Assessment of the functional impact on the pre-mRNA splicing process of 28 nucleotide variants associated with Pompe disease in GAA exon 2 and their recovery using antisense technology.

Glycogen storage disease II (GSDII), also called Pompe disease, is an autosomal recessive inherited disease caused by a defect in glycogen metabolism due to the deficiency of the enzyme acid alpha-glucosidase (GAA) responsible for its degradation. So far, more than 500 sequence variants of the GAA gene have been reported but their possible involvement on the pre-messenger RNA splicing mechanism has not been extensively studied. In this work, we have investigated, by an in vitro functional assay, all putative splicing variants within GAA exon 2 and flanking introns. Our results show that many variants falling in the canonical splice site or the exon can induce GAA exon 2 skipping. In these cases, therefore, therapeutic strategies aimed at restoring protein folding of partially active mutated GAA proteins might not be sufficient. Regarding this issue, we have tested the effect of antisense oligonucleotides (AMOs) that were previously shown capable of rescuing splicing misregulation caused by the common c.-32-13T>G variant associated with the childhood/adult phenotype of GSDII. Interestingly, our results show that these AMOs are also quite effective in rescuing the splicing impairment of several exonic splicing variants, thus widening the potential use of these effectors for GSDII treatment.

Clinical characteristics and genotypes in the ADVANCE baseline data set, a comprehensive cohort of US children and adolescents with Pompe disease.

PURPOSE: To characterize clinical characteristics and genotypes of patients in the ADVANCE study of 4000 L-scale alglucosidase alfa (NCT01526785), the largest prospective United States Pompe disease cohort to date. METHODS: Patients aged ≥1 year with confirmed Pompe disease previously receiving 160 L alglucosidase alfa were eligible. GAA genotypes were determined before/at enrollment. Baseline assessments included histories/physical exams, Gross Motor Function Measure-88 (GMFM-88), pulmonary function tests, and cardiac assessments. RESULTS: Of 113 enrollees (60 male/53 female) aged 1-18 years, 87 had infantile-onset Pompe disease (IOPD) and 26 late-onset (LOPD). One hundred eight enrollees with GAA genotypes had 215 pathogenic variants (220 including combinations): 118 missense (4 combinations), 23 splice, 35 nonsense, 34 insertions/deletions, 9 duplications (1 combination), 6 other; c.2560C>T (n = 23), c.-32-13T>G (n = 13), and c.525delT (n = 12) were most common. Four patients had previously unpublished variants, and 14/83 (17%) genotyped IOPD patients were cross-reactive immunological material-negative. All IOPD and 6/26 LOPD patients had cardiac involvement, all without c.-32-13T>G. Thirty-two (26 IOPD, 6 LOPD) were invasively ventilated. GMFM-88 total %scores (mean ± SD, median, range): overall 46.3 ± 33.0% (47.9%, 0.0-100.0%), IOPD 41.6 ± 31.64% (38.9%, 0.0-99.7%), LOPD: 61.8 ± 33.2 (70.9%, 0.0-100.0%). CONCLUSION: ADVANCE, a uniformly assessed cohort comprising most US children and adolescents with treated Pompe disease, expands understanding of the phenotype and observed variants in the United States.

Efficacy, safety profile, and immunogenicity of alglucosidase alfa produced at the 4,000-liter scale in US children and adolescents with Pompe disease: ADVANCE, a phase IV, open-label, prospective study.

PURPOSE: Pompe disease results from lysosomal acid α-glucosidase (GAA) deficiency and its associated glycogen accumulation and muscle damage. Alglucosidase alfa (recombinant human GAA (rhGAA)) received approval in 2006 as a treatment for Pompe disease at the 160 L production scale. In 2010, larger-scale rhGAA was approved for patients up to 8 years old without cardiomyopathy. NCT01526785 evaluated 4,000 L rhGAA efficacy/safety in US infantile- or late-onset Pompe disease (IOPD, LOPD) patients up to 1 year old transitioned from 160 L rhGAA. METHODS: A total of 113 patients (87 with IOPD; 26 with LOPD) received 4,000 L rhGAA for 52 weeks dosed the same as previous 160 L rhGAA. Efficacy was calculated as the percentage of patients stable/improved at week 52 (without death, new requirement for invasive ventilation, left ventricular mass z-score increase >1 if baseline was >2, upright forced vital capacity decrease ≥15% predicted, or Gross Motor Function Measure-88 decrease ≥8 percentage points). Safety evaluation included an extension ≤20 months. RESULTS: Week 52 data was available for 104 patients, 100 of whom entered the extension. At week 52, 87/104 (83.7%) were stable/improved. Overall survival was 98.1% overall, 97.6% IOPD, 100% LOPD; 92.4% remained invasive ventilator-free (93.4% IOPD, 88.7% LOPD). Thirty-five patients had infusion-associated reactions. Eight IOPD patients died of drug-unrelated causes. CONCLUSIONS: Most Pompe disease patients were clinically stable/improved after transitioning to 4,000 L rhGAA. Safety profiles of both rhGAA forms were consistent.

