BRCA1 C-terminal Domain Research Articles

Study on the Molecular Structure and Bio-Activity (DNA Polymerase Inhibitory Activity, Anti-Inflammatory Activity and Anti-Oxidant Activity) Relationship of Curcumin Derivatives

We found that phenolic compounds, petasiphenol and curcumin (diferuloylmethane), were selective inhibitors of DNA polymerase λ (pol λ) in vitro. In this review, we described the molecular structure and bio-activity (i.e., pol inhibitory activity, antiinflammatory activity and anti-oxidant activity) relationship of phenolic compounds such as petasiphenol, curcumin and 13 chemically synthesized derivatives of curcumin. The inhibitory effect on pol λ (full-length, i.e. intact pol λ including the BRCA1 C-terminus (BRCT) domain) by some derivatives was stronger than that by curcumin, and monoacetylcurcumin (compound (13)) was the strongest pol λ inhibitor of all the compounds tested, achieving 50% inhibition at a concentration of 3.9 μM. Compound (13) did not inhibit the activity of the C-terminal catalytic domain of pol λ including the pol β-like core, in which the proline-rich region and BRCT domain were deleted from its N-terminal region. These curcumin derivatives had anti-12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammatory activity with the same tendency as pol λ inhibitory activity, but compound (13) had no anti-oxidant activity (i.e., DPPH radical-scavenging activity). This compound did not influence the activities of replicative pols such as α, δ and η, and had no effect on repair-related pol activity such as pol β, although the three-dimensional structure of pol β is thought to be highly similar to that of pol λ. Based on these results, the pol λ-inhibitory mechanism of compound (13) and the structure-activity relationship of anti-inflammation are discussed. Keywords: Curcumin, Monoacetylcurcumin, DNA polymerase λ, DNA polymerase inhibitory activity, Anti-inflammatory activity, Antioxidant activity, Enzyme inhibitor, TPA (12-O-tetradecanoylphorbol-13-acetate)

BRCA1 Haploinsufficiency, but not Heterozygosity for a BRCA1-truncating Mutation, Deregulates Homologous Recombination

Humans heterozygous for BRCA1 mutations have a high risk of losing the remaining wild-type BRCA1 allele and developing breast/ovarian cancer, but a molecular basis for this has not yet been determined. It is thought that heterozygosity status — reduced wild-type BRCA1 protein dosage (haploinsufficiency) and/or the presence of a mutant BRCA1 protein — may affect BRCA1 functions and heighten the risk of cancer promoting mutations. BRCA1 maintains genome stability, at least in part, by regulating homologous recombination according to the type of DNA damage. To investigate whether this BRCA1 function is affected by heterozygosity status, we employed, as recombination reporters, human breast cancer MCF-7 cells known to have a single wild-type BRCA1 allele and reduced BRCA1 protein dosage. These cells revealed: 1) a spontaneous hyper-recombination phenotype; 2) reduced efficiency in homologous recombination repair of DNA double-strand breaks (DSBs); and 3) sensitivity to the DSB-inducing chemotherapeutic agent mitomycin C. Correction of BRCA1 protein dosage to the wild-type level reversed all these phenotypes, whereas physiological expression of the cancer-eliciting BRCA1 5382insC mutant allele had no effect on either phenotype. These findings implicate BRCA1 C-terminal domain in recombination control, and indicate that BRCA1 haploinsufficiency alone, which is also a feature of sporadic breast/ovarian cancer, is sufficient to compromise genome stability by triggering spontaneous recombination events that are likely to account for the loss of the remaining wild-type BRCA1 allele and increased cancer risk. Our observations may also have implications for the medical management of cancer patients and cancer prevention.

