Molecular analysis of Sanfilippo syndrome type C in Spain: seven novel HGSNAT mutations and characterization of the mutant alleles.

The Sanfilippo syndrome type C [mucopolysaccharidosis IIIC (MPS IIIC)] is caused by mutations in the HGSNAT gene, encoding an enzyme involved in heparan sulphate degradation. We report the first molecular study on several Spanish Sanfilippo syndrome type C patients. Seven Spanish patients, one Argentinean and three Moroccan patients were analysed. All mutant alleles were identified and comprised nine distinct mutant alleles, seven of which were novel, including four missense mutations (p.A54V, p.L113P, p.G424V and p.L445P) and three splicing mutations due to two point mutations (c.633+1G>A and c.1378-1G>A) and an intronic deletion (c.821-31_821-13del). Furthermore, we found a new single nucleotide polymorphism (SNP) (c.564-98T>C). The two most frequent changes were the previously described c.372-2A>G and c.234+1G>A mutations. All five splicing mutations were experimentally confirmed by studies at the RNA level, and a minigene experiment was carried out in one case for which no fibroblasts were available. Expression assays allowed us to show the pathogenic effect of the four novel missense mutations and to confirm that the already known c.710C>A (p.P237Q) is a non-pathogenic SNP. Haplotype analyses suggested that the two mutations (c.234+1G>A and c.372-2A>G) that were present in more than one patient have a common origin, including one (c.234+1G>A) that was found in Spanish and Moroccan patients.

Multi O- and S-isotopes as tracers of black crusts formation under volcanic and non-volcanic atmospheric conditions in Sicily (Italy).

The deterioration of monument or building stone materials is mostly due to the growth of black crusts that cause blackening and disaggregation of the exposed surface. This study reports on new oxygen (δ17O, δ18O and Δ17O) and sulphur (δ33S, δ34S, δ36S, Δ33S and Δ36S) isotopic analyses of black crust sulphates formed on building stones in Sicily (Southern Italy). The measurements are used to identify the possible influence of volcanic emissions on black crust formation. Black crusts were mostly sampled on carbonate stone substrate in different locations subject to various sulphur emission sources (marine, anthropogenic and volcanic). Unlike atmospheric sulphate aerosols that mostly exhibit Δ33S > 0‰, here most of the analysed black crust sulphates show negative Δ33S. This confirms that black crust sulphates do not result from deposition of sulphate aerosols or of rainwater but mostly from the oxidation of dry deposited SO2 onto the stone substrate. The δ34S and δ18O values indicate that most of black crust sulphate originates from anthropogenic activities. Δ17O values are found to be related to the sampling location. The largest 17O-anomalies (up to ~4‰) are measured in black crust from areas highly influenced by volcanic emissions, which demonstrates the strong involvement of ozone in the formation of black crusts in volcanically influenced environments.

A new infantile case of alpha-N-acetylgalactosaminidase deficiency. Cardiomyopathy as a presenting symptom.

alpha-N-Acetylgalactosaminidase deficiency is a lysosomal disorder with clinically very different infantile and adult forms. To date, 12 patients from eight families are known. Neuroaxonal dystrophy or moderate psychomotor retardation, without visceral involvement, have been reported in the infantile form. We describe a new Spanish patient with Schindler disease who presented with hepatomegaly and cardiomyopathy, traits not previously associated with this disease. There was no dysmorphism or neurological involvement in the patient, who died at the age of 8 months. alpha-N-Acetylgalactosaminidase activity was reduced in fibroblasts and liver to 1.6% and 0.57% of controls, respectively. Several lysosomal enzyme activities associated with infantile cardiomyopathy were found in the normal ranges. The patient was a compound heterozygote for the novel mutation p.D217N (c.649G>A) in exon 6 and the already reported mutation p.E325K (c.973G>A) in exon 8. The description of this new case broadens the clinical spectrum of the infantile forms and indicates that Schindler disease should be considered in the diagnosis of metabolic cardiomyopathies.

Sudden deterioration in nonclassical infantile-onset Pompe disease responding to alglucosidase alfa infusion therapy: a case report.

A patient with atypical infantile Pompe disease suffered acute respiratory insufficiency at the age of 8 years which resulted in complete immobilization and dependence on assisted ventilation. Shortly after initiation of enzyme replacement therapy, she regained her mobility and, after 20 months of treatment, she now leads an almost normal life with limited restrictions.

Modeling of soiling based on silica-soda-lime glass exposure at six European sites.

