Fig 1 - uploaded by Anver Kuliev
Content may be subject to copyright.
Schematic diagram of multiplex PCR for the delta F-508 mutation and intron 6 polymorphism marker in the CFTR gene. Top left: Intron 6, 4-bp polymorphism (six or seven repeats). Primer sequences described in Ref. 4. Top right: Exon 10 delta-F508 mutation. Primer sequences described in Ref. 4. Middle: Expected sizes of the second-round PCR products for both loci. Bottom: Diagram of scheme. Initially four outside primers are added to the single cell and 25 rounds of PCR performed. Then 2-to 5-u aliquots are distributed into two separate tubes. In one tube the inside primers for the intron 6 polymorphism are added prior to performing 25 additional PCR cycles (second-round PCR). In the second tube, inside primers for the delta F-508 mutation are added prior to performing 25 additional PCR cycles second-round PCR).
Source publication
Because allele dropout (ADO) is frequently observed in single-cell polymerase chain reaction analysis, it is important to develop a method for efficient detection of ADO, in order to avoid possible misdiagnosis in preimplantation diagnosis.
We introduced a simultaneous amplification of mutant genes and linked polymorphic markers, such as a 4-bp rep...
Context in source publication
Context 1
... multiplex PCR experiments the first-round reac- tion buffer contained a mixture of outside primers for all systems. After 25 cycles, 2-5 (xl was removed and placed in separate tubes containing the inside primers for each individual locus (Fig. 1). PCR products were analyzed by heteroduplex detection for delta F-508, by Ddel (Promega) restriction digestion for sickle cell disease, and by fragment size assignment for short tandem repeats ...
Similar publications
We have identified a DNA-binding activity with specificity for the beta DRE, an evolutionarily conserved transcriptional regulatory
element in mammalian adult beta-globin promoters. This binding activity, which we term beta DRf, for beta-globin direct repeat
factor, was detected in fractionated nuclear extracts from the murine erythroleukemia cell...
Citations
... The ADO rate for detecting genetic markers in PB1 was reported to be approximately 5.9%-9.6% [22] . Given the relatively low ADO rate, the corresponding embryos of oocyte 2 and 6 probably inherited the wildtype allele. ...
Background Trophectoderm (TE) cell biopsy at the blastocyst stage is currently the predominant method used in preimplantation genetic testing for monogenic disorders (PGT-M). However, this approach may result in the discarding of genetically unaffected embryos due to the fact that not all embryos progress to the blastocyst stage. This study aimed to investigate the advantages of utilizing polar body (PB) biopsy in females with disease-causing mutations and a limited number of oocytes undergoing PGT-M. Methods Three couples with females carrying autosomal dominant or X-linked mutations in IRF6, FMR1, and EDA were recruited. The number of retrieved oocytes was no more than six per cycle. The first and second PBs (PB1 and PB2) of each oocyte were individually biopsied and subjected to multiple displacement amplification (MDA). The genotype of each embryo was deduced by analyzing the MDA products of corresponding PB1 and PB2 using a novel strategy that combined direct mutation testing and single nucleotide polymorphism linkage analysis. Mutation-free embryos cryopreserved before the blastocyst stage were selected to transfer. Results In total, four cycles were conducted resulting in the retrieval of 15 oocytes for the three couples. The genotype of each embryo was successfully determined. Seven mutation-free embryos were identified. Three of them were transferred and resulted in two clinical pregnancies, culminating in the birth of two healthy babies. The accuracy of the embryo genotypes was validated through genetic testing of the fetuses in the second trimester or at birth. Conclusions The PB-based strategy is feasible and effective to detect the mutation carrier statuses of embryos in PGT-M for maternal mutations. Compared with blastocyst stage detection, this method may preserve a higher number of genetically unaffected embryos for the patients. Further clinic trials are warranted to verify whether PB biopsy offers greater benefits than TE cell biopsy for females with disease-causing mutations and a limited number of oocytes in PGT-M.
... Fetal cells and free fetal DNA are also present in the circulation of the pregnant mother and provided a potential source for "non-invasive" fetal sampling, but reliable protocols have yet to be established for clinical application [20,21]. As data have accumulated from chromosomal analysis of human pre-implantation embryos, it has become apparent that there is higher rate of chromosomal abnormalities in cleavage stage embryos and blastocyst detected by FISH [22,23]. Reported pregnancy rates vary, but rarely surpass about one third of all cycles initiated [24,25]. ...
