Objectives: This study aims to demonstrate the role of case-level American College of Medical Genetics (ACMG) criteria, such as familial segregation and pathology data, in providing conclusive evidence for the pathogenicity of ultrarare GLA variants causing Anderson–Fabry disease when gene-level and variant-level criteria provide ambiguous or discrepant results. Case/family description: A 52-year-old woman presented with new-onset shortness of breath, chest pain, and palpitations. Echocardiography revealed mild left ventricular wall thickening (14 mm) and mild diastolic dysfunction. She was the second of three siblings born to unrelated parents, both of whom died from malignancies. Family screening identified brothers, one affected 55-year-old with hypertension and asthma and one unaffected 47-year-old. The 15-year-old son of the proband complained of exercise-induced burning feet acral pain his electrocardiogram showed a short PR interval and signs of early hypertrophy. Results: Endomyocardial biopsies of the proband and the affected sibling demonstrated substrate accumulation (globotriaosylceramide). The anti-α-galactosidase-A immunostain showed a total loss of the enzyme in the hemizygous male and a mosaic pattern in the heterozygous female. The next-generation sequencing short-read multigene panel identified the c.547+3A>G variant in the GLA gene and excluded variants in other genes; Oxford-Nanopore long-read sequencing excluded known pathogenic deep intronic variants. A Multiplex-Ligation-dependent-Probe-Amplification assay excluded copy number variations. Based on the variant-level and gene-level ACMG criteria, the variant was classified as a Variant of Uncertain Significance or Likely Benign using different bioinformatic tools. By adding case-level functional data (endomyocardial biopsy, PS3_VeryStrong) and familial data (segregation of genotype with phenotype, PP2_Moderate), the variant was classified as Likely Pathogenic/Pathogenic. Conclusion: ACMG case-level data can unambiguously resolve uncertain interpretations of GLA variants.
