In vitro effect of genistein on DNA damage in leukocytes from mucopolysaccharidosis IVA patients.

Mucopolysaccharidosis IVA is a lysosomal storage disorder leading to an increase in glycosaminoglycans storage. Genistein is an isoflavone capable to inhibit glycosaminoglycans production. The objective of this study was to analyze the in vitro effect of different concentrations of genistein on DNA injury in mucopolysaccharidosis IVA patients. The lower concentration tested (10 µM) showed a significant increase on DNA injury in vitro, although higher concentrations (30 µM and 50 µM) showed higher DNA damage.

Assay of heparan-N-sulfamidase in dried leukocytes impregnated in filter paper: a new tool for the identification of mucopolisaccharidosis IIIA and potentially other lysosomal disorders.

Diagnosis of lysosomal storage disorders (LSDs) is mainly based on specific enzyme assays in leucocytes. Dried blood spots have also been used as sample for the enzyme assays. However, some lysosomal enzymes such as heparan-N-sulfamidase (HNS) and others cannot be assayed by this material. We developed an assay for HNS using dried leukocytes impregnated in filter paper (DLFP) as source of enzyme, and the results allowed the correct identification of Mucopolisaccharidosis IIIA. From this proof of concept we predict that the assay of lysosomal enzymes in DLFP samples, which still needs further development, could be a useful tool for the diagnosis of LSDs, especially in regions where transportation of liquid blood samples in appropriate conditions for long distances and/or across country borders is challenging.

Expert recommendations for the laboratory diagnosis of MPS VI.

Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disease caused by a deficiency of N-acetylgalactosamine 4-sulfatase (arylsulfatase B, ASB). This enzyme is required for the degradation of dermatan sulfate. In its absence, dermatan sulfate accumulates in cells and is excreted in large quantities in urine. Specific therapeutic intervention is available; however, accurate and timely diagnosis is crucial for maximal benefit. To better understand the current practices for diagnosis and to establish diagnostic guidelines, an international MPS VI laboratory diagnostics scientific summit was held in February of 2011 in Miami, Florida. The various steps in the diagnosis of MPS VI were discussed including urinary glycosaminoglycan (uGAG) analysis, enzyme activity analysis, and molecular analysis. The following conclusions were reached. Dilute urine samples pose a significant problem for uGAG analysis and MPS VI patients can be missed by quantitative uGAG testing alone as dermatan sulfate may not always be excreted in large quantities. Enzyme activity analysis is universally acknowledged as a key component of diagnosis; however, several caveats must be considered and the appropriate use of reference enzymes is essential. Molecular analysis supports enzyme activity test results and is essential for carrier testing, subsequent genetic counseling, and prenatal testing. Overall the expert panel recommends caution in the use of uGAG screening alone to rule out or confirm the diagnosis of MPS VI and acknowledges enzyme activity analysis as a critical component of diagnosis. Measurement of another sulfatase enzyme to exclude multiple sulfatase deficiency was recommended prior to the initiation of therapy. When feasible, the use of molecular testing as part of the diagnosis is encouraged. A diagnostic algorithm for MPS VI is provided.

[Critical reflections concerning euthanasia for persons with dementia]. / Réflexions critiques à propos de l'euthanasie de personnes atteintes de démence.

In the public debate on the extension of euthanasia for people with dementia, in addition to ethical considerations and arguments, other issues have to be kept in mind. The diagnosis of dementia is difficult and the clinical picture is very fluctuating. The assessment and especially the operationalization of legal capacity and the use of advance directives are complex problems. The discussion should be conducted against the backdrop of a cultural framework in which the interpretation and development of palliative care is crucial. The development of a framework like advance care planning creates opportunities. The question remains whether the legal issues can be clarified and whether a legal approach generates solutions for the problems described.

Clinical and biochemical studies in mucopolysaccharidosis type II carriers.

The aim of the study was to characterize clinically and biochemically mucopolysaccharidosis type II (MPS II) heterozygotes. Fifty-two women at risk to be a carrier, with a mean age of 34.1 years (range 16-57 years), were evaluated through pedigree analysis, medical history, physical examination, measurement of iduronate sulfatase (IDS) activities in plasma and in leukocytes, quantification of glycosaminoglycans (GAGs) in urine, and analysis of the IDS gene. Eligibility criteria for the study also included being 16 years of age or older and being enrolled in a genetic counselling programme. The pedigree and DNA analyses allowed the identification of 40/52 carriers and 12/52 non-carriers. All women evaluated were clinically healthy, and their levels of urinary GAGs were within normal limits. Median plasma and leukocyte IDS activities found among carriers were significantly lower than the values found for non-carriers; there was, however, an overlap between carriers' and non-carriers' values. Our data suggests that MPS II carriers show lower plasma and leukocyte IDS activities but that this reduction is generally associated neither with changes in levels of urinary GAGs nor with the occurrence of clinical manifestations.

