Molecular Analysis Of Mutations Research Articles

Molecular Characterization and Mutational Analysis of Fluoroquinolones and Tetracycline Resistant Genes of Escherichia coli Isolated from UTI Patients

HIGHLIGHTS Mutational analysis of antibiotic resistant genes of E. coli in UTI Patients. These genes were detected by PCR and sequenced using NGS. E. coli isolates were positive for GyrA gene, GyrB gene, tetB and tetR gene. The novel mutation was detected in gyrB gene at codon554.

A Genotyped Case of Townes-Brocks Syndrome with Absent Pulmonary Valve Syndrome from Turkey.

Townes-Brocks syndrome (TBS) is a rare syndrome characterized by triad of anal, ear, and thumb anomalies. Further malformations/anomalies include congenital heart diseases, foot malformations, sensorineural and/or conductive hearing impairment, genitourinary malformations, and anomalies of eye and nervous system. Definitive diagnosis for TBS is confirmed by molecular analysis for mutations in the SALL1 gene. Only one known case of TBS with absent pulmonary valve syndrome (APVS) has been previously described to our knowledge. Here, we report a newborn diagnosed with TBS with APVS and tetralogy of Fallot (TOF) who was found to carry the most common pathogenic SALL1 gene mutation c.826C > T (p.R276X), with its surgical repair and postoperative follow-up. To our knowledge, this is the first genotyped case of TBS from Turkey to date. TBS should be suspected in the presence of ear, anal, and thumb malformations in a neonate. If a patient with TBS and TOF-APVS needs preoperative ventilation within the first months of life, this implies prolonged postoperative intubation and increased risk of mortality.

Macroglobulinemia and Autoinflammatory Disease.

Macroglobulinemia is associated with Schnitzler syndrome (SchS) and Waldenstrom macroglobulinemia (WM). The aim of this article was to review the above-mentioned two diseases from clinical aspects and their potential genetic links. We performed a PubMed search using the following keywords: "SchS," "WM," "autoinflammatory disease," "periodic fever syndrome," and "nucleotide-binding oligomerization domain containing protein 2 (NOD2)." A case is exemplified. Both SchS and WM share some clinical phenotypes, and SchS can evolve into WM. Though no genetic link to SchS has been established, myeloid differentiation primary response gene 88 (MyD88) mutations are detected in one-third of SchS patients and 86% WM patients. Genetic analysis of periodic fever syndrome genes has detected NOD2 mutations in 18% SchS patients and rarely NLRP3 mutations. The literature data suggest that both MyD88 and NOD2 mutations may contribute to SchS. Both MyD88 and NOD2 are known to play important roles in innate immune response, and they may be cooperative in certain autoinflammatory diseases. Molecular analysis of NOD2 mutations may be incorporated into genetic testing for patients with suspected SchS or SchS/WM.

Single Centre Analysis of JAK2 V617F and Exon 12 Negative Idiopathic Erythrocytosis

