Acid Alpha-glucosidase Activity Research Articles

Efficacy and safety of enzyme replacement therapy with alglucosidase alfa for the treatment of patients with infantile-onset Pompe disease: a systematic review and metanalysis.

Pompe disease (PD) is a glycogen disorder caused by the deficient activity of acid alpha-glucosidase (GAA). We sought to review the latest available evidence on the safety and efficacy of recombinant human GAA enzyme replacement therapy (ERT) for infantile-onset PD (IOPD). We systematically searched the MEDLINE (via PubMed) and Embase databases for prospective clinical studies evaluating ERT for IOPD on pre-specified outcomes. Meta-analysis was also performed. Of 1,722 articles identified, 16 were included, evaluating 316 patients. Studies were heterogeneous and with very low certainty of evidence for most outcomes. A moderate/high risk of bias was present for most included articles. The following outcomes showed improvements associated with alglucosidase alfa, over natural history of PD/placebo, for a mean follow-up of 48.3 months: left ventricular (LV) mass {mean change 131.3 g/m2 [95% confidence interval (CI) 81.02, 181.59]}, time to start ventilation (TSV) [HR 0.21 (95% CI: 0.12, 0.36)], and survival [HR 0.10 (95% CI: 0.05, 0.19)]. There were no differences between the pre- and post-ERT period for myocardial function and psychomotor development. Adverse events (AEs) after ERT were mild in most cases. Our data suggest that alglucosidase alfa potentially improves LV mass, TSV, and survival in IOPD patients, with no important safety issues. PROSPERO identifier (CRD42019123700).

Hepatic ChREBP orchestrates intrahepatic carbohydrate metabolism to limit hepatic glucose 6-phosphate and glycogen accumulation in a mouse model for acute Glycogen Storage Disease type Ib

ObjectiveCarbohydrate Response Element Binding Protein (ChREBP) is a glucose 6-phosphate (G6P)-sensitive transcription factor that acts as a metabolic switch to maintain intracellular glucose and phosphate homeostasis. Hepatic ChREBP is well-known for its regulatory role in glycolysis, the pentose phosphate pathway, and de novo lipogenesis. The physiological role of ChREBP in hepatic glycogen metabolism and blood glucose regulation has not been assessed in detail, and ChREBP's contribution to carbohydrate flux adaptations in hepatic Glycogen Storage Disease type 1 (GSD I) requires further investigation. MethodsThe current study aimed to investigate the role of ChREBP as a regulator of glycogen metabolism in response to hepatic G6P accumulation, using a model for acute hepatic GSD type Ib. The immediate biochemical and regulatory responses to hepatic G6P accumulation were evaluated upon G6P transporter inhibition by the chlorogenic acid S4048 in mice that were either treated with a short hairpin RNA (shRNA) directed against ChREBP (shChREBP) or a scrambled shRNA (shSCR). Complementary stable isotope experiments were performed to quantify hepatic carbohydrate fluxes in vivo. ResultsShChREBP treatment normalized the S4048-mediated induction of hepatic ChREBP target genes to levels observed in vehicle- and shSCR-treated controls. In parallel, hepatic shChREBP treatment in S4048-infused mice resulted in a more pronounced accumulation of hepatic glycogen and further reduction of blood glucose levels compared to shSCR treatment. Hepatic ChREBP knockdown modestly increased glucokinase (GCK) flux in S4048-treated mice while it enhanced UDP-glucose turnover as well as glycogen synthase and phosphorylase fluxes. Hepatic GCK mRNA and protein levels were induced by shChREBP treatment in both vehicle- and S4048-treated mice, while glycogen synthase 2 (GYS2) and glycogen phosphorylase (PYGL) mRNA and protein levels were reduced. Finally, knockdown of hepatic ChREBP expression reduced starch domain binding protein 1 (STBD1) mRNA and protein levels while it inhibited acid alpha-glucosidase (GAA) activity, suggesting reduced capacity for lysosomal glycogen breakdown. ConclusionsOur data show that ChREBP activation controls hepatic glycogen and blood glucose levels in acute hepatic GSD Ib through concomitant regulation of glucose phosphorylation, glycogenesis, and glycogenolysis. ChREBP-mediated control of GCK enzyme levels aligns with corresponding adaptations in GCK flux. In contrast, ChREBP activation in response to acute hepatic GSD Ib exerts opposite effects on GYS2/PYGL enzyme levels and their corresponding fluxes, indicating that GYS2/PYGL expression levels are not limiting to their respective fluxes under these conditions.

