Diagnosis and management of glycogen storage disease type IV, including adult polyglucosan body disease: A clinical practice resource.

Glycogen storage disease type IV (GSD IV) is an ultra-rare autosomal recessive disorder caused by pathogenic variants in GBE1 which results in reduced or deficient glycogen branching enzyme activity. Consequently, glycogen synthesis is impaired and leads to accumulation of poorly branched glycogen known as polyglucosan. GSD IV is characterized by a remarkable degree of phenotypic heterogeneity with presentations in utero, during infancy, early childhood, adolescence, or middle to late adulthood. The clinical continuum encompasses hepatic, cardiac, muscular, and neurologic manifestations that range in severity. The adult-onset form of GSD IV, referred to as adult polyglucosan body disease (APBD), is a neurodegenerative disease characterized by neurogenic bladder, spastic paraparesis, and peripheral neuropathy. There are currently no consensus guidelines for the diagnosis and management of these patients, resulting in high rates of misdiagnosis, delayed diagnosis, and lack of standardized clinical care. To address this, a group of experts from the United States developed a set of recommendations for the diagnosis and management of all clinical phenotypes of GSD IV, including APBD, to support clinicians and caregivers who provide long-term care for individuals with GSD IV. The educational resource includes practical steps to confirm a GSD IV diagnosis and best practices for medical management, including (a) imaging of the liver, heart, skeletal muscle, brain, and spine, (b) functional and neuromusculoskeletal assessments, (c) laboratory investigations, (d) liver and heart transplantation, and (e) long-term follow-up care. Remaining knowledge gaps are detailed to emphasize areas for improvement and future research.

Development of a clinically validated in vitro functional assay to assess pathogenicity of novel GAA variants in patients with Pompe disease identified via newborn screening.

Purpose: The addition of Pompe disease (Glycogen Storage Disease Type II) to the Recommended Uniform Screening Panel in the United States has led to an increase in the number of variants of uncertain significance (VUS) and novel variants identified in the GAA gene. This presents a diagnostic challenge, especially in the setting of late-onset Pompe disease when symptoms are rarely apparent at birth. There is an unmet need for validated functional studies to aid in classification of GAA variants. Methods: We developed an in vitro mammalian cell expression and functional analysis system based on guidelines established by the Clinical Genome Resource (ClinGen) Sequence Variant Interpretation Working Group for PS3/BS3. We validated the assay with 12 control variants and subsequently analyzed eight VUS or novel variants in GAA identified in patients with a positive newborn screen for Pompe disease without phenotypic evidence of infantile-onset disease. Results: The control variants were analyzed in our expression system and an activity range was established. The pathogenic controls had GAA activity between 0% and 11% of normal. The benign or likely benign controls had an activity range of 54%-100%. The pseudodeficiency variant had activity of 17%. These ranges were then applied to the variants selected for functional studies. Using the threshold of <11%, we were able to apply PS3_ supporting to classify two variants as likely pathogenic (c.316C > T and c.1103G > A) and provide further evidence to support the classification of likely pathogenic for two variants (c.1721T > C and c.1048G > A). One variant (c.1123C > T) was able to be reclassified based on other supporting evidence. We were unable to reclassify three variants (c.664G > A, c.2450A > G, and c.1378G > A) due to insufficient or conflicting evidence. Conclusion: We investigated eight GAA variants as proof of concept using our validated and reproducible in vitro expression and functional analysis system. While additional work is needed to further refine our system with additional controls and different variant types in order to apply the PS3/BS3 criteria at a higher level, this tool can be utilized for variant classification to meet the growing need for novel GAA variant classification in the era of newborn screening for Pompe disease.

Beyond predicting diagnosis: Is there a role for measuring biotinidase activity in liver glycogen storage diseases?

