A novel mutation in the glycogen synthase 2 gene in a child with glycogen storage disease type 0

Background: Glycogen storage disease type 0 is an autosomal recessive disease presenting in infancy or early childhood and characterized by ketotic hypoglycemia after prolonged fasting and postprandial hyperglycemia and hyperlactatemia. Sixteen different mutations have been identified to date in the gene which encodes hepatic glycogen synthase, resulting in reduction of glycogen storage in the liver. Case Presentation: Biochemical evaluation as well as direct sequencing of exons and exon-intron boundary regions of the GYS2 gene were performed in a patient presenting fasting hypoglycemia and postprandial hyperglycemia and her parents. The patient was found to be compound heterozygous for one previously reported nonsense mutation (c. 736 C>T; R243X) and a novel frameshift mutation (966_967delGA/insC) which introduces a stop codon 21 aminoacids downstream from the site of the mutation that presumably leads to loss of 51% of the COOH-terminal part of the protein. The glycemia and lactatemia of the parents after an oral glucose tolerance test were evaluated to investigate a possible impact of the carrier status on the metabolic profile. The mother, who presented a positive family history of type 2 diabetes, was classified as glucose intolerant and the father, who did not exhibit metabolic changes after the glucose overload, had an antecedent history of hypoglycemia after moderate alcohol ingestion. Conclusion: The current results expand the spectrum of known mutations in GYS2 and suggest that haploinsufficiency could explain metabolic abnormalities in heterozygous carriers in presence of predisposing conditions.

Fabry Disease in Hemodialysis Patients in Southern Brazil: Prevalence Study and Clinical Report

Background. Fabry disease (FD) is a lysosomal storage disorder caused by a deficiency of -Galactosidase A (-Gal A). Fabry nephropathy typically progresses throughout the fifth decade to end-stage renal disease (ESRD), requiring hemodialysis and/or kidney transplantation. Objective. To estimate the prevalence of FD among ESRD males on hemodialysis treatment in Rio Grande do Sul, the southernmost state of Brazil. Methods. Screening for -Gal A activity was performed by a dried blood spot (normal reference value: 1.5 nmoles/hour/mL). Positive screening results were confirmed by plasma -Gal A activity assay (reference value: 3.3 nmoles/hour/mL). Results. Five hundred fifty-eight male patients on hemodialysis were evaluated. Of these, only two had low -Gal A activity and were diagnosed with Fabry disease (0.36%). One of these, age 42, had left ventricular hypertrophy and renal manifestations of Fabry disease without the classic symptoms. The other, age 46, had the classical manifestations of angiokeratomas, acroparesthesias, hypohidrosis, and ocular opacities. Conclusions. Although the prevalence of Fabry disease was very low in our study (0.36%), routine screening of male hemodialysis patients would enable earlier identification of many other affected relatives in their families who might benefit from specific clinical treatment.

Mucopolysaccharidoses in Brazil: What happens from birth to biochemical diagnosis?

Mucopolysaccharidoses (MPS) form a group of inherited metabolic disorders characterized by intralysosomal storage of glycosaminoglycans. This study aimed to investigate the path followed by Brazilian patients from birth to diagnosis. An interview was conducted with patient`s parents or guardians with subsequent review of patient`s medical records. One hundred thirteen patients with MPS were included (MPS I: 18, MPS II: 43, MPS III: 2, MPS IIIB: 3, MPS IIIC: 1, MPS IVA: 15, MPS IVB: 1, MPS VI: 29, MPS VII: 1) from 97 families. Median age at the onset of sings/symptoms was 18 months (MPS I: 18, MPS II: 24, MPS IVA: 8, MPS IV: 8). Skeletal abnormalities (for MPS IVA and MPS VI), joint contracures (for MPS II), and typically first signs/symptoms. Several health professionals were involved in patient`s care as of the onset of symptoms until biochemical diagnosis was established. Median age at diagnosis was 76 months (MPS I: 75, MPS II: 95, MPS IVA: 75, MPS VI: 52). Considering the group as a whole, there was a 4.8-year delay between the onset of signs/symptoms and establishment of the diagnosis. Considering that specific therapies are available for some of these disorders and that early treatment is likely to change more favorably the natural history of the disease, efforts should be made to minimize this delay. We believe that this situation can be improved by measures that both increase awareness of health professionals about MPS and improve access to diagnostic tests. (C) 2008 Wiley-Liss, Inc.

