Gamma Gene Research Articles

Prediction of Alzheimer’s disease-specific phospholipase c gamma-1 SNV by deep learning-based approach for high-throughput screening

Exon splicing triggered by unpredicted genetic mutation can cause translational variations in neurodegenerative disorders. In this study, we discover Alzheimer's disease (AD)-specific single-nucleotide variants (SNVs) and abnormal exon splicing of phospholipase c gamma-1 (PLCγ1) gene, using genome-wide association study (GWAS) and a deep learning-based exon splicing prediction tool. GWAS revealed that the identified single-nucleotide variations were mainly distributed in the H3K27ac-enriched region of PLCγ1 gene body during brain development in an AD mouse model. A deep learning analysis, trained with human genome sequences, predicted 14 splicing sites in human PLCγ1 gene, and one of these completely matched with an SNV in exon 27 of PLCγ1 gene in an AD mouse model. In particular, the SNV in exon 27 of PLCγ1 gene is associated with abnormal splicing during messenger RNA maturation. Taken together, our findings suggest that this approach, which combines in silico and deep learning-based analyses, has potential for identifying the clinical utility of critical SNVs in AD prediction.

Association between peroxisome proliferator-activated receptor-alpha, -delta and -gamma gene (PPARA, PPARD, PPARG) polymorphisms and overweight parameters in physically active men.

Peroxisome proliferator-activated receptors (PPARs) have unique functions in energy metabolism regulation but are also involved in regulation of the inflammatory process and obesity. The aim of this study was to analyse potential associations between polymorphisms of PPARA (rs1800206), PPARD (rs1053049; rs2267668) and PPARG (rs1801282) and overweight parameters. One hundred and sixty-six males, unrelated Caucasian military professionals, were recruited in the genetic case-control study conducted in the period 2016–2019. All the participants were aged 21–41 and had similar levels of physical activity. Body mass, height and body composition were measured. The participants were divided into two groups depending on their BMI (body mass index) and FMI (fat mass index). The control group consisted of people with BMI between 20.0 and 25.0 or FMI values ≤ 6, while the overweight group consisted of people with BMI of ≥ 25.0 or FMI values > 6. Genomic DNA was isolated from extracted buccal cells. All samples were genotyped using real-time polymerase chain reaction (real-time PCR). It was found that two polymorphisms rs2267668 and rs1053049 of the PPARD gene were significantly associated with BMI: SNP rs2267668 for the dominant (OR = 2.04, 95%CI 1.01–4.11, p-value = 0.04) model (A/G-G/G vs A/A). The likelihood of being overweight was over 2 times smaller for allele A. A relationship between the polymorphism of PPARG (rs1801282) and BMI was found for the overdominant (OR = 2.03, 95%CI 1.03–4.00, p-value = 0.04) model (C/G vs C/C-G/G). Significant associations were found in different models for PPARD, PPARG and PPARA genes with BMI. In SNP rs2267668 for the codominant genetic model (G/G vs A/A) (p-value = 0.04) and in SNP rs1053049 for the codominant (C/C vs T/T) (p-value = 0.01) and the recessive genetic model (C/C vs T/T–C/T) (p-value = 0.004) all polymorphisms were associated with BMI. In conclusion, it was found that three of the four polymorphisms (rs1053049, rs2267668, rs1801282) selected are associated with the risk of being overweight. Having said that, one has to bear in mind that DNA variants do not fully explain the reasons for being overweight. Therefore more research is needed to make a thorough assessment using the latest genomic methods in sequencing and genotyping, combined with epigenomics, proteomics, transcriptomics, and metabolomics.

Adenine Base Editor Creates Novel Substitution Mutations in eIF4G Gene of Rice

Two single nucleotide polymorphic mutations and deletion affecting Y1059V1060V1061 amino acid residues in a host translation initiation factor four gamma (eIF4G) gene in rice are reported to confer resistance to rice tungrospherical virus in resistant genotypes. A CRISPR-based adenine base editing vector was used to target these residues in a susceptible indica cultivar, ASD16.Agrobacterium-mediated transformation of ASD16 generated 16 missense mutants and two deletion mutants. Substitution mutations occurred at A5> G5 and A4> G4, where 5.5 % and 3.37 % of adenosines got converted to guanosines, respectively. The mutantsgenerated had missense mutations affecting the YVV residues and the residues immediately adjacent to YVV.Thus,these novel mutationsare promising candidates in imparting resistance against rice tungro disease.

