RESUMEN
Tyrosinemia type I is a genetic disorder characterized by accumulation in the blood and urine of the toxic metabolite succinylacetone (SUAC), not detectable in healthy samples. In many countries, newborns are screened for tyrosinemia type I using tyrosine as a primary marker. Unfortunately, tyrosine accumulation may take longer to occur and it may be not obvious when specimens are collected, in the first few days of life, as for newborn screening. In 2008, we reported changes to simultaneously measure acylcarnitines, amino acids, and SUAC during expanded newborn screening. We established the usefulness of this method after identifying a first asymptomatic newborn affected by tyrosinemia type I. Now we report a second infant with positive SUAC screening result (14.1 µmol/L, n.v. < 2) and normal tyrosine concentration (74 µmol/L; n.v. < 250). We also performed molecular analysis of FAH gene in both patients after diagnosis at newborn screening. They had consanguineous parents and were both homozygous for two known disease-causing mutations of the FAH gene. The outcome of patients detected in the MS/MS screening is significantly favorable. We also report our results of newborn screening for tyrosinemia type I before and after inclusion of SUAC as a primary marker for this disease.
RESUMEN
The aim of this study was to set up a robust method suitable for large-scale studies (screening) with a minimized preparation process and with reduced running costs, for measuring five enzyme activities on dried blood spots by a new and simplified tandem mass spectrometry-based method. After incubation, all 5 reaction mixtures, carried out separately, were stopped, combined together, and centrifuged. The cleaning-up of the injected mixture was performed through a fast online trapping step preceding a liquid chromatography/tandem mass-spectrometry measurement. This method takes only 4 min as analysis run time and without any purification following the enzymatic reaction. We assessed the effectiveness of this approach in assaying the enzymatic activities on dried blood spots from 10 patients affected by "Pompe", 6 by "Gaucher", 12 by "Fabry", 3 by "Niemann-Pick" A/B, and 2 by "Krabbe" diseases. Reference values were established on 5000 healthy newborns and 300 healthy adults. All affected patients showed enzymatic activities below the normal range. In heterozygous carriers (18 for Fabry, 10 for Pompe, and 4 for Gaucher disease) the activities were slightly lower than in control subjects. The results show that the method set out in its simplicity, low costs, and low processes preparations can be fully applicable to a mass screening.
Asunto(s)
Cromatografía Liquida , Enfermedades por Almacenamiento Lisosomal/diagnóstico , Tamizaje Neonatal , Espectrometría de Masas en Tándem , Adulto , Recolección de Muestras de Sangre , Humanos , Recién Nacido , Enfermedades por Almacenamiento Lisosomal/sangre , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
BACKGROUND: The glycosphingolipid storage disorder GM1-gangliosidosis is a severe neurodegenerative condition for which no therapy is currently available. Protein misfolding in lysosomal defects may have the potential to be corrected by chemical chaperones: in vitro and clinical approaches are being investigated. AIMS: We investigated the in vitro effect of galactose on some lysosomal hydrolases, and its in vitro efficacy as a chemical chaperone in GM1-gangliosidosis. METHODS: Galactose was added to the culture medium of fibroblasts from patients, controls and transfected COS-1 cells. Enzyme assays of lysosomal hydrolases, beta galactosidase in particular, were performed. RESULTS: Our data show that galactose alters selectively alpha and beta galactosidases. A significant increase (2,5 fold) in beta galactosidase activity occurred when galactose was added to the cultured fibroblasts of an adult patient. Chemical chaperone therapy requires the presence of residual enzyme activity. The adult patient here reported is heterozygous for the p.T329A mutation that showed no beta galactosidase activity, and for the p.R442Q mutation with residual enzyme activity. The p.R442Q mutation was therefore selected as a potential target for the galactose chaperone; after the addition of galactose, COS-1 cells transfected with this mutation showed an increase in beta galactosidase activity from 6.9% to 12% of control values. CONCLUSIONS: These results suggest that galactose or its derivatives with potential chaperone properties could be used in the development of non-invasive therapies for GM1-gangliosidosis.
