Silent Mutations Research Articles

Update of the current knowledge on genetics, evolution, immunopathogenesis, and transmission for coronavirus disease 19 (COVID-19).

In December 2019, an acute respiratory disease caused by novel species of coronavirus (SARS-CoV-2), emerged in China and has spread throughout the world. On 11th March 2020, the World Health Organization (WHO) officially declared coronavirus disease 19 (COVID-19) a pandemic, severe coronavirus-mediated human disease. Based on genomic and phylogenetic studies, SARS-CoV-2 might originate from bat coronaviruses and infects humans directly or through intermediate zoonotic hosts. However, the exact origin or the host intermediate remains unknown. Genetically, SARS-CoV-2 is similar to several existing coronaviruses, particularly SARS-CoV, but differs by silent and non-silent mutations. The virus uses different transmission routes and targets cells and tissues with angiotensin-converting enzyme 2 (ACE2) protein, which makes it contagious. COVID-19 shares both the main clinical features and excessive/dysregulated cell responses with the two previous Middle East respiratory syndrome coronavirus (MERS) and severe acute respiratory syndrome coronavirus (SARS) epidemics. In this review, we provide an update of the current knowledge on the COVID-19 pandemic. Gaining a deeper understanding of SARS-CoV-2 structure, transmission routes, and molecular responses, will assist in the prevention and control of COVID-19 outbreaks in the future.

Molecular characterization of heat shock protein 70 gene in Iraqi buffalo

The heat shock protein 70 (HSP 70) has important roles in protecting cells and keeping them alive when exposed to different stress conditions. The polymorphism of the hsp 70 gene could be linked with the ability of stress tolerance. This study aimed to determine the polymorphism of the hsp 70 gene in Iraqi buffaloes and study bits effects on the resistance to stress. This study was conducted during from November 2018 to February 2019. The number of buffalo females used was 35 at the age of 4 - 6 years, which belonged to the local farmers from Basra city, Iraq. The DNA was extraction from the blood samples then the polymerase chain reaction (PCR) amplification was performed. The DNA sequences were analyzed by using bioinformatics analysis. The results of the molecular analysis showed that there were two groups of the hsp 70 gene as a compare with the same genes in GenBank due to silent and missense mutations. Based on these findings, it can be concluded that the Iraqi buffaloes have adapted to the surrounding environmental conditions as a result of the action of HSP 70 proliferation. The hsp 70 gene was a useful biomarker of stress tolerance in buffaloes.

Human genes differ by their UV sensitivity estimated through analysis of UV-induced silent mutations in melanoma

Human genes differ by their UV sensitivity estimated through analysis of UV-induced silent mutations in melanoma

Analysis of Codon Usage and Nucleotide Bias in Middle East Respiratory Syndrome Coronavirus Genes

The Middle East Respiratory Syndrome (MERS) is an emerging disease caused by a recently identified human coronavirus (CoV). Over 2494 laboratory-confirmed cases and 858 MERS-related deaths have been reported from 27 countries. MERS-CoV has been associated with a high case fatality rate, especially in patients with pre-existing conditions. Despite the fatal nature of MERS-CoV infection, a comprehensive study to explore its evolution and adaptation in different hosts is lacking. We performed codon usage analyses on 4751 MERS-CoV genes and determined underlying forces that affect the codon usage bias in the MERS-CoV genome. The current analyses revealed a low but highly conserved, gene-specific codon usage bias in the MERS-CoV genome. The codon usage bias is mainly shaped by natural selection, while mutational pressure emerged as a minor factor affecting codon usage in some genes. Other contributory factors included CpG dinucleotide bias, physical and chemical properties of encoded proteins and gene length. Results reported in this study provide considerable insights into the molecular evaluation of MERS-CoV and could serve as a theoretical basis for optimizing MERS-CoV gene expression to study the functional relevance of various MERS-CoV proteins. Alternatively, an attenuated vaccine strain containing hundreds of silent mutations could be engineered. Codon de-optimization will not affect the amino acid sequence or antigenicity of a vaccine strain, but the sheer number of mutations would make viral reversion to a virulent phenotype extremely unlikely.

Specific targeting of PDGFRβ in the stroma inhibits growth and angiogenesis in tumors with high PDGF-BB expression.

PDGF-BB/PDGFRβ signaling plays an important role during vascularization by mediating pericyte recruitment to the vasculature, promoting the integrity and function of vessels. Until now it has not been possible to assess the specific role of PDGFRβ signaling in tumor progression and angiogenesis due to lack of appropriate animal models and molecular tools.Methods: In the present study, we used a transgenic knock-in mouse strain carrying a silent mutation in the PDGFRβ ATP binding site that allows specific targeting of PDGFRβ using the compound 1-NaPP1. To evaluate the impact of selective PDGFRβ inhibition of stromal cells on tumor growth we investigated four tumor cell lines with no or low PDGFRβ expression, i.e. Lewis lung carcinoma (LLC), EO771 breast carcinoma, B16 melanoma and a version of B16 that had been engineered to overexpress PDGF-BB (B16/PDGF-BB).Results: We found that specific impairment of PDGFRβ kinase activity by 1-NaPP1 treatment efficiently suppressed growth in tumors with high expression of PDGF-BB, i.e. LLC and B16/PDGF-BB, while the clinically used PDGFRβ kinase inhibitor imatinib did not suppress tumor growth. Notably, tumors with low levels of PDGF-BB, i.e. EO771 and B16, neither responded to 1-NaPP1 nor to imatinib treatment. Inhibition of PDGFRβ by either drug impaired tumor vascularization and also affected pericyte coverage; however, specific targeting of PDGFRβ by 1-NaPP1 resulted in a more pronounced decrease in vessel function with increased vessel apoptosis in high PDGF-BB expressing tumors, compared to treatment with imatinib. In vitro analysis of PDGFRβ ASKA mouse embryo fibroblasts and the mesenchymal progenitor cell line 10T1/2 revealed that PDGF-BB induced NG2 expression, consistent with the in vivo data.Conclusion: Specific targeting of PDGFRβ signaling significantly inhibits tumor progression and angiogenesis depending on PDGF-BB expression. Our data suggest that targeting PDGFRβ in the tumor stroma could have therapeutic value in patients with high tumor PDGF-BB expression.

