Null Mutation Research Articles

Tyrosine phosphorylation controlled poly(A) polymerase I activity regulates general stress response in bacteria.

RNA 3'-end polyadenylation that marks transcripts for degradation is implicated in general stress response in Escherichia coli Yet, the mechanism and regulation of poly(A) polymerase I (PAPI) in stress response are obscure. We show that pcnB (that encodes PAPI)-null mutation widely stabilises stress response mRNAs and imparts cellular tolerance to multiple stresses, whereas PAPI ectopic expression renders cells stress-sensitive. We demonstrate that there is a substantial loss of PAPI activity on stress exposure that functionally phenocopies pcnB-null mutation stabilising target mRNAs. We identify PAPI tyrosine phosphorylation at the 202 residue (Y202) that is enormously enhanced on stress exposure. This phosphorylation inhibits PAPI polyadenylation activity under stress. Consequentially, PAPI phosphodeficient mutation (tyrosine 202 to phenylalanine, Y202F) fails to stimulate mRNA expression rendering cells stress-sensitive. Bacterial tyrosine kinase Wzc phosphorylates PAPI-Y202 residue, and that wzc-null mutation renders cells stress-sensitive. Accordingly, wzc-null mutation has no effect on stress sensitivity in the presence of pcnB-null or pcnB-Y202F mutation. We also establish that PAPI phosphorylation-dependent stress tolerance mechanism is distinct and operates downstream of the primary stress regulator RpoS.

A simple high-throughput method for automated detection of Drosophila melanogaster light-dependent behaviours

BackgroundLike most living organisms, the fruit fly Drosophila melanogaster exhibits strong and diverse behavioural reactions to light. Drosophila is a diurnal animal that displays both short- and long-term responses to light, important for, instance, in avoidance and light wavelength preference, regulation of eclosion, courtship, and activity, and provides an important model organism for understanding the regulation of circadian rhythms both at molecular and circuit levels. However, the assessment and comparison of light-based behaviours is still a challenge, mainly due to the lack of a standardised platform to measure behaviour and different protocols created across studies. Here, we describe the Drosophila Interactive System for Controlled Optical manipulations (DISCO), a low-cost, automated, high-throughput device that records the flies’ activity using infrared beams while performing LED light manipulations.ResultsTo demonstrate the effectiveness of this tool and validate its potential as a standard platform, we developed a number of distinct assays, including measuring the locomotor response of flies exposed to sudden darkness (lights-off) stimuli. Both white-eyed and red-eyed wild-type flies exhibit increased activity after the application of stimuli, while no changes can be observed in Fmr1 null allele flies, a model of fragile X syndrome. Next, to demonstrate the use of DISCO in long-term protocols, we monitored the circadian rhythm of the flies for 48 h while performing an alcohol preference test. We show that increased alcohol consumption happens intermittently throughout the day, especially in the dark phases. Finally, we developed a feedback-loop algorithm to implement a place preference test based on the flies’ innate aversion to blue light and preference for green light. We show that both white-eyed and red-eyed wild-type flies were able to learn to avoid the blue-illuminated zones.ConclusionsOur results demonstrate the versatility of DISCO for a range of protocols, indicating that this platform can be used in a variety of ways to study light-dependent behaviours in flies.

Dll1 haploinsufficiency causes brain abnormalities with functional relevance.

