Screening data from 19 patients with late-onset Pompe disease for a phase I clinical trial of AAV8 vector-mediated gene therapy.

Late-onset Pompe disease (LOPD) is a multisystem disorder with significant myopathy. The standard treatment is enzyme replacement therapy (ERT), a therapy that is lifesaving, yet with limitations. Clinical trials have emerged for other potential treatment options, including adeno-associated virus (AAV) gene therapy. We present clinical parameters and AAV antibody titers for 19 individuals with LOPD undergoing screening for a Phase I clinical trial with an AAV serotype 8 vector targeting hepatic transduction (AAV2/8-LSPhGAA). Reported clinical parameters included GAA genotype, assessments of muscle function, upright and supine spirometry, anti-recombinant human GAA antibody titers, and biomarkers. Variability in measured parameters and phenotypes of screened individuals was evident. Eligibility criteria required that all participants have six-minute walk test (6MWT) and upright forced vital capacity (FVC) below the expected range for normal individuals, and were stably treated with ERT for >2 years. All participants had Pompe disease diagnosed by enzyme deficiency, and all had the common c.-32-13T>G LOPD pathogenic variant. Screening identified 14 patients (74%) with no or minimal detectable neutralizing antibodies against AAV8 (titer ≤1:5). 6MWT distance varied significantly (percent of expected distance ranging from 24% to 91% with an average of 60 and standard deviation of 21). Upright FVC percent predicted ranged from 35% predicted to 91% predicted with an average of 66 and standard deviation of 18. None of the participants had significantly elevated alanine transaminase, which has been associated with LOPD and could complicate screening for hepatitis related to AAV gene therapy. We review the parameters considered in screening for eligibility for a clinical trial of AAV8 vector-mediated gene therapy.

Phase I study of liver depot gene therapy in late-onset Pompe disease.

Gene therapy with an adeno-associated virus serotype 8 (AAV8) vector (AAV8-LSPhGAA) could eliminate the need for enzyme replacement therapy (ERT) by creating a liver depot for acid α-glucosidase (GAA) production. We report initial safety and bioactivity of the first dose (1.6 × 1012 vector genomes/kg) cohort (n = 3) in a 52-week open-label, single-dose, dose-escalation study (NCT03533673) in patients with late-onset Pompe disease (LOPD). Subjects discontinued biweekly ERT after week 26 based on the detection of elevated serum GAA activity and the absence of clinically significant declines per protocol. Prednisone (60 mg/day) was administered as immunoprophylaxis through week 4, followed by an 11-week taper. All subjects demonstrated sustained serum GAA activities from 101% to 235% of baseline trough activity 2 weeks following the preceding ERT dose. There were no treatment-related serious adverse events. No subject had anti-capsid T cell responses that decreased transgene expression. Muscle biopsy at week 24 revealed unchanged muscle glycogen content in two of three subjects. At week 52, muscle GAA activity for the cohort was significantly increased (p < 0.05). Overall, these initial data support the safety and bioactivity of AAV8-LSPhGAA, the safety of withdrawing ERT, successful immunoprophylaxis, and justify continued clinical development of AAV8-LSPhGAA therapy in Pompe disease.

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.

Chronic liver disease and impaired hepatic glycogen metabolism in argininosuccinate lyase deficiency.

