Petra Oliva

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 We identified 723 Pompe cases with 283 different GAA alterations, and 98 variants have been unpublished so far.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.

Pompe disease (GSD II) is an autosomal recessive disorder caused by deficiency of the lysosomal enzyme acid-α-glucosidase (GAA, EC 3.2.1.20), leading to generalized accumulation of lysosomal glycogen especially in the heart, skeletal, and smooth muscle, and the nervous system. It is generally classified based on the age of onset as infantile (IOPD) presenting during the first year of life, and late onset (LOPD) when it presents afterwards. In our study, a cohort of 13,627 samples were tested between January 2017 and December 2018 for acid-α-glucosidase (GAA, EC 3.2.1.20) deficiency either by fluorometry or tandem mass spectrometry (MS). Testing was performed for patients who displayed conditions of unknown etiology, e.g., CK elevations or cardiomyopathy, in the case of infantile patients. On average 8% of samples showed activity below the reference range and were further assessed by another enzyme activity measurement or molecular genetic analysis. Pre-analytical conditions, like proper drying, greatly affect enzyme activity, and should be assessed with measurement of reference enzyme(s). In conclusion, at-risk testing can provide a good first step for the future introduction of newborn screening for Pompe disease. It yields immediate benefits for the patients regarding the availability and timeliness of the diagnosis. In addition, the laboratory can introduce the required methodology and gain insights in the evaluation of results in a lower throughput environment. Finally, awareness of such a rare condition is increased tremendously among local physicians which can aid in the introduction newborn screening.

A total of 11 948 females suspicious of Fabry disease were tested by a combined biochemical and genetic approach. The enzyme activity, together with the concentration of lyso‐GL‐3 (lyso‐Gb3) biomarker in dried blood spots (DBS), substantially improved the diagnostic detection of Fabry disease in females compared to the enzyme activity alone. Abnormal values for both were highly suspicious of Fabry disease (97% positive predictive value [PPV], similar to PPV in males). In cases with one abnormal biochemical value, elevated lyso‐GL‐3 is a far more important indicator than low enzyme activity (39% PPV vs 6% PPV). Cases with clearly negative results for both biochemical parameters are unlikely to have Fabry disease, even in clinically highly suspicious cases.