Introduction: Biotinidase synthesis is needed to recycle biotin for essential metabolic reactions. Biotinidase activity is lower than normal levels in advanced liver disease but is higher in hepatic glycogen storage disorders (GSDs), however the cause of this association remains unclear. Methods: In this study, biotinidase activity was measured in plasma samples from 45 individuals with hepatic GSDs; GSDI (a, b; n = 25) and GSD III (a, b; n = 20), complemented by a chart review to associate biotinidase activity levels with clinical laboratory and imaging findings known to be implicated in these GSDs. Results: Our findings showed variation in biotinidase activity levels among subjects with GSD I and III; biotinidase activity correlated positively with hypertriglyceridemia in subjects with GSD I (r = 0.47, P = 0.036) and GSD III (r = 0.58, P = 0.014), and correlated negatively with age (r = -0.50, P = 0.03) in patients with GSD III. Additionally, biotinidase activity was reduced, albeit within the normal range in subjects with evidence of fibrosis/cirrhosis, as compared to subjects with hepatomegaly with or without steatosis (P = 0.002). Discussions: These findings suggest that abnormal lipid metabolism in GSD I and III and progressive liver disease in GSD III may influence biotinidase activity levels. We suggest that a prospective, multi-center, longitudinal study designed to assess the significance of monitoring biotinidase activity in a larger cohort with hepatic GSDs is warranted to confirm this observation. Take-home message: Altered lipid metabolism and advancing liver fibrosis/cirrhosis may influence biotinidase activity levels in patients with hepatic glycogen storage disease. Thus, longitudinal monitoring of biotinidase activity, when combined with clinical and other biochemical findings may be informative.

A comprehensive testing algorithm for the diagnosis of Fabry disease in males and females.

PURPOSE: Successful diagnosis of Fabry disease is often delayed or missed in patients, especially females, due to clinical heterogeneity and a lack of disease awareness. We present our experience testing for Fabry disease in high risk populations and discuss the relative sensitivities of α-galactosidase A (α-Gal A) enzyme activity in blood, plasma lyso-globotriaosylceramide (lyso-Gb3) biomarker, and GLA gene sequencing as diagnostic tests for Fabry disease in both males and females. METHODS: Patients with a clinical suspicion of Fabry disease were evaluated with enzyme analysis, biomarker analysis, and GLA sequencing. All three assays were performed from a single tube of EDTA blood. α-Gal A activity was determined in dried blood spots using a fluorometric assay, plasma lyso-Gb3 by UPLC-MS/MS, and GLA analysis by Sanger sequencing. RESULTS: Peripheral blood samples were received from 94 males and 200 females, of which 29% of males and 22% of females had a positive family history of Fabry disease. A likely pathogenic or pathogenic variant was identified in 87 (30%) patients (50 males, 37 females), confirming a diagnosis of Fabry disease. Of the remaining patients, 178 (61%) were determined to be unaffected based on normal enzyme activity (males) or normal lyso-Gb3 and negative sequencing results (females). A VUS was identified in 29 (10%) patients. The positive and negative predictive value of plasma lyso-Gb3 was 100% and 97% in males and 100% and 99% in females, respectively. This compares with 84% and 100% in males, and 58% and 50% in females for α-Gal A activity testing, respectively. CONCLUSIONS: Plasma lyso-Gb3 has high sensitivity and specificity for Fabry disease in males and females, and provides supportive diagnostic information when gene sequencing results are negative or inconclusive. α-Gal A activity in dried blood spots (DBS) has high sensitivity, but lower specificity for Fabry disease in males, as not all males with low α-Gal A activities were confirmed to have Fabry disease. Therefore, reflexing to gene sequencing and plasma lyso-Gb3 is useful for disease confirmation in males. For females, we found that first tier testing consisting of GLA sequencing and plasma lyso-Gb3 analysis provided the greatest sensitivity and specificity. Enzyme testing has lower sensitivity in females and is therefore less useful as a first-tier test. Enzyme analysis in females may still be helpful as a second-tier test in cases where molecular testing and plasma lyso-Gb3 analysis are uninformative and in vitro enzyme activity is low. SUMMARY: Sex-specific testing algorithms that prioritize tests with high specificity and sensitivity offer an effective means of identifying individuals with Fabry disease.