BACKGROUNDLiver disease in urea cycle disorders (UCDs) ranges from hepatomegaly and chronic hepatocellular injury to cirrhosis and end-stage liver disease. However, the prevalence and underlying mechanisms are unclear.METHODSWe estimated the prevalence of chronic hepatocellular injury in UCDs using data from a multicenter, longitudinal, natural history study. We also used ultrasound with shear wave elastography and FibroTest to evaluate liver stiffness and markers of fibrosis in individuals with argininosuccinate lyase deficiency (ASLD), a disorder with high prevalence of elevated serum alanine aminotransferase (ALT). To understand the human observations, we evaluated the hepatic phenotype of the AslNeo/Neo mouse model of ASLD.RESULTSWe demonstrate a high prevalence of elevated ALT in ASLD (37%). Hyperammonemia and use of nitrogen-scavenging agents, 2 markers of disease severity, were significantly (P < 0.001 and P = 0.001, respectively) associated with elevated ALT in ASLD. In addition, ultrasound with shear wave elastography and FibroTest revealed increased echogenicity and liver stiffness, even in individuals with ASLD and normal aminotransferases. The AslNeo/Neo mice mimic the human disorder with hepatomegaly, elevated aminotransferases, and excessive hepatic glycogen noted before death (3-5 weeks of age). This excessive hepatic glycogen is associated with impaired hepatic glycogenolysis and decreased glycogen phosphorylase and is rescued with helper-dependent adenovirus expressing Asl using a liver-specific (ApoE) promoter.CONCLUSIONOur results link urea cycle dysfunction and impaired hepatic glucose metabolism and identify a mouse model of liver disease in the setting of urea cycle dysfunction.TRIAL REGISTRATIONThis study has been registered at ClinicalTrials.gov (NCT03721367, NCT00237315).FUNDINGFunding was provided by NIH, Burroughs Wellcome Fund, NUCDF, Genzyme/ACMG Foundation, and CPRIT.

Variable clinical features and genotype-phenotype correlations in 18 patients with late-onset Pompe disease.

BACKGROUND: Pompe disease is a lysosomal storage disorder caused by the deficiency of enzyme acid alpha-glucosidase (GAA) which results in accumulation of glycogen, particularly in the skeletal, cardiac, and smooth muscles. The late-onset form with symptoms presenting in childhood through adulthood, is characterized by proximal muscle weakness, respiratory insufficiency, and unlike the infantile-onset form often with no cardiac involvement. METHODS: We report our experience with 18 adult patients (14 males/4 females) with Pompe disease, several of whom had unique findings and novel pathogenic variants. Patients ranged in ages from 22-74 years (mean 53.7 years) and were diagnosed at an age range of 11-65 years (mean 43.6 years), often after a history of progressive muscle disease of several years' duration. All 18 patients were treated with alglucosidase alfa (Lumizyme) and their response to treatment was monitored by measurements of their pulmonary function and muscle weakness, six-minute walk test (6MWT), and other functional studies. RESULTS: Genetic sequencing revealed that 16 out of 18 individuals had the common c.-32-13T>G splicing variant, and six patients, including two sibships had four novel pathogenic variants: c.1594G>A, c.2655_2656delCG, c.1951-1952delGGinsT, and c.1134C>G. A male with the c.1594G>A variant developed an intracerebral aneurysm at the age of 43 years treated with surgery. Two siblings with the c.2655_2656delCG developed very high antibody titers, one of whom developed a severe infusion reaction. Other clinical features included BiPAP requirement in twelve, tinnitus in seven, scoliosis in five, cardiomyopathy in three, one individual was diagnosed with a cerebral aneurysm who underwent successful Penumbra coil placement, and another individual was diagnosed with both Graves' disease and testicular cancer. CONCLUSIONS: Our study illustrates significant variability in the range of clinical features, and the variable clinical response to enzyme replacement therapy. It also alerts us to the importance of careful monitoring and early management of complications. Possible genotype-phenotype associations with the novel mutations identified may emerge with larger studies.

Liver fibrosis during clinical ascertainment of glycogen storage disease type III: a need for improved and systematic monitoring.