▪IntroductionThe molecular categorisation of myeloproliferative neoplasms (MPNs) has changed the landscape of diagnosis and treatment. Polycythaemia vera (PV) is characterised by red blood cell proliferation and JAK2 V617F or Exon 12 mutations in up to 98% of patients 1. However, some patients without such mutations have an arduous diagnostic course with varying management and prognostic outcomes 2. We present our experience in managing this challenging cohort and aim to illuminate a potential diagnostic pathway for patients.MethodWe searched electronic records of patients attending haematology clinics over the last 20 years at University College Hospital with a prior diagnosis of PV / erythrocytosis (presenting with raised Haemoglobin (Hb) &/ Haematocrit (Hct)) with no evidence of JAK2 exon 12 or 14 mutations on bone marrow or peripheral blood molecular analysis (multiplex PCR sensitivity 0.1%). We reviewed their diagnostic workup, which included full blood count & where available, bone marrow myeloid (Illumina TruSight Myeloid) & erythroid next generation sequencing (NGS) panels.Results37 patients with JAK2 V617F & Exon 12 mutation negative erythrocytosis were identified. Patients were categorised in to 3 groups 2; idiopathic erythrocytosis (IE), secondary polycythaemia (SP) & high affinity haemoglobinopathies (HAH); patient characteristics are summarised in Table 1. The median age of IE & HAH was younger, their presenting Hb/Hct levels was higher compared to SP, with a male predominance. Constitutional symptoms were only reported in the IE cohort. Erythropoietin (EPO) was elevated in HAH & IE patients but within normal range in SP. Thrombotic events occurred in all cohorts, most frequently in IE. Splenomegaly was reported in 4/21 IE, 1/13 SP, but was not a feature in HAH patients. When performed, IE red cell mass (RCM) studies were raised but within normal range in SP patients. Table 2 details IE cohort erythroid mutations. Myeloid NGS only identified MPL and BCOR mutations of pathogenic significance and multiple single nucleotide polymorphisms of no known significance. No abnormalities were demonstrated in 15% of SP patients that underwent bone marrow myeloid mutational analysis. Venesections (VS) were instigated in 95% of the IE cohort, antiplatelets (AP) in 52%, anticoagulation (AC) in 14% and cytoreductive therapy (CT) in 19% due to intolerance/failure of VS. VS programme was instigated in 46% of SP patients, AP in 7% and AC in 47%.DiscussionThe median age of our IE cohort was 48 yrs with a 19% incidence of thrombosis. Where performed, bone marrow histology demonstrated hypercellularity but was not consistent with MPN diagnostic criteria. Myeloid NGS panel mutations such as BCOR may represent clonal haematopoieis of indeterminate potential. Heterozygous VHL C598T & C376A mutations, in keeping with Chuvash polycythaemia, were demonstrated. Mutations in EGLN1 &BPGM,were detected in our patients, however there was an absence of correlating haematological parameters or family history to support a diagnosis of congenital erythrocytosis (2). Variants of unknown significance were also detected in SH2B3, BMP6 &EGLN3 gene duplication. SP patients were older (median age 68 years) and where performed had normal RCM and no myeloid mutations identified.The initial approach adopted at our centre for diagnosing and managing JAK2 V617F & Exon 12 negative erythocytosis begins with clinical evaluation for secondary causes. This is followed by assessment of EPO level, RCM study, extended molecular mutational analysis involving screening for high affinity haemoglobins, congenital erythrocytosis and bone marrow histology. Where patients are symptomatic or considered at high risk for thrombosis, we venesect to a personalised target and patients are offered AP and/or AC. CT is instigated where VS is not tolerated or ineffective, however this approach is not corroborated in the literature. Our experience highlights the clinical heterogeneity of JAK2 negative erythrocytosis and the need to develop a robust and systematic diagnostic and treatment algorithm with further clarification of the role of molecular profiling.1. William W, Kralovics R. Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms. Blood. 2017 Feb; 129(6):667-6792. McMullin MF. Idiopathic erythrocytosis: a disappearing entity. Hematology Am Soc Educ Program. 2009; 2009(1):629-635 [Display omitted] DisclosuresSekhar: Novartis: Consultancy, Research Funding.

Identification and Characterization of a Novel Single Domain Antibody Against Ebola Virus.

Ebola virus (EBOV) belongs to the Filoviridae family and causes severe illnesses such as hemorrhagic fever with a high mortality rate up to 90%. Now two antibody drugs termed Inmazeb and Ebanga have been approved for treating EBOV infection. However, clinical studies have demonstrated that the mortality rate of the patients who received these two antibody drugs remains above 30%. Therefore, novel therapeutics with better efficacy is still desired. The isolated human IgG1 constant domain 2 (CH2 domain) has been proposed as a scaffold for the development of C-based single domain antibodies (C-sdAbs) as therapeutic candidates against viral infections and other diseases. Here, we screened and identified a novel C-sdAb termed M24 that targets EBOV glycoprotein (GP) from a C-sdAb phage display library. M24 neutralizes the pseudotype EBOV with IC50 of 0.8 nmol/L (12 ng/mL) and has modest neutralizing activity against authentic EBOV. Epitope determination, including molecular docking and site mutation analysis, discloses that M24 binds to the internal fusion loop (IFL) within GP2, a transmembrane subunit of GP. Interestingly, we found that the binding of M24 to GP at pH 5.5 has dramatically decreased compared to the binding at pH 7.5, which may lead to weak efficacy in the neutralization of authentic EBOV. Since no sdAb against EBOV infection has been reported to date, our results not only give a proof of concept that sdAbs could be utilized for the development of potential therapeutic candidates against EBOV infection, but also provide useful information for the discovery and improvement of anti-EBOV agents.

Computational modeling reveals key molecular properties and dynamic behavior of disruptor of telomeric silencing 1-like (DOT1L) and partnering complexes involved in leukemogenesis.