Evaluation of X-Linked Adrenoleukodystrophy Newborn Screening in North Carolina.

Importance: X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal genetic disorder in which an accumulation of very long-chain fatty acids leads to inflammatory demyelination in the central nervous system and to adrenal cortex atrophy. In 2016, X-ALD was added to the US Recommended Uniform Screening Panel. Objective: To evaluate the performance of a single-tier newborn screening assay for X-ALD in North Carolina. Design, Setting, and Participants: This diagnostic screening study was of all newborn dried blood spot specimens received in the North Carolina State Laboratory of Public Health between January 2 and June 1, 2018, excluding specimens of insufficient quantity or quality. A total of 52 301 specimens were screened for X-ALD using negative ionization high-performance liquid chromatography tandem mass spectrometry to measure C24:0- and C26:0-lysophosphatidylcholine concentrations. Sanger sequencing of the adenosine triphosphate-binding cassette subfamily D member 1 (ABCD1) gene was performed on screen-positive specimens. Exposures: A medical and family history, newborn physical examination, sequencing of ABCD1 on dried blood spot samples, and plasma analysis of very long-chain fatty acids were obtained for all infants with screen-positive results. Main Outcomes and Measures: The prevalence of X-ALD in North Carolina and the positive predictive value and false-positive rate for the first-tier assay were determined. Results: Of 52 301 infants tested (47.8% female, 50.6% male, and 1.7% other or unknown sex), 12 received screen-positive results. Of these 12 infants, 8 were confirmed with a genetic disorder: 3 male infants with X-ALD, 3 X-ALD-heterozygous female infants, 1 female infant with a peroxisome biogenesis disorder, and 1 female infant with Aicardi-Goutières syndrome. Four infants were initially classified as having false-positives results, including 3 female infants who were deemed unaffected and 1 male infant with indeterminate results on confirmatory testing. The positive predictive value for X-ALD or other genetic disorders for the first-tier assay was 67%, with a false-positive rate of 0.0057%. Conclusions and Relevance: This newborn screening pilot study reported results on 2 lysophosphatidylcholine analytes, identifying 3 male infants with X-ALD, 3 X-ALD-heterozygous female infants, and 3 infants with other disorders associated with increased very long-chain fatty acids. These results showed successful implementation in a public health program with minimal risk to the population. The findings will support other state laboratories planning to implement newborn screening for X-ALD and related disorders.

Characterization of immune response in Cross-Reactive Immunological Material (CRIM)-positive infantile Pompe disease patients treated with enzyme replacement therapy.

Enzyme replacement therapy (ERT) with rhGAA has improved clinical outcomes in infantile Pompe disease (IPD). A subset of CRIM-positive IPD patients develop high and sustained antibody titers (HSAT; ≥51,200) and/or sustained intermediate titer (SIT; ≥12,800 and <51,200), similar to CRIM-negative patients. To date there has been no systematic study to analyze the extent of IgG antibody response in CRIM-positive IPD. Such data would be critical and could serve as a comparator group for potential immune modulation approaches. A retrospective analysis of the dataset from the original rhGAA clinical trials final reports was conducted. CRIM-positive patients who received ERT monotherapy and had >6 months of antibody titer data available, were included in the study. Patients were classified based on their longitudinal antibody titers into HSAT, SIT, and low titer (LT; <12,800) groups. Of the 37 patients that met inclusion criteria, five (13%), seven (19%), and 25 (68%) developed HSAT, SIT, and LT, respectively. Median peak titers were 204,800 (51,200-409,600), 25,600 (12,800-51,200), and 800 (200-12,800) for HSAT, SIT, and LT groups, respectively. Median last titers were 102,400 (51,200-409,600), 1600 (200-25,600), and 400 (0-12,800) at median time since ERT initiation of 94 weeks (64-155 weeks), 104 weeks (86-144 weeks), and 130 weeks (38-182 weeks) for HSAT, SIT, and LT groups, respectively. 32% (12/37) of CRIM-positive IPD patients developed HSAT/SIT which may lead to limited ERT response and clinical decline. Further Studies are needed to identify CRIM-positive IPD patients at risk of developing HSAT/SIT, especially with the addition of Pompe disease to the newborn screening.

