Ispitivanje povezanosti promena u mitohondrijskom genomu sa fenotipskim karakteristikama pacijenata sa Leberovom hereditarnom optičkom neuropatijom

Abstract

Cilj ove doktorske disertacije bila je detaljna genotipska i fenotipska karakterizacija grupe pacijenata sa kliničkom slikom Leberove hereditarne optičke neuropatije (LHON) u akutnoj ili hroničnoj fazi bolesti i funkcionalna analiza mitohondrijske funkcije kod nosilaca mogućih novih patogenih mutacija. Metodologija: U grupi od 82 pacijenata sa kliničkom slikom LHON-a urađena je genetska analiza (MLPA, sekvenciranje nove generacije mitohondrijske DNK [mtDNK], celog egzoma i genoma), kao i detaljna fenotipska karakterizacija (vidna oštrina, kolorni vid, optička koherentna tomografija [OCT] i elektrofiziologija). Karakteristike genetski različitih grupa međusobno su poređene. Kod nosilaca novih mogućih patogenih mutacija izvedeni su testovi mitohondrijske funkcije (respirometrija visoke rezolucije Oroboros i protočna citometrija). Metodom spekularne mikroskopije, utvrđivali smo i broj endotelnih čelija rožnjače. Rezultati: Kod 17 pacijenata je identifikovan genetski uzrok LHON-a, a kod 6 pacijenata promene u mitohondrijskoj DNK (retke mutacije) koje su mogući uzročnici pojave LHON-a. Testovi mitohondrijske funkcije su kod ovih pacijenata pokazali smanjenu aktivnost kompleksa I lanca prenosilaca elektrona, povećanu produkciju slobodnih kiseoničnih radikala (ROS) i depolarizaciju unutrašnje membrane miohondrija, što ukazuje na patogenu prirodu identifikovanih promena. Kod 2 pacijenta, čije su fenotipske karakteristike prikazane zasebno, potvrđen je autozomno recesivni LHON. Kod nekoliko pacijenata sa genetski potvrđenim LHONom došlo je do poboljšanja vidne oštrine. Poređenjem fenotipskih karakteristika ovih pacijenata sa pacijentima bez poboljšanja utvrđena je bolja očuvanost pojedinih slojeva retine u srednjem ETDRS prstenu, kao i bolje elektrofiziološke karakteristike u akutnoj i u hroničnoj fazi bolesti. Kod izvesnog broja pacijenata i pored jasnog fenotipa LHON-a nije identifikovana genetska osnova, ali su upoređivane fenotipske karakteristike ovih pacijenata sa grupom genetski potvrđenih LHON pacijenata radi identifikovanja kliničkih biomarkera bolesti. Utvrđeno je postojanje bolje očuvanosti debljine unutrašnje retine kod LHON pacijenata što se stoga može smatrati potencijalnim biomarkerom bolesti, kao i smanjen broj endotelnih ćelija rožnice. Zaključak: Rezultati prikazani u okviru ove teze pomogli su boljoj karakterizaciji pacijenata sa LHON-om, kao i identifikaciji potencijalnih biomarkera bolesti. Takođe su identifikovane nove promene u mtDNK, i potvrđena je njihova potencijalna patogenost.

The goal of this doctoral dissertation was to genotypically and phenotypically characterize a group of patients with a clinical presentation of Leber’s Hereditary Optic Neuropathy (LHON) in the acute and chronic phase of the disease and perform functional analysis of the mitochondrial function in carriers of the possible pathogenic novel changes in mitochondrial DNA. Methodology: Genetic analysis (MLPA, next-generation sequencing of the mtDNK, whole exome, and genome) and detailed clinical work-up (visual acuity testing, color vision, optical coherent tomography (OCT), and electrophysiology) were performed in a group of 82 patients with phenotypic characteristics of LHON. Patients were divided into groups based on the genetic testing results and their phenotypic characteristics were compared. In patients with identified novel mtDNK changes testing of mitochondrial function was performed. We have also determined the count of endothelial cells in patients with confirmed LHON. Results: We have identified the genetic cause of LHON in 17 patients, and possible causative pathogenic mtDNK changes (rare mutations) in 6 patients. Mitochondrial function testing in these patients showed decreased complex I-based respirometry, membrane depolarizationand increased ROS production. These results indicate the pathogenic nature of the novel changes. Autosomal recessive LHON as a distinct entity has been identified in two patients and presented separately. The improvement of the visual function has been noticed in 4 LHON patients. By comparing the phenotypic characteristics of LHON patients with and without visual function improvement we have concluded that there is better preservation of the retinal layers in the middle ETDRS ring and that these patients have better electrophysiology results both in the acute and the chronic phase in patients with improvement. A certain number of patients remained unclarified regardless of the typical LHON phenotype. We have compared the phenotypic characteristics of these patients with a group of patients with genetically confirmed LHON in order to identify clinical biomarkers of the disease and concluded that in genetically confirmed LHON patients there is a better preservation of the ETDRS center which could be a novel biomarker of the disease. We have also found a small decrease in the endothelial cell count in the cornea of LHON patients. Conclusions: The results presented in this doctoral dissertation have attributed to a better characterization of LHON patients and the identification of novel biomarkers of the disease. We have also identified novel changes in the mtDNK whose pathogenicity has been proven by the serial of the mitochondrial function tests.