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OBJECTIVE: Epalrestat, an aldose reductase inhibitor increases phosphomannomutase (PMM) enzyme activity in a PMM2-congenital disorders of glycosylation (CDG) worm model. Epalrestat also decreases sorbitol level in diabetic neuropathy. We evaluated the genetic, biochemical, and clinical characteristics, including the Nijmegen Progression CDG Rating Scale (NPCRS), urine polyol levels and fibroblast glycoproteomics in patients with PMM2-CDG. METHODS: We performed PMM enzyme measurements, multiplexed proteomics, and glycoproteomics in PMM2-deficient fibroblasts before and after epalrestat treatment. Safety and efficacy of 0.8 mg/kg/day oral epalrestat were studied in a child with PMM2-CDG for 12 months. RESULTS: PMM enzyme activity increased post-epalrestat treatment. Compared with controls, 24% of glycopeptides had reduced abundance in PMM2-deficient fibroblasts, 46% of which improved upon treatment. Total protein N-glycosylation improved upon epalrestat treatment bringing overall glycosylation toward the control fibroblasts' glycosylation profile. Sorbitol levels were increased in the urine of 74% of patients with PMM2-CDG and correlated with the presence of peripheral neuropathy, and CDG severity rating scale. In the child with PMM2-CDG on epalrestat treatment, ataxia scores improved together with significant growth improvement. Urinary sorbitol levels nearly normalized in 3 months and blood transferrin glycosylation normalized in 6 months. INTERPRETATION: Epalrestat improved PMM enzyme activity, N-glycosylation, and glycosylation biomarkers in vitro. Leveraging cellular glycoproteome assessment, we provided a systems-level view of treatment efficacy and discovered potential novel biosignatures of therapy response. Epalrestat was well-tolerated and led to significant clinical improvements in the first pediatric patient with PMM2-CDG treated with epalrestat. We also propose urinary sorbitol as a novel biomarker for disease severity and treatment response in future clinical trials in PMM2-CDG. ANN NEUROL 20219999:n/a-n/a. ispartof: ANNALS OF NEUROLOGY vol:90 issue:6 pages:887-900 ispartof: location:United States status: published

Congenital disorders of glycosylation (CDGs) are a continuously expanding group of monogenic disorders of glycoprotein and glycolipid biosynthesis that cause multisystem diseases. Individuals with ALG3-CDG frequently exhibit severe neurological involvement (epilepsy, microcephaly, and hypotonia), ocular anomalies, dysmorphic features, skeletal anomalies, and feeding difficulties. We present 10 unreported individuals diagnosed with ALG3-CDG based on molecular and biochemical testing with 11 novel variants in ALG3, bringing the total to 40 reported individuals. In addition to the typical multisystem disease seen in ALG3-CDG, we expand the symptomatology of ALG3-CDG to now include endocrine abnormalities, neural tube defects, mild aortic root dilatation, immunodeficiency, and renal anomalies. N-glycan analyses of these individuals showed combined deficiencies of hybrid glycans and glycan extension beyond Man(5)GlcNAc(2) consistent with their truncated lipid-linked precursor oligosaccharides. This spectrum of N-glycan changes is unique to ALG3-CDG. These expanded features of ALG3-CDG facilitate diagnosis and suggest that optimal management should include baseline endocrine, renal, cardiac, and immunological evaluation at the time of diagnosis and with ongoing monitoring.

Glycosylation is a critical post/peri-translational modification required for the appropriate development and function of the immune system. As an example, abnormalities in glycosylation can cause antibody deficiency and reduced lymphocyte signaling, although the phenotype can be complex given the diverse roles of glycosylation. Human MGAT2 encodes N-acetylglucosaminyltransferase II, which is a critical enzyme in the processing of oligomannose to complex N-glycans. Complex N-glycans are essential for immune system functionality, but only one individual with MGAT2-CDG has been described to have an abnormal immunologic evaluation. MGAT2-CDG (CDG-IIa) is a congenital disorder of glycosylation (CDG) associated with profound global developmental disability, hypotonia, early onset epilepsy, and other multisystem manifestations. Here, we report a 4-year old female with MGAT2-CDG due to a novel homozygous pathogenic variant in MGAT2, a 4-base pair deletion, c.1006_1009delGACA. In addition to clinical features previously described in MGAT2-CDG, she experienced episodic asystole, persistent hypogammaglobulinemia, and defective ex vivo mitogen and antigen proliferative responses, but intact specific vaccine antibody titers. Her infection history has been mild despite the testing abnormalities. We compare this patient to the 15 previously reported patients in the literature, thus expanding both the genotypic and phenotypic spectrum for MGAT2-CDG.

