Non-Lysosomal Glycogen Storage Disorders Biochemical Diagnosis - PowerPoint PPT Presentation

Non-Lysosomal Glycogen Storage Disorders Biochemical Diagnosis Katie Bainbridge Enzyme Laboratory Great Ormond Street Hospital

Glycogen Metabolism Lysosome Glycogen Glycogen -glucosidase Glucose Phosphorylase b inactive Brancher Phosphorylase b kinase Phosphorylase a active Glycogen synthase Glycogen Debrancher UDP-Glucose Glucose-1-P Glucose-6-P Glucose Glucose Glucose-6- Fructose-6-phosphate phosphatase GLUT 2 Phosphofructokinase ER Fructose-1,6-bisphosphate Aldolase A Circulating Phosphoglyerate mutase Glucose Lactate β -enolase Lactate dehydrogenase TCA Pyruvate Acetyl CoA Fatty acids Trigs Cycle

Glycogen Metabolism & Glycogen Storage Disorders Lysosome GSD IV Glycogen (GSD IV) Glycogen -glucosidase Glucose Phosphorylase b inactive Brancher Phosphorylase b kinase Phosphorylase a active GSD VI Glycogen synthase GSD II (GSD V) Glycogen Debrancher GSD III (GSD IIIb) GSD 0 GSD IX UDP-Glucose Glucose-1-P (GSD IX) Glucose-6-P Glucose Glucose Glucose-6- phosphatase Fructose-6-phosphate Phosphofructokinase GLUT 2 (GSD VII) Fructose-1,6-bisphosphate ER (GSD XII) Aldolase A GSD I Phosphoglyerate mutase (GSD XI) (GSD X) Lactate Fanconi- β -enolase Bickel Lactate dehydrogenase (GSD XIII) syndrome TCA Pyruvate Acetyl CoA Fatty acids Trigs Cycle

Glycogen Metabolism & Hepatic Glycogen Storage Disorders Glycogen Phosphorylase b inactive Phosphorylase b kinase GSD VI Phosphorylase a active GSD IX Glycogen Debrancher GSD III Glucose-1-P Pentose P Pathway Ribose-6-P Glucose-6-P Glucose Glucose Glucose-6- GSD I Fanconi- phosphatase Urate Bickel Pyruvate ER Syndrome GLUT 2 Acetyl CoA Fatty acids Trigs TCA Circulating Lactate Cycle Glucose

Glycogen Metabolism & Hepatic Glycogen Storage Disorders Glycogen Phosphorylase b inactive Brancher Phosphorylase b kinase Phosphorylase a active Glycogen synthase Glycogen Debrancher GSD 0 UDP-Glucose Glucose-1-P Glucose-6-P Glucose Glucose Glucose-6- phosphatase GLUT 2 Pyruvate ER Circulating Glucose Acetyl CoA Fatty acids Trigs TCA Cycle

Glycogen Metabolism & Hepatic Glycogen Storage Disorders Glycogen Phosphorylase b inactive Brancher Phosphorylase b kinase Phosphorylase a active Glycogen synthase Glycogen Debrancher GSD 0 UDP-Glucose Glucose-1-P Dietary glucose Glucose-6-P Glucose Glucose Glucose-6- phosphatase GLUT 2 Pyruvate ER Lactate Circulating TCA Glucose Acetyl CoA Fatty acids Trigs Cycle

Glycogen Metabolism & Hepatic Glycogen Storage Disorders Abnormal Fibrosis Glycogen Phosphorylase b inactive Brancher Phosphorylase b kinase GSD IV Phosphorylase a active Glycogen synthase UDP-Glucose Glucose-1-P Glucose-6-P Glucose Glucose Glucose-6- phosphatase Pyruvate ER GLUT 2 TCA Acetyl CoA Fatty acids Trigs Circulating Cycle Glucose

Glycogen Metabolism & Muscle Glycogen Storage Disorders Lysosome Glycogen Glycogen -glucosidase Glucose Phosphorylase b inactive Brancher GSD II Phosphorylase b kinase GSD IV Phosphorylase a active GSD V GSD IX Glycogen Debrancher GSD IIIb UDP-Glucose Glucose-1-P Abnormal Glucose-6-P glycogen Fructose-6-phosphate GSD VII Phosphofructokinase Fructose-1,6-bisphosphate Aldolase A ATP FFA GSD XII Phosphoglycerate mutase GSD X β -enolase GSD XIII Lactate TCA Pyruvate Lactate dehydrogenase GSD XI Cycle

Glycogen Storage Diseases Predominately Hepatic GSDs: GSD I – glucose-6-phosphatase or transport systems in ER GSD III – debranching enzyme GSD IX – liver phosphorylase b kinase GSD VI – liver phosphorylase GSD IV – branching enzyme GSD 0 – glycogen synthase Rare Muscular forms: Predominately Muscle GSDs: GSD X - phosphoglycerate mutase GSD II – acid a-glucosidase GSD XI - LDH GSD V – muscle phosphorylase GSD XII – Aldolase A GSD XIII – β -enolase GSD VII - muscle phosphofructokinase

Glycogen Storage Diseases Muscle variant forms of (hepatic) GSDs: GSD IXd – muscle phosphorylase b kinase GSD IIIb - debranching enzyme GSD IV – branching enzyme, neuromuscular form Liver and muscle affected: GSD III – debranching enzyme GSD IV – branching enzyme GSD IX – phosphorylase b kinase

