The c.533A>G variant in PAH is a missense variant predicted to cause substitution of glutamine by glycine at amino acid 178 (p.Glu178Gly). At least 5 patients with this variant displayed serum Phe levels > 6.5–10 mg/dl which is highly specific for PAH deficiency; BH4 deficiency was excluded (PP4_moderate, PMID: 18299955, PMID:9634518). Of those individuals, 4 probands were compound heterozygous for the variant and a pathogenic variant and 1 proband was compound heterozygous with a likely pathogenic variant (phase unknown, 2.25 points). The population allele frequency in gnomAD v4.1 is 0.00003965 which is lower than the ClinGen PAH VCEP threshold (<0.0002) for PM2_Supporting, meeting this criterion (PM2_Supporting). The results from in silico predictors [REVEL=0.841], predict a damaging effect on PAH function (PP3_moderate). Enzyme activity assay showed 39% residual phenylalanine hydroxylase activity indicating that this variant impacts protein function (PMID:17935162)(PS3_supporting). In summary, this variant meets criteria to be classified as pathogenic for PAH deficiency in an autosomal recessive manner based on the ACMG/AMP criteria applied, as specified by the ClinGen PAH Expert Panel: PM2_supporting, PP3_moderate, PS3_supporting, PP4_Moderate, PM3_Strong (version 2.0, 11/16/2024).