Abstract
Case–control studies have shown an association of BARD1 with hereditary breast and/or ovarian cancer (HBOC) predisposition. BARD1 alternatively spliced isoforms are abundant and some are highly expressed in different cancer types. In addition, a number of BARD1 germline pathogenic variants have been reported among HBOC patients. In previous reports, BARD1 c.1977A>G variant has been classified as pathogenic since it produces a frameshift transcript lacking exons 2 to 9. In the present study, we sought to validate the mRNA splicing results previously published and to contribute with new evidence to refine the classification of this substitution according to ACMG/AMP guidelines. The presence of the variant was screened in patients and controls. RT-PCR was performed in order to compare the transcriptional profiles of two variant carriers and ten non-carrier controls. In addition, allele-specific expression was assessed. No differences in variant frequency were detected between patients and controls. The RNA assay confirmed the presence of the shorter transcript lacking exons 2–9, but it was detected both in carriers and non-carriers. Furthermore, allelic imbalance was discarded and no significant differences in the proportion of full-length and shorter transcript were detected between carriers and controls. The shorter transcript detected corresponds to BARD1 isoform η, constituted by exons 1, 10 and 11. Our results support that this transcript is a constitutive splicing product rather than an aberrant transcript caused by BARD1 c.1977A>G variant, and for this reason this variant should be considered as likely benign following ACMG/AMP guidelines.
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Introduction
BRCA1-associated RING domain 1 (BARD1) was first identified in 1996 as a BRCA1-interacting protein1. Full-length (FL) BARD1 transcript comprises 11 exons and encodes a 777 amino acid protein that consists of one N-terminal RING-finger domain, four ankyrin (Ank) repeats and two C-terminal tandem BRCT domains2,3. It is through their RING domains that BARD1 and BRCA1 directly interact, mediating double-strand break (DSB) repair as a heterodimer1,4. For this reason, BARD1 has been regarded as a potential breast and/or ovarian cancer predisposing gene and its association with cancer risk has been deeply investigated (reviewed in5).
BARD1 acts as a tumor suppressor, and its expression is necessary to maintain genomic stability and control the cell cycle6. However, several oncogenic BARD1 isoforms have been discovered in different types of cancer with antagonistic effects. The first BARD1 isoforms were described by Li et al. in 2007 associated to human cytotrophoblast invasion and gynecological malignancies7,8. Since then, other isoforms have been identified in breast cancer9, colorectal cancer10, non-small cell lung cancer11 and neuroblastoma12. These spliced isoforms have been detected both in tumoral and non-tumoral tissues8,9,10,11,12, suggesting that BARD1 alternative splicing is a common feature in human cells10,13. Of note, some of these isoforms have been related with a poor prognosis, as they were overexpressed in tumor tissues while FL transcript was underrepresented or absent8.
Isoform η is composed of exons 1, 10 and 11. The open reading frame (ORF) is disrupted, but translation can be initiated in an alternative reading frame upstream of the new splice junction7,8. Interestingly, the synonymous BARD1 c.1977A>G has been previously reported as a pathogenic variant that affects splicing14, generating an aberrant transcript characterized by the skipping of exons 2 to 9, coinciding with isoform η. The aim of our study was to validate the mRNA splicing results published by Ratajska et al. and to provide with new evidence to refine its clinical interpretation according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines.
Materials and methods
Patients
A total of 4168 index patients with a personal or family history suggestive of hereditary cancer (HC) referred to the genetic counseling unit at Catalan Institute of Oncology (ICO) hospital were included in the present study. Genetic testing was performed in peripheral blood DNA using our ad hoc NGS custom panel I2HCP, which comprises 122–135 HC-associated genes, depending on the version used15. Library preparation methods and bioinformatics pipeline were previously described15. The analysis of the panel for diagnostics is phenotype-driven and includes a reduced number of clinically valid and actionable genes associated with a specific tumor type16. Informed written consent for both diagnostic and research purposes was obtained from all patients, and the study protocol was approved by the ethics committee of Bellvitge Biomedical Research Institute (IDIBELL; PR278/19). All experiments were performed in accordance with the relevant guidelines and regulations.