The Notch pathway is fundamental for the generation of neurons during development. We previously reported that adult mice heterozygous for the null allele of the gene encoding the Delta-like ligand 1 for Notch (Dll1lacZ ) have a reduced neuronal density in the substantia nigra pars compacta. The aim of the present work was to evaluate whether this alteration extends to other brain structures and the behavioral consequences of affected subjects. Brains of Dll1 +/lacZ embryos and mice at different ages were phenotypically compared against their wild type (WT) counterpart. Afterwards, brain histological analyses were performed followed by determinations of neural cell markers in tissue slices. Neurological deficits were diagnosed by applying different behavioral tests to Dll1 +/lacZ and WT mice. Brain weight and size of Dll1 +/lacZ mice was significantly decreased compared with WT littermates (i.e., microcephaly), a phenotype detected early after birth. Interestingly, enlarged ventricles (i.e., hydrocephalus) was a common characteristic of brains of Dll1 haploinsufficient mice since early ages. At the cell level, general cell density and number of neurons in several brain regions, including the cortex and hippocampus, of Dll1 +/lacZ mice were reduced as compared with those regions of WT mice. Also, fewer neural stem cells were particularly found in the adult dentate gyrus of Dll1 +/lacZ mice but not in the subventricular zone. High myelination levels detected at early postnatal ages (P7-P24) were an additional penetrant phenotype in Dll1 +/lacZ mice, observation that was consistent with premature oligodendrocyte differentiation. After applying a set of behavioral tests, mild neurological alterations were detected that caused changes in motor behaviors and a deficit in object categorization. Our observations suggest that Dll1 haploinsufficiency limits Notch signaling during brain development which, on one hand, leads to reduced brain cell density and causes microcephaly and hydrocephalus phenotypes and, on the other, alters the myelination process after birth. The severity of these defects could reach levels that affect normal brain function. Therefore, Dll1 haploinsufficiency is a risk factor that predisposes the brain to develop abnormalities with functional consequences.

The CUL3 adaptor protein SPOP plays a role in tauopathy mechanisms

AbstractBackgroundThe pathological accumulation of the microtubule binding protein tau drives age‐related neurodegeneration in a variety of disorders, collectively called tauopathies. In the most common tauopathy, Alzheimer’s disease (AD), the accumulation of pathological tau strongly correlates with cognitive decline. The underlying molecular mechanisms that drive neurodegeneration in tauopathies remain unknown and no effective disease modifying pharmacological interventions for tauopathy currently exist.MethodsWe employed classical forward genetic approaches and identified multiple loss of function alleles in the C. elegans spop‐1 gene that ameliorate tauopathy, suggesting SPOP is required for tau mediated neurodegeneration. CRISPR based genome editing methodology enabled the generation of customized SPOP‐1 loss of function and null alleles. Molecular genetics, behavioral, neuronal reporter assays, and biochemical analyses were also employed to characterize the consequences of spop‐1 loss of function on tauopathy related phenotypes in model systems.ResultsKnockout of SPOP‐1 rescues tau mediated behavioral deficits caused by neuronal dysfunction in tau transgenic C. elegans. Biochemical analysis revealed that SPOP‐1 loss of function promotes clearance of phosphorylated and total tau species from C. elegans neurons, but no change in tau transgene mRNA levels. Tau transgenic animals exhibit obvious neurodegeneration of GABAergic neurons, but loss of spop‐1 rescues neurodegeneration. While SPOP functions as an CUL3 E3 ligase adaptor protein, CUL3 function is not required for SPOP loss of function rescue of tauopathy. Genetic epistasis analysis suggests the nuclear speckle resident poly(A) RNA binding protein sut‐2 and spop‐1 function in a parallel molecular pathway.ConclusionsSPOP is a novel modifier of tauopathy phenotypes. Combined with previous findings investigating ALYREF, PARN/TOE1, SUT‐1 and SUT‐2/MSUT2, this work suggests phase‐separated nuclear speckles are an important cellular site controlling susceptibility to pathological tau. Recent work showing SPOP modification of PR dipeptides derived from C9orf72 expansion suggests common pathways may be at work in the neurodegenerative molecular mechanisms of tauopathy and repeat dipeptides.

Mitochondrial cristae architecture protects against mtDNA release and inflammation.

Mitochondrial damage causes mitochondrial DNA (mtDNA) release to activate the type I interferon (IFN-I) response via the cGAS-STING pathway. mtDNA-induced inflammation promotes autoimmune- and aging-related degenerative disorders. However, the global picture of inflammation-inducing mitochondrial damages remains obscure. Here, we have performed a mitochondria-targeted CRISPR knockout screen for regulators of the IFN-I response. Strikingly, our screen reveals dozens of hits enriched with key regulators of cristae architecture, including phospholipid cardiolipin and protein complexes such as OPA1, mitochondrial contact site and cristae organization (MICOS), sorting and assembly machinery (SAM), mitochondrial intermembrane space bridging (MIB), prohibitin (PHB), and the F1Fo-ATP synthase. Disrupting these cristae organizers consistently induces mtDNA release and the STING-dependent IFN-I response. Furthermore, knocking out MTX2, a subunit of the SAM complex whose null mutations cause progeria in humans, induces a robust STING-dependent IFN-I response in mouse liver. Taken together, beyond revealing the central role of cristae architecture to prevent mtDNA release and inflammation, our results mechanistically link mitochondrial cristae disorganization and inflammation, two emerging hallmarks of aging and aging-related degenerative diseases.