Light Chain 1 of Microtubule-associated Protein 1B Can Negatively Regulate the Action of Pes1

Pes1 was first identified as the locus affected in the zebrafish mutant pescadillo, which exhibits severe defects in gut and liver development. It has since been demonstrated that loss of Pes1 expression in mammals and yeast affects ribosome biogenesis, resulting in a block in cell proliferation. Pes1 contains a BRCA1 C-terminal domain, a structural motif that has been shown to facilitate protein-protein interactions, suggesting that Pes1 has binding partners. We used a yeast two-hybrid screen to identify putative interacting proteins. We found that light chain 1 of the microtubule-associated protein 1B (Mtap1b-LC1) could partner with Pes1, and deletion analyses revealed a specific interaction of Mtap1b-LC1 with the Pes1 BRCA1 C-terminal domain. We confirmed the integrity of the interaction between Pes1 and Mtap1b-LC1 by co-immunoprecipitation experiments. Protein localization studies in NIH3T3 cells revealed that exogenously expressed Pes1 was typically restricted to nuclei and nucleoli. However, exogenous Pes1 was found predominantly in the cytoplasm in cells that were forced to express Mtap1b-LC1. We also observed that the expression of endogenous Pes1 protein was significantly reduced or undetectable in nuclei when Mtap1b-LC1 was overexpressed, implying that a dynamic interaction exists between the two proteins and that Mtap1b-LC1 has the potential to negatively impact Pes1 function. Finally, we demonstrated that, as is the case when Pes1 expression is depleted by shRNA, overexpression of Mtap1b-LC1 resulted in diminished proliferation of NIH3T3 cells, suggesting that Mtap1b-LC1 has the potential to repress cell proliferation by modulating the nucleolar levels of Pes1.

The DNA topoisomerase IIβ binding protein 1 (TopBP1) interacts with poly (ADP‐ribose) polymerase (PARP‐1)

We investigated the physical association of the DNA topoisomerase IIbeta binding protein 1 (TopBP1), involved in DNA replication and repair but also in regulation of apoptosis, with poly(ADP-ribose) polymerase-1 (PARP-1). This enzyme plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. It was shown that the sixth BRCA1 C-terminal (BRCT) domain of TopBP1 interacts with a protein fragment of PARP-1 in vitro containing the DNA-binding and the automodification domains. More significantly, the in vivo interaction of endogenous TopBP1 and PARP-1 proteins could be shown in HeLa-S3 cells by co-immunoprecipitation. TopBP1 and PARP-1 are localized within overlapping regions in the nucleus of HeLa-S3 cells as shown by immunofluorescence. Exposure to UVB light slightly enhanced the interaction between both proteins. Furthermore, TopBP1 was detected in nuclear regions where poly(ADP-ribose) (PAR) synthesis takes place and is ADP-ribosylated by PARP-1. Finally, cellular (ADP-ribosyl)ating activity impairs binding of TopBP1 to Myc-interacting zinc finger protein-1 (Miz-1). The results indicate an influence of post-translational modifications of TopBP1 on its function during DNA repair.

Functional Impact of Missense Variants in BRCA1 Predicted by Supervised Learning

Many individuals tested for inherited cancer susceptibility at the BRCA1 gene locus are discovered to have variants of unknown clinical significance (UCVs). Most UCVs cause a single amino acid residue (missense) change in the BRCA1 protein. They can be biochemically assayed, but such evaluations are time-consuming and labor-intensive. Computational methods that classify and suggest explanations for UCV impact on protein function can complement functional tests. Here we describe a supervised learning approach to classification of BRCA1 UCVs. Using a novel combination of 16 predictive features, the algorithms were applied to retrospectively classify the impact of 36 BRCA1 C-terminal (BRCT) domain UCVs biochemically assayed to measure transactivation function and to blindly classify 54 documented UCVs. Majority vote of three supervised learning algorithms is in agreement with the assay for more than 94% of the UCVs. Two UCVs found deleterious by both the assay and the classifiers reveal a previously uncharacterized putative binding site. Clinicians may soon be able to use computational classifiers such as those described here to better inform patients. These classifiers can be adapted to other cancer susceptibility genes and systematically applied to prioritize the growing number of potential causative loci and variants found by large-scale disease association studies.

“Similarity Trap” in Protein-Protein Interactions Could Be Carcinogenic: Simulations of p53 Core Domain Complexed with 53BP1 and BRCA1 BRCT Domains

Similar binding sites often imply similar protein-protein interactions and similar functions; however, similar binding sites may also constitute traps for nonfunctional associations. How are similar sites distinguished to prevent misassociations? BRCT domains from breast cancer-susceptibility gene product BRCA1 and protein 53BP1 have similar structures yet different binding behaviors with p53 core domain. 53BP1-BRCT domain forms a stable complex with p53. In contrast, BRCA1-p53 interaction is weak or other mechanisms operate. To delineate the difference, we designed 13 BRCA1-BRCT mutants and computationally investigated the structural and stability changes compared to the experimental p53-53BP1 structure. Interestingly, of the 13, the 2 mutations that are cancerous and involve nonconserved residues are those that enforced p53 core domain binding with BRCA1-BRCT in a way similar to p53-53BP1 binding. Hence, falling into the "similarity trap" may disrupt normal BRCA1 and p53 functions. Our results illustrate how this trap is avoided in the native state.

