Osteoblasts from a mandibuloacral dysplasia patient induce human blood precursors to differentiate into active osteoclasts.

Mandibuloacral dysplasia type A (MADA) is a rare disease caused by mutations in the LMNA gene encoding A type lamins. Patients affected by mandibuloacral dysplasia type A suffer from partial lipodystrophy, skin abnormalities and accelerated aging. Typical of mandibuloacral dysplasia type A is also bone resorption at defined districts including terminal phalanges, mandible and clavicles. Little is known about the biological mechanism underlying osteolysis in mandibuloacral dysplasia type A. In the reported study, we analyzed an osteoblast primary culture derived from the cervical vertebrae of a mandibuloacral dysplasia type A patient bearing the homozygous R527H LMNA mutation. Mandibuloacral dysplasia type A osteoblasts showed nuclear abnormalities typical of laminopathic cells, but they proliferated in culture and underwent differentiation upon stimulation with dexamethasone and beta-glycerophosphate. Differentiated osteoblasts showed proper production of bone mineral matrix until passage 8 in culture, suggesting a good differentiation activity. In order to evaluate whether mandibuloacral dysplasia type A osteoblast-derived factors affected osteoclast differentiation or activity, we used a conditioned medium from mandibuloacral dysplasia type A or control cultures to treat normal human peripheral blood monocytes and investigated whether they were induced to differentiate into osteoclasts. A higher osteoclast differentiation and matrix digestion rate was obtained in the presence of mandibuloacral dysplasia type A osteoblast medium with respect to normal osteoblast medium. Further, TGFbeta 2 and osteoprotegerin expression were enhanced in mandibuloacral dysplasia type A osteoblasts while the RANKL/osteoprotegerin ratio was diminished. Importantly, inhibition of TGFbeta 2 by a neutralizing antibody abolished the effect of mandibuloacral dysplasia type A conditioned medium on osteoclast differentiation. These data argue in favor of an altered bone turnover in mandibuloacral dysplasia type A, caused by upregulation of bone-derived stimulatory cytokines, which activate non-canonical differentiation stimuli. In this context, TGFbeta 2 appears as a major player in the osteolytic process that affects mandibuloacral dysplasia type A patients.

Splicing mutations in glycogen-storage disease type II: evaluation of the full spectrum of mutations and their relation to patients' phenotypes.

Glycogen-storage disease type II is an autosomal recessive-inherited disorder due to the deficiency of acid α-glucosidase. A large number of mutations in the acid α-glucosidase gene have been described to date. Among them, ~15% are variations that may affect mRNA splicing process. In this study, we have for the first time comprehensively reviewed the available information on splicing mutations of the acid α-glucosidase gene and we have evaluated their possible impact on the splicing process using different in silico approaches. Out of the 39 different GAA-sequence variations described, an in silico analysis using seven different programs showed that 97% of them are predicted to have an impact on the splicing process. Moreover, this analysis showed a quite good correlation between the impact of the mutation on the splicing process and the clinical phenotype. In addition, we have performed the functional characterization of three novel sequence variants found in Italian patients and still uncharacterized. Using a minigene system, we have confirmed their pathogenic nature. In conclusion, this study has shown that in silico analysis represents a useful tool to select mutations that affect the splicing process of the acid α-glucosidase gene and provides an updated picture of all this kind of mutations reported till now.

Long-term observational, non-randomized study of enzyme replacement therapy in late-onset glycogenosis type II.

OBJECTIVES: Type II glycogenosis (GSDII) is a lysosomal storage disorder due to acid alpha-glucosidase (GAA) deficiency. Enzyme replacement therapy (ERT) with human recombinant alpha-glucosidase (rhGAA) has been demonstrated to be effective in the treatment of infantile forms of GSDII, but little information is available concerning late-onset phenotypes. Long-term follow-up studies are not available at present. The aim of this study was to evaluate the ERT long-term effects in late-onset GSDII. METHODS: Twenty-four patients, including 7 juveniles and 17 adults, received bi-weekly infusion of rhGAA (20 mg/kg) for at least 36 months. Clinical conditions, muscular function (6-min walking test, 6MWT; Walton scale, WS), respiratory function (vital capacity, VC; forced expiratory volume, FEV1; arterial pCO(2)), and muscle enzymes were assessed every 6 months. RESULTS: The 6MWT improved in both juvenile and adult patients (p = 0.01, p = 0.0002, respectively), as well as in patients with moderate to severe muscle function impairment (WS >3.5; p = 0.002). An overall improvement in WS was also observed (p = 0.0003). VC and FEV1 remained unchanged, while pCO(2) decreased (p = 0.017). Muscle enzymes decreased significantly (p < 0.0001). Two patients (8%) showed transient secondary events during ERT. CONCLUSIONS: Long-term ERT with rhGAA was shown to be safe, well tolerated, and effective in improving motor function and in stabilizing respiratory function in late-onset GSDII. The response pattern showed a progressive clinical improvement during the follow-up period in juvenile patients, while in adults it reached and maintained a plateau after the first year of treatment.