Abstract PR011: Histone demethylase KDM5D drives sex-specific differences in colorectal cancer

Abstract Gender exerts a profound impact on cancer incidence, spectrum and outcomes, yet the molecular genetic bases of such sex differences are ill-defined and presumptively ascribed to X-chromosome genes and sex hormones. Such sex differences are particularly prominent in colorectal cancer (CRC) where men experience higher metastases and mortality. A murine CRC model, engineered with an inducible oncogenic Kras transgene (KRAS*) and conditional null alleles of Apc and Trp53 tumor suppressors (designated iKAP), revealed higher metastases and worse outcomes specifically in males with KRAS* CRC. Integrated cross-species molecular and transcriptomic analyses identified Y-chromosome gene histone demethylase KDM5D as a transcriptionally up-regulated gene driven by KRAS*-mediated activation of the STAT4 transcription factor. KDM5D-dependent chromatin mark and transcriptome changes showed repression of regulators of epithelial cell tight junction and MHC class I complex components. Deletion of KDM5D in iKAP cancer cells increased tight junction integrity, decreased cell invasiveness, and enhanced cancer cell killing by CD8+ T cells. Conversely, iAP mice engineered with a Kdm5d transgene to provide constitutive KDM5D expression specifically in iAP cancer cells exhibited an increased propensity for more invasive tumors in vivo. Thus, KRAS*-STAT4-mediated upregulation of Y chromosome KDM5D contributes significantly to the sex differences in KRAS* CRC via its disruption of cancer cell adhesion properties and tumor immunity, thus providing an actionable therapeutic strategy for metastasis risk reduction for men afflicted with KRAS* CRC. Citation Format: Jiexi Li, Zhengdao Lan, Wenting Liao, James W. Horner, Jielin Liu, Shan Jiang, Hong S. Shim, Max Slotnik, Kyle A. LaBella, Wen-Hao Hsu, Denise J. Spring, Y. Alan Wang, Ronald A. DePinho. Histone demethylase KDM5D drives sex-specific differences in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr PR011.

Epithelial morphogenesis in the Drosophila egg chamber requires Parvin and ILK.

Integrins are the major family of transmembrane proteins that mediate cell-matrix adhesion and have a critical role in epithelial morphogenesis. Integrin function largely depends on the indirect connection of the integrin cytoplasmic tail to the actin cytoskeleton through an intracellular protein network, the integrin adhesome. What is currently unknown is the role of individual integrin adhesome components in epithelia dynamic reorganization. Drosophila egg chamber consists of the oocyte encircled by a monolayer of somatic follicle epithelial cells that undergo specific cell shape changes. Egg chamber morphogenesis depends on a developmental array of cell-cell and cell-matrix signalling events. Recent elegant work on the role of integrins in the Drosophila egg chamber has indicated their essential role in the early stages of oogenesis when the pre-follicle cells assemble into the follicle epithelium. Here, we have focused on the functional requirement of two key integrin adhesome components, Parvin and Integrin-Linked Kinase (ILK). Both proteins are expressed in the developing ovary from pupae to the adult stage and display enriched expression in terminal filament and stalk cells, while their genetic removal from early germaria results in severe disruption of the subsequent oogenesis, leading to female sterility. Combining genetic mosaic analysis of available null alleles for both Parvin and Ilk with conditional rescue utilizing the UAS/Gal4 system, we found that Parvin and ILK are required in pre-follicle cells for germline cyst encapsulation and stalk cell morphogenesis. Collectively, we have uncovered novel developmental functions for both Parvin and ILK, which closely synergize with integrins in epithelia.

Nephrotic Syndrome Gene TBC1D8B Is Required for Endosomal Maturation and Nephrin Endocytosis in Drosophila.

