THE LIM ITATIONS OF INDIRECT A NALYSIS

This study has investigated the efficacy o f direct mutation detection in 2 X-linked disorders: ornithine carbamoyl transferase deficiency and Pelizaeus-Merzbacher disease.

Indirect analysis using restriction fragment length polymorphisms (RPLPs) or variable number tandem repeats (VNTRs) has proved very useful in carrier detection and prenatal diagnosis of genetic disorders. However, its usefulness is limited by the need for family studies and the requirement for DNA from an affected individual to establish phase. Individuals may also be uninformative; the disorder may be heterogeneous with one or more genes involved; analysis can be complicated by the possibility of nonpaternity; and recombination between the marker and the mutation is possible, especially for large genes and intergenic probes.

Germline mosaicism (Hall. 1988), such that a proportion of germ cells bear a mutation while the rest are normal, can result in uncharacteristic patterns of inheritance and may severely complicate analysis. This has been reported, for example, in several cases of Duchenne muscular dystrophy (van Essen et al. 1992).

In X-linked disorders analysis is complicated by the high proportion of new mutations, estimated to be at least 1/3 and 2/3 in sporadic males and females respectively (Section 1.1.2). In the absence of a family history this can result in the misdiagnosis o f a noncarrier as a carrier and the termination of unaffected pregnancies. Additional biochemical, clinical or genetic analyses are generally required to identify carriers before gene tracking is used.

5.1.1 ORNITHINE CARBAMOYL TRANSFERASE DEFICIENCY

Carrier detection and prenatal diagnosis in OCT deficiency are typically provided by a combination of family history and biochemical and linkage based analyses.

in only 60-80% of women (Rozen et al. 1985, Fox et al. 1986.) and for this reason, as well as the need for family studies and DNA from affected individuals to establish phase, linkage based analysis is typically not possible in a proportion of OCT deficiency families. Further due to the possibility of germline mosaicism and the high incidence of new mutations, it should only be used for carrier exclusion or after a woman has been diagnosed as a carrier by biochemical analysis or family history.

As the number of cases with a positive family history are limited, carrier detection is predominantly by biochemical analysis. This, though significantly improved, is not 100% accurate (Hauser et al. 1990, Pellet et al. 1990) and while a positive test result strongly suggests carrier status, a negative result does not eliminate it. Pelet et al. (1990) calculated that despite a negative allopurinol test result, the mother o f an affected boy retains a significant 30% risk of being a carrier.

Biochemical analysis is limiting in four main groups. These are carriers o f the mild form of late onset OCT deficiency who may give normal or borderline test results; young girls in whom the test has not been completely verified and who represent the predominant group presenting for testing; and favourably lyonised carriers. Carrier females exhibit a wide variation in the proportion of mutant X-chromosome which is active and, in cases where the normal X is predominantly active, this may result in borderline or even normal protein load results. The final category are mosaics whose tissues contain cells with either the normal or the mutant genotype. If the hepatic tissues contain few or no mutant cells but these are present in the germ cell population, protein load results may be normal, but the female will still be at risk of passing on the mutant gene to her offspring.

In several cases o f late onset OCT deficiency asymptomatic males have been identified (Finkelstein et al. 1990a, Hata et al. 1991, this report) who, without direct mutation detection, can be identified only by an invasive liver biopsy and enzyme analysis. Diagnosis of such males is especially important as any daughters are obligate carriers for a potentially lethal disorder.

Direct mutation detection in these situations would enable unequivocal diagnosis, carrier detection and prenatal diagnosis.

5.1.2 PELIZAEUS-M ERZBACHER DISEASE

In Pelizaeus-Merzbacher disease carrier females are generally asymptomatic and no biochemical means of carrier detection exists. Magnetic imaging has been used (Boltshauser et al. 1988) but has been shown to be inaccurate in young girls, the group presenting predominantly for testing (Pratt et al. 1991).

Carrier detection has been carried out in families with X-linked PMD using RFLP analysis, based on the assumption that the disorder was caused by a mutation at the PLP locus (Maenpaa et al. 1990, Bridge et al. 1991). But this approach has been thrown into question by the failure to find mutations in 75% of PMD cases in the PLP coding, control and splice site regions (Hudson et al. 1989a, Pham-Dinh et al. 1991a, Pratt et al. 1991, Doll et al. 1992) and hence the possibility that a second X-linked gene involved in PMD exists. Further as many cases are sporadic and manifesting females have been reported, the possibility of autosomal recessive inheritance remains (Begleiter and Harris. 1989).

Diagnosis of this disorder is complicated by the fact that both the clinical symptoms and the MR! evidence o f a dysmyelinating disorder are common to a range of diseases and as the limits of clinical presentation have not been determined, uncharacteristic cases may be missed. The only definitive diagnostic element was, until recently, considered to be a scarcity of mature oligodendrocytes but with the discovery of the rumpshaker mouse mutant (Schneider et al. 1992), in which defective myelination and oligodendrocyte maturation are independent, the clinical criteria have been brought into question.

The direct identification of the causative mutation in each family provides the only accurate means of diagnosis, carrier detection and prenatal diagnosis in this disorder. Further this will enable the determination of the range of phenotypes associated with a mutation at the PLP locus.