Controls
A set of 194 Spanish cancer-free individuals from the GCAT cohort (GenomesForLife—Cohort Study of the Genomes of Catalonia)17 were analyzed with I2HCP panel. In addition, the gnomAD non-Finnish European population, non-cancer data set (Genome Aggregation Database, v2.1.1, http://gnomad.broadinstitute.org/)18 was used as a control population.
Variant nomenclature
Human Genome Variation Society (HGVS) approved guidelines19 (http://varnomen.hgvs.org/) were used for BARD1 variant nomenclature using NM_000465.2 (LRG_297). For variant numbering, nucleotide 1 is the A of the ATG-translation initiation codon.
In silico analysis
The spliceogenic effect of BARD1 c.1977A>G variant was evaluated using Alamut Visual software v2.11.0 (Interactive Biosoftware, Rouen, France), which integrates the following prediction methods: Splice Site Prediction by Neural Network20 (NNSPLICE), SpliceSiteFinder-like21 (SSF), MaxEntScan22 (MES) and GeneSplicer23 (GS). All algorithms were used with the default settings. In addition, SpliceAI software was also used24.
RNA analysis
Lymphocytes were isolated by Ficoll gradient centrifugation of peripheral blood samples from two patients who harbored BARD1 c.1977A>G variant and ten non-carrier controls. Cells were cultured in PB-Max medium for 5–7 days in two subcultures, one of which was treated with puromycin 4–6 h before RNA extraction in order to prevent the potential degradation of unstable transcripts by nonsense-mediated decay (NMD). Total RNA was isolated using TRIzol reagent and reverse transcribed with iScript cDNA Synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA). cDNA amplification was performed with the following primers: exon 1 forward primer 5′-CCATGGAACCGGATGGTC-3′ and exon 11 reverse primer 5′-AGGTTGTCCTTTGGATGGTG-3′ (Fig. 1A). Transcriptional profiles from variant carriers were compared with those derived from ten control lymphocyte cultures by agarose gel analysis, TapeStation Assay analysis (Agilent 4200 TapeStation System, Agilent Technologies, Santa Clara, CA, USA) (Fig. 1B) and Sanger sequencing (Fig. 1C)25.
Quantification of full-length transcripts produced by the variant allele
In order to evaluate the extent of the splicing effect, bands corresponding to the FL transcript were excised from agarose gels and single-nucleotide primer extension (SNuPE) was performed to assess allele-specific expression (ASE) using BARD1 c.1519G>A polymorphism as tag-SNP (Fig. 1D). Briefly, gene-specific primers were used to amplify the region enclosing the targeted variant, both in gDNA and cDNA of one carrier and one control. gDNA amplification was performed with 5′-TCCATTGCTCTTTCTTATCACTTC-3′ forward primer and 5′-TCTGCTTTATCACACACCTTGA-3′ reverse primer, whereas cDNA amplification was performed with 5′-CCATGGAACCGGATGGTC-3′ forward primer and 5′-AGGTTGTCCTTTGGATGGTG-3′ reverse primer. PCR products were purified using GFX PCR DNA and Gel Band Purification kit (GE Healthcare, Chicago, IL, USA), and then used in the SNaPshot quantitative primer extension assay according to the manufacturer’s protocol (SNaPshot Multiplex kit, ThermoFisher Scientific, Waltham, MA, USA). The primer used for the SNuPE assay was: 5′-AACAGCTTGACTATATCCA-3′. Following cleanup with SAP (shrimp alkaline phosphatase), products of the primer extension reaction were separated on an ABI 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Data were analyzed using GeneMapper v4.0 (Applied Biosystems) and ASE was calculated by dividing the proportion of variant/wild-type allele in cDNA by the proportion of variant/wild-type allele in gDNA25. All experiments were performed with three replicates.
