Identification of different classes of genome instability suppressor genes through analysis of DNA damage response markers.

Cellular pathways that detect DNA damage are useful for identifying genes that suppress DNA damage, which can cause genome instability and cancer predisposition syndromes when mutated. We identified 199 high-confidence and 530 low-confidence DNA damage-suppressing (DDS) genes in Saccharomyces cerevisiae through a whole-genome screen for mutations inducing Hug1 expression, a focused screen for mutations inducing Ddc2 foci, and data from previous screens for mutations causing Rad52 foci accumulation and Rnr3 induction. We also identified 286 high-confidence and 394 low-confidence diverse genome instability-suppressing (DGIS) genes through a whole-genome screen for mutations resulting in increased gross chromosomal rearrangements and data from previous screens for mutations causing increased genome instability as assessed in a diversity of genome instability assays. Genes that suppress both pathways (DDS+ DGIS+) prevent or repair DNA replication damage and likely include genes preventing collisions between the replication and transcription machineries. DDS+ DGIS- genes, including many transcription-related genes, likely suppress damage that is normally repaired properly or prevent inappropriate signaling, whereas DDS- DGIS+ genes, like PIF1, do not suppress damage but likely promote its proper, nonmutagenic repair. Thus, induction of DNA damage markers is not a reliable indicator of increased genome instability, and the DDS and DGIS categories define mechanistically distinct groups of genes.

The discovery of NLRP3 and its function in cryopyrin-associated periodic syndromes and innate immunity.

From studies of individual families to global collaborative efforts, the NLRP3 inflammasome is now recognized to be a key regulator of innate immunity. Activated by a panoply of pathogen-associated and endogenous triggers, NLRP3 serves as an intracellular sensor that drives carefully coordinated assembly of the inflammasome, and downstream inflammation mediated by IL-1 and IL-18. Initially discovered as the cause of the autoinflammatory spectrum of cryopyrin-associated periodic syndrome (CAPS), NLRP3 is now also known to play a role in more common diseases including cardiovascular disease, gout, and liver disease. We have seen cohesion in results from clinical studies in CAPS patients, ex vivo studies of human cells and murine cells, and in vivo murine models leading to our understanding of the downstream pathways, cytokine secretion, and cell death pathways that has solidified the role of autoinflammation in the pathogenesis of human disease. Recent advances in our understanding of the structure of the inflammasome have provided ways for us to visualize normal and mutant protein function and pharmacologic inhibition. The subsequent development of targeted therapies successfully used in the treatment of patients with CAPS completes the bench to bedside translational loop which has defined the study of this unique protein.

Insights into DNA cleavage by MutL homologs from analysis of conserved motifs in eukaryotic Mlh1.

MutL family proteins contain an N-terminal ATPase domain (NTD), an unstructured interdomain linker, and a C-terminal domain (CTD), which mediates constitutive dimerization between subunits and often contains an endonuclease active site. Most MutL homologs direct strand-specific DNA mismatch repair by cleaving the error-containing daughter DNA strand. The strand cleavage reaction is poorly understood; however, the structure of the endonuclease active site is consistent with a two- or three-metal ion cleavage mechanism. A motif required for this endonuclease activity is present in the unstructured linker of Mlh1 and is conserved in all eukaryotic Mlh1 proteins, except those from metamonads, which also lack the almost absolutely conserved Mlh1 C-terminal phenylalanine-glutamate-arginine-cysteine (FERC) sequence. We hypothesize that the cysteine in the FERC sequence is autoinhibitory, as it sequesters the active site. We further hypothesize that the evolutionary co-occurrence of the conserved linker motif with the FERC sequence indicates a functional interaction, possibly by linker motif-mediated displacement of the inhibitory cysteine. This role is consistent with available data for interactions between the linker motif with DNA and the CTDs in the vicinity of the active site.

Variant STAT4 and Response to Ruxolitinib in an Autoinflammatory Syndrome.

