Identification and Functional Testing of ERCC2 Mutations in a Multi-national Cohort of Patients with Familial Breast- and Ovarian Cancer

Abstract

The increasing application of gene panels for familial cancer susceptibility disorders will probably lead to an increased proposal of susceptibility gene candidates. Using ERCC2 DNA repair gene as an example, we show that proof of a possible role in cancer susceptibility requires a detailed dissection and characterization of the underlying mutations for genes with diverse cellular functions (in this case mainly DNA repair and basic cellular transcription). In case of ERCC2, panel sequencing of 1345 index cases from 587 German, 405 Lithuanian and 353 Czech families with breast and ovarian cancer (BC/OC) predisposition revealed 25 mutations (3 frameshift, 2 splice-affecting, 20 missense), all absent or very rare in the ExAC database. While 16 mutations were unique, 9 mutations showed up repeatedly with population-specific appearance. Ten out of eleven mutations that were tested exemplarily in cell-based functional assays exert diminished excision repair efficiency and/or decreased transcriptional activation capability. In order to provide evidence for BC/OC predisposition, we performed familial segregation analyses and screened ethnically matching controls. However, unlike the recently published RECQL example, none of our recurrent ERCC2 mutations showed convincing co-segregation with BC/OC or significant overrepresentation in the BC/OC cohort. Interestingly, we detected that some deleterious founder mutations had an unexpectedly high frequency of > 1% in the corresponding populations, suggesting that either homozygous carriers are not clinically recognized or homozygosity for these mutations is embryonically lethal. In conclusion, we provide a useful resource on the mutational landscape of ERCC2 mutations in hereditary BC/OC patients and, as our key finding, we demonstrate the complexity of correct interpretation for the discovery of "bonafide" breast cancer susceptibility genes.

Fig 1. Domain structure and modeling of the ERCC2 mutations.

(A) Mutations in the XPD/ERCC2 protein domains. The diagram shows the ERCC2 protein with the four XPD domains shown as HD1 (blue), HD2 (green), FeS (Orange) and Arch (purple). The human enzyme has a C-terminal (grey) extension (CTE) that probably forms an interaction surface with the p44 protein. Disease-relevant ERCC2 mutation sites are indicated in boxes (blue or red frame: missense or truncating mutation, respectively; fillings: light-gray, cases with breast cancer (BC); pink, case with ovarian cancer only (OC); dark-gray: cases with either breast- or ovarian cancer (BC or OC); dark-green, patients with both breast- and ovarian cancer (BC + OC)). Numbers in brackets indicate recurrent mutations. (B) Structural placement of mutations on a C-alpha trace model of human ERCC2. The residues targeted by HBOC-causing mutations are represented as space-filled red spheres. Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) disease causing mutations sites as reported in ClinVar are shown in yellow and black spheres. Missense variants at residue position 423, 461, 487, 568, 461 and 722 have been found in both BC/OC as well as XP (red-yellow spheres) and TTD (red-black spheres) patients.

Fig 2. Nucleotide excision repair (NER) capacity and Transcriptional activity of breast cancer associated XPD/ERCC2 variants.

(A) Several XPD/ERCC2 variants cloned into an expression vector were analyzed regarding to complementation of ERCC2-defective XP6BE cells overexpressing the NER-deficient R601W XPD mutant [15] (normalization for overexpression artifacts). Black bars indicate the mean relative repair capacity (in %, WT-XPD was set to 100%) of an UV irradiated firefly luciferase reporter gene plasmid (UVC 1000 J/m²) obtained by host cell reactivation (n>6 in triplicates). Red lines mark the range between DNA-repair levels of empty vector, i.e. residual repair activity of the cells, and WT-XPD, i.e. 100% repair capacity. (B) Dominant modulation of firefly luciferase reporter gene expression (without irradiation) via overexpression of XPD/ERCC2 BC/OC-associated variants was estimated in the transcriptionally-proficient but repair-deficient XPD/ERCC2-defective XP6BE cells. Black bars indicate the mean relative reporter gene expression (in %, empty vector control was set to 100%), obtained by CMV-promotor driven basal transcription (n>6 in triplicates). Error bars indicate the standard error of the mean. Significance levels were calculated, after pairwise testing for normal distribution of the values, using appropriate statistical tests for comparison of two groups (T-Test or U-Test, # = reference group, *** = p<0.001, ** = p<0.01, * = p<0.05, n.s. = not significant). (C) Additional characteristics of the mutations tested for repair efficiency and transcriptional activity.

Fig 3. ERCC2 frameshift mutation c.1703_1704delTT (p.Phe568fs) in familial breast and ovarian cancer pedigrees.

Individuals with breast cancer (BC), ovarian cancer (OC) or both (BC, OC) are shown as circles filled in black. Individuals tested positive for the familial mutation are indicated in detail; those with WT (wild-type) have been tested negative. All affected individuals with BC or OC not tested for germline mutations in ERCC2 were either deceased or refused testing. (A) German, (B) Lithuanian and (C-E) Czech pedigrees.