Pompe disease is a lysosomal and neuromuscular disorder caused by deficiency of acid alpha-glucosidase (GAA), and causes classic infantile, childhood onset, or adulthood onset phenotypes. The biochemical diagnosis is based on GAA activity assays in dried blood spots, leukocytes, or fibroblasts. Diagnosis can be complicated by the existence of pseudodeficiencies, i.e., GAA variants that lower GAA activity but do not cause Pompe disease. A large-scale comparison between these assays for patient samples, including exceptions and borderline cases, along with clinical diagnoses has not been reported so far. Here we analyzed GAA activity in a total of 1709 diagnostic cases over the past 28 years using a total of 2591 analyses and we confirmed the clinical diagnosis in 174 patients. We compared the following assays: leukocytes using glycogen or 4MUG as substrate, fibroblasts using 4MUG as substrate, and dried blood spots using 4MUG as substrate. In 794 individuals, two or more assays were performed. We found that phenotypes could only be distinguished using fibroblasts with 4MUG as substrate. Pseudodeficiencies caused by the GAA2 allele could be ruled out using 4MUG rather than glycogen as substrate in leukocytes or fibroblasts. The Asian pseudodeficiency could only be ruled out in fibroblasts using 4MUG as substrate. We conclude that fibroblasts using 4MUG as substrate provides the most reliable assay for biochemical diagnosis and can serve to validate results from leukocytes or dried blood spots.

庞贝病是一种由酸性 α-葡萄糖苷酶 (GAA) 缺乏引起的溶酶体和神经肌肉疾病，可导致典型的婴儿、儿童期或成年期发病表型。生化诊断基于干血斑、白细胞或成纤维细胞中的 GAA 活性测定。假性缺陷的存在可能会使诊断变得复杂，即GAA降低 GAA 活性但不会引起庞贝病的变体。迄今为止，尚未报道这些针对患者样本（包括异常和临界病例）的检测与临床诊断之间的大规模比较。在这里，我们使用总共 2591 次分析分析了过去 28 年中总共 1709 例诊断病例中的 GAA 活性，我们确认了 174 例患者的临床诊断。我们比较了以下测定：使用糖原或 4MUG 作为底物的白细胞、使用 4MUG 作为底物的成纤维细胞和使用 4MUG 作为底物的干血斑。在 794 名个体中，进行了两次或多次检测。我们发现只能使用以 4MUG 作为底物的成纤维细胞来区分表型。GAA2引起的伪缺陷可以使用 4MUG 而不是糖原作为白细胞或成纤维细胞中的底物来排除等位基因。仅以4MUG为底物，才能排除成纤维细胞中的亚洲假性缺陷。我们得出结论，使用 4MUG 作为底物的成纤维细胞为生化诊断提供了最可靠的检测方法，并可用于验证白细胞或干血斑的结果。