Turbulence and jet-driven zonal flows: Secondary circulation in rotating fluids due to asymmetric forcing.

We report on experiments and modeling on a rotating confined liquid that is forced by circumferential jets coaxial with the rotation axis, wherein system-scale secondary flows are observed to emerge. The jets are evenly divided in number between inlets and outlets and have zero net mass transport. For low forcing strengths the sign of this flow depends on the sign of a sloped end cap, which simulates a planetary ß plane. For increased forcing strengths the secondary flow direction is insensitive to the slope sign, and instead appears to be dominated by an asymmetry in the forcing mechanism, namely, the difference in radial divergence between the inlet and outlet jet profiles. This asymmetry yields a net radial velocity that is affected by the Coriolis force, inducing secondary zonal flow.

Epstein-Barr virus-induced transformation of B cells for the diagnosis of genetic metabolic disorders--enumerative conditions for cryopreservation.

Epstein-Barr virus (EBV) infection in vitro causes transformation of B cells and generates B lymphoblastoid cell lines (LCLs). These LCLs have been widely used for the diagnostic of several genetic metabolic disorders. However, up to now, efficiency of LCL generation has been based on misleading subjective analysis. In this study, quantitative analyses have been performed to indicate efficiency of B-cell transformation to measuring human lysosomal acid hydrolases associated with: GM1-gangliosidosis type I, Gaucher disease and mucopolysaccharidosis type I. Peripheral blood mononuclear cells were isolated from 13 subjects, and LCLs were produced by culturing them with EBV for 12 days. Activities of the enzymes beta-galactosidase, beta-glucosidase and alpha-iduronidase were measured before and after cryopreservation in liquid nitrogen for 30 days. Efficiency of the B-cell transformation was screened every 4 days by the enumeration of cell proliferation, cell counts and changes in granularity estimated by flow cytometry. We observed the generation of 13 LCLs. Cell transformation was confirmed by the gradual increase of cellular clusters, cell size and granularity. In addition, we determined that the activity of the enzymes mentioned above did not change following cryopreservation. These data suggest that our enumerative approach for screening of EBV-LCLs is efficient for the enzymatic determination of human lysosomal acid hydrolases and may thus replace misleading subjective analyses.

Diagnosis and molecular characterization of non-classic forms of Tay-Sachs disease in Brazil.

Molecular analysis of five Brazilian families, including eight patients presenting with nonclassic Tay-Sachs disease, was performed to identify frequent causative mutations and their correlation with clinical course. Three patients were affected by the B1 subacute variant and were shown to carry the R178H mutation (the DN allele), which is also common among Portuguese patients. Two of them were compound heterozygotes, whereas the third presented with the mutation in both alleles. Since Brazil was a Portuguese colony for over two centuries, common ancestry might be the probable explanation. The fourth patient presented with a juvenile phenotype and carries the R499H mutation, which has been reported only once worldwide and is associated with residual enzyme activity, responsible for a slower clinical course. The fifth family, of an Ashkenazi Jewish background, showed an extensive intrafamilial clinical variability among three affected sibs presenting with muscle atrophy, ataxia, and psychiatric symptoms. They were first diagnosed as having atypical spinal muscular atrophy and, subsequently, spinocerebellar ataxia, but, recently, the diagnosis of late-onset Tay-Sachs disease was confirmed based on reduced plasma hexosaminidase A activity and the G269S/InsTATC1278 genotype. It is therefore highly recommended to test patients with a similar clinical history for Tay-Sachs disease. In the same family, one first cousin committed suicide at the age of 24 years, presenting with a clinical phenotype that suggested an undiagnosed case and highlighting the effect of the intrafamilial clinical variability in delaying a prompt diagnosis. It is now recognized that his parents are, in fact, a carrier couple. Additionally, another relative had been previously identified as a heterozygote in a Tay-Sachs disease screening program, but the information was not shared among the family. Since this information might anticipate diagnosis and genetic counseling, it is advisable that heterozygote screening programs encourage families to share genetic information.

Features of spectral properties of Sm(3+) complexes with dithia- and diselenophosphinate ligands.