A Comprehensive Update on Late-Onset Pompe Disease.

Pompe disease (PD) is an autosomal recessive disorder caused by mutations in the GAA gene that lead to a deficiency in the acid alpha-glucosidase enzyme. Two clinical presentations are usually considered, named infantile-onset Pompe disease (IOPD) and late-onset Pompe disease (LOPD), which differ in age of onset, organ involvement, and severity of disease. Assessment of acid alpha-glucosidase activity on a dried blood spot is the first-line screening test, which needs to be confirmed by genetic analysis in case of suspected deficiency. LOPD is a multi-system disease, thus requiring a multidisciplinary approach for efficacious management. Enzyme replacement therapy (ERT), which was introduced over 15 years ago, changes the natural progression of the disease. However, it has limitations, including a reduction in efficacy over time and heterogeneous therapeutic responses among patients. Novel therapeutic approaches, such as gene therapy, are currently under study. We provide a comprehensive review of diagnostic advances in LOPD and a critical discussion about the advantages and limitations of current and future treatments.

Pompe disease, a rare condition in two patients, case reports

Pompe disease, or type II glycogen storage disease, is a lysosomal storage disorder in which a deficiency in alpha-glucosidase results in the accumulation of glycogen, which eventually causes weakness to progressively increase and heart enlargement.
 Infantile-onset and late-onset forms of Pompe illness are distinguished. The heart is the organ most impacted by glycogen buildup in infantile-onset Pompe illness. A late-onset form, however, frequently presents as a weakening of the skeletal muscles that worsens over time.
 The key factor used to make the diagnosis of Pompe illness is enzymology, which shows a lack of lysosomal acid alpha-glucosidase (GAA) activity, although molecular genetic testing for GAA mutations can also be used to confirm the diagnosis.
 Recombinant human a glucosidase alfa and a large multidisciplinary team are required for the treatment of patients with Pompe disease (rhGAA, MyozymeR).
 Two cases of Pompe disease are presented in this case-report. A 13-year-old female patient who is still alive and receiving enzyme replacement therapy, and a 5-month-old newborn who died from cardiomyopathy.

Prevalence of acid alpha-glucosidase (GAA) pseudodefiency allele and it's clinical significance among patients with cardiomyopathy

Pompe disease is an autosomal recessive lysosomal storage disorder caused by deficiency of lysosomal acid alpha-glucosidase (GAA) activity, leading to the progressive accumulation of glycogen in lysosomes of the skeletal and cardiac muscles. An alpha-glucosidase (GAA) pseudodeficiency allele is a change in the GAA gene sequence that results in GAA enzyme activity reduction, but does not cause Pompe disease. In Japan and Taiwan, there is high prevalence of pseudodeficiency allele (c.1725G > A and c.2065G > A) detected from their newborn screening.

GFPT1-Associated Congenital Myasthenic Syndrome Mimicking a Glycogen Storage Disease - Diagnostic Pitfalls in Myopathology Solved by Next-Generation-Sequencing.

GFPT1-related congenital myasthenic syndrome (CMS) is characterized by progressive limb girdle weakness, and less prominent involvement of facial, bulbar, or respiratory muscles. While tubular aggregates in muscle biopsy are considered highly indicative in GFPT1-associated CMS, excessive glycogen storage has not been described. Here, we report on three affected siblings with limb-girdle myasthenia due to biallelic pathogenic variants in GFPT1: the previously reported missense variant c.41G > A (p.Arg14Gln) and the novel truncating variant c.1265_1268del (p.Phe422TrpfsTer26). Patients showed progressive proximal atrophic muscular weakness with respiratory involvement, and a lethal disease course in adulthood. In the diagnostic workup at that time, muscle biopsy suggested a glycogen storage disease. Initially, Pompe disease was suspected. However, enzymatic activity of acid alpha-glucosidase was normal, and gene panel analysis including 38 genes associated with limb-girdle weakness (GAA included) remained unevocative. Hence, a non-specified glycogen storage myopathy was diagnosed. A decade later, the diagnosis of GFPT1-related CMS was established by genome sequencing. Myopathological reexamination showed pronounced glycogen accumulations, that were exclusively found in denervated muscle fibers. Only single fibers showed very small tubular aggregates, identified in evaluation of serial sections. This family demonstrates how diagnostic pitfalls can be addressed by an integrative approach including broad genetic analysis and re-evaluation of clinical as well as myopathological findings.

