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R Giugliani

Publications and source records attributed to R Giugliani.

At least 55 records · Page 3Linked to original sources
PubMed1999

Six novel beta-galactosidase gene mutations in Brazilian patients with GM1-gangliosidosis.

GM1-gangliosidosis is a lysosomal storage disease caused by a deficiency of acid beta-galactosidase. Three clinical forms are recognized-infantile, juvenile, and adult-based on age of onset and severity of the symptoms. We have performed molecular analysis of a large cohort of GM1 patients (19 Brazilian and one Uruguayan), using nonradioactive single-strand conformation polymorphism (SSCP) and restriction enzyme analysis of genomic DNA. Six novel mutations (R121S, V240M, D491N, 638-641insT, 895-896insC, 1622-1627insG) and two previously described point mutations (R59H, R208C) were identified. Together they accounted for 90% of the disease alleles of the patients. Two mutations, 1622-1627insG and R59H, were present in 18 of 20 patients. In addition, four polymorphisms (L10P, L12L, R521C, S532G) were identified. All cases reported are infantile GM1 gangliosidosis. This report constitutes the most comprehensive molecular study to date of this disorder in infantile patients. Since GM1-gangliosidosis is the most common lysosomal storage disorder in Southern Brazil, molecular diagnosis will be important for genetic counseling, carrier detection and prenatal diagnosis in index families.

Brazil↗
PubMed1999

Biochemical studies on leukocyte and fibroblast human beta-galactosidase.

OBJECTIVES: Some biochemical characteristics of the human leukocyte and fibroblast beta-galactosidase were studied. DESIGN AND METHODS: Leukocyte and fibroblast enzyme activity was determined fluorometricaly using 4-methylumbelliferyl-beta-D-galactoside as artificial substrate. Optimum pH, Km, Vmax and thermostability of the enzyme at 42 degrees C were determined. RESULTS: The leukocyte and fibroblast enzyme has an optimum pH at 4.2, which is in agreement with the lysosomal origin of the enzyme. The Km of the enzyme was 0.62 in leukocytes and 0.67 in fibroblasts, and Vmax was 289.9 nmol/h/mg of protein and 1779.2 nmol/h/mg of protein in the two tissues, respectively. When fibroblast or leukocyte beta-galactosidase was pre-incubated at 42 degrees C, it did not retain its activity because the residual activity after 80 minutes of pre-incubation at this temperature was lower than 30% of the initial activity both in leukocytes and fibroblasts. CONCLUSIONS: This was the first study of Km, Vmax and thermostability of beta-galactosidase performed on leukocytes and provided data for a better characterization of the enzyme beta-galactosidase, allowing the improvement of the analytical conditions.

Fibroblasts↗
PubMed1999

Effect of dimethylsulfoxide on sphingomyelinase activity and cholesterol metabolism in Niemann-Pick type C fibroblasts.

Niemann-Pick type C (NPC) fibroblasts present a large concentration of cholesterol in their cytoplasm due to a still unidentified deficiency in cholesterol metabolism. The influence of dimethylsulfoxide (DMSO) on the amount of intracellular cholesterol was measured in 8 cultures of normal fibroblasts and in 7 fibroblast cultures from NPC patients. DMSO was added to the fibroblast cultures at three different concentrations (1, 2 and 4%, v/v) and the cultures were incubated for 24 h. Sphingomyelinase activity was significantly increased in both groups of cells only when incubated with 2% DMSO (59.4 +/- 9.1 and 77.0 +/- 9.1 nmol h-1 mg protein-1, controls without and with 2% DMSO, respectively: 47.7 +/- 5.2 and 55.8 +/- 4.1 nmol h-1 mg protein-1. NPC without and with 2% DMSO, respectively). However, none of the DMSO concentrations used altered the amount of cholesterol in the cytoplasm of NPC cells (0.704 +/- 0.049, 0.659 +/- 0.041, 0.688 +/- 0.063 and 0.733 +/- 0.088 mg/mg protein, without DMSO, 1% DMSO, 2% DMSO and 4% DMSO, respectively). This finding suggests that sphingomyelinase deficiency is a secondary defect in NPC and shows that DMSO failed to remove the stored cholesterol. These data do not support the use of DMSO in the treatment of NPC patients.

Analysis of Variance↗
PubMed1999

Arylsulfatase A pseudodeficiency in healthy Brazilian individuals.

Molecular alterations associated with arylsulfatase A pseudodeficiency (ASA-PD) were characterized by PCR and restriction endonuclease analysis in a sample of healthy individuals from Brazil. ASA activity was also assayed in all subjects. Two individuals homozygous for the N350S and 1524+95A<--G mutations were detected, corresponding to a frequency of 1.17% (4 of 324 alleles). The individual frequency of the N350S mutation was 20.7% (71 of 342 alleles) and 7.9% (27 of 342 alleles) for the 1524+95A<--G mutation. The frequency of the ASA-PD allele in our population was estimated to be 7.9%. This is the first report of ASA-PD allele frequency in a South American population. In addition, the methods used are effective and suitable for application in countries with limited resources. All patients with low ASA activity should be screened for ASA-PD as part of the diagnostic protocol for metachromatic leukodystrophy.