BACKGROUND: Disabling pansclerotic morphea (DPM) is a rare systemic inflammatory disorder, characterized by poor wound healing, fibrosis, cytopenias, hypogammaglobulinemia, and squamous-cell carcinoma. The cause is unknown, and mortality is high. METHODS: We evaluated four patients from three unrelated families with an autosomal dominant pattern of inheritance of DPM. Genomic sequencing independently identified three heterozygous variants in a specific region of the gene that encodes signal transducer and activator of transcription 4 (STAT4). Primary skin fibroblast and cell-line assays were used to define the functional nature of the genetic defect. We also assayed gene expression using single-cell RNA sequencing of peripheral-blood mononuclear cells to identify inflammatory pathways that may be affected in DPM and that may respond to therapy. RESULTS: Genome sequencing revealed three novel heterozygous missense gain-of-function variants in STAT4. In vitro, primary skin fibroblasts showed enhanced interleukin-6 secretion, with impaired wound healing, contraction of the collagen matrix, and matrix secretion. Inhibition of Janus kinase (JAK)-STAT signaling with ruxolitinib led to improvement in the hyperinflammatory fibroblast phenotype in vitro and resolution of inflammatory markers and clinical symptoms in treated patients, without adverse effects. Single-cell RNA sequencing revealed expression patterns consistent with an immunodysregulatory phenotype that were appropriately modified through JAK inhibition. CONCLUSIONS: Gain-of-function variants in STAT4 caused DPM in the families that we studied. The JAK inhibitor ruxolitinib attenuated the dermatologic and inflammatory phenotype in vitro and in the affected family members. (Funded by the American Academy of Allergy, Asthma, and Immunology Foundation and others.).

The unstructured linker of Mlh1 contains a motif required for endonuclease function which is mutated in cancers.

Eukaryotic DNA mismatch repair (MMR) depends on recruitment of the Mlh1-Pms1 endonuclease (human MLH1-PMS2) to mispaired DNA. Both Mlh1 and Pms1 contain a long unstructured linker that connects the N- and carboxyl-terminal domains. Here, we demonstrated the Mlh1 linker contains a conserved motif (Saccharomyces cerevisiae residues 391-415) required for MMR. The Mlh1-R401A,D403A-Pms1 linker motif mutant protein was defective for MMR and endonuclease activity in vitro, even though the conserved motif could be >750 Å from the carboxyl-terminal endonuclease active site or the N-terminal adenosine triphosphate (ATP)-binding site. Peptides encoding this motif inhibited wild-type Mlh1-Pms1 endonuclease activity. The motif functioned in vivo at different sites within the Mlh1 linker and within the Pms1 linker. Motif mutations in human cancers caused a loss-of-function phenotype when modeled in S. cerevisiae. These results suggest that the Mlh1 motif promotes the PCNA-activated endonuclease activity of Mlh1-Pms1 via interactions with DNA, PCNA, RFC, or other domains of the Mlh1-Pms1 complex.

Mlh1 interacts with both Msh2 and Msh6 for recruitment during mismatch repair.

Eukaryotic DNA mismatch repair (MMR) initiates through mispair recognition by the MutS homologs Msh2-Msh6 and Msh2-Msh3 and subsequent recruitment of the MutL homologs Mlh1-Pms1 (human MLH1-PMS2). In bacteria, MutL is recruited by interactions with the connector domain of one MutS subunit and the ATPase and core domains of the other MutS subunit. Analysis of the S. cerevisiae and human homologs have only identified an interaction between the Msh2 connector domain and Mlh1. Here we investigated whether a conserved Msh6 ATPase/core domain-Mlh1 interaction and an Msh2-Msh6 interaction with Pms1 also act in MMR. Mutations in MLH1 affecting interactions with both the Msh2 and Msh6 interfaces caused MMR defects, whereas equivalent pms1 mutations did not cause MMR defects. Mutant Mlh1-Pms1 complexes containing Mlh1 amino acid substitutions were defective for recruitment to mispaired DNA by Msh2-Msh6, did not support MMR in reconstituted Mlh1-Pms1-dependent MMR reactions in vitro, but were proficient in Msh2-Msh6-independent Mlh1-Pms1 endonuclease activity. These results indicate that Mlh1, the common subunit of the Mlh1-Pms1, Mlh1-Mlh2, and Mlh1-Mlh3 complexes, but not Pms1, is recruited by Msh2-Msh6 through interactions with both of its subunits.

Rad5 and Its Human Homologs, HLTF and SHPRH, Are Novel Interactors of Mismatch Repair.