Disruptor of telomeric silencing 1‐like (DOT1L) is the only non‐SET domain histone lysine methyltransferase (KMT) and writer of H3K79 methylation on nucleosomes marked by H2B ubiquitination. DOT1L has elicited significant attention because of its interaction or fusion with members of the AF protein family in blood cell biology and leukemogenic transformation. Here, our goal was to extend previous structural information by performing a robust molecular dynamic study of DOT1L and its leukemogenic partners combined with mutational analysis. We show that statically and dynamically, D161, G163, E186, and F223 make frequent time‐dependent interactions with SAM, while additional residues T139, K187, and N241 interact with SAM only under dynamics. Dynamics models reveal DOT1L, SAM, and H4 moving as one and show that more than twice the number of DOT1L residues interacts with these partners, relative to the static structure. Mutational analyses indicate that six of these residues are intolerant to substitution. We describe the dynamic behavior of DOT1L interacting with AF10 and AF9. Studies on the dynamics of a heterotrimeric complex of DOT1L1‐AF10 illuminated describe coordinated motions that impact the relative position of the DOT1L HMT domain to the nucleosome. The molecular motions of the DOT1L–AF9 complex are less extensive and highly dynamic, resembling a swivel‐like mechanics. Through molecular dynamics and mutational analysis, we extend the knowledge previous provided by static measurements. These results are important to consider when describing the biochemical properties of DOT1L, under normal and in disease conditions, as well as for the development of novel therapeutic agents.

Clinical application of whole transcriptome sequencing for the classification of patients with acute lymphoblastic leukemia

BackgroundConsidering the clinical and genetic characteristics, acute lymphoblastic leukemia (ALL) is a rather heterogeneous hematological neoplasm for which current standard diagnostics require various analyses encompassing morphology, immunophenotyping, cytogenetics, and molecular analysis of gene fusions and mutations. Hence, it would be desirable to rely on a technique and an analytical workflow that allows the simultaneous analysis and identification of all the genetic alterations in a single approach. Moreover, based on the results with standard methods, a significant amount of patients have no established abnormalities and hence, cannot further be stratified.MethodsWe performed WTS and WGS in 279 acute lymphoblastic leukemia (ALL) patients (B-cell: n = 211; T-cell: n = 68) to assess the accuracy of WTS, to detect relevant genetic markers, and to classify ALL patients.ResultsDNA and RNA-based genotyping was used to ensure correct WTS-WGS pairing. Gene expression analysis reliably assigned samples to the B Cell Precursor (BCP)-ALL or the T-ALL group. Subclassification of BCP-ALL samples was done progressively, assessing first the presence of chromosomal rearrangements by the means of fusion detection. Compared to the standard methods, 97% of the recurrent risk-stratifying fusions could be identified by WTS, assigning 76 samples to their respective entities. Additionally, read-through fusions (indicative of CDKN2A and RB1 gene deletions) were recurrently detected in the cohort along with 57 putative novel fusions, with yet untouched diagnostic potentials. Next, copy number variations were inferred from WTS data to identify relevant ploidy groups, classifying an additional of 31 samples. Lastly, gene expression profiling detected a BCR-ABL1-like signature in 27% of the remaining samples.ConclusionAs a single assay, WTS allowed a precise genetic classification for the majority of BCP-ALL patients, and is superior to conventional methods in the cases which lack entity defining genetic abnormalities.

Molecular Docking Analysis of the T450A Mutation of the Gene rpoB Mycobacterium leprae from Leprosy Patients in Papua, West Papua and North Maluku, Indonesia

