Small pool PCR (SPPCR) reveals allelic distortion in normal tissues and in tumors of FCCX patients thought microsatellite stable.

539 Might attenuated MMR defects be present, making MSI unrecognizable, in tumor tissue of the high proportion (\u003e40%) of families meeting all the criteria for HNPCC except MSI detection - so called Familial Colorectal Cancer Type X or FCCX (JAMA 293:1979, 2005)? Could these defects contribute to or interact with microchromosomal alterations such as copy number variation (CNV)?\u2028 We address both questions here using PCR in multiple pools of DNA diluted to single genome equivalents (SPPCR) - a procedure shown to be so sensitive as to quantify low, but significant levels of MSI increasing with age in the PBLs of normal individuals (Mech Aging Dev 126:1051, 2005). Studied were the DNAs from paired PBL and tumor samples from 7 HNPCC MSI-H having null mutations in major MMR genes (MLH1 and MSH2) as well as from 10 FCCX patients. PBLs from age matched normal individuals were run simultaneously as controls for both sets. D2S123, D5S356, and D17S518 loci were amplified using hemi-nested PCR in \u003e112 replicates of ~0.75 genome equivalents per locus/per tissue/per patient to determine allele and mutant frequencies (f), 95% confidence intervals (CI). Frequencies of allele 1 versus allele 2 at each locus were compared by Chi-square test, p-values less than 0.05 were considered significant and an indication of allelic distortion at that locus.\u2028 Normal control PBL DNA had f = 0.012, CI +/- 0.012. MSI-H PBL and tumor showed f = 0.107, CI +/- 0.052 and f = 0.236, CI +/- 0.133, respectively. Syndrome X PBL and tumor had f = 0.016, CI +/- 0.011 and f = 0.090, CI +/- 0.029, respectively. Allele distribution in normal control PBL at D2S123, D5S356, and D17S518 had chi-square p-values of 0.51, 0.58, and 0.69, respectively, suggesting that there is no allele drop-out nor allelic distortion in normal blood. In comparison, the same loci in the FCCX tumors had p-values of 5e-03, 2e-07, and 2e-06, and blood had p-values of 0.13, 0.06, and 0.03, respectively. Neither HNPCC blood nor tumor showed significant distortion at the loci, with p-values ranging from 0.25 - 0.94.\u2028 Conclusions. Significant MSI in FCCX tumors but not in their PBLs suggest: (1) FCCX might be due to mutations in MMR pathways that have less severe consequences than those seen in the MSI-H patients; (2) SPPCR analysis of tumors identifies patients carrying such mutations; (3) Such quantitative analysis of FCCX tumor reveals significant allelic distortion at D2, D5, and D17 loci. FCCX PBL DNA also has significant allelic distortion at 2 of the 3 loci, though not as high levels as seen in tumor. NC PBL and HNPCC PBL and tumor DNA do not show allelic distortion by SPPCR. FCCX allele distortion may be due to rearrangement or CNV of genomic architecture features such as alu repeats that flank the microsatellite loci on 17q12.CGH array chips may reveal microchromosomal CNV in these regions, supporting the SP observations. Such CNV may be either a mechanism for disease or diagnostic for FCCX.