Characterization of Substitution Mutations of eIF4G Gene Generated through Adenine Base Editors in Rice

Adenine base editor (ABE) creates A to G transitions within its editing window. In the present study, an ABE was used to target a stretch of six amino acid residues, VLFPNL in translation initiation factor four gamma (eIF4G) gene of rice. Agrobacterium-mediated transformation of rice cultivar ASD16 resulted in T0 events with high mutation efficiency of 89.29 %. Substitution mutations of A > G occurred within the editing window of four to eight bases at A7 > G7 (74.67 %) and A4 > G4 (2.46 %). Non-canonical substitutions of G > C/A was also observed at G15 > C15 (9.29 %) and G8 > A8 (1.15 %). A total of 15 missense base substitution events affecting the target residue was identified. Taken together, the present study showed that ABEs create unexpected base substitutions besides efficient canonical editing of A > G in the rice genome

Targeted mutation in eIF4G GENE in rice

 Rice tungro disease (RTD), caused by the interaction between rice tungro spherical virus (RTSV) and rice tungro bacilliform virus (RTBV) is a serious constraint in rice production. Natural RTSV resistance is due to a mutation in the translation initiation factor 4 gamma (eIF4G) gene. To develop novel alleles for resistance to RTD through CRISPR/Cas9 mediated genome editing, a guide RNA sequence, having higher GC content and fewer off -targets, targeting exon 7 of eIF4G was designed and cloned into a binary vector, pRGEB32. This construct was used for generating mutations in rice cultivar ASD16 through Agrobacterium-mediated transformation. A target region in the thirteen putative mutants generated in this experiment was sequenced, of which six were found to possess indels. The analysis of the predicted amino acid sequence identified deletion mutants that possess near full -length eIF4G gene which can impart resistance to RTSV.   &nbsp

Prenatal Diagnosis of β-Thalassemias by Amniocentesis: Linkage Analysis Using Multiple Polymorphic Restriction Endonuclease Sites

In order to assess the applicability of multiple restriction endonuclease analyses of amniocyte DNA to the prenatal diagnosis of beta-thalassemias in general, we studied 12 consecutive couples at risk. DNA of both members of the 12 couples and a previous offspring of each was analyzed for the presence of 4 polymorphic restriction endonuclease sites: the Hpa I site 3' to the beta-globin gene, the Hind III site in the G gamma gene, the Hind III site in the A gamma gene, and the Bam HI site 3' to the beta-gene. Linkage disequilibrium between these sites and beta A or beta thal genes was not found, presumably due to the heterogeneity of beta thal genes. However, the high frequency of polymorphism at these sites allowed differentiation of beta A-bearing chromosomes from beta thal or beta S-bearing chromosomes in both members of 6 couples. In these couples, complete prenatal diagnosis by linkage analysis of amniocyte DNA would be possible. In the remaining 6 couples, beta A and beta thal chromosomes could be discriminated in one member. In about 50% of the pregnancies of these couples, exclusion of beta-thalassemia is possible by this analysis. These data suggest that when linkage analysis of polymorphic restriction endonuclease sites is carried out, prenatal diagnosis of beta-thalassemia states can be accomplished by amniocentesis alone in 75% of pregnancies at risk.

T Cell Receptor (α, β, γ) Gene Rearrangements and Expression in Normal and Leukemic Large Granular Lymphocytes/Natural Killer Cells

The large granular lymphocyte (LGL) population, which effects a natural killer (NK) function, consists of cells whose lineage derivation has not been clearly established on the basis of phenotypic and functional properties. To clarify the relationship of LGL/NK cells to T cells we studied patterns of rearrangement and expression of the T cell receptor (Ti) genes alpha, beta, and gamma in normal human LGLs; in CD8+, CD8-, Mol+, and Mol- LGL subsets; and in 17 cases of leukemic LGL proliferations (T gamma LPD). T alpha, T beta, and T gamma genes were not expressed, nor were T beta and T gamma genes rearranged in normal LGLs or LGL subsets. The T gamma LPD were divided into two groups. One group (15/17 cases) was characterized as CD3+ and displayed Ti gene rearrangements. Seven of these cases were reactive with monoclonal antibody WT31, which suggested expression of an alpha/beta heterodimer on the cell surface. The other group (2/17 cases) was CD3- with unrearranged Ti genes. These results indicate that the normal LGL/NK population is homogeneous and distinct from the normal T cell population because it does not express, and as a result, cannot effect its immune function through the T cell receptor molecules. Conversely, T gamma LPDs represent a heterogeneous group of lymphoproliferative diseases within which the CD3-, Ti- cases most likely represent the neoplastic counterpart of normal LGL cells. The more frequent CD3+ cases may be related to recently described NK-like T cells. The observations that normal LGLs maintain germline T gamma genes and that many CD3+ T gamma LPD display an alpha/beta heterodimer suggest that a T gamma-containing receptor may not be necessary for NK or NK-like cytotoxicity.

