GAA variants and phenotypes among 1,079 patients with Pompe disease: Data from the Pompe Registry.

Identification of variants in the acid α-glucosidase (GAA) gene in Pompe disease provides valuable insights and systematic overviews are needed. We report on the number, nature, frequency, and geographic distribution of GAA sequence variants listed in the Pompe Registry, a long-term, observational program and the largest global repository of Pompe disease data. Variant information was reviewed and compared with publicly available GAA databases/resources. Among 1,079 eligible patients, 2,075 GAA variants (80 unique novel) were reported. Variants were listed by groups representing Pompe disease phenotypes. Patients were classified as Group A: Symptom onset ≤ 12 months of age with cardiomyopathy; Group B: Symptom onset ≤ 12 years of age (includes patients with symptom onset ≤ 12 months of age without cardiomyopathy); or Group C: Symptom onset > 12 years of age. Likely impact of novel variants was predicted using bioinformatics algorithms. Variants were classified by pathogenicity using ACMG guidelines. Data reported from the Pompe Registry provide new information about the distribution of GAA variants globally and across the clinical spectrum, add to the number and diversity of GAA variants registered in public databases through published data sharing, provide a first indication of the severity of novel variants, and assist in diagnostic practice and outcome prediction.

Quality of life and participation in daily life of adults with Pompe disease receiving enzyme replacement therapy: 10 years of international follow-up.

BACKGROUND: Pompe disease is an inheritable metabolic disorder for which enzyme replacement therapy (ERT) has been available since 2006. Effects of ERT have been shown on distance walked, pulmonary function and survival. We investigated whether it also improves quality of life and participation in daily life in adult patients with the disease. METHODS: In an international survey, we assessed quality of life (Short Form 36, SF-36) and participation (Rotterdam Handicap Scale, RHS) annually between 2002 and 2012. Repeated measurements mixed effects models were used to describe the data over time. RESULTS: Responses were available for 174 adult patients. In the periods before and after start of ERT, the median follow-up times were 4 years each (range 0.5-8). The SF-36 Physical Component Summary measure (PCS) deteriorated before ERT (-0.73 score points per year (sp/y); CI 95 % -1.07 to -0.39), while it improved in the first 2 years of ERT (1.49 sp/y; CI 0.76 to 2.21), and remained stable thereafter. The Mental Component Summary measure (MCS) remained stable before and during ERT. After declining beforehand (-0.49 sp/year; CI -0.64 to-0.34), the RHS stabilized under ERT. CONCLUSION: In adult patients with Pompe disease, ERT positively affects quality of life and participation in daily life. Our results reinforce previous findings regarding the effect of ERT on muscle strength, pulmonary function and survival.

Enzyme therapy and immune response in relation to CRIM status: the Dutch experience in classic infantile Pompe disease.

BACKGROUND: Enzyme-replacement therapy (ERT) in Pompe disease--an inherited metabolic disorder caused by acid α-glucosidase deficiency and characterized in infants by generalized muscle weakness and cardiomyopathy--can be complicated by immune responses. Infants that do not produce any endogenous acid α-glucosidase, so-called CRIM-negative patients, reportedly develop a strong response. We report the clinical outcome of our Dutch infants in relation to their CRIM status and immune response. METHODS: Eleven patients were genotyped and their CRIM status was determined. Antibody formation and clinical outcome were assessed for a minimum of 4 years. RESULTS: ERT was commenced between 0.1 and 8.3 months of age, and patients were treated from 0.3 to 13.7 years. All patients developed antibodies. Those with a high antibody titer (above 1:31,250) had a poor response. The antibody titers varied substantially between patients and did not strictly correlate with the patients' CRIM status. Patients who started ERT beyond 2 months of age tended to develop higher titers than those who started earlier. All three CRIM-negative patients in our study succumbed by the age of 4 years seemingly unrelated to the height of their antibody titer. CONCLUSION: Antibody formation is a common response to ERT in classic infantile Pompe disease and counteracts the effect of treatment. The counteracting effect seems determined by the antibody:enzyme molecular stoichiometry. The immune response may be minimized by early start of ERT and by immune modulation, as proposed by colleagues. The CRIM-negative status itself seems associated with poor outcome.

