Sanger Sequencing Technique Research Articles

A novel causative functional mutation in GATA6 gene is responsible for familial dilated cardiomyopathy as supported by in silico functional analysis

Dilated cardiomyopathy (DCM), one of the most common types of cardiomyopathies has a heterogeneous nature and can be seen in Mendelian forms. Next Generation Sequencing is a powerful tool for identifying novel variants in monogenic disorders. We used whole-exome sequencing (WES) and Sanger sequencing techniques to identify the causative mutation of DCM in an Iranian pedigree. We found a novel variant in the GATA6 gene, leading to substituting Histidine by Tyrosine at position 329, observed in all affected family members in the pedigree, whereas it was not established in any of the unaffected ones. We hypothesized that the H329Y mutation may be causative for the familial pattern of DCM in this family. The predicted models of GATA6 and H329Y showed the high quality according to PROCHECK and ERRAT. Nonetheless, simulation results revealed that the protein stability decreased after mutation, while the flexibility may have been increased. Hence, the mutation led to the increased compactness of GATA6. Overall, these data indicated that the mutation could affect the protein structure, which may be related to the functional impairment of GATA6 upon H329Y mutation, likewise their involvement in pathologies. Further functional investigations would help elucidating the exact mechanism.

Investigation of CDH1 germline mutations in Turkish patients with Kaposi’s sarcoma

Abstract Objectives Kaposi’s sarcoma (KS) develops from the lining cells of blood or lymphatic vessels and may appear as red, purple, brown, or black lesions. E-cadherin, CDH1, is a cell adhesion molecule located on the surface of epithelial cells. CDH1 gene expression is downregulated in several cancers and is considered a tumor suppressor gene involved in epithelial-mesenchymal transition in carcinomas. Loss of CDH1 gene expression is observed in many carcinomas, mainly diffuse gastric carcinomas and lobular breast carcinomas, as well as skin tumors. This study investigates the CDH1 germline mutations in HIV-negative (Human Immunodeficiency) Virus KS patients in the Turkish population. Methods The study examined 25 peripheral blood mononuclear cells from KS patients using the Sanger sequencing technique. Results Sixteen exons of the CDH1 gene were sequenced, and a pathogenic functional germline mutation, HET c.2245C > T, p.(Arg749Trp) rs776975632, NM _004360.5, was identified in a patient with a family history of gastric and breast cancer with a high number of lesions compared to other KS patients. Discussion: KS patients with a family history of cancer could be screened for CDH1 gene and cancer-related-gen variants in the future. Conclusions KS is a rare malignancy, and genetic analysis will benefit KS patients. Further studies are needed to describe better the variations detected in a large number of KS patients in this study.

Frequency of JAK2 and MPL Mutation in BCR/ABL Negative Myelofibrosis in KPK

Introduction: BCR-ABL1-negative myeloproliferative disorders are a sub-group of myeloproliferative neoplasms (MPNs) that consist of polycythemia Vera (PV), Essential thrombocythemia (ET) and Primary Myelofibrosis (PMF). Over the past decade, the morphological and clinical division of myeloproliferative neoplasms (MPN) has changed to a classification that describes the molecular heterogeneity and is unique to this subgroup of haematological malignancies. This includes alterations in Janus kinase 2 (JAK2), and MPL genes. Objective: To determine the frequency of JAK2 (p.V617F) and MPL (p.W515L) mutation in primary myelofibrosis in KPK province of Pakistan. Materials and Methods: Fifty patients with PMF were enrolled in the study. JAK2 mutation status was determined using allele specific primers and MPL mutation was detected by direct Sanger sequencing technique. The data was analyzed using BioEdit by aligning the sequence data with reference genome hg19 assembly. Results: Among 50 patients, 41 patients were diagnosed with PMF, while 9 patients had secondary myelofibrosis i.e. Post PV-MF and Post ET-MF. Out of the 41 PMF patient 2 patients had MPL gene variation, while one of the Post ET –MF had a MPL gene variation. Forty eight (96%) were positive for JAK2 mutation. Five patients who had MPL mutation also showed JAK 2 mutation. Two of the MPL positive patients were also positive for JAK2 mutation. Conclusion: We reported rate of recurrence of JAK2 mutation in 96% of the cases and MPL exon 10 mutations in 6% of the cases.

