Loss Of Gene Function Research Articles

Bilateral Calcification of Basal Ganglia in a Patient with Duplication of Both 11q13.1q22.1 and 4q35.2 with New Phenotypic Features

We report on a female patient who presented with severe intellectual disability and autistic behavior, dysmorphic features, orodental anomalies, and bilateral calcification of basal ganglia. Using a high-density oligonucleotide microarray, we have identified a de novo duplication of 11q13.1q22.1 involving the dosage sensitive genes FGF3 and FGF4, genes related to autosomal dominant disorders KMT5B, GAL, SPTBN2, and LRP5, susceptibility loci SCZD2, SLEH1, and SHANK2, mitochondrial genes NDUFV1, NDUFS8, and TMEM126B, and many loss of function genes, including PHOX2A, CLPB, MED17, B3GNT1, LIPT2, and CLPB. However, the duplication did not involve Ribonuclease H2, subunit C (RNASEH2C) which is considered to be located in the critical region for Aicardi-Goutières syndrome. In combination with the duplication at 11q13.1, a 1.849-Mb heterozygous duplication at 4q35.2 was also identified. Although this duplicated region does not contain causative genes related to brain calcification, the duplication at 4q35 was reported previously in a patient with basal ganglia calcification, coats' like retinopathy, and glomerulosclerosis. Our patient's presentation and genomic findings indicate that duplication of 4q35.2 could be a novel genetic cause of calcification of basal ganglia. Our report also underscores the clinical significance of rearrangements in 11q13.1q22.1 in the pathogenesis of basal ganglia calcification.

Efficacy of OR21, a Novel Oral Demethylating Agent in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disease induced by t(9;22)(q34;q11) translocation. The prognosis of patients with CML has dramatically improved since tyrosine kinase inhibitors (TKIs) were introduced, and recent studies show that approximately 40 to 50 % of CML patients achieved deep molecular response (DASISION and ENESTnd trial) within several years. However, therapeutic options for patients with CML who are resistant for TKIs are limited. Besides BCR-ABL kinase domain mutation, somatic mutations associated with epigenetic gene alteration (e.g. TET, DNMT3A) are reportedly involved in TKI resistance and disease progression. Thus, we investigated the efficacy of DNA demethylating agents in CML. Azacitidine (AZA) and decitabine (DAC), currently available as DNA demethylating agents, have low bioavailability for oral administration because they are easily degraded by cytidine deaminase. Then, we are developing a novel demethylating agent, OR21, with possible oral absorbability as a prodrug of DAC. In vivo analysis using cynomolgus monkeys, the area under the curve of DAC after intraduodenally administration of DAC (1.5mg/kg, 6.6µmol/kg) or OR21 (2.25mg/kg, 6.6µmol/kg) was 0.01 µM·h, 0.298 µM·h respectively, indicating OR21 have high oral absorbability. To assess the demethylating activity of OR21 for CML, we performed western blot analysis and bisulfite pyrosequencing assay to measure LINE1 methylation, using CML cell-lines (K562, BV173). OR21 decreased DNMT1 protein level as a result of demethylating and LINE1 methylation in K562 and BV173 comparable to DAC. Next, we performed cell growth inhibition and cell apoptosis assay after 72 hours exposure of OR21 to assess anti-tumor effect in vitro. OR21 inhibited cell growth comparable to DAC in CML cell-lines (K562, BV173, KCL22, MYL) in a dose-dependent manner. Notably, OR21 inhibited the cell growth and apoptosis against BV173 with an extremely low concentration (IC50; 5nM) than that of AZA (IC50; 122nM) via significant accumulation of reactive oxygen species. Whereas, OR21 weakly induced cell apoptosis against K562. OR21 induced G2/M phase cell-cycle arrest in CML cell-lines except for BV173 via pRb downregulation. These results indicated mechanisms of anti-tumor effect in OR21 were induction of cell apoptosis (BV173) or cell cycle arrest (K562, KCL22, MYL). Because of the different mechanism of action, we assessed whether OR21 and TKIs combination can enhance the anti-tumor effect of CML. We investigated the combination effects of OR21 with imatinib (IM) or dasatinib (DAS) in K562. Combination index values at IC80 calculated by Calucusyn software showed 0.642±0.129 (with IM), 1.182±0.2 (with DAS), respectively. Accordingly, OR21 combined with TKIs showed at least additive or synergistic effect for K562. TKI resistance, which can be associated with somatic mutations leading to epigenetic gene alteration or loss of function of p53, is an obstacle for molecular remission in patients with CML, thus we examined the effects of OR21 in TKI resistant cell lines or efficacy on p53 mutational status. OR21 inhibited the cell growth in IM-resistant cell-line MYL-R, a derivative of MYL, which had overexpression of Lyn, and Ba/F3 BCR-ABLT315I, which exogenously expressed Bcr-Abl (T315I), indicating OR21 could overcome TKI resistance in CML. OR21 or cytarabine did not enhance cell apoptosis against K562, MYL and KCL22 (p53 deficient or mutant cell lines) combination with nutlin-3a (MDM-2 inhibitor), while increased cell apoptosis was observed in BV173 (p53 wild type) treated with cytarabine and nutlin-3a, but not with OR21 and nutlin-3a. These results suggested the effects of OR21 did not depend on p53 mutational status. Finally, we used a mouse xenograft model to evaluate anti-tumor effect of OR21 in vivo. BALB/c Rag-2/JAK3 double-deficient (BRJ) mice were injected intravenously via tail vein with 5 ×106 BV173 cells. OR21 were administered at a dose of 2.7mg/kg (equivalent to DAC 1.0mg/kg in AUC) and PBS (vehicle) twice weekly. OR21 significantly prolonged survival in a xenograft mice model (median 35 days vs not reached, P<0.01). In conclusion, a novel orally available demethylating agent OR21 is effective for CML cells including TKI resistant clones. The efficacy and safety of OR21 for CML is expected to be verified by early-phase clinical trials. Disclosures Kamachi: Ohara Pharmaceutical Co.: Research Funding. Ureshino:OHARA Pharmaceutical Co.: Research Funding. Yoshida:OHARA Pharmaceutical Co., Ltd.: Research Funding. Kurahashi:Ohara Pharmaceutical Co.: Employment. Watanabe:Ohara Pharmaceutical Co.: Research Funding. Okada:Japan Agency for Medical Research and Development: Research Funding; Bristol-Myers Squibb: Research Funding. Kimura:Ohara Pharmaceutical Co.: Research Funding; Novartis: Honoraria, Research Funding.