Diagnosis and management of glycogen storage diseases type VI and IX: a clinical practice resource of the American College of Medical Genetics and Genomics (ACMG).

PURPOSE: Glycogen storage disease (GSD) types VI and IX are rare diseases of variable clinical severity affecting primarily the liver. GSD VI is caused by deficient activity of hepatic glycogen phosphorylase, an enzyme encoded by the PYGL gene. GSD IX is caused by deficient activity of phosphorylase kinase (PhK), the enzyme subunits of which are encoded by various genes: ɑ (PHKA1, PHKA2), ß (PHKB), É£ (PHKG1, PHKG2), and δ (CALM1, CALM2, CALM3). Glycogen storage disease types VI and IX have a wide spectrum of clinical manifestations and often cannot be distinguished from each other, or from other liver GSDs, on clinical presentation alone. Individuals with GSDs VI and IX can present with hepatomegaly with elevated serum transaminases, ketotic hypoglycemia, hyperlipidemia, and poor growth. This guideline for the management of GSDs VI and IX was developed as an educational resource for health-care providers to facilitate prompt and accurate diagnosis and appropriate management of patients. METHODS: A national group of experts in various aspects of GSDs VI and IX met to review the limited evidence base from the scientific literature and provided their expert opinions. Consensus was developed in each area of diagnosis, treatment, and management. Evidence bases for these rare disorders are largely based on expert opinion, particularly when targeted therapeutics that have to clear the US Food and Drug Administration (FDA) remain unavailable. RESULTS: This management guideline specifically addresses evaluation and diagnosis across multiple organ systems involved in GSDs VI and IX. Conditions to consider in a differential diagnosis stemming from presenting features and diagnostic algorithms are discussed. Aspects of diagnostic evaluation and nutritional and medical management, including care coordination, genetic counseling, and prenatal diagnosis are addressed. CONCLUSION: A guideline that will facilitate the accurate diagnosis and optimal management of patients with GSDs VI and IX was developed. This guideline will help health-care providers recognize patients with GSDs VI and IX, expedite diagnosis, and minimize adverse sequelae from delayed diagnosis and inappropriate management. It will also help identify gaps in scientific knowledge that exist today and suggest future studies.

Current State of the Art of Newborn Screening for Lysosomal Storage Disorders.

Prospective full-population newborn screening for multiple lysosomal storage disorders (LSDs) is currently practiced in a few NBS programs, and several others are actively pursuing this course of action. Two platforms suitable for multiple LSD screening-tandem mass spectrometry (MS/MS) and digital microfluidic fluorometry (DMF)-are now commercially available with reagent kits. In this article, we review the methods currently used for prospective NBS for LSDs and objectively compare their workflows and the results from two programs in the United States that screen for the same four LSDs, one using MS/MS and the other DMF. The results show that the DMF platform workflow is simpler and generates results faster than MS/MS, enabling results reporting on the same day as specimen analysis. Furthermore, the performance metrics for both platforms while not identical, are broadly similar and do not indicate the superior performance of one method over the other. Results show a preponderance of inconclusive results for Pompe and Fabry diseases and for Hurler syndrome, due to genetic heterogeneity and other factors that can lead to low enzyme activities, regardless of the screening method. We conclude that either platform is a good choice but caution that post-analytical tools will need to be applied to improve the positive predictive value for these conditions.

