CRISPR gene-drive systems based on Cas9 nickases promote super-Mendelian inheritance in Drosophila

ABSTRACTCRISPR-based gene drive systems can be used to modify entire wild populations due to their ability to bias their own inheritance towards super-Mendelian rates (>100%). Current gene drives contain a Cas9 and a gRNA gene inserted at the location targeted by the gRNA. These gene products are able to cut the opposing wildtype allele, and lead to its replacement with a copy of the gene drive through the homology-directed DNA repair pathway. When this allelic conversion occurs in the germline it leads to the preferential inheritance of the engineered allele — a property that has been proposed to disseminate engineered traits for managing disease-transmitting mosquito populations. Here, we report a novel gene-drive strategy relying on Cas9 nickases which operates by generating staggered paired-nicks in the DNA to promote propagation of the gene drive allele. We show that only when 5’ overhangs are generated, the system efficiently leads to allelic conversion. Further, the nickase gene-drive arrangement produces large stereotyped deletions, providing potential advantages for targeting essential genes. Indeed, the nickase-gene-drive design should expand the options available for gene drive designs aimed at applications in mosquitoes and beyond.

Changing pattern of Plasmodium falciparum multi-drug resistance-1 gene polymorphisms in southern Rwanda

Plasmodium falciparum multidrug resistance-1 gene ( pfmdr1 ) polymorphisms associate with altered antimalarial susceptibility. Between 2010 and 2018/19, we observed that the prevalence of the wildtype allele N86 and the wildtype combination NYD increased ten-fold (4% versus 40%) and more than two-fold (18% versus 44%), respectively. Haplotypes other than NYD or NFD declined by up to >90%. Our molecular data suggest the pfmdr1 pattern to have shifted towards one associated with artemether-lumefantrine resistance.

Characteristics of Adrenocortical Carcinoma Associated With Lynch Syndrome

Context Lynch syndrome (LS) is the most common inherited colorectal and endometrial cancer syndrome, caused by germline mutations in DNA mismatch repair (MMR) genes. It is also characterized by an increased risk of other tumors with lower prevalence, such as adrenal cortical carcinoma (ACC), an endocrine tumor with an incidence of <2 cases/million individuals/year. Most ACC developed during childhood are associated with hereditary syndromes. In adults, this association is not as well established as in children. Previous studies showed a 3.2% prevalence of LS among patients with ACC. Evidence Acquisition The objective of this study is to determine the prevalence of ACC in a Spanish LS cohort and their molecular and histological characteristics. This retrospective study includes 634 patients from 220 LS families registered between 1999 and 2018. Evidence Synthesis During the follow-up period, 3 patients were diagnosed with ACC (0.47%); all were carriers of a MSH2 germline mutation. The 3 ACC patients presented loss of expression of MSH2 and MSH6 proteins. One tumor analysis showed loss of heterozygosity of the MSH2 wildtype allele. Our findings support previous data that considered ACC as a LS spectrum tumor. Conclusion MMR protein immunohistochemistry screening could be an efficient strategy to detect LS in patients with ACC.

A TAC3 Missense Variant in a Domestic Shorthair Cat with Testicular Hypoplasia and Persistent Primary Dentition

A single male domestic shorthair cat that did not complete puberty was reported. At four years of age, it still had primary dentition, testicular hypoplasia, and was relatively small for its age. We hypothesized that the phenotype might have been due to an inherited form of hypogonadotropic hypogonadism (HH). We sequenced the genome of the affected cat and compared the data to 38 genomes from control cats. A search for private variants in 40 candidate genes associated with human HH revealed a single protein-changing variant in the affected cat. It was located in the TAC3 gene encoding tachykinin 3, a precursor protein of the signaling molecule neurokinin B, which is known to play a role in sexual development. TAC3 variants have been reported in human patients with HH. The identified feline variant, TAC3:c.220G>A or p.(Val74Met), affects a moderately conserved region of the precursor protein, 11 residues away from the mature neurokinin B sequence. The affected cat was homozygous for the mutant allele. In a cohort of 171 randomly sampled cats, 169 were homozygous for the wildtype allele and 2 were heterozygous. These data tentatively suggest that the identified TAC3 variant might have caused the suppression of puberty in the affected cat.

