Mutation In Codon Research Articles

Abstract 7082: Epidemiology meets epitranscriptomics: Exploring the cancer-related role of a novel TERT-antisense transcript and its regulation by RNA methylation

Abstract Human telomerase reverse transcriptase (TERT) maintains telomeres at chromosome ends and its dysregulation is implicated in various human health conditions. Multiple GWAS signals are detected within the TERT genomic region at chr 5p15.33. As a potential proxy for these signals, we investigated a highly polymorphic variable number tandem repeat (VNTR)2-1 within intron 2 of TERT, with 57-143 copies of a 42 bp repeat unit per allele. By functional annotation of VNTR2-1, we predicted that it generates an intronic transcript antisense to TERT with a variable-size open reading frame (ORF) of 2502-5211 bp (834-2038 aa). Specific to the TERT-antisense transcript, we predicted N6-methyladenosine (m6A) consensus motifs within each 42 bp unit of VNTR2-1, with longer VNTR2-1 regions carrying more m6A motifs. In a stranded RNA-seq dataset of African pediatric Burkitt lymphoma (BL) tumors, which have high TERT expression, we detected the VNTR2-1-containing TERT-antisense transcript in a subset of tumors (n=35/113, 30.9%), confirming the expression of this novel transcript. VNTR2-1 expression was also experimentally validated by 5’-RACE/Nanopore-seq in Raji (BL cell line) and UMUC3 (bladder cancer cell line). Additionally, the VNTR2-1 transcript was visualized in UMUC3 cells using RNA-FISH. The consensus 42 bp VNTR2-1 repeat sequence and its m6A-deficient variants were cloned into a m6A-deficient dual-luciferase reporter vector (m-psiCHECK-2) and expressed in UMUC3 cells, showing an m6A-motif dose-dependent response driving luciferase protein expression. Transient treatment with STM2457, an inhibitor of the m6A writer METTL3, reduced the expression of the luciferase reporter, confirming that m6A motifs within VNTR2-1 are methylated by METTL3. Additionally, Long-term cigarette smoke condensate (CSC) treatment of UMUC3 reduced luciferase expression, suggesting alterations of VNTR2-1 m6A due to smoke exposure. We explored potential peptides produced from VNTR2-1 transcript by analysis of public mass spectrometry datasets using PepQuery and identified several unique VNTR2-1 peptide fragments in human tumors. Next, we cloned a synthetic FLAG-tagged gene containing three VNTR2-1 consensus repeats with native Kozak sequence and translation start/stop codons. Western blotting confirmed the translation of the VNTR2-1 peptide that was ablated by start codon mutation, suggesting that the native Kozak sequence is sufficient for VNTR2-1 translation. These results support VNTR2-1 as a peptide-producing transcript. In conclusion, we identified a novel intronic transcript produced by VNTR2-1 antisense to TERT. This m6A-regulated transcript could function through several mechanisms, such as a lncRNA or translated peptide, potentially regulating TERT expression and telomerase function in health and disease conditions, including cancer. Citation Format: Brenen W. Papenberg, Oscar Florez-Vargas, Michelle Ho, Kaitlin Forsythe, Chi Zhang, Chia-Han Lee, Sam Mbulaiteye, Pedro J. Batista, Ludmila Prokunina-Olsson. Epidemiology meets epitranscriptomics: Exploring the cancer-related role of a novel TERT-antisense transcript and its regulation by RNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7082.

Abstract 5915: ADT-1004: A promising pan-RAS inhibitor for targeting KRAS mutations in pancreatic ductal adenocarcinoma

