Hereditary Breast and Ovarian Cancer (HBOC) represents 5–10% of all breast cancers. Nowadays, more than 25 HBOC susceptibility genes have been identified, most of them involved in DNA repair pathways (Nielsen et al., 2016). Deleterious variants of the most prevalent genes BRCA1 (MIM# 113705) and BRCA2 (MIM# 600185) confer up to 87% of risk to develop breast cancer by the age of 70 years (Petrucelli et al., 2013). Apart from specific founder deleterious mutations (Levy-Lahad et al., 1997; Infante et al., 2013), there have been described thousands of different BRCA1/2 variants at the mutation databases. According to Universal Mutation Database (UMD, http://www.umd.be; date last accessed 2017/06/16) 2,495 and 3,454 different variants have been detected in BRCA1 and BRCA2, respectively, where a relevant fraction of them has been classified as variants of uncertain significance (VUS). These pose a challenge in clinical genetics since mutation carriers could benefit from preventive and prophylactic measures as well as new targeted therapies such as the Poly-ADP Ribose Polymerase Inhibitors (Ricks et al., 2015).

Standard approaches tend to classify DNA variants from the protein point of view. In this way, nonsense variants and frameshift insertions and deletions are automatically classified as pathogenic if they truncate critical protein domains [Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) class 5¹]. However, upstream gene expression mechanisms, such as splicing, can be disrupted by DNA changes. In fact, splicing is a critical highly regulated process involved in many cell functions whose disruption has been directly related with disease, being common in cancer (Wang and Cooper, 2007; Douglas and Wood, 2011). Likewise, spliceogenic variants are more common than they are thought, and they are not restricted to the sequences of the canonical donor and acceptor sites since it has been suggested that up to 50% of exon variants could also affect splicing (López-Bigas et al., 2005). This can be explained by the wide range of splicing regulatory elements (SREs) that control this process, which include the conserved splice sites (5’ss and 3’ss), the branch point, polypyrimidine track, exonic/intronic splicing enhancers (ESEs/ISEs) and exonic/intronic splicing silencers (ESSs/ISSs) (Grodecká et al., 2017), as well as other regulatory components or the RNA secondary structure (Soemedi et al., 2017). Thus, all these factors cooperate with splicing factors and the spliceosome, to accurately remove introns (Will and Lührmann, 2011).

Interestingly, spliceogenic variants are often found in BRCA2. Our previous results showed that more than a half of tested BRCA2 variants impaired splicing (Acedo et al., 2012, 2015; Fraile-Bethencourt et al., 2017). Moreover, the minigene technology was confirmed as a reliable tool to functionally assay potential splicing variants. Here, we aimed to check BRCA2 exon 16 candidate variants to characterize the splicing effects using the pSAD-based minigene MGBR2_14-20, previously employed to assay DNA variants of exons 17 and 18 (Fraile-Bethencourt et al., 2017). We have assayed 12 likely spliceogenic variants from HBOC patients reported in databases and selected after bioinformatics predictions. Wild-type (wt) and mutant minigenes assays showed that eight variants altered the splicing. Thus, we provide valuable information of spliceogenic BRCA2 exon 16 variants that could be classified following ENIGMA and American College of Medical Genetics and Genomics (ACMG) guidelines (Richards et al., 2015).

Ethical approval for this study was obtained from the Ethics Review Committee of the Hospital Universitario Río Hortega de Valladolid (6/11/2014).

Nowadays, with the advent of new generation sequencing technologies and, namely, cancer-gene panels (Slavin et al., 2015), thousands of variants are being described. However, their classifications as neutral or deleterious variants pose a challenge in Human Genetics. In fact, some deleterious variants can be missed because they are synonymous or intronic. Moreover, a significant fraction of BRCA2 variants are considered VUS and require additional proofs to be reclassified, including functional tests. Here, we have shown that the minigene MGBR2_14-20 is a robust tool to functionally assay candidate spliceogenic variants of the BRCA2 exon 16. Until now, we have comprehensively studied candidate splicing variants from 20 out of 27 BRCA2 exons (Sanz et al., 2010; Acedo et al., 2012, 2015; Fraile-Bethencourt et al., 2017). Thus, we have found six intronic and two missense BRCA2 variants which alter the splicing and could confer cancer risk.

