Enzymatic diagnosis of Pompe disease: lessons from 28 years of experience

Abstract Pompe disease is a lysosomal and neuromuscular disorder caused by deficiency of acid alpha-glucosidase (GAA), and causes classic infantile, childhood onset, or adulthood onset phenotypes. The biochemical diagnosis is based on GAA activity assays in dried blood spots, leukocytes, or fibroblasts. Diagnosis can be complicated by the existence of pseudodeficiencies, i.e., GAA variants that lower GAA activity but do not cause Pompe disease. A large-scale comparison between these assays for patient samples, including exceptions and borderline cases, along with clinical diagnoses has not been reported so far. Here we analyzed GAA activity in a total of 1709 diagnostic cases over the past 28 years using a total of 2591 analyses and we confirmed the clinical diagnosis in 174 patients. We compared the following assays: leukocytes using glycogen or 4MUG as substrate, fibroblasts using 4MUG as substrate, and dried blood spots using 4MUG as substrate. In 794 individuals, two or more assays were performed. We found that phenotypes could only be distinguished using fibroblasts with 4MUG as substrate. Pseudodeficiencies caused by the GAA2 allele could be ruled out using 4MUG rather than glycogen as substrate in leukocytes or fibroblasts. The Asian pseudodeficiency could only be ruled out in fibroblasts using 4MUG as substrate. We conclude that fibroblasts using 4MUG as substrate provides the most reliable assay for biochemical diagnosis and can serve to validate results from leukocytes or dried blood spots.

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

Journal of Clinical Medicine ◽

10.3390/jcm10173868 ◽

2021 ◽

Vol 10 (17) ◽

pp. 3868

Author(s):

Fanny Thuriot ◽

Elaine Gravel ◽

Katherine Hodson ◽

Jorge Ganopolsky ◽

Bojana Rakic ◽

...

Keyword(s):

Enzymatic Activity ◽

Pompe Disease ◽

Dried Blood Spots ◽

Gene Panel ◽

Muscle Disease ◽

Molecular Diagnostic ◽

Blood Spots ◽

Glucosidase Activity ◽

Gene Panel Testing ◽

Alpha Glucosidase

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.

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Newborn screening for Pompe disease with dried blood spots in a Japanese region: a pilot study

Molecular Genetics and Metabolism ◽

10.1016/j.ymgme.2013.12.183 ◽

2014 ◽

Vol 111 (2) ◽

pp. S79

Author(s):

Ken Momosaki ◽

Shirou Matsumoto ◽

Kimitoshi Nakamura ◽

Hiroshi Mitsubuchi ◽

Toshika Okumiya ◽

...

Keyword(s):

Pilot Study ◽

Newborn Screening ◽

Pompe Disease ◽

Dried Blood Spots ◽

Blood Spots

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Measurement of Glycated Hemoglobin A1c from Dried Blood by Turbidimetric Immunoassay

Journal of Diabetes Science and Technology ◽

10.1177/193229680900300527 ◽

2009 ◽

Vol 3 (5) ◽

pp. 1203-1206 ◽

Cited By ~ 23

Author(s):

Ramakrishnan Lakshmy ◽

Ruby Gupta

Keyword(s):