Highly Efficient CRISPR-Cas9-Based Methods for Generating Deletion Mutations and F0 Embryos that Lack Gene Function in Zebrafish.

Inconsistent activity limits the use of CRISPR-Cas9 in zebrafish. We show supernumerary guanine nucleotides at the 5' ends of single guide RNAs (sgRNAs) account for diminished CRISPR-Cas9 activity in zebrafish embryos. Genomic sequences can be targeted consistently with extremely high efficiency using Cas9 ribonucleoproteins (RNPs) containing either a sgRNA molecule or a synthetic crRNA:tracrRNA duplex that perfectly matches the protospacer target site. Following injection of zebrafish eggs with such RNPs, virtually every copy of a targeted locus harbors an induced indel mutation. Loss of gene function is often complete, as F0 embryos closely resemble true null mutants without detectable non-specific effects. Mosaicism is sufficiently low in F0 embryos that cell non-autonomous gene functions can be probed effectively and redundant activities of genes can be uncovered when two genes are targeted simultaneously. Finally, heritable deletion mutations of at least 50 kbp can be readily induced using pairs of duplex guide RNPs targeted to a single chromosome.

Abstract 2916: Delineating the radiogenomic landscape of cancer through systematic annotation of genetic variants

Abstract Radiation is a fundamental pillar of treatment for many cancer patients. However, there are still no established means by which to identify patients who are more or less likely to respond to treatments. In addition, there are few synergistic drug-radiotherapy combinations currently in clinical use. To address these needs, we sought to discover genetic alterations that can predict the vulnerability of cancer to radiation. Here, we report on a large-scale study to identify and classify tumor associated genetic variants that determine survival to radiation. To gain insight into the genetic landscape of radioresistance, we previously established a high-throughput platform that measures cellular survival to radiation. We then used this technology to analyze the radiosensitivity of more than 500 cancer cell lines and identified mutant alleles associated with radiation resistance. To functionally annotate a multitude of these variants, mutant constructs were generated using a high-throughput, site-directed mutagenesis approach and shuffled into lentiviral vectors for stable expression in immortalized BEAS2B lung cells. The effect of individual variants on radiation survival was assessed using our high-throughput platform and benchmarked against the radio-protective NRF2 E79K gain-of-function mutation and validated by colony forming assay. Radioresistant variants were further profiled in TP53 deficient BEAS2B cells and in cells across multiple lineages. For a subset of variants predicted to confer resistance by a loss of gene function, expression of the endogenous gene was knocked out by CRISPR directed to an exon-intron junction, thus allowing for the expression of the intron-less variant ORF. We successfully generated and profiled over 550 tumor associated genetic variants. Top radioresistant conferring alleles were comprised of both rare and recurrent mutations. In many instances, rare variants were more impactful than common mutations in the same domain or even the same amino acid position. Therapeutically targetable radioresistant variants were discovered within ARAF, BRAF, CTNNB1, EGFR, MAP2K1 and PIK3CA, among others. Loss of gene function variants were profiled in KEAP1, STK11, and TP53. A subset of KEAP1 loss of gene function variants were found to confer a dominant-negative phenotype by stabilizing NRF2 levels. Determining the impact of gene variants remains a major obstacle in the implementation of personalized radiotherapy. Here, we report on a large-scale profiling effort to identify and classify mutant alleles that govern the sensitivity of cells to radiation. We demonstrate that new genetic features responsible for mediating cellular survival to DNA damage can be discovered. We also show that a substantial proportion of variants that confer resistance to radiation are druggable, advancing drug-radiation combinations in “baskets” of patients with these alterations. Citation Format: Brian Yard, Aaron Petty, Mohamed Abazeed. Delineating the radiogenomic landscape of cancer through systematic annotation of genetic variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2916.

Immediate Dosage Compensation Is Triggered by the Deletion of Y-Linked Genes in Silene latifolia

SummaryThe loss of functional genes from non-recombining sex-specific chromosomes [1, 2], such as the Y chromosomes in mammals [3] or W chromosomes in birds [4], should result in an imbalance of gene products for sex-linked genes [5]. Different chromosome-wide systems that rebalance gene expression are known to operate in organisms with relatively old sex chromosomes [6]; e.g., Drosophila overexpress X-linked genes in males [7], while mammals shut down one of the X chromosomes in females [8]. It is not known how long it takes for a chromosome-wide dosage compensation system to evolve. To shed light on the early evolution of dosage compensation, we constructed a high-density Y-deletion map and used deletion mutants to manipulate gene dose and analyze gene expression in white campion (Silene latifolia), which evolved dioecy and sex chromosomes only 11 million years ago [9]. We demonstrate that immediate dosage compensation can be triggered by deletions in a large portion of the p arm of the Y chromosome. Our results indicate that dosage compensation in S. latifolia does not have to evolve gene by gene because a system to upregulate gene expression is already operating on part of the X chromosome, which likely represents an intermediate step in the evolution of a chromosome-wide dosage compensation system in this species.

