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Combined use of MLPA and nonfluorescent multiplex PCR analysis by high performance liquid chromatography for the detection of genomic rearrangements

Large genomic rearrangements are recognized as playing a pathogenic role in an increasing number of human genetic diseases. It is important to develop efficient methods for the routine detection and confirmation of these germline defects. Multiplex ligation‐dependent probe amplification (MLPA) is considered an early step for molecular diagnosis of several genetic disorders. However, artifacts might hamper the interpretation of MLPA analysis, especially when rearrangements involve a single exon. Therefore, rearrangements must be verified by two independent methods. In this study, we developed nonfluorescent multiplex‐PCR coupled to high‐performance liquid chromatography (NFMP‐HPLC) and analyzed whether the use of this method combined with MLPA could be helpful in the detection and confirmation of gross MSH2 and MLH1 genomic rearrangements. A total of nine nonfluorescent multiplex‐PCRs were developed to analyze the 16 MSH2 and 19 MLH1 exons. Reliable multiplex amplifications and nonfluorescent peak quantitation were obtained with a limited number of cycles (≤25) using a denaturing HPLC (DHPLC) instrument under nondenaturing conditions. The results obtained by NFMP‐HPLC were highly reproducible. The combined use of MLPA and NFMP‐HPLC identified and independently confirmed putative MLH1 and MSH2 deletions in eight out of 50 unrelated patients with hereditary nonpolyposis colorectal cancer (HNPCC). In five cases, the deletions affected a single exon and in three cases multiple contiguous exons. These results were in agreement with breakpoint and complementary DNA (cDNA) analyses. Considering that MLPA and NFMP‐HPLC are unlikely to be affected by the same artifacts, their combined use could also provide a robust and cost‐effective strategy for routine screening and confirmation of putative rearrangements in other genes, especially when a single exon is involved or a precise characterization of breakpoints is not achieved. Hum Mutat 27(10), 1047–1056, 2006. © 2006 Wiley‐Liss, Inc.