DNA mismatch repair (MMR) repairs replication errors, and MMR defects play a role in both inherited cancer predisposition syndromes and in sporadic cancers. MMR also recognizes mispairs caused by environmental and chemotherapeutic agents; however, in these cases mispair recognition leads to apoptosis and not repair. Although mutation avoidance by MMR is fairly well understood, MMR-associated proteins are still being identified. We performed a bioinformatic analysis that implicated Saccharomyces cerevisiae Rad5 as a candidate for interacting with the MMR proteins Msh2 and Mlh1. Rad5 is a DNA helicase and E3 ubiquitin ligase involved in post-replicative repair and damage tolerance. We confirmed both interactions and found that the Mlh1 interaction is mediated by a conserved Mlh1-interacting motif (MIP box). Despite this, we did not find a clear role for Rad5 in the canonical MMR mutation avoidance pathway. The interaction of Rad5 with Msh2 and Mlh1 is conserved in humans, although each of the Rad5 human homologs, HLTF and SHPRH, shared only one of the interactions: HLTF interacts with MSH2, and SHPRH interacts with MLH1. Moreover, depletion of SHPRH, but not HLTF, results in a mild increase in resistance to alkylating agents although not as strong as loss of MMR, suggesting gene duplication led to specialization of the MMR-protein associated roles of the human Rad5 homologs. These results provide insights into how MMR accessory factors involved in the MMR-dependent apoptotic response interact with the core MMR machinery and have important health implications into how human cells respond to environmental toxins, tumor development, and treatment choices of tumors with defects in Rad5 homologs.

Rad27 and Exo1 function in different excision pathways for mismatch repair in Saccharomyces cerevisiae.

Eukaryotic DNA Mismatch Repair (MMR) involves redundant exonuclease 1 (Exo1)-dependent and Exo1-independent pathways, of which the Exo1-independent pathway(s) is not well understood. The exo1Δ440-702 mutation, which deletes the MutS Homolog 2 (Msh2) and MutL Homolog 1 (Mlh1) interacting peptides (SHIP and MIP boxes, respectively), eliminates the Exo1 MMR functions but is not lethal in combination with rad27Δ mutations. Analyzing the effect of different combinations of the exo1Δ440-702 mutation, a rad27Δ mutation and the pms1-A99V mutation, which inactivates an Exo1-independent MMR pathway, demonstrated that each of these mutations inactivates a different MMR pathway. Furthermore, it was possible to reconstitute a Rad27- and Msh2-Msh6-dependent MMR reaction in vitro using a mispaired DNA substrate and other MMR proteins. Our results demonstrate Rad27 defines an Exo1-independent eukaryotic MMR pathway that is redundant with at least two other MMR pathways.

Ligation of newly replicated DNA controls the timing of DNA mismatch repair.

Mismatch repair (MMR) safeguards genome stability through recognition and excision of DNA replication errors.1-4 How eukaryotic MMR targets the newly replicated strand in vivo has not been established. MMR reactions reconstituted in vitro are directed to the strand containing a preexisting nick or gap,5-8 suggesting that strand discontinuities could act as discrimination signals. Another candidate is the proliferating cell nuclear antigen (PCNA) that is loaded at replication forks and is required for the activation of Mlh1-Pms1 endonuclease.7-9 Here, we discovered that overexpression of DNA ligase I (Cdc9) in Saccharomyces cerevisiae causes elevated mutation rates and increased chromatin-bound PCNA levels and accumulation of Pms1 foci that are MMR intermediates, suggesting that premature ligation of replication-associated nicks interferes with MMR. We showed that yeast Pms1 expression is mainly restricted to S phase, in agreement with the temporal coupling between MMR and DNA replication.10 Restricting Pms1 expression to the G2/M phase caused a mutator phenotype that was exacerbated in the absence of the exonuclease Exo1. This mutator phenotype was largely suppressed by increasing the lifetime of replication-associated DNA nicks, either by reducing or delaying Cdc9 ligase activity in vivo. Therefore, Cdc9 dictates a window of time for MMR determined by transient DNA nicks that direct the Mlh1-Pms1 in a strand-specific manner. Because DNA nicks occur on both newly synthesized leading and lagging strands,11 these results establish a general mechanism for targeting MMR to the newly synthesized DNA, thus preventing the accumulation of mutations that underlie the development of human cancer.

Immunodeficiency and bone marrow failure with mosaic and germline TLR8 gain of function.

