Sanger DNA Sequencing Method Research Articles

Prevalence, risk factors, and BRAF mutation of colorectal sessile serrated lesions among Vietnamese patients.

Sessile serrated lesions (SSLs) are considered precancerous colorectal lesions that should be detected and removed to prevent colorectal cancer. Previous studies in Vietnam mainly investigated the adenoma pathway, with limited data on the serrated pathway. To evaluate the prevalence, risk factors, and BRAF mutations of SSLs in the Vietnamese population. This is a cross-sectional study conducted on patients with lower gastrointestinal symptoms who underwent colonoscopy at a tertiary hospital in Vietnam. SSLs were diagnosed on histopathology according to the 2019 World Health Organization classification. BRAF mutation analysis was performed using the Sanger DNA sequencing method. The multivariate logistic regression model was used to determine SSL-associated factors. There were 2489 patients, with a mean age of 52.1 ± 13.1 and a female-to-male ratio of 1:1.1. The prevalence of SSLs was 4.2% [95% confidence interval (CI): 3.5-5.1]. In the multivariate analysis, factors significantly associated with SSLs were age ≥ 40 [odds ratio (OR): 3.303; 95%CI: 1.607-6.790], male sex (OR: 2.032; 95%CI: 1.204-3.429), diabetes mellitus (OR: 2.721; 95%CI: 1.551-4.772), and hypertension (OR: 1.650, 95%CI: 1.045-2.605). The rate of BRAF mutations in SSLs was 35.5%. The prevalence of SSLs was 4.2%. BRAF mutations were present in one-third of SSLs. Significant risk factors for SSLs included age ≥ 40, male sex, diabetes mellitus, and hypertension.

Plasma TNF-α Elevation in Biologic Naive Rheumatoid Arthritis Patients Belonging to a Population with New Mutations in TLR4 and CYP51A1 genes without Association with Disease-Related Antibodies Levels.

In a system biology-based study, we previously reported that IL-6 and IL6R -specific m-RNA levels were elevated in leukocytes of patients with Rheumatoid arthritis (RA). Here, the association of toll-like receptor4 (TLR4) rs 141534085 and cytochrome P450 family 51 subfamily A member 1(CYP51A1) rs6 with tumor necrosis factor-α (TNF- α), rheumatoid factor (RF)- and Anti- cyclic citrullinated peptide (anti-CCP) antibody -positivity was investigated in almost the same subjects. Forty-six patients and 48 normal subjects were recruited in this study. The blood leucocytes TLR4 rs 141534085 and CYP51A1 rs6 -comprising DNA sequences were amplified by using tetra-primer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) technique and the PCR products were checked by Sanger DNA sequencing method. ELISA method was used to determine plasma levels of TNF- α, anti-CCP antibody and RF positivity of plasma was evaluated through a latex agglutination test. The TNF- α level was significantly higher in the patient group than control subjects (p= 0.001). Moreover, we were not able to find any correlation between TNF-α levels and RF as well as anti-CCP antibodies when we used the K2/ Fisher's exact test and Pearson test respectively. Our DNA sequencing data revealed the following new mutations in TLR4 rs141534085 comprising regions: A>T in position 1050, T>A in position 1052, and C>A in position 1054; and for CYP51A1 rs6 encompassing region, the new mutations were; G>A in position 21680, the T nucleotide was inserted in position 21762 and the G nucleotide was inserted in position 21763, G>T in position 21764. The data of this study showed that both TLR4 rs141534085 and CYP51A1 rs6 related DNA regions should be considered as hotspot areas in RA pathogenicity. Moreover, these data indicated that, TNF- α did not alter the production of anti-CCP and RF pathogenic antibodies in patients with long-term RA.

The polymorphism of the ovine insulin like growth factor-2 (IGF2) gene and their associations with growth related traits in Tibetan sheep.

