113  705 ) predispose carriers to early - onset of breast and ovarian cancer with a risk of 6571% for breast cancer and 3959% for ovarian cancer by age 70 years .
variants in brca1 are scattered throughout the gene , and to date ( until 1 october 2013 ) , approximately 14  900 variants ( listed online at the breast cancer information core ( bic ) database : http://research.nhgri.nih.gov/bic/ ) have been identified by mutational screening since the cloning and characterization of brca1 in the mid-1990s .
many of these variants are pathogenic , such as frameshift and nonsense variants , as well as variants affecting splicing , of which several have been identified in danish breast and/or ovarian cancer patients . however , a large number of the brca1 variants , including small in - frame insertions or deletions , missense , silent , and intron variants , are variants of unknown clinical significance ( vus ) , which makes genetic counseling of patients and their families complicated . to characterize the biological effect of these variants and to provide clinicians with better evidence about treatment and preventive care , functional studies are required .
it has been previously shown that a large portion of brca1 variants induce splicing defects .
ideally , rna from a patient should be examined by rt - pcr analysis to establish if a variant has an effect on splicing . however , in many cases , rna is not available from the patient .
alternatively , the variant can be examined by mini - gene splicing analysis . to confidently interpret the obtained splicing results
, a mini - gene splicing assay should be validated by assessment of the sensitivity and specificity using a panel of variants classified previously . here ,
we report the in silico and functional examination of 37 brca1 variants using a mini - gene splicing assay , of which 24 were used to validate the use of the mini - gene splicing assay by comparing data to rt - pcr results using rna from blood samples / lymphoblastoid cell lines .
brca1 variants are numbered according to the guidelines from the human genome variation society ( http://www.hgvs.org/mutnomen ) using ncbi reference sequence ng_005905.2 , as well as according to genbank accession number u14680.1 , in which the a in the aug start codon has number 120 .
all data have been submitted to the leiden open variation database v.2.0 ( http://chromium.liacs.nl/lovd2/cancer/home.php ) .
the following five splice site prediction programs were used to predict the effect of variants on the efficiency of splicing : splicesitefinder ( http://www.interactive-biosoftware.com ) ; genesplicer ( http://www.cbcb.umd.edu/software/genesplicer ) ; splice site prediction by neural network ( http://www.fruitfly.org/seq_tools/splice.html ) ; maxentscan ( http://genes.mit.edu/burgelab/maxent/xmaxentscan_scoreseq.html ) ; and human splicing finder ( http://www.umd.be/hsf/ ) .
the analysis was performed by the integrated software alamut v.2.3 ( http://www.interactive-biosoftware.com ) using default settings in all predictions .
a variation of more than 10% in at least two algorithms was considered as having an effect on splicing .
wild - type brca1 exons ( 2 , 3 , 5 , 6 , 7 , 8 , 9 , 10 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , and 23 ) along with at least 200  bp of 5 and 3 intronic sequences were pcr amplified from human genomic dna using phusion dna polymerase ( roche , hvidovre , denmark ) and forward and reverse primers carrying restriction sites for either ecori , bamhi , noti , or xhoi ( primer sequences are available on request ) .
single - nucleotide substitutions or deletions were introduced using finnzymes ' phusion high - fidelity dna polymerase according to the accompanying instructions .
wild - type and mutant constructs were transfected in duplicate into cos-7 cells as described recently , to account for differences in transfection efficiencies .
cells were harvested after 48  h and total rna was extracted using trizol reagent ( invitrogen , naerum , denmark ) .
cdna was synthesized using 1  g of total rna , m - mulv reverse transcriptase polymerase ( new england biolabs , hitchin , uk ) , and 20  m of nucleotide oligo(dt)15 primer .
cdna was amplified with phusion dna polymerase using the primers dusd2 ( 5-tctgagtcacctggacaacc-3 ) and dusa4 ( 5-atctcagtggtatttgtgagc-3 ) .
each dna band was gel purified using ge healthcare 's ( bronby , denmark ) illustra gfx pcr dna and gel band purification kit and sequenced with dusd2 and dusa4 primers .
brca1 variants are numbered according to the guidelines from the human genome variation society ( http://www.hgvs.org/mutnomen ) using ncbi reference sequence ng_005905.2 , as well as according to genbank accession number u14680.1 , in which the a in the aug start codon has number 120 .
all data have been submitted to the leiden open variation database v.2.0 ( http://chromium.liacs.nl/lovd2/cancer/home.php ) .