Leprosy persists to be a health problem in Indonesia, especially in the provinces of North Maluku, West Papua and Papua. Early diagnosis and complete treatment with multidrug therapy (MDT) remain the key strategy for reducing the disease burden. One of the major components of MDT is rifampicin which in certain cases in several countries, M. leprae resistance to this drug issue has been reported albeit only a few. This research aimed to detect and analyze polymorphism in M. leprae rpoB gene that was isolated from leprosy patients in three provinces: North Maluku Province, West Papua Province and Papua Province, Indonesia. The identification of mutations in the M. leprae rpoB gene was carried out by aligning the results of DNA sequencing with the reference strain. The 3D structure of rpoB was derived using the Swiss Model. The T450A, S456L, and H451Y variants of RNA Polymerase B subunits were constructed using FoldX based on the wild-type structure. The structures were repaired, and protein stability was evaluated using foldX under the Yasara viewer. The QC of the rpoB M. leprae homology models was conducted with Ramachandran Plot modeling using PROCHECK. The difference in binding affinity between native protein and T450A, S456L, and H45I variants were analyzed using molecular docking. rpoB gene of M. leprae contains a mutation found in nucleotide of 1348 bp. The mutation triggered the conversion of the amino acid Threonine to Alanine in the amino acid to 450 rpoB subunit B. The structure of 3D RNA Polymerase Subunit B was constructed using rpoB Mycobacterium tuberculosis with PDB code 5UH5 as template. According to Ramachandran Plot, the percentage of residues in the most favored regions are 91.9%, and there was no significant number of residues in the disallowed regions. The results of molecular docking showed that the T450A variant had the same binding affinity with the native protein which was -8.9 kcal. Binding affinity on the S456L and H451Y variants increased by -7.3 kcal and -8.2 kcal, respectively. According to Molecular Docking analysis, T450A variant did not affect the energy binding between RNA polymerase and rifampicin.

RUNX1 mutation and elevated FLT3 gene expression cooperates to induce inferior prognosis in cytogenetically normal acute myeloid leukemia patients

BackgroundAcute myeloid leukemia (AML) is a bone marrow malignancy having multiple molecular pathways driving its progress. In recent years, the main causes of AML considered all over the world are genetic variations in cancerous cells. The RUNX1 and FLT3 genes are necessary for the normal hematopoiesis and differentiation process of hematopoietic stem cells into mature blood cells, therefore they are the most common targets for point mutations resulting in AML.MethodsWe screened 32 CN-AML patients for FLT3-ITD (by Allele-specific PCR) and RUNX1 mutations (by Sanger sequencing). The FLT3 mRNA expression was assessed in all AML patients and its subgroups.ResultsEight patients (25%) carried RUNX1 mutation (K83E) while three patients (9.37%) were found to have internal tandem duplications in FLT3 gene. The RUNX1 mutation data were correlated with clinical parameters and FLT3 gene expression profile. The RUNX1 mutations were observed to be significantly prevalent in older males. Moreover, RUNX1 and FLT3-mutated patients had lower complete remission rate, event-free survival rate, and lower overall survival rate than patients with wild-type RUNX1 and FLT3 gene. The RUNX1 and FLT3 mutant patients with up-regulated FLT3 gene expression showed even worse prognosis. Bradford Assay showed that protein concentration was down-regulated in RUNX1 and FLT3 mutants in comparison to RUNX1 and FLT3 wild-type groups.ConclusionThis study constitutes the first report from Pakistan reporting significant molecular mutation analysis of RUNX1 and FLT3 genes including FLT3 expression evaluation with follow-up. This provides an insight that aforementioned mutations are markers of poor prognosis but the study with a large AML cohort will be useful to further investigate their role in disease biology of AML.

Diagnosis and Molecular Profiles of Large Cell Neuroendocrine Carcinoma With Potential Targets for Therapy

Large cell neuroendocrine carcinoma (LCNEC) together with small cell carcinoma (SCLC) and typical and atypical carcinoids form the group of pulmonary neuroendocrine tumors. LCNEC and SCLC are high-grade carcinomas. Although both can be found outside the thoracic cavity, they are most common in the lung. LCNEC differs from SCLC by morphologic pattern, and by cytological features such as nuclear size, nucleoli, chromatin pattern, but also by genetic differences. Originally thought to represent a single entity, it became evident, that three subgroups of LCNEC can be identified at the molecular level: a SCLC-like type with loss of retinoblastoma 1 gene (RB1) and TP53 mutations; a non-small cell lung carcinoma (NSCLC)-like type with wildtype RB1, TP53 mutation, and activating mutations of the phosphoinositol-3 kinase (PI3K-CA), or loss of PTEN; and a carcinoid-like type with MEN1 gene mutation. These subtypes can be identified by immunohistochemical staining for RB1, p53, and molecular analysis for PI3K and MEN1 mutations. These subtypes might also respond differently to chemotherapy. Immuno-oncologic treatment has also been applied to LCNEC, however, in addition to the evaluation of tumor cells the stroma evaluation seems to be important. Based on personal experiences with these tumors and available references this review will try to encompass our present knowledge in this rare entity and provoke new studies for better treatment of this carcinoma.

