RT-PCR results for <i>BRCA1</i> c.4484G>C(p.Arg1495Thr) and <i>BRCA2:</i>c.7828G>A (p.Val2610Met).

<p><i>M</i> - 100bp DNA marker (New England Biolabs). A) <i>BRCA2</i>:c.7828G>A (p.Val2610Met). Lane 1: RT-PCR products from variant carrier derived cycloheximide treated LCL. Lane 2–7: Cycloheximide treated LCLs from unaffected female controls. There is no evidence for a predicted loss of 149bp from exon 17 as a result of a <i>de novo</i> donor site. The Δexon 18 (540bp) and Δexon 17/18 (369bp) are detected in the variant carrier and all but one control samples. B) <i>BRCA1</i> c.4484G>C(p.Arg1495Thr). Lane 1: RT-PCR products from whole blood derived RNA from the variant carrier showing the Δexon 14 and Δexon 14/15 splicing aberration. Lane 2: RT-PCR carried out on whole blood derived RNA from an unaffected female control (collection and extraction methods as per the variant carrier). Lane 3–7: Cycloheximide treated LCLs from unaffected female controls.</p

BRCA1 RING-domain variants with reported loss of function on the basis of <i>in-vitro</i> functional assays and/or (likely) clinically significant from multifactorial likelihood analysis.

*<p>p.Met18Thr, p.Cys61Gly and p.Cys64Gly are also shown to have abrogated function using mouse embryonic stem cell assays <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086836#pone.0086836-Bouwman1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086836#pone.0086836-Chang1" target="_blank">[49]</a>. (No other variants listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086836#pone-0086836-t003" target="_blank">Table 3</a> were assayed using this method).</p

Classification of <i>BRCA1</i> and <i>BRCA2</i> variants on the basis of multifactorial and splicing information.

<p>Classifications for multifactorial likelihood as described in Plon et al. (3) and splicing as described in Spurdle et al. (32). Frequency data from 1000 Genomes and EVS datasets is available for a subset of the variants studied (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086836#pone.0086836.s002" target="_blank">Table S2</a>). Information used to determine tumor pathology LRs was as follows: <i>BRCA1</i>c.122A>G(p.His41Arg) - one ER-positive Grade 3 tumor; <i>BRCA2</i> variants c.440A>G (p.Gln147Arg) and c.1514T>C (p.Ile505Thr) - tubule formation present in <10% of tumor; <i>BRCA2</i>:c.5278T>G (p.Ser1760Ala) – tubule formation in >75% tumor.</p

Bioinformatic splice prediction scores^* and <i>in-vitro</i> splicing assay results.

*<p>Bracketed percentages refer to the difference between variant and wild-type scores as a proportion of the wild-type score. NSC, no sites created (no scores provided by bioinformatic program output). Positive values for HSF matrices and MaxEntScan represent an increased likelihood of creating a <i>de novo</i> site when compared with the wild-type sequence where the variant occurs. Negative values represent a decreased likelihood. Positive values for ESEfinder represent an increase in strength for the enhancer motif as a result of the variant. The proximal consensus site is taken as the donor or acceptor site of the exon in which the variant occurs. Variant scores for NNsplice are for splice sites created by the variant, except for <i>BRCA1</i>:c.4484G>C (p.Arg1495Thr) for which the variant score is for the consensus splice junction in the presence of the variant.</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