Pre-implantation genetic diagnosis (PIGD) is the genetic profiling of the embryos prior to implantation and sometimes even of oocytes prior to fertilization. PGD is considered in a similar fashion to prenatal diagnosis. Initially offered for diagnosis in couples at risk for single gene genetic disorders, such as cystic fibrosis, spinal muscular atrophy and Huntington’s disease, preimplantation genetic diagnosis (PGD) has most frequently been employed in assisted reproduction for detection of chromosome aneuploidy from advancing maternal age or structural chromosome rearrangements. Major improvements have been seen in PGD analysis with movement away from older, less effective technologies, such as fluorescence in situ hybridization (FISH), to newer molecular tools, such as DNA microarrays and next generation sequencing. Discussions regarding the scientific, ethical, legal and social issues surrounding the use of sequence data from embryo biopsy have begun and must continue to avoid concern regarding eugenic or inappropriate use of this technology.
... NP_000483.3:p.(Leu467Phe)) в составе комплексного аллеля с мутацией F508del, доказанно приводящего к резистентности к терапии [83][84][85][86][87][88] (УУР -С УДД -4). ...
The problem of timely diagnosis and proper management of patients with cystic fibrosis is crucial not only in our country, but throughout the world. Experts of the Union of Pediatricians of Russia have considered various issues of etiology, pathogenesis, epidemiology, diagnosis, and treatment of this genetic disease in a modern light. Particular attention was paid to screening methods for early diagnosis of cystic fibrosis. The principles of complex therapy were justified, including rational use of antibacterial and mucolytic drugs and enzyme replacement therapy that significantly determine the disease prognosis.
... PGD has introduced a valuable and sometimes irreplaceable procedure and brings several technically challenging areas together like in vitro fertilization, embryo biopsy and culture, and molecular genetic diagnosis on a single cell. Using multiplex PCR in PGD which simultaneously amplify many linked informative polymorphic markers to a specific gene, was found to be quite helpful for the detection of both contamination and allele dropouts (ADOs) [13]. ADO is defined as the failure or preferential amplification of only one allele and is the primary cause of misdiagnosis in PGD especially if a single approach like mutation detection is applied [14]. ...
Background
Preimplantation genetic diagnosis (PGD) has been developed to detect genetic disorders before pregnancy which is usually done on blastomeres biopsied from 8-cell stage embryos obtained from in vitro fertilization method (IVF).
Here we report molecular PGD results for diagnosing of beta thalassemia (beta-thal) which are usually accompanied with evaluating chromosomal aneuploidies, HLA typing and sex selection.
Methods
In this study, haplotype analysis was performed using short tandem repeats (STRs) in a multiplex nested PCR and the causative mutation was detected by Sanger sequencing.
Results
We have performed PGDs on 350 blastomeres from 55 carrier couples; 142 blastomeres for beta-thal only, 75 for beta-thal and HLA typing, 76 for beta-thal in combination with sex selection, and 57 for beta-thal and aneuploidy screening. 150 blastomeres were transferable, 15 pregnancies were happened, and 11 babies born.
We used 6 markers for beta-thal, 36 for aneuploidy screening, 32 for sex selection, and 35 for HLA typing. To our knowledge combining all these markers together and the number of STR markers are much more than any other studies which have ever done.
Conclusions
PGD is a powerful diagnostic tool for carrier couples who desire to have a healthy child and wish to avoid medical abortion.
... PGD has introduced a valuable and sometimes irreplaceable procedure and brings several technically challenging areas together like in vitro fertilization, embryo biopsy and culture, and molecular genetic diagnosis on a single cell. Usingmultiplex PCR in PGDwhich simultaneously amplify many linked informative polymorphic markers to a speci c gene,wasfound to be quite helpful for the detection of both contamination and allele dropouts (ADOs) (13). ADO is de ned as the failure or preferential ampli cation of only one allele and is the primary cause of misdiagnosis in PGD especially if a single approach like mutation detection is applied (14). ...
Background: Preimplantation genetic diagnosis (PGD) has been developed to detect genetic disorders before pregnancy which is usually done on blastomeres biopsied from 8-cell stage embryos obtained from in vitro fertilization method (IVF).
Here we report molecular PGD results for diagnosing of beta thalassemia (beta-thal) which are usually accompanied with evaluating chromosomal aneuploidies, HLA typing and sex selection.
Methods: In this study, haplotype analysis was performed using short tandem repeats (STRs) in a multiplex nested PCR and the causative mutation was detected by Sanger sequencing.
Results: We have performed PGDs on 350 blastomeres from 55 carrier couples; 142 blastomeres for beta-thal only, 75 for beta-thal and HLA typing, 76 for beta-thal in combination with sex selection, and 57 for beta-thal and aneuploidy screening. 150 blastomeres were transferable, 15 pregnancies were happened, and 11 babies born.
We used 6 markers for beta-thal, 36 for aneuploidy screening, 32 for sex selection, and 35 for HLA typing. To our knowledge combining all these markers together and the number of STR markers are much more than any other studies which have ever done.
