[CANCER RESEARCH 63, 1449 –1453, April 1, 2003] Advances in Brief Large Genomic Deletions and Duplications in the BRCA1 Gene Identified by a Novel Quantitative Method Frans B. L. Hogervorst, Petra M. Nederlof, Johan J. P. Gille, Cathal J. McElgunn, Maartje Grippeling, Roelof Pruntel, Rein Regnerus, Tibor van Welsem, Resie van Spaendonk, Fred H. Menko, Irma Kluijt, Charlotte Dommering, Senno Verhoef, Jan P. Schouten, Laura J. van’t Veer, and Gerard Pals 1 Family Cancer Clinic, Netherlands Cancer Institute, Amsterdam [F. B. L. H., P. M. N., R. P., R. R., T. v. W., I. K., C. D., S. V., L. J. v. V.]; Family Cancer Clinic, Department of Clinical Genetics and Human Genetics, VU Medical Center, 1081 BT Amsterdam [J. J. P. G., M. G., R. v. S., F. H. M., C. D., G. P.]; MRC-Holland, Amsterdam [C. J. M., T. v. W., J. P. S.]; and Department of Clinical Genetics, Amsterdam Medical Center, Amsterdam [I. K., S. V.], the Netherlands Abstract We applied a novel method to detect single or multiple exon deletions and amplifications in the BRCA1 gene. The test, called multiplex ligation- dependent probe amplification (MLPA), uses probes designed to hybridize adjacently to the target sequence. After ligation, the joined probes are amplified and quantified. Our two diagnostic laboratories have tested in the recent years 805 families by conventional PCR-based techniques, and found 116 BRCA1 and 28 BRCA2 mutation-positive families. Using MLPA, we have tested the remaining 661 noninformative breast cancer families and identified five distinct BRCA1 germ-line mutations in five families: a deletion of exon 8, a deletion of exons 20 –22, a duplication of exon 13 and exons 21–23, respectively, and a triplication, encompassing exons 17–19. Genomic deletions of BRCA1 constitute a substantial fraction of mutations in Dutch breast cancer families. If MLPA had been included in our initial BRCA1 testing, 33 families with a deletion or duplication would have been identified, representing 27% of the total 121 BRCA1 mutation-positive families. The MLPA test for BRCA1 ensures a sensitive and comprehensive high-throughput screening test for genomic rear- rangement and can easily be implemented in the molecular analysis of BRCA1. Introduction Germ-line mutations in the BRCA1 gene cause a hereditary predis- position to breast and ovarian cancer. Methods used to screen for mutations focus on genomic DNA and are usually PCR-based, ena- bling the detection of sequence alterations such as point mutations, and small deletions and insertions. By this approach, thousands of BRCA1 families have been identified worldwide (the Breast Cancer Information Core on the Internet). 2 An increasing number of large genomic alterations have been described recently as laboratories put more effort into the detection of such alterations. At present, 18 different large genomic rearrangements have been characterized; they include both deletions and duplications of one or more exons in BRCA1 (1– 6). Although for some recurrent mutations a simple PCR test has been developed (4, 5), the gold standard to search for aberrant copy numbers of one or more exons is Southern blotting. However, this technique is time consuming and laborious and, therefore, screen- ing is usually restricted to selected families (1– 4, 7, 8). Ideally, all of the individuals eligible for BRCA1 mutation screening should be screened for the presence of large genomic deletions and duplications. Therefore, we applied a new method, called MLPA, 3 which enables us to determine the relative copy number of all of the BRCA1 exons simultaneously with high sensitivity in a high-throughput format (9). We have shown recently that this method is very successful in the identification of large genomic deletions in the hMLH1 and hMSH2 genes in hereditary nonpolyposis colorectal carcinoma families (10). The principle of this technique is shown in Fig. 1. In brief, only adjacently hybridized and subsequently ligated probes can be ampli- fied by PCR. Because up to 40 –50 target sequences can be analyzed simultaneously, the complete BRCA1 gene can be screened in one single reaction. Fragment analysis is carried out on an automated, preferably capillary, sequencer identifying each of the fragments based on the specific PCR fragment length. The measured peak area is used to calculate the relative quantity of each probe. In this report we show the results of the MLPA-BRCA1 analyses of 660 breast and/or ovarian cancer families, reveal alterations not reported before, and conclude that the method is reliable and very suitable to be included in the routine molecular analysis of predisposed families. Materials and Methods Family Ascertainment. Self-referred or physician-referred breast and/or ovarian cancer-prone families investigated at the family cancer clinics of the Netherlands Cancer Institute and the VU University Medical Center, which were opting for mutation screening, were the subject of this study. Between 1995 and 2001, individuals from 805 families have been tested in our labora- tories of which 79% were breast cancer only families, 18% were breast and ovarian cancer families, and 3% were ovarian cancer only families. In general, these families had an a priori chance of 10% of harboring a deleterious BRCA1 or BRCA2 mutation (11). MLPA Reaction and Fragment Analysis. The BRCA1-MLPA test was developed and manufactured by MRC-Holland in close collaboration with one of the authors (G. P.). The preparation and sequences of the probes has been described elsewhere (9). 4 The BRCA1-MLPA test itself is commercially avail- able at MRC-Holland. In short, 50 –500 ng target DNA/5 l of 10 mM Tris (pH 8)-0.1 mM EDTA was denatured for 5 min at 98°C after which 3 l of the probe mix was added. The mixture was heated at 95°C for 1 min and incubated at 60°C overnight (16 h). Ligation was performed with the temperature-stable Ligase-65 enzyme (MRC-Holland) for 15 min at 54°C. Next, the ligase was inactivated by incubation for 5 min at 98°C. Ten l of this ligation mix was premixed with 30 l of PCR buffer and put in a PCR machine at 60°C. Subsequently, a 10-l mix was added containing deoxynucleoside triphos- phate, Taq polymerase, and one unlabeled and one carboxyfluorescein-labeled PCR primer, which are complementary to the universal primer sequences. PCR was carried out for 33 cycles (30 s at 95°C, 30 s at 60°C, and 60 s at 72°C). The fragments were analyzed on an ABI model 310 or 3700 capillary se- quencer (Applied Biosystems) using Genescan-ROX 500 size standards (Ap- Received 11/11/02; accepted 2/18/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Department of Clinical and Human Genetics, VU Medical Center, van der Boechorststraat 7, 1081 BT, Amsterdam, the Netherlands. Phone: 31-20-4448278; Fax: 31-20-4448293; E-mail: g.pals@VUmc.nl. 2 Internet address: http://www.nhgri.nih.gov/Intramural_research/Lab_transfer/Bic/. 3 The abbreviations used are: MLPA, multiplex ligation-dependent probe amplifica- tion; nt, nucleotide; RT-PCR, reverse transcription-PCR. 4 Internet address: http://www.mrc-holland.com. 1449 Research. on February 16, 2016. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from