Ljiljana Bulat-Kardum

Interferon-γ Gene (T874A and G2109A) Polymorphisms Are Associated With Microscopy-positive Tuberculosis

Genetic susceptibility to tuberculosis includes several unknown yet different loci each contributing to a small extent. Intronic polymorphisms within the interferon‐γ (IFN‐γ) gene IFNG T+874A and IFNG G+2109A correlate with the IFN‐γ production in vitro, and the frequency of potential high IFN‐γ producers was previously reported by others to be lower in patients than in controls from Sicily. The aim of this study was to determine whether there is an association between polymorphisms in the IFN‐γ gene and predisposition to tuberculosis. We analysed two IFNG SNPs (T+874A and G+2109A) in patients (n = 253) hospitalized in Rijeka (Croatia) and controls (n = 519) from the same area. One‐fifth of the controls were healthy contacts of the diseased, and the rest were blood donors. IFNG alleles, their predicted haplotypes or genotypes were not associated with disease susceptibility. Thus, we could not reproduce results from Sicilian case‐control study. However, T/T+874 (possible high IFN‐γ producer) and +874A/A (putative low producer) genotypes were associated with microscopically positive–negative forms of disease. Haplotypes (T+874A and G+2109A) based on a prediction by software phase and subsequent genotype analysis corroborated these findings. Patients had significantly higher frequency of genotypes without T at +874 (AA/AA; AA/AG and AG/AG) in microscopy‐ or bacterial culture‐positive groups compared with their negative counterparts. These data suggest an association with disease severity rather than susceptibility to tuberculosis in Croatian Caucasian population.

Interferon-gamma receptor-1 gene polymorphism in tuberculosis patients from Croatia.

Recent studies have indicated that the interleukin‐12/interferon‐γ (IFN‐γ) axis is important in mycobacterial infection susceptibility. Using an intronic (CA)n polymorphic microsatellite marker within the IFN‐γ receptor‐1 (IFNGR1) gene, we have compared the allelic frequencies of this marker in hospitalized tuberculosis patients (n = 120) with that of controls (n = 87) from Rijeka, Croatia. We identified 13 (CA)n alleles in the tuberculosis patients, whereas only 10 were found in the controls. A significant difference between one allelic marker and the control group was observed (P = 0.02, 95% confidence interval 0.14–0.94), suggesting a possible protective association. In contrast, several other allelic markers showed a trend towards association with the disease. We also found a trend towards an increased frequency in homozygosity of one allelic marker in patients (11.7%) as compared with controls (4.6%). We conclude that there is no evidence for disease association of the IFNGR1 gene marker in Mendelian‐type (single‐allele) inheritance. However, our results also suggest that unidentified allelic variations in the IFNGR1 gene might elevate or decrease the risk in this ethnic population, as a part of the multigenic predisposition to tuberculosis.

Interferon-γ Receptor-1 Gene Promoter Polymorphisms (G-611A; T-56C) and Susceptibility to Tuberculosis

We analysed frequencies of two single‐nucleotide polymorphisms (SNP) in the interferon‐γ (IFN‐γ) receptor‐1 (IFNGR1) gene promoter (G‐611A, T‐56C) in tuberculosis patients (n = 244) and compared them with controls (n = 521). These frequencies were not significantly different, whether analysed independently or as haplotypes. Because these SNP affect transcription, the results suggest that the expression of the IFNGR1 gene does not confer susceptibility to disease in patients from Croatia. Further analysis revealed a significant association between the protective (CA)n polymorphism (22 repeats, 192 FA1), located in the fifth intron of the IFNGR1 gene (+16682), and GT promoter haplotype (−611; −56) that showed the strongest expression capacity. In addition to this cis relationship, the (CA)22 allele was correlated in trans with an IFN‐γ SNP (IFNG G + 2109A), which might affect the transcription of the IFNG gene. These results suggest that a particular combination of IFNG and IFNGR1 SNP might offer a better protection against tuberculosis in this population.

Toll-like receptor 2 (P631H) mutant impairs membrane internalization and is a dominant negative allele.

