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.

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.

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.

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.

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.

Calidad en genética bioquímica prenatal / Quality in prenatal biochemical genetics

Para diagnosticar enfermedades metabólicas hereditarias(EMH), tanto postnatales como prenatalmente, se empleanlos recursos de la genética bioquímica, que se basanmayoritariamente en el estudio en fluidos biológicos y tejidos de productos génicos (proteínas) y de metabolitos específicos que sean demostrativos o estén directamente relacionados con la pérdida de función de un gen. Las EMH, debido a sus bajas prevalencias individuales, entran en la categoría de enfermedades raras o minoritarias, pero se han descrito centenares de ellas y en general son enfermedades abrumadoras y a menudo letales. Debido a ello y a las dificultadesde tratamiento, su diagnóstico prenatal es muy relevante.En el presente artículo se resumen unos conocimientosmínimos indispensables sobre su complejidad y losmedios del laboratorio para llegar al diagnóstico, a fin de demostrar la importancia que tiene en el diagnóstico prenatal la labor de reunir la máxima información posible acerca del caso índice y de los padres (heterocigotos). Seguidamente se trata el tema de la calidad de los laboratorios y de laspruebas o ensayos de genética bioquímica, incluyendo elcontrol europeo específico de la ERNDIM, el proyecto Eurogentest de la CE para la armonización, validación y estandarización de pruebas genéticas, la Ley 14/2007 de 3 de julio de 2007 de investigación biomédica que define y regula el marco para la realización de pruebas genéticas en investigación y en asistencia y las Normas ISO para la certificación y acreditación de los laboratorios. El artículo finaliza con unrecordatorio de las técnicas y materiales fetales utilizados para el diagnóstico prenatal de las EMH

For the diagnosis of Inherited disorders of metabolism(IDM), postnatal as well as prenatal, we need the resources of Biochemical genetics, which are mainly based in the studies in biological fluids and tissues of the gene products (proteins) and specific metabolites, directly related or demonstratives of a gene function impairment. Because of the very low individual prevalence of IDM, they are consideredrare diseases, but it had been described hundreds ofthem, being moreover overwhelming and often lethal diseases. These facts, together with difficulties for treatment made prenatal diagnosis very relevant. In this paper, there are summarised some essential knowledge on the complexity of IDM and the resources of the laboratory for its diagnosis, in order to show how it is important for the prenatal diagnosis, to collect all the possible information about the index case and the parents (heterozygous). Next, issues are the quality of the biochemical genetics tests and laboratories,including the specific ERNDIM QAP; the FP6 projectEurogentest for the harmonization, validation andstandardization of diagnostic genetic testing; the Spanish bill 14/2007 on biomedical research, that among other issues provides for genetic testing for research as well as for medical care purposes and some concepts on ISO norms for the certification/accreditation of laboratories. Finally, there is a reminder of the technology and foetal materials suitable for the prenatal diagnosis of IDM

Identification of a novel pseudodeficiency allele in the GLB1 gene in a carrier of GM1 gangliosidosis.

The term 'pseudodeficiency' is used in lysosomal storage diseases to denote the situation in which individuals show greatly reduced enzyme activity but remain clinically healthy. Pseudodeficiencies have been reported for several lysosomal hydrolases. GM1 gangliosidosis is a rare autosomal recessive lysosomal storage disorder caused by beta-galactosidase hydrolase deficiency as a result of mutations in the GLB1 gene. Until now, two variants altering the beta-galactosidase activity have been described, p.Arg521Cys and p.Ser532Gly. Here we report the new variant p.Arg595Trp in the GLB1 gene, which markedly reduces beta-galactosidase activity when expressed in COS-1 cells. The variant was identified in the healthy father of a girl with GM1 gangliosidosis. He was a heterozygous compound with p.Arg595Trp in trans with one of the disease-causing mutations identified in his daughter; in leukocytes and plasma he showed lower beta-galactosidase activity than that observed in GM1 gangliosidosis carriers. As this family originated from the Basque Country in the north of Spain, we decided to analyse individuals of Basque and non-Basque origin, finding the p.Arg595Trp allele in 3.2% of Basque and in 0.8% of non-Basque alleles. The detection of the presence of alterations resulting in pseudodeficient activity in leukocytes and plasma is important for the correct diagnosis of GM1 gangliosidosis.

Identification of 14 novel GLB1 mutations, including five deletions, in 19 patients with GM1 gangliosidosis from South America.

GM1 gangliosidosis is a lysosomal storage disorder caused by the absence or reduction of lysosomal beta-galactosidase activity because of mutations in the GLB1 gene. Three major clinical forms have been established: type I (infantile), type II (late infantile/juvenile) and type III (adult). A mutational analysis was performed in 19 patients with GM1 gangliosidosis from South America, mainly from Argentina. Two of them were of Gypsy origin. Main clinical findings of the patients are presented. All 38 mutant alleles were identified: of the 22 different mutations found, 14 mutations are described here for the first time. Among the novel mutations, five deletions were found. Four of them are relatively small (c.435_440delTCT, c.845_846delC, c.1131_1145del15 and c.1706_1707delC), while the other one is a deletion of 1529 nucleotides that includes exon 5 and is caused by an unequal crossover between intronic Alu sequences. All the described patients with GM1 gangliosidosis were affected by the infantile form, except for four unrelated patients classified as type II, III, and II/III (two cases). The two type II/III patients bore the previously described p.R201H mutation, while the adult patient bore the new p.L155R. The juvenile patient bore two novel mutations: p.S434L and p.G554E. The two Gypsy patients are homozygous for the p.R59H mutation as are all Gypsy patients previously genotyped.

