Opredelitev sprememb v številu kopij gena za stereocilin (STRC) pri bolnikih z izgubo sluha

Damjanović, Diana (avtor), Trebušak Podkrajšek, Katarina (mentor), Battelino, Saba (komentor)

Najpogostejši vzrok senzorinevralne izgube sluha so spremembe v dednem zapisu, zato je genetsko testiranje bolnikov z izgubo sluha ključnega pomena pri dokončni opredelitvi bolezni. V populaciji bolnikov z izgubo sluha so vzroki povezani s spremembami v genih GJB2 in GJB6 dobro raziskani. Z razvojem sekvenciranja naslednje generacije pa se obsežneje analizirajo tudi drugi geni povezani z izgubo sluha. To pa ne velja za vzročne spremembe v genu STRC, čeprav po navedbah raziskav predstavljajo signifikanten delež vzrokov nesindromske izgube sluha. Analiza STRC je namreč s klasičnim sekvenciranjem ali sekvenciranjem naslednje generacije izredno otežena, delno zaradi prisotnega psevdogena STRCP1, ki kaže skoraj 100% podobnost z genom, delno pa tudi zato, ker s sekvenčno analizo težko določimo prisotnost sprememb v številu kopij. Namen magistrske naloge je v populaciji bolnikov z izgubo sluha, kjer genetski vzrok še ni bil dokončno razjasnjen, opredeliti spremembe v številu kopij gena STRC in sicer z vzpostavitvijo stopenjskega analitskega pristopa. V raziskavo smo vključili 174 bolnikov z različnimi vrstami izgube sluhe in različnih starosti. Pri vseh je bilo predhodno že opravljeno sekvenciranje po Sangerju gena GJB2 in GJB6 ter sekvenciranje kliničnega eksoma s sekvenciranjem naslednje generacije. Določanje sprememb v številu kopij gena smo opravili stopenjsko z uvedbo kvantitativne fluorescenčne verižne reakcije (QF-PCR), ki je služila kot presejalna analiza, dobljene rezultate smo nato potrdili z metodo hkratnega pomnoževanja od ligacije odvisnih sond (MLPA). Spremembe v številu kopij gena STRC smo opredelili pri 7 preiskovancih, kar predstavlja 4% vseh preiskovancev vključenih v magistrsko nalogo. Izgubo sluha kot posledico prisotne bolezenske spremembe v STRC pa smo določili le pri dveh preiskovankah s homozigotno delecijo, kar predstavlja le 1,15% naše raziskovane skupine. Ostalim 5 preiskovancem smo določili heterozigotno spremembo, od tega 1 duplikacijo in 4 heterozigotne delecije, ki pa ne morejo biti samostojen vzrok izgube sluha. Pojavnost sprememb v številu kopij gena STRC v preiskovani skupina je tako nižja od pričakovane. Za dokončno opredelitev pomena gena STRC za izgubo sluha pri teh bolnikih pa bi bilo potrebno izvesti še analizo točkovnih sprememb ter majhnih delecij in duplikacij gena STRC.

• Jezik: Slovenski jezik
• Ključne besede: izguba sluha, genetika, gen STRC, MLPA
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2021
• PID: 20.500.12556/RUL-131506
• Datum objave v RUL: 29.09.2021

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Stereocilin (STRC) gene copy number variations in patients with hearing loss

Izvleček

The most common cause of sensorineural hearing loss is hereditary in nature, thus genetic testing of patients with hearing loss is crucial to genetically clarify the disease. In the population of patients with hearing loss, the genetic causes associated with variants in the GJB2 and GJB6 genes are well researched. Nevertheless, with the development of new generation sequencing (NGS) also other genes associated with hearing loss are being numerously analyzed. However, this does not apply to genetic variants in the STRC gene, eventhouhg they represent a significant proportion of the non-syndromic hearing loss causes. Namely, STRC analysis is difficult when using Sanger or next generation sequencing. This is partly because of the pseudogene STRCP1, which is highly homologous, and partly because sequencing makes it difficult to determine the presence of copy number variants. The aim of this master's thesis is to identify changes in the copy number variations of the STRC gene in the population of patients with hearing loss with not definitively determined genetic cause of the disease by establishing a step-by-step analytical approach. The study included 174 patients with different degree of hearing loss and of different ages. All of them had previously sequenced GJB2 and GJB6 genes by Sanger sequencing, and had already sequenced clinical exons by next generation sequencing. Determination of the copy number variations was performed step-by-step, by introducing the quantitative fluorescent polymerase chain reaction (QF-PCR), which was performed as a screening approach. The results were then confirmed by multiplex ligation-dependent probe amplification (MLPA). Copy number variations in the STRC gene were identified in 7 subjects, representing 4% of all subjects included in the master's thesis. However, we identified the STRC deletion as a cause of hearing loss only in 2 subjects which were homozygous for the deletion, therefore only in 1.15% of our study group. The other 5 subjects were heterozygous carriers, of which 1 was a heterozygous carrier of the STRC duplication and 4 were heterozygous carriers of the STRC deletion. The frequency of the copy number variants of the STRC gene in the analysed group of patients is thus lower than expected. In order to definitively determine the significance of the STRC gene for hearing loss in these patients, it would be necessary to perform an analysis of substitution and small indels in the STRC gene.

• Ključne besede: hearing loss, genetics, gene STRC, MLPA