Table 4.

Points allocated for different types of evidence for PP4.

Description of evidence	Points
Deficient GAA activitya, documented as either 1) <10% of normal mean control level of GAA activity in leukocytes, lymphocytes, or muscleb samples, and/or <30% in cultured fibroblastsc, or 2) Activity in the affected range (which must be provided in the publication) in any appropriate tissue (muscle, cultured skin fibroblasts, leukocytes, lymphocytes, whole blood or dried blood spot diagnostic assay; newborn screening methodologies not appropriate).	2
Patient reported to have Infantile Onset Pompe disease (IOPD) AND documentation of symptomsd of that condition. At a minimum, cardiomegaly, hypertrophic cardiomyopathy, left ventricular hypertrophy or a related term, and hypotonia, muscle weakness, or a related term, must be reported.	1
Cross reactive immunological material (CRIM) study of cultured skin fibroblasts or peripheral blood mononuclear cells	1
reported to show absence of the 76 Da and 70 kDa bands, which represent the mature, active GAA enzymee. This includes patients described as CRIM-negative (with no detectable GAA protein on Western blot), or those who are CRIM-positive but do not make the mature protein (e.g. only 110 kDa and 95 kDa bands are present).
The patient is reported to be on enzyme replacement therapyf for Pompe disease.	1
GAA activity is reported to be deficienta but the data are not provided (i.e. values for the patient and normal range as determined by the testing laboratory)	0.5
Individual identified by positive newborn screening results	0.25
Urinary Glc4g is elevated above the normal range.	0.25
Muscle MRI shows evidence of Pompe disease.	0.25
Muscle histologyh is consistent with Pompe disease; there is glycogen storage in the lysosomes of muscle cells appearing as vacuoles that stain positively with periodic acid-Schiff.	0.25

For a total of 2 points or more, PP4_Moderate is applied; for 1 point, PP4 is applied.

Criterion not applied if the variant meets BA1, or otherwise meets criteria for benign or likely benign classification.

^aIf either of the pseudodeficiency variants c.1726G>A - p.(Gly576Ser) or c.2065G>A - p.(Glu689Lys) are present, whether heterozygous or homozygous, deficiency of GAA activity cannot be used to apply PP4. If c.271G>A - p.(Asp91Asn) is present, deficiency of GAA activity cannot be used to apply PP4 if glycogen was the assay substrate, but the data can be used if 4-MU was the substrate³⁶.

^b<10% activity in muscle is used because activity of GAA in muscle samples can overlap in patients with LOPD and carriers.

^cTo account for higher residual GAA activity in patients with late onset Pompe disease (LOPD)

^dSymptoms in patients with IOPD are fairly specific with few other conditions mimicking this disorder. Therefore, documentation of symptoms is not considered to be sufficient for application of PP4

^eFor further details on the synthesis and intracellular processing and transport of GAA, and CRIM analysis in patients with Pompe disease, please see refs 35 and 63.

^fIf a patient is receiving Enzyme Replacement Therapy, the assumption is that their diagnosis of Pompe disease is well supported by clinical and laboratory evaluations

^gElevated urinary glucose tetrasaccharide (Glc4, also known as hexose tetrasaccharide or Hex4), which is derived from the amylolytic degradation of glycogen, has a high sensitivity and specificity for Pompe disease^{64, 65}.

^hWhile histochemical evidence of glycogen storage in muscle is supportive of a glycogen storage disorder it is not specific for Pompe disease.