Haplotype analysis suggests a single Balkan origin for the Gaucher disease [D409H;H255Q] double mutant allele.

Gaucher disease is an autosomal recessive lysosomal storage disease that is mainly due to mutations in the GBA gene. Most of the mutant alleles described so far bear a single mutation. However, there are a few alleles bearing two or more DNA changes. It has been reported that patients homozygous for the [D409H;H255Q] double mutant allele (HGVS-approved nomenclature, p.[D448H;H294Q]) present a more severe phenotype than patients homozygous for the relatively common D409H mutation. In this study, we confirmed the detrimental cumulative effect of these two mutations at the enzymatic activity level by the heterologous expression of the single and double mutant alleles. Additionally, we found a high frequency of the [D409H;H255Q] allele in patients from the Balkans and the Adriatic area of Italy. This prompted us to perform a haplotype analysis, using five microsatellite polymorphisms close to the GBA gene, to determine the origin of this allele. The results of the 37 chromosomes analysed showed that most of them share a common haplotype and are consistent with a single origin in the Balkans and the Adriatic area of Italy for the [D409H;H255Q] allele.

Altered intracellular redox status in Gaucher disease fibroblasts and impairment of adaptive response against oxidative stress.

Gaucher disease (GD) is a lysosomal storage disorder, due to glucosylceramide (GlcCer) accumulation in several body tissues, which causes cellular failure by yet unidentified mechanisms. Several evidence indicates that GD pathogenesis is associated to an impairment in intracellular redox state. In fibroblast primary cultures, reactive oxygen species (ROS) levels and protein carbonyl content resulted significantly increased in GD patients compared to healthy donors, suggesting that GD cells, facing a condition of chronic oxidative stress, have evolved an adaptive response to survive. The ROS rise is probably due to NAD(P)H oxidase activity, being inhibited by the treatment with diphenylene iodonium chloride. Interestingly, GD cells are more sensitive to H(2)O(2) induced cell death, suggesting a dysregulation in the adaptive response to oxidative stress in which APE1/Ref-1 plays a central role. We found that the cytoplasmic amounts of APE1/Ref-1 protein were significantly higher in GD fibroblasts with respect to controls, and that GD cells failed to upregulate its expression upon H(2)O(2) treatment. Both ROS and APE1/Ref-1 increases are due to GlcCer accumulation, being prevented by treatment of GD fibroblasts with Cerezyme and induced in healthy fibroblasts treated with conduritol-beta-epoxide. These data, suggesting that GD cells display an impairment in the cellular redox state and in the adaptive cellular response to oxidative stress, may open new perspectives in the comprehension of GD pathogenesis.

Characterization of two novel GBA mutations causing Gaucher disease that lead to aberrant RNA species by using functional splicing assays.

The correct identification of disease-causing mutations from the background of harmless nucleotide polymorphisms/substitutions has become a critical issue in the investigation of human genetic diseases. Here, we describe two novel disease-causing splicing mutations in the glucocerebrosidase gene, g.4252C>G and g.4426A>G, that have been found in two patients affected by Gaucher disease. The g.4252C>G substitution occurred in intron 5 and was located 12 nucleotides upstream of exon 6 acceptor site whilst the g.4426A>G mutation was located within this exon, 12 nucleotides upstream of the donor site. An in silico analysis suggested that both mutations could have altered the splicing process of exon 6 by creating a novel acceptor and donor site, respectively. However, because the wild-type acceptor and donor sites of exon 6 were not apparently affected, the severity of both mutations could not be established by simple sequence analysis alone. Nonetheless, the use of minigene functional assays to complement transcript analysis of patient fibroblasts shows that both mutations cause the almost complete switch of splice site usage from the wild-type to the newly-created ones, thus providing a functional explanation for the appearance of disease.

Pulmonary involvement in an adult female affected by type B Niemann Pick disease.

