A high-throughput functional complementation assay for classification of BRCA1 missense variants.

UNLABELLED Mutations in BRCA1 and BRCA2 account for the majority of hereditary breast and ovarian cancers, and therefore sequence analysis of both genes is routinely conducted in patients with early-onset breast cancer. Besides mutations that clearly abolish protein function or are known to increase cancer risk, a large number of sequence variants of uncertain significance (VUS) have been identified. Although several functional assays for BRCA1 VUSs have been described, thus far it has not been possible to conduct a high-throughput analysis in the context of the full-length protein. We have developed a relatively fast and easy cDNA-based functional assay to classify BRCA1 VUSs based on their ability to functionally complement BRCA1-deficient mouse embryonic stem cells. Using this assay, we have analyzed 74 unclassified BRCA1 missense mutants for which all predicted pathogenic variants are confined to the BRCA1 RING and BRCT domains. SIGNIFICANCE BRCA1 VUSs are frequently found in patients with hereditary breast or ovarian cancer and present a serious problem for clinical geneticists. This article describes the generation, validation, and application of a reliable high-throughput assay for the functional classification of BRCA1 sequence variants of uncertain significance.

[1]  Mo Li,et al.  Function of BRCA1 in the DNA damage response is mediated by ADP-ribosylation. , 2013, Cancer cell.

[2]  L. Tessarollo,et al.  Human BRCA1 gene rescues the embryonic lethality of Brca1 mutant mice , 2001, Genesis.

[3]  Suhwan Chang,et al.  Expression of human BRCA1 variants in mouse ES cells allows functional analysis of BRCA1 mutations. , 2009, The Journal of clinical investigation.

[4]  Laura S. Itzhaki,et al.  Toward Classification of BRCA1 Missense Variants Using a Biophysical Approach , 2010, The Journal of Biological Chemistry.

[5]  Junjie Chen,et al.  PALB2 is an integral component of the BRCA complex required for homologous recombination repair , 2009, Proceedings of the National Academy of Sciences.

[6]  Alan Ashworth,et al.  Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy , 2005, Nature.

[7]  F. Couch,et al.  Stable interaction between the products of the BRCA1 and BRCA2 tumor suppressor genes in mitotic and meiotic cells. , 1998, Molecular cell.

[8]  J. Jonkers,et al.  Mouse models of BRCA1 and BRCA2 deficiency: past lessons, current understanding and future prospects , 2006, Oncogene.

[9]  J. Glover,et al.  Comprehensive analysis of missense variations in the BRCT domain of BRCA1 by structural and functional assays. , 2010, Cancer research.

[10]  M. Vidal,et al.  Multimodal assessment of protein functional deficiency supports pathogenicity of BRCA1 p.V1688del. , 2009, Cancer research.

[11]  Peter Devilee,et al.  Comparative genomic hybridization profiles in human BRCA1 and BRCA2 breast tumors highlight differential sets of genomic aberrations. , 2005, Cancer research.

[12]  Jens C. Brüning,et al.  Single copy shRNA configuration for ubiquitous gene knockdown in mice , 2005, Nucleic acids research.

[13]  T. Katagiri,et al.  Genetic analysis of BRCA 1 ubiquitin ligase activity and its relationship to breast cancer susceptibility , 2006 .

[14]  Peter Bouwman,et al.  BRCA1 RING function is essential for tumor suppression but dispensable for therapy resistance. , 2011, Cancer cell.

[15]  A. Berns,et al.  p107 is a suppressor of retinoblastoma development in pRb-deficient mice. , 1998, Genes & development.

[16]  Sue Healey,et al.  ENIGMA—Evidence‐based network for the interpretation of germline mutant alleles: An international initiative to evaluate risk and clinical significance associated with sequence variation in BRCA1 and BRCA2 genes , 2012, Human mutation.

[17]  T. Ludwig,et al.  BRCA1 Tumor Suppression Depends on BRCT Phosphoprotein Binding, But Not Its E3 Ligase Activity , 2011, Science.

[18]  Thomas Helleday,et al.  Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase , 2007, Nature.

[19]  B. Karlan,et al.  Secondary BRCA1 mutations in BRCA1-mutated ovarian carcinomas with platinum resistance. , 2008, Cancer research.

[20]  D. Adams,et al.  53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers , 2010, Nature Structural &Molecular Biology.

[21]  Jean-Philippe Vert,et al.  Guidelines for splicing analysis in molecular diagnosis derived from a set of 327 combined in silico/in vitro studies on BRCA1 and BRCA2 variants , 2012, Human mutation.