Variant classification
The ACMG/AMP guidelines were used for variant interpretation26.
Results
Prevalence of BARD1 c.1977A>G variant in HC and control cohorts
Multi-gene panel testing was performed in a total of 4168 HC patients, and BARD1 carrier status was retrieved from all individuals in a research context for this study. Twenty patients harboring BARD1 c.1977A>G variant were identified, representing a carrier frequency of 0.5%. Of these, twelve patients were recruited with a clinical suspicion of hereditary breast and/or ovarian cancer, seven with a clinical suspicion of hereditary colorectal cancer and one with a clinical suspicion of melanoma (Table 1). In addition to BARD1 c.1977A>G, only one patient also harbored BRCA1 c.68_69del; p.(Glu23Valfs*17) pathogenic variant. Among 194 Spanish control samples, one carrier of the variant was identified (carrier frequency = 0.5%). Furthermore, 354 individuals from gnomAD non-cancer database harbored BARD1 c.1977A>G variant (carrier frequency = 0.6%), one of them in homozygosis. Therefore, the frequency of the variant in the HC cohort was comparable to that of the Spanish control cohort and gnomAD non-cancer cohort (Table 1).
mRNA splicing assay results
Total RNA was isolated from cultured lymphocytes of two patients who harbored BARD1 c.1977A>G variant and ten non-carrier controls, and their transcriptional profiles were compared by agarose and capillary electrophoresis analyses (Fig. 1B,C)25. As previously reported by Ratajska et al.14, a transcript lacking exons 2 to 9 was detected (r.159_1903del), but it was observed in both variant carriers and controls. This transcript presumably leads to a truncated protein (p.(Cys53Trpfs*12)) (Fig. 1A). However, only minor traces of this shorter transcript were detected in comparison to the FL (Fig. 1C). Contrarily to the study published by Ratajska et al., the presence of this shorter transcript was confirmed in all controls (Fig. 1B).
In order to elucidate whether the splicing effect was total or partial, allele-specific expression (ASE) was evaluated. To this aim we analyzed the expression of one patient and one control that harbored the BARD1 c.1519G>A polymorphism, located in the skipped region (exon 6). The proportion of variant and wild-type allele in cDNA did not differ from that detected in gDNA, and no significant differences were detected between patient and control samples. Furthermore, biallelic expression was detected after excision of the FL band (Fig. 1D). Therefore, allele-specific imbalance was discarded.
Although other transcripts were also detected, their size distribution and expression levels were equivalent between HC patients and controls (Fig. 1B,C). Therefore, they were disregarded as aberrant transcripts associated with c.1977A>G variant.
Variant interpretation following ACMG/AMP guidelines
The guidelines published by ACMG/AMP26 are based on scoring 28 different criteria of pathogenicity (P) or benignity (B) to interpret sequence variants. Each criterion is in turn ascribed with different strength levels: very strong (VS), strong (S), moderate (M) or supporting (P).
PS3 criterion can be used when well-established in vitro or in vivo functional studies are supportive of a damaging effect on the gene or gene product. In a previous publication by our group25, we devised a proposal on how to weight this evidence according to the results observed in in vitro RNA assays. The predominance of the FL transcript from the variant allele detected in HC patients, the low proportion of the shorter transcript observed, and the same splicing pattern detected in ten controls, suggested the alternative transcript r.159_1903del as a constitutive splicing product. BARD1 c.1977A>G variant is a synonymous substitution without impact at the protein level, p.Arg659=. Our results further discard a potential splicing alteration, r.1977a>g, since no damaging effect on the gene or gene product was detected and allele-specific silencing was dismissed. Therefore, the variant was weighted with BS3 criterion.
PP3 criterion is used when computational evidence supports a deleterious effect, and when no impact on gene or gene product is suggested BP4 is used instead. In this case, the variant was evaluated using five different in silico algorithms, and no deleterious effect was computationally predicted (Supplemental Table 1). Therefore, BARD1 c.1977A>G was weighted with BP4 criterion.
