Higher precision, first tier newborn screening for metachromatic leukodystrophy using 16:1-OH-sulfatide.

Newborn screening (NBS) for metachromatic leukodystrophy (MLD) is based on first-tier measurement of sulfatides in dried blood spots (DBS) followed by second-tier measurement of arylsulfatase A in the same DBS. This approach is very precise with 0-1 false positives per ∼30,000 newborns tested. Recent data reported here shows that the sulfatide molecular species with an α-hydroxyl, 16­carbon, mono-unsaturated fatty acyl group (16:1-OH-sulfatide) is superior to the original biomarker 16:0-sulfatide in reducing the number of first-tier false positives. This result is consistent across 4 MLD NBS centers. By measuring 16:1-OH-sulfatide alone or together with 16:0-sulfatide, the estimated false positive rate is 0.048% and is reduced essentially to zero with second-tier arylsulfatase A activity assay. The false negative rate is predicted to be extremely low based on the demonstration that 40 out of 40 newborn DBS from clinically-confirmed MLD patients are detected with these methods. The work shows that NBS for MLD is extremely precise and ready for deployment. Furthermore, it can be multiplexed with several other inborn errors of metabolism already tested in NBS centers worldwide.

Changes in plasma arylsulfatase A level as a compensatory biomarker of early Parkinson's disease.

Lysosomal dysfunction has been associated with Parkinson's disease (PD). However, the activity of lysosomal enzymes is heterogeneously observed in PD. We investigated whether arylsulfatase A (ARSA) level can be used as a fluid biomarker of PD and can reflect disease progression. Plasma ARSA level was measured in 55 patients with early and drug-naïve PD, 13 patients with late PD, and 14 healthy controls. We compared the plasma ARSA level among the groups and assessed its correlation to clinical parameters and striatal dopamine transporter (DAT) activity. Plasma ARSA level was not correlated with age. The early PD group had higher plasma ARSA level than the control and late PD groups. In a generalized additive model including all patients with PD, the plasma ARSA level showed an inverted U-shape according to disease duration, peaking at 2.19 years. In patients with early PD, plasma ARSA level was positively correlated to parkinsonian motor score and negatively to striatal DAT activity. In summary, plasma ARSA level was elevated in early stage of PD, and elevated plasma ARSA level was correlated to the clinical and imaging markers of nigrostriatal degeneration. These results suggest that ARSA level is a potential biomarker of compensation in early PD.

Arylsulfatase A, a genetic modifier of Parkinson's disease, is an α-synuclein chaperone.

Mutations in lysosomal genes increase the risk of neurodegenerative diseases, as is the case for Parkinson's disease. Here, we found that pathogenic and protective mutations in arylsulfatase A (ARSA), a gene responsible for metachromatic leukodystrophy, a lysosomal storage disorder, are linked to Parkinson's disease. Plasma ARSA protein levels were changed in Parkinson's disease patients. ARSA deficiency caused increases in α-synuclein aggregation and secretion, and increases in α-synuclein propagation in cells and nematodes. Despite being a lysosomal protein, ARSA directly interacts with α-synuclein in the cytosol. The interaction was more extensive with protective ARSA variant and less with pathogenic ARSA variant than wild-type. ARSA inhibited the in vitro fibrillation of α-synuclein in a dose-dependent manner. Ectopic expression of ARSA reversed the α-synuclein phenotypes in both cell and fly models of synucleinopathy, the effects correlating with the extent of the physical interaction between these molecules. Collectively, these results suggest that ARSA is a genetic modifier of Parkinson's disease pathogenesis, acting as a molecular chaperone for α-synuclein.

Clinical significance of serum DRAM1 mRNA, ARSA mRNA, hsa-miR-2053 and lncRNA-RP1-86D1.3 axis expression in malignant pleural mesothelioma.

AIM AND BACKGROUND: Malignant pleural mesothelioma (MPM) is a lethal cancer mainly caused by chronic exposure of asbestos. In this pilot study, we aimed to assess the expression of serum RNA-based biomarker panel exploring their clinical utility as diagnostic and prognostic biomarkers for MPM. METHODS: We have selected an MPM-specific RNA-based biomarker panel through bioinformatics analysis based on the integration of DNA damage regulated autophagy modulator 1 (DRAM1) and arylsulfatase A ( ARSA) gene expression with their epigenetic regulators microRNA ( miR-2053) and long noncoding RNA ( lncRNA-RP1-86D1.3). Then, quantitative real-time polymerase chain reaction (qPCR) validation in sera of 60 MPM patients, 20 chronic asbestos exposure patients, and 20 healthy volunteers was done. Lastly, the prognostic power of the selected panel was assessed. RESULTS: The expression of serum DRAM1 messenger RNA (mRNA), ARSA mRNA, hsa-miR-2053 and lncRNA-RP1-86D1.3 were positive in 78.3%, 90%, 85%, and 83.3% of MPM patients, respectively. The RNA-based biomarker panel was able to discriminate between MPM patients and controls with high accuracy and their combined sensitivity reached 100% for the diagnosis of MPM. Kaplan-Meier analysis showed that hsa-miR-2053 is an independent prognostic factor of MPM. CONCLUSION: Our preliminary data revealed that the chosen RNAs play an important role in driving MPM development and progression.