Recombination and darwinian selection as drivers of genetic diversity and evolution of sweet potato leaf curl viruses in Tanzania

Using High-throughput Sequencing (HTS) and Sanger sequencing techniques, we detected sweet potato leaf curl viruses (SPLCVs; Genus Begomovirus) and determined their genetic diversities, recombination, and evolutionary selection pressure imposed on their genes. Eleven complete (2767–2824 nucleotides) and fifty partial (V1 and C1 proteins) sequences of SPLCVs were generated, and one complete genome of sweet potato pakakuyu virus was also generated. The detected SPLCVs were genetically diverse, with complete nucleotide sequence identities ranging from 78.7 to 100%. These sequences were closely related (>89% identical) to the sweet potato leaf curl virus, the sweet potato Sao Paulo virus, and the sweet potato leaf curl Uganda virus. The diversity of SPLCVs indicated the occurrence of their strains and variants in Tanzania. Intra- and interspecies recombination events were detected in four and two complete and partial sequences of SPLCVs, respectively. The genes of SPLCVs, except the C4 gene, are evolving under purifying selection pressure. In the C4 gene, numerous codon sites are evolving under positive selection pressure. High genetic diversity and diversifying selection imply an increased chance of SPLCVs to interact and adapt to diverse sweet potato genotypes and their wild relatives in Tanzania.

Association study identifies genetic determinants and non-genetic factors on steady-state plasma and therapeutic outcome of galantamine in mixed dementia.

This study aimed to evaluate the influence of genetic polymorphisms of drug-metabolizing enzyme genes, transporter gene, pathological gene (APOE), and non-genetic factors on therapeutic outcomes as well as steady-state plasma concentrations (Cpss) of galantamine in Thai patients with mixed dementia. Fifty-one Thai patients with mixed dementia who received galantamine for at least 6months were recruited. CYP2D6, CYP3A5, and ABCB1 polymorphisms were detected by TaqMan® Genotyping Assay. UGT1A1 and APOE polymorphism was detected by direct Sanger sequencing technique and restriction fragment length polymorphism technique. Cpss of galantamine was measured by ultra-performance liquid chromatography. Associations of genetic and non-genetic factors with Cpss and clinical outcomes (change in cognitive function as measured by the Thai Mental State Examination (ΔTMSE) scores) were determined by using univariate and multivariate analysis. The multivariate regression model revealed that patients who carried one or more detrimental allelic variant (CYP2D6, CYP3A5, and UGT1A1) showed a tendency toward a higher galantamine adjusted Cpss (B = 34.559, 95% CI = 0.741-68.377, p value = 0.045). Logistic regression analysis also revealed CYP2D6*10 carriers were significantly associated with higher ΔTMSE (B = 5.227, 95% CI = 2.395-8.060, p value = 0.001). UGT1A1 mutant alleles and non-genetic factors including concomitant use of statin drugs and higher education level can attenuate therapeutic outcomes of galantamine. Pharmacokinetic-related genes including CYP2D6*10 and UGT1A1 mutant alleles were significantly associated with galantamine adjusted Cpss and cognitive function. Determination of Cpss and genotype could be an adjunct examination to provide further explanation in interindividual variability of galantamine therapeutic outcome.

Mutation analysis reveals novel and known mutations in SAG gene in first two Egyptian families with Oguchi disease

BackgroundOguchi disease is a rare type of congenital stationary night blindness associated with an abnormal fundus appearance. It is inherited in an autosomal recessive manner where two types exist according to the gene affected; type 1 associated with S-antigen (SAG) gene mutations and type 2 associated with rhodopsin kinase (GRK1) gene mutations.PurposeThe aim of this work was to describe the clinical and genetic findings of the first two reported families of Oguchi disease in Egypt and African region.MethodsFour members of two consanguineous Egyptian families with history of night blindness since childhood underwent complete ophthalmological examination, standard automated static perimetry, fundus color photography, fundus autofluorescence (FAF), fundus fluorescein angiography (FFA) in light-adapted state and spectral-domain optical coherence tomography (SD-OCT) of both the macula and the optic nerve head as well as central corneal thickness with repeated fundus photography following prolonged dark adaptation. Mutation screening of 7 coding exons of GRK1 gene and 15 coding exons of SAG gene as well as some flanking regions were performed using Sanger sequencing technique. The variants were tested for pathogenicity using different in silico functional analysis tools.ResultsThe clinical examination and investigations confirmed Oguchi disease phenotype. One patient showed p.R193* (c.577C > T) which is a previously reported SAG gene mutation in a homozygous form. The other three patients from a different family showed (c.649–1 G > C), a novel canonical splice site SAG gene mutation in a homozygous form.ConclusionThe identification of the novel canonical splice site SAG gene variant in three members of the same family with clinically confirmed Oguchi disease reinforces its pathogenicity. A fourth patient from another family carried a previously reported mutation in the same gene. SAG gene variants may be the underlying genetic cause for Oguchi disease in Egypt. Our findings have expanded the spectrum of Oguchi disease-associated mutations in SAG gene and may serve as a basis for genetic diagnosis for Oguchi disease.

