Biomedical subjects

S Rechitsky

Publications and source records attributed to S Rechitsky.

36 records · Page 2Linked to original sources

PubMed1998

Sequence analysis of libraries from individual human blastocysts.

PURPOSE: It has recently become possible to construct cDNA libraries from individual human blastocysts to investigate the expression of embryonic genes in human preimplantation development. We have previously reported the expression of beta-actin, CD-59, and homeobox OCT-3 and identified almost-complete homology of sequences to human histone 3.1 and human ribosome protein S25. In the present paper, our further sequencing analysis of cDNA libraries from single human blastocysts is described. METHODS: cDNA libraries were constructed from 13 blastocysts. Sequence analysis was performed in 120 clones from one of these cDNA libraries with fragments of 50 to 1000 bp. Their sequence identity was analyzed using the expressed sequence tag (EST) database. RESULTS: The presence of two housekeeping genes, hexokinase I and serine/threonine phosphorylase, and four other ESTs was demonstrated, the identity of which, with particular gene expression in preimplantation development, has not yet been established. CONCLUSIONS: The data demonstrate the usefulness of constructing cDNA libraries from individual human blastocysts and their values in the analysis of genetic expression in human preimplantation development.

Blastocyst↗

PubMed1998

Allele dropout in polar bodies and blastomeres.

PURPOSE: 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. METHODS: We introduced a simultaneous amplification of mutant genes and linked polymorphic markers, such as a 4-bp repeat (GATT) at the 3' end of intron 6 in the cystic fibrosis (CF) gene and a short tandem repeat at the 5' end of the beta-globin gene. Three types of single heterozygous cells were studied for the amplification of both alleles, including 150 blastomeres, 1615 fibroblasts, and 170 first polar bodies, obtained from patients at risk for having children with cystic fibrosis (delta F-508 mutation) or sickle cell disease. RESULTS: ADO rates of as high as 33.3% for delta F-508 mutation and 22.8% for beta-globin gene were observed in single blastomeres, compared to 7.1 and 7.7% in single fibroblasts and 5.9 and 9.6% in first polar bodies, respectively. The application of simultaneous amplification of the above linked polymorphic markers allowed detection of more than half of the cases of ADO in blastomeres (19.4% for cystic fibrosis and 12.3% for beta-globin gene) and almost all ADOs in polar bodies, particularly when the two-step sequential analysis of the first and second polar body was applied in preimplantation diagnosis of single gene disorders. CONCLUSIONS: Simultaneous amplification of linked polymorphic markers in single-cell DNA analysis of single-gene defects is an efficient method for avoiding the risk of misdiagnosis in preimplantation diagnosis.

Alleles↗

PubMed1998

Preimplantation diagnosis of thalassemias.

PURPOSE: Preimplantation genetic diagnosis (PGD) is an important option for couples at risk of having children with beta-globin mutations to avoid selective abortions of affected fetuses following prenatal diagnosis. METHODS: We performed PGD for thalassemia in 12 clinical cycles (IVS1-110, and IVS-745 mutations) using biopsy of the first and second polar bodies (PBs) extruded from oocytes during maturation and fertilization, coupled with nested polymerase chain reaction analysis and restriction digestion. RESULTS: A total of 118 oocytes was obtained, of which 78 had results for both the first and the second PBs. This resulted in the selection and transfer of 30 unaffected embryos (2.5 embryos per cycle). To avoid a possible misdiagnosis due to allele dropout (ADO), we have also introduced simultaneous detection of two highly polymorphic linked markers, a short tandem repeat immediately at the 5' end of the globin gene and HUMTH01 which is a syntenic short tandem repeat. The application of multiplex polymerase chain reaction of the beta-globin gene and linked polymorphic markers enabled detection of ADO in five first PBs, thus avoiding the transfer of potentially affected embryos resulting from their corresponding oocytes. CONCLUSIONS: Confirmation studies of the embryos resulting from the oocytes predicted to contain an affected gene confirmed the diagnosis in 98% of the cases, thus demonstrating the accuracy and reliability of PB PGD of thalassemia mutations. The application of PB analysis in six patients resulted in two ongoing pregnancies with a thalassemia-free fetus already confirmed in both of them by prenatal diagnosis.

Alleles↗

PubMed1998

Preimplantation diagnosis of autosomal dominant retinitis pigmentosum using two simultaneous single cell assays for a point mutation in the rhodopsin gene.

A couple requested preimplantation genetic analysis for a dominant form of retinitis pigmentosum caused by a C-->A transversion in the rhodopsin gene. Since this point mutation does not alter a restriction endonuclease site we designed two separate analytical systems, one involving site-specific mutagenesis and the other involving allele-dependent length polymorphism. After establishing the accuracy and robustness of these assay systems we utilized both systems simultaneously in a heminested polymerase chain reaction (PCR) system. This allowed accurate preimplantation diagnosis to be performed. One embryo was transferred but a pregnancy did not occur.