A Patient With Atypical Multiple Sulfatase Deficiency.

BACKGROUND: Multiple sulfatase deficiency is an autosomal recessive lysosomal storage disorder characterized by the absence of several sulfatases and resulting from mutations in the gene encoding the human C (alpha)-formylglycine-generating enzyme. There have been a variety of biochemical and clinical presentations reported in this disorder. PATIENT DESCRIPTION: We present a 4-year-old girl with clinical findings of microcephaly, spondylolisthesis and neurological regression without ichthyosis, coarse facies, and organomegaly. RESULTS: The child's magnetic resonance imaging demonstrated confluent white matter abnormalities involving the periventricular and deep cerebral white matter with the U-fibers relatively spared. Biochemical testing showing low arylsulfatase A levels were initially thought to be consistent with a diagnosis of metachromatic leukodystrophy. The diagnosis of multiple sulfatase deficiency was pursued when genetic testing for metachromatic leukodystrophy was negative. CONCLUSION: This child illustrates the clinical heterogeneity of multiple sulfatase deficiency and that this disorder can occur without the classic clinical features.

Hematopoietic SCT: a useful treatment for late metachromatic leukodystrophy.

In metachromatic leukodystrophy (MLD), the deficiency of the lysosomal enzyme arylsulfatase A (ARSA) leads to demyelination in the central and peripheral nervous system and ultimately to death. Allogeneic hematopoietic SCT (HSCT) is currently the only treatment for adult and late-onset juvenile MLD, although it is still in question because of insufficient follow-up. We wanted to determine whether HSCT could halt the progression of adult and late-onset juvenile MLD. Four treated unrelated patients and three untreated siblings were included in the study, and followed regularly for up to 18 years after transplantation. The patients were assessed from clinical examination, ARSA enzyme levels, magnetic resonance imaging of the brain and neuropsychological and neurophysiological tests. In the treated patients, ARSA levels were normal up to 18 years after transplantation. The parameters evaluated stabilized and remained stable after a latency period of 12-24 months. Two patients live normal lives, partially in a protected environment. The other two patients stabilized at a low cognitive and functional level. One of the controls is demented, one is in a vegetative state and one died. We conclude that, in comparison with their untreated siblings, HSCT halted the progression of the disease in our treated patients.

Critical issues for the proper diagnosis of Metachromatic Leukodystrophy.

Metachromatic Leukodystrophy is a lysosomal storage disorder caused by Arylsulfatase A deficiency. Diagnosis is usually performed by measurement of enzymatic activity and/or characterization of the gene mutations. Here we describe a family case in which the determination of enzyme activity alone did not allow diagnosis of the pre-symptomatic sibling of the index case. Only combination of gene sequencing with thorough biochemical analysis allowed the correct diagnosis of the sibling, who was promptly directed to treatment.

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.

Haematopoietic stem cell transplantation does not retard disease progression in the psycho-cognitive variant of late-onset metachromatic leukodystrophy.

Haematopoietic stem cell transplantation has an unproven role in the management of late-onset metachromatic leukodystrophy: theoretically justified through the engraftment of enzyme-replete haematopoietic progenitors and restoration of capacity for sulphatide catabolism in neural tissue through enzyme recapture, the long-term outcome is unknown. The rarity of the psycho-cognitive variant and slow progression of late-onset disease impairs evaluation of treatment. We report detailed clinical and neuropsychological assessments after haematopoietic stem-cell transplantation in a patient with a late-onset psycho-cognitive form of metachromatic leukodystrophy. Cognitive decline, indistinguishable from the natural course of the disease, was serially documented over 11 years despite complete donor chimaerism and correction of leukocyte arylsulphatase A to wild type values; subtle motor deterioration was similarly noted and progressive cerebral volume loss was evident upon magnetic resonance imaging. Sensory nerve conduction deteriorated 17 months post-transplantation with apparent stabilisation at 11-year review. Haematopoietic stem-cell transplantation was ineffective for this rare attenuated variant of metachromatic leukodystrophy. In the few patients identified pre-symptomatically or with early-phase disease, clear recommendations are lacking; when transplantation is considered, umbilical cord blood grafts from enzyme-replete donors with adjunctive mesenchymal stem cell infusions from the same source may be preferable. Improved outcomes will depend on enhanced awareness and early diagnosis of the disease, so that promising interventions such as genetically modified, autologous stem cell transplantation have the best opportunity of success.