PURPOSE: In glycogen storage disease type III (GSD III), liver aminotransferases tend to normalize with age giving an impression that hepatic manifestations improve with age. However, despite dietary treatment, long-term liver complications emerge. We present a GSD III liver natural history study in children to better understand changes in hepatic parameters with age. METHODS: We reviewed clinical, biochemical, histological, and radiological data in pediatric patients with GSD III, and performed a literature review of GSD III hepatic findings. RESULTS: Twenty-six patients (median age 12.5 years, range 2-22) with GSD IIIa (n = 23) and IIIb (n = 3) were enrolled in the study. Six of seven pediatric patients showed severe fibrosis on liver biopsy (median [range] age: 1.25 [0.75-7] years). Markers of liver injury (aminotransferases), dysfunction (cholesterol, triglycerides), and glycogen storage (glucose tetrasaccharide, Glc4) were elevated at an early age, and decreased significantly thereafter (p < 0.001). Creatine phosphokinase was also elevated with no significant correlation with age (p = 0.4). CONCLUSION: Liver fibrosis can occur at an early age, and may explain the decrease in aminotransferases and Glc4 with age. Our data outlines the need for systematic follow-up and specific biochemical and radiological tools to monitor the silent course of the liver disease process.

The North Carolina Experience with Mucopolysaccharidosis Type I Newborn Screening.

OBJECTIVE: To evaluate the performance of a 2-tiered newborn screening method for mucopolysaccharidosis type I (MPS I) in North Carolina. STUDY DESIGN: The screening algorithm included a flow injection analysis-tandem mass spectrometry assay as a first-tier screening method to measure α-L-iduronidase (IDUA) enzyme activity and Sanger sequencing of the IDUA gene on dried blood spots as a second-tier assay. The screening algorithm was revised to incorporate the Collaborative Laboratory Integrated Reports, an analytical interpretive tool, to reduce the false-positive rate. A medical history, physical examination, IDUA activity, and urinary glycosaminoglycan (GAG) analysis were obtained on all screen-positive infants. RESULTS: A total of 62 734 specimens were screened with 54 screen-positive samples using a cut-off of 15% of daily mean IDUA activity. The implementation of Collaborative Laboratory Integrated Reports reduced the number of specimens that screened positive to 19 infants. Of the infants identified as screen-positive, 1 had elevated urinary GAGs and a homozygous pathogenic variant associated with the severe form of MPS I. All other screen-positive infants had normal urinary GAG analysis; 13 newborns had pseudodeficiency alleles, 3 newborns had variants of unknown significance, and 2 had heterozygous pathogenic variants. CONCLUSIONS: An infant with severe MPS I was identified and referred for a hematopoietic stem cell transplant. Newborn IDUA enzyme deficiency is common in North Carolina, but most are due to pseudodeficiency alleles in infants with normal urinary GAG analysis and no evidence of disease. The pilot study confirmed the need for second-tier testing to reduce the follow-up burden.

Rescue of Pompe disease in mice by AAV-mediated liver delivery of secretable acid α-glucosidase.

Glycogen storage disease type II or Pompe disease is a severe neuromuscular disorder caused by mutations in the lysosomal enzyme, acid α-glucosidase (GAA), which result in pathological accumulation of glycogen throughout the body. Enzyme replacement therapy is available for Pompe disease; however, it has limited efficacy, has high immunogenicity, and fails to correct pathological glycogen accumulation in nervous tissue and skeletal muscle. Using bioinformatics analysis and protein engineering, we developed transgenes encoding GAA that could be expressed and secreted by hepatocytes. Then, we used adeno-associated virus (AAV) vectors optimized for hepatic expression to deliver the GAA transgenes to Gaa knockout (Gaa-/-) mice, a model of Pompe disease. Therapeutic gene transfer to the liver rescued glycogen accumulation in muscle and the central nervous system, and ameliorated cardiac hypertrophy as well as muscle and respiratory dysfunction in the Gaa-/- mice; mouse survival was also increased. Secretable GAA showed improved therapeutic efficacy and lower immunogenicity compared to nonengineered GAA. Scale-up to nonhuman primates, and modeling of GAA expression in primary human hepatocytes using hepatotropic AAV vectors, demonstrated the therapeutic potential of AAV vector-mediated liver expression of secretable GAA for treating pathological glycogen accumulation in multiple tissues in Pompe disease.