Role of BRCT motif containing proteins in Chk1 activation

Chk1, along with Chk2, regulates processes such as DNA replication, cell cycle control, chromatin restructuring and apoptosis. DNA damage/replication stress activates Chk1 by phosphorylation from the PI3/PI4 family of kinases. Activation of Chk1 is thought to be mediated by proteins containing the BRCA1 C-terminal domain (BRCT). We previously identified a potential complex of four Chk1-associated proteins by immunoprecipitation, western blotting and mass spectrometry, one of which is BRCA1. Germline mutations in BRCA1 are responsible for many cases of hereditary breast cancer, and cells deficient in BRCA1 sustain spontaneous aberrations in chromosome structure. Such findings indicate that BRCA1 is essential for suppressing genome instability.

The tandem BRCT domain of 53BP1 is not required for its repair function.

53BP1 plays an important role in cellular response to DNA damage. It is thought to be the mammalian homologue of budding yeast Rad9 and/or fission yeast Crb2. Rad9/Crb2 are bona fide checkpoint proteins whose activation requires their corresponding C-terminal tandem BRCT (BRCA1 C-terminal) motifs, which mediate their oligomerization and phosphorylation at multiple sites following DNA damage. Here we show that the function of human 53BP1 similarly depends on its oligomerization and phosphorylation at multiple sites but in a BRCT domain-independent manner. Moreover, unlike its proposed yeast counterparts, human 53BP1 only has limited checkpoint functions but rather acts as an adaptor in the repair of DNA double strand breaks. This difference in function may reflect the higher complexity of the DNA damage response network in metazoa including the evolution of other BRCT domain-containing proteins that may have functions redundant or overlapping with those of 53BP1.

Role of Single-stranded DNA in Targeting REV1 to Primer Termini

Cellular functions of the REV1 gene have been conserved in evolution and appear important for maintaining genetic integrity through translesion DNA synthesis. This study documents a novel biochemical activity of human REV1 protein, due to higher affinity for single-stranded DNA (ssDNA) than the primer terminus. Preferential binding to long ssDNA regions of the template strand means that REV1 is targeted specifically to the included primer termini, a property not shared by other DNA polymerases, including human DNA polymerases alpha, beta, and eta. Furthermore, a mutant REV1 lacking N- and C-terminal domains, but catalytically active, lost this function, indicating that control is not due to the catalytic core. The novel activity of REV1 protein might imply a role for ssDNA in the regulation of translesion DNA synthesis.

REV1 Protein Interacts with PCNA: Significance of the REV1 BRCT Domain In Vitro and In Vivo

REV1 protein, a eukaryotic member of the Y family of DNA polymerases, is involved in the tolerance of DNA damage by translesion DNA synthesis. It is unclear how REV1 is recruited to replication foci in cells. Here, we report that mouse REV1 can bind directly to PCNA and that monoubiquitylation of PCNA enhances this interaction. The interaction between REV1 protein and PCNA requires a functional BRCT domain located near the N terminus of the former protein. Deletion or mutational inactivation of the BRCT domain abolishes the targeting of REV1 to replication foci in unirradiated cells, but not in UV-irradiated cells. In vivo studies in both chicken DT40 cells and yeast directly support the requirement of the BRCT domain of REV1 for cell survival and DNA damage-induced mutagenesis.

