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Comparison of whole genome amplification and nested-PCR methods for preimplantation genetic diagnosis for BRCA1 gene mutation on unfertilized oocytes-a pilot study.

ABSTRACT: 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.

SUBMITTER: Michalska D

PROVIDER: S-EPMC3751838 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Comparison of whole genome amplification and nested-PCR methods for preimplantation genetic diagnosis for BRCA1 gene mutation on unfertilized oocytes-a pilot study.

Michalska Danuta D Jaguszewska Kinga K Liss Joanna J Kitowska Kamila K Mirecka Agata A Łukaszuk Krzysztof K

Hereditary cancer in clinical practice 20130813 1

<h4>Background</h4>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. Th ...[more]

PMID: 23941236

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Project description:PURPOSE:To compare multiple displacement amplification and OmniPlex whole genome amplification technique performance during array comparative genome hybridization (aCGH), Sanger sequencing, SNaPshot and fragment size analysis downstream applications in frame of multifactor embryo preimplantation genetic testing. METHODS:Preclinical workup included linked short tandem repeat (STR) marker selection and primer design for loci of interest. It was followed by a family haplotyping, after which an in vitro fertilization preimplantation genetic testing (IVF-PGT) cycle was carried out. A total of 62 embryos were retrieved from nine couples with a confirmed single gene disorder being transmitted in their family with various inheritance traits-autosomal dominant (genes-ACTA2, HTT, KRT14), autosomal recessive (genes-ALOX12B, TPP1, GLB1) and X-linked (genes-MTM1, DMD). Whole genome amplification (WGA) for the day 5 embryo trophectoderm single biopsies was carried out by multiple displacement amplification (MDA) or polymerase chain reaction (PCR)-based technology OmniPlex and was used for direct (Sanger sequencing, fragment size analysis, SNaPshot) and indirect mutation assessment (STR marker haplotyping), and embryo aneuploidy testing by array comparative genome hybridization (aCGH). RESULTS:Family haplotyping revealed informative/semi-informative microsatellite markers for all clinical cases for all types of inheritance. Indirect testing gave a persuasive conclusion for all embryos assessed, which was confirmed through direct testing. The overall allele dropout (ADO) rate was higher for PCR-based WGA, and MDA shows a better genomic recovery scale. Five euploid embryos were subjected to elective single embryo transfer (eSET), which resulted in four clinical pregnancies and birth of two healthy children, which proved free of disease causative variants running in the family postnataly. CONCLUSIONS:A developed multifactor PGT protocol can be adapted and applied to virtually any genetic condition and is capable of improving single gene disorder preimplantation genetic testing in a patient-tailored manner thus increasing pregnancy rates, saving costs and increasing patient reliability.

Project description:BACKGROUND: WGA (Whole Genome Amplification) in forensic genetics can eliminate the technical limitations arising from low amounts of genomic DNA (gDNA). However, it has not been used to date because any amplification bias generated may complicate the interpretation of results. Our aim in this paper was to assess the applicability of MDA to forensic SNP genotyping by performing a comparative analysis of genomic and amplified DNA samples. A 26-SNPs TaqMan panel specifically designed for low copy number (LCN) and/or severely degraded genomic DNA was typed on 100 genomic as well as amplified DNA samples. RESULTS: Aliquots containing 1, 0.1 and 0.01 ng each of 100 DNA samples were typed for a 26-SNPs panel. Similar aliquots of the same DNA samples underwent multiple displacement amplification (MDA) before being typed for the same panel. Genomic DNA samples showed 0% PCR failure rate for all three dilutions, whilst the PCR failure rate of the amplified DNA samples was 0% for the 1 ng and 0.1 ng dilutions and 0.077% for the 0.01 ng dilution. The genotyping results of both the amplified and genomic DNA samples were also compared with reference genotypes of the same samples obtained by direct sequencing. The genomic DNA samples showed genotype concordance rates of 100% for all three dilutions while the concordance rates of the amplified DNA samples were 100% for the 1 ng and 0.1 ng dilutions and 99.923% for the 0.01 ng dilution. Moreover, ten artificially-degraded DNA samples, which gave no results when analyzed by current forensic methods, were also amplified by MDA and genotyped with 100% concordance. CONCLUSION: We investigated the suitability of MDA material for forensic SNP typing. Comparative analysis of amplified and genomic DNA samples showed that a large number of SNPs could be accurately typed starting from just 0.01 ng of template. We found that the MDA genotyping call and accuracy rates were only slightly lower than those for genomic DNA. Indeed, when 10 pg of input DNA was used in MDA, we obtained 99.923% concordance, indicating a genotyping error rate of 1/1299 (7.7 x 10(-4)). This is quite similar to the genotyping error rate of STRs used in current forensic analysis. Such efficiency and accuracy of SNP typing of amplified DNA suggest that MDA can also generate large amounts of genome-equivalent DNA from a minimal amount of input DNA. These results show for the first time that MDA material is suitable for SNP-based forensic protocols and in general when samples fail to give interpretable STR results.

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Comparison of whole genome amplification and nested-PCR methods for preimplantation genetic diagnosis for BRCA1 gene mutation on unfertilized oocytes-a pilot study.

Publications

Comparison of whole genome amplification and nested-PCR methods for preimplantation genetic diagnosis for BRCA1 gene mutation on unfertilized oocytes-a pilot study.

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