Variants in TBC1D8B cause nephrotic syndrome. TBC1D8B is a GTPase-activating protein for Rab11 (RAB11-GAP) that interacts with nephrin, but how it controls nephrin trafficking or other podocyte functions remains unclear. We generated a stable deletion in Tbc1d8b and used microhomology-mediated end-joining for genome editing. Ex vivo functional assays utilized slit diaphragms in podocyte-like Drosophila nephrocytes. Manipulation of endocytic regulators and transgenesis of murine Tbc1d8b provided a comprehensive functional analysis of Tbc1d8b. A null allele of Drosophila TBC1D8B exhibited a nephrocyte-restricted phenotype of nephrin mislocalization, similar to patients with isolated nephrotic syndrome who have variants in the gene. The protein was required for rapid nephrin turnover in nephrocytes and for endocytosis of nephrin induced by excessive Rab5 activity. The protein expressed from the Tbc1d8b locus bearing the edited tag predominantly localized to mature early and late endosomes. Tbc1d8b was required for endocytic cargo processing and degradation. Silencing Hrs, a regulator of endosomal maturation, phenocopied loss of Tbc1d8b. Low-level expression of murine TBC1D8B rescued loss of the Drosophila gene, indicating evolutionary conservation. Excessive murine TBC1D8B selectively disturbed nephrin dynamics. Finally, we discovered four novel TBC1D8B variants within a cohort of 363 patients with FSGS and validated a functional effect of two variants in Drosophila, suggesting a personalized platform for TBC1D8B-associated FSGS. Variants in TBC1D8B are not infrequent among patients with FSGS. TBC1D8B, functioning in endosomal maturation and degradation, is essential for nephrin trafficking.

Stable ARMADILLO REPEAT KINESIN 2 in light inhibits hypocotyl elongation and facilitates light-induced cortical microtubule reorientation in Arabidopsis.

Hypocotyls undergo different morphogenesis in light and dark conditions, with cortical microtubules being reoriented in response to light to coordinate cell growth status. Kinesins are microtubule-based motor proteins that are mostly responsible for transporting organelles and vesicles, although some can also regulate microtubule organization; however, it is currently not known whether they are involved in microtubule reorientation and hypocotyl elongation. In this study, we found that ARMADILLO REPEAT KINESIN 2 (ARK2) negatively regulated the hypocotyl elongation of Arabidopsis. The hypocotyl cells of plants with the ark2 null allele were longer than those of the wild type and had relatively more transversely arranged cortical microtubules. In addition, ARK2 co-localized with cortical microtubules and facilitated the light-induced reorientation of the cortical microtubule arrays. Interestingly, the ARK2 protein is stable in the light and degraded through the 26S proteasome pathway in the dark. Furthermore, we determined that ARK2 could interact with the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), which contributed to down-regulation of ARK2 in darkness that might benefit hypocotyl growth in the dark.

The Caenorhabditis elegans ARIP-4 DNA helicase couples mitochondrial surveillance to immune, detoxification, and antiviral pathways

Surveillance of Caenorhabditis elegans mitochondrial status is coupled to defense responses such as drug detoxification, immunity, antiviral RNA interference (RNAi), and regulation of life span. A cytochrome p540 detoxification gene, cyp-14A4, is specifically activated by mitochondrial dysfunction. The nuclear hormone receptor NHR-45 and the transcriptional Mediator component MDT-15/MED15 are required for the transcriptional activation of cyp-14A4 by mitochondrial mutations, gene inactivations, or toxins. A genetic screen for mutations that fail to activate this cytochrome p450 gene upon drug or mutation-induced mitochondrial dysfunction identified a DNA helicase ARIP-4 that functions in concert with the NHR-45 transcriptional regulatory cascade. In response to mitochondrial dysfunction, ARIP-4 and NHR-45 protein interaction is enhanced, and they relocalize from the nuclear periphery to the interior of intestinal nuclei. NHR-45/ARIP-4 also regulates the transcriptional activation of the eol-1 gene that encodes a decapping enzyme required for enhanced RNAi and transgene silencing of mitochondrial mutants. In the absence of arip-4, animals were more susceptible to the mitochondrial inhibitor antimycin. Thus, ARIP-4 serves as a transcriptional coactivator of NHR-45 to promote this defense response. A null mutation in arip-4 extends the life span and health span of both wild type and a mitochondrial mutant, suggesting that the activation of detoxification pathways is deleterious to health when the mitochondrial dysfunction is caused by mutation that cannot be cytochrome p450-detoxified. Thus, arip-4 acts in a pathway that couples mitochondrial surveillance to the activation of downstream immunity, detoxification, and RNAi responses.