In the current study, the role of the ovine IGF2 as a potential candidate gene was investigated as though marker-assisted selection in Chinese Tibetan sheep. The Sanger DNA sequencing method explored five single nucleotide polymorphisms (SNPs) in 5'UTR of the ovine IGF2 gene (C15640T, G15801A, G15870A, C15982G and G15991A) in Chinese Tibetan sheep. The frequencies of four SNPs were within the Hardy-Weinberg Equilibrium (chi-square test) except C15982G. The statistical analysis indicated that the C15640T and G15801A were significantly associated with body height, body length, chest circumference, and body weight (P < 0.05 or P < 0.01). Furthermore, C15982G variant exhibited significant correlation with the body weight (P < 0.01). These findings suggests that the promoter variants of IGF2 gene could be used as a candidate gene through marker-assisted selection for the body weight and body measurement traits in Tibetan sheep breeding program.

Identification and Integrative Bioinformatics Analysis of Common and Rare Mutations in the Patients with Transfusion Dependent HbE/b and b-thalassemia in Chittagong, Bangladesh

Background: Thalassemia is an inherited blood disorder that affects hemoglobin’s structure and functions. Among several forms of this life-threatening disorder, HbE/β and β-thalassemia are most common in Bangladesh and worldwide as well. But the molecular and clinical data are not adequate regarding the underlying cause of this genetic disorder in Bangladesh. So, we aimed to identify the genetic mutations within β-globin gene (HBB) and to investigate the correlation of the mutations with HBB mRNA structure, gene transcription and hematological status among the patients with blood transfusion dependent HbE/β and β-thalassemia in Bangladesh. Methods: A total of 40 blood samples were collected from the patients with blood transfusion dependent HbE/β and β-thalassemia prior to taking their consent. Detection of mutations within HBB gene was carried out by polymerase chain reaction followed by Sanger DNA sequencing method. Identification and characterization of mutations along with their effects on HBB gene were analyzed by various bioinformatics approaches. In addition, complete blood count (CBC), and hemoglobin electrophoresis were done for hematological analysis. Results: c.92+5G&gt;C, c.79G&gt;A and c.9T&gt;C genetic mutations were identified within the HBB gene, where c.92+5G&gt;C was the most common mutation among the study patients. Mutations along with hematological status and putative transcription factor binding sites revealed that the severity of the disease depends upon the mutation type and its location in the HBB gene sequence. In addition, mRNA structure analysis showed that the identified mutations contribute to its structural diversity by altering folding mechanism that ultimately affects the stability and function of the HBB protein among the patients with blood transfusion dependent HbE/β and β-thalassemia. Conclusions: The study showed the underlying cause of HbE/β and β-thalassemia in genetic level by identifying rare and common mutations within HBB gene and their effects on with HBB gene transcription and mRNA structure. We hope study will contribute in designing effective molecular medicine and other therapeutics for the patients with HbE/β and β-thalassemia to improve their health condition. Bangladesh Medical Res Counc Bull 2022; 48(3): 180-188

Validation of a Novel Rac1 Inhibitor As Potent Secdrug Against Activating Ras Mutant and Drug-Resistant Multiple Myeloma

