Opredelitev sprememb v genu in psevdogenih PKD1 pri bolnikih z avtosomno dominantno policistično boleznijo ledvic

Snoj, Lara (avtor), Trebušak Podkrajšek, Katarina (mentor), Jenko Bizjan, Barbara (komentor)

Avtosomna dominantna policistična bolezen ledvic je ena izmed najpogostejših življenje ogrožajočih genetskih motenj. Povzročajo jo spremembe v genih PKD1 in PKD2, ki sledijo avtosomno dominantnemu načinu dedovanja. Policistin-1 in policistin-2, proteinska produkta genov PKD1 in PKD2, sta integralna membranska proteina, odgovorna za transport Ca2+ ionov preko celične membrane. Motnje v njunem delovanju povzročijo motnje v homeostazi Ca2+, kar vodi v povečano proliferacijo tubulnih epitelijskih celic ter nastanek cist v ledvicah. Posledica sta simetrično povečani ledvici ter okvare njune funkcije, ki lahko privedejo do končne ledvične odpovedi. Diagnostika ADPKD trenutno temelji na rezultatih ultrazvočne preiskave ter kliničnih znakih bolnikov. Genetska diagnostika je otežena zaradi velike genetske in alelne heterogenosti, kompleksne strukture ter velike podobnosti PKD1 s 6 psevdogeni, ki se nahajajo v njegovi neposredni bližini. Metode sekvenciranja naslednje generacije (NGS) namreč niso dovolj specifične, da bi lahko zanesljivo ločile med spremembami v genu in psevdogenih, kar se odraža v velikem številu lažno pozitivnih rezultatov. Za detekcijo sprememb v PKD1 se uporablja dvostopenjski pristop. V prvi stopnji metoda pomnoževanja dolgih odsekov (LR-PCR) zagotovi tarčno pomnožitev aktivnega gena, v drugi stopnji pa se določi zaporedje z NGS, na primer s sekvenciranjem celotnega eksoma (WES), ali s sekvenciranjem po Sangerju. V okviru magistrske naloge smo želeli dokazati, da je metoda WES dovolj zanesljiva za uporabo v rutinski diagnostiki ADPKD. Zato smo rezultate pridobljene z WES ali z analizo kliničnega eksoma (TSO), primerjali z rezultati pridobljenimi z novo vpeljano, dvostopenjsko metodo LR-PCR in NGS. Pri 29 preiskovancih smo z WES/TSO določili 13 patogenih ali verjetno patogenih sprememb, z novo metodo smo jih potrdili le 8. Za 4 spremembe, ki so se nahajale v eksonih 41, 44 in 46, smo predpostavljali 100 % zanesljivost WES, zato jih v potrjevanje nismo vključili. S tem pristopom smo določili tudi novo spremembo, ki smo jo predhodno s TSO panelom spregledali. Prav tako pristop z WES ne omogoča primerne kvalitete analize eksona 1, zato bi bilo pri vsakemu preiskovancu z negativnim rezultatom WES, potrebno rezultat potrditi še z LR-PCR za to področje.

• Jezik: Slovenski jezik
• Ključne besede: Avtosomna dominantna policistična bolezen ledvic, verižna reakcija s polimerazo za pomnoževanje dolgih odsekov, sekvenciranje naslednje generacije, občutljivost, psevdogeni.
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2023
• PID: 20.500.12556/RUL-147346
• Datum objave v RUL: 02.07.2023

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Detection of variants in PKD1 gene and pseudogenes in patients with autosomal dominant polycystic kidney disease

Izvleček

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common life-threatening genetic disorders. It is caused by genetic variants in PKD1 and PKD2, which follow an autosomal dominant inheritance pattern. The proteins encoded by PKD1 and PKD2, namely polycystin-1 and polycystin-2, are integral membrane proteins that form an ion-channel complex responsible for the transport of Ca2+ across the cell membrane. Disruptions in their function lead to imbalances in Ca2+ homeostasis, resulting in increased proliferation of tubular epithelial cells and cyst growth. This results in symetrically enlarged kidneys and impaired renal function, which can progress to end-stage renal failure. Currently, the diagnosis of ADPKD relies on ultrasound examination and the presence of clinical symptoms in the patient. Genetic testing for ADPKD is somewhat challenging due to the high genetic and allelic heterogeneity, the complex structure, and the significant homology of PKD1 with 6 pseudogenes in its vicinity. Next-generation sequencing (NGS) methods are not sufficiently specific to distinguish between variants in the gene and pseudogenes, leading to a high number of false-positive results. In routine diagnostics, a two-step approach is currently used to detect variants in PKD1. The first step involves specific amplification of the functional gene using long-range polymerase chain reaction (LR-PCR), followed by NGS or Sanger sequencing. We aimed to evaluate the reliability of whole-exome sequencing (WES) for use in a routine diagnostics by comparing the results obtained with WES or clinical exome analysis (TSO) to those obtained with the newly introduced two-step approach. We identified 13 pathogenic or likely pathogenic variants using WES/TSO in 29 subjects, of which only 8 were confirmed with the new method. We assumed 100 % reliability of WES for 4 variants indetified in exons 41, 44 and 46, so we did not include them in the confirmation. We identified one new variant with the novel approach that was missed previously with TSO panel. Additionally, sequencing of the exon 1 with WES is not reliable, so in subjects with negative WES result, a confirmation with LR-PCR of exon 1 needs to be performed.

• Ključne besede: Autosomal dominant polycystic kidney disease, long range polymerase chain reaction, next generation sequencing, sensitivity, pseudogenes.