Diagnostic and prognostic value of polymorphism of candidate genes of secondary osteoporosis in patients with chronic pancreatitis and hypertension

Study of features of the combined course of a number of diseases of internal organs is caused by their mutually enhancing negative influence and need to correct diagnostic and therapeutic measures. Among such nosologies, attention is drawn to chronic pancreatitis (CP) and hypertension. Their comorbidity increases a risk of atypical clinical manifestations, torpid to conventional therapy and early development of complications. One of these complications is secondary osteoporosis. Developing structural and functional changes in the bone tissue are not only a compensatory response to an increased need for calcium ions, but also an independent factor in further disease progression when these pathologies are combined. An important role in the diagnosis and prediction of impaired metabolism of bone tissue is played by the study of the polymorphism of candidate genes, which can affect not only the development of osteoporosis, but also determine the timing of this complication to certain extent. Vitamin D (VDR) and lactase (LCT) genes are considered among them.
 Aim of research is to study the role of gene polymorphism of vitamin D receptors (VDR) and lactase gene (LCT) in the risk of developing osteopenic conditions in patients with comorbidity of CP and hypertension.
 Materials and methods. 110 patients with CP were examined, which made it possible to create two groups: main group — 70 people with comorbid CP and hypertension, and comparison group — 40 patients with isolated CP. Control group included 78 healthy individuals. All patients were representative by age and sex. The state of the bone tissue was determined by conducting double-energy X-ray absorptiometry (DEXA). Polymerase chain reaction was used to study the polymorphism of the vitamin D receptor (VDR) and lactase (LCT) genes.
 Results. Majority of patients in the main group (84.3%) had an unfavorable B-allele, in contrast to the comparison group, where this index was equal to 77.5% of cases. Changes in the VDR gene polymorphism, which influenced the frequency of lesions of the osteo-articular system, were stated. Lactose insufficiency (LI) was found out in more than half of the patients with CP (57.5%). Upon comorbidity of CP and hypertension, number of such patients increased (68.6%), which could be considered as a result of impaired vascular pancreatic regulation. At the same time, LI occurred against the background of normal (C/C) polymorphic variants of the LCT gene. Almost a third of patients (35.7%) had osteopenic states, but they were not associated with the lactase gene polymorphism. Comparing the pathological VDR and LCT genes of the entire sample of patients (188 people), we obtained the frequency distribution of a statistically significant nature (CCP, χ2=21.92547, df=4, p=0.00021). Namely, the coincidence of Bb and CT heterozygotes was 37.88%, and that of BB and CC homozygotes was 45.57%. The coincidence of Bb and CC was 21.21%, BB and CT — 31.65%. Frequency distribution in patients with isolated CP (40) also had a statistically significant character (CCP, χ2=10.69637, df=4, p=0.03020). Coincidence in the heterozygote of Bb and CT was 25%, and that of homozygotes for BB and CC — 60%. Distribution of BB and CC corresponded to 31.25%, BB and CT — 33.33%.
 Conclusion. It was stated that upon CP, as well as in its comorbidity with hypertension, osteoporotic conditions might be formed. Combination of calcium-dependent diseases (CP and hypertension) and the vitamin D receptors gene polymorphism with a predominance of unfavorable B-allele could be the cause of such conditions. At the same time, the risk of developing osteoporotic conditions increases 4 times. Course of CP is often accompanied by LI, which may be the result of both gene aberrations and loss of lactase-secreting function in a given disease.

Loss and Gain in the Evolution of the Salmonella enterica Serovar Gallinarum Biovar Pullorum Genome

Salmonella enterica subspecies enterica serovar Gallinarum biovar Pullorum (S Pullorum) is the etiological agent of pullorum disease, causing white diarrhea with high mortality in chickens. There are many unsolved issues surrounding the epidemiology of S Pullorum, including its origin and transmission history as well as the discordance between its phenotypic heterogeneity and genetic monomorphism. In this paper, we report the results of whole-genome sequencing of a panel of 97 S Pullorum strains isolated between 1962 and 2014 from four countries across three continents. We utilized 6,795 core genome single nucleotide polymorphisms (SNPs) to reconstruct a phylogenetic tree within a spatiotemporal Bayesian framework, estimating that the most recent common ancestor of S Pullorum emerged in ∼914 CE (95% confidence interval [95%CI], 565 to 1273 CE). The extant S Pullorum strains can be divided into four distinct lineages, each of which is significantly associated with geographical distribution. The intercontinental transmissions of lineages III and IV can be traced to the mid-19th century and are probably related to the "Hen Fever" prevalent at that time. Further genomic analysis indicated that the loss or pseudogenization of functional genes involved in metabolism and virulence in S Pullorum has been ongoing since before and after divergence from the ancestor. In contrast, multiple prophages and plasmids have been acquired by S Pullorum, and these have endowed it with new characteristics, especially the multidrug resistance conferred by two large plasmids in lineage I. The results of this study provide insight into the evolution of S Pullorum and prove the efficiency of whole-genome sequencing in epidemiological surveillance of pullorum disease.IMPORTANCE Pullorum disease, an acute poultry septicemia caused by Salmonella Gallinarum biovar Pullorum, is fatal for young chickens and is a heavy burden on poultry industry. The pathogen is rare in most developed countries but still extremely difficult to eliminate in China. Efficient epidemiological surveillance necessitates clarifying the origin of the isolates from different regions and their phylogenic relationships. Genomic epidemiological analysis of 97 S Pullorum strains was carried out to reconstruct the phylogeny and transmission history of S Pullorum. Further analysis demonstrated that functional gene loss and acquisition occurred simultaneously throughout the evolution of S Pullorum, both of which reflected adaptation to the changing environment. The result of our study will be helpful in surveillance and prevention of pullorum disease.

