BACKGROUND

Various cytokines and inflammatory mediators are known to be involved in the pathogenesis of rheumatoid arthritis (RA). We hypothesized that polymorphisms in selected inflammatory response and tissue repair genes contribute to the susceptibility to and severity of RA.

METHODS

Polymorphisms in TNFA, IL1B, IL4, IL6, IL8, IL10, PAI1, NOS2a, C1INH, PARP, TLR2 and TLR4 were genotyped in 376 Caucasian RA patients and 463 healthy Caucasian controls using single base extension. Genotype distributions in patients were compared with those in controls. In addition, the association of polymorphisms with the need for anti-TNF-α treatment as a marker of RA severity was assessed.

RESULTS

The IL8 781 CC genotype was associated with early onset of disease. The TNFA -238 G/A polymorphism was differentially distributed between RA patients and controls, but only when not corrected for age and gender. None of the polymorphisms was associated with disease severity.

CONCLUSIONS

We here report an association between IL8 781 C/T polymorphism and age of onset of RA. Our findings indicate that there might be a role for variations in genes involved in the immune response and in tissue repair in RA pathogenesis. Nevertheless, additional larger genomic and functional studies are required to further define their role in RA.

The density of genetic markers required for successful association mapping of complex diseases depends on linkage disequilibrium (LD) between non-functional markers and functional variants. The haplotypic relationship between stable markers and potentially unstable but highly informative markers (e.g. microsatellites) indicates that LD might be maintained over considerable genetic distance in non-African populations, supporting the use of such 'mixed marker haplotypes' in LD-based mapping, and allowing inferences to be drawn about human origins. We investigated sequence variation in the proximal 2.6 kb of the inducible nitric oxide synthase (NOS2A) promoter and the relationship between SNP haplotypes and a pentanucleotide microsatellite (the 'NOS2A(-2.6) microsatellite') in Gambians and UK Caucasians. UK Caucasians exhibited a subset of sequence diversity observed in Gambians, sharing four of 11 SNPs and a similar haplotypic structure. Five SNPs were found in the sequence of interspersed repetitive DNA elements. In both populations, there was dramatic loss of LD between SNP haplotypes and microsatellite alleles across a very short physical distance, suggesting a high intrinsic mutation rate of the NOS2A(-2.6) microsatellite, the SNP haplotypes are relatively ancient, or that this was a region of frequent recombination. Understanding locus- and population-specific LD is essential when designing and interpreting genetic association studies.

BACKGROUND

Allele frequencies reported from public databases or articles are mostly based on small sample sizes. Differences in genotype frequencies by age, race and sex have implications for studies designed to examine genetic susceptibility to disease. In a community-based cohort of 9,960 individuals, we compared the allele frequencies of 49 single nucleotide polymorphisms (SNPs) of genes involved in inflammatory pathways to the frequencies reported on public databases, and examined the genotypes frequencies by age and sex. The genes in which SNPs were analyzed include CCR2, CCR5, COX1, COX2, CRP, CSF1, CSF2, IFNG, IL1A, IL1B, IL2, IL4, IL6, IL8, IL10, IL13, IL18, LTA, MPO, NOS2A, NOS3, PPARD, PPARG, PPARGC1 and TNF.

RESULTS

Mean(SD) age was 53.2(15.5); 98% were Caucasians and 62% were women. Only 1 out of 33 SNPs differed from the SNP500Cancer database in allele frequency by >10% in Caucasians (n = 9,831), whereas 12 SNPs differed by >10% (up to 50%) in African Americans (n = 105). Two out of 15 SNPs differed from the dbSNP database in allele frequencies by >10% in Caucasians, and 5 out of 15 SNPs differed by >10% in African Americans. Age was similar across most genotype groups. Genotype frequencies did not differ by sex except for TNF(rs1799724), IL2(rs2069762), IL10(rs1800890), PPARG(rs1801282), and CRP(rs1800947) with differences of less than 4%.

CONCLUSIONS

When estimating the size of samples needed for a study, particularly if a reference sample is used, one should take into consideration the size and ethnicity of the reference sample. Larger sample size is needed for public databases that report allele frequencies in non-Caucasian populations.