the following five splice site prediction programs were used to predict the effect of variants on the efficiency of splicing : splicesitefinder ( http://www.interactive-biosoftware.com ) ; genesplicer ( http://www.cbcb.umd.edu/software/genesplicer ) ; splice site prediction by neural network ( http://www.fruitfly.org/seq_tools/splice.html ) ; maxentscan ( http://genes.mit.edu/burgelab/maxent/xmaxentscan_scoreseq.html ) ; and human splicing finder ( http://www.umd.be/hsf/ ) .
the analysis was performed by the integrated software alamut v.2.3 ( http://www.interactive-biosoftware.com ) using default settings in all predictions .
a variation of more than 10% in at least two algorithms was considered as having an effect on splicing .
wild - type brca1 exons ( 2 , 3 , 5 , 6 , 7 , 8 , 9 , 10 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , and 23 ) along with at least 200  bp of 5 and 3 intronic sequences were pcr amplified from human genomic dna using phusion dna polymerase ( roche , hvidovre , denmark ) and forward and reverse primers carrying restriction sites for either ecori , bamhi , noti , or xhoi ( primer sequences are available on request ) .
single - nucleotide substitutions or deletions were introduced using finnzymes ' phusion high - fidelity dna polymerase according to the accompanying instructions .
wild - type and mutant constructs were transfected in duplicate into cos-7 cells as described recently , to account for differences in transfection efficiencies .
cells were harvested after 48  h and total rna was extracted using trizol reagent ( invitrogen , naerum , denmark ) .
cdna was synthesized using 1  g of total rna , m - mulv reverse transcriptase polymerase ( new england biolabs , hitchin , uk ) , and 20  m of nucleotide oligo(dt)15 primer .
cdna was amplified with phusion dna polymerase using the primers dusd2 ( 5-tctgagtcacctggacaacc-3 ) and dusa4 ( 5-atctcagtggtatttgtgagc-3 ) .
each dna band was gel purified using ge healthcare 's ( bronby , denmark ) illustra gfx pcr dna and gel band purification kit and sequenced with dusd2 and dusa4 primers .
we have previously used a mini - gene splicing assay to examine the effect of brca1/brca2 variants on splicing . to validate this assay ,
24 variants previously examined by rt - pcr using rna from blood samples / lymphoblastoid cell lines were introduced into the pspl3 vector ( figure 1a ) .
variants present in brca1 exon 11 were excluded as we were unable to achieve the expression of wild - type exon 11 , probably owing to its large size .
however , variants in the exon 2 , 3 , 5 , 6 , 7 , 8 , 9 , 10 , 13 , 14 , 15 , 16 , 17 , 19 , 20 , 21 , 22 , and 23 vector constructs were examined in the validation setup ( supplementary figures 1a  u ) .
as summarized in table 1 , all major effects identified in the rt - pcr analyses on rna from blood samples / lymphoblastoid cell lines were also identified by the mini - gene splicing assays .
thus , there is 100% concordance between the mini - gene splicing assay results and the results from rna from blood samples / lymphoblastoid cell lines , taking into account that the position of the cryptic splice sites activated by two brca1 variants ( c.5074 + 1g > t and c.5277 + 1g > a ) were wrongly annotated in the original publications but are identical to our findings upon sequence comparison .
moreover , in our hands , a minor increased skipping of exon 17 was observed for the c.5074 + 6c > g variant that was not described in the literature , although splicing data were not presented in the studies . following this successful validation of the mini - gene splicing assay
, we investigated 13 brca1 variants not previously examined by functional assays ( table 2 ) .
first , the potential pathogenicity of the variants was investigated using five different in silico splice site prediction programs , which predict changes in splice site strength .
the applicable threshold was a variation between the wild - type and the variant score of more than 10% in at least two different algorithms . according to this criterion , seven brca1 variants , c.80 + 1g > a , c.132c > t ( p.= ) , c.2131g > a
, c.30215c > g , c.670 + 1delg , c.4185 + 1g > a , and c.50751g > c ( table 2 ) , were suggested to have an effect on splicing , whereas no splicing alterations were predicted for the remaining six variants ( c.-19 - 22_-19 - 21dupat , c.547 + 14delg , c.670 + 16g > a , c.467620a > g , c.498721g > t , and c.527814c >
the functional effects of all 13 brca1 variants were subsequently examined by mini - gene splicing assays .
then , mrna was purified , analyzed by rt - pcr , and finally pcr products were visualized on 1% agarose gels ( figure 1b  m ) . in accordance with the in silico results , six brca1 variants ( c.80 + 1g > a , c.132c > t ( p.= ) , c.213 - 1g > a , c.670 + 1delg , c.4185 + 1g > a , and c.50751g >
c ) revealed the presence of alternative gel bands compared with the corresponding wild types .
the wild - type brca1 exon 2 construct generated one transcript comprising the expected 276  bp , whereas the c.80 + 1g > a mutant yielded one strong band of 177  bp lacking exon 2 ( figure 1b ) .
