Additional file 1 of Performance of in silico prediction tools for the classification of rare BRCA1/2 missense variants in clinical diagnostics

Table S1. List of all variants (n=236) from the Classified Variant Set, including reference, functional impact and number of families affected within the cohort of patients from the German Consortium of Hereditary Breast and Ovarian Cancer (as of September 2016). (XLSX 28 kb

Additional file 3 of Performance of in silico prediction tools for the classification of rare BRCA1/2 missense variants in clinical diagnostics

Table S2. The table lists previous studies investigating the performance of Align-GVGD, SIFT, MutationTaster or PolyPhen-2, the characteristics of the data sets utilized, and the observed values. (PDF 79 kb

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

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

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> 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

Ocular phenotypic characteristics of patient IV-7.

<p>Spectral domain optic coherence tomography (SD-OCT) of the left eye shows a normal foveal contour (A).Fundus photographies show discreet palor of the optic nerve (<b>B</b>) and irregular pigmentation in the macular area (<b>C</b>). Ocular phenotypic characteristics of patient IV-8: Spectral domain optic coherence tomography (SD-OCT) of the left eye shows a normal foveal contour (<b>D</b>). Fundus photographies show discreet palor of the optic nerve (<b>E</b>) and irregular pigmentation in the macular area especially in the right eye (<b>F</b>). Ocular phenotypic characteristics of patient III-5: Fundus fluorescence and indocyanine green angiography of the left eye show no leakage (<b>G</b>, <b>H</b>). Fundus photographies show myopic changes: posterior staphyloma, lacquer cracks, Fuchs’ spot of the macula and chorioretinal and parapapillary atrophy (<b>I</b>). Electrophysiologic examination (<b>J</b>): Full-field ERG shows normal scotopic and photopic answers in the female carrier (III-6). In the school boy (IV-7) ERG showed slightly reduced scotopic a- and b-waves with photopic answers below noise level. In the two adults (III-5, II-8), scotopic a- and b-waves were severely reduced with non-recordable photopic ERG.</p

Graphical representation of <i>CACNA1F</i> NGS coverage (exons 14 to 35) for individual III-6 (conductor) versus pooled DNA samples derived from affected males.

<p>The genomic structure of <i>CACNA1F</i> (exons 14-35) is shown below. NGS coverage data suggest reduced signals of the <i>CACNA1F</i> exons 18-26 for individual III-6 while signals for those exons are absent in the pooled DNA sample (<b>A</b>). Analysis of genomic DNA shows junction fragment PCR-products only in patients and carriers but not in controls (<b>B</b>). Exons 18-26 were absent in all patients tested. NTC = no template control. Sequencing of the junction fragment product revealed breakpoints within two AluSx repeats located in intron 17 and 26 (<b>C</b>).</p

Pedigree of the family compatible with an x-linked recessive mode of inheritance.

<p>Four of the 10 affected males (IV-7, IV-8, III-5 and II-8) and two female carriers (III-6 and III-9) have been examined.</p

Analysis of cDNA derived from EBV-transformed lymphoblastoid cell lines (LCLs) of individuals IV-7 (affected), III-6 (female carrier) and II-5 (affected) shows expression of the truncated transcript in all samples while the WT-transcript was present only in the female carrier and the positive control (Human Retina Marathon-Ready™ cDNA).

<p>NTC = no template control.</p