Norfolk Island is a human genetic isolate, possessing unique population characteristics that could be utilized for complex disease gene localization. Our intention was to evaluate the extent and strength of linkage disequilibrium (LD) in the Norfolk isolate by investigating markers within Xq13.3 and the NOS2A gene encoding the inducible nitric oxide synthase. A total of six microsatellite markers spanning approximately 11 Mb were assessed on chromosome Xq13.3 in a group of 56 men from Norfolk Island. Additionally, three single nucleotide polymorphisms (SNPs) localizing to the NOS2A gene were analyzed in a subset of the complex Norfolk pedigree. With the exception of two of the marker pairs, one of which is the most distantly spaced marker, all the Xq13.3 marker pairs were found to be in significant LD indicating that LD extends up to 9.5-11.5 Mb in the Norfolk Island population. Also, all SNPs studied showed significant LD in both Norfolk Islanders and Australian Caucasians, with two of the marker pairs in complete LD in the Norfolk population only. The Norfolk Island study population possesses a unique set of characteristics including founder effect, geographical isolation, exhaustive genealogical information and phenotypic data of use to cardiovascular disease risk traits. With LD extending up to 9.5-11 Mb, the Norfolk isolate should be a powerful resource for the localization of complex disease genes.

The great amount of nitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS) exerts deleterious effects, and iNOS expression is raised in the colonic mucosa of inflammatory bowel disease (IBD) patients. This is the first association analysis of polymorphisms within the NOS2A extended gene with IBD susceptibility. We analyzed 336 patients of Crohn's disease (CD), 355 of ulcerative colitis (UC), and 536 healthy controls from a Spanish population. We tested a (CCTTT)n microsatellite, a (-/TAAA) insertion, and two single nucleotide polymorphisms (SNPs) flanking them (rs2779251 and rs2779248) in the NOS2A promoter, together with two SNPs in the coding region: one within exon 10, D385D (rs1137933), and another mapping to exon 16, S608L (rs2297518). Analysis of these markers evidenced differences among IBD patients and healthy controls. Allele (CCTTT) 13 is related to higher UC risk (p = 0.001; odds ratio [OR] [95% confidence interval, CI] = 1.64 [1.20-2.23]). Carriers of minor alleles of the two promoter SNPs analyzed showed an association with UC predisposition, and common allele homozygotes of the two exonic SNPs were more frequent among CD patients than among controls. Concordantly, one out of the three haplotypes carrying both exonic risk alleles was found to increase CD susceptibility (p = 0.007; OR [95%CI] = 1.74 [1.13-2.67]). Therefore, the NOS2A gene seems to be involved in IBD aetiology.

Resolution of inflammation is an active process involving a novel category of lipid factors known as specialized pro-resolving lipid mediators, which includes Resolvin D1 (RvD1). While accumulating evidence suggests that RvD1 counteracts proinflammatory signaling and promotes resolution, the specific cellular targets and mechanisms of action of RvD1 remain largely unknown. In the present study, we investigated the role and molecular mechanisms of RvD1 in ischemia/reperfusion (IR)-induced sterile liver inflammation. Male C57BL/6 mice underwent 70% hepatic ischemia for 60min, followed by reperfusion. RvD1 (5, 10, and 15μg/kg, i.p.) was administered to the mice 1h before ischemia and then immediately prior to reperfusion. RvD1 attenuated IR-induced hepatocellular damage and the proinflammatory response. In purified Kupffer cells (KCs) from mice exposed to IR, the levels of M1 marker genes (Nos2a and Cd40) increased, while those of M2 marker genes (Arg1, Cd206, and Mst1r) decreased, demonstrating a proinflammatory shift. RvD1 markedly attenuated these changes. Depletion of KCs by liposome clodronate abrogated the effects of RvD1 on proinflammatory mediators and macrophage polarization. In addition, RvD1 attenuated increases in myeloperoxidase activity and Cxcl1 and Cxcl2 mRNA expression. RvD1 markedly augmented the efferocytic activity of KCs, as indicated by increases in F4/80(+)Gr-1(+) cells in the liver. However, antagonist pretreatment or gene silencing of the RvD1 receptor, ALX/FPR2, abrogated the anti-inflammatory and pro-resolving actions of RvD1. These data indicate that RvD1 ameliorates IR-induced liver injury, and this protection is associated with enhancement of M2 polarization and efferocytosis via ALX/FPR2 activation.