wild - type brca1 exon 3 generated one transcript at the expected 231  bp , whereas the c.132c > t ( p.= ) mutant resulted in one strong band of 227  bp excluding the last four bases of exon 3 ( figure 1c ) .
wild - type brca1 exon 6 generated one transcript at the expected size of 266  bp , whereas the c.2131g > a mutant resulted in one strong band of 325  bp , including 59  bp of intron 5 ( figure 1d ) .
wild - type brca1 exon 10 revealed the presence of two bands , one of 254  bp including exon 10 and one of 177  bp excluding exon 10 .
the c.670 + 1delg mutant resulted in a 253-bp transcript lacking 1  bp of exon 10 ( figure 1e ) .
wild - type brca1 exon 12 revealed the presence of one band at the expected size of 266  bp , whereas the c.4185 + 1g > a mutant resulted in one strong band of 177  bp lacking exon 12 ( figure 1f ) .
wild - type brca1 exon 18 exhibited one band with the expected size of 255  bp , whereas the c.50751g > c mutant revealed a single band of 177  bp lacking exon 18 ( figure 1 g ) .
in contrast , the c.-19 - 22_-19 - 21dupat ( figure 1h ) , c.30215c > g ( figure 1i ) , c.547 + 14delg ( figure 1j ) , c.467620a > g ( figure 1k ) , c.498721g > t ( figure 1l ) , and c.527814c > g ( figure 1 m ) variants showed no band size or intensity differences between wild - type and mutant constructs .
this was in agreement with the in silico data except for the c.30215c > g variant .
the c.670 + 16g > a variant ( figure 1e ) , however , appeared to make the consensus splice donor site stronger .
our results revealed a major band of 254  bp including exon 10 and a minor 177-bp band lacking exon 10 , which is the opposite to the wild - type pattern . in all cases ,
all variants were classified according to the 5-tier splicing classification scheme proposed recently ( tables 1 and 2 ) .
to ensure patients with hereditary breast cancer receive optimal genetic counseling and thereby preventive care , it is important to classify all identified brca1 sequence variants .
it has previously been established that all variant types in brca1 can lead to splicing abnormalities , and therefore it is important to investigate all variants at the rna level before classification .
splicing analysis is preferably carried out using rna from blood samples / lymphoblastoid cell lines . however , in many cases , rna is not available from the patient .
alternatively , the variant can be examined by mini - gene splicing analysis , which has previously been shown to be a valid method for investigating the impact of variants on the splicing pattern .
the mini - gene splicing assay validation performed in the current study showed a 100% concordance with the 24 brca1 variants previously classified using rt - pcr analysis on rna from blood samples / lymphoblastoid cell lines and therefore supports the previous findings .
the position of two of the cryptic splice sites activated was incorrectly annotated in the original publications . the brca1 c.5074 + 1g > t variant
our mini - gene splicing assay showed that 153  bp of intron 17 were included in the transcript , which is in agreement with the sequence reported in the original publication .
moreover , the brca1 c.5277 + 1g > a variant was reported to result in an out - of - frame inclusion of 85  bp of intron 20 , leading to a premature stop codon .
our mini - gene splicing assay results revealed that the variant induced an in - frame inclusion of 87  bp of intron 20 , which also is in agreement with the sequence reported in the original publication .
therefore , the variant results in the inclusion of an extra 29 amino acids in the protein instead of a premature stop codon , indicating that the variant should be reclassified as a vus .
in addition , our validation analysis revealed that the brca1 c.5074 + 6c > g variant showed a minor increase in the transcripts lacking exon 17 compared with the wild type . in two previous studies ,
this variant was classified as neutral based on rt - pcr using rna from blood samples .
splicing data were not presented in these publications , and as they used rna purified from blood samples , the weak band observed here could have been degraded by nonsense - mediated decay .
on the basis of the partial skipping of exon 17 , we classify this variant as a vus .
we also examined the effect of 13 ( 12 intronic and one exonic ) uncharacterized brca1 variants on splicing by in silico analysis and mini - gene splicing assay .
the in silico analysis predicted alterations in the splicing pattern for seven brca1 variants ( c.80 + 1g > a , c.132c > t ( p.= ) , c.2131g > a , c.30215c > g , c.670 + 1delg , c.4185 + 1g > a , and c.50751g > c ) , whereas six brca1 variants ( c.-19 - 22_-19 - 21dupat , c.547 + 14delg , c.670 + 16g > a , c.467620a > g , c.498721g > t , and c.527814c >
however , mini - gene splicing analysis revealed that the c.30215c > g variant had no effect on splicing , suggesting that the criterion used ( > 10% difference between wild - type and mutant scores in at least two programs ) results in false - positive predictions as shown previously .
