Cyclooxygenase-2 gene polymorphisms in an Australian population: association of the -1195G > A promoter polymorphism with mild asthma.

BACKGROUND: Cyclooxygenase (COX)-2 is an inducible enzyme responsible for catalysing the formation of prostaglandins (PGs) in settings of inflammation. Single nucleotide polymorphisms (SNPs) of the COX-2 gene may influence gene transcription and PG production in the asthmatic airway. OBJECTIVE: To evaluate the frequencies of COX-2 SNPs in an Australian Caucasian population, and determine potential associations between common COX-2 promoter SNPs and asthma, asthma severity and aspirin-intolerant asthma (AIA). METHODS: The frequencies of 25 COX-2 SNPs were determined in a random population (n=176). The SNPs with a minor allele frequency of >10% were then studied in asthmatic (n=663), non-asthmatic controls (n=513) and AIA subjects (n=58). Genotype, allele and haplotype associations were assessed. Functional assessment of SNPs was performed by transfection into HeLa cells measured using the luciferase dual-reporter assay system. RESULTS: Eighteen COX-2 SNPs were not detected, five were rare and two promoter SNPs, -1195G>A (rs689465), and -1290A>G (rs689466), were further studied. The A allele of the -1195 SNP was present at a significantly higher frequency among all asthmatic subjects (P=0.012). Over 60% of the asthmatic individuals were -1195A homozygotes compared with 54.6% of the control subjects (odds ratio, 1.35; 95% CI, 1.06-1.72, P=0.03). After classifying for severity, the mild asthmatics represented 64.6% of -1195AA individuals, the highest of all the asthma groups compared with 54.6% of the control subjects (odds ratio, 1.5; 95% CI, 1.12-2.02, P=0.02). The -1290A/-1195G/-765G haplotype was associated with a reduced incidence of asthma (odds ratio, 0.76; 95% CI, 0.61-0.95, P=0.017). CONCLUSION: The -1195G>A polymorphism appears to be associated with asthma, and in particular with mild asthma.

A novel tissue inhibitor of metalloproteinase-1 (TIMP-1) polymorphism associated with asthma in Australian women.

BACKGROUND: Airway remodelling is a characteristic feature of chronic asthma and there is evidence that an airway imbalance between levels of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1) is associated with airway remodelling. On this basis, we hypothesised that polymorphisms in the MMP-9 and TIMP-1 genes were associated with the disease process. METHODS: A number of MMP-9 and TIMP-1 gene polymorphisms were examined in an adult white Australian population of mild (n = 259), moderate (n = 213) and severe (n = 71) asthmatics and non-asthmatic controls (n = 406) using PCR-RFLP and PCR-SSCP analyses. RESULTS: MMP-9 -1562C>T and 836G>A (Arg279Gln) were not associated with asthma (p> or =0.15) or asthma severity (p> or =0.13), and TIMP-1 434T>C (Phe124Phe) was not associated with asthma in women (p = 0.094) or men (p = 0.207). In this population, MMP-9 -861C>T and TIMP-1 323C>T (Pro87Pro) were not informative (with minor allele frequencies of <1%), and MMP-9 -1702T>A and TIMP-1 595C>T (Ser178Phe) were not detectable. However, a novel polymorphism was detected in the TIMP-1 gene 536C>T (Ile158Ile) which was significantly associated with asthma in women (p = 0.011; OR = 5.54, 95% CI 1.66 to 34.4) but not in men (p = 1.0). 536C>T was found to be in linkage disequilibrium with 434T>C, and haplotype analysis supported an association with asthma (p = 0.014). CONCLUSIONS: This is the first reported association between a polymorphism in the TIMP-1 gene and asthma, and supports the hypothesis that the protease/antiprotease balance has an important role in this common disease.

Cyclooxygenase-1 gene polymorphisms in patients with different asthma phenotypes and atopy.