Knowledge of genetic polymorphisms in gene-environment studies may contribute to more accurate identification of avoidable risks and to developing tailor-made preventative measures. The aim of this study was to describe the allele frequencies of single nucleotide polymorphisms (SNPs) of select genes, which may be included in future gene-environment studies on cancer in Japan. SNP typing was performed on middle-aged Japanese men randomly selected from the general population in five areas of Japan. We genotyped and calculated allele frequencies of 153 SNPs located on 40 genes: CYP1A1, CYP1B1, CYP2C9, CYP2C19, CYP2E1, CYP17A1, CYP19A1, AHR, ESR1, ESR2, ERRRG, PGR, EPHX1, EPHX2, HSD17B2, HSD17B3, GSTM2, GSTM3, GSTT2, GSTP1, NAT1, NAT2, COMT, ADH1A, ADH1B, ADH1C, ALDH2, NOS2A, NOS3, IL1A, IL1B, OGG1, NUDT1 [MTH1], DRD2, DRD3, DRD4, SLC6A4, NR3C1 [GCCR], MTHFR, and NQO1. In the present study, the Japanese allele frequencies were verified by using nationwide population samples.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of autoantibodies against intracellular components, the formation of immune complexes, and inflammation in various organs, typically the skin and kidney glomeruli. The etiology of the disease is not well understood but is most likely the result of the interaction between genetic and environmental factors. In order to identify susceptibility loci for SLE, we have performed genome scans with microsatellite markers covering the whole genome in families from Argentina, Italy, and Europe. The results reveal a heterogeneous disease with different susceptibility loci in different family sets. We have found significant linkage to chromosome 17p12-q11 in the Argentine set of families. The maximum LOD score was given by marker D17S1294 in combination with D17S1293, when assuming a dominant inheritance model (Z = 3.88). We also analyzed a repeat in the promoter region of the NOS2A gene, a strong candidate gene in the region, but no association was found. The locus on chromosome 17 has previously been identified in genetic studies of multiple sclerosis families. Several other interesting regions were found at 1p35, 1q31, 3q26, 5p15, 11q23 and 19q13, confirming previously identified loci for SLE or other autoimmune diseases.

The aim of this study was to assess whether prematurely born infants have a genetic predisposition to respiratory syncytial virus (RSV) infection-related respiratory morbidity. One hundred and forty-six infants born at less than 36 weeks of gestation were prospectively followed. Nasopharygeal aspirates were obtained on every occasion the infants had a lower respiratory tract infection (LRTI) regardless of need for admission. DNA was tested for 11 single-nucleotide polymorphisms (SNPs). Chronic respiratory morbidity was assessed using respiratory health-related questionnaires, parent-completed diary cards at a corrected age of 1 year and review of hospital notes. Lung function was measured at a post menstrual age (PMA) of 36 weeks and corrected age of 1 year. A SNP in ADAM33 was associated with an increased risk of developing RSV LRTIs, but not with significant differences in 36-week PMA lung function results. SNPs in several genes were associated with increased chronic respiratory morbidity (interleukin 10 (IL10), nitric oxide synthase 2A (NOS2A), surfactant protein C (SFTPC), matrix metalloproteinase 16 (MMP16) and vitamin D receptor (VDR)) and reduced lung function at 1 year (MMP16, NOS2A, SFTPC and VDR) in infants who had had RSV LRTIs.

CONCLUSIONS

Our results suggest that prematurely born infants may have a genetic predisposition to RSV LRTIs and subsequent respiratory morbidity which is independent of premorbid lung function.