An Epidemiological Study of Drug Resistance and Resistance Genes in Bovine Escherichia coli Isolates in Heilongjiang Province of China

Background: To explore the epidemiology of bovine multidrug-resistant Escherichia coli isolates and resistance genes in Heilongjiang province of China. This study examined the prevalence of genes in bovine E. coli isolates, which confer resistance to antibiotics that are commonly used in the clinic, in regions of Baiquan, Shangzhi, and Songbei of Harbin. The purpose of the study was to investigate the epidemiology of the main resistance genes of bovine E. coli isolates in clinical veterinary medicine, and to provide a theoretical basis for preventing the spread of drug-resistant bacteria, as well as for rational drug use.Materials, Methods & Results: The sensitivity of 105 isolates to 22 antibiotics was determined using the KirbyBauer disk diffusion method, and the distribution of 19 kinds of common drug resistance genes was investigated using Polymerase Chain Reaction. The results showed that the resistance rate to nine antibiotics was over 50%, including rifampin (84.76%), ampicillin (73.58%), tetracycline (69.52%), and sulfisoxazole (59.05%). In total, 105 strains of bovine E. coli presented 21 spectra of drug resistance, including eight strains (7.62%, 8/105) that were resistant to one antibiotic and four strains (3.81%, 4/105) that were resistant to 21 antibiotics. The resistance gene detection results showed that the streptomycin-resistance gene strA was found in 73 isolates, accounting for 69.52% of the isolates, followed by the sulfanilamide-resistance genes sul3/sul2 and the aminoglycoside-resistance gene aphA, which accounted for 57.14%, 51.43%, and 50.48%, respectively, of the isolates.Discussion: This study revealed serious drug resistance of bovine E. coli isolates in some areas of Heilongjiang province. Of 105 E. coli isolates, more than 50% were resistant to the following antibacterial drugs: rifampicin, ampicillin, tetracycline, sulfisoxazole, and cephalothin. The isolates were the most sensitive to amikacin, with a sensitivity of 84.76%, followed by sensitivity to ofloxacin, ciprofloxacin, norfloxacin, cefoxitin, and tobramycin. Drug sensitivity tests showed that the drug resistance spectra of the bovine E. coli isolates was different in different regions, indicating that there were multidrug-resistant bovine E. coli isolates in different regions of Heilongjiang province, and that drug resistance differed among different regions. This may be due to prolonged use or overuse of antibiotics in a particular locality. Additionally, because of different management modes of livestock farms, the application of antimicrobial drugs in some farms may have imposed selective pressure on the intestinal flora including E. coli, resulting in the horizontal transmission of drug resistance among the bacteria. The study found that some strains had a resistance phenotype, but no resistance gene, while some had a resistance gene without expressing a resistance phenotype, which is consistent with relevant reports in the literature. This may be related to the same genotype corresponding to different resistance phenotypes, or different levels of gene expression, or different drug metabolic rates. In our study, some strains with certain drug resistance genes were sensitive to the corresponding drug, which may be due to mutations of drug-resistance genes, the loss of a strains resistance phenotype, or the loss of gene function. These issues require further study. This study revealed serious drug resistance of bovine E. coli isolates in some areas of Heilongjiang province. Of 105 E. coli isolates, more than 50% were resistant to the following antibacterial drugs: rifampicin, ampicillin, tetracycline, sulfisoxazole, and cephalothin. The isolates were the most sensitive to amikacin, with a sensitivity of 84.76%, followed by sensitivity to ofloxacin, ciprofloxacin, norfloxacin, cefoxitin, and tobramycin.