Clinical and biochemical study of 29 Brazilian patients with metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a lysosomal disorder caused by arylsulfatase A (ARSA) deficiency. It is classified into three forms according to the age of onset of symptoms (late infantile, juvenile, and adult). We carried out a cross-sectional and retrospective study, which aimed to determine the epidemiological, clinical, and biochemical profile of MLD patients from a national reference center for Inborn Errors of Metabolism in Brazil. Twenty-nine patients (male, 17) agreed to participate in the study (late infantile form: 22; juvenile form: 4; adult form: 1; asymptomatic: 2). Mean ages at onset of symptoms and at biochemical diagnosis were, respectively, 19 and 39 months for late infantile form and 84.7 and 161.2 months for juvenile form. The most frequently reported first clinical symptom/sign of the disease was gait disturbance and other motor abnormalities (72.7%) for late infantile form and behavioral and cognitive alterations (50%) for juvenile form. Leukocyte ARSA activity level did not present significant correlation with the age of onset of symptoms (r = -0.09, p = 0.67). Occipital white matter and basal nuclei abnormalities were not found in patients with the late infantile MLD. Our results suggest that there is a considerable delay between the age of onset of signs and symptoms and the diagnosis of MLD in Brazil. Correlation between ARSA activity and MLD clinical form was not found. Further studies on the epidemiology and natural history of this disease with larger samples are needed, especially now when specific treatments should be available in the near future.

Adult metachromatic leukodystrophy: a new mutation in the schizophrenia-like phenotype with early neurological signs.

OBJECTIVES: The adult type of metachromatic leukodystrophy can manifest itself as motor or as psycho-cognitive form, the latter is very similar to schizophrenia. We report on two sisters with adult metachromatic leukodystrophy who display symptoms of both forms. METHODS: Presented are genotype analyses and 4-year follow-up data regarding clinical manifestations as well as neurocognitive and neuroimaging results for two adult sisters with metachromatic leukodystrophy. RESULTS: Whereas the younger sister developed disorganized schizophrenia-like symptoms, the other exhibited schizophrenia-like, negative symptoms. In both sisters, neurological signs were already present at the onset of the disease and progression towards dementia was documented within 1-2 years. In peripheral leukocytes, the activity of arylsulphatase A was reduced to 2 and 5% of the mean normal activity in both women. Genotype analysis revealed compound heterozygosity for a known severe splice site mutation, (c.459+1G>A) together with two known polymorphisms, [(c.937G>T), (p.Trp193Asp)] and [(c.1530C>G), (p.Thr391Ser)], and a novel missense mutation, (c.1194C>T). The latter results in the exchange of a conserved polar amino acid, threonine 279, to hydrophobic isoleucine (Thr279Ileu), which could not be found among >100 control alleles. A family analysis identified T279I as the paternal allele, whereas (c.459+1G>A) as well as the two polymorphisms were inherited from the mother. This is consistent with a disease-causing effect of the novel mutation. CONCLUSIONS: The novel mutation, T279I detected in our patients, correlates with a specific phenotype with schizophrenia-like symptoms, neurological signs and cognitive impairment early in the course of the disease and a relatively fast progression towards dementia. This is in contrast to previous reports on adult metachromatic leukodystrophy patients with the psycho-cognitive phenotype who did not show any neurological signs for decades, however, most of these patients were heterozygous for another specific missense mutation, I179S.

[Carbon monoxide contamination: an environmental health problem]. / Contaminación por monóxido de carbono: un problema de salud ambiental.