A Chromosome With Five γ-Globin Genes

Globin gene mapping of DNA from a Black newborn resulted in the detection of a chromosome with five gamma-globin genes. Based on results from digests with enzymes EcoRI and PstI, we concluded that the three genes between the 5'G gamma and 3'A gamma genes are G gamma genes with a possible 5' segment derived from A gamma. The high G gamma level in the fetal hemoglobin (Hb F) of the baby is consistent with this view. Family relationships were such that speculation as to the mechanism causing this quintuplication of the gamma-globin genes was not possible.

Synergistic Effect of Omega-3 Fatty Acids and Oral-Hypoglycemic Drug on Lipid Normalization through Modulation of Hepatic Gene Expression in High Fat Diet with Low Streptozotocin-Induced Diabetic Rats.

Type 2 diabetes mellitus, which an outcome of impaired insulin action and its secretion, is concomitantly associated with lipid abnormalities. The study was designed to evaluate the combinational effect of omega-3 fatty acids (flax and fish oil) and glibenclamide on abnormal lipid profiles, increased blood glucose, and impaired liver and kidney functions in a high fat diet with low streptozotocin (STZ)-induced diabetic rats, including its probable mechanism of action. The male Wistar rats (n = 48) were distributed into eight groups. All animal groups except the healthy received a high fat diet (HFD) for 90 days. Further, diabetes was developed by low dose STZ (35 mg/kg). Diabetic animals received, omega-3 fatty acids (500 mg/kg), along with glibenclamide (0.25 mg/kg). Both flax and fish oil intervention decreased (p ≤ 0.001) serum triglycerides and very low density lipoprotein and elevated (p ≤ 0.001) high density lipoprotein levels in diabetic rats. Total cholesterol and low-density lipoprotein level was decreased (p ≤ 0.001) in fish oil-treated rats. However, it remained unaffected in the flax oil treatment group. Both flax and fish oil intervention downregulate the expression of fatty acid metabolism genes, transcription factors (sterol regulatory element-binding proteins-1c and nuclear factor-κβ), and their regulatory genes i.e., acetyl-coA carboxylase alpha, fatty acid synthase, and tumor necrosis factors-α. The peroxisome proliferator-activated receptor gamma gene expression was upregulated (p ≤ 0.001) in the fish oil treatment group. Whereas, carnitine palmitoyltransferase 1 and fatty acid binding protein gene expression were upregulated (p ≤ 0.001) in both flax and fish oil intervention group.

Leonotis Leonurus improves the crosstalk between peripheral tissues both in vivo and in vitro

Ethnopharmacological relevanceUnravelling the anti-diabetic mechanism of action of L. leonurus at adipose, liver, muscle and pancreatic level. AimsTo investigate the mechanism of action of an organic extract of L. leonurus and marrubiin at the gene level in adipose, liver and muscle tissues of an obese rat model and in a co-culture model. Materials and methodsObese Wistar rats were fed a cafeteria diet for eight weeks, treated with an extract of L. Leonurus, marrubiin, sulfonylurea and aspirin for two weeks and the level of gene expression of selected markers were investigated across different tissues. The effects mediated by the different treatments were investigated in co-culture cell models involving 3T3-L1 (fat), Chang (liver), C2C12 (muscle) and INS-1 (pancreatic) cells under both normal and hyperglycemic conditions. ResultsL. leonurus extract mediated a significant increase in PPAR gamma, glucokinase, FAS and UCP2 gene expression in adipose tissue, whilst the opposite was observed in the liver. At the muscle level, a significant increase in FAS gene expression was observed relative to the obese control rats. Furthermore, the extract as well as marrubiin, modulated improvements in the adipokine profile. The co-culture models showed that the effect mediated by the extract was dependent on, the tissue type as well as the glycemic conditions. ConclusionsL. Leonurus extract as well as marrubiin exhibit anti-diabetic properties where the mechanism of action is mainly at the adipose tissue level. The increase in expression of the genes of interest mentioned above potentially play a protective role towards the liver and possibly towards the muscle tissues as well.

Comparative study on the effect of dietary β-carotene and phycocyanin extracted from Spirulina platensis on immune-oxidative stress biomarkers, genes expression and intestinal enzymes, serum biochemical in Nile tilapia, Oreochromis niloticus