DNA Gyrase and Topoisomerase IV Mutations and their effect on Quinolones Resistant Proteus mirabilis among UTIs Patients.

Objective:This study aimed to highlight the importance of mutations within Proteus mirabilis genome that are related to fluoroquinolone resistance.Methods:This is a cross sectional study performed in different teaching hospitals in Khartoum State from June 2016 to May 2017. A total of (120) P mirabilis isolates from patients with symptoms of UTIs attending different hospitals in Khartoum State were examined. First, modified Kurby Bauer method was performed for phenotypical detection of resistant isolates. Then polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by sequencing were applied for detection of mutations in GyrA, GyrB, ParC and ParE genes of isolates.Results:P. mirabilis showed 30% resistance to ciprofloxacin. All samples revealed mutation at (serine 83) of GyrA and (serine 84) of ParC by Hinf1 restriction endonuclease digestion. Sequencing was performed for 12 samples. For each gene, two resistant and one susceptible strains were randomly selected. The mutations associated with ciprofloxacin resistant P. mirabilis were as follows; (1/3) GyrA (Ser 83 to Ile) and (2/3) ParC (Ser 81 to Ile). Also it revealed silent mutations at codons of GyrB 474 leucine (3/3), 585 valine (2/3), 612 histidine (1/3) and 639 asparagine (1/3) and ParE 469 isoleucine (2/3), 531 aspartic (2/3) and 533 glycine (1/3).Conclusions:Ciprofloxacin resistance in P. mirabilis could be monitored through detection of mutations within DNA gyrase (encoded by gyrA and gyrB) and topoisomerase IV (encoded by parC and parE).

Forensic Relationship between ATP6 Gene and Sperm Motility in a Saudi Population

Approximately 600 bp of ATP synthase subunit 6 mitochondrial gene (ATP6) were amplified and sequenced for 110 semen samples collected from infertile and normal Saudi men inhabiting Riyadh province. These data were analyzed in order to identify the Single Nucleotide Polymorphisms (SNPs) associated with male infertility. Six silent substitutions G8697A, G8856A, T8889C, T8952C, C9075T and C9060A were recorded with one novel site (G8856A) in the infertile men. Seven non- synonymous substitutions were also obtained with 4 novel sites (C8684T, G8860A, C8876T and G9055A) in the infertile men. Nine haplogroups (H2a, H1a, H5a, T, J2a, K1a, N1b, M25 and U7) were predicted. H5a, M25 and U7 were found in the oligoasthenoteratospermic (AOT) infertile men only with a percentage of 4.5%. It could be concluded that there was a correlation between ATP6 substitutions/haplogroups and male infertility since specific SNPs and haplogroups appeared in the AOT infertile men only. The present results are considered preliminary for using the mitogenome of the infertile men in a forensic perspective. More mtDNA data from infertile men are necessary to assess mtDNA forensic contributions in male fertility. Examination of autosomal Short Tandem Repeats (STRs) and Y chromosome -STR profiles for infertile men should be conducted to ascertain any changes in the genetic loci.

Molecular Characterization of BK Polyomavirus’ Large T Antigen Gene Sequences Detected in Prostate Cancer Tissues of Sudanese Patients

Background: BK virus, which is associated with Prostate Cancer (PCa), have a global seroprevalence in humans. Based on the sequences of VP1 and the Large Antigen (LTAg) genes, there are four subtypes of BKV. Each subtype has its own subgroups. Objective: The aim of this study was to identify the BKV subtype that circulates among Sudanese patients with PCa. Materials and Methods: A total of 8 samples from our previous work on BKV were studied in this investigation. The LTAg gene was partially amplified (176nt) by a homemade PCR. All the amplicons were purified and subjected to sequencing. Bioedit version 7.0 and Mega X version 6.0 were used to analyze the sequence and compare the results with the BKV sequences and build a phylogenetic tree. Results: All the BKV LTAg gene sequences derived from Sudanese patients were classified with Subtype-1 BKV strains from Iran and Japan. Translated protein alignment showed that some isolates had identical amino acids with Iranian and Japanese strains, whereas others had a silent mutation. Interestingly, a point mutation was identified in the sequences of isolate 5 and 8 where adenine nucleotide (A) was replaced with Cytosine (C) at position 276, resulting in amino acid substitution. Conclusion: It was concluded that all the BKV isolates which circulated among Sudanese prostate tumor patients belonged to subtype 1. These findings only highlighted the need for the molecular detection and subtyping of BKV strains in Sudanese patients in order to better demonstrate the relationship between BKV infection and PCa.