Discovery of a novel HLA-C*04 null allele, HLA-C*04:279N, in a Singaporean individual.

A nucleotide mutation in codon 75 of HLA-C*04:01:01:01 results in a novel allele HLA-C*04:279N.

MYB44-ENAP1/2 restricts HDT4 to regulate drought tolerance in Arabidopsis.

Histone acetylation has been shown to involve in stress responses. However, the detailed molecular mechanisms that how histone deacetylases and transcription factors function in drought stress response remain to be understood. In this research, we show that ENAP1 and ENAP2 are positive regulators of drought tolerance in plants, and the enap1enap2 double mutant is more sensitive to drought stress. Both ENAP1 and ENAP2 interact with MYB44, a transcription factor that interacts with histone deacetylase HDT4. Genetics data show that myb44 null mutation enhances the sensitivity of enap1enap2 to drought stress. Whereas, HDT4 negatively regulates plant drought response, the hdt4 mutant represses enap1enap2myb44 drought sensitive phenotype. In the normal condition, ENAP1/2 and MYB44 counteract the HDT4 function for the regulation of H3K27ac. Upon drought stress, the accumulation of MYB44 and reduction of HDT4 leads to the enrichment of H3K27ac and the activation of target gene expression. Overall, this research provides a novel molecular mechanism by which ENAP1, ENAP2 and MYB44 form a complex to restrict the function of HDT4 in the normal condition; under drought condition, accumulated MYB44 and reduced HDT4 lead to the elevation of H3K27ac and the expression of drought responsive genes, as a result, plants are drought tolerant.

293 Application of nanopore sequencing in identifying null mutations and intragenic copy number variations in filaggrin gene

Mutations in the gene encoding filaggrin (FLG) have been identified as the cause of ichthyosis vulgaris (IV) and shown to be major predisposing factors for atopic dermatitis. The profilaggrin protein consists of 10-12 filaggrin monomer repeats arranged in tandem, which arise from an ultra-long sequence of exon 3 comprised of near-indistinguishable repeats, demonstrating intragenic copy number variations (CNVs). As such, it was not until 2006 that null mutations in FLG could be detected using the newly developed long-range polymerase chain reaction (PCR) technique.

186 Dissecting the role of the common neurotrophin receptor CD271 in the skin: generation and characterization of a novel mouse model with keratinocyte-specific conditional deletion

The common neurotrophin receptor CD271, also called p75NTR, is the transmembrane low-affinity receptor for neurotrophins, belonging to the TNF superfamily. In the skin, CD271 is involved in the differentiation process and seems like to be implicated in cutaneous pathogenesis. To date, different animal models have been developed to obtain null mutations of CD271, but they defy to explain CD271 precise role in keratinocytes. By using a Keratin14-Cre mediated excision of the CD271 gene flanked by loxP sites, we generated a novel mouse model with a conditional deletion of CD271 in the epidermis.

A novel HLA-C null allele, HLA-C*08:236N, identified by next-generation sequencing in a Chinese individual.

HLA-C*08:236N differs from C*08:01:01 by a single nucleotide exchange in exon 5 at position 1991.

Testis-specific serine kinase 3 is required for sperm morphogenesis and male fertility.

