Sexing for X-linked disease is one of the major indications for PGD (Verlinsky et al., 1994; ESHRE PGD Consortium, 1999). X-linked recessive diseases account for 6-7% of single gene defects and include conditions such as Duchenne muscular dystrophy (DMD), haemophilia, and various mental retardation syndromes. Table 11.1 lists some of the common X-linked diseases for which PGD sexing has been offered.
The mother carries the mutation on one of her X chromosomes and so transmits the defective gene to half of her offspring (see Chapter 2; p. 17). The females inheriting the mutation are carriers, like their mother. Male children inheriting the mutation are affected as the Y chromosome, which they inherit from their father, does not carry the same genes. There are over 400 X-linked diseases (McKusick, 1994). For some of these, the molecular nature of the mutation is known and a specific diagnosis of the disease is possible, either prenatally or postnatally. However, for many X-linked diseases, the location and mutation is unknown and so no genetic test is available. In these cases, unless linkage is possible, all that can be offered prenatally is sexing of the fetus, with the option of terminating male pregnancies, knowing that there is only a 50% risk that the fetus will be affected. Therefore, for such X-linked diseases, PGD has an added benefit.
FISH AND EMBRYO SEXING 195
Table 11.1 X-linked diseases for which PGD been requested
Duchenne and Becker's muscular dystrophy
Fragile X syndrome
Hypohidrotic ectodermal dysplasia
Vitamin D resistant rickets
Adapted from the ESHRE PGD Consortium (1999).
The first clinical application of PGD was the diagnosis of embryonic sex for couples who carried X-linked disorders (Handyside et al., 1990). This was achieved by PCR amplification of a repeated sequence on the long arm of the Y chromosome. Embryos were diagnosed as female if no Y-specific band was seen on a gel. This carried some risks, as the same result would be obtained if the amplification failed, the cell was lost or an anucleate fragment was inadvertently biopsied from the embryo. In later studies these problems were overcome by the use of the homologous ZFX and ZFY loci (Chong et al., 1993), the amelogenin gene (Nakagome et al., 1991), and the steroid sulphatase gene (Liu et al., 1994) (see Chapter 10; 169).
In addition to amplification failure, there are two drawbacks that apply when amplifying DNA from single blastomeres in order to sex the embryo. First the risk of amplifying contaminating material is high, which applies to any reaction involving PCR. The second is that the presence of an amplified band only indicates that an X or Y chromosome is present and gives no information about chromosome copy number. Hence XO (Turner's syndrome) cannot be distinguished from a normal XX female, and XXY (Klinefelter's syndrome) cannot be distinguished from a normal XY male. This can be overcome using FISH where, not only can sex be diagnosed, but it is possible to determine the copy number of the sex chromosomes. The importance of this became apparent as data accumulated from PGD cases. In one series of five couples undergoing embryo sexing by FISH to avoid X-linked disease, no transfers took place for two of them despite the identification of X signals and no Y signals (Delhanty et al., 1993). In the first, three X signals were seen in the biopsied cell; this was shown as likely to be due to mitotic non-disjunction by the reciprocal finding of a cell with a single X in the remainder of the embryo, on a background of XX cells. In the second case, two independent biopsied cells from one embryo had only a single X signal; investigation of the remainder of the embryo confirmed that the abnormality was present throughout. Transfer of a potentially 45, X embryo could be disastrous when the aim is to avoid X-linked disease from the carrier mother, as the great majority of such embryos lack the paternal sex chromosome (Hassold et al., 1988). Since 45, X conceptuses are thought to occur with a frequency approaching 1% such a finding will not be rare when carrying out PGD. Information on chromosome copy number has recently allowed us to avoid the transfer of a triploid embryo and one with multinucleate blastomeres (Delhanty et al., 1997). It is possible that use of information gained in this way to avoid transferring embryos that will later miscarry could account for the higher proportion of ongoing pregnancies obtained as a result of using dual FISH for sexing rather than PCR. An extra advantage of FISH is that the risk of contamination is minimal as the nucleus can be observed at all stages of the process.