Asunto(s)
Fibroblastos/efectos de los fármacos , Galactosa/farmacología , Gangliosidosis GM1/tratamiento farmacológico , Chaperonas Moleculares/farmacología , alfa-Galactosidasa/metabolismo , beta-Galactosidasa/metabolismo , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Fibroblastos/enzimología , Gangliosidosis GM1/enzimología , Heterocigoto , Humanos , Mutación , Transfección , alfa-Galactosidasa/genética , beta-Galactosidasa/genéticaRESUMEN
Total or partial deficiency of the human lysosomal hydrolase alpha-galactosidase A is responsible for Fabry disease, the X-linked inborn error of glycosphingolipid metabolism. Together with the predominant alpha-galactosidase A gene mRNA product encoding the lysosomal enzyme, a weakly regulated alternatively spliced alpha-galactosidase A transcript is expressed in normal tissues, but its overexpression, due to the intronic g.9331G>A mutation, leads to the cardiac variant. We report the molecular characterization of five Fabry patients including two siblings. Sequencing analysis of the alpha-galactosidase A gene coding region and intron/exon boundaries identified the new c.124A>G (p.M42V) genetic lesion as well as a known deletion in three patients, whereas in the two remaining patients, no mutations were identified. To evaluate possible alpha-galactosidase A gene transcription alterations, both predominant and alternatively spliced mRNAs were quantified by absolute real-time PCR on total RNA preparations from the patients' fibroblasts. An impressive reduction in the predominant alpha-galactosidase A transcript was detected in the last patients (Pt 4 and Pt 5). However, the alternatively spliced mRNA was dramatically overexpressed in one of them, carrying a new intronic lesion (g.9273C>T). These findings strongly suggest a correlation between this new intronic mutation and the unbalanced alpha-galactosidase A mRNAs ratio, which could therefore be responsible for the reduced enzyme activity causing Fabry disease. The real-time assay developed here to investigate the two alpha-galactosidase A mRNAs might play a crucial role in revealing possible genetic lesions and in confirming the pathogenetic mechanisms underlying Fabry disease.
Asunto(s)
Enfermedad de Fabry/enzimología , Enfermedad de Fabry/genética , Fibroblastos/enzimología , Mutación Puntual , alfa-Galactosidasa/biosíntesis , alfa-Galactosidasa/genética , Empalme Alternativo/genética , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Masculino , Sistemas de Lectura Abierta/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/genéticaRESUMEN
In expanded newborn screening programs by liquid chromatography/tandem mass spectrometry false negatives for tyrosinemia type I are a significant problem. We describe a method for inclusion of succinylacetone in order to avoid false negatives. We studied spots from 13,000 neonates born in Tuscany (January-May 2007) and ten spots from six patients with tyrosinemia type I. The traditional screening method was modified by adding dioxooctanoid acid (or 13C2-succinylacetone) as an internal standard to the methanolic solution of deuterated acylcarnitines and amino acids. A hydrazine solution was added to the mixture. The times of extraction, butylation and drying were only slightly prolonged. Specific multiple reaction monitoring for derivatized and labelled succinylacetone and dioxooctanoic acid was carried out. The assays were linear up to 100 micromol/L for succinylacetone. Intra- and inter-day imprecision data were in the range of 1.34% to 7.09% and 3.50% to 4.49%. Limits of detection and of quantification were 0.2 micromol/L and 0.4 micromol/L, respectively. Recovery ranged from 97.02% to 100.29%. Succinylacetone levels in samples from unaffected neonates were very close to the detection limit. Of the 46 recalls, eight (17.4%) were for abnormal tyrosine levels and all these cases had succinylacetone levels within the normal range (<2.4 micromol/L). In ten spots from six affected patients succinylacetone values ranged from 3.3 to 35.0 micromol/L. Including succinylacetone in newborn screening programs for amino acids and acylcarnitines avoids false-negative results for tyrosinemia type I. Newborn screening laboratories should consider implementing these modifications.