Newborn screening for pompe disease? a qualitative study exploring professional views.

BACKGROUND: Developments in enzyme replacement therapy have kindled discussions on adding Pompe disease, characterized by progressive muscle weakness and wasting, to neonatal screening. Pompe disease does not fit traditional screening criteria as it is a broad-spectrum phenotype disorder that may occur in lethal form in early infancy or manifest in less severe forms from infancy to late adulthood. Current screening tests cannot differentiate between these forms. Normally, expanding screening is discussed among experts in advisory bodies. While advisory reports usually mention the procedures and outcome of deliberations, little is known of the importance attached to different arguments and the actual weighing processes involved. In this research we aim to explore the views of a wide range of relevant professionals to gain more insight into the process of weighing pros and cons of neonatal screening for Pompe disease, as an example of the dilemmas involved in screening for broad-spectrum phenotype disorders. METHODS: We conducted 24 semi-structured interviews with medical, lab, insurance and screening professionals, and executive staff of patient organisations. They were asked about their first reaction to neonatal screening for Pompe disease, after which benefits and harms and requirements for screening were explored in more detail. RESULTS: Advantages included health gain by timely intervention, avoiding a diagnostic quest, having a reproductive choice and gaining more knowledge about the natural course and treatment. Being prepared was mentioned as an advantage for the later manifesting cases. Disadvantages included treatment costs and uncertainties about its effect, the timing of treatment in later manifesting cases, the psychological burden for the patient-in-waiting and the family. Also the downsides of having prior knowledge as well as having to consider a reproductive option were mentioned as disadvantages. CONCLUSION: When weighing pros and cons, interviewees attach different importance to different arguments, based on personal and professional views. Professionals expect benefits from neonatal screening for Pompe disease, especially for early-onset cases. Some interviewees valued screening in later manifesting cases as well, while stressing the need for adequate support of pre-symptomatic patients and their families. Others considered the psychological burden and uncertainties regarding treatment as reasons not to screen.

Enzyme replacement therapy and fatigue in adults with Pompe disease.

BACKGROUND: Pompe disease is a hereditary metabolic myopathy, for which enzyme replacement therapy (ERT) has been available since 2006. We investigated whether ERT reduces fatigue in adult patients with Pompe disease. METHODS: In this prospective international observational survey, we used the Fatigue Severity Scale (FSS) to measure fatigue. Repeated measures ANOVA was used to analyze the data over time. In a subgroup of patients, we also evaluated muscle strength using the Medical Research Council Scale, measured pulmonary function as Forced Vital Capacity, and assessed depression using the Hospital Anxiety and Depression Scale. RESULTS: We followed 163 patients for a median period of 4 years before ERT and for 3 years during ERT. Before ERT, the mean FSS score remained stable at around 5.3 score points; during ERT, scores improved significantly by 0.13 score points per year (p < 0.001). Fatigue decreased mainly in women, in older patients and in those with shorter disease duration. Patients' improvements in fatigue were moderately correlated with the effect of ERT on depression (r 0.55; CI 95% 0.07 to 0.70) but not with the effect of ERT on muscle strength or pulmonary function. CONCLUSIONS: Fatigue is a common and disabling problem in patients with early and advanced stages of Pompe disease. Our finding that ERT helps to reduce fatigue is therefore important for this patient population, irrespective of the mechanisms underlying this effect.

The genotype-phenotype correlation in Pompe disease.

