Lysosomal storage disorders: Molecular basis and laboratory testing (original) (raw)
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Clinica Chimica Acta, 2012
Background-We sought to modify a previously published tandem mass spectrometry method of screening for 5 lysosomal storage disorders (LSDs) in order to make it better suited for highthroughput newborn screening. Methods-Two 3-mm dried blood spot (DBS) punches were incubated, each with a different assay solution. The quadruplex solution was used for screening for Gaucher, Pompe, Krabbe and Fabry diseases, while a separate solution was used for Niemann-Pick A/B disease. Results-The mean activities of acid-β-glucocerebrosidase (ABG), acid sphingomyelinase (ASM), acid glucosidase (GAA), galactocerebroside-β-galactosidase (GALC) and acidgalactosidase A (GLA) were measured on 5055 unidentified newborns. The mean activities (compared with their disease controls) were, 15.1 (0.35), 22.2 (1.34), 16.8 (0.51), 3.61 (0.23), and 20.7 (1.43) (μmol/L/h), respectively. The number of specimens that fell below our retest level cutoff of <20% daily mean activity (DMA) for each analyte is: ABG (6), ASM (0), GAA (5), GALC (17), and GLA (2). Conclusions-This method provides a simplified and reliable assay for screening for five LSDs with clear distinction between activities from normal and disease samples. Advantages of this new method include significant decreases in processing time and the number of required assay solutions and overall decreased complexity.
Inherited metabolic disorders: prenatal diagnosis of lysosomal storage disorders
Prenatal Diagnosis, 2015
Objective To offer accurate prenatal diagnosis of lysosomal storage disorders in early pregnancy. Method Prenatal enzymatic diagnoses of Gaucher, Fabry, Pompe, Niemann Pick A/B, Tay Sach, Sandoff, GM1, mucoplysaccharidoses, Wolman, Krabbe, Metachromatic leukodystrophy and Batten diseases were made in uncultured chorionic villi samples by fluorometric/spectrophotometric methods. Results Of 331 prenatal enzymatic diagnosis, 207 fetuses (67%) were normal and 124 (37%) were affected. The interpretation of affected, normal and carrier fetuses was done using their respective reference ranges as well as % enzyme activity of normal mean. The prenatal molecular confirmation was feasible in 43 biochemically diagnosed fetuses. Of the 207 normal reported fetuses, post natal enzymatic confirmation was done in 23 babies, clinical status of another 165 babies was assessed as unaffected via questionnaire on telephone and 19 were lost to follow-up. In affected pregnancies, 123 opted for termination of which 44 were confirmed enzymatically after abortion. A single false positive was determined to be a carrier by prenatal mutation analysis and carried to term. Conclusion We recommend uncultured chorionic villi for reliable prenatal enzymatic diagnosis of various lysosomal storage disorders on account of the low rate of false positive (0.5%) and false negative (2.2%) results.
Prenatal diagnosis of lysosomal storage disorders: our experience in 120 cases
Molecular Cytogenetics, 2014
The reported prevalence of lysosomal storage disorder (LSD) is 1:5,000-7,000 live births and with the limited availability of therapeutic option; prenatal diagnosis (PD) remains the only preventable cure for storage disorders. One hundred forty pregnancies having confirmed diagnosis of LSDs in index case were selected for enzymes study during PD from uncultured and/or cultured chorionic villus (CV/CCV) and cultured amniotic fluid (CAF) cells. In seven pregnancies, molecular analysis was additionally carried out where mutation was known in an index case. Of 140 pregnancies, 60 (42.9 %) were diagnosed as affected, 13 (9.3 %) had an intermediate enzyme activity and 67 (47.8 %) had normal enzyme activity. Results were confirmed in 83 cases whereas 57 cases were lost from the follow-up. In one case, enzyme b-galactose-6 sulphate sulphatase specific for Morquio-A disorder [mucopolysaccharidosis-IVA (MPS-IVA)] had shown 30 % reduced activity in CV cells and the case was diagnosed as carrier for MPS-IVA while it delivered an affected child. Further molecular analysis in seven cases that included six with Tay-Sachs and one with Gaucher disease, confirmed the results obtained by enzymatic study during PD. PD of LSDs can be carried out by enzymes study from CV/CCV and CAF with an accuracy of molecular method. However, in cases of MPS and mucolipidosis, Amniotic fluid (AF) is preferred over CV/CCV. In addition, special care is needed while interpreting enzyme results encompassing carrier status and they need to be further evaluated by molecular studies.