Molecular characterization of colorectal cancer patients

ResumenIntroducción. El cáncer colorrectal tiene una alta incidencia en la población mundial. Diversas vías moleculares están involucradas en su desarrollo, entre ellas, la inestabilidad cromosómica, la inestabilidad microsatelital y la epigenética.Objetivo. Hacer la caracterización molecular de 44 individuos con cáncer colorrectal esporádico.Materiales y métodos. El análisis de mutaciones en los genes APC, KRAS, TP53 y BRAF se hizo mediante secuenciación de Sanger; la inestabilidad microsatelital se determinó mediante electroforesis capilar utilizando cinco marcadores de repetición corta en tándem (Short Tandem Repeat) y el estado de metilación del promotor del gen MLH1 se hizo con la técnica MS-PCR (Methylation-Specific PCR).Resultados. La frecuencia de mutación de los genes APC, KRAS y TP53 fue del 18,1, 25 y 4,5 %, respectivamente; las mutaciones detectadas se localizaron con mayor frecuencia en el colon derecho. La frecuencia de inestabilidad microsatelital fue del 27,2 % y el 73,1 % en los tumores con metilación en el gen MHL1, y el 91,6 % de los tumores con inestabilidad microsatelital presentaba metilación en el gen MLH1. En el grupo de tumores con estabilidad microsatelital, las mutaciones en los genes APC, KRAS y TP53 fueron más frecuentes que en el grupo de tumores con inestabilidad microsatelital. La metilación del gen MLH1 fue la alteración más predominante.Conclusiones. En los pacientes con cáncer colorrectal evaluados se demostró la presencia de alteraciones moleculares en las diferentes vías genéticas, las cuales son comunes en su carcinogénesis. Los pacientes presentaron un perfil de mutaciones diferente al de otras poblaciones. Los hallazgos obtenidos en este estudio confirman la heterogeneidad molecular descrita en el desarrollo del cáncer colorrectal.

ANALYSIS OF CALRETICULIN (CALR) MUTATION IN MYELOFIBROSIS PATIENTS IN KHYBER PAKHTUNKHWA

Objective: To analyze the prevalence of CALR and JAK2 mutation co-occurrence in MF patients in KPK.
 Methods: This cross-sectional study included MF patients (n = 50) enrolled in the Hematology/Oncology department of Hayatabad Medical Complex (HMC), IRNUM (Institute of Radiotherapy and Nuclear Medicine), Peshawar and Blood Diseases Clinic, Peshawar. Non-probability convenience sampling technique was used. All Patients with JAK2 V617F positive and negative primary or secondary myelofibrosis were included. After taking blood samples, DNA was extracted manually and analysed for JAK2 mutations using conventional PCR. Sanger sequencing technique was implied to analyse the samples for CALR mutations. Data were recorded & analysed statistically.
 Results: Among the 50 patients of myelofibrosis under study, 48(96%) were harboring JAK2 mutations while it was not present in 2 (4%) patients. The typical CALR mutations was not identified in the current study. However, 02 specific genetic variants were identified in 30 patients i.e., a single nucleotide polymorphism (c.1381 G > T) in the 3? UTR and a novel insertion-deletion variant (c. 1099 CTT > AC). Seven patients had the Indel frameshift variant (p.Leu 367 Thr Fx 63), whereas 23 had SNPs. In JAK-2 positive patients (n=28), 06 had the Indel variant, and 22 had the SNPs. Two patients were JAK2 negative, one with SNP and the other with a frameshift mutation.
 Conclusion: Majority of the MF patients had a novel frameshift variant (p. Leu367Thr Fx63) (n = 07) and an SNP in the 3? UTR region (G > T) (n = 23) in CALR gene.

