Article Figures & Data
Figures
- Figure 1.
Pedigrees of families with BRCA1 p.Val1736Ala. A, pedigree of the index family. Circles indicate females and squares indicate males. Slashes indicate death. The proband is indicated by an arrow. Shading in the left lower quadrant indicates ovarian cancer. Shading in the left top quadrant indicates unilateral breast cancer, and in both left and right top quadrants, bilateral breast cancer. Shading in the right bottom quadrant indicates cancer that is not breast or ovarian. Current ages or age at death and age at cancer diagnosis are listed below each individual, as is genetic status if known. B, a second representative pedigree with the BRCA1 p.Val1736Ala alteration. B1, BRCA1; B2, BRCA2; unk, unknown; dx, diagnosis.
- Figure 2.
Analysis of the BRCA1 p.Val1736Ala mutation. A, partial sequence alignment of a BRCA1 BRCT domain from different species showing that BRCA1 V1736 and P1749 residues (highlighted in red) are completely conserved across all vertebrate species. Numbers on top of the alignment indicate amino acid positions of the human BRCA1 protein. Conservation below describes sequence conservation (*, identical; :, ≥80% conservation; ., ≥60% conservation). B, modeling [based on protein data bank (pdb) code 1t15] of the interaction between the BRCA1 BRCT domains and a peptide of BACH1. BRCA1 is gray, with disease-causing mutants of the conserved residues in red. The BACH1 peptide is purple. C, WT BRCA1 (shown as a green focus) but not the p.Val1736Ala or p.Pro1749Arg mutant efficiently colocalized with mcherryLacIFokI fusion endonuclease-induced DNA DSBs. D, percentage of cells with BRCA1 (WT or mutant) colocalizing to FokI was quantified. At least 100 cells were assessed for each data point (n > 100). Measurements were obtained in triplicate and reported as means of 3 replicates. P values were calculated using Student t test; P < 0.05 for all comparisons. Error bars, SEM. E, Coimmunoprecipitation of epitope-tagged BRCA1 (Myc-BRCA1) WT or mutants from 293T cells at room temperature for 2 hours followed by immunoblot for RAP80.
- Figure 3.
Resequencing of BRCA1 c.2681_2682delAA mutation carrier. A, chromatogram showing the WT BRCA1 sequence in lymphocyte DNA from a noncarrier individual. B, chromatogram from lymphocyte DNA showing the heterozygous status of the Scottish woman previously reported as homozygous for the BRCA1 c.2681_2682delAA mutation. Both the mutant and WT alleles are clearly present.
Tables
- Table 1.
Loss of heterozygosity from the index family
Sample ID Description Germline BRCA1 Taqman result Interpretation S2504 Cell line WT/WT A/A No LOH S2366 Tumor (breast) WT/WT A/A No LOH Ego 1 Lymphoblasts WT/p.Val1736Ala A/G No LOH Ego 10 Lymphoblasts WT/p.Val1736Ala A/G No LOH Ego 1 Tumor (Breast) WT/p.Val1736Ala G/G LOH at WT Ego 9 Tumor (Ovarian) WT/p.Val1736Ala G/G LOH at WT Ego 28 Tumor (Ovarian) p.2576delC/p.Val1736Ala A/G No LOH NOTE: Ovarian/primary peritoneal cancer tumor blocks from Family A egos 1 and 9 showed that LOH had occurred at the wild-type BRCA1 allele with retention of the p.Val1736Ala allele. “A” at position 5207 represents the wild-type allele and “G” at position 5207 represents the Val1736Ala allele. Conversely, in ego 28, who carried germline BRCA1 2576delC and p.Val1736Ala alterations in trans, the ovarian tumor did not display LOH at either allele.
Abbreviation: LOH, loss of heterozygosity; WT, wild type.
Supplementary Data
Files in this Data Supplement:
- Supplementary Methods - Supplementary Methods PDF file 61K, This document describes the methods for all supplemental data
- Supplementary Figure 1 - Supplementary Figure 1 PDF file 108K, This document provides the raw data for V1736A genotyping used in LOH experiments
- Supplementary Figure 2 - Supplementary Figure 2 PDF file 78K, This figure depicts the assay used to visualize repair proteins at DNA double-strand breaks
- Supplementary Figure 3 - Supplementary Figure 3 PDF file 2065K, This figure provides functional data for BRCA1 V1736A and an example of tumor for LOH studies
- Supplementary Table 1 - Supplementary Table 1 PDF file 38K, This table summarizes all additional V1736A pedigrees
Volume 3, Issue 4