Background The prognosis of patients with Hereditary Tyrosinemia Type 1 (HT-1) has greatly improved with early detection through newborn screening and the introduction of nitisinone (NTBC) therapy. A recent guideline calls for periodic monitoring of biochemical markers and NTBC levels to tailor treatment; however, this is currently only achieved through a combination of clinical laboratory tests. We developed a multiplexed assay measuring relevant amino acids, succinylacetone (SUAC), and NTBC in dried blood spots (DBS) to facilitate treatment monitoring. Methods Tyrosine, phenylalanine, methionine, NTBC and SUAC were eluted from DBS with methanol containing internal standards for each analyte and analyzed by liquid chromatography tandem mass spectrometry over 6.5 min in the multiple reaction monitoring positive mode. Results Pre-analytical and analytical factors were studied and demonstrated a reliable assay. Chromatography resolved an unknown substance that falsely elevates SUAC concentrations and was present in all samples. To establish control and disease ranges, the method was applied to DBS collected from controls (n = 284) and affected patients before (n = 2) and after initiation of treatment (n = 29). In the treated patients SUAC concentrations were within the normal range over a wide range of NTBC levels. Conclusions This assay enables combined, accurate measurement of revelevant metabolites and NTBC in order to simplify treatment monitoring of patients with HT-1. In addition, the use of DBS allows for specimen collection at home to facilitate more standardization in relation to drug and dietary treatment.

Newborn screening (NBS) for Krabbe disease (KD) is performed by measurement of galactocerebrosidase (GALC) activity as the primary test. This revealed that GALC activity has poor specificity for KD. Psychosine (PSY) was proposed as a disease marker useful to reduce the false positive rate for NBS and for disease monitoring. We report a highly sensitive PSY assay that allows identification of KD patients with minimal PSY elevations. PSY was extracted from dried blood spots or erythrocytes with methanol containing d5-PSY as internal standard, and measured by liquid chromatography–tandem mass spectrometry. Analysis of PSY in samples from controls (N = 209), GALC pseudodeficiency carriers (N = 55), GALC pathogenic variant carriers (N = 27), patients with infantile KD (N = 26), and patients with late-onset KD (N = 11) allowed for the development of an effective laboratory screening and diagnostic algorithm. Additional longitudinal measurements were used to track therapeutic efficacy of hematopoietic stem cell transplantion (HSCT). This study supports PSY quantitation as a critical component of NBS for KD. It helps to differentiate infantile from later onset KD variants, as well as from GALC variant and pseudodeficiency carriers. Additionally, this study provides further data that PSY measurement can be useful to monitor KD progression before and after treatment.

Purpose To describe an efficient and effective multiplex screening strategy for sulfatide degradation disorders and mucolipidosis type II/III (MLII/III) using 3 mL of urine. Methods Glycosaminoglycans were analyzed by liquid chromatography-tandem mass spectrometry. Matrix assisted laser desorption/ionization-time of flight tandem mass spectrometry was used to identify free oligosaccharides and identify 22 ceramide trihexosides and 23 sulfatides, which are integrated by 670 calculated ratios. Collaborative Laboratory Integrated Reports (CLIR; https://clir.mayo.edu ) was used for post-analytical interpretation of the complex metabolite profile and to aid in the differential diagnosis of abnormal results. Results Multiplex analysis was performed on 25 sulfatiduria case samples and compiled with retrospective data from an additional 15 cases revealing unique patterns of biomarkers for each disorder of sulfatide degradation (MLD, MSD, and Saposin B deficiency) and for MLII/III, thus allowing the formulation of a novel algorithm for the biochemical diagnosis of these disorders. Conclusions Comprehensive and integrated urine screening could be very effective in the initial workup of patients suspected of having a lysosomal disorder as it covers disorders of sulfatide degradation and narrows down the differential diagnosis in patients with elevated glycosaminoglycans.