A locus for essential hypertension has been found recently on chromosome 17 in the general vicinity of the inducible nitric oxide synthase (iNOS) gene (NOS2A at 17cen-q11.2). We therefore tested NOS2A markers for association and linkage with hypertension in affected Australian Anglo-Caucasians. Patients for the association study (n=112) were from our cohort of hypertensives (systolic/diastolic=175+/-25 SD/112+/-19 mm Hg) who were the offspring of 2 hypertensive parents; control subjects (n=164) were normotensives whose parents were both normotensive. The linkage study involved 156 hypertensive sib-pairs. Genotypes for an 8-allele pentameric repeat located 2.6 kb upstream of NOS2A and of a biallelic tetranucleotide repeat 0.7 kb upstream were determined by polymerase chain reaction and automated gene scan analysis. In the association study, the frequency of the minor allele of the biallelic marker was 0.18 in the hypertensives and 0.14 in the normotensives (chi21 df=1.1, P=0.3). Allele frequencies for the multiallelic marker were also similar in each group (chi2 7 df=9.8, P=0.2). Furthermore, no genotypic differences in blood pressure were apparent. In the sib-pair study, SPLINK APM, and MAPMAKERS/SIBS did not indicate excess allele sharing. We also examined genotype as a function of age. In the younger (< 60 years) hypertensives as well as younger or older normotensives, genotype and allele frequency of the biallelic marker was similar (0.12 to 0.14). However, in hypertensives>>/=60 years of age, frequency of the minor allele was 0.28 (chi2=7.4, P=0.006). Homozygotes for this allele were rare. Frequency of heterozygotes was 0.19 for normotensives but 0.39 for the older hypertensives (chi2=8.0, P=0.018) and was 0.40 for hypertensive sibs>>/=60 years of age with a diastolic pressure>>/=100 mm Hg. Furthermore, homozygotes for the major allele were 7 years younger than heterozygotes (P=0.05 by ANOVA). In conclusion, the present study shows (1) no evidence for a role of NOS2A in hypertension and (2) a genotypic difference in frequency of a NOS2A promoter variant in older hypertensives, seen in 2 different cohorts. A possible interpretation of the latter observation is that NOS2A genotype could affect longevity, at least in patients at high risk by having moderate to severe hypertension.

Tuberculosis (TB) treatment can cause serious sequelae including adverse effects such as anti-TB drug-induced hepatotoxicity (ATDH). We performed a candidate gene-based association study between single nucleotide polymorphisms (SNPs) in 10 genes in the antioxidant pathway and ATDH susceptibility. The subjects comprised 100 Japanese patients with pulmonary TB who received a treatment regimen including isoniazid and rifampicin. Out of them, 18 patients had ATDH. Thirty-four tag SNPs in 10 genes were analyzed by PCR-restriction fragment length polymorphism or PCR-direct DNA sequencing. The frequencies of alleles and genotypes between patients with and without ATDH were compared in three different genetic models. Statistical analyses revealed that a C/C genotype at rs11080344 in NOS2A, a C/C genotype at rs2070401 in BACH1, and a G/A or A/A genotype at rs4720833 in MAFK independently conferred ATDH susceptibility. Remarkably, the association of the latter two tag SNPs with ATDH susceptibility was highly statistically significant (P = 0.0006) with an odds ratio of 9.730. This study is the first report to demonstrate that NOS2A, BACH1, and MAFK appear to be genetic determinants of ATDH in Japanese patients with TB. Furthermore, a combination of BACH1 and MAFK polymorphisms may be useful as new biomarkers to identify high-risk Japanese TB patients for ATDH.

Nitric oxide synthase (NOS) genes are candidates for Parkinson's disease (PD) because NOS enzymes produce nitric oxide (NO), a pro-oxidant that can damage neurons. Widely used organophosphate (OP) pesticides can induce oxidative stress and are reported to increase PD risk. Additionally, two single nucleotide polymorphisms (SNPs) from the PON1 (paraoxonase 1) gene influence the ability to metabolize OPs.

Here, we investigated contributions of NOS genes and OP pesticides to PD risk, controlling for PON1 status.

In 357 incident PD cases and 495 population controls, we investigated eight NOS SNPs and interactions with both household and ambient agricultural OP exposures assessed with geographic information system (GIS).

In comparing PD in homozygous variant carriers of NOS2A rs1060826 versus homozygous wild-type or heterozygotes, we estimate an adjusted odds ratio (OR) of 1.51 (95% CI: 0.95, 2.41). When considering interactions between NOS1 rs2682826 and OP exposure from household use, the OR for frequent OP use alone was 1.30 (95% CI: 0.72, 2.34) and for the CT+TT genotype alone was 0.89 (95% CI: 0.58, 1.39), and for frequent OP use combined with the CT+TT genotype the OR was 2.84 (95% CI: 1.49, 5.40) (interaction p-value 0.04). Similar results were seen for ambient OP exposure. Interactions between OP exposure and three other NOS1 SNPs and a genetic risk score combining all NOS1 SNPs reached statistical significance.