Wolman Disease: A Mimic of Infant Leukemia.

BACKGROUND: Infant leukemia most commonly present with pallor and hepatosplenomegaly. The possibility of other differential diagnosis also has to be kept in mind during evaluation, as identifying the precise etiology for this clinical presentation is crucial for management. OBSERVATION: An infant, was referred to us with suspected infant leukemia and was subsequently diagnosed to have lysosomal acid lipase deficiency/Wolman disease with a novel 5 bp deletion "c.1180_1184del" in the last exon (exon 10) of the lipase A (LIPA) gene. CONCLUSIONS: Hepatosplenomegaly and pallor resulting from nutritional deficiency or bone marrow involvement in Wolman disease can mimic infant leukemia.

A Recurrent De Novo Variant in NACC1 Causes a Syndrome Characterized by Infantile Epilepsy, Cataracts, and Profound Developmental Delay.

Whole-exome sequencing (WES) has increasingly enabled new pathogenic gene variant identification for undiagnosed neurodevelopmental disorders and provided insights into both gene function and disease biology. Here, we describe seven children with a neurodevelopmental disorder characterized by microcephaly, profound developmental delays and/or intellectual disability, cataracts, severe epilepsy including infantile spasms, irritability, failure to thrive, and stereotypic hand movements. Brain imaging in these individuals reveals delay in myelination and cerebral atrophy. We observe an identical recurrent de novo heterozygous c.892C>T (p.Arg298Trp) variant in the nucleus accumbens associated 1 (NACC1) gene in seven affected individuals. One of the seven individuals is mosaic for this variant. NACC1 encodes a transcriptional repressor implicated in gene expression and has not previously been associated with germline disorders. The probability of finding the same missense NACC1 variant by chance in 7 out of 17,228 individuals who underwent WES for diagnoses of neurodevelopmental phenotypes is extremely small and achieves genome-wide significance (p = 1.25 × 10-14). Selective constraint against missense variants in NACC1 makes this excess of an identical missense variant in all seven individuals more remarkable. Our findings are consistent with a germline recurrent mutational hotspot associated with an allele-specific neurodevelopmental phenotype in NACC1.

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.

Adjunctive albuterol enhances the response to enzyme replacement therapy in late-onset Pompe disease.

Effective dosages for enzyme replacement therapy (ERT) in Pompe disease are much higher than for other lysosomal storage disorders, which has been attributed to low cation-independent mannose-6-phosphate receptor (CI-MPR) in skeletal muscle. We have previously demonstrated the benefit of increased CI-MPR-mediated uptake of recombinant human acid-α-glucosidase during ERT in mice with Pompe disease following addition of albuterol therapy. Currently we have completed a pilot study of albuterol in patients with late-onset Pompe disease already on ERT for >2 yr, who were not improving further. The 6-min walk test (6MWT) distance increased in all 7 subjects at wk 6 (30±13 m; P=0.002), wk 12 (34±14 m; P=0.004), and wk 24 (42±37 m; P=0.02), in comparison with baseline. Grip strength was improved significantly for both hands at wk 12. Furthermore, individual subjects reported benefits; e.g., a female patient could stand up from sitting on the floor much more easily (time for supine to standing position decreased from 30 to 11 s), and a male patient could readily swing his legs out of his van seat (hip abduction increased from 1 to 2+ on manual muscle testing). Finally, analysis of the quadriceps biopsies suggested increased CI-MPR at wk 12 (P=0.08), compared with baseline. With the exception of 1 patient who succumbed to respiratory complications of Pompe disease in the first week, only mild adverse events have been reported, including tremor, transient difficulty falling asleep, and mild urinary retention (requiring early morning voiding). Therefore, this pilot study revealed initial safety and efficacy in an open label study of adjunctive albuterol therapy in patients with late-onset Pompe disease who had been stable on ERT with no improvements noted over the previous several years.

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.