Abstract Background/Objective: Pancreatic ductal adenocarcinoma (PDAC) is a highly challenging cancer with a dismal 5-year survival rate of under 12%. More than 90% of PDAC cases involve mutations in the GTPase KRAS. Unfortunately, existing drugs targeting the RAS family, like sotorasib, exclusively address the KRAS-G12C mutation, mainly found in NSCLC. In PDAC, most patients are diagnosed at advanced stages with unresectable tumors that exhibit various genetic mutations. Approximately 70% of these cases have KRAS-G12D mutations, often co-occurring with KRAS-G12V, KRAS-G12C, and KRAS-G12R mutations. This highlights the urgent need for innovative treatments that effectively address the complex mutation landscape in PDAC and avoid resistance mechanisms associated with mutant-specific or pan-KRAS inhibitors. Methods: KPC-Luc (1 × 105) and 2838C3-Luc (1.5 × 105) PDAC cells carrying the KRAS-G12D mutation were injected into the pancreas of C57BL/6J mice. After one week, the mice were randomly divided into treatment groups (n = 7 per group) and received oral ADT-1004 at a dose of 40mg/kg body weight five times a week for four weeks. Tumor burden was assessed by bioluminescence signals using the IVIS Xenogen imaging system for patient-derived xenografts from PDAC patients with the KRAS-G12V mutation who were subcutaneously implanted in NSG mice. A week later, mice were randomized (n=7 per group) and treated orally with 40mg/kg of ADT-1004 five times a week for six weeks. Body weight and tumor size were measured twice weekly. Results: ADT-1004, a prodrug of ADT-007 that exhibits potent inhibitory effects on the proliferation of both human and murine RAS mutant PDAC cell lines with single-digit nM IC50 values, compared with µM IC50 in PDAC cells with WT RAS, was well tolerated in mice at a dose up to 175 mg/kg bid orally with sustained plasma levels of ADT-007 that exceeded IC50 values by 50-fold and even higher concentrations in pancreatic tumors. When administered orally at 40mg/kg body weight, ADT-1004 resulted in substantial growth inhibition of the KRAS-G12D and KRAS-G12V tumors, leading to profound tumor regression without any accompanying loss in body weight. ADT-1004 also displayed a significant inhibitory effect on ERK phosphorylation. Notably, this inhibition is effective regardless of the specific RAS mutational codon or isozyme, as it specifically targets activated RAS and disrupts downstream KRAS signaling pathways. Conclusion: These results highlight the promise of ADT-1004 as a potential breakthrough for treating PDAC. This novel pan-RAS inhibitor demonstrates potent inhibition of PDAC growth, irrespective of the specific KRAS mutation. Furthermore, it shows an excellent safety profile, with profound tumor regression and no impact on body weight in animal models. These findings open new possibilities for addressing the complex genetic landscape of PDAC. Citation Format: Dhana Sekhar Reddy Bandi, Purnachandra Nagaraju Ganji, Sujith Sarvesh, Yulia Maxuitenko, Jeremy B Foote, Adam B Keeton, Xi Chen, Donald Buchsbaum, Gary Piazza, Bassel El-Rayes. ADT-1004: A promising pan-RAS inhibitor for targeting KRAS mutations in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5915.

Probable Novel APP Met671Leu Mutation in a Chinese Han Family with Early-Onset Alzheimer's Disease.

Familial Alzheimer's disease (AD) is a rare disease caused by autosomal-dominant mutations. APP (encoding amyloid precursor protein), PSEN1 (encoding presenilin 1), and PSEN2 (encoding presenilin 2) are the most common genes cause dominant inherited AD. This study aimed to demonstrate a Chinese early-onset AD pedigree presenting as progressive memory impairment, apraxia, visual-spatial disorders, psychobehavioral disorders, and personality changes with a novel APP gene mutation. The family contains four patients, three carries and three normal family members. The proband underwent brain magnetic resonance imaging (MRI), 18F-fludeoxyglucose positron emission tomography (18F-FDG-PET), cerebrospinal fluid amyloid detection, 18F-florbetapir (AV-45) Positron Emission Computed Tomography (PET) imaging, whole-exome sequencing and Sanger sequencing. Brain MRI images showed brain atrophy, especially in the entorhinal cortex, temporal hippocampus, and lateral ventricle dilation. The FDG-PET showed hypometabolism in the frontotemporal, parietal, and hippocampal regions. 18F-florbetapir (AV-45) PET imaging showed cerebral cortex Aβ protein deposition. The cerebrospinal fluid amyloid protein test showed Aβ42/Aβ40 ratio decreases, pathological phosphor-tau level increases. Whole-exome sequencing detected a new missense mutation of codon 671 (M671L), which was a heterozygous A to T point mutation at position 2011 (c.2011A > T) in exon 16 of the amyloid precursor protein, resulting in the replacement of methionine to Leucine. The co-separation analysis was validated in this family. The mutation was found in 3 patients, 3 clinical normal members in the family, but not in the other 3 unaffected family members, 100 unrelated normal subjects, or 100 sporadic patients with AD. This mutation was probably pathogenic and novel in a Chinese Han family with early-onset AD.

Human TP53 gene polymorphisms among patients with Hepatocellular carcinoma and chronic hepatitis B infection in Kenya

Background Human TP53 is the gatekeeper for generation of human cells and is highly conserved. Some alteration/mutation in TP53 adversely affects the regulatory function of the protein, potentially resulting in cancer. This study investigated mutations in codons 72 and 249 of TP53, among patients with hepatocellular carcinoma (HCC) and chronic hepatitis B virus (HBV) infection at the Moi Teaching and Referral Hospital (MTRH), Eldoret, Kenya. Methods In total, 33 HBV-positive patients attending MTRH hospital between September 2013 and July 2017 were purposely selected from medical records for the study; those with HCC were confirmed from the cancer registry. The patients were aged between 25-67 years, with a male-to-female ratio of 1.1:1. Blood samples were collected from the patients. DNA was extracted, amplified and sequenced using TP53 forward and reverse primers. Gene mutation detection and analysis was done on exons 4 codon 72 and exon 7 codon 249. Results Of the 33 patients, 75.8% were chronically infected with HBV and had HCC; the rest were HBsAg positive without HCC. Homozygous proline was prevalent (54.5%) at exon 4 codon 72, followed by heterozygous Arg/Pro (33.3%) and lastly homozygous Arg/Arg (12.1%). Pro/Pro allele was frequent in HCC group while Arg/Arg allele was common in patients without HCC. There was no significant association between the HCC and codon polymorphisms (P=0.12). In exon 7, codon 249, 24.2% of patients had an Arg/Ser mutation of which, 75.0% had HCC and 25.0% did not. There was no significant association between HCC patients and codon 249 mutation (P=0.15). Conclusion TP53 is a gene gate keeper, the mutations under study may dependently play a role in HCC development. This study did not find any association between TP53 mutations and presence of HCC. Therefore, TP53 Arg-72 and Ser-249 mutation is not a clear prognosis indicator for hepatocellular carcinoma among HBV infected patients in Kenya.

