Development, validation and application of single molecule molecular inversion probe based novel integrated genetic screening method for 29 common lysosomal storage disorders in India.

BACKGROUND: Current clinical diagnosis pathway for lysosomal storage disorders (LSDs) involves sequential biochemical enzymatic tests followed by DNA sequencing, which is iterative, has low diagnostic yield and is costly due to overlapping clinical presentations. Here, we describe a novel low-cost and high-throughput sequencing assay using single-molecule molecular inversion probes (smMIPs) to screen for causative single nucleotide variants (SNVs) and copy number variants (CNVs) in genes associated with 29 common LSDs in India. RESULTS: 903 smMIPs were designed to target exon and exon-intron boundaries of targeted genes (n = 23; 53.7 kb of the human genome) and were equimolarly pooled to create a sequencing library. After extensive validation in a cohort of 50 patients, we screened 300 patients with either biochemical diagnosis (n = 187) or clinical suspicion (n = 113) of LSDs. A diagnostic yield of 83.4% was observed in patients with prior biochemical diagnosis of LSD. Furthermore, diagnostic yield of 73.9% (n = 54/73) was observed in patients with high clinical suspicion of LSD in contrast with 2.4% (n = 1/40) in patients with low clinical suspicion of LSD. In addition to detecting SNVs, the assay could detect single and multi-exon copy number variants with high confidence. Critically, Niemann-Pick disease type C and neuronal ceroid lipofuscinosis-6 diseases for which biochemical testing is unavailable, could be diagnosed using our assay. Lastly, we observed a non-inferior performance of the assay in DNA extracted from dried blood spots in comparison with whole blood. CONCLUSION: We developed a flexible and scalable assay to reliably detect genetic causes of 29 common LSDs in India. The assay consolidates the detection of multiple variant types in multiple sample types while having improved diagnostic yield at same or lower cost compared to current clinical paradigm.

[Importance of lysosomal storage diseases in rheumatology]. / Bedeutung lysosomaler Speicherkrankheiten in der Rheumatologie.

Lysosomal storage diseases are a group of rare hereditary metabolic diseases. Due to a deficiency of lysosomal enzymes, complex substrates accumulate in the lysosomes of various organs. Depending on the affected enzyme, this results in clinically variable and chronic progressive multiorgan diseases. Diagnosis is often delayed. As clinical symptoms include the musculoskeletal system, an awareness of lysosomal storage diseases is of relevance to (pediatric) rheumatologists. This article is focused on Mucopolysaccharidosis type I­S, Mucolipidosis type III, Gaucher disease and Fabry disease. When suspecting a lysosomal storage disease, enzyme activity should be determined in dried blood spots or leukocytes. For some diseases, specific biomarkers can additionally be analyzed. Diagnosis should be confirmed by genetic testing. As causal treatment options are available for three of the presented diseases, a timely diagnosis is very important.

Newborn Screening for 6 Lysosomal Storage Disorders in China.