Cyclooxygenase-1 (COX-1) regulates the biosynthesis of prostaglandins, which are important mediators in asthma. The possible association of COX-1 gene polymorphisms with asthma has not been investigated. The allele frequencies of 20 COX-1 polymorphisms were determined in a random Australian Caucasian population using MassARRAY technology. Informative and potentially functional promoter (c.8592C > T, c.1676C > T) and coding region (c.22C > T, c.50C > T) polymorphisms were investigated in carefully phenotyped patients with mild (n = 316), moderate (n = 241), severe (n = 86) or aspirin-intolerant asthma (AIA) (n = 58), and in nonasthmatic subjects (n = 477). There were no allelic, genotypic or haplotypic associations between these four polymorphisms and asthma or asthma severity. Over-representation of the c.50TT genotype among AIA patients (3.4%) compared with aspirin-tolerant patients (0.8%), and a global haplotype association with AIA did not reach statistical significance. The c.22TT genotype was less frequent among atopic (0.1%) rather than nonatopic individuals (1.2%; odds ratio = 9.05, 95% confidence interval 1.01-81.29). In conclusion, the present investigation of cyclooxygenase-1 polymorphisms in asthma indicates that they do not appear to play a substantial role in genetic pre-disposition for asthma or asthma severity. However, the c.22TT genotype confers a small protective effect against atopy. Potential associations with aspirin-intolerant asthma were identified and warrant further investigation in a larger population of aspirin-intolerant asthma patients.

A polymorphism in the promoter region of the human interleukin-16 gene is not associated with asthma or atopy in an Australian population.

BACKGROUND: IL-16 is an immunomodulatory cytokine whose expression is increased in the bronchial mucosa, bronchoalveolar lavage fluid and induced sputum of asthmatic patients. It has been suggested that IL-16 has a regulatory role in the pathophysiology of asthma. A single-nucleotide polymorphism (T(-295)C) has been described in the promoter region of the gene and it has been hypothesized that this polymorphism may be associated with altered levels of IL-16 expression, and account for the increased levels of IL-16 seen in the asthmatic airway. OBJECTIVE: To determine the association between the T(-295)C promoter polymorphism and asthma, disease severity and atopy in a large Australian Caucasian population. METHODS: We used PCR and restriction fragment length polymorphism analysis to establish the allele frequency of the T(-295)C promoter polymorphism in a random Australian Caucasian population (n=176) and to characterize the polymorphism in a large Australian Caucasian population of mild (n=273), moderate (n=230) and severe (n=77) asthmatic patients, and non-asthmatic controls (n=455). Genotype association analyses were performed using chi(2) tests. RESULTS: The polymorphic C allele was present in 19% of the asthmatic population and 21% of the non-asthmatic population. There was no association between the polymorphism and asthma (P=0.153) nor with asthma severity (P=0.417) or atopy (P=0.157) in this population. CONCLUSION: Although it has been hypothesized that the T(-295)C promoter polymorphism may be associated with increased IL-16 gene expression, it is not associated with asthma, disease severity or atopy in this Australian population.

Polymorphisms in the 5-lipoxygenase activating protein (ALOX5AP) gene are not associated with asthma in an Australian population.

BACKGROUND: The cysteinyl-leukotrienes (cys-LTs) are important pro-inflammatory mediators in asthma, and have been shown to have a role in specific disease subtypes, including asthma severity. Few studies have investigated the role of polymorphisms in the ALOX5AP gene, encoding 5-lipoxygenase activating protein (FLAP), and asthma. We hypothesized that polymorphisms in this gene are associated with asthma and in particular, with asthma severity, in an Australian population. OBJECTIVE: To screen the coding region of the ALOX5AP gene for polymorphisms and to determine the association between previously described polymorphisms and asthma and asthma severity in an Australian population. METHODS: We used PCR-SSCP and PCR-RFLP analysis to examine a previously described promoter polyA variable repeat polymorphism and two intronic polymorphisms (IVS2+12C>A, IVS2+105T>C), and to screen all five exons of the gene for new polymorphisms, in a large Australian population of randomly selected, non-asthmatic controls (n=457), mild asthmatics (n=274), moderate asthmatics (n=231) and severe asthmatics (n=79). RESULTS: We confirmed the presence of two polymorphisms in intron 2 and found no association between these polymorphisms and asthma or asthma severity, nor between a promoter polymorphism in the ALOX5AP gene and asthma or asthma severity. Gene fragment analysis of the promoter polymorphism revealed novel, conserved repeat numbers in our population, and no new polymorphisms were found in the coding region of the gene. CONCLUSION: These findings in a large, well characterized asthma population, reveal that, while FLAP is an important enzyme in cys-LTs biosynthesis, polymorphisms in the ALOX5AP gene are not likely to be functionally associated with the asthma phenotype.