We have sequenced 416 Toll‐like receptor‐2 (TLR2) alleles in 208 subjects in a tuberculosis case–control study in Croatian Caucasian population. We found ten single nucleotide polymorphisms (SNP) among which three were novel (S97S, T138I and L266F). The genotype containing TLR2‐P631H SNP was significantly overrepresented in patients with tuberculosis when compared to contact controls, suggesting a small yet increased risk to disease. The causative agent of tuberculosis is Mycobacterium tuberculosis, which can bind to TLR2 with its lipoprotein coat. The TLR2‐P631H mutant has a dominant negative effect on the wild type TLR2 signalling in transfected HEK293 kidney cells using the NF‐κB‐driven luciferase as a reporter gene with ligands like M. avium extracts, Pam3CysSK4 or FSL‐1 that bind TLR2/TLR1 or TLR2/TLR6 heterodimers, respectively. Studies on internalization from the Regular Madine Darby Canine Kidney cell surface into the early endosomal compartments showed a lower rate of the mutant compared to the wild type. Our data, in combination with a report by others show that the TLR2‐P631H allele could be associated with protection to meningococcal meningitis, suggest that by dominantly inhibiting the response of cells important in the immune response this mutant might confer either protection or susceptibility to meningitis or tuberculosis, respectively.

Genetic Polymorphisms in the Toll-like Receptor 10, Interleukin (IL)17A and IL17F Genes Differently Affect the Risk for Tuberculosis in Croatian Population.

Proinflammatory conditions leading to activation of macrophages via interferon‐γ bear an important role in host defence against intracellular bacteria such as Mycobacterium tuberculosis (Mt). Interleukin‐17 plays a similar role, as it appears to be also an activator of macrophages. Recently, the TLR‐10 was identified as an anti‐inflammatory factor that exerts its action via association with the TLR‐2 chain at the cell surface of macrophages, the latter being an Mt‐binding protein. We have previously found that gene polymorphisms that either inactivate the TLR2 gene product or have a dominant‐negative role are associated with tuberculosis (TB) in Croatian population. We have now extended our survey and found that single nucleotide polymorphism (SNP) in TLR10 (rs11096957) is associated with risk for TB. Homozygotes carrying the A allele are associated with predisposition to disease as analysed by the dominant model of inheritance. In contrast, SNPs in the proinflammatory IL17A and IL17F genes (rs2275913 and rs763780, respectively), found previously to correlate with the disease occurrence in Chinese population, were not significantly associated with tuberculosis in the Croatian population.

Association of variable number of tandem repeats in the coding region of the FAM46A gene, FAM46A rs11040 SNP and BAG6 rs3117582 SNP with susceptibility to tuberculosis.

We analyzed for association between the Family with sequence similarity 46, member A (FAM46A) gene (located on chromosome 6q14.1), BCL2-Associated Athanogene 6 (BAG6) gene (located on chromosome 6p21.3) and tuberculosis in Croatian Caucasian. We genotyped the FAM46A rs11040 SNP, FAM46A VNTR and BAG6 rs3117582 polymorphisms in a case-control study with 257 tuberculosis patients and 493 healthy individuals in a Croatian Caucasian population. We found that genotype FAM46A 3/3 (three VNTR repeats homozygote) was associated with susceptibility to tuberculosis (p<0.0015, Pcorr.<0.029, Odds ratio = 2.42, 95% Confidence Interval = 1.34–4.3). This association suggests that the protein domain encoded by the VNTR might be important for the function of the FAM46A protein, which, in turn, could be relevant in developing tuberculosis. In addition, we found that FAM46A rs11040 SNP:FAM46A VNTR:BAG6 haplotype 132 (G-3-C) is associated with susceptibility to tuberculosis (p<0.012, pcorr.<0.024, Odds ratio 3.45, 95% Confidence Interval = 1.26–9.74). This may suggests that the interaction between the FAM46A and BAG6 proteins may be involved in tuberculosis etiology. We found also that infection of human macrophages with heat-killed M. tuberculosis (H37Rv) led to over-expression of FAM46A (VNTR 3/4) transcript. This is the first study to show associations between the FAM46A gene VNTR polymorphisms, FAM46A rs11040 SNP:FAM46A VNTR:BAG6 haplotypes and any disease.

Tumor necrosis factor-alpha gene promoter -308 and -238 polymorphisms in patients with lung cancer as a second primary tumor

Background Lung cancer is the most common second primary cancer. We investigated whether the TNF-α-308 and TNF-α-238 polymorphisms were associated with the susceptibility and severity of lung cancer as the second primary cancer (LC2). Material/Methods This study included 104 patients from the group LC2. The control subjects included 2 groups. The first control group (LC1) comprised 201 unrelated patients with lung cancer as a first primary cancer. The second control group (HC) comprised 230 healthy blood donors, matched for sex and age to the study group. Results The frequencies of the TNF-α-238 polymorphism GG genotype and the G allele were higher in the LC2 group than in the LC1 group, but the differences did not reach significance (p=0.054 and p=0.057, respectively). Similar differences were found in the TNF-α-238 polymorphism GG genotype and G allele between the LC2 group and the HC group (p=0.054 and p=0.057, respectively). In terms of the different types of lung cancer, patients with a second primary NSCLC (non-small cell lung cancer) more frequently had TNF-α-238 polymorphism GG genotypes and G alleles than patients with a first primary NSCLC (the differences approached statistical significance: p=0.060, p=0.064, respectively). All (100%) patients of group LC2 (n=104) had the GG genotype and the G allele. GG genotype was exclusive and no A allele was found in group LC2. Conclusions TNF-α-238 polymorphism GG genotype and the G allele could have a promotional effect on the development of NSCLC in the group of patients with LC2.

