The development of "upQMPFS" and the study of large genomic rearrangements (LGRs) in familial ovarian cancer susceptibility genes
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Genetic mutations responsible for hereditary diseases, such as familial breast and ovarian cancers, occur in a variety of forms, including non-sense and missense point mutations, deleterious small deletions/insertions, and Large Genomic Rearrangements (LGRs) involving deletions or duplications of one or more exons of a gene or even the entire gene and gross structural abnormalities. Current mutation detection methodologies suffer certain limitations by being able to detect only a single type of mutations and/or at insufficient sensitivity and many with tedious procedure. To overcome these limitations, we have developed an improved version of Q uantitative M ultiplex P CR S hort fluorescent F ragment (QMPSF), named u niversally p rimed QMPSF (upQMPSF). The improvements include replacing the individually labeled fluorescent primers with one universal fluorescent primer and maximized multiplex PCR capacity to allow up to 15 products per set. Furthermore, the primers were designed to cover entire exon and splicing junction regions, such that the method can be used also to detect small deletions and insertions in these regions. These modifications significantly improve the mutation detection spectrum and the cost efficiency, upQMPSF, to our knowledge, is the only currently available method that has the capacity of simultaneously detecting genomic rearrangements and small insertions/deletions. Using upQMPSF, we screened 88 familial ovarian cancer patient samples and detected and confirmed a novel ~2.8 kb duplication spanning exon 15 of BRCA1 . The efficiency of upQMPSF in detecting insertions and deletions as small as 1 bp was demonstrated by its capacity in detecting all previous identified mutations in tested samples. The method has a broad application in genetic diagnosis/testing and in research laboratories as a method for validating array comparative genome hybridization (aCGH) data and as an alternative method for quantitative expression profiling of one or multiple gene(s). Because of these advantages, it is possible to use upQMPSF as a first-round screening method for small deletions/insertions and LGRs in one test. Furthermore, our study showed that LGRs occur less frequently in BRCA1/2 genes compared to other mutation types. Nevertheless, our data support the recommendation that accurate and complete BRCA mutation susceptibility screening should include testing for LGRs in hereditary cancer syndrome.