Background and Aims Vacuolar H+-ATP complex (V-ATPase) is a multisubunit protein complex required for acidification of intracellular compartments. At least five different factors are known to be essential for its assembly in the endoplasmic reticulum (ER). Genetic defects in four of these V-ATPase assembly factors show overlapping clinical features, including steatotic liver disease and mild hypercholesterolemia. An exception is the assembly factor vacuolar ATPase assembly integral membrane protein (VMA21), whose X-linked mutations lead to autophagic myopathy.Approach and Results Here, we report pathogenic variants in VMA21 in male patients with abnormal protein glycosylation that result in mild cholestasis, chronic elevation of aminotransferases, elevation of (low-density lipoprotein) cholesterol and steatosis in hepatocytes. We also show that the VMA21 variants lead to V-ATPase misassembly and dysfunction. As a consequence, lysosomal acidification and degradation of phagocytosed materials are impaired, causing lipid droplet (LD) accumulation in autolysosomes. Moreover, VMA21 deficiency triggers ER stress and sequestration of unesterified cholesterol in lysosomes, thereby activating the sterol response element-binding protein-mediated cholesterol synthesis pathways.Conclusions Together, our data suggest that impaired lipophagy, ER stress, and increased cholesterol synthesis lead to LD accumulation and hepatic steatosis. V-ATPase assembly defects are thus a form of hereditary liver disease with implications for the pathogenesis of nonalcoholic fatty liver disease.

Key Clinical Message We report a patient with developmental delay, autism, epilepsy, macrocephaly, facial dysmorphism, gastrointestinal, and behavioral issues due to EXT2 compound heterozygous likely pathogenic variants. This case report expands the EXT2 gene mutation database and the clinical spectrum of patients with deficiencies in the heparan sulfate pathway.

BACKGROUND Spinal muscular atrophy (SMA) is a progressive neuromuscular disorder with neuronal degeneration leading to muscular atrophy and respiratory failure. SMA is frequently caused by homozygous deletions that include exon 7 of the survival motor neuron gene SMN1, and its clinical course is influenced by the copy number of a nearby 5q SMN1 paralog, SMN2. Multiple ligation probe amplification (MLPA) and real-time quantitative PCR (qPCR) can detect SMN1 deletions. Yet, qPCR needs normalization or standard curves, and MLPA demands DNA concentrations above those obtainable from dried blood spots (DBSs). We developed a multiplex, droplet digital PCR (ddPCR) method for the simultaneous detection of SMN1 deletions and SMN2 copy number variation in DBS and other tissues. An SMN1 Sanger sequencing process for DBS was also developed. METHODS SMN1, SMN2, and RPP30 concentrations were simultaneously measured with a Bio-Rad AutoDG and QX200 ddPCR system. A total of 1530 DBSs and 12 SMA patients were tested. RESULTS Population studies confirmed 1 to 5 SMN1 exon 7 copies detected in unaffected specimens, whereas patients with SMA revealed 0 SMN1 copies. Intraassay and interassay imprecisions were CONCLUSIONS This ddPCR method is sensitive, specific, and applicable to newborn screening and carrier status determination for SMA. It can also be incorporated with a parallel ddPCR T-cell excision circles assay for severe combined immunodeficiencies.

Genomics methodologies have significantly improved elucidation of Mendelian disorders. The combination with high-throughput functional-omics technologies potentiates the identification and confirmation of causative genetic variants, especially in singleton families of recessive inheritance. In a cohort of 99 individuals with abnormal Golgi glycosylation, 47 of which being unsolved, glycomics profiling was performed of total plasma glycoproteins. Combination with whole-exome sequencing in 31 cases revealed a known genetic defect in 15 individuals. To identify additional genetic factors, hierarchical clustering of the plasma glycomics data was done, which indicated a subgroup of four patients that shared a unique glycomics signature of hybrid type N-glycans. In two siblings, compound heterozygous mutations were found in SLC10A7, a gene of unknown function in human. These included a missense mutation that disrupted transmembrane domain 4 and a mutation in a splice acceptor site resulting in skipping of exon 9. The two other individuals showed a complete loss of SLC10A7 mRNA. The patients' phenotype consisted of amelogenesis imperfecta, skeletal dysplasia, and decreased bone mineral density compatible with osteoporosis. The patients' phenotype was mirrored in SLC10A7 deficient zebrafish. Furthermore, alizarin red staining of calcium deposits in zebrafish morphants showed a strong reduction in bone mineralization. Cell biology studies in fibroblasts of affected individuals showed intracellular mislocalization of glycoproteins and a defect in post-Golgi transport of glycoproteins to the cell membrane. In contrast to yeast, human SLC10A7 localized to the Golgi. Our combined data indicate an important role for SLC10A7 in bone mineralization and transport of glycoproteins to the extracellular matrix. ispartof: HUMAN MOLECULAR GENETICS vol:27 issue:17 pages:3029-3045 ispartof: location:England status: published