Regarding population data, BARD1 c.1977A>G variant is present in approximately 1 in 170 individuals with European ancestry, being more common than most cancer-predisposing variants. This could explain the reason why most ClinVar submissions classify this variant as (likely) benign, disregarding the functional assay published by Ratajska et al.14. BS1 criterion can be applied when population data is not consistent with disease prevalence. Despite the association of BARD1 with breast cancer risk27, gnomAD data suggest a greater tolerance for loss-of function variants in BARD1 (observed/expected ratio = 1.03 (90% Confidence Interval = 0.79–1.36)). Consequently, due to difficulty in establishing a threshold for BS1 criterion for this gene, it was disregarded for BARD1 c.1977A>G variant.
Following ACMG/AMP guidelines, the combination of all the evidences collected (BS3 + BP4) supports the classification of BARD1 c.1977A>G substitution as a likely benign variant.
Discussion
BARD1 was postulated as a hereditary breast and ovarian cancer (HBOC) predisposing gene shortly after it was first described, due to its relationship with BRCA1 in terms of shared structural homology and functional association for the development of their tumor-suppressor roles1. However, its role in cancer susceptibility remains inconclusive, and the results obtained in several case–control studies have been controversial so far5. We have recently published a case–control study investigating the role of BARD1 in cancer predisposition in a Spanish HBOC cohort of 4015 individuals. The results supported a significant association of BARD1 PVs with hereditary breast cancer (OR = 4.18; CI = 2.10–7.70; p = 5.45 × 10–5), particularly among triple-negative tumors (OR = 5.40; CI = 1.77–18.15; p = 0.001)28. However, it should be noted that only truncating and canonical splice site variants were considered for the risk calculations, whose prevalence in breast and/or ovarian cancer cases is very limited. As a result of the lack of bona fide protein functional studies, missense, synonymous and intronic variants have not yet been contemplated in most association studies. These three types of variants can be potentially spliceogenic, as the introduction of a DNA sequence alteration can lead to the disruption of canonical splice sites, as well as the activation or creation of other cryptic splice donors or acceptors29. As a consequence of an aberrant splicing pattern, transcripts carrying loss-of-function (LoF) alterations associated with the disease could be generated.
This article is focused on the characterization of BARD1 c.1977A>G variant. A previous study published by Ratajska et al.14 identified a shorter transcript characterized by the skipping of exons 2 to 9 (r.159_1903del; p.(Cys53Trpfs*12)) in an ovarian cancer patient harboring this variant. The presence of this transcript was discarded in one non-carrier control, thus it was attributed to the variant studied. Nevertheless, the analysis of ten non-carrier controls of our cohort has evidenced the presence of this shorter transcript in all of them, and therefore it cannot be associated to BARD1 c.1977A>G variant. Due to the inconsistencies in variant classification across different laboratories, the ENIGMA Consortium Splicing Working group defined some reporting guidelines to ensure the standardization of splicing assay protocols, as well as the interpretation of the data obtained30. Accordingly, mRNA assays from patient samples should be compared to at least ten reference controls, in order to facilitate the identification of naturally occurring isoforms. Besides, our study has evidenced the utility of highly sensitive electrophoretic analyses, since the limited resolution of conventional agarose gels could hinder the detection of less abundant alternative transcripts. Sanger sequencing should always be performed in order to assess the presence and to accurately characterize all transcripts, not only in variant carriers, but also in controls25.
We have also performed an ASE assay using BARD1 c.1519G>A polymorphism, which is located in the skipped region. Both alleles were equally expressed in total cDNA and in the FL band in carriers and controls, which points out to a physiologic alternative splicing and rules out an allelic imbalance. Furthermore, the shorter transcript does not appear to be enriched in patients, as no differences were detected between carriers and controls. In this shorter transcript, translation could be initiated in an alternative ORF8 that would result in an in frame deletion. Consequently, NMD would not be triggered, which is supported by our results as no differences were detected between samples treated with puromycin and untreated samples.