Importance: Newborn screening (NBS) for lysosomal storage disorders (LSDs) is becoming an increasing concern in public health. However, the birth prevalence of these disorders is rarely reported in the Chinese population, and subclinical forms of diseases among patients identified by NBS have not been evaluated. Objective: To evaluate the birth prevalence of the 6 LSDs in the Shanghai population and determine subclinical forms based on clinical, biochemical, and genetic characteristics. Design, Setting, and Participants: This cohort study included 50 108 newborns recruited from 41 hospitals in Shanghai between January and December 2021 who were screened for 6 LSDs using tandem mass spectrometry (MS/MS). Participants with screen-positive results underwent molecular and biochemical tests and clinical assessments. Data were analyzed from January 2021 through October 2022. Exposures: All participants were screened for Gaucher, acid sphingomyelinase deficiency (ASMD), Krabbe, mucopolysaccharidosis type I, Fabry, and Pompe diseases using dried blood spots. Main Outcomes and Measures: Primary outcomes were the birth prevalence and subclinical forms of the 6 LSDs in the Shanghai population. Disease biomarker measurements, genetic testing, and clinical analysis were used to assess clinical forms of LSDs screened. Results: Among 50 108 newborns (26 036 male [52.0%]; mean [SD] gestational age, 38.8 [1.6] weeks), the mean (SD) birth weight was 3257 (487) g. The MS/MS-based NBS identified 353 newborns who were positive. Of these, 27 newborns (7.7%) were diagnosed with 1 of 6 LSDs screened, including 2 newborns with Gaucher, 5 newborns with ASMD, 9 newborns with Krabbe, 8 newborns with Fabry, and 3 newborns with Pompe disease. The combined birth prevalence of LSDs in Shanghai was 1 diagnosis in 1856 live births, with Krabbe disease the most common (1 diagnosis/5568 live births), followed by Fabry disease (1 diagnosis/6264 live births), and ASMD (1 diagnosis/10 022 live births). Biochemical, molecular, and clinical analysis showed that early-onset clinical forms accounted for 3 newborns with positive results (11.1%), while later-onset forms represented nearly 90% of diagnoses (24 newborns [88.9%]). Conclusions and Relevance: In this study, the combined birth prevalence of the 6 LSDs in Shanghai was remarkably high. MS/MS-based newborn screening, combined with biochemical and molecular genetic analysis, successfully identified and characterized newborns who were screen-positive, which may assist with parental counseling and management decisions.

What should rheumatologists know about Gaucher disease and Fabry disease? Connecting the dots for an overview.

Gaucher and Fabry diseases are lysosomal storage disorders in which deficient enzyme activity leads to pathological accumulation of sphingolipids. These diseases have a broad phenotypic presentation. Musculoskeletal symptoms and pain complaints are frequently reported by patients. Thus, rheumatologists can be contacted by these patients, contributing to the correct diagnosis, earlier indication of appropriate treatment and improvement of their prognosis. This review describes important concepts about Gaucher and Fabry diseases that rheumatologists should understand to improve patients' quality of life and change the natural history of these diseases.

Investigation on lysosomal accumulation by a quantitative analysis of 2D phase-maps in digital holography microscopy.

Lysosomes are the terminal end of catabolic pathways in the cell, as well as signaling centers performing important functions such as the recycling of macromolecules, organelles, and nutrient adaptation. The importance of lysosomes in human health is supported by the fact that the deficiency of most lysosomal genes causes monogenic diseases called as a group Lysosomal Storage Diseases (LSDs). A common phenotypic hallmark of LSDs is the expansion of the lysosomal compartment that can be detected by using conventional imaging methods based on immunofluorescence protocols or overexpression of tagged lysosomal proteins. These methods require the alteration of the cellular architecture (i.e., due to fixation methods), can alter the behavior of cells (i.e., by the overexpression of proteins), and require sample preparation and the accurate selection of compatible fluorescent markers in relation to the type of analysis, therefore limiting the possibility of characterizing cellular status with simplicity. Therefore, a quantitative and label-free methodology, such as Quantitative Phase Imaging through Digital Holographic (QPI-DH), for the microscopic imaging of lysosomes in health and disease conditions may represent an important advance to study and effectively diagnose the presence of lysosomal storage in human disease. Here we proof the effectiveness of the QPI-DH method in accomplishing the detection of the lysosomal compartment using mouse embryonic fibroblasts (MEFs) derived from a Mucopolysaccharidosis type III-A (MSP-IIIA) mouse model, and comparing them with wild-type (WT) MEFs. We found that it is possible to identify label-free biomarkers able to supply a first pre-screening of the two populations, thus showing that QPI-DH can be a suitable candidate to surpass fluorescent drawbacks in the detection of lysosomes dysfunction. An appropriate numerical procedure was developed for detecting and evaluate such cellular substructures from in vitro cells cultures. Results reported in this study are encouraging about the further development of the proposed QPI-DH approach for such type of investigations about LSDs.

Alpha-mannosidosis: a case with novel ultrastructural and light microscopy findings.