MDS Diagnosis Using cfRNA Profiling By Targeted Enrichment Next Generation Sequencing

Introduction: The confirmation of a diagnosis of myelodysplastic syndrome (MDS) can be very difficult based on morphology alone. The detection of cytogenetic abnormalities or the presence of mutations in one or more of the myeloid-related genes can provide significant help in confirming this diagnosis. However, some patients with MDS may lack mutations or cytogenetic abnormalities. Cell free DNA (cfDNA) and microRNA are used extensively in testing for molecular abnormalities in hematologic and solid tumors, but little is known about the value of cell free mRNA (cfRNA) in liquid biopsy. We explored the use of targeted enrichment and Next Generation Sequencing (NGS) of cfRNA in the RNA profiling and diagnosis of MDS.Methods: We used NucliSenS EasyMAG (bioMérieux, Marcy-l'Étoile, France) automated platform for extracting total nucleic acid from peripheral blood plasma collected in EDTA. We used the TruSight RNA Pan-Cancer Panel (Illumina, San Diego, CA) for detecting fusion, expression, and mutations in 1385 genes. The Pan-Cancer Panel is a hybridization based targeted panel, therefore alignments and gene expression are calculated only to the 1385 genes as per manufacturer recommendations. Each sample was deeply sequenced (>55M reads) using Illumina paired-end 75 bp sequencing and duplicate reads were removed before fragment per kilobase of transcript per million mapped reads (FPKMs) were calculated for each gene. Samples from 23 patients were analyzed including 19 patients confirmed by molecular, cytogenetic, and morphologic methods to have MDS or chronic myelomonocytic leukemia (CMML) and 4 individuals who had one or more cytopenia, but MDS was ruled out by molecular mutation analysis, cytogenetics, as well as morphology. Expression levels were first normalized using ABL1 and PAX5 (i.e., regressing each gene on ABL1 and PAX5 jointly and using the residuals instead of the raw data) and models for distinguishing MDS from non-MDS were developed.Results: In this study, only expression levels were considered and not the mutational profile. We used t-tests and examined the statistical significance of each gene separately. Then we selected genes with P-values < 0.05 to filter out non-significant genes, resulting in 49 significant genes. After adjusting for multiple hypothesis testing, only three genes remained significant with false discovery rate set at 0.1. The three genes were: CBFB, RASGRF2, and SP1 . Using a logistic regression model based on these three variables, we were able to obtain accuracy rate of 87.0% and AUC (area under the ROC curve) of 92.1% based on the leave-one-out testing. This model identified all the normal cases correctly, but misclassified 3 MDS cases as normal. Alternatively, using the top 3 principal components based on the selected 49 significant genes in a linear regression model, we can improve prediction and AUC to 95.6% and 96.7%, respectively, based on the leave-one-out testing. In this model, only one MDS case (out of 19) was misclassified as normal, while all the normal cases were classified correctly.Conclusion: Although additional studies and validation are needed, RNA profiling using targeted enrichment NGS of cfRNA is potentially useful in the classification and diagnosis of MDS. This model can be expanded to incorporate an entire mutational profile assessment. This approach has the potential to be used not only for MDS diagnosis, but also in the diagnosis, prognostic prediction and response to therapy of a variety of myeloid and lymphoid neoplasms. [Display omitted] DisclosuresWong:NeoGenomics: Employment. Funari:NeoGenomics: Employment. Ma:NeoGenomics: Employment. De Dios:NeoGenomics: Employment. Blocker:NeoGenomics: Employment. Albitar:NeoGenomics: Employment.

Gastrodin relieves Vibrio harveyi infection by blocking hemolysin active centers

Vibrio harveyi hemolysin (VHH) is an important virulence determinant of V. harveyi. It is an extracellular protein with phospholipase activity and plays a key role in the process of pathogen infection of the host. The key role of VHH in the virulence of V. harveyi makes it an ideal target for the design of new antiviral drugs. We found that gastrodin is a natural flavonoid with no antibacterial activity and an effective antagonist of VHH-mediated hemolysis. The compound can effectively inhibit the damage caused by V. harveyi in cell culture and animal infection models. Gastrodin can down-regulate the transcription of the vhh gene, and molecular modeling and mutation analysis show that it directly blocks the amino acid residues in the active center of the protein, thereby inhibiting its cytotoxicity. Our research results indicate that gastrodin is an effective antitoxin agent for VHH, which can be further developed into a new drug for the treatment of Vibrio infection.