JIMD Reports, 2016
High consanguinity rates, poor access to accurate diagnostic tests, and costly therapies are the main causes of increased burden of lysosomal storage disorders (LSDs) in developing countries. Therefore, there is a major unmet need for accurate and economical diagnostic tests to facilitate diagnosis and consideration of therapies before irreversible complications occur. In crosscountry study, we utilized dried blood spots (DBS) of 1,033 patients clinically suspected to harbor LSDs for enzymatic diagnosis using modified fluorometric assays from March 2013 through May 2015. Results were validated by demonstrating reproducibility, testing in different sample types (leukocytes/plasma/skin fibroblast), mutation study, or measuring specific biomarkers. Thirty percent (307/1,033) were confirmed to have one of the LSDs tested. Reference intervals established unambiguously identified affected patients. Correlation of DBS results with other biological samples (n ¼ 172) and mutation studies (n ¼ 74) demonstrated 100% concordance in Gaucher, Fabry, Tay Sachs, Sandhoff, Niemann-Pick, GM1, Neuronal ceroid lipofusci-nosis (NCL), Fucosidosis, Mannosidosis, Mucopolysaccharidosis (MPS) II, IIIb, IVa, VI, VII, and I-Cell diseases, and 91.4% and 88% concordance in Pompe and MPS-I, respectively. Gaucher and Pompe are the most common LSDs in India and Pakistan, followed by MPS-I in both India and Sri Lanka. Study demonstrates utility of DBS for reliable diagnosis of LSDs. Diagnostic accuracy (97.6%) confirms veracity of enzyme assays. Adoption of DBS will overcome significant hurdles in blood sample transportation from remote regions. DBS enzymatic and molecular diagnosis should become the standard of care for LSDs to make timely diagnosis, develop personalized treatment/monitoring plan, and facilitate genetic counseling. Abbreviations CK Creatine phosphokinase DBS Dried blood spot EQAS External Quality Assurance Scheme ERNDIM European Research Network for evaluation and improvement of screening, diagnosis, and treatment of inborn errors of metabolism GAGs Glycosaminoglycans LSDs Lysosomal storage disorders MPS Mucopolysaccharidosis Communicated by: Gajja Salomons
The Korean Journal of Laboratory Medicine, 2011
We evaluated the performance of multiplex tandem mass spectrometry (MS/MS) in newborn screening for detection of 6 lysosomal storage disorders (LSDs), namely, Niemann-Pick A/B, Krabbe, Gaucher, Fabry, and Pompe diseases and Hurler syndrome. Methods: We revised the conditions and procedures of multiplex enzyme assay for the MS/MS analysis and determined the precision of our enzyme assay and the effects of sample amounts and incubation time on the results. We also measured the degree of correlation between the enzyme activities in the dried blood spots (DBSs) and those in the leukocytes. DBSs of 211 normal newborns and 13 newborns with various LSDs were analyzed using our revised methods. Results: The intra-and inter-assay precisions were 2.9-18.7% and 8.1-18.1%, respectively. The amount of product obtained was proportional to the DBS eluate volume, but a slight flattening was observed in the product vs. sample volume curve at higher sample volumes. For each enzyme assay, the amount of product obtained increased linearly with the incubation period (range, 0-24 hr). Passing and Bablok regression analysis revealed that the enzyme activities in the DBSs and those in the leukocytes were favorably correlated. The enzyme activities measured in the DBSs were consistently lower in patients with LSDs than in normal newborns. Conclusions: The performance of our revised techniques for MS/MS detection and enzyme assays was of the generally acceptable standard. To our knowledge, this is the first report on the use of MS/MS for newborn screening of LSDs in an Asian population.
Characterization of 42 Egyptian Children with Lysosomal Storage Disorders
The Egyptian Journal of Hospital Medicine
Background: Lysosomal storage disorders (LSDs) are a heterogeneous family of genetic diseases with a broad phenotypic spectrum. There is a paucity of data on LSDs from developing countries. Objective: We aimed to study the pattern, relative frequency, and phenotypic spectrum of LSDs in children at an Egyptian medical center. Patients and Methods: This study included children < 18 years with LSDs diagnosed and followed up at an Egyptian medical center from January 2018 to December 2021. Data were collected on patients' demographics, clinical features, characteristic metabolites, specific enzyme assay, and genetic testing. Results: Forty-two children (62% males, 74% parental consanguinity and 26% positive family history) were diagnosed with 10 different LSDs, representing 14% of all cases with inborn errors of metabolism (IEMs). The most frequent LSDs groups were mucopolysaccharidosis (MPS) (52.4%) and sphingolipidosis (40.8%). The most common individual diseases were MPS I (26.2%), Gaucher disease (23.8), MPS III (16.7%), and acid sphingomyelinase deficiency (11.9%). The median age at presentation was two years with a median diagnostic delay of 12 months. The most common clinical manifestations were delayed development, intellectual disability, visceromegaly, coarse facial features, and skeletal abnormalities. Finally, genetic data were available for only 12 patients (8 Gaucher disease, 3 MPS-III, and 1 MPS-VI). Conclusion: LSDs (most commonly MPS and Gaucher disease) represent an important part of IEMs at our medical center, and the diagnosis seems challenging and often delayed.