Amplification of Mutant NRAS in Melanocytic Tumors With Features of Spitz Tumors

NRAS activating mutations are prevalent in melanocytic neoplasia, occurring in a subset of common acquired melanocytic nevi and ∼30% of cutaneous melanomas. In this study, we described a cohort of 7 distinctive melanocytic tumors characterized by activating point mutations in codon 61 of NRAS with amplification of the mutant NRAS allele and shared clinicopathologic features. These tumors occurred predominantly in younger patients, with a median age of 20 years (range, 6-56 years). They presented as papules on the helix of the ear (4 cases) or extremities (3 cases). Microscopically, the tumors were cellular, relatively well-circumscribed, compound, or intradermal proliferations. The tumor cells often extended into the deep reticular dermis and involved the superficial subcutaneous fat in some cases. The melanocytes were epithelioid to spindled with moderate amounts of cytoplasm and conspicuous nucleoli. They were arranged in short plexiform fascicles, nests, and cords. Some cases had occasional pleomorphic and multinucleated melanocytes. Rare dermal mitotic figures were present in all cases. The dermis contained thick collagen bundles and minimal solar elastosis. Follow-up data were available for 5 patients, with a median period of 4.2 years (range, 1-9 years), during which no recurrences or metastases were reported. Our series highlights a clinicopathologically and molecularly distinctive subset of NRAS-mutated tumors with amplification of the mutant NRAS allele.

Abstract B101: Characterizing somatic TP53 mutations in non-cancerous and high-risk fallopian tubes using ultra-deep sequencing

Abstract High-grade serous carcinoma (HGSC), the most common and pervasive subtype of ovarian cancer originates in the fallopian tube epithelium from precursor lesions carrying somatic TP53 mutations. These lesions may evolve into carcinomas, eventually spreading to the ovary and peritoneum1,2. Recent research in non-cancerous tissues and gynecologic liquid biopsies of individuals with and without HGSC have demonstrated that somatic TP53 mutations accumulate and clonally expand with age3–9. However, TP53 somatic evolution has not been characterized in the normal fallopian tube epithelium. In addition, it is not known whether TP53 mutations are more abundant in individuals with germline BRCA1/2 mutations (BRCA carriers), who are at a high risk of HGSC10. The goals of this study were to characterize age-related TP53 somatic mutations in non-cancer fallopian tube epithelium across the human lifespan and to determine whether the TP53 mutational landscape is altered in BRCA carriers. Our cohort consisted of 1) autopsy cases, spanning the ages of 0-91 years, from individuals without ovarian cancer (N=46) and 2) prophylactic surgery cases from BRCA carriers and non-carriers (N=26, ongoing analyses). Fallopian tube tissues were collected, frozen, and macrodissected using a 1mm biopsy punch, enriching for the fallopian tube epithelium. DNA was extracted from each sample, the sequenced for TP53 using ultra-deep duplex sequencing (mean depth: ~12,000x). In the normal fallopian tube epithelium collected across human lifespan, we found that the number of unique TP53 mutations and the size of pathogenic clones increased with age, showcasing somatic evolution and clonal expansion of TP53 in non-cancerous fallopian tube epithelium with age. We also found that the number of TP53 mutations in hotspot codons increased with age, suggesting that cancer-like mutations are positively selected as individuals age. We compared the normal fallopian tube TP53 mutations with HGSC TP53 mutations and demonstrated that the types of mutations (e.g. indels, splice, nonsense, missense, and silent mutations) in the aging fallopian tube closely mirrored the mutations types observed in HGSC. Data analysis from BRCA carriers is ongoing, but based on preliminary data, we anticipate a higher burden of pathogenic TP53 mutations, indicating that predisposition to HGSC in BRCA carriers may be related to an excess of TP53 somatic evolution in the fallopian tube. Altogether, our research demonstrates that TP53 somatic evolution is a normal aging phenotype in the fallopian tube, with more and larger pathogenic clones occurring later in life. We expect to determine whether this effect is more prominent in BRCA carriers, which would provide a much needed biological explanation to understand the increased risk of HGSC in these patients, opening new venues for better cancer prediction and prevention. Citation Format: Coleen Ann Coombes, Elizabeth U. Parker, Brendan F. Kohrn, Jeanne Uy Fredrickson, Elena Latorre-Esteves, Emma Hazard, Shreya Suresh, Marc R. Radke, Anh P. Vo, Barbara S. Norquist, Rosana Risques. Characterizing somatic TP53 mutations in non-cancerous and high-risk fallopian tubes using ultra-deep sequencing [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B101.

C and G are frequently mutated into T and A in coding regions of human genes.