Whole Blood ◽

Large Scale ◽

Hemoglobin A1c ◽

Glycated Hemoglobin ◽

Intraclass Correlation ◽

Dried Blood Spots ◽

Sample Collection ◽

Blood Spots ◽

Glycated Hemoglobin A1c ◽

The Impact

Background: Glycated hemoglobin A1c (A1C) is an important marker in the diagnosis and treatment of diabetes. Dried blood measurement of A1C is useful in large scale epidemiological evaluation of A1C, especially to assess the impact of intervention programs. The possibility of using dried blood for measurement of A1C by the immunoturbidimetric method was explored in the present study. Method: Blood was collected from 30 patients, and blood spots were prepared and dried. The dried blood spot samples were kept for different lengths of time at 4°C to assess stability. Glycated hemoglobin was measured in whole blood and dried blood on the day of collection as well as on days 10 and 15 by immunoturbidimetric method. Results: The A1C values of 30 samples analyzed for comparison between whole blood estimation and dried blood ranged from 4.6% to 9.9%. The mean A1C on the day of sample collection was 6.01% ± 1.58% in fresh whole blood samples and 5.94% ± 1.58 % in dried blood spots. A linear and highly correlated relationship was observed between dried blood A1C values and those in whole blood ( r = 0.986 and intraclass correlation value = 0.993). Glycated hemoglobin values on day 10 and day 15 were comparable with the values on day 1 with a shift in mean of just 1% on day 10 and 3.04% on day 15. Conclusion: In conclusion, dried blood can be used for measurement of A1C by immunoturbidimetric method, and further stability of A1C measurement from dried blood for up to 15 days at 4°C makes it an ideal matrix for transportation in developing countries like India.

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Comparison of Dried Blood Spots Versus Conventional Plasma Collection for the Characterization of Efavirenz Pharmacokinetics in a Large-Scale Global Clinical Trial—The ENCORE1 Study

Therapeutic Drug Monitoring ◽

10.1097/ftd.0000000000000448 ◽

2017 ◽

Vol 39 (6) ◽

pp. 654-658 ◽

Cited By ~ 5

Author(s):

Alieu B. Amara ◽

Laura J. Else ◽

Dianne Carey ◽

Saye Khoo ◽

David J. Back ◽

...

Keyword(s):

Clinical Trial ◽

Large Scale ◽

Dried Blood Spots ◽

Blood Spots ◽

Global Clinical Trial ◽

Plasma Collection

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Determination of Acid α-Glucosidase Protein: Evaluation as a Screening Marker for Pompe Disease and Other Lysosomal Storage Disorders

Clinical Chemistry ◽

10.1093/clinchem/46.9.1318 ◽

2000 ◽

Vol 46 (9) ◽

pp. 1318-1325 ◽

Cited By ~ 36

Author(s):

Kandiah Umapathysivam ◽

Alison M Whittle ◽

Enzo Ranieri ◽

Colleen Bindloss ◽

Elaine M Ravenscroft ◽

...

Keyword(s):

Pompe Disease ◽

Screening Program ◽

Dried Blood Spots ◽

Lysosomal Storage Disorders ◽

Lysosomal Storage ◽

Total Acid ◽

Blood Spots ◽

Enzyme Replacement ◽

Storage Disorders

Abstract Background: In recent years, there have been significant advances in the development of enzyme replacement and other therapies for lysosomal storage disorders (LSDs). Early diagnosis, before the onset of irreversible pathology, has been demonstrated to be critical for maximum efficacy of current and proposed therapies. In the absence of a family history, the presymptomatic detection of these disorders ideally can be achieved through a newborn screening program. One approach to the development of such a program is the identification of suitable screening markers. In this study, the acid α-glucosidase protein was evaluated as a marker protein for Pompe disease and potentially for other LSDs. Methods: Two sensitive immunoquantification assays for the measurement of total (precursor and mature) and mature forms of acid α-glucosidase protein were used to determine the concentrations in plasma and dried blood spots from control and LSD-affected individuals. Results: In the majority of LSDs, no significant increases above control values were observed. However, individuals with Pompe disease showed a marked decrease in acid α-glucosidase protein in both plasma and whole blood compared with unaffected controls. For plasma samples, this assay gave a sensitivity of 95% with a specificity of 100%. For blood spot samples, the sensitivity was 82% with a specificity of 100%. Conclusions: This study demonstrates that it is possible to screen for Pompe disease by screening the concentration of total acid α-glucosidase in plasma or dried blood spots.

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Experience of a Brazilian laboratory on acid alpha-glucosidase enzyme assay in dried blood spots on filter paper (DBS) and leukocytes samples

Molecular Genetics and Metabolism ◽

10.1016/j.ymgme.2012.11.139 ◽

2013 ◽

Vol 108 (2) ◽

pp. S57

Author(s):

Sandra Kyosen ◽

Karen Muller ◽

Marion Braga ◽

Joyce Yamamoto ◽

Ana Maria Martins ◽

...

