| Abstract | Cilj istraživnja: Rezultati nedavnih studija sugeriraju da interleukin-12 / interferon-gama osovina ima ulogu u sklonosti obolijevanju od tuberkuloze. Željeli smo ustanoviti da li je polimorfizam u genu koji kodira interferon-gama receptor 1 (IFNyR1) povezan sa sklonošću tuberkulozi. Zato smo analizirali polimorfizam mikrosatelitnog markera (CA)n iz petog introna IFNGR1 gena i dva točkasta polimorfizma (SNP-a) G-611A i T-56C unutar promotorne regije IFNGR1. Kako je aktivnost IFN-y posredovana receptorom (IFNyR1), ispitali smo stoga i polimorfizam u genu za IFN-y (/FNG).
Ispitanici i metode: Ispitanici su bolesnici liječeni od tuberkuloze (n=245) na Pulmološkom odjelu KBC-a Rijeka, uspoređeni su sa zdravim kontrolama (n=519). Srednja dob pacijenata bila je 51,0±15,8 (SD) godina, 75,5 % su bili muškaraci. U kontrolnoj skupini srednja dob je bila 41,6±11,7 (SD) godina, od kojih je bilo 80,7% muškaraca. Analiza IFNGR1 i IFNG SNP-a je Taqman PCR metodom i dodatno provjerena sa SNP specifičnom metodom baziranom na elektroforezi na agaroznom gelu nakon digestije restriktivnim enzimom. (CA)n polimorfizam je ispitan PCR metodom, a veličina PCR fragmenata analizirana je na poliakrilamidnom gelu.
Rezultati: Identificirali smo 13 (CA)n alela u naših ispitanika što je više od do sada izvještavanog. U oboljelih smo našli 12 alela, dok ih je u zdravih ispitanika identificirana 11. Značajna razlika je zapažena između alela 192 (CA)n i kontrolne grupe, sugerirajući povezanost s protekcijom za obolijevanje. Kada su analizirane neovisno (frekvencije alela i genotipova) ili kao haplotipove, frekvencije SNP-a IFNGR1 i IFNG nisu se statistički značajnorazlikovale u oboljelih i kontrola. To da nema jednostavne (Mendelove) udruženosti s tuberkulozom. Dodatne analize otkrile su značajnu udruženost protektivnog alela 192 (CA)n iz petog introna IFNGR1 gena (+16682) i GT haplotipa (-611,-56) koji fenotip snažnog promotora. Našli smo korelaciju alela 192 (CA)n sa SNP gena IFNG (G+2109A), što može mjenjati transkripciju IFNG gena. Analizirali smo zatim mikroskopski
pozitivnu i negativnu, te u kulturama pozitivnu i negativnu tuberkulozu, kao i radiološku proširenost tuberkuloznih lezija pluća. Nismo našli statistički značajnu razliku za polimorfizme IFNGR1 , osim za SNP IFNG T +87 4A koji je udružen sa mikroskopski pozitivnom tuberkulozom. U sklopu populacijske genetike, usporedili smo Hrvate sa Kavkaskim Amerikancima od kojih se nismo razlikovali. Statistički smo se značajno razlikovali od Afroamerikanaca i Amerikanaca korejskog porijekla za SNP IFNGR1 G-611A i T-56C u grupi zdravih kontrola.
Zaključak: Nema dokaza za udruženost markera IFNGR1 gena i Mendelovog tipa naslijeđivanja sklonosti ka obolijevanju od tuberkuloze. Naši rezultati također sugeriraju postojanje dosad neindentificirane varijacije u IFNGR1 genu, a što može povećati ili smanjiti rizik za obolijevanje u našoj populaciji. Kako polimorfizmi promatra IFNGR1 utječu na transkripciju, rezultati sugeriraju da ekspresija gena IFNGR1 ne utječe na sklonost obolijevanju u grupi pacijenata. Rezultati sugeriraju da pojedine kombinacije IFNGR1 i IFNG alela mogu omogućiti bolju protekciju protiv tuberkuloze u našoj populaciji. |
| Abstract (english) | Objectives: Recent studies have indicated that the interleukin-12/interferon-y (IFN-y) axis is important in susceptibility to diseases caused by mycobacterial infections. We wanted to investigate whether polymorphisms in the gene encoding interferon-gamma receptor-1(IFNyR1) were associated with susceptibility to tuberculosis. We thus analysed (CA)n polymorphic microsatellite marker in an intron of the IFNGR1, and two single nucleotide polymorphisms (SNPs), G-611A and T-56C in the IFNGR1 gene promoter. Since the action of the IFN-y is mediated through its receptor (IFNyR1), we also investigated the polymorphisms in the interferon-gamma gene (/FNG).
Material and Metodes: The tuberculosis patients (n=245) hospitalised at the Department of pulmology, KBC Rijeka, Croatia were compared with the healthy controls (n=521). The mean age of patients was 48,5±12(SD) years, and 75,5% were male. ln the control group 90,7% were male with a mean age of 42,4±9 (SD) years. Detection of the IFNGR1 and IFNG SNPs were dane by Taqman-based allele-specific PCR assay, and, additionally,checked by SNP specific methods based on agarose gel electrophoresis after restriction enzyme digestions. (CA)n polymorphism was detected by PCR and analyzed for size on polyacrylamide gels.
Results: We identified 13 (CA)n alleles in all participants, and this is more
then previously reported . ln tuberculosis patients were found 12 alleles,
whereas 11 were identified in the control group. A significant difference
between patients and controls was observed for the allele (CA)22 , suggesting a posible protective association. Whether analyzed independently (frequencies of alleles and genotypes) or as haplotypes, neither IFNGR1 nor IFNG SNP frequencies of were significantly different indicating no simple (Mendelian) association with tuberculosis. However, further analysis revealed a significant association between the protective (CA)22 allele of the IFNGR1 gene (+16682), and the strongest IFNGR1 promoter haplotype (G-611 ;T-56) .. ln addition , the (CA)22 allele was correlated with the IFNG SNP (G+2109A), which might affect the transcription of the IFNG gene. Subsequently, we correlated microscopy- and bacterial-culture positive and negative forms of tuberculosis with all SNPs , as well as the spread of radiologyc pulmonary lesions. None were statstically significant, except the IFNG SNP at +874 which was associated with microscopy-positive tuberculosis. From population genetics viewpoint, Croats are similar to Caucasian Americans, and are significantly different trom African Americans and Koreans trom America for IFNGR1 G-611A and T-56C polymorphisms, in healthy controls.
Conclusion: There is no evidence for association of the IFNGR1 gene
markers with disease in Mendelian type (single-allele) inheritance. Since the IFNGR1 promoter SNPs affect transcription, the results suggest that the expression of the IFNGR1 gene does not confer susceptibility to tuberculosis. However, an unidentified allelic variation in the IFNGR1 gene might decrease the risk for disease in our ethnic population. Results also suggest that a particular combination of IFNG and IFNGR1 alleles might offer a better protection against tuberculosis in this population. |