CAR T Cell Cytotoxicity Is Dependent on Death Receptor-Driven Apoptosis

▪Background: T cells engineered to express chimeric antigen receptors targeting the B-cell antigen CD19 (CART19) have demonstrated impressive results in the treatment of lymphoid cancers. Despite these promising outcomes, a significant subset of patients relapse after initial response. To investigate the molecular pathways that drive relapse, we performed an unbiased, CRISPR/Cas9-mediated genome-wide knockout screen in the acute lymphoblastic leukemia (ALL) cell line Nalm6, and found that loss of CD19 was the primary driver of relapse after initial response. This finding is consistent with clinical observations that antigen loss is a primary driver of late disease recurrence, however it fails to address the molecular etiology of intrinsic resistance, which affects ~50% of patients with non-Hodgkin lymphoma and ~20% of patients with ALL, or of late antigen-independent relapse, which accounts for ~40% of relapses in ALL. Identification of the mechanisms regulating CART19 susceptibility is an essential first step in overcoming resistance to this powerful therapy. We hypothesized that genetic alteration in ALL cells were responsible for mediating intrinsic, CD19-independent resistance. To investigate this, we conducted a genome-wide loss of function screen in a model designed to evaluate intrinsic resistance to CART19.Methods: Using a lentiviral guide RNA (gRNA) library containing four distinct gRNAs targeting each human gene (~80,000 gRNAs total), we enabled genome-wide knockout in Nalm6, whereby each target cell lost function of only one gene. This gene-modified cell pool was then exposed to either CART19 or control T cells at a low effector:target ratio (0.25:1) to model the expected in vivo E:T ratio. At 24h, surviving Nalm6 cells were collected and gRNA from these cells underwent next-generation sequencing. Sequenced samples were processed using three distinct genome-scale knockout screen algorithms (MAGeCK, permutation-based non-parametric analysis and ScreenBeam). This pipeline allowed identification of (i) significantly enriched gRNA, postulated to mediate loss of gene function that confers resistance to CART19, and (ii) significantly depleted guides, postulated to mediate loss of gene function that confers sensitivity to CART19. The role of identified genes was then validated in in vitro and in vivo studies.Results: Analysis of gRNA sequencing data from our screen (Figure 1) revealed that the three genes whose loss of function most significantly promoted resistance to CART19 were BID, FADD and CASP8, all of which are key regulators of death receptor-driven apoptosis. TNFRSF10B, encoding the death receptor TRAIL-R2, was also significantly enriched. Interestingly, amongst the 10 genes whose loss most significantly sensitized to CART19 were TRAF2, BIRC2 and CFLAR, all negative regulators of death receptor activity. Pathway analysis of the top 50 genes (25 enriched, 25 depleted) demonstrated significant enrichment in the death receptor pathway, with a false discovery rate of 3.79x10-7.We proceeded to functionally validate the role of BID and FADD in mediating resistance to CART19 by deleting these genes in Nalm6 using de novo designed gRNAs. Strikingly, BIDKO and FADDKO cells were highly resistant to CART19 cytotoxicity in vitro as compared to wild-type Nalm6. Resistance was evident as early as 6 hours after co-culture and was maintained for at least 7 days. Observed resistance to CART19 directly correlated to fraction of KO cells present, suggesting that gene loss was mechanistically responsible for failed CART19 cytotoxicity. We further evaluated the impact of BID or FADD loss on anti-leukemic activity of CART19 in our Nalm6 xenograft model. We observed that BIDKO or FADDKO significantly impaired the anti-leukemic activity of CART19 in vivo.Conclusions: CART19 can cure select patients with B-cell cancers, while others experience transient or no clinical benefit. Using a genome-wide loss of function screen, we identified that death receptor-associated proteins are centrally involved in regulating CART19 cytotoxicity, and that loss of these molecules leads to intrinsic resistance to CART19. These findings are, to our knowledge, the first to characterize the role of death receptors as critical regulators of CART19 cytotoxicity, and suggest that tumor cell modulation of death receptor signaling may drive both inherent resistance and antigen-independent relapse. DisclosuresJune:Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Gill:Novartis: Research Funding; Carisma Therapeutics: Equity Ownership; Extellia: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ruella:University of Pennsylvania: Patents & Royalties.

An Integrated Genomics Analysis Identifies Recurrent Gain- and Loss-of-Function Events and in Distinct Subtypes of Multiple Myeloma

Multiple myeloma (MM) is a pathological description for a plasma cell malignancy that exhibits a high degree of genetic diversity between patients. Whole genome analysis methods have elucidated multiple distinct subtypes of disease, however, we have a very limited understanding of what drives each independent subtype. To address this issue, we developed a comprehensive analysis approach to identify MM subtypes and associated genetic features in untreated patient samples from the MMRF CoMMpass study (NCT01454297). In this study we attempt to characterize each sample using whole genome (WGS), exome (WES), and RNA (RNAseq) sequencing. For this analysis we have limited the baseline cohort from the 982 patients with at least one assay completed to the 591 which are completely characterized.Most cohort level analyses focus on specific genetic aberrations such as copy number (CN) (eg. GISTIC) or somatic mutations (eg. MutSig) to identify genes which are important in the cancer. However, it is well known that cancer cells will dysregulate genes and pathways through a diverse array of mechanisms. We developed an integrated model, leveraging WGS, WES, and RNAseq data, that simultaneously considers; translocations, inversions, CN, copy neutral loss-of-heterozygosity, somatic mutations, constitutional inherited gene defects, gene expression, and in-frame genomically validated gene fusions; in a comprehensive analysis to aid in the discovery of putative oncogenes or tumor suppressor genes.We first defined two independent subtype classes within the cohort using consensus clustering on the gene expression and copy number data, which identified 12 and 14 subtypes, respectively. We also identified 59 significantly mutated genes using MutSig with a robust resampling approach. Then using our integrated approach we identified 64 putative loss-of-function (LOF) genes which have complete, bi-allelic loss, through anyone of 11 possible combinations of genetic defects in at least 1% of the cohort. These include expected tumor suppressors; TRAF3 (10.1%), DIS3 (6.9%), FAM46C (5.1%), TP53 (4.1%), RB1 (3.2%), and NF1 (1.0%). On chromosome 13 alone we identified 17 LOF genes with LOF events occuring in 15.4% of patients with the most common genes being PSPC1, BRCA2, RB1, DIS3, and TGDS. This process also identified LOF events in genes associated with 5-FU sensitivity, DPYD, and PARP inhibitor sensitivity, BRCA1, BRCA2, and STAG2 suggesting some patients may benefit from these agents. We identified 19 putative gain-of-function (GOF) genes, which had recurrent mutations at the same amino acid position, high level CN gains (6 or more copies), overexpression associated with a structural rearrangement, or inframe fusion transcripts in at least 1% of the cohort. These include expected oncogenes; NRAS, KRAS, BRAF, and myeloma specific genes like WHSC1. Interestingly this approach identified a series of genes that were not detected by MutSig such as IRF4, HIST1H1E, DUSP2, and MAP3K14 plus a number with recurrent suspect activating mutations; BMP2K, PTPN11, ALOX15, and ANKLE1.Few subtypes of MM have defined genetic events outside of gene expression subtypes defined by the common IgH translocations. Comparing genes identified in our LOF and GOF analysis with our gene expression or CN defined subtypes identified a number of associations. For instance, in the MS gene expression subtype, characterized by t(4;14), we detected the expected associations with WHSC1 and FGFR3 along with a significant enrichment of TRAF3 and DIS3 LOF events and an under representation of NRAS mutations. A gene expression group associated with a hyperdiploid karyotype and 1q gains has significantly fewer NRAS and KRAS GOF events but is enriched for TRAF3 LOF events. GOF events in IRF4 were exclusively found in the CD2 gene expression groups associated with cyclin D translocations. Interestingly, the LOF events in TGDS were exclusively found in a single CN defined subtype characterized by a hyperdiploid karyotype with 1q gains but lacking chromosome 11 trisomies. Finally, the gene expression defined proliferation (PR) subtype, which exhibits poor OS (HR = 3.996, 95% CI = 2.632 - 6.067, p < 0.001) is highly enriched for bi-allelic LOF events in RB1 and MAX. This finding provides the first genetic explanation for why patients with such diverse genetic backgrounds cluster in this high risk subtype. DisclosuresLonial:Amgen: Research Funding.

