Lúcia Lacerda

Glycosaminoglycan Storage Disorders: A Review

Impaired degradation of glycosaminoglycans (GAGs) with consequent intralysosomal accumulation of undegraded products causes a group of lysosomal storage disorders known as mucopolysaccharidoses (MPSs). Characteristically, MPSs are recognized by increased excretion in urine of partially degraded GAGs which ultimately result in progressive cell, tissue, and organ dysfunction. There are eleven different enzymes involved in the stepwise degradation of GAGs. Deficiencies in each of those enzymes result in seven different MPSs, all sharing a series of clinical features, though in variable degrees. Usually MPS are characterized by a chronic and progressive course, with different degrees of severity. Typical symptoms include organomegaly, dysostosis multiplex, and coarse facies. Central nervous system, hearing, vision, and cardiovascular function may also be affected. Here, we provide an overview of the molecular basis, enzymatic defects, clinical manifestations, and diagnosis of each MPS, focusing also on the available animal models and describing potential perspectives of therapy for each one.

Evidence for a link between sphingolipid metabolism and expression of CD1d and MHC-class II: monocytes from Gaucher disease patients as a model.

Gaucher disease (GD) is an autosomal recessive inherited defect of the lysosomal enzyme glucocerebrosidase (GluCerase) that leads to glucosylceramide (GluCer) accumulation. We previously demonstrated the existence of imbalances in certain lymphocyte populations in GD patients. We now show that GluCerase‐deficient monocytes from GD patients or monocytes from healthy subjects treated with conduritol‐B‐epoxide (CBE), an irreversible inhibitor of GluCerase activity, display high levels of surface expression of the lipid‐binding molecule CD1d. GluCerase‐deficient monocytes from GD patients also showed increased surface expression of major histocompatibility complex (MHC)‐class II, but not of other lysosomal trafficking molecules, such as CD63 and MHC‐class I. However, CD1d and MHC‐class II mRNA levels were not increased. GluCerase‐deficient monocytes from GD patients undergoing enzyme replacement therapy also exhibited increased levels of CD1d and MHC‐class II and imbalances in the percentage of CD4+, CD8+, and Vα24+ T cells. Interestingly, follow‐up studies revealed that enzyme replacement therapy induced a decrease in MHC‐class II expression and partial correction of the CD4+ T cell imbalances. These results reveal a new link between sphingolipid accumulation in monocytes and the expression of certain MHC molecules that may result in imbalances of regulatory T cell subsets. These immunological anomalies may contribute to the clinical heterogeneity in GD patients.

Molecular analysis of the GNPTAB and GNPTG genes in 13 patients with mucolipidosis type II or type III – identification of eight novel mutations

Mucolipidosis II (ML II) and mucolipidosis III (ML III) are diseases in which the activity of the uridine diphosphate (UDP)‐N‐acetylglucosamine:lysosomal enzyme N‐acetylglucosamine‐1‐phosphotransferase (GlcNAc‐phosphotransferase) is absent or reduced, respectively. In the absence of mannose phosphorylation, trafficking of lysosomal hydrolases to the lysosome is impaired. In these diseases, mistargeted lysosomal hydrolases are secreted into the blood, resulting in lysosomal deficiency of many hydrolases and a storage‐disease phenotype. GlcNAc‐phosphotransferase is a multimeric transmembrane enzyme composed of three subunits (α, β and γ) encoded by two genes –GNPTAB and GNPTG. Defects in GNPTAB result in ML II and III whereas mutations in GNPTG were only found in ML III patients. We have performed a molecular analysis of the GNPTAB and GNPTG genes in 13 mucolipidosis II and III patients (10 Portuguese, one Finnish, one Spanish of Arab origin and one Indian). Mutations were identified by the study of both cDNA and gDNA. The GNPTAB and GNPTG mRNA expressions were determined by quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR). The study led to the identification of 11 different mutations. Eight of these mutations are novel, six in the GNPTAB gene [c.121delG (V41FfsX42), c.440delC (A147AfsX5), c.2249_50insA (N750KfsX8), c.242G>T (W81L), c.1208T>C (I403T) and c.1999G>T (p.E667X)] and two in the GNPTG gene [c.610‐1G>T and c.639delT (F213LfsX7)]. With regard to the mRNA expression studies, the values obtained by qRT‐PCR indicate the possible existence of feedback regulation mechanisms between α/β and the γ subunits.

Molecular characterization of Portuguese patients with mucopolysaccharidosis type II shows evidence that the IDS gene is prone to splicing mutations

SummaryMucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal storage disease caused by a defect in the iduronate-2-sulfatase gene (IDS). Alternative splicing of the IDS gene can occur and the underlying regulatory mechanism may be rather complex. Nevertheless, little information is available on the role of variations at the IDS locus in the splicing process. Here we report that splice mutations at the IDS locus are an important source of MPS II pathogenicity, accounting for almost 56% of Portuguese cases. Among 16 unrelated Portuguese MPS II patients, 15 different mutations were identified: six intronic splice mutations (c.104−2AG, c.241−2A>G, c.241−1G>A, c.418+1G>A, c.880−8AG and c.1181−1G>C); two exonic splice mutations (c.1006G>lC and c.1122C>T); five missense mutations (D269V, D69V, D148N, R88C and P86L); one nonsense mutation (Q465Ter); one total IDS gene deletion; and one rearrangement involving a IDS gene inversion. Furthermore, nine of the 15 detected mutations affected the usual splicing pattern at the locus. Some of them are responsible for dramatic changes in the splicing mechanism. For example, the substitution mutation, c.418+1G>A, revealed the presence of an exonic sequence inside intron 3. Our study provides evidence that the IDS locus is prone to splicing mutations and that such susceptibility is particularly high in exon 3 and neighbouring regions. Consequently, mutation screening of the IDS gene cannot be restricted to gDNA examination. Unless cDNA analysis is also conducted, misclassifications as silent or missense mutations can be produced and even uncharacteristic splice-site mutations can be misinterpreted as classic splicing defects that may generate severe, unconventional splicing alterations.