Multiple myeloma is the second-most common hematopoietic malignancy in the United States. Although several drugs have so far been approved by the FDA, myeloma still remains an incurable disease with dose-limiting toxicities and resistance to primary drugs like proteasome inhibitors (PIs) and Immunomodulatory drugs (IMiDs). Furthermore, activating Ras gene (NRAS, KRAS, and HRAS) mutations are known to occur in >50% of myeloma patients, which are also associated with greater tumor burden, cancer aggressiveness, high recurrence rates, and poor survival. We have recently established a novel drug development/re-purposing pipeline called secDrug that incorporates a pharmacogenomics data-driven computational algorithm to introduce several new drug candidates (secDrugs), including EHT1864 (a Rac1 (Ras superfamily GTPase) inhibitor), CCT018159 (Hsp90 inhibitor), and CP466722 (a reversible ATM inhibitor) as synergistic partners of primary drugs against drug-resistant myeloma. The overall objective of this study is to validate EHT1864 using in vitro and ex vivo models of relapsed and refractory myeloma (RRMM) patients. First, we used single-cell RNA sequencing (scRNAseq) as a novel screening tool to demonstrate that EHT1864 is potentially effective against myeloma subclones based on the enrichment of target genes (Figure 1). For in vitro validation, we used a human myeloma cell line (HMCL) panel representing drug-sensitive (FLAM76), innate/refractory resistance (LP1), and acquired/relapsed resistance (parental and clonally derived PI-resistant and IMiD-resistant HMCL pairs U266 P/VR, RPMI P/VR, MM1S P/VR/LenR). FLAM76 KRas (FLAM76-K12) and FLAM76 Nras (FLAM76-N12) cell lines were generated using Adeno-associated viral (AAV) vector-mediated delivery of CRISPR-Cas9 for genome editing in humans. Ras mutations were confirmed using Sanger DNA sequencing method. Cell viability measured using CellTiter-Glo luminescent cell viability assay showed the potency of EHT1864 as a single-agent (IC50 range 15-40µM) as well as in combination with PIs (represented by Ixazomib) and IMiDs (represented by Lenalidomide). Combination index (CI) values calculated using Chou-Talalay's CI theorem were consistently less than 0.9, while all the four synergy scores (HSA, Bliss, Loewe, and ZIP) were higher than 5, demonstrating high synergy. Cell death due to apoptosis was confirmed using flow cytometry (Annexin-V-Propidium Iodide staining), Caspase 3/7 activity assay, and immunoblotting. Interestingly, EHT1864 was most potent against drug-resistant and Ras-mutant HMCLs. Further, we showed that EHT1864 treatment also affects ROS generation and mitochondrial membrane potential, indicating that EHT1864 induces apoptosis via mitochondria-mediated pathway. Finally, we performed pre-vs-post-treatment genome-wide transcriptome analysis (tumor mRNAseq) and single-cell multi-ome analysis (single-cell RNA sequencing/scRNAseq + single-cell Assay of Transposase Accessible Chromatin sequencing/scATACseq) analysis to derive molecular signatures and pathways representing the basis of (on-target and off-target) efficacy of EHT1864 and drug synergy at the bulk tumor and subclonal levels. Finally, we will perform single-cell proteomics (high-dimensional immunophenotyping using mass cytometry/CyTOF/Cytometry time of flight) in CD138+ bone marrow cells derived from newly-diagnosed and relapsed myeloma patients (ex vivo) to demonstrate efficacy in RRMM. Thus, our work lays a framework to establish EHT1864 as a clinical trial-ready therapeutic option for the management of drug-resistant myeloma. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Investigation of neuraminidase 1 gene association in Henoch-Schönlein Purpura (HSP) with renal involvement

Objectives: HSP is a common small vessel vasculitis. It is the most common cause of non-thrombocytopenic purpura in childhood. The role of genes in etiopathogenesis of the disease, which has not yet been clearly elucidated, is being emphasized. Many genes called sialidases are being studied and is thought that the NEU1 gene may be particularly important in the etiopathogenesis of HSP. The aim of this study is to investigate the role of the NEU1 gene in the etiopathogenesis of HSP and its relation to renal involvement. Materials and methods: Fifty patients followed in the Celal Bayar University Hafsa Sultan Hospital Pediatric Nephrology Department, with the diagnosis of HSP renal involvement were included into the study. For the control group, age and gender matched 50 cases were accepted among the outpatients admitted to Pediatric Department without any chronic diseases. NEU1 gene mutation analysis was performed in blood samples of both patient and control groups by using the Sanger DNA sequencing method. Results: NEU1 genetic mutation was not detected in any HSP patient with renal involvement and control group. Conclusion: In our study, the NEU 1 gene was not found to be associated with HSP nephritis. No changes were detected in the investigated regions of the NEU1 gene.

