Janice Carneiro Coelho

Guidelines for the Management of Mucopolysaccharidosis Type I

Mucopolysaccharidosis type I (MPS I) is the prototype of the MPS disorders, a subgroup of lysosomal storage diseases. The incidence of MPS I in Brazil is unknown, but a retrospective population study in Australia conducted between 1980 and 1996 yielded an overall prevalence of 1 in 22 500 for all MPS types. 1 In British Columbia, cases ascertained between 1952 and 1986 determined that the frequency of MPS type I Hurler, the most severe form of the disease, was approximately 1 in 144 000 newborns, 2 a result similar to that found in The Netherlands. 3 A recent analysis of data collected by the Society for Mucopolysaccharides in the United Kingdom in patients with MPS I found a prevalence of 1.07 per 100 000 births. 4 MPS I is characterized by a deficiency in a-L-iduronidase enzyme activity, leading to buildup and urinary excretion of high levels of glycosaminoglycans (GAGs), specifically dermatan and heparan sulfates. The disease is genetically determined and shows autosomal recessive inheritance. 5,6 MPS

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 β‐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. Hum Mutat 13:401–409, 1999.

Oxidative stress in Niemann-Pick type C patients: a protective role of N-butyl-deoxynojirimycin therapy.

Niemann‐Pick type C (NPC) is a rare neurodegenerative disorder biochemically characterized by the accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes of the affected patients. N‐butyl‐deoxynojirimycin is the only approved drug for patients with NPC disease. It inhibits glycosphingolipid synthesis, therefore reducing intracellular lipid storage. Although the mechanisms underlying the neurologic damage in the NPC disease are not yet well established, in vitro and in vivo studies suggest an involvement of reactive species in the pathophysiology of this disease. In this work we aimed to evaluate parameters of lipid and protein oxidation, measured by thiobarbituric acid‐reactive species (TBA‐RS) and protein carbonyl formation, respectively, as well as the enzymatic and non‐enzymatic antioxidant defenses in plasma, erythrocytes and fibroblasts from NPC1 patients, at diagnosis and during treatment with N‐butyl‐deoxynojirimycin. We found a significant increase of TBA‐RS in plasma and fibroblasts, as well as increased protein carbonyl formation and decreased total antioxidant status (TAS) in plasma of untreated NPC1 patients as compared to the control group. In addition, erythrocyte glutathione peroxidase (GSH‐Px) activity was increased, whereas CAT and SOD activities were normal in these patients. We also observed that patients treated with N‐butyl‐deoxynojirimycin normalized plasma TBA‐RS and TAS, as well as erythrocyte GSH‐Px activity. Taken together, the present data indicate that oxidative stress is increased in patients with NPC1 disease and that treatment with N‐butyl‐deoxynojirimycin is able to confer protection against this pathological process.

Clinical and laboratorial study of 19 cases of mucopolysaccharidoses

UNLABELLED The mucopolysaccharidoses (MPS) are a heterogeneous group of inborn errors of lysosomal glycosaminoglycan (GAG) metabolism. The importance of this group of disorders among the inborn errors of metabolism led us to report 19 cases. METHOD We performed clinical, radiological, and biochemical evaluations of the suspected patients, which allowed us to establish a definite diagnosis in 19 cases. RESULTS Not all patients showed increased GAG levels in urine; enzyme assays should be performed in all cases with strong clinical suspicion. The diagnosis was made on average at the age of 48 months, and the 19 MPS cases, after a full clinical, radiological, and biochemical study, were classified as follows: Hurler - MPS I (1 case); Hunter - MPS II (2 cases); Sanfilippo - MPS III (2 cases); Morquio - MPS IV (4 cases); Maroteaux-Lamy - MPS VI (9 cases); and Sly - MPS VII (1 case). DISCUSSION The high relative frequency of Maroteaux-Lamy disease contrasts with most reports in the literature and could express a population variability.

Fibroblasts of skin fragments as a tool for the investigation of genetic diseases: technical recommendations

Skin biopsies are frequently indicated for investigation and/or confirmation of genetic disorders. Although relatively simple a nd noninvasive, these procedures require care in order to increase probability of success and to avoid patient discomfort and unnecessary repea ted analyses and associated laboratory fees. The present report highlights the importance of skin biopsies in genetic disorder diag nosis and presents general rules for collecting, storing, transporting and processing samples. We recommend its reading to professionals intending to use this important and sometimes fundamental diagnostic tool.