Pompe disease is an autosomal recessive lysosomal glycogen storage disorder that is caused by acid α-glucosidase (GAA) deficiency and is due to pathogenic sequence variations in the corresponding GAA gene. The correlation between genotypes and phenotypes is strict, in that patients with the most severe phenotype, classic infantile Pompe disease, have two pathogenic mutations, one in each GAA allele, that prevent the formation of GAA or totally obliterates its function. All patients with less progressive phenotypes have at least one sequence variation that allows normal or low level synthesis of GAA leading to the formation of analytically measurable, low level GAA activity in most cases. There is an overall trend of finding higher GAA enzyme levels in patients with onset of symptoms in adulthood when compared to patients who show clinical manifestations in early childhood, aged 0-5 years, with a rapidly progressive course, but who lack the severe characteristics of classic infantile Pompe disease. However, several cases have been reported of adult-onset disease with very low GAA activity, which in all those cases corresponds with the GAA genotype. The clinical diversity observed within a large group of patients with functionally the same GAA genotype and the same c.-32-13C > T haplotype demonstrates that modifying factors can have a substantial effect on the clinical course of Pompe disease, disturbing the GAA genotype-phenotype correlation. The present day challenge is to identify these factors and explore them as therapeutic targets.

Rapid ultraperformance liquid chromatography-tandem mass spectrometry assay for a characteristic glycogen-derived tetrasaccharide in Pompe disease and other glycogen storage diseases.

BACKGROUND: Urinary excretion of the tetrasaccharide 6-α-D-glucopyranosyl-maltotriose (Glc4) is increased in various clinical conditions associated with increased turnover or storage of glycogen, making Glc4 a potential biomarker for glycogen storage diseases (GSD). We developed an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay to detect Glc4 in urine without interference of the Glc4 isomer maltotetraose (M4). METHODS: Urine samples, diluted in 0.1% ammonium hydroxide containing the internal standard acarbose, were filtered, and the filtrate was analyzed by UPLC-MS/MS. RESULTS: We separated and quantified acarbose, M4, and Glc4 using the ion pairs m/z 644/161, 665/161, and 665/179, respectively. Response of Glc4 was linear up to 1500 µmol/L and the limit of quantification was 2.8 µmol/L. Intra- and interassay CVs were 18.0% and 18.4% (10 µmol/L Glc4), and 10.5% and 16.2% (200 µmol/L Glc4). Glc4 in control individuals (n = 116) decreased with increasing age from a mean value of 8.9 mmol/mol to 1.0 mmol/mol creatinine. M4 was present in 5% of urine samples. Mean Glc4 concentrations per age group in untreated patients with Pompe disease (GSD type II) (n = 66) were significantly higher, ranging from 39.4 to 10.3 mmol/mol creatinine (P < 0.001-0.005). The diagnostic sensitivity of Glc4 for GSD-II was 98.5% and the diagnostic specificity 92%. Urine Glc4 was also increased in GSD-III (8 of 9), GSD-IV (2 of 3) and GSD-IX (6 of 10) patients. CONCLUSIONS: The UPLC-MS/MS assay of Glc4 in urine was discriminative between Glc4 and M4 and confirmed the diagnosis in >98% of GSD-II cases.

Remarkably low fibroblast acid α-glucosidase activity in three adults with Pompe disease.

INTRODUCTION: Most adults with Pompe disease are compound heterozygotes in which one acid α-glucosidase (GAA) allele harbors the c.-32-13T>G mutation, causing partial loss of GAA, and the other allele harbors a fully deleterious mutation. The fibroblast GAA activity in these patients is usually between 5% and 25% of the average in healthy individuals. In some adult patients, however, the fibroblast GAA activity is much lower and is in the range that is normally observed in classic-infantile Pompe disease. We investigated the genotype-phenotype correlation in three such adult patients and measured the GAA activity as well as the glycogen content in muscle and fibroblasts in order to better understand the clinical course. METHODS: DNA was sequenced and GAA activity and glycogen content were measured in leukocytes, fibroblasts and muscle. Muscle biopsies were microscopically analyzed and the biosynthesis of GAA in fibroblasts was analyzed by immunoblotting. GAA activity and glycogen content in fibroblasts and muscle tissue in healthy controls, adult patients with Pompe disease and classic-infantile patients were compared with those of the three index patients. RESULTS: One patient had genotype c.525delT/c.671G>A (r.0/p.Arg224Gln). Two affected brothers had genotype c.569G>A/c.1447G>A (p.Arg190His/p.Gly483Arg). In all three cases the GAA activity and the glycogen content in fibroblasts were within the same range as in classic-infantile Pompe disease, but the activity and glycogen content in muscle were both within the adult range. In fibroblasts, the first step of GAA synthesis appeared unaffected but lysosomal forms of GAA were not detectable with immunoblotting. CONCLUSION: Some adult patients with mutations other than c.-32-13T>G can have very low GAA activity in fibroblasts but express higher activity in muscle and store less glycogen in muscle than patients with classic-infantile Pompe disease. This might explain why these patients have a slowly progressive course of Pompe disease.