We describe the case of a 33-year-old woman who presented with a pattern of diffuse micronodular opacities with centrolobular distribution at high resolution chest tomography (HRCT) performed after exposure to the smoke of a home fire. An abdominal CT scan showed the presence of 3 rounded hypodense lesions in the spleen parenchyma. A bronchoalveolar lavage (BAL) was performed, showing the presence of lipid laden cells in the aspirated fluid. A video-assisted thoracoscopic biopsy confirmed the presence of the foamy cells filling the alveolar spaces and showed a preserved background pulmonary architecture; the bronchiolar epithelium presented a cytoplasm with a particularly clear colour. Foam cells were present in the bone marrow aspirate too, that contained also sea-blue histiocytes. These data induced us to consider the diagnosis of a metabolic storage disorder, in the first instance Niemann-Pick type B, that was confirmed by low acid lysosomal sphingomyelinase activity on cultured fibroblasts: 5.22 nmol/mg prot/h (n.v. 92 +/- 18.2). Lung involvement is an important complication of Niemann-Pick disease, presenting with the clinical and radiological features of a diffuse lung disease.

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.

Functional in vitro characterization of 14 SMPD1 mutations identified in Italian patients affected by Niemann Pick Type B disease.

Niemann Pick disease (NPD) is an autosomal recessive lysosomal storage disorder caused by the deficient activity of acid sphingomyelinase due to mutations in the SMPD1 gene. We functionally characterized three novel SMPD1 mutations and 11 already reported in the Italian population. Mutant alleles were studied for enzyme activity and protein processing in transiently transfected COS-1 cells. The c.96G>A, c.100delG, c.565dupC, and c.575dupC (p.W32X, p.G34fsX42, p.P189fsX1, and p.P192fs14) alleles expressed no immunoreactive protein and consequently no enzyme activity. In contrast, cells transfected with mutants c.308T>C, c.389T>C, c.674T>C, c.732G>C, c.841G>A, c.1687G>A, c.1799G>A, and c.1799G>C (p.L103P, p.V130A, p.L225P, p.W244C, p.A281T, p.D563Y, p.R600H, p.R600P) expressed protein levels comparable to wild-type ASM expressing cells. Only three of these constructs, c.389T>C, c.1687G>A, and c.1799G>A (p.V130A, p.D563Y, p.R600H), retained residual activity while the other five expressed very low or no enzyme activity. As expected, the c.1669underscore;1670delGT (p.V557fsX18) mutant expressed a completely inactive truncated protein. Interestingly, the c.2T>G (p.M1_W32del) mutant expressed 26.9% of the wild type activity, even though no ASM protein was detected by Western blot analysis, suggesting that the amount of produced enzyme is below detection levels. The results presented in this study are consistent with the wide phenotype variability found in NP type B patients and provide valuable insights into the molecular basis of the disease.

Identification and characterization of five novel MAN2B1 mutations in Italian patients with alpha-mannosidosis.

Mutation analysis performed on six Italian families with alpha-mannosidosis type II allowed the identification of five new mutations in the MAN2B1 gene: c.157G>T, c.562C>T, c.599A>T, c.293dupA, c.2402G>A (p.E53X, p.R188X, p.H200L, p.Y99VfsX61, p.G801D). Protein residues G801 and H200 are conserved among the four mammalian alpha-mannosidases cloned to date: human, cattle, cat and mouse. In vitro expression studies demonstrated that both missense mutations expressed no residual alpha-mannosidase activity indicating that they are disease-causing mutations. Modelling into the three-dimensional structure revealed that the p.H200L could involve the catalytic mechanism, whereas p.G801D would affect the correct folding of the enzyme.

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.

Comparative in vitro expression study of four Fabry disease causing mutations at glutamine 279 of the alpha-galactosidase A protein.

Fabry disease is an X-linked lysosomal storage disorder caused by the deficiency of alpha-galactosidase A that results in the accumulation of neutral sphingolipids. We report a novel point mutation in exon 6, Q279K, carried by an asymptomatic child with a family history of classic Fabry disease. Moreover, we comparatively study the in vitro expression and enzyme activity of Q279K and three other already described mutants in glutamine 279. The Q279K, Q279H and Q279R mutants transfected in COS-1 cells expressed no activity while the residual enzyme activity of the Q279E mutant represented 10% of wild type value. Western blot analysis demonstrated a differential behavior of the mutant proteins: Q279K and Q279H persisted as the inactive 50-kD precursor, indicating that these mutations may affect the normal processing of the enzyme, while the Q279R mutant was not detected probably due to an unstable protein which is rapidly degraded. The in vitro expression studies of the novel Q279K mutation were confirmed by Western blot analysis performed in the patient's lymphocytes which revealed the alpha-galactosidase A precursor of 50 kD but not the processed form.

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.

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.