Pompe disease in a Brazilian series: clinical and molecular analyses with identification of nine new mutations

Pompe disease (glycogen storage disease type II or acid maltase deficiency) is an inherited autosomal recessive deficiency of acid alpha-glucosidase (GAA), with predominant manifestations of skeletal muscle weakness. A broad range of studies have been published focusing on Pompe patients from different countries, but none from Brazil. We investigated 41 patients with either infantile-onset (21 cases) or late-onset (20 cases) disease by muscle pathology, enzyme activity and GAA gene mutation screening. Molecular analyses identified 71 mutant alleles from the probands, nine of which are novel (five missense mutations c.136T > G, c.650C > T, c.1456G > C, c.1834C > T, and c.1905C > A, a splice-site mutation c.1195-2A > G, two deletions c.18_25del and c.2185delC, and one nonsense mutation c.643G > T). Interestingly, the c.1905C > A variant was detected in four unrelated patients and may represent a common Brazilian Pompe mutation. The c.2560C > T severe mutation was frequent in our population suggesting a high prevalence in Brazil. Also, eight out of the 21 infantile-onset patients have two truncating mutations predicted to abrogate protein expression. Of the ten late-onset patients who do not carry the common late-onset intronic mutation c.-32-13T > G, five (from three separate families) carry the recently described intronic mutation, c.-32-3C > A, and one sibpair carries the novel missense mutation c.1781G > C in combination with known severe mutation c.1941C > G. The association of these variants (c.1781G > C and c.-32-3C > A) with late-onset disease suggests that they allow for some residual activity in these patients. Our findings help to characterize Pompe disease in Brazil and support the need for additional studies to define the wide clinical and pathological spectrum observed in this disease.

Generation of Polyclonal Antibodies Against Recombinant Human Glucocerebrosidase Produced in Escherichia coli

Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher`s disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59-66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.

Echocardiographic study of paediatric patients with mucopolysaccharidosis

Principle Mucopolysaccharidosis is an inborn error of metabolism causing glucosaminoglycans tissue storage. Cardiovascular involvement is variable but contributes significantly towards the morbidity and mortality of the patients. Objective To characterise the echocardiographic abnormalities in children and adolescents with different types of mucopolysaccharidosis. Method Echocardiograms and medical records of 28 patients aged 2–14 years, seen from 2003 to 2005, were revised. At that time, the enzymatic replacement therapy was still not available in our institution.Results Echocardiographic alterations were detected in 26 patients (93 per cent), whereas 16 (57 per cent) had abnormal auscultation, and only 6 (21 per cent) presented with cardiovascular complaint. Mitral valve thickening with dysfunction (regurgitation, stenosis, or double lesion) was diagnosed in 60.8 per cent, left ventricular hypertrophy in 43 per cent and aortic valve thickening with regurgitation in 35.8 per cent of the patients. There was no systolic dysfunction and mild left diastolic dysfunction was shown in 21.5 per cent of the patients. Pulmonary hypertension was present in 36 per cent of the patients, causing the only two deaths recorded. There was a strong association between the accumulation of dermatan sulphate and the presence of mitral valve dysfunction (p = 0.0003), aortic valve dysfunction (p = 0.006), and pulmonary hypertension (p = 0.006). Among individuals with two or more examinations, 82 per cent had a worsening evolution. Conclusions Echocardiographic alterations in children with Mucopolysaccharidosis are frequent and have a progressive character Left valve lesions, ventricular hypertrophy, and pulmonary hypertension were the most common findings and there was an association between the accumulation of dermatan sulphate and cardiovascular involvement. Unlike in adults, pulmonary hypertension was the main cause of death, not left ventricle systolic dysfunction

MR Spectroscopy Detects Lipid Peaks in Cerebrotendinous Xanthomatosis

CTX is a rare lipid-storage disease. Novel MRS findings from 3 patients, using a short TE, were the presence of lipid peaks at 0.9 and 1.3 ppm in the depth of the cerebellar hemisphere; this might represent an additional marker of disease that is CNS-specific and noninvasive. A decrease in NAA concentration was also detected and attributed to neuroaxonal damage. One patient presented an increase in mlns concentration, pointing to gliosis and astrocytic proliferation.