the mini - gene splicing analysis revealed that three of the six deleterious variants resulted in the use of a cryptic splice site .
the silent variant c.132c > t ( p.= ) was predicted to introduce a new strong splice site located 4  bp upstream from the consensus splice site , which was confirmed by the mini - gene splicing assay .
moreover , the c.213 - 1g > a variant destroyed the exon 5 consensus acceptor splice site , resulting in the use of a cryptic splice site located 59  bp upstream of exon 5 .
this is in agreement with another variant ( c.21311t > g ) , which uses the same cryptic splice site .
finally , the c.670 + 1delg variant disrupted the exon 10 consensus donor splice site , creating a new splice site using the last base of exon 10 .
all three variants ultimately lead to the disruption of the reading frame and are classified as pathogenic .
interestingly , 10 of the 20 pathogenic variants investigated in the present study result in the use of a cryptic splice site .
this emphasizes the importance of functional evaluation of all potential variants affecting splicing , including variants in the consensus splice sites , as cryptic splice sites can result in small in - frame deletions or insertions that may not be pathogenic .
the analysis moreover emphasizes the need for sequencing of all gel bands observed in the mini - gene splicing assay as some variants induce the use of cryptic splice sites in close proximity of the splice donor or acceptor sites ( eg , c.132c > t and c.51531g > a ) .
c.80 + 1g > a , c.4185 + 1g > a , and c.50751g > c  resulted in skipping of exons 2 , 12 , and 18 , respectively .
all three variants result in disruption of the reading frame and are classified as pathogenic .
furthermore , our results showed that the brca1 c.670 + 16g > a variant induced almost complete inclusion of exon 10  an exon known to be involved in alternative splicing .
the brca1 c.670 + 8c > t variant has previously been found , by both mini - gene splicing assay and rt - pcr using rna from blood samples , to result in complete inclusion of exons 9 and 10 .
currently , knowledge is lacking regarding the functions of the different brca1 isoforms and their roles in cancer protection .
some experiments indicate that the balance between the different isoforms is important , whereas others have shown that the balance between the isoforms changes during the cell cycle and differs between different tissues . until a better understanding of the different isoforms
is achieved , variants affecting the ratio of splicing isoforms can not be classified properly . in conclusion , based on a validated mini - gene splicing assay , we classified six brca1 variants ( c.80 + 1g > a , c.132c > t ( p.= ) , c.2131g > a , c.670 + 1delg , c.4185 + 1g > a , and c.50751g > c ) as pathogenic , whereas six brca1 variants ( c.-19 - 22_-19 - 21dupat , c.30215c > g , c.547 + 14delg , c.467620a > g , c.498721g > t , and c.527814c >
one variant ( c.670 + 16g > a ) was shown to increase the inclusion of exon 10 compared with the wild type and was therefore classified as a vus .
this study supports the use of in silico prediction tools and mini - gene splicing assays in the assessment of the pathogenicity of brca1 variants .
 mutational screening of the breast cancer susceptibility gene brca1 leads to the identification of numerous pathogenic variants such as frameshift and nonsense variants , as well as large genomic rearrangements . 
 the screening moreover identifies a large number of variants , for example , missense , silent , and intron variants , which are classified as variants of unknown clinical significance owing to the lack of causal evidence . 
 variants of unknown clinical significance can potentially have an impact on splicing and therefore functional examinations are warranted to classify whether these variants are pathogenic or benign . 
 here we validate a mini - gene splicing assay by comparing the results of 24 variants with previously published data from rt - pcr analysis on rna from blood samples / lymphoblastoid cell lines . 
 the analysis showed an overall concordance of 100% . 
 in addition , we investigated 13 brca1 variants of unknown clinical significance or putative variants affecting splicing by in silico analysis and mini - gene splicing assay . 
 both the in silico analysis and mini - gene splicing assay classified six brca1 variants as pathogenic ( c.80 + 1g > a , c.132c > t ( p.= ) , c.2131g > a , c.670 + 1delg , c.4185 + 1g > a , and c.50751g > c ) , whereas six brca1 variants were classified as neutral ( c.-19 - 22_-19 - 21dupat , c.30215c > g , c.547 + 14delg , c.467620a > g , c.498721g > t , and c.527814c > 
 g ) and one brca1 variant remained unclassified ( c.670 + 16g > a ) . in conclusion , our study emphasizes that in silico analysis and mini - gene splicing assays are important for the classification of variants , especially if no rna is available from the patient . 
 this knowledge is crucial for proper genetic counseling of patients and their family members . 
Introduction
Materials and methods
Variant nomenclature
None
Mini-gene splicing assay
Results
Discussion
Supplementary Material