N-glycanase deficiency (NGLY1 deficiency, NGLY1-CDDG), the first autosomal recessive congenital disorder of N-linked deglycosylation (CDDG), is caused by pathogenic variants in NGLY1. The majority of affected individuals have been identified using exome or genome sequencing. To date, no reliable, clinically available biomarkers have been identified. Urine oligosaccharide analysis was included as part of a routine evaluation for possible biomarkers in patients with confirmed NGLY1-CDDG. During the qualitative review of oligosaccharide profiles by an experienced laboratory director an abnormal analyte with a proposed structure of Neu5Ac1Hex1GlcNAc1-Asn was identified in NGLY1-CDDG patient urine samples. The same species has been observed in profiles from individuals affected with aspartylglucosaminuria, although the complete spectra are not identical. Additional studies using tandem mass spectrometry confirmed the analyte's structure. In addition to the known NGLY1-CDDG patients identified by this analysis, a single case was identified in a population referred for clinical testing who subsequently had a diagnosis of NGLY1-CDDG confirmed by molecular testing. Urine oligosaccharide screening by MALDI-TOF MS can identify individuals with NGLY1-CDDG. In addition, this potential biomarker might also be used to monitor the effectiveness of therapeutic options as they become available.

Transport of activated nucleotide-sugars into the Golgi is critical for proper glycosylation and mutations in these transporters cause a group of rare genetic disorders termed congenital disorders of glycosylation. We performed exome sequencing on an individual with a profound neurological presentation and identified rare compound heterozygous mutations, p.Thr156Arg and p.Glu196Lys, in the CMP-sialic acid transporter, SLC35A1. Patient primary fibroblasts and serum showed a considerable decrease in the amount of N- and O-glycans terminating in sialic acid. Direct measurement of CMP-sialic acid transport into the Golgi showed a substantial decrease in overall rate of transport. Here we report the identification of the third patient with CMP-sialic acid transporter deficiency, who presented with severe neurological phenotype, but without hematological abnormalities.

Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS) is a recently described autosomal dominant syndrome of developmental delay, cortical vision loss with optic nerve atrophy, epilepsy, and autism spectrum disorder. Due to its many overlapping features with congenital disorders of glycosylation (CDG), the differential diagnosis between these disorders may be difficult and relies on molecular genetic testing. We report on a 31-year-old female initially diagnosed with ALG6-CDG based on glycosylation abnormalities on transferrin isoelectrofocusing and targeted genetic testing, and later diagnosed with BBSOAS by whole-exome sequencing (WES). Functional studies on cultured fibroblasts including Western blotting and RT-qPCR, as well as mass spectrometry of glycosylated transferrin and MALDI-TOF glycan analysis in serum, demonstrated normal glycosylation in this patient. In this report, we extend the phenotype of BBSOAS with ataxia and protein-losing enteropathy. This case is illustrative of the utility of whole exome sequencing in the diagnostic odyssey, and the potential pitfalls of relying on focused genetic testing results for diagnosis of conditions with complex overlapping phenotypes.

