Aims We sought to use PCR followed by high-resolution melting analysis to develop a single closed-tube screening panel to screen for Lynch syndrome. This comprises tests for microsatellite instability (MSI), MLH1 methylation promoter and BRAF mutation.
Methods For MSI testing, five mononucleotide markers (BAT25, BAT26, BCAT25, MYB, EWSR1) were developed. In addition, primers were designed to interrogate Region C of the MLH1 promoter for methylation (using bisulphite-modified DNA) and to test for mutations in codon 600 of BRAF. Two separate cohorts from Nottingham (n=99, 46 with MSI, 53 being microsatellite stable (MSS)) and Edinburgh (n=88, 45 MSI, 43 MSS) were tested.
Results All the cases (n=187) were blind tested for MSI and all were correctly characterised by our panel. The MLH1 promoter and BRAF were tested only in the Nottingham cohort. Successful blinded analysis was performed on the MLH1 promoter in 97 cases. All MSS cases showed a pattern of non-methylation while 41/44 cases with MSI showed full methylation. The three cases with MSI and a non-methylated pattern had aberrations in MSH2 and MSH6 expression. BRAF mutation was detected in 61% of MSI cases and 11% of MSS cases.
Finally, 12 cases were blind screened by using the whole panel as a single test. Of these, five were identified as MSS, four as MSI/non-LS and three as MSI/possible LS. These results were concordant with the previous data.
Conclusion We describe the Nottingham Lynch Syndrome Test (N_LyST). This is a quick, simple and cheap method for screening for Lynch syndrome.
- colorectal cancer
- cancer genetics
- molecular pathology
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Handling editor Runjan Chetty.
Contributors Conception or design of the work: SS, HOE, WF, MI. Data collection: SS, HOE, WF, AA, AN, EH, HAH-K, JF, KF. Data analysis and interpretation: SS, HOE, WF, JCH. Drafting the article: SS, HOE, WF, MI. Critical revision of the article: BM-B, AS, AO, MI. Approval of the version to be published: all authors.
Funding This research received financial support from the EC Marie Curie Actions, AIDPATH project (Contract No. 612471) and Nottingham Molecular Pathology Node (NMPN).
Competing interests MI was appointed as specialist committee member to the Diagnostics Assessment Committee of the National Institute for Health and Care Excellence (NICE), which produced the guidance DG27 on Lynch syndrome testing. Other authors have no competing interest to declare.
Patient consent Not required.
Ethics approval Access to tissues and ethics approval were granted by Nottingham Health Sciences Biobank, which has approval as an IRB from North West—Greater Manchester Central Research Ethics Committee (REC reference: 15/NW/0685). Access to anonymised use of tissues was granted by Tissue Governance NHS Lothian under ethics approval number SR783.
Provenance and peer review Not commissioned; externally peer reviewed.