Kidney Function and 24-Hour Proteinuria in Patients with Fabry Disease during 36 Months of Agalsidase Alfa Enzyme Replacement Therapy: A Brazilian Experience

Background. Prior to the introduction of enzyme replacement therapy (ERT), management of Fabry disease (FD) consisted of symptomatic and palliative measures. ERT has been available for several years using recombinant human agalsidase alfa, an analogue of alpha-galactosidase A (GALA). However, the limitations of ERT in improving kidney function have not been established. This study evaluates the safety and therapeutic effect of agalsidase alfa replacement in terms of kidney function and reduction in 24-hour proteinuria. Methods. During the period between January 1, 2002, and August 1, 2005, nine Fabry patients (7 male, 2 female) were treated according to protocol, receiving 0.2 mg/kg agalsidase alfa IV every two weeks. Kidney function was evaluated by measuring the glomerular filtration rate (GFR) using chromium ethylene diamine tetra-acetate clearance (51Cr-EDTA mL/min/ 1.73 m2) at baseline, 12, 24, and 36 months. 24-hour proteinuria was measured at baseline, 3, 6, 12, 18, 24, and 36 months of ERT. Kidney disease was classified according to National Kidney Foundation Disease Outcome Quality Initiative (NKF/DOQI) Advisory Board criteria, which define stage I chronic kidney disease (CKD) as GFR ≥ 90mL/min/1.73 m2, stage II as 60–89 mL/min/1.73m2, stage III as 30–59 mL/min/1.73 m2, stage IV as 15–29 mL/min/1.73m2, and stage V as < 15 mL/min/1.73m2. Results. Six patients completed 36 months of therapy, 2 patients completed 18 months, and 1 patient completed 12 months. Mean patient age at baseline was 34.6 ± 11.3 years. During the study period, kidney function remained stable in patients with stages I, II, or III CKD. One patient, who entered the study with stage IV CKD, progressed to end-stage chronic kidney disease, beginning hemodialysis after 7 months and receiving a kidney transplant after 12 months of ERT. Proteinuria also remained stable in the group of patients with pathologic proteinuria. The use of agalsidase alfa was well tolerated in 99.5% of the infusions administered. Conclusion. Over the course of 36 months of ERT, there was no change in kidney function and 24-hour proteinuria. This suggests that agalsidase alfa may slow or halt the progression of kidney disease when used before extensive kidney damage occurs. No significant side effects were observed with ERT during the course of the study.

High frequency of type 1 GM1 gangliosidosis in southern Brazil.

To the Editor: GM1 gangliosidosis (McKusick 230500) is an inborn error of metabolism (IEM) caused by the deficiency of a specific lysosomal hydrolase, bgalactosidase (EC 3.2.1.23). It is considered to be a very rare disease without ethnic preference (1). GM1 gangliosidosis was the lysosomal storage disease (LSD) most frequently diagnosed in a previous survey conducted at the Regional Laboratory of Inborn Errors of Metabolism (RLIEM) of the Medical Genetics Service of University ‘Hospital de Clı́nicas de Porto Alegre’ (2). In that study, most of the cases corresponded to the infantile type and were originated from Rio Grande do Sul (RS), the southern-most state in Brazil, located along the border with Uruguay and Argentina. In the present study, we collected demographic, clinical and laboratory data from all type 1 GM1 gangliosidosis (GM1-1) patients diagnosed between January 1982 and December 1996 at the RLIEM. Laboratory diagnosis was based on bgalactosidase deficiency in leukocytes and/or a typical chromatographic pattern for urinary oligosaccharides (and no increase in sialic acid) in patients with clinical signs and symptoms compatible with GM1-1. To calculate the incidence of the disease, population data from the National Live Born System of the Health Department of Porto Alegre municipality were used. The calculation of the proportion of heterozygotes in the population was based on the Hardy–Weinberg law. The chisquare test was used to compare the defined characteristics. A total of 13000 high-risk patients were investigated at the RLIEM from 1982 to 1996 and 962 cases of IEM were diagnosed. More than half of these cases (493, 51.3%) presented with LSD. GM1 was the condition most frequently diagnosed (73 cases, 14.8% of the total number of IEM cases). Of these 73 cases, 67 were GM1-1, with a predominance of patients from the state of RS (43 cases, 68.2% of the 63 cases for which the information was available). Parental consanguinity was present in 7 of the 56 cases for which this information was obtained, 4 of them being first cousins and 3 first cousins once removed. This frequency (12.5%), although higher than the usual rate for the region (4%) (3), was half that reported for the remaining cases of GM1-1 described in the literature (25%). The incidence of GM1-1 for the Porto Alegre city was estimated at 1:17000 live births. This rate was obtained by considering that 118609 births occurred in this municipality from 1992 to 1996, 7 of which presented with GM1-1, corresponding to 6 cases per 100000 live births for this period in this region. The proportion of GM1-1 heterozygotes in the population of Porto Alegre was calculated to be 1:67 on the basis of the Hardy–Weinberg law. The incidence of GM1-1 was elevated compared to most figures reported in the literature of 1:100000 (4, 5), 1:200000 (6) and 1:320000 (7). As far as we know, a higher incidence of GM1-1 than that observed in the present study was only reported in Malta (1:3700) (8). A significantly higher proportion of patients with GM1-1 was from RS (43/63, 68.2%) than from other Brazilian states. This could be explained, in part, by the fact that the RLIEM is located in the capital of RS and in a reference hospital. However, of the various other cases of LSD whose biochemical diagnosis was also made at the same laboratory, only 34.8% (139/399) were from RS. This difference between GM1-1 and the other LSD is statistically significant (pB0.001). We believe that a considerable number of individuals bearing mutations for GM1-1 may be concentrated in this state, with a consequent larger number of births of affected infants. A result that supports this idea is the estimate of the proportion of carriers for GM1-1 in the population of Porto Alegre (1:67). This proportion is quite high compared to the information available in the literature with respect to other lysosomal diseases in unselected populations, such as Sanfilippo A, 1:290 (9); Mórquio A, 1:233 (10); Niemann–Pick A, 1:200 (11); and Hurler’s syndrome, 1:100 (12). Another

