A comprehensive custom panel evaluation for routine hereditary cancer testing: improving the yield of germline mutation detection

Abstract

Background: In the context of our Regional Program of Hereditary Cancer, individuals fulfilling the criteria are tested for germline mutations to subsequently establish the clinical management. Our standard diagnostic approach focuses on sequencing a few classic high-risk genes, a method that frequently renders uninformative genetic results. This study aims to examine the improved yield offered by an On-Demand panel.

Methods: We designed an On-Demand panel for the analysis of 35-genes associated with inherited cancer susceptibility in a total of 128 cases of Hereditary Breast and Ovarian Cancer (HBOC) and Hereditary Nonpolyposis Colorectal Cancer (HNPCC).

Results: Eighteen deleterious mutations were detected, in both routinely (BRCA2, MLH1, MSH2, PMS2) and non-routinely (ATM, BLM, BRIP1, CHEK2, MUTYH) tested genes. The screening extended to 35 genes rendered by patients carrying several- up to 6-Variants of Unknown Significance (VUS). Moreover, we confirmed the splicing disruption at RNA level for a not previously reported BRIP1 splicing mutation. Using an On-Demand panel, we identified 18 pathogenic mutation carriers, seven of which would have gone unnoticed with traditional analysis.

Conclusions: Our results reinforce the utility of NGS gene panels in the diagnostic routine to increase the performance of genetic testing, especially in individuals from families with overlapping cancer phenotypes.

Keywords: Genetic counselling; Germline mutation; Hereditary Cancer syndrome; Next generation sequencing; On-Demand gene panel.

Fig. 1

Representation of the different percentages of patients defined by the different types of mutation according to the Hereditary Cancer Syndrome. a For HBOC, the mutation rates for patients with PV in BRCA1 and BRCA2 genes (3%) was significantly lower than the 11% represented by PV in ATM, BLM, BRIP1 and MUTYH genes, which were not screened routinely for HBOC. A total of 53 of the cases carried 1, 2, 3 or more (up to 6) VUS. In the 33% of the analyzed samples, we did not find any relevant variant (negatives); b For HNPCC, the mutation rates for patients with PV in the MMR genes (9%) was significantly higher than the PV identified in other different genes, which in this case only refers to CHEK2. A total of 58% of the cases carried 1, 2, 3 or more (up to 5) VUS. In 30% of the analyzed samples, we did not find any relevant variant

Fig. 2

Distribution of the PV and VUS along the different genes according to the Hereditary Cancer Syndrome. The bar diagram represents the number of variants identified in the different genes. The bars define the number of VUS detected: pink bars correspond to HBOC and blue bars to HNPCC. The number of PVs is indicated in the upper part of the bar for the respective gene

Fig. 3

Segregation analysis of VUS. The segregation of some VUS was evaluated in three different families. The pedigrees show the different types of tumor in the affected probands, represented with an inner black square, and the diagnosis and current age when available. The index case is marked with an arrowhead. The individuals tested for the indicated mutations are surrounded by a square. Positive results are represented by “+” and negative ones by “−”

Fig. 4

Correlation between cancer history and genetic results for high CADD score variants. Representation of the personal and family history of the samples carrying two or more VUS with high CADD scores. For each variant, the CADD-score and the gene affected is indicated. The different tumor types for the index case and the family members are coded as following: Br, breast; Ov, ovarian; Col, colon; Pan, pancreas; Gas, gastric; Thy, thyroid; Pr, porstatic; End, endometrial; H&N, head and neck; Liv, liver; Kid, kidney; CNS, central nervous system; Skin, skin; Lun, lung and CUP, cancer of unknown primary origin. When multiple cancer cases in the family or several variants in the same gene existed, we used the “x” symbol to represent the multiplicative condition: 1x (1), 2x (2), 3x (3), etc

Fig. 5

Association between the age of diagnoses of the index cases and tumor diversity in the respective families with the number of VUS identified in the sample. The upper plot shows how the condition of carrying 3 or more VUS could be determining early cancer onset. The lower plot represents the possible association of the categorical variable “tumor diversity in the family” defined as ‘yes’ when the pedigree harbored more than 3 tumor types. In particular, for the condition of carrying 4, 5 and 6 VUS, the tumor diversity in the pedigrees tends to increase

Fig. 6

Characterization of the c.1140 + 1G > C variant in BRIP1 at RNA level. The amplified PCR products were separated by a 2% agarose gel electrophoresis to detect the possible aberrant transcripts. Then, Sanger sequencing confirmed the different exon exclusions. The c.1140 + 1G > C variant in BRIP1 caused the skipping of exon 8, resulting in a product which is 222 nucleotides shorter than the full-length product (879 nucleotides)