Optimal polymerase chain reaction amplification for preimplantation diagnosis in cystic fibrosis ((Delta)F508)

BMJ 1995;311:536-540 (26 August)

Papers

Optimal polymerase chain reaction amplification for preimplantation diagnosis in cystic fibrosis ((Delta)F508)

Ke-Hui Cui, scientist in charge of preimplantation diagnosis,^a Eric A Haan, director of department,^b Ling-Jia Wang, scientist,^a Colin D Matthews, professor ^a

^a Department of Obstetrics and Gynaecology, University of Adelaide, Queen Elizabeth Hospital, Woodville, Adelaide, SA5011, Australia, ^b Department of Medical Genetics, Women's and Children's Hospital, North Adelaide, SA 5006

Correspondence to: Dr Cui.

Abstract

Objective: To evaluate direct polymerase chain reaction amplificationof mutation on single embryo cells for the routine preimplantationdiagnosis of cystic fibrosis.
Design: Direct polymerase chain reaction amplification of mutationwas performed to identify the cystic fibrosis {delta} F508 mutationin human blood DNA, single lymphocytes, embryos, and embryocells obtained by biopsy. Preimplantation diagnosis was performedfor a couple who were heterozygous carriers of the {delta} F508 mutation.
Setting: Laboratory for preimplantation diagnosis in a reproductivemedicine unit.
Main outcome measure: Correct diagnosis of homozygous normal,heterozygous, and homozygous abnormal DNA of the cystic fibrosis {delta} F508 mutation.
Results: 45 blood samples (18 homozygous normal, 17 heterozygous,and 10 homozygous abnormal) and 204 single lymphocytes fromknown sources showed 100% amplification and were diagnosed correctly.17 human embryos and 52 normal nucleated embryo cells obtainedby single cell embryo biopsy also showed 100% amplification.After a miscarriage of the initial pregnancy (diagnosed at preimplantationto be homozygous normal) in the heterozygous carrier couple,fetal tissue was confirmed to be homozygous normal.
Conclusion: Direct polymerase chain reaction amplification ofmutation is a simple, fast, reliable test for the common cysticfibrosis mutation ( {delta} F508) in blood DNA and single cells and shouldbe applicable to routine programmes of general screening, maternalblood examination, and preimplantation diagnosis.

Key messages

Key messages
Misdiagnoses in sex linked diseases and cystic fibrosis are mainly due to limitations of methods
A basic experimental design with 100% correct results when testing blood DNA, single lymphocytes, embryos, and single embryo cells combined with good clinical practice and success in an animal model were the key factors in good outcome of this study
Applying direct polymerase chain reaction amplification of mutation in general screening, preimplantation, and prenatal diagnosis should lead to a substantial reduction in cystic fibrosis in the future

Add to CiteULike CiteULike Add to Complore Complore Add to Connotea Connotea Add to Del.icio.us Del.icio.us Add to Digg Digg Add to Reddit Reddit Add to Technorati Technorati What's this?

This article has been cited by other articles:

Phillipson, G.T.M., Petrucco, O.M., Matthews, C.D. (2000). Congenital bilateral absence of the vas deferens, cystic fibrosis mutation analysis and intracytoplasmic sperm injection. Hum Reprod 15: 431-435 [Abstract] [Full text]
Hussey, N. D., Donggui, H., Froiland, D. A.H., Hussey, D. J., Haan, E. A., Matthews, C. D., Craig, J. E. (1999). Analysis of five Duchenne muscular dystrophy exons and gender determination using conventional duplex polymerase chain reaction on single cells. Mol Hum Reprod 5: 1089-1094 [Abstract] [Full text]
Bogardus, S. T. Jr, Concato, J., Feinstein, A. R. (1999). Clinical Epidemiological Quality in Molecular Genetic Research: The Need for Methodological Standards. JAMA 281: 1919-1926 [Abstract] [Full text]
Raeburn, J A (1995). Commentary: Preimplantation diagnosis raises a philosophical dilemma. BMJ 311: 540-540 [Full text]