The importance of phosphorylation in sperm during spermatogenesis has not been pursued extensively. Testis-specific serine kinase 3 (Tssk3) is a conserved gene, but TSSK3 kinase functions and phosphorylation substrates of TSSK3 are not known. The goals of our studies were to understand the mechanism of action of TSSK3. We analyzed the localization of TSSK3 in sperm, used CRISPR/Cas9 to generate Tssk3 knockout (KO) mice in which nearly all of the Tssk3 open reading frame was deleted (ensuring it is a null mutation), analyzed the fertility of Tssk3 KO mice by breeding mice for 4 months, and conducted phosphoproteomics analysis of male testicular germ cells. TSSK3 is expressed in elongating sperm and localizes to the sperm tail. To define the essential roles of TSSK3 in vivo, heterozygous (HET) or homozygous KO male mice were mated with wild-type females, and fertility was assessed over 4 months; Tssk3 KO males are sterile, whereas HET males produced normal litter sizes. The absence of TSSK3 results in disorganization of all stages of testicular seminiferous epithelium and significantly increased vacuolization of germ cells, leading to dramatically reduced sperm counts and abnormal sperm morphology; despite these histologic changes, Tssk3 null mice have normal testis size. To elucidate the mechanisms causing the KO phenotype, we conducted phosphoproteomics using purified germ cells from Tssk3 HET and KO testes. We found that proteins implicated in male infertility, such as GAPDHS, ACTL7A, ACTL9, and REEP6, showed significantly reduced phosphorylation in KO testes compared to HET testes, despite unaltered total protein levels. We demonstrated that TSSK3 is essential for male fertility and crucial for phosphorylation of multiple infertility-related proteins. These studies and the pathways in which TSSK3 functions have implications for human male infertility and nonhormonal contraception.

Understanding Mendelian errors in SNP arrays data using a Gochu Asturcelta pig pedigree: genomic alterations, family size and calling errors

Up to 478 Gochu Asturcelta pig parents-offspring trios (61 different families) were genotyped using the Axiom_PigHDv1 Array to identify the causes of Mendelian errors (ME). Up to 545,364 SNPs were retained. Up to 40,540 SNPs gathering 292,297 allelic mismatches were identified and were overlapped with SINEs and LINEs (Sscrofa genome 11.1). Copy number variations (CNV) were called using PennCNV. ME were classified into eight different classes according to the trio member (“Trio” meaning no assignment) and the allele on which ME was identified: TrioA/B, FatherA/B, MotherA/B, OffspringA/B. Most ME occurred due to systematic causes: (a) those assigned to the Father, Mother or Offspring occurred by null or partial null alleles characterized by heterozygote deficiency, varied with family size, involved a low number of loci (6506), and gathered most mismatches (228,145); (b) TrioB errors varied with family size, covaried with SINEs, LINEs and CNV, and involved most ME loci (33,483) and mismatches (65,682); and (c) TrioA errors were non-systematic ME with no sampling bias involving 1.2% of mismatches only and a low number of loci (1939). The influence of TrioB errors on the overall genotyping quality may be low and, since CNV vary among populations, their removal should be considered in each particular dataset. ME assignable to the Father, Mother or Offspring may be consistent within technological platforms and may bias severely linkage or association studies. Most ME caused by null or partial null alleles can be removed using heterozygote deficiency without affecting the size of the datasets.

Prospective and Retrospective Observational Study in Adult and Pediatric Patients with GATA2 Variants (GATA2 Deficiency)

