Improved multiplex ligation‐dependent probe amplification analysis identifies a deleterious PMS2 allele generated by recombination with crossover between PMS2 and PMS2CL

Abstract

Heterozygous PMS2 germline mutations are associated with Lynch syndrome. Up to one third of these mutations are genomic deletions. Their detection is complicated by a pseudogene (PMS2CL), which – owing to extensive interparalog sequence exchange – closely resembles PMS2 downstream of exon 12. A recently redesigned multiplex ligation-dependent probe amplification (MLPA) assay identifies PMS2 copy number alterations with improved reliability when used with reference DNAs containing equal numbers of PMS2- and PMS2CL-specific sequences. We selected eight such reference samples – all publicly available – and used them with this assay to study 13 patients with PMS2-defective colorectal tumors. Three presented deleterious alterations: an Alu-mediated exon deletion; a 125-kb deletion encompassing PMS2 and four additional genes (two with tumor-suppressing functions); and a novel deleterious hybrid PMS2 allele produced by recombination with crossover between PMS2 and PMS2CL, with the breakpoint in intron 10 (the most 5′ breakpoint of its kind reported thus far). We discuss mechanisms that might generate this allele in different chromosomal configurations (and their diagnostic implications) and describe an allele-specific PCR assay that facilitates its detection. Our data indicate that the redesigned PMS2 MLPA assay is a valid first-line option. In our series, it identified roughly a quarter of all PMS2 mutations. © 2012 Wiley Periodicals, Inc.

Agenesis of the corpus callosum and gray matter heterotopia in three patients with constitutional mismatch repair deficiency syndrome

“…PMS2 MLPA was performed with SALSA kits P008-A1 and/or P008-B1 (MRC-Holland, Amsterdam, The Netherlands) 15,16 according to the manufacturer's instructions. MLH1 MLPA was performed with the SALSA kit P003 (MRC-Holland, Amsterdam, The Netherlands).…”

PMS2 inactivation by a complex rearrangement involving an HERV retroelement and the inverted 100-kb duplicon on 7p22.1

“…9 Both primer pairs used to amplify the entire coding sequence of the PMS2 gene in two overlapping RT-PCR fragments (Supplementary Table S1) failed to generate a PCR product from cDNA of the patient (Supplementary Figure S2A). MLPA analysis using SALSA MLPA kit P008 (MRC-Holland) showed amplification loss of the first, that is, the most 5′, of three MLPA probes hybridising to PMS2 exon 11 (probe-no.…”

“…Recombination-based sequence exchanges between the paralogs are thought to be a still ongoing mechanism, which is also responsible for the generation of deleterious PMS2 alleles. 8,9 Furthermore, a known inversion polymorphism of~0.7 Mb (Supplementary Figure S1B) is thought to result from intrachromosomal NAHR with cross-over between the two copies of the LCR. 10,11 A close proximity of the LCRs in the three dimensional space, which is a prerequisite for recombination to take place, might also bring replication forks together 12 and thereby facilitate template switching events that may create other chromosomal rearrangements.…”

Lynch Syndrome Presenting as Endometrial Cancer

“…Furthermore, the current clinical guidelines (both Revised Bethesda and Amsterdam II) will miss a substantial number of LS cases (65 ).…”

“…Analysis of the PMS2 gene has historically been hampered by the presence of 15 pseudogenes located on the same chromosome (65 ). One pseudogene in particular, PMS2CL, contains 6 of the PMS2 exons (9,(11)(12)(13)(14)(15)67 ).…”