Nucleotide mutations in human genes have long been a hot subject for study because some of them may lead to severe human diseases. Understanding the general mutational process and evolutionary trend of human genes could help answer such questions as why certain diseases occur and what challenges we face in protecting human health. In this study, we conducted statistics on 89,895 single-nucleotide variations identified in coding regions of 18,339 human genes. The results show that C and G are frequently mutated into T and A in human genes. C/G (C or G)-to-T/A mutations lead to reduction of hydrogen bonds in double-stranded DNA because C-G and T-A base pairs are maintained by three and two hydrogen bonds respectively. C-to-T and G-to-A mutations occur predominantly in human genes because they not only reduce hydrogen bonds but also belong to transition mutation. Reduction of hydrogen bonds could reduce energy consumption not only in separating double strands of mutated DNA for transcription and replication but also in disrupting stem-loop structure of mutated mRNA for translation. It is thus considered that to reduce hydrogen bonds (and thus to reduce energy consumption in gene expression) is one of the driving forces for nucleotide mutation. Moreover, codon mutation is positively correlated to its content, suggesting that most mutations are not targeted on changing any specific codons (amino acids) but are merely for reducing hydrogen bonds. Our study provides an example of utilizing single-nucleotide variation data to infer evolutionary trend of human genes, which can be referenced to conduct similar studies in other organisms.

Distribution of Voltage Gated Sodium Channel (VGSC) Gene Mutational Variation And Acetylcholinesterase-1 (ACE-1) as A Marker For Insecticide Resistance In Culex spp. Mosquitoes In Surabaya

Culex spp. is a vector for Japanese Encephalitis (JE) and filariasis in Indonesia. JE is caused by Japanese Encephalitis Virus (JEV). JEV attacks the central nervous system and causes inflammation of the brain, lifelong disability, and even death. Filariasis is a chronic infectious disease that can cause permanent disability. Transmission of JE and filariasis can increase due to the density of Culex spp mosquitoes. The purpose of this study was to identify the distribution of mutation variations in the Voltage Gated Sodium Channel (VGSC) and acetylcholinesterase-1 (ace-1) genes in Culex spp. mosquitoes in Surabaya. Mutation variations of the VGSC Culex spp. gene show that there are three genotypes, namely wildtype (LL), heterozygous (LS, FS), and homozygous (FF, SS). Heterozygous (FS) and homozygous (FF, FS) genotypes caused by point mutations in codon 1014 of the VGSC gene are found in the Culex spp. mosquito population in Surabaya. Heterozygous (LF) genotype was not detected in the Culex spp. mosquito population in Surabaya. Another study in Nigeria showed that homozygous/FF genotypes (45.1%) were more prevalent than heterozygotes/LF (36.3%) and wildtypes/LL (18.6%).

Определение соматических мутаций в гене EZH2 и оценка их прогностической значимости при фолликулярной лимфоме 1–3А цитологического типа

Aim. To determine the incidence and prognostic value of mutations in exon 16 of EZH2 as well as those of polymorphism с.1582-21А>G (rs2072407) in EZH2 in patients with follicular lymphoma (FL) grades 1–3А in relation to morphologic and cytogenetic tumor characteristics.
 Materials & Methods. The prospective cohort study conducted by the National Research Center for Hematology from January 2017 to April 2021 enrolled 80 patients with newly diagnosed FL grades 1/2 and 3А. The median follow-up was 53 months. Molecular and cytogenetic analyses were based on biopsy samples of lymph nodes obtained before chemotherapy. The mutation status of exon 16 in EZH2 and the presence of intronic polymorphism rs2072407 in EZH2 were examined by Sanger sequencing method. Translocation t(14;18)(q32;q21) was detected by karyotyping or FISH.
 Results. Mutations in exon 16 of EZH2 (mutEZH2) were identified in 10/80 (13 %) patients. All patients showed missense mutation in codon 646 of EZH2. Translocation t(14;18) was detected in 45/80 (56 %) cases. Poor outcome in the cohort with no t(14;18) was observed 3 times more often than in the group of patients with t(14;18) (p = 0.0001). The presence of t(14;18) was associated with favorable prognosis irrespective of either the mutation status of exon 16 in EZH2 or the FL grade. The analysis of the polymorphism rs2072407 status yielded the following genotypes: AA in 24 % (n = 19), AG in 42 % (n = 34), and GG in 34 % (n = 27) of cases. The variants АА and AG were associated with higher risk of death (hazard ratio 2.9; 95% confidence interval 1.2–10.6; p = 0.01), whereas the genotype GG was associated with wtEZH2 (10 % vs. 37 %) and favorable prognosis (p = 0.065).
 Conclusion. Significant biological markers for favorable prognosis in FL appeared to be the presence of t(14;18)(q32;q21) and GG genotype of polymorphism rs2072407 in EZH2. The previously identified prognostic factors (grade 3А, bulky tumor lesions > 6 cm, Ki-67 > 35 %, and a short interval between symptom onset and chemotherapy start) were incorporated into a new unified personalized predictive (index PPI) FL model by supplementing it with two additional biological markers: the presence of t(14;18)(q32;q21) and GG genotype of polymorphism rs2072407. This approach may increase the prognostic value of the new personalized design which will provide the basis for risk-adapted algorithms for FL treatment.