Severely impaired health status at diagnosis of Pompe disease: a cross-sectional analysis to explore the potential utility of neonatal screening.

Since the introduction of enzyme replacement therapy for Pompe disease, awareness and early diagnosis have gained importance. Because the therapy is most effective when started early and methods for dried bloodspot screening for Pompe disease are currently being explored, neonatal screening is getting increased attention. The objective of this study was to investigate the gains that might be achieved with earlier diagnosis by neonatal screening. For this purpose we analyzed the health and functional status of non-screened patients with Pompe disease at the time of diagnosis. Previously collected clinical data and results of an international patient-reported questionnaire were used. Cross-sectional data of 53 patients with Pompe disease diagnosed between 1999 and 2009 (aged 0-64 years) were analyzed. According to the World Health Organization's International Classification of Functioning, Disability and Health the following domains are described: body function, activity, participation and contextual factors. In all patients with classic infantile Pompe disease cardiac function, hearing, muscle strength and motor development were considerably impaired at the time of clinical diagnosis. The use of oxygen and/or nasogastric tube-feeding was reported in more than 70% of these cases. Most children, adolescents and adults had advanced muscle weakness and impaired respiratory function at the time of their diagnosis, causing varying degrees of handicap. About 12% of them used a walking device and/or respiratory support at the time of diagnosis. The severely impaired health status reported here provides a strong argument for earlier diagnosis and to further explore the potential of neonatal screening for Pompe disease.

Lentiviral gene therapy of murine hematopoietic stem cells ameliorates the Pompe disease phenotype.

Pompe disease (acid alpha-glucosidase deficiency) is a lysosomal glycogen storage disorder characterized in its most severe early-onset form by rapidly progressive muscle weakness and mortality within the first year of life due to cardiac and respiratory failure. Enzyme replacement therapy prolongs the life of affected infants and supports the condition of older children and adults but entails lifelong treatment and can be counteracted by immune responses to the recombinant enzyme. We have explored the potential of lentiviral vector-mediated expression of human acid alpha-glucosidase in hematopoietic stem cells (HSCs) in a Pompe mouse model. After mild conditioning, transplantation of genetically engineered HSCs resulted in stable chimerism of approximately 35% hematopoietic cells that overexpress acid alpha-glucosidase and in major clearance of glycogen in heart, diaphragm, spleen, and liver. Cardiac remodeling was reversed, and respiratory function, skeletal muscle strength, and motor performance improved. Overexpression of acid alpha-glucosidase did not affect overall hematopoietic cell function and led to immune tolerance as shown by challenge with the human recombinant protein. On the basis of the prominent and sustained therapeutic efficacy without adverse events in mice we conclude that ex vivo HSC gene therapy is a treatment option worthwhile to pursue.

Hearing in adults with Pompe disease.

Hearing loss has been recognized as an important cause of morbidity in infants with Pompe disease, a metabolic disorder caused by deficiency of acid α-glucosidase. It is unknown whether hearing is also affected in adult Pompe patients. We have studied the prevalence, severity, and type of hearing loss in 58 adult patients using tympanometry and pure-tone audiometry. Compared to normative data (International Organisation for Standardisation standard 7029), 72% of patients had impaired hearing thresholds at one or more frequencies in at least one ear. All measured frequencies were equally affected. All patients had a sensorineural type of hearing loss, pointing to cochlear or retrocochlear pathology. Categorised according to the standards of the World Health Organisation 21% of patients had a clinically relevant hearing loss (16% slight, 3% moderate, 2% profound). Though this suggests that hearing loss occurs in a considerable number of patients with Pompe disease, this prevalence is similar to that in the general population. Therefore, we conclude that hearing loss is not a specific feature of Pompe disease in adults.