BACKGROUND Newborn screening for lysosomal storage disorders (LSD) has revealed that late-onset variants of these conditions are unexpectedly frequent and therefore may evade diagnosis. We developed an efficient and cost-effective multiplex assay to diagnose six LSDs and several peroxisomal disorders in patients presenting with diverse phenotypes at any age. METHODS Three 3-mm dried blood spot (DBS) punches were placed into individual microtiter plates. One disc was treated with a cocktail containing acid sphingomyelinase-specific substrate and internal standard (IS). To the second DBS we added a cocktail containing substrate and IS for β-glucosidase, acid α-glucosidase, α-galactosidase A, galactocerebrosidase, and α-L-iduronidase. The third DBS was extracted with methanol containing d4-C26 lysophosphatidylcholine as IS and stored until the enzyme plates were combined and purified by liquid–liquid and solid-phase extraction. The extracts were evaporated, reconstituted with the extract from the lysophosphatidylcholine plate, and analyzed by flow injection tandem mass spectrometry. RESULTS Reference intervals were determined by analysis of 550 samples from healthy controls. DBS from confirmed patients with 1 of the 6 LSDs (n = 33), X-adrenoleukodystrophy (n = 9), or a peroxisomal biogenesis disorder (n = 5), as well as carriers for Fabry disease (n = 17) and X-adrenoleukodystrophy (n = 5), were analyzed for assay validation. Prospective clinical testing of 578 samples revealed 25 patients affected with 1 of the detectable conditions. CONCLUSIONS Our flow injection tandem mass spectrometry approach is amenable to high-throughput population screening for Hurler disease, Gaucher disease, Niemann–Pick A/B disease, Pompe disease, Krabbe disease, Fabry disease, X-adrenoleukodystrophy, and peroxisomal biogenesis disorder in DBS.

Congenital disorders of glycosylation (CDG) arise from pathogenic mutations in over one hundred genes leading to impaired protein or lipid glycosylation. ALG1 encodes a β1,4 mannosyltransferase that catalyzes the addition of the first of nine mannose moieties to form a dolichol-lipid linked oligosaccharide intermediate (DLO) required for proper N-linked glycosylation. ALG1 mutations cause a rare autosomal recessive disorder termed ALG1-CDG. To date thirteen mutations in eighteen patients from fourteen families have been described with varying degrees of clinical severity. We identified and characterized thirty-nine previously unreported cases of ALG1-CDG from thirty-two families and add twenty-six new mutations. Pathogenicity of each mutation was confirmed based on its inability to rescue impaired growth or hypoglycosylation of a standard biomarker in an alg1-deficient yeast strain. Using this approach we could not establish a rank order comparison of biomarker glycosylation and patient phenotype, but we identified mutations with a lethal outcome in the first two years of life. The recently identified protein-linked xeno-tetrasaccharide biomarker, NeuAc-Gal-GlcNAc2, was seen in all twenty-seven patients tested. Our study triples the number of known patients and expands the molecular and clinical correlates of this disorder.

Newborn screening (NBS) for Krabbe disease (KD) in New York and Missouri is conducted by measuring galactocerebrosidase (GALC) activity using tandem mass spectrometry (MS/MS). These NBS efforts have shown that the incidence of KD is unexpectedly low (1:400,000) while many individuals (ca. 1:6000) with reduced GALC activity and genotypes of uncertain significance are detected and subjected to follow up testing. Psychosine (PSY) is a putative marker of KD progression and can be measured in dried blood spots (DBS). We sought to determine the role that PSY levels play in NBS for KD, follow up, and treatment monitoring. PSY was eluted from DBS with methanol containing N,N-dimethyl-D-erythro-sphingosine as internal standard (IS). Liquid chromatography-MS/MS was conducted over 17 minutes in the multiple reaction monitoring positive mode to follow the precursor to product species transitions for PSY and IS. Separation of the structural isomers PSY and glucosylsphingosine was accomplished by hydrophilic interaction liquid chromatography. Pre-analytical and analytical factors were studied and revealed satisfactory results. PSY was also measured in DBS collected from controls (range

The mucopolysaccharidoses (MPSs) are a group of disorders caused by a deficiency of enzymes involved in the stepwise degradation of dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), or chondroitin sulfate (CS). We describe a LC-MS/MS method modification for routine determination of MPS which improves upon the existing method with respect to sample volume, sample preparation, and reduces the potential for false positive and false negative diagnosis when relying on a qualitative method. DS, HS and KS are enzymatically digested to disaccharides by the addition of chondroitinase B, heparinase I, II, III, and keratanase, while CS is enzymatically digested to disaccharides by chondroitinase AC prior to LC-MS/MS analysis. Intra- and inter-assay precision was assessed using urine samples (N=3) of varying concentrations. Intra-assay precision CVs ranged from 4.7 - 8.4% for DS 4.9 - 10.4% for HS 4.1 - 9.1% for KS and 4.9 - 5.3% for CS, respectively (N=20). Inter-assay precision CVs ranged from 10.9 - 14.2% for DS 8.9 - 14.9% for HS 9.2 - 14.8% for KS and 6.9 - 8.8% for CS, respectively in the same specimens (N=20). Three urine specimens spiked with MPS standard solutions at 2 levels exhibited recoveries ranging from 88% - 112%. Pediatric urine (age 1 day