Samples of silica-soda-lime float glass, a material selected as a pertinent soiling sensor, were exposed for up to 28 months at four urban sites (Athens, Krakow, London and Prague) and at a semi-urban one (Monte Libretti, near Rome), sheltered from rain. This exhaustive experience permitted to complete and to test the generalisation ability of the results previously obtained on the same material, exposed according to the same protocol, during 24 months, at a single site (Paris). The model previously fitted for the Paris experiment, the Hill equation, could be successfully generalised for four other sites of exposure (Athens, Krakow, Prague and Rome). The analytical form of this model was interpreted in terms of a physical description of the soiling phenomenon. Some of the model coefficients were different from one site to another, depending on the specific environment of exposure (air pollution levels, meteorological factors), while the other ones were rather equivalent. The analysis of the model coefficients, on the one hand, led to an estimation of the period after which soiling is close to its saturation level, and on the other hand, it will permit to correlate these coefficients to the environmental factors, in order to select the most appropriate ones for building dose-response functions for soiling, with broad geographical application.

[Outcome of two patients with Hurler's syndrome under enzyme replacement therapy with human recombinant alpha-L-iduronidase]. / Evolución de dos pacientes con síndrome de Hurler en tratamiento con enzima recombinante humana alpha-L-iduronidasa.

We performed a prospective study of two patients with Hurler's syndrome (aged 4.8 years and 17 months at the beginning of the intervention) under enzyme replacement therapy with human recombinant alpha-L-iduronidase for 452 and 28 weeks respectively. The aim of this study was to analyze the safety and efficacy of the intervention during the treatment periods. Several diagnostic imaging tests, clinical examinations, and serial laboratory determinations were performed to demonstrate the effectiveness of the therapy in both patients. In patient 1 (a boy aged 4.8 years, homozygote W402X), the treatment was always intended to be palliative because of the advanced stage of the disease. In patient 2 (a 17-month-old girl, heterozygote W402X) the treatment was initiated early with subsequent clinical stabilization without acquisition of regressive factors. Bone marrow transplantation from an unrelated donor was successful. Currently, because of the lack of histocompatible bone marrow donors, transplantation of hematopoietic stem cells from umbilical cord blood or peripheral blood are being performed with satisfactory results. In the future, gene therapy may be able to prevent the diseases associated with Hurler's syndrome and halt the neurocognitive deterioration characteristic of these patients.

Characterisation of CIME, an experimental chamber for simulating interactions between materials of the cultural heritage and the environment.

An approach consisting in combining in situ and laboratory experiments is often favoured for investigating the mechanisms involved in the weathering of the materials of the cultural heritage. However, the realistic simulation in the laboratory of the environmental conditions ruling the interactions of atmospheric compounds with materials is a very complex task. The aim of this work is to characterise CIME, a new chamber specially built to simulate the interactions between materials of the cultural heritage and the environment. The originality of this instrument is that beside the usual climatic parameters (temperature, relative humidity, solar radiation) and gaseous pollutants, it also allows the controlled injection of different types of particulate matter such as terrigenous, marine and anthropogenic. Therefore, varied realistic atmospheric environments (marine or urban) can be easily simulated within CIME. In addition to the technical description of CIME, this paper shows the first results obtained by the impact of gaseous pollutants on non-durable glass, bronze and limestone. The first experiments for the deposition of different particles (calcite, clays, soot and halite) are also presented.

Two new mild homozygous mutations in Gaucher disease patients: clinical signs and biochemical analyses.

Gaucher disease (GD) is a lysosomal storage disorder resulting from impaired activity of lysosomal beta-glucocerebrosidase. More than 60 mutations have been described in the GBA gene. They have been classified as lethal, severe, and mild on the basis of the corresponding phenotype. The fact that most GD patients are compound heterozygous and that most type 1 patients bear the N370S allele, which by itself causes a mild phenotype, make it difficult to correlate the clinical signs with the mutations. Besides N370S, about 10 mild mutations have been described, but only one undoubtedly classified as mild was found at homozygosity. Here we report 2 novel mutations, I402T and V375L, at homozygosity in 2 adult Italian type 1 GD patients. Some properties of the I402T fibroblast enzyme have been compared to those of the enzyme from cells of several N370S/N370S patients. Analysis of the catalytic properties and heat stability as well as the response to phosphatidylserine and sphingolipid activator protein indicate a marked similarity between the 2 enzymes. The finding of another, unrelated patient bearing the I402T mutation (in this case as a compound heterozygote with mutation N370S) suggests that this allele might be quite frequent in the area of Sicily from where both patients originated. In conclusion, the phenotypic expression in the 2 homozygous patients presented here and the biochemical data for one of them allowed the classification of these mutations as mild thus extending the group of mild mutations found at homozygosity.