Analysis of clinicopathologic characteristics of gastric cancer in patients ≤40 and ≥40 years of age

Objectives: Gastric cancer (GC) in young patients is a troubling clinical problem. The aim of this study was to analyze whether patients ≤40 years of age with GC differ from patients (age >40 years) in terms of clinicopathological and selected genetic factors.Materials and methods: Between 1984 and 2011, data were collected for 840 GC patients diagnosed and treated for GC at the Department of Gastroenterology at Pomeranian Medical University. The following clinicopathological features were compared between two age groups: sex, symptom duration, family history of cancer, tumor site, stage (early vs. advanced), blood group, histology, Helicobacter pylori infection and BRCA2 C572T silent mutation status.Results: A total of 65 (7.7%) patients were age 40 years or younger. GC was predominant in women in the younger group (p < .001). Patients (≤40 years) more frequently reported a positive family history of cancer (p = .01) and a diffuse tumor type was more common in this group (p < .001). The two age groups did not differ significantly regarding symptom duration, tumor location or stage, H. pylori infection, blood group, or BRCA2 C572T silent mutation status. A comparison of male and female patients aged 40 years or less did not reveal sex-based differences in any analyzed features.Conclusion: Patients ≤40 years of age with GC differ from patient >40 years of age in having a predominance of women, diffuse tumor type, and positive family history of cancer. These results offer openings for further investigation of the relevance of these differences.

Association of phylogenetic distribution and presence of integrons with multidrug resistance in Escherichia coli clinical isolates from children with diarrhoea

BackgroundEscherichia coli strains include both commensal and virulent clones distributed in different phylogenetic groups. Antimicrobial resistance is an increasingly serious public health threat at the global level and integrons are important mobile genetic elements involved in resistance dissemination. This paper aims to determine the phylogenetic groups and presence of class 1 (intl1) and 2 (intl2) integrons in E. coli clinical isolates from children with diarrhoea, and to associate these characteristics with their antimicrobial resistance. MethodsPhylogeny and presence of integrons (intl1 and intl2) were analysed by PCR and amplicon sequencing in 70 E. coli isolates from children with and without diarrhoea (35 of each group) from Sinaloa, Mexico; these variables were analysed for correlation with the antimicrobial resistance profile of the isolates. ResultsThe most frequent phylogroups were A (42.9%) and B2 (15.7%). The E. coli isolates from children with diarrhoea were distributed in all phylogroups; while strains from children without diarrhoea were absent from phylogroups C, E, and clade I. The 17.1% of the isolates carried integrons (15.7% intI1 and 1.4% intI2); 28.6% of the isolates from children with diarrhoea showed the class 1 integron. Strains of phylogroup A showed the highest frequency of integrons (33.3%). The association of multidrug resistance and the presence of integrons was identified in 58.3% of strains isolated from children with diarrhoea included in phylogroups A and B2. The sequence analysis of intl1 and intl2 showed silent point mutations and similarities with plasmids of some APEC and AIEC strains. ConclusionCommensal E. coli strains are potential disseminators of antimicrobial resistance, and the improvement in the use of antimicrobials to treat childhood diarrhoea is essential for the control of such resistance.

Hair growth-promoting effect of recombinant human sonic hedgehog proteins

BackgroundHair loss, known as alopecia, causes psychological stress and influences social interactions. At present, treatment for hair loss is somewhat limited, transient, and unpredictable. Based on the critical role of sonic hedgehog (Shh) in hair follicle development, Shh itself would be a good therapeutic candidate for the prevention of hair loss as well as promotion of hair growth.MethodsTo investigate its potential use for hair treatment, we overexpressed Shh protein in E.coli by making silent mutations in 12 amino acids from N-terminus of Shh and purified it with Ni-affinity chromatography.ResultsPurified Shh proteins were able to activate Gli-responsive luciferase reporter gene in a dose-dependent manner and induce the alkaline phosphatase (AP) activity in the presence of bone morphogenic protein 4 (BMP-4). In addition, we confirmed that the treatment of recombinant Shh proteins to human dermal papilla (hDP) cells significantly increased the expressions of Shh-responsive genes as well as hair growth-related genes. Finally, we found that the administration of Shh proteins into the dorsal skin of the mouse could promote the hair density and length.ConclusionExogenous Shh proteins induced hair-related gene expression by stimulating both Shh signaling pathway and Wnt/β-catenin pathway in DP cells, and might therefore be a potential hair promoting agent.

Leptin Receptor Gene Polymorphisms in Some Turkish Donkey Populations

Leptin receptor is a fundamental regulator in physiological functions of the regulation of food intake, energy homeostasis, immune function, and reproduction as well as on ovarian follicular cells on the placenta and lactating mammary glands. The aim of this study was to investigate the LEPR gene polymorphism in 60 donkeys reared in Thrace region of Turkey. A 585 bp long partial intron 6, exon 7, intron 7, and exon 8 regions of LEPR gene were amplified, and polymerase chain reaction products analyzed via DNA sequencing. A novel single-nucleotide polymorphism (SNP) was identified as g.713668A>G in the seventh exon region of LEPR gene. This novel SNP was first identified, and the partial DNA sequence of LEPR gene in donkeys was reported for the first time in this study, and these sequences were deposited to NCBI Genbank database with the accession number: MK807114-MK807115. The A>G transition revealed a silent mutation (CAA-CAG) in glutamine amino acid. This nucleotide mutation could cause the changes of secondary structure of protein and expression level of LEPR hormone. For this reason, additional studies are needed to reveal new SNPs and in the LEPR gene that may affect economic traits and structure of protein in donkey breeds.

