| Sažetak | Nedostatna aktivnost S-adenozilhomocistein hidrolaze (SAHH) je nasljedni poremećaj u ciklusu metabolizma metionina koji se, u do sada opisanih bolesnika, uglavnom očitovao psihomotoričkim zaostajanjem, zakašnjelom mijelinizacijom mozga, mišićnom slabošću, znakovima hepatopatije s koagulopatijom. Biokemijska obilježja bolesti su povišeni metaboliti ciklusa metionina S-adenozilmetionin (AdoMet) i S-adenozilhomocistein (AdoHcy), hipermetioninemija (koja ne mora uvijek biti prisutna), te trajno povišena aktivnost kreatin kinaze (CK). Enzim SAHH zbog uloge u razgradnji AdoHcy, glavnog inhibitora transmetilacijskih reakcija u stanicama, ima ključnu ulogu u održavanju homeostaze procesa metilacije, jednog od najvažnijih epigenetskih fenomena u organizmu. AdoMet je u tim reakcijama donor metilne skupine. Upravo je bolest zbog nedostatne aktivnosti SAHH idealan biološki model za proučavanje posljedica narušenih transmetilacijskih reakcija.
U ovoj je disertaciji prva hipoteza bila da ćemo među osobama s etiološki nerazjašnjeno trajno povišenom aktivnošću CK, sa simptomima slabosti mišića ili bez njih, otkriti osobe s nedostatnom aktivnošću SAHH. Druga hipoteza je bila da će ispitanici imati različit metilacijski potencijal u odnosu na referentnu skupinu, a treća hipoteza je bila da će ispitanici imati različitu učestalost polimorfizama rs13043752 i rs41301825 gena AHCY u odnosu na zdrave ispitanike.
Da bismo provjerili hipoteze i ispunili ciljeve istraživanja okupili smo 100 ispitanika sa slabošću mišića nerazjašnjene etiologije i trajno povišenom aktivnošću CK, odnosno osobe s trajno povišenom aktivnošću CK, a bez simptoma, i u njih u punoj krvi izmjerili AdoMet i AdoHcy, te potom izračunali metilacijski potencijal (omjer AdoMet/AdoHcy).
Nismo otkrili bolesnike s nedostatnom aktivnošću SAHH. Također, nije nađena statistički značajna razlika u metilacijskom potencijalu između ispitanika i referentne skupine. Međutim, neki su ispitanici, gledano pojedinačno, imali snižen metilacijski potencijal (manji od 10). Snižen metilacijski potencijal u tih bolesnika je pokazatelj narušenih metilacijskih procesa, koji su moguće negativno utjecali na ekspresiju njihove bolesti, odnosno stanja.
Ispitivana skupina je imala statistički značajno viši raspon koncentracija i viši medijan AdoMet i AdoHcy u punoj krvi. Jedan dio ispitanika je imao višestruko povišene koncentracije oba metabolita, koje su vjerojatno dovele do narušenih metilacijskih procesa, neovisno o tome što je metilacijski potencijal u tih ispitanika bio normalan. U disertaciji je obrazloženo na koji način povišene koncentracije AdoMet i AdoHcy mogu utjecati na metilacijske procese unutar stanice, neovisno o promjeni metilacijskog potencijala. Koncentracije AdoMet i AdoHcy su u ispitanika jasno korelirale, dok je samo koncentracija AdoHcy korelirala, i to negativno, s metilacijskim potencijalom. Nismo dokazali korelaciju koncentracije AdoMet, AdoHcy, niti metilacijskog potencijala s aktivnošću CK u krvi, dobi ispitanika niti sa spolom.
U svrhu ispunjenja zadnjeg cilja smo u skupini ispitanika ispitali učestalost polimorfizama rs13043752 i rs41301825 gena AHCY koji su povezani s promijenjenom funkcijom proteina u uvjetima in vitro. Na ova dva polimorfizma testirano je 77 ispitanika. Nije nađena statistički značajna razlika u prevalenciji navedenih polimorfizama, u odnosu na zdrave ispitanike. Zbog toga smo zaključili da polimorfizmi sami za sebe, u heterozigotnom obliku nisu povezani s češćom pojavom slabosti mišića, odnosno povišenom aktivnosti CK u krvi. Međutim, potencijalni utjecaj navedenih polimorfizma na težinu simptoma ili na aktivnost CK u krvi, ne možemo sa sigurnošću isključiti.