Alleles↗
PubMed1999

Persistent tyrosinemia detected by thin-layer chromatography.

Screening for tyrosinemia is not routinely performed worldwide. Using a low expense thin-layer chromatography (TLC) for amino acids we detected a high frequency of transient tyrosinemia with secondary hyperphenylalaninemia in some newborns. Serum follow up showed the need to introduce adequate therapy in these babies.

Chromatography, Thin Layer↗
PubMed1999

A comprehensive screening program in South Brazil.

We present the experience and figures about a screening program in South Brazil carried on in Porto Alegre, capital of the Southern Brazilian State. We present the tests performed routinely in our laboratory, the prevalence of some diseases and tests for infectious diseases to be added in the most comprehensive regional program in our country.

Brazil↗
PubMed1999

Geographic heterogeneity of 4 common worldwide cystic fibrosis non-DF508 mutations in Brazil.

Cystic fibrosis (CF) is an autosomal recessive disease caused by at least 750 different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The frequency of the most common mutation (DF508) in Brazilian patients of European origin is 47%. To determine the frequency of 4 other common CF mutations (G542X, G551D, R553X, and N1303K) in Brazilian patients of European origin, we used direct polymerase chain reaction (PCR) amplification of DNA obtained from dried blood spots on Guthrie cards. The DNA came from 247 non-DF508 chromosomes from 172 Brazilian CF patients ascertained from 5 different states of Brazil. The results show that the 4 mutations account for 17% of the non-DF508 alleles and only 9% of the total number of Brazilian CF alleles. Overall, the frequency of each mutation is different from northern European and North American populations but similar to southern European populations, mainly the Italian and Spanish populations. When Brazilian patients of European origin are grouped according to state of birth, the frequencies of the mutations are significantly different between southern and southeastern states of Brazil. Therefore there are serious implication for risk assessment of DNA-based tests in heterogeneous populations such as Brazilians. Further studies are needed to identify the remaining 44% of CF mutations for the different populations and regions of Brazil.

Adolescent↗
PubMed1997

Selective screening of 10,000 high-risk Brazilian patients for the detection of inborn errors of metabolism.

UNLABELLED: The number of diagnosed inborn errors of metabolism (IEM) is growing constantly due to the improvement and widespread availability of analytical techniques. In 1982, a laboratory for the detection of IEM was set up in Porto Alegre, Brazil, and became a national reference centre for the diagnosis of these disorders. Ten thousand patients with signs and symptoms suggestive of IEM were investigated in our laboratory from 1982 to 1995 using specific protocols which included tests for the detection of glucosaminoglycans (GAGS), amino acids, sugars, oligosaccharides, sialyloligosaccharides, organic acids, as well as various metabolite. The biochemical investigation was completed in 9,901 patients and an IEM was detected in 647 cases (6.5%). Groups of IEM of higher incidence in our sample were lysosomal storage disorders (59.8%) and aminoacidopathies (21.2%). The disorders most frequently diagnosed were classical phenylketonuria, GM1 gangliosidosis, mucopolysaccharidosis type I, mucopolysaccharidosis type VI and metachromatic leukodystrophy. CONCLUSION: This study shows that the establishment of reference centres for the investigation of rare genetic diseases is a suitable approach to the study of IEM in developing countries such as Brazil.

Brazil↗
PubMed1997

Evaluation of an aspartame loading test for the detection of heterozygotes for classical phenylketonuria.

Classical phenylketonuria (PKU) is an inborn error of metabolism of autosomal recessive inheritance characterized by the accumulation of phenylalanine (Phe) in tissues due to Phe-4-hydroxylase deficiency. Several methods have been developed for the detection of PKU heterozygotes based on the determination of plasma Phe and tyrosine (Tyr) levels, on the analysis of the Phe/Tyr and Phe2/Tyr ratios and on the use of discriminant functions. The objective of the present study was to test the value of loading with aspartame (a sweetener consisting of Phe, aspartate and methanol) for the identification of PKU carriers. The study was conducted on 22 obligate heterozygotes and 27 controls. Two blood samples were collected (under fasting conditions and 30 min after the loading) for fluorometric determination of Phe and Tyr. Phe, Phe/Tyr and Phe2/Tyr values were higher in heterozygotes, whereas Tyr was higher in controls in both situations investigated. Linear discriminant function was considered to be the best parameter for differentiation of the individuals in the two groups. Under the conditions employed in the present study, aspartame loading did not show any advantages in discriminating between PKU carriers and normal individuals when compared to the same analysis performed under fasting conditions.

Aspartame↗
PubMed1997

Detection of metabolic disorders in high-risk patients. A pilot study in Salvador, Bahia.