Relationships between serum markers of monocyte/macrophage activation in type 1 Gaucher's disease

Abstract We studied 44 patients with type 1 Gauchers disease (16 non-treated patients and 28 treated with enzyme replacement therapy). We measured serum levels of chitotriosidase (ChT), neopterin, angiotensin-converting enzyme (ACE), adenosine deaminase (ADA) and β-hexosaminidase (Hex) and its major isoenzymes Hex A and Hex B. In the untreated group of patients, the increase in serum levels was ChT>neopterin>ACE>ADA>Hex, with all decreasing significantly in treated patients (p<0.001). Highly significant correlations were obtained between the markers of monocyte/macrophage activation which were tested (p<0.001). However, partial correlations between serum Hex B (with Hex A constant) and ChT, ACE, neopterin and ADA did not reach statistical significance. This suggests that hepatocytes are the major cellular source of this isoenzyme. Similarly, partial correlation of ChT with neopterin, with the other variables constant, was not significant, which would suggest a different expression of these two markers in Gauchers disease.

Gaucher disease : N370S glucocerebrosidase gene frequency in the portuguese population

In the Portuguese population the most frequent form of Gaucher disease is type 1. The N370S glucocerebrosidase gene mutation accounts for 63% of mutated alleles. The frequency of this mutation was accurately determined in the Portuguese population, which does not present an Ashkenazi Jewish genetic background. A gene frequency of 0.0043, with 95% confidence limits between 0.0023 and 0.0063, was obtained studying the genomic DNA of 2000 blood cards randomly sampled from the national neonatal screening program. On the basis of this frequency a significantly high number of homozygotes for the N370S mutation should be expected in the Portuguese population. This finding supports the idea that the majority of homozygotes for this mutation present a very mild clinical phenotype and remain undiagnosed.

Activity of glucocerebrosidase in extracts of different cell types from type 1 Gaucher disease patients

Glucocerebrosidase activity in extracts of leukocytes, Epstein‐Barr virus transformed lymphocytes and fibroblasts from Portuguese Type 1 Gaucher disease patients was studied. The residual glucocerebrosidase activity in all extracts from patients was less than 25% if measured in the presence of bile salt taurocholate. However, if measured in the absence of bile salt the residual enzyme activity in extracts from patients was cell type specific: it was severely reduced in the case of fibroblasts, mildly reduced in the case of lymphoblasts and not significantly reduced in the case of leukocytes. The glucocerebrosidase activity in extracts from all control cell types was stimulated by taurocholate. In the patients the enzyme activity in fibroblasts extracts was also stimulated but that in lymphoblasts and leukocytes was inhibited by the bile salt. The differences in glucocerebrosidase activity (in the absence of taurocholate) in extracts from different cell types from Gaucher disease patients are attributable to differences in the proportion of glucocerebrosidase present as a monomer with low activity (form I) and as a highly active aggregate (form II) that may also contain sphingolipid activator protein 2 (SAP‐2). In extracts from leukocytes and lymphocytes from Type 1 Gaucher disease patients, but not in those from fibroblasts, a relatively high proportion of enzyme is present in aggregated form with near normal specific activity.

Clinical, biochemical and molecular characterization of Cystinuria in a cohort of 12 patients

Barbosa M, Lopes A, Mota C, Martins E, Oliveira J, Alves S, De Bonis P, do Céu Mota M, Dias C, Rodrigues‐Santos P, Fortuna AM, Quelhas D, Lacerda L, Bisceglia L, Cardoso ML. Clinical, biochemical and molecular characterization of Cystinuria in a cohort of 12 patients.

Molecular characterisation of type 1 Gaucher disease families and patients: intrafamilial heterogeneity at the clinical level.

Type 1 Gaucher disease families were studied in an attempt to establish a phenotype/genotype correlation in affected persons and also to identify carriers accurately. In the Portuguese type 1 Gaucher patients, screening for mutations N370S, L444P, R463C, and 1066 + 1 G-->A allowed the identification of 85% of the alleles among unrelated patients. A subclinical case with genotype N370S/1066 + 1 G-->A was identified in one family in which there were three other symptomatic sibs. To our knowledge this is the first subclinical case with a genotype other than N370S/N370S. No genotype-phenotype correlation could be established and considerable clinical heterogeneity was found even among sibs with the same genotype. The data collected on the origins of the Gaucher families indicated two areas in northern Portugal where a higher frequency of the disease may be expected to exist.

The N370S mutation in the glucocerebrosidase gene of Portuguese type 1 gaucher patients : linkage to the PvuII polymorphism

SummaryThe mutation N370S accounts for 63% of the mutated glucocerebrosidase alleles of Portuguese type 1 Gaucher patients. It has been shown previously that this mutation is linked to the Pv1.1− form of the PvuII polymorphism and suggested that the N370S mutation in glucocerebrosidase alleles has an Ashkenazi Jewish origin. We have found that in Portuguese type 1 Gaucher patients this mutation is also invariably associated with the Pv1.1− haplotype, despite the fact that there is no evidence of Ashkenazi Jewish background in this population.