Analysis of 30 Putative <em>BRCA1</em> Splicing Mutations in Hereditary Breast and Ovarian Cancer Families Identifies Exonic Splice Site Mutations That Escape <em>In Silico</em> Prediction

<div><p>Screening for pathogenic mutations in breast and ovarian cancer genes such as <em>BRCA1/2</em>, <em>CHEK2</em> and <em>RAD51C</em> is common practice for individuals from high-risk families. However, test results may be ambiguous due to the presence of unclassified variants (UCV) in the concurrent absence of clearly cancer-predisposing mutations. Especially the presence of intronic or exonic variants within these genes that possibly affect proper pre-mRNA processing poses a challenge as their functional implications are not immediately apparent. Therefore, it appears necessary to characterize potential splicing UCV and to develop appropriate classification tools. We investigated 30 distinct <em>BRCA1</em> variants, both intronic and exonic, regarding their spliceogenic potential by commonly used <em>in silico</em> prediction algorithms (HSF, MaxEntScan) along with <em>in vitro</em> transcript analyses. A total of 25 variants were identified spliceogenic, either causing/enhancing exon skipping or activation of cryptic splice sites, or both. Except from a single intronic variant causing minor effects on <em>BRCA1</em> pre-mRNA processing in our analyses, 23 out of 24 intronic variants were correctly predicted by MaxEntScan, while HSF was less accurate in this cohort. Among the 6 exonic variants analyzed, 4 severely impair correct pre-mRNA processing, while the remaining two have partial effects. In contrast to the intronic alterations investigated, only half of the spliceogenic exonic variants were correctly predicted by HSF and/or MaxEntScan. These data support the idea that exonic splicing mutations are commonly disease-causing and concurrently prone to escape <em>in silico</em> prediction, hence necessitating experimental <em>in vitro</em> splicing analysis.</p> </div

Classification of putative <i>BRCA1</i> splicing mutations.

<p><b>A:</b> Variants that severely affect splicing, <b>B:</b> Variants having a partial effect only, and <b>C:</b> Variants that do not affect processing of <i>BRCA1</i> pre-mRNA species in PBL.</p>*<p>Variants with severe impact on splicing are considered as likely pathogenic (class 4) according to the classification system proposed by Plon et al., <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050800#pone.0050800-Plon1" target="_blank">[42]</a>, while variants with only partial effects on splicing remain of uncertain clinical significance (class 3). Variant descriptions (BIC nomenclature, HGVS nomenclature), consequences on transcript- and protein levels, family IDs, analyzed index patients (phenotypes, age at onset), family histories (phenotypes, age at onset) and ethnic backgrounds are given. Family members carrying the same <i>BRCA1</i> variant are indicated (asterisk). All other listed family members were not available for analysis. Abbreviation: BC = breast cancer; OC = ovarian cancer; n.a. = not affected; bil = bilateral; ProC = prostate carcinoma; MTX = mastectomy; DCIS = Ductal carcinoma <i>in situ</i>.</p

RT-PCR analyses of <i>BRCA1</i> exon 19 and flanking sequences.

<p><b><i>A</i></b><i>)</i> Compared with controls C (1) to C (5), the variants <i>IVS18-2delA</i> and <i>IVS19+2T>G</i> elevate exclusion of exon 19 (Δex19). Regarding the variant <i>IVS18-2delA</i>, two mRNA samples derived from two unrelated mutation carriers were analyzed. Effects of <i>IVS18-6C>A</i> on <i>BRCA1</i> pre-mRNA processing were not observed. <i>IVS19+1delG</i> did not associate with a suspicious splicing pattern as shown by RT-PCR followed by gel electrophoresis. * Note that <i>IVS19+1delG</i> causes a 1 nt deletion on transcript level not detectable by agarose gel electrophoresis. <b><i>B</i></b><i>)</i> Direct sequencing of <i>IVS19+1delG</i> samples following RT-PCR revealed the deletion of the last nucleotide of exon 19 on mRNA level due to the activation of a cryptic splice site, which incorporates the last nucleotide of exon 19. NTC = no template control.</p

RT-PCR analyses of <i>BRCA1</i> exons 5 (A, B), 9 (C, D), and flanking sequences.