Identification of population of bacteria from culture negative surgical site infection patients using molecular tool

BackgroundManaging surgical site infections, with negative culture report in routine diagnosis is a common dilemma in microbiology accounting more than 30% worldwide. The present study attempted to identify the presence of bacterial spp. if any in wound aspirates/swabs of culture negative surgical site infections of hospitalised patients using molecular tools.MethodsNinety-seven patients with post-operative SSI whose wound swabs/aspirate were negative in the conventional aerobic culture after 72 h of incubation were analysed by 16S rRNA gene specific broad range PCR. The amplified DNA fragments were sequenced by Sanger DNA sequencing method and homology of the sequence were matched using NCBI BLAST (NCBI, USA)ResultsOf the 97 patients, 16S rRNA based broad range PCR assay could identify the presence of bacterial pathogen in 53(54.63%) cases, of which 29 isolates were supposed to be of viable but non-culturable bacteria (VBNC), 07 were of obligatory anaerobes and 13 were of unculturable bacteria, 04 were with poly bacterial infections.ConclusionsOur study highlights the usefulness of PCR assay in detecting the presence of any VBNC, anaerobes and unculturable bacteria in SSI patients regardless of how well the bacteria may or may not grow in culture. Measures should be taken to use anaerobic culture system and PCR diagnosis along with conventional culture to detect the VBNC and unculturable bacteria where Gram stain is positive for better patient care.

Antibiotic susceptibility and phylogenetic analyses for the origins and serotypes of Listeriamonocytogenes strains isolated from ice cream and cream cakes

Listeria monocytogenes is a zoonotic bacterium which also infects humans. The aim of this study was to isolate this organism from cream cakes and ice cream and obtain 16S rRNA and hlyA gene sequences from isolates in order to perform phylogenetic analyses. Serotypes and antibiotic susceptibility were also determined. The cream cake and ice cream samples were examined for L. monocytogenes according to ISO 11290-1 and using the mini Vidas LMO 2 kit procedure. Antibiotic susceptibilities were investigated using the disc diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI) standards. The Sanger DNA sequencing method for phylogenetic analysis was used for L. monocytogenes isolates. A total of 16 (n =128, 12.50%) L. monocytogenes strains, 9 (12.16%) from cream cake samples and 7 (12.96%) from ice cream samples, were isolated. This was the first investigation involving sequencing of L. monocytogenes isolated from cream cakes and ice creams in Turkey. All isolates were susceptible to sulfamethoxazole/trimethoprim, tetracycline, streptomycin, gentamicin, meropenem, and erythromycin. The numbers of isolates resistant to sulbactam/ampicillin, penicillin G, chloramphenicol, and ampicillin were 16, 2, 1, and 1, respectively. Moreover, 3 isolates showed intermediate resistance to amikacin and 2 to ciprofloxacin. The hlyA gene sequences of 11 of the L. monocytogenesisolates isolated from milk were closely related to the hlyA gene sequences of the GenBank reference strains. The comparison of the 16s rRNA gene sequences of the L. monocytogenes strains with the GenBank reference serotypes identified 1 isolate as serotype 1/2c, 1 as serotype 1/2a, and 1 as serotype 4. Nucleic acid sequencing is useful for identification of L. monocytogenes. The 16S rRNAand hlyA sequences can be used to determine the origin and relationship between L. monocytogenesisolates, as well as the serotype.