OBJECTIVES: Alpha-mannosidosis is a rare genetic lysosomal storage condition leading to the systemic buildup of oligomannoside. Clinical presentation and associated conditions, as well as the full extent of histopathologic changes associated with this disease process, are not fully understood. CASE PRESENTATION: We present the case of an 8-year-1-month old patient with persistent anemia and who was initially diagnosed with Celiac disease before ultimately being diagnosed with alpha-mannosidosis. As part of his diagnostic work-up, duodenal and bone marrow biopsies were examined by pathology. Duodenal biopsies showed foamy plasma cells expanding the lamina propria which triggered a workup for a genetic storage disease; features suggestive of Celiac disease which resolved on gluten-free diet were also noted by pathology. Bone marrow analysis via electron microscopy showed cytoplasmic granules and inclusions in multiple immune cell lines. CONCLUSIONS: Alpha-mannosidosis can occur with Celiac disease and milder forms may only be suspected from incidental pathology findings. The ultrastructural bone marrow findings from this case, the first to be reported from human, show numerous disease-associated changes in multiple immune cell lines whose contribution to disease-associated immunodeficiency is unclear.

Long-range PCR amplification-based targeted enrichment & next generation sequencing: A cost-effective testing strategy for lysosomal storage disorders.

Background & objectives: Lysosomal storage disorders (LSDs) are genetic metabolic disorders which result from deficiency of lysosomal enzymes or defects in other lysosomal components. Molecular genetic testing of LSDs is required for diagnostic confirmation when lysosomal enzyme assays are not available or not feasible to perform, and for the identification of the disease causing genetic variants. The aim of this study was to develop a cost-effective, readily customizable and scalable molecular genetic testing strategy for LSDs. Methods: A testing method was designed based on the in-house creation of selective amplicons through long range PCR amplification for targeted capture and enrichment of different LSD genes of interest, followed by next generation sequencing of pooled samples. Results: In the first phase of the study, standardization and validation of the study protocol were done using 28 samples of affected probands and/or carrier parents (group A) with previously identified variants in seven genes, and in the second phase of the study, 30 samples of enzymatically confirmed or biopsy-proven patients with LSDs and/or their carrier parents who had not undergone any prior mutation analysis (group B) were tested and the sequence variants identified in them through the study method were validated by targeted Sanger sequencing. Interpretation & conclusions: This testing approach was found to be reliable, easily customizable and cost-effective for the molecular genetic evaluation of LSDs. The same strategy may be applicable, especially in resource poor settings, for developing cost-effective multigene panel tests for other conditions with genetic heterogeneity.

Lysosomal storage disorders: Clinical and therapeutic aspects.

The lysosomal storage disorders are hereditary metabolic disorders characterized by autosomal recessive inheritance, mainly caused by deficiency of an enzyme responsible for the intra-lysosomal breakdown of various substrates and products of cellular metabolism. This chapter examines the underlying defects, clinical manifestations, and provides context for the expected clinical outcome of various available therapy options employing enzyme replacement therapy, hematopoietic stem cell transplantation, substrate reduction, and enzyme enhancement therapies.

Screening for lysosomal diseases in a selected pediatric population: the case of Gaucher disease and acid sphingomyelinase deficiency.

BACKGROUND: GD and ASMD are lysosomal storage disorders that enter into differential diagnosis due to the possible overlap in their clinical manifestations. The availability of safe and effective enzymatic therapies has recently led many investigators to develop and validate new screening tools, such as algorithms, for the diagnosis of LSDs where the lack of disease awareness or failure to implement newborn screening results in a delayed diagnosis. RESULTS: the proposed algorithm allows for the clinical and biochemical differentiation between GD and ASMD. It is based on enzyme activity assessed on dried blood spots by multiplexed tandem mass spectrometry (MS/MS) coupled to specific biomarkers as second-tier analysis. CONCLUSIONS: we believe that this method will provide a simple, convenient and sensitive tool for the screening of a selected population that can be used by pediatricians and other specialists (such as pediatric hematologists and pediatric hepatologists) often engaged in diagnosing these disorders.

Perspectives of adult patients with lysosomal storage diseases on the transition from pediatric to adult healthcare in Turkey.