The p.A2215D Thyroglobulin Gene Mutation Leads to Deficient Synthesis and Secretion of the Mutated Protein and Congenital Hypothyroidism with Wide Phenotype Variation

Context: Thyroglobulin (TG) is a large glycoprotein and functions as a matrix for thyroid hormone synthesis. TG gene mutations give rise to goitrous congenital hypothyroidism (CH) with considerable phenotype variation. Objectives: The aim of the study was to report the genetic screening of 15 patients with CH due to TG gene mutations and to perform functional analysis of the p. A2215D mutation. Design: Clinical evaluation and DNA sequencing of the TG gene were performed in all patients. TG expression was analyzed in the goitrous tissue of one patient. Human cells were transfected with expression vectors containing mutated and wild-type human TG cDNA. Results: All patients had an absent rise of serum TG after stimulation with recombinant human TSH. Sequence analysis revealed three previously described mutations (p. A2215D, p. R277X, and g. IVS30 + 1G > T), and two novel mutations (p. Q2142X and g. IVS46-1G > A). Two known (g. IVS30 + 1G/p. A2215D and p. A2215D/p. R277X) and one novel (p. R277X/g. IVS46-1G > A) compound heterozygous constellations were also identified. Functional analysis indicated deficiency in TG synthesis, reduction of TG secretion, and retention of the mutant TG within the cell, leading to an endoplasmic reticulum storage disease, whereas small amounts of mutant TG were still secreted within the cell system. Conclusion: All studied patients were either homozygous or heterozygous for TG gene mutations. Two novel mutations have been detected, and we show that TG mutation p. A2215D promotes the retention of TG within the endoplasmic reticulum and reduces TG synthesis and secretion, causing mild hypothyroidism. In the presence of sufficient iodine supply, some patients with TG mutations are able to compensate the impaired hormonogenesis and generate thyroid hormone. (J Clin Endocrinol Metab 94: 2938-2944, 2009)

Updated Clinical Classification of Pulmonary Hypertension

The aim of a clinical classification of pulmonary hypertension (PH) is to group together different manifestations of disease sharing similarities in pathophysiologic mechanisms, clinical presentation, and therapeutic approaches. In 2003, during the 3rd World Symposium on Pulmonary Hypertension, the clinical classification of PH initially adopted in 1998 during the 2nd World Symposium was slightly modified. During the 4th World Symposium held in 2008, it was decided to maintain the general architecture and philosophy of the previous clinical classifications. The modifications adopted during this meeting principally concern Group 1, pulmonary arterial hypertension (PAH). This subgroup includes patients with PAH with a family history or patients with idiopathic PAH with germline mutations (e. g., bone morphogenetic protein receptor-2, activin receptor-like kinase type 1, and endoglin). In the new classification, schistosomiasis and chronic hemolytic anemia appear as separate entities in the subgroup of PAH associated with identified diseases. Finally, it was decided to place pulmonary venoocclusive disease and pulmonary capillary hemangiomatosis in a separate group, distinct from but very close to Group 1 (now called Group 1`). Thus, Group 1 of PAH is now more homogeneous. (J Am Coll Cardiol 2009;54:S43-54) (C) 2009 by the American College of Cardiology Foundation

Hepatic Hemosiderosis in Red-Spectacled Amazons (Amazona pretrei) and Correlation with Nutritional Aspects

Alimentary habits of free-living Psittaciformes vary significantly among different species. Amazona pretrei is under risk of extinction and has very specific free-living dietary habits, which are based on Parana pine seeds. Hemosiderosis is a pathologic process characterized by intracellular accumulation of iron without other evident lesions. It is associated with increased prevalence of infections, neoplasms, and hepatopathies. The purpose of this study was to quantify hepatic hemosiderin deposits in captive A. pretrei and verify their association with nutritional parameters. Liver samples were processed for histopathology and stained with Prussian blue. The sections were analyzed by computerized morphometry to quantify the hepatic hemosiderin deposits. The hepatic hemosiderosis rates showed positive correlation with age and time in captivity. These results suggest that the menus and commercial rations for Psittacidae must be carefully revised.