The current trial investigated the roles of β-carotene and phycocyanin extracted from Spirulina platensis on growth, serum biochemical, digestive enzymes, antioxidant defense, immune responses, and immune gene expression in Nile tilapia (Oreochromis niloticus). Fish (1.52 ± 0.10 g) were randomly stocked to three treatments with three replicates (12 fish per replicate) in nine aquaria (60 L glass aquarium for each), and reared for 70-days. Three tested diets were formulated to be isonitrogenous and isolipidic, and were offered for experimental fish until ad-libitum three times daily at 09:00 a.m., 11.00 a.m. and 3:00 p.m. The first diet (control) was without supplementation. About 50 mg β-carotene and 50 mg phycocyanin kg−1 were supplemented to the other experimental diets, respectively. Results indicated that feed intake was not (P > 0.05) differ among experimental diets. Compared to control diet wight gain and specific growth rate were significantly (P < 0.05) in fish fed diet containing β-carotene, while, the highest weight gain and the best FCR were detected in phycocyanin diet. Survival fish among treatments was significantly (P < 0.05) differ and the highest survival rate was showed in fish fed diet supplemented with phycocyanin. Either β-carotene or phycocyanin significantly (P < 0.05) improved the intestinal digestive enzymes compared with control diet, where the highest values of chymotrypsin, trypsin, lipase and amylase were noticed in fish fed phycocyanin. Diets supplemented with β-carotene and phycocyanin significantly (P < 0.05) improved hematology parameters contents compared with to the control diet, and the best contents were detected in fish fed diet supplemented with phycocyanin. The highest significant (P < 0.05) phagocytic, lysozyme, immunoglobulin M (IgM), superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and total antioxidant capacity (T-AOC) activities were recorded in diet supplemented with phycocyanin. The transcripts of interferon gamma and interleukin 1β genes were (P < 0.05) up-regulated in the liver of fish fed diet supplemented with β-carotene and phycocyanin, but expression of HSP70 gene down-regulated in fish fed β-carotene and phycocyanin containing diet compared control. The highest gene expression of the interferon gamma and interleukin 1β was observed in fish fed phycocyanin.

Immunophenotypic Characterization of Canine Nodal T-Zone Lymphoma

T-zone lymphoma (TZL) is an indolent nodal T-cell lymphoma most commonly observed in submandibular lymph nodes in dogs. The diagnosis is based on its distinct morphology and expression of CD3. TZL has been reported to have a low Ki67 index and to lack expression of CD45. The latter feature has been used to diagnose this type of lymphoma via fine needle aspirate and flow cytometry without confirmation of the characteristic tissue architecture. The goal of this study was to characterize the immunophenotype of canine nodal TZL in greater detail. Twenty-seven TZLs were selected based on their characteristic morphology. A tissue microarray was generated, and immunohistochemical expression of CD3, CD5, CD20, CD21, CD25, CD45, Bcl-6, and Ki67 was evaluated. Neoplastic T cells in all cases were positive for CD3, CD5, and CD25, and negative for CD20, CD21, and Bcl-6. Positive labelling for CD45 was detected in 2 of the 27 cases with the remaining 25 cases being negative. All cases had a low Ki67 index with an average index of 19.56%. For the CD45-positive TZLs, clonality of the T-cell antigen receptor gamma gene was confirmed in only one of these cases. The observed immunophenotype of canine TZL is similar to previous publications with the exception that 2 cases expressed CD45. Expression of CD45 in TZLs in this study emphasizes the importance of interpreting immunophenotypic findings in conjunction with histopathology to reach an accurate diagnosis and not to use lack of expression of a particular antigen as the sole diagnostic criterion.

T-Cell Monoclonality As Assessed By TCR-Sequencing: A Potential Biomarker of Response in Patients with MDS with Autoimmune Cytopenias Treated with IVIG