Human phosphoglucomutase 3 (PGM3) catalyzes the conversion of N-acetyl-glucosamine (GlcNAc)-6-phosphate into GlcNAc-1-phosphate during the synthesis of uridine diphosphate (UDP)-GlcNAc, a sugar nucleotide critical to multiple glycosylation pathways. We identified three unrelated children with recurrent infections, congenital leukopenia including neutropenia, B and T cell lymphopenia, and progression to bone marrow failure. Whole-exome sequencing demonstrated deleterious mutations in PGM3 in all three subjects, delineating their disease to be due to an unsuspected congenital disorder of glycosylation (CDG). Functional studies of the disease-associated PGM3 variants in E. coli cells demonstrated reduced PGM3 activity for all mutants tested. Two of the three children had skeletal anomalies resembling Desbuquois dysplasia: short stature, brachydactyly, dysmorphic facial features, and intellectual disability. However, these additional features were absent in the third child, showing the clinical variability of the disease. Two children received hematopoietic stem cell transplantation of cord blood and bone marrow from matched related donors; both had successful engraftment and correction of neutropenia and lymphopenia. We define PGM3-CDG as a treatable immunodeficiency, document the power of whole-exome sequencing in gene discoveries for rare disorders, and illustrate the utility of genomic analyses in studying combined and variable phenotypes.

Congenital disorders of glycosylation (CDG) are rare genetic defects mainly in the post-translational modification of proteins via attachment of carbohydrate chains. We describe an infant with the phenotype of a congenital muscular dystrophy, with borderline microcephaly, hypotonia, camptodactyly, severe motor delay, and elevated creatine kinase. Muscle biopsy showed muscular dystrophy and reduced α-dystroglycan immunostaining with glycoepitope-specific antibodies in a pattern diagnostic of dystroglycanopathy. Carbohydrate deficient transferrin testing showed a pattern pointing to a CDG type I. Sanger sequencing of DPM1 (dolichol-P-mannose synthase subunit 1) revealed a novel Gly > Val change c.455G > T missense mutation resulting in p.Gly152Val) of unknown pathogenicity and deletion/duplication analysis revealed an intragenic deletion from exons 3 to 7 on the other allele. DPM1 activity in fibroblasts was reduced by 80%, while affinity for the substrate was not depressed, suggesting a decrease in the amount of active enzyme. Transfected cells expressing tagged versions of wild type and the p.Gly152Val mutant displayed reduced binding to DPM3, an essential, non-catalytic subunit of the DPM complex, suggesting a mechanism for pathogenicity. The present case is the first individual described with DPM1-CDG (CDG-Ie) to also have clinical and muscle biopsy findings consistent with dystroglycanopathy.

BACKGROUND Friedreich ataxia (FRDA) is caused by reduced frataxin (FXN) concentrations. A clinical diagnosis is typically confirmed by DNA-based assays for GAA-repeat expansions or mutations in the FXN (frataxin) gene; however, these assays are not applicable to therapeutic monitoring and population screening. To facilitate the diagnosis and monitoring of FRDA patients, we developed an immunoassay for measuring FXN. METHODS Antibody pairs were used to capture FXN and an internal control protein, ceruloplasmin (CP), in 15 μL of whole blood (WB) or one 3-mm punch of a dried blood spot (DBS). Samples were assayed on a Luminex LX200 analyzer and validated according to standard criteria. RESULTS The mean recovery of FXN from WB and DBS samples was 99%. Intraassay and interassay imprecision (CV) values were 4.9%–13% and 9.8%–16%, respectively. The FXN limit of detection was 0.07 ng/mL, and the reportable range of concentrations was 2–200 ng/mL. Reference adult and pediatric FXN concentrations ranged from 15 to 82 ng/mL (median, 33 ng/mL) for DBS and WB. The FXN concentration range was 12–22 ng/mL (median, 15 ng/mL) for FRDA carriers and 1–26 ng/mL (median 5 ng/mL) for FRDA patients. Measurement of the FXN/CP ratio increased the ability to distinguish between patients, carriers, and the reference population. CONCLUSIONS This assay is applicable to the diagnosis and therapeutic monitoring of FRDA. This assay can measure FXN and the control protein CP in both WB and DBS specimens with minimal sample requirements, creating the potential for high-throughput population screening of FRDA.

