The c.532-8G>A variant in APC is an intronic variant which is located 8 nucleotides upstream of the splice acceptor site for exon 6. This variant has been reported in 3 families meeting phenotypic criteria, resulting in a total phenotype score of 2.5 (PS4_Moderate; Ambry internal data; Invitae internal data; Leiden, Bonn, PMID 24599579; PMID19196998). This variant is absent from gnomAD v2.1.1 (PM2_supporting). The results from ≥2 in silico splicing predictors (SpliceAI, MaxEntScan, VarSeak) indicate that this variant may affect splicing by disrupting the acceptor splice site of intron 5 of APC (PP3). RT-PCR and minigene assay demonstrate that this variant impacts splicing by creating a novel splice acceptor site six nucleotides upstream of the regular splice acceptor site of exon 6 which results in the inclusion of six nucleotides including a premature termination codon. A complete splice defect was confirmed using transcript-specific PCR and SNP analysis of c.1458T>C (PS3_VeryStrong; PMID 19196998). In summary, this variant meets the criteria to be classified as Pathogenic for FAP based on the ACMG/AMP criteria applied, as specified by the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel: PS3_VeryStrong; PS4_moderate; PM2_supporting; PP3 (VCEP specifications version 1; date of approval: 12/12/2022).