Background: We have previously reported 75% of patients (pts) achieved clinical responses to treatment with intravenous immunoglobulin (IVIG) in pts with myelodysplastic syndromes (MDS) that exhibit autoimmune cytopenias in the context of T-cell clonality. T-cell clonality was previously determined via peripheral blood (PB) gel-based testing of T-cell receptor (TCR) beta (TRB) and/or gamma (TRG) gene rearrangements or flow cytometry. IVIG was shown to be variably effective in normalizing hemolytic parameters and cytopenias (Feld et al, Blood 2019). Since next generation sequencing of TRG and TRB (TCR-seq) offers better resolution as to the identity of T-cell clones, we reassessed T-cell clonality in a subset of our cohort and longitudinally evaluated the persistence of clones with IVIG treatment. Methods: Among 27 pts who were previously treated with IVIG +/- steroids, we processed the PB of 9 pts who had serial samples of leftover DNA available for TCR-Seq analysis (7 had samples available prior to treatment). Clinical responses were per MDS IWG 2006 criteria, whereas hemolytic responses were established by either a complete normalization or a mild response if there was &amp;gt;50% improvement in hemolytic parameters from baseline. TCR-Seq was performed on 100ng DNA using the Lymphotrack® TRG and TRB Assays on Illumina® MiSeq platform according to manufacturer's instructions (Invivoscribe, Inc.). Using the LymphoTrackMiseq2.4.3 software T-cell clonality was interpreted as monoclonal (MC), oligoclonal, small clone(s) of uncertain significance (SCUS) or polyclonal (PC), based on percent total merged clonal reads and polyclonal background. Results: Baseline characteristics of our 9 pt cohort included 8 males and 1 female, a median age of 70 years (range 45-82), and a median duration of treatment with IVIG of 23 months. Six pts received steroids in addition to IVIG. Diagnosis included 8 pts with MDS and one with large granular lymphocytic (LGL) leukemia. Of the MDS pts measurable by IPSS criteria, 6 were Int-1 and one was low risk. Expanded lymphocyte populations were detected in 7 pts by PB flow cytometry and all pts exhibited T-cell clonality by gel-based assessment. All were TRB positive (+) and 7 were TRG+ by PCR. In 3 pts, 4 serial samples were available for TCR-seq; 4 pts had 3 samples available and 2 had only 2 serial samples. Samples were collected over a period of 5 months up to 4-5 years. With the better resolution afforded by TCR-Seq, 3 pts exhibited only PC populations of T-cells for TRG and TRB at all time points, and 1 pt exhibited SCUS in 1 of 3 samplings for TRG. Of the four pts, only one had an erythroid response; importantly, this sample was tested after treatment. One pt had a normalization in their bilirubin, otherwise only mild responses in hemolytic parameters were noted in the remaining 3 PC pts. Median duration of treatment was 1.5 months. Five pts had a MC TRB and/or TRG population by TCR-Seq. Two pts had MC TRG, two had MC TRG &amp; TRB, and one had MC TRB populations. Four of the 5 pts had hematologic improvement (HI): 1 had an erythroid and platelet response, 2 had an erythroid response, and 1 had a platelet response. More significant hemolytic responses were also observed in 3/5 pts, with a mild response in the 2 remaining pts. Only 1 TCR-Seq+ pt had no HI, and this pt had a monoclonal population detected by TCR-Seq after finishing IVIG treatment. Median duration of treatment was 45 months. For all these cases, the same clonal sequences were found across all samples up to 4-5 years. Of 4 evaluable patients, 3 had reductions in their clonal read percentage burden over their treatment period. Conclusions: TCR-Seq of the TRG and TRB genes affords better resolution of T-cell clones and permits tracking of clones over time. On a further subgroup analysis of 9 pts from our cohort of IVIG-treated pts, we found that pts who exhibited monoclonality as established by TCR-Seq were more likely to respond to IVIG, with more significant erythroid and hemolytic responses, associated with a consequential (45+ months) longer treatment duration. Thus, TCR-Seq appears to be a superior option to PCR-based TCR gene rearrangement assays to reliably identify T cell clones in pts with MDS, and may be a useful biomarker to prospectively identify a subset of pts that can respond to IVIG. Further studies are ongoing to test this hypothesis. Disclosures Navada: Onconova Therapeutics Inc: Research Funding. Silverman:Onconova Therapeutics Inc: Patents &amp; Royalties, Research Funding; Medimmune: Research Funding; Celgene: Research Funding.

Recurrent and Refractory Hemophagocytic Lymphohistiocytosis in an Elderly. Role of Immune Aberration Due to Myeloid Gene Mutation

Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal hyper-inflammatory disease induced by aberrant immune activation and subsequent proliferation of macrophages, histiocytes and T-helper cells. In this abstract we present a case of HLH, which relapsed twice despite ongoing treatment, and we hypothesize on possible causes and mechanisms. A 77 year old female presented to our hospital with ongoing fevers and worsening cytopenia. Blood counts from three years before the current presentation showed Hb 120g/L, WBC 4.0 x 10^9/L, Neutrophil count 1.8 x 10^9/L, Lymphocytes count 1.8 x 10^9/L and Platelet count 104 x 10^9/L. A bone marrow examination at that time revealed a normocellular marrow with 28% lymphocytes of which70% were CD 4+, CD 3+, CD5+ and CD 7-. Molecular studies confirmed T cell receptor (TCR) gamma gene rearrangement. The karyotype on the bone marrow was normal. In the absence of clinical symptoms, the patient was regularly followed up without specific therapy. During the current admission, however, the patient was febrile, had progressive pancytopenia and biochemistry suggestive of HLH (Fig 1). She was extensively evaluated which ruled out infective and malignant causes. A bone marrow aspirate and biopsy was performed and treatment initiated as per HLH-94 protocol. The bone marrow examination showed marked features of haemophagocytosis on a normocellular background. A small clone of T-lymphocytes was again noted with similar features as in the first biopsy. In addition, a prominent population (10%) of promonocytes was apparent with an uncertain significance. Karyotype was normal. Next Generation Sequencing showed TET2 frame shift mutation at low variant allele frequency (5%). Patient responded well to treatment. While on tapering dose of steroids, the disease flared up (Fig 1) and the patient was restarted on high dose steroids with etoposide. After a quick initial response, while still on active treatment, she again relapsed within 3 weeks, coupled with sepsis and acute myocardial ischemia, followed by sudden death. We were unable to identify a cause for HLH. There were 10% promonocytes in bone marrow and evidence of aberrant T-cells on flow cytometry. Although there was no obvious evidence of dysplasia on microscopy, the flow cytometry showed up- regulation of CD 64 and CD 14, down regulation of CD13 and 11b, and CD 34 expression in granulocytes possible indicating dysplasia as per the Wells criteria. (Wells et al., Blood 2003; 102(1):393) The Next generation sequencing showed TET2 mutation as mentioned above. Mutations in TET2 have been found to have overrepresented in chronic myelomonocytic leukemia in as much as 50% of patients and around 20-35% of patients with myelodysplastic syndrome (MDS). These patients with TET2 mutation have been found to have altered methylation. Recently TET2 has been implicated in immune regulation with evidence of abnormal CD 4 T cell proliferation (present in our patient) and disruption of T cell homeostasis. In addition, patients with TET2 mutation associated myelodysplastic syndrome are known to have auto-immune manifestations (Yimei Feng et al., Frontiers in Oncology, 2019 (9):1). Alyssa H et al have shown that TET2 mutant in patients with MDS (Myelodysplasia) leads to alteration of immune environment in the macrophage differentiation (Alyssa et al., Experimental Hematology, 2017:55; 56). Whether these immune aberration caused recurrent florid relapse of hemophagocytosis in our patient within a span of 2-3 months remained unclear and it could be considered in future research. Even though the occurrence of hemophagocytic syndrome has been described in acute leukemias, the association of the same with myeloid gene aberrations with or without overt myelodysplastic features is unknown. However since hemophagocytosis itself is not common, a careful evaluation to look for uncommon associations which may be a triggering factor may pave the way for identifying their possible role in the pathogenesis. And therapeutic options such as hypo methylating agents can evolve when such associations are confirmed in future studies. Figure 1 Disclosures No relevant conflicts of interest to declare.