First report of sweet potato chlorotic stunt virus infecting sweet potatoes in Hungary.

Sweet potato chlorotic stunt virus (SPCSV), a crinivirus in the family Closteroviridae, is a quarantine pest in Europe and one of the most economically important viruses of sweet potato (Ipomoea batatas (L.) Lam) crops globally. It forms synergies with other viruses in sweet potato, leading to yield loss of 30-100% (Qin et al., 2014). In summer 2020, 62 symptomatic and 38 symptomless sweet potato vines were randomly collected in farmers' fields in the south (Ásotthalom, Szeged) and central (Galgahévíz) parts of Hungary and transplanted in an insect-proof greenhouse. Six of the plants expressed SPCSV-like symptoms, including stunting, vein clearing and leaf purpling (Suppl1). To check for common viruses of sweet potato (Suppl2), total RNA and DNA were extracted from leaves of each of the 100 plants using Trizolate reagent (UD-GenoMed, Debrecen, Hungary) and Zenogene kit (Zenon Bio, Szeged, Hungary), respectively. Primer pair Ch2N (Suppl2) was designed using Primer3 (v. 0.4.0) to amplify a 194 bp fragment of SPCSV RNA1. Presence of the RNA viruses was checked by qPCR using qPCRBIO SyGreen 1-step qPCR kit (PCR Biosystems, London, UK), while DNA viruses were checked by PCR using DreamTaq DNA Polymerase (Thermo Scientific, Vilnius, Lithuania), followed by 1% agarose gel electrophoresis. Four samples (labelled A5.1, A6.1, A6V9-1, A6V9-2) out of the 100 tested positive for SPCSV. Two of them (A6V9-1 and A6V9-2) were co-infected with SPCSV, a badnavirus sweet potato pakakuy virus (SPPV) and a potyvirus sweet potato virus 2 (SPV2), while the other two (A5.1 and A6.1) lacked SPV2. Plants infected with SPCSV, SPV2 and SPPV displayed more severe symptoms. To confirm the results, cDNA synthesized from the four SPCSV positive samples using RevertAid first strand cDNA synthesis kit (Thermo Scientific, Vilnius, Lithuania) underwent PCR (94oC 4 min, 94oC 1 min, 53oC 30 s, 72oC 70 s and 72oC 10 min for a total of 30 cycles) using primers CL43U and CL43L for the viral heat shock protein 70 gene (Maliogka et al., 2020). An expected band size of 486 bp was obtained in all cases. The amplicon from sample A6.1 was sequenced and found to be identical to SPCSV Guatemalan isolate GT:B3:08 (acc. JF699628). RNA1 and RNA2 complete sequences from sample A6.1 were obtained via PCR amplifications of cDNA using primers (Suppl2) designed (from acc. KC888966 for RNA1 and acc. KC888963 for RNA2) to amplify overlapping fragments of West African strain of SPCSV. QIAquick gel extraction kit (QIAGEN, Hilden, Germany) was used to purify the PCR fragments, which were then cloned into pGEM-T Easy Vector (Promega, Madison, USA) and sequenced using Sanger sequencing technique (Biomi, Gödöllő, Hungary). BLASTn search revealed that RNA1 of our isolate Hun_01 (acc. MW892835) had 99.63% sequence identity to SPCSV isolate su-17-10 (acc. MK802073), while RNA2 of Hun_01 (acc. MW892836) was 99.68% similar to SPCSV isolate min-17-1 (acc. MK802078) and isolate 24-1 (acc. MK802080). Phylogenetic analysis using MegAlign (v. 7.1.0, 44.1) showed a close relationship between our isolate and those isolated in China, suggesting that they may have a common origin (Suppl1). Severe stunting and leaf yellowing symptoms developed in I. setosa indicator plants grafted with SPCSV infected sweet potato scions. qPCR test for the virus confirmed its presence in the I. setosa leaves. To the best of our knowledge, this is the first report on the occurrence of SPCSV in Hungary and the third in Europe (Valverde et al. 2004; EPPO 2021).

A murine Skint6 W168X allele contributes to autoimmune disease in a transgenic model.