Modeling of shotgun sequencing of DNA plasmids using experimental and theoretical approaches

BackgroundProcessing and analysis of DNA sequences obtained from next-generation sequencing (NGS) face some difficulties in terms of the correct prediction of DNA sequencing outcomes without the implementation of bioinformatics approaches. However, algorithms based on NGS perform inefficiently due to the generation of long DNA fragments, the difficulty of assembling them and the complexity of the used genomes. On the other hand, the Sanger DNA sequencing method is still considered to be the most reliable; it is a reliable choice for virtual modeling to build all possible consensus sequences from smaller DNA fragments.ResultsIn silico and in vitro experiments were conducted: (1) to implement and test our novel sequencing algorithm, using the standard cloning vectors of different length and (2) to validate experimentally virtual shotgun sequencing using the PCR technique with the number of cycles from 1 to 9 for each reaction.ConclusionsWe applied a novel algorithm based on Sanger methodology to correctly predict and emphasize the performance of DNA sequencing techniques as well as in de novo DNA sequencing and its further application in synthetic biology. We demonstrate the statistical significance of our results.Graphical abstract

A Study on the Effect of Prolactin Gene Variants on Milk Production Traits of Holstein Cattle

Recent studies aiming to improve the daily milk production of cattle using both genetic and environmental factors are extremely important. The aim of this study is to determine the possible relationships between daily milk production and polymorphic distribution of exon 3 region of the prolactin (PRL) gene, which is an important marker for selection. With this purpose, 155 Holstein cows were included in the study, and the demographic data of the cattle were recorded. Venous blood was collected from the jugular vein of cows in 2 tubes of 2 mL each containing EDTA for DNA isolation. Genomic DNA were isolated from these whole blood samples. The prolactin gene region of the cattle were identified using PCR/RFLP and Sanger DNA sequencing method. The genotype, allele frequencies and Hardy–Weinberg equilibrium were calculated using Arlequin version 3.5.1.3 package software. Accordingly, it was found that cows with genotype AA had statistically higher milk yield as compared to those with genotype AB and BB. Missense mutation, frameshift and silent mutations were detected in the prolactin gene exon 3. It was also found that cows with missense mutation had statistically lower milk fat percentage as compared to the others. Consequently, the effects of genotypic distribution and mutation in the prolactin gene on the milk production of Holstein cows, which is a special breed in terms of milk productivity, have been revealed.

A Systematic Investigation of PIK3CA Mutations in Isolated Macrodactyly: Indication for Accurate Classification, Diagnosis and Potential Novel Therapeutics

Grant received from: Beijing Talent Funding There is no financial information to disclose. Macrodactyly, a rare congenital digital anomaly, has been challenging the hand surgery society. The recent discovery of somatic PIK3CA mutations in a few isolated macrodactyly patients shed light on the fundamental understanding in pathogenesis of this disabling deformity. We set up to systematically test the association between PIK3CA mutations with isolated macrodactyly in order to establish a molecular pathophysiology-based diagnosis, classification and possibly a novel therapeutic strategy. Overgrowth tissues including skin, nerve, adipose tissues from clinically diagnosed isolated macrodactyly patients (N = 12) were removed during routine surgical debulking or correction procedures and preserved as formalin-fixed-paraffin-embedded (FFPE) blocks. DNA recovered from these FFPE samples were tested for PIK3CA mutation status using a modified and targeted Sanger DNA sequencing method with greatly improved sensitivity for detecting low level somatic mosaic mutations. PIK3CA mutation level, tissue(s) of occurring, type of mutations and their genomic locations were analyzed. PIK3CA mutations were detected from affected tissues in 9 out of the 12 patients studied, with mutation level ranging from 8 to 27%. The mutations detected include p.His1047Arg (N = 4), p.His1047Leu (N = 2), p.545Glu > Lys (N = 2) and p.542Glu > Lys (N = 1). These are codons in the PIK3CA gene that are frequently mutated in cancers. In terms of the tissue sources in which a mutation was found, adipose tissue has the highest mutation detection rate (100%), followed by nerve (83%) and skin (71%). No mutations found in bone tissues from two patients with PIK3CA mutations detected in other tissues of the same patients. Patients and/or tissues negative for PIK3CA mutations are subjected to downstream analysis using high throughput next generation DNA sequencing. (Figs. 40-1, 40-2) •A high proportion (75%) of isolated macrodactyly patients harbors activating PIK3CA mutations that lead to digital overgrowth as a result of abnormally activated Akt-PI3K signaling.•Adipose and nerve tissues provide the highest PIK3CA mutation detection yield among all the sources of affected tissues.•A modified Sanger DNA sequencing method is a cost-effective way to exam PIK3CA mutations in isolated macrodactyly patients clinically.•Patients negative for PIK3CA mutations may harbor other PIK3CA mutations beyond the detection of our assay or due to different molecular mechanism.•To our knowledge, our study is the largest study linking PIK3CA mutations to isolated macrodactyly.Figure 40-2PIK3CA mutations in isolated macrodactyly patients. PIK3CA gene structure with critical domains, targeted regions, and mutations identified in isolated macrodactyly patients are summarized. Red arrows indicate three targeted regions (p.418-435, p.527-555, and p.1009-1058) by Sanger DNA sequencing. Mutations previously reported and numbers of patients are depicted above the gene structure and those found in our patients are shown under. Cancer hotspot mutations are highlighted in red.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Investigation of Distribution of Beta -Thalassemia Hereditary Mutations in Gaziantep and the Surrounding Areas