Biochemical analysis and whole-exome sequencing identified mutations in the Golgi-localized UDP-galactose transporter SLC35A2 that define an undiagnosed X-linked congenital disorder of glycosylation (CDG) in three unrelated families. Each mutation reduced UDP-galactose transport, leading to galactose-deficient glycoproteins. Two affected males were somatic mosaics, suggesting that a wild-type SLC35A2 allele may be required for survival. In infancy, the commonly used biomarker transferrin showed abnormal glycosylation, but its appearance became normal later in childhood, without any corresponding clinical improvement. This may indicate selection against cells carrying the mutant allele. To detect other individuals with such mutations, we suggest transferrin testing in infancy. Here, we report somatic mosaicism in CDG, and our work stresses the importance of combining both genetic and biochemical diagnoses.

Fabry disease, metachromatic leukodystrophy (MLD), multiple sulfatase deficiency (MSD), saposin B deficiency (SAPB) and mucolipidosis II (MLII) are disorders caused by enzyme deficiencies leading to accumulation of ceramide trihexosides and/or sulfatides in tissues and increased excretion in urine. Ceramide trihexosides and sulfatides are extracted in chloroform from urine using a simple extraction method (modified from Alharbi et al, 2016) and analyzed in both positive mode for ceramide trihexosides and negative mode for sulfatides using MALDI-TOF mass spectrometry. Validation included specimens from confirmed patients affected with Fabry disease (N=16 males N=11 females), MLD (N=23), MSD (N=7), SAPB (N=2) and MLII (N=5). Fabry disease has a specific ceramide trihexoside profile pattern without presence of sulfatides. Overlapping sulfatide profiles, without ceramide trihexosides, for MLD and MSD can be confirmed by glucosaminoglycan analysis. SAPB has both the ceramide trihexoside profile pattern similar to Fabry disease and the sulfatide profile pattern similar to MLD and MSD, but reduced peak intensities. Overlapping profiles to MLII, which can have both ceramide trihexosides and sulfatides as well, can be confirmed by oligosaccharide analysis. To further delineate profile patterns, peak ratios to respective internal standards in positive and negative modes (N=30) are analyzed using a custom-made automated post-analytical multivariate pattern recognition software, Collaborative Laboratory Integrated Reports (CLIR https://clir.mayo.edu). This software generates additional ratios (N=248) between the analyte/IS ratios to aid in reliable pattern recognition.

BACKGROUND Newborn screening (NBS) for inborn errors of propionate, methionine, and cobalamin metabolism relies on finding abnormal concentrations of methionine and propionylcarnitine. These analytes are not specific for these conditions and lead to frequent false-positive results. More specific markers are total homocysteine (tHCY), methylmalonic acid (MMA), and methylcitric acid (MCA), but these markers are not detected by current NBS methods. To improve this situation, we developed a method for the detection of tHCY, MMA, and MCA in dried blood spots (DBSs) by liquid chromatography–tandem mass spectrometry (LC-MS/MS). METHODS The analytes were extracted from a single 4.8-mm DBS punch with acetonitrile:water:formic acid (59:41:0.42) containing dithiothreitol and isotopically labeled standards (d3-MMA, d3-MCA, d8-homocystine). The extract was dried and treated with 3 N HCl in n-butanol to form butylesters. After evaporation of the butanol, the residue was reconstituted and centrifuged and the supernatant was subjected to LC-MS/MS analysis. Algorithms were developed to apply this method as an efficient and effective second-tier assay on samples with abnormal results by primary screening. RESULTS The 99th percentiles determined from the analysis of 200 control DBSs for MMA, MCA, and HCY were 1.5, 0.5, and 9.8 μmol/L, respectively. Since 2005, prospective application of this second-tier analysis to 2.3% of all NBS samples led to the identification of 13 affected infants. CONCLUSIONS Application of this assay reduced the false-positive rate and improved the positive predictive value of NBS for conditions associated with abnormal propionylcarnitine and methionine concentrations.