CdsA, a CDP-diacylglycerol synthase involved in phospholipid and glycolipid MPIase biosynthesis, possesses multiple initiation codons.

CdsA is a CDP-diacylglycerol synthase essential for phospholipid and glycolipid MPIase biosynthesis, and therefore for growth. The initiation codon of CdsA has been assigned as "TTG," while methionine at the 37th codon was reported to be an initiation codon in the original report. Since a vector containing the open reading frame starting with "TTG" under a controllable promoter complemented the cdsA knockout, "TTG" could function as an initiation codon. However, no evidence supporting that this "TTG" is the sole initiation codon has been reported. We determined the initiation codon by examining the ability of mutants around the N-terminal region to complement cdsA mutants. Even if the "TTG" was substituted with a stop codon, the clear complementation was observed. Moreover, the clones with multiple mutations of stop codons complemented the cdsA mutant up to the 37th codon, indicating that cdsA possesses multiple codons that can function as initiation codons. We constructed an experimental system in which the chromosomal expression of cdsA can be analyzed. By means of this system, we found that the cdsA mutant with substitution of "TTG" with a stop codon is fully functional. Thus, we concluded that CdsA contains multiple initiation codons.

VEXAS and Myelodysplastic Syndrome: An Interdisciplinary Challenge.

VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a recently recognized systemic autoinflammatory disease caused by somatic mutations in hematopoietic progenitor cells. This case series of four patients with VEXAS syndrome and comorbid myelodysplastic syndrome (MDS) aims to describe clinical, imaging, and hematologic disease presentations as well as response to therapy. Four patients with VEXAS syndrome and MDS are described. A detailed analysis of imaging features, hemato-oncological presentation including bone marrow microscopy and clinical-rheumatological disease features and treatment outcomes is given. All patients were male; ages ranged between 64 and 81 years; all were diagnosed with MDS. CT imaging was available for three patients, all of whom exhibited pulmonary infiltrates of varying severity, resembling COVID-19 or hypersensitivity pneumonitis without traces of scarring. Bone marrow microscopy showed maturation-disordered erythropoiesis and pathognomonic vacuolation. Somatic mutation in the UBA1 codon 41 were found in all patients by next-generation sequencing. Therapy regimes included glucocorticoids, JAK1/2-inhibitors, nucleoside analogues, as well as IL-1 and IL-6 receptor antagonists. No fatalities occurred (observation period from symptom onset: 18-68 months). Given the potential underreporting of VEXAS syndrome, we highly recommend contemporary screening for UBA1 mutations in patients presenting with ambiguous signs of systemic autoinflammatory symptoms which persist over 18 months despite treatment. The emergence of cytopenia, especially macrocytic hyperchromic anemia, should prompt early testing for UBA1 mutations. Notably conspicuous, pulmonary alterations in CT imaging of patients with therapy-resistant systemic autoinflammatory symptoms should be discussed in interdisciplinary medical teams (Rheumatology, Hematology, Radiology and further specialist departments) to facilitate timely diagnosis during the clinical course of the disease.

Isopropanol production using engineered Corynebacterium glutamicum from waste rice straw biomass

Isopropanol, a well-known biofuel, is a widely used precursor for chemical products that can replace nonrenewable petroleum energy. Here, engineered Corynebacterium glutamicum that can effectively utilize all xylose and glucose in agricultural waste rice straw to produce isopropanol was described. First, codon mutations were introduced into transporters and glycolytic-related genes to decrease the glucose preference of C. glutamicum. A more energetically favorable xylose oxidative pathway was constructed that replaced traditional xylose isomerization pathways, saving twice the number of enzymatic steps. A succinate auxiliary module was incorporated into the tricarboxylic acid cycle (TCA), connecting the xylose-utilized pathway with the isopropanol pathway to maximize xylose orientation towards the product. The final engineered strain successfully consumed 100 % of the xylose from NaOH-pretreated, enzyme-hydrolyzed rice straw and effectively synthesized 4.91 g/L isopropanol. This study showcases the successful conversion of agricultural waste into renewable energy, unveiling new possibilities for advancing biological fermentation technology.

A lncRNA Dleu2-encoded peptide relieves autoimmunity by facilitating Smad3-mediated Treg induction

Micropeptides encoded by short open reading frames (sORFs) within long noncoding RNAs (lncRNAs) are beginning to be discovered and characterized as regulators of biological and pathological processes. Here, we find that lncRNA Dleu2 encodes a 17-amino-acid micropeptide, which we name Dleu2-17aa, that is abundantly expressed in T cells. Dleu2-17aa promotes inducible regulatory T (iTreg) cell generation by interacting with SMAD Family Member 3 (Smad3) and enhancing its binding to the Foxp3 conserved non-coding DNA sequence 1 (CNS1) region. Importantly, the genetic deletion of Dleu2-17aa in mice by start codon mutation impairs iTreg generation and worsens experimental autoimmune encephalomyelitis (EAE). Conversely, the exogenous supplementation of Dleu2-17aa relieves EAE. Our findings demonstrate an indispensable role of Dleu2-17aa in maintaining immune homeostasis and suggest therapeutic applications for this peptide in treating autoimmune diseases.