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Análisis Químico de la Sangre/métodos , Cromatografía Líquida de Alta Presión/métodos , Heptanoatos/sangre , Espectrometría de Masas/métodos , Tamizaje Neonatal/métodos , Tirosinemias/sangre , Tirosinemias/diagnóstico , Biomarcadores/sangre , Femenino , Humanos , Recién Nacido , Masculino , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
OBJECTIVES: Hereditary fructose intolerance is caused by a deficiency of the aldolase B enzyme, which is expressed in the liver, small intestine and kidneys. Patients usually show a marked aversion to fruits and sweets; if, however, it is not diagnosed, persistent or incidental ingestion of fructose might be lethal. Our paper aims at improving the clinical and molecular characterizations of these patients, to avoid dangerous misdiagnoses. METHODS: Here we report the molecular results in an Italian cohort: on the occurrence of aldolase B mutations and, in particular, on the clinical and molecular characterization of a large family with recurrent hereditary fructose intolerance. RESULTS: Patients included in our cohort showed the three most common mutations (p.A150P, p.A175D and p.N335K). Such molecular tests were enough to cover all the mutated alleles of hereditary fructose intolerance found in our patients. The allele frequencies of hereditary fructose intolerance mutations detected were 69.2% for p.A150P, 23.1% for p.A175D and 7.7% for p.N335K. The proband of the family with recurrence of the disease was heterozygous for the known p.A150P and p.A175D mutated alleles of the aldolase B gene. Molecular characterization of at-risk family members also identified the p.N335K mutation. In addition, the oldest affected patients exhibited mild clinical impairment. CONCLUSIONS: Our results indicate that the diagnosis of hereditary fructose intolerance can be complicated by clinical and genetic intrafamilial variability. A knowledge of the clinical and geographical history of each family member is thus essential, to reduce potentially lethal misdiagnoses and to facilitate such patients to receive appropriate genetic counselling.
Asunto(s)
Intolerancia a la Fructosa/genética , Adulto , Preescolar , Estudios de Cohortes , Intolerancia a la Fructosa/diagnóstico , Fructosa-Bifosfato Aldolasa/genética , Frecuencia de los Genes , Genotipo , Humanos , Lactante , Italia , Mutación , Linaje , RecurrenciaRESUMEN
BACKGROUND: The expansion of newborn screening programs has increased the number of newborns diagnosed with inborn errors of metabolism in the presymptomatic phase, but it has also increased the number of costly, stress-producing false-positive results. Because propionylcarnitine (C3) is one of the analytes most frequently responsible for false-positive results, we aimed to develop a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to identify free methylmalonic (MMA) and 3-OH propionic (3OH-PA) acids in blood spots. METHODS: We studied newborn screening spots from 250 healthy controls; 124 from infants with abnormal C3, of whom only 5 (4%) were truly affected; 124 from infants with altered isolated methylmalonylcarnitine; and 4 from clinically diagnosed patients. Whole blood was eluted from a 3.2-mm dried blood spot by a CH(3)CN/H(2)O 7:3 and 5 mL/L formic. This extract was injected into a LC-MS/MS equipped with pneumatically assisted electrospray without derivatization. Total analysis time was 5 min per sample. RESULTS: The assays were linear up to 3300 nmol/L for both metabolites. Intra- and interassay imprecision data were 3.6%-8% and 3.1%-6%, respectively, for MMA and 5.2%-20% and 3.6%-17% for 3OH-PA. Limit of detection and limit of quantitation were 1.95 and 4.2 micromol/L, respectively, for MMA and 8 and 10 micromol/L for 3OH-PA. The recoveries were 92.9%-106.1%. No deterioration was noted on the columns after 500 chromatographic runs. If the new method had been used as a 2nd-tier test for the 124 samples, only the 5 true positives would have been recalled for additional samples, and the positive predictive value would have been 100%. CONCLUSIONS: This method has the potential to markedly reduce false-positive results and the associated costs and anxiety. It may also be suitable for diagnosing and routinely monitoring blood spots for methylmalonic aciduria and propionic acidemia.
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Recolección de Muestras de Sangre , Carnitina/análogos & derivados , Ácido Láctico/análogos & derivados , Ácido Metilmalónico/sangre , Tamizaje Neonatal/métodos , Carnitina/sangre , Cromatografía Liquida , Reacciones Falso Positivas , Humanos , Recién Nacido , Ácido Láctico/sangre , Valor Predictivo de las Pruebas , Espectrometría de Masas en TándemRESUMEN
The human GLB1 gene produces two alternatively spliced transcripts that encode the lysosomal enzyme beta-galactosidase (GLB1) and the elastin binding protein (EBP). Mutations at the GLB1 locus, which are responsible for the storage disorder GM1 gangliosidosis, may affect either both proteins or GLB1 only. The EBP, when affected, contributes to specific features of GM1 gangliosidosis patients, such as cardiomyopathy and connective-tissue abnormalities. Here we report the development of reliable and quantitative assays based on real-time PCR for assessing the levels of GLB1 and EBP transcripts in patients' samples. We also report the characterisation of GLB1 gene mutations in nine GM1 gangliosidosis patients in order to correlate the genetic lesions with mRNA levels and phenotypes. Mutation analysis identified four new (c.1835_1836delCC; p.Arg148Cys; c.1068+1G>T; and p.Pro549Leu), five known (p.Arg59His; p.Arg201His; p.Gly123Arg; c.245+1G>A; and c.75+2insT) mutations and one new polymorphism (c.1233+8T>C). Comparative analysis of the patients' phenotypes enabled a more thorough correlation between GLB1 mutations and specific clinical manifestations. GLB1 and EBP mRNA levels were both reduced in three patients carrying the splicing defects. The accurate and fast method for the detection of alternatively spliced transcripts of the GLB1 gene could be applied to other disease-causing lysosomal genes that encode multiple mRNAs.