Up-regulation of the Fidelity of Human DNA Polymerase λ by Its Non-enzymatic Proline-rich Domain

DNA repair pathways are essential for maintaining genome stability. DNA polymerase beta plays a critical role in base-excision repair in vivo. DNA polymerase lambda, a recently identified X-family homolog of DNA polymerase beta, is hypothesized to be a second polymerase involved in base-excision repair. The full-length DNA polymerase lambda is comprised of three domains: a C-terminal DNA polymerase beta-like domain, an N-terminal BRCA1 C-terminal domain, and a previously uncharacterized proline-rich domain. Strikingly, pre-steady-state kinetic analyses reveal that, although human DNA polymerase lambda has almost identical fidelity to human DNA polymerase beta, the C-terminal DNA polymerase beta-like domain alone displays a dramatic, up to 100-fold loss in fidelity. We further demonstrate that the non-enzymatic proline-rich domain confers the increase in fidelity of DNA polymerase lambda by significantly lowering incorporation rate constants of incorrect nucleotides. Our studies illustrate a novel mechanism, in which the DNA polymerase fidelity is controlled not by an accessory protein or a proofreading exonuclease domain but by an internal regulatory domain.

The critical mutagenic translesion DNA polymerase Rev1 is highly expressed during G 2 /M phase rather than S phase

The Rev1 protein lies at the root of mutagenesis in eukaryotes. Together with DNA polymerase zeta (Rev3/7), Rev1 function is required for the active introduction of the majority of mutations into the genomes of eukaryotes from yeast to humans. Rev1 and polymerase zeta are error-prone translesion DNA polymerases, but Rev1's DNA polymerase catalytic activity is not essential for mutagenesis. Rather, Rev1 is thought to contribute to mutagenesis principally by engaging in crucial protein-protein interactions that regulate the access of translesion DNA polymerases to the primer terminus. This inference is based on the requirement of the N-terminal BRCT (BRCA1 C-terminal) domain of Saccharomyces cerevisiae Rev1 for mutagenesis and the interaction of the C-terminal region of mammalian Rev1 with several other translesion DNA polymerases. Here, we report that S. cerevisiae Rev1 is subject to pronounced cell cycle control in which the levels of Rev1 protein are approximately 50-fold higher in G(2) and throughout mitosis than during G(1) and much of S phase. Differential survival of a rev1Delta strain after UV irradiation at various points in the cell cycle indicates that this unanticipated regulation is physiologically relevant. This unexpected finding has important implications for the regulation of mutagenesis and challenges current models of error-prone lesion bypass as a process involving polymerase switching that operates mainly during S phase to rescue stalled replication forks.

Mapping and conformational characterization of the DNA-binding region of the breast cancer susceptibility protein BRCA1

The breast cancer susceptibility gene, BRCA1, encodes a large nuclear phosphoprotein, the major isoform of which is 1863 amino acids in size. Structure-function studies have been largely restricted to the only two domains identified by homology searches: the RING (really interesting new gene) and BRCT (BRCA1 C-terminus) domains. However, we have recently reported the identification of a large central soluble region of BRCA1 (residues 230-534) that binds specifically to four-way junction DNA, a property that potentially facilitates its role in the repair of DNA lesions by homologous recombination. We have now used a combination of limited proteolysis and extension cloning to identify more accurately the DNA-binding region of BRCA1. Limited trypsinolysis of BRCA1-(230-534) resulted in the production of a soluble domain identified as residues 230-339. However, after cloning, expression and purification of this region, studies revealed that it was unable to bind to four-way junctions, suggesting that the DNA-binding activity, in part, resides within residues 340-534. A series of fragments extending from residue 340 were produced, and each was tested for its ability to bind to four-way junction DNA in gel retardation assays. In these experiments, residues 340-554 of BRCA1 were identified as the minimal DNA-binding region. We then went on to characterize the conformation of this region using CD spectroscopy and analytical centrifugation.

5-(Hydroxymethyl)-2-furfural: A selective inhibitor of DNA polymerase λ and terminal deoxynucleotidyltransferase

5-(Hydroxymethyl)-2-furfural (HMF), a pyrolysate of carbohydrate isolated from instant coffee ( Coffea arabica L.), selectively inhibits the activities of mammalian DNA polymerase λ (pol λ) and terminal deoxynucleotidyltransferase (TdT) which are family X pols, in vitro. The compound influenced neither the activities of replicative DNA polymerases such as α, δ, and ε, nor even the activity of pol β which is from the same family and thought to have a very similar three-dimensional structure to the pol β-like region of pol λ. Since parts of HMF such as furan, furfuryl alcohol, and 2-furaldehyde did not influence the activities of any enzymes tested, the substituted form of furan with a hyroxymethyl group and a formyl group might be important for the inhibition of pol λ and TdT. The inhibitory effect of HMF on intact pol λ (i.e., residues 1–575), a truncated pol λ lacking the N-terminal BRCA1 C-terminus domain (133–575, del-1 pol λ) and another truncated pol λ lacking the N-terminal proline-rich region (245–575, del-2 pol λ) was dose-dependent, and 50% inhibition was observed at a concentration of 26.1, 10.3, and 4.6 μM, respectively. The IC 50 value of HMF for TdT was the same as that for del-2 pol λ (5.5 μM). The HMF-induced inhibition of both pol λ and TdT activities was competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, HMF was suggested to bind to the pol β-like region of pol λ and TdT.