The CD14 C-159T polymorphism is not associated with asthma or asthma severity in an Australian adult population.

BACKGROUND: CD14 functions as a multifunctional receptor for bacterial cell wall components including endotoxin and lipopolysaccharide and is likely to play a role in the polarisation of T lymphocytes into Th1 and Th2 subsets, thereby influencing the cytokine profile and subsequent IgE production in response to antigen/allergen contact in allergic phenotypes. A functional C-159T polymorphism has been described in the promoter region of the gene and has been associated with increased gene expression, atopy, and non-atopic asthma in different ethnic populations. A study was undertaken to examine the association between the C-159T polymorphism and asthma, asthma severity, and atopy in a large Australian white population. METHODS: PCR-RFLP analysis was used to characterise the C-159T polymorphism in mild (n = 264), moderate (n = 225) and severe (n = 79) asthmatic patients and non-asthmatic controls (n = 443), including atopic (n = 688) and non-atopic (n = 323) individuals. Association analyses were performed using chi(2) tests. RESULTS: There was no association between the polymorphism and asthma (p = 0.468) or asthma severity (p = 0.727), and only a very weak association with atopy (p = 0.084). A meta-analysis of all studies conducted to date revealed similar genotypic frequencies in white ethnic populations and confirmed that there was no overall association with atopy (p = 0.52) or asthma (p = 0.23), although there was significant between study heterogeneity (p = 0.01). CONCLUSIONS: This study confirms that there is no association between the CD14 C-159T polymorphism and asthma or asthma severity and a weak association between this polymorphism and atopy in an adult population.

A functional polymorphism in the promoter region of the cyclooxygenase-2 gene is not associated with asthma and atopy in an Australian population.

BACKGROUND: Cyclooxygenase (COX)-2 is a key inducible enzyme that regulates the production of anti-inflammatory prostaglandin E(2). A single-nucleotide polymorphism, -765G>C, located within a stimulatory protein-1 binding site in the COX-2 promoter region, has been shown to have significantly lower promoter activity in vitro compared with the wild-type and was associated with decreased plasma levels of C-reactive protein after coronary artery bypass surgery. We hypothesized that this polymorphism, which may result in decreased COX-2 transcription, could be associated with more severe asthma, and/or aspirin-intolerant asthma (AIA). OBJECTIVE: To determine the association between the -765G>C COX-2 polymorphism and asthma, disease severity and AIA in a large, well-phenotyped Australian population. METHODS: PCR and restriction fragment length polymorphism analysis was used to characterize the polymorphism in an Australian Caucasian population of patients with mild (n=322), moderate (n=254) or severe (n=88) asthma and in non-asthmatic control subjects (n=512), as well as in patients with AIA (n=58). Genotype and allele association analyses were performed using chi(2) tests. RESULTS: The polymorphic -765C allele was present in approximately 30% of asthmatic patients and non-asthmatic controls. There was no association between the -765G>C polymorphism and asthma (P=0.920), disease severity (P=0.840), atopy (P=0.655) or AIA (P=0.841) in this population. CONCLUSION: Although the -765G>C polymorphism may have lower promoter activity and result in decreased COX-2 expression, it is not associated with asthma, disease severity, AIA or atopy in this Australian population.

Susceptibility of chacma baboons (Papio ursinus orientalis) to infection by hepatitis B virus.