We found that OP pesticides were more strongly associated with PD among participants with variant genotypes in NOS1, consistent with the importance of oxidative stress-inducing mechanisms. Our data provide evidence for NOS1 modifying PD risk in OP exposed populations.

Paul KC, Sinsheimer JS, Rhodes SL, Cockburn M, Bronstein J, Ritz B. 2016. Organophosphate pesticide exposures, nitric oxide synthase gene variants, and gene-pesticide interactions in a case-control study of Parkinson's disease, California (USA). Environ Health Perspect 124:570-577; http://dx.doi.org/10.1289/ehp.1408976.

Airway inflammation and pulmonary disease are heterogeneous phenotypes in cystic fibrosis (CF) patients, even among patients with the same cystic fibrosis transmembrane conductance regulator (CFTR) genotype. Endothelin, a proinflammatory peptide and smooth muscle agonist, is increased in CF airways, potentially contributing to the pulmonary phenotype. Four cohorts of CF patients were screened for variants in endothelin pathway genes to determine whether any of these variants associated with pulmonary function. An initial cohort of 808 CF patients homozygous for the common CF mutation, DeltaF508, showed significant association for polymorphisms in the endothelin receptor A gene, EDNRA (P = 0.04), but not in the related endothelin genes (EDN1, EDN2, EDN3, or EDNRB) or NOS1, NOS2A, or NOS3. Variants within EDNRA were examined in three additional cohorts of CF patients, 238 patients from Seattle, WA, 303 from Ireland and the U.K., and 228 from Cleveland, OH, for a total of 1,577 CF patients. The three additional groups each demonstrated a significant association between EDNRA 3'-untranslated region (UTR) variant rs5335 and pulmonary function (P = 0.002). At the molecular level, single nucleotide primer extension assays suggest that the effect of the variants is quantitative. EDNRA mRNA levels from cultured primary tracheal smooth muscle cells are greater for the allele that appears to be deleterious to lung function than for the protective allele, suggesting a mechanism by which increased receptor function is harmful to the CF airway. Finally, cell proliferation studies using human airway smooth muscle cells demonstrated that cells homozygous for the deleterious allele proliferate at a faster rate than those homozygous for the protective allele.

Chronic inflammation induced by Helicobacter pylori is a key process in gastric carcinogenesis. We hypothesized that genetic polymorphisms in important mediators of H. pylori-induced inflammation may influence the risk of developing various grades of precancerous lesions. We studied the associations between single nucleotide polymorphisms (SNPs) in cyclooxygenase 1 and 2 (PTGS1 and PTGS2), inducible nitric oxide synthase (NOS2A), interferon gamma (IFNG) and its receptor (IFNGR1), and risk of gastric precancerous lesions in a Venezuelan population characterized by high rates of H. pylori infection. We found no association of precancerous lesions with SNPs in PTGS1 and in IFNG. A nonsynonymous SNP of NOS2A (Ser608Leu) and an SNP located in the promoter of IFNGR1 (C-56T) were associated with higher risk of atrophic gastritis [odds ratio (OR)=1.37, 95% confidence interval (CI)=1.01-1.86, and OR=1.49, 95% CI=1.01-2.19, respectively]. Two SNPs of PTGS2 were associated with risk of dysplasia (OR=1.60, 95% CI=1.01-2.54, and OR=0.66, 95% CI=0.43-0.99). We conclude that genetic variability in the genes we studied does not play a major role in the early stages of gastric carcinogenesis.

Macrophages infiltrate peripheral nerves and may contribute to neural damage in the Guillain-Barré syndrome (GBS). We determined whether single nucleotide polymorphisms (SNP) in genes encoding macrophage-mediators are related to the susceptibility and severity of GBS. The frequencies of SNP in the TNFA, MMP9, IL10, and NOS2a genes did not differ between 263 GBS patients and 210 healthy subjects. The MMP9 C(-1562)T and TNFA C(-863)A SNP were associated with severe weakness and poor outcome, indicating that these SNP may be one of the factors predisposing to a severe form of GBS.