Wwox deletion leads to reduced GABA-ergic inhibitory interneuron numbers and activation of microglia and astrocytes in mouse hippocampus

The association of WW domain-containing oxidoreductase WWOX gene loss of function with central nervous system (CNS) related pathologies is well documented. These include spinocerebellar ataxia, epilepsy and mental retardation (SCAR12, OMIM: 614322) and early infantile epileptic encephalopathy (EIEE28, OMIM: 616211) syndromes. However, there is complete lack of understanding of the pathophysiological mechanisms at play. In this study, using a Wwox knockout (Wwox KO) mouse model (2 weeks old, both sexes) and stereological studies we observe that Wwox deletion leads to a significant reduction in the number of hippocampal GABA-ergic (γ-aminobutyric acid) interneurons. Wwox KO mice displayed significantly reduced numbers of calcium-binding protein parvalbumin (PV) and neuropeptide Y (NPY) expressing interneurons in different subfields of the hippocampus in comparison to Wwox wild-type (WT) mice. We also detected decreased levels of Glutamic Acid Decarboxylase protein isoforms GAD65/67 expression in Wwox null hippocampi suggesting lower levels of GABA synthesis. In addition, Wwox deficiency was associated with signs of neuroinflammation such as evidence of activated microglia, astrogliosis, and overexpression of inflammatory cytokines Tnf-a and Il6. We also performed comparative transcriptome-wide expression analyses of neural stem cells grown as neurospheres from hippocampi of Wwox KO and WT mice thus identifying 283 genes significantly dysregulated in their expression. Functional annotation of transcriptome profiling differences identified ‘neurological disease’ and ‘CNS development related functions’ to be significantly enriched. Several epilepsy-related genes were found differentially expressed in Wwox KO neurospheres. This study provides the first genotype-phenotype observations as well as potential mechanistic clues associated with Wwox loss of function in the brain.

Genetics of blood lipids among ~300,000 multi-ethnic participants of the Million Veteran Program.

The Million Veteran Program (MVP) was established in 2011 as a national research initiative to determine how genetic variation influences the health of U.S. military veterans. We genotyped 312,571 MVP participants using a custom biobank array and linked the genetic data to laboratory and clinical phenotypes extracted from electronic health records covering a median of 10.0 years of follow-up. Among 297,626 veterans with at least 1 blood lipid measurement including 57,332 blacks and 24,743 Hispanics, we tested up to ~32 million variants for association with lipid levels and identified 118 novel genome-wide significant loci after meta-analysis with data from the Global Lipids Genetics Consortium (total N > 600,000). Through a focus on mutations predicted to result in a loss of gene function and a phenome-wide association study, we propose novel indications for pharmaceutical inhibitors targeting PCSK9 (abdominal aortic aneurysm), ANGPTL4 (type 2 diabetes), and PDE3B (triglycerides and coronary disease).

Principles and characteristics of the Arabidopsis WRKY regulatory network during early MAMP-triggered immunity.

During microbe-associated molecular pattern-triggered immunity more than 5000 Arabidopsis genes are significantly altered in their expression, and the question arises, how such an enormous reprogramming of the transcriptome can be regulated in a safe and robust manner? For the WRKY transcription factors (TFs), which are important regulators of numerous defense responses, it appears that they act in a complex regulatory sub-network rather than in a linear fashion, which would be much more vulnerable to gene function loss either by pathogen-derived effectors or by mutations. In this study we employed RNA-seq, mass spectrometry and chromatin immunoprecipitation-seq to find evidence for and uncover principles and characteristics of this network. Upon flg22-treatment, one can distinguish between two sets of WRKY genes: constitutively expressed and induced WRKY genes. Prior to elicitation the induced WRKY genes appear to be maintained in a repressed state mainly by the constitutively expressed WRKY factors, which themselves appear to be regulated by non-WRKY TFs. Upon elicitation, induced WRKYs rapidly bind to induced WRKY gene promoters and by auto- and cross-regulation build up the regulatory network. Maintenance of this flg22-induced network appears highly robust as removal of three key WRKY factors can be physically and functionally compensated for by other WRKY family members.