Tijekom ispunjavanja ciljeva ove disertacije i obrade ispitanika u nekih smo postavili dijagnozu rijetkih nasljednih bolesti mišića.
Zaključno, rezultati koje smo dobili ukazuju da dio osoba s mišićnom slabošću nejasne etiologije i trajno povišenom aktivnošću CK, te onih s izolirano trajno povišenom aktivnošću CK, ima narušene metilacijske procese. Daljnja istraživanja su potrebna da se utvrde točni patološki mehanizmi, stupanj poremećaja metilacije gena odgovornih za strukturu i funkciju mišića te dismetilacije proteina mišića u tih osoba, kao i utjecaj opisanih promjena na primarni patološki proces, odnosno ekspresiju same bolesti. |
| Sažetak (engleski) | S-adenosylhomocysteine hydrolase (SAHH) deficiency is a rare autosomal recessive methylation disorder. Clinical presentation includes psychomotor retardation, muscle weakness, delayed myelination of cerebral white matter, hepatopathy and coagulopathy. Biochemical hallmarks of this disease are increased S-adenosylmethionine (AdoMet), S-adenosylhomocysteine (AdoHcy) and hypermethioninemia (which is not a constant finding). Great majority of patients had muscle weakness with permanently elevated activity of creatine kinase (CK). The SAHH hydrolyzes AdoHcy, which is the major inhibitor of various methyltransferases, and, as a consequence, represents an important regulator of cellular transmethylation reactions - keeping the ratio between AdoMet (an universal methyl donor) and AdoHcy, known as methylation potential, within the optimal range. The pathogenesis of SAHH deficiency is rather complex. However, the decreased AdoMet/AdoHcy ratio and inhibition of various methyltransferases probably contributes to this complexity. Therefore, this disease is a unique biological model for studying disturbed methylation processes.
The first hypothesis of this thesis was that we would be able to diagnose patients suffering from SAHH deficiency among subjects with myopathy of unknown origin with permanently elevated CK, or with asymptomatically permanently elevated CK only. The second hypothesis was that this group of subjects would have changed methylation ratio when compared to referral group. The third hypothesis was that this group would have a different prevalence of the AHCY rs13043752 and rs41301825 polymorphisms, when compared to healthy subjects.
In order to reach goals of this study, we included 100 examinees with permanently elevated CK. The majority of them had muscle weakness, but some were asymptomatic. Known muscular disease or confirmed other condition which could cause elevated CK were exclusion criteria. AdoMet and AdoHcy were measured in the whole blood, their ratio was calculated for obtaining the value of the methylation potential in order to compare it with the values obtained in the refferal group.
We did not identify any patient with SAHH deficiency, in the group of examinees. There was no statistical significance in the difference between methylation potential between these two groups. However, certain examinees (5% of them) had low methylation potential, which is probably an indicator of disturbed methylation processes.
Statistically significant difference between these two groups was found in relation to concentrations of AdoMet and AdoHcy. We found that studied subjects had higher concentrations of AdoMet and AdoHcy. Certain examinees had significantly increased concentrations of both metabolites, which may be considered as an indicator of changed methylation reactions, unrelated to methylation potential. Evidences obtained from the literature and from our own unpublished data, were used for explaining how increased AdoMet and AdoHcy, even without changes in methylation potential, may disturb transmethylation processes within the cells, and how these processes can cause muscle pathology.
In our group of examinees, AdoMet and AdoHcy concentrations were in strong correlation, as was concentration of AdoHcy and methylation potential (that particular correlation was negative). There were no statistically significant correlations attributed to metabolites or methylation index with the activity of CK in the blood, nor was any correlation between the age or gender of the subjects.
In order to reach the last goal of this thesis, the prevalences of polymorphisms rs13043752 and rs41301825 of the AHCY gene were studied in examined group. We tested 77 subjects and didn't find higher prevalence of these polymorphisms, when compared to healthy subjects. Although the prevalences did not differ between the groups, we cannot exclude the possibility that this polymorphisms may be associated with the higher risk for muscle weakness or elevated CK in the blood.
During this study and clinical workup of the subjects we diagnosed several rare inherited muscular diseases.
In conclusion, our results show that, among subjects with myopathy of unknown origin with permanently elevated CK, or asymptomatically permanently elevated CK, some subjects had disturbed methylation processes. Further studies are needed to elucidate pathogenesis of those changes and consequences of disturbed methylation to the primary pathological processes, and expression of the disease. |
