822 DNA numbera NA07348 NA10842 NA07019 NA12853 NA17002 NA17005 NA17008 NA17009 WERNSTEDT ET AL. TABLE 2. Coriell Cell Repository DNA Samples Identiﬁed as Valid References for Use with the P008-B1 PMS2 MLPA Kit Catalog ID GM07348 GM10842 GM07019 GM12853 GM17002 GM17005 GM17008 GM17009 Description CEPH/UTAH pedigree 1345 CEPH/UTAH pedigree 1423 CEPH/UTAH pedigree 1340 CEPH/UTAH pedigree 1400 Human variation panel – Northern European Human variation panel – Northern European Human variation panel – Northern European Human variation panel – Northern European aEach sample listed was MLPA-veriﬁed to contain two copies of PMS2-speciﬁc and two copies of PMS2CL-speciﬁc sequences. MLPA proﬁles of all 24 Coriell DNAs investigated in this study are available on request. Sequence Pilot algorithm, version 3.3 (JSI Medi- cal Systems, Kippenheim, Germany). A relative probe signal of 1 (¼ 100%) in a sample DNA reﬂects to two copies of target sequence of PMS2- and PMS2CL-speciﬁc probes and four cop- ies of target sequence of universal probes which bind to both PMS2 and PMS2CL sequences. Hence, relative probe signals of 0.5 (50%) and 1.5 (150%) of PMS2- and PMS2CL-speciﬁc probes indicate one and three DNA copies, respectively, of the respective target sequences. A reduction to 0.75 (75%) indicates a copy number change from four to three copies of target sequence of univer- sal probes. cDNA Sequencing of PMS2 and PMS2CL Transcripts This approach was used to conﬁrm MLPA ﬁndings of a PMS2 exon 8 deletion in patient CH11 and those for patient TR13 showing a del- eterious hybrid PMS2 allele containing a PMS2CL-derived sequence in exons 11-15. In the former case, we extracted RNA from the patient’s freshly collected blood lymphocytes and reverse- transcribed it using the High Capacity RNA-to- cDNA kit (Applied Biosystems, Foster City, CA). cDNA containing PMS2 exons 6–9 was PCR- ampliﬁed with primers PMS2/11 (forward) and PMS2/4 (reverse) (Supporting Information Table S1). These primers were also used to sequence the resulting RT-PCR product. In case TR13, we extracted RNA from a short-term culture of the patient’s lymphocytes, which had been treated with puromycin before harvest (Etzler et al., 2008) to prevent nonsense-mediated decay (Andreutti-Zaugg et al., 1997). The entire coding sequence of PMS2 was ampliﬁed in two partially overlapping RT-PCR products, as previously described (Etzler et al., 2008). The amplicons were sequenced with internal primers (Support- ing Information Table S1), directly and after Genes, Chromosomes & Cancer DOI 10.1002/gcc cloning into the pCR4-TOPO vector (Invitrogen, Carlsbad, CA). In the latter case, 10 clones were sequenced after ampliﬁcation of the plasmid inserts by colony PCR performed under standard conditions with Taq DNA polymerase (New England Biolabs GmbH, Frankfurt am Main, Germany) and M13 forward and reverse primers. PMS2CL transcripts from the same patient were RT-PCR-ampliﬁed and subsequently sequenced as previously described (Ganster et al., 2010). All sequence reactions were performed with Big Dye Terminator chemistry V1.1 (Applied Biosystems) on the Applied Biosystems 3130 Genetic Analyzer. Before sequencing, PCR prod- ucts were treated with ExoSAP-IT (GE Health- care, Vienna, Austria), and sequences were analyzed with the Sequence Pilot algorithm Ver- sion 3.3 (JSI Medical Systems, Kippenheim, Germany). Determination of the Extent of the Exon 8 Deletion at the gDNA Level The exon 8-deleted allele from patient CH11 was ampliﬁed from gDNA with primers PMS2_7f (forward) and PMS2/4 (reverse), which are located in PMS2 intron 6 and exon 9, respectively (Supporting Information Table S1). Phusion High-Fidelity DNA polymerase (Finnzymes, Espoo, Finland) was used in a reaction containing 3% DMSO under the PCR conditions recom- mended by the manufacture. The resulting PCR product (i.e., a single band presumably derived from the deletion-containing allele) was sequenced with forward primer PMS2/3 (Support- ing Information Table S1) located within exon 7. Genomic Array Analysis of the Deletion Encompassing the Entire PMS2 Gene and Flanking Sequences The Affymetrix Genome-Wide Human SNP 6.0 array platform (Affymetrix, Santa Clara, CA)