patients. In diabetic patients from Northern Ireland, the 14-repeat allele of the NOS2A marker was significantly associated with the absence of diabetic retinopathy. Carriers of this repeat had 0.21-fold the relative risk of developing diabetic retinopathy than noncarriers of this allele. They also had significantly fewer renal and cardiovascular complications. The ability of differing numbers of (CCTTT)(n) pentanucleotide repeats to induce transcription of the NOS2A gene was analyzed using a luciferase reporter gene assay in transfected colonic carcinoma cells. Interleukin 1beta (IL-1beta) induction was most effective in constructs carrying the 14-repeat allele. When cells were incubated in 25 mM glucose to mimic the diabetic state, IL-1beta induction was inhibited in all cases, but to a significantly lesser extent with the 14-repeat allele. These unique properties of the 14-repeat allele may confer selective advantages in diabetic individuals, which may delay or prevent microvascular complications of diabetes.

BACKGROUND

Air pollutants have been associated with childhood asthma and wheeze. Epigenetic regulation of nitric oxide synthase--the gene responsible for nitric oxide production--may be affected by air pollutants and contribute to the pathogenesis of asthma and wheeze.

OBJECTIVE

Our goal was to investigate the association between air pollutants, DNA methylation, and respiratory outcomes in children.

METHODS

Given residential address and buccal sample collection date, we estimated 7-day, 1-month, 6-month, and 1-year cumulative average PM₂.₅ and PM₁₀ (particulate matter ≤ 2.5 and ≤ 10 µm aerodynamic diameter, respectively) exposures for 940 participants in the Children's Health Study. Methylation of 12 CpG sites in three NOS (nitric oxide synthase) genes was measured using a bisulfite-polymerase chain reaction Pyrosequencing assay. Beta regression models were used to estimate associations between air pollutants, percent DNA methylation, and respiratory outcomes.

RESULTS

A 5-µg/m³ increase in PM₂.₅ was associated with a 0.20% [95% confidence interval (CI): -0.32, -0.07] to 1.0% (95% CI: -1.61, -0.56) lower DNA methylation at NOS2A position 1, 0.06% (95% CI: -0.18, 0.06) to 0.58% (95% CI: -1.13, -0.02) lower methylation at position 2, and 0.34% (95% CI: -0.57, -0.11) to 0.89% (95% CI: -1.57, -0.21) lower methylation at position 3, depending on the length of exposure and CpG locus. One-year PM2.5 exposure was associated with 0.33% (95% CI: 0.01, 0.65) higher in average DNA methylation of 4 loci in the NOS2A CpG island. A 5-µg/m³ increase in 7-day and 1-year PM₂.₅ was associated with 0.6% (95% CI: 0.13, 0.99) and 2.8% (95% CI: 1.77, 3.75) higher NOS3 DNA methylation. No associations were observed for NOS1. PM₁₀ showed similar but weaker associations with DNA methylation in these genes.

CONCLUSIONS

PM₂.₅ exposure was associated with percent DNA methylation of several CpG loci in NOS genes, suggesting an epigenetic mechanism through which these pollutants may alter production of nitric oxide.

In the 1970s and 1980s, analysis of recombinant inbred, congenic and recombinant haplotype mouse strains permitted us to effectively 'scan' the murine genome for genes controlling resistance and susceptibility to leishmanial infections. Five major regions of the genome were implicated in the control of infections caused by different Leishmania species which, because they show conserved synteny with regions of the human genome, immediately provides candidate gene regions for human disease susceptibility genes. A common intramacrophage niche for leishmanial and mycobacterial pathogens, and a similar spectrum of immune response and disease phenotypes, also led to the prediction that the same genes/candidate gene regions might be responsible for genetic susceptibility to mycobacterial infections such as leprosy and tuberculosis. Indeed, one of the murine genes (Nramp1) was identified for its role in controlling a range of intramacrophage pathogens including leishmania, salmonella and mycobacterium infections. In recent studies, multicase family data on visceral leishmaniasis and the mycobacterial diseases, tuberculosis and leprosy, have been collected from north-eastern Brazil and analysed to determine the role of these candidate genes/regions in determining disease susceptibility. Complex segregation analysis provides evidence for one or two major genes controlling susceptibility to tuberculosis in this population. Family-based linkage analyses (combined segregation and linkage analysis; sib-pair analysis), which have the power to detect linkage between marker loci in candidate gene regions and the putative disease susceptibility genes over 10-20 centimorgans, and transmission disequilibrium testing, which detects allelic associations over 1 centimorgan (ca. 1 megabase), have been used to examine the role of four regions in determining disease susceptibility and/or immune response phenotype. Our results demonstrate: (i) the major histocompatibility complex (MHC: H-2 in mouse, HLA in man: mouse chromosome 17/human 6p; candidates class II and class III including TNF alpha/beta genes) shows both linkage to, and allelic association with, leprosy per se, but is only weakly associated with visceral leishmaniasis and shows neither linkage to nor allelic association with tuberculosis; (ii) no evidence for linkage between NRAMP1, the positionally cloned candidate for the murine macrophage resistance gene Ity/Lsh/Bcg (mouse chromosome 1/human 2q35), and susceptibility to tuberculosis or visceral leishmaniasis could be demonstrated in this Brazilian population; (iii) the region of human chromosome 17q (candidates NOS2A, SCYA2-5) homologous with distal mouse chromosome 11, originally identified as carrying the Scl1 gene controlling healing versus nonhealing responses to Leishmania major, is linked to tuberculosis susceptibility; and (iv) the 'T helper 2' cytokine gene cluster (proximal murine chromosome 11/human 5q; candidates IL4, IL5, IL9, IRF1, CD14) controlling later phases of murine L. major infection, is not linked to human disease susceptibility for any of the three infections, but shows linkage to and highly significant allelic association with ability to mount an immune response to mycobacterial antigens. These studies demonstrate that the 'mouse-to-man' strategy, refined by our knowledge of the human immune response to infection, can lead to the identification of important candidate gene regions in man.

Here, we report on the discovery of a locus in the human genome, which evolved by gene duplication followed by an internal DNA inversion. This locus exhibits high sequence similarity to the gene for the inducible isoform of NOS protein (NOS2A) and is transcribed into a noncoding RNA containing a region of significant antisense homology with the NOS2A mRNA. We show that this antisense transcript (anti-NOS2A RNA) is expressed in different types of brain tumors, including meningiomas and glioblastomas. More importantly, we demonstrate that the expression profiles of the anti-NOS2A RNA and the NOS2A mRNA exhibit concurrent reciprocal changes in undifferentiated human embryonic stem cells (hESCs) and in hESCs induced to differentiate into neurogenic precursors such as neurospheres. As NOS2A has a role in neurogenesis, our results suggest that the anti-NOS2A RNA is involved in the regulation of neuronal differentiation of hESCs through the modulation of NOS2A gene expression.