The samarium complexes Sm(S2PPh2)3(THF)2 (1) and Sm(Se2PPh2)3(THF)2 (2) with soft-donor dithia- and diselenophosphinate ligands were synthesized and their photophysical properties were studied in detail. Both complexes displayed the metal-centered photoluminescence (PL) in visible and NIR regions corresponding to (4)G5/2→(6)HJ (J=5/2, 7/2, 9/2, 11/2, 13/2, 15/2), (6)FJ (J=1/2, 3/2, 5/2, 7/2, 9/2, 11/2) f-f transitions of Sm(3+). Luminescence decay curves exhibit an initial short build-up region and can be described by double or triple exponential function owing to multiphonon relaxation from the (4)F3/2 energy level to the (4)G5/2 one and reversible energy transfer from the Sm(3+) excited states to the triplet ((3)T1) state of phosphinate ligand. A Judd-Ofelt analysis was performed to estimate PL quantum efficiency (QE), branching ratios (ß) and induced-emission cross section (σem) of the compounds obtained. It was found that the Judd-Ofelt parameter Ω2 of 1 is significantly greater than that of 2. This feature is responsible for large values of ß (50.98%) and σem (4.29×10(-21)cm(2)) which suggest 1 as a good candidate for the development of samarium doped polymethylmethacrylate (PMMA) laser medium acting on the (4)G5/2→(6)H9/2 transition at 645nm. The estimated room-temperature PL QE of 1 and 2 equals to 1.9 and 0.17%, respectively.

Synthesis, structure and luminescent properties of lanthanide fluoroalkoxides.

Alkoxides [Ln(OR)3(DME)]2 (R = CH(CF3)2, Ln = Sm (1), Yb (2)), [Ce(OR)3(Phen)]2 (3) (Phen = 1,10-phenanthroline), [Ce(OR')3(DME)2]2 (R' = C(CF3)3) (4), {Gd(OR')3(DME)2} (5), {Ln2[O(CF3)2C­C(CF3)2O]3} (Ln = Ce (6), Gd (7)), {Ce2[O(CF3)2C­C(CF3)2O]3(Phen)2} (8), and {Ce[O(CF3)2C­C(CF3)2O][O(CF3)2­C(CF3)2OH](Phen)2} (9) were synthesized by the reactions of silylamides Ln[N(SiMe3)2]3 with respective fluorinated alcohols. The heterovalent trinuclear complex {Sm2(µ2-OR)3(µ3-OR)2Sm(OR)2(THF)2.5(Et2O)0.5} (10) was obtained by treatment of SmI2(THF)2 with ROK. The reaction of europium(II) and yttrium(III) silylamides with ROH afforded the heterobimetallic alkoxide {Eu2(µ2-OR)3(µ3-OR)2Y(OR)2(DME)2} (11) containing divalent europium. The molecular structures of 1, 2, 3, 9, 10 and 11 were determined by X-ray analysis. All the prepared cerium derivatives as well as the europium­yttrium isopropoxide upon UV excitation exhibited photoluminescence in the regions of 370­425 (for Ce3+) and 485 nm (for Eu2+) which was assigned to 4d→5f transitions.

Galactocerebrosidase assay on dried-leukocytes impregnated in filter paper for the detection of Krabbe disease.

BACKGROUND: Krabbe disease (KD) is an inherited lysosomal storage disease (LSD) caused by the deficiency of galactocerebrosidase (GALC) and is characterized by a severe and progressive leukodystrophy with death frequently before one year of life in the classical early-onset form. As a consequence of the enzyme defect, globoid cells containing undigested galactosylceramide are observed and are characteristic of the disease. Hematopoietic stem cell transplantation is the current treatment for this disease, with some success in the classical cases if performed very early in life. Definitive diagnosis of KD is generally accessed by determination of GALC in leukocytes or fibroblasts. For the last few years, dried-blood filter paper (DBFP) samples have been increasingly used for lysosomal enzyme assays. Originally, some lysosomal enzymes could not be tested in DBFP samples using fluorometric assays, including GALC, heparan-sulfamidase and a few others. Recently, we reported successful results using dried-leukocytes filter paper (DLFP) samples for heparan sulfamidase and ß-galactosidase. Extending these studies, we present now a new GALC assay on these type of samples. METHODS: Adapted leukocyte fluorometric assay was used for the evaluation of GALC in DLFP samples. RESULTS: Our results using this method showed a clear discrimination between GALC levels observed in KD patients and healthy controls. CONCLUSIONS: The assay is robust and reliable and could be adopted by reference laboratories for diagnosis of LSDs. It is expected that the use of DLPF would make it possible to diagnose patients living in isolated areas, where liquid samples usually have to be transported over several days and sometimes across country borders before reaching reference laboratories.