Sudemycin Selectively Inhibits Growth of Primary Murine Hematopoietic Cells Expressing Mutant U2AF1

Abstract 554 Core components of the pre-mRNA splicing complex (the “spliceosome”) are targets of recurrent somatic mutation in patients with myelodysplastic syndrome (MDS). Using whole genome and targeted resequencing, we previously identified recurrent missense mutations in U2AF1 (encoding U2AF35, the 35 kDa U2 accessory factor) at serine 34 (S34F or S34Y) or glutamine 157 (Q157R) in 8.7% of de novo MDS patients. The mechanism by which mutations in U2AF1 and other recurrently mutated spliceosome components contribute to MDS pathogenesis is unknown, but recurrent mutations in this pathway suggest that it may provide a novel therapeutic target. Pladienolide B and FR901464 are two bacterial products that modulate activity of the spliceosome. The derivatives, E7107 and spliceostatin A, and synthetic analogs (sudemycins), bind SF3B, a complex that includes SF3B1 and six other subunits of the spliceosome. Treatment of cell lines with these agents causes accumulation of aberrantly spliced gene products. Sudemycins have favorable pharmacokinetic properties (improved solubility, chemical stability, and plasma half-life) and induce cell cycle arrest and apoptosis in low nanomolar concentrations in cell lines in vitro. They also inhibit growth in xenograft models in vivo, with no evidence of toxicity in mice treated with pharmacologically active doses. We hypothesize that splicing gene mutations cause alterations in splice isoforms that provide a selective advantage to tumor cells, and that perturbation of this environment with small molecule spliceosome modulators may cause a selective disadvantage for cells with these mutations. To test this hypothesis, we generated MSCV-based retroviruses encoding the wildtype (MSCV-WT-ires-GFP) or S34F mutant (MSCV-MT- ires-GFP) U2AF1 alleles. We infected c-kit+ primary C57BL/6J murine bone marrow cells with these recombinant retroviruses (or empty vector) and cultured them in vitro with cytokine-supplemented media. Consistent with previous reports, vehicle-treated cells (0.1% DMSO) expressing the mutant allele had reduced growth over 5 days, compared to cells expressing the wildtype allele or empty vector (P<0.0001, n=5 technical replicates, two experiments). At doses between 10–150 nM, sudemycin (but not an inactive sudemycin analog) induced a reproducible dose-dependent decrease in cell growth, most significant at 75 nM (p<0.0001, n=5 technical replicates, two experiments), and an increase in apoptosis (p=<0.05, n=3 technical replicates), as measured by annexin V positivity, in cells expressing the mutant, compared to the wildtype allele or empty vector. To study the effect of the U2AF1 mutation and sudemycin exposure on splicing, we analyzed RNA isoforms of Mdm2 and Fmr1, two genes known to undergo alternative splicing. After retroviral transduction, GFP+ cells were sorted and RNA was isolated for RT-PCR analysis at baseline or 2h–6h after exposure to sudemycin (75 nM) or vehicle. The U2AF1 S34F mutation increased the abundance of Fmr1 transcripts utilizing a cryptic splice acceptor, compared to cells transduced with empty vector or wildtype U2AF1 (P<0.05, n=3 technical replicates). Sudemycin treatment decreased Fmr1 cryptic splice site utilization (p<0.05, n=3 technical replicates) and increased Mdm2 exon skipping (p<0.05, n=3 technical replicates) at 2h and 6hr, compared to vehicle-treated cells, with the most significant differences in cells expressing mutant U2AF1. These experiments provide proof-of-concept that cells expressing mutant U2AF1 have increased sensitivity to spliceosome modulators and warrant further investigation as a possible treatment for patients with these mutations. Ongoing experiments include in vivo experiments in mice transplanted with transduced cells, and in vitro treatment of primary clinical samples. Small molecule modulators of the spliceosome may provide a novel therapeutic strategy for MDS/AML patients with spliceosome mutations. Disclosures: No relevant conflicts of interest to declare.

