Development of a clinically validated in vitro functional assay to assess pathogenicity of novel GAA variants in patients with Pompe disease identified via newborn screening

Front Genet. 2022 Sep 30;13:1001154. doi: 10.3389/fgene.2022.1001154.

PMID: 36246652

Shelly Goomber, Erin Huggins, Catherine W Rehder

Highlights: There is an unmet need for validated functional studies to aid in classification of GAA variants.

Abstract

Purpose: The number of new and variants of unknown significance (VUS) in the GAA gene has increased as a result of Pompe disease (Glycogen Storage Disease Type II) being added to the Recommended Uniform Screening Panel in the United States. This poses a diagnostic challenge, particularly in the case of late-onset Pompe disease, where symptoms are rarely visible at birth. Validated functional investigations are still needed to help categorize GAA variations.

Methods: Based on the recommendations issued by the Clinical Genome Resource (ClinGen) Sequence Variant Interpretation Working Group for PS3/BS3, an in vitro mammalian cell expression and functional analysis system is created. Eight VUS or novel variants were investigated in GAA found in patients with a positive neonatal screen for Pompe disease without phenotypic evidence of infantile-onset disease after validating the technique with 12 control variations.

Results: In the expression system, the control variants were examined, and an activity range was developed. The GAA activity in the pathogenic controls ranged from 0% to 11% of normal. The activity range for the benign or probably benign controls was 54%–100%. Activity in the pseudodeficiency variant was 17%. The variants chosen for functional studies were then subjected to these ranges. PS3_ supporting was applied and two variants were categorized as likely pathogenic (c.316C > T and c.1103G > A) using the threshold of 11%. Also additional information was provided to support the categorization of two more variants (c.1721T > C and c.1048G > A) as likely pathogenic. Based on additional supporting data, one variant (c.1123C > T) was able to receive a new classification. Due to insufficient or contradictory evidence, three variants could not be reclassified (c.664G > A, c.2450A > G, and c.1378G > A).

Conclusion: Eight GAA variants were investigated as proof of concept using the validated and reproducible in vitro expression and functional analysis system. This tool can be used for variant classification to meet the expanding demand for novel GAA variant classification in the era of newborn screening for Pompe disease, even though more work is required to further refine our system with more controls and different variant types in order to apply the PS3/BS3 criteria at a higher level.

Keywords: functional studies, in vitro assay, lysosomal storage disease (LSD), newborn screening, Pompe disease, variant classification