Improved assay for differential diagnosis between Pompe disease and acid α-glucosidase pseudodeficiency on dried blood spots.

The high frequency (3.3-3.9%) of acid α-glucosidase pseudodeficiency, c.[1726G>A; 2065G>A] homozygote (AA homozygote), in Asian populations complicates newborn screening for Pompe disease (glycogen storage disease type II or acid maltase deficiency) on dried blood spots, since AA homozygotes have a considerably low enzyme activity. We observed that hemoglobin in the enzyme reaction solution strongly interferes with the fluorescence of 4-methylumbelliferone released from 4-methylumbelliferyl α-D-glucopyranoside (4MU-αGlc) by acid α-glucosidase. Therefore, we have searched for a method to effectively eliminate hemoglobin in the reaction solution. Hemoglobin precipitation with barium hydroxide and zinc sulfate (Ba/Zn method) carried out after the enzyme reaction considerably enhances the fluorescence intensity while it does not reduce the intensity to any extent as can occur with conventional deproteinization agents like trichloroacetic acid. The Ba/Zn method greatly improved the separation between 18 Japanese patients with Pompe disease and 70 unaffected AA homozygotes in a population of Japanese newborns in the assay with 4MU-αGlc on dried blood spots. No overlap was observed between both groups. We further examined acid α-glucosidase activity in fibroblasts from 11 Japanese patients and 57 Japanese unaffected individuals including 31 c.[1726G; 2065G] homozygotes, 18 c.[1726G; 2065G]/[1726A; 2065A] heterozygotes and 8 AA homozygotes to confirm that fibroblasts can be used for definitive diagnosis. The patients were reliably distinguished from three control groups. These data provide advanced information for the development of a simple and reliable newborn screening program with dried blood spots for Pompe disease in Asian populations.

First experience with enzyme replacement therapy during pregnancy and lactation in Pompe disease.

Enzyme replacement therapy (ERT) with alglucosidase alfa was registered as a treatment for Pompe disease in 2006. It is as yet unknown whether ERT can be safely applied during pregnancy and lactation. A primiparous 40-year-old woman diagnosed with Pompe disease continued receiving ERT during pregnancy and lactation. Before pregnancy, she had moderate limb-girdle weakness and used nocturnal ventilation. During pregnancy, her clinical condition remained fairly stable until the 25th gestational week. Thereafter she experienced more problems with mobility and respiration. Fetal growth was normal as monitored by regular ultrasound investigations. A healthy boy was born at a gestational age of 37 weeks and 5 days by elective Cesarean section. There were no maternal complications and the child developed normally. One year after delivery the mother's physical condition was similar as prior to her pregnancy. Pharmacokinetic studies following enzyme infusion showed that alglucosidase alfa was secreted into the breast milk. Activity levels in the milk (245 nmol/ml.h) peaked at 2.5h after the end of the infusion; which was 2h later than in the plasma (80 µmol/ml.h). Twenty-four hours after start of the infusion, the enzyme activity in the breast milk was back to the pre-infusion level. In this case report, the continuation of treatment with alglucosidase alfa during pregnancy and lactation has been safe for the mother and the child.

Design and validation of a metabolic disorder resequencing microarray (BRUM1).

The molecular genetic diagnosis of inherited metabolic disorders is challenging. The diseases are rare, and most show locus heterogeneity. Hence, testing of the genes associated with IMDs is time consuming and often not easily available. We report a resequencing array that allows the simultaneous resequencing of up to 92 genes associated with IMDs. To validate the array, DNA samples from 51 patients with 52 different known variants (including point variants, small insertion, and deletions [indels]) in seven genes (C14ORF133, GAA, NPC1, NPC2, VPS33B, WFS1, and SLC19A2) were amplified by PCR and hybridized to the array. A further patient cohort with 48 different mutations in NPC1 were analyzed blind. Out of 76 point variants, 73 were identified using automated software analysis followed by manual review. Ten insertion and deletion variants were detected in the extra tiling using mutation specific probes, with 11 heterozygous deletions and 3 heterozygous insertions. In summary, we identified 96% (95% confidence interval [CI] 89-99%) of point variants added to the array, but the pickup rate reduced to 83% (95% CI 75-89%) when insertions/deletions were included. Although the methodology has strengths and weaknesses, application of this technique could expedite diagnosis in most patients with multilocus IMDs.