Mutation and haplotype analyses in 26 Spanish Sanfilippo syndrome type A patients: possible single origin for 1091delC mutation.

Mucopolysaccharidosis IIIA, also known as Sanfilippo syndrome type A, is an autosomal recessive storage disorder caused by deficiency of sulfamidase. The disease results in severe central nervous system degeneration often with mild somatic features that may delay the clinical diagnosis. Molecular analyses would allow early and unequivocal heterozygote detection, providing a useful tool for genetic counselling. About 40 mutations have been reported in the sulfamidase gene, with a very uneven distribution in different patient populations. We have previously described the high prevalence of mutation 1091delC in a small number of Spanish Sanfilippo A patients. The aim of the present work is to extend the mutational study to a total of 26 unrelated patients and perform haplotype analysis in order to study the origin of some mutations. The whole coding region of the gene was scanned by SSCP analysis and sequencing. This allowed the identification of 14 different mutations, corresponding to 90% of the mutant alleles. Seven of these mutations were only found in this Spanish group of patients, three of which, R150W, R433Q and R433W, are described here for the first time. We have also analyzed four internal polymorphisms and constructed the corresponding haplotypes. Chromosomes bearing mutation 1091delC show a conserved haplotype suggesting a common origin for this mutation. Moreover, all other mutations found twice or more also have conserved haplotypes for those polymorphic markers.

Mutation analysis of Gaucher disease patients from Argentina: high prevalence of the RecNciI mutation.

Gaucher disease (GD) is caused by a deficiency of beta-glucocerebrosidase activity mainly due to mutations in the gene coding for the enzyme. More than 100 mutations have been identified to date and their frequencies have been established in several populations, including Ashkenazi Jews, among whom the disease is particularly prevalent. In order to study the molecular pathology of the disease in patients from Argentina, we conducted a systematic search for mutations in the glucocerebrosidase gene. Genomic DNA from 31 unrelated GD patients was screened for seven previously described mutations: N370S (1226A-->G), L444P (1448T-->C), D409H (1342G-->C), R463C (1504C-->T), 1263de155, RecNciI, and RecTL. This allowed the identification of 77.4% of the GD alleles: N370S and RecNciI were the most prevalent mutations found (46.8% and 21% respectively). Southern analysis demonstrated three distinct patterns for the RecNciI alleles. In order to identify the remaining alleles, the full coding region of the gene, all the splice sites, and part of the promoter region were analyzed by single-strand conformational polymorphism analysis (SSCP) after polymerase chain reaction amplification. This extensive screening allowed the identification of 13 different mutations, accounting for 93% of the total number of GD alleles. Three novel missense mutations, I161S (599T-->G), G265D (911G-->A), and F411I (1348T-->A), were detected. Twelve polymorphic sites within the glucocerebrosidase gene are in complete linkage disequilibrium and define two major haplotypes, "-" and "+". Mutation N370S was always associated with the "-" haplotype, as described in other populations. Interestingly, the RecNciI alleles with the same Southern-blot pattern were always associated with the same haplotype.

Chloroquine stabilizes pancreatic lysosomes and improves survival of mice with diet-induced acute pancreatitis.

Activation of digestive zymogens by lysosomal enzymes has been suggested as a triggering event in acute pancreatitis (AP). chloroquine (CQ), a weak base that accumulates in the lysosomes and increases their pH, can inhibit the activity of lysosomal enzymes. In the present study, we examined the effect of CQ on choline-deficient, ethionine-supplemented (CDE) diet-induced AP. CQ-diphosphate (15-50 mg.kg-1) or vehicle was given intraperitoneally at 0, 24, and 48 h to female CD1 mice that were fed with either normal diet or CDE diet. For mortality studies, animals were observed for 168 h. Serum and pancreas samples were collected from animals sacrificed 56 h after the start of the CDE diet. Treatment with CQ at 50 mg.kg-1 significantly (p < 0.05) improved the survival of mice with CDE diet-induced AP. In the normal pancreas, CQ decreased the specific activity of lysosomal enzymes cathepsin B1, beta-hexosaminidase, beta-glucuronidase, and acid phosphatase. In the pancreas with AP, CQ did not modify the activity of cathepsin B1, whereas it increased the latency of all enzymes. In conclusion, our results confirm the beneficial effect of CQ on survival of mice with CDE diet-induced AP and suggest that this effect of CQ may be due to its stabilizing action on lysosomes.

Angiokeratoma corporis diffusum associated with beta-mannosidase deficiency.