Carbon monoxide is considered to be a major factor contaminating earth's atmosphere. The main sources producing this contamination are cars using gasoline or diesel fuel and industrial processes using carbon compounds; these two are responsible for 80% of carbon monoxide being emitted to the atmosphere. This substance has a well-known toxic effect on human beings and its acute poisonous effects (including death) have been widely studied; however, its long-term chronic effects are still not known. During the last few years, experimental research on animals and studies of human epidemiology have established the relationship between chronic exposure to low and middle levels of carbon monoxide in breathable air and adverse effects on human health, especially on organs consuming large amounts of oxygen such as the heart and brain. Harmful cardiovascular and neuropsychological effects have been documented in carbon monoxide concentration in air of less than 25 ppm and in carboxyhaemoglobin levels in blood of less than 10%. The main cardiac damage described to date has been high blood pressure, cardiac arrhythm and electrocardiograph signs of ischemia. Lack of memory, attention, concentration and Parkinson-type altered movement are the neuropsychological changes most frequently associated with chronic exposure to low levels of carbon monoxide and carboxyhaemoglobin.

Kinetics and activity of arylsulfatase A in leukocytes derived from patients with cerebral palsy.

Activity and kinetics of arylsulfatase A (ASA, EC 3.1.6.8) were analyzed in leukocyte homogenates derived from patients suffering from cerebral palsy. Lower ASA activity was found in the patients' leukocytes than in controls, as determined by spectrophotometry using chromogenic substrate p-nitrocatechol sulfate (p-NCS). Kinetic parameters, K(m) and v(max), for leukocyte ASA were determined from the dependence of initial reaction velocities on the p-NCS concentrations. A slight difference in K(m) values was found for leukocyte enzyme in cerebral palsy (0.26 mmol L(-1)) compared to the control (0.21 mmol L(-1)), whereas v(max) value for leukocyte ASA in disease reached only 58% of the control value. In addition, the presence of the most common mutations associated with ASA pseudo-deficiency (N350S, 1524+95 A>G) and metachromatic leukodystrophy (P426L) was detected in all investigated patients. Changes in activity and kinetic parameters of leukocyte ASA in cerebral palsy are most probably related to the decrease of enzyme concentration; the detected mutations might at least partially contribute to the observed changes.

Contaminación por monóxido de carbono: un problema de salud ambiental / Carbon monoxide contamination: an environmental health problem

El monóxido de carbono es considerado uno de los mayores contaminantes de la atmósfera terrestre. Sus principales fuentes productoras responsables de aproximadamente 80 por ciento de las emisiones, son los vehículos automotores que utilizan como combustible gasolina o diesel y los procesos industriales que utilizan compuestos del carbono. Esta sustancia es bien conocida por su toxicidad para el ser humano. Sus efectos tóxicos agudos incluida la muerte han sido estudiados ampliamente; sin embargo, sus potenciales efectos adversos a largo plazo son poco conocidos. En los últimos años, los estudios de investigación experimentales en animales y epidemiológicos en humanos han evidenciado relación entre población expuesta en forma crónica a niveles medios y bajos de monóxido de carbono en aire respirable y la aparición de efectos adversos en la salud humana especialmente en órganos de alto consumo de oxígeno como cerebro y corazón. Se han documentado efectos nocivos cardiovasculares y neuropsicológicos en presencia de concentraciones de monóxido de carbono en aire inferiores a 25 partes por millón y a niveles de carboxihemoglobina en sangre inferiores a 10 por ciento. Las alteraciones cardiovasculares que se han descrito son hipertensión arterial, aparición de arritmias y signos electrocardiográficos de isquemia. Déficit en memoria, atención, concentración y alteraciones del movimiento tipo parkinsonismo, son los cambios neuropsicológicos con mayor frecuencia asociados a exposición crónica a bajos niveles de monóxido de carbono y carboxihemoglobina.

Carbon monoxide is considered to be a major factor contaminating earths atmosphere. The main sources producing this contamination are cars using gasoline or diesel fuel and industrial processes using carbon compounds; these two are responsible for 80 percent of carbon monoxide being emitted to the atmosphere. This substance has a well-known toxic effect on human beings and its acute poisonous effects (including death) have been widely studied; however, its long-term chronic effects are still not known. During the last few years, experimental research on animals and studies of human epidemiology have established the relationship between chronic exposure to low and middle levels of carbon monoxide in breathable air and adverse effects on human health, especially on organs consuming large amounts of oxygen such as the heart and brain. Harmful cardiovascular and neuropsychological effects have been documented in carbon monoxide concentration in air of less than 25 ppm and in carboxyhaemoglobin levels in blood of less than 10 percent. The main cardiac damage described to date has been high blood pressure, cardiac arrhythm and electrocardiograph signs of ischemia. Lack of memory, attention, concentration and Parkinson-type altered movement are the neuropsychological changes most frequently associated with chronic exposure to low levels of carbon monoxide and carboxyhaemoglobin.