A CASE OF INFANTILE POMPE DISEASE: A CASE REPORT AND REVIEW OF INDIAN LITERATURE.

Background: Pompe disease is a metabolic disorder due to deciency of lysosomal acid alpha-glucosidase enzyme. Deciency of this enzyme leads to lysosomal glycogen accumulation in multiple tissues and cell types, predominantly affecting cardiac, skeletal, and smooth muscle cells, eventually causing progressive muscle destruction. The pattern of inheritance is autosomal recessive. Our case describes a Clinical Description: four-month-old female child, rst born baby to a non-consanguineous marriage, who presented to our out-patient department with respiratory tract infection, and difculty in swallowing for last 10 days. Clinical examination revealed hypotonia along with hepatosplenomegaly. Management: There was cardiomegaly evident on routine chest X ray. This signicant nding was further dwelled upon and an ECG with echocardiography was planned. ECG showed short PR interval with tall QRS complex, and Echocardiography revealed a hypertrophic cardiomyopathy. Based on the above ndings, the importance of ruling out possible storage disorders was considered eminent. Further tests revealed elevated CPK and LDH. Based on the clinical ndings and laboratory results, the differentials of storage disorders were narrowed to Pompe disease. Dried blood spots for quantitative enzyme assay of acid alpha glucosidase (GAA) activity were tested. The result conrmed that the GAA activity was lower than the normal range, leading to a diagnosis of Pompe disease. Infantile pompe disease Conclusion: is fatal without specic enzyme replacement therapy. The subtle clinical features or routine ndings heralding this ominous disease need to be dealt with a high index of suspicion for the same, the advantage being early diagnosis and initiation of enzyme replacement therapy.

A Systematic Review and Meta-Analysis of Enzyme Replacement Therapy in Late-Onset Pompe Disease.

Pompe disease (PD) is a glycogen storage disorder caused by deficient activity of acid alpha-glucosidase (GAA). We sought to review the latest available evidence on the safety and efficacy of recombinant human GAA enzyme replacement therapy (ERT) for late-onset PD (LOPD). Methods: We systematically searched the MEDLINE (via PubMed), Embase, and Cochrane databases for prospective clinical studies evaluating ERT for LOPD on pre-specified outcomes. A meta-analysis was also performed. Results: Of 1601 articles identified, 22 were included. Studies were heterogeneous and with very low certainty of evidence for most outcomes. The following outcomes showed improvements associated with GAA ERT, over a mean follow-up of 32.5 months: distance walked in the 6-min walking test (6MWT) (mean change 35.7 m (95% confidence interval [CI] 7.78, 63.75)), physical domain of the SF-36 quality of life (QOL) questionnaire (mean change 1.96 (95% CI 0.33, 3.59)), and time on ventilation (TOV) (mean change −2.64 h (95% CI −5.28, 0.00)). There were no differences between the pre- and post-ERT period for functional vital capacity (FVC), Walton and Gardner-Medwin Scale score, upper-limb strength, or total SF-36 QOL score. Adverse events (AEs) after ERT were mild in most cases. Conclusion: Considering the limitations imposed by the rarity of PD, our data suggest that GAA ERT improves 6MWT, physical QOL, and TOV in LOPD patients. ERT was safe in the studied population. PROSPERO register: 135102.

Molecular Diagnosis of Pompe Disease in the Genomic Era: Correlation with Acid Alpha-Glucosidase Activity in Dried Blood Spots.

Measurement of alpha-glucosidase activity on dried blood spots has been the main method to screen for Pompe disease, but a paradigm shift has been observed in recent years with the incorporation of gene panels and exome sequencing in molecular diagnostic laboratories. An 89-gene panel has been available to Canadian physicians since 2017 and was analyzed in 2030 patients with a suspected muscle disease. Acid alpha-glucosidase activity was measured in parallel in dried blood spots from 1430 patients. Pompe disease was diagnosed in 14 patients, representing 0.69% of our cohort. In 7 other patients, low enzyme activities overlapping those of Pompe disease cases were attributable to the presence of pseudodeficiency alleles. Only two other patients had enzymatic activity in the Pompe disease range, and a single heterozygous pathogenic variant was identified. It is possible that a second variant could have been missed; we suggest that RNA analysis should be considered in such cases. With gene panel testing increasingly being performed as a first-tier analysis of patients with suspected muscle disorders, our study supports the relevance of performing reflex enzymatic activity assay in selected patients, such as those with a single GAA variant identified and those in whom the observed genotype is of uncertain clinical significance.