Heterozygous germline GATA2 variants are associated with a broad spectrum of manifestations, encompassing immuno-haematological alterations, severe/recurrent infections and extra-haematological phenomena. We here describe 25 patients (11 males) with molecular diagnosis of GATA2 deficiency identified in 9 AIEOP (Associazione Italiana di Ematologia Oncologia Pediatrica) centers in Italy (Table 1). Patients' median age at onset of symptoms was 12.5 years. The main signs/symptoms at onset were severe/recurrent infections (40%), cytopenias (20%) and myelodysplasia (MDS)/acute myeloid leukemia (AML) (20%). The prevalence of these manifestations increased during the follow-up and further clinical manifestations, i.e.hepatosplenomegaly, haemorrhagic diathesis, thrombosis, bone marrow failure, lymphedema, pulmonary alveolar proteinosis and sensorineural deafness, occurred. Once the diagnosis was established, congenital deafness was considered part of the clinical picture of GATA2 deficiency in two individuals. We identified a rare (myelofibrosis) and two possible new manifestations (pilonidal cyst/sacro-coccygeal fistula and cholangiocarcinoma). Complete blood count with differential and lymphocyte subsests revealed that monocytopenia (83%) and CD19+ lymphocytopenia (70%) were the most frequent and distinctive changes in patients without hematological malignancies or in whom we obtained data before the onset of MDS/AML (Table 2). Different myeloid neoplasms were observed: refractory cytopenia in childhood (RCC) (24%), AML (12%), MDS with excess blasts (MDS-EB) (12%), MDS-EB in transformation (MDS-EBt) (4%) and MDS with multilineage dysplasia(MDS-MLD) (8%). Monosomy 7 (8%) and trisomy 8 (16%) were the two most frequent karyotype abnormalities. Targeted next-generation sequencing (NGS) analysis for somatic mutations associated with myeloid neoplasms was performed in 48% of the patients, resulting positive in 8% of cases (i.e. EZH2 and MECOM in one individual each). The majority of GATA2 variants were located at zinc-finger 2 (ZF2) domain of the GATA2 protein. Missense and null mutations were the most frequent (46% each). Intronic variants were found in 8% of the cohort. We report 3 missense mutations out of the 4 cases of lymphedema described.Median age at molecular diagnosis was 18 years. The diagnostic delay (time between age at onset of symptoms and molecular diagnosis) had a median of 4 years, but, excluding who had onset of the disease before the first description of GATA2 deficiency (2011) the median was 1 year. Hematopoietic stem cell transplantation (HSCT) was the most widely used therapeutic option (52%). HSCT was performed in case of myeloid neoplasia (8/13) and bone marrow failure (3/13), but also in patients with immunodeficiency (i.e. hypogammaglobulinemia with immunoglobulin replacement therapy, immunodeficiency with previous severe infections). Time between molecular diagnosis and HSCT was1 year. The event free survival (EFS) was 20% at the age of 20 and 15% at the age of 40. The overall survival (OS) was 90% at the age of 20 and 70% at the age of 40. Family screening was essential to identify asymptomatic patients (8% in our series). In conclusion, our series represents the third largest national cohort after the ones described by Donadieu and Spinner (79 and 57 patients, respectively)1,2. GATA2 deficiency should be included in targeted gene panels for congenital deafness and we recommend to perform the sequencing of the regulatory region. Our data disproves the hypothesis that lymphedema is exclusively associated with null and regulatory mutations3, as we here describe three patients with lymphedema and missense mutations. HSCT should be considered in case of patients with immunodeficiency without myeloid neoplasms. Family screening should be offered to first degree relatives, as identification of asymptomatic GATA2 deficient patients could allow to exclude potential HSC donors and to investigate risk factors that may explain the phenotypic difference.

Efficacy of Vcp/p97 Inhibitor, CB-5339, Alone and in Combinations Against High-Risk AML, Including Those with Genetic Lesion in TP53