Giant Cell Tumor of Bone in Patients under 16 Years Old: A Single-Institution Case Series.

Simple SummaryGiant cell tumor of the bone is a locally aggressive, rarely metastasizing tumor that accounts for about 5% of bone tumors; it generally occurs in patients between 20 and 45 years old. Sporadic cases (less than 140) have been described as occurring in the first two decades of life. A histone 3.3 (H3.3) gene, H3F3A, has been recently identified in as many as 96% of giant cell tumors of bone. These mutations are useful in the differential diagnosis of giant cell tumor of bone with its mimickers. The immunohistochemical expression of H3F3A resulted comparable to molecular analysis as reported in a recent investigation. In the present study, we describe our series of giant cell tumors of bone in pediatric patients <16 years old.Background: Giant cell tumor of bone is a locally aggressive, rarely metastasizing tumor that accounts for about 5% of bone tumors and generally occurs in patients between 20 and 45 years old. A driver mutation in the histone 3.3 (H3.3) gene H3F3A has been identified in as many as 96% of giant cell tumors of bone. The immunohistochemical expression of H3F3A H3.3 G34 expression was found in 97.8% of cases. In the present study, we describe our series of cases of giant cell tumor of bone in pediatric patients <16 years old. Methods: All cases of giant cell tumor of bone in pediatric patients <16 years old treated in our institute between 1982 and 2018 were reviewed. Immunohistochemistry and/or molecular analysis for H3F3A gene mutations was performed to confirm the diagnosis. A group of aneurysmal bone cysts in patients <16 years old was used as a control group. Results: Fifteen cases were retrieved. A pronounced female predominance (93%) was observed. A pure metaphyseal central location occurs in 2 skeletally immature patients. Conclusions: Giant cell tumor of bone should be distinguished from its mimickers due to differences in prognosis and treatment. Immunohistochemical and molecular detection of H3F3A gene mutation represents a reliable diagnostic tool.

Molecular analysis of CFTR gene mutations among Iraqi cystic fibrosis patients

BackgroundCystic fibrosis (CF) is an autosomal recessive multisystem disease that results from mutation(s) of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. More than 2100 mutations and polymorphisms have been reported in this gene so far. Incidence and genotyping of CF are under-identified in Iraq. This study aims to determine the types and frequencies of certain CFTR mutations among a sample of Iraqi CF patients. Two groups of patients were included: 31 clinically confirmed CF patients in addition to 47 clinically suspected patients of CF. All confirmed patients had typical, moderate-severe clinical presentation and course of the disease. Molecular analysis was performed on the majority of enrolled patients using the CF-stripAssay® kit supplied by ViennaLab diagnostics, GmbH, Austria.ResultsThe mutation-detection rate from the tested 34 mutations in this study was 19.5% and the 8 detected mutations were as follows: 3120+1G>A and W1282X were found in 3 (4.17%) patients each; F508del and R1162X were found in 2 (2.78%) patients each; 3272-26A>G, R347P, I507del, and 2183AA>G were found in 1 (1.38%) patient each. Polymorphic variants of IVS8, namely 5T, 7T, and 9T, were detected in ~ 70%. These results were nearly similar to what was reported in regional countries.ConclusionCystic fibrosis seems to be not rare as previously thought. 3120+1G>A and W1282X are the two most commonly detected mutations. F508del needs to be included in all future tests, while the I507del mutation was uniquely reported in this study but not in regional studies.

Molecular and computational analysis of natural drug resistance mutations among Moroccan chronic hepatitis B carriers

Despite the effectiveness of nucleotide analogs (NAs), drug resistance and cccDNA inaccessibility are still significant obstacles to long-term efficacy of antiviral therapy in chronic hepatitis B infection (CHB). Mutations pre-exist spontaneously in Hepatitis B Virus (HBV) reverse transcriptase (RT) gene in minor viral populations and can emerge to confer drug resistance and treatment failure. This study aimed to analyze mutations within the HBV polymerase gene in treatment-naive Moroccan patients with CHB.The HBV polymerase gene was analyzed by nested-PCR and direct sequencing in 123 subjects. A computational analysis was performed to confirm the effect of detected mutations on targeted NAs, using homology and docking tools.Sixty samples were successfully amplified by PCR. Analyses revealed an intermediate rate (13%) of pre-treatment NA resistance mutations and a high frequency of putative resistance mutations (65%). Among these, 56% were lamivudine resistance mutations and 44% adefovir resistance mutations. Two pretreatment resistance mutations, “Y124N” and “N139D/K/H/Q” were detected in 8/60 patients, and 11 putative resistance mutations (N53S/T/D, L91I, H126Y, T128I, V207I, V214A, Q215P/S, L217R, F221Y, L229V, N238H/T) in 39/60 patients, where two have a combination of F221Y and A194T “a classical NA drug resistance mutation.” The computational analysis demonstrated that the combination of A194T + F221Y mutations on HBV RT sequence does not affect its susceptibility to ADV.These results confirm that HBV mutations conferring resistance against currently available anti-HBV NA predate treatment in naïve Moroccan patients. The high rate of non-classical NA resistance mutations needs further in vitro phenotype analysis.

