Novel GAA sequence variant c.1211 A>G reduces enzyme activity but not protein expression in infantile and adult onset Pompe disease

Pompe disease is a clinically and genetically heterogeneous autosomal recessive disorder caused by lysosomal acid α-glucosidase (GAA) deficiency. We report on two affected members of a non-consanguineous Caucasian family, including a classical infantile-onset patient with severe cardiomyopathy (IO) and his paternal grandmother with the adult-onset (AO) form. Two compound heterozygous sequence variants of the GAA gene were identified in each patient by mutation analyses (IO=c.1211A>G and c.1798C>T; AO=c.1211A>G and c.692+5G>T). For this study, the biochemical phenotype resulting from the missense mutation c.1211A>G in exon 8, which converts a highly conserved aspartate to glycine (p.Asp404Gly), was of specific interest because it had not been reported previously. Western blotting revealed a robust expression of all GAA isoforms in quadriceps muscle of both patients (fully CRIM positive), while enzymatic activity was 3.6% (IO) and 6.6% (AO) of normal controls. To further validate these findings, the c.1211A>G sequence variant was introduced in wild type GAA cDNA and over-expressed in HEK293T cells. Site-directed mutagenesis analyses confirmed that the mutation does not affect processing or expression of GAA protein, but rather impairs enzyme function. Similar results were reported for c.1798C>T (p.Arg600Cys), which further supports the biochemical phenotype observed in IO. The third mutation (c.692+5G>T, in intron 3) was predicted to affect normal splicing of the GAA mRNA, and qPCR indeed verified a 4-fold lower mRNA expression in AO. It is concluded that the novel sequence variant c.1211A>G results in full CRIM but significantly lower GAA activity, which in combination with c.1798C>T leads to infantile-onset Pompe disease. We surmise that the difference in disease severity between the two family members in this study is due to a milder effect of the intronic mutation c.692+5G>T (vs. c.1798C>T) on phenotype, partially preserving GAA activity and delaying onset in the proband (paternal grandmother).