Pompe disease is a rare genetic disorder occurring due to mutations in the GAA gene, which encodes for the information required to produce the acid alpha-glucosidase enzyme. This enzyme normally breaks down glycogen into glucose. In the absence or reduced activity of this enzyme, excessive amounts of glycogen accumulates throughout the cells, damaging them and leading to symptoms, including muscle weakness, breathing problems, and eating difficulties.1

The diagnosis of Pompe disease is dependent on detailed patient and family history, complete clinical evaluation, and various biochemical tests with measurement of GAA activity as being the most important.2

Enzyme Activity Tests

Laboratory diagnosis is based on the measurement of GAA activity in blood or tissues.3 The confirmation of clinical diagnosis is done by the absence (infantile-onset) or severely reduced (late-onset) GAA activity in tissues like cultured fibroblasts from a skin biopsy, muscle biopsy, purified lymphocytes, mononuclear cells, and lymphoid cell lines.4 Acid alpha-glucosidase (GAA) shows an optimum activity at pH 3.7 to 4.5 and causes hydrolysis of alpha 1–4 and alpha 1–6 bonds in glucose polymers. In a typical assay, measurement of GAA activity is done at an acidic pH (3.7 or 4.5) and comparison is made with the activity of neutral glucosidase at pH 7.0, with the use of maltose and glycogen or the maltose fluorescent synthetic analogue, 4-methylumbelliferyl-α-D-glucoside (4-MUG) as substrates.3,4 The current gold standard is measurement of GAA activity in skin fibroblasts.4

Dried Blood Spot (DBS) Test

The evaluation of acid alpha-glucosidase activity in dried blood spots is a rapid, non-invasive and beneficial diagnostic test for the diagnosis of Pompe disease.5 New techniques have been developed that assay GAA activity in DBS extracts.4 Isolation of GAA from DBS extracts by immunocapture5 or competitive inhibition of  maltase-glucoamylase (MGA) activity using acarbose6 have been done to remove the interfering MGA activity. DBS may be easily collected with the heel or finger-stick method and then shipped from locations distant from the analytical center.6 The DBS methods are also useful for newborn screening of Pompe disease. 

The ease of collection and shipping dried blood specimens in addition to rapid turnaround time makes this method a good alternative to previously established techniques.6

Urine Biomarker 

Glucose tetrasaccharide (Glc4) has been found to be a useful biomarker for the diagnosis of Pompe disease.7,8 Increase in levels of Glc4 in urine is seen in Pompe disease. It has a sensitivity close to 100% for cases of infantile Pompe disease.8 Moreover, it shows a correlation with the clinical response to enzyme replacement therapy (ERT).7 Analysis of urinary Glc4 has been recommended as a noninvasive ancillary diagnostic test for this disease.

In combination with the GAA enzyme assay in DBS, the diagnostic sensitivity reaches near to 100% for infantile Pompe disease. However, it is crucial to note that negative results from both tests definitely excludes a diagnosis of infantile Pompe disease.4

Mutation Analysis

Though enzyme activity analysis is the diagnostic test of choice for Pompe disease, mutation testing is very beneficial for identifying carriers in cases of known familial mutation.4 Molecular genetic testing for GAA gene copy analysis is done as a confirmation test for Pompe disease.2 The GAA gene is situated on chromosome 17q25.3 The genetic testing aim is to recognize disease-causing mutations in the GAA gene with a blood or saliva sample.1

Muscle Biopsy

Muscle biopsy findings in cases of Pompe disease depict the presence of vacuoles which stain positive for glycogen. In the advanced disease forms, accumulation of glycogen accumulation is observed both in the lysosomes and dispersed in the cytosol. Quantitatively, levels of muscle glycogen content are increased up to tenfold above normal values in infantile disease and relatively lesser increase is seen in late-onset disease.4

Chest X-Ray

Chest X-ray is useful as a screening test in the diagnosis of infantile Pompe disease. Marked cardiomegaly is noted on a chest x-ray. However, in cases of late-onset disease, it is rarely seen.4 

Electrocardiogram (ECG)

Electrocardiogram (ECG) is another useful screening test for Pompe disease. ECG shows a short PR interval and very tall QRS complexes. In Infantile Pompe disease, ECG typically depicts hypertrophic cardiomyopathy with or without left ventricular outflow tract obstruction in the early disease phase.4

Electromyogram  (EMG)

An electromyogram (EMG) can be used to look for presymptomatic myopathy in cases presenting with cardiomyopathy.4 

Newborn Screening

Newborn screening (NBS) with the use of dried blood spots for first-tier testing has a significant contribution for early detection and treatment of Pompe disease. 2

Other Tests

Levels of Creatine kinase (CK) are elevated in Pompe disease. It is a sensitive but a nonspecific marker for this disease.The higher levels are commonly observed in infantile-onset cases (close to 2000 UI/L).4 Around  95% of late-onset cases also have raised CK values,9 however, some of the affected adults can have CK levels within the normal range. 

Serum enzymes like aspartate aminotransferase (AST), alanine aminotransferase (ALT), or lactate dehydrogenase (LDH) can also be increased and might reflect the enzymes released from muscle.10 Pulmonary function testing can also be used to identify respiratory compromise in both forms of disease.4


  1. Sedef Iskit, PhD. “Diagnosis of pompe disease.” Pompe Disease News, May 20  2021. 
  2. NORD  Pompe disease – NORD (National Organization for Rare Disorders). Accessed July 17 2021.
  3. Chien YH, Hwu WL. A review of treatment of pompe disease in infants. Biologics. 2007;1(3):195-201.
  4. Kishnani PS, Steiner RD, Bali D, et al. Pompe disease diagnosis and management guideline [published correction appears in Genet Med. 2006 Jun;8(6):382. ACMG Work Group on Management of Pompe Disease [removed]; Case, Laura [corrected to Case, Laura E]]. Genet Med. 2006;8(5):267-288. doi:10.1097/01.gim.0000218152.87434.f3
  5. Umapathysivam K, Hopwood JJ, Meikle PJ. Determination of acid alpha-glucosidase activity in blood spots as a diagnostic test for Pompe disease. Clin Chem. 2001;47(8):1378-1383.
  6. ​​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(5):302-306. doi:10.1097/01.gim.0000217781.66786.9b
  7. An Y, Young SP, Kishnani PS, et al. Glucose tetrasaccharide as a biomarker for monitoring the therapeutic response to enzyme replacement therapy for Pompe disease. Mol Genet Metab. 2005;85(4):247-254. doi:10.1016/j.ymgme.2005.03.010
  8. Young SP, Stevens RD, An Y, Chen YT, Millington DS. Analysis of a glucose tetrasaccharide elevated in pompe disease by stable isotope dilution-electrospray ionization tandem mass spectrometry. Anal Biochem. 2003;316(2):175-180. doi:10.1016/s0003-2697(03)00056-3
  9. ​​Ausems MG, Lochman P, van Diggelen OP, Ploos van Amstel HK, Reuser AJ, Wokke JH. A diagnostic protocol for adult-onset glycogen storage disease type II. Neurology. 1999;52(4):851-853. doi:10.1212/wnl.52.4.851
  10. Di Fiore MT, Manfredi R, Marri L, Zucchini A, Azzaroli L, Manfredi G. Elevation of transaminases as an early sign of late-onset glycogenosis type II. Eur J Pediatr. 1993;152(9):784. doi:10.1007/BF01954008

Reviewed by Harshi Dhingra, MD, on 7/27/2021.