Overview of the Genetic Causes of Hereditary Breast and Ovarian Cancer Syndrome in a Large French Patient Cohort

Abstract

The use of multigene panel testing for patients with a predisposition to Hereditary Breast and Ovarian Cancer syndrome (HBOC) is increasing as the identification of mutations is useful for diagnosis and disease management. Here, we conducted a retrospective analysis of BRCA1/2 and non-BRCA gene sequencing in 4630 French HBOC suspected patients. Patients were investigated using a germline cancer panel including the 13 genes defined by The French Genetic and Cancer Group (GGC)-Unicancer. In the patients analyzed, 528 pathogenic and likely pathogenic variants (P/LP) were identified, including BRCA1 (n = 203, 38%), BRCA2 (n = 198, 37%), PALB2 (n = 46, 9%), RAD51C (n = 36, 7%), TP53 (n = 16, 3%), and RAD51D (n = 13, 2%). In addition, 35 novel (P/LP) variants, according to our knowledge, were identified, and double mutations in two distinct genes were found in five patients. Interestingly, retesting a subset of BRCA1/2-negative individuals with an expanded panel produced clinically relevant results in 5% of cases. Additionally, combining in silico (splicing impact prediction tools) and in vitro analyses (RT-PCR and Sanger sequencing) highlighted the deleterious impact of four candidate variants on splicing and translation. Our results present an overview of pathogenic variations of HBOC genes in the southeast of France, emphasizing the clinical relevance of cDNA analysis and the importance of retesting BRCA-negative individuals with an expanded panel.

Keywords: BRCA gene; HBOC; NGS; RNA analysis; multigene panel.

Figure 1

Distribution of pathogenic or likely pathogenic (P/LP) variants detected by NGS. (A) Distribution and percentages of the 530 (P/LP) variants detected in all HBOC cases. (B) Frequencies of (P/LP) variants in all HBOC cases. (C) Genes with (P/LP) variants detected in the 177 patients with breast cancer diagnosed before 31 years of age. (D) Genes with (P/LP) variants detected in the 759 patients with ovarian cancer. (E) Genes with (P/LP) variants detected in 91 patients with male breast cancer (F). Genes with (P/LP) variants detected in the 492 patients who had previously tested negative for BRCA pathogenic variants and retested using the GGC-HBOC gene panel.

Figure 2

The PALB2 c.1631_1684+1846del variant identified in the proband of the HBOC family. (A) In silico splicing analysis using Alamut Visual plus v1.7.1 (Interactive Biosoftware). (B) RT-PCR from lymphocyte-derived RNA. Automated gel electrophoresis using the TapeStation detection system from patient and control samples. Two additional bands were observed in patient samples which were absent in the negative control. (C) Electropherogram related to Sanger sequencing (reverse sequence) of these amplicons demonstrates the abnormal structure of the two corresponding transcripts reflecting by exon 4 or exons 3–4 skipping during splicing. The sequence of the wild-type transcript is represented in black while sequences of aberrant transcripts are represented in red.

Figure 3

The RAD51C c.145+3A>C variant identified in the proband of HBOC family. (A) In silico splicing analysis using Alamut Visual Plus v1.7.1 (Interactive Biosoftware). (B) RT-PCR of lymphocyte-derived RNA gel electrophoresis from patient and control samples in triplicate. Electrophoresis of RT-PCR products demonstrates an additional band (820 bp), as well as the expected band (923 bp), in the proband. The density of the aberrant band is much stronger in the proband than in the controls. (C) Sequencing of the RT-PCR products confirmed the significant increase in the aberrant transcript Δ1q’ (r.43_145del). (D) Schematic representation of the two transcripts observed. (E) Tapestation analysis of the two transcripts observed in the patient and a normal control sample.

Figure 4

The RAD51C: c.905-2delA variant identified in the proband of HBOC family. (A) In silico splicing analysis using Alamut Visual Plus v1.7.1 (Interactive Biosoftware). (B) RT-PCR of lymphocyte-derived RNA. Electrophoresis of RT-PCR products demonstrates an additional band (604 bp), as well as the expected wild-type band (664 bp), in the proband and in the positive control (carrier of the c.965+5G>A). The density of the aberrant band is comparable to the wild-type band. (C) Electropherogram showing that the variant causes an aberrant transcript corresponding to exon 7 skipping in the patient sample (forward). The sequence of the wild-type transcript is represented in black while the sequence of the aberrant transcript is represented in red.

Figure 5

The PALB2 c.3350+4A>G variant identified in the proband of HBOC family. (A) In silico splicing analysis using Alamut Visual Plus v1.7.1 (Interactive Biosoftware). (B) Schematic representation of the aberrant transcripts observed. (C) RT-PCR of lymphocyte-derived RNA in the absence and in the presence of an NMD inhibitor (puromycin). Electrophoresis of RT-PCR products demonstrates an additional band (754 bp), as well as the expected wild-type band (903 bp), in the proband. The density of the aberrant band is comparable to the wild-type band. (D) Electropherogram showing that the variant causes an aberrant transcript corresponding to the transcript with exon 12 skipping in the patient sample (forward). The sequence of the wild-type transcript is represented in black while the sequence of the aberrant transcript is represented in red.