Identification of specific codon 201 mutation of the DCC Gene in the colonoscopic specimen of colorectal cancer.

To identify the mutation in codon 201 of the deleted in colorectal cancer gene in colorectal cancer, and to correlate that mutation to the histopathological grading of colorectal cancer. The cross-sectional study was conducted from February 2019 to February 2021 after approval from the ethics review board of the Dow University of Health Sciences, Karachi, and comprised biopsy-proven colorectal cancer patients regardless of age and gender. After histopathological reporting, formalin-fixed paraffin-embedded tissue blocks of colorectal cancer were used for deoxyribonucleic acid extraction, followed by polymerase chain reaction optimisation and deoxyribonucleic acid Sanger sequencing for mutational analysis. Data was analysed using SPSS 25. Of the 100 biopsy specimens assessed, 45(45%) were selected. Of them, 13(29%) samples failed to show any band on gel electrophoresis. The remaining 32(71%) samples were used for Sanger sequencing. Of these, 1(3%) sample did not sequence, while 31(97%) showed sequencing. All the sequenced samples identified a mutation in codon 201 of exon 3 in the deleted in colorectal cancer gene; 30(97%) showed homozygosity, and 1(3%) showed heterozygosity. No significant association of point mutation was noted with various demographic and clinicopathological parameters (p>0.05). The deleted in colorectal cancer gene's missense mutation in codon 201 was frequently observed in colorectal cancer patients.

Genetic diversity, population structure analysis and codon substitutions of Indicine Badri cattle using ddRAD sequencing.

The present study was carried out on 96 animals representing three distinct colour variants of Badri cattle to investigate the genetic diversity, population structure and substitution mutations in the genetic codons due to single nucleotide variations. The DNA samples of 96 Badri cows were genotyped using a double digestion restriction associated DNA (ddRAD) sequencing approach. A standardized bioinformatics pipeline was employed to identify single nucleotide polymorphisms (SNPs), initially detecting 7,168,552 SNPs through alignment with the Bos indicus reference genome assembly. Subsequent stringent quality filtration yielded 65,483 high-confidence SNPs for downstream analysis. Genetic diversity analysis of the Badri cattle population resulted in average values of 0.145, 0.088, and 0.091 for Shannon's diversity Index (I), Simpson's Diversity (h), and Simpson's Unbiased Diversity (uh), respectively. Genetic similarities between the black and brown, black and grey, and brown and grey Badri variantswere found to be 0.9972, 0.9980 and 0.9970, respectively. Tajima's D diversity value was observed to be significant and positive for 99.29% of high-confidence SNPs (65,483). STRUCTURE analysis showed admixture among the three Badri colour variants, suggesting a lack of genetic differentiation. Annotation of high-confidence SNPs regarding genetic codon changes indicated maximum substitutions in the GGC with GGT (22 occurrences), followed by AAC to AGC (20 occurrences), GAA to TAA (19 occurrences) and CAA to CAG (19 occurrences). The study concludes there are genetic similarities among colour variants, lack of rare alleles, balancing selection, sudden population contraction and genetic codon substitutions within the Badri cattle population. Insights derived from SNP data analysis hold potential significance for conservation initiatives and breed improvement programs for indicine cattle.

Abstract A012: The Spanish Familial Pancreatic Cancer Registry (PANGENFAM): Genetic testing and follow-up of high risk individuals

