Abstract
BackgroundPreimplantation 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.MethodsAnonymous 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).ResultsRepeatability 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%.ConclusionsOur 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.
Highlights
Preimplantation genetic diagnosis (PGD) remains nowadays a valid alternative for couples at high-risk of having a child with monogenetic diseases, i.e. cystic fibrosis, β-talasemia, Huntington’s disease, myotonic dystrophy and for women with the high risk of chromosomal aneuploidies in the embryos [1,2].Large body of literature documented the use of PGD for high penetrance recessive, dominant and X-liked disorders occurring in early life
whole genome amplification (WGA) performed on the single cells did not reflect expected results
The present study reports a comparison of two PGD techniques: WGA and nested-PCR
Summary
Large body of literature documented the use of PGD for high penetrance recessive, dominant and X-liked disorders occurring in early life. It was used as well in case of high penetrance cancer syndromes that appear later [3,4]. 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. 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. We sought to assess the different molecular techniques used in PGD for detecting three most frequent BRCA1 gene mutations: 5382insC, 185delAG and C61G
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