Pompe Disease Screening in a Sample of Iranian Patients

The reason why the experiments were done: previous studies have shown that the incidence rates vary in different populations. Now we report the preliminary results of the screening study. Pompe disease is a rare but potentially treatable disorder caused by the deficiency of the lysosomal enzyme acid-α-glucosidase (GAA). GAA activity was measured on DBS in 65 patients with undiagnosed myopathies presenting to the hospital of the research setting in Isfahan, Iran, from 2016 to 2017 and then was confirmed by genetic analysis. Of the total of 65 patients, 29 (44.6%) were male, and 36 (55.4%) were female. The mean age of the patients was 29±12.55 years, and their mean age at the disease onset was 17±12.75. Two patients (one male and one female) were diagnosed with a low acid alpha-glucosidase activity. Only one patient (female) showed a compound heterozygotic mutation of the GAA gene (c.-32-13T>G). Early diagnosis of Pompe disease is important for improving the outcome.

The long-term follow-up of enzyme replacement treatment in late onset Pompe disease

Pompe disease (PD) is a rare lysosomal disease caused by the deficient activity of acid alpha-glucosidase (GAA) enzyme due to mutations in the GAA gene. The enzymatic deficiency leads to the accumulation of glycogen within the lysosomes. Clinically, the disease has been classically classified in infantile and childhood/adult forms. Presently cc. close to 600 mutations distributed throughout the whole gene have been reported. The c.-32-13T>G splice mutation that is very common in patients of Caucasian origin affected by the childhood/adult form of the disease, with an allelic frequency close to 70%. Enzyme replacement treatment (ERT) is available for the patients with Pompe disease (Myozyme). In this paper, we are presenting the long term follow up of 13 adult onset cases treated more than 5 years. The longest follow up was 15 years. To evaluate the treatment efficacy, the 6 minutes walking test (6MWT) and the respiratory functions were monitored annually. The analysis revealed that at the beginning of ERT for 3-4 years the 6MWT had been generally increasing, then it declined, and after 10 years it was lower in 77% of the cases than it had been at the start of the treatment. In 23% of the cases the 6MWT increased during the follow up time. Only one of the patients become wheelchair dependent during the follow-up period. The respiratory function showed similar results especially in supine position. A high degree of variability was observed among patients in their responses to the treatment, which only partially associated with the antibody titer against the therapeutic protein. The efficacy of the ERT was associated with the type of the disease causing mutation, the baseline status of the disease, the lifestyle and the diet of the patient. The long-term follow up of the patients with innovative orphan drugs is necessary to really understand the value of the treatment and the need of the patients.

Late onset of pompe disease, involvement of three brothers: A case report

A 49-year-old Brazilian male, experienced progressive weakness to walk, especially to climb stairs and get up since 2013. He denied any instances of myalgias, paresthesia, dysphagia, dysarthria, or dysautonomia. His two brothers reported difficulty getting up from the ground. Increased lordosis, bilaterally winged scapulae, quadriceps atrophy, Gowers sign, and hypoactive tendon reflexes globally. He showed tongue weakness, muscular strength in the lower limbs grade 3/5 during flexion. The muscle strength in the upper limbs was of grade 4/5 during the abduction. He showed normal sensitivity. Creatine kinase, lactate dehydrogenase, and aldolase were increased. An electrocardiogram revealed an incomplete right bundle branch block and left atrial hypertrophy. His acid alpha-glucosidase activity measured was 1.4 μmol/L/h (normal > 4), while his sister’s (62 years old ) enzyme level was 0.98 μmol/L/h and his brother’s (51 years old) was 1.31 mol/L/h. Enzyme replacement was initiated and the patient showed a slight improvement in muscle strength. Pompe disease (PD) is caused by the partial or total deficiency of the enzyme acid alpha-glucosidase activity causing glycogen accumulation in the cells, with muscular involvement.1 In the United States, the prevalence of PD is one in every 28,000 births.2 It is an autosomal recessive disease. The risk of recurrence in the offspring is approximately 25%. The diagnosis of PD justifies the active investigation of the disease among the siblings.1 We report a case of late-onset PD with involvement of two relatives in addition to the indexed patient.

