J Assist Reprod Genet

Preimplantation genetic diagnosis of inherited cancer: familial adenomatous polyposis coli.

Year 1998
Ao A. Wells D. Handyside AH. Winston RM. Delhanty JD.
Institute of Obstetrics and Gynaecology, RPMS, Hammersmith Hospital, London, UK.
PURPOSE: Our purpose was to achieve preimplantation genetic diagnosis (PGD) of the dominant cancer predisposition syndrome, familial adenomatous polyposis coli (FAPC), as an alternative to prenatal diagnosis. METHODS: The affected patient was superovulated and oocytes were retrieved and fertilized by intracytoplasmic sperm injection (ICSI). Two cells were biopsied from each embryo and the whole genome was amplified by primer extension preamplification (PEP). Nested PCR was then used to amplify two APC fragments: one including the APC mutation site and the other an informative intragenic polymorphism. Both were detected by simultaneous single-strand conformation polymorphism and heteroduplex analysis. RESULTS: Four normally fertilized embryos were biopsied on day 3 post ICSI, and two cells were successfully removed from each embryo. Following PEP the APC mutation was successfully amplified in 7 of 8 cells, and the polymorphism in 6 of 8 cells. The APC mutation was detected in three embryos. This result was confirmed by identification of the mutation associated polymorphism in two cases. A single embryo was diagnosed as homozygous normal for the mutation and the polymorphism in both cells sampled. This unaffected embryo was transferred to the mother, but no pregnancy resulted. CONCLUSIONS: We report here the first diagnosis of a cancer predisposition syndrome in human preimplantation embryos. Our results indicate that difficulties associated with single-cell PCR, allele-specific amplification failure in particular, need not prevent preimplantation diagnosis of diseases with a dominant mode of inheritance, provided appropriate strategies are applied.

Allele dropout in polar bodies and blastomeres.

Year 1998
Rechitsky S. Strom C. Verlinsky O. Amet T. Ivakhnenko V. Kukharenko V. Kuliev A. Verlinsky Y.
Reproductive Genetics Institute, Chicago, Illinois 60657, USA.
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.

Chromosomal mosaicism in cleavage-stage human embryos and the accuracy of single-cell genetic analysis.

Year 1998
Kuo HC. Ogilvie CM. Handyside AH.
Department of Obstetrics and Gynaecology, St. Thomas' Hospital, London, UK.
PURPOSE: Our purpose was to assess the effect of chromosomal mosaicism in cleavage-stage human embryos on the accuracy of single-cell analysis for preimplantation genetic diagnosis. METHODS: Multicolor fluorescence in situ hybridization with X, Y, and 7 or X, Y, 7, and 18 chromosome-specific probes was used to detect aneuploidy in cleavage-stage human embryos. RESULTS: Most nuclei were diploid for the chromosomes tested but there was extensive mosaicism including monosomic, double-monosomic, nullisomic, chaotic, and haploid nuclei. CONCLUSIONS: Identification of sex by analysis of a single cleavage-stage nucleus is accurate but 7% of females are not identified. One or both parental chromosomes 7 were absent in at least 6.5% of the nuclei. With autosomal recessive conditions such as cystic fibrosis, carriers would be misdiagnosed as normal or affected. With autosomal dominant conditions, failure to analyze the affected parents allele (1.6-2.5%) would cause a serious misdiagnosis and analysis of at least two nuclei is necessary to reduce errors.

Sequence analysis of libraries from individual human blastocysts.

Year 1998
Morozov G. Verlinsky O. Rechitsky S. Kukharenko V. Goltsman E. Ivakhnenko V. Gindilis V. Strom C. Kuliev A. Verlinsky Y.
Reproductive Genetics Institute, Chicago, Illinois 60657, USA.
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.