Extended use of dried-leukocytes impregnated in filter paper samples for detection of Pompe, Gaucher, and Morquio A diseases.

BACKGROUND: Lysosomal storage diseases (LSD) are a group of genetic conditions which could present a vast spectrum of abnormalities that may include skeletal abnormalities, organ dysfunction, neuronal involvement, and tissue accumulation of complex molecules, among other manifestations. Definitive diagnosis of LSD is generally obtained by specific enzyme assays performed in leukocytes, fibroblasts, or more recently, dried-blood filter paper (DBFP) samples. METHODS: We recently introduced dried-leukocytes filter paper (DLFP) as an alternative source of enzyme to assay heparan sulfamidase and galactocerebrosidase activities, which could not be measured in DBFP samples using fluorometric methods. We present a new fluorometric methods on DLFP samples, for evaluation of α-glucosidase (GAA), ß-glucosidase (GBA), and N-acetylgalactosamine-6-sulfatase (GALNS) activities, key enzyme assays for the identification of patients with Pompe disease (PD), Gaucher disease (GD), and Morquio A disease (MD), respectively. RESULTS: We show a clear discrimination between confirmed PD, GD, and MD patients and healthy controls. CONCLUSIONS: We conclude that the assays of GAA, GBA, and GALNS on DLFP are reliable and useful methods for the identification of PD, GD, and MD diseases, respectively. As sample preparation is feasible in standard biochemical laboratories and transportation is very simple, it could enable patients living in remote areas to be investigated, diagnosed and eventually treated with the specific therapies available for these diseases.

Genotypic characterization of Brazilian patients with infantile and juvenile forms of metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder inherited as an autosomal recessive trait. MLD is caused by the deficiency of arylsulfatase A (ARSA), a lysosomal enzyme that catalyzes the first step in the degradation of sulfated glycolipids, which are essential components of the myelin sheet. Notably, between 7% and 15% of healthy individuals show in vitro deficiency of ARSA, a condition called ARSA pseudodeficiency (ARSA-PD). To date, 151 ARSA-MLD mutations have been reported in the gene encoding ARSA (ARSA), among which IVS2+1G>A and P426L occur at high frequencies in most of the studied populations. The aim of this work was to identify ARSA mutant alleles in a cohort of 27 unrelated Brazilian MLD patients. The most frequent ARSA-MLD mutation, IVS2+1G>A, and the ARSA-PD polymorphisms, N350S and 1524+95A>G, were detected using real-time PCR, while the remaining mutations were detected using direct sequencing of ARSA. In concordance with previous reports, IVS2+1G>A and P426L were the most common ARSA-MLD mutations in our cohort of MLD patients, found at frequencies of 0.05 and 0.08, respectively. Interestingly, two mutations previously reported as rare, 103_110del8 and 1190_1191insC, were found at higher frequencies in our cohort of MLD patients, 0.08 and 0.06, respectively. Additionally, 11 other rare ARSA-MLD mutations were found at lower frequencies in our cohort of MLD patients. To our knowledge, this is the first systematic genotypic characterization of MLD patients from Latin America. This work highlights the genetic heterogeneity of MLD, and supports genotype-phenotype associations, which become more important as specific treatments are being developed for this devastating disorder.

Exome sequencing for mucolipidosis III: Detection of a novel GNPTAB gene mutation in a patient with a very mild phenotype.

Mucolipidosis II and III alpha/beta (ML II/III alpha/beta) are rare autosomal recessive lysosomal storage diseases that are caused by a deficiency of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the enzyme responsible for the synthesis of the mannose 6-phosphate targeting signal on lysosomal hydrolases. A Brazilian patient suspected of having a very mild ML III was investigated using whole next-generation sequencing (NGS). Two mutations in the GNPTAB gene were detected and confirmed to be in trans status by parental analysis: c.1208T>C (p.Ile403Thr), previously reported as being pathogenic, and the novel mutation c.1723G>A (p.Gly575Arg). This study demonstrates the effectiveness of using whole NGS for the molecular diagnosis of very mild ML III alpha/beta patients.

Protracted course of Krabbe disease in an adult patient bearing a novel mutation.

