| Abstract | Cilj istraživanja: S obzirom da nedavni dokazi upućuju na moguću ulogu disregulacije
željeza u patogenezi multiple skleroze (MS), cilj istraživanja bio je testirati sljedeću hipotezu:
polimorfizmi HFE gena (C282Y i H63D), TF gena (C1/C2) i TNF- gena (–308 i –238), kao i
njihova međusobna interakcija, utječu na predispoziciju za razvoj MS i/ili na kliničku
ekspresiju bolesti.
Ispitanici i metode: U ispitivanje je uključeno 368 osoba oboljelih od MS (266 žena i 102
muškarca), od toga njih 79 (46 žena i 33 muškarca) iz područja većeg rizika (autohtoni
stanovnici Gorskog kotra i Kočevja) i 289 (220 žena i 69 muškaraca) iz podrucja niže
prevalencije MS (populacije Hrvatske i Slovenije). Bolesnici su udovoljavali dijagnostičkoj
kategoriji sigurne MS prema Poserovim kriterijima, a klinički su obrađeni sukladno EDMUS
protokolu. Kontrolnu skupinu cinilo je 368 zdravih dobrovoljnih davatelja krvi (86 autohtonih
stanovnika Gorskog kotara i Kočevja; 282 ispitanika s područja nižeg rizika) čija se
prosječna dob i spol nisu statistički znacajno razlikovali od MS bolesnika. Prisutnost
polimorfizama u HFE, TF i TNF- genu utvrđena je lančanom reakcijom polimeraze i
restrikcijom s odgovarajućim restrikcijskim endonukleazama.
Rezultati: Učestalost nositelja HFE C282Y mutacije veća je (7,1%) u MS bolesnika negoli u
kontrolnih ispitanika (3,8%), a statistički značajna razlika utvrđena je usporedbom MS
bolesnika s SP+RR tijekom bolesti i kontrole (p=0,026; OR=2,09; 95% CI=1,07-4,08), te je
prisutna između MS bolesnika i kontrole izvan žarišnog područja (p=0,019; OR=2,33; 95%
CI=1,13-4,84). Bolest se javlja ranije u bolesnika s C282Y mutacijom (p=0,035), dok utjecaj
mutacije na daljnu ekspresiju bolesti (trajanje, EDSS i PI) nije primijećen (p>0,05).
Frekvencija HFE H63D alela veća je u kontroli (15,6%) negoli u MS bolesnika (13,3%), a
razlika u učestalosti H63D homozigota između dvije skupine pokazuje graničnu vrijednost
statističke značajnosti (p=0,050; OR=0,29; 95 %CI=0,08-1,08). H63D mutacija s obzirom na
blagi učinak u taloženju željeza ne korelira znacajno (p>0,05) s težinom klinicke slike MS.
Nije utvrđena razlika (p>0,05) u frekvenciji TF C1/C2 genotipova i alela između MS
bolesnika i kontrole, kao ni razlika u ispitivanim kliničkim parametrima s obzirom na status
nositelja TF C2 alela. Usporedbom različitih kombinacija polimorfizama HFE i TF gena
između MS bolesnika i kontrole nije utvrđena značajna razlika (p>0,05), niti je utvrđen
učinak zajedničkog djelovanja ovih gena na ekspresiju bolesti. Broj ispitanika s TNF- -308 A
alelom veci je u kontroli (26,6%) negoli u MS bolesnika (20,9%) (p=0,069), dok je razlika u
žarištu statistički značajna (p=0,016; OR=0,41; 95 %CI=0,19-0,86). Polimorfizam TNF- -308
ne korelira statistički značajno (p>0,05) s težinom kliničke slike MS. Distribucije TNF- -238
genotipova i alela između MS bolesnika i kontrole statistički se značajno razlikuju (p<0,001),
pri čemu je veća učestalost TNF- -238 A alela utvrdena u MS bolesnika (10%) negoli u
kontroli (3,8%) (p=0,0008; OR=2,83; 95% CI=1,50-5,32). Nije utvrđena statistički značajna
razlika (p>0,05) u dobi nastupa, trajanju bolesti, EDSS i PI između bolesnika s TNF- -238 A
alelom, odnosno bez tog alela. Kombinacije potencijalno protektivnih alela HFE i TNF-
gena (C282Y-/TNF- -308AG+AA, C282Y-/TNF- -238GG) smanjuju rizik za MS, a
kombinacije rizičnih alela povećavaju taj rizik (H63D-/TNF- -238AG+AA) (p<0,05). TNF- -
308 i –238 polimorfizmi ne pokazuju značajnu interakciju s mutacijama HFE gena za većinu
promatranih kliničkih parametara. Statistički značajna razlika (p<0,05) međudjelovanja H63D
i TNF- -238, odnosno -308 polimorfizama, na trajanje bolesti odnosno PI može biti
posljedica manjeg broja ispitanika i njihovog progresivnog tijeka bolesti.