Abstract Introduction: The Spanish familial pancreatic cancer registry (PANGENFAM) was established in 2009, with the main objective to study the genetic and phenotypic characteristics of familial pancreatic cancer families and offer screening of high-risk individuals for early detection. Methods: Targeted panel and exome sequencing of 52 FPC and 33 sporadic PDAC cases was performed to identify genes and novel variants related to PDAC risk. Healthy high-risk relatives of FPC cases enter into a screening that consists of an annual MRI, echo-endoscopy and routine blood tests, which is followed by a biopsy if a suspicious lesion is identified. Results: The registry and associated biobank is maintained thanks to a stable collaboration between the departments of Medical Oncology, Gastroenterology, Radiology General Surgery and Pathology. The registry currently includes over 150 families and 350 individuals, including PDAC cases and high risk individuals. Targeted sequencing analysis of DNA repair genes involved in familial cancer identified pathogenic variants in 7.5% of FPC cases in the MLH1 and CDKN2A genes, likely pathogenic variants were found in 5.7% of cases in the genes MUTYH, TERT and ATM. Likely pathogenic variants were found in 9.4% of sporadic cases in the genes POLQ, CHEK2 and FANCM and no pathogenic variants were identified. By exome sequencing, missense variants with a damaging effect were found in 11 FPC cases, including stop gain, stop loss or frameshift variants in 5 FPC cases (45.45%), affecting WWOX, C2orf83, CYP3A5 and TANGO2. KRAS somatic mutations in codon 12 and 13 were more frequently detected in sporadic PDAC cases (70%) compared to familial cases (16%) via the liquid biopsy. Interestingly, the median overall survival for sporadic and familial PDAC cases in our cohort was 10.2 vs. 21.7 months (p⇒0.01), respectively, even when adjusted for stage at diagnosis and first line adjuvant therapy. From all the families included in the registry, 185 were offered the high-risk screening program for early detection. Currently 129 individuals are actively participating in the screening program, of which, 72% of these individuals have a non-normal looking pancreas via MRI or EUS. The most commonly detected lesions were cysts and IPMN found in 32% and 12% individuals, respectively. Screening has successfully detected and treated solid lesions in 4 high-risk individuals, including 3 early PDAC and 1 neuroendocrine tumor. An additional 11 individuals with lesions with worrisome features are under close follow-up. Conclusions: Panel sequencing has identified pathogenic variants in known familial cancer genes that are also associated with FPC risk. Exome sequencing in panel negative FPC cases has identified novel risk associated genes. The molecular and clinical profile of FPC and sporadic cases are distinct which should be taken into account in the clinic. Citation Format: Emma Melian, Ana García García de Paredes, Mercedes Rodríguez, Jorge Villalón López, María Muñoz, Javier Blaquez, Ignacio Ruz, Alfonso Sanjuanbenito, Eduardo Lobo, Victoria Lopéz, Carmen Guillen Ponce, José Ramón Foruny Olcina, Sergio López Durán, Alfredo Carrato, Julie Earl. The Spanish Familial Pancreatic Cancer Registry (PANGENFAM): Genetic testing and follow-up of high risk individuals [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A012.

Abstract B089: Oncogenic signaling and responses to treatment in RAS mutant cancers are mutation-specific

Abstract The KRAS, NRAS, and HRAS genes are frequently mutated in cancer, often at hotspots altering codons 12, 13, and 61. Yet how different RAS isoforms and mutations precisely regulate oncogenic signaling is still largely unknown. For example, the role of mutant RAS in driving overactivation of ERK signaling in cancer has long been elucidated, but its involvement in the oncogenic activation of PI3K is still controversial, and different studies have reported conflicting findings regarding the requirement of RAS for physiological and oncogenic PI3K signaling. One of the reasons for this lack of understanding has been the inability to directly target RAS, which has historically been circumvented through genomic perturbation and murine models. However, these approaches are limited because it is not possible to distinguish between the direct effects of RAS on PI3K and compensatory events that occur as an adaptation to the oncogene. With the discovery of RAS inhibitors, we can now directly interrogate the biological effects of RAS isoforms and hotspot mutations in cancer cells with improved time resolution. Here, we use RM-042, a first-in-class broad-spectrum RAS·GTP inhibitor (RASMULTI(ON)), to characterize the features and therapeutic vulnerabilities of mutant RAS signaling in human cancers. Through a detailed biochemical analysis of the signaling response to RAS and MEK inhibition, we reveal that RAS-mediated oncogenic signaling is largely independent of the mutant RAS isoform, rather depending on the mutant codon. Specifically, RASQ61X is exquisitely sensitive to MAPK pathway inhibition, confers near-complete independence of upstream regulation, and is impaired in its signaling to PI3K/Akt/mTOR. Conversely, RASG12X enables oncogenic signaling to both MAPK and PI3K/Akt/mTOR by amplifying upstream input from receptor tyrosine kinases (RTKs). Simultaneous RAS and RTK inhibition selectively impairs the growth of RASG12X-mutant tumors, irrespective of RAS isoform and tumor type, and we investigate the molecular mechanisms behind this synergistic effect. Additionally, we have generated a genetically modified mouse model (GEMM) harboring liver tumors induced by RASG12D or RASQ61R. In cell lines derived from these respective models, RASG12D but not RASQ61R activates PI3K upon growth-factor stimulation. Furthermore, in this system, RASQ61R is less oncogenic than RASG12D, an effect that can be rescued by the concurrent deletion of PTEN. These findings provide mechanistic insights into how mutant RAS interacts with upstream and downstream partners to maximize oncogenic signaling. Furthermore, our results shed light on the selective pressure driving the emergence of RASQ61X mutations as a mechanism of resistance against RTK inhibition: we show that colorectal cancer cells that acquire RASQ61X mutations to overcome EGFR inhibition become more independent of upstream signaling than those that acquire RASG12X. Citation Format: Michelangelo Marasco, Dinesh Kumar, Riccardo Mezzadra, Wei Luan, Ilinca Aricescu, Rona Yaeger, Vladimir Markov, Yu Zhu, Elisa de Stanchina, Scott Lowe, Sandra Misale, Neal Rosen. Oncogenic signaling and responses to treatment in RAS mutant cancers are mutation-specific [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B089.