Abstract 423: Investigating the germline deleterious rare variants for genetic susceptibility of esophagus squamous cell carcinomas by target sequencing

Abstract Purpose: The genetic basis of esophageal squamous cell carcinoma (ESCC) still remains unclear. Several genome-wide association studies identified some common variants associated with small effects. Here we aimed to identify some rare deleterious variants in cancer predisposition genes, which may contribute to the genetic susceptibility of ESCC with a large effect, using family history-positive (FH+) samples from the Mainland China Henan high-risk region by targeted sequencing. Methods: A two-phase study was conducted, the discovery phase and the validation phase. In the discovery phase, we sequenced the blood DNAs of 214 independent family history ESCC cases and 123 non-ESCC hospital controls by Illumina HiSeq platform using a custom capture kit. The capture kit included more than 1000 genes, which were cancer-related genes, DNA repair genes, and other genes reported in some ESCC functional studies. For each gene, the exonic region plus 3'-UTR, 5'-UTR, upstream and downstream regions were designed for targeted capture with a total capture size of ~11 Mb. The sequencing reads were aligned to the human reference genome (GRCh37/hg19) using burrows-wheeler aligner (bwa). PCR duplication was marked by Picards. Local realignment was performed with genome analysis toolkit (GAKT). Annovar was used to do the annotation. Loss-of-function (LoF) variants, including splicing, stopgain, and frameshift InDel variants in the cases and controls were identified. The damaging effect of the missense variants was predicted by the combination of 4 different bioinformatics tools (CADD score, MutationTaster, SIFT, Polyphen2). We also used the 1000genomes or ExAC dataset as public controls. All the identified LoF variants in BRCA2 were validated by Sanger sequencing. Results: There was a total of 110 LoF genes identified. Pathway enrichment analysis of these genes found that they were enriched in the Fanconi Anemia (FA) pathway after the background correction of all captured genes. BRCA2 is in the FA pathway and was found to have the most LoF variants; this included 6 individuals from the 214 FH+ cases, and only 1 in the control cohort. We did not find many somatic mutations in ESCC tissues in BRCA2. BRCA2 expression was also significantly upregulated in ESCC tumor tissues, as compared to adjacent normal tissues from in-house and 3 other publicly available RNA-seq datasets. The variant spectra of candidate genes were compared with the public controls to filter out some genes. Conclusions: In the discovery study, we identified some LoF variants in BRCA2 and other genes in the FA pathway that may contribute to ESCC genetic susceptibility. Further validation with a larger sample size is now underway. Acknowledgements: This work was supported by the Research Grants Council Collaborative Research Fund grant C7031-15G and the Asian Fund for Cancer Research to MLL. Citation Format: Lvwen Ning, Josephine Mun Yee Ko, Lisa Chan Lei, Li Dong Wang, Maria Li Lung. Investigating the germline deleterious rare variants for genetic susceptibility of esophagus squamous cell carcinomas by target sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 423.

Conceptual frameworks of synthetic lethality in clear cell carcinoma of the ovary.

Targeting non-oncogenes may result in the selective death of cancer cells. Clear cell carcinoma of the ovary (CCC) may exhibit resistance against conventional chemotherapy and is associated with poor prognosis. The aim of the present report was to review synthetic lethality-based therapies for CCC. Previous English-language studies were reviewed to accumulate preclinical and clinical data on targeting synthetic lethal partners. Synthetic lethal interactions have a variety of types, involving components of a backup or parallel pathway with overlapping functions, components encoded by paralogous pairs, subunit components that form heteromeric complexes and components that are arranged in a single linear pathway. A set of candidate gene targets potentially resulting in synthetic lethality have been previously identified. HNF class homeobox, AT-rich interaction domain 1A, ATR serine/threonine kinase, ATM serine/threonine kinase, checkpoint kinase 1 and phosphatase and tensin homolog may be the key partner genes. A variety of loss of function genes in CCC are driver or passenger events and may function as synthetic lethal pairs under replication stress conditions. Further clinical studies will be required to investigate the safety and therapeutic effect of synthetic lethality pairs in CCC tumor types with replication stress.

Intraspecific variation in the petal epidermal cell morphology of Vicia faba L. (Fabaceae)

At a microscopic scale, the shape and fine cell relief of the petal epidermal cells of a flower play a key role in its interaction with pollinators. In particular, conical shaped petal epidermal cells have been shown to have an important function in providing grip on the surface of bee-pollinated flowers and can influence bee visitation rates. Previous studies have explored interspecific variation in this trait within genera and families, but naturally-occurring intraspecific variation has not yet been comprehensively studied. Here, we investigate petal epidermal cell morphology in 32 genotypes of the crop Vicia faba, which has a yield highly dependent on pollinators. We hypothesise that conical cells may have been lost in some genotypes as a consequence of selective sweeps or genetic drift during breeding programmes. We find that 13% of our lines have a distribution of conical petal epidermal cells that deviates from that normally seen in V. faba flowers. These abnormal phenotypes were specific to the ad/abaxial side of petals, suggesting that these changes are the result of altered gene expression patterns rather than loss of gene function.