BRCA2 exon 16 codifies from Leucine 2540 to Arginine 2602 (p.2540_2602). Interestingly, according to the International Agency for Research on Cancer (IARC⁶), this is a conserved region, since there is ∼22% of ultra-conserved aminoacids from human to sea urchin and ∼54% between mammals. Furthermore, this protein segment belongs to FANCD2- and DSS1-binding domains. Fanconi Anemia group D2 (FANCD2) protein binds to aminoacids from position p.2350 to p.2545 of BRCA2 and it has been suggested to have a role in the repair process (Hussain et al., 2004). DSS1 (Delete in Split hand/Split foot) protein, which binds to BRCA2 at positions p.2467_2957 (Marston et al., 1999), is an essential element of BRCA2 stability, since its loss supposes a dramatic decrease of BRCA2 levels (Li et al., 2006). Altogether, this highlights the value of exon 16 in BRCA2 function. Moreover, exon 16 skipping supposes a frame-shift deletion and the generation of a PTC (p.L2540Gfs^∗4), which would truncate the protein and subsequently loss the C-terminal region that would compromise BRCA2 function.

This study, based on minigene technology, provides detailed information about the impact on splicing of 12 BRCA2 exon 16 variants. Aberrant splicing outcomes were found in eight of these variants, six intronic and two missense changes. Intriguingly, none of the aberrant transcripts described here was previously reported as natural alternative splicing events of the BRCA2 gene (Fackenthal et al., 2016). Among them, seven (c.7618-2A>T, c.7618-2A>G, c.7618-1G>A, c.7618-1G>C, c.7805G>C, c.7805+1G>A, and c.7805+3A>C) provoked more than ∼92% of frameshift transcripts. Interestingly, previous studies of variant c.7618-1G>A in lymphoblastoid cells showed that 3′ss disruption induced transcripts Δ16p₄₄ and Δ16,17p₆₉ (Whiley et al., 2011). Here we found both transcripts, but also other minor ones: Δ16p₅₅, Δ16p₉₃, Δ16 (Table 3). Additionally, according to our data Δ16p₄₄ is the main transcript (∼91%) that other authors also identified but described as a minor transcript in agarose gels (Whiley et al., 2011). These differences could be due to: (i) the cell line; (ii) the use of cycloheximide to inhibit the NMD; (iii) the fact that we work with a single-mutant allele, avoiding the wt counterpart effect; and (iv) the high sensitivity of fluorescent capillary electrophoresis, which can detect rare transcripts versus agarose electrophoresis. In any case, both results show that c.7618-1G>A severely disrupted splicing. On the other hand, variant c.7805G>C was previously reported to result in Δ16 and Δ16q₁₀₀, with the total absence of the canonical transcript (Bonnet et al., 2008). This outcome matches our results (Table 3): Δ16 as the main transcript (∼78%), followed by Δ16q₁₀₀ (∼14%), and the lack of the full-length transcript. It is also worthy to mention that we detected other minor transcripts due to the high sensitivity of fluorescent capillary electrophoresis (▾16q²⁰ at ∼6.5% and Δ16,17p₆₉ at ∼1.5%) that otherwise could not be easily detected on agarose gels. In any case, the spliceogenic effects of variants c.7618-1G>A and c.7805G>C were supported by our data.

Variant c.7802A>G probably generated the most conflicting result since it triggered ∼54% of canonical transcript and ∼46% of Δ16q₄, so that its interpretation is more complex. The transcript Δ16q₄, caused by the use of a new 5′ss, generated a frameshift deletion and the protein truncation by a PTC 46 codons downstream (p.Y2601Wfs^∗46). However, it is still unclear if ∼54% of full-length transcript can preserve BRCA2 function, given that, for example, 20–30% of BRCA1 transcript is able to maintain BRCA1 activity (de la Hoya et al., 2016). It is also important to keep in mind that full-length transcript carries a missense variant (p.Y2601W) that, according to IARC alignment⁷, Tyrosine 2601 is highly conserved from human to sea urchin, suggesting an important function in the protein. Moreover, PolyPhen-2 (Adzhubei et al., 2010) predicted that this aminoacid change is damaging with the maximum score (1.0). Curiously, c.7802A>G was reported a family with a significant history of primary cancers (colorectal, lymphoma, and breast cancers) which carried biallelic BRCA2 mutations (c.7802A>G and c.1845_1856delCT). However, patients did not present the typical FA phenotype, which suggested that p.Y2601W BRCA2 maintained at least enough BRCA2 activity to prevent early childhood FA features (Degrolard-Courcet et al., 2014). Nevertheless, this missense change remains classified as VUS in ClinVar⁸.