Mutation Profile of PPM1D and TP53 in Patients with Myelodysplastic Syndromes and Complex Chromosome Abnormalities

Introduction: Complex (≥3) abnormalities (cA) are associated with an inferior outcome in myelodysplastic syndromes (MDS). About 50% of MDS with cA show mutations in TP53 that might contribute to the formation of the cA and worsen prognosis (Haase et al., Leukemia, 2019). In former single nucleotid polymorphism (SNP) analysis we found chromosome 17q being affected in several patients with cA with a higher incidence as by chance. In just this region is a gene called PPM1D located which already has been observed as one of the most frequently mutated genes in pts./individuals with clonal hematopoiesis with indetermined significance (CHIP). PPM1D is encoding for a protein named Wip1. This protein acts as an inhibitor of p53. About 5% of MDS with 5q deletions show mutations in PPM1D (Panagiota et al., ASH 2017). Mutations in PPM1D are even more common among pts with therapy-related MDS (15%, Lindsley et al., 2017). The aim of our study was to determine the frequency of PPM1D mutations in MDS with cA and to shed light upon their possible contribution to the formation of cA. Methods and patients: We included 100 patients characterized by conventional cytogenetics in our analysis (67x MDS; 30x secondary acute myeloid leukemia, AML; 3x chronic myelomonocytic leukemia, CMML). 20 pts had a therapy-related MDS. All the included pts had cA with a median number of aberrations of 8 (range: 3-50). The median age at first diagnosis of MDS with cA was 72 (range 29-95). A deletion of 5q was found in 71 patients (71%). The TP53 status was known for all pts by fluorescence in situ hybridization (FISH) and/or molecular karyotyping (TP53 deletion status) and sequencing (TP53 mutation status). 68 of 100 pts had an alteration on TP53 (68%, 4 deletions, 34 mutations, 30 biallelic changes). All pts were subjected to next generation sequencing of PPM1D. Amplicons for exons 1 to 6 were generated by multiplex polymerase chain reaction (PCR). The pooled amplicons were processed using the Nextera XT2 sample preparation kit (Illumina, San Diego, Ca, USA) followed by sequencing on a MiniSeq platform (Illumina, San Diego, Ca, USA). We used our local bioinformatics pipeline to identify single-nucleotide variants (SNVs) and indels. Results: In ten pts (10%) we found single-nucleotide variants of PPM1D. The median number of aberrations was 8 (range: 5-15). Six of those PPM1D variants have already been described as very rare SNPs. Three of them were located in the 3'UTR (untranslated region), the other three seem to be silent mutations. The other four are not listed in common databases. Three of those four are potential missense mutations, one is a potential nonsense mutation. Two variants are located at the same -previously undescribed- position (c.230A&gt;C, p.D77A). Two of those four patients showed an additional TP53 mutation, one of them biallelic. A deletion of 5q was identified in two of them. One pt had therapy related MDS. At a clone size of the complex karyotype of 94% and 90%, the VAF of three of the recurrent mutation was just 7% and 8%, indicating that the PPM1D mutation arised in a subclone in these pts. In one pt the VAF was 33,6%. The VAF of 30-38% in the other cases implies PPM1D being an ancestral or co-dominant mutation. Conclusion: We were able to show that PPM1D is mutated in MDS with cA in a relevant fraction of pts. In our cohort, 10% of MDS pts with cA are affected. 4% may have a deleterious mutation of PPM1D. Although PPM1D mutations were described to preferentially occur in therapy related diseases (Lindsley et al., 2017), in our cohort three of four patients with potential PPM1D mutation had no known prior chemo-/radiation therapy. Mutations in PPM1D might contribute to the formation or toleration of cA alternatively to TP53 mutations as two of four patients with PPM1D mutations did not show TP53 mutations and the PPM1D mutations could be the ancestral or co-dominant mutation in two of four cases. Our data imply that also mutations in PPM1D may be important for prognosis and therapy decisions in MDS patients with cA. We will continue observing our patients in order to enlarge the database and to find out which impact mutations in PPM1D may have on overall survival and whether they can affect the prognosis of patients with cA. Disclosures Germing: Jazz Pharmaceuticals: Honoraria; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria. Hertenstein:RS Media: Research Funding. Platzbecker:Novartis: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Efficient Correction of ELANE mutations in Primary HSPCs of Severe Congenital Neutropenia Patients Using CRISPR/Cas9 and rAVV6 HDR Repair Templates