Cells, Cultured↗

PubMed1998

Isolation of cDNA libraries from individual human preimplantation embryos.

Although available data from the mouse model suggest that morphogenesis during preimplantation development is dependent on the expression of embryonic genes, convincing data for human preimplantation embryos are missing. To investigate the expression of genes involved in human preimplantation development we constructed cDNA libraries from human individual blastocysts and screened them for the expression of beta-actin, CD59, homeoboxes OCT-3 and HOXA4, and HLA-G and hMLH-1 genes. Beta-actin, CD59, and OCT-3 were detected by reverse transcription-polymerase chain reaction (RT-PCR), while HOXA4, HLA-G and hMLH-1 were undetected. Sequencing of 48 random clones from two libraries revealed a different identity to the known genes including 99% identity to human histone 3.1 and human ribosome protein S25 complete cDNA. These data demonstrate the feasibility of constructing cDNA libraries from individual human preimplantation embryos and their potential usefulness in the assessment of the relevance of specific gene expression in the failures of preimplantation development.

Blastocyst↗

PubMed1998

Use of nested PCR to identify charred human remains and minute amounts of blood.

Reliable single cell PCR requires nested or heminested PCR and careful optimization of conditions. This report describes the successful use of nested PCR for gender identification and reverse paternity testing in a forensic case where the only available materials consisted of charred human remains and a minute quantity of blood that were unsuitable for standard PCR. Use of nested PCR allowed the blood and burned tissue to be identified as human female. Analysis of two PCR length polymorphisms (AMPFLP) was successful on the blood sample and reverse paternity testing yielded a 98% probability that the blood spot was from the victim. The defendant was convicted of murder following a bench trial and the verdict was upheld by the Appellate court.

Amelogenin↗

PubMed1997

Preimplantation diagnosis of single gene disorders by two-step oocyte genetic analysis using first and second polar body.

Previous work on preimplantation genetic diagnosis (PGD) of single gene disorders by the first polar body (IPB) analysis has demonstrated that the genotype of a considerable number of embryos resulting from heterozygous oocytes cannot be predicted without testing their second PB (IIPB). To overcome this limitation we introduce a two-step DNA analysis of oocytes using both IPB and IIPB to identify hemizygous mutation-free oocytes following the second meiotic division. In the application of the approach to PGD of cystic fibrosis (CF) Delta F-508 mutation, sickle cell disease, and hemophilia B, 80 oocytes were studied by both PBs, resulting in the identification and transfer of 32 homozygous normal embryos. A follow-up genotyping of 52 embryos, resulting from oocytes tested by both IPB and IIPB demonstrated the accuracy of the predicted genotypes. In addition to a nested PCR analysis of the mutant genes in PBs and resulting embryos, simultaneous amplification of different polymorphic markers was performed, demonstrating the reliability of the two-step polar body analysis of oocytes.

Alleles↗

PubMed1996

Allele dropout in sequential PCR and FISH analysis of single cells (cell recycling).

PURPOSE: Our purpose was to investigate the feasability of using sequential PCR and FISH analysis of single cells for preimplantation diagnosis. METHODS: Protocols for sequential PCR and FISH analysis of a single fibroblast (cell recycling) were optimized for six loci and the rates of allele specific dropout (ADO) were determined. RESULTS: Conditions that allow reliable genotyping of single cells in lysis buffer were not optimal for amplifying fibroblasts fixed to coverslips. After optimizing conditions, we observed a success rate of 85% for both analyses in sequential PCR-FISH experiments in single cells for the four loci studied. The individual success rates for each technique revealed a slightly higher rate for FISH (91-95%) than for PCR (85-87%) for single cells on coverslips. The presence of two hybridization signals in FISH experiments demonstrated that the failure to amplify both alleles from heterozygous cells on coverslips was due to true ADO, and not the loss of chromosomal material. The ADO rate observed on coverslips varied between 10 and 14%, which is significantly higher than that observed in solution, even after meticulous optimization. CONCLUSIONS: Sequential PCR and FISH analysis of single cells remains an attractive possibility. However, until the problem of the increased rate of ADO is resolved, cell recycling should be applied to clinical preimplantation genetic analysis.

Alleles↗

PubMed1996

Expression of homebox-containing genes in human preimplantation development and in embryos with chromosomal aneuploidies.

