Mutation analysis of Krabbe Disease and Metachromatic Leukodystrophy in Portugal. A.M.L. Marcao, O.M.O. Amaral, E.M. Pinto, M.C. Sa Miranda. Genetic Neurobiology, IBMC- Univ.of Porto, Porto, Portugal.

   The molecular characterisation of Portuguese MLD and KD patients was carried out in order to identify the molecular lesions underlying the biochemical deficits. In the case of MLD, the application of PCR-RFLP and PCR-SSCP analysis allowed a 100% rate of mutation identification; eight different mutations were identified. The I allele was found at a very high frequency ( 64%), whereas the A allele represented only 3,5% of the mutated MLD alleles. As expected, all the homozygotes for the I allele presented the late infantile form, making it much more frequent than the juvenile and adult forms. Two previously described missense mutations (I179S; D255H), here associated to adult and late infantile MLD were both found in heterozygosity with allele I. Two new missense mutations (C300F; P425T) were found to be associated to late infantile and juvenile forms and two novel microlesions (g.2408delC and g.1190-1191insC) were identified in two late infantile patients. The haplotypes previously proposed for I, A, I179S and D255H mutations were confirmed, suggesting the possible existence of common ancestors. With regard to KD, although all patients presented the infantile form, large molecular heterogeneity was found. In this case, the approach followed consisted on RT-PCR-SSCP analysis. In addition to the five mutations previously identified, in a collaborative study with Dr. Wenger, four other mutations and several polymorphic changes were found. Of the nine mutations identified four led to deletions, one was nonsense and four were missense mutations, three alleles remain undefined. It should be noted that only one of the deletions (del30-kb, ex11-17) and one of the missense mutations (T513M) are known to be common in North European KD patients, all other mutations seem to be private and appear in various polymorphic backgrounds. As described for patients with North European ancestry, mutations del30Kb and T513M appeared associated with polymorphisms 502T and 694A, respectively. The molecular findings here reported may provide useful information for the study of other populations, since different mutation profiles were found in Portuguese patients.