Expression analysis of gamma globin gene switching associated micro RNAs, flowing BCL11A gene inhibition

Background/aimMicroRNAs are small, single-stranded RNA molecules that are targeting between 10% and 30% of the human genome, also a gene that binds to several miRNAs. By connecting them to targets gene, the silencing occurs after the translation. Materials and methodsThe aim of this study was to investigate changes in the expression of miRNAs before and after inhibiting the gamma-beta gene switch by siRNA against the BCL11A gene. MiR- 15a, miR- 16, miR-486-3p, miR- 23a and miR-27a, important miRs in switching gamma to beta gene, were investigated. Peripheral blood mononuclear cells were isolated by Ficoll-Paque. They were cultured in the IMDM medium containing supplementary materials for differentiation to erythroid series for a period of 28 days and stored in a 37-degree incubator containing 5% CO2. Every 2–3 days, identification of the cells was done. On the 21st day, cells were introduced by insertion of siRNA against BCL11A. ResultsThe expression of miR- 15a in the cultured cells gradually increased from the first day to the eighteenth day and decreased on 21 day. Expression pattern of miR- 16 was increased on the 8th day and decreased on the 22th day. MiR-23a decreased on day 8, increased on day 21, and remained unchanged on days 18 and 22. miR-27a increased the expression on day 8, decreased on day 21, and remained unchanged on days 18 and 22. Expression of miR-486-3p increased on the 8th, 18th and 21th days; also, on the 22thday, there was a decrease in expression of miR-486-3p. ConclusionOur data showed that changes in the expression of miRNAs were significant. The results of this study suggest that inhibition of the gamma gene switch to beta, keeps hemoglobin F high, therefore, this could lead to improved symptoms in patients with hemoglobinopathy.

Leukemia Cutis in a Patient with Rare Indolent T-Prolymphocytic Leukemia - A Case Report and Review of the Literature

Abstract Introduction/Objective T-cell prolymphocytic leukemia (T-PLL) is rare, comprising 2% of mature lymphocytic leukemias in adults. Clinical course is usually aggressive, however, 20-30% patients can have an indolent early phase. Most patients present with extensive disease with peripheral blood lymphocytosis, hepatosplenomegaly, and lymphadenopathy. T-PLL has a higher incidence in patients with ataxia-telangiectasia. Methods We present a case of a 67-year old man with history of somatic mutation of ataxia-telangiectasia gene (ATM) and multiple malignancies, including melanoma, skin and laryngeal squamous cell carcinomas, adrenal adenoma and spindle cell lung tumor (diagnosed on biopsy), who developed leukemia cutis during an indolent phase of T-PLL. Results Initial diagnosis of T-PLL was on routine complete blood count when patient had mild atypical lymphocytosis (5.5 x 109/L). Flow-cytometry immunophenotyping detected CD4 + 8 positive mature T-cells with no aberrant loss of T- cell antigens, and molecular studies confirmed clonal T-cell receptor beta and gamma gene rearrangements. T-PLL associated leukemia cutis was diagnosed on a leg skin nodule biopsy 10 months later. Subsequent bone marrow biopsy demonstrated low level involvement by T-PLL. The patient’s T-PLL followed an indolent trajectory for three years and treatment was withheld until symptomatic presentation. Rapid disease progression was heralded with emergence of widespread T-PLL leukemia cutis, rapidly increasing lymphocytosis, extensive lymphadenopathy, splenomegaly and increasing LDH levels. Conclusion Skin involvement is reportedly identified in 20% to 50% of T-PLL cases in some series. Skin manifestations can be heterogenous with a broad clinical differential. T-PLL associated leukemia cutis may not be recognized owing to the overall low incidence and in skin lesions appearing during an undiagnosed indolent early phase of T-PLL.We highlight the importance of accurate evaluation of non-specific skin lesions in early detection of T- PLL. Being aware of this diagnosis and early engagement in an interdisciplinary approach may allow for better outcomes of this aggressive malignancy.