High antibody titer in an adult with Pompe disease affects treatment with alglucosidase alfa.

Clinical trials have demonstrated beneficial effects of enzyme replacement therapy (ERT) with alglucosidase alfa in infants, children and adults with Pompe disease. Recent studies have shown that high antibody titers can occur in patients receiving ERT and counteract the effect of treatment. This particularly occurs in those patients with classic-infantile Pompe disease that do not produce any endogenous acid α-glucosidase (CRIM-negative). It is still unclear to what extent antibody formation affects the outcome of ERT in adults with residual enzyme activity. We present the case of a patient with adult-onset Pompe disease. He was diagnosed at the age of 39years by enzymatic testing (10.7% residual activity in fibroblasts) and DNA analysis (genotype: c.-32-13T>G/p.Trp516X). Infusion-associated reactions occurred during ERT and the patient's disease progressed. Concurrently, the antibody titer rose to a similarly high level as reported for some CRIM-negative patients with classic-infantile Pompe disease. Using newly developed immunologic-assays we could calculate that approximately 40% of the administered alglucosidase alfa was captured by circulating antibodies. Further, we could demonstrate that uptake of alglucosidase alfa by cultured fibroblasts was inhibited by admixture of the patient's serum. This case demonstrates that also patients with an appreciable amount of properly folded and catalytically active endogenous acid α-glucosidase can develop antibodies against alglucosidase alfa that affect the response to ERT.

PAS-positive lymphocyte vacuoles can be used as diagnostic screening test for Pompe disease.

Screening of blood films for the presence of periodic acid-Schiff (PAS)-positive lymphocyte vacuoles is sometimes used to support the diagnosis of Pompe disease, but the actual diagnostic value is still unknown. We collected peripheral blood films from 65 untreated Pompe patients and 51 controls. Lymphocyte vacuolization was quantified using three methods: percentage vacuolated lymphocytes, percentage PAS-positive lymphocytes, and a PAS score depending on staining intensity. Diagnostic accuracy of the tests was assessed using receiver operating characteristic (ROC) curves. All three methods fully discerned classic infantile patients from controls. The mean values of patients with milder forms of Pompe disease were significantly higher than those of controls, but full separation was not obtained. The area under the ROC curve was 0.98 for the percentage vacuolated lymphocytes (optimal cutoff value 3; sensitivity 91%, specificity 96%) and 0.99 for the percentage PAS-positive lymphocytes and PAS score (optimal cutoff value 9; sensitivity 100%, specificity 98%). Our data indicate that PAS-stained blood films can be used as a reliable screening tool to support a diagnosis of Pompe disease. The percentage of PAS-positive lymphocytes is convenient for use in clinical practice but should always be interpreted in combination with other clinical and laboratory parameters.

Lentiviral Hematopoietic Stem Cell Gene Therapy Corrects Murine Pompe Disease.

Pompe disease is an autosomal recessive lysosomal storage disorder characterized by progressive muscle weakness. The disease is caused by mutations in the acid α-glucosidase (GAA) gene. Despite the currently available enzyme replacement therapy (ERT), roughly half of the infants with Pompe disease die before the age of 3 years. Limitations of ERT are immune responses to the recombinant enzyme, incomplete correction of the disease phenotype, lifelong administration, and inability of the enzyme to cross the blood-brain barrier. We previously reported normalization of glycogen in heart tissue and partial correction of the skeletal muscle phenotype by ex vivo hematopoietic stem cell gene therapy. In the present study, using a codon-optimized GAA (GAAco), the enzyme levels resulted in close to normalization of glycogen in heart, muscles, and brain, and in complete normalization of motor function. A large proportion of microglia in the brain was shown to be GAA positive. All astrocytes contained the enzyme, which is in line with mannose-6-phosphate receptor expression and the key role in glycogen storage and glucose metabolism. The lentiviral vector insertion site analysis confirmed no preference for integration near proto-oncogenes. This correction of murine Pompe disease warrants further development toward a cure of the human condition.