Assessment of toxicity and biodistribution of recombinant AAV8 vector-mediated immunomodulatory gene therapy in mice with Pompe disease.

A preclinical safety study was conducted to evaluate the short- and long-term toxicity of a recombinant adeno-associated virus serotype 8 (AAV2/8) vector that has been developed as an immune-modulatory adjunctive therapy to recombinant human acid α-glucosidase (rhGAA, Myozyme) enzyme replacement treatment (ERT) for patients with Pompe disease (AAV2/8-LSPhGAApA). The AAV2/8-LSPhGAApA vector at 1.6 × 10(13) vector particles/kg, after intravenous injection, did not cause significant short- or long-term toxicity. Recruitment of CD4(+) (but not CD8(+)) lymphocytes to the liver was elevated in the vector-dosed male animals at study day (SD) 15, and in group 8 animals at SD 113, in comparison to their respective control animals. Administration of the vector, either prior to or after the one ERT injection, uniformly prevented the hypersensitivity induced by subsequent ERT in males, but not always in female animals. The vector genome was sustained in all tissues through 16-week postdosing, except for in blood with a similar tissue tropism between males and females. Administration of the vector alone, or combined with the ERT, was effective in producing significantly increased GAA activity and consequently decreased glycogen accumulation in multiple tissues, and the urine biomarker, Glc4, was significantly reduced. The efficacy of the vector (or with ERT) was better in males than in females, as demonstrated both by the number of tissues showing significantly effective responses and the extent of response in a given tissue. Given the lack of toxicity for AAV2/8LSPhGAApA, further consideration of clinical translation is warranted in Pompe disease.

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.

Identification of differentially expressed microRNAs in human hepatocellular adenoma associated with type I glycogen storage disease: a potential utility as biomarkers.

BACKGROUND: It is known that malignant transformation to hepatocellular carcinoma (HCC) occurs at a higher frequency in hepatocellular adenoma (HCA) from type I glycogen storage disease (GSD I) compared to HCA from other etiologies. In this study, we aimed to identify differentially expressed miRNAs in GSD Ia HCA as candidates that could serve as putative biomarkers for detection of GSD Ia HCA and/or risk assessment of malignant transformation. METHODS: Utilizing massively parallel sequencing, the miRNA profiling was performed for paired adenomas and normal liver tissues from seven GSD Ia patients. Differentially expressed miRNAs were validated in liver tumor tissues, HCC cell lines and serum using quantitative RT-PCR. RESULTS: miR-34a, miR-34a, miR-224, miR-224, miR-424, miR-452 and miR-455-5p were found to be commonly deregulated in GSD Ia HCA, general population HCA, and HCC cell lines at compatible levels. In comparison with GSD Ia HCA, the upregulation of miR-130b and downregulation of miR-199a-5p, miR-199b-5p, and miR-214 were more significant in HCC cell lines. Furthermore, serum level of miR-130b in GSD Ia patients with HCA was moderately higher than that in either GSD Ia patients without HCA or healthy individuals. CONCLUSION: We make the first observation of distinct miRNA deregulation in HCA associated with GSD Ia. We also provide evidence that miR-130b could serve as a circulating biomarker for detection of GSD Ia HCA. This work provides prominent candidate miRNAs worth evaluating as biomarkers for monitoring the development and progress of liver tumors in GSD Ia patients in the future.

Adjunctive ß2-agonists reverse neuromuscular involvement in murine Pompe disease.

