Sensitivity and specificity of loss of heterozygosity analysis for the classification of rare germline variants in BRCA1/2: results of the observational AGO-TR1 study (NCT02222883)

J Med Genet. 2022 Mar;59(3):248-252. doi: 10.1136/jmedgenet-2020-107353. Epub 2020 Dec 3.


Variant-specific loss of heterozygosity (LOH) analyses may be useful to classify BRCA1/2 germline variants of unknown significance (VUS). The sensitivity and specificity of this approach, however, remains unknown. We performed comparative next-generation sequencing analyses of the BRCA1/2 genes using blood-derived and tumour-derived DNA of 488 patients with ovarian cancer enrolled in the observational AGO-TR1 trial (NCT02222883). Overall, 94 pathogenic, 90 benign and 24 VUS were identified in the germline. A significantly increased variant fraction (VF) of a germline variant in the tumour indicates loss of the wild-type allele; a decreased VF indicates loss of the variant allele. We demonstrate that significantly increased VFs predict pathogenicity with high sensitivity (0.84, 95% CI 0.77 to 0.91), poor specificity (0.63, 95% CI 0.53 to 0.73) and poor positive predictive value (PPV; 0.71, 95% CI 0.62 to 0.79). Significantly decreased VFs predict benignity with low sensitivity (0.26, 95% CI 0.17 to 0.35), high specificity (1.0, 95% CI 0.96 to 1.00) and PPV (1.0, 95% CI 0.85 to 1.00). Variant classification based on significantly increased VFs results in an unacceptable proportion of false-positive results. A significantly decreased VF in the tumour may be exploited as a reliable predictor for benignity, with no false-negative result observed. When applying the latter approach, VUS identified in four patients can now be considered benign. Trial registration number NCT02222883.

Keywords: genetic predisposition to disease; genetic research; genetic testing; germ-line mutation.

Publication types

  • Observational Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • BRCA1 Protein / genetics
  • BRCA2 Protein / genetics
  • Breast Neoplasms* / diagnosis
  • Breast Neoplasms* / genetics
  • Female
  • Genes, BRCA1
  • Genetic Predisposition to Disease
  • Germ Cells / pathology
  • Germ-Line Mutation / genetics
  • Humans
  • Loss of Heterozygosity / genetics
  • Ovarian Neoplasms* / diagnosis
  • Ovarian Neoplasms* / genetics
  • Ovarian Neoplasms* / pathology


  • BRCA1 Protein
  • BRCA1 protein, human
  • BRCA2 Protein

Associated data