Gut microbiota and metabolic marker alteration following dietary isoflavone-photoperiod interaction.

IntroductionThe interaction between isoflavones and the gut microbiota has been highlighted as a potential regulator of obesity and diabetes. In this study, we examined the interaction between isoflavones and a shortened activity photoperiod on the gut microbiome.MethodsMale mice were exposed to a diet containing no isoflavones (NIF) or a regular diet (RD) containing the usual isoflavones level found in a standard vivarium chow. These groups were further divided into regular (12L:12D) or short active (16L:8D) photoperiod, which mimics seasonal changes observed at high latitudes. White adipose tissue and genes involved in lipid metabolism and adipogenesis processes were analysed. Bacterial genomic DNA was isolated from fecal boli, and 16S ribosomal RNA sequencing was performed.ResultsNIF diet increased body weight and adipocyte size when compared to mice on RD. The lack of isoflavones and photoperiod alteration also caused dysregulation of lipoprotein lipase (Lpl), glucose transporter type 4 (Glut‐4) and peroxisome proliferator‐activated receptor gamma (Pparg) genes. Using 16S ribosomal RNA sequencing, we found that mice fed the NIF diet had a greater proportion of Firmicutes than Bacteroidetes when compared to animals on the RD. These alterations were accompanied by changes in the endocrine profile, with lower thyroid‐stimulating hormone levels in the NIF group compared to the RD. Interestingly, the NIF group displayed increased locomotion as compared to the RD group.ConclusionTogether, these data show an interaction between the gut bacterial communities, photoperiod length and isoflavone compounds, which may be essential for understanding and improving metabolic health.

Alkaloid-based regimen is beneficial for acute myeloid leukemia resembling acute promyelocytic leukemia with NUP98/RARG fusion and RUNX1 mutation

Rationale:Some acute myeloid leukemia (AML) patients present with features mimicking the classical hypergranular subtype of acute promyelocytic leukemia (APL) but without the typical promyelocytic leukemia/retinoic acid receptor α (PML/RARα) rearrangement. Herein, we report an AML patient resembling APL but with nucleoporin 98/retinoid acid receptor gamma gene (NUP98/RARG) fusion transcript and Runt-related transcription factor 1 (RUNX1) mutation.Patient concerns:An 18-year-old male presented at the hospital with a diagnosis of AML.Diagnoses:The patient was diagnosed with bone marrow examination. Bone marrow smear displayed 90.5% promyelocytes. Fluorescence in situ hybridization analysis failed to detect the PML/RARα fusion transcript or RARα amplification. While real-time polymerase chain reaction showed positivity for the NUP98/RARG fusion transcript. G-banding karyotype analysis showed a normal karyotype.Interventions:The patient showed resistance to arsenic trioxide and standard 3 + 7 chemotherapy, but eventually achieved complete remission through the Homoharringtonine, Cytarabine, and Aclarubicin chemotherapy.Outcomes:These measures resulted in a rapid response and disease control.Lessons:Acute myeloid leukemia with the NUP98/RARG fusion gene and the RUNX1 mutation may be a special subtype of AML and may benefit from the alkaloid-based regimen.

Abstract A04: Analysis of molecular and immune features that correlate with serum lactate dehydrogenase (LDH) levels in patients (pts) with metastatic melanoma