Inborn errors of immunity (IEI) are a genetically heterogeneous group of disorders with a broad clinical spectrum. Identification of molecular and functional bases of these disorders is important for diagnosis, treatment, and an understanding of the human immune response. We identified 6 unrelated males with neutropenia, infections, lymphoproliferation, humoral immune defects, and in some cases bone marrow failure associated with 3 different variants in the X-linked gene TLR8, encoding the endosomal Toll-like receptor 8 (TLR8). Interestingly, 5 patients had somatic variants in TLR8 with <30% mosaicism, suggesting a dominant mechanism responsible for the clinical phenotype. Mosaicism was also detected in skin-derived fibroblasts in 3 patients, demonstrating that mutations were not limited to the hematopoietic compartment. All patients had refractory chronic neutropenia, and 3 patients underwent allogeneic hematopoietic cell transplantation. All variants conferred gain of function to TLR8 protein, and immune phenotyping demonstrated a proinflammatory phenotype with activated T cells and elevated serum cytokines associated with impaired B-cell maturation. Differentiation of myeloid cells from patient-derived induced pluripotent stem cells demonstrated increased responsiveness to TLR8. Together, these findings demonstrate that gain-of-function variants in TLR8 lead to a novel childhood-onset IEI with lymphoproliferation, neutropenia, infectious susceptibility, B- and T-cell defects, and in some cases, bone marrow failure. Somatic mosaicism is a prominent molecular mechanism of this new disease.

Mechanisms underlying genome instability mediated by formation of foldback inversions in Saccharomyces cerevisiae.

Foldback inversions, also called inverted duplications, have been observed in human genetic diseases and cancers. Here, we used a Saccharomyces cerevisiae genetic system that generates gross chromosomal rearrangements (GCRs) mediated by foldback inversions combined with whole-genome sequencing to study their formation. Foldback inversions were mediated by formation of single-stranded DNA hairpins. Two types of hairpins were identified: small-loop hairpins that were suppressed by MRE11, SAE2, SLX1, and YKU80 and large-loop hairpins that were suppressed by YEN1, TEL1, SWR1, and MRC1. Analysis of CRISPR/Cas9-induced double strand breaks (DSBs) revealed that long-stem hairpin-forming sequences could form foldback inversions when proximal or distal to the DSB, whereas short-stem hairpin-forming sequences formed foldback inversions when proximal to the DSB. Finally, we found that foldback inversion GCRs were stabilized by secondary rearrangements, mostly mediated by different homologous recombination mechanisms including single-strand annealing; however, POL32-dependent break-induced replication did not appear to be involved forming secondary rearrangements.

FEN1 endonuclease as a therapeutic target for human cancers with defects in homologous recombination.

Synthetic lethality strategies for cancer therapy exploit cancer-specific genetic defects to identify targets that are uniquely essential to the survival of tumor cells. Here we show RAD27/FEN1, which encodes flap endonuclease 1 (FEN1), a structure-specific nuclease with roles in DNA replication and repair, and has the greatest number of synthetic lethal interactions with Saccharomyces cerevisiae genome instability genes, is a druggable target for an inhibitor-based approach to kill cancers with defects in homologous recombination (HR). The vulnerability of cancers with HR defects to FEN1 loss was validated by studies showing that small-molecule FEN1 inhibitors and FEN1 small interfering RNAs (siRNAs) selectively killed BRCA1- and BRCA2-defective human cell lines. Furthermore, the differential sensitivity to FEN1 inhibition was recapitulated in mice, where a small-molecule FEN1 inhibitor reduced the growth of tumors established from drug-sensitive but not drug-resistant cancer cell lines. FEN1 inhibition induced a DNA damage response in both sensitive and resistant cell lines; however, sensitive cell lines were unable to recover and replicate DNA even when the inhibitor was removed. Although FEN1 inhibition activated caspase to higher levels in sensitive cells, this apoptotic response occurred in p53-defective cells and cell killing was not blocked by a pan-caspase inhibitor. These results suggest that FEN1 inhibitors have the potential for therapeutically targeting HR-defective cancers such as those resulting from BRCA1 and BRCA2 mutations, and other genetic defects.

Essential Saccharomyces cerevisiae genome instability suppressing genes identify potential human tumor suppressors.

