PROTOCOLO H.261 H.263 H.264 VELOCIDAD

a)

b)

c)

d)

Biopsy o f a human cleavage stage embryo three days after fertilisation. The embryo is held against a polished pipette by the application o f a gentle suction (a). A second pipette is introduced and a stream o f acidified Tyrode’s solution is passed through it and over a small region o f the zona pellucida that encapsulates the embryo. This cause a hole to dissolve in the zona pellucida (b). Another polished pipette can then be pushed through the opening and cells removed from inside by gentle suction (c). Scanning electron micrograph o f an embryo after biopsy (d).

biopsied are shown to be unaffected by a genetic disorder then it can be inferred that the rest o f the embryo is also free o f the disease. Only unaffected embryos are transferred back to the mother's uterus, and consequently any pregnancy resulting from the procedure must be unaffected.

The first clinical application o f preimplantation genetic diagnosis was for X- linked disorders such as Duchenne muscular dystrophy (DMD) and retinitis pigmentosa. For this purpose the sex o f the embryos was determined by PCR using primers specific for DNA sequences on the Y chromosome. A blastomere giving amplification was indicative of a male embryo at a high (50%) risk o f developing disease (Handyside et a i, 1989; Handyside et a l, 1990). Several normal girls were bom following PCR sexing, however the possibility o f a misdiagnosis, resulting from amplification failure, remained a concern. In all eight women underwent a total of thirteen cycles of IVF treatment, with five o f the patients becoming pregnant. Chorionic villus sampling and subsequent chromosome studies revealed two sets of female twins and three singletons, two female but critically one male (Handyside and Delhanty, 1993).

Current PCR sexing protocols use one set o f primers to amplify related sequences present on both the X- and Y-chromosome. The sequences amplified are identical at the site o f primer annealing, but differ internally being o f different size or containing a restriction site only present on one o f the two sex chromosomes, examples include the homologous ZFX and ZFY (Chong et al., 1993) and the amelogenin gene (Nakagome et al., 1991). As all cells sampled should have at least one X chromosome an X-specific product should always be produced. Absence o f X- product is indicative of a failed PCR, and consequently any data on Y-specific amplification from the same reaction should not be trusted. Despite this improvement contamination with exogenous DNA coupled with simultaneous amplification failure of the biopsied cell could still produce an erroneous diagnosis, and consequently a new strategy for approaching X-linked disease was desirable. This coincided with the increasing use o f fluorescent in situ hybridisation (FISH) for the detection of specific chromosomes (Pmkel et al., 1986). The hybridisation o f X- or Y-chromosome specific repetitive DNA probes to the nuclei o f human blastomeres was successfully accomplished in 1991 (Griffin et al., 1991). However, the method was limited by the possibility of hybridisation failure which could theoretically lead to misdiagnosis.

Dual FISH using both X- and Y-chromosome probes, detected in different colours, was reported a year later (Griffin et a l, 1992). The use of two probes in tandem provides a useful in built control that excludes cells with abnormal chromosome complements or failed hybridisation. For example, a male (XY) cell in which the Y- chromosome specific probe failed to hybridise to the nucleus would appear to be derived from a Tumer’s syndrome (XO) embryo and would not therefore be transferred. By September 1995 49 patients had undergone preimplantation sexing using FISH in the course o f 70 IVF cycles, resulting in a total of 11 normal girls bom and no misdiagnoses (Harper, 1996).

Since the initial application to sexing FISH has also been applied to autosomal chromosome anomahes. Chromosome copy number has been assessed in patients predisposed to the production o f aneuploid gametes. Such patients include carriers o f balanced translocations and those in which an aneuploid cell lineage is restricted to the germinal tissue (Conn et al., 1997).

Although FISH has superseded PCR for the preimplantation analysis o f sex, the specific diagnosis o f single-gene defects remains dependent on DNA amplification. Cystic fibrosis, a disease which represents the most common autosomal recessive disorder amongst people of Caucasian origin, was the first single gene disorder to be approached in preimplantation embryos. Cystic fibrosis is caused by a heterogeneous group o f mutations in the cystic fibrosis transmembrane regulator gene (CFTR). However, the three base pair deletion AF508 accounts for the majority (approximately 75%) o f cystic fibrosis alleles in the UK population (McIntosh et al., 1989). Because o f the prevalence o f AF508 a PGD strategy was designed for couples in which both partners are carriers of AF508. Following embryo biopsy a ‘nested’ PCR protocol was applied to provide specific amplification o f the CFTR gene, and heteroduplex analysis was employed for mutation detection. O f the three couples initially treated two had unaffected embryos available for transfer back to the mother. In each case two embryos were returned, leading in one case to pregnancy and the subsequent birth o f an unaffected baby girl (Handyside et al, 1992). Heteroduplex formation has since been applied to PGD for Tay Sachs disease, a severe neurodegenerative disorder resulting from deficiency of the lysosomal hydrolase p- hexosaminidase-A (Gibbons et a l, 1995). The target of heteroduplex analysis in this case was the common 4 bp insertion in exon 11 of the p-hexosaminidase-A gene

which accounts for approximately 80% of Tay Sachs alleles in the Ashkenazi Jewish population (Peleg et a l, 1994).

A different mutation detection system was used for the preimplantation diagnosis o f Lesch-Nyhan syndrome, an X-linked disorder caused by mutation in the hypoxanthine phosphoribosyl transferase (HPRT) gene. Disease causing mutations in H P RT tend to be unique to individual families and consequently it is difficult to develop a single strategy for all families segregating a mutation. Despite this problem two couples have received preimplantation diagnosis for this disorder, and a healthy girl has been delivered (Hughes et a i, unpublished). In these cases the mutations were detected in PCR amplified fragments by enzymatic digestion using restriction endonucleases specific to the mutation sites, followed by electrophoresis to reveal cleaved DNA fragments.

Since these early successes diseases with an autosomal dominant mode o f inheritance have also been approached by PCR and preimplantation genetic diagnosis. These include Marfan syndrome a disease o f the connective tissue caused by inheritance o f a defective fibrillin gene (Harton et al., 1996), and familial adenomatous polyposis (FAP) a disorder predisposing to colorectal neoplasia (undertaken during this study). Embryos affected with Marfan syndrome were identified by virtue o f a linked dinucleotide repeat polymorphism. This protocol has been successful in the single treatment cycle attempted so far, leading to the birth o f a healthy male. The preimplantation diagnosis o f FAP is discussed fully in chapter four of this thesis. Protocols for PGD of a variety of other diseases have also been reported, including myotonic dystrophy and p-thalassaemia (El Hashemite et a i, 1996; Ray et al., 1996; Sermon et al., 1997).

Chapter 2