BACKGROUND: Krabbe disease, or globoid cell leukodystrophy, is an autosomal recessive disorder caused by the deficiency of galactocerebrosidase (GALC) activity. Although most cases are diagnosed in infancy and show a fatal outcome in childhood, adult patients have been identified, showing progressive spastic hemiparesis to tetraparesis, followed by optic atrophy, dementia, and neuropathy. The disease can be diagnosed by detecting the deficiency of GALC activity (less than 5% of normal) in any available tissue sample. The cloning of the human GALC gene allowed the molecular characterization of newly diagnosed patients. More than 75 disease-causing mutations and polymorphisms in this gene have been identified. OBJECTIVE: To describe a 28-year-old woman with Krabbe disease, correlating clinical and biochemical abnormalities to a novel mutation on the GALC gene. METHODS: Clinical investigation was enriched by neurophysiological and neuroimaging data. The activity of GALC was assayed in white blood cells using radiolabeled natural substrate. Genomic DNA was isolated from peripheral blood, and the GALC gene was sequenced. The mutated gene was expressed and GALC activity was measured in transfected COS-1 cells. RESULTS: The patient had progressive and bilateral amaurosis starting at 8 years of age. Although she was experiencing weakness in all her extremities, her intellect remained intact. She was found to be homozygous for a previously unreported missense mutation (T1886G), which leads to low, but not totally deficient, GALC activity. CONCLUSIONS: Expression of this mutation in COS-1 cells using the pcDNA3 expression vector (Invitrogen, Carlsbad, Calif) resulted in low, although not null, GALC activity, which can explain the protracted clinical course in this patient. Patients carrying the mutation described herein might be potential candidates for therapeutic trials, such as bone marrow transplantation or gene therapy.

Effect of collection, transport, processing and storage of blood specimens on the activity of lysosomal enzymes in plasma and leukocytes.

This study was designed to evaluate the effect of different conditions of collection, transport and storage on the quality of blood samples from normal individuals in terms of the activity of the enzymes ss-glucuronidase, total hexosaminidase, hexosaminidase A, arylsulfatase A and ss-galactosidase. The enzyme activities were not affected by the different materials used for collection (plastic syringes or vacuum glass tubes). In the evaluation of different heparin concentrations (10% heparin, 5% heparin, and heparinized syringe) in the syringes, it was observed that higher doses resulted in an increase of at least 1-fold in the activities of ss-galactosidase, total hexosaminidase and hexosaminidase A in leukocytes, and ss-glucuronidase in plasma. When the effects of time and means of transportation were studied, samples that had been kept at room temperature showed higher deterioration with time (72 and 96 h) before processing, and in this case it was impossible to isolate leukocytes from most samples. Comparison of heparin and acid citrate-dextrose (ACD) as anticoagulants revealed that ss-glucuronidase and hexosaminidase activities in plasma reached levels near the lower normal limits when ACD was used. In conclusion, we observed that heparin should be used as the preferable anticoagulant when measuring these lysosomal enzyme activities, and we recommend that, when transport time is more than 24 h, samples should be shipped by air in a styrofoam box containing wet ice.

[Diagnosis and treatment of nerve injuries in fractures and bone diseases in children]. / Diagnostika i lechenie povrezhdenii nervov pro perelomakh i zabolevaniiakh kostei u detei.

The author observed 78 children with injuries to the nerves. In 49 patients the injury was due to fractures or bone diseases. In 29 patients the nerve injuries were associated with the bone surgery (osteosynthesis, open reposition of fragments etc.) Peculiarities of the diagnosis and treatment of nerve injuries in children are described.

Extended use of a selective inhibitor of acid lipase for the diagnosis of Wolman disease and cholesteryl ester storage disease.

Lysosomal acid lipase (LAL) deficiency produces two well defined inborn disorders, Wolman disease (WD) and cholesteryl ester storage disease (CESD). WD is a severe, early-onset condition involving massive storage of triglycerides and cholesteryl esters in the liver, with death usually occurring before one year of life. CESD is a more attenuated, later-onset disease that leads to a progressive and variable liver dysfunction. Diagnosis of LAL deficiency is mainly based on the enzyme assay of LAL activity in fibroblasts. Recently, a selective acid lipase inhibitor was used for the determination of enzyme activity in dried-blood filter paper (DBFP) samples. To extend and to validate these studies, we tested LAL activity with selective inhibition on DBFP samples, leukocytes and fibroblasts. Our results showed a clear discrimination between patients with LAL deficiency and healthy controls when using DBFP, leukocytes or fibroblasts (p<0.001). Deficiency of LAL was also demonstrated in individuals referred to our laboratory with suspected clinical diagnosis of WD, CESD, and Niemann-Pick type B. We conclude that the assay of LAL using selective inhibitor is a reliable and useful method for the identification of LAL deficiency, not only in DBFP samples but also in leukocytes and fibroblasts. This is important as enzyme replacement therapy for LAL deficiency is currently being developed, making the correct diagnosis a critical issue.