Introduction:
Peroxisomal disorders encompass a clinically and genetically heterogeneous group of disorders caused by impaired peroxisome biogenesis or function. Among them, ABCD1 defcency a.k.a Adrenoleukodystrophy (ALD) is the most common, caused by pathogenic variants in ABCD1 gene (OMIM: 300371) on the X chromosome. ALD manifests as early-onset cerebral adrenoleukodystrophy, late-onset adrenomyeloneuropathy, or isolated adrenal insufficiency. Disease expression varies among affected individuals, and approximately half of the female individuals with ABCD1 deficiency manifest with a late-onset myeloneuropathy. The presymptomatic detection of ALD and peroxisome biogenesis disorders has increased significantly since 2013, primarily enabled by newborn screening programs. Follow-up molecular testing with next-generation sequencing has led to the identification of an increasing number of variants that need to be classified. Therefore, the National Institutes of Health (NIH)-funded Clinical Genome Resource (ClinGen) convened the Peroxisomal Disorders Variant Curation Expert Panel (Peroxisomal VCEP) for variant curation in genes causing peroxisomal disorders.

Methods:
We, the Peroxisomal VCEP, are an international collaboration of expert clinicians, genetic counselors, clinical laboratory diagnosticians and researchers working toward the goal of precise and consistent sequence variant interpretation for genes causing peroxisomal disorders. We prioritized the ABCD1 gene, the main target of the newborn screening program since therapies are available for the childhood cerebral form and for adrenal insufficiency, to adapt the 2015 ACMG/AMP guidelines for sequence variant interpretation by laboratories. We modified the 28 criteria codes, considering information specific to the ABCD1 gene and the disease’s clinical presentation. We tested these criteria on 40 ClinVar variants, ranging from benign to pathogenic in classification, chosen for the pilot study. 

Results:
The variability in disease expression and the presence of pseudogenes for ABCD1 complicates the application of phenotype-related ACMG/AMP criteria and the use of molecular data, respectively. Therefore, we developed a phenotype chart to guide variant curators in evaluating individual cases. We incorporated all the reported phenotypic, family and laboratory data, and assigned supporting or moderate strength levels to the PP4 code. Additional unrelated probands with the variant who meet at least the supporting phenotypic criteria can be counted in the PS4 code, with strength levels adjusted based on the number of probands. Similar adjustments were made to the other codes, after careful evaluation of the available evidence. 

We then applied the modified rules to provisionally classify 40 variants with existing ClinVar classifications. Currently, we are finalizing curation classifications based on expert review. Among the 13 pathogenic/likely pathogenic variants from ClinVar, we classified 2 as pathogenic and 2 as VUS. The remaining 9 were classified likely pathogenic, and we are currently engaged in efforts to obtain more case-level information from unpublished sources, which may upgrade these variants. Of the 12 VUS from ClinVar, 3 were upgraded to likely pathogenic and 2 downgraded to likely benign, leading to reclassification of 42% of VUS. Except 1 likely benign variant that was classified as VUS, all 12 benign/likely benign variants retained their classification. Importantly, we reclassified all 3 variants with conflicting interpretations (100%): 1 as likely pathogenic and 2 as likely benign. Overall, our modified curation rules have enabled reclassification of 57% of variants of uncertain significance or with conflicting interpretations. 

Conclusion:
Once our pilot variant curations are approved, our rule specifications will become available for the community to use. Our efforts to classify ABCD1 variants and submit them to ClinVar will continue, prioritizing variants with conflicting classifications. The standardized use of evidence criteria for the evaluation of variants in ABCD1 will reduce the number of VUS and conflicting interpretations, making genetic testing results more informative for providers and patients.