Patients with the rare pre-leukemia bone marrow failure syndrome severe congenital neutropenia (CN) have reduced numbers of neutrophils in peripheral blood (&lt;500/µl) leading to frequent infections and requiring chronic granulocyte stimulating factor (G-CSF) treatment. The majority of patients harbor heterogenous mutations in ELANE, coding for Neutrophil Elastase. Up to now, the only curative therapy for CN patients that do not respond to G-CSF or with overt AML remains hematopoietic stem cells transplantation with its associated risks. A clinical need for gene therapy for these patients is imminent. We recently described the CRISPR/Cas9 mediated ELANE knockout as a possible gene therapy approach for CN patients with ELANE mutations (ELANE-CN) (Nasri et al. 2019). As an alternative, we wanted to test if specific target therapy for individual ELANE-CN patients could be an option. Here we describe the correction of ELANE mutations using CRISPR/Cas9 to edit the ELANE gene and recombinant adeno-associated virus 6 (rAAV6) to deliver a template for homology directed repair (HDR). We selected ELANE mutations p.A57V or p.A57T in exon 2, and p.G214R or p.G214RV in exon 5, both known hot spot mutations observed in G-CSF non-responders or in CN/AML patients (Makaryan et al. 2015). We used SpCas9 V3 and chemically modified sgRNA. For exon 2, we choose the highly efficient sgRNA (Nasri et al. 2019) yielding the benefit, that double-strand breaks (DB) that do not result in HDR correction are producing ELANE knockout. For exon 5, we established a sgRNA that produced average 87% (± 6%) editing in healthy donor cells. Two HDR donor template backbones (DTB) were generated. DTB1 is spanning exons 1-3 and DTB2 exons 4-5 of ELANE. Silent mutations were introduced in the repair templates for both ELANE mutations between the cut site and mutation to enhance HDR. To test the knock-in efficacy, we electroporated healthy donor CD34+cells with CRISPR/Cas9 RNP and transduced them with rAAV6 containing the templates at MOI 105. We achieved 34,5% (± 4,5%) knock-in (KI) and 35,6% (± 2,5%) indels for exon 2, or 39,2% KI (± 12,8%) and 18,85% indels (± 4,25%) for exon 5. Edited cells showed high viability, expanded and differentiated well into neutrophils in vitro. We further applied this approach to primary HSPCs from 4 CN patients harboring selected ELANE mutations. For p.A57, we achieved 14% (±2,3%) KI and 44,7% (±1,9%) indels. For p.G214, the KI was 59,9% (± 0,1%) and indels 28,8% (± 0,6%). To assess the effect of ELANE correction on the neutropenic phenotype in vitro, we performed CFU and liquid culture neutrophilic differentiation assays. We compared the corrected cells to cells from the same patient that were edited in the AAVS1 safe harbor, as isogenic controls. We observed a significant (p &lt; 0,05) increase in number of CFU-GMs for CRISPR/Cas9 edited HSPCs from two CN patients with p.A57V/T mutations and of CFU-G or CFU-GM for two CN patients with p.G214R/V ELANE mutation. Morphological assessment of Wright-Giemsa stained cytospins of cells derived on day 14 of differentiation revealed significant increases of mature neutrophils for all four edited patient samples ascompared to the respective controls. Further we performed live cell imaging of neutrophil extracellular trap (NET) formation after PMA stimulation and chemotaxis. NET formation was either improved or comparable between control- and ELANE- edited cells. Chemotaxis showed no difference between control- and ELANE-edited cells. For a patient with p.G214V ELANE mutation, we were able to evaluate chemotaxis and phagocytosis in vivo in zebrafish embryos at 48hpf, as described in Nasri et al 2019. This showed a qualitative improvement of ELANE- corrected cells ascompared to control AAVS1 edited cells. This indicates that our manipulation does not alter the functionality of produced neutrophils while increasing the number of mature cells being produced. Taken together, we established a protocol for efficient correction of ELANE mutations in primary HSPCs using CRISPR/Cas9 and rAVV6 HDR repair templates. We reached high enough editing to correct the dominant negative effects of mutations, as assessed by markedly improved neutrophilic differentiation in vitro. Generated repair constructs allow fast adaptation to patient-specific mutations in all exons of ELANE. This approach is enticing to be investigated further for clinical translation. Disclosures No relevant conflicts of interest to declare.

Disease Modeling of Severe Congenital Neutropenia Using CRISPR/Cas9 Gene Correction or Knockout of ELANE in Patients Derived Induced Pluripotent Stem Cells

