Analiza sprememb v številu kopij gena NF1 z od ligacije odvisnim hkratnim pomnoževanjem sond pri bolnikih s kliničnim sumom na nevrofibromatozo tipa 1

Gartner, Meta (author), Trebušak Podkrajšek, Katarina (mentor), Hovnik, Tinka (comentor)

Nevrofibromatoza tipa 1 (NF1) je avtosomno dominantna bolezen, za katero so značilni številni madeži barve bele kave (CALMs) na koži, aksilarna in ingvinalna pegavost, kožni nevrofibromi in Lischevi noduli. Pri približno 50 % posameznikov so prisotne tudi učne težave. Vzrok za pojav bolezni je heterozigotna bolezenska sprememba gena NF1, ki je z 61 eksoni eden večjih človeških genov in se nahaja v pericentromerni regiji dolge ročice 17. kromosoma, na citogenetskem področju 17q11.2. Pri skoraj polovici prizadetih posameznikov je vzrok za pojav bolezni nova (t. i. de novo) bolezenska sprememba gena NF1. Posledica je delna ali popolna odsotnost proteina nevrofibromina v telesu. Omenjeni protein je prisoten v velikih koncentracijah zlasti v celicah perifernega in centralnega živčnega sistema (nevroni, Schwannove celice, oligodendrociti in levkociti). Posledično se začnejo na teh mestih pretirano tvoriti tumorji, t. i. nevrofibromi. Diagnoza NF1 navadno temelji na kliničnih ugotovitvah in uporabi diagnostičnih kriterijev, ki jih je določil ameriški Nacionalni inštitut za zdravje (NIH). Najpogostejše genetske spremembe gena NF1 so točkovne spremembe, ki jih z metodo hkratnega pomnoževanja od ligacije odvisnih sond (MLPA) ne moremo detektirati, zato se za diagnosticiranje bolezni priporoča uporaba metode MLPA v kombinaciji s sekvenciranjem naslednje generacije (NGS). Namen magistrske naloge je bil z metodo MLPA določiti večje spremembe v številu kopij (CNV) gena NF1 v populaciji pediatričnih bolnikov s sumom ali diagnozo na NF1, ki še nimajo odkrite točkovne ali druge spremembe v genu. Dobljene rezultate smo primerjali z rezultati predhodno uporabljenih metod in jih prikazali še z računalniškim programom CNVkit. V raziskavo je bilo vključenih 82 otrok z diagnozo ali sumom na NF1, ki so vodeni v ambulanti Kliničnega oddelka za endokrinologijo, diabetes in bolezni presnove ter v genetski ambulanti Pediatrične klinike UKC Ljubljana. Večje spremembe v številu kopij gena NF1 smo odkrili pri šestih preiskovancih, kar je 7 % raziskovane skupine. Pri enem preiskovancu je bila dokazana že znana heterozigotna delecija celotnega gena, pri dveh sta bili dokazani že znani deleciji, ki zajemata več eksonov, pri treh preiskovancih pa je bila na novo odkrita delecija enega eksona (eksoni 3, 23 oziroma 25). Na podlagi naše raziskave lahko trdimo, da je metoda MLPA v kombinaciji z uporabljeno mešanico sond SALSA P081 MIX1 in P082 MIX2 bolj zmogljiva od v laboratoriju predhodno uporabljene metode MLPA v kombinaciji z mešanico sond SALSA P122 NF1 area.

• Language: Slovenian
• Keywords: nevrofibromatoza tipa 1, MLPA, gen NF1, CNVkit
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2021
• PID: 20.500.12556/RUL-129908
• Publication date in RUL: 09.09.2021

GARTNER, Meta, 2021, Analiza sprememb v številu kopij gena NF1 z od ligacije odvisnim hkratnim pomnoževanjem sond pri bolnikih s kliničnim sumom na nevrofibromatozo  tipa 1 [online]. Master’s thesis. [Accessed 22 August 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=129908

Secondary language

• Language: English
• Title: Analysis of copy number variation in the NF1 gene by the multiplex ligation-dependent probe amplification in patients with clinical suspicion for type 1 neurofibromatosis

Abstract

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease characterized by multiple café-au-lait macules (CALMs) on the skin, axillary and inguinal freckling, cutaneous neurofibromas and Lisch nodules. About 50% of children also have learning disabilities. The disease is caused by a heterozygous mutation of the NF1 gene. This is one of the major human genes. It has 61 exons and is located in the pericentromeric region of the long arm of chromosome 17, in the cytogenetic region 17q11.2. In almost half of individuals with NF1, the disorder is caused by de novo disease-causing variant of gene NF1. Its consequence is partial or complete absence/non-function of protein neurofibromin in the body. The highest expression levels of neurofibromin are in peripheral cells and in the central nervous system (neurons, Schwann cells, oligodendrocytes and leukocytes). They cause excessive tumours (i.e. neurofibromas) that start to form in these cells. The NF1 diagnosis is usually based on clinical findings and diagnostic criteria established by the U.S. National Institutes of Health. The most common genetic changes of the NF1 gene are point mutations, which cannot be detected by the multiplex ligation-dependent probe amplification (MLPA). For diagnostic reasons, the MLPA method in combination with new generation sequencing (NGS) is thus recommended. The aim of this study was to use MLPA method to identify large copy number variations (CNV) of NF1 gene in paediatric patients with suspected or diagnosed NF1 for whom point mutation or any other change in the gene has not been detected. The results were compared with results of previously used methods and presented with the computer program CNVkit. The study included 82 children diagnosed with or suspected of having NF1. All were referred either to the Department of Pediatric Endocrinology, Diabetes and Metabolic Diseases or to the Clinical Institute of Medical Genetics at the University Children’s Hospital Ljubljana. Major changes in the CNV of the NF1 gene were detected in 6 patients, or 7% of the study group. In one patient the study proved a previously known heterozygous deletion of the whole gene. In two patients it proved previously known deletions involving multiple exons. In three other patients deletion of one exon (exons 3, 23 and 25, respectively) was discovered de novo. Taking into account these results we can say that MLPA method in combination with mixture of probes SALSA P081 MIX1 and P082 MIX2 is more powerful than the one (SALSA P122 NF1 area) previously used in the laboratory.

• Keywords: neurofibromatosis type 1, MLPA, NF1 gene, CNVkit