A Modified Enzymatic Method for Measurement of Glycogen Content in Glycogen Storage Disease Type IV

  • Haiqing Yi
  • Quan Zhang
  • Chunyu Yang
  • Priya S. Kishnani
  • Baodong SunEmail author
Research Report
Part of the JIMD Reports book series (JIMD, volume 30)


Deficiency of glycogen branching enzyme in glycogen storage disease type IV (GSD IV) results in accumulation of less-branched and poorly soluble polysaccharides (polyglucosan bodies) in multiple tissues. Standard enzymatic method, when used to quantify glycogen content in GSD IV tissues, causes significant loss of the polysaccharides during preparation of tissue lysates. We report a modified method including an extra boiling step to dissolve the insoluble glycogen, ultimately preserving the glycogen content in tissue homogenates from GSD IV mice. Muscle tissues from wild-type, GSD II and GSD IV mice and GSD III dogs were homogenized in cold water, and homogenate of each tissue was divided into two parts. One part was immediately clarified by centrifugation at 4°C (STD-prep); the other part was boiled for 5 min then centrifuged (Boil-prep) at room temperature. When glycogen was quantified enzymatically in tissue lysates, no significant differences were found between the STD-prep and the Boil-prep for wild-type, GSD II and GSD III muscles. In contrast, glycogen content for GSD IV muscle in the STD-prep was only 11% of that in the Boil-prep, similar to wild-type values. Similar results were observed in other tissues of GSD IV mice and fibroblast cells from a GSD IV patient. This study provides important information for improving disease diagnosis, monitoring disease progression, and evaluating treatment outcomes in both clinical and preclinical clinical settings for GSD IV. This report should be used as an updated protocol in clinical diagnostic laboratories.


Glycogen quantitation method Glycogen storage disease type IV Lafora disease Polyglucosan body 



We thank Dr. Craigen and Dr. Akman of Baylor College of Medicine for sharing their new mouse model of GSD IV (Gbe1 ys/ys mice). We would also like to thank Cecelia Mizelle for helping edit the manuscript. This work was supported by the Alice and Y.T. Chen Research Center for Genetics and Genomics at Duke University.


