Imunogenetické aspekty v etiopatogenezi revmatoidní artritidy

MUDr. Petr Němec, Ph.D.

Doctoral thesis

Immunogenetic aspects in etiopathogenesis of rheumatoid arthritis

Abstract:

RevmatoidnŞ artritida (RA) je modelem polygenn§ podmŞn§nŚho onemocn§nŞ, kde genetickŚ faktory mohou ovlivžovat jak vnŞmavost ke vzniku choroby, tak zvanost jejŞho prb§hu. TNF je pleiotropnŞ prozn§tlivý cytokin, který hraje významnou roli v indukci T bun§nŚ imunitnŞ odpov§di, pi stimulaci produkce proteolytických enzym, prostaglandin, chemokin, pi zvýenŚ expresi adhezivnŞch molekul i HLA antigen II. tŞdy v patogenezi RA. Opakovan§ byla prokzna asociace RA s n§kterými alelami HLA-DRB1. Jeliko gen pro TNF leŞ v jejich t§snŚ blŞzkosti, ve vysoce variabilnŞ oblasti oznaovanŚ jako oblast HLA gen III. tŞdy na krtkŚm ramŚnku 6. chromozomu (6p21.3), vyvolv studium polymorfism tohoto genu znaný zjem. ZvýenŚ koncentrace TNF prokazovanŚ v plazm§ a synovilnŞ tekutin§ pacient s RA mohou být asociovny s polymorfismy v genu pro TNF. Choroby kardiovaskulrnŞho systŚmu jsou zodpov§dny za 35 50 % pŞpad mrtŞ pacient s RA, akoliv v b§nŚ dosp§lŚ populaci jsou pŞinou mrtŞ piblin§ ¼ mu a 1/5 en. Toto zvýenŚ riziko me být zapŞin§no pŞtomnostŞ klasických rizikových faktor, morbiditou spojenou s farmakoterapiŞ RA nebo me být spojeno s pŞtomnostŞ rizikových faktor specifických pro RA, jako jsou hyperhomocysteinemie, s chorobou souvisejŞcŞ dyslipidŚmie, zvýenŚ hladiny TNF a zn§t cŚvnŞ st§ny. Byla ji prokzna asociace polymorfismu - 308 G/A TNF s rizikovými faktory ICHS: Obezitou, dyslipidŚmiŞ, inzulinovou rezistencŞ a hypertenzŞ. CŞlem mŚ prce bylo prokzat moný vztah mezi polymorfismem - 308 G/A v promotorovŚ oblasti genu pro TNF a vnŞmavostŞ ke vzniku a zvanostŞ RA. Celkem 130 pacient (98 en, 32 mu, rozp§tŞ v§ku 20 - 79 rok, medin 56,2 rok), kteŞ splnili revidovan diagnostick kritŚria ACR pro RA, s dŚlkou trvnŞ choroby minimln§ 2 roky, bylo rozd§leno do 4 skupin podle stupn§ rentgenovŚho postienŞ kloub rukou a zp§stŞ, podle Steinbrockerovy klasifikace: stdium I: neerozivnŞ RA - 15 pacient, stdium II - IV: erozivnŞ - 14 pacient. KontrolnŞ soubor zahrnoval 150 zdravých dobrovolnŞk obdobnŚ distribuce v§ku a pohlavŞ. Provd§nŞ studie bylo schvleno Etickou komisŞ LŚkaskŚ fakulty Masarykovy Univerzity v Brn§. Pacientm i kontrolm byla odebrna perifernŞ krev a z nŞ izolovna DNA z leukocyt pomocŞ metody s vyuitŞm proteinzy. Polymorfismus - 308 G/A genu pro TNF byle detekovn pomocŞ metody polymerase chain reaction (PCR) a restriction fragment length polymorphism (RFLP) analýzou pomocŞ restriknŞ endonuklezy BsmFI (New England Biosystem, USA). RozdŞly v distribuci genotyp a alelických frekvencŞch polymorfismu - 308 G/A genu pro TNF mezi pacienty a kontrolnŞm souborem a mezi skupinami pacient byly kalkulovny pomocŞ chi2 testu a Fisher exact testu. Shoda genotypových distribucŞ s Hardy-Weinbergovou rovnovhou byla testovna pomocŞ chi2 testu. Odds ratio (OR) a 95 % konfidennŞ interval (CI) byl vyuit k výpotu rizika polymorfismu - 308 G/A TNF ve vztahu k onemocn§nŞ. K výpotu signifikance OR byl pouit Fisher exact test. Nebyl zjit§n rozdŞl v genotypových distribucŞch ani alelických frekvencŞch polymorfismu - 308 G/A TNF mezi pacienty s RA a kontrolnŞm souborem. SignifikantnŞ rozdŞl byl pozorovn v alelických frekvencŞch polymorfismu - 308 G/A TNF v rmci souboru pacient s RA, a to mezi neerozivnŞ (stdium I) a erozivnŞ formou (stdium II-IV) tohoto onemocn§nŞ (Pa = 0,05). Mezi pacienty s erozivnŞ formou RA bylo patrnŚ vŞce ne trojnsobn§ vyŞ zastoupenŞ genotypu GG oproti ostatnŞm genotypm AA + AG (47,0 % vs. 74,6 %; OR = 3,35; 95 % CI 0,99-11,45; P = 0,03). Jestlie jsem proti sob§ srovnal pacienty s nejt§Şm stupn§m postienŞ (st