Molecular Genetics and Metabolism, 2017
To evaluate the results of a lysosomal newborn screening (NBS) program in a cohort of 20,018 Mexican patients over the course of 3 years in a closed Mexican Health System (Petróleos Mexicanos [PEMEX] Health Services). Study design: Using dried blood spots (DBS), we performed a multiplex tandem mass spectrometry enzymatic assay for six lysosomal storage disorders (LSDs) including Pompe disease, Fabry disease, Gaucher disease, mucopolysaccharidosis type I (MPS-I), Niemann-Pick type A/B, and Krabbe disease. Screen-positive cases were confirmed using leukocyte enzymatic activity and DNA molecular analysis. Results: From July 2012 to April 2016, 20,018 patients were screened; 20 patients were confirmed to have an LSD phenotype (99.9 in 100,000 newborns). Final distributions include 11 Pompe disease, five Fabry disease, two MPS-I, and two Niemann-Pick type A/B patients. We did not find any Gaucher or Krabbe patients. A final frequency of 1 in 1001 LSD newborn phenotypes was established. Discussion: NBS is a major public health achievement that has decreased the morbidity and mortality of inborn errors of metabolism. The introduction of NBS for LSD presents new challenges. This is the first multiplex Latin-American study of six LSDs detected through NBS.
Serum protein profile analysis in lysosomal storage disorders patients
Clinica Chimica Acta, 2020
Serum protein electrophoresis (SPE) is a well-established technique to identify alterations in plasma protein profiles, caused by diseases as multiple myeloma (MM). In addition, it could be a cost-effective technique to discover new plasma biomarkers. Relation between MM and lysosomal storage diseases (LSDs) as Gaucher disease has been set out but, it has not been evaluated on other LSDs nor the utility of the SPE as first step on LSDs biomarkers discovery projects. Materials and methods: Stored plasma samples at diagnosis from several LSDs patients underwent analysis. Quality control was checked prior to the SPE was analyzed by capillary electrophoresis. The analysis for monoclonal spikes and the differences between each fraction on patients' samples vs the control data previously published, were evaluated. Furthermore, immunoprotein quantification and free light chains ratio were done by nephelometry and turbidimetry. Results: Seventy-five samples of LSD patients at diagnosis, were assessed. The frequency of the MGUS on LSDs patients was not higher than in general population whereas one lysosomal acid lipase deficiency infant showed increased IgA and kappa deviation. Regarding to the usefulness of SPE in biomarkers discovery, statistically significant differences were observed on SPE fractions between LSDs and healthy population. Discussion: The evaluation of SPE fractions can be a useful tool to understand pathophysiologic aspects in LDSs and, to simplify new marker discovery projects. In some of them, the MGUS appearance is a risk factor for the MM development despite its frequency is not increased on the studied LSDs at diagnosis.
2014
Background: With over 50 different disorders and a combined incidence of up to 1/3000 births, lysosomal storage diseases (LSDs) constitute a major public health problem and place an enormous burden on affected individuals and their families. Many factors make LSD diagnosis difficult, including phenotype and penetrance variability, shared signs and symptoms, and problems inherent to biochemical diagnosis. Developing a powerful diagnostic tool could mitigate the protracted diagnostic process for these families, lead to better outcomes for current and proposed therapies, and provide the basis for more appropriate genetic counseling. Methods: We have designed a targeted resequencing assay for the simultaneous testing of 57 lysosomal genes, using in-solution capture as the enrichment method and two different sequencing platforms. A total of 84 patients with high to moderate-or low suspicion index for LSD were enrolled in different centers in Spain and Portugal, including 18 positive controls. Results: We correctly diagnosed 18 positive blinded controls, provided genetic diagnosis to 25 potential LSD patients, and ended with 18 diagnostic odysseys. Conclusion: We report the assessment of a next-generation-sequencing-based approach as an accessory tool in the diagnosis of LSDs, a group of disorders which have overlapping clinical profiles and genetic heterogeneity. We have also identified and quantified the strengths and limitations of next generation sequencing (NGS) technology applied to diagnosis.
Newborn screening for lysosomal storage disorders
Acta Paediatrica, 2008
Background Lysosomal storage diseases (LSDs) are inborn errors of metabolism resulting from 50 different inherited disorders. The increasing availability of treatments and the importance of early intervention have stimulated newborn screening (NBS) to diagnose LSDs and permit early intervention to prevent irreversible impairment or severe disability. We present our experience screening newborns in North East Italy to identify neonates with Mucopolysaccharidosis type I (MPS I) and Pompe, Fabry, and Gaucher diseases. Methods Activities of acid β-glucocerebrosidase (ABG; Gaucher), acid α-glucosidase (GAA; Pompe), acid αgalactosidase (GLA; Fabry), and acid α-L-iduronidase (IDUA; MPS-I) in dried blood spots (DBS) from all newborns during a 17-month period were determined by multiplexed tandem mass spectrometry (MS/MS) using the NeoLSD ® as-Robert J. Desnick and Alessandro P. Burlina contributed equally to this work.