Gene conversion rates between PMS2, and its pseudogene PMS2CL; impact on the reliability of current mutation scanning

H. van der Klift1, C. Tops2, E. Bik2, M. Boogaard2, K. Pavlikova3, H. Morreau4, P. Devilee1,4, J. Wijnen1,2
Departments of Human Genetics1, Clinical genetics2 and Pathology4, Leiden University Medical Center; Institute of Biology and Medical Genetics, Praha, Czech Republic3

Mutation detection in PMS2, a Mismatch Repair gene involved in Lynch Syndrome (formerly Hereditary Non Polyposis Colorectal Cancer or HNPCC), is complicated by the presence of the pseudogene PMS2CL.  It has been shown that gene conversion can occur between the 3’ part of PMS2 and this 700 kb proximally located pseudogene. We investigated 83 familial colorectal cancer cases and 25 healthy controls by semi-quantification of paralogous sequence variants (nucleotides that differ between  PMS2 and PMS2CL) in sequence chromatograms of non-specific PCR products (amplification of PMS2 and PMS2CL sequences simultaneously) and show that, from exon 12 onwards, all PMS2 specific nucleotides on which commonly used MLPA and PMS2 specific PCR primers are based, can be subject to gene conversion with an increasing frequency of up to 50 % at the 3’ end of the gene. Current mutation scanning designs are thus extremely prone to generate false-negatives or even worse false-positives in this part of the gene.

With a newly developed protocol including non-specific sequencing, cDNA analysis and Southern Blot analysis that excludes false-negative and false-positive results due to gene conversion events, we scanned colorectal cancer patients including strong candidates for a PMS2 germline mutation based on immunohistochemistry analysis of their tumours. We show that several mutations detected in the 3’ part of PMS2 using the current mutation scanning designs are actually false-positives and that also mutations can be missed.