Molecular Diagnosis of Pompe Disease in the Genomic Era: Correlation with Acid Alpha-Glucosidase Activity in Dried Blood Spots.

Fanny Thuriot,Paula J Waters,Sébastien Lévesque,Serge Gravel,Elaine Gravel,Katherine Hodson,Jorge Ganopolsky,Bojana Rakic

doi:10.3390/jcm10173868

Abstract

Measurement of alpha-glucosidase activity on dried blood spots has been the main method to screen for Pompe disease, but a paradigm shift has been observed in recent years with the incorporation of gene panels and exome sequencing in molecular diagnostic laboratories. An 89-gene panel has been available to Canadian physicians since 2017 and was analyzed in 2030 patients with a suspected muscle disease. Acid alpha-glucosidase activity was measured in parallel in dried blood spots from 1430 patients. Pompe disease was diagnosed in 14 patients, representing 0.69% of our cohort. In 7 other patients, low enzyme activities overlapping those of Pompe disease cases were attributable to the presence of pseudodeficiency alleles. Only two other patients had enzymatic activity in the Pompe disease range, and a single heterozygous pathogenic variant was identified. It is possible that a second variant could have been missed; we suggest that RNA analysis should be considered in such cases. With gene panel testing increasingly being performed as a first-tier analysis of patients with suspected muscle disorders, our study supports the relevance of performing reflex enzymatic activity assay in selected patients, such as those with a single GAA variant identified and those in whom the observed genotype is of uncertain clinical significance.

Highlights

Pompe disease is an autosomal recessive disorder caused by pathogenic variants in the GAA gene, which encodes an acid alpha-glucosidase enzyme [1]
14 patients were diagnosed with Pompe disease, representing 5.1% of all diagnoses, 1.15% of our limb-girdle muscular dystrophies (LGMD) patients, and 0.69% of our entire cohort
All 14 Pompe disease patients were found to be compound heterozygous for two variants, each considered pathogenic or likely pathogenic [20], in the GAA gene

Summary

Introduction

Pompe disease is an autosomal recessive disorder caused by pathogenic variants in the GAA gene, which encodes an acid alpha-glucosidase enzyme [1] This lysosomal glycogen storage disorder has a prevalence of 1:40,000 in individuals in the United States, with increased incidence in African Americans [2,3]. With decreasing cost of sequencing technologies, targeted and whole-exome sequencing have increasingly been used to diagnose patients with Pompe disease, especially in adults with suggestive symptoms or in populations where newborn screening is not yet available. These methods have been shown to have a high diagnostic yield in contexts where differential diagnoses, such as other limb-girdle muscular dystrophies (LGMD), cannot be discounted [16–18]. We present data supporting the use of acid alpha-glucosidase enzymatic assay on dried blood spot as a reflex test following molecular analysis, for the confirmation of diagnosis of Pompe disease in symptomatic patients

Materials and Methods

Acid Alpha-Glucosidase Enzymatic Activity Assays

Gene Panel and Next Generation Sequencing Method

Splicing Analysis

Bioinformatics

Patients with Pompe Disease