AIM: In many countries, adult clinics specifically dedicated to adult patients with lysosomal storage diseases (LSDs) do not exist. In Turkey, these patients are managed either by pediatric metabolic specialists or adult physicians who do not specifically specialize in LSDs. In this study, we aimed to identify the unmet clinical needs of these adult patients and their suggestions. METHODS: The focus group participants were 24 adult LSD patients. Interviews were conducted in person. RESULTS: A total of 23 LSD patients and parents of a patient with mucopolysaccharidosis type-3b with intellectual deficit were interviewed, with 84.6% of patients diagnosed after the age of 18 years and 18% of patients diagnosed before the age of 18 years desiring management by adult physicians. Patients with particular physical characteristics or severe intellectual deficit declined the transition. Patients reported structural problems in the hospital and social problems associated with pediatric clinics. They made suggestions to facilitate the possible transition. CONCLUSION: With improved care, more patients with LSDs survive into adulthood or receive the diagnosis in adulthood. Children with chronic diseases need to transition to the care of adult physicians when they reach adulthood. Thus, there is an increasing need for adult physicians to manage these patients. In this study, most LSD patients accepted a well-planned and organized transition. Problems were related to stigmatization and social isolation in the pediatric clinic or adult issues with which pediatricians are not familiar. There is a need for adult metabolic physicians. Thus, health authorities should adopt necessary regulations for training of physicians in this field.

New Perspectives in Dried Blood Spot Biomarkers for Lysosomal Storage Diseases.

Dried blood spots (DBSs) biomarkers are convenient for monitoring for specific lysosomal storage diseases (LSDs), but they could have relevance for other LSDs. To determine the specificity and utility of glycosphingolipidoses biomarkers against other LSDs, we applied a multiplexed lipid liquid chromatography tandem mass spectrometry assay to a DBS cohort of healthy controls (n = 10) and Gaucher (n = 4), Fabry (n = 10), Pompe (n = 2), mucopolysaccharidosis types I-VI (n = 52), and Niemann-Pick disease type C (NPC) (n = 5) patients. We observed no complete disease specificity for any of the markers tested. However, comparison among the different LSDs highlighted new applications and perspectives of the existing biomarkers. We observed elevations in glucosylceramide isoforms in the NPC and Gaucher patients relative to the controls. In NPC, there was a greater proportion of C24 isoforms, giving a specificity of 96-97% for NPC, higher than 92% for the NPC biomarker N-palmitoyl-O-phosphocholineserine ratio to lyso-sphingomyelin. We also observed significantly elevated levels of lyso-dihexosylceramide in Gaucher and Fabry disease as well as elevated lyso-globotriaosylceramide (Lyso-Gb3) in Gaucher disease and the neuronopathic forms of Mucopolysaccharidoses. In conclusion, DBS glucosylceramide isoform profiling has increased the specificity for the detection of NPC, thereby improving diagnostic accuracy. Low levels of lyso-lipids can be observed in other LSDs, which may have implications in their disease pathogenesis.

Expert-opinion-based guidance for the care of children with lysosomal storage diseases during the COVID-19 pandemic: An experience-based Turkey perspective.

This expert-opinion-based document was prepared by a group of specialists in pediatric inherited metabolic diseases and infectious diseases including administrative board members of Turkish Society for Pediatric Nutrition and Metabolism to provide guidance for the care of children with lysosomal storage disorders (LSDs) during the COVID-19 pandemic in Turkey. The experts reached consensus on key areas of focus regarding COVID-19-based risk status in relation to intersecting immune-inflammatory mechanisms and disease patterns in children with LSDs, diagnostic virus testing, particularly preventive measures and priorities during the pandemic, routine screening and diagnostic interventions for LSDs, psychological and socioeconomic impact of confinement measures and quarantines and optimal practice patterns in managing LSDs and/or COVID-19. The participating experts agreed on the intersecting characteristics of immune-inflammatory mechanisms, end-organ damage and prognostic biomarkers in LSD and COVID-19 populations, emphasizing the likelihood of enhanced clinical care when their interaction is clarified via further studies addressing certain aspects related to immunity, lysosomal dysfunction and disease pathogenesis. In the context of the current global COVID-19 pandemic, this expert-opinion-based document provides guidance for the care of children with LSDs during the COVID-19 pandemic based on the recent experience in Turkey.