Enzyme replacement improves nervous system pathology and function in a mouse model for metachromatic leukodystrophy.

A deficiency of arylsulfatase A (ASA) causes the lysosomal storage disease metachromatic leukodystrophy, which is characterized by accumulation of the sphingolipid 3-O-sulfogalactosylceramide (sulfatide). Sphingolipid storage results in progressive demyelination and severe neurologic symptoms. The disease is lethal, and curative therapy is not available. To assess the therapeutic potential of enzyme replacement therapy (ERT), ASA knockout mice were treated by intravenous injection of recombinant human ASA. Plasma levels of ASA declined with a half-time of approximately 40 min, and enzyme was detectable in tissues within minutes after injection. The uptake of injected enzyme was high into liver, moderate into peripheral nervous system (PNS) and kidney and very low into brain. The apparent half-life of endocytosed enzyme was approximately 4 days. A single injection led to a time- and dose-dependent decline of the excess sulfatide in PNS and kidney by up to 70%, but no reduction was seen in brain. Four weekly injections with 20 mg/kg body weight not only reduced storage in peripheral tissues progressively, but also were surprisingly effective in reducing sulfatide storage in brain and spinal cord. The histopathology of kidney and central nervous system was ameliorated. Improved neuromotor coordination capabilities and normalized peripheral compound motor action potential demonstrate the benefits of ERT on the nervous system function. Enzyme replacement may therefore be a promising therapeutic option in this devastating disease.

Determination of diagnostic and prognostic values of urinary interleukin-8, tumor necrosis factor-alpha, and leukocyte arylsulfatase-A activity in patients with bladder cancer.

OBJECTIVES: This study was planned to evaluate the feasibility of the assay of leukocyte arylsulfatase-A (AS-A) activity, and some urinary cytokine levels (tumor necrosis factor-alpha [TNF-alpha] and interleukin-8 [IL-8]), as noninvasive diagnostic tools in different stages of bladder cancer patients. DESIGN AND METHODS: Blood and urine samples of 79 subjects were analyzed, including 28 healthy volunteers, 27 patients with superficial bladder cancer (SBC), and 24 patients with muscle invasive bladder cancer (MIBC). RESULTS: In SBC patients, the mean leukocyte AS-A activity was slightly higher (11.4%) than healthy subjects without reaching statistical significance. On the other hand, the enzyme activity in MIBC patients was significantly higher than those of controls (38.9%) and SBC patients (18.3%). Urinary TNF-alpha levels in both cancer groups were not significantly different from the control group. Urinary IL-8 levels of MIBC patients were significantly increased when compared with the levels of SBC patients and healthy subjects (P < 0.001). CONCLUSIONS: Based on our results, it may be concluded that measurement of leukocyte AS-A activity is not a sufficiently reliable noninvasive diagnostic test in distinguishing early stage bladder cancer from healthy subjects as well as detecting disease progression. Whereas measurement of urinary IL-8 may be valuable as a noninvasive diagnostic and prognostic test especially in patients with advanced bladder cancer. It also appears that complementary biochemical information may be obtained about the prognosis of the disease by monitoring urinary IL-8 profile. However, further confirmatory clinical trials about the prognostic value of the measurement of urinary IL-8 are needed.

Biochemical characteristics of a Korean patient with mucolipidosis III (pseudo-Hurler polydystrophy).

We performed a biochemical study on the patient with mucolipidosis III (ML-III, pseudo-Hurler polydystrophy) in Korea. Confluent fibroblasts from the patient and from normal controls were cultured for 4, 12, 24, 48, and 72 hr, respectively. Lysosomal enzyme activities in culture media after different incubation times and in plasma, leuko-cytes, and fibroblasts were determined. Most of the leukocyte lysosomal enzymes were within normal limits or slightly lowered; however, plasma lysosomal enzyme activities such as those of hexosaminidase and arylsulfatase A were markedly increased. Numerous phase-dense inclusions were present in the cytoplasm of cultured fibroblasts. Lysosomal enzyme activities of fibroblasts were markedly decreased except for beta-glucosidase. The rates of increase of the lysosomal enzyme activities with incubation time were greater in the culture medium of the patient than in normal control, whereas no difference in the beta-glucosidase activity of the culture media of the patient and the control was found. This study describes the first case of ML-III in Korea, with its typical biochemical characteristics, i.e., a problem with targeting and transporting of lysosomal enzymes which results in a marked increase in plasma lysosomal enzyme activities and a high ratio of extracellular to intracellular lysosomal enzyme activities in cultured fibroblasts.