Analiza celotnega genoma bolnikov s predhodno genetsko neopredeljeno izgubo sluha

Zadravec, Anja (author), Trebušak Podkrajšek, Katarina (mentor), Battelino, Saba (co-mentor)

Izguba sluha je ena najpogostejših senzoričnih pomanjkljivosti, za katero trpi več kot 1,5 milijarde ljudi po celem svetu. Glavni vzroki za izgubo sluha so prirojena ali zgodnja izguba sluha v otroštvu, kronične okužbe srednjega ušesa, izguba sluha zaradi hrupa, s starostjo povezana izguba sluha in ototoksična zdravila, ki poškodujejo notranje uho. Obstajajo tri glavne vrste izgube sluha in sicer prevodna (konduktivna) izguba sluha, senzorično nevralna izguba sluha in mešana izguba sluha. Z leti so odkrili genetske variante v več kot 200 genih, ki prispevajo h klinično heterogenim oblikam izgube sluha, kar je osnova za genetsko testiranje in povezano genetsko svetovanje. Namen magistrske naloge je bil opredeliti genetski vzrok izgube sluha v družinah z več člani z izgubo sluha, kjer predhodno uporabljeni genetski pristopi niso bili uspešni. Uporabili smo sekvenciranje celotnega genoma, ki omogoča analizo vseh genomskih regij in določanje vseh vrst genetskih sprememb, tudi sprememb v številu kopij. Kot potrditveno metodo smo uporabili sekvenciranje po Sangerju ali metodo MLPA. V raziskavo je bilo vključenih 15 preiskovancev s klinično diagnozo izgube sluha oziroma naglušnosti. Od tega je 8 preiskovancev moškega spola in 7 preiskovank ženskega spola. Preiskovanci prihajajo iz 15 različnih družin in imajo povprečno starost 19 let. S sekvenciranjem naslednje generacije smo pri šestih preiskovancih opredelili 9 različnih sprememb v genih AIFM1, COL4A3, GATA3, MYH14, PEX6, OTOA, OTOG in TECTA. Med njimi smo opredelili šest sprememb, ki predhodno še niso bile opisane v bazi podatkov o bolezenskih spremembah v humanih genih (HGMD). Pri še enem od preiskovancev je bila po sekvenciranju naslednje generacije opredeljena večja delecija v genu EYA4. S sekvenciranjem po Sangerju smo potrdili šest genetskih sprememb. S potrditveno metodo smo ovrgli prisotnost delecije v genu EYA4. Metoda sekvenciranja naslednje generacije kot pristop genetske diagnostike prirojene izgube sluha omogoča zanesljivo in predvsem hitro opredelitev vzročnih genetskih sprememb v več genih hkrati. To smo uspeli dokazati, saj je bil delež preiskovancev z opredeljenimi vzročnimi spremembami v tej raziskavi primerljiv tistemu v literaturi.

• Language: Slovenian
• Keywords: izguba sluha, genetika, sekvenciranje naslednje generacije, sekvenciranje po Sangerju, analiza celotnega genoma
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-145209
• Publication date in RUL: 13.04.2023

Secondary language

• Language: English
• Title: Whole-genome analysis in patients with previously genetically un-characterised hearing loss

Abstract

Hearing loss is one of the most common sensory deficits, affecting more than 1.5 billion people worldwide. The main causes of hearing loss are congenital or early childhood hearing loss, chronic middle ear infections, noise-induced hearing loss, age-related hearing loss, and ototoxic drugs that damage the inner ear. There are three main types of hearing loss: conductive hearing loss, sensorineural hearing loss, and mixed hearing loss. Over the years, genetic variants in more than 200 genes have been discovered that contribute to clinically heterogeneous forms of hearing loss, enabling genetic testing and genetic counseling. The aim of this study was to define the genetic cause of hearing loss in families with multiple members with hearing loss, where previously used genetic approaches have not been successful. We will use whole genome sequencing, which will allow the analysis of all genomic regions and determination of all types of genetic changes, including copy number variations. We will use Sanger sequencing or MLPA as a confirmatory method. Fifteen subjects with a clinical diagnosis of hearing loss were enrolled in this study. Of these, eight subjects are male and seven are female. The subjects come from 15 different families and have an average age of 19 years. Next-generation sequencing identified 9 different genetic variants in the genes AIFM1, COL4A3, GATA3, MYH14, PEX6, OTOA, OTOG and TECTA in six of the people included in the study. Among them, we identified six genetic variants that had not been previously described in the Human Gene Mutation Database (HGMD). One of the subjects was found to have a large deletion in the EYA4 gene after next-generation sequencing. Six genetic variants were confirmed by Sanger sequencing. The presence of a deletion in the EYA4 gene was excluded by the confirmatory method. The next-generation sequencing approach to genetic diagnostics of congenital hearing loss allows rapid and reliable identification of causal genetic variants in many genes simultaneously, as the proportion of subjects studied here with identified causal variants in genes was comparable to that reported in the literature.

• Keywords: hearing loss, genetics, next generation sequencing, Sanger sequencing, whole genome analysis