The genetic factor is a great driver of systemic lupus erythematosus. A Skint6 W168X allele was previously identified in the murine lupus susceptibility rec1d1 sublocus. The purpose of this study is to investigate the pathogenic role and mechanism of the Skint6 W168X allele in lupus autoimmune disease. The gene-editing CRISPR/Cas9 system was used to generate transgenic models with the Skint6 W168X allele. PCR and Sanger's sequencing techniques were applied to mRNA quantification and DNA sequence detection. Flow cytometry was adopted for immunophenotyping. Pathological evaluation of kidneys and lungs was performed using several immunopathological approaches. The transgenic models with the Skint6 W168X allele were created, including B6.Skint6X/X and B6.lpr.Skint6X/X strains. The B6.lpr.Skint6X/X mice showed bigger spleen and lymph nodes, more lymphocytes and effector T cell populations, more severe nephritis with more IgG and C3 deposit in glomeruli as well as worse proteinuria, and more severe lung inflammation than control B6.lpr mice. In addition, a skint6 receptor binding Skint6 peptide was identified from T and B lymphocytes. B6.Skint6X/X mice have lower percentages of skint6 receptor+ T and B cells in spleen than B6 mice. The Skint6 W168X allele in murine lupus rec1d1 sublocus was validated to be a pathogenic mutant gene and contributes to autoimmune disease through producing a truncated Skint6 peptide of binding the skint6 receptors on lymphocytes.

Review of genome sequencing technologies in molecular characterization of influenza A viruses in swine.

The rapidly evolving antigenic diversity of influenza A virus (IAV) genomes in swine makes it imperative to detect emerging novel strains and track their circulation. We analyzed in our review the sequencing technologies used for subtyping and characterizing swine IAV genomes. Google Scholar, PubMed, and International Nucleotide Sequence Database Collaboration (INSDC) database searches identified 216 studies that have utilized Sanger, second-, and third-generation sequencing techniques to subtype and characterize swine IAV genomes up to 31 March 2021. Sanger dideoxy sequencing was by far the most widely used sequencing technique for generating either full-length (43.0%) or partial (31.0%) IAV genomes in swine globally; however, in the last decade, other sequencing platforms such as Illumina have emerged as serious competitors for the generation of whole-genome sequences of swine IAVs. Although partial HA and NA gene sequences were sufficient to determine swine IAV subtypes, whole-genome sequences were critical for determining reassortments and identifying unusual or less frequently occurring IAV subtypes. The combination of Sanger and second-generation sequencing technologies also greatly improved swine IAV characterization. In addition, the rapidly evolving third-generation sequencing platform, MinION, appears promising for on-site, real-time sequencing of complete swine IAV genomes. With a higher raw read accuracy, the use of the MinION could enhance the scalability of swine IAV testing in the field and strengthen the swine IAV disease outbreak response.

Diagnosis and detection of VicK gene in Streptococcus mutans isolated from the saliva of patients with diabetic type 2 with tooth decay in the Iraqi population.

Type 2 diabetes mellitus (T2DM) is gradually becoming more common in Iraq. Salivary changes and proliferation of specific bacterial communities cause oral disease that can adversely affect systemic conditions such as diabetes. Fifty saliva samples were collected from people with T2DM suffering from tooth decay and twenty-five people without T2DM suffering from tooth decay. The periodontal status, the extent of the root surface, and coronal caries were evaluated. Saliva was cultured for investigating Streptococcus mutans. The results showed that patients with type 2 diabetes had significantly more severe Periodontitis and a higher prevalence and magnitude of bacterial caries. Diabetic subjects had higher levels of Hemoglobin A1c (HbA1c) and Random Blood Sugar (R.B.S.). The S. mutans diagnosis by PCR for Sanger Sequencing technique by using VicK gene sequences (1300bp). The PCR products of the isolate were submitted to Macrogen Company for sequencing. Selected seven isolates as new isolates registered in global gene bank as locally S. Mutans isolates in Bagdad city/Iraq and their accepted accession numbers include LOCUS MT603520, MT603521, MT603522, MT603523, MT603524, MT603525,and MT603526 of nucleotide sequence. The VicK genes isolates' phylogenetic trees revealed a genotype that was closely connected to other isolates in GenBank. Furthermore, gene sequencing demonstrated a success rate of 99 percent. resemblance to other isolates in the GenBank database The likelihood of a link between S. Mutans and dental carries was determined by these findings.

Sustained Replication of Synthetic Canine Distemper Virus Defective Genomes In Vitro and In Vivo.