Clinicopathological Significance of BRAF (V600E), NRAS (Q61K) and TERT (C228T, C250T and SNP Rs2853669) Mutations in Bulgarian Papillary Thyroid Carcinoma Patients

Abstract Introduction: Thyroid carcinoma is the most common endocrine cancer. Some somatic mutations in genes (BRAF, NRAS and TERT) involved in key signaling pathways and genome stability have been recently identified to play an important role in its development. Very little research has been done on their frequency and clinical relevance in Bulgarian patients with papillary thyroid cancer (PTC). This study is focused on investigating somatic mutation frequency in Bulgarian patients with PTC and their association with clinicopathologic features. Material and Methods: The study included 50 PTC from Bulgarian patients analyzed for mutations in BRAF (V600E), NRAS (Q61K), single nucleotide polymorphism (SNP) rs2853669 and TERT (C228T and C250T) genes by Sanger sequencing. The results were interpreted using Benchling and SeqScape software, and statistical analysis performed with SPSS. Results: In the studied PTC group BRAF(V600E) and TERT (C228T) mutations were found with frequency of 24% and 2%, respectively. Co-occurrence of both mutations was found in 1 patient (2%). The mutations Q61K (NRAS), and C250T (TERT) were not detected. The SNP rs2853669 was found in 18 patients (52.9%). Correlation analysis with the clinical characteristics of the patients revealed statistically significant association with larger size of the tumor for BRAF(V600E) and smaller tumor size for rs2853669. Conclusion: In the present pilot study, we found that BRAF(V600E) and rs2853669 in TERT are common among PCT patients. While the presence of BRAF V600E mutation was associated with large tumors, the presence of rs2853669 in TERT was found in the majority of PCT below 2 cm. More extensive molecular genetic analysis of TERT, BRAF or RAS mutations in larger sample is needed to further elucidate the clinically important diagnostic and prognostic biomarkers for thyroid cancer.

Mutational analysis of p53 gene in cervical cancer and useful polymorphic variants in exons 3 and 4

BackgroundFactors contributing to the pathogenesis and progression of cervical cancer include poor attitude to screening and health intervention, late presentation, among others. Mutations in p53 gene have been attributed to several cancer cases. The present study was designed to find relationships between the mutation patterns in p53 gene and cervical carcinoma staging. Such knowledge could contribute to early diagnosis of cervical cancer.ResultsFrom the sequence analysis of p53 gene fragment isolated by polymerase chain reactions (PCR), nineteen (19) polymorphic variants were identified. Missense mutations occurred in 47% of the samples, 32% were silent mutations, 16% were frameshift mutations and 5% nonsense mutations. Socio-biological characteristics of the study participants revealed that 60% have husbands with multiple sexual partners and that only 23.3% of the participants have ever had the Papanicolaou (Pap) smear test prior to diagnosis, whilst 20% were unaware of the screening test.ConclusionsIncreased severity of cervical carcinoma staging as revealed from the histopathological analysis was found to be associated with accumulation of higher levels of mutations in the p53 gene. Molecular analysis of p53 gene mutations may prove useful as a screening biomarker for cervical cancer.