Phylogenetic comparison of enteroinvasive Escherichia coli isolated from cases of diarrhoeal disease in England, 2005-2016.

We compared the genomes of 60 isolates of enteroinvasive Escherichia coli (EIEC) in order to better understand the step-wise evolutionary process from non-pathogenic to host-adapted pathogenic E. coli. All isolates belonged to either sequence type (ST) 6, ST99 or ST270. Each ST was located on different branches of the E. coli phylogeny and had invasion plasmids (pINVs) belonging to FII-21 (ST99, ST270), FII-27 (ST270) or FII-28 (ST6, ST270) incompatibility groups. A higher number of insertion sequence (IS) elements were identified in ST6 and ST270 than in ST99, and appeared to be driving the loss of functional genes. Comparison of the pINV from each ST revealed different degrees of gene loss, with pINV from ST270 being most similar to that found in Shigella species. We captured three EIEC STs at different stages of patho-adaptation, with ST270 being the most 'shigella-like' and the most divergent from non-pathogenic E. coli, and ST99 being the least divergent.

Interdisciplinary Models for Research and Clinical Endeavors in Genomic Medicine: A Scientific Statement From the American Heart Association.

The completion of the Human Genome Project has unleashed a wealth of human genomics information, but it remains unclear how best to implement this information for the benefit of patients. The standard approach of biomedical research, with researchers pursuing advances in knowledge in the laboratory and, separately, clinicians translating research findings into the clinic as much as decades later, will need to give way to new interdisciplinary models for research in genomic medicine. These models should include scientists and clinicians actively working as teams to study patients and populations recruited in clinical settings and communities to make genomics discoveries-through the combined efforts of data scientists, clinical researchers, epidemiologists, and basic scientists-and to rapidly apply these discoveries in the clinic for the prediction, prevention, diagnosis, prognosis, and treatment of cardiovascular diseases and stroke. The highly publicized US Precision Medicine Initiative, also known as All of Us, is a large-scale program funded by the US National Institutes of Health that will energize these efforts, but several ongoing studies such as the UK Biobank Initiative; the Million Veteran Program; the Electronic Medical Records and Genomics Network; the Kaiser Permanente Research Program on Genes, Environment and Health; and the DiscovEHR collaboration are already providing exemplary models of this kind of interdisciplinary work. In this statement, we outline the opportunities and challenges in broadly implementing new interdisciplinary models in academic medical centers and community settings and bringing the promise of genomics to fruition.

Uncommon Filaggrin Variants Are Associated with Persistent Atopic Dermatitis in African Americans

Atopic dermatitis (AD) is a common illness that has been associated with filaggrin gene (FLG) loss of function (LoF) variation. In African Americans, a group that commonly has AD and has not been well studied, FLG LoF variation is rarely found. Our objective was to use massively parallel sequencing to evaluate FLG LoF variation in children of African ancestry to evaluate the association between FLG LoF variation and AD and AD persistence. We studied 262 African American children with AD. Nine unique FLG exon 3 LoF variants were identified for an overall minor variant frequency of 6.30% (95% confidence interval [CI]= 4.37-8.73). The most common variants were p.R501X (1.72%, 95% CI= 0.79-3.24), p.S3316X (1.34%, 95% CI= 0.54-2.73), and p.R826X (0.95%, 95% CI= 0.31-2.2). Over an average follow-up period of 96.4 (95% CI= 92.0-100.8) months, African American children with FLG LoF were less likely to be symptom free (odds ratio= 0.36, 95% CI= 0.14-0.89, P= 0.027) compared with a FLG wild-type child. In contrast to previous reports, uncommon FLG LoF variants in African American children exist and are associated with AD and more persistent AD. In contrast to Europeans, no FLG LoF variants predominate in African American children. Properly determining FLG LoF status requires advanced sequencing techniques.

The tumor suppressor Hic1 maintains chromosomal stability independent of Tp53

Hypermethylated-in-Cancer 1 (Hic1) is a tumor suppressor gene frequently inactivated by epigenetic silencing and loss-of-heterozygosity in a broad range of cancers. Loss of HIC1, a sequence-specific zinc finger transcriptional repressor, results in deregulation of genes that promote a malignant phenotype in a lineage-specific manner. In particular, upregulation of the HIC1 target gene SIRT1, a histone deacetylase, can promote tumor growth by inactivating TP53. An alternate line of evidence suggests that HIC1 can promote the repair of DNA double strand breaks through an interaction with MTA1, a component of the nucleosome remodeling and deacetylase (NuRD) complex. Using a conditional knockout mouse model of tumor initiation, we now show that inactivation of Hic1 results in cell cycle arrest, premature senescence, chromosomal instability and spontaneous transformation in vitro. This phenocopies the effects of deleting Brca1, a component of the homologous recombination DNA repair pathway, in mouse embryonic fibroblasts. These effects did not appear to be mediated by deregulation of Hic1 target gene expression or loss of Tp53 function, and rather support a role for Hic1 in maintaining genome integrity during sustained replicative stress. Loss of Hic1 function also cooperated with activation of oncogenic KRas in the adult airway epithelium of mice, resulting in the formation of highly pleomorphic adenocarcinomas with a micropapillary phenotype in vivo. These results suggest that loss of Hic1 expression in the early stages of tumor formation may contribute to malignant transformation through the acquisition of chromosomal instability.