Novel Mutations in the Glucocerebrosidase Gene of Brazilian Patients with Gaucher Disease

Gaucher disease (GD) is an autosomal recessive disorder resulting from glucocerebrosidase (GC) deficiency due to mutations in the gene (GBA) coding for this enzyme. We have developed a strategy for analyzing the entire GBA coding region and applied this strategy to 48 unrelated Brazilian patients with GD. We used long-range PCR, genotyping based on the Taqman® assay, nested PCR, and direct DNA sequencing to define changes in the gene. We report here seven novel mutations that are likely to be harmful: S125N (c.491G>A), F213L (c.756T>G), P245T (c.850C>A), W378C (c.1251G>C), D399H (c.1312G>C), 982-983insTGC (c.980_982dupTGC), and IVS10+1G>T (c.1505+1G>T). The last alteration was found as a complex allele together with a L461P mutation. We also identified 24 different mutations previously reported by others. G377S was the third most frequent mutation among the patients included in this study, after N370S and L444P. Therefore, this mutation needs be included in preliminary screens of Brazilian GD patients. The identification of mutant GBA alleles is crucial for increasing knowledge of the GBA mutation spectrum and for better understanding of the molecular basis of GD.

Influence of pre-analytical factors on α-galactosidase A, arylsulfatase B and α-glucosidase activities measured on dried blood spots on filter paper

OBJECTIVES To analyze the effect of blood collection and storage conditions on activity of α-galactosidase A, arylsulfatase B and α-glucosidase. DESIGN AND METHODS Blood was collected in EDTA, heparin, or direct spotting on filter paper and stored at different temperatures (-20, 4, 25 and 37°C) and storage times (3, 10, 17 and 180 days). The influence of filter paper size was also assessed (3.0 and 1.2mm). RESULTS No statistically significant difference was observed between the three collection methods. α-Glucosidase A activity significantly decreased after the 10th day, while arylsulfatase B activity only differed significantly after the 180th day, and α-galactosidase A activity remained constant throughout this storage time. Excellent correlation coefficients were observed for the two filter paper sizes used. CONCLUSIONS Both paper sizes may be employed. Filter paper specimens should be transported under refrigeration as soon as possible after blood collection.

Detection of organic acidemias in Brazil.

BACKGROUND Organic acidurias or organic acidemias are inherited metabolic disorders in which organic acids (carboxylic acids) accumulate in tissues and physiologic fluids of affected individuals. They are considered the most frequent metabolic disorders among severely ill children. Patients frequently present acute symptoms in early life. Metabolic acidosis and neurologic symptoms are the most common signs. METHODS Urine specimens obtained from 1,926 children from January 1994 to July 2001 were used in analyses. Venous blood specimens were also collected from some patients. Samples were initially submitted to screening tests for detection of inborn errors of metabolism. Identification and semi-quantitation of organic acids in urine were performed by gas chromatography or gas chromatography coupled to mass spectrometry using capillary column (DB-5) and flame ionization detection. RESULTS Ninety three (4.8%) cases of organic acidemias were diagnosed among 1,926 patients investigated from January 1994 to July 2001. Prompt therapy was instituted after diagnosis in a considerable number of patients and resulted in rapid improvement in their symptomatology, distinct from our previous cases diagnosed abroad where patients representing index cases died before any measure could be taken. CONCLUSIONS Results demonstrate the importance of diagnosing organic acidurias in loco in developing countries despite implied extra costs.