Molecular genetics of Pompe disease: a comprehensive overview.

Pompe disease (PD) is an autosomal recessive lysosomal disorder caused by the deficient activity of acid alpha-glucosidase (GAA) enzyme due to mutations in the GAA gene. The enzymatic deficiency leads to the accumulation of glycogen within the lysosomes. Clinically, the disease has been classically classified in infantile and childhood/adult forms. The GAA gene has been localized to chromosome 17q25.2-q25.3 and to date, 582 mutations distributed throughout the whole gene have been reported (HGMD: http://www.hgmd.cf.ac.uk/ac/). All types of mutations have been described; missense variants are the most frequent type followed by small deletions. Most GAA mutations are private or found in a small number of families. However, an exception is represented by the c.-32-13T>G splice mutation that is very common in patients of Caucasian origin affected by the childhood/adult form of the disease, with an allelic frequency ranging from 40% to 70%. In this article, we review the spectrum of GAA mutations, their distribution in different populations, and their classification according to their impact on GAA splicing process, protein expression and activity. In addition, whenever possible, we discuss the phenotype/genotype correlation. The information collected in this review provides an overview of the molecular genetics of PD and can be used to facilitate diagnosis and genetic counseling of families affected by this disorder.

Newborn screening for Pompe disease in Japan: report and literature review of mutations in the GAA gene in Japanese and Asian patients.

A newborn screening program for Pompe disease using dried blood spots (DBSs) was initiated in Japan. Here, we summarized this screening program and described the results of the GAA gene analysis. From April 2013 to November 2016, 103,204 newborns were screened; 71 had low acid alpha-glucosidase (AαGlu) activity. GAA sequencing showed that 32 (45.1%) and 37 (52.1%) of these newborns were homozygous and heterozygous for pseudodeficiency alleles c.[1726G>A; 2965G>A], respectively. Moreover, 24 of 32 newborns with homozygous c.[1726G>A; 2965G>A] alleles had no mutations, and the other eight had one mutation each. Thirty-five of 37 newborns with heterozygous c.[1726G>A; 2965G>A] alleles had one mutation, and the other two had two mutations each. Only one newborn who had two mutations did not harbor c.[1726G>A; 2965G>A] alleles. Thus, it was difficult to distinguish newborns with c.[1726G>A; 2965G>A] alleles from newborns with pre-symptomatic Pompe disease using AαGlu assays in DBSs or fibroblasts; GAA gene sequencing was necessary. Seventy-one newborns had 50 variants, including 21 mutations or predictably pathogenic variants, and 29 polymorphisms or predictably non-pathogenic variants. Four of 21 mutations or predictably pathogenic variants and four of 29 polymorphisms or predictably non-pathogenic variants were novel. No infantile-onset Pompe disease was detected, and three newborns were diagnosed with potential late-onset Pompe disease. In the literature, 156 variants have been reported for 296 patients from 277 families in 41 articles from Japan, Korea, Taiwan, and China. Our results provide insights into GAA gene mutation profiles and the relationship between GAA and Pompe disease in Asian populations.

Clinical and Technological Dependence Characteristics on a Series of Brazilian Cases with Infantile Onset Pompe Disease in Enzyme Replacement Therapy

Pompe disease (PD) is a rare inborn error of metabolism due to an abnormal acid alpha-glucosidase (GAA) activity that comprises glycogen breakdown mainly in the lysosomes. Since the introduction of enzyme replacement therapy (ERT), with recombinant human GAA for the early onset PD patient, a relevant field of clinical research due to the benefits regarding survival rate has been widely documented worldwide. Objective: To describe the clinical characteristics and the ERT effects in a series of Brazilian patients with infantile onset PD (IOPD) under ERT. Methods: Brazilian patients diagnosed with IOPD under ERT were recruited through their physicians participating in the International Pompe Disease Registry from 2009 to 2017. Data were collected by an online survey. Results: 10 IOPD patients were identified through the survey with a death rate of 30% and technology dependency rate reported as 80% (motor, respiratory or nutritional fields) of the patients. After the third year of ERT, motor disabilities were lost in 50% of ambulated patients. The overall characteristics were similar to international studies. Conclusion: Despite ERT benefits in cardiac involvement, motor disabilities seem to be much more compromised in IOPD patients, with high technology dependence, especially after three years of age.

