Multigene testing of moderate-risk genes: be mindful of the missense

Abstract

Background: Moderate-risk genes have not been extensively studied, and missense substitutions in them are generally returned to patients as variants of uncertain significance lacking clearly defined risk estimates. The fraction of early-onset breast cancer cases carrying moderate-risk genotypes and quantitative methods for flagging variants for further analysis have not been established.

Methods: We evaluated rare missense substitutions identified from a mutation screen of ATM, CHEK2, MRE11A, RAD50, NBN, RAD51, RINT1, XRCC2 and BARD1 in 1297 cases of early-onset breast cancer and 1121 controls via scores from Align-Grantham Variation Grantham Deviation (GVGD), combined annotation dependent depletion (CADD), multivariate analysis of protein polymorphism (MAPP) and PolyPhen-2. We also evaluated subjects by polygenotype from 18 breast cancer risk SNPs. From these analyses, we estimated the fraction of cases and controls that reach a breast cancer OR≥2.5 threshold.

Results: Analysis of mutation screening data from the nine genes revealed that 7.5% of cases and 2.4% of controls were carriers of at least one rare variant with an average OR≥2.5. 2.1% of cases and 1.2% of controls had a polygenotype with an average OR≥2.5.

Conclusions: Among early-onset breast cancer cases, 9.6% had a genotype associated with an increased risk sufficient to affect clinical management recommendations. Over two-thirds of variants conferring this level of risk were rare missense substitutions in moderate-risk genes. Placement in the estimated OR≥2.5 group by at least two of these missense analysis programs should be used to prioritise variants for further study. Panel testing often creates more heat than light; quantitative approaches to variant prioritisation and classification may facilitate more efficient clinical classification of variants.

Keywords: Cancer: breast; Clinical genetics; Genetics; Screening.

Figure 1

Observed OR and thresholds for each missense substitution analysis program. The observed OR for the carriers of key domain rare missense substitutions (rMS) of both the ‘above’ and ‘below’ groups for each severity threshold tested with (A) PolyPhen-2, (B) CADD, (C) MAPP and (D) Align-GVGD, adjusting for race/ethnicity and study centre for just the combined ensemble of ATM, CHEK2 and NBN. Vertical lines are indicative of the threshold for which the observed OR≥2.5. (E) A four-way Venn diagram detailing the number of rMS for which CADD, PolyPhen-2, Align-GVGD and MAPP placed in the ‘above threshold’ group for key domain rMS observed in all nine moderate-risk genes. (F) A four-way Venn diagram detailing the number of individuals for which CADD, PolyPhen-2, Align-GVGD and MAPP were placed in the ‘above threshold’ group for key domain rMS observed in all nine moderate-risk genes. Mammals=score obtained from using protein multiple sequence alignments (pMSA) containing sequences from human through platypus; 3S/P=scores are from gene-specific 3S/P depth pMSAs.

Figure 2

The normalised polygene score (NPS): distribution, NPS-OR correlation and threshold for medically actionable. (A) NPS distribution for all subjects. Comparison of observed OR and NPS for each decile for (B) all subjects and (C) only Caucasians, with the corresponding equations derived from linear regressions, excluding the central quintile. The observed OR when dividing subjects based on NPS, using the middle quintile as reference for (D) all subjects and (E) only Caucasians, adjusting for race/ethnicity and study centre. Vertical lines are indicative of the threshold for which the observed OR≥2.5, as well as the 1, 5, 95 and 99 percentiles. Bubble sizes are proportional to the number of subjects in each decile.

Figure 3

Combined normalised polygene score (NPS) and rare variant OR. (A) Distribution of subjects by NPS when carriers of rare missense substitutions (rMS) in the ‘above threshold’ group had their NPS increased by the factor of their OR. Stacked histograms of the expansion of the combined contribution of NPS and rMS for carriers with a variant conferring an average OR≥2.5 with (B) PolyPhen-2, (C) Align-GVGD, (D) MAPP, (E) CADD as well as (F) carriers of variants that were placed in the ‘above threshold’ group for two or more missense analysis programs and (G) truncation and splice junction variant (T+SJV) carriers.