Nitric oxide synthase (NOS) produces nitric oxide (NO) from arginine. Three NOS isoforms have been identified in mammals, namely a neuronal (NOS1), an inducible (NOS2) and an endothelial (NOS3) enzyme. In zebrafish genome, one nos1 gene and two nos2 genes (nos2a and nos2b) were observed. We cloned zebrafish nos2a cDNA and compared nos2a and nos2b sequences, expression and inducibility. When analyzed by reverse transcription-PCR, the expression of nos2a remained very low during initial development, then increased at 96 hpf, while nos2b was expressed from 6 hpf and subsequently remained stable. Expression of nos2a is detected in the head, eye and gut regions by WISH experiments performed at 48, 72 and 96 hpf larvae. In adults, nos2a expression varies from one tissue to another whereas nos2b is expressed in all studied tissues. Both nos2 isoforms can be induced by pro-inflammatory or mechanical stresses (tissue injury). In vitro as in vivo stimulations with Poly I:C and lipopolysaccharides (LPS) enhanced more dramatically nos2a than nos2b expression. After tail transection in 4 dpf larvae a strong increase of nos2a and nos2b expression was evidenced in the regeneration site, skin cells and for the nos2b gene in neuromasts. Phylogenetic and syntenic analyses show that nos2b gene was associated with syntenic genes identified for nos2 genes in vertebrate. This is not the case for the nos2a gene, despite zebrafish nos2a presenting the inducible property of a classical vertebrate nos2 isoform. A myristoylation consensus site was detected at the N-terminal extremity of zebrafish Nos2b, a property shared with mammal NOS3 isoforms. Thus, the evolution of nos2 genes in zebrafish provides a typical example of gene divergence after duplication.

Allelic frequencies of a (CCTTT)(n) pentanucleotide repeat in the NOS2A promoter region were determined in a total of 1393 unrelated individuals from five specific population groups in four continents: Africa, Europe, Asia, and the Caribbean. There were highly significant differences in allele frequencies between the ethnically diverse populations. The repeat variation may have implications for the selective pressure of malaria or other infectious diseases that may operate at the NOS2 locus.

Approx. 4% of patients experiencing chronic infection of human HCV (hepatitis C virus) ultimately develop HCC (hepatocellular carcinoma). The NS5A (non-structural protein 5A) encoded by HCV has been reported to have an oncogenic role during HCV infection, but the precise mechanism remains largely unclear. The aim of this study is to investigate the signal transduction pathways that mediate the role of NS5A in hepatocarcinogenesis. HepG2 cells were transfected with a plasmid expressing HCV NS5A protein. Subsequently, cell proliferation was analysed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and cell counting, apoptosis was analysed by Hoechst 33342 staining, and the gene expression profile was identified by microarray and subsequently validated by RT-PCR (reverse transcription-PCR). The protein levels of survivin, p53, NOS2A (nitric oxide synthase 2A), cyclin D1 and NF-κB (nuclear factor κB) were monitored by Western blotting. Our results showed that transfection of HCV NS5A expression plasmid significantly down-regulated the expression of nine genes and up-regulated the expression of ten genes among the 104 genes detectable by the microarray associated with signalling transduction. The increased expression of survivin mRNA and protein, down-regulated p53 protein levels and increased NOS2A, cyclin D1 and NF-κB protein levels were further identified. Our results suggested that HCV NS5A protein can enhance survivin transcription by increasing p53 degradation and stimulating NOS2A expression as well as NF-κB relocation to the nucleus. The functions of survivin in anti-apoptosis and regulation of cell division might mediate the role of NS5A in HCV-induced HCC.

BACKGROUND

Nitric oxide (NO) plays an important role in cardiovascular health by maintaining and regulating vascular tone and blood flow. Epigenetic regulation of NO synthase (NOS), the genes responsible for NO production, may affect cardiovascular disease, including the development of atherosclerosis in children.