Clinical and Molecular Variability in Patients with PHKA2 Variants and Liver Phosphorylase b Kinase Deficiency.

Glycogen storage disease (GSD) type IX is a rare disease of variable clinical severity affecting primarily the liver tissue. Individuals with liver phosphorylase b kinase (PhK) deficiency (GSD IX) can present with hepatomegaly with elevated serum transaminases, ketotic hypoglycemia, hyperlipidemia, and poor growth with considerable variation in clinical severity. PhK is a cAMP-dependent protein kinase that phosphorylates the inactive form of glycogen phosphorylase, phosphorylase b, to produce the active form, phosphorylase a. PhK is a heterotetramer; the alpha 2 subunit in the liver is encoded by the X-linked PHKA2 gene. About 75% of individuals with liver PhK deficiency have mutations in the PHKA2 gene; this condition is also known as X-linked glycogenosis (XLG). Here we report the variability in clinical severity and laboratory findings in 12 male patients from 10 different families with X-linked liver PhK deficiency caused by mutations in PHKA2. We found that there is variability in the severity of clinical features, including hypoglycemia and growth. We also report additional PHKA2 variants that were identified in 24 patients suspected to have liver PhK deficiency. The basis of the clinical variation in GSDIX due to X-linked PHKA2 gene mutations is currently not well understood. Creating systematic registries, and collecting longitudinal data may help in better understanding of this rare, but common, glycogen storage disorder. SYNOPSIS: Liver phosphorylase b kinase (PhK) deficiency caused due to mutations in X-linked PHKA2 is highly variable.

Severe Cardiomyopathy as the Isolated Presenting Feature in an Adult with Late-Onset Pompe Disease: A Case Report.

Many inborn errors of metabolism can cause cardiomyopathy. Cardiomyopathy associated with glycogen storage includes PRKAG2-associated glycogen storage disease (GSD), Danon disease, infantile-onset Pompe disease (GSD II), GSD III, GSD IV, and phosphofructokinase deficiency (Tarui disease or GSD VII).We present a 35-year-old female who presented with cardiomyopathy after a pregnancy complicated by primary hyperparathyroidism. She had enjoyed excellent health until her first pregnancy at age 33. One week postpartum, she developed dyspnea and an echocardiogram revealed left ventricular ejection fraction (LVEF) of 35%. A cardiac MRI was consistent with nonischemic cardiomyopathy with an infiltrative process. Endomyocardial biopsy showed striking sarcoplasmic vacuolization, excess glycogen by PAS staining, and frequent membrane-bound glycogen by electron microscopy, consistent with lysosomal GSD. Acid alpha-glucosidase (GAA) activity in skin fibroblasts was in the affected range for Pompe disease. Sequencing of the GAA gene revealed a paternally inherited pathogenic c.525delT (p.Glu176Argfs*45) and a de novo c.309C>G (p.Cys103Trp) with unknown pathogenicity. Testing of the familial mutations in her daughter indicated that the variants in the proband were in trans. 26-gene cardiomyopathy sequencing panel had normal results thereby excluding GSD III, Danon disease, Fabry disease, and PRKAG2-associated cardiomyopathy. Therefore, results strongly suggest a diagnosis of Pompe disease.Pompe disease has a broad disease spectrum, including infantile-onset (IOPD) and late-onset (LOPD) forms. LOPD typically presents with proximal muscle weakness and respiratory insufficiency in childhood or late adulthood. Our case may represent a very unusual presentation of adult LOPD with isolated cardiomyopathy without skeletal muscle involvement or respiratory failure.

Clinical Laboratory Experience of Blood CRIM Testing in Infantile Pompe Disease.