Asunto(s)
Análisis Mutacional de ADN/métodos , Gangliosidosis GM1/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , beta-Galactosidasa/genética , Empalme Alternativo , Animales , Células COS , Chlorocebus aethiops , Gangliosidosis GM1/genética , Pruebas Genéticas/métodos , Humanos , Lactante , Mutación Missense , Fenotipo , Polimorfismo Genético , ARN Mensajero/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Purines and pyrimidines are the basic constituents of DNA and RNA and constitute the basis of at least 50 other important compounds that serve equally vital but separate roles as integral components of intracellular mononucleotide pools. They maintain the supply of these basic components to the different nucleotide pools through an extremely efficient mechanism involving the degradation and recycling of the daily waste products of normal cell turnover. We have developed an LC-MS/MS diagnostic and routine monitoring method for known defects due to both purine and pyrimidine metabolism in a single analysis. Precision tests were made by spiking several urine samples with different creatinine concentrations. For nonspiked low-creatinine urine, intraday precision was in the range of 0.1-9.8% and interday precision was between 1.6 and 14.1%. For nonspiked high-creatinine urine, intraday precision was in the range 0.5-17.2% and interday precision was between 1.5 and 29%. Limit-of-detection (LOD) was in the range 0.1-10 micromol/l and limit-of-quantification (LOQ) in the range of 0.2-15 micromol/l. The current 'dilute and shoot' approach monitors many metabolites, and utilizes a reverse phase chromatographic analysis with a detection requiring 17 min of analysis time. Tandem mass spectrometry and isotope dilution technique enable the accurate quantitation of more than 30 metabolites in one analysis.
Asunto(s)
Purinas/metabolismo , Purinas/orina , Pirimidinas/metabolismo , Pirimidinas/orina , Espectrometría de Masas en Tándem/métodos , Adolescente , Adulto , Calibración , Preescolar , Femenino , Humanos , Recién Nacido , Masculino , Estándares de ReferenciaRESUMEN
We describe two patients affected by Barth syndrome. Their symptoms became manifest on respectively the third and first day of their lives. Clinical presentation included poor sucking, lethargy, hypotonia, hypothermia and cardiomyopathy. Laboratory findings such as hypoglycaemia, metabolic acidosis, elevated transaminases, hyperlactacidaemia and mild hyperammonaemia pointed to an inborn error of energy metabolism with possible mitochondrial involvement. Molecular analysis of the TAZ (G4.5) gene showed the c.877G > A mutation leading to the G197R amino acid substitution in patient 1, and the new splice donor c.829 + 1G > A genetic lesion in patient 2.
