Detection of haemoglobinopathies in single cells: An approach for preimplantation genetic diagnosis and early prenatal diagnosis

β thalassaemia is defined as a group of heterogeneous anaemias in which the β globin peptide synthesis is either absent or reduced by 30% or more. It is the world's most widespread autosomal recessive single gene disorder and represents a major health problem. More than 200 million individuals world-wide carry the β thalassaemia gene with an estimated 75,000 thousands of affected individuals born annually. Because of this β thalassaemia has attracted a great deal of research interest both at the molecular and clinical levels, which has led to the understanding of the complex molecular pathogenesis. Recent clinical management has improved and increased the life expectancy of affected individuals, however, the treatment required is very expensive and unsatisfactory. Therefore, the aim is to control the incidence of the disorder through the provision of carrier detection and prenatal diagnosis. Although prenatal diagnosis has reduced the number of β thalassaemia births, potential risks to the fetus have been associated with the available procedures. Furthermore, pregnancy termination is still unacceptable for many families, and couples at risk are looking for a safer, less emotionally traumatic way to have normal offspring. An alternative is offered by preimplantation genetic diagnosis carried out on the third day after in vitro fertilisation. With the application of nested PCR, amplification of the minute amount of DNA in a single cell has become possible and DNA analysis of single gene disorders for preimplantation diagnosis can be achieved. To enable preimplantation diagnosis of β thalassaemia by direct detection of mutant β globin genes at the single cell level nested PCR and silver stained single stranded conformation polymorphism (SSCP) analysis were employed. However, two main problems with single cell PCR, contamination and allele specific amplification failure in addition to mosaicism can lead to misdiagnosis and the transfer of affected embryos. In this study, the reduction of allele specific amplification failure by the use of SDS/proteinsae K as a lysis buffer, and the application of fluorescence based PCR to reduce the incidence of contamination which becomes apparent when reamplification is done, are reported. Furthermore, this study has also focused on optimising DNA amplification procedures using standard or quantitative multiplex fluorescent PCR in single cells for the detection of mosaicism and age related trisomy 21 along with specific diagnosis of single gene disorder diagnosis and developing mutation analysis techniques such as fluorescent SSCP and automated fluorescent DGGE for either early prenatal diagnosis using transcervical cells or preimplantation diagnosis. Finally, the first step of applying any prevention program for haemoglobinopathies is the identification of mutations within the β globin gene. The molecular characterisation of β thalassaemia has been studied in samples from Jordan and Egypt to facilitate screening and prenatal programmes based on DNA methodology