Diagnosis Of Glycogen Storage Disease Type Ⅲa By Detecting Glycogen Debranching Enzyme Activity, Glycogen Content And Morphous In Muscle

BackgroundGlycogen storage diseases(GSDs) are hereditary metabolic disorders caused by deficiency of several glycogen metabolism enzymes.There are over 12 types classified based on different deficient enzymes and different tissues involved.GSD typeâ…¢(GSD-â…¢) is an autosomal recessive inherited disease,which results from deficient glycogen debranching enzyme(GDE) activity.GSD-â…¢accounts for approximately 24%of all GSD cases. There are 4 GSD-â…¢subtypes,â…¢a,â…¢b,â…¢c andâ…¢d.Gene of GDE was named as AGL. Deficiency in GDE results in an excessive accumulation of abnormal glycogen,which is harmful for hepatocytes and/or myocytes.GSD-â…¢is charactered by hepatomegaly, hypoglycemia,short stature,dyslipidemia,muscle and liver involvement.A few cases of liver cirrhosis and hepatocellular carcinoma(HCC) have been reported.Slowly progressive weakness and distal muscle wasting are observed.In the majority ofâ…¢a patients,there is a cardiac involvement with different severity,even causing death.It is difficult to diagnose by clinical characters only,and many diseases and other types of GSD should be make differential diagnosis with it,especially GSD-â… .An identified diagnosis could be obtained by AGL gene analysis,which is too expensive to make use in clinic,because there are 35extrons and rarely mutational hot spots.A definitive diagnosis,could be obtained by demonstrating abnormal glycogen(limit dextrin with short outer branches) in liver and/or muscle and deficiency of debranching enzyme activity,which is considered as an international diagnostic criteria but there is no report in our country up to now.ObjectivesTo establish enzymologic diagnosis method of GSD-â…¢for clinic use by detecting debranching enzyme activity,glycogen content and morphous in muscle,and determine the range of normal values in Chinese population.Methods1.Clinical expected GSD-â…¢patients were screened by AGLgene analysis to obtain identified GSD-â…¢a patients.2.Fifty-six providers were divided into 3 groups:normal controls(35),identified patients (12) and other myopathy patients(9).Prepared muscle homogenate.3.Glycogen in the homogenate was degraded into glucose by amyloglucosidase.Identify glycogen content by glucose yield determination.4.Glycogen in the homogenate was degraded into glucose by phosphorylase.Identify glycogen morphous by glucose yield determination.5.Determined debranching enzyme activity using limit dextrin as substrate.6.Reviewed clinical date of 12 GSD-â…¢a patients.7.Using the method to check four un-â…¢a patients.8.Detected muscle samples which contained lots of lipoids.Observe the influence of lipid to the experiment results.9.Sum up the clinical date to make a diagnosis guidelines of GSD-â…¢.Results1.Twelve GSD-â…¢a patients were identified by AGL gene analysis,and 16 mutations were found.2.There was significant difference between GSD-â…¢a patients and other two groups (p=0.000).3.There was no significant difference between normal controls and other myopathy patients.4.The range of normal values:glycogen content(0.31%-0.43%,g/g),G1P/G ratio (22.37%-26.43%),GDE activity(0.234U-0.284U).5.GDE activity is out of the range of normal values(higher) in un-â…¢a type GSD(with muscle involved) patients.6.Lipid could make a significant influence to experiment results.Conclusions1.There was an equal value in identified diagnosis of GSD-â…¢a between AGL gene analysis and enzymologic assay,which was specially an available method for GSD-â…¢a diagnosis used in clinic and would not be affected by other myopathies.2.Enzymologic diagnosis method of GSD-â…¢a was first established in our country.The range of normal values was determined in the Chinese population.3.The quality of muscle samples should be strictly controled,all of adipose tissue should be rejected as clean as possible.Attention should be paid on strange results,if muscle fibers contain more lipoids.