RESULTS

We measured percentage DNA methylation using bisulfite conversion and pyrosequencing assays on DNA from buccal cells provided by 377 participants of the Children's Health Study on whom carotid artery intima-media thickness (CIMT) measurements were also collected. We examined a total of 16 CpG loci located within NOS1, NOS2A, NOS3, ARG1, and ARG2 genes responsible for NO production. CIMT was measured using high-resolution B-mode carotid ultrasound. The association between percentage DNA methylation in ARG and NOS genes with CIMT was evaluated using linear regression adjusted for sex, ethnicity, body mass index, age at CIMT, town of residence, and experimental plate for pyrosequencing reactions. Differences in the association by ethnicity and ancestral group were also evaluated. For a 1% increase in average DNA methylation of NOS1, CIMT increased by 1.2 μm (P=0.02). This association was greater in Hispanic children of Native American descent (β=2.3; P=0.004) than in non-Hispanic whites (β=0.3; P=0.71) or Hispanic whites (β=1.0; P=0.35).

CONCLUSIONS

DNA methylation of NOS1 has a plausible role in atherogenesis through regulation of NO production, although ancestry may alter the magnitude of this association.

The aetiology of cluster headache is still not yet completely understood, but the potential relevance of genetic factors has been recognized during recent years. Nitric oxide (NO) plays a critical role in the regulation of vasodilation, neurotransmission, inflammation and many other events throughout the body. NO also appears to be an important mediator of vascular headache pathophysiology. In this study we have performed an association analysis of five polymorphic microsatellite markers in the three different NO synthase (NOS) genes; nNOS (NOS1), iNOS (NOS2A) and eNOS (NOS3). Ninety-one cluster headache patients diagnosed according to International Headache Society criteria and 111 matched controls were studied. Phenotype and allele frequencies were similarly distributed in patients and controls except for an iNOS (NOS2A) pentanucleotide repeat allele which was significantly more common in controls. We observed a higher phenotype frequency of this allele in our control group compared with rates in control groups of other studies, whereas the frequency in our patients was similar to that in controls from previous reports. Thus, we conclude that it is unlikely that genetic variations within the NOS genes contribute greatly to cluster headache susceptibility.

OBJECTIVE

To assess the contribution of 2 polymorphisms within the inducible nitric oxide (NOS2A) promoter region to the susceptibility to Henoch-Schönlein purpura (HSP), and to determine if implications exist with severe systemic complications of HSP, in particular with severe renal involvement and permanent renal dysfunction (renal sequelae).

METHODS

Fifty-eight patients from Northwest Spain with primary cutaneous vasculitis classified as HSP were studied. All patients were required to have had at least 2 years' followup. Patients and ethnically matched controls (n=251) were genotyped by PCR based techniques for a multiallelic (CCTTT)n and for the biallelic TAAA repeat in the promoter region of the NOS2A gene.

RESULTS

HSP patients exhibited a significantly increased frequency of the NOS2A short (8-11) CCTTTn alleles (OR 1.64, 95% CI 1.09-2.47, p=0.017) and genotypes (OR 3.59, 95% CI 1.79-7.20, p=0.0002) compared to controls, particularly when patients with nephritis were compared with controls. However, when the NOS2A TAAA repeat polymorphism was assessed, no differences were found.

CONCLUSIONS

Significant differences in the NOS2A promoter polymorphism allele and genotype frequency between HSP patients and controls suggest a potential role for this gene in the susceptibility to HSP and in the development of nephritis.