Analysis of urinary oligosaccharide excretion patterns by UHPLC/HRAM mass spectrometry for screening of lysosomal storage disorders.

Oligosaccharidoses, sphingolipidoses and mucolipidoses are lysosomal storage disorders (LSDs) in which defective breakdown of glycan-side chains of glycosylated proteins and glycolipids leads to the accumulation of incompletely degraded oligosaccharides within lysosomes. In metabolic laboratories, these disorders are commonly diagnosed by thin-layer chromatography (TLC) but more recently also mass spectrometry-based approaches have been published. To expand the possibilities to screen for these diseases, we developed an ultra-high-performance liquid chromatography (UHPLC) with a high-resolution accurate mass (HRAM) mass spectrometry (MS) screening platform, together with an open-source iterative bioinformatics pipeline. This pipeline generates comprehensive biomarker profiles and allows for extensive quality control (QC) monitoring. Using this platform, we were able to identify α-mannosidosis, ß-mannosidosis, α-N-acetylgalactosaminidase deficiency, sialidosis, galactosialidosis, fucosidosis, aspartylglucosaminuria, GM1 gangliosidosis, GM2 gangliosidosis (M. Sandhoff) and mucolipidosis II/III in patient samples. Aberrant urinary oligosaccharide excretions were also detected for other disorders, including NGLY1 congenital disorder of deglycosylation, sialic acid storage disease, MPS type IV B and GSD II (Pompe disease). For the latter disorder, we identified heptahexose (Hex7), as a potential urinary biomarker, in addition to glucose tetrasaccharide (Glc4), for the diagnosis and monitoring of young onset cases of Pompe disease. Occasionally, so-called "neonate" biomarker profiles were observed in young patients, which were probably due to nutrition. Our UHPLC/HRAM-MS screening platform can easily be adopted in biochemical laboratories and allows for simple and robust screening and straightforward interpretation of the screening results to detect disorders in which aberrant oligosaccharides accumulate.

Utility of morphologic assessment of bone marrow biopsy in diagnosis of lysosomal storage disorders.

Introduction: Lysosomal storage disorders (LSDs) are rare disorders and pose a diagnostic challenge for clinicians owing to their generalized symptomatology. In this study, we aim to classify LSDs into two broad categories, namely, Gaucher disease (GD) and Niemann-Pick/Niemann-Pick-like diseases (NP/NP-like diseases) based on the morphology of the storage cells in the bone marrow (BM) aspiration smears and trephine biopsy sections. Materials and Method: This retrospective study includes 32 BM specimens morphologically diagnosed as LSDs at our institute, in the last 10 years. Subsequently, they were subclassified into GD and NP/NP-like diseases. Further, we have compared and analyzed the clinical, hematological, and biochemical parameters for the two groups of LSDs. Results: Based on BM morphology, 59.4% (n = 19) cases were diagnosed as NP/NP-like diseases and 40.6% (n = 13) cases as GD. Abdominal distension and failure to thrive were the most common clinical manifestations in both groups of LSDs. Anemia and thrombocytopenia were frequently seen in either of the LSDs. On the assessment of metabolic profile, elevated total/direct bilirubin and liver enzymes were more commonly seen in NP/NP-like diseases when compared with GD. Conclusion: We have classified LSDs into GD and NP/NP-like diseases based on the morphology of the storage cells in the BM specimen. The hallmark findings on BM biopsy annexed with the comparative features of the two proposed categories can aid the clinician in clinching the diagnosis. Formulation of such a methodology will prove instrumental for patient care in an underresourced setting.

Influence of initial clinical suspicion on the diagnostic yield of laboratory enzymatic testing in lysosomal storage disorders. Experience from a multispecialty hospital.

