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INTERNATIONAL CONGRESS OF

CancerGenetics

• Low-Allele-Fraction Pathogenic/Likely Pathogenic Calls in Diagnostic Whole-Exome Sequencing for Hereditary Breast Cancer: A Cautionary Case Series of Sanger Discordance

• Sara Jamali,¹ Mirsalar Kahaei,² Toktam Dehghani,³ Nasrin Mojaver,⁴ Pegah Moosavi,⁵ Majid Mojarrad,^6,*
  1. Department of Medical Genetics, Faculty of Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran
  2. Department of Medical Genetics, Faculty of Medicine, Hormozgan University of Medical Sciences
  3. Faculty of Medicine, Department of Medical Genetics, Mashhad University of Medical Sciences
  4. Department of Medical Informatics, Faculty of Medicine, Mashhad University of Medical Sciences
  5. Genetic Laboratory, Genetic Foundation of Khorasan Razavi, Mashhad, Iran
  6. Endocrinology and Metabolism Research Center, Hormozgan University of Medical Sciences, Bandar Abbas, Iran
  
  
  
• Introduction: Whole-exome sequencing (WES) has become an important tool for germline variant detection in hereditary cancer testing, but its broad genomic scope also introduces challenges in distinguishing clinically relevant variants from technically unreliable calls. A variant may meet Pathogenic/Likely Pathogenic (P/LP) criteria based on clinical and biological evidence while the underlying sequencing evidence remains insufficient for confident germline assignment. Low variant allele fraction (VAF), limited alternate-read support, restricted sequencing depth, and challenging genomic context may compromise analytical confidence and therefore warrant assessment at the variant level before clinical interpretation and reporting rather than only after a variant has been selected for Sanger confirmation. Current guidance emphasizes that laboratories should establish assay-specific depth and allele-fraction parameters and investigate ambiguous allele fractions using appropriate orthogonal approaches.²,⁶ Importantly, low allele-fraction (LAF) findings are not necessarily technical artifacts: recent large-scale hereditary cancer data demonstrate that variants in the 10–30% range may include both pathogenic variants and constitutional mosaicism, underscoring the need for appropriate follow-up rather than automatic biological interpretation.⁵ Accordingly, laboratory-specific, empirically validated quality-control criteria and manual read-level review are essential for evaluating low-confidence calls, while universal VAF or alternate-read thresholds should be avoided in the absence of platform-specific validation. Integrating sequencing depth, alternate-read support, allele fraction, genomic context, variant consequence, and orthogonal confirmation may therefore improve the analytical safety of WES-based hereditary cancer testing and reduce the risk of inappropriate clinical reporting.
• Methods: Three unrelated patients undergoing diagnostic WES for hereditary/suspected hereditary breast cancer were retrospectively evaluated. Three variants—NF1 c.569T>A (p.Leu190Ter), BRCA2 c.9113delT (p.Leu3038Hisfs24), and APC c.6715dup (p.Ser2239Lysfs10)—were classified as P/LP by independent variant-interpretation resources and selected for Sanger confirmation based on their potential clinical relevance. WES read-level metrics, including total depth, alternate-read count, allele fraction, genotype information, and variant consequence, were reviewed and compared with Sanger findings.
• Results: All three variants demonstrated discordance between WES and Sanger sequencing, with Sanger showing the reference sequence and no detectable alternate allele. The NF1 stop-gain variant was detected at 24× depth with four alternate reads (VAF ≈17%). The BRCA2 frameshift variant was detected at 33× depth with five alternate reads (VAF ≈15%). The APC frameshift variant was detected at 17× depth with four alternate reads (VAF ≈24%). Thus, all three findings had limited alternate-read support and allele fractions substantially below the approximately 50% expected for a conventional heterozygous germline variant. The APC call additionally showed a non-resolved genotype field (GT = “.”) in the reviewed output. Retrospective assessment of these metrics would have raised concern regarding the analytical confidence of the calls before clinical interpretation and Sanger referral. The BRCA2 finding was particularly relevant because of the established role of pathogenic BRCA2 variants in hereditary breast and ovarian cancer risk and their potential implications for risk-reduction management. The observed pattern is consistent with the recognized challenge of interpreting LAF findings in hereditary cancer testing; importantly, LAF variants are not necessarily technical artifacts and may represent mosaic or other biologically relevant events in some cases.
• Conclusion: These cases highlight a practical limitation of diagnostic WES: a variant may meet P/LP criteria based on clinical and biological evidence while the underlying sequencing evidence remains insufficient for confident germline assignment. Low VAF, limited alternate-read support, and restricted sequencing depth should therefore be incorporated into variant-level analytical quality assessment before clinical interpretation and not considered only after a variant has been selected for Sanger confirmation. Current guidance emphasizes that laboratories should establish assay-specific depth and allele-fraction parameters and investigate ambiguous allele fractions using orthogonal approaches. Importantly, the present cases do not establish that the three variants were technical artifacts, nor do they support a universal VAF or alternate-read cutoff. Instead, they demonstrate the value of laboratory-specific, empirically validated QC criteria and manual read-level review for low-confidence calls. Recent large-scale hereditary cancer data further demonstrate that LAF variants in the 10–30% range can include both pathogenic findings and constitutional mosaicism, underscoring the need for appropriate follow-up rather than automatic biological interpretation. Accordingly, systematic integration of sequencing depth, alternate-read support, allele fraction, genomic context, variant consequence, and orthogonal confirmation may improve the analytical safety of WES-based hereditary cancer testing and reduce the risk of inappropriate clinical reporting.
• Keywords: Hereditary breast cancer; Whole-exome sequencing; Low-allele-fraction variants; Variant allele fract