Molecular analysis of the HEXA gene in Italian patients with infantile and late onset Tay-Sachs disease: detection of fourteen novel alleles.

Tay-Sachs disease (TSD) is a recessively inherited disorder caused by the hexosaminidase A deficiency. We report the molecular characterization performed on 31 Italian patients, 22 with the infantile, acute form of TSD and nine patients with the subacute juvenile form, biochemically classified as B1 Variant. Of the 29 different alleles identified, fourteen were due to 15 novel mutations, two being in-cis on a new complex allele. The new alleles caused four frameshifts, three premature stop codons, three amino acid changes, two amino acid deletions and two splicing alterations. As previously reported, the c.533G>A (p.R178H) mutation was present either in homozygosity or as compound heterozygote, in all the patients with the late onset TSD form (B1 Variant); the allele frequency in this group is discussed by comparison with that found in infantile TSD.

Identification and functional characterization of five novel mutant alleles in 58 Italian patients with Gaucher disease type 1.

Gaucher disease (GD) is the most frequent lysosomal glycolipid storage disorder due to an autosomal recessive deficiency of acid beta-glucosidase characterized by the accumulation of glucocerebroside. In this work we carried out the molecular analysis of the glucocerebrosidase gene (GBA) in 58 unrelated patients with GD type 1. We identified five novel genetic alterations: three missense changes c.187G>A (p.D63N), c.473T>G (p.I158S), c.689T>A (p.V230E), a gene-pseudogene recombinant allele and a non-pseudogene-derived complex allele [c.1379G>A;c.1469A>G] encoding [p.G460D;p.H490R]. All mutant alleles were present as compound heterozygotes in association with c.1226A>G (p.N409S), the most common mutation in GD1. The missense mutant proteins were expressed in vitro in COS-1 cells and analyzed by enzyme activity, protein processing and intracellular localization. Functional studies also included the c.662C>T (p.P221L) mutation recently reported in the Spanish GD population (Montfort et al., 2004). The missense mutant alleles retained an extremely low residual enzyme activity with respect to wild type; the complex allele expressed no activity. Processing of the mutant proteins was unaltered except for c.473T>G which was differently glycosylated due to the exposition of an additional glycosylation site. Immunofluorescence studies showed that protein trafficking into the lysosomes was unaffected in all cases. Finally, the characterization of the novel recombinant allele identified a crossover involving the GBA gene and pseudogene between intron 5 and exon 7.

Identification of four novel mutations in the alpha glucosidase gene in five Italian patients with infantile onset glycogen storage disease type II.

Glycogen storage disease type II (GSDII) is an autosomal recessive disorder due to the deficiency of the lysosomal enzyme acid alpha glucosidase. Four novel mutations (C670T, G989A, G2188T, and Delta 23 nt 828-850) were identified in five Italian patients with the infantile form of the disease. The C670T mutation was present in two unrelated patients in heterozygosity; the effect on enzyme activity was assessed by in vitro expression. COS-1 cells expressing the C670T allele had a twofold higher activity than the negative control cells. The G989A and G2188T point mutations lead to the introduction of premature stop signals that results in truncated forms of alpha glucosidase. The in vitro expression of G2188T allele demonstrated no increment in activity compared to negative control. The frame shifting deletion of nucleotides 828-850 was identified in one patient in heterozygosity. The shift in the reading frame introduces a stop codon 135 nucleotides downstream the deletion junction that results in a truncated protein without catalytic activity. Nested PCR screening showed that the mutation was carried by the mother and was absent in the other members of the family. The four novel severe mutations herein described concerned only infantile onset GSDII patients; the loss of enzyme activity is correlated with the severity of the disease.

Glycogenosis type II: identification and expression of three novel mutations in the acid alpha-glucosidase gene causing the infantile form of the disease.

Glycogenosis type II (GSDII) is an autosomal recessive disorder due to the deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). We identified three novel point mutations, C399A, T1064C, and C2104T, in three unrelated Italian patients with the infantile form of the disease. The C399A mutation was present in homozygosity in proband 1. The C >A transition introduces a premature stop signal in exon 2 resulting in no enzyme production that is correlated with the severe clinical phenotype in this patient. The other two nucleotide changes were missense mutations. The T1064C mutation, which changes Leu in position 355 into Pro, was carried in homozygosity by proband 2. The C2104T nucleotide change, which substitutes Arg 702 into Cys, was present in proband 3 in combination with a known severe mutation DeltaI17-18. The in vitro expression in COS-1 cells of T1064C and C2104T constructs demonstrated no enzymatic activity with respect to the negative control cells. Western blot analysis revealed that both T1064C and C2104T mutant proteins produced in COS-1 cells migrated in SDS-PAGE as the GAA inactive precursor of 110kDa. Immunofluorescence detection of mutant alpha-glucosidases showed enzyme localization primarily in the ER-Golgi compartment, suggesting that T1064C and C2104T mutations could affect the normal processing and stability of the enzyme. In vitro studies demonstrated that the same degree of deficiency in T1064C and C2104T mutations, which is in contrast with patient phenotype. A better correlation was observed with the in vivo studies since proband 2, with a less severe phenotype, presented with low residual enzyme activity while in proband 3, with a classic severe infantile onset GSDII, fibroblast enzyme activity was completely absent.