| Abstract | Cilj istraživanja: S obzirom na dokazanu reduciranu fibrinolizu u bolesnika, slučajeva multiple skleroze (MS) posljednjih se godina kao mogući rizični čimbenici istražuju geni koji posredno ili neposredno reguliraju taj proces. Stoga je osnovni cilj ovog rada istraživanje specifičnih polimorfizama gena za tkivni plazminogen aktivator (TPA), inhibitor plazminogen aktivatora-1 (PAI-1) i angiotenzin-konvertirajući enzim I (ACE) u MS bolesnika, te utvrđivanje njihovog pojedinačnog i međusobnog utjecaja na predispoziciju i kliničku ekspresiju bolesti.
Ispitanici i metode: U ispitivanje je uključeno 170 MS bolesnika koji žive na području Bosne i Hercegovine (BiH) i liječe se u KB Mostar i KUC Tuzla. Bolesnici, slučajevi MS prema McDonald-ovim kriterijima, klinički su obrađeni sukladno EDMUS protokolu. Za svakog bolesnika utvrđena je dob početka bolesti, početni simptomi/znaci, familijarna pojavnost bolesti, stupanj invaliditeta (EDSS), dužina trajanja bolesti, te indeks progresije (PI). Kontrolnu skupinu činilo je 170 zdravih dobrovoljnih davatelja krvi, koji u svojim obiteljima nemaju neurodegenerativnih bolesti. Svakom bolesniku određen je kontrolni ispitanik istog spola, dobi, mjesta rođenja i življenja, te etničke pripadnosti. Za identifikaciju indel polimorfizama u TPA i ACE genima korištena je alel-specifična PCR metoda, a za polimorfizam 4G/5G u promotorskoj regiji PAI-1 gena PCR-RFLP.
Rezultati: U ukupnom uzorku bolesnika uočena je značajna razlika u dobi nastupa bolesti (p=0,023), koja je za tri godine bila niža u muškaraca (27,57,0) negoli žena (30,58,3). Isti trend je zamijećen u relapsno-remitirajućim (RR) (p=0,012) i relapsno-remitirajućim (RR) + sekundarno progresivnim (SP) (p=0,023) podskupinama bolesnika, dok su primarno-progresivni (PP) bolesnici imali kasniji početak bolesti (34 godine) i jednak u oba spola. Prosječna dužina trajanja bolesti iznosila je 8,2 godine (SD 4,7) i bila značajno kraća (p=0,004) u žena (5,95,1) negoli u muškaraca (9,17,0) s RR tipom MS, dok kod SP i PP bolesnika nije bilo razlike među spolovima. Srednja vrijedost EDSS u ukupnom uzorku bolesnika iznosila je 3,4 (SD 2,8), a značajna razlika u skali invalidnosti (p=0,031) opažena je samo kod SP bolesnika kod kojih je EDSS bio viši u žena (8,34,3) negoli muškaraca (5,12,0). Ispitivanje TPA I/D polimorfizma je ukazalo da su MS bolesnici imali veću učestalost II genotipa (31,8%) i I alela (53,5%) u odnosu na kontrolne ispitanike (II=24,7%; I=50,3%), dok je kod PP bolesnika frekvencija I alela (40,0%) bila niža od one u kontrolnoj skupini (50,3%), ali razlike nisu bile značajne (p0,05). Distribucije PAI-1 i ACE I/D genotipova i alela su bile podjednako raspoređene u skupinama bolesnika i kontrolnih ispitanika, kao i podskupinama formiranim prema spolu i tijeku bolesti. Uočeno je da su PP bolesnici imali veću učestalost rizičnog 4G4G genotipa (30,0%) i 4G alela (60,0%) u odnosu na kontrolne ispitanike (4G4G=25,9%; 4G=51,5%), kao i veću učestalost DD genotipa (50,0%) i D alela (60,0%) u odnosu na kontrolu, ali razlike ipak nisu dosegle razinu statističke značajnosti (p0,05). Također, u PP bolesnika opažen je trend kasnijeg nastupa bolesti od 10 godina (p=0,095), kao i kraćeg trajanja za 6 godina (p=0,057) u bolesnika s 4G4G genotipom u odnosu na ostale PAI-1 genotipove. Kod RR+SP bolesnika EDSS je značajno ovisio o interakciji TPA I/D i PAI-1 polimorfizma kod muških bolesnika (p=0,007), od kojih su najvišu vrijednost imali oni s TPA II i PAI-1 4G5G genotipovima, a također se pokazao ovisnim o međudjelovanju sva tri polimorfizma, premda s manjom značajnošću (p=0,023). U bolesnica je zamijećeno da je na trajanje bolesti značajno utjecala interakcija PAI-1 i ACE I/D polimorfizama (p=0,012), pri čemu su one s 4G4G i DD genotipovima ispoljavale kraće trajanje bolesti od 2-6 godina u odnosu na ostale kombinacije genotipova.
