Primjena MLPA metode u molekularnoj dijagnostici Lynch sindroma

master's thesis

• Title (english): Application of the MLPA method in molecular diagnostics of Lynch syndrome
• Author: Ema Vodolšak
• Mentor: Dunja Rogić (mentor)
• Mentor: Ivana Rako (mentor)
• Committee member: Dunja Rogić (predsjednik povjerenstva)
• Committee member: Ivana Rako (član povjerenstva)
• Committee member: Karmela Barišić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2023-06-21, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Kolorektalni karcinom (CRC) jedan je od najčešćih zloćudnih tumora. Sa stajališta nasljeđivanja
razlikujemo sporadični, obiteljski i nasljedni oblik raka pri čemu je svega 5% nasljedno. Lynch
sindrom (LS) nasljedni je CRC sindrom uzrokovan nasljednom promjenom u jednom od gena
zaduženih za popravak krivo sparenih baza (MMR) - MLH1, MSH2, MSH6 i PMS2 ili EPCAM genu.
Iako su točkaste mutacije najčešći tip mutacije, >20% otpada i na velike genomske promjene
(delecije i duplikacije). Upravo zbog prisutnosti različitih genskih promjena u LS, u dijagnostici je
neophodna upotreba komplementarnih metoda koje će detektirati sve moguće promjene u genima.
Hipoteza ovog istraživanja je da korištenje MLPA metode, kao komplementarne metode NGS
metodi, može povećati dijagnostičku osjetljivost genetičkog testiranja pacijenata sa kliničkom
sumnjom na LS. U istraživanje je uključeno 22 ispitanika koji su prošli genetičko savjetovanje i kod
kojih je postavljena klinička sumnja na Lynch sindrom. Kod 6 pacijenata pronađena je točkasta
promjena NGS metodom pa ti uzorci nisu analizirani MLPA metodom. U istraživanju su detektirane
dvije velike delecije karakterizirane kao patogene - delecija MSH2 eksona 1 i regije upstream te
delecija eksona 9-15 gena MLH1. Primjenom MLPA u kombinaciji sa NGS metodom nasljedna
promjena u MMR genima detektirala se kod 36,4% pacijenata uključenih u istraživanje, dok bi
primjenom samo sekvenciranja taj postotak iznosio 27,3%. Dakle, MLPA se pokazala korisnom
nadopunom NGS metodi i njeno uvođenje u rutinsku dijagnostiku bilo bi opravdano.

Abstract (english)

Colorectal cancer (CRC) is one of the most common malignancies. From a genetic point of view, it
comes in sporadic, familial or hereditary forms. Hereditary forms of cancer are found in only 5% of
all cancer cases. Lynch syndrome (LS) is a cancer-susceptibility syndrome caused by a germline
mutation in one of the mismatch repair genes - MLH1, MSH2, MSH6 and PMS2 or EPCAM gene.
Genetic testing focuses on determing germline alteration in MMR genes. Although point mutations
are the most common change in MMR genes, >20% are large genomic rearrangments (deletions and
duplications). It is therefore essential to use different commplementary molecular methods to detect
all potential changes in MMR genes. Hypothesis of this research is that combined usage of MLPA
and NGS can increase diagnostic sensitivity of genetic testing. The research included 22 LSsuspected cases. Point mutations were detected by sequencing in 6 cases and patogenic large
genomic rearrangments were detected in two cases – MSH2 del ex1 and upstream and MLH1 del
ex9-15. Combined usage of sequencing and MLPA method resulted in detecting alterations in 36,4%
of patients, whereas using sequencing by itself, alterations would be detected in 27,3% of patients.
In conclusion, MLPA has shown to be very usful in diagnostics of LS and its implementation in
rutine testing would benefit both patients and health care providers.

• Keywords: Lynch sindrom; MLPA; MMR; NGS; nasljedne mutacije; nasljedni rak
• Keywords (english): Lynch syndrome; MLPA; MMR; hereditary cancer; germline mutations
• Language: croatian
• URN:NBN: urn:nbn:hr:163:898662
• Study programme: Title: Medical biochemistry; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra medicinske biokemije (magistar/magistra medicinske biokemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2023-11-21 15:18:48