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Many rearrangement mutations in the BRCA1 gene have been identified. It is becoming clear that some of these mutations are prevalent, and therefore their detection is necessary in order for clinical genetic tests to have high sensitivity. Published information on particular rearrangements is frequently limited to a single patient, small groups of patients, or patients of a particular ethnicity. The objectives of this work included characterizing the prevalence of five specific rearrangement mutations in a large North American patient population. A mutation-specific multiplex PCR assay was used for determining the prevalence of five BRCA1 rearrangement mutations that previously had been reported to occur in unrelated patients. The mutation status of these rearrangements, which came from 20,712 patients at high risk for hereditary breast and/or ovarian cancers who had submitted specimens for clinical genetic testing, is presented. The results, obtained from 2,634 mutation carriers, showed a 6-kb duplication of exon 13, identified in 53 patients (2.01%); a 26-kb deletion encompassing exons 14-20, detected in seven patients (0.27%); a 510-bp deletion of exon 22, detected in 5 patients (0.19%); and a 3.4-kb deletion of exon 13, detected in one patient (0.04%). A previously reported 7.1-kb deletion of exons 8-9 was not found. The high frequency of the exon 13 duplication makes it the fourth most prevalent mutation in these patients. These results provide an accurate picture of the prevalence of these mutations in hereditary breast/ovarian cancer patients undergoing genetic testing in North America., ((c) 2005 Wiley-Liss, Inc.)

Base-calling precision of short tandem repeat (STR) allelic bands on dynamic slab-gel electrophoresis systems was evaluated. Data was collected from over 6000 population database allele peaks generated from 468 population database samples amplified with the AmpF/STR Profiler Plus (PP) kit and electrophoresed on ABD 377 DNA sequencers. Precision was measured by way of standard deviations and was shown to be essentially the same, whether using fixed or floating bin genotyping. However, the allelic ladders have proven more sensitive to electrophoretic variations than database samples, which have caused some floating bins of D18S51 to shift on occasion. This observation prompted the investigation of polyacrylamide gel formulations in order to stabilize allelic ladder migration. The results demonstrate that, although alleles comprised in allelic ladders and questioned samples run on the same gel should migrate in an identical manner, this premise needs to be verified for any given electrophoresis platform and gel formulation. We show that the compilation of base-calling data is a very informative and useful tool for assessing the performance stability of dynamic gel electrophoresis systems, stability on which depends genotyping result quality.

As part of the validation of the AmpFlSTR Profiler Plus short tandem repeat (STR) system, under reduced polymerase chain reaction (PCR) volume conditions (i.e., 25 microL), a total of 275 casework samples were processed. Examples of profiles are presented along with amplification conditions to improve the odds of obtaining balanced and complete profiles for samples showing partial results or profiles with a descending slope. Data collected and used to develop our interpretation guidelines are included. From the mixture studies, full profiles were obtained for minor contributors, using 2 ng of DNA, with ratios of 10:1 or 1:20 and using 1 ng of DNA, with ratios of 10:1 and 1:8. The specificity of the Profiler Plus amplification reaction performed in 25 microL was examined and confirmed using a large spectrum of nonhuman DNAs. This report supports the use of the AmpFlSTR Profiler Plus STR system for casework DNA typing under reduced PCR volume conditions.