On the other hand, variant c.7625C>G was previously computed to disrupt one SRp55 motif (Pettigrew et al., 2008), although functional mapping by microdeletions indicated that exon 16 is likely not regulated by splicing enhancers. Nevertheless, this change was selected because it presumably created new strong 3′ and 5′ss as well, both with a NNSplice score >0.9 (Table 2). However, c.7625C>G only produced the full-length transcript without any splicing anomaly. The protein would even carry the missense variant p.T2542R. However, consistent with PolyPhen, this change might be considered as benign with a score of 0.0, which could be explained by the low conservation of the affected threonine. Anyway, further functional and association studies must be performed to interpret this variant. Other variant that resulted in a normal splicing pattern was the nonsense variant c.7738C>T (p.Q2580X), that a priori had been classified as pathogenic. In this case, the protein would be truncated at codon 2580 losing 839 aminoacids of the C-terminal where the DSS1-binding site, the DNA-binding domain, the RAD51C-binding site, and the cyclin-dependent kinase (CDK) phosphorylation site are located (Roy et al., 2012). Interestingly, this variant was found in an Italian non-Ashkenazi BRCA1 and BRCA2 double heterozygote family (Musolino et al., 2005).

According to the ACMG guidelines (Table 4; Richards et al., 2015), five variants (c.7618-2A>T, c.7618-2A>G, c.7618-1G>A, c.7618-1G>C, and c.7805+1G>A) can be classified as pathogenic as they match criteria PVS1 (very strong evidence of pathogenicity: null variant – nonsense, frameshift, canonical ±1 or 2 ss, initiation codon, single or multiexon deletion – in a gene where LOF is a known mechanism of disease), PS3 (strong evidence: well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product), PM2 (moderate evidence: absent from controls in Exome Sequencing Project, 1000 Genomes Project, or Exome Aggregation Consortium), PP3 (supporting evidence: multiple lines of computational evidence support a deleterious effect on the gene or gene product: conservation, evolutionary, splicing impact, etc.), and PP5 (reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation). On the other hand, variants c.7802A>G, c.7805G>C, and c.7805+3A>C were classified as likely pathogenic as they match criteria PS3, PM2, PP3, and PP5.

Similarly, following the ENIGMA rules for variant classification⁹, all variants, except for c.7802A>G, should be reclassified as class 4 (likely pathogenic) because they are “considered extremely likely to alter splicing based on position” and are “predicted bioinformatically to alter the use of the native donor/acceptor site.” Conversely, minigenes are not considered robust approaches to functionally test these variants yet (“… results from construct-based mRNA assays alone are not considered sufficiently robust to be used as evidence for variant classification …”). However, this specific minigene with BRCA2 exons 14–20 was confirmed as a robust tool since it reproduced patient RNA results from eight variants (Fraile-Bethencourt et al., 2017), and also c.7618-1G>A and c.7805G>C of this study, so that these seven class 4 variants could be even reclassified as class 5. Finally, c.7802A>G was classified as class 3 because it did not meet the above standards and induce a partial aberrant outcome with more than 50% of the canonical transcript.

In summary, we detected eight spliceogenic BRCA2 exon 16 variants that should be classified as pathogenic or likely pathogenic according to the ACMG guidelines (Table 4). Moreover, they account for 22% of causal variants of exon 16 and 11% of all recorded variants of this exon at the mutation databases. Taken together this and our previous studies, we have tested 283 BRCA1/2 variants under the splicing perspective, 154 of which induced anomalous patterns and 111 could be classified as pathogenic or likely pathogenic. These data remark the importance of variants of splicing regulatory sequences, which are often underestimated because most of them are placed in non-coding regions of the protein. Until now, genetic family-based studies have set up the impact of some variants on cancer risk. However, because of the exponential increment in the number of variants, their low frequencies and different nature, functional assays are strictly required. In this context, minigene technology constitutes a robust tool which can be used to functionally test spliceogenic candidate variants of any disease-gene without the interference of the counterpart wt allele. Certainly, pSAD-based minigenes represented valuable tools to functionally check variants of the SERPINA1 (severe alpha-1 antitrypsin deficiency) and CHD7 (Charge Syndrome) genes (Lara et al., 2014; Villate et al., 2018). RNA assays provide essential data for the initial characterization of VUS and improve the genetic counseling of hereditary diseases.

EF-B contributed to the bioinformatics analysis, minigene construction, manuscript writing, and performed most of the splicing functional assays. BD-G and AV-P participated in minigene construction, mutagenesis experiments, and functional assays. AA participated in minigene construction and functional mapping experiments. EV conceived the study and the experimental design, supervised all the experiments, and wrote the manuscript. All authors contributed to data interpretation, revisions of the manuscript, and approved the final version of the manuscript.

The authors declare that theresearch was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.