Conclusions: PGD is a powerful diagnostic tool for carrier couples who desire to have a healthy child and wish to avoid medical abortion.
... Através desta técnica, é possível também minimizar o fenômeno "allelic dropout", este que pode levar a não detecção de um alelo mutante em uma célula heterozigótica e é o fenômeno mais comum associado às técnicas de PCR. Para isto, junto com o gene de interesse são amplificados marcadores polimórficos associados, podendo diminuir pela metade o erro diagnóstico pelo fenômeno (Rechitsky, 1998). ...
O desenvolvimento das tecnologias genéticas pré-implantacionais nos permite, hoje, identificar anomalias genéticas hereditárias, realizar triagem em busca de mutações e analisar previamente a viabilidade de um embrião, inclusive conhecer a compatibilidade genética embrionária com a de uma pessoa já nascida e doente, que necessite de um doador compatível. Os testes genéticos pré-implantacionais consistem em um conjunto de técnicas, realizadas após o processo de Fertilização in vitro (FIV) e antes da implantação do embrião. Devido à escassez de artigos na literatura brasileira sobre o assunto, foi desenvolvida uma revisão de literatura, que tem como objetivo explanar sobre os avanços que ocorreram nos últimos 30 anos nos testes utilizados para o diagnóstico genético pré-implantacional (DGPI), esclarecendo e apresentando seus protocolos. Para isso, foram selecionadas as principais técnicas adotadas, sendo elas Nested PCR, Multiplex PCR, qPCR, FISH, aCGH, SNP’s, NGS Ion Torrent e Illumina, além de contextualizar a cronologia dos testes e apresentar todo o contexto ético que os envolve. Para tanto, foram utilizadas 48 referências de língua inglesa, portuguesa e espanhola, em sua maioria datadas entre 2017 a 2021. É fato que o DGPI confere um grande avanço no campo da reprodução assistida e possibilita mulheres com idade avançada ou histórico recorrente de aborto, além de pessoas com anomalias genéticas, terem o DNA de seus embriões analisados antes de sua implantação, permitindo identificar mutações que poderão afetar a saúde do bebê e garantindo a sua compatibilidade com a vida.
... New methods have been developed to eliminate or at least minimize some of the issues noted above. Many groups started adding linked markers, typically short tandem repeat (STR) DNA markers that were found inside of the gene or closely linked to the gene of interest [5]. Utilizing these tightly linked markers allows for a secondary analysis of inheritance of the gene in question and also allows the laboratory to track contamination of DNA from exogenous sources including maternal, paternal, IVF lab staff, and molecular lab staff. ...
Purpose
The purpose of this research is to study the clinical outcomes using a next-generation sequencing-based protocol allowing for simultaneous testing of mutations in the beta thalassemia (HBB) gene, including single nucleotide polymorphism (SNP) markers for PGT-M along with low-pass whole genome analysis of chromosome aneuploidies for PGT-A.
Methods
A combined PGT-M (thalassemia) plus PGT-A system was developed for patients undergoing IVF in Vietnam. Here we developed a system for testing numerous thalassemia mutations plus SNP-based testing for backup mutation analysis and contamination control using next-generation sequencing (NGS). Low -pass next-generation sequencing was used to assess aneuploidy in some of the clinical PGT cases. Patients underwent IVF followed by embryo biopsy at the blastocyst stage for combined PGT-A/M.
Results
Two cases have completed the entire process including transfer of embryos, while a further nine cases have completed the IVF and PGT-M/A analysis but have not completed embryo transfer. In the two cases with embryo transfer, both patients achieved pregnancy with an unaffected, euploid embryo confirmed through prenatal diagnosis. In the further nine cases, 39 embryos were biopsied and all passed QC for amplification. There were 8 unaffected embryos, 31 carrier embryos, and 11 affected embryos. A subset of 24 embryos also had PGT-A analysis with 22 euploid embryos and 2 aneuploid embryos.
Conclusions
Here we report the development and clinical application of a combined PGT-M for HBB and PGT-A for gross chromosome aneuploidies from 11 patients with detailed laboratory findings along with 2 cases that have completed embryo transfer.
... It is noteworthy that there was no conclusive result on the blastomere biopsies of patient #100648 due to failure in the amplification of der [10]. One possible reason for this inconclusive result was the high allelic dropout (failed to amplify one of the two alleles) rate in blastomeres [28,29]. Another possibility is that the large deletion (3673 bases) on der [10] may have adversely affected amplification. ...