Valosin-containing protein (VCP)/p97 or p97 is an AAA+ type ATPase involved in quality control of cellular proteins in normal and transformed cells, especially under cellular stress. VCP/p97 alleviates proteotoxic stress by facilitating bulk protein degradation through the ubiquitin-proteasome system (UPS), harnessing the energy of ATP hydrolysis to unfold and extract its ubiquitylated protein substrates from protein complexes or organelles and targeting them for proteasomal degradation, e.g., ER-associated degradation (ERAD). Targeted inhibition of p97 induces lethal ER stress. The vital role of p97 in protein homeostasis has been recognized as one of the key non-oncogenic addictions in transformed cells. Targeting p97 has been previously shown to yield preferential lethality in cancer versus normal cells and exhibit in vivo efficacy against xenograft models of cancers. By probing the CRISPR-screen dependency map (DepMap), we found that p97 is a dependency across AML cell lines, including those harboring alterations in genes that indicate an increased risk or poor prognosis. CB-5339 is a selective and potent, ATP-competitive inhibitor of p97, with only low inhibitory activity against other ATPases, AAA+ ATPases, and kinases. CB-5339 was previously shown to induce accumulation of polyubiquitylated proteins, retention of ERAD substrates, and lethal ER stress in cancer cells mediated by CHOP, DR5 and NOXA. In present studies, we determined that treatment with CB-5339 dose-dependently (200 to 1000 nM for 48 hours) induces in vitro loss of viability in AML cell lines (OCI-AML3, MOLM13, SET2 and HEL92.1.7 cells), as well as in fresh, patient-derived (PD) AML cells, including those with TP53 mutations and/or TP53 allelic loss, or with 3q26 (MECOM locus) lesions and EVI1 overexpression. Utilizing MOLM13 cells edited to express the missense mutation R175H or R248Q, or the null allele of TP53, introduced via CRISPR/Cas9 into the endogenous TP53 locus, we determined that CB-5339 was active against MOLM13 cells (with MLL-AF9, FLT3-ITD and wild-type TP53) and retained significant activity against MOLM13 cells with TP53-R175H, TP53-R248Q and MOLM13-TP53-/-. Comparing ATAC-Seq and RNA-Seq findings in CB-5339-treated versus -untreated MOLM13 cells we found concordant, significantly altered chromatin accessibility and mRNA expressions of gene-loci, e.g., increase in ATF4, ATF3, HSPA5 (GRP78), NOXA, BBC3 (PUMA), FBXO32, ITGAM, and CD86, but decrease in FOXA1, TLR4, CLEC12A, CXCR4 and IL7R. Following CB-5339 treatment, gene-set enrichment analyses (GSEA) showed significantly increased normalized enrichment scores of the gene-set involved in ER stress response, not only in MOLM13 with WT-TP53 but also in those with the TP53 mutations or allelic loss of TP53. However, treatment with CB-5339 increased levels of p-ATM, p-TP53 and TP53 only in MOLM13 and OCI-AML3 (each expressing only WT-TP53), without altering the protein levels of MDM2/4. In contrast, CB-5339-induced loss of viability correlated with augmented levels of ATF4, CHOP, as well as of pro-death proteins NOXA, DR5 and active BAX in AML cells regardless of TP53 status. Importantly, similar effects were also noted in PD AML cells with or without TP53 mutation. Co-treatment with relatively lower concentrations of CB-5339 (100 to 225 nM) and venetoclax (5 to 20 nM) exerted synergistic in vitro lethality in not only MOLM13 but also PD AML cells with WT-TP53, as well as in SET2 and PD AML cells harboring mutant TP53. Co-treatment with CB-5339 and pan-BET inhibitor OTX015 was also synergistically lethal against MOLM13 and PD AML cells with or without mutant TP53. Finally, in a tail-vein infused, luciferase transduced, aggressive xenograft model of MOLM13 cells, after AML engraftment, co-treatment for 3 weeks with CB-5339 (50 mg/kg/day, PO) and either venetoclax (30 mg/kg/day, PO) or OTX015 (30 mg/kg/day, PO), as compared to treatment with vehicle or each drug alone, significantly reduced the AML burden and improved median and overall survival of the NSG mice, without inducing significant toxicity. Taken together, these findings highlight that CB-5339 induces lethal ER stress in AML cells regardless of the TP53 status, and underscore the promise of CB-5339 treatment alone and in rational combinations in exerting efficacy against AML, including those with high-risk genetic alterations in TP53 or chromosome 3q26 lesions and EVI1 overexpression.

Ancestral reconstruction and correlation of the frequencies of the hemoglobin S allele and the Duffy blood group alleles in human populations.

Malaria is an important selective force for human genetic adaptation due to the sustained, lethal impact it has had on populations worldwide. High frequencies of both hemoglobin S and the null allele FYBES of the Duffy blood group have been found in areas where this disease is endemic, attributed to the protective action of the carriers of these variants against malaria infection. The objective of this work was to perform ancestral reconstruction and analyze the correlation of the frequencies of these alleles throughout the phylogeny of 24 human populations. A tree topology and the allelic frequencies reported in the literature for the 24 populations were used. The ancestral frequencies for the two alleles were reconstructed using the maximum likelihood method and the Brownian model of evolution (CI=95%), and the correlation analysis was performed using phylogenetically independent contrasts (PICs). Statistical analyses were performed with the statistical software R version 3.4.1. For both alleles, a correspondence was found in the reconstruction of the ancestral frequencies, and a significant statistical correlation (p=.001) was observed between the S and FYBES alleles. These results provide evidence of an epistatic relationship between the two alleles, which may influence the fitness of the individuals who present with them when they are subjected to a selective force such as malaria.