METABOLIC MYOPATHIES II: P.352A novel recombinant human acid alpha-glucosidase, ATB200, co-administered with a pharmacological chaperone, leads to greater substrate reduction and improvement in Pompe disease-relevant markers compared to alglucosidase alfa in Gaa KO mice

Pompe disease is an inherited lysosomal storage disorder caused by a deficiency in acid alpha-glucosidase (GAA) activity, and is characterized by progressive accumulation of lysosomal glycogen in cardiac and skeletal muscles. Enzyme replacement therapy using recombinant human GAA (rhGAA), alglucosidase alfa, is currently the only approved treatment for Pompe disease. However, alglucosidase alfa may have limitations in delivery to skeletal muscles due to sub-optimal levels of mannose-6-phosphate (M6P), a carbohydrate that binds cation-independent M6P receptors at the cell surface to mediate enzyme internalization and lysosomal delivery. We have developed a novel rhGAA (designated ATB200) with a significantly higher M6P content compared to alglucosidase alfa; ATB200 is further stabilized by the small molecule pharmacological chaperone, AT2221 (miglustat). In this study, we compared the effects of ATB200, orally co-administered with AT2221 (ATB200/AT2221; co-administration), to those of alglucosidase alfa in Gaa knockout (KO) mice. Repeat administration of ATB200/AT2221 resulted in substantially greater enzyme levels in lysosomes and significant glycogen reduction in key skeletal muscles. Importantly, extensive histological analysis of Gaa KO mouse muscle showed that ATB200/AT2221 is more effective compared to alglucosidase alfa in correcting overall cellular dysfunction in the endocytic-lysosomal-autophagic pathways, including lysosomal proliferation and autophagic buildup, the two known hallmarks of Pompe disease. Notably, these positive changes correlated well with improvements in muscle strength and muscle fiber size. Collectively, these data indicate that ATB200, stabilized by the pharmacological chaperone AT2221, offers potential advantages over the standard of care, and warrants further investigation as a next-generation treatment in patients with Pompe disease.

Alpha galactosidase A activity in Parkinson's disease

Glucocerebrosidase (GCase, deficient in Gaucher disease) enzymatic activity measured in dried blood spots of Parkinson's Disease (PD) cases is within healthy range but reduced compared to controls. It is not known whether activities of additional lysosomal enzymes are reduced in dried blood spots in PD. To test whether reduction in lysosomal enzymatic activity in PD is specific to GCase, we measured GCase, acid sphingomyelinase (deficient in Niemann–Pick disease types A and B), alpha galactosidase A (deficient in Fabry), acid alpha-glucosidase (deficient in Pompe) and galactosylceramidase (deficient in Krabbe) enzymatic activities in dried blood spots of PD patients (n = 648) and controls (n = 317) recruited from Columbia University. Full sequencing of glucocerebrosidase (GBA) and the LRRK2 G2019S mutation was performed. Enzymatic activities were compared between PD cases and controls using t-test and regression models adjusted for age, gender, and GBA and LRRK2 G2019S mutation status. Alpha galactosidase A activity was lower in PD cases compared to controls both when only non-carriers were included (excluding all GBA and LRRK2 G2019S carriers and PD cases with age-at-onset below 40) [2.85 μmol/l/h versus 3.12 μmol/l/h, p = 0.018; after controlling for batch effect, p = 0.006 (468 PD cases and 296 controls)], and when including the entire cohort (2.89 μmol/l/h versus 3.10 μmol/l/h, p = 0.040; after controlling for batch effect, p = 0.011). Because the alpha galactosidase A gene is X-linked, we stratified the analyses by sex. Among women who were non-carriers of GBA and LRRK2 G2019S mutations (PD, n = 155; control, n = 194), alpha galactosidase A activity was lower in PD compared to controls (2.77 μmol/l/h versus 3.10 μmol/l/h, p = 0.044; after controlling for a batch effect, p = 0.001). The enzymatic activity of acid sphingomyelinase, acid alpha-glucosidase and galactosylceramidase was not significantly different between PD and controls. In non-carriers, most lysosomal enzyme activities were correlated, with the strongest association in GCase, acid alpha-glucosidase, and alpha galactosidase A (Pearson correlation coefficient between 0.382 and 0.532). In a regression model with all five enzymes among non-carriers (adjusted for sex and age), higher alpha galactosidase A activity was associated with lower odds of PD status (OR = 0.54; 95% CI:0.31–0.95; p = 0.032). When LRRK2 G2019S PD carriers (n = 37) were compared to non-carriers with PD, carriers had higher GCase, acid sphingomyelinase and alpha galactosidase A activity. We conclude that alpha galactosidase A may have a potential independent role in PD, in addition to GCase.