The Role of Different Genetic Subtypes In CEBPA Mutated AML

Abstract 752 To evaluate the role of CEBPA mutations (CEBPAmut) in the context of other molecular mutations and cytogenetic aberrations we have analyzed 1567 AML cases for CEBPAmut. The patients were selected according to cytogenetics excluding the following karyotypes: t(15;17)/PML-RARA, t(8;21)/AML1-ETO, inv(16)/t(16;16)/CBFB-MYH11, inv(3)/t(3;3)/EVI1, t(6;9)/DEK-CAN and 11(q23)/MLL, and complex aberrations. The cohort was composed of 697 females and 870 males. Age ranges from 16.7 to 88.3 years (y) (median: 71.0 y). CEBPAmut were detected in 126/1567 cases (8.0%). The biologic characteristics of the CEBPAmut patients (age range 16.7 to 87.6 y, median: 64.8 y) were further investigated. Three different CEBPAmut patterns were observed: 1) in 50/126 cases (39.7%) one mutation and one wildtype allele were detected (monoallelic pattern), 2) 61 cases (48.4%) had two different mutations (biallelic pattern), 3) 15 cases (11.9%) had one mutation without detectable wildtype allele due to loss of heterozygosity (LOH). Overall we found 186 different mutations of following types: 1) 108 led to a premature N-terminal stop of the protein (6 due to a nonsense and 102 due to a frameshift mutation), 2) 60 were inframe mutations in the b-ZIP region, 3) 8 were frameshifts in the b-ZIP region and, 4) 2 were frameshifts 3`of the b-ZIP region, and 8 were C-terminal point mutations. Correlation to cytogenetics shows a normal karyotype (NK) in 86 (68.3%) of the 126 CEBPAmut patients whereas in 40 pts (31.7%) at least one cytogenetic aberration was detected (-7: n=7; +8: n=7, 9q-: n=2; 11q-: n=3, other trisomies: n=11; other non recurrent translocations: n=4, all others: n=6). Cytogenetic aberrations were more frequent in the monoallelic group (55%) compared to the biallelic (35%) (p=0.001) and to cases with LOH (10%) (p=0.047). Interestingly in the total cohort of 13 pts with monosomy 7 seven pts (53.8%) were CEBPA mutated (53.8%) and of these 6 were in the monoallelic group. Additional mutations were detected in 48 cases (RUNX1: n=11, NPM1: n=10, FLT3-ITD: n=20, FLT3-TKD: n=3, MLL-PTD: n=5, NRAS: n=9, IDH1: n=2, IDH2: n=7; 15 pts showed 2 and 2 pts 3 of these mutations). Similar to the cytogenetic aberrations the molecular mutations were more frequent in the monoallelic group (61.9%) compared to the biallelic (31.0%) and the LOH group (7.1%) (p=0.001). NPM1 mutations were mutually exclusive of biallelic CEBPAmut. As previously described we also detected a significantly higher expression of CD7 in the CEBPAmut compared to the CEBPAwt group (71.2% vs. 18.9%, p<0.001). Furthermore, CD7 was higher expressed in biallelic cases as compared to the monoallelic ones (86.2% vs. 43.8%, p=0.011). It was similar in the LOH group (71.4%) compared to the biallelic group. There was no influence of cytogenetic aberrations or any additional mutation on EFS and OS. Solely the presence of high FLT3-ITD load (>0.5 FLT3-ITD/FLT3wt) was correlated with a shorter EFS (EFS at 2 y: 20.3% vs. 44.8%; p=0.020) and OS (OS at 2 y 54% vs 68%, p=0.047) when compared to the combined group of FLT3wt and those with an FLT3-ITD load of <0.5. Regarding the different CEBPA groups the biallelic cases had a slightly better OS compared to monoallelic cases (OS at 2 y: 75.0% vs. 60.8%; n.s.). The 2-year OS in the LOH group was significantly lower (33.8%; p=0.023, compared to the biallelic group; and p=0.043 compared to 69.7% in the combined biallelic + monoallelic group). In addition, the different functional mutation types were analyzed. Out of frame mutations in b-ZIP had no specific impact on survival within the CEBPAmut cohort. N-terminal stop mutations and in frame mutations in b-ZIP were associated with favourable outcome (OS at 2 y: 72.1% vs. 43.9% all others mutations, p=0.007 and 2 y OS at 2 y: 76.3% vs. 46.9%; p=0.043, respectively), whereas all other mutations were extremely unfavourable (OS at 2 y: 0% vs. 70.5% compared to N-terminal and b-ZIP mutations, p<0.001). In summary, 1) the biology and prognostic impact varies depending on distinct CEBPAmut patterns. 2) Cytogenetic and molecular alterations had no prognostic impact with the exception of FLT3-ITD with a mutation load of >0.5 FLT3-ITD/FLT3wt. 3) Our data for the first time demonstrate that CEPBAmut with LOH are associated with an even inferior outcome than monoallelic mutations. In conclusion, these data show that CEBPA should be analyzed in detail in all NPM1wt NK AML and in those with unfavourable but non-complex karyotypes. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Prognostic Impact of FLT3 Mutation Load in NPM1 Mutated AML.