Severe congenital neutropenia (CN) is a monogenic bone marrow failure syndrome with the frequency of 1:200,000 and is characterized by an absolute neutrophil count below 500 cells per microliter. Patients with CN suffer from severe life-threatening bacterial infections starting early after birth due to the absent or very low numbers of neutrophils in peripheral blood. While CN is a heterogeneous disease caused by many different gene mutations, autosomal-dominant ELANE mutations are the most common cause of CN. Although the majority of CN patients respond to daily treatment with granulocyte colony-stimulating factor (G-CSF), approximately 15 % do not respond at doses up to 20 μg/kg/day and approximately 20 % of G-CSF treated patients develop myelodysplasia (MDS) or acute myeloid leukemia (AML). In the present study, we first established an efficient gene-editing platform for induced pluripotent stem cells (iPSC) of CN patients using CRISPR/Cas9 technology. The platform uses ribonucleoprotein form of CRISPR/Cas9 making the editing approach safer as it is virus- or DNA free. Also, any further selection step or introducing extra modifications in the genome of edited cells such as silent mutation are not required. We generated and characterized iPSCs from ELANE-CN patients harboring p.A57V, p.C151Y, and p.G214R mutations, that are more severe hot-spot mutations associated with G-CSF non-response or MDS/AML. We corrected each mutation followed by EB-based hematopoietic differentiation, to evaluate and compare granulocytic differentiation of CN-patient specific iPSCs, with or without ELANE mutation, in an isogenic model. To study granulocytic differentiation, we performed live cell counts, flow cytometry analysis of myeloid-specific surface marker expression, CFU assay, cell morphology of cytospin preparations and neutrophil functional tests. Our isogenic model showed that correction of ELANE mutations led to fully normalized granulocytic differentiation. We have recently shown that CRISPR/Cas9 mediated ELANE knockout (KO) enables neutrophilic maturation of primary HSPCs and iPSCs of CN patients. We observed that granulocytic differentiation of ELANE KO iPSCs and primary HSPCs were comparable to healthy individuals. Phagocytic functions, ROS production, and chemotaxis of the ELANE KO neutrophils were also normal. To model CN in silico and to reveal the key driving pathomechanisms, we designed an isogenic patient-specific disease modeling system by comparing RNA-sequencing results of CN-ELANE corrected- or CN-ELANE KO hematopoietic stem and progenitor cells (HSPCs) to the original CN-ELANE patient cells. HSPCs were derived from iPSC lines. Our analysis showed a degree of similarity in enriched pathways upon ELANE correction or ELANE KO in a patient-specific manner. Thus, upon correction of p.C151Y mutation, TNF, IL4 and IL13 signaling pathways as well as MAPK signaling, PD-1 signaling and IL10 signaling were down-regulated. Interestingly, the same pathways were down-regulated upon ELANE KO in HSPCs of the same CN patient. Correction of p.A57V mutation led to down-regulation of IL12 expression which activates STAT family. Upon ELANE KO in the cells from the same patient, IL12, IL18, and IL1-beta expression were down-regulated. We also identified common pathways enriched in most of the isogenic samples upon ELANE correction or ELANE KO like down-regulation of MAPK or IFN α/β signaling as well as down-regulation of the Rap-1 signaling pathway leading to the Erk pathway activation. Analysis of putative transcription factor binding sites (TFBSs) that are enriched in the differentially expressed gene list upon ELANE mutation correction or ELANE KO showed that transcription factors GKLF (KLF4), MAZ, Kaiso (ZBTB33) and CHURCHILL are highly enriched in UP-regulated genes, for both, correction and KO samples. Taken together, we established a safe and efficient CRISPR/Cas9-RNP based ELANE gene-correction/knockout platform of iPSCs of ELANE-CN patients that may be used to establish an isogenic disease modeling system or provide novel stem cell-based therapy for CN patients with a high risk of leukemia development as well as for G-CSF-non-responsive patients. This platform could be also applied for other monogenic bone marrow failure syndromes. Disclosures No relevant conflicts of interest to declare.

Landscape of TP53 Mutations in MPN

Landscape of TP53 Mutations in MPN

Pediatric Acute Promyelocytic Leukemia in Brazil: Epidemiology, Molecular Features, and Importance of GST-Theta 1 in Chemotherapy Response and Outcome

Background. Previous studies have suggested a variation in the incidence of acute promyelocytic leukemia (APL) among geographic regions with relatively higher percentages within the Latin American population. We aimed to describe the population burden of pediatric APL (p-APL) in Brazil assessing the incidence rates according to a hospital-based and population-based cancer (PBCR) registries. We also explored mutations in genes of the RAS pathway and the association of polymorphisms in genes of glutathione S-transferases (GSTs) with outcome. Our goal was to provide insights into the distribution of clinical-demographic data and the molecular epidemiology characteristics associated with APL outcome. Methods. One hundred and sixty-four p-APL cases (&lt;19 years old) were identified from a dataset of a hospital-based registry based at a leukemia diagnostic central reference laboratory (2002-2018) and from 15 PBCR (2002-2009). Diagnostic criteria included morphological, immunophenotypic, and cytogenetic-molecular features. The PML-RARafusion gene was detected by FISH and/or RT-PCR. Additionally, mutations in FLT3 [D835 and internal tandem duplications (ITD)], KRAS, NRAS, and PTPN11 mutations were analyzed. We also evaluated the risk association of pharmacogenetically important GST deletion polymorphisms (GSTT1 and GSTM1) by multiplex-PCR, in a case-case analysis to test the effect of genetic susceptibility on overall survival (OS). Patients were treated with the inclusion of all-trans retinoic acid in chemotherapy with anthracyclines and cytarabine. Kaplan-Meier survival analysis was used to calculate the 5-year probabilities of OS (pOS), and estimated survival values were compared using the log-rank test. Cox proportional-hazard regression model estimated the hazard ratio (HR) and 95% confidence intervals (CI). All p-values were two-sided using significance level of 0.05. Results. From the hospital-based registry, APL patients represented 17.6% of the cohort (164 out of 933 acute myeloid leukemia cases), while in the PBCR, they represented 4.4% (35 out of 805 acute myeloid leukemia cases registered). The age-adjusted incidence rate was 0.32 per million persons during 2000-2009 based on PBCR. According to the hospital-based registry, we found similar distribution of patients among age ranges &gt;2-10 and &gt;10-21 years old (43.3% and 51.8%, respectively) and sex. RAS mutations were observed in 51.7% of APLs, including FLT3 (43.0%), NRAS (6.5%), and KRAS (2.2%). Variants in PTPN11 were silent amino acid substitutions (rs61736914; 4.9%). We observed a statistically significant association between FLT3 mutations and high white blood cell count at diagnosis (&gt;10x109/L; 72.6%), low platelet count (&lt;40×109/L; 83.0%), and the PML-RARa breakpoint cluster region 3 (90.5%). Death in the first ten days after diagnosis (early death) affected 17.5% (24/137) patients and these cases were excluded from the survival analysis. The mean of overall survival was 45.1 months (95% CI 40.1-50.1 months; pOS 66.9±5.8%). Univariate analysis did not show an association between variables and OS rates, except for APL patients carrying the GSTT1 polymorphism. GSTT1 null genotype conferred adverse prognosis compared to wild-type genotype (pOS 48.2±13.4% and 79.0±6.3%, p=0.024; hazard ratio 2.6, 95% CI 1.1-6.2, p=0.030; Figure 1). Conclusions. APL represented 17.6% of acute myeloid leukemia in our cohort of Latino patients, which is a higher proportion compared to Northern European countries and the United States (5-10%). GSTT1 polymorphism modulated outcome, suggesting that lower enzyme activity may impact response to therapy. Decreasing early death and inclusion of GSTT1 polymorphism in therapeutic protocols for chemotherapy dose modulation may allow patients to reach the same overall survival observed in the United States (&gt;70%). Disclosures No relevant conflicts of interest to declare.