Keyword(s):

Filter Paper ◽

Enzyme Assay ◽

Dried Blood Spots ◽

Blood Spots ◽

Alpha Glucosidase

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Determination of Oligosaccharides in Pompe Disease by Electrospray Ionization Tandem Mass Spectrometry

Clinical Chemistry ◽

10.1093/clinchem/48.1.131 ◽

2002 ◽

Vol 48 (1) ◽

pp. 131-139 ◽

Cited By ~ 40

Author(s):

Tina Rozaklis ◽

Steven L Ramsay ◽

Phillip D Whitfield ◽

Enzo Ranieri ◽

John J Hopwood ◽

...

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Electrospray Ionization ◽

Pompe Disease ◽

Dried Blood Spots ◽

Tandem Mass ◽

Lysosomal Storage ◽

Blood Spots ◽

Limited Application ◽

Ionization Tandem Mass Spectrometry

Abstract Background: The development of therapies for lysosomal storage disorders has created a need for biochemical markers to monitor the efficacy of therapy and methods to quantify these markers in biologic samples. In Pompe disease, the concentration of a tetrasaccharide, consisting of four glucose residues, is reputedly increased in urine and plasma, but faster and more sensitive methods are required for the analysis of this, and other oligosaccharides, from biologic fluids. Methods: We optimized the derivatization of storage oligosaccharides with 1-phenyl-3-methyl-5-pyrazolone for the measurement, by electrospray ionization tandem mass spectrometry, of oligosaccharide concentrations in urine (n = 6), plasma (n = 11), and dried-blood spots (n = 17) from Pompe-affected individuals. Age-matched control samples of urine (n = 10), plasma (n = 28), and blood spots (n = 369) were also analyzed. Results: The mean tetrasaccharide concentration was increased in urine from infantile-onset (0.69–12 mmol/mol of creatinine) and adult-onset (0.22–3.0 mmol/mol of creatinine) Pompe individuals compared with age-matched controls. In plasma samples, an increased tetrasaccharide concentration was observed in some infantile patients (up to 22 μmol/L) compared with age-matched controls (mean, 2.2 μmol/L). The method developed was sensitive enough to determine oligosaccharide concentrations in a single 3-mm blood spot, but no differences were observed between blood spots from control and Pompe-affected individuals. Conclusions: Measurements of oligosaccharide concentrations in urine by this new method have potential application for the diagnosis and monitoring of patients with Pompe disease. Plasma analysis may have limited application for infantile patients, but analysis of blood spots does not discriminate between controls and affected individuals.

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Late-onset Pompe disease: what is the prevalence of limb-girdle muscular weakness presentation?

Arquivos de Neuro-Psiquiatria ◽

10.1590/0004-282x20180018 ◽

2018 ◽

Vol 76 (4) ◽

pp. 247-251 ◽

Cited By ~ 3

Author(s):

Paulo José Lorenzoni ◽

Cláudia Suemi Kamoi Kay ◽

Nádia Sugano Higashi ◽

Vânia D'Almeida ◽

Lineu Cesar Werneck ◽

...

Keyword(s):

Enzyme Activity ◽

Muscle Biopsy ◽

Pompe Disease ◽

Late Onset ◽

Dried Blood Spots ◽

Muscular Weakness ◽

Inherited Disease ◽

Blood Spots ◽

Vacuolar Myopathy ◽

Gaa Gene

ABSTRACT Pompe disease is an inherited disease caused by acid alpha-glucosidase (GAA) deficiency. A single center observational study aimed at assessing the prevalence of late-onset Pompe disease in a high-risk Brazilian population, using the dried blood spot test to detect GAA deficiency as a main screening tool. Dried blood spots were collected for GAA activity assay from 24 patients with “unexplained” limb-girdle muscular weakness without vacuolar myopathy in their muscle biopsy. Samples with reduced enzyme activity were also investigated for GAA gene mutations. Of the 24 patients with dried blood spots, one patient (4.2%) showed low GAA enzyme activity (NaG/AaGIA: 40.42; %INH: 87.22%). In this patient, genetic analysis confirmed two heterozygous mutations in the GAA gene (c.-32-13T>G/p.Arg854Ter). Our data confirm that clinicians should look for late-onset Pompe disease in patients whose clinical manifestation is an “unexplained” limb-girdle weakness even without vacuolar myopathy in muscle biopsy.

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