Hepato- Glucose GSD Muscle symptoms Other Biochemistry megaly homeostasis Fasting ketotic Post-prandial hyperglycaemia, GSD 0 No None hypoglycaemia and raised lactate Raised lipids, urate, lactate, Severe (ketotic) GSD I Yes None AST/ALT, proteinuria, anaemia, hypoglycaemia +/- neutopenia Truncal & proximal muscle weakness. Raised CK,vacuolated GSD II No No overt effect More severe infantile form. lymphocytes Fasting ketotic Raised lipids, AST/ALT, CK may GSD III Yes Myopathy can occur hypoglycaemia be raised GSD IV Normal until end Raised AST/ALT, CK can be Yes Myopathy can occur Hepatic stage liver disease raised Exertional muscle weakness with risk GSD V No No effect Raised CK of rhabdomyolysis Fasting ketotic GSD VI Yes None Raised AST/ALT, lipids hypoglycaemia Exertional muscle weakness with risk GSD VII No No effect Raised CK of rhabdomyolysis Fasting ketotic GSD IX liver Raised AST/ALT, lipids, CK can Yes Myopathy can occur hypoglycaemia can form be raised occur Fanconi- Raise AST/ALT, Abnormal renal Ketotic Bickel Syn. Yes None biochemistry including tubular hypoglycaemia (GSD XI) markers.

Laboratory Tests for the Investigation of Suspected GSD  Blood glucose  Initial differential diagnostic tests:  Pre and post feed  If hypoglycaemia include insulin,  Bloodspot carnitine FFA, ketones etc  RBC Gal-1-PUT  Blood lactate  PAA  Pre and post feed  UOAs  Urate  Further Tests  LFTs  Vacuolated lymphocytes  Lipids  Tissue Histology  CK  Blood GSD screen  FBC (including WBCs)  Bloodspot glucosidase  U&E, tubular proteins,  Glucagon stimulation test protein/albumin, phosphate  Tissue Enzymology  LDH  Genetics

Glycogen storage disease screen:  Minimum 5ml blood in lithium heparin  Red cells – glycogen and phosphorylase b kinase  White cells – debrancher and phosphorylase - (brancher)  Batch consists of 8 samples (manageable no. of assay tubes)  Screen takes operator one a week to complete

RBC glycogen  Relatively non invasive assessment of glycogen storage  Not elevated in GSD I, II or IV  Most useful for confirmation of GSD III  GSD IX – may be elevated to a lesser degree.  Assay takes 3 days to complete  Relatively stable  Available in RBCs, liver and muscle

Glycogen levels in GSDs GSD RBC Tissue glycogen Histology Glycogen GSD 1 Normal Raised liver glycogen PAS pos cyoplasmic glycogen, significant lipid accumulation GSD II Normal Raised muscle PAS pos lysosomal glycogen glycogen GSD III Significantly Significantly raised PAS pos cyoplasmic abnormal raised liver glycogen glycogen, some lipid accumulation GSD IV Normal Muscle glycogen PAS positive amylopectin like conc may be normal cytoplasmic glycogen (polyglucosan) GSD V Normal Muscle glycogen may PAS pos cyoplasmic glycogen be normal GSD VI Normal Raised liver glycogen PAS pos cyoplasmic glycogen, GSD VII Normal Muscle glycogen PAS pos cyoplasmic glycogen, may be normal GSD IX Often mild/mod Usually raised liver PAS pos cyoplasmic glycogen, raised glycogen

GSD IV: Liver Histology  Liver biopsy showing diffuse deposition of PAS positive amylopectin like material in hepatocytes (PAS stain).

Iodine spectrum of glycogen 1.4 1.2 1 Absorbance 0.8 0.6 Glycogen 0.4 GSD IV GSD III 0.2 0 320 340 360 390 400 420 440 460 480 500 520 540 Wavelength (nm)

Glycogen Debrancher  Glycogen debrancher: amyloglucosidase activity & oligoglucanotransferase activity  GSD IIIa: Glycogen debrancher deficiency. Liver & muscle involvement  GSD IIIb: : Glycogen debrancher deficiency. muscle involvement  GSD IIIc: Amyloglucosidase only  Type IIId Oligoglucanotransferase only  All due to mutations in AGL (1p21)

Glycogen Debrancher  Available in WBCs, fibroblasts and liver  Relatively stable  Reliable for diagnosis of GSD III in WBCs  Assay required phosphorylase limit dextrin substrate (not commercially available, takes 7 days to synthesise in house)

Glycogen Phosphorylase  Liver and muscle isoenzymes  Liver glycogen phosphorylase deficiency: GSD VI, PYGL (14q21-q22) mutations  Myophosphorylase deficiency: GSD V, PYGM (11q13) mutations  Total phosphorylase and phosphorylase a in WBCs, liver, muscle and fibroblasts available

Glycogen Phosphorylase  GSD V will have normal phosphorylase activity in WBCs, fibroblasts and liver.  Confirmed GSD VI cases described with very high residual enzyme activity in leucocytes  Very labile enzyme  WBCs must be prepared within 24 hours  Storage at -80°C improves stability  Heterozygotes for GSD V increased risk of statin induced myopathy

Phosphorylase b Kinase  Four Subunit subunit: regulatory, X allele , muscle & liver  forms  subunit: regulatory subunit: catalytic  subunit: Calcium binding 