Zaključak: U ovoj studiji su se po prvi puta kao mogući rizični faktori u MS analizirali pojedinačni i međusobni utjecaj specifičnih polimorfizama u genima koji direktno (TPA i PAI-1) ili indirektno (ACE) reguliraju proces fibrinolize u MS bolesnika. Dobiveni rezultati ukazuju da ne postoji značajan utjecaj polimorfizama TPA, PAI-1 i ACE gena, niti njihovog međudjelovanja, na predispoziciju za MS u ispitanih bolesnika. Uočeni trendovi u povezanosti pojedinih genotipova i njihovih kombinacija s pojedinim kliničkim karakteristikama, od kojih su neki dosegli razinu statističke značajnosti, govore o mogućem učinku zajedničkog djelovanja TPA, PAI-1 i ACE gena na kliničku ekspresiju bolesti u BiH. Osim što predstavlja prvo genetičko istraživanje MS u BiH, studija donosi i značajne podatke o genetičkoj strukturi navedene populacije, te doprinosi istraživanju drugih multifaktorijalnih bolesti u ovoj regiji. |
| Abstract (english) | Objectives: Given the proved reduced fibrinolysis in MS patients, in the recent years the genes which regulate this process have been studied as possible risk factors. Therefore, the main goal of the present study is the research of specific polymorfisms of the genes for tissue plasminogen activator (TPA), inhibitor of plasminogen activator -1 (PAI-1) and angiotensin-converting enzyme I (ACE) in MS patients, and establishment of their individual and mutual influence on the predisposition and clinical expression of the disease.
Patients and methods: The research included 170 patients living in Bosnia and Herzegovina (BiH) and being treated in KB Mostar and KUC Tuzla. The patients were recruited using the the criteria of McDonald, and their clinical status was evaluated using the EDMUS protocol. For each patient the age of the onset of the disease was established, the initial symptoms/marks, familial incidence of the disease, the degree of disability (EDSS), duration of the disease, and the progression index (PI). The control group consisted of 170 healthy voluntary blood donors, who do not have neurodegenerative diseases in their families. Each patients was matched with a control of the same sex, age, place of birth and living, and ethnicity. Genotyping of TPA I/D, PAI-1 4G/5G and ACE I/D polymorphisms was performed according to the allele-specific PCR and PCR-RFLP methods.
Results: In the total sample of the BiH patients a significant difference was observed in the age of the disease onset (p=0,023), which was three years lower in male patients (27,5+7,0) than in female patients (30,5+8,3). The same trend was observed in RR (p=0,012) and RR+SP (p=0,023) subgroups of the patients, while PP patients had later
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onset of the disease (34 years of age) and it was the same in both sexes. The average duration of the disease was 8,2 years (SD 4,7) and was significantly shorter (p=0,004) in female patients (5,9+5,1) than in male patients (9,1+7,0) with RRMS, while SP and PP patients did not show any difference between the sexes. The mean EDSS in the total sample of the patients was 3,4 (SD 2,8), and a significant difference in the degree of disability (p=0,031) was observed only in SP patients whose EDSS was higher in female patients (8,3+4,3) than in male patients (5,1+2,0). The study of TPA I/D polymorphism showed that MS patients had higher frequency of II genotype (31,8%) and I alleles (53,3%) in comparison with the control group (II=24,7%;I=50,3%), while the PP patients had the frequency of I alleles (40,0%) lower than controls (50,3%), but the difference was not significant (p<0,05). The distribution of PAI-1 and ACE I/D genotypes and alleles was equally distributed in the groups of the patients and the control group, as well as in the subgroups stratified by sex and the disease course. It was observed that PP patients had higher frequency of risk 4G4G genotype (30,0%) and 4G alleles (60%) in comparison with the control group (4G4G=25,9%, 4G=51,5%), as well as higher frequency of DD genotype (50,0%) and D alleles (60,0%) in comparison with the controls, but the differences did not reach the level of statistical significance (p<0,05). Also, in the PP patients it was observed the trend of later onset of the disease of 10 years (p=0,095), as well as shorter duration by 6 years (p=0,057), in the patients with 4G4G genotype in comparison with the other PAI-1 genotypes. In RR+SP patients EDSS significantly depended on the interaction of TPA I/D and PAI-1 polymorphisms in the male patients (p=0,007) of whom the ones with TPA II and PAI-1 4G5G genotypes had the highest value, and also it proved to be dependant on the interaction of all three polymorphisms, although with lower significance (p=0,023). In the female patients it was
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noticed that the duration of the disease was significantly influenced by the interaction of PAI-1 and ACE I/D polymorphisms (p=0,012), while the ones with 4G4G and DD genotypes had shorter duration of the disease by 2-6 years in comparison with the other combinations of genotypes.
Conclusion: This study represents the first report describing the individual and mutual influence of specific polymorphisms in genes which directly (TPA and PAI-1) or indirectly (ACE) regulate the process of fibrinolysis in MS patients. The obtained results show that there is no significant influence of TPA I/D, PAI-1 4G/5G and ACE I/D polymorphisms, nor their interaction on the predisposition for MS in BiH patients. The observed trends in the connection of individual genotypes and their combinations with certain clinical characteristics, of which some have reached the level of statistical significance, suggest the possible effects of the joint influence of TPA, PAI-1 and ACE genes on the clinical expression of the disease in BiH. In addition to being the first genetic investigation of MS in BiH, the study brings significant data on the genetic structure of the stated population, and contributes to the research of other multifactorial diseases in this region. |