BACKGROUND: Because baboons are being considered as a source of xenografts for human liver transplantation in patients with hepatitis B virus- (HBV) induced cirrhosis to forestall infection of the graft by the virus, we undertook a study to ascertain if baboons are resistant to HBV infection. METHODS: Six chacma baboons were inoculated with serum containing HBV and were followed for 52 weeks to detect transmission of infection. RESULTS: Anti-HBc was detected in the serum of four baboons 16 weeks after inoculation. Virions, small spherical particles, and tubular forms were seen at this time in the serum of the one baboon studied by transmission electron microscopy. HBV DNA was detected by polymerase chain reaction in the serum of the same four baboons throughout the period of follow-up, as well as in liver tissue obtained after 52 weeks. The specificity of the DNA was confirmed by Southern hybridization. Nucleotide sequences showed complete sequence identity between the HBV DNA in each of the baboon sera and one of the two HBV genotypes inoculated. Serum transaminase levels tested at 4-weekly intervals were always normal and histological examination of liver tissue after 52 weeks showed no evidence of chronic hepatitis. Examination of squash preparations of liver tissue by electron microscopy in one baboon revealed core-like particles. CONCLUSIONS: Chacma baboons are susceptible to HBV infection and appear to develop a chronic carrier state. The use of xenografts from baboons should preferably be avoided, but if they are used again for HBV-infected patients it would be prudent to treat the patients as if they had received an organ from a human donor.

Characterization of six tumor suppressor genes and microsatellite instability in hepatocellular carcinoma in southern African blacks.

AIM:To analyse cumulative loss of heterozygosity (LOH) of chromosomal regions and tumor suppressor genes in hepatocellular carcinomas (HCCs) from 20 southern African blacks.METHODS: p53,RB1, BRCA1, BRCA2, WT1 and E-cadherin genes were analysed for LOH, and p53 gene was also analysed for the codon 249 mutation, in tumor and adjacent non tumorous liver tissues using molecular techniques and 10 polymorphic microsatellite markers.RESULTS:p53 codon 249 mutation was found in 25% of the subjects,as was expected, because many patients were from Mozambique, a country with high aflatoxin B-1 exposure. LOH was found at the RB1, BRCA2 and WT1 loci in 20%(4/20) of the HCCs, supporting a possible role of these genes in HCC. No LOH was evident in any of the remaining genes. Reports of mutations of p53 and RB1 genes in combination, described in other populations, were not confirmed in this study. Change in microsatellite repeat number was noted at 9/10 microsatellite loci in different HCCs, and changes at two or more loci were detected in 15%(3/20) of subjects.CONCLUSION:We propose that microsatellite/genomic instability may play a role in the pathogenesis of a subset of HCCs in black Africans.

Non-specificity of messenger RNA of alpha-fetoprotein in peripheral blood in detecting early spread of hepatocellular carcinoma in black Africans.

Awareness of early spread of hepatocellular carcinoma is crucial in selecting patients for surgical intervention. Alpha-fetoprotein is widely used as a serum marker for hepatocellular carcinoma. Our aim was to evaluate the specificity of alpha-fetoprotein-mRNA transcription in cells in the peripheral blood for diagnosing early spread of hepatocellular carcinoma in black Africans. Alpha-fetoprotein-, albumin- and prothrombin-mRNA were detected in peripheral blood mononuclear cells by reverse transcription-polymerase chain reaction. Alpha-fetoprotein-mRNA was shown in peripheral blood mononuclear cells in 53% (35/66) of patients with hepatocellular carcinoma, but also in 45% (10/22) of healthy blacks, 64% (14/22) of black patients with acute hepatitis, 55% (11/20) of those with chronic hepatitis or cirrhosis and 75% (9/12) of those with hepatic metastases (from a number of primary sites). Specificity of albumin- and prothrombin-mRNA was better than that of alpha-fetoprotein-mRNA, although the sensitivity was reduced. The corresponding prevalence of albumin-mRNA for each group of patients or controls was 30% (20/66), 9% (2/22), 41% (9/22), 10% (2/20), and 17% (2/12), respectively, and for prothrombin-mRNA 27% (18/66), 4.5% (1/22), 27% (6/22), 20% (4/20) and 17% (2/12), respectively. We conclude that the non-specificity of alpha-fetoprotein-mRNA transcription in peripheral blood in recognizing malignant hepatocytes in the circulation severely limits its usefulness in diagnosing the early spread of hepatocellular carcinoma in black Africans.

