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. 2013 Dec;21(12):1361-8.
doi: 10.1038/ejhg.2013.50. Epub 2013 Mar 27.

PGD for Hereditary Breast and Ovarian Cancer: The Route to Universal Tests for BRCA1 and BRCA2 Mutation Carriers

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Free PMC article

PGD for Hereditary Breast and Ovarian Cancer: The Route to Universal Tests for BRCA1 and BRCA2 Mutation Carriers

Marion Drüsedau et al. Eur J Hum Genet. .
Free PMC article

Abstract

Preimplantation Genetic Diagnosis (PGD) is a method of testing in vitro embryos as an alternative to prenatal diagnosis with possible termination of pregnancy in case of an affected child. Recently, PGD for hereditary breast and ovarian cancer caused by BRCA1 and BRCA2 mutations has found its way in specialized labs. We describe the route to universal single-cell PGD tests for carriers of BRCA1/2 mutations. Originally, mutation-specific protocols with one or two markers were set up and changed when new couples were not informative. This route of changing protocols was finalized after 2 years with universal tests for both BRCA1 and BRCA2 mutation carriers based on haplotyping of, respectively, 6 (BRCA1) and 8 (BRCA2) microsatellite markers in a multiplex PCR. Using all protocols, 30 couples had a total of 47 PGD cycles performed. Eight cycles were cancelled upon IVF treatment due to hypostimulation. Of the remaining 39 cycles, a total of 261 embryos were biopsied and a genetic diagnosis was obtained in 244 (93%). In 34 of the 39 cycles (84.6%), an embryo transfer was possible and resulted in 8 pregnancies leading to a fetal heart beat per oocyte retrieval of 20.5% and a fetal heart beat per embryonic transfer of 23.5%. The preparation time and costs for set-up and validation of tests are minimized. The informativity of microsatellite markers used in the universal PGD-PCR tests is based on CEPH and deCODE pedigrees, making the tests applicable in 90% of couples coming from these populations.

Figures

Figure 1
Figure 1
PGD protocols. Time-line overview of the developed single-cell PGD-PCR protocols for BRCA1 and BRCA2 mutation carriers. (a) Developed protocols for BRCA1, (b) developed protocols for BRCA2.
Figure 2
Figure 2
BRCA1 and BRCA2 loci and markers. Schematic overview of the BRCA1 and BRCA2 locus on chromosomes 17 and 13, respectively. The large gene arrows indicate the direction of the loci with respect to chromosome orientation. Marker names and their genetic (cM) and physical distances (Mb) to the BRCA loci are indicated in the figure. Genetic distance is based on the Genethon genetic map. Heterozygosity of marker alleles (if known) are indicated between brackets. Underlined markers are the markers used in the described universal tests.
Figure 3
Figure 3
Haplotype analysis. (a) Genescan analysis example of a BRCA1 PGD index (patient) and a BRCA2 PGD index (partner). Absolute allele lengths are indicated in boxes, marker names above the diagrams. Colored peaks (see online colored version) indicate fluorescent labeling of VIC=green, NED=black, FAM=blue and PET=red. (b) Examples of a BRCA1 and BRCA2 family with haplotypes used in PGD cycles. Big black arrows represent patients corresponding to the genescan analysis in (a). Alleles in black represent the risk haplotypes co-segregating with the mutation. Asterisk indicates the position of the familial mutation.

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