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.

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

Estudio prospectivo de 2 pacientes con enfermedad de Hurler (de 4,8 años y 17 meses de edad al inicio de la intervención) en tratamiento enzimático sustitutivo con enzima recombinante humana α -L-iduronidasa durante 52 y 28 semanas, respectivamente, con el objetivo de analizar la eficacia y seguridad de la intervención durante dicho período de tiempo. Se realizaron para ello diversas pruebas diagnósticas por imagen, exámenes clínicos y analíticos seriados que demostraron en ambos casos ser una terapia efectiva. En el caso 1 (varón de 4,8 años, homocigoto W402X) la intervención se planteó con fines paliativos desde el comienzo debido a su estado clínico muy evolucionado. En el caso 2 (mujer de 17 meses, heterocigota para W402X) la intervención se realizó de forma precoz y se observó una estabilización clínica posterior sin la adquisición de factores regresivos. Posteriormente se realizó con éxito un trasplante de médula ósea de donante no emparentado. Actualmente, ante la carencia de donantes de médula ósea histocompatibles, se está realizando trasplante de células madre hematopoyéticas procedentes de cordón o de sangre periférica con resultados satisfactorios. La terapia génica se considera el tratamiento futuro capaz de prevenir la enfermedad asociada al síndrome de Hurler y detener el deterioro neurocognitivo característico de estos pacientes.

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 α -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

Angiokeratoma corporis diffusum in a Spanish patient with aspartylglucosaminuria.

Angiokeratoma corporis diffusum (ACD), initially considered to be synonymous with Fabry's disease, represents a well-known cutaneous marker of some other lysosomal enzyme disorders. Aspartylglucosaminuria (AGU) is a rare hereditary disorder mostly affecting the Finnish population, with only a few sporadic patients of non-Finnish origin. To date, only three patients with AGU have been reported with cutaneous lesions of ACD. A 19-year-old Spanish woman presented with a 10-year history of progressive ACD affecting the limbs, buttocks and trunk. After the age of 6 years she had developed progressive mental deterioration, coarse facies and macroglossia with a scrotal appearance. Peripheral blood smears showed many vacuolated lymphocytes. Enzyme analysis in cultured fibroblasts revealed a decreased activity of aspartylglucosaminidase. By the age of 31 years the patient had developed a bipolar psychosis, polycystic ovarian disease and severe impairment of cognitive skills. This is the first case of AGU detected in a Spanish patient presenting with cutaneous lesions of ACD. To our knowledge, macroglossia with a scrotal appearance and polycystic ovarian disease have not been reported in previous cases of AGU.

Biochemical and molecular studies in 26 Spanish patients with congenital disorder of glycosylation type Ia.

We present our experience with the diagnosis of 26 patients (19 families) with congenital disorders of glycosylation classified as type Ia due to PMM deficiency. In all but one of these CDG Ia families the patients are compound heterozygous for mutations in PMM2. Eighteen different mutations were detected. In contrast to other series in which R141H represents 43-50% of the alleles, only 9/36 (25%) alleles have this mutation. Two mutations (R123Q and T237M) have been found on three disease chromosomes, four (V44A, Y64C, P113L and F207S) on two disease chromosomes and 12 mutations (D65Y, Y76C, IVS3+2C>T, E93A, R123X, V129M, I153T, F157S, E197A, N216I, T226S, C241S) only on one disease chromosome. V44A and D65Y probably originated in the Iberian peninsula, as they have only been reported in Portuguese and Latin-American patients; Y64C, Y76C, R123X and F207S have not been detected in other patients. R123X is the only stop codon mutation so far described in PMM2. The common European F119L mutation has not been found in our patients, although it is very frequent in other populations (43% allele frequency in Danish patients). Probably because of this genetic heterogeneity, Spanish patients show very diverse phenotypes that are, in general, milder than in other series. This points to the necessity of widening the criteria for CDG in the routine screening for inborn metabolic diseases.

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.

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.

New insights into the origin of the Gaucher disease-causing mutation N370S: extended haplotype analysis using the 5GC3.2, 5470 G/A, and ITG6.2 polymorphisms.

Gaucher disease is a lysosomal storage disorder inherited as an autosomal recessive trait. It is highly prevalent among Ashkenazi Jews but also present in other populations. Mutations in the glucocerebrosidase gene are the main cause of the disorder. One of these gene defects, N370S, is the most prevalent disease allele in the Ashkenazi Jewish patient population and also frequent in others, such as the Spanish and Portuguese Gaucher disease populations. Previous results based on haplotype analysis support the hypothesis of a single origin for this mutation. We have extended the haplotype analysis to include three newly described polymorphisms, 5GC3.2, ITG6.2 (very close to the gene), and 5470 G/A (in intron 7 of the GBA gene) in a sample of Spanish and Ashkenazi Jewish patients. The results confirm the single origin of the mutation in these two populations. The 5470A allele is only found in N370S chromosomes and was believed to be limited to the Portuguese population. Here we describe that it is also present with a similar frequency in Spain. Moreover, most of the 5470A alleles are found within particular haplotypes, which have some differences from the common N370S haplotype.