La garantía del derecho a la igualdad en la aplicación de las

Several lines of evidence support the hypothesis that disease-causing mutations have been identified in this study. Analysis of the protein-coding region and splice sites did not find more than one m utation in each patient and all of these m utations were predicted to alter the amino-acid sequence of the protein. Further evidence was provided by in vitro expression studies, for 6 of the missense m utations.

4.3.2.1 Evidence from genetic analysis

The mutations detected in Fabiy patients were believed to be disease-causing for the following reasons. Firstly, all but two mutations, R49S and D92H, were not found in DNA from at least 100 normal chromosomes. R49S and D92H were not detected by the standard method for SSCP analysis and normal chromosomes were not screened for these mutations. Secondly, no other SSCP changes were seen in the remaining 6 exons, other than polymorphisms, and finally, the exon containing the unique change was fully sequenced and did not contain any other mutations. However, not all regions of the gene were anafysed and because we and others (Orlta et oL, 1989; Hayashi, 1991, Spinardi et oL, 1991; Fan et oL, 1993; Sheffield

et oL. 1993) have shown that SSCP is not 100% efficient, it is possible that an undetected, second m utation is responsible for the Fabry phenotype.

The sequence change may be a rare polymorphic allele that is present in the normal population but is not in the 100 chromosomes anafysed in this study. If a two allele system is considered, then the probability that an allele is not present in a sample population is given by:

Probability = (1- allele frequency)” ,

or, log (probability) = n log (1-allele firequency)

where ‘n’ is the number of chromosomes analysed. Therefore, if a rare polymorphism has a frequency of 1 allele in 100 in a sample size of n=100, there is a 37% chance that the allele w ill not be detected. By the same calculation for the same sample size, an allele w ith a frequency of 0.03 or greater w ill have a probability of less than 5% that it is not present. This relationship is illustrated in

the graph below, which shows the probability that an allele of a certain frequency is not present in a sample of 100 chromosomes:

« o « 8

- . E g

it:

Rare allele frequency

Further support for the disease-causing status of these mutations comes from the fact that SSCP analysis detected a rare sequence change in 26/30 (87%) of the Fabiy patients and that no other variants were detected in the protein-coding region. If it is assumed that 87% of all mutations are detected then the probability that the mutation is a rare polymorphism (with a frequency of 0.01) and that a m utation was not detected by SSCP analysis is calculated by:

probability = [(l-O.Ol)ioo ] x (1-0.87) = 0.05

Therefore, there is a 95% probability that each mutation detected by SSCP analysis is not present in the normal population with a frequency of 1/100 and that it is the only m utation in the protein-coding or splice sites of the a-galactosidase A gene. For 8 mutaüons, M42V, R49S, C56Y, D92H, W287G, R342Q, 717del2 and 1087dell, sequencing proved conclusively that these mutations were the only ones present in the protein-coding region and splice sites.

Further evidence for the disease-causing effect of mutations comes from finding recurrent or de novo mutations. The N215S, R227X, R342Q and 717del2 mutations have been found in more than one unrelated family, showing segregation with the disease phenotype and absence in the normal population. The C56Y m utation was present in a clinically typical Fabry hemizygote but was

absent in lymphocyte DNA from his mother. Since no other affected famlty members were known, this de novo m utation was believed to cause Fabiy disease. Other evidence comes from the fact that analysis of the protein-coding region and splice sites by us and others (table 4, pg 56) has identified more than one sequence change in only 1/106 Fabry patients, indicating that in the m ajority of cases, only one m utation is present in this region. In this patient, expression studies showed that the disease-causing m utation was P40S, while a silent base change at codon 8, did not reduce the a-galactosidase activity (Koide et oL, 1990). Complete sequencing of either the cDNA or the protein-coding regions and splice sites of the gene has provided evidence that in at least 57 patients there were no other m utations in this region of the a-galactosidase A gene.

4.3.2 2 Biochemical evidence for the disease-causing nature of mutations

A ll of the mutations detected in Fabiy patients were predicted to alter the protein sequence indicating that they are likely to cause disease (tables 13 and 14, section 3.1.2.1). In addition, the G35R, R49L, V269A, V316E, Q327K and G361R m utations found in this study were transiently expressed in COS-1 cells. A ll of them caused a decrease in a-galactosidase A activity, compared with the wild-type cDNA, supporting the hypothesis that they were disease-causing. R49L, V269A, V316E and Q327K produced no detectable activity w ithin the cells or secreted into the medium. G361R did not produce intracellular activity but did secrete catafytically active protein into the medium. These results indicated that there would be no activity in the lysosomes and would therefore cause disease. However, the G35R m utation did produce low, intracellular activity and in four transfection experiments it was in the range of 12-45% of the normal (tables 19 and 22(B), pgs 161 and 166). The presence of residual activity was in agreement w ith finding 15% and 5% of the normal activity in cultured fibroblasts and leukocytes, respectively, from the hemizygote in fam ily 1 (Dr H. Christomanou, personal communication).

In conclusion, the above lines of evidence support the hypothesis that all 26 m utations identified in this study are causative for Fabry disease.