Improved sensitivity to detect recombination using qPCR for Dyskeratosis Congenita PGD

Gueye, Ndeye-Aicha; Jalas, Chaim; Tao, Xin; Taylor, Deanne; Scott, Richard T.; Treff, Nathan R.

doi:10.1007/s10815-014-0298-9

Cited by 10 publications

(5 citation statements)

References 17 publications

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“…In general, obtaining micrograms of DNA through WGA of day-5 embryo biopsies allowed us to perform embryo haplotype analysis, aneuploidy screening by aCGH and direct mutation testing through SNaPshot, Sanger sequencing or fragment size analysis. As shown before, direct variant locus testing boldly complements the indirect one [ 18 , 21 ], since crossover events cannot be completely ruled out and ignored [ 11 ] especially in the case of ADO, which was also true in our cohort. We designed hemi-nested primers for all assessed loci following PGDIS guidelines for good practice in PGD [ 16 , 24 ].…”

Section: Discussionmentioning

confidence: 84%

Performance comparison of two whole genome amplification techniques in frame of multifactor preimplantation genetic testing

Voložonoka

Perminov

Kornejeva

et al. 2018

J Assist Reprod Genet

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PurposeTo 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.MethodsPreclinical 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).ResultsFamily 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.ConclusionsA 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.Electronic supplementary materialThe online version of this article (10.1007/s10815-018-1187-4) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 84%

Performance comparison of two whole genome amplification techniques in frame of multifactor preimplantation genetic testing

Voložonoka

Perminov

Kornejeva

et al. 2018

J Assist Reprod Genet

View full text Add to dashboard Cite

show abstract

“…Although haplotype analysis with short tandem repeat (STR) may reduce the effects of ADO, the number of STR loci is limited. Further, recombination between STR loci and target genes may affect the diagnostic accuracy [ 17 , 18 ]. Hence, single-nucleotide polymorphisms (SNPs) linked to the mutated genes are increasingly being used to establish haplotype linkages in clinical practice [ 19 ].…”

Section: Discussionmentioning

confidence: 99%

Using affected embryos to establish linkage phase in preimplantation genetic testing for thalassemia

Deng

Liang

et al. 2022

Reprod Biol Endocrinol

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Background This study aimed to evaluate the ability of next-generation sequencing (NGS) to conduct preimplantation genetic testing (PGT) for thalassemia using affected embryos. Methods This study included data from 36 couples who underwent PGT for thalassemia without probands and relative pedigrees. NGS results were compared with prenatal diagnosis results. Results Thirty-six couples (29 α-thalassemia and 7 β-thalassemia) underwent 41 PGT cycles (31 α-thalassemia and 10 β-thalassemia). Analysis using NGS produced conclusive results for all biopsied blastocysts (100%, 217/217). One hundred and sixty (73.7%, 160/217) were unaffected by thalassemia. Preimplantation genetic testing for aneuploidy revealed that 112 (70.0%, 112/160) were euploid. Single blastocysts were transferred into the uteri of 34 women (53 frozen embryo transfer [FET] cycles). Thirty-two cycles resulted in clinical pregnancies, with a clinical pregnancy rate of 60.1% (32/53) per FET cycle. Twenty-two cycles (22 couples) resulted in 23 live births, with a live birth rate of 43.4% (23/53; 3 cycles were ongoing pregnancies). All 25 embryos’ prenatal diagnosis results and/or thalassemia gene analyses after delivery were concordant with the NGS-PGT results. Seven embryos (21.9%, 7/32) were miscarried before 12 weeks’ gestation, and the abortion villus in four showed a normal karyotype and thalassemia results consistent with the NGS-PGT results. Aborted fetus samples from 3 cycles were not available because the pregnancy lasted less than 5 weeks. Conclusion NGS can be used to conduct PGT for thalassemia using affected embryos as a reference. Trial registration Retrospectively registered.

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“…Although haplotype analysis with short tandem repeat (STR) may reduce the effects of ADO, the number of STR loci is limited. Further, recombination between STR loci and target genes may affect the diagnosis accuracy (16,17). Hence, single-nucleotide polymorphisms (SNPs) linked to the mutated genes are being used more and more to establish haplotype linkages in clinical practice (18).…”

Section: Discussionmentioning

confidence: 99%

Preimplantation Genetic Testing for Thalassemia Through Next- Generation Sequencing of Affected-embryos

Deng

Liang

et al. 2021

Preprint

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Background: To evaluate the ability of next-generation sequencing (NGS) to conduct preimplantation genetic testing (PGT) for thalassemia using affected embryos. Methods: This study included data from 36 couples who underwent PGT for thalassemia without proband and relative pedigrees. NGS results were compared with prenatal diagnosis results.Results: Thirty-six couples (29 α-thalassemia and 7 β-thalassemia) underwent 41 PGT cycles (31 α-thalassemia and 10 β-thalassemia). All biopsied blastocysts received conclusive results from NGS analysis (100%, 217/217). One hundred and sixty (73.7%, 160/217) were determined to be unaffected by thalassemia. PGT-A (PGT for aneuploidy) results showed that 112 (70.0%, 112/160) were euploid. Thirty-four couples were transferred with a single blastocyst (53 frozen embryo transfer (FET) cycles). Thirty-two cycles resulted in clinical pregnancies, and the clinical pregnancy rate was 60.1% (32/53) per FET cycle. Twenty-two cycles (22 couples) resulted in 23 live births and the live birth rate was 43.4% (23/53, 3 cycles were ongoing pregnancy). All 25 cycles’ prenatal diagnosis results and/or thalassemia gene analysis after the delivery were concordant with the NGS-PGT results. Seven cycles were miscarried before 12 weeks’ gestation, and the abortion villus in four cycles showed a normal karyotype and thalassemia results consistent with the NGS-PGT results. Aborted fetus samples from 3 cycles were not available because the pregnancy was less than 5 weeks.Conclusion: NGS can be used to conduct PGT for thalassemia using affected embryos as a reference.Trial registration: Retrospectively registered.

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Improved sensitivity to detect recombination using qPCR for Dyskeratosis Congenita PGD

Cited by 10 publications

References 17 publications

Performance comparison of two whole genome amplification techniques in frame of multifactor preimplantation genetic testing

Performance comparison of two whole genome amplification techniques in frame of multifactor preimplantation genetic testing

Using affected embryos to establish linkage phase in preimplantation genetic testing for thalassemia

Preimplantation Genetic Testing for Thalassemia Through Next- Generation Sequencing of Affected-embryos

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