Comparison of whole genome amplification and nested-PCR methods for preimplantation genetic diagnosis for BRCA1 gene mutation on unfertilized oocytes-a pilot study.
BACKGROUND
Preimplantation genetic diagnosis (PGD) remains nowadays a valid alternative for couples at high-risk of having a child with a genetic disease and for women older than 37-40 years with the high risk of chromosomal aneuploidies in the embryos. However the use of PGD for high penetrance recessive, dominant and X-liked disorders occurring in early life is documented, debate exists regarding its appropriateness in lower penetrance and late-onset cancer susceptibility syndromes. The data regarding the efficacy of different molecular techniques used in PGD are still lacking. We therefore sought to assess the different molecular techniques used in PGD for detecting three most frequent BRCA1 gene mutations: 5382insC, 185delAG and C61G.


METHODS
Anonymous donors of the oocytes and control healthy blood samples were extracted and analyzed in the Fertility and Reproductive Center Invicta in Gdansk. Preimplantation genetic diagnosis for the most frequent mutations: 185delAG, 5382insC, C61G in BRCA 1 gene was carried out on single, unfertilized oocytes, in metaphase of second meiotic division, not qualified to IVF. Positive mutation controls were represented by cell lines from the Coriell Institute for Medical Research: GM14090 (185delAG), GM14097 (C61G), GM13715 (5382insC).


RESULTS
Repeatability of the results acquired from the WGA analysis for the mutation 5382insC was 38%. The repeatability of the nested-PCR analysis in the second round of the amplification was labile for the mutation 5382insC and 185delAG and was ranged from 47% to 57%. However, the repeatability for the mutation C61G was 100%.


CONCLUSIONS
Our results suggest that the nested-PCR technique remains more sensitive and specific method as compared to WGA. WGA performed on the single cells did not reflect expected results. The repeatability of the WGA methodology remains questionable, and any analysis attempt does not guarantee reliable results. Further evaluation is strongly needed to propose the most accurate molecular technique used in PGD for detecting three most frequent BRCA1 gene mutations: 5382insC, 185delAG and C61G.
INVICTA Fertility and Reproductive Center, Rajska 10, Gdansk, 80-850, Poland.|Faculty of Biology, University of Gdansk, Gdansk, Poland.
Danuta
D
Michalska
INVICTA Fertility and Reproductive Center, Rajska 10, Gdansk, 80-850, Poland.
Kinga
K
Jaguszewska
INVICTA Fertility and Reproductive Center, Rajska 10, Gdansk, 80-850, Poland.
Joanna
J
Liss
INVICTA Fertility and Reproductive Center, Rajska 10, Gdansk, 80-850, Poland.
Kamila
K
Kitowska
INVICTA Fertility and Reproductive Center, Rajska 10, Gdansk, 80-850, Poland.
Agata
A
Mirecka
INVICTA Fertility and Reproductive Center, Rajska 10, Gdansk, 80-850, Poland.|Department of Obstetrics, Gynecology and Endocrinology, University of Varmia and Masuria, Olsztyn, Poland.|Department of Obstetrics and Gynecology Nursing, Medical University of Gdansk, Gdansk, Poland.
Krzysztof
K
Łukaszuk