Preimplantation diagnosis for Fanconi anemia combined with HLA matching.

CONTEXT: The advent of single-cell polymerase chain reaction (PCR) has presented the opportunity for combined preimplantation genetic diagnosis (PGD) and HLA antigen testing. This is a novel and useful way to preselect a potential donor for an affected sibling requiring stem cell transplantation. OBJECTIVE: To perform in vitro fertilization (IVF) and preimplantation HLA matching combined with PGD for Fanconi anemia (FA). DESIGN: DNA analysis for the IVS 4 + 4 A-->T (adenine to thymine) mutation in the FA complement C (FANCC) gene in single blastomeres, obtained by biopsy of embryos, to identify genetic status and HLA markers of each embryo before intrauterine transfer. SETTING: In vitro fertilization programs at large medical centers in Chicago, Ill, and Denver, Colo. PARTICIPANTS: A couple, both carriers of the IVS 4 + 4 A-->T mutation in the FANCC gene with an affected child requiring an HLA-compatible donor for cord blood transplantation. MAIN OUTCOME MEASURES: DNA analysis of single blastomeres to preselect unaffected embryos representing an HLA match for the affected sibling. RESULTS: Of 30 embryos tested in 4 IVF attempts, 6 were homozygous affected and 24 were unaffected. Five of these embryos were also found to be HLA-compatible, of which 2 were transferred in the first and 1 in each of the other 3 cycles, resulting in a pregnancy and birth of an unaffected child in the last cycle. CONCLUSION: To our knowledge, this is the first PGD with HLA matching, demonstrating feasibility of preselecting unaffected embryos that can also be an HLA-compatible source for stem cell transplantation for a sibling.     

Evolutionary Classification of Homeodomains

Purpose and Methods: We performed multiple comparisons between available amino acid (aa) sequences of homeodomain(HOM)-containing proteins from a wide spectrum of animals to create an evolutionary classification of the proteins. Results: Based on results of statistical and special computational analyses of over 500 homeodomain aa sequences (HOMs) a novel system of concepts describing complex structural correlations between homologous proteins is proposed. This system includes such notions as differentiated isofunctionality of aa, chemotype, stereotype, local functional motifs, gradual conservativeness of aa positions, and group-specific domain patterns, as well as major categories of the evolutionary classification of HOMs (Division, Type, Branch, Class, Family, Series, Variety, Sort). Using this approach, a complete structural systematics of HOMs belonging to proteomes of eukaryotic animals is proposed. Conclusions: The proposed structural classification of HOMs is in full agreement with the bulk of experimental data revealing complex functional similarities and differences among HOMs in terms of their expression patterns in developing embryos. It turn, this classification can provide answers regarding homology among homeodomains when experimental data are conflicting.     

Substandard application of preimplantation genetic screening may interfere with its clinical success

The intent of this study was to evaluate a recent randomized clinical trial evaluating the effect of preimplantation genetic screening (PGS) that reports a negative effect on pregnancy outcome. This article reviews appropriate PGS techniques and how they differ from the trial in question. A closer look at the clinical trial in question reveals significant lack of expertise in biopsy, cell fixation, genetic analysis, and patient selection. At most, this trial demonstrates that in inexperienced hands, PGS can be detrimental. No other conclusions concerning the effect of PGS on pregnancy results can be drawn from the trial.     

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 discoses. We analyzed the gender of 141 consecutive single cells in a blinded manner without a single incorrect gender assignment     

Visualization and cytogenetic analysis of second polar body chromosomes following its fusion with a one-cell mouse embryo

Purpose This study was designed to visualize the second polar body (2PB) chromosomes using its electrofusion with a one-cell-stage mouse embryo to approach preconception diagnosis of chromosomal disorders. Results Eighty to 90% hybridization efficiency has been achieved by electrofusion of 2PB with mouse zygotes. 2PB chromosomes were visualized in 40–50% of hybrids. Sixty-five percent of 2PB chromosomes were visualized when fused with the cytoplast obtained microsurgically by removing pronuclei from a one-cell embryo. As much as 33–43% of these resulting metaphases appeared to contain chromosomal aberrations. The follow-up of the development of the reconstructed one cell-stage hybrids in vitro revealed a significant decrease in their viability. The hybrid embryos resulting from 2PB electrofusion with enucleated zygotes did not develop beyond the two-cell stage. Conclusion Electrofusion is an efficient approach for hybridization of 2PB with a one-cell mouse embryo and may be useful for visualization and cytogenetic analysis of 2PB chromosomes. The visualization rate of 2PB chromosomes is higher if 2PB is fused with enucleated zygotes. However, the method induces over 30% of chromosomal aberrations and may lead to a significant decrease in the viability of the resulting one-cell embryos.     

Birth of Healthy Children After Preimplantation Diagnosis of Thalassemias

Background: Preimplantation genetic diagnosis (PGD) allows couples at risk of having children with thalassemia to ensure the healthy outcome of their pregnancy. Methods: Seventeen PGD clinical cycles were initiated for Cypriot couples at risk of having children with different thalassemia mutations, including IVS1-110, IVSI-6, and IVSII-745. Unaffected embryos for transfer were selected by testing oocytes, using first and second polar body (PB) removal and nested polymerase chain reaction analysis followed by restriction digestion. Results: Unaffected embryos were selected in 16 of 17 PGD cycles. Of 166 oocytes studied from these cycles, 110 were analyzed by sequential analysis of both the first and the second PB, resulting in preselection and transfer of 45 unaffected embryos. This resulted in seven pregnancies and in the birth of five healthy thalassemia-free children. The embryos predicted to have inherited the affected allele were not transferred. Analysis of these embryos confirmed the PB diagnosis. Conclusions: Sequential first and second PB testing of oocytes is reliable for PGD of thalassemia and is a feasible alternative to prenatal diagnosis in high-risk populations.     

Preimplantation genetic diagnosis for early-onset torsion dystonia

Early-onset primary torsion dystonia (DYT1) is the most severe and common form of hereditary movement disorders, characterized by sustained twisting contractures that begin in childhood, which is caused in majority of cases by a 3-bp deletion of the DYT1 gene on chromosome 9q34 at the heterozygote state. As there is no effective treatment of this disease, preimplantation genetic diagnosis (PGD) may be a useful option for at-risk couples to establish an DYT1 mutation-free pregnancy. PGD was performed for two obligate carriers of the DYT1 3-bp deletion, using blastomere testing to preselect the mutation-free embryos, based on mutation analysis with simultaneous testing of the three closely linked markers, D9S62, D9S63 and ASS. Of 19 tested blastomeres in three cycles, 17 had conclusive information about the mutation and linked markers, of which eight were predicted to be free of 3-bp deletion. Six of these embryos were transferred back to patients, two in each cycle, yielding singleton DYT1 3-bp deletion-free clinical pregnancies in two. One of these pregnancies was terminated due to severe anencephaly and the other resulted in birth of a mutation-free child. This is the first PGD for primary torsion dystonia, providing an alternative for those at-risk couples who cannot accept prenatal diagnosis and termination of pregnancy as an option for avoiding early onset torsion dystonia.