Zaključak: Dobiveni rezultati istraživanja ukazuju na moguć utjecaj HFE C282Y i TNF- -
238 A alela kao rizičnih čimbenika u predispoziciji za MS, dok je potencijalni protektivni efekt
HFE H63D i TNF- -308 A alela slabije izražen. TF C1/C2 polimorfizam ne utječe na
podložnost ni na kliničku ekspresiju bolesti. Utjecaj ovih polimorfizama na kliničke parametre
pokazuje da je HFE C282Y mutacija dobar prediktor ranijeg javljanja bolesti. Istraživanje
zajedničkog djelovanja HFE, TF i TNF- gena na predispoziciju i težinu klinicke slike u MS
ukazuje na mogućnost da TNF- -308 i -238 polimorfizmi u ovisnosti o prisutnosti mutacija
HFE gena djelomično modificiraju podložnost za bolest. |
| Abstract (english) | Objectives: Recent evidence has indicated a role for iron dysregulation in disease
pathogenesis. Thus, in our investigation, we tested the hypothesis that polymorphisms in
HFE (C282Y and H63D), TF (C1 and C2), and TNF- (-308 and -238), and interactions
among these polymorphisms, influence predisposition to and clinical presentation (age of
onset, duration, EDSS, and PI) of multiple sclerosis (MS).
Patients and methods: The study included 368 MS patients (266 females; 102 males).
Seventy-nine patients (46 females; 33 males) were from high-risk regions in Gorski kotar
and Kocevje, and 289 (220 females;69 males) patients were from other parts of Croatia and
Slovenia. The patients were recruited using the the criteria of Poser, and their clinical status
was evaluated using the EDMUS protocol. The control group comprised 368 unrelated
healthy blood donors, matched for geographic region, age, and gender. We analyzed the
HFE, TF, and TNF- gene polymorphisms using the PCR-RFLP method.
Results: The HFE C282Y carrier mutation frequency was higher (7.1%) in the MS patient
group than in the control group (3.8%). A statistically significant difference was observed
after exluding the high-risk group from Gorski kotar and Kocevje (p=0.019; OR=2.33; 95%
CI=1.13–4.84). We detected significant differences in the HFE C282Y carrier mutation
frequency between SP+RR MS patients and controls (p=0.026; OR=2.09; 95% CI=1.07–
4.08). A significantly earlier age of onset was found in carriers of the C282Y mutation
(p=0.035). The frequency of the HFE H63D mutation was higher (15.6%) in controls than in
MS patients (13.3%), and the difference in the frequency of H63D homozygotes between
MS patients and controls was of borderline significance (p=0.050; OR=0.29; 95% CI=0.08–
1.08). The H63D mutation exhibits a mild effect on iron overload and we found no corelation
between its presence and disease behavior (p>0.05). We were unable to detect significant
differences (p>0.05) in the frequencies of the TF C1/C2 genotypes and alleles between MS
patients and controls, and we found no differences in clinical parameters in carriers of the
TF C2 allele. We did not observe that interactions among HFE and TF gene polymorphisms
had any effect on either predisposition to MS or on on disease progression (p>0.05). The frequency of carriers with the TNF--308 A alelle was higher (26.6%) in the control group
than in the MS patient group (20.9%) (p=0.069), and we found a statistically significant
difference in the high-risk group from Gorski kotar and Kocevje (p=0.016; OR=0.41; 95%
CI=0.19–0.86). We found no evidence of a correlation between the TNF-308- gene
polymorphism and MS clinical severity. The distribution of the TNF-238- genotypes and
alleles in MS patients and controls was statistically significantly different (p < 0.001). The
TNF--238 A allele occurred at a higher frequency in MS patients (10.0%) than in controls
(3.8%) (p=0.0008; OR=2.83; 95% CI=1.50–5.32). However, there was no statistically
significant difference regarding disease progression in patients with or without the TNF-238-
A allele (p>0.05). Several combinations of HFE and TNF- alleles that have been
suggested to have a protective effect (C282Y-/TNF--308AG+AA, C282Y-/TNF--238GG)
were correlated with a diminished a risk for MS. The combination of the possible risk alleles
of the HFE i TNF- genes (H63D-/TNF--238AG+AA) did correlate with a higher risk of MS
(p<0.05). We did not observe any significant effects of TNF--308 or -238 polymorphisms
on most of the clinical parameters. However, there was a statistically significant association
(p<0.05) between interactions between HFE H63D and both TNF--308 and TNF--238
polymorphisms and disease duration and PI. But this could be the consequence of a lower
number of patients with these polymorphisms and progressive form of disease.
Conclusion: Our results indicate that HFE C282Y and TNF--238 gene polymorphisms
may be risk factors with respect to MS susceptibility. The possible protective effects of the
HFE H63D and TNF--308 gene polymorphisms were less pronounced. The TF C1/C2 gene
polymorphism had no effect on MS development. Furthermore, our results suggest that the
C282Y mutation is a good predictor for early onset of MS. Overall, our investigation of the
gene–gene interactions among HFE, TF, and TNF- indicates that they may have an effect
on MS susceptibility. |