Phenotypic resistance not associated with knockdown mutations (kdr) in Anopheles albimanus exposed to deltamethrin in southern coastal Ecuador

BackgroundDecrease in malaria rates (e.g. incidence and cases) in Latin America maintains this region on track to achieve the goal of elimination. During the last 5 years, three countries have been certified as malaria free. However, the region fails to achieve the goal of 40% reduction on malaria rates and an increase of cases has been reported in some countries, including Ecuador. This scenario has been associated with multiple causes, such as decrease of funding to continue anti-malarial programmes and the development of insecticide resistance of the main malaria vectors. In Ecuador, official reports indicated phenotypic resistance in Aedes aegypti and Anopheles albimanus to deltamethrin and malathion, particularly in the coastal areas of Ecuador, however, information about the mechanisms of resistance have not been yet elucidated. This study aims to evaluate phenotypic response to deltamethrin and its relationship with kdr mutations in An. albimanus from two localities with different agricultural activities in southern coastal Ecuador.MethodsThe CDC bottle assay was carried out to evaluate the phenotypic status of the mosquito’s population. Sequencing the voltage gated sodium channel gene (VGSC) sought knockdown mutations (kdr) in codons 1010, 1013 and 1014 associated with resistance.ResultsPhenotypic resistance was found in Santa Rosa (63.3%) and suspected resistance in Huaquillas (82.1%); with females presenting a higher median of knockdown rate (83.7%) than males (45.6%). No statistical differences were found between the distributions of knockdown rate for the two localities (p = 0.6048) which indicates no influence of agricultural activity. Although phenotypic resistance was confirmed, genetic analysis demonstrate that this resistance was not related with the kdr mechanism of the VGSC gene because no mutations were found in codons 1010 and 1013, while in codon 1014, 90.6% showed the susceptible sequence (TTG) and 7.3% ambiguous nucleotides (TKK and TYG).ConclusionsThese results highlighted the importance of continuous monitoring of resistance in malaria vectors in Ecuador, particularly in areas that have reported outbreaks during the last years. It is also important to elucidate the mechanism involved in the development of the resistance to PYs to propose alternative insecticides or strategies for vector control in areas where resistance is present.

LsMybW-encoding R2R3-MYB transcription factor is responsible for a shift from black to white in lettuce seed

Key messageWe identified LsMybW as the allele responsible for the shift in color from black to white seeds in wild ancestors of lettuce to modern cultivars.Successfully selected white seeds are a key agronomic trait for lettuce cultivation and breeding; however, the mechanism underlying the shift from black—in its wild ancestor—to white seeds remains uncertain. We aimed to identify the gene/s responsible for white seed trait in lettuce. White seeds accumulated less proanthocyanidins than black seeds, similar to the phenotype observed in Arabidopsis TT2 mutants. Genetic mapping of a candidate gene was performed with double-digest RAD sequencing using an F2 population derived from a cross between “ShinanoPower” (white) and “Escort” (black). The white seed trait was controlled by a single recessive locus (48.055–50.197 Mbp) in linkage group 7. Using five PCR-based markers and numerous cultivars, eight candidate genes were mapped in the locus. Only the LG7_v8_49.251Mbp_HinfI marker, employing a single-nucleotide mutation in the stop codon of Lsat_1_v5_gn_7_35020.1, was completely linked to seed color phenotype. In addition, the coding region sequences for other candidate genes were identical in the resequence analysis of “ShinanoPower” and “Escort.” Therefore, we proposed Lsat_1_v5_gn_7_35020.1 as the candidate gene and designated it as LsMybW (Lactuca sativaMyb White seeds), an ortholog encoding the R2R3-MYB transcription factor in Arabidopsis. When we validated the role of LsMybW through genome editing, LsMybW knockout mutants harboring an early termination codon showed a change in seed color from black to white. Therefore, LsMybW was the allele responsible for the shift in seed color. The development of a robust marker for marker-assisted selection and identification of the gene responsible for white seeds have implications for future breeding technology and physiological analysis.

Altered larval activation response associated with multidrug resistance in the canine hookworm Ancylostoma caninum.

Parasitic gastrointestinal nematodes pose significant health risks to humans, livestock, and companion animals, and their control relies heavily on the use of anthelmintic drugs. Overuse of these drugs has led to the emergence of resistant nematode populations. Herein, a naturally occurring isolate (referred to as BCR) of the dog hookworm, Ancylostoma caninum, that is resistant to 3 major classes of anthelmintics is characterized. Various drug assays were used to determine the resistance of BCR to thiabendazole, ivermectin, moxidectin and pyrantel pamoate. When compared to a drug-susceptible isolate of A. caninum, BCR was shown to be significantly resistant to all 4 of the drugs tested. Multiple single nucleotide polymorphisms have been shown to impart benzimidazole resistance, including the F167Y mutation in the β-tubulin isotype 1 gene, which was confirmed to be present in BCR through molecular analysis. The frequency of the resistant allele in BCR was 76.3% following its first passage in the lab, which represented an increase from approximately 50% in the founding hookworm population. A second, recently described mutation in codon 134 (Q134H) was also detected at lower frequency in the BCR population. Additionally, BCR exhibits an altered larval activation phenotype compared to the susceptible isolate, suggesting differences in the signalling pathways involved in the activation process which may be associated with resistance. Further characterization of this isolate will provide insights into the mechanisms of resistance to macrocyclic lactones and tetrahydropyrimidine anthelmintics.