Molecular Characterization and Novel Therapeutic Strategy for X-Linked Sideroblastic Anemia Associated with ALAS2 Missense Variants

Background: Congenital sideroblastic anemia (CSA) is an inherited anemia characterized by the presence of ring sideroblasts (RSs) in bone marrow. X-linked sideroblastic anemia (XLSA) is the most common form of CSA and is caused by germline mutations in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene. Each ALAS2 mutation has a variable effect on the protein's enzymatic function, and only half of XLSA patients are responsive to pyridoxal 5′-phosphate (PLP) treatment. Thus, novel therapeutic strategies should be explored. We have established an in vitro XLSA model in human induced pluripotent stem cell-derived erythroid progenitors (HiDEP-1) by disrupting GATA-1-binding intronic enhancer region of ALAS2, which accounts for a minor XLSA subtype with PLP refractoriness (Saito and Ono et al. MCB 2019; ASH 2018). However, evidence regarding molecular characterization of ALAS2 missense variants is lacking, presumably due to difficulties in introducing the desired missense mutations in primary erythroblasts. In this study, we optimized a protocol for introducing point mutations in human umbilical cord blood-derived erythroid progenitor (HUDEP)-2 cells and subsequently characterized XLSA models harboring ALAS2 missense variants. Methods: Based on homology-directed CRISPR/Cas9 system, we introduced an ALAS2 mutation from arginine at amino acid residue 170, one of the XLSA hot spots, into leucine (R170L) or histidine (R170H). A twenty-nucleotide targeted genomic sequence, located upstream of protospacer adjacent motif (PAM) near ALAS2 R170, was cloned into plasmid vectors expressing Cas9 nuclease and green fluorescent protein (GFP). Each homologous recombinant template was designed as a 127 single-stranded oligodeoxynucleotide (ssODN) asymmetrically distributed from the Cas9-mediated cleavage site. PAM silent mutations were added to avoid continuous cleavage after successful mutation. HUDEP-2 cells were co-transduced with gRNA/Cas9/GFP-expressing vectors and ssODNs using Amaxa Nucleofector 2b (Lonza). After 48 hours, cells were isolated by GFP-positive expression; subsequently, single-cell dilution was performed, followed by clonal analysis. Results: Both XLSA clones (ALAS2 R170L and R170H) appeared pink/pale colored, reflecting impaired hemoglobin biosynthesis. Consistently, quantitative RT-PCR analysis demonstrated decreased globin gene (HBB) expression. On the other hand, XLSA clones did not show RSs and were morphologically similar to wild-type controls. When XLSA clones were induced to undergo erythroid differentiation by co-culturing with OP9 cells in a 100 mM sodium ferrous citrate (SFC)-supplemented medium, increased numbers of RSs were observed in mutant clones, mimicking findings in XLSA patients. Electron microscopy confirmed an aberrant mitochondrial iron deposit in XLSA clones. Also, HBB expression and intracellular heme concentration were significantly lower than those in wild-type controls. Intriguingly, while OP9 co-culture and SFC addition promoted erythroid differentiation in both wild-type and XLSA clones, XLSA clones exhibited a more immature morphological phenotype than wild-type controls. Expression profiling revealed that 317 and 86 genes were commonly up- and downregulated &amp;gt;2-fold in XLSA clones compared with those in wild-type controls. Gene ontology analysis showed significant (p &amp;lt; 0.01) enrichment of genes associated with mitochondrial gene expression and organization, suggesting that mitochondrial alteration is involved in XLSA pathogenesis. On the other hand, 188 genes in total were differentially expressed between ALAS2 R170L and R170H. Genes associated with ferroptosis, such as GPX4 and GCLC, showed a significantly lower expression in ALAS2 R170H clones, possibly accounting for phenotypic differences among patients with ALAS2 R170L or R170H mutation. Finally, supplementation with 5-aminolevulinic acid (ALA) significantly improved the compromised heme biosynthesis in XLSA clones, suggesting ALA treatment to be a promising therapeutic option for XLSA. Conclusion: The established models may act as useful tools for exploring the precise molecular mechanisms of XLSA harboring missense mutations and for drug testing. Also, our homology-directed CRISPR/Cas9-based protocol can be applied to establish a wide variety of CSA as well as congenital anemia models. Disclosures Fukuhara: Zenyaku: Honoraria; Eisai: Honoraria, Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Janssen Pharma: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Gilead: Research Funding; Ono Pharmaceutical Co., Ltd.: Honoraria; Nippon Shinkyaku: Honoraria; Celgene Corporation: Honoraria, Research Funding; Mundi: Honoraria; Kyowa-Hakko Kirin: Honoraria; Mochida: Honoraria; AbbVie: Research Funding; Bayer: Research Funding; Solasia Pharma: Research Funding. Onishi:Celgene: Honoraria; Kyowa-Hakko Kirin: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma: Honoraria; Pfizer Japan Inc.: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen Pharmaceutical K.K.: Honoraria; MSD: Honoraria, Research Funding; Astellas Pharma Inc.: Honoraria; Nippon Shinyaku: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria.

