Sanger Sequencing Method Research Articles

DMD mutations in pediatric patients with phenotypes of Duchenne/Becker muscular dystrophy

Abstract Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are common X-inherited neuromuscular diseases. The genetic diagnosis has been used as the diagnostic choice for DMD/BMD. The study subjects consisted of 37 patients from Southwest China. Peripheral blood was collected for the extraction of genomic DNA. DMD mutation was sequenced using the next-generation sequencing approach. The detected mutation was validated using the multiplex ligation-dependent probe amplification or Sanger sequencing methods. Variation annotation and pathogenicity prediction were performed using the online databases. Pathogenic mutations were identified 3 splicing site, 7 single nucleotide, 1 indel, 23 deletion, and 3 duplication mutations. Novel DMD variants were discovered, including two novel splicing variations (c.1890 + 1G>T; c.1923 + 1G>A), one missense mutation (c.1946G>T), one nonsense mutation (c.7441G>T), one indel mutation (INDEL EX20), and one duplication mutation (DUP EX75-78). The current study provides mutation information of DMD for the genetic diagnosis of DMD/BMD.

Prevalence, genotyping and phylogenetic analysis of HPV infecting Palestinian women.

Human Papillomavirus (HPV) is a significant global public health concern due to its association with cervical, other anogenital, and oropharyngeal cancers. This study aimed to evaluate the prevalence and the phylogenetic relationships of HPV among Palestinian in order to inform public health strategies. A cross-sectional study was conducted between September 2023 and April 2024 involving 379 Palestinian women over the age of 18 from 11 governorates in the West Bank. Cervical swabs were collected and analyzed using nested PCR and Sanger sequencing methods to detect and genotype HPV. The study also included phylogenetic analysis to understand the genetic relationships between HPV strains. The overall HPV prevalence was 14.5%. The highest prevalence was observed in the 20-29 age group (19.6%), the Middle region of the West Bank (19.0%), and lower educational attainment. Genotyping revealed a diverse distribution of HPV types, with HPV 11 and HPV 6 being the most common low-risk types, while HPV 16 was the most common high-risk type. About 21.8% of the detected strains were high-risk strains. Phylogenetic analysis indicated significant regional clustering of HPV strains. The study highlights the need for targeted public health interventions, including vaccination and regular screening, particularly for younger women and those with lower educational attainment. Continued surveillance and research are essential to reduce the burden of HPV-related diseases in the West Bank, Palestine.

Relationships Between Polymorphisms in HLA-G 3'UTR Region and COVID-19 Disease Severity.

The objective of this study is to investigate the the relationships between HLA-G gene variants and sHLA-G with susceptibility to SARS-CoV-2 infection. In this case-control study, 65 Patients with COVID-19 were and 67 healthy controls were genotyped for their main functional polymorphisms namely, the 14-bp Ins/Del (rs371194629), +3003C/T (rs1707), +3010C/G (rs1710), +3027A/C (rs17179101), +3035C/T (rs17179108), +3142C/G (rs1063320), +3187A/G (rs9380142) and +3196C/G (rs1610696) in the exon 8 of the 3' untranslated regions (3' UTRs) using sanger sequencing method. Associations were assessed for five inheritance models (codominant, dominant, recessive, over-dominant and log-additive). Moreover, the levels of plasma soluble HLA-G (sHLA-G) were explored using ELISA method. Our results revealed that the 14-bp INS/DEL polymorphism was strongly associated with COVID-19 symptoms development for almost all tested inheritance models (p < 0.001). Inversely, the (+3196C/G) polymorphism exhibited a protective effect against COVID-19. In addition, three haplotypes; UTR-1, UTR-3, and UTR-5 were found associated with COVID-19 symptoms (p < 0.05), The level of HLA-G in the serum was significantly higher in COVID-19 individuals than in healthy individuals (p < 0.001).These findings suggest that HLA-G gene polymorphisms in the regulatory 3'UTR region of the HLA-G gene may influence the host immune response to SARS-CoV-2 infection. A deeper comprehension of the functional effect of these associated polymorphisms could be useful in identifying high-risk individuals and in developing adaptive treatments for patients.