Pompe's disease.

Pompe's disease, glycogen-storage disease type II, and acid maltase deficiency are alternative names for the same metabolic disorder. It is a pan-ethnic autosomal recessive trait characterised by acid alpha-glucosidase deficiency leading to lysosomal glycogen storage. Pompe's disease is also regarded as a muscular disorder, but the generalised storage of glycogen causes more than mobility and respiratory problems. The clinical spectrum is continuous and broad. First symptoms can present in infants, children, and adults. Cardiac hypertrophy is a key feature of classic infantile Pompe's disease. For a long time, there was no means to stop disease progression, but the approval of enzyme replacement therapy has substantially changed the prospects for patients. With this new development, the disease is now among the small but increasing number of lysosomal storage disorders, for which treatment has become a reality. This review is meant to raise general awareness, to present and discuss the latest insights in disease pathophysiology, and to draw attention to new developments about diagnosis and care. We also discuss the developments that led to the approval of enzyme replacement therapy with recombinant human alpha-glucosidase from Chinese hamster ovary cells (alglucosidase alfa) by the US Food and Drug Administration and European Medicines Agency in 2006, and review clinical practice.

High frequency of acid alpha-glucosidase pseudodeficiency complicates newborn screening for glycogen storage disease type II in the Japanese population.

To investigate the feasibility of newborn screening for glycogen storage disease type II (GSDII; Pompe disease or acid maltase deficiency) in the Japanese population, we assayed the acid alpha-glucosidase activity in dried blood spots from 715 Japanese newborns and 18 previously diagnosed patients using a fluorometric procedure. The enzyme activity of apparently healthy newborns showed a bimodal distribution. The median activity of the minor group (31 individuals, 4.3% of the samples) was 6.5 times lower than that of the major group. Four of the 715 control samples (0.56%) fell in the patient range. We then analyzed genomic DNA, extracted from the same blood spots, for the occurrence of two sequence variants, c.1726G>A and c.2065G>A, known to cause "pseudodeficiency". This analysis revealed that 27 of 28 individuals homozygous for c.[1726A; 2065A] belonged to the minor group. One c.[1726A; 2065A] homozygote had just slightly higher activity. Twelve of the 18 patients with GSDII either had one (9 cases) or two (3 cases) c.[1726A; 2065A] alleles. The frequency of this allele was double in the patient compared to the control group (0.42 vs 0.19) at the expense of a lower frequency of the c.[1726G; 2065G] and c.[1726G; 2065A] alleles (0.58 vs 0.71 and 0 vs 0.1). These findings illustrate that c.[1726A; 2065A] homozygosity among apparently healthy individuals (3.9 per 100) complicates newborn screening for GSDII in Japan, and further that one or more pathogenic mutations are associated with the c.[1726A; 2065A] allele.

Structural modeling of mutant alpha-glucosidases resulting in a processing/transport defect in Pompe disease.

To elucidate the mechanism underlying transport and processing defects from the viewpoint of enzyme folding, we constructed three-dimensional models of human acid alpha-glucosidase encompassing 27 relevant amino acid substitutions by means of homology modeling. Then, we determined in each separate case the number of affected atoms, the root-mean-square distance value and the solvent-accessible surface area value. The analysis revealed that the amino acid substitutions causing a processing or transport defect responsible for Pompe disease were widely spread over all of the five domains comprising the acid alpha-glucosidase. They were distributed from the core to the surface of the enzyme molecule, and the predicted structural changes varied from large to very small. Among the structural changes, we paid particular attention to G377R and G483R. These two substitutions are predicted to cause electrostatic changes in neighboring small regions on the molecular surface. The quality control system of the endoplasmic reticulum apparently detects these very small structural changes and degrades the mutant enzyme precursor (G377R), but also the cellular sorting system might be misled by these minor changes whereby the precursor is secreted instead of being transported to lysosomes (G483R).