Cross-reactive immunological material (CRIM) status is an important prognostic factor in patients with infantile Pompe disease (IPD) being treated with enzyme replacement therapy. Western blot analysis of cultured skin fibroblast lysates has been the gold standard for determining CRIM status. Here, we evaluated CRIM status using peripheral blood mononuclear cell (PBMC) protein. For 6 of 33 patients (18%) CRIM status determination using PBMC was either indeterminate or discordant with GAA genotype or fibroblast CRIM analysis results. While the use of PBMCs for CRIM determination has the advantage of a faster turnaround time, further evaluation is needed to ensure the accuracy of CRIM results.

CRIM-negative infantile Pompe disease: characterization of immune responses in patients treated with ERT monotherapy.

PURPOSE: Enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) prolongs survival in infantile Pompe disease (IPD). However, the majority of cross-reactive immunologic material (CRIM)-negative (CN) patients have immune responses with significant clinical decline despite continued ERT. We aimed to characterize immune responses in CN patients with IPD receiving ERT monotherapy. METHODS: A chart review identified 20 CN patients with IPD treated with ERT monotherapy for ≥6 months. Patients were stratified by anti-rhGAA antibody titers: high sustained antibody titers (HSAT; ≥51,200) at least twice; low titers (LT; <6,400) throughout treatment; or sustained intermediate titers (SIT; 6,400-25,600). RESULTS: Despite early initiation of treatment, the majority (85%) of CN patients developed significant antibody titers, most with HSAT associated with invasive ventilation and death. Nearly all patients with HSAT had at least one nonsense GAA mutation, whereas the LT group exclusively carried splice-site or frameshift mutations. Only one patient in the HSAT group is currently alive after successful immune modulation in the entrenched setting. CONCLUSION: Immunological responses are a significant risk in CN IPD; thus induction of immune tolerance in the naive setting should strongly be considered. Further exploration of factors influencing immune responses is required, particularly with the advent of newborn screening for Pompe disease.

Diagnosis and management of glycogen storage disease type I: a practice guideline of the American College of Medical Genetics and Genomics.

PURPOSE: Glycogen storage disease type I (GSD I) is a rare disease of variable clinical severity that primarily affects the liver and kidney. It is caused by deficient activity of the glucose 6-phosphatase enzyme (GSD Ia) or a deficiency in the microsomal transport proteins for glucose 6-phosphate (GSD Ib), resulting in excessive accumulation of glycogen and fat in the liver, kidney, and intestinal mucosa. Patients with GSD I have a wide spectrum of clinical manifestations, including hepatomegaly, hypoglycemia, lactic acidemia, hyperlipidemia, hyperuricemia, and growth retardation. Individuals with GSD type Ia typically have symptoms related to hypoglycemia in infancy when the interval between feedings is extended to 3­4 hours. Other manifestations of the disease vary in age of onset, rate of disease progression, and severity. In addition, patients with type Ib have neutropenia, impaired neutrophil function, and inflammatory bowel disease. This guideline for the management of GSD I was developed as an educational resource for health-care providers to facilitate prompt, accurate diagnosis and appropriate management of patients. METHODS: A national group of experts in various aspects of GSD I met to review the evidence base from the scientific literature and provided their expert opinions. Consensus was developed in each area of diagnosis, treatment, and management. RESULTS: This management guideline specifically addresses evaluation and diagnosis across multiple organ systems (hepatic, kidney, gastrointestinal/nutrition, hematologic, cardiovascular, reproductive) involved in GSD I. Conditions to consider in the differential diagnosis stemming from presenting features and diagnostic algorithms are discussed. Aspects of diagnostic evaluation and nutritional and medical management, including care coordination, genetic counseling, hepatic and renal transplantation, and prenatal diagnosis, are also addressed. CONCLUSION: A guideline that facilitates accurate diagnosis and optimal management of patients with GSD I was developed. This guideline helps health-care providers recognize patients with all forms of GSD I, expedite diagnosis, and minimize adverse sequelae from delayed diagnosis and inappropriate management. It also helps to identify gaps in scientific knowledge that exist today and suggests future studies.