ABSTRACTDefective interfering (DI) genomes restrict viral replication and induce type I interferon. Since DI genomes have been proposed as vaccine adjuvants or therapeutic antiviral agents, it is important to understand their generation, delineate their mechanism of action, develop robust production capacities, assess their safety and in vivo longevity, and determine their long-term effects. To address this, we generated a recombinant canine distemper virus (rCDV) from an entirely synthetic molecular clone designed using the genomic sequence from a clinical isolate obtained from a free-ranging raccoon with distemper. rCDV was serially passaged in vitro to identify DI genomes that naturally arise during rCDV replication. Defective genomes were identified by Sanger and next-generation sequencing techniques, and predominant genomes were synthetically generated and cloned into T7-driven plasmids. Fully encapsidated DI particles (DIPs) were then generated using a rationally attenuated rCDV as a producer virus to drive DI genome replication. We demonstrate that these DIPs interfere with rCDV replication in a dose-dependent manner in vitro. Finally, we show sustained replication of a fluorescent DIP in experimentally infected ferrets over a period of 14 days. Most importantly, DIPs were isolated from the lymphoid tissues, which are a major site of CDV replication. Our established pipeline for detection, generation, and assaying DIPs is transferable to highly pathogenic paramyxoviruses and will allow qualitative and quantitative assessment of the therapeutic effects of DIP administration on disease outcome.IMPORTANCE Defective interfering (DI) genomes have long been considered inconvenient artifacts that suppressed viral replication in vitro. However, advances in sequencing technologies have led to DI genomes being identified in clinical samples, implicating them in disease progression and outcome. It has been suggested that DI genomes might be harnessed therapeutically. Negative-strand RNA virus research has provided a rich pool of natural DI genomes over many years, and they are probably the best understood in vitro. Here, we demonstrate the identification, synthesis, production, and experimental inoculation of novel CDV DI genomes in highly susceptible ferrets. These results provide important evidence that rationally designed and packaged DI genomes can survive the course of a wild-type virus infection.

Study on TYR gene variant from a pedigree with oculocutaneous albinism

To analyze gene variants in a Chinese pedigree with oculocutaneous albinism (OCA). Gene sequencing of the proband and his parents was performed using chip capture high-throughput sequencing and Sanger sequencing techniques, and PolyPhen-2, SIFT, MutationTaster, and FATHMM software were used to predict the function of new variants. At the same time,the pedigree and variant genes of 4 albinism patients from this pedigree were analyzed. Sequencing results showed that the proband's TYR gene (NM_000372) has c.230G>A (p.Arg77Gln) and c.120_121insG (p.Asp42GlyfsTer35) compound heterozygous variants. The proband's father carries c.230G>A heterozygous variant, and the mother carries c.120_121insG heterozygous variant, indicating that the proband's two variants are from his father and mother. The former is a known missense variant, which can cause abnormal or loss of the original function of the protein polypeptide chain. The latter c.120_121insG(p.Asp42GlyfsTer35) is an unreported frameshift variant of the TYR gene subregion (EX1; CDS1). PolyPhen-2, SIFT, MutationTaster and FATHMM predictions are all prompted as "harmful variants". This variant caused the amino acid encoded protein to terminate prematurely, producing a truncated protein, which eventually formed a 76-amino acid short-type TYR protein instead of the 529-amino acid wild-type TYR protein. Through the pedigree analysis, the four patients in the pedigree are all of the same type of compound heterozygous variants, and the disease-causing genes are all from the patient's parents. They belong to a special form of consanguineous marriage within 5 generations. The compound heterozygous variants of c.230G>A (p.Arg77Gln) and c.120_121insG (p.Asp42GlyfsTer35) of the TYR gene may underlie the disease in this pedigree. The gene sequencing results enrich the variant spectrum of the TYR gene, and has facilitated molecular diagnosis for the patient.

Prevalence of Naturally Occurring Resistance Associated Substitutions in NS3/4AProtease Inhibitors in Iranian HCV/HIV Infected Patients.