Abstract:

dium IV) a pacienty s neerozivnŞ formou RA (stdium I), byla prevalence GG genotypu oproti AA + AG genotypm vŞce ne estinsobn§ vyŞ u pacient s nejt§Şm stupn§m postienŞ (stdium IV) (40,0 % vs. 87,0 %; OR = 6,29; 95 % CI 1,19-36,43; P = 0,01). Statisticky významný byl rovn§ rozdŞl v alelických frekvencŞch polymorfismu - 308 G/A TNF mezi t§mito dv§ma skupinami (Pa = 0.02). NavŞc se mi podailo prokzat asociaci polymorfismu - 308 G/A TNF s výŞ plazmatickŚ hladiny LDL-cholesterolu u pacient s RA. Statisticky významný rozdŞl jsem zaznamenal v alelických frekvencŞch mezi pacienty s normlnŞ a se zvýenou plazmatickou hladinou LDL-cholesterolu (Pa = 0,047). Prevalence genotypu GG oproti AA + AG genotypm byla vŞce ne dvojnsobn§ vyŞ mezi pacienty se zvýenou plazmatickou hladinou LDL-cholesterolu (60,7 % vs. 77,9 %; OR = 2,29; 95 % CI 0,94 5,63; P = 0,035). Výsledky mŚ prce naznaujŞ asociaci polymorfismu 308 G/A v genu pro TNF se zvanostŞ prb§hu RA. ZŞskanŚ výsledky navŞc naznaily monou asociaci mezi tŞmto polymorfismem a plazmatickou hladinou LDL-cholesterolu u pacient s RA.

Abstract:

Rheumatoid arthritis (RA) is recognized as a polygenic disorder, with genetic polymorpfisms contributing to both the sesceptibility and the severity of the disease. TNF is one of the most potent pro-inflammatory cytokine known to regulate cell survival, death, and/or growth, depending on cell types. Several alleles from the HLA-DRB1 locus have been associated with RA, thus the interest in polymorphisms of the TNF gene, which is located within the highly polymorphic HLA class III region on chromosome 6 (6p21.3). TNF is detected at highly enhanced concentrations in the plasma and synovial fluids of patients with RA. Over-expression of this protein is probably related to polymorphisms in the TNF gene. Cardiovascular disease is responsible for 35 50 % of RA deaths, whereas, in the general adult population, coronary heart disease is responsible for 1/4 deaths in males and 1/5 deaths in female. This increased risk may be attributable to RA-specific risk factors such as hyperhomocysteinemia, disease-related dyslipidemia or vascular inflammation, or to morbidity related to medications and high levels of TNF. The 308 G/A TNF gene variant has been associated with obesity, dyslipidemia, insulin resistance, and hypertension, a risk factors for coronary heart disease. The aim of the study was to associate the promoter polymorphism - 308 G/A in TNF gene with RA. A total of 130 patients with RA according to the ACR revised criteria (98 women and 32 men, age range 20 - 79 years, median 56,2 years) were consecutively recruited into the study during a 3 years period. All patients had a disease duration of at least 2 years. Patients were classified into four groups according to the grade of radiographic involvement of the hand and wrist (grade I - IV according to Steinbrocker radiographic score: 15 patients of grade I: non-erosive RA and 114 patients of grade II - IV: erosive RA), and control group of 150 subjects of similar age and sex distribution were available for this study. The study was approved by the Committee for Ethics of Medical Experiment on Human Subjects, Faculty of Medicine, Masaryk University, Brno. Genomic DNA was extracted from peripheral leukocytes using standard proteinase K technique. The polymorphism 308 G/A of TNF gene was detected by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis using restriction enzyme BsmFI (New England Biolabs). Within all groups of subjects, distributions of genotype and allelic frequencies and their differences were calculated using chi2-test and Fischers exact test. Consistency of genotype frequencies with the Hardy - Weinberg equilibrium was tested using chi2 test. Odds ratio (OR) and 95 % confidence interval were calculated to estimate the relative risks related to RXRA polymorphism and ABO blood groups. To calculate the significance of OR, Fischers exact test was used. The program package Statistica v. 6.0 (Statsoft Inc., Tulsa, UK) was used to analyse data. The power of test was calculated by EpiInfo6. We observed no differences in genotype distributions and/or allelic frequencies of 308 G/A TNF polymorphism between RA patients and control group. Significant association to - 308 G/A TNF polymorphism was found between non-erosive and erosive RA in allelic frequencies (Pa = 0.05). Compared to GG genotype with AA and AG genotypes, the prevalence of GG genotype was more than 3-fold higher within patients with erosive RA (47.0 % vs. 74.6 %; OR = 3.35; 95 % CI 0.99-11.45; P = 0.03). When the worst grade of erosive RA (grade IV) was compared to non-erosive RA (grade I) the prevalence of GG genotype was more than 6-fold higher within patients with the worst grade of erosive RA (40.0 % vs. 87.0 %; OR = 6.29; 95 % CI 1.19-36.43; P = 0,01). Significant association to - 308 G/A TNF polymorphism was also found between these two RA groups in allelic frequencies (Pa = 0.02). In adition significant association to - 308 G/A TNF polymorphism was found between RA pat

Abstract:

ients with normal and enhanced LDL-cholesterol plasma concentration in allelic frequencies (Pa = 0.047). Compared to GG genotype with AA and AG genotypes, the prevalence of GG genotype was more than 2-fold higher within patients with enhanced LDL-cholesterol plasma concentration (60,7 % vs. 77,9 %; OR = 2.29; 95 % CI 0.94 5.63; P = 0.035). In conclusion, results obtained in this study suggest an association of - 308 G/A TNF polymorphism with the severity of RA. In adition our results suggest a possible association of this polymorphism with LDL-cholesterol plasma concentration in RA patients.

Keywords

RevmatoidnŞ artritida, gen, polymorfismus, TNFŚÁ Rheumatoid arthritis, gene, polymorphism, TNFŚÁ

Language used: Czech

Date on which the thesis was submitted / produced: 17. 5. 2006

Thesis defence

• Date of defence: 29. 6. 2006
• Supervisor: prof. MUDr. Anna Vašků, CSc.

Masaryk University

Faculty of Medicine

Doctoral programme / field:
General Medicine / Physiology and Pathological Physiology