Introduction: Beta-thalassemia is the most common autosomal recessive disease. More than 200 different mutations determined in beta-globin gene. Beta thalassemia disease has a severe clinical picture. Treatment of the diseases should be maintained properly otherwise quality of life and life period can be affected. In this study, we tried to reveal inherited beta-thalassemia mutations in surrounding areas of Gaziantep. Material and Methods: In this study, we included 208 patients who applied with beta -thalassemia suspicion to the pediatric hematology clinic (age range 4-14). In that study, 138 patients with homozygous mutations and 70 patients’ heterozygous mutations were identified. HBB gene was sequenced by Sanger DNA Sequencing method. Results: Most common homozygote mutations are IVS I-110 G>A, IVS I-1 G>A, IVS II-1 G>A, the heterozygote mutations are IVS I-110 G>A, IVS II-1 G>A, codon 8 (AA) del. The incidence of IVS 1.110 (G>A) mutation is 24%, 28 in homozygote patients, 29.7% in heterozygote patients. The 8% homozygote patients and 19.7% heterozygote patients were effected with IVS 1.110 (G>A) mutation. Conclusion: The consanguinity marriages are very abundant particularly in our region due to fact that we are able to see many autosomal recessive diseased in our region. Reducing incidence of autosomal recessive disease by given genetic counseling could help the solution but it is still a major problem. J Clin Exp Invest 2016; 7(4): 265-268

Disagreement in genotyping results of drug resistance alleles of the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) gene by allele-specific PCR (ASPCR) assays and Sanger sequencing.

The rapid spread of antimalarial drug resistance in Plasmodium falciparum over the past few decades has necessitated intensive monitoring of such resistance for an effective malaria control strategy. P. falciparum dihydropteroate synthase (Pfdhps) and P. falciparum dihydrofolate reductase (Pfdhfr) genes act as molecular markers for resistance against the antimalarial drugs sulphadoxine and pyrimethamine, respectively. Resistance to pyrimethamine which is used as a partner drug in artemisinin combination therapy (ACT) is associated with several mutations in the Pfdhfr gene, namely A16V, N51I, C59R, S108N/T and I164L. Therefore, routine monitoring of Pfdhfr-drug-resistant alleles in a population may help in effective drug resistance management. Allele-specific PCR (ASPCR) is one of the commonly used methods for molecular genotyping of these alleles. In this study, we genotyped 55 samples of P. falciparum for allele discrimination at four codons of Pfdhfr (N51, C59, S108 and I164) by ASPCR using published methods and by Sanger's DNA sequencing method. We found that the ASPCR identified a significantly higher number of mutant alleles as compared to the DNA sequencing method. Such discrepancies arise due to the non-specificity of some of the allele-specific primer sets and due to the lack of sensitivity of Sanger's DNA sequencing method to detect minor alleles present in multiple clone infections. This study reveals the need of a highly specific and sensitive method for genotyping and detecting minor drug-resistant alleles present in multiple clonal infections.

