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

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.

Molecular diagnosis of muscular diseases in outpatient clinics: A Canadian perspective.

OBJECTIVE: To evaluate the diagnostic yield of an 89-gene panel in a large cohort of patients with suspected muscle disorders and to compare the diagnostic yield of gene panel and exome sequencing approaches. METHODS: We tested 1,236 patients from outpatient clinics across Canada using a gene panel and performed exome sequencing for 46 other patients with sequential analysis of 89 genes followed by all mendelian genes. Sequencing and analysis were performed in patients with muscle weakness or symptoms suggestive of a muscle disorder and showing at least 1 supporting clinical laboratory. RESULTS: We identified a molecular diagnosis in 187 (15.1%) of the 1,236 patients tested with the 89-gene panel. Diagnoses were distributed across 40 different genes, but 6 (DMD, RYR1, CAPN3, PYGM, DYSF, and FKRP) explained about half of all cases. Cardiac anomalies, positive family history, age <60 years, and creatine kinase >1,000 IU/L were all associated with increased diagnostic yield. Exome sequencing identified a diagnosis in 10 (21.7%) of the 46 patients tested. Among these, 3 were attributed to genes not included in the 89-gene panel. Despite differences in median coverage, only 1 of the 187 diagnoses that were identified on gene panel in the 1,236 patients could have been potentially missed if exome sequencing had been performed instead. CONCLUSIONS: Our study supports the use of gene panel testing in patients with suspected muscle disorders from outpatient clinics. It also shows that exome sequencing has a low risk of missing diagnoses compared with gene panel, while potentially increasing the diagnostic yield of patients with muscle disorders.

Clinical validity of phenotype-driven analysis software PhenoVar as a diagnostic aid for clinical geneticists in the interpretation of whole-exome sequencing data.

PURPOSE: We sought to determine the diagnostic yield of whole-exome sequencing (WES) combined with phenotype-driven analysis of variants in patients with suspected genetic disorders. METHODS: WES was performed on a cohort of 51 patients presenting dysmorphisms with or without neurodevelopmental disorders of undetermined etiology. For each patient, a clinical geneticist reviewed the phenotypes and used the phenotype-driven analysis software PhenoVar (http://phenovar.med.usherbrooke.ca/) to analyze WES variants. The prioritized list of potential diagnoses returned was reviewed by the clinical geneticist, who selected candidate variants to be confirmed by segregation analysis. Conventional analysis of the individual variants was performed in parallel. The resulting candidate variants were subsequently reviewed by the same geneticist, to identify any additional potential diagnoses. RESULTS: A molecular diagnosis was identified in 35% of the patients using the conventional analysis, and 17 of these 18 diagnoses were independently identified using PhenoVar. The only diagnosis initially missed by PhenoVar was rescued when the optional "minimal phenotypic cutoff" filter was omitted. PhenoVar reduced by half the number of potential diagnoses per patient compared with the conventional analysis. CONCLUSION: Phenotype-driven software prioritizes WES variants, provides an efficient diagnostic aid to clinical geneticists and laboratories, and should be incorporated in clinical practice.

Diagnosis of late-onset Pompe disease and other muscle disorders by next-generation sequencing.

BACKGROUND: Late-onset Pompe disease (LOPD) is a rare treatable lysosomal storage disorder characterized by progressive lysosomal glycogen accumulation and muscle weakness, with often a limb-girdle pattern. Despite published guidelines, testing for LOPD is often overlooked or delayed in adults, owing to its low frequency compared to other muscle disorders with similar muscle patterns. Next-generation sequencing has the capability to test concurrently for several muscle disorders. This could potentially lead to increased diagnosis of LOPD, disorders with non-specific muscle weakness or atypical patients. METHODS: We developed a gene panel to further study its clinical utility in a cohort of patients with suspected muscle disorders. We designed a gene panel to analyze the coding sequences and splice site junctions of GAA causing LOPD, along with 77 other genes causing muscle disorders with overlapping phenotypes. RESULTS: At a median coverage of ~200X (sequences per base), all GAA exons were successfully covered with >20X and only 0.3 % of exons across all genes were <20X. The panel showed an excellent sensitivity (100 %) and specificity (98 %) across all selected genes, using known variations in Pompe patients and controls. We determined its clinical utility by analyzing 34 patients with suspected muscle disorders of undetermined etiology and various muscle patterns, who were referred or followed in neuromuscular and genetics clinics. A putative diagnosis was found in up to 32 % of patients. The gene panel was instrumental in reaching a diagnosis in atypical patients, including one LOPD case. Acid alpha-glucosidase activity was used to confirm the molecular results in all patients. CONCLUSION: This work highlights the high clinical utility of gene panels in patients with suspected muscle disorders and its potential to facilitate the diagnosis of patients showing non-specific muscle weakness or atypical phenotypes. We propose that gene panels should be used as a first-tier test in patients with suspected muscle disorders of undetermined etiology, which could further increase overall diagnosis of muscle conditions, and potentially reduce diagnostic delay. Further studies are necessary to determine the impact of first-tier gene panels on diagnostic delay and on treatment outcome for LOPD.