The use of interphase FISH using probes for chromosomes X and Y was developed to sex embryos for patients at risk of transmitting X-linked diseases (Griffin et al., 1991, 1992, 1993). In these early studies indirectly labelled probes were used and the FISH efficiency was relatively low with approximately 70% of interphase blastomeres giving clear signals (Griffin et al., 1992). The biopsied cells were prepared using methanokacetic acid as the spreading solution (Tarkowski, 1966; Kola & Wilton, 1991). This traditional spreading method was not ideal for the preparation of blastomeres as these cells contain high quantities of cytoplasm. In some cases blastomeres were lost or nuclei were found to be covered in cytoplasm. To remove the cytoplasm and make the nuclei accessible to the probes, proteinase K and RNase digestion steps were performed. The probes were added and the samples denatured to make the DNA single stranded to allow probe hybridization. To avoid the risk of hybridization failure, two probes were used for the Y chromosome and one for the X. These DNA probes were indirectly labelled with digoxigenin and biotin, and so an immunochemical detection step was required (Griffin et al., 1994). This method took approximately seven hours to perform. Using this approach, 27 cycles of PGD were performed, resulting in 21 embryo transfers, and 7 clinical pregnancies of which 2 spontaneously aborted and 5 delivered (3 singletons and 2 twins). All pregnancies were normal females (Griffin et al., 1994).
Due to the problems observed with the blastomere fixing procedure, a novel and more efficient method of spreading blastomeres was developed in mouse embryos (Coonen et al., 1994a) using HC1 and Tween 20 as the spreading solution. This method was successfully applied to whole human embryos (Coonen et al., 1994b) and single blastomeres (Harper et al., 1994). The spreading solution lyses the cell membrane and dissolves the cytoplasm, leaving the nuclei clear and dried onto the slide. A short pepsin digestion is then performed instead of the longer proteinase K and RNase steps. The use of directly labelled DNA probes has been possible, which reduces the time of the FISH procedure from seven to two hours (Harper et al., 1994). Most laboratories use three-colour FISH for PGD of sex with probes for chromosomes X, Y and an autosome (usually 18) to provide additional information concerning aneuploidy and polyploidy (Staessen et al., 1999) (Figure 11.2; see Plate IV). More recently five-colour FISH with additional probes for chromosomes 13 and 21 has been used for polar body and blastomere analysis (Figure 11.3; see Plate IV) (Verlinsky et al., 1999; Gianaroli et al., 1997). With reprobing even more chromosomes can be analysed (Munné et al., 1998a), but the efficiency of the FISH procedure decreases with additional probes and steps.
The first report of the high levels of chromosome abnormalities observed using FISH to sex human preimplantation embryos for preimplantation diagnosis was by Delhanty et al. (1993). In this report, both mitotic non-disjunction and the presence of a Turner's syndrome (45,X) embryo produced XO cells. Four groups of chromosomes patterns have been classified in the preimplantation embryo: normal, abnormal, mosaic and chaotic (Harper & Delhanty, 1996; Delhanty et al., 1997) (see 7; p. 110). This has important implications for preimplantation diagnosis of sex. To date, there have been no reports of an XX nucleus in a male embryo, and so these abnormalities should not lead to a misdiagnosis of sex, but care needs to be taken as cumulus cells could become attached to the slide and would be normal, female cells. However, XO nuclei have been observed in male embryos (Harper et al., 1995). Therefore only an embryo where there are two X signals in the absence of a Y should be considered for transfer and an embryo with an XO nucleus should never be considered. This situation highlights the need to use FISH for sexing as the use of non-quantitative PCR cannot differentiate between an XO and XX nucleus. Therefore an XO nucleus from an XY or XO embryo, both of which should not be transferred back to the patient, will be diagnosed as female.
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