Analysis of Sequence and Copy Number Variants in Canadian Patient Cohort With Familial Cancer Syndromes Using a Unique Next Generation Sequencing Based Approach

Abstract

Background: Hereditary cancer predisposition syndromes account for approximately 10% of cancer cases. Next generation sequencing (NGS) based multi-gene targeted panels is now a frontline approach to identify pathogenic mutations in cancer predisposition genes in high-risk families. Recent evolvement of NGS technologies have allowed simultaneous detection of sequence and copy number variants (CNVs) using a single platform. In this study, we have analyzed frequency and nature of sequence variants and CNVs, in a Canadian cohort of patients, suspected with hereditary cancer syndrome, referred for genetic testing following specific genetic testing guidelines based on patient's personal and/or family history of cancer.

Methods: A 2870 patients were subjected to a single NGS based multi-gene targeted hereditary cancer panel testing algorithm to identify sequence variants and CNVs in cancer predisposition genes at our reference laboratory in Southwestern Ontario. CNVs identified by NGS were confirmed by alternative techniques like Multiplex ligation-dependent probe amplification (MLPA).

Results: A 15% (431/2870) patients had a pathogenic variant and 36% (1032/2870) had a variant of unknown significance (VUS), in a cancer susceptibility gene. A total of 287 unique pathogenic variant were identified, out of which 23 (8%) were novel. CNVs identified by NGS based approach accounted for 9.5% (27/287) of pathogenic variants, confirmed by alternate techniques with high accuracy.

Conclusion: This study emphasizes the utility of NGS based targeted testing approach to identify both sequence and CNVs in patients suspected with hereditary cancer syndromes in clinical setting and expands the mutational spectrum of high and moderate penetrance cancer predisposition genes.

Keywords: breast cancer; colorectal cancer; copy number variants; familial cancer syndromes; next generation sequencing.

FIGURE 1

Number of patients tested for hereditary cancer predisposition with multi gene targeted panel test: This chart describes number of patients tested with each sub-panel and versions. Each sub-panel and its respective version constitute of a set of genes associated with increased predisposition to specific cancers (List of genes included is described in Supplementary Table 2).

FIGURE 2

Frequency of pathogenic variants and variants of unknown significance identified in the study. This figure gives information about frequency of different types of variants (A) Pie chart showing number of pathogenic variants (ACMG 1 and 2) and ACMG 3 (variants of unknown significance), (B) Pie chart showing frequency of different types of pathogenic variants (ACMG 1 and 2), (C) Table showing frequency of copy number variants (CNVs) and sequence variants.

FIGURE 3

Graph showing distribution of pathogenic variants (PVs) in patients with breast and/or ovarian cancer: Patients presenting with Breast and/or Ovarian cancer (n = 2003). 13.7% (276/2003) patients tested positive for a PV. Genes with PVs are listed on the X-axis; Number of patients positive for a pathogenic variant is shown on Y-axis.

FIGURE 4

Graph showing distribution of pathogenic variants (PVs) in patients with ovarian cancer: Patients presenting with Ovarian cancer (n = 455). 18%(82/455) patients tested positive for a PV. Genes with PVs are listed on the X-axis; Number of patients positive for a pathogenic variant is shown on Y-axis.

FIGURE 5

Normalized copy number variant (CNV) plots demonstrating deletions and duplications in genes on the hereditary cancer panel. Y-axis represents quantile normalized copy number data (for unique autosomal genes, 0.5 indicates 1 copy; 1 indicates 2 copies; and 1.5, 3 copies; for homologous autosomal genes with their pseudogene, 0.75 indicates 3 copies; 1, 4 copies; and 1.25, 5 copies). Constitutional deletions are defined by a mean ratio of ≤0.65, and duplications are defined by a ratio of ≥1.35. Homologous region PMS2/PMS2CL deletions and duplications are assessed by a ratio of <0.8 and >1.2, respectively. X-axis indicates gene locations. From left to right, arrows represent patients with the following CNVs. (A) single patient with deletions in MSH2 and MSH6 genes [MSH2:c.(?_-21)_(*21_?)del/MSH6:c.(?_-21)_(*21_?)del] (represented by 2 arrows), pathogenic deletions of MLH1, PMS2, and BRCA2 [MLH1:c.(116 + 21_11721)_(545 + 21_546-21)del, PMS2:c.(537 + 21_538-21)_(903 + 21_904-21)del, BRCA2:c.(?_-21)_(67 + 21_68-21)del], novel VUS with four copies of a region of ATM [ATM:c.(8850 + 21_8851-21)_(*21_?) (Schon and Tischkowitz, 2018)], pathogenic deletions of PALB2 and BRCA1 [PALB2:c.(2586 + 21_2587-21)_(2748 + 21_2749-21)del, BRCA1:c.(5406 + 21_5407-21)_(*21_?)del, BRCA1:c.(5332 + 21_5333-21)_(5406 + 21_5407-21)del, BRCA1:c.(4357 + 21_4358-21)_(4484 + 21_4485-21)del], duplication of unknown significance in BRCA1 [BRCA1:c.(?_-21)_(80 + 21_81-21) (Li et al., 2019)], novel pathogenic deletion in RAD51C [RAD51C: c.(571 + 21_572-21)_(*21_?)del], pathogenic deletion and duplication of CHEK2 [CHEK2:c.(908 + 21_909-21)_(1095 + 21_1096-21)del, CHEK2:c.(319 + 21_320-21)_(592 + 21_593-21)dup], respectively. (B) Pathogenic deletions in MSH2, MSH6, BARD1, and APC genes [MSH2:c.(366 + 21_367-21)_(1076 + 21_1077-21)del, MSH6:c.(?_-21)_(260 + 21_261-21)del, BARD1:c.(?_-21)_(*21_?)del, APC:c.1958 + 241_4457del], duplications of unknown significance in PMS2 and ATM (novel variant) [PMS2:c.(2006 + 21_2007-21)_(*21_?)dup, ATM:c.(662 + 21_663-21)_(9171 + 21_9172-21)dup], pathogenic deletions in BRCA2, RAD51D (novel variant) and BRCA1 [BRCA2:c.(8487 + 21_8488-21)_(8632 + 21_8633-21)del, RAD51D:c.(?_-21)_(*21_?)del, BRCA1:c.(5277 + 21_5278-21)_(*21_?)del, BRCA1:c.(4986 + 21_4987-21)_(5074 + 21_5075-21)del], pathogenic duplication of BRCA1 [BRCA1:c.(4185 + 21_4186-21)_(4357 + 21_4358-21)dup], pathogenic deletion in BRCA1 [BRCA1:c.(?_-21)_(80 + 21_81-21)del], pathogenic duplication of RAD51C [RAD51C:c.(837 + 21_838-21)_(965 + 21_966-21)dup], pathogenic deletion of CHEK2 [CHEK2:c.(1461 + 21_1462-21)_(*21_?)del].