Pan-India novel coronavirus SARS-CoV-2 genomics and global diversity analysis in spike protein

The mortality rates due to COVID-19 have been found disproportionate globally and are currently being researched. India mortality rate with a population of 1.3 billion people is relatively lowest to other countries with high infection rates. Genetic composition of circulating isolates continues to be a key determinant of virulence and pathogenesis. This study aimed to analyse the extent of divergence between genomes of Indian isolates (n = 2525 as compared to reference Wuhan-1 strain and isolates from countries showing higher fatality rates including France, Italy, Belgium, and the USA. The study also analyses the impact of key mutations on interactions with angiotensin converting enzyme 2 (ACE2) and panel of neutralizing monoclonal antibodies. Using 1,44,605 spike protein sequences, global prevalence of mutations in spike protein was observed. The study suggests that SARS-CoV-2 genomes from India share consensus with global trends with respect to D614G as most prevalent mutational event (81.66% among 2525 Indian isolates). Indian isolates did not reported prevalence of N439K mutation in receptor binding motif (RBM) as compared to global isolates (0.54%). Computational docking and molecular dynamics simulation analysis of N439K mutation with respect to ACE 2 binding and reactivity with RBM targeted antibodies viz., B38, BD23, CB6, P2B–F26 and EY6A suggests that variant have relatively higher affinity with ACE 2 receptor which may support higher infectivity. The study warrants large scale monitoring of Indian isolates as SARS-CoV-2 virus is expected to evolve and mutations may appear in unpredictable way.

The Correlation of Mutations and Expressions of Genes within the PI3K/Akt/mTOR Pathway in Breast Cancer-A Preliminary Study.

There is an urgent need to seek new molecular biomarkers helpful in diagnosing and treating breast cancer. In this elaboration, we performed a molecular analysis of mutations and expression of genes within the PI3K/Akt/mTOR pathway in patients with ductal breast cancer of various malignancy levels. We recognized significant correlations between the expression levels of the studied genes. We also performed a bioinformatics analysis of the data available on the international database TCGA and compared them with our own research. Studies on mutations and expression of genes were conducted using High-Resolution Melt PCR (HRM-PCR), Allele-Specific-quantitative PCR (ASP-qPCR), Real-Time PCR molecular methods in a group of women with ductal breast cancer. Bioinformatics analysis was carried out using web source Ualcan and bc-GenExMiner. In the studied group of women, it was observed that the prevalence of mutations in the studied PIK3CA and AKT1 genes was 29.63%. It was stated that the average expression level of the PIK3CA, PIK3R1, PTEN genes in the group of breast cancer patients is lower in comparison to the control group, while the average expression level of the AKT1 and mTOR genes in the studied group was higher in comparison to the control group. It was also indicated that in the group of patients with mutations in the area of the PIK3CA and AKT1 genes, the PIK3CA gene expression level is statistically significantly lower than in the group without mutations. According to our knowledge, we demonstrate, for the first time, that there is a very strong positive correlation between the levels of AKT1 and mTOR gene expression in the case of patients with mutations and without mutations.

The mutation of BCOR is highly recurrent and oncogenic in mature T-cell lymphoma

BackgroundBCOR acts as a corepressor of BCL6, a potent oncogenic protein in cancers of the lymphoid lineage. We have found the recurrent somatic mutation of BCOR occurred in mature T-cell lymphoma (TCL). The role of BCOR mutation in lymphoid malignancies is unknown.MethodsLymphoma patient samples were analyzed to identify missense mutations in BCOR using Sanger sequencing. Transfection, RNA interference, immunoprecipitation, western blotting, cell proliferation, cytokine assays and quantitative real-time PCR were employed to determine the functional relevance of the novel K607E mutation in BCOR. The significant transcriptional changes were analyzed by performing DNA microarray profiling in cells expressing BCOR K607E mutant.ResultsOne hundred thirty-seven lymphoma patient samples were analyzed to identify K607E mutation of the BCOR gene. The BCOR K607E mutation was identified in 15 of 47 NK/T cell lymphoma cases (31.9%), 2 of 18 angioimmunoblastic T-cell lymphoma cases (11.1%), 10 of 30 peripheral T-cell lymphoma, not otherwise specified cases (33.3%), and 13 of 42 diffuse large B-cell lymphoma cases (30.9%). Molecular analysis of BCOR K607E mutation revealed that compared to the wild-type BCOR, the mutant BCOR bound to the BCL6, PCGF1, and RING1B proteins with lesser affinity. Ectopic expression of BCOR K607E mutant significantly enhanced cell proliferation, AKT phosphorylation and the expression of interleukin-2 (IL-2) with up-regulated expression of HOX and S100 protein genes in T cells. BCOR silencing also significantly enhanced cell proliferation, AKT phosphorylation, and IL-2 production.ConclusionsFunctional analyses indicated that K607E mutation of BCOR is oncogenic in nature and can serve as a genetic marker of T-cell lymphoma.