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Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Mutación , Enfermedad Aguda , Humanos , Recién Nacido , Enfermedades Musculares/genética , Síndrome , Transaminasas/biosíntesisAsunto(s)
Carnitina/análisis , Carnitina/sangre , Glutamato Formimidoiltransferasa/deficiencia , Tamizaje Neonatal , Artefactos , Consanguinidad , Reacciones Falso Positivas , Femenino , Humanos , Recién Nacido , Masculino , Errores Innatos del Metabolismo/enzimología , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
G(M1)-gangliosidosis is a lysosomal storage disorder caused by acid beta-galactosidase deficiency. Aside from the lysosomal beta-galactosidase enzyme, the beta-galactosidase gene also encodes the elastin-binding protein (EBP), deficiency in which impairs elastogenesis. Using expression studies and Western blots of COS-1 cells, we identified and characterized four new and two known beta-galactosidase gene mutations detected in G(M1)-gangliosidosis patients with infantile, juvenile, or adult forms of disease. We then focused on impaired elastogenesis detected in fibroblasts from patients with infantile and juvenile disease. The juvenile patient showed connective-tissue abnormalities, unusual urinary keratan sulfate excretion, and an EBP reduction, despite mutations affecting only beta-galactosidase. Because galactosugar-bearing moieties may alter EBP function and impair elastogenesis, we assessed infantile and juvenile patients for the source of altered elastogenesis. We confirmed that the infantile patient's impaired elastogenesis arose from a primary EBP defect, according to molecular analysis. We examined the juvenile's fibroblasts by immunohistochemistry, addition of keratanase, soluble/insoluble elastin assay, and radiolabeling of tropoelastin. These experiments revealed that the juvenile's impaired elastogenesis likely arose from secondary EBP deficiency caused by keratan sulfate accumulation. Thus, impaired elastogenesis in G(M1)-gangliosidosis can arise from primary or secondary EBP defects in fibroblasts from infantile and juvenile patients, respectively.
Asunto(s)
Fibroblastos/fisiología , Gangliosidosis GM1/patología , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/fisiología , Adolescente , Adulto , Edad de Inicio , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Niño , Chlorocebus aethiops , Tejido Conectivo/patología , Cartilla de ADN , Elasticidad , Exones , Cara/anomalías , Fibroblastos/patología , Gangliosidosis GM1/genética , Gangliosidosis GM1/orina , Humanos , Lactante , Sulfato de Queratano/orina , Mutación , Fenotipo , Receptores de Superficie Celular/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Tropoelastina/genéticaRESUMEN
OBJECTIVES: We report on the first prenatal molecular diagnosis of holocarboxylase synthetase (HLCS) deficiency in the fourth pregnancy of an at-risk family. This disorder is a rare autosomal recessive inborn error of metabolism, leading to a multiple carboxylase defect (MCD). HLCSD diagnosis was performed postmortem in the proband on DNA from autoptic biological material. Molecular analysis of the proband's entire HLCS gene by direct sequencing identified the R508W amino acid change, at the homozygous status. METHODS: Fetal DNA was isolated from chorionic villus sampling at 11 weeks of gestation. Direct sequencing of exon 6 of the fetal HLCS gene was performed. RESULTS: The R508W mutation was identified in the fetal DNA at the homozygous level. The genetic lesion was confirmed on abortive tissue. CONCLUSION: Molecular diagnosis has several advantages over enzymatic activity assay of carboxylases in chorionic villi or amniocytes. It can be performed earlier, is faster, and the response time is shorter.
Asunto(s)
Ligasas de Carbono-Nitrógeno/genética , Familia , Deficiencia de Holocarboxilasa Sintetasa/diagnóstico , Deficiencia de Holocarboxilasa Sintetasa/genética , Diagnóstico Prenatal , Consanguinidad , Análisis Mutacional de ADN , Femenino , Homocigoto , Humanos , Recién Nacido , Masculino , Mutación , Linaje , Embarazo , Atención PrenatalRESUMEN
GM1 gangliosidosis is a lysosomal storage disorder caused by deficiency of beta-galactosidase. It is mainly characterized by progressive neurodegeneration, and in its most severe infantile form, it leads to death before the age of 4. The GLB1 gene gives rise to two alternatively spliced mRNAs that encode the beta-galactosidase and the elastin binding protein (EBP). The diagnosis of two patients with the infantile form of GM1 gangliosidosis and 11 carriers in a small mountainous village in Cyprus prompted us to carry out a study in order to establish the frequency of carriers in the village and identify the mutations involved. Carrier detection was initially based on the measurement of beta-galactosidase activity in leucocytes. Among 85 random samples from the village, 10 were classified as carriers. Sequencing of the GLB1 gene in a Cypriot patient identified the missense mutation c.1445G>A (p.Arg482His) in the homozygous state. Seven of the 10 carriers identified using the enzyme assay were found to carry the same mutation by NspI restriction enzyme analysis. The three individuals who were negative for the c.1445G>A had borderline enzyme results and were probably wrongly classified as carriers. The frequency of GM1 gangliosidosis carriers in this village is approximately 8% (1:12). Western blot analysis showed a marked decrease of the 64-kDa mature form of the enzyme protein and a similar reduction of the 67-kDa EBP. Our results indicate that the c.1445G>A mutation, which appears to be responsible for all GM1 gangliosidosis alleles in this Cypriot village, affects protein conformation.