Abstract 826 According to the new WHO classification NPM1 mutated AML is defined as a provisional entity. Those AML without FLT3-ITD (also referred to as FLT3-LM) are regarded as prognostically favorable whereas those with detectable FLT3-ITD in addition are defined as unfavorable. However, some previous studies not taking NPM1 status into account have shown that the prognostic impact of the FLT3 mutation is highly dependent on the load of the mutation. To evaluate a possible impact of FLT3 load in NPM1 mutated AML we have quantified the FLT3 load in 641 NPM1 mutated AML.The cohort was composed of 341 females and 300 males with a median age of 65.2 years (range 17.8-88 years). The NPM1 mutation was detected by melting curve analysis whereas the FLT3-ITD was quantitatively analysed by fragment analysis (Gene Scan, 3130 sequence detection system, ABI). The FLT3-ITD load was quantified as the ratio of the mutation compared to the wildtype allele. All ratios >1 are indicative for allelic loss of the wildtype allele (wt). Overall in 242/641 (37.8%) of the NPM1 mutated AML an FLT3- ITD was detected. The length of the mutation (LM) varied between 9 and 579 bp (median: 51 bp). The ratio of FLT3- ITD was in the range between 0.016 and 44.85 (median: 0.565). Using Cox regression analysis we demonstrated that increasing FLT3-ITD/wt ratio has a significantly unfavorable influence on EFS (p=0.028) . At next, for Kaplan Meier analysis 3 groups were defined according to FLT3-ITD/wt ratio: 1) only FLT3wt (n=398); 2) FLT3-ITD/wt ratio ≤1 (n=189) and 3) FLT3-ITD/wt ratio ≥1 (n=53). It was shown that the FLT3wt group had the best median EFS of 230 days compared to 203 day in group 2 (p=0.032) and only 86 days in group 3 (p<0.001). In a next step the FLT3 ratios were further subdivided into smaller groups 1) only WT (n=398), 2) ratio <0.25 (n=63) 3) ratio > 0.25 but <0.5 (n=44); 4) ratio ≥0.5 but <1 (n=82) 5) ratio ≥1 (n=53). Although the median EFS was continuously decreasing from group 1 to 5 (230 vs 200 vs 198 vs 96 vs 86 days, respectively) a significant difference compared to the FLT3wt group was only detected for group 4 (p=0.014) and group 5 (p<0.001). This analysis demonstrated that only FLT3-ITD/wt ratios ≥0.5 led to inferior outcome in NPM1 mutated AML. The impact of the FLT3/wt ratio on overall survival (OS) could also be detected only for group 5 with a median OS of 142 days compared to 293 days in the FLT3-WT group (p<0.002) and for group 4 (median 147 days) (p=0.033), respectively. In addition other known prognostic factors were analysed for EFS: karyotype (p=0.788; n.s.), gender (p=0.042; better for female) age (p<0.001), WBC (p<0.001), FLT3-status irrespective of ratio (p=0.001) and FLT3-ITD length (p=0.002). In a multivariate analysis only age (p<0.001), WBC (p=0.001) and FLT3-ITD/wt ratio (p=0.042) came out to be independent prognostic factors in NPM1 mutated AML. For OS ratios ≥0.5 came out to be more important than FLT3 status per se (0=0.044 vs. p=0.359). Subsequently ratios '0.5 were multivariately tested against age and WBC. All three parameters came out to be of independent significance for OS (p<0.001, p=0.001, and p=0.008, respectively). In conclusion, this study clearly demonstrates that not FLT3-ITD status per se is predictive for survival in NPM1 mutated AML but ratios of FLT3-ITD load has to be taken into account. Only ratios of ≥0.5 demonstrated to have a significant impact on prognosis in NPM1 mutated AML. This data has enormous implication on clinical decision making in AML including the option of allogeneic transplantation in first CR. Disclosures: Schnittger: MLL Munich Leukemia Lab: Equity Ownership. Weiss:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Lab: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Chromosome Homology in Tetraploid Southern Highbush × Vaccinium elliottii Hybrids

A yellow-leaf seedling marker, r, was used to determine if there was preferential chromosome pairing in a group of tetraploid southern highbush blueberry hybrids. Plants with four copies of r (no copies of R) fail to develop anthocyanins, and cotyledons, hypocotyls, leaves, stems, and other vegetative tissues have a bright yellow-green color. In the hybrids that were studied, two genomes were from the diploid wild species, V. elliottii Chapman, and both carried the recessive marker r. The other two genomes were from southern highbush cultivars and both carried the dominant wildtype allele, R. When RRrr hybrids were intercrossed or crossed to rrrr yellow-leaf plants, the number of yellowleaf rrrr seedlings obtained usually equalled or exceeded the number predicted from nonpreferential chromosome pairing. Since rr gametes can only be produced by RRrr plants when R and r chromosomes pair at Meiosis I, there was no evidence that the chromosomes derived from V. elliottii were pairing at a higher-than-random rate.