Test ID: GAABS
Acid Alpha-Glucosidase, Blood Spot

Evaluation of patients of any age with a clinical presentation suggestive of Pompe disease (muscle hypotonia, weakness, and/or cardiomyopathy)

• Informed Consent for Genetic Testing

Fluorometric Enzyme Assay

Container/Tube:

Preferred: Whatman Protein Saver 903 Paper

Acceptable: Ahlstrom 226 Filter Paper, Supplemental Newborn Screening Card (Supply T493)

Specimen Volume: 2 blood spots

Collection Instructions:

1. Do not use device or capillary tube containing EDTA to collect specimen.

2. An alternative blood collection option for a patient >1 year of age is fingerstick.

3. Let blood dry on the filter paper at ambient temperature in a horizontal position for 3 hours.

4. Do not expose specimen to heat or direct sunlight.

5. Do not stack wet specimens.

6. Keep specimen dry.

Forms:

1.   1. New York Clients-Informed consent is required. Please document on the request form or electronic order that a copy is on file. An Informed Consent for Genetic Testing (Supply T576) is available in Special Instructions.

2.   2. If not ordering electronically, submit a Biochemical Genetics Request Form (Supply T439) with the specimen.

Pompe disease, also known as glycogen storage disease type II, is an autosomal recessive disorder caused by a deficiency of the lysosomal enzyme alpha-glucosidase (GAA) leading to an accumulation of glycogen in the lysosome causing swelling, cell damage, and progressive organ dysfunction. Pompe disease is caused by mutations in the GAA gene, and it is characterized by muscle hypotonia, weakness, cardiomyopathy, and eventually death due to either cardiorespiratory or respiratory failure. The clinical phenotype, in general, appears to be dependent on residual enzyme activity, with complete loss of activity causing onset in infancy leading to death, typically within the first year of life. Juvenile and adult-onset forms are characterized by later onset and longer survival. Treatment by enzyme replacement therapy, recently available, makes early diagnosis of Pompe disease desirable, as early initiation of treatment may improve the prognosis. The estimated incidence is 1 in 40,000 live births.

Since Pompe disease is considered a rare condition that progresses rapidly in infancy, the disease, in particular the juvenile and adult-onset forms, is often considered late, if at all, during the evaluation of patients presenting with muscle hypotonia, weakness, and/or cardiomyopathy. Testing traditionally required a skin or muscle biopsy to establish cultures for enzyme testing. More recently, molecular genetic testing of the GAA gene (GAAMS/89898 Pompe Disease, Full Gene Sequencing) became clinically available. Determination of the enzyme assay in dried blood spot specimens can be performed in a timely fashion and provide better guidance in the decision to submit samples for further confirmatory testing by molecular genetic analysis (GAAMS/89898 Pompe Disease, Full Gene Sequencing).

Normal >7.4 pmol/punch/h

Normal results (>7.4 pmol/dried blood spot punch/hour) in properly submitted specimens are not consistent with classic Pompe disease. Affected individuals typically show <2.5 pmol/dried blood spot punch/hour; however, some later onset cases may show higher enzyme activity.

Results <7.5 pmol/dried blood spot punch/hour can be followed up by molecular genetic analysis of the GAA gene (GAAMS/89898 Pompe Disease, Full Gene Sequencing) to determine carrier or disease status.

Specimens exposed to heat >25 degrees C for more than 48 hours will yield higher activity levels than properly submitted specimens. This may cause false-normal (false-negative) results in affected patients.

1. Kishnani PS, Howell RR: Pompe disease in infants and children. J Pediatr 2004;144:S35-S43

2. Winkel LP, Hagemans ML, van Doorn PA, et al: The natural course of non-classic Pompe's disease; a review of 225 published cases. J Neurol 2005;252:875-884

3. Katzin LW, Amato AA: Pompe disease: a review of the current diagnosis and treatment recommendations in the era of enzyme replacement therapy. J Clin Neuromuscul Dis 2008 Jun;9(4):421-431

4. Enns GM, Steiner RD, Cowan TM: Lysosomal disorders. In Pediatric Endocrinology and Inborn Errors of Metabolism. Edited by K Sarafoglou, GF Hoffmann, KS Roth. McGraw-Hill, Medical Publishing Division, 2009, pp 750-751

Whole blood is collected on Protein Saver 903 (Whatman) filter paper and dried at room temperature. A one-eighth inch (3-mm) disk is punched out of the dried blood spots into a microcentrifuge tube. The extraction of blood from the blood spot using water takes place on an orbital shaker for 1 hour at 2 to 8 degrees C in a chilled shaker or cool room. A total of 40 mcL of the extracted sample is combined with 50 mcL of substrate A (40 mM sodium acetate, pH 3.8 with 1.4 mM 4-methylumbelliferyl-alpha-D-glucopyranoside [4-MUG]) and 10 mcL of 8 mcmol acarbose (acts as an inhibitor of leukocyte-derived alpha-glucosidase) in a black 96-well plate, in duplicate. Blanks that contain substrate and inhibitor only are also set up in duplicate with 2 wells used for each patient. The plate is sealed with aluminum sealer and incubated at 37 degrees C for 20 hours. The remaining extracts are also incubated at 37 degrees C for 20 hours. The plate is centrifuged at 600 g for 5 minutes to collect any condensation on the sealer. A total of 40 mcL of the leftover extracts are added to each of the corresponding "blank" wells for each patient. Finally, 200 mcL of stop buffer (150 mM EDTA, pH 11.4) is added to all wells. A calibration curve is set up on every plate to calculate results. (Chamoles NA, Niizawa G, Blanco M, et al: Glycogen storage disease type II: enzymatic screening in dried blood spots on filter paper. Clin Chim Acta 2004;347:97-102; Zhang H, Kallwass H, Young SP, et al: Comparison of maltose and acarbose as inhibitors of maltase-glucoamylase activity in assaying acid alpha-glucosidase activity in dried blood spots for the diagnosis of infantile Pompe disease. Genet Med 2006;8:302-306)

Thursday; morning

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