Lysosomal storage disorders (LSD) are a group of inherited metabolic diseases mainly caused by a deficiency of lysosomal hydrolases, resulting in a gradual accumulation of non-degraded substrates in different tissues causing the characteristic clinical manifestations of such disorders. Confirmatory tests of suspected LSD individuals include enzymatic and genetic testing. A well-oriented clinical suspicion can improve the cost-effectiveness of confirmatory tests and reduce the time expended to achieve the diagnosis. Thus, this work aims to retrospectively study the influence of clinical orientation on the diagnostic yield of enzymatic tests in LSD by retrieving clinical, biochemical, and genetic data obtained from subjects with suspicion of LSD. Our results suggest that the clinical manifestations at the time of diagnosis and the initial clinical suspicion can have a great impact on the diagnostic yield of enzymatic tests, and that clinical orientation performed in specialized clinical departments can contribute to improve it. In addition, the analysis of enzymatic tests as the first step in the diagnostic algorithm can correctly guide subsequent confirmatory genetic tests, in turn increasing their diagnostic yield. In summary, our results suggest that initial clinical suspicion plays a crucial role on the diagnostic yield of confirmatory enzymatic tests in LSD.

Adults with lysosomal storage diseases in the undiagnosed diseases network.

OBJECTIVES: To review the referral and clinical characteristics of adult patients diagnosed with lysosomal storage diseases (LSD) through the Undiagnosed Diseases Network (UDN). METHODS: Retrospective review of both application and evaluation records for adults admitted to the UDN with a final diagnosis of a lysosomal storage disease. RESULTS: Ten patients were identified. Final diagnoses included late onset Tay Sachs, attenuated MPS I, MPS IIIA, MPS IIIB, and MPS IIIC. Most patients presented with neurocognitive changes. Prior to referral, all patients had been evaluated by neurology, four patients underwent phenotype specific panel testing that did not include the causative gene, and four patients had non-diagnostic clinical exome sequencing. CONCLUSIONS: LSDs figure highly in the differential diagnosis of neurometabolic disorders in pediatric onset progressive diseases. In adults, their subtle initial presentations overlap with symptoms of more common disorders and less practitioner awareness may lead to prolonged diagnostic challenges.

Inborn Errors of Metabolism: Becoming Ready for Rare.

Inborn errors of metabolism (IEMs) are a large group of disorders that can present in any age group and must be considered in the differential diagnosis for a variety of signs and symptoms appearing in infants and children. The rarity and complexity of these conditions often make them difficult to recognize, as they may mimic more common conditions. This review article discusses some of the more commonly presenting IEMs that are important for the general pediatrician to understand when evaluating a sick patient. Many of these diseases are also on the newborn screen, which pediatricians often encounter as first-line providers. Disorders that are discussed in detail herein include disorders of amino acid metabolism, including amino acidopathies and organic acidurias; urea cycle disorders; defects in fatty acid ß-oxidation; disorders of carbohydrate metabolism, including the glycogen storage diseases and galactosemia; and lysosomal storage diseases.

Real-world patient data on immunity and COVID-19 status of patients with MPS, Gaucher, and Pompe diseases from Turkey.

BACKGROUND: COVID-19 and lysosomal storage disorders (LSDs) share a common immunological pathway as they cause the release of cytokines in a similar pattern. We aimed to evaluate the immunity status and reveal the course of COVID-19 in patients with LSDs. RESULTS: The median age of 110 patients with LSDs was 129 months (range: 21-655), and all but one patient with mucopolysaccharidosis (MPS) type III were regularly receiving enzyme replacement therapy (ERT). In 53.6% (n = 56) of the patients (23 patients with Gaucher disease [10 type III, 13 type I], 26 patients with MPS [8 type VI, 11 type IVA, 1 type III, 3 type II, and 3 type I], and 7 patients with Pompe disease), an abnormality in at least one of the autoimmunity or immunodeficiency parameters was reported. Furthermore, 12 (57%) of 21 Gaucher cases (7 type III, 5 type I), 18 (40.9%) of 44 MPS cases (9 type IVA, 5 type VI, 1 type I, 2 type II, and 1 type III), and six (66%) of nine Pompe cases were reported to involve abnormalities in at least one of the parameters related to immunodeficiency. Immunoglobulin (Ig) M and IgA levels were reported to be lower, and there were abnormalities in the lymphocyte counts and subgroups in the MPS group. ANA was reported to be positive in one patient with Gaucher type III, anti-DNA in two patients with Gaucher type I and one patient with MPS type VI, antithyroglobulin in two patients with Gaucher type I, anti-TPO in one patient with Gaucher type I, TRAB in one patient with Gaucher type I, antiphospholipid IgM in three patients with Gaucher type III and one patient with Gaucher type I, anticardiolipin IgM in one patient with Gaucher type I, one patient with Gaucher type III, and one patient with MPS type II. However, no clinical presentation was consistent with the laboratory results except for one patient with Gaucher type I disease with Hashimoto thyroiditis. Two of the four patients who survived the COVID-19 infection with mild symptoms had a diagnosis of Gaucher type I, and no abnormality was detected in their laboratory tests. The other two patients had a diagnosis of MPS types VI and II. Immune dysfunction was detected in the patient with a diagnosis of MPS type II. Four of our patients were discharged without any sequelae. CONCLUSION: Problems with immunity did not cause any noticeable clinical results. Being well protected by reducing social contact might have played a role. However, we believe that it should be borne in mind that cardiac and pulmonary involvement, as well as immune dysfunction in LSDs, may cause an increased need for intensive care because of secondary bacterial infections.

