Área: Gestión de Vulnerabilidades

amplification of a highly polymorphic locus (HUMTHOl) in addition to the p-globin gene described here is very useful in reducing misdiagnosis caused by contamination and clarifies the diagnosis of cells affected by ADO. The two PGD protocols designed for P-thalassaemia with different types of mutations, i.e. single base pair substitution and deletion, reflect the potential for clinical application to a wide range of P-globin gene mutations. Therefore, these protocols may also be useful for other p-globin gene mutations that reside between these sets of primers with or without minor modification.

3,4,4 PGD strategy fo r a-thalassaemia, mutation

The PGD protocol for a-thalassaemia, — mutation (Section 3.3.4) was

designed and optimised using a single step multiplex gap F-PCR strategy. Gap PCR

(Section 2.2.S.6) is a cleverly modified PCR technique for detecting a very large deletion,

i.e. 20kb, without a reduction in amplification efficiency (Ko et al, 1992). This study has

the advantages of F-PCR and integrated F-PCR with gap PCR. The labelling of primers amplifying the normal and mutant alleles with different dyes allows the normal and mutant alleles to be clearly distinguished during the analysis. The addition of polymorphic HUMTHOl analysis is useful for contamination identification. In the case of an un-informative HUMTHOl locus, other informative microsatellites can be substituted. The study in single buccal cells of a heterozygote subject gave acceptable amplification efficiency and ADO rates, 90% and 22.2%, respectively. However, the higher ADO rate compared with other studies could be due to the quality of the samples. As for the study of P-thalassaemia, codon 41-42 mutation (Section 3.3.3), the buccal cell samples used in

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that their DNA was degraded. The results from the study on fresh single blastomeres where the ADO rates of the HUMTHOl locus was lower, from 22.2% to 8.7%, confirmed this hypothesis. The previous PGD protocol for a-thalassaemia, — mutation using semi-nested gap PCR and the traditional gel electrophoresis showed an amplification efficiency of 76.1% (Chang et al, 1996). Therefore, this PGD protocol is comparable or

better than the previous report. This is probably due to the different DNA extraction technique and the use of F-PCR technology. The promising results from the study in human blastomeres show that this PGD protocol can be clinically applied.

3,4,5 Single cell sequencing

It is essential that new PGD protocols be rigorously tested prior to any clinical application. In most cases this involves the isolation, amplification and testing of a significant number of cells of various genotypes. The significant investment of time and resources that this requires limits the number of new protocols that a PGD centre can develop in a given time. Because of this restriction, and also financial constraints, many PGD centres are forced to prioritise, only developing protocols that will be applicable to a large population of patients.

The number of centres offering PGD increases each year, however the range of diseases for which PGD is available expands at a disappointingly slow rate. Review of the literature reveals that PGD is applied to approximately four new diseases per year and that this rate of increase has remained static since 1998. Paradoxically there has been an increase in the number of new PGD protocols published, but most of these simply detail alternative strategies to existing PGD protocols. The reason for this is that most PGD

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centres have concentrated on developing their own protocols for the most common mutations in their local population. For example, more than 10 different methods for detecting the common cystic fibrosis mutation, AF508, have been reported. The main reason for the slow increase in the number of diseases diagnosed at the preimplantation stage is the amount of time and resources required for design and testing of new PGD protocols. This makes it uneconomical to develop protocols for rare mutations. Any methodology that is applicable to multiple diseases/mutations, with little optimisation required, will accelerate the rate at which new methods can be developed.

For PGD the most popular have been restriction enzyme digestion, heteroduplex analysis and single strand conformational polymorphism (SSCP). Enzyme digestion is the most simple of these and requires the least work-up. However, many mutations do not disrupt enzyme recognition sites and therefore cannot be detected in this way. Heteroduplex analysis and SSCP have the advantage that a single protocol can detect multiple mutations within a given DNA fragment. Over 50% of DNA sequence alterations can be detected using heteroduplex analysis while for SSCP detection rates can be as high as 90%. However, this increased detection rate comes at the price of increased complexity. To achieve maximum detection rates for SSCP technical know-how is necessary and a significant number of experimental parameters must be optimised. This is a process that can only be achieved by trial and error. A protocol that allows detection of

100% of mutations within a DNA fragment, but requires little or no change of conditions for each mutation, would reduce the amount of time taken to develop new protocols. The greatest saving in time would come from PGD for diseases characterised by a heterogeneous spectrum of mutations. One such example is p-thalassaemia, for which several published PGD protocols already exist. In this study the bthalwl primers used

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