  1. Akman HO, Emmanuele V, Kurt YG, Kurt B, Sheiko T, DiMauro S, Craigen WJ (2015) A novel mouse model that recapitulates adult onset glycogenosis type 4. Hum Mol Genet 24:6801–6810CrossRefGoogle Scholar
  2. Brown BI, Brown DH (1966) Lack of an alpha-1,4-glucan: alpha-1,4-glucan 6-glycosyl transferase in a case of type IV glycogenosis. Proc Natl Acad Sci U S A 56:725–729CrossRefPubMedPubMedCentralGoogle Scholar
  3. Chen Y-T, Kishnani PS, Koeberl DD (2009) Glycogen storage diseases. In: Valle D, Beaudet A, Vogelstein B, Kinzler K, Antonarakis S, Ballabio A (eds) Scriver’s online metabolic & molecular bases of inherited disease. McGraw-Hill, New YorkGoogle Scholar
  4. Fernandes J, Huijing F (1968) Branching enzyme-deficiency glycogenosis: studies in therapy. Arch Dis Child 43:347–352CrossRefPubMedPubMedCentralGoogle Scholar
  5. Huijing F (1970) A rapid enzymic method for glycogen estimation in very small tissue samples. Clin Chim Acta 30:567–572CrossRefPubMedGoogle Scholar
  6. Huijing F (1975) Glycogen metabolism and glycogen-storage diseases. Physiol Rev 55:609–658PubMedGoogle Scholar
  7. Kakhlon O, Glickstein H, Feinstein N, Liu Y, Baba O, Terashima T, Akman HO, Dimauro S, Lossos A (2013) Polyglucosan neurotoxicity caused by glycogen branching enzyme deficiency can be reversed by inhibition of glycogen synthase. J Neurochem 127:101–113PubMedGoogle Scholar
  8. Kikuchi T, Yang HW, Pennybacker M, Ichihara N, Mizutani M, Van Hove JL, Chen YT (1998) Clinical and metabolic correction of pompe disease by enzyme therapy in acid maltase-deficient quail. J Clin Invest 101:827–833CrossRefPubMedPubMedCentralGoogle Scholar
  9. Koeberl DD, Bottema CD, Sommer SS (1990) Comparison of direct and indirect methods of carrier detection in an X-linked disease. Am J Med Genet 35:600–608CrossRefPubMedGoogle Scholar
  10. Koeberl DD, Sun BD, Damodaran TV, Brown T, Millington DS, Benjamin DK, Bird A, Schneider A, Hillman S, Jackson M et al (2006) Early, sustained efficacy of adeno-associated virus vector-mediated gene therapy in glycogen storage disease type Ia. Gene Ther 13:1281–1289CrossRefPubMedGoogle Scholar
  11. Lossos A, Meiner Z, Barash V, Soffer D, Schlesinger I, Abramsky O, Argov Z, Shpitzen S, Meiner V (1998) Adult polyglucosan body disease in Ashkenazi Jewish patients carrying the Tyr329Ser mutation in the glycogen-branching enzyme gene. Ann Neurol 44:867–872CrossRefPubMedGoogle Scholar
  12. Mercier C, Whelan WJ (1970) The fine structure of glycogen from type IV glycogen-storage disease. Eur J Biochem/FEBS 16:579–583CrossRefGoogle Scholar
  13. Minassian BA (2001) Lafora’s disease: towards a clinical, pathologic, and molecular synthesis. Pediatr Neurol 25:21–29CrossRefPubMedGoogle Scholar
  14. Mochel F, Schiffmann R, Steenweg ME, Akman HO, Wallace M, Sedel F, Laforêt P, Levy R, Powers JM, Demeret S et al (2012) Adult polyglucosan body disease: natural history and key magnetic resonance imaging findings. Ann Neurol 72:433–441CrossRefPubMedPubMedCentralGoogle Scholar
  15. Murat JC, Serfaty A (1974) Simple enzymatic determination of polysaccharide (glycogen) content of animal tissues. Clin Chem 20:1576–1577PubMedGoogle Scholar
  16. Pederson BA, Csitkovits AG, Simon R, Schroeder JM, Wang W, Skurat AV, Roach PJ (2003) Overexpression of glycogen synthase in mouse muscle results in less branched glycogen. Biochem Biophys Res Commun 305:826–830CrossRefPubMedGoogle Scholar
  17. Pederson BA, Chen H, Schroeder JM, Shou W, DePaoli-Roach AA, Roach PJ (2004) Abnormal cardiac development in the absence of heart glycogen. Mol Cell Biol 24:7179–7187CrossRefPubMedPubMedCentralGoogle Scholar
  18. Raben N, Nagaraju K, Lee E, Kessler P, Byrne B, Lee L, LaMarca M, King C, Ward J, Sauer B et al (1998) Targeted disruption of the acid alpha-glucosidase gene in mice causes an illness with critical features of both infantile and adult human glycogen storage disease type II. J Biol Chem 273:19086–19092CrossRefPubMedGoogle Scholar
  19. Raben N, Danon M, Lu N, Lee E, Shliselfeld L, Skurat AV, Roach PJ, Lawrence JC Jr, Musumeci O, Shanske S et al (2001) Surprises of genetic engineering: a possible model of polyglucosan body disease. Neurology 56:1739–1745CrossRefPubMedGoogle Scholar
  20. Raben N, Danon M, Gilbert AL, Dwivedi S, Collins B, Thurberg BL, Mattaliano RJ, Nagaraju K, Plotz PH (2003) Enzyme replacement therapy in the mouse model of Pompe disease. Mol Genet Metab 80:159–169CrossRefPubMedGoogle Scholar
  21. Sun B, Zhang H, Franco LM, Brown T, Bird A, Schneider A, Koeberl DD (2005) Correction of glycogen storage disease type II by an adeno-associated virus vector containing a muscle-specific promoter. Mol Ther J Am Soc Gene Ther 11:889–898CrossRefGoogle Scholar
  22. Sun B, Bird A, Young SP, Kishnani PS, Chen YT, Koeberl DD (2007) Enhanced response to enzyme replacement therapy in Pompe disease after the induction of immune tolerance. Am J Hum Genet 81:1042–1049CrossRefPubMedPubMedCentralGoogle Scholar
  23. Suzuki Y, Lanner C, Kim JH, Vilardo PG, Zhang H, Yang J, Cooper LD, Steele M, Kennedy A, Bock CB et al (2001) Insulin control of glycogen metabolism in knockout mice lacking the muscle-specific protein phosphatase PP1G/RGL. Mol Cell Biol 21:2683–2694CrossRefPubMedPubMedCentralGoogle Scholar
  24. Van Hove JL, Yang HW, Wu JY, Brady RO, Chen YT (1996) High-level production of recombinant human lysosomal acid alpha-glucosidase in Chinese hamster ovary cells which targets to heart muscle and corrects glycogen accumulation in fibroblasts from patients with Pompe disease. Proc Natl Acad Sci U S A 93:65–70CrossRefPubMedPubMedCentralGoogle Scholar
  25. Yi H, Thurberg BL, Curtis S, Austin S, Fyfe J, Koeberl DD, Kishnani PS, Sun B (2012) Characterization of a canine model of glycogen storage disease type IIIa. Dis Model Mech 5:804–811CrossRefPubMedPubMedCentralGoogle Scholar
  26. Yi H, Brooks ED, Thurberg BL, Fyfe JC, Kishnani PS, Sun B (2014) Correction of glycogen storage disease type III with rapamycin in a canine model. J Mol Med (Berl) 92:641–650CrossRefGoogle Scholar

Copyright information

© SSIEM and Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Haiqing Yi
    • 1
  • Quan Zhang
    • 1
    • 2
  • Chunyu Yang
    • 1
  • Priya S. Kishnani
    • 1
  • Baodong Sun
    • 1
    Email author
  1. 1.Division of Medical Genetics, Department of PediatricsDuke University School of MedicineDurhamUSA
  2. 2.College of Veterinary Medicine, Yangzhou UniversityYangzhouChina

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