DIMENSIÓN DESARROLLO DEL CLIMA SOCIOFAMILIAR Y

In Fabiy families it is important to diagnose hemizygotes and heterozygotes for counselling because both can be clinically affected and can transm it the defect in a single chromosome to produce an affected child.

1.3.5.1 The problem of heterozygote detection bv clinical and biochemical methods

Hemizygotes for Fabiy disease can be identified conclusively by enzyme assay and their diagnosis is not a problem. Heterozygotes can be identified when the symptoms are clearty observed and the a-galactosidase A activities are below the normal range. However, the diagnosis of heterozygotes that are clinically asymptomatic and have enzyme activities in the normal range is a major problem in Fabiy families. Phenotypic variability is caused by the process of random X - inactivation (Lyon, 1961; Migeon, 1994). In all somatic cells, only one X - chromosome is genetically active and the other is inactivated, for most genes. Random inactivation occurs early during embryonic development and before tissue differentiation. Once this occurs, the same chromosome, either m aternal or paternal, is the onfy active chromosome in all daughter cells. This causes mosaicism in the pattern of enzyme expression and some cells w ill produce the normal protein while others express the defective gene. The extent of accumulation of the storage product and therefore the phenotype and disease progression in carrier females depends on the proportion and location of cells expressing the m utant. This phenomenon accounts for the variant phenotypes in female monozygotic twins, in which one twin was clinically normal and the other was affected (Levade et oL, 1991; Winchester et a l, 1992) and highlights the problem of predicting the clinical effect in hetero^gotes and in diagnosis of asymptomatic females.

Another potential, complicating factor in the detection of Fabry heterozygotes is the possibility of age-related reactivation of the inactive X-chromosome. The ornithine transcarbamylase gene on the inactive X chromosome has been shown to be reactivated in older mice (Wareham et a l, 1987). This occurrence may explain the observation that older heterozygotes of Fabry disease tend to have an a-

galactosidase activity nearer to the normal range than younger heterozygotes (Desnick et a l, 1987) and may also account for the increase in fibroblast a- galactosidase A activity w ith cell age (Hozumi et a l, 1990).

The problem of X-inactlvation and diagnosis by enzyme assay has led to the development of modified biochemical assays. The heterozygote detection frequency was improved by measuring the activity of other tysosomal enzymes such as the p- galactosidase, in addition to a-galactosidase, as a control (Spence et aL, 1977; Sheth et aL, 1981). Others have measured enzyme activity in single cell fibroblast clones (Romeo et aL, 1970; Jongkind et aL, 1983) or in hair roots (Spence et aL,

1977) of hetero^gotes to detect the presence of single cells w ith no enzyme activity. H air follicles originate from a small cell population and so a significant proportion are likely to have cells w ith the same inactivated chromosome. Therefore, by testing several hair follicles for enzyme activity, diagnosis may be made if a significant number of enzyme-deficient follicles are found and a trim odal distribution of activities is observed (Ropers et aL, 1977). However, these methods are difficult, very labour intensive and may s till yield inconclusive results. Therefore, no satisfactory biochemical methods for heterozygote detection have been developed.

1,3.5.2 Molecular genetic detection of heterozygotes

Genetic detection methods can identify heterozygotes (Desnick et aL, 1987). Analysis of closely linked polymorphisms can be used to track the inheritance of the defective gene. The polymorphic markers DXS17, DXS87 and DXS88 have been used in analysis of Fabry families, using Southern blot analysis of RFLPs (MacDermot et aL, 1987; Morgan et aL, 1987). These three regions cem also be amplified by PCR for RFLP analysis and have been used for carrier detection in some fam ilies (Komreich et aL, 1992). The closest known m arker to the a- galactosidase A gene is DXS178, which is about lOOkb telomeric to the gene (Sweatman et aL, 1994; Vetrle et aL, 1994). CA. repeat markers have also been identified at the DXS178 locus (Allen et aL, 1992; de Weers et aL, 1992) and may prove useful for carrier detection in Fabry families.

In addition to polymorphic markers located outside of the a-galactosidase A gene, two other polymorphisms have been found w ithin, or close to the GLA locus. A Sac

I RFLP in intron 4 of the gene was observed at a firequency of 0.08 in normals and 0.02 in Fabry families. A rare Nco I RFLP, located about lOkb downstream from the last gene exon, has been found at a frequency of 0.13 in normals and 0.12 in Fabry families (Desnick et aL, 1987). The Nco I RFLP was used for diagnosis in a Fabry

family w ith 60 members. In this famlfy, 7 obligate heterozygotes that were undetected by enzyme assay were identified (Kirkillonis et oL, 1991). No intragenic polymorphisms, except for the Sac I RFLP were known prior to this study.

Two problems exist for the identification of hetero:^gotes by polymorphism analysis. One is that the polymorphisms are not always informative. The second problem is that in the absence of a family history of Fabiy disease, the presence of a new disease-causing m utation cannot be determined. Detection of a linked polymorphism in an affected child and subsequent detection of the polymorphism in the mother does not conclusively show that the mother has the m utation as it may have arisen de novo in the child. Therefore, direct detection of the specific defect for heterozygote identification is desirable.

The cloning of the gene and the lack of informative, intragenic polymorphic markers for hetero^gote detection has prompted the study of the family-specific mutations for use in carrier detection. This allows direct analysis of inheritance of the genetic defect. One problem exists with this type of analysis. If the defect is a new m utation and the individual is a mosaic then it is possible that analysis of DNA from somatic cells w ill not show the presence of a m utation which is in the germ- line cells (Hall, 1988). In this case, although the individual appears to be genetically normal, the children are at risk from inheriting the defect and should be considered for genetic counselling.