c.510C>T is associated with symptom onset and worsening of splicing outcome in homozygous IVS1 patients. (A) Genotypes and symptom onset in 10 homozygous IVS1 patients. (B) RT-PCR analysis of GAA exon 1 to 3 in fibroblasts from homozygous IVS1 patients with and without c.510C>T. Products N, SV6, SV5, SV3 and SV2 are indicated on the right. *: structural variant (see Suppl. Fig. S4). (C) Quantification using RT-qPCR of normal splicing (product N) in fibroblasts from two homozygous IVS1 patients, one carrying the c.510C>T variant homozygously, and one without c.510C>T. For comparison, expression in fibroblasts from a healthy control is shown (individual 14). (D) As (C), but now of aberrant splice products SV2 and SV3. In fibroblasts from healthy controls, SV2 and SV3 expression was undetectable and could not therefore be quantified. (E) Ratio of SV2 and SV3. (F) Enzymatic activity of GAA. Data in C-D and F represent means ± SD (n= 3 biological replicates).