A versatile strategy for preimplantation genetic diagnosis of haemophilia A based on F8-gene sequencing

Summary

Preimplantation genetic diagnosis (PGD) of hemophilia A (HA) and other X-linked diseases through sex selection implies that male embryos will be systematically discarded, even though 50% are unaffected. The objective of the present work was to develop a PGD protocol for direct mutation identification that could be applied to first polar bodies (1PBs) in several HA clinical cases. Single buccal cells from controls and patients, and 1PBs were subjected to primer extension preamplification (PEP) PCR followed by amplification of F8 gene coding and intronic flanking regions, and direct sequencing. Moreover, multiplex fluorescent amplification of four short tandem repeats was adapted to a single cell preamplification in order to rule out contamination and allele drop-out, and for confirmatory indirect diagnosis. A couple at risk of HA transmission, with a familial mutation characterized as a 41-bp duplication in exon 14 of the F8 gene, was selected for the first clinical study. After optimizing the protocol, the complete F8 gene coding sequence was obtained from single cells to demonstrate the sensitivity of our methodology although in any clinical case only the relevant region, not the whole gene, must be amplified. The woman enrolled in the first clinical case has completed the first in-vitro fertilization cycle, and seven oocytes were analyzed with concordant results by both linkage analysis and direct sequencing method. Only one oocyte, among those diagnosed as mutation free, developed to embryo at day 3. It was transferred but pregnancy was not achieved. This PGD procedure enables non-affected and noncarrier embryo selection in families with any point or smallrange mutation in the F8 gene, without the need for further custom-made modifications.

Keywords

ADO - STR - haemophilia

• References

• 1 Verlinsky Y, Kuliev A. Preimplantation polar body diagnosis. Biochem Mol Med 1996; 58: 13-7.
• 2 Verlinsky Y, Kuliev A. Micromanipulation of gametes and embryos in preimplantation genetic diagnosis and assisted fertilization. Curr Opin Obstet Gynecol 1992; 04: 720-5.
• 3 Handyside AH, Pattinson JK, Penketh RJ. et al. Biopsy of human preimplantation embryos and sexing by DNA amplification. Lancet 1989; 01: 347-9.
• 4 Thornhill AR, Snow K. Molecular diagnostics in preimplantation genetic diagnosis. J Mol Diagn 2002; 04: 11-29.
• 5 Sermon K. Current concepts in preimplantation genetic diagnosis (PGD):a molecular biologist’s view. Hum Reprod Update 2002; 08: 11-20.
• 6 Staessen C, Van Assche E, Joris H. et al. Clinical experience of sex determination by fluorescent in-situ hybridization for preimplantation genetic diagnosis. Mol Hum Reprod 1999; 05: 382-9.
• 7 Thornhill AR, deDie-Smulders CE, Geraedts JP. et al. ESHRE PGD Consortium ‘Best practice guidelines for clinical preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS)’. Hum Reprod 2005; 20: 35-48.
• 8 Snabes MC, Chong SS, Subramanian SB. et al. Preimplantation single-cell analysis of multiple genetic loci by whole-genome amplification. Proc Natl Acad Sci USA 1994; 91: 6181-5.
• 9 Fiorentino F, Magli MC, Podini D. et al. The minisequencing method: an alternative strategy for preimplantation genetic diagnosis of single gene disorders. Mol Hum Reprod 2003; 09: 399-410.
• 10 Tomi D, Griesinger G, Schultze-Mosgau A. et al. Polar body diagnosis for hemophilia A using multiplex PCR for linked polymorphic markers. J Histochem Cytochem 2005; 53: 277-80.
• 11 Michaelides K, Tuddenham EG, Turner C. et al. Live birth following the first mutation specific pre-implantation genetic diagnosis for haemophilia A. Thromb Haemost 2006; 95: 373-9.
• 12 Kaufman RJ. Advances toward gene therapy for hemophilia at the millennium. Hum Gene Ther 1999; 10: 2091-107.
• 13 Vidal F, Farssac E, Altisent C. et al. Rapid Hemophilia A Molecular Diagnosis by a Simple DNA Sequencing Procedure: Identification of 14 Novel Mutations. Thromb Haemost 2001; 85: 580-3.
• 14 Sanchez-Garcia JF, Benet J, Gutierrez-Mateo C. et al. Multiple mutation analysis of the cystic fibrosis gene in single cells. Mol Hum Reprod 2005; 11: 463-8.
• 15 Olerup O, Zetterquist H. HLA-DR typing by PCR amplification with sequence-specific primers (PCRSSP) in 2 hours: an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation. Tissue Antigens 1992; 39: 225-35.
• 16 Wells D, Sherlock JK. Strategies for preimplantation genetic diagnosis of single gene disorders by DNA amplification. Prenat Diagn 1998; 18: 1389-401.
• 17 Zhang L, Cui X, Schmitt K. et al. Whole genome amplification from a single cell: implications for genetic analysis. Proc Natl Acad Sci USA 1992; 89: 5847-51.
• 18 Sanchez-Garcia JF, Gallardo D, Ramírez L. et al. Multiplex fluorescent analysis of four short tandem repeats for rapid Hemophilia A molecular diagnosis. Thromb Haemost 2005; 94: 1099-103.
• 19 Kolibianakis E, Bourgain C, Albano C. et al. Effect of ovarian stimulation with recombinant follicle-stimulating hormone, gonadotropin releasing hormone antagonists, and human chorionic gonadotropin on endometrial maturation on the day of oocyte pick-up. Fertil Steril 2002; 78: 1025-9.
• 20 Sermon K, Van Steirteghem A, Liebaers I. Preimplantation genetic diagnosis. Lancet 2004; 363: 1633-41.
• 21 Gitlin SA, Gibbons WE, Gosden RG. Oocyte biology and genetics revelations from polar bodies. Reprod Biomed Online 2003; 06: 403-9.
• 22 Bermudez MG, Piyamongkol W, Tomaz S. et al. Single-cell sequencing and mini-sequencing for preimplantation genetic diagnosis. Prenat Diagn 2003; 23: 669-77.
• 23 Kokkali G, Vrettou C, Traeger-Synodinos J. et al. Moving towards blastocyst biopsy: trophectoderm biopsy for PGD of beta-thalassaemia. Reprod Biomed Online 2005; 10: 23.