Gross Chromosomal Rearrangements (GCRs) play an important role in human diseases, including cancer. Although most of the nonessential Genome Instability Suppressing (GIS) genes in Saccharomyces cerevisiae are known, the essential genes in which mutations can cause increased GCR rates are not well understood. Here 2 S. cerevisiae GCR assays were used to screen a targeted collection of temperature-sensitive mutants to identify mutations that caused increased GCR rates. This identified 94 essential GIS (eGIS) genes in which mutations cause increased GCR rates and 38 candidate eGIS genes that encode eGIS1 protein-interacting or family member proteins. Analysis of TCGA data using the human genes predicted to encode the proteins and protein complexes implicated by the S. cerevisiae eGIS genes revealed a significant enrichment of mutations affecting predicted human eGIS genes in 10 of the 16 cancers analyzed.

EPCAM mutation update: Variants associated with congenital tufting enteropathy and Lynch syndrome.

The epithelial cell adhesion molecule gene (EPCAM, previously known as TACSTD1 or TROP1) encodes a membrane-bound protein that is localized to the basolateral membrane of epithelial cells and is overexpressed in some tumors. Biallelic mutations in EPCAM cause congenital tufting enteropathy (CTE), which is a rare chronic diarrheal disorder presenting in infancy. Monoallelic deletions of the 3' end of EPCAM that silence the downstream gene, MSH2, cause a form of Lynch syndrome, which is a cancer predisposition syndrome associated with loss of DNA mismatch repair. Here, we report 13 novel EPCAM mutations from 17 CTE patients from two separate centers, review EPCAM mutations associated with CTE and Lynch syndrome, and structurally model pathogenic missense mutations. Statistical analyses indicate that the c.499dupC (previously reported as c.498insC) frameshift mutation was associated with more severe treatment regimens and greater mortality in CTE, whereas the c.556-14A>G and c.491+1G>A splice site mutations were not correlated with treatments or outcomes significantly different than random simulation. These findings suggest that genotype-phenotype correlations may be useful in contributing to management decisions of CTE patients. Depending on the type and nature of EPCAM mutation, one of two unrelated diseases may occur, CTE or Lynch syndrome.

The Swr1 chromatin-remodeling complex prevents genome instability induced by replication fork progression defects.

Genome instability is associated with tumorigenesis. Here, we identify a role for the histone Htz1, which is deposited by the Swr1 chromatin-remodeling complex (SWR-C), in preventing genome instability in the absence of the replication fork/replication checkpoint proteins Mrc1, Csm3, or Tof1. When combined with deletion of SWR1 or HTZ1, deletion of MRC1, CSM3, or TOF1 or a replication-defective mrc1 mutation causes synergistic increases in gross chromosomal rearrangement (GCR) rates, accumulation of a broad spectrum of GCRs, and hypersensitivity to replication stress. The double mutants have severe replication defects and accumulate aberrant replication intermediates. None of the individual mutations cause large increases in GCR rates; however, defects in MRC1, CSM3 or TOF1 cause activation of the DNA damage checkpoint and replication defects. We propose a model in which Htz1 deposition and retention in chromatin prevents transiently stalled replication forks that occur in mrc1, tof1, or csm3 mutants from being converted to DNA double-strand breaks that trigger genome instability.

The properties of Msh2-Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair.

DNA mismatch repair (MMR) corrects mispaired DNA bases and small insertion/deletion loops generated by DNA replication errors. After binding a mispair, the eukaryotic mispair recognition complex Msh2-Msh6 binds ATP in both of its nucleotide-binding sites, which induces a conformational change resulting in the formation of an Msh2-Msh6 sliding clamp that releases from the mispair and slides freely along the DNA. However, the roles that Msh2-Msh6 sliding clamps play in MMR remain poorly understood. Here, using Saccharomyces cerevisiae, we created Msh2 and Msh6 Walker A nucleotide-binding site mutants that have defects in ATP binding in one or both nucleotide-binding sites of the Msh2-Msh6 heterodimer. We found that these mutations cause a complete MMR defect in vivo The mutant Msh2-Msh6 complexes exhibited normal mispair recognition and were proficient at recruiting the MMR endonuclease Mlh1-Pms1 to mispaired DNA. At physiological (2.5 mm) ATP concentration, the mutant complexes displayed modest partial defects in supporting MMR in reconstituted Mlh1-Pms1-independent and Mlh1-Pms1-dependent MMR reactions in vitro and in activation of the Mlh1-Pms1 endonuclease and showed a more severe defect at low (0.1 mm) ATP concentration. In contrast, five of the mutants were completely defective and one was mostly defective for sliding clamp formation at high and low ATP concentrations. These findings suggest that mispair-dependent sliding clamp formation triggers binding of additional Msh2-Msh6 complexes and that further recruitment of additional downstream MMR proteins is required for signal amplification of mispair binding during MMR.

