A genetic modifier of symptom onset in Pompe disease

Abstract

Background: Neonatal screening for Pompe disease is complicated by difficulties in predicting symptom onset in patients with the common c.-32-13T>G (IVS1) variant/null (i.e. fully deleterious) acid α-glucosidase (GAA) genotype. This splicing variant occurs in 90% of Caucasian late onset patients, and is associated with a broad range of symptom onset.

Methods: We analyzed a cohort of 143 compound heterozygous and 10 homozygous IVS1 patients, and we assessed ages at symptom onset, the presence of cis-acting single nucleotide variants (SNVs), and performed splicing analysis and enzyme activity assays.

Findings: In compound heterozygous IVS1 patients, the synonymous variant c.510C>T was uniquely present on the IVS1 allele in 9/33 (27%) patients with childhood onset, but was absent from 110 patients with onset in adulthood. GAA enzyme activity was lower in fibroblasts from patients who contained c.510C>T than it was in patients without c.510C>T. By reducing the extent of leaky wild-type splicing, c.510C>T modulated aberrant splicing caused by the IVS1 variant. The deleterious effect of c.510C>T was also found in muscle cells, the main target cells in Pompe disease. In homozygous IVS1 patients, the c.510C>T variant was absent in 4/4 (100%) asymptomatic individuals and present in 3/6 (50%) symptomatic patients. In cells from homozygous IVS1 patients, c.510C>T caused reduced leaky wild-type splicing.

Interpretation: c.510C>T is a genetic modifier in compound heterozygous and homozygous IVS1 patients. This finding is important for neonatal screening programs for Pompe disease. FUND: This work was funded by grants from Sophia Children's Hospital Foundation (SSWO, grant S17-32) and Metakids (2016-063).

Keywords: Lysosomal storage disease; Modifying factor; Pompe disease; Pre-mRNA splicing; c.510C>T.

Fig. 1

Allele frequencies of SNVs around GAA IVS1. (A) Cartoon of the genomic region of GAA spanning exon 1–3. Primers used for allele-specific amplification of the IVS1 allele are highlighted in red (Supplementary Table S4). Exonic regions are highlighted as red boxes, and introns are indicated by lines. (B) List of 21 SNVs in GAA exons 1–3 with RS-numbers and allele frequencies (MAF was obtained from gnomAD: http://gnomad.broadinstitute.org/).

Fig. 2

Association of c.510C>T with age at symptom onset and GAA enzymatic activity in compound heterozygous IVS1 patients. (A) Distribution of ages at symptom onset in compound heterozygous IVS1 patients with c.510C>T (red symbols) and without c.510C>T (green symbols). Each dot in the graph represents one patient. The dashed red line indicates the cut-off age of 18 years between patients with childhood onset and adult onset of symptoms. (B) Median age at symptom onset in compound heterozygous IVS1 patients with and without c.510C>T. *** p < 0.001. (C) GAA enzymatic activity in fibroblasts from compound heterozygous IVS1 patients with and without c.510C>T. ** p < 0.01.

Fig. 3

c.510C>T worsens splicing outcome in compound heterozygous IVS1 patients. (A) Left: RT-PCR analysis of GAA exons 1–3 of fibroblasts from a healthy control (WT) and a compound heterozygous IVS1 patient (IVS1). The second allele in this patient did not produce mRNA. M indicates the DNA size marker in base pairs (bp). Right: Cartoons of the major splice products. Numbered boxes represent canonical exons. Unnumbered boxes represent facultative exons or parts of exons: a pseudoexon in intron 1 (in red) or the C-terminal part of exon 2, which is derived from utilization of the cryptic splice site at c.487 (in brown). Dotted lines represent introns. Continuous lines represent splicing events. *: structural variant (see Suppl. Fig. S4). (B) Position of c.510C>T in GAA exon 2. Positions of the cryptic and canonical splice sites of exon 2 are also indicated. Numbered boxes represent canonical exons. The unnumbered gray box represent the pseudoexon in intron 1. Dotted lines represent introns. (C) RT-PCR analysis of GAA exon 1 to 3 in fibroblasts from compound heterozygous IVS1 patients with and without c.510C>T. Splice products N, SV6, SV5, SV3 and SV2 are indicated on the right. *: structural variant (see Suppl. Fig. S4). The patients or individuals analyzed are indicated and listed in Supplementary Tables S2 and S3. (D) As (C), but now analyzed using RT-qPCR for the wildtype splice product N. (E) As (C), but now analyzed using RT-qPCR for the aberrant splice products SV2 and SV3. (F) Ratio of SV2/SV3. In healthy control cells, expression of SV2 and SV3 was too low to allow quantification. Data in d-e represent means ± SD (n= 3 biological replicates). * = p < 0.05, *** = p < 0.001.

Fig. 4

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).

Fig. 5

c.510C>T worsens splicing outcome in skeletal muscle cells from compound heterozygous and homozygous IVS1 patients. (A) Flanking exon RT-PCR of GAA exon 2 in myoblasts. Patient 16: compound heterozygous for IVS1, c.510C>T absent; Patient 4: compound heterozygous for IVS1, c.510C>T present on IVS1 allele; Patient 18: homozygous for IVS1, homozygous for c.510C>T. Splice products N, SV6, SV5, SV3 and SV2 are indicated. *: structural variant (see Suppl. Fig. S4). Splice product N (normally spliced product) is highly expressed in patient 16 due to the presence of the c.2481+102_2646+31del variant on the second allele (which allows normal expression of GAA product N). (B) Quantification of splice product N using RT-qPCR. (C) Quantification of splice products SV2 and SV3. (D) Ratio of SV2 and SV3. (E) GAA enzymatic activity. Data in C-D and F represent means ± SD (n= 3 biological replicates).