Prevalence of adult Pompe disease in patients with proximal myopathic syndrome and undiagnosed muscle biopsy

We examined patients with limb-girdle muscle weakness and/or hyper-CKaemia and undiagnosed muscle biopsy for late onset Pompe disease (LOPD). Patients with an inconclusive limb-girdle muscle weakness who presented at our neuromuscular centre between 2005 and 2015 with undiagnosed muscle biopsies were examined by dry blood spot testing (DBS) including determination of the enzyme activity of acid alpha-glucosidase (GAA). In the case of depressed enzyme activity, additional gene testing of the GAA gene was carried out. Of the 340 evaluated muscle biopsies, 69 patients fulfilled the inclusion criteria and were examined with DBS. Among those patients, 76% showed a limb-girdle muscle weakness and 14% showed a hyper-CKaemia. A diagnosis of LOPD could be established in the case of two patients (2.9%) with reduced GAA enzyme activity and proof of mutations in the GAA gene. One of the two patients presents in the muscle biopsy suggestive features of Pompe disease including vacuoles with positive acid phosphatase reaction. In summary, our results show that a muscle biopsy can be helpful in identifying LOPD patients, but vacuolation with glycogen storage can also be absent. An inconspicuous muscle biopsy does not rule out Pompe disease. Consequently, all patients with limb-girdle muscle weakness should be examined by DBS before conducting a muscle biopsy.

Muscle ultrasound

Our study aimed to evaluate the utility of muscle ultrasound in newborn screening of infantile-onset Pompe disease (IOPD) and to establish a system of severity grading. We retrospectively selected 35 patients with initial low acid alpha-glucosidase (GAA) activity and collected data including muscle ultrasound features, GAA gene mutation, activity/performance, and pathological and laboratory findings. The echogenicity of 6 muscles (the bilateral vastus intermedius, rectus femoris, and sartorius muscles) was compared to that of epimysium on ultrasound and rated either 1 (normal), 2 (mildly increased), or 3 (obviously increased). These grades were used to divide patients into 3 groups. IOPD was present in none of the grade-1 patients, 5 of 9 grade-2 patients, and 5 of 5 grade-3 patients (P < .001). Comparing grade-2 plus grade-3 patients to grade-1 patients, muscle ultrasound detected IOPD with a sensitivity and specificity of 100.0% (95% confidence interval [CI]: 69.2%–100%) and 84.0% (95% CI: 63.9%–95.5%), respectively. The mean number of affected muscles was larger in grade-3 patients than in grade-2 patients (4.2 vs. 2.0, P = .005). Mean alanine transaminase (ALT), aspartate transaminase (AST), creatine kinase (CK), and lactate dehydrogenase (LDH) levels were differed significantly different between grade-3 and grade-1 patients (P < .001). Because it permits direct visualization of injured muscles, muscle ultrasound can be used to screen for IOPD. Our echogenicity grades of muscle injury also correlate well with serum levels of muscle-injury biochemical markers.

Glycogen Reduction in Myotubes of Late-Onset Pompe Disease Patients Using Antisense Technology

Glycogen storage disease type II (GSDII) is a lysosomal disorder caused by the deficient activity of acid alpha-glucosidase (GAA) enzyme, leading to the accumulation of glycogen within the lysosomes. The disease has been classified in infantile and late-onset forms. Most late-onset patients share a splicing mutation c.-32-13T > G in intron 1 of the GAA gene that prevents efficient recognition of exon 2 by the spliceosome. In this study, we have mapped the splicing silencers of GAA exon 2 and developed antisense morpholino oligonucleotides (AMOs) to inhibit those regions and rescue normal splicing in the presence of the c.-32-13T > G mutation. Using a minigene approach and patient fibroblasts, we successfully increased inclusion of exon 2 in the mRNA and GAA enzyme production by targeting a specific silencer with a combination of AMOs. Most importantly, the use of these AMOs in patient myotubes results in a decreased accumulation of glycogen. To our knowledge, this is the only therapeutic approach resulting in a decrease of glycogen accumulation in patient tissues beside enzyme replacement therapy (ERT) and TFEB overexpression. As a result, it may represent a highly novel and promising therapeutic line for GSDII.