Identification of Exo1-Msh2 interaction motifs in DNA mismatch repair and new Msh2-binding partners.

Eukaryotic DNA mismatch repair (MMR) involves both exonuclease 1 (Exo1)-dependent and Exo1-independent pathways. We found that the unstructured C-terminal domain of Saccharomyces cerevisiae Exo1 contains two MutS homolog 2 (Msh2)-interacting peptide (SHIP) boxes downstream from the MutL homolog 1 (Mlh1)-interacting peptide (MIP) box. These three sites were redundant in Exo1-dependent MMR in vivo and could be replaced by a fusion protein between an N-terminal fragment of Exo1 and Msh6. The SHIP-Msh2 interactions were eliminated by the msh2M470I mutation, and wild-type but not mutant SHIP peptides eliminated Exo1-dependent MMR in vitro. We identified two S. cerevisiae SHIP-box-containing proteins and three candidate human SHIP-box-containing proteins. One of these, Fun30, had a small role in Exo1-dependent MMR in vivo. The Remodeling of the Structure of Chromatin (Rsc) complex also functioned in both Exo1-dependent and Exo1-independent MMR in vivo. Our results identified two modes of Exo1 recruitment and a peptide module that mediates interactions between Msh2 and other proteins, and they support a model in which Exo1 functions in MMR by being tethered to the Msh2-Msh6 complex.

Analyzing Genome Rearrangements in Saccharomyces cerevisiae.

Genome rearrangements underlie different human diseases including many cancers. Determining the rates at which genome rearrangements arise and isolating unique, independent genome rearrangements is critical to understanding the genes and pathways that prevent or promote genome rearrangements. Here, we describe quantitative S. cerevisiae genetic assays for measuring the rates of accumulating genome rearrangements including deletions, translocations, and broken chromosomes healed by de novo telomere addition that result in the deletion of two counter-selectable genes, CAN1 and URA3, placed in the nonessential regions of the S. cerevisiae genome. The assays also allow for the isolation of individual genome rearrangements for structural studies, and a method for analyzing genome rearrangements by next-generation DNA sequencing is provided.

Pathways and Mechanisms that Prevent Genome Instability in Saccharomyces cerevisiae.

Genome rearrangements result in mutations that underlie many human diseases, and ongoing genome instability likely contributes to the development of many cancers. The tools for studying genome instability in mammalian cells are limited, whereas model organisms such as Saccharomyces cerevisiae are more amenable to these studies. Here, we discuss the many genetic assays developed to measure the rate of occurrence of Gross Chromosomal Rearrangements (called GCRs) in S. cerevisiae These genetic assays have been used to identify many types of GCRs, including translocations, interstitial deletions, and broken chromosomes healed by de novo telomere addition, and have identified genes that act in the suppression and formation of GCRs. Insights from these studies have contributed to the understanding of pathways and mechanisms that suppress genome instability and how these pathways cooperate with each other. Integrated models for the formation and suppression of GCRs are discussed.

A genetic network that suppresses genome rearrangements in Saccharomyces cerevisiae and contains defects in cancers.

Gross chromosomal rearrangements (GCRs) play an important role in human diseases, including cancer. The identity of all Genome Instability Suppressing (GIS) genes is not currently known. Here multiple Saccharomyces cerevisiae GCR assays and query mutations were crossed into arrays of mutants to identify progeny with increased GCR rates. One hundred eighty two GIS genes were identified that suppressed GCR formation. Another 438 cooperatively acting GIS genes were identified that were not GIS genes, but suppressed the increased genome instability caused by individual query mutations. Analysis of TCGA data using the human genes predicted to act in GIS pathways revealed that a minimum of 93% of ovarian and 66% of colorectal cancer cases had defects affecting one or more predicted GIS gene. These defects included loss-of-function mutations, copy-number changes associated with reduced expression, and silencing. In contrast, acute myeloid leukaemia cases did not appear to have defects affecting the predicted GIS genes.