Asunto(s)
Sustitución de Aminoácidos , Gangliosidosis GM1/genética , Heterocigoto , beta-Galactosidasa/genética , Adulto , Animales , Arginina/genética , Células COS , Chlorocebus aethiops , Chipre , Femenino , Feto , Técnica del Anticuerpo Fluorescente , Gangliosidosis GM1/fisiopatología , Humanos , Lactante , Recién Nacido , Masculino , Mutación , EmbarazoRESUMEN
UNLABELLED: We studied the genotype/phenotype correlation in a cohort of glycogen storage disease type (GSD) 1b patients. A total of 25 GSD1b patients, 13 females and 12 males, age range: 4.3-28.4 years, mean:14.6+/-6.8 years; median: 15 years, representing the entire case load of Italian GSD1b patients, were enrolled in the study. Molecular analysis of the glucose 6-phosphate translocase (G6PT1) gene was performed in all patients. We analysed the presence of a correlation among both the clinical features associated with GSD1b (neutropenia, frequency of admission to the hospital for severe infections) and the presence of systemic complications (liver adenomas, nephropathy, bone mineral density defect, polycystic ovaries, short stature, inflammatory bowel disease) and the mutations detected in each patient. Nine patients were homozygous or compound heterozygous for mutations causing stop codons. In particular, three patients were homozygous for the same mutation (400X); of these patients, one showed chronic neutropenia with severe and frequent infections and severe inflammatory bowel disease, another patient cyclic neutropenia associated with rare bacterial infections and mild bowel involvement and the last one normal neutrophil count. Two patients were homozygous for the mutation 128X; one of these patients did not show neutropenia, whereas the other one had severe neutropenia needing frequent hospital admission and was under granulocyte-colony stimulating factor treatment. In three patients no mutations were detected. CONCLUSION: No correlation was found between individual mutations and the presence of neutropenia, bacterial infections and systemic complications. These results suggest that different genes and proteins modulate neutrophil differentiation, maturation and apoptosis and thus the severity and frequency of infections. The absence of detectable mutations in three patients could suggest that a second protein plays a role in microsomal phosphate transport.
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Antiportadores/genética , Enfermedad del Almacenamiento de Glucógeno Tipo I/complicaciones , Enfermedad del Almacenamiento de Glucógeno Tipo I/genética , Proteínas de Transporte de Monosacáridos/genética , Mutación , Adolescente , Adulto , Niño , Preescolar , Estudios de Cohortes , Análisis Mutacional de ADN , Femenino , Genotipo , Humanos , Italia , Masculino , Neutropenia/genética , Neutropenia/terapia , FenotipoRESUMEN
G(M1)-gangliosidosis is a lysosomal storage disorder caused by a deficiency of beta-galactosidase (GLB1). The GLB1 gene gives rise to the GLB1 lysosomal enzyme and to the elastin binding protein (EBP), involved in elastic fiber deposition. GLB1 forms a complex with protective protein cathepsin A (PPCA), alpha neuraminidase (NEU1), and galactosamine 6-sulphate sulfatase (GALNS) inside lysosomes, while EBP binds to PPCA and NEU1 on the cell surface. We investigated the function of the GLB1 and EBP mutated proteins by analyzing the clinical, genetic, and cellular data of 11 G(M1)-gangliosidosis patients. Their molecular analysis, followed by expression studies, lead to the identification of four new and 10 known GLB1 mutations. Some common amino acid substitutions [c.1445G>A (p.Arg482H), c.622C>T (p.Arg208His), c.175C>T (p.Arg59Cys) and c.176G>A (p.Arg59His)] were present in the GLB1 enzyme of several patients, all of Mediterranean origin, suggesting a common origin. Western blotting analyses against GLB1, EBP, and PPCA proteins showed that the identified mutations affect GLB1 enzyme activity and/or stability. The c.1445G>A (p.Arg482His), c.175C>T (p.Arg59Cys), c.733+2T>C, c.1736G>A (p.Gly579Asp), and c.1051C>T (p.Arg351X) GLB1 mutations, affect the stabilization of PPCA probably because they hamper the interaction between GLB1/EBP and PPCA within the multiprotein complex. The amount of EBP was normal, but the detection of impaired elastogenesis in such patients suggests an alteration in its function. We conclude that the presence of genetic lesions in both GLB1 and EBP coding region does not directly predict impaired elastogenesis and that elastic fiber assembly has to be evaluated specifically in each case. Nevertheless, the degree of EBP involvement may be linked to specific clinical findings.