Label-free multiplex electrochemical immunosensor for early diagnosis of lysosomal storage disorders.

Pompe, Gaucher and Krabbe disease are lysosomal storage disorders (LSDs) which are a group of genetic diseases that causes the accumulation of lipids in tissues and cells. Pompe, Gaucher and Krabbe are characterized by the deficiency of acid α-glucosidase (GAA), ß-Glucocerebrosidase (GBA) and galactocerebrosidase (GALC), and treatable if detected in their early stages. Here, we present the fabrication of an electrochemical immunosensor for the multiplexed quantification and simultaneous detection of GAA, GBA and GALC. The sensor was developed by electrodepositing gold nanoparticles (AuNPs) on an array of carbon electrodes, followed by the immobilization of GAA, GBA and GALC specific antibodies via functionalization with cysteamine and glutaraldehyde. The multiplexed immunosensor was able to successfully detect GAA, GBA and GALC at the femtomolar level with respective low detection limits of 0.12 pg/ml, 0.31 pg/ml and 0.18 pg/ml. The immunosensor showed good selectivity, sensitivity and good recovery when spiked in human serum, which confirms its possible applicability in point-of-care testing for the early diagnosis of LSDs.

Cut-off values of neonatal lysosomal storage disease-related enzymes detected by tandem mass spectrometry.

OBJECTIVE: To establish cut-off values of lysosomal storage disease (LSD)-related enzymes by tandem mass spectrometry. METHODS: A total of 26 689 newborns and 7 clinically confirmed LSD children underwent screening for LSDs (glycogen storage disease typeⅡ, Fabry disease, mucopolysaccharidosis type Ⅰ, Krabbe disease, Niemann-Pick disease A/B and Gaucher disease). The activities of LSD-related enzymes were detected by tandem mass spectrometry. The 20% of the median enzyme activity of each batch of acid ß-glucocerebrosidase, acid sphingomyelinase, ß-galactocerebroside, α- L-iduronidase and acid α-glucosidase, and the 30% of the median enzyme activity of α-galactosidase were taken as cut-off values of corresponding enzymes. The genetic diagnosis was performed in neonates whose enzyme activity was lower than 70% of the cut-off value. RESULTS: The enzyme activities of 7 clinically confirmed cases were all lower than the cut-off values. Among 26 689 newborns, 142 cases (0.53%) were suspected positive for LSDs, including 25 cases of ß-galactocerebroside deficiency, 1 case of α- L-iduronidase deficiency, 19 cases of α-galactosidase deficiency, and 97 cases of acid α-glucosidase deficiency. Eight infants were genetically diagnosed with LSDs, including 3 cases of glycogen storage disease type Ⅱ, 3 cases of Krabbe disease, and 2 cases of Fabry disease, with a positive predictive value of about 5.6%. Cut-off values ​​of the 6 LSD enzyme activities all showed a downward trend from March to August, and an upward trend from September to December. There was a statistically significant difference in LSD enzyme activity among different months ( P<0.05). CONCLUSION: The established cut-off values of LSD-related enzyme activities detected by tandem mass spectrometry can be used for screening LSDs in neonates, and the enzyme activity would be affected by temperature and humidity.