Pompe disease has resisted enzyme replacement therapy with acid α-glucosidase (GAA), which has been attributed to inefficient cation-independent mannose-6-phosphate receptor (CI-MPR) mediated uptake. We evaluated ß2-agonist drugs, which increased CI-MPR expression in GAA knockout (KO) mice. Clenbuterol along with a low-dose adeno-associated virus vector increased Rotarod latency by 75% at 4 wk, in comparison with vector alone (P<2×10(-5)). Glycogen content was lower in skeletal muscles, including soleus (P<0.01), extensor digitorum longus (EDL; P<0.001), and tibialis anterior (P<0.05) following combination therapy, in comparison with vector alone. Glycogen remained elevated in the muscles following clenbuterol alone, indicating an adjunctive effect with gene therapy. Elderly GAA-KO mice treated with combination therapy demonstrated 2-fold increased wirehang latency, in comparison with vector or clenbuterol alone (P<0.001). The glycogen content of skeletal muscle decreased following combination therapy in elderly mice (P<0.05). Finally, CI-MPR-KO/GAA-KO mice did not respond to combination therapy, indicating that clenbuterol's effect depended on CI-MPR expression. In summary, adjunctive ß2-agonist treatment increased CI-MPR expression and enhanced efficacy from gene therapy in Pompe disease, which has implications for other lysosomal storage disorders that involve primarily the brain.

Predicting cross-reactive immunological material (CRIM) status in Pompe disease using GAA mutations: lessons learned from 10 years of clinical laboratory testing experience.

Enzyme replacement therapy (ERT) for Pompe disease using recombinant acid alpha-glucosidase (rhGAA) has resulted in increased survival although the clinical response is variable. Cross-reactive immunological material (CRIM)-negative status has been recognized as a poor prognostic factor. CRIM-negative patients make no GAA protein and develop sustained high antibody titers to ERT that render the treatment ineffective. Antibody titers are generally low for the majority of CRIM-positive patients and there is typically a better clinical outcome. Because immunomodulation has been found to be most effective in CRIM-negative patients prior to, or shortly after, initiation of ERT, knowledge of CRIM status is important before ERT is begun. We have analyzed 243 patients with infantile Pompe disease using a Western blot method for determining CRIM status and using cultured skin fibroblasts. Sixty-one out of 243 (25.1%) patients tested from various ethnic backgrounds were found to be CRIM-negative. We then correlated the CRIM results with GAA gene mutations where available (52 CRIM-negative and 88 CRIM-positive patients). We found that, in most cases, CRIM status can be predicted from GAA mutations, potentially circumventing the need for invasive skin biopsy and time wasted in culturing cells in the future. Continued studies in this area will help to increase the power of GAA gene mutations in predicting CRIM status as well as possibly identifying CRIM-positive patients who are at risk for developing high antibody titers.

Digital microfluidic platform for multiplexing enzyme assays: implications for lysosomal storage disease screening in newborns.

BACKGROUND: Newborn screening for lysosomal storage diseases (LSDs) has been gaining considerable interest owing to the availability of enzyme replacement therapies. We present a digital microfluidic platform to perform rapid, multiplexed enzymatic analysis of acid α-glucosidase (GAA) and acid α-galactosidase to screen for Pompe and Fabry disorders. The results were compared with those obtained using standard fluorometric methods. METHODS: We performed bench-based, fluorometric enzymatic analysis on 60 deidentified newborn dried blood spots (DBSs), plus 10 Pompe-affected and 11 Fabry-affected samples, at Duke Biochemical Genetics Laboratory using a 3-mm punch for each assay and an incubation time of 20 h. We used a digital microfluidic platform to automate fluorometric enzymatic assays at Advanced Liquid Logic Inc. using extract from a single punch for both assays, with an incubation time of 6 h. Assays were also performed with an incubation time of 1 h. RESULTS: Assay results were generally comparable, although mean enzymatic activity for GAA using microfluidics was approximately 3 times higher than that obtained using bench-based methods, which could be attributed to higher substrate concentration. Clear separation was observed between the normal and affected samples at both 6- and 1-h incubation times using digital microfluidics. CONCLUSIONS: A digital microfluidic platform compared favorably with a clinical reference laboratory to perform enzymatic analysis in DBSs for Pompe and Fabry disorders. This platform presents a new technology for a newborn screening laboratory to screen LSDs by fully automating all the liquid-handling operations in an inexpensive system, providing rapid results.