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Lynch syndrome (LS) is caused by germline mutations in one of the four mismatch repair (MMR) genes. Defects in this pathway lead to microsatellite instability (MSI) in DNA tumors, which constitutes the molecular hallmark of this disease. Selection of patients for genetic testing in LS is usually based on fulfillment of diagnostic clinical criteria (i.e. Amsterdam criteria or the revised Bethesda guidelines). However, following these criteria PMS2 mutations have probably been underestimated as their penetrances appear to be lower than those of the other MMR genes. The use of universal MMR study-based strategies, using MSI testing and immunohistochemical (IHC) staining, is being one proposed alternative. Besides, germline mutation detection in PMS2 is complicated by the presence of highly homologous pseudogenes. Nevertheless, specific amplification of PMS2 by long-range polymerase chain reaction (PCR) and the improvement of the analysis of large deletions/duplications by multiplex ligation-dependent probe amplification (MLPA) overcome this difficulty. By using both approaches, we analyzed 19 PMS2-suspected carriers who have been selected by clinical or universal strategies and found five large deletions and one frameshift mutation in PMS2 in six patients (31%). Owing to the high incidence of large deletions found in our cohort, we recommend MLPA analysis as the first-line method for searching germline mutations in PMS2., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Lynch syndrome is caused by germline mutations in the mismatch repair genes MLH1, MSH2, MSH6 or PMS2. The novel MSH2 c.[2635-3T>C; 2635-5C>T] mutation was identified in 4 Lynch families, cosegregating with the disease. This mutation, located in intron 15, was predicted to alter the correct mRNA processing by in silico analysis. Our aim was to perform the c.[2635-3T>C; 2635-5C>T] mutation screening in high risk CRC cases and control populations, to evaluate the founder effect in our population by haplotype analysis and to confirm the pathogenic effect of the mutation by MSH2 expression studies. Mutation screening was performed by SSCP and CSCE in genomic DNA from 323 high risk CRC cases and 289 controls. Haplotyping was performed analysing 4 MSH2 extragenic microsatellite markers (D2S288, D2S2227, D2S1247 and D2S1248) in 50 controls and mutation carriers by using the PHASE program. We analysed the effect of the mutation in mRNA processing by RT-PCR and in MSH2 expression by qRT-PCR using RNA from 5 mutation carriers and 18 controls. None of the remaining high risk CRC cases or controls analysed harboured the mutation. We identified a common telomeric haplotype and two centromeric haplotypes, both rare in our population. Although we were not able to identify any abnormal transcript by RT-PCR with the design used, we observed a significant reduction of mRNA MSH2 expression in carriers when compared with controls. Haplotype analyses suggest a founder effect of the c.[2635-3T>C; 2635-5C>T] MSH2 mutation and expression studies support a pathogenic role of this mutation.