Ispitivanje senzitivnosti genoma kod pacijenata sa bolestima hromozomske nestabilnosti .

Abstract

U cilju ispitivanja senzitivnosti genoma i potvrde klinički postavljene dijagnoze bolesti hromozomske nestabilnosti, korišćeni su uzorci dve grupe pacijenata sa kliničkom slikom: Fankonijeve anemije (FA) i Nijmegenovog sindroma nestabilnosti (NSN) (eng. Nijmegen Breakage syndrome, NBS). Specifična hipersenzitivnost ćelija obolelih od FA na diepoksibutan (DEB), odnosno kod obolelih od NSN na bleomicin (BLC), iskorišćena je u diferencijalno dijagnostičke svrhe. Studija je obuhvatila ukupno 100 pacijenata: 90/100 sa kliničkom slikom FA i 10/100 pacijenata sa kliničkim znacima NSN, koji su dijagnostikovani na Institutu za zdravstvenu zaštitu majke i deteta Srbije „Dr Vukan Čupić“. U detekciji DEB-om indukovane hromozomske nestabilnosti obolelih od FA primenjene su standardne citogenetičke metode. Za potvrdu ili isključenje mozaičnog fenotipa FA korišćene su dodatne metode: analiza oštećenja DNK komet testom i protočno citometrijska analiza zastoja ćelija u G2 fazi ćelijskog ciklusa. Hromozomska osetljivost ćelija obolelih od NSN je analizirana primenom bleomicinovog testa (BLC test). Molekularna analiza prisustva ili odsustva c.657_661del5 mutacije u NBN genu obolelih od NSN, urađena je korišćenjem modifikovane PCR metode uz analizu heterodupleksa. Kod 11,11% (10/90) pacijenata pod sumnjom da boluju od FA (FA grupa), otkrivena je povećana hromozomska osetljivost na DEB u odnosu na kontrolnu grupu (Mann-Whitneyev test: p < 0,05), dok su kod 3,33% (3/90) pacijenata dobijene granične vrednosti iste analize (FA* grupa). U cilju preciznijeg klasifikovanja pacijenata u FA grupi, primenjen je indeks hromozomske fragilnosti. Na ovaj način je u FA grupi, kod 40% (4/10) pacijenata otkriven ćelijski fenotip FA nemozaičnog tipa, dok je kod 60% (6/10) pacijenata dobijen mozaični odgovor na DEB. Kod svih pacijenata sa mozaičnim fenotipom FA, kao i kod dva pacijenta sa graničnim vrednostima diepoksibutanovog testa (DEB testa), daljim analizama primenom komet testa i analizom zastoja ćelija u G2 fazi, potvrđena je dijagnoza FA. U drugoj grupi od 10 bolesnika sa sumnjom da boluju od NSN analizirana je BLC-om indukovana hromozomska nestabilnost, koja je bila povećana kod 40% (4/10) pacijenata (NBS grupa). Kod 30% (3/10) pacijenata dobijeni rezultati su se preklapali sa rezultatima kontrolne grupe (ne-NBS grupa), dok je kod preostala tri pacijenta analiza bila neuspešna zbog slabog mitotskog indeksa. Molekularnim analizama je otkriveno homozigotno prisustvo c.657_661del5 mutacije u NBN genu kod sva četiri pacijenta iz NBS grupe, kao i kod tri pacijenta kod kojih je citogenetička analiza bila neuspešna, i tako potvrđena konačna dijagnoza NSN. Na osnovu dobijenih rezultata može se zaključiti da su analize korišćene u ovoj studiji omogućile potvrdu klinički postavljene dijagnoze FA i NSN, što opravdava njihovu dalju primenu u diferencijalnoj dijagnostici ovih oboljenja.

In order to determine the sensitivity of the genome and to confirm a clinical diagnosis of chromosomal instability disorders, blood samples from patients with clinical suspicion of Fanconi’s anemia (FA) and Nijmegen Breakage syndrome (NBS), were collected for chromosome fragility evaluation by the diepoxybutane (DEB) and bleomycin (BLC) tests. The study considered a total of 100 patients: 90/100 with the hematological and/or congenital phenotypic symptoms reminiscent of FA and 10/100 patients with clinical features of NBS, all diagnosed at the Mother and Child Health Care Institute of Serbia "Dr Vukan Čupić”. The DEB-induced chromosomal fragility analysis in patients with FA symptoms was carried out by using standard cytogenetic methods. The additional methods, such as: DNA damage analysis by comet assay and flow cytometric analysis of G2 phase cell cycle arrest, were used in order to confirm FA phenotype in patients with mosaic cytogenetic response to DEB. Chromosomal sensitivity of cells in patients with NBS symptoms was analyzed using a BLC test. Molecular analysis for the presence of the c.657_661del5 mutation in NBN gene in patients with NBS was carried out using modified PCR method and heteroduplex analysis on PAGE gel. In this study 11.11% (10/90) of patients with clinical suspicion of FA were found to have an increased chromosomal sensitivity to DEB (FA group), comparing to healthy controls (Mann-Whitney test: p < 0.05), while 3.33% (3/90) of patients showed borderline results of the same analysis (FA* group). The chromosome fragility index was used in order to provide a clear cut-off diagnostic level distinguishing FA mosaic from other patients in the FA group. In this group, 40% (4/10) of FA patients revealed non-mosaic FA phenotype, while the remaining 60% (6/10) of FA patients showed a mosaic response to DEB. In all of FA mosaic patients, and two patients with borderline DEB-sensitivity, further analyses were performed using comet assay and analysis of G2 phase cell cycle arrest, in order to confirm the diagnosis of FA. In the second group of 10 patients with clinical suspicion of NBS, the cytogenetic analysis revealed an increased chromosomal sensitivity to BLC in 40% (4/10) of them (NBS group). The BLC-induced chromosome fragility values in 30% (3/10) of these patients were overlapping those in the control group (non-NBS group). In the remaining of three patients the cytogenetic analysis was unsuccessful, due to the low mitotic index. Molecular analysis confirmed the presence of homozygous c.657_661del5 mutation in all seven NBS patients, including three patients with unsuccessful cytogenetic analysis, which confirmed the final diagnosis of NBS. Based on these results it can be concluded that the analyses used in this study are useful in confirmation of clinical diagnosis of FA and NBS, which justifies their further application in the differential diagnosis of these two diseases.