PURPOSE: The purpose of the study was to investigate homeobox gene expression in human oocytes and preembryos and in postimplantation embryos with impaired embryonic development determined by chromosomal abnormalities. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) with intron spanning primer sets for Homeobox gene sequences was used. RESULTS: The homeobox genes HoxA4, HoxA7, HoxB4, and HoxB5 were present in human oocytes and cleaving normal and triploid embryos. The expression pattern was different between chromosomally abnormal and normal first-trimester embryos. Of four homeobox transcripts (HoxA7, HoxB4+ ++, HoxB5, and HoxC6) that are expressed in diploid embryos, only HoxA7, HoxB4 and HoxC6 were present in a trisomy 7 embryo, and only HoxB4 and HoxB 5 in triploid embryos and an embryo with trisomy 9. Cloning experiments revealed differences in the number of homeobox clones obtained from trisomy 7 and control embryos. CONCLUSIONS: The transcripts of homeobox genes, HoxA4, HoxA7, HoxB4, and HoxB5, were present in oocytes and cleaving embryos. The pattern of expression of homeobox genes in cultured fibroblasts derived from spontaneously aborted embryos with aneuploidies was different from that in control diploid cells.

Abortion, Spontaneous↗

PubMed1996

Prenatal paternity testing with deoxyribonucleic acid techniques.

OBJECTIVES: Our purpose was to determine the feasibility and optimal techniques for prenatal paternity testing. STUDY DESIGN: Since January 1989 we have offered prenatal paternity testing by deoxyribonucleic acid testing. We analyzed the ability to complete the testing and the time required to complete the testing and developed polymerase chain reaction-based tests to speed test results. RESULTS: Before April 1990 only five of nine cases could be completed. Since that time 28 consecutive cases were successfully completed before delivery. Introduction of polymerase chain reaction-based testing has allowed us to perform testing on uncultured chorionic villi and to derive results within 3 weeks. CONCLUSION: Analysis of uncultured chorionic villi allows prenatal paternity testing to be completed within the first trimester of pregnancy. Prenatal paternity testing can also be performed on cultured amniocytes and chorionic villi.

Abortion, Therapeutic↗

PubMed1996

Birth of a healthy girl after preimplantation gender determination using a combination of polymerase chain reaction and fluorescent in situ hybridization analysis. Preimplantation Genetics Group.

OBJECTIVE: To perform preimplantation gender determination by a combination of polymerase chain reaction (PCR) sexing and fluorescent in situ hybridization technique using the directly labeled fluorescent alpha-satellite centromeric DNA probes for X and Y chromosomes. SETTING: The IVF program of Illinois Masonic Medical Center. PATIENTS: A couple requested preimplantation diagnosis because the mother is a carrier for hemophilia A. RESULTS: Two blastomeres were aspirated from each of the four- to eight-cell embryos, and only the embryos with both fluorescent in situ hybridization and PCR results indicating female sex chromosomal complement were transferred, resulting in a singleton pregnancy and delivery of a healthy female infant, after prenatal confirmation of the diagnosis as female. The male embryos or embryos diagnosed as females only by PCR were followed up by confirmatory fluorescent in situ hybridization analysis demonstrating a discrepancy of PCR and fluorescent in situ hybridization results in four embryos, presumably because of a possible sperm contamination of the PCR reaction or chromosomal mosaicism. CONCLUSION: The analysis of two blastomeres from the same embryo by a combination of PCR sexing and fluorescent in situ hybridization increases the reliability of preimplantation gender identification at the cleavage stage.

Adult↗

PubMed1995

Homeobox gene expression in human oocytes and preembryos.

Homeobox gene expression in human preimplantation development has not been established. We used reverse transcriptase-polymerase chain reaction (RT-PCR) with intron spanning primer sets to investigate the presence of mRNA of homeobox genes in human oocytes and preembryos. RT-PCR products obtained from normal and unfertilized oocytes, from cleaving normal and triploid embryos, and from morulae and blastocysts were cloned, sequenced, and analyzed for the presence of homeobox sequences. The presence of mRNA of homeoboxes HoxA4 and HoxA7 was demonstrated; HoxA4 was present in normal and unfertilized oocytes and also in a 4-cell embryo. HoxA7 was present in normal oocytes and cleaving triploid embryos.

Amino Acid Sequence↗

PubMed1994

Nonisotopic identification of two point mutations in the CYP21 gene responsible for nonclassic 21-hydroxylase deficiency.

A simple nonradioactive method was developed for identification of the Pro-30-Leu and Val-281-Leu mutant alleles in the CYP21B gene. Not only does this approach improve mutation analysis for patients with the late onset form of 21-hydroxylase deficiency, but it also decreases problems with interference by the CYP21A pseudogene sequence.