Complications of diabetes have a genetic influence. Since increased inducible nitric oxide synthase (iNOS) gene ( NOS2A) expression can contribute to tissue damage, NOS2A is a worthy candidate for such a role. We therefore tested a 4-bp insertion/deletion (+/-) polymorphism 0.7 kb upstream of NOS2A for association with complications in type 2 diabetes patients, and also performed transient transfection experiments to examine the effect of this variant on promoter activity in kidney cells in culture. We investigated 379 Caucasian type 2 diabetes patients of British/European descent, 93 of whom had microalbuminuria, 26 overt nephropathy, 46 retinopathy, and 73 clinical neuropathy. Genotyping for the variant was carried out by PCR and automated Genescan analysis. Transient transfection studies involved the renal HEK 293 cell line and luciferase reporter gene constructs containing 1.1 kb of 5'-flanking DNA from '+' or '-' allele homozygotes. We found that the '+' allele frequency in patients without microalbuminuria was 12%, but was 23% in those with microalbuminuria ( P=0.0005), and was 26% in those with nephropathy ( P=0.0007), 22% in those with retinopathy ( P=0.037), and 23% in those with neuropathy ( P=0.045). The odds ratios for homozygote +/+ to have microalbuminuria or nephropathy were 2.4 (95% CI 1.4-4.2, P=0.0023) and 5.4 (95% CI 1.8-16, P=0.0009), respectively. Luciferase reporter gene constructs containing 1 kb of NOS2A promoter DNA for each allele were made and sequence analysis confirmed that the +/- variation was the only sequence difference present. Transient transfection of these into HEK 293 cells revealed 25 times higher reporter gene activity for the '+' allele compared with the '-' allele. Gel shift analysis with 30mer oligonucleotides corresponding to each allele showed specific binding to nuclear extracts, being greater for the '+' allele. Thus the '+' allele of the NOS2A promoter variant may confer higher iNOS expression, and could contribute to complications of type 2 diabetes, especially in the approximately 5% of patients homozygous for this variant.

BACKGROUND

Surgery for trachomatous trichiasis (TT) is a key component of the SAFE Strategy for trachoma control. Unfortunately, recurrent TT following surgery is common, probably due to various surgical and disease factors. To develop strategies to reduce recurrence rates it is necessary to understand its pathological basis. In this study we investigated the relationship between recurrent trichiasis and the expression of various cytokines and fibrogenic genes during a two-year follow-up period.

RESULTS

Individuals undergoing surgery for TT were examined at baseline (pre-operative), 6, 12, 18 and 24 months. Conjunctival swab samples were collected from the tarsal conjunctiva for RNA isolation on each occasion. Individuals who developed recurrent TT with at least 3 lashes touching the eye on one or more occasion were designated "cases" and an equal number of "controls" were randomly selected from those without recurrent TT, frequency matched for age and baseline TT severity. The expression of the following genes was measured by quantitative RT-PCR: S100A7, IL1B, CXCL5, TNFA, NOS2A, CTGF, MMP7, MMP9 and MMP12. Thirteen hundred individuals were enrolled and underwent surgery. By two years 122 had developed recurrent TT with at least 3 lashes touching the eye. Recurrent TT was consistently associated across multiple time points with about a 2-fold increase in S100A7 expression (p = 0.008). Clinically visible conjunctival inflammation was associated with increased S100A7, IL1B, CXCL5, MMP9 and MMP12 expression.

CONCLUSIONS

Increased S100A7 expression was associated with trachomatous conjunctival scarring and may be linked to the pathophysiology of recurrent TT. S100A7 expression could be a potential biomarker for this disease process. As part of the epithelial innate immune response S100A7 has multiple actions, potentially contributing to a chronic pro-inflammatory response, which may lead to ongoing tissue damage and increased scarring.

Basic information on the association between lifestyle factors and candidate genes is valuable for genetic-environmental study. We screened the association of habitual smoking or drinking with polymorphism in 40 candidate genes for a total of 153 single nucleotide polymorphisms (SNPs) using a sample of 339 middle-aged, randomly selected Japanese men. Smoking and drinking statuses were elicited during questionnaire-based interviews. Genes were selected based on their possible involvement in genetic-environmental, life-style interactions and constitute the genes expressing xenobiotic metabolism enzymes, DNA repair enzymes, and other stress-related proteins. The P values of odds ratios to habitual smoking for CYP17A1, ESR1, EPHX1, GSTT2, ALDH2, NOS2A, OGG1, and SLC6A4 and those of odds ratios to habitual drinking for CYP1B1, ESR1, HSD17B3, GSTM3, COMT, ADH1C, ALDH2, NOS3, and NUDT1 were under 0.05. These variables were included in a stepwise logistic analysis in order to develop a predictive model for smoking or drinking behavior. In the final model, the only significant variables selected for smoking were OGG1, SLC6A4, EPHX1, ESR1, and CYP17A1, and for drinking, ALDH2 and NUDT1. The findings of the present study suggest that polymorphism in associated candidate genes plays a role in the habitual use of tobacco and alcohol among Japanese men.