Molecular Characterization of Portuguese Patients with Pathologies Related to the Lysosomal Multienzymatic Complex: Sialidosis, Galactosialidosis and GM1 Gangliosidosis.

The functional activity of lysosomal enzymes sialidase, beta-galactosidase and N-acetylaminogalacto-6-sulfate in the cell depends on their association in a multienzyme complex with the lysosomal carboxipeptidase, cathepsin A. Mutations in any of these complex components results in their functional deficiency causing severe lysosomal storage disorders. Here we report the molecular defects underlying sialidosis (mutations in sialidase; gene NEU1), galactosialidosis (mutations in cathepsin A; gene PPGB) and GM1 gangliosidosis (mutations in beta-galactosidase; gene GLB1) in the Portuguese population. Methods: Using gDNA extracted from patient’s fibroblasts, we performed a molecular study of the PPGB, NEU1 and GLB1 genes in the biochemically diagnosed Portuguese patients with galactosialidosis, sialidosis and GM1 gangliosidosis, respectively. The expression of these genes was determined by qRT-PCR. The effect of each mutation was evaluated at protein levels using bioinformatic tools. Results: In the PPGB gene, we identified two missense mutations, one novel (p.G85V) and one previously reported (p.V132M) as well as two new deletions (c.228-229delC and c.1075-1076delT) both giving origin to transcripts that lead to the synthesis of truncated non-functional proteins. In the NEU1 gene, we found two novel missense mutations (p.P200L and p.D234N). At protein levels, these mutations result in the substitution of two aminoacids located in a surface region of the molecule, already proposed to be involved in the interface sialidase/cathepsin A. Finally, in the GLB1 gene, we found four different mutations, all of them previously described: one missense mutation (R59H), one nonsense (W527X), one insertion (1572-1577InsG) and one deletion (845-846delC). Interestingly, in the Portuguese population the missense mutation R59H has a higher prevalence among the other ones. This is totally in accordance with which has been described for Brazilian, Iberian and Italian populations. Conclusion: Seven novel mutations are here reported for the first time, which contributes to enrich the knowledge on the mutational spectrum of these diseases and, by extension, to understand better the genetics of the lysosomal multienzymatic complex (LMC). The knowledge of the sialidosis, galactosialidosis and GM1 gangliosidosis mutational spectrum is also an important contribute to a better diagnosis, as well as to allow carrier detection in affected families and prenatal molecular diagnosis, leading to the improvement of genetic counseling with great benefits for the affected families. The existence of a molecular approach to the diagnosis is also of particular important strategy since it helps to overcome the difficulties associated to the neuraminidase enzymatic assay