Hepatitis C virus (HCV) acts in the host as a complicated mixture of related variants with the potency to genetically escape host immune responses. Direct acting antivirals (DAAs) have been approved for HCV treatment with shorter duration, better cure rates and lower side effects. However, naturally occurring resistance associated substitutions (RASs) create some obstacles to this antiviral therapy success. In this study, we aimed at the determination of the naturally occurring NS3/4A RASs in HCV/human immunodeficiency virus (HIV)infected patients. A total of 120 DAA-naïve HCV-HIV co-infected patients were included. HCV NS3/4Agenome region was amplified with PCR and mutation analysis was performed by Sanger sequencing technique. The amino acid sequence diversity of the region was analyzed using geno2pheno HCV. Phylogenetic analysis showed that 73 cases were infected by 3a and 47 subjects by subtype1a. The overall RASs among studied subjects were observed in 6 (5%) individuals from 120 studied cases who were infected with HCV 1a. V36M/L, Q80L, S122G/L, R155T/G, A156S, D168Y/N and S174A/N/T mutations were detected in this study. Although the prevalence of RASs was totally low in this study, the presence of several cases of double and triple mutants among this population suggests prior evaluation of protease inhibitors related mutations before initiation of standard treatment and also an investigation on a large population could be of high value.

Vertical structure of the bacterial diversity in meromictic Fayetteville Green Lake.

The permanently stratified water columns in euxinic meromictic lakes produce niche environments for phototrophic sulfur oxidizers and diverse sulfur metabolisms. While Green Lake (Fayetteville, New York, NY) is known to host a diverse community of ecologically important sulfur bacteria, analyses of its microbial communities, to date, have been largely based on pigment analysis and smaller datasets from Sanger sequencing techniques. Here, we present the results of next‐generation sequencing of the eubacterial community in the context of the water column geochemistry. We observed abundant purple and green sulfur bacteria, as well as anoxygenic photosynthesis‐capable cyanobacteria within the upper monimolimnion. Amidst the phototrophs, we found other sulfur‐cycling bacteria including sulfur disproportionators and chemotrophic sulfur oxidizers, further detailing our understanding of the sulfur cycle and microbial ecology of euxinic, meromictic lakes.

A Review on the Methods for Identification of Mutations in the Tumour Suppressor Gene Retinoblastoma RB1

Retinoblastoma is the most common intraocular cancer of childhood. RB1 is the gene responsible for causing retinoblastoma, spans more than 180 kilobases (kb) located on chromosome 13q14, which consist of 27 exons. Retinoblastoma in children may either be hereditary or non-hereditary. Mutations in RB1 gene are mostly point mutations of non-sense or missense type but could also be of frameshift type. These mutations can be identified from both blood and tumour samples by Sanger sequencing and other molecular identification techniques such as Multiplex Ligation-dependent Probe Amplification (MLPA). ‘Fragile’ codons are codons which gets point mutated to form stop codons so that the resulting protein will be incomplete or immature. In RB1, fragile codons get mutated predominantly and lead to the truncation of RB1 protein. The frequent mutations that predominantly occur in the arginine (CGA) codon, wherein changes in the single nucleotide results in the stop (UGA) codon, than any other fragile codon. The present paper reviews the role of RB1 mutations in retinoblastoma and the methods to identify it. We also make an attempt to identify the fragile codons in the RB genome based on the NCBI reference sequence NM_000321.2

Screening for antifolate and artemisinin resistance in Plasmodium falciparum clinical isolates from three hospitals of Eritrea

Background: Antimalarial drug resistance is a major challenge hampering malaria control and elimination. Plasmodium falciparum, the leading causative parasite species, has developed resistance to basically all antimalarials. Continued surveillance of drug resistance using genetic markers provides important molecular data for treatment policies. This study sought to verify the genetic mechanism of resistance to sulfadoxine-pyrimethamine and assess the occurrence of point mutations associated with artemisinin resistance in P. falciparum clinical isolates from Eritrea. Methods: Nineteen dried blood spot samples were collected from patients visiting Adi Quala, Keren and Gash Barka Hospitals, Eritrea. The patients were followed up after receiving treatment with first line artesunate-amodiaquine. Nested polymerase chain reaction and Sanger sequencing techniques were employed to genotype point mutations in the P. falciparum bifunctional dihydrofolate reductase-thymidylate synthase (Pfdhfr, PF3D7_0417200), dihydropteorate synthase (Pfdhps, PF3D7_0810800) and kelch 13 (PfK13, PF3D7_1343700) genes. Results: Eight of nineteen (42%) of the dried blood spot samples were successful for PCR-amplification. Data analyses of the PCR-positive isolates revealed the following point mutations: Pfdhfr N51I in four isolates, C59R in one isolate, S108N in four isolates, a rare non-synonymous substitution V45A in four isolates and Pfdhps K540E in four isolates. No PfK13 point mutations were reported. Conclusions: Pfdhfr C59R and Pfdhps K540E point mutations are reliable markers for the sulfadoxine-pyrimethamine quintuple mutant haplotype combination. These findings highlight first reports in Eritrea, which verify the underlying genetic mechanism of antifolate resistance. Continuous monitoring of the PfK13 marker is recommended.

