Fig 2: PALB2 mutations in the affected family.Electropherograms of PALB2 mutations in DNA in the affected siblings. A) Top panel, c.1676_1677delAAinsG in genomic DNA; lower panel, wt sequence at that position. B) Top panel mutant c.2586+1G>A in genomic DNA; lower panel, wt sequence at that position. C) cDNA sequencing showing that the c.2586+1G>A mutation causes exon 6 to be omitted during RNA splicing. D) Diagramatic representation of the PALB2 protein and predicted effects of mutations. Diagram of PALB2 showing the epitopes to which the two anti-PALB2 antibodies bind and also the locations of the mutations in the patients. Note that because the epitope for the PALB2 antibody 246A is in exon 4, the exon revertant protein (Exon4delRev) deletion mutant cannot be detected with this antibody. Similarly, because the Y551Ter and Q559RfsTer2 mutants truncate at residues 551 and 559 respectively, they do not possess the epitopes for the 247A antibody. Only wild type and T839_K862del exon six deletion PALB2 are detectable with both antibodies.

Fig 3: Co-immunoprecipitation of BRCA2 with mutant PALB2 proteins.A) Cultures of U2OS cell lines that inducibly express FLAG-tagged WT or mutant PALB2 proteins were transfected with PALB2 siRNA and induced to express the tagged PALB2 proteins by treatment with doxycycline. Samples were incubated with anti-FLAG antibody and immunoprecipitates electrophoresed and blotted serially with mouse anti-BRCA2 (top panel), rabbit anti-PALB2 246A antibody (middle panel) and then rabbit anti-PALB2 247A antibody (bottom panel). 246A antibody was not stripped before blotting with 247A. A band approximating to 460kDa and detected with anti-BRCA2 antibody was immunoprecipitated with FLAG-tagged WT, T839_K862del (exon 6 deleted) and PALB2 revertant (Exon4delRev) proteins, in those samples that were incubated with anti-FLAG antibody. Anti-PALB2 antibodies identified FLAG-tagged WT protein at ~170kDa as expected (lane 1), with the exon 6 deleted protein appearing to be slightly smaller (lane 2). The FLAG-tagged Q559RfsTer2 and Y551Ter proteins were found at their expected sizes (middle panel). The FLAG-tagged PALB2 revertant protein (Exon4delRev) was evident with Ab 247A in bottom panel. B) Samples of the NETN lysates were taken as ‘input’ samples before immunoprecipitation, and 5% of total lysate immunoprecipitated was subjected to PAGE and Western blotting. The filters were blotted serially with different antibodies as in A. These blots show BRCA2 in all samples before immunoprecipitation. Anti-PALB2 antibodies identify FLAG-tagged PALB2 protein in all WT and mutant expressing cell lines. The aprataxin protein indicates the similarity of loading.

Fig 4: Analysis of expression of PALB2 protein in a family with previously unrecognised FA-N patients.A) Western blot showing loss of full length PALB2 in the affected patient II-5 (Ab 301-246A)(lane 3). However, there is the presence of a faint signal for a slightly smaller protein, probably corresponding to the exon six deletion mutation. Also present in the patient lane is a much smaller band at 90kDa. Lane 4 is lysate of cells from a FA-D1 (biallelic mutation of BRCA2) patient. B) All relatives of the patient II-5 show bands for WT PALB2. Also found in the lanes for the mother (I-2) and sibling II-3, (lanes 3 and 7) is the smaller dense 90kD protein band consistent with I-2 and II-3 carrying the c.1676_1677delAAinsG mutation. In addition, a less dense band of the same size, 90kD, appears as a consequence of the antibody cross reacting with another protein. This cross reacting band is visible in the normal control (both left and right), I-1, II-7 and II-6. The father carries the c.2586+1G>A, although the exon 6 deleted protein cannot be seen against the background of the normal full length protein from his WT allele.

Fig 5: Suggested model of the mutant PALB2 WD40-repeat.A) Amino acid sequence of the region immediately upstream of the PALB2 WD40-repeat. The in-frame deletion, created by the skipping of exon 6, is indicated by the black box. The position of beta-strand 7D and the upstream region of helical propensity are indicated by the magenta arrow and yellow box respectively. The amino acids visible in the X-ray crystal structure of the wild-type PALB2 WD40-repeat (PDB: 2W18) are also indicated. B) In the wild-type protein Leu861 of beta-strand 7D sits in a small hydrophobic pocket lined by the indicated amino acids. C) Molecular cartoons showing the N-terminal part of the WD40-repeat (cyan). (Left) the labelled amino acids (coloured asterisks), on one face of the upstream helical element (yellow), resemble structurally and spatially, those of the 7D beta-strand (Right, magenta).