A novel mutation in the PAX3 gene causes Waardenburg syndrome type I in an Iranian family

ObjectivesSensorineural hearing impairment (HI) is one of the most frequent congenital defects, with a prevalence of 1 in 500 among neonates. Although there are over 400 syndromes involving HI, most cases of HI are nonsyndromic (70%), 20% of which follow autosomal dominant mode of inheritance. Waardenburg syndrome (WS) ranks first among autosomal dominant syndromic forms of HI. WS is characterized by sensorineural hearing impairment, pigmentation abnormalities of hair and skin and hypoplastic blue eyes or heterochromia iridis. WS is subdivided into four major types, WS1–WS4. WS1 is diagnosed by the presence of dystopia canthorum and PAX3 is the only gene involved. This study aims to determine the pathogenic mutation in a large Iranian pedigree affected with WS1 in order to further confirm the clinical diagnosis. MethodsIn the present study, a family segregating HI was ascertained in a genetic counseling center. Upon clinical inspection, white forelock, dystopia canthorum, broad high nasal root and synophrys, characteristic of WS1 were evident. In order to clarify the genetic etiology and confirm the clinical data, primers were designed to amplify exons and exon–intron boundaries of the responsible gene, PAX3 with 10 exons, followed by the Sanger DNA sequencing method. ResultsGenetic analysis of PAX3 revealed a novel mutation in PAX3 (c.1024_1040 del AGCACGATTCCTTCCAA). Our data provide genotype–phenotype correlation for the mutation in PAX3 and WS1 in the studied family, with implications for genetic counseling, which necessitates detailed clinical inspection of HI patients to distinguish syndromic HI from the more common non-syndromic cases. ConclusionOur results reveal the value of phenotype-directed genetic analysis and could further expand the spectrum of PAX3 mutations.

A novel splice site deletion in the OFD1 gene is responsible for oral–facial–digital syndrome type 1 in an Emirati child

Oral–facial–digital syndrome type 1 (OFD1) is a male-lethal X-linked dominant disorder characterized by a dysmorphic face, associated with oral cavity and digital anomalies. Polycystic kidney disease is another typical feature of this syndrome. Heterozygous mutations in the OFD1 gene are responsible for this condition. This gene encodes a centrosomal and basal body cilia protein that plays an important role in the early development of the brain, face, limbs and kidneys. In this study we clinically evaluated an affected Emirati female child exhibiting features of OFD1 syndrome. Screening of the OFD1 gene was carried out using a Sanger DNA sequencing method. Moreover, bioinformatics tools were used to predict the pathogenicity of the identified mutation. As a result, we identified a heterozygous single-nucleotide deletion in the donor splice site of exon 20 (c.2757+1delG). Both parents were homozygous for the wild-type alleles. The deletion might be a de novo mutation in nature, but we cannot exclude the possibility of mosaicism in the mother. Prediction analyses showed that the deletion abolishes the authentic splice site leading to the generation of a cryptic splice site. Subsequently, this mutation will result in a frameshift and premature termination codon (p.Lys920ArgfsX2). In this report, we describe the clinical features and molecular studies of an Emirati child with OFD1 syndrome. To our knowledge this is the first report of the clinical and molecular aspect of OFD1 from the UAE.