Asunto(s)
Gangliosidosis GM1/genética , Receptores de Superficie Celular/fisiología , beta-Galactosidasa/fisiología , Adulto , Empalme Alternativo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Células COS , Catepsina A/química , Células Cultivadas/metabolismo , Chlorocebus aethiops , Tejido Elástico/ultraestructura , Femenino , Fibroblastos/metabolismo , Gangliosidosis GM1/clasificación , Gangliosidosis GM1/patología , Humanos , Lactante , Recién Nacido , Lisosomas/enzimología , Masculino , Datos de Secuencia Molecular , Complejos Multiproteicos , Mutación Missense , Fenotipo , Unión Proteica , Pliegue de Proteína , Mapeo de Interacción de Proteínas , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Transfección , beta-Galactosidasa/química , beta-Galactosidasa/genéticaAsunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Hidroxiprolina/sangre , Tamizaje Neonatal/métodos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/deficiencia , Espectrometría de Masa por Ionización de Electrospray/métodos , Errores Innatos del Metabolismo de los Aminoácidos/sangre , Humanos , Recién Nacido , Masculino , Errores Innatos del MetabolismoRESUMEN
Multiple sulfatase deficiency (MSD) is a rare disorder characterized by impaired activity of all known sulfatases. The gene mutated in this disease is SUMF1, which encodes a protein involved in a post-translational modification at the catalytic site of all sulfatases that is necessary for their function. SUMF1 strongly enhances the activity of sulfatases when coexpressed with sulfatase in Cos-7 cells. We performed a mutational analysis of SUMF1 in 20 MSD patients of different ethnic origin. The clinical presentation of these patients was variable, ranging from severe neonatal forms to mild phenotypes showing mild neurological involvement. A total of 22 SUMF1 mutations were identified, including missense, nonsense, microdeletion, and splicing mutations. We expressed all missense mutations in culture to study their ability to enhance the activity of sulfatases. Of the predicted amino acid changes, 11 (p.R349W, p.R224W, p.L20F, p.A348P, p.S155P, p.C218Y, p.N259I, p.A279V, p.R349Q, p.C336R, p.A177P) resulted in severely impaired sulfatase-enhancing activity. Two (p.R345C and p.P266L) showed a high residual activity on some, but not all, of the nine sulfatases tested, suggesting that some SUMF1 mutations may have variable effects on the activity of each sulfatase. This study compares, for the first time, clinical, biochemical, and molecular data in MSD patients. Our results show lack of a direct correlation between the type of molecular defect and the severity of phenotype.
Asunto(s)
Mutación , Esfingolipidosis/genética , Sulfatasas/genética , Animales , Células COS , Humanos , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro , Esfingolipidosis/enzimología , Sulfatasas/deficienciaRESUMEN
We describe the clinical findings, and the molecular and biochemical studies in an Italian family with recurrent hydrops fetalis due to galactosialidosis (GS). GS is a rare lysosomal storage disorder caused by a deficiency of the protective protein/cathepsin A (PPCA). This protein forms a high-molecular-weight complex with the hydrolases beta-galactosidase (GLB1) and neuraminidase (NEU1). By virtue of this association these two enzymes are correctly compartmentalized in lysosomes and protected against rapid proteolytic degradation. Controversial data show that PPCA is also present in a second complex, including the Elastin Binding Protein (EBP) the EBP-receptor, which is involved in elastogenesis, and NEU1. We investigated the potential role of the PPCA in both complexes. Two new genetic lesions (c60delG and IVS2+1 G > T) that lead to a frameshift and a premature stop codon were detected in the PPCA cDNA and genomic DNA of the patient. The deleterious effect of such mutations was confirmed by the complete absence of the PPCA protein on Western blots. Thus, we examined the effect of the loss of PPCA on the two protein complexes in the patient's fibroblasts. Interestingly, a reduced amount of both GLB1 and EBP proteins was detected. These data confirm that PPCA is present in two functional complexes one with GLB1 and NEU1 in the lysosomal lumen and the other with EBP at the cell surface. The reduction in GLB1 and EBP confirms that PPCA is essential for their integrity.