[22]  Marcel J T Reinders,et al.  Molecular classification of breast carcinomas by comparative genomic hybridization: a specific somatic genetic profile for BRCA1 tumors. , 2002, Cancer research.

[23]  D. Adams,et al.  A High-Throughput Pharmaceutical Screen Identifies Compounds with Specific Toxicity against BRCA2-Deficient Tumors , 2009, Clinical Cancer Research.

[24]  J. Peterse,et al.  Somatic loss of BRCA1 and p53 in mice induces mammary tumors with features of human BRCA1-mutated basal-like breast cancer , 2007, Proceedings of the National Academy of Sciences.

[25]  Rochelle L. Garcia,et al.  Secondary somatic mutations restoring BRCA1/2 predict chemotherapy resistance in hereditary ovarian carcinomas. , 2011, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[26]  Qiang Yu,et al.  BRCA1-deficient mammary tumor cells are dependent on EZH2 expression and sensitive to Polycomb Repressive Complex 2-inhibitor 3-deazaneplanocin A , 2009, Breast Cancer Research.

[27]  Dominique Stoppa-Lyonnet,et al.  Evaluation of in silico splice tools for decision‐making in molecular diagnosis , 2008, Human mutation.

[28]  T. Katagiri,et al.  Genetic analysis of BRCA1 ubiquitin ligase activity and its relationship to breast cancer susceptibility. , 2006, Human molecular genetics.

[29]  A. Zharkikh,et al.  Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral , 2005, Journal of Medical Genetics.

[30]  David M. Livingston,et al.  The BRCA1/BARD1 Heterodimer Modulates Ran-Dependent Mitotic Spindle Assembly , 2006, Cell.

[31]  Thomas Helleday,et al.  Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase , 2005, Nature.

[32]  Chikashi Ishioka,et al.  Identification of breast tumor mutations in BRCA1 that abolish its function in homologous DNA recombination. , 2010, Cancer research.

[33]  L. Shulman,et al.  A Systematic Genetic Assessment of 1,433 Sequence Variants of Unknown Clinical Significance in the BRCA1 and BRCA2 Breast Cancer–Predisposition Genes , 2008 .

[34]  Hong Wu,et al.  A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2. , 2012, Cancer cell.

[35]  Etienne Rouleau,et al.  A guide for functional analysis of BRCA1 variants of uncertain significance , 2012, Human mutation.

[36]  A. Vincent-Salomon,et al.  High frequency of TP53 mutation in BRCA1 and sporadic basal-like carcinomas but not in BRCA1 luminal breast tumors. , 2009, Cancer research.

[37]  F. Couch,et al.  Biallelic deleterious BRCA1 mutations in a woman with early-onset ovarian cancer. , 2013, Cancer discovery.

[38]  M. Jasin,et al.  BRCA2 is required for homology-directed repair of chromosomal breaks. , 2001, Molecular cell.

[39]  P. Angrand,et al.  Improved properties of FLP recombinase evolved by cycling mutagenesis , 1998, Nature Biotechnology.

[40]  T. Ludwig,et al.  BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair. , 2012, Molecular cell.

[41]  Fred H. Gage,et al.  BRCA1 tumor suppression occurs via heterochromatin mediated silencing , 2011, Nature.

[42]  P. Nederlof,et al.  High incidence of protein-truncating TP53 mutations in BRCA1-related breast cancer. , 2009, Cancer research.

[43]  B. Henderson,et al.  The BRCA1 RING and BRCT Domains Cooperate in Targeting BRCA1 to Ionizing Radiation-induced Nuclear Foci* , 2005, Journal of Biological Chemistry.

[44]  Jeremy M. Stark,et al.  53BP1 Inhibits Homologous Recombination in Brca1-Deficient Cells by Blocking Resection of DNA Breaks , 2010, Cell.

[45]  Fergus J Couch,et al.  A review of a multifactorial probability‐based model for classification of BRCA1 and BRCA2 variants of uncertain significance (VUS) , 2012, Human mutation.

[46]  S. Sharan,et al.  Mouse embryonic stem cell–based functional assay to evaluate mutations in BRCA2 , 2008, Nature Medicine.

[47]  B. Koller,et al.  Brca1 controls homology-directed DNA repair. , 1999, Molecular cell.

[48]  L. Thompson,et al.  XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. , 1999, Genes & development.

[49]  F. Couch,et al.  BRCA1 R1699Q variant displaying ambiguous functional abrogation confers intermediate breast and ovarian cancer risk , 2012, Journal of Medical Genetics.

[50]  C. Deng,et al.  A selective requirement for 53BP1 in the biological response to genomic instability induced by Brca1 deficiency. , 2009, Molecular cell.