Biochemical and Genetic Testing of GAA in Over 30.000 Symptomatic Patients Suspected to Be Affected With Pompe Disease

Abstract

Pompe disease (PD) is a rare autosomal recessive lysosomal disorder caused by loss-of-function of the α-glucosidase (GAA) gene. The deficient GAA enzyme activity may result in potential life-threatening muscle weakness, thus requiring a rapid diagnosis to initiate therapeutic interventions. In this large retrospective study, we analyzed 30.836 PD suspect samples from 57 countries using a two-step approach utilizing dried blood spots (DBSs): biochemical testing of GAA activity followed by complementary genetic sequencing of GAA in biochemically conspicuous cases. Of these 30.836 samples, 2% (n = 639) were excluded; accordingly, this study consisted of 30.193 cases. Biochemical testing of GAA enzyme activity showed normal values in 28.354 (93.90%) and enzyme activity below the cut-off in 1843 (6.10%) cases. These biochemically suspicious cases were genetically analyzed. We identified 723 Pompe cases with 283 different GAA alterations, and 98 variants have been unpublished so far. The most common variant was the splice variant c.-32-13T>G (IVS1). Looking at the IVS1-genotype, the majority was compound heterozygous (n = 169) and identified in late-onset cases (n = 162). Comparison of early- versus late-onset cases to evaluate whether certain genotypes correlate with the age of onset revealed that homozygosity was predominantly found in infantile (85.65%) and compound heterozygosity in late-onset (76.9%) cases. Analysis of homozygous cases revealed 61% nonsense variants in the early stages and 87% missense variants in the late stages. Mapping of disease-associated (homozygous) missense variants to functional GAA protein domains showed that missense variants were found throughout GAA, but we identified enrichment in the catalytic domain. A strict genotype-phenotype correlation cannot be established; nevertheless, a phenotypic implication of some GAA variants could be drawn (e.g., c.896T>C/p.L299P, c.2015G>A/p.R672Q, and c.-32-13T>G). The combined enzyme activity and genetic testing from DBS cards can reliably identify PD and significantly accelerate diagnosis. We identified new genetic variants that contribute to the spectrum of pathogenic variants of the GAA gene.

Keywords: GAA variants; Pompe disease; dried blood spots (DBSs); genotype–phenotype; α-glucosidase (GAA) enzyme.

Figure 1

Flowchart of the study population. ⁣^∗In four cases, the enzyme activity revealed borderline values, and the variants were classified as variants of unknown significance (VUS). Because no further workup was possible, these four cases were excluded.

Figure 2

Schematic representation of the found (a) missense variants, (b) stop point variants (nonsense), and (c) frameshift/small in-frame variants along the GAA protein. Any position with a variant obtains a circle. The missense variants are shown in green, truncation variants (stop point, frameshift) in black, and in-frame variants in brown. The thin grey bar represents the entire protein with the different amino acid (aa) positions. The light and dark grey boxes are specific functional domains. The structure of human GAA is adapted from [22, 23]. For reasons of simplification, gross deletions, splice variants, and major in-frame deletions were not shown in this figure.

Figure 3

Pie charts showing the percentage of patients with PD ((a) late-onset and (b) early-onset) identified with IVS1 variants. The cases in which we identified IVS1 homozygously or heterozygously were included in this figure.

Figure 4

Lollipop plot illustrating all GAA variants in our (a) homozygous early-onset cases and (b) homozygous late-onset cases along the protein structure. Any position with a variant obtains a circle; the length of the line depends on the number of variants detected at that codon. The missense variants are shown in green, truncation variants (stop point and frameshift) in black, and in-frame deletions in brown. The thin grey bar represents the entire protein with the different amino acid (aa) positions. The light and dark grey boxes are specific functional domains. The structure of human GAA is adapted from [22, 23]. For reasons of simplification, gross deletions, splice variants, and in-frame deletions were not shown in this figure.