<p><b><i>A</i></b><i>)</i> Compared with controls C (1) to C (5), the variants <i>IVS4-18T>G</i> and <i>IVS4-1G>C</i> elevate exon 5 exclusion (Δex5), while <i>IVS5+1G>T</i>, <i>IVS5+1G>C</i> and <i>IVS5+1G>A</i> promote the usage of an upstream cryptic splice site, resulting in a 22 bp deletion on mRNA level (Δ22nt ex5). Regarding the variant <i>IVS5+1G>T</i>, two mRNA samples derived from two related mutation carriers were analyzed. NTC = no template control. Effects of the variant <i>IVS5+23T>A</i> on <i>BRCA1</i> pre-mRNA processing were not observed. <b><i>B</i></b><i>)</i> Compared with two control samples, enhanced exon 5 skipping in <i>IVS4-18T>G</i> and <i>IVS4-1G>C</i> samples was confirmed by quantitative real-time RT-PCR analyses. Expression data are given as mean ± standard deviation (s.d.). Relative to an internal <i>BRCA1</i> control set to 100% (amplicon spanning exon 6 and 7 sequences, <i>BRCA1</i> ex6/7), the relative amounts of transcripts lacking exon 5 (<i>BRCA1</i> ex2/3/6) account for 3.49% (+1.01, −0.78) and 3.03% (+1.11, −0.81) in control samples, respectively, while the relative amounts of <i>BRCA1</i> ex2/3/6 transcripts are approximately 3fold increased in <i>IVS4-18T>G</i> samples (10.83%, +0.76, −0.71). <i>IVS4-1G>C</i> increases the relative amount of <i>BRCA1</i> ex2/3/6 transcripts to 56.21% (+13.77, −11.06), while the share of transcripts harbouring exon 5 sequences is significantly reduced. Three levels of statistical significance were discriminated: * = P<0.05, ** = P<0.01, *** = P<0.001 (t-test). <b><i>C</i></b><i>)</i> The variant <i>710C>T,C197C</i> elevates skipping of exon 9 (Δex9) compared with controls. Total mRNA samples derived from two unrelated <i>710C>T, C197C</i> mutation carriers were analyzed. <b><i>D</i></b><i>)</i> Enhanced exon 9 skipping was confirmed by quantitative real-time analysis. While transcripts lacking exon 9 (<i>BRCA1</i> ex8/10) account for 2.51% (+0.23, −0.21) and 2.14% (+0.35, −0.30) relative to the respective internal controls, the amounts of <i>BRCA1</i> ex8/10 mRNA species are approximately 2fold increased in samples derived from two independent patients carrying the <i>710C>T, C197C</i> variant (5.23%, +0.70, −0.62; 6.92%, +0.55, −0.51). Similar results were observed when analyzing the relative amounts of transcripts lacking exons 9 and 10 (<i>BRCA1</i> ex8/11). In controls, relative <i>BRCA1</i> ex8/11 levels account for 7.69% (+0.70, −0.64) and 8.04% (+1.30, −1.12) and 16.73% (+2.25, −1.98) and 15.48% (+1.23, −1.14) in samples derived from two independent <i>710C>T, C197C</i> carriers.</p

Additional file 1: Table S1. of BRIP1 loss-of-function mutations confer high risk for familial ovarian cancer, but not familial breast cancer

Inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer (GC-HBOC) for BRCA1 and BRCA2 germline testing. Table S2. Heterozygous protein-truncating mutations identified in the BRIP1 gene. Figure S1. Characterization of the c.507G > A variant within the BRIP1 gene (rs876660937) on transcript level. Table S3. Genotypes and phenotypes of heterozygous BRIP1 mutation carriers identified within the BC/OC index patient cohorts. Table S4. Potentially damaging missense variants identified in the BRIP1 gene. (PDF 215 kb

The <i>HMMR</i> locus and breast cancer risk in <i>BRCA1</i> mutation carriers.