Precursor system of liquid crystalline phase containing propolis microparticles for the treatment of periodontal disease: Development and characterization

Precursor systems of liquid crystalline phase were prepared using the surfactant PPG-5-Ceteth-20, isopropyl myristate, and water; gelatin microparticles containing propolis were then added into these systems. Homogeneity of dispersion, the in-system microparticle morphology, and sedimentation behavior of each formulation were evaluated. The rheological and mechanical properties (hardness, compressibility, and adhesiveness), the work of syringing, and the propolis release profile were also evaluated. All the formulations exhibited pseudoplastic flow and thixotropy, and they displayed storage modulus, loss modulus, dynamic viscosity, and loss tangent that depended on temperature, frequency, and composition. Mechanical properties varied significantly among the formulations being affected by changes in the composition and temperature. Raising the concentration of surfactant and adding propolis microparticles significantly decreased the work of syringing. The drug release was non-Fickian (anomalous) and there was no significant difference between the tested systems in the times required for 10%, 30%, and 50% release of the initial drug loading.

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 ((51)Cr-EDTA mL/min/1.73 m(2)) 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 m(2), stage II as 60-89 mL/min/1.73m(2), stage III as 30-59 mL/min/1.73 m(2), stage IV as 15-29 mL/min/1.73m(2), and stage V as < 15 mL/min/1.73m(2). 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 thatagalsidase 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.

Increase of Cholesterol Oxidation and Decrease of PUFA as a Result of Thermal Processing and Storage in Eggs Enriched with n-3 Fatty Acids

In this work, cholesterol oxide formation and alteration of fatty acid composition were analyzed in n-3 enriched eggs under different storage periods and two temperatures. The eggs enriched with n-3 fatty acids were stored at 5 or 25 degrees C for 45 days and subsequently boiled or fried. For each treatment, 12 yolks were analyzed every 15 days including time zero. The concentrations of the cholesterol oxides 7-ketocholesterol, 7 beta-hydroxycholesterol, and 7 alpha-hydroxycholesterol increased during the storage period and were higher in fried eggs. Only the 7-ketocholesterol was affected by the storage temperature, and its concentration was highest in eggs stored at 25 degrees C. There was no significant difference in the contents of cholesterol and vitamin E at the different storage periods; however, the concentration of vitamin E decreased with thermal treatment. In addition, the n-3 polyunsaturated fatty acids, especially 18:3, 20:5, and 22:6, were reduced throughout the storage at 5 and 25 degrees C.

Knowledge and attitude of parents or caretakers regarding transmissibility os caries disease

Dental caries is a transmissible infectious disease in which mutans streptococci are generally considered to be the main etiological agents. Although the transmissibility of dental caries is relatively well established in the literature, little is known whether information regarding this issue is correctly provided to the population. The present study aimed at evaluating, by means of a questionnaire, the knowledge and usual attitude of 640 parents and caretakers regarding the transmissibility of caries disease. Most interviewed adults did not know the concept of dental caries being an infectious and transmissible disease, and reported the habit of blowing and tasting food, sharing utensils and kissing the children on their mouth. 372 (58.1%) adults reported that their children had already been seen by a dentist, 264 (41.3%) answered that their children had never gone to a dentist, and 4 (0.6%) did not know. When the adults were asked whether their children had already had dental caries, 107 (16.7%) answered yes, 489 (76.4%) answered no, and 44 (6.9%) did not know. Taken together, these data reinforce the need to provide the population with some important information regarding the transmission of dental caries in order to facilitate a more comprehensive approach towards the prevention of the disease.

Effect of cement type and water storage time on the push-out bond strength of a glass fiber post

This study investigated the effects of the cement type and the water storage time on the push-out bond strength of a glass fiber post. Glass fiber posts (Fibrekor, Jeneric Pentron) were luted to post spaces using a self-cured resin cement (C&B Cement [CB]), a glass ionomer cement (Ketac Cem [KC]) or a resin-modified glass ionomer cement (GC FujiCEM [FC]) according to the manufacturers’ instructions. For each luting agent, the specimens were exposed to one of the following water storage times (n=5): 1 day (T1), 7 days (T7), 90 days (T90) and 180 days (T180). Push-out tests were performed after the storage times. Control specimens were not exposed to water storage, but subjected to the push-out test 10 min after post cementation. Data (in MPa) were analyzed by Kruskal-Wallis and Dunn`s test (α=0.05). Cement type and water storage time had a significant effect (p<0.05) on the push-out bond strength. CB showed significantly higher values of retention (p<0.05) than KC and FC, irrespective of the water storage time. Water storage increased significantly the push-out bond strength in T7 and T90, regardless of the cement type (p<0.05). The results showed that fiber posts luted to post spaces with the self-cured resin cement exhibited the best bonding performance throughout the 180-day water storage period. All cements exhibited a tendency to increase the bond strength after 7 and 90 days of water storage, decreasing thereafter.