Calreticulin mutational analysis in patients with myelofibrosis

Objective Our study aimed to determine the occurrence of CALR and JAK-2 mutation in patients with myelofibrosis. Patients and methods This cross-sectional study was conducted in the Department of Hematology, Lady Reading Hospital, Peshawar, and Princess Nourah Bint AbdulRehman University, Saudi Arabia. Blood samples and data were collected from patients in the Hematology Department of Lady Reading Hospital, Peshawar, Pakistan. Nonprobability convenience sampling technique was used. All patients of JAK-2 V617F-positive and JAK-2 V617F-negative primary or secondary myelofibrosis due to polycythemia vera or essential thrombocythemia were included. After taking blood and bone marrow sampling, DNA extraction was done manually, and these samples were analyzed for CALR mutations by Sanger sequencing technique. Data were recorded and analyzed in SPSS, version 20. Results Of 118 patients of myelofibrosis, we have found two types of genetic variations. One is single nucleotide polymorphism (SNP) in the 3’ UTR variant and the other is a novel indel frameshift mutation in the form of p.Leu 367 Thr Fx 63. Among these 118 patients, 14.40% patients had the indel frameshift mutation, whereas 46.61% patients had SNPs. The remaining patients did not harbor any significant changes. Conclusion Our study concluded that most patients had a SNP in the 3’ UTR variant and a novel frameshift mutation. Further large-scale studies should be organized to determine the co-occurrence of calreticulin mutations in the JAK-2-positive patients of myelofibrosis. The influence of this coexistence on the phenotype and clinical course of myelofibrosis should also be studied to better understand the diagnosis and prognosis of the patients with myelofibrosis.

Recurrent KRAS, KIT and SF3B1 mutations in melanoma of the female genital tract

BackgroundMalignant melanoma of the female genital tract is relatively uncommon and accounts for 3–7% of all melanoma localizations. This study aimed to identify driver genes in melanoma of the female genital tract with the purpose of enhancing understanding of disease pathogenesis and identifying potential new therapeutic targets to develop effective therapies.MethodsKIT (CD117) and BRAF expression were detected immunohistochemically. Polymerase Chain Reaction (PCR) and Sanger sequencing techniques were performed to identify the mutational status of BRAF, NRAS, KRAS, NF1, KIT, PDGFRA and SF3B1 on 19 melanomas of the female genital tract, paired with 25 cutaneous melanomas, 18 acral melanomas and 11 melanomas of nasal cavity.ResultsSomatic variant analysis identified KRAS (6/19; 32%) as the most commonly mutated gene, followed by KIT (4/19; 21%), SF3B1 (3/19; 16%) and NRAS (1/19; 5%). None of the cases were found to harbor BRAF, NF1 and PDGFRA mutations in melanomas of the female genital tract. However, none of the cases were found to harbor SF3B1 and KIT mutations in cutaneous melanomas, acral melanomas and melanomas of nasal cavity. Recurrent KIT mutations, as well as mutations in the less frequently mutated genes NRAS and SF3B1, were exclusively detected in vulvovaginal melanomas, but not in tumors arising in the cervix. However, recurrent KRAS mutations were detected in similar frequencies in tumors of the vulva, vagina, and cervix. Additionally, recurrent KRAS and KIT mutations occurred predominantly in polygonal and epithelioid cell types of melanoma in the female genital tract. Immunohistochemistry revealed moderate or strong cytoplasmic CD117 expression in 6 of the 19 cases (31.6%).ConclusionsWe observed that gynecologic melanoma harbored distinct mutation rates in the KIT, BRAF, SF3B1, KRAS, and NRAS genes. Our findings support the notion that gynecologic melanoma is a distinct entity from non-gynecologic melanoma, and these findings offer insights into future therapeutic options for these patients.