Visual DNA microarray for detection of epidermal growth factor receptor (EGFR) gene mutations

Objective. To develop a gold nanoparticle-based visual DNA microarray for simple and rapid screening of EGFR gene mutations. Methods. The DNA fragments contain epidermal growth factor receptor (EGFR) exons 18, 19, 20 and 21 were amplified by polymerase chain reaction (PCR) using biotin-modified primers. The amino-modified oligonucleotides were immobilized on glass surface, which were used as the capturing probes to bind the complement biotinylated target DNA. After the PCR product has been hybridized to the immobilized capture probe DNA on the glass slides, the Streptavidin-conjugated gold nanoparticles were introduced to the microarray via specific binding to 5ʹ-end biotin of the PCR products. The hybridization signal on array spots was enhanced and visualized by silver amplification. The EGFR mutation in 286 clinical samples from cancer patients were tested using the gold nanoparticle-based microarray and verified with Sanger DNA sequencing method. Results. A novel visual DNA microarray has been developed to detect EGFR mutations in tumor tissue specimens rapidly; its limit of detection (LOD) is up to 10−9 mol/L and distinguishes power to detect 5% gene mutation in the mixture samples. Conclusion. For its high specificity and sensitivity, simplicity, lower price and higher speed, the present visual mutation detecting technique has potential application in clinical fields.

Molecular tests for human papillomavirus (HPV), Chlamydia trachomatis and Neisseria gonorrhoeae in liquid-based cytology specimen.

BackgroundLaboratory detection of Human papillomavirus (HPV), Chlamydia trachomatis and Neisseria gonorrhoeae in liquid-based cervicovaginal cytology specimens is now based on identification of the DNA sequences unique to these infectious agents. However, current commercial test kits rely on nucleotide probe hybridization to determine DNA sequences, which may lead to diagnostic errors due to cross-reactivity. The aim of this study was to find a practical approach to perform automated Sanger DNA sequencing in clinical laboratories for validation of the DNA tests for these three infectious agents.MethodsA crude proteinase K digestate of 5% of the cells collected in a liquid-based cervicovaginal cytology specimen was used for the detection of DNA molecules specific for HPV, C trachomatis and N gonorrhoeae, and for preparation of materials suitable for direct automated DNA sequencing. Several sets of commercially available polymerase chain reaction (PCR) primers were used to prepare nested PCR amplicons for direct DNA sequencing.ResultsSome variants of HPV-16 and HPV-31 were found to share an at least 34-base long sequence homology downstream of the GP5+ binding site, and all HPV-6 and HPV-11 variants shared an upstream 34-base sequence including part of the GP5+ primer. Accurate HPV genotyping frequently required more than 34-bases for sequence alignments to distinguish some of the HPV genotype variants with closely related sequences in this L1 gene hypervariable region. Using the automated Sanger DNA sequencing method for parallel comparative studies on split samples and to retest the residues of samples previously tested positive for C trachomatis and/or for N gonorrhoeae, we also found false-negative and false-positive results as reported by two commercial nucleic acid test kits.ConclusionIdentification of a signature DNA sequence by the automated Sanger method is useful for validation of HPV genotyping and for molecular testing of C trachomatis and N gonorrhoeae in liquid-based cervicovaginal Papanicolaou (Pap) cytology specimens for clinical laboratories with experience in molecular biology to increase the specificity of these DNA-based tests. However, even a highly specific test for high-risk HPV genotyping may have unacceptably low positive predictive values for precancer lesion in populations with a low cervical cancer prevalence rate.

The recognition of DNA cleavage sites by porcine spleen topoisomerase II.

The cutting sites specificity of topoisomerase II from porcine spleen were determined by a modified Sanger's DNA sequencing method. The topoisomerase II prefers to cut DNA at the 3' side of A and leave 5' protruding end with two staggering bases. Through the free energy analysis for DNA duplex, we also found that the topoisomerase II seemed cut DNA preferably at energetically unstable regions. So it is concluded that the specific DNA cutting by porcine spleen topoisomerase II has two structural recognition factors: one is to localize around the energetically unstable region and another is to act at the 3' side of A base.