Bent spine syndrome as the initial symptom of late-onset Pompe disease.

INTRODUCTION: Late-onset Pompe disease (LOPD) is a rare disorder characterized by progressive proximal muscle weakness and early respiratory insufficiency, for which enzyme replacement therapy (ERT) is available. METHODS: Having diagnosed a case of LOPD presenting with bent spine syndrome, we conducted a brief survey in the French centers involved in management of Pompe disease, from which we collected data on 3 other cases. RESULTS: The patients (3 women and 1 man) had a mean age of 64 years (range 51-77 years) and a delay in diagnosis of approximately 10 years (range 8-42 years). At diagnosis, 3 patients already had respiratory symptoms. All had normal or very mildly raised creatine kinase levels and magnetic resonance imaging abnormalities in the paraspinal muscles. They exhibited the most frequent mutation in Pompe disease (c.-32-13 T>G). CONCLUSION: Clinicians should be aware of this atypical presentation of LOPD to enable earlier diagnosis and treatment. Muscle Nerve 56: 167-170, 2017.

Evaluating avalglucosidase alfa for the management of late-onset Pompe disease.

INTRODUCTION: Glycogenosis type II (GSDII) is a rare autosomal disorder that is caused by the deficiency of alpha-glucosidase, a lysosomal enzyme that hydrolyzes glycogen to glucose. Autophagy dysregulation plays a critical role. Importantly, since 2006, both patients with infantile (classic Pompe disease) and adult GSDII (late-onset Pompe disease or LOPD) have been treated with enzyme replacement therapy (ERT). To support this use, several double-blind and observational studies including large cohorts of GSDII patients have been undertaken and have shown ERT to be effective in modifying the natural course of disease. Indeed, most LOPD cases improve in the first 20 months of treatment in a six-minute walk test (6MWT), while those who are untreated do not; instead, their response declines over time. AREAS COVERED: The author reviews avalglucosidase alpha, a therapy approved by both the FDA and European regulatory agencies. Herein, the author considers the pathophysiological approaches such as the role of enzyme entry, autophagy, and the response to ERT treatment of motor and respiratory components. EXPERT OPINION: There has been a notable drive toward the research of various aspects of this disease regarding the role of new enzyme penetration and immune adverse events. Consequently, avalglucosidase alpha might be a further step forward.

Deferoxamine mesylate improves splicing and GAA activity of the common c.-32-13T>G allele in late-onset PD patient fibroblasts.

Pompe disease (PD) is an autosomal recessive lysosomal storage disorder due to deficient activity of the acid alpha glucosidase enzyme (GAA). As a consequence of the enzymatic defect, undigested glycogen accumulates within lysosomes. Most patients affected by the late-onset (LO) phenotype carry in at least one allele the c.-32-13T>G variant, which leads to exon 2 exclusion from the pre-mRNA. These patients display a variable and suboptimal response to enzyme replacement therapy. To identify novel therapeutic approaches, we developed a fluorescent GAA exon 2 splicing assay and screened a library of US Food and Drug Administration (FDA)-approved compounds. This led to the identification of several drugs able to restore normal splicing. Among these, we further validated the effects of the iron chelator deferoxamine (Defe) in c.-32-13T>G fibroblasts. Defe treatment resulted in a 2-fold increase of GAA exon 2 inclusion and a 40% increase in enzymatic activity. Preliminary results suggest that this effect is mediated by the regulation of iron availability, at least partially. RNA-seq experiments also showed that Defe might shift the balance of splicing factor levels toward a profile promoting GAA exon 2 inclusion. This work provides the basis for drug repurposing and development of new chemically modified molecules aimed at improving the clinical outcome in LO-PD patients.

Diagnostic challenges in metabolic myopathies.

INTRODUCTION: Metabolic myopathies comprise a clinically etiological diverse group of disorders caused by defects in cellular energy metabolism including the breakdown of carbohydrates and fatty acids, which include glycogen storage diseases and fatty acid oxidation disorders. Their wide clinical spectrum ranges from infantile severe multisystemic disorders to adult-onset myopathies. To suspect in adults these disorders, clinical features such as exercise intolerance and recurrent myoglobinuria need investigation while another group presents fixed weakness and cardiomyopathy as a clinical pattern. AREAS COVERED: In metabolic myopathies, clinical manifestations are important to guide diagnostic tests used in order to lead to the correct diagnosis. The authors searched in literature the most recent techniques developed. The authors present an overview of the most common phenotypes of Pompe disease and what is currently known about the mechanism of ERT treatment. The most common disorders of lipid metabolism are overviewed, with their possible dietary or supplementary treatments. EXPERT COMMENTARY: The clinical suspicion is the clue to conduct in-depth investigations in suspected cases of metabolic myopathies that lead to the final diagnosis with biochemical molecular studies and often nowadays by the use of Next Generation Sequencing (NGS) to determine gene mutations.