The purpose of this pilot-study was to evaluate the applicability of a screening protocol for the detection of inborn errors of metabolism (IEM) in high-risk patients. The protocol was applied in 65 patients referred to the Medical Genetics Laboratory of the University Hospital Professor Edgard Santos due to the suspicion of an IEM. Eight of these patients (12.3%) displayed an abnormal result in the screening protocol. These patients, along with 22 who displayed normal results in the screening protocol but who presented clinical symptoms or signs suggestive of an IEM not detectable by the tests applied, were selected for a further diagnostic investigation. In 5 of these 30 patients (7.7% of the total sample) it was possible to establish the diagnosis of an specific IEM. The results indicate that the designed screening protocol was successfully applied, allowing the detection of affected patients in a frequency comparable to that observed in larger studies performed elsewhere. The continuation of this study and the enlargement of the sample will help to delineate the profile of IEM in northeast of Brazil and will allow the identification of a significative number of patients and families, who could benefit from the therapeutic and preventive measures available for these diseases.

Amino Acids↗
PubMed1997

Regional distribution of cystic fibrosis-linked DNA haplotypes in Brazil: multicenter study.

The restriction fragment length polymorphism (RFLP) haplotypes of cystic fibrosis (CF) alleles vary between populations. To determine the distribution of two RFLPs (XV-2C and KM-19) that are tightly linked to the CF locus, we analyzed a white sample from five different states of Brazil. The haplotypes of 314 CF- and 237 non-CF-bearing chromosomes were uniformly distributed over the five states. The XV-2C allele and haplotype frequencies and the degree of linkage disequilibrium were determined. These were similar to values previously reported in southern European countries but different from results reported for northern and central Europe and North America. In contrast, although KM-19 allele frequencies differed between Brazilian states and European and North American countries, these frequencies were similar to values reported in black Americans. A significant proportion of Brazilian CF-bearing chromosomes had less common haplotypes, suggesting a heterogeneous distribution of CF gene mutations among Brazilians. Further studies are needed to identify the mutations affecting the Brazilian CF patients with various haplotypes.

Adolescent↗
PubMed1997

Cystic fibrosis in the Brazilian population: DF508 mutation and KM-19/XV-2C haplotype distribution.

We have used PCR amplification of DNA obtained from Guthrie cards to identify the DF508 mutation and correlate it with the allele frequencies at two polymorphic loci (XV-2C and KM-19) closely linked to the cystic fibrosis gene. The DNA came from 193 white Brazilian families affected by cystic fibrosis and living in five different states of Brazil. The distribution of the haplotypes derived from the DF508 and non-DF508 XV-2C/KM-19 genotypes indicates that 88% of the DF508 alleles are linked to haplotype B and suggests that high heterogeneity exists among the non-DF508 cystic fibrosis alleles occurring in different states. Our data can be used to compare linkage disequilibrium between Brazilians and other heterogeneous populations where the DF508 mutation frequency is low and where many different rare mutations account for the remaining recessive cystic fibrosis alleles.

Adolescent↗
PubMed1996

Three missense mutations in the galactose-1-phosphate uridyltransferase gene of three families with mild galactosaemia.

UNLABELLED: Classical galactosaemia caused by deficiency of galactose-1-phosphate uridyltransferase (GALT) is characterized by acute symptoms of hepatocellular dysfunction, sepsis, cataracts and failure to thrive. Galactose limitation reverses these complications immediately, however, most of these children have a long-term complication of verbal dyspraxia mental retardation and ovarian failure. The GALT gene was cloned and several mutations including the common Q188R have been reported. In this study the coding region of GALT was amplified by polymerase chain reaction from genomic DNA of classical galactosaemic individuals and characterized by direct sequencing of the products. Three missense mutations were identified in three patients with a mild galactosaemic variant: (1) replacement of threonine-138 by methionine (T138M); (2) replacement of arginine by tryptophan (R259W); and (3) replacement of threonine by alanine (T350A). All three galactosaemic individuals, one girl and two boys, have varying degrees of residual GALT activity in RBC and their galactose-1-phosphate levels decreased much faster than in other galactosaemic patients. These missense mutations occur in regions that are not highly conserved domains. CONCLUSION: The study of the molecular basis related to the phenotype variation may indeed help to prognosticate the outcome of patients with classical galactosaemia.

Amino Acid Sequence↗
PubMed1996

Study on possible increase in twinning rate at a small village in south Brazil.

A high frequency of twin births has been observed in Linha São Pedro, a small settlement which belongs to the city of Cãndido Godói, located 524 km Northwest from Porto Alegre, Rio Grande do Sul, Brazil, in an ethnically homogeneous population of German descent restricted to a small geographic region. From 1990 to 1994, the proportion of twin births in Linha São Pedro was 10%, significantly higher than the 1.8% rate for the state of Rio Grande do Sul as a whole. Genealogical analysis showed a high recurrence of multiple births within families, as well as a high level of inbreeding in the community. Zygosity data indicated that 9 of the 17 pairs of twins studied (53%) were dizygotic. No external environmental factors were detected that could be influencing the appearance of this characteristic. This preliminary investigation confirmed the presumed existence of a high twinning rate in the community. The high familial recurrence and the high inbreeding rate suggests the presence of genetic twinning factors. Complementary studies of twins that have yet to be evaluated and the search for additional risk factors, as well as linkage studies, should contribute to a further understanding of the biological factors related to twin births in the human species.

Adult↗