To validate a 2-tier approach for newborn screening (NBS) of remethylation defects.The original NBS dried blood spots of 5 patients with a proven diagnosis of a remethylation disorder and 1 patient with biochemical evidence of such disorder were analyzed retrospectively to determine disease ranges for methionine (Met; 4.7-8.1 micromol/L; 1 percentile of healthy population, 11.1 micromol/L), the methionine/phenylalanine ratio (Met/Phe; 0.09-0.16; 1 percentile of healthy population, 0.22), and total homocysteine (tHcy; 42-157 micromol/L; 99 percentile of normal population, 14.7 micromol/L). These preliminary disease ranges showed a sufficient degree of segregation from healthy population data, allowing the selection of cutoff values. A simple algorithm was then developed to reflex cases to a second-tier testing for tHcy, which has been applied prospectively for 14 months.A total of 86 333 NBS samples were tested between January 2007 and March 2008, and 233 of them (0.27%) met the criteria for second-tier testing of tHcy. All cases revealed concentrations of tHcy15 micromol/L and were considered unaffected. No false-negative results have been reported with a state-wide system based on 2 combined metabolic clinics and laboratories that cover the entire Minnesota population and border areas of neighboring states.Pending more conclusive evidence from the prospective identification of additional true-positive cases, NBS for remethylation disorders appears to be feasible with existing methodologies, with only a marginal increase of the laboratory workload.

In this issue of Clinical Chemistry , a group led by Miao He at Emory University describes a new method for the qualitative profiling of free oligosaccharides and other glycoaminoacids in urine samples (1). The analytical platform is MALDI-TOF mass spectrometry, a technology that is making a rapid transition from an elite proteomics research tool to a diagnostic tool broadly adopted in laboratory medicine, particularly in clinical microbiology (2). The authors have successfully targeted 11 different lysosomal disorders that have very similar clinical presentations and are virtually indistinguishable in the first few months of life, when a diagnostic evaluation is likely to take place. Notably, the authors have provided a clear and objective road map to the interpretation of abnormal profiles. Table 1 of this report is a truly fundamental contribution that should pave the way to rapid implementation of this method as well as objective and consistent interpretation of clinical results. This outcome has been hardly the case for the last 4 decades. Indeed, the significance of this report cannot be fully appreciated by …

We report the artifactual elevation of homogentisic acid (HGA) in urine from alkaptonuric patients after replacing the creatinine method (Jaffe reaction) in our laboratory with an automated enzymatic method. Samples with elevated HGA by GC-MS had lower creatinine values as determined by the enzymatic method than by the Jaffe reaction. The low creatinine values were due to interference by HGA in the enzymatic method. The enzymatic method is unsuitable for creatinine determination in urine of patients with alkaptonuria.

Congenital disorders of glycosylation (CDG) are inherited autosomal-recessive diseases that impair N-glycosylation. Approximately 20% of patients do not survive beyond the age of 5 years old as a result of widespread organ dysfunction. Although most patients receive a CDG diagnosis based on abnormal glycosylation of transferrin, this test cannot provide a genetic diagnosis; indeed, many patients with abnormal transferrin do not have mutations in any known CDG genes. Here, we combined biochemical analysis with whole-exome sequencing (WES) to identify the genetic defect in an untyped CDG patient, and we found a 22 bp deletion and a missense mutation in DDOST, whose product is a component of the oligosaccharyltransferase complex that transfers the glycan chain from a lipid carrier to nascent proteins in the endoplasmic reticulum lumen. Biochemical analysis with three biomarkers revealed that N-glycosylation was decreased in the patient's fibroblasts. Complementation with wild-type-DDOST cDNA in patient fibroblasts restored glycosylation, indicating that the mutations were pathological. Our results highlight the power of combining WES and biochemical studies, including a glyco-complementation system, for identifying and confirming the defective gene in an untyped CDG patient. This approach will be very useful for uncovering other types of CDG as well.