Recognizing Lynch Syndrome by DNA Mismatch Repair Deficiency

The most common inherited cancer syndrome, Lynch syndrome (LS), is caused by a defected post-replicative DNA mismatch repair (MMR) pathway. Human MMR is initiated by the binding of a heterodimeric mismatch recognition factor MutSα (MSH2+MSH6) or MutSβ (MSH2+MSH3), followed by the assembly of the repairosome by MutLα (MLH1+PMS2). In addition to sharing a common heterodimerisation protein, the roles of MutSα and MutSβ have been suggested to overlap in the repair of small insertion deletion loops. A germline mutation in MLH1, MSH2, MSH6 or PMS2 is most commonly the cause of LS. Mutation carriers have a significantly increased risk for colon and endometrial cancers in particular, although tumorigenesis is thought to only commence upon losing the healthy allele. The clinical importance of identifying LS patients is reflected by the significantly increased cancer risk of LS mutation carriers and the effectiveness of LS associated cancer surveillance. The clinical diagnosis of LS relies on tumour pathological analyses, the identification of an MMR gene variation by mutation analysis and the subsequent pathogenicity assessment of the variation. However, the clinical significance of non-truncating genetic alterations can be difficult to interpret as they are associated to a variety of clinical phenotypes, ranging from the lack of adverse effects to a highly increased cancer risk. Due to the unknown functional significance of such variants, functional assessment is required for their pathogenicity assessment. Hence, biological tools used to assess the pathogenicity of MMR gene variations can be central to identifying LS patients. The aim of the studies in this thesis was to understand how MMR proteins and MMR gene alterations affect the MMR mechanism and contribute to LS. The role and significance of the wild type and variant MMR proteins were analysed in a homologous human MMR system by an in vitro MMR assay. Three different substrate molecules consisting of a GT mismatch, a single or a two nucleotide loop were used to study the substrate specificities and MMR efficiencies of the MutS protein complexes. Even though MutSβ does not participate in mononucleotide loop repair, it was shown to exceed MutSα in dinucleotide loop repair indicating that dinucleotide microsatellite instability in the absence of mononucleotide instability is indicative of MSH3 defects. Furthermore, the introduction of a dinucleotide loop substrate to our assay allowed the novel pathogenicity assessment of an MSH3 variation. Functional variant pathogenicity assessment of MMR gene variations linked to atypical clinical features also confirmed the pathogenicity of two novel MSH6 variations and one biallelic MLH1 variation. The compound contribution of MMR gene VUS pairs, as found in LS cancer patients, was assessed and a subtle compound effect of two MSH2 variations that appear MMR proficient when assayed individually was shown. Also, the application of MMR gene variants with known molecular effects to verify a variation pathogenicity assessment model helped describe the model appropriate for MLH1 and MSH2 variations. Finally, we established MMR gene specific knockdown cell lines to investigate the effect of reduced MMR gene expression on the MMR efficiency. The knockdown clones retaining 50% of MSH2 or MSH6 mRNA expression demonstrated significantly reduced in vitro MMR efficiencies whilst a decrease was also detectable in MLH1 knockdown extracts. The knowledge of the gene specific mRNA expression levels that can be detected as MMR deficient, presents the opportunity to develop the assay to recognise LS from non-cancerous cells.

Grantor dc:publisher

Helsingin yliopisto

Year dc:date.issued

2014

Author dc:creator

• Kansikas, Minttu

• perinnöllisyystiede

Statement dc:rights

• Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.
• This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.
• Publikationen är skyddad av upphovsrätten. Den får läsas och skrivas ut för personligt bruk. Användning i kommersiellt syfte är förbjuden.

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/10138/42476

OAI record oai:identifier

https://helda.helsinki.fi/server/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Ahelda.helsinki.fi%3A10138%2F42476