GENES, CHROMOSOMES & CANCER 51:819–831 (2012) RESEARCH ARTICLES Improved Multiplex Ligation-Dependent Probe Amplification Analysis Identifies a Deleterious PMS2 Allele Generated by Recombination with Crossover Between PMS2 and PMS2CL Annekatrin Wernstedt,1 Emanuele Valtorta,2 Franco Armelao,3 Roberto Togni,4 Salvatore Girlando,4 Michael Baudis,5 Karl Heinimann,6 Ludwine Messiaen,7 Noemie Staehli,2 Johannes Zschocke,1 Giancarlo Marra,2† and Katharina Wimmer1†* 1Division of Human Genetics, Medical University Innsbruck, Austria 2Institute of Molecular Cancer Research, Faculty of Medicine,University of Zurich, Switzerland 3Department of Gastroenterology,Ospedale Santa Chiara, Azienda Provinciale per I Servizi Sanitari,Trento,Italy 4Department of Pathology,Ospedale Santa Chiara, Azienda Provinciale per I Servizi Sanitari,Trento,Italy 5Institute of Molecular Life Sciences, Faculty of Science,University of Zurich, Switzerland 6Department of Biomedicine,Research Group Human Genetics,University of Basel, Switzerland 7Department of Genetics, Medical Genomics Laboratory,University of Alabama at Birmingham,Birmingham, AL 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 refer- ence 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 addi- tional 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 50 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 muta- tions. VC 2012 Wiley Periodicals, Inc. INTRODUCTION Lynch syndrome (MIM#120435) is the most common heritable cause of neoplastic disease in the large intestine. Formerly referred to as hered- itary nonpolyposis colorectal cancer, this syn- drome accounts for approximately 2-3% of all colorectal malignancies and is associated with an increased risk for cancer at other sites as well (Cunningham et al., 2001; Lynch and de la Chap- elle, 2003; Hampel et al., 2008; Lynch et al., 2009). It is caused mainly by heterozygous germ- line mutations in the mismatch repair (MMR) genes, in most cases MSH2 ( 40% of cases) or MLH1 ( 50%) (Liu et al., 1996; Peltomaki, 2003). A third MMR gene, MSH6, has been found mutated in 10% of cases (Berends et al., 2002; Hendriks et al., 2004; Plaschke et al., 2004). Another essential MMR gene, PMS2, was VC 2012 Wiley Periodicals, Inc. previously thought to play only a minor role in colorectal cancer predisposition. However, in a large series of unselected colorectal carcinomas, evidence of primary alteration of PMS2 (i.e., im- munohistochemical ﬁndings of isolated loss of PMS2 protein expression with normal expression Additional Supporting Information may be found in the online version of this article. Supported by: Austrian science funds, FWF, Grant number: P21172-B12 (A. W. and K. W.); Union Bank of Switzerland (G. M. and E. V.); Cancer League of Central Switzerland (N. S.). Tel.: þ43 512 9003 70513, Fax: þ43 512 9003 73080. yThe authors equally contributed to this work. *Correspondence to: Katharina Wimmer, Division of Human Genetics, Medical University Innsbruck, Schoepfstraße 41, AT-6020 Innsbruck, Austria. E-mail: Katharina.wimmer@i-med.ac.at Received 15 February 2012; Accepted 2 April 2012 DOI 10.1002/gcc.21966 Published online 14 May 2012 in Wiley Online Library (wileyonlinelibrary.com).