Balanced reciprocal translocation carriers are usually phenotypically normal but are at an increased risk of infertility, recurrent miscarriage or having affected children. Preimplantation genetic testing on chromosomal structural rearrangement (PGT-SR) offers a way to screen against unbalanced embryos. Here, we demonstrated a new method to distinguish carrier from noncarrier embryos. Translocation breakpoints were first delineated by nanopore sequencing followed by polymerase chain reaction (PCR) across breakpoints. High-resolution breakpoint mapping was successful in all (9/9) balanced reciprocal translocation carriers. Retrospective analysis of their embryo biopsies with breakpoint PCR showed 100% concordant results with PGT-SR on trophectoderm biopsies (40/40) and 53% concordance on blastomere biopsies (8/15). The low concordant rate in blastomeres was due to failure in the amplification of derivative chromosomes involving large deletions. Breakpoint PCR also showed 100% concordant results with prenatal/postnatal outcomes on 5 pregnancies, indicating that our new method can accurately distinguish carrier from noncarrier embryos.
... 23,24 Dessa maneira é um dos principais problemas da PCR de célula única, dificulta a interpretação dos resultados. 25 Para condiç ões de doenças autossômicas recessivas, quando ambos os pais carregam a mesma mutação, ADO não deve resultar na transferência de um embrião afetado. Contudo, quando os pais são heterozigotos ou em caso de doença autossômica dominante, o ADO pode ter consequências graves, como a transferência de um embrião portador da doença. ...
... 26 Além disso, estudos tem demostrado que a amplificação simultânea de um ou mais marcadores polimórficos, localizados no mesmo cromossomo e perto do gene causador da doença em estudo, pode assegurar uma análise sem ADO. 25,27 Essa metodologia é denominada multiplex PCR e também usada para melhorar a detecção de mutação características de doenças (ex. fibrose cística). ...
O diagnóstico genético pré‐implantacional (PGD) é uma ferramenta que permite a seleção do embrião saudável por meio da análise gênica e cromossômica. Beneficia casais no grupo de risco, como, por exemplo, casais com histórico de aborto de repetição ou com histórico familiar de doença hereditária. Diante do avanço das metodologias empregadas no PGD, esta revisão tem como objetivo reunir as informações acerca das técnicas de biópsia, de biologia molecular e aspectos bioéticos dessa prática. Assim, foi possível agregar as informações como vantagens, desvantagens, restrições e indicações referentes ao estágio embrionário em que é retirado o material genético e as técnicas de biologia molecular usadas na análise genética. Além disso, foi possível especificar alguns questionamentos bioéticos que surgem com a prática do PGD, como, por exemplo, a possível eugenia. Concluiu‐se que a análise da trofoectoderme dos embriões e da aplicação da tecnologia de NGS é promissora para o futuro do PGD, bem como a primordialidade da criação de leis que regem e completam as lacunas no sentido ético e moral dessa prática.
... Allele drop out (ADO) and amplification failure of both alleles are well known phenomena in PCR on single cells. This can depend on several reasons: the cell type employed for the PCR (Rechitsky et al. 1998), genes and markers (Dreesen et al. 1996), lysis and PCR conditions (Levinson et al. 1992;Gitlin et al. 1996;Ray et al. 1996). ...
Introduction:
Preimplantation genetic diagnosis and/or screening (PGD/PGS) allow the assessment of the genetic health of an embryo before transferring it into the uterus. These techniques require the removal of cellular material (polar bodies, blastomere(s) or trophectoderm cells) in order to perform the proper genetic analysis. We report the implantation and live birth outcome of a vitrified-warmed blastocyst developed after triple biopsy and double vitrification procedures at oocyte, cleavage embryo and blastocyst stage.
Case description:
An infertile couple, with family history of β-thalassemia, searched for IVF procedure and PGD. First polar bodies biopsy with subsequent vitrification was uninformative due to meiotic crossing-over, so oocytes were inseminated after warming. Two embryos were obtained and blastomere biopsy was performed on day 3 with inconclusive results on their genetic status. Their culture resulted in one expanded blastocyst stage on day 7 that underwent trophectoderm biopsy and vitrification. This embryo showed to be normal. It was then warmed and transferred in an artificial cycle.
Discussion and evaluation:
Preconception genetic analysis by removal and analysis of the first polar body is technically possible, but the genetic information that we can obtain at this stage may be limited and the oocytes to be inseminated is not predictable. Compared to blastomere biopsy, trophectoderm biopsy has more diagnostic efficiency with respect to both chromosomal mosaicism and PCR accuracy, reducing the problems of amplification failure and allele drop out. Moreover, embryos biopsied at the cleavage stage seem to have lower implantation rate than biopsied blastocyst.
Conclusions:
This is the first case report of a live birth obtained from a three step biopsy and double vitrification procedures of a blastocyst. This case report seems also to suggest the harmlessness of all these procedures if carefully performed by a skilled biologist in an IVF lab with quality management system. Finally, our study highlight that blastocyst cryopreserved on day 7 have clinically important potential and embryos that not reach blastocyst stage on day 6 should not to be discharged because they may result in an ongoing pregnancy.
