RNA assay identifies a previous misclassification of BARD1 c.1977A>G variant

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.

BRCA1-associated RING domain 1 (BARD1) was first identified in 1996 as a BRCA1-interacting protein 1 . Fulllength (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 domains 2,3 . It is through their RING domains that BARD1 and BRCA1 directly interact, mediating double-strand break (DSB) repair as a heterodimer 1,4 . 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 in 5 ).
BARD1 acts as a tumor suppressor, and its expression is necessary to maintain genomic stability and control the cell cycle 6 . 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 malignancies 7,8 . Since then, other isoforms have been identified in breast cancer 9 , colorectal cancer 10 , non-small cell lung cancer 11 and neuroblastoma 12 . These spliced isoforms have been detected both in tumoral and non-tumoral tissues [8][9][10][11][12] , suggesting that BARD1 alternative splicing is a common feature in human cells 10,13 . 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 absent 8 .
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 junction 7,8 . Interestingly, the synonymous BARD1 c.1977A>G has been previously reported as a pathogenic variant that affects splicing 14 , 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  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′-CCA TGG AAC CGG ATG GTC -3′ and exon 11 reverse primer 5′-AGG TTG TCC TTT GGA TGG TG-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 singlenucleotide 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′-TCC ATT GCT CTT TCT TAT CAC TTC -3′ forward primer and 5′-TCT GCT TTA TCA CAC ACC TTGA-3′ reverse primer, whereas cDNA amplification was performed with 5′-CCA TGG AAC CGG ATG GTC -3′ forward primer and 5′-AGG TTG TCC TTT GGA TGG TG-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   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/
AMP 26 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 group 25 , 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 www.nature.com/scientificreports/ 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 risk 27 , 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 roles 1 . However, its role in cancer susceptibility remains inconclusive, and the results obtained in several case-control studies have been controversial so far 5 . 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 acceptors 29 . 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 obtained 30 . 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 controls 25 .
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 ORF 8 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 splicing 31 , it is essential to differentiate between naturally occurring isoforms and aberrant splicing events. BARD1 splice variants are abundant 13 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 cancers 7-12 . Since isoform η also lacks exons 2 to 9 and taking into account all the evidence Scientific Reports | (2021) 11:22948 | https://doi.org/10.1038/s41598-021-02465-y www.nature.com/scientificreports/ 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 noncarrier 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.