Figure 1
Analysis of the large mutations in BARD1, conducted with the use of a homemade MLPA assay. (a) A schematic map of the BARD1 gene and the flanking genomic regions, with the positions and IDs of the MLPA probes indicated. The exons are presented as vertical rectangles with proportional size and spacing based on the NM_000465 BARD1 sequence (reverse complement) retrieved from the UCSC Genome Browser (human genome reference sequence Mar 2006 NCBI36/hg18 assembly). The upper and lower rectangles correspond to the protein coding and untranslated sequences, respectively. In panels (b–e) there are representative results of the following control samples and samples with different point mutations (from the top): (i) the representative negative result without any mutations; (ii) the positive control sample with duplication of 2q34-37 in which BARD1 is located; (iii) artificial positive control sample, composed of 1:1 mixture of HindIII digested and undigested genomic DNA sample; (iv) artificial positive control sample, generated by masking the target sequences of BARD1_e06 and BARD1_e07 probes with probe-specific masking-oligonucleotides; (v) samples #53 and #4031 with the mutation c.1690C >T in exon 8; (vi) samples #4163 and #4349 with the mutation c.1972C >T in exon 10 and (vii) samples #4062, #4217 and #4321 with the mutation c.1977A >G in exon 10. (b) The MLPA electropherograms of the representative MLPA results. The probe IDs are shown under the electropherograms. An arrowhead indicates a reduced signal of the MLPA probe. (c) The bar plots (corresponding to the electropherograms shown in panel b) representing the normalized copy number value (y-axis) of each probe (x-axis). The gray bar plot (above) indicates standard deviation values (SD; ranged between 0.066 and 0.086) of test MLPA probes, calculated based on signal variation of particular probes in each analyzed sample (except the samples with mutation). (d) Sequencing results of the exons showing a reduced signal in the MLPA analysis. (e) The target sequences of the affected probes. A schematic representation of the MLPA probe is shown above (for details see37,41). The 5’- and 3’-target sequences are indicated in yellow and green, respectively. The positions of HindIII sites, the masking-oligonucleotides and corresponding mutations are indicated in red.
