The American College of Medical Genetics and Genomics (ACMG) guideline facilitates prompt identification of patients with GSDs VI and IX, expedite diagnosis, and minimise adverse sequelae from delayed diagnosis and inappropriate management.

Glycogen storage disease (GSD) types VI and IX are rare diseases of variable clinical severity affecting primarily the liver. GSD VI is caused by deficient activity of hepatic glycogen phosphorylase, whereas GSD IX is caused by deficient activity of phosphorylase kinase (PhK).

Individuals with GSDs VI and IX may present with hepatomegaly with elevated serum transaminases, ketotic hypoglycemia, hyperlipidemia, and poor growth. The plethora of clinical manifestations observed with GSD types VI and IX makes it difficult to distinguish it from other liver GSDs just on the basis of clinical presentation.

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The ACMG guideline can help health-care providers to facilitate prompt and accurate diagnosis, which, in turn, may lead to appropriate management of patients.

Differential diagnosis: GSD VI and liver GSD IX

Due to the overlapping features, it is important to differentiate GSD VI and liver GSD IXs with other forms of GSD associated with hepatomegaly and hypoglycaemia, especially GSD I and III.

In ultrasound imaging of the liver, the presence of isolated hepatomegaly and nephromegaly with hypoglycemia, hypertriglyceridaemia, hyperuricaemia, and lactic acidosis, suggests diagnosis of GSD I.

In liver biopsy, GSD I demonstrates extensive hepatic steatosis with some glycogen accumulation, whereas GSD III, VI, and IX have extensive glycogen accumulation in the liver due to which swollen hepatocytes are observed on electron microscopy. GSD III and IX also show periportal liver fibrosis in the initial stages of the disease and extensive fibrosis and cirrhosis in the later stages.

Hepatomegaly, hypoglycaemia, and hyperlipidaemia are commonly observed in GSDs I, III, VI, and IX; however, some key differences can help differential diagnosis.

1. GSD I patients present with severe fasting hypoglycaemia in the first few months of life, which is associated with lactic acidosis and hyperuricaemia (not observed in other GSDs).
2. As gluconeogenesis is intact, hypoglycemia is not very severe in patients with GSDs VI and IX; however, some patients may present with severe recurrent hypoglycaemia.
3. Hyperketonaemia with fasting hypoglycaemia is commonly observed in GSDs III, VI, and IX; however, blood ß-hydroxybutyrate levels increase only modestly in GSD I.
4. Hepatic transaminase levels are significantly higher in GSD III, VI, and IX as compared with GSD I. The extent of hepatomegaly is comparable in GSDs VI and IX and both disorders may be associated with hyperketonaemia after an overnight fast.

GSD VI is an autosomal recessive condition and GSD IX has subtypes that are autosomal recessive and X-linked. Males are more likely to have GSD IX due to a PHKA2 pathogenic variant, but they can also be affected with GSD VI or other GSD IX subtypes. Females can be affected with either the autosomal recessive subtypes or the X-linked subtype (rare cases).

GSD IV can be distinguished from GSDs VI and IX by absence of hypoglycaemia and ketosis with progressive liver dysfunction leading to liver cirrhosis. In GSD IV there is an accumulation of abnormally structured glycogen, resembling plant-like fibers of amylopectin, whereas in GSD III the abnormally structured glycogen resembles limit dextrin.

Secondary PhK deficiency is observed with Fanconi–Bickel syndrome (GSD XI) and cardiac/muscle glycogenosis caused by PRKAG2 deficiency.

Due to the presence of severe hepatomegaly, Gaucher disease and Niemann–Pick type B disease is sometimes confused with GSDs VI and IX. However, the presence of striking splenomegaly and absence of hypoglycemia helps in distinguishing the both.

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Clinical and Laboratory Evaluation

Initial workup in patients presenting with hepatomegaly and hypoglycemia include the following:

• Liver ultrasound
• Serum transaminases (AST, ALT)
• ɣ-glutamyl transferase (GGT)
• Liver function tests (prothrombin time, albumin)
• Blood glucose, lactate, uric acid
• Basic chemistry
• Creatine kinase (CK)
• Plasma total and free carnitine, acylcarnitine profile
• Urinalysis, urine organic acids
• Cholesterol, triglycerides
• Complete blood count (CBC) with manual differential white cell count

The detection of plasma ketones is important as the presence of serum β-OHB during episodes of hypoglycaemia helps to separate ketotic hypoglycaemia from nonketotic or hypoketotic hypoglycaemia conditions.

Measurement of insulin, growth hormone, cortisol, and free fatty acids during a critical sample of hypoglycaemia helps to rule out endocrine causes. This is particularly useful when hepatomegaly is not a significant feature.

Patients with GSDs VI and IX have elevated transaminases, with the levels typically being higher in patients with GSD IX compared with GSD VI. GGT levels may vary from normal to elevated.

CK concentration is usually normal in GSDs VI and IX as there is no overt muscle involvement; slight elevations may sometimes be observed in cases with profound protein deficiency. The uric acid level and lactate are also normal; lactate can be elevated in rare cases postprandially.

The levels of triglyceride and cholesterol are often observed to be elevated. Abdominal ultrasound shows mild to marked diffuse hepatomegaly, often with increased liver echogenicity.

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Liver histology

Usually if GSD VI or IX are suspected, a liver biopsy is not recommended to establish the diagnosis; however when no definitive diagnosis can be made noninvasively, liver biopsy can be performed. The following liver histology features are found in GSD VI and GSD IX.

• Presence of periportal fibrosis with thin septa in between lobules in GSD IX (early stages)
• Liver parenchyma with a mosaic of hepatocytes that are distended because of excessive glycogen accumulation in GSD VI and IX
• Coarse cell membranes with an undulated appearance; scattered cytoplasmic vacuoles
• Glycogen staining with periodic acid–Schiff (PAS) stain is diastase digestion–sensitive
• Electron microscopy shows glycogen structure with excessive glycogen accumulation; glycogen has a frayed or burst appearance and is found to be less compact than GSD I or III
• Cytoplasmic lipid bodies are more likely to be found in hepatocytes in GSD IX
• Children with GSD IX often show fibrosis of the portal tracts that may also be associated with inflammation
• Liver cirrhosis is more likely to be detected in individuals with PHKG2 pathogenic variants

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Biochemical analysis: glycogen content and enzyme activity

Snap frozen liver biopsies show markedly elevated glycogen content with normal structure in patients affected with GSD VI and GSD IX. GSD III is associated with elevated glycogen content of abnormal structure.

Laboratory diagnostic testing recommendations

1. Instead of considering liver biopsy, DNA testing should be done first. As multiple genes are involved, use of next-generation sequencing panels is recommended. The limitations of sequencing should however, be recognized, and in cases with a strong clinical suspicion, the diagnosis should be done by other methods.
2. PhK enzyme activity may be either normal or elevated in blood cells in the patients. If variants of unknown significance are observed during genetic testing, liver biopsy is recommended to confirm diagnosis
3. In GSD VI and GSD IX, a marked elevation of glycogen (structurally normal) content is observed in the liver. Phosphorylase and PhK enzyme activity can also be measured. As both enzymes are highly labile, samples must be handled carefully. It should be noted that in certain individuals with GSD IX, the PhK activity can be normal or not clearly deficient in liver.
4. PhK enzyme activity can also be reduced due to a different, primary metabolic defect such as GLUT2 pathogenic variant in Fanconi–Bickel syndrome, PRKAG2 cardiomyopathy syndrome, or mitochondrial complex 1 deficiency.