Dietary iron overload as a risk factor for hepatocellular carcinoma in Black Africans.

Although the iron-loading disease, hereditary hemochromatosis, has a strong causal association with hepatocellular carcinoma (HCC), the carcinogenic potential of dietary iron overload in Black Africans is not known. We investigated this potential by evaluating iron status, alcohol consumption, markers for hepatitis B (HBV) and C virus (HCV) infections, and exposure to dietary aflatoxin B1 in 24 rural patients with this tumor, 48 race-, sex-, and age-matched hospital-based controls, and 75 related or unrelated close family members of the cancer patients. Iron overload was defined as a raised serum ferritin concentration in combination with a transferrin saturation > or = 60%, and was confirmed histologically when possible. Among 24 patients and 48 hospital controls, the risk of developing HCC in the iron-loaded subjects was 10.6 (95% confidence limits of 1.5 and 76.8) relative to individuals with normal iron status, after adjusting for alcohol consumption, chronic HBV and HBC infections, and exposure to aflatoxin B1. The risk of HCC in subjects with HBV infection was 33.2 (7.2, 153.4) (odds ratio [95% confidence limits]), HCV infection 6.4 (0.3, 133.5), and alcohol consumption 2.0 (0.5, 8.2). Aflatoxin B1 exposure did not appear to increase the risk of HCC. The population attributable risk of iron overload in the development of HCC was estimated to be 29%. Among 20 cancer patients and 75 family members, the risk of developing HCC with iron overload was 4.1 (0.5, 32.2). We conclude that dietary iron overload may contribute to the development of HCC in Black Africans.

Does hepatitis GB virus-C infection cause hepatocellular carcinoma in black Africans?

The newly cloned and characterized hepatitis GB virus-C (HGBV-C), which is the same virus as the independently discovered hepatitis G virus, has a global distribution, is transmitted parenterally, and causes chronic viremia. The pathological consequences of infection with HGBV-C are uncertain, and its hepatocarcinogenic potential is unknown. We used a case-control format to compare the prevalence of HGBV-C infection in 167 southern African blacks with hepatocellular carcinoma (HCC) and 167 race-, age-, and sex-matched hospital-based control subjects, and to test for possible interactive effects between this virus and hepatitis B and C viruses in the development of the tumor. The presence of HGBV-C ribonucleic acid was detected in serum samples by reverse transcription, amplification of the resulting complementary deoxyribonucleic acid by the polymerase chain reaction (PCR), and Southern hybridization using a probe from the NS3/helicase region of the genome. Serum samples were also tested for the presence of hepatitis B virus surface antigen, antibodies to hepatitis C virus, and hepatitis C virus ribonucleic acid. Individuals infected with HGBV-C did not have an increased relative risk of developing HCC (relative risk 0.9; 95% confidence limits 0.5, 1.7). Moreover, co-infection with HGBV-C did not further increase the risk of tumor development in patients who were chronically infected with hepatitis B and/or C viruses. HGBV-C is unrelated to hepatocellular carcinoma development in black Africans.

Improved glucose tolerance after effective lipid-lowering therapy with bezafibrate in a patient with lipoatrophic diabetes mellitus: a putative role for Randle's cycle in its pathogenesis?

This report describes a patient with lipoatrophic diabetes mellitus (LDM), which is a rare clinical syndrome characterized by lipoatrophy and severe insulin resistance. Although a genetic abnormality is suspected in the development of LDM, no functional mutations in key domains of the insulin receptor gene were detected. Therapy was directed primarily at decreasing the availability of non-esterified fatty acids (NEFA), and thereby improving glucose tolerance (Randle's cycle), by the administration of a lipid-lowering drug, bezafibrate. Serial changes in fasting levels of the hormones of glucose homeostasis and lipids were measured, as well as glucose and insulin responses to a 75-g oral glucose challenge at onset and following 3 and 6 months of fibrate therapy. Progressive reductions in the patient's levels of triglycerides and NEFA were paralleled by an improvement in beta-cell function, a decrease in insulin resistance, and the attainment of normal glucose homeostasis. We conclude that the pathogenesis of LDM may be related primarily to abnormal regulation of lipid, rather than glucose, metabolism.