Variability of disease spectrum in children with liver phosphorylase kinase deficiency caused by mutations in the PHKG2 gene.

Liver phosphorylase b kinase (PhK) deficiency (glycogen storage disease type IX), one of the most common causes of glycogen storage disease, is caused by mutations in the PHKA2, PHKB, and PHKG2 genes. Presenting symptoms include hepatomegaly, ketotic hypoglycemia, and growth delay. Clinical severity varies widely. Autosomal recessive mutations in the PHKG2 gene, which cause about 10-15% of cases, have been associated with severe symptoms including increased risk of liver cirrhosis in childhood. We have summarized the molecular, biochemical, and clinical findings in five patients, age 5-16 years, diagnosed with liver PhK deficiency caused by PHKG2 gene mutations. We have identified five novel and two previously reported mutations in the PHKG2 gene in these five patients. Clinical severity was variable among these patients. Histopathological studies were performed for four of the patients on liver biopsy samples, all of which showed signs of fibrosis but not cirrhosis. One of the patients (aged 9 years) developed a liver adenoma which later resolved. All patients are currently doing well. Their clinical symptoms have improved with age and treatment. These cases add to the current knowledge of clinical variability in patients with PHKG2 mutations. Long term studies, involving follow-up of these patients into adulthood, are needed.

Abnormalities in glycogen metabolism in a patient with alpers' syndrome presenting with hypoglycemia.

Intermittent hypoglycemia has been described in association with Alpers' syndrome, a disorder caused by mutations in the mitochondrial DNA polymerase gamma gene. In some patients hypoglycemia may define the initial disease presentation well before the onset of the classical Alpers' triad of psychomotor retardation, intractable seizures, and liver failure. Correlating with the genotype, POLG pathogenicity is a result of increased mitochondrial DNA mutability, and mitochondrial DNA depletion resulting in energy deficient states. Hypoglycemia therefore could be secondary to any metabolic pathway affected by ATP deficiency. Although it has been speculated that hypoglycemia is due to secondary fatty acid oxidation defects or abnormal gluconeogenesis, the exact underlying etiology is still unclear. Here we present detailed studies on carbohydrate metabolism in an Alpers' patient who presented initially exclusively with intermittent episodes of hypoglycemia and ketosis. Our results do not support a defect in gluconeogenesis or fatty acid oxidation as the cause of hypoglycemia. In contrast, studies performed on liver biopsy suggested abnormal glycogenolysis. This is shown via decreased activities of glycogen brancher and debrancher enzymes with normal glycogen structure and increased glycogen on histology of the liver specimen. To our knowledge, this is the first report documenting abnormalities in glycogen metabolism in a patient with Alpers' syndrome.

Development of a fluorometric microtiter plate based enzyme assay for MPS IVA (Morquio type A) using dried blood spots.

Mucopolysaccharidosis type IVA or Morquio type-A disease is a hereditary lysosomal storage disorder caused by deficient activity of the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). The disease is caused by lysosomal accumulation of unprocessed glycosaminoglycans (GAGs) that manifests with severe to mild skeletal and cardiopulmonary abnormalities. We have developed a modified microtiter plate-based enzyme activity assay using dried blood spots and a fluorescent substrate for measuring specific GALNS activity to identify patients with MPS IVA.

Development of a fluorometric microtiter plate-based enzyme assay for arylsulfatase B (MPS VI) using dried blood spots.

Mucopolysaccharidosis type VI or Maroteaux-Lamy syndrome is an autosomal recessive lysosomal storage disorder caused by deficiency of arylsulfatase B (ARS-B) enzyme activity. It results in mild to severe multi-organ system failure from accumulation of undigested glycosaminoglycans (GAGs); dermatan sulfate and chondroitin-4-sulfate. We have developed a single-step enzyme assay using a fluorescent substrate and dried blood spots to measure ARS-B activity to identify disease patients. This assay is robust, reproducible, specific and convenient to perform.