FBXO11 Is a Candidate Tumor Suppressor in the Transformation of MDS to Secondary AML

Myelodysplastic syndrome (MDS) is a heterogenous myeloid lineage malignancy characterized by blood cell morphological dysplasia, ineffective clonal hematopoiesis, and risk of secondary transformation to acute myeloid leukemia (sAML). Genomic sequencing of large MDS cohorts has led to the identification of recurrent genetic abnormalities that carry independent prognostic significance and overlap with mutational changes in sAML. However, no set of mutations is sufficient to predict the transformation of MDS raising the question of how an identical genotype produces MDS in one patient and sAML in another? We hypothesize there are therapeutically targetable cellular processes altered by the initiating genetic changes in MDS that predict transformation to sAML. To uncover novel cellular pathways involved in MDS transformation, we performed an unbiased genome-wide CRISPR/Cas9 in the human MDS-L cell line. MDS-L was established from bone marrow mononuclear cells in a 52-year-old male patient and requires IL3-containing media for growth in vitro (Figure 1A). GFP expressing MDS-L cells were transduced with Cas9 and a sgRNA against GFP to confirm functional Cas9 expression in MDS-L (Figures 1B and C). In Cas9 expressing MDS-L cells, we then transduced the Brunello sgRNA CRISPR library and subjected the cells to IL-3 starvation for 4 weeks. Cells surviving IL-3 starvation were then expanded and harvested for genomic DNA. High throughput sequencing of the barcoded DNA produced raw reads that were analyzed using the PinAPL-Py web-based software. sgRNAs appearing in duplicate with absolute read counts over 1000 or in triplicate over 100 were considered significant. We identified 5 genes that conferred resistance to IL-3 starvation, which included FBXO11 (Figure 1D). The Fbox protein FBXO11 is a component of the SCF ubiquitin ligase complex and regulates its substrates via ubiquitination and proteasomal degradation. FBXO11 is mutated in up to 20% of diffuse large B-cell lymphomas and its loss in breast cancer models leads to increased metastases. Therefore, we hypothesized FBXO11 may also function as a tumor suppressor in the transformation of MDS to AML. We confirmed in the Bloodspot gene expression database that there are decreased levels of FBXO11 in a variety of AML samples, including complex karyotype, compared to normal HSCs. To validate the results of the screen, we synthesized two sgRNAs targeting FBXO11, transduced these into MDS-L cells, and detected reduced FBXO11 expression (Figure 1E). Loss of FBXO11 expression promoted survival in IL-3 free media, confirming the selection readout of the screen (Figure 1F). We then designed a silent mutation in the shorter isoform of FBXO11 (FBXO11v1sm1) that rendered resistance to CRISPR/Cas9 (Figure 1G) and observed that overexpression of FBXO11v1sm1 re-sensitized cells to cytokine starvation (Figure 1H). Whether there are different functions between FBXO11 variant 1 and 4 are currently being explored. We are actively performing RNA sequencing and ubiquitin proteomics in FBXO11 knockout cells to identify its downstream targets and assaying for reduced expression of FBXO11 in primary patient MDS and AML samples. Based on our studies, we predict that SCF ubiquitin ligase component FBXO11 is a tumor suppressor regulating the transformation of MDS to secondary AML. Figure 1 Disclosures Crispino: Sierra Oncology: Consultancy; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Forma Therapeutics: Research Funding; Scholar Rock: Research Funding.

Thermosensitive pilus production by FCT type 3 Streptococcus pyogenes controlled by Nra regulator translational efficiency.

Summary Streptococcus pyogenes produces a diverse variety of pili in a serotype‐dependent manner and thermosensitive expression of pilus biogenesis genes was previously observed in a serotype M49 strain. However, the precise mechanism and biological significance remain unclear. Herein, the pilus expression analysis revealed the thermosensitive pilus production only in strains possessing the transcriptional regulator Nra. Experimental data obtained for nra deletion and conditional nra‐expressing strains in the background of an M49 strain and the Lactococcus heterologous expression system, indicated that Nra is a positive regulator of pilus genes and also highlighted the importance of the level of intracellular Nra for the thermoregulation of pilus expression. While the nra mRNA level was not significantly influenced by a temperature shift, the Nra protein level was concomitantly increased when the culture temperature was decreased. Intriguingly, a putative stem‐loop structure within the coding region of nra mRNA was a factor related to the post‐transcriptional efficiency of nra mRNA translation. Either deletion of the stem‐loop structure or introduction of silent chromosomal mutations designed to melt the structure attenuated Nra levels, resulting in decreased pilus production. Consequently, the temperature‐dependent translational efficacy of nra mRNA influenced pilus thermoregulation, thereby potentially contributing to the fitness of nra‐positive S. pyogenes in human tissues.