<p>(<b>A</b>) Forest plots showing rs299290 HRs and 95% CIs (retrospective likelihood trend estimation) for participating countries (relatively small sample sets are not shown) ordered by sample size. Left and right panels show results for <i>BRCA1</i> and <i>BRCA2</i> mutation carriers, respectively. The sizes of the rectangles are proportional to the corresponding country/study precision. (<b>B</b>) The rs299290-containing region, including the genes, variation and regulatory evidence mentioned in HMECs. Exons are marked by black-filled rectangles and the direction of transcription is marked by arrows in the genomic structure. The chromosome 5 positions (base pairs (bp)) and linkage disequilibrium structure from Caucasian HapMap individuals are also shown.</p

Gene expression interactions in breast cancer survival.

<p>(<b>A</b>) Kaplan–Meier survival curves based on categorization of <i>HMMR</i> (probe NM_012484) and <i>AURKA</i> (NM_003600) expression in tertiles (low, medium or high expression). For simplicity, only the tertiles for “high” <i>AURKA</i> are shown. The tumours with high expression levels for both genes were not those with the poorest prognosis. (<b>B</b>) Kaplan–Meier survival curves based on categorization of <i>HMMR</i> (NM_012484) and <i>TUBG1</i> (NM_016437) expression in tertiles (low, medium or high expression). For simplicity, only the tertiles for “high” <i>HMMR</i> are shown. The cases with high expression levels for both genes were those with the poorest prognosis.</p

Potential GxG associated with breast cancer risk in <i>BRCA1/2</i> mutation carriers.

<p>*Each estimate is derived from the interaction term of a Cox regression model.</p><p>Potential GxG associated with breast cancer risk in <i>BRCA1/2</i> mutation carriers.</p

p-values of association (−log10 scale) with breast cancer risk in <i>BRCA2</i> carriers for genotyped and imputed SNPs in the <i>NEIL2</i> gene.

<p>SNP rs1466785 is indicated with a purple arrow and the best causal imputed SNPs, rs804276 and rs804271 are indicated with a red arrow. Colors represent the pariwise r². Plot generated with LocusZoom <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004256#pgen.1004256-Pruim1" target="_blank">[42]</a> (<a href="http://csg.sph.umich.edu/locuszoom/" target="_blank">http://csg.sph.umich.edu/locuszoom/</a>).</p

Associations with breast and ovarian cancer risk for SNPs observed at p-trend<0.05 in stage II of the experiment.

a<p>Hazard Ratio per allele (1 df) estimated from the retrospective likelihood analysis.</p>b<p>Hazard Ratio under the genotype specific models (2df) estimated from the retrospective likelihood analysis.</p>c<p>p-values were based on the score test.</p>d<p>HR per allele of 1.69 and p-trend of 1×10⁻⁴ for <i>BRCA2</i> mutation carriers in stage I of the study.</p>e<p>HR per allele of 1.43 and p-trend of 0.01 for <i>BRCA1</i> mutation carriers in stage I of the study.</p>f<p>HR per allele of 1.30 and p-trend of 0.03 for <i>BRCA1</i> mutation carriers in stage I of the study.</p>g<p>HR per allele of 0.64 and p-trend of 0.057 for <i>BRCA2</i> mutation carriers in stage I of the study.</p>h<p>HR per allele of 1.25 and p-trend of 0.04 for <i>BRCA1</i> mutation carriers in stage I of the study.</p>i<p>HR per allele of 1.25 and p-trend of 0.058 for <i>BRCA2</i> mutation carriers in stage I of the study.</p>j<p>rs3093926 did not yield results under the genotype specific model due to the low minor allele frequency.</p><p>Complete description of results from stage I are included in Supplementary <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004256#pgen.1004256.s002" target="_blank">Table S1</a>.</p><p>Highlighted in bold are those SNPs showing strongest associations with breast or ovarian cancer risk (p<0.01).</p