MDC1 interacts with Rad51 and facilitates homologous recombination

Mediator of DNA damage checkpoint protein-1 (MDC1) is a recently identified nuclear protein that participates in DNA-damage sensing and signaling. Here we report that knockdown of MDC1 by RNA interference results in cellular hypersensitivity to the DNA cross-linking agent mitomycin C and ionizing radiation and in impaired homology-mediated repair of double-strand breaks in DNA. MDC1 forms a complex with Rad51 through a direct interaction with the forkhead-associated domain of MDC1, not the BRCA1 C-terminal domain. Depletion of MDC1 results in impaired formation of Rad51 ionizing radiation-induced foci, reduced amounts of nuclear and chromatin-bound Rad51, and a corresponding increase in Rad51 protein degradation. Together, our findings suggest that MDC1 functions in Rad51-mediated homologous recombination by retaining Rad51 in chromatin.

Inhibition of Aurora-B kinase activity by poly(ADP-ribosyl)ation in response to DNA damage

The cell cycle-regulated Aurora-B kinase is a chromosomal passenger protein that is implicated in fundamental mitotic events, including chromosome alignment and segregation and spindle checkpoint function. Aurora-B phosphorylates serine 10 of histone H3, a function that has been associated with mitotic chromatin condensation. We find that activation of poly(ADP-ribose) polymerase (PARP) 1 by DNA damage results in a rapid block of H3 phosphorylation. PARP-1 is a NAD(+)-dependent enzyme that plays a multifunctional role in DNA damage detection and repair and maintenance of genomic stability. Here, we show that Aurora-B physically and specifically associates with the BRCT (BRCA-1 C-terminal) domain of PARP-1. Aurora-B becomes highly poly(ADP-ribosyl)ated in response to DNA damage, a modification that leads to a striking inhibition of its kinase activity. The highly similar Aurora-A kinase is not regulated by PARP-1. We propose that the specific inhibition of Aurora-B kinase activity by PARP-1 contributes to the physiological response to DNA damage.

BRCA1 Interaction with Human Papillomavirus Oncoproteins

Previously, we reported that BRCA1 strongly represses the transcriptional activity of estrogen receptor-alpha (ER-alpha) in human breast and prostate cancer cells but only weakly inhibits ER-alpha in cervical cancer cells. We now report that introduction of the human papillomavirus E7 or E6 oncogenes into human papillomavirus-negative cells rescues the BRCA1 repression of ER-alpha activity and that the E7 and E6 oncoproteins interact directly with BRCA1 in vitro and associate with BRCA1 in vivo in cultured cells. This interaction involves at least two contact points on BRCA1, one within an N-terminal site shown previously to interact with ER-alpha and the other in a C-terminal region of BRCA1 containing the first BRCA1 C-terminal domain. Point mutations within the zinc finger domains of E7 and E6 inactivated the binding to the N terminus of BRCA1 and reduced their ability to rescue BRCA1 inhibition of ER-alpha. E6 and E7 also antagonized the ability of BRCA1 to inhibit c-Myc E-box-mediated transactivation and human telomerase reverse transcriptase promoter activity, in a manner dependent upon the zinc finger domains. Finally, the ability of E6 and E7 to antagonize BRCA1 did not involve proteolytic degradation of BRCA1. These findings suggest functional interactions of BRCA1 with E7 and E6. The potential significance of these findings is discussed.