BACKGROUND: Angiokeratoma corporis diffusum (ACD) was at one time thought to be synonymous with Anderson-Fabry disease. However, it is well known that widespread angiokeratomas may also be found in other lysosomal enzyme disorders, as well as in patients with normal enzyme activities. beta-Mannosidase deficiency was first described in humans in 1986; since then, only 11 cases of beta-mannosidase deficiency, which occurred in 8 families, have been reported. Although the clinical manifestations are varied, mental retardation and neurologic disorders are present in practically all patients. OBSERVATIONS: We describe a 22-year-old woman who, since the age of 12 years, presented with progressive ACD affecting the lower limbs and the buttocks. Enzymatic studies revealed beta-mannosidase deficiency in cultured fibroblasts and in samples of serum and leukocytes. The patients's parents in turn exhibited intermediate enzyme levels, thus confirming the recessive autosomal hereditary nature of the disease. With the exception of an introverted character, the patient demonstrated no other anomalies. CONCLUSIONS: This is the first case of beta-mannosidase deficiency diagnosed as a result of purely dermatologic findings, in the form of ACD. beta-Mannosidase deficiency should therefore be included when screening for enzyme abnormalities in patients with ACD, even in the absence of neurologic disorders or mental retardation.

Congenital disorders of glycosylation (CDG) may be underdiagnosed when mimicking mitochondrial disease.

Congenital disorders of glycosylation (CDG) and mitochondrial diseases are multisystem disorders with clinical characteristics that may overlap. We present four patients with CDG whose phenotypes suggested the diagnosis of a mitochondrial disease. Patients 1 and 2 are siblings with hemiplegic headache, stroke-like episodes, lactic acidaemia and history of maternal migraine; their initial clinical diagnosis was MELAS syndrome (mitochondrial encephalopathy, lactic acidosis and stroke-like episodes). Patient 3 suffers from ataxia, neuropathy, ophtalmoplegia and retinitis pigmentosa suggestive of NARP (neuropathy, ataxia, and retinitis pigmentosa) syndrome. Patient 4 presented with neurological regression mimicking Leigh disease, with ptosis, myoclonus, ataxia and brainstem and cerebellar atrophy. Screening for mitochondrial disease including enzyme and mtDNA investigations on muscle biopsy were performed on Patients 1, 2 and 4 with normal results. However, evidence for a glycosylation disorder was substantiated by an increased carbohydrate deficient transferrin (CDT). The isoelectric focussing pattern of serum sialotransferrin was typical of CDG type I in Patients 1, 2 and 3 and was shifted towards the less sialylated bands in case 4. A deficiency of phosphomanomutase (PMM) confirmed the diagnosis of CDG-Ia in Patients 1, 2 and 3, who are compound heterozygous for mutations R141H/T237M (Patients 1 and 2) and R141H/P113L (Patient 3). In Patient 4, PMM activity was normal, and further enzymatic and molecular studies are underway. As the search for the primary defect in mitochondrial diseases is often unsuccessful, the pool of mitochondrial patients that remain without definite diagnosis might include CDG cases. Routine screening for CDG may avoid precocious invasive investigations.

[Activity and subcellular distribution of lysosomal enzymes in acute pancreatitis induced by CDE diet in mice]. / Actividad y distribución subcelular de enzimas lisosomales en la pancreatitis aguda inducida por dieta CDE en el ratón.

The aim of the present study was to analyze the activity and subcellular distribution of the lisosomal enzymes and the stability of the lisosomes in acute pancreatitis induced by CDE diet in mice. The activity and the latency of the catepsin-B1 enzymes, acid phosphatase, beta-hexosaminidase and beta-glucuronidase in normal pancreas and in pancreatitis induced by CDE diet were determined. The distribution of the acid phosphatase lisosomal marker was determined in subcellular fractions obtained by differential centrifugation. The activity of the catepsin-B1 enzyme increased 47% in the pancreas of mice with pancreatitis induced by CDE diet. The acid phosphatase activity was not modified and the beta-hexosaminidase and beta-glucuronidase was decreased. The specific activity of the acid phosphatase lisosomal marker also increased in the subcellular fraction containing the zimogene granules and decreased the latency (parameter indicative of lisosome stability) of all the lisosomal enzymes analyzed in the pancreatic homogenate. These results suggest that the lisosomal enzymes, specially the catepsin-B1, and the decrease in the stability of the lisosomes may play a role in the pathogenesis of acute pancreatitis.

[Late infantile and juvenile form of GM2-gangliosidosis variant B1]. / Fenotipo infantil tardío y juvenil de la variante B1 de gangliosidosis GM2.