Adrenal Hyperplasia, Congenital↗

PubMed1994

Reliability of polymerase chain reaction (PCR) analysis of single cells for preimplantation genetic diagnosis.

PURPOSE: We investigated the reliability of polymerase chain reaction (PCR) genotype analyses performed on single cells for the purposes of preimplantation genetic analysis. METHODS: We performed blind analysis of 130 single skin fibroblasts heterozygous for the delta-F508 mutation in the cystic fibrosis transmembrane regulator (CFTR) gene and 73 single skin fibroblasts from an individual heterozygous for the XbaI polymorphic site of the Factor VIII gene. RESULTS: Amplification was successful for 116 cells and 52 cells respectively and in all but one case (a CFTR analysis) both alleles were amplified. The incidence of diagnostic error was 1 out of 203 analyses or 0.0043. We conclude that PCR is a reliable method for determining the genotype of single cells for the purposes of preimplantation genetic analysis.

Alleles↗

PubMed1992

Preconception and preimplantation diagnosis for cystic fibrosis.

Preimplantation diagnosis provides couples at high genetic risk the possibility of avoiding genetic disease without the need for prenatal diagnosis and selective abortion of the affected pregnancy. Following extensive background work on the reliability of genetic diagnosis in a single cell, we offered on a research basis preimplantation diagnosis to five couples at risk for offspring with the delta-F508 mutation (the major mutation causing cystic fibrosis). There was no detrimental effect from polar body removal on either fertilization or preimplantation development. Genetic analysis, undertaken in 22 polar bodies and 15 corresponding blastomeres, identified 21 embryos of which ten were transferred.

Biopsy↗

PubMed1991

Reliability of gender determination using the polymerase chain reaction (PCR) for single cells.

Contamination with extraneous DNA sequences is a frequent problem when performing PCR analysis of single cells. This report describes our experience with eliminating contaminating DNA sequences from PCR reagents for the purposes of gender identification. We have used amplification of Y-specific sequences to identify the gender of single human amniocytes. Female cells consistently showed no Y-specific bands but only 80% of male cells showed the expected intense Y-specific band. This phenomenon could lead to incorrect gender identification of single cells. We developed a technique of simultaneous amplification of X- and Y-specific sequences to prevent misdiagnosis because of failed PCR, which allows accurate preimplantation gender determination for women at risk for conceiving children with X-linked genetic diseases. We analyzed the gender of 141 consecutive single cells in a blinded manner without a single incorrect gender assignment.

Blastomeres↗

PubMedDate not supplied

Polar body-based preimplantation diagnosis for X-linked disorders.

Preimplantation diagnosis for X-linked disorders has been performed predominantly by gender determination, which, however, leads to the discarding of 50% unaffected male embryos. In an attempt to identify X-linked mutation-free embryos for transfer, the present authors introduced preimplantation genetic diagnosis (PGD), using a sequential first and second polar body analysis, as an alternative to gender determination. This method was offered to eight couples at risk for having children with X-linked disorders, including haemophilia B, fragile-X syndrome (FMR1), myotubular myotonic dystrophy (MTMD), ornithine transcarbamylase (OTC) deficiency and X-linked hydrocephalus. The first and second polar bodies were removed following maturation and fertilization of oocytes in a standard IVF protocol and analysed using a multiplex nested polymerase chain reaction (PCR), involving testing for mutations simultaneously with linked markers. Overall, 13 PGD cycles were performed, resulting in the detection of 25 embryos with the predicted mutation-free maternal contribution; these embryos were transferred back to the patients in all cycles, yielding four clinical pregnancies. Four children were born following these pregnancies, including three unaffected and one with misdiagnosis as a result of allele dropout (ADO), which was predictable in the case of FMR1. Presented results demonstrate the clinical usefulness of the specific polar body testing for X-linked disorders as an alternative to PGD by gender determination.

Female↗

PubMedDate not supplied

Preimplantation diagnosis of single disorders.

Preimplantation diagnosis of inherited and chromosomal diseases provides an option for couples at risk for conceiving genetically abnormal fetus to avoid a birth of an affected child without the need for a prenatal diagnosis and selective abortion of affected fetus (1-3). In some countries this might be the only way to the prevention of genetic disease, as abortion is not acceptable procedure. Even in those countries where prenatal diagnosis is practiced for many years, there is also concern that existing genetic programs based on prenatal screening will lead to increasing number of abortions. On the other hand, the possibilities for genotyping oocytes and cleaving embryos open a new prospect for genetic diagnosis before pregnancy, making genetic programs more ethically acceptable in any social setting. This will make preimplantation diagnosis the method of choice in the community based programs for prevention of genetic disease in the future, as well as a useful addition to assisted reproduction technologies, at least for IVF patients of advanced maternal age.

Female↗