The relative roles of hepatitis B and C viruses in the etiology of hepatocellular carcinoma in southern African blacks.

BACKGROUND & AIMS: Epidemiological studies have shown the relative roles of hepatitis B and C viruses in hepatocarcinogenesis to vary considerably among populations. The aim of this study was to define the independent and interactive roles of the two viruses in the genesis of hepatocellular carcinoma in southern African blacks. METHODS: Blood samples were taken from 231 black patients with hepatocellular carcinoma and matched controls treated at four Johannesburg hospitals. These were tested for hepatitis B surface antigen, antibodies to hepatitis C virus, and hepatitis C virus RNA. RESULTS: Relative to individuals without serological evidence of hepatitis B or C infection, those positive for hepatitis B surface antigen alone had a statistically significant 23.3-fold increased risk for hepatocellular carcinoma, whereas those positive for hepatitis C serology alone had a statistically significant risk of 6.6. A synergistic effect on risk was evident when both hepatitis B and C markers were present (relative risk, 82.5). Hepatitis B virus alone is estimated to cause 43% of hepatocellular carcinoma in southern African blacks, hepatitis C alone 5%, and coinfection with the two viruses 20%. CONCLUSIONS: Hepatitis B virus plays a predominant role in hepatocellular carcinogenesis in southern African blacks, with hepatitis C virus responsible for a smaller proportion of cases. Coinfection with the two viruses carries a synergistic risk of hepatocellular carcinoma formation.

Hepatocarcinogenic potential of genotype 5 of hepatitis C virus.

Evidence is emerging that hepatitis C virus genotypes have different carcinogenic potentials. The hepatocarcinogenicity of genotype 5, the predominant subtype in hepatitis C virus isolates in South Africa, is not known. We have compared the prevalence of genotype 5 of hepatitis C virus in 44 southern African blacks with hepatitis C virus-related hepatocellular carcinoma with that in a comparable group of patients with hepatitis C virus-induced chronic liver disease (cirrhosis or chronic hepatitis) in the absence of cancer. Hepatitis C virus serotypes 1 to 6 were identified by measuring type-specific antibodies to NS4-derived peptide antigens. Serotype 5 was present in 48% (21/44) of the patients with hepatocellular carcinoma and 37% (15/41) of those with liver disease in the absence of hepatocellular carcinoma, an insignificant difference. Although the numbers of the other genotypes were small, the differences in the prevalence rates of these serotypes between the two groups of patients were also not significant. We conclude that genotype 5 of the hepatitis C virus is neither more nor less carcinogenic than other genotypes found in isolates in South Africa.

Kaposi's sarcoma-associated herpesvirus in Kaposi's sarcoma occurring in immunosuppressed renal transplant recipients.

Kaposi's sarcoma-associated herpesvirus (KSHV) has been implicated in the genesis of Kaposi's sarcoma and other tumors occurring in immunosuppressed individuals. Using amplification by the polymerase chain reaction and nucleotide sequencing of extracted DNA, we have detected the KS330(233) sequence of KSHV DNA in Kaposi's sarcoma tissue from 4 immunosuppressed renal transplant recipients. The sequences shared a greater than 98% homology with those described in KSHV DNA from Kaposi's sarcoma in patients with acquired immunodeficiency syndrome. In another 2 renal transplant recipients KSHV DNA could not be detected in scar tissue at the site of previous Kaposi's sarcoma that had resolved after immunosuppression was discontinued and haemodialysis recommenced. These findings support the hypothesis that KSHV may be the infectious agent concerned in the genesis of Kaposi's sarcoma.

SSCP analysis of the tyrosine kinase domain of the insulin receptor gene: polymorphisms detected in South African black and white subjects.

The frequency of DNA polymorphisms in the tyrosine kinase domain (exons 17-21) of the insulin receptor gene was assessed in 30 black and 30 white South Africans, using single-stranded conformation polymorphism and direct sequencing analysis. A comparison of the frequencies of the normal versus the combined polymorphic alleles, found only in exon 17, showed a significant difference between black and white groups (P = 0.037).