Another remarkable feature is that there do not seem to be differences in the prevalence of BARD1 c.1977A>G variant between patients and the general population. On one side, we identified BARD1 c.1977A>G substitution as the most prevalent BARD1 variant in our HC cohort, with 20 carriers among 4168 individuals (carrier frequency = 0.5%). Interestingly, its presence was not restricted to HBOC patients, as one would expect considering the association of BARD1 with breast and/or ovarian tumors. Besides, one out of 194 geographically-matched controls (GMCs) also harbored the same variant (carrier frequency = 0.5%). The comparison with a large-scale dataset evidenced that there were no significant differences between patients and controls (carrier frequency in gnomAD non-cancer cohort = 0.6%), indicating that this variant does not appear to be particularly associated with cancer. Unfortunately, the lack of association between a variant and disease does not constitute a criterion in the current ACMG/AMP classification guidelines.
Although nearly 95% of mammalian genes undergo alternative splicing31, it is essential to differentiate between naturally occurring isoforms and aberrant splicing events. BARD1 splice variants are abundant13 and several isoforms (including α, β, κ, γ, δ, φ, ε, η, π and ω) with antagonistic effects have been found to be highly expressed in various cancers7,8,9,10,11,12. Since isoform η also lacks exons 2 to 9 and taking into account all the evidence collected in this study, we believe that the skipped transcript detected corresponds to the constitutive splicing product named isoform η, equally expressed in variant carriers and controls. According to ACMG/AMP guidelines, our results reinforce the reinterpretation of this variant as (likely) benign, in consistency with most of its current ClinVar classifications.
In conclusion, our data does not support that BARD1 c.1977A>G variant promotes any aberrant splicing that could be associated with disease, as we have detected the same constitutive transcript in variant carriers and non-carrier controls. Furthermore, the frequency of this variant is similar in patients and in the general population, ruling out an increased risk of cancer associated to this variant.
Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files.
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Acknowledgements
We thank CERCA Program/Generalitat de Catalunya for their institutional support. We also wish to thank all the members of the ICO Hereditary Cancer Program. This study makes use of data generated by IGTP, specifically by its research group GCAT-Genomes for Life, Cohort study of the Genomes of Catalonia. A full list of the investigators who contributed to the generation of the data is available from http://www.genomesforlife.com/. IDIBELL and IGTP are part of the CERCA Program/Generalitat de Catalunya.
Funding
Supported by Carlos III National Health Institute and Ministerio de Ciencia e Innovación, funded by FEDER funds—a way to build Europe—[PI19/00553; PI16/00563; PID2019-111254RB-I00]; CIBERONC [CB16/12/00234]; and the Government of Catalonia—Pla estratègic de recerca i innovació en salut (PERIS_MedPerCan and URDCat projects) [2017SGR1282 and 2017SGR496]. GCAT is supported by Acción de Dinamización del ISCIII-MINECO [ADE 10/00026]; and the Government of Catalonia and Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) [2017SGR529]. GCAT Cession Research Project PI-2018-09 GCAT_CM.
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Conceptualization: P.R., J.V., C.L.; Data curation: P.R., M.P., L.F., M.M., R.C., G.C, J.B., J.V., C.L.; Formal analysis: P.R., M.P., L.F., M.M., R.C., J.V.; Funding acquisition: G.C., J.B., C.L.; Methodology: P.R., C.G., E.M.; Supervision: J.V., C.L.; Validation: M.P., L.F., M.M., J.V., C.L.; Writing—original draft: P.R., C.L.; Writing—review and editing: all authors critically reviewed the manuscript for intellectual content and approved the final version.
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Rofes, P., Pineda, M., Feliubadaló, L. et al. RNA assay identifies a previous misclassification of BARD1 c.1977A>G variant. Sci Rep 11, 22948 (2021). https://doi.org/10.1038/s41598-021-02465-y
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DOI: https://doi.org/10.1038/s41598-021-02465-y
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