Abstract Background: Elevated serum LDH is a poor prognostic factor in pts with MM. It is currently unknown if serum LDH levels correlate with molecular or immune differences in tumors, including LDH expression. We analyzed resected melanoma metastases to identify features that correlate with serum LDH elevation. Methods: Metastases with serum LDH levels measured within 30 days of surgery were included in the study: (1) a TMA of resected stage IV metastases from MD Anderson (MDACC) (n=207); (2) resected stage IV metastases from MDACC with previously acquired RNA-sequencing (RNA-seq) and CD8+ immunohistochemistry (IHC) data (n=24); (3) publicly available data for regional metastases from the melanoma TCGA (n=104) from MDACC and the Melanoma Institute of Australia. IHC was performed for LDHA, LDHB, PTEN, PD-L1, MITF, and Ki67 on the stage IV TMA, and publicly available data for DNA, RNA, and proteins for the TCGA samples were downloaded. Data were analyzed to identify features that differed between tumors with elevated and normal serum LDH levels. TCGA and stage IV tumors expressing the 25% highest and lowest LDHA and LDHB by RNA-seq were also compared. Results: IHC of the stage IV TMA identified no significant associations with serum LDH levels, including tumor LDHA (p=0.69) and LDHB (p=0.93) expression. Serum LDH was elevated in 33.3% of stage IV metastases with RNA-seq data and 21.6 % of TCGA regional metastases, which by Ensemble of Gene Set Enrichment Analyses (EGSEA) correlated with decreased expression of interferon alpha (q=8.26 × 10−14 and q=2.31 × 10−14), interferon gamma (q=9.46 × 10−14 and q=2.34 × 10−19), and inflammatory response (q= 1.26 × 10−20 and q=4.17 × 10−28) gene sets. Fewer (p=0.0464) CD8+ T cells were detected in stage IV tumors from pts with elevated serum LDH. Comparison of stage IV and TCGA tumors with high vs. low LDHA and LDHB mRNA expression levels did not correlate with serum LDH status, but significant enrichment of oxidative phosphorylation (q=6.32 × 10−21 and q=5.69 × 10−19), pyruvate metabolism (q= 7.90 × 10−16 and q=1.78 × 10−14), and citrate cycle (q= 1.51 × 10−06 and q=5.44 × 10−06) gene sets were detected in LDHA-high tumors. Increased LDHB expression correlated with enrichment of citrate cycle (q= 4.04 × 10−16 and q= 1.68 × 10−14), pyruvate metabolism (q= 2.34 × 10−11 and q=1.50 × 10−10), and glycolysis (q= 2.92 × 10−9 and q=1.55 × 10−8) gene sets. Conclusions: Elevated serum LDH levels correlated with decreased expression of immune response genes, but not LDHA or LDHB expression, in melanoma regional and distant metastases. Elevated serum LDH also correlated with decreased CD8+ T cell infiltrates by IHC. LDHA and LDHB expression levels in regional metastases correlated with distinct metabolic pathways, but not serum LDH levels. Citation Format: Fernando Cintra Lopes Carapet, Grant Fischer, Aron Joon, Huiqin Chen, Lauren Haydu, Sandra Lee, Melissa Saul, Savi Appana, John Thompson, Graham Mann, Jennifer McQuade, Alexander Lazar, Michael Tetzlaff, John Kirkwood, Richard Scolyer, Georgina Long, Michael Davies. Analysis of molecular and immune features that correlate with serum lactate dehydrogenase (LDH) levels in patients (pts) with metastatic melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A04.

High Expression of CD39 is Associated with Poor Prognosis and Immune Infiltrates in Clear Cell Renal Cell Carcinoma.

IntroductionThe cell-surface ectonucleotidase CD39 is a key molecule of the immunosuppressive adenosine pathway within the tumor microenvironment. However, the relationship between CD39 and clear cell renal cell carcinoma (ccRCC) is rarely reported and still remains unclear.MethodsCD39 expression was first analyzed using the Oncomine and the Tumor IMmune Estimation Resource (TIMER) databases, and then examined in ccRCC patients (n=367) who had undergone radical nephrectomy using immunohistochemistry (IHC) and real-time quantitative PCR analysis (qPCR). The prognosis value of CD39 in ccRCC was evaluated by Cox proportional hazards analysis. Functional and gene set enrichment analysis (GSEA) was performed using transcriptomic data of ccRCC from TCGA. Correlation analysis between CD39 and tumor-infiltrating lymphocytes (TILs) was performed using the TISIDB database. The impact of CD39 on immune checkpoint therapy (ICT) was evaluated by two public cohorts.ResultsCD39 mRNA and protein expression was upregulated in tumor tissues from ccRCC patients and aberrant expression of CD39 was associated with advanced tumor stage and poor prognosis in ccRCC patients. EMT, IL-2/STAT5, inflammatory response, interferon gamma and KRAS hallmark gene sets were identified as CD39-related signaling pathway. The expression level of CD39 was significantly and positively correlated with high abundance of the regulatory TILs including NK cells, macrophages, Th cells and Treg cells. CD39 was correlated with expression of several immune checkpoints and higher CD39 expression was associated with better OS of ccRCC patients who received ICT.ConclusionCD39 is a powerful prognostic marker of ccRCC patients. Increased tumor expression of CD39 mRNA is significantly correlated with infiltrating levels of TILs, and better efficacy of ICT to ccRCC. CD39 could be a novel therapeutic target for ccRCC.