Low Allele Frequency Variants Identified on Germline Multi-Gene Panel Testing for Cancer Predisposition Can Suggest the Presence of Constitutional Mosaicism.

Purpose: Enabled by advancements in next-generation sequencing for hereditary cancer conditions, low allele frequency variants (LAFV) are detected by testing laboratories. This study describes the frequency and clinical factors associated with LAFVs and reports results of follow-up testing when available.

Experimental design: This retrospective study analyzed LAFV cases identified through multi-gene panel testing (MGPT) at a single high-volume germline genetic testing laboratory. LAFVs were defined as variant allele frequencies (VAF) between 10% and 30% as confirmed by Sanger sequencing. Comparative analyses were conducted between pathogenic variants with a VAF of 30% to 60% (inferred heterozygous) and LAFV cohorts. Clinical characteristics were analyzed using descriptive statistics and logistic regression models. Ancillary testing was performed on alternative specimens or family members to determine whether LAFVs were due to constitutional mosaicism.

Results: Among 363,405 individuals undergoing MGPT, 965 (1.8%) had variants with a VAF between 10% and 30%. Sanger sequencing confirmed 463 (0.1%) as LAFVs. Among the confirmed LAFVs, 262 (57.6%) were classified as pathogenic variants. The LAFV cohort compared with the control heterozygous cohort was significantly older, with a higher proportion of individuals >50 years (84.1% vs. 54.9%; P < 0.001). LAFVs were present in the following genes: TP53 (110; 64.7%), NF1 (23; 13.5%), CHEK2 (13; 7.6%), ATM (12; 7.1%), and BRCA1 (4; 2.4%). Ancillary testing performed on 62 cases with LAFVs confirmed 17.7% (11/62) as constitutional mosaicism.

Conclusions: LAFVs were infrequently detected in MGPT, representing 0.8% of the total variants and 0.1% of total tested. Ancillary testing is needed to understand the origins and clinical implications of LAFVs in patients and families.