Structural Basis for Cell Cycle Checkpoint Control by the BRCA1−CtIP Complex,

The breast and ovarian tumor suppressor BRCA1 has important functions in cell cycle checkpoint control and DNA repair. Two tandem BRCA1 C-terminal (BRCT) domains are essential for the tumor suppression activity of BRCA1 and interact in a phosphorylation-dependent manner with proteins involved in DNA damage-induced checkpoint control, including the DNA helicase BACH1 and the CtBP-interacting protein (CtIP). The crystal structure of the BRCA1 BRCT repeats bound to the PTRVSpSPVFGAT phosphopeptide corresponding to residues 322-333 of human CtIP was determined at 2.5 A resolution. The peptide binds to a cleft formed by the interface of the two BRCTs in a two-pronged manner, with phospho-Ser327 and Phe330 anchoring the peptide through extensive contacts with BRCA1 residues. Several hydrogen bonds and salt bridges that stabilize the BRCA1-BACH1 complex are missing in the BRCA1-CtIP interaction, offering a structural basis for the approximately 5-fold lower affinity of BRCA1 for CtIP compared to that of BACH1, as determined by isothermal titration calorimetry. Importantly, the side chain of Arg1775 in the cancer-associated BRCA1 mutation M1775R sterically clashes with the phenyl ring of CtIP Phe330, disrupting the BRCA1-CtIP interaction. These results provide new insights into the molecular mechanisms underlying the dynamic selection of target proteins involved in DNA repair and cell cycle control by BRCA1 and reveal how certain cancer-associated mutations affect these interactions.

Boundaries and physical characterization of a new domain shared between mammalian 53BP1 and yeast Rad9 checkpoint proteins

Eukaryotic cells have evolved DNA damage checkpoints in response to genome damage. They delay the cell cycle and activate repair mechanisms. The kinases at the heart of these pathways and the accessory proteins, which localize to DNA lesions and regulate kinase activation, are conserved from yeast to mammals. For Saccharomyces cerevisiae Rad9, a key adaptor protein in DNA damage checkpoint pathways, no clear human ortholog has yet been described in mammals. Rad9, however, shares localized homology with both human BRCA1 and 53BP1 since they all contain tandem C-terminal BRCT (BRCA1 C-terminal) motifs. 53BP1 is also a key mediator in DNA damage signaling required for cell cycle arrest, which has just been reported to possess a tandem Tudor repeat upstream of the BRCT motifs. Here we show that the major globular domain upstream of yeast Rad9 BRCT domains is structurally extremely similar to the Tudor domains recently resolved for 53BP1 and SMN. By expressing several fragments encompassing the Tudor-related motif and characterizing them using various physical methods, we isolated the independently folded unit for yeast Rad9. As in 53BP1, the domain corresponds to the SMN Tudor motif plus the contiguous HCA predicted structure region at the C terminus. These domains may help to further elucidate the structural and functional features of these two proteins and improve knowledge of the proteins involved in DNA damage.

A Multi-Exonic BRCA1 Deletion Identified in Multiple Families through Single Nucleotide Polymorphism Haplotype Pair Analysis and Gene Amplification with Widely Dispersed Primer Sets

The identification of intragenic rearrangements is important for a comprehensive understanding of mutations that occur in some clinically important genes. Single nucleotide polymorphism haplotypes obtained from clinical sequence data have been used to identify patients at high risk for rearrangement mutations. Application of this method identified a novel 26-kb deletion of BRCA1 exons 14 through 20 in patients from multiple families with hereditary breast and ovarian cancer. Clinical sequence data from 5911 anonymous patients were screened for genotypes that were inconsistent with known pairs of canonical haplotypes in BRCA1 that could be explained by hemizygous deletions involving exon 16. Long-range polymerase chain reaction demonstrated that two of six samples identified by this search contained a deletion in the expected region encompassing exons 14 through 20. The breakpoint was fully characterized by DNA sequencing and demonstrated that the deletion resulted from Alu-mediated recombination. This mutation was also identified twice in a set of 982 anonymous specimens that had negative clinical test results, but uninformative haplotypes. Three additional occurrences of this mutation were found by testing 10 other patients with the indicative genotype. An assay for this mutation was added to a comprehensive clinical breast/ovarian cancer test and eight more instances were found in 20,649 probands. This multiexon deletion has therefore been detected in 15 different North American families with hereditary breast/ovarian cancer. In conclusion, this primarily computational approach is highly effective and identifies specimens using existing data that are enriched for deletion mutations.