INTRODUCTION: Variant B1 is a rare form of GM2-gangliosidosis characterized by the presence of a mutation in the hexosaminidase A gene (HEXA) leading to a defect in the catalytic region of the alpha-subunit of beta-hexosaminidase A (alpha beta heterodymer). The mutated Hex A has almost normal activity against the natural synthetic substrates (4-methylumbelliferyl-N-acetyl-beta-D-glucosamine, 4MU-NAG) but is unable to hydrolyse GM2-ganglioside and the sulphated synthetic substrates (4MU-NAGS). The first and more frequent mutation described in the alpha-subunit gene associated to B1 variant GM2-gangliosidosis was a G533-->A transition (DN allele) resulting in Arg178His substitution. CLINICAL CASES: Here, we report the clinical, enzymatic and molecular characterization in two variant B1 late infantile and juvenile cases. Both cases presented regression of mental skills leading to dementia, epilepsy and severe motor impairment with dystonic involuntary movements and quadriplegia. In the late infantile case (death at 5 years and 8 months), cherry-red spot was also present. Enzymatic assays were performed in fibroblasts, leukocytes and serum and confirmed the abnormally low beta-hexosaminidase A activity against sulphated substrate despite a normal or nearly normal total hexosaminidase activity (unsulphated substrates). The patient with the late infantile phenotype was found to be compound heterozygote for the DN allele whilst the juvenile form was homozygote for that mutation. CONCLUSION: Variant B1 form of GM2-gangliosidosis is a rare and heterogeneous condition that must be kept in mind when evaluating neurodegenerative disorders associated with speech or gait disturbances, dystonia, seizures and pyramidal features.

[Evolutive neuroradiological alterations in Sandhoff's disease]. / Alteraciones neurorradiológicas evolutivas en la enfermedad de Sandhoff.

Sandhoff's disease is a severe form of gangliosidosis GM2 which presents in the first year of life, basically as progressive psychomotor retardation and/or a macular red cherry spot. Our patient presented the clinical picture characteristic of the disease. Diagnosis was confirmed by determining the activity of hexosaminidases A and B in serum and of beta-N-acetil hexosaminases in fibroblast culture. In view of the fatal prognosis of the disease, in 1991 a transplant of alogenic bone marrow (TMO) was carried out to try to replace the enzymes. This required exhaustive radiological follow-up to determine the possible neuro-radiological changes seen in this storage disease. Although treatment was not successful, the neuro-radiological findings may be of interest as perhaps being characteristic of the GM2 gangliosidosis: 1. Bilateral thalamic hyperecogenity in the cerebral ecography. 2. Differences between the thalamo-putamen densities due to bilateral homogeneous thalamic hyperdensity on the CT scan. 3. Thalamic hypointensity both on T2 sequences and in proton density on MR with the cerebral white matter being progressively affected. In conclusion, we suggest that bilateral symmetrical thalamic changes are an early finding which is probably specific to the GM2 gangliosidoses and may be useful from the point of view of carrying out more specific investigations in infants suspected of having a degenerative neurological disorder.

Physico-chemical characterisation of glass soiling in rural, urban and industrial environments.

Glass materials are broadly used in the built environment (windows, facades, roofs, museum showcases, and solar panels) due to their optical (transparency) and thermal properties. Their interaction with the multiphase atmospheric medium results in a more or less pronounced transparency loss called soiling. This phenomenon leads to a loss of amenity of artefacts; consequently, high cleaning costs have to be supported by public and private entities. Complete understanding of the nature of surface deposit appears thus extremely important for addressing strategies to control it. The present research is based on the sheltered exposure, in different environments, of durable glass panels during 1 year. At these different locations, airborne pollutant concentrations have also been monitored. Three environments have been investigated: rural (R), urban (U) and industrial (I). Results show that the mass of the deposit and the optical impairment of the glass (haze) are too spread to allow discriminating between different environments. However, the analyses of soluble species and particulate organic matter allow identifying factors responsible for soiling and highlighted the reactivity of deposit to relative humidity which favours post-deposit evolution.

Dose-response function for the soiling of silica-soda-lime glass due to dry deposition.

Several exposure campaigns of silica-soda-lime window glass have been performed in 30 European sites and 1 in Canada in order to understand, quantify and model the phenomenon of soiling. In this purpose samples were exposed sheltered from the rain. Parallel to exposure, several meteorological parameters and pollution concentrations have been monitored. This paper shows first results on the establishment of a dose-response function for glass soiling. Statistical analyses show that PM(10) is not the only parameter, but also SO(2) and NO(2) atmospheric concentrations seem to be responsible for the optical impairment of glass surfaces, expressed as haze.