Heterozygous carriage of a dysfunctional Toll-like receptor 9 allele affects CpG oligonucleotide responses in B cells.

Background: Human B cells respond to Toll-like receptor (TLR) 9 stimulation by cytokine production. Results: A rare, novel TLR9 allele fails to activate NF-κB in HEK293 cells. Heterozygous carrier B cells show defective IL-6 and IL-10 production. Conclusion: Heterozygosity for this TLR9 allele modifies CpG oligonucleotide responsiveness. Significance: This is the first analysis of human TLR9 variants in primary cells. Toll-like receptors (TLR) are employed by the innate immune system to detect microbial pathogens based on conserved microbial pathogen molecules. For example, TLR9 is a receptor for CpG-containing microbial DNA, and its activation results in the production of cytokines and type I interferons from human B cells and plasmacytoid dendritic cells, respectively. Both are required for mounting an efficient antibacterial or antiviral immune response. These effects are mimicked by synthetic CpG oligodeoxynucleotides (ODN). Although several hyporesponsive TLR9 variants have been reported, their functional relevance in human primary cells has not been addressed. Here we report a novel TLR9 allele, R892W, which is hyporesponsive to CpG ODN and acts as a dominant-negative in a cellular model system. The R892W variant is characterized by increased MyD88 binding and defective co-localization with CpG ODN. Whereas primary plasmacytoid dendritic cells isolated from a heterozygous R892W carrier responded normally to CpG by interferon-α production, carrier B cells showed impaired IL-6 and IL-10 production. This suggests that heterozygous carriage of a hyporesponsive TLR9 allele is not associated with complete loss of TLR9 function but that TLR9 signals elicited in different cell types are regulated differently in human primary cells.

Pneumocephalus after an uneventful scuba dive.

Scuba diving has become increasingly popular in the last 20 yr. Although it is considered safe, accidents, sometimes with fatal outcomes, do occur. The incidence of diving-related CNS barotrauma is low and it has been reported very infrequently. The clinical presentation may range from minimal dysesthesias to complete quadriplegia, encephalopathy, or death. In this paper we present a case of pneumocephalus in a 36-yr-old male scuba diver that presented with minor neurologic symptoms. A discussion, including a review of the literature, is also presented. The authors recommend that diving-induced neurologic dysbarism syndromes, including pneumocephalus, should be considered a possible cause when a scuba diver presents with neurologic symptoms, even minor ones.

Association of the FAM46A gene VNTRs and BAG6 rs3117582 SNP with non small cell lung cancer (NSCLC) in Croatian and Norwegian populations

We analyzed for associations between a variable number of tandem repeat (VNTR) polymorphism in the Family with sequence similarity 46, member A (FAM46A) gene and a single nucleotide polymorphism (rs3117582) in the BCL2-Associated Athanogene 6 (BAG6) with non small cell lung cancer in Croatian and Norwegian subjects. A total of 503 (262 Croatian and 241Norwegian) non small cell lung cancer patients and 897 controls (568 Croatian and 329 Norwegian) were analyzed. We found that the frequency of allele b (three VNTR repeats) of FAM46A gene was significantly increased in the patients compared to the healthy controls in the Croatian and the combined Croatian and Norwegian subjects. Genotype frequencies of cd (four and five VNTR repeats) and cc (four VNTR repeats homozygote) of the FAM46A gene were significantly decreased in the patients compared to the healthy controls in the Croatian and Norwegian subjects, respectively. Logistic regression analyses revealed FAM46A genotype cc to be an independent predictive factor for non small cell lung cancer risk in the Norwegian subjects after adjustment for age, gender and smoking status. This is the first study to suggest an association between the FAM46A gene VNTR polymorphisms and non small cell lung cancer. We found also that BAG6 rs3117582 SNP was associated with non small cell lung cancer in the Norwegian subjects and the combined Croatian-Norwegian subjects corroborating the earlier finding that BAG6 rs3117582 SNP was associated with lung cancer in Europeans. Logistic regression analyses revealed that genotypes and alleles of BAG6 were independent predictive factor for non small cell lung cancer risk in the Norwegian and combined Croatian-Norwegian subjects, after adjustment for age and gender.