Sequencing and Analysis of Wolbachia Strains from A and B Supergroups Detected in Sylvatic Mosquitoes from Brazil

Wolbachia are endosymbiotic bacteria that infect a wide range of arthropods and filarial nematodes, often manipulating host reproduction. The efficacy of Wolbachia-based interventions for dengue and chikungunya control has been validated through numerous field studies in recent years. This study aimed to investigate the diversity and prevalence of Wolbachia infections in sylvatic mosquitoes from two locations in Recife, Brazil. Multiple mosquito species were screened for Wolbachia using both target marker gene amplification coupled with Sanger sequencing and whole-genome sequencing (WGS) approaches. Phylogenetic analyses were conducted to classify Wolbachia strains into supergroups and assess their evolutionary relationships. Results revealed the presence of Wolbachia in eleven mosquito species examined, with different infection rates. Both supergroups A and B of Wolbachia strains were identified, with Aedes albopictus showing co-infection by both supergroups through the WGS approach. We also detected indirect evidence of Wolbachia horizontal transmission among mosquitoes and other distant host orders. This study provides valuable insights into the distribution and diversity of Wolbachia in sylvatic mosquitoes from Brazil and adds new important data about Wolbachia detection through target marker gene amplicon coupled with Sanger sequencing and WGS methods, highlighting its complementarity to ascertain the presence of Wolbachia in mosquito samples.

Genetic polymorphism of IL-40 in male patients with ankylosing spondylitis

Background: Ankylosing spondylitis (AS) is a kind of arthritis marked by persistent inflammation in the spine's joints, usually at the point where the spine and pelvis meet. Such a disease has different causes, including immunological causes. Therefore, it is necessary to investigate the role of the immune system in this disease to improve a potential treatment strategy. In this context, the present study examined the single nucleotide polymorphisms (SNPs) of the IL-40 gene of IL-40 in a group of Iraqi males with AS to determine their associations in AS pathogenesis. Method: The study included a total of 200 Iraqi male participants (100 with AS and 100 healthy subjects). The detection of SNPs of IL-40 was performed using polymerase chain reaction (PCR) techniques followed by the Sanger sequencing method. Multinomial logistic regression analysis was used to analyze the SNPs under five genetic models (allele, recessive, dominant, and co-dominant). Results: Detected that the heterozygous genotype (GA and AA) and alleles (G and A) of rs72896265, the heterozygous genotype (AG and GG) and alleles (A and G) of rs60316778 and both heterozygous genotype (CC) and alleles (C and T) of rs9915090 were significantly associated with AS risk (p &lt; 0.001).

Association Between the Risk of Dental Caries and DLX3 Gene Polymorphisms in Chinese Children.

To explore the association between the risk of dental caries and distal-less homeobox 3 (DLX3) gene in Chinese children. Based on the decayed, missing, and filled teeth (dmft) score, the children were divided into a control group (dmft &#61; 0) and a case group (dmft ≥ 1). DLX3 gene (rs11656951 and rs2278163) polymorphisms were genotyped by polymerase chain reaction (PCR) and Sanger sequencing methods. Possible association of DLX3 gene (rs11656951 and rs2278163) polymorphisms with dental caries risk was assessed using the chi-squared test. Subgroup analysis of association was assessed by logistic regression analysis for the potential risk factors. The age at which toothbrushing was started, the brushing frequency, brushing with fluoride toothpaste, and regular dental visits were statistically significantly different between case and control groups (p 0.05). The frequencies of rs11656951 TT genotype and T allele were statistically significantly higher in the control group than in the case group. The chi-squared test showed that CT genotype (p &#61; 0.026, OR &#61; 0.613, 95%CI &#61; 0.398-0.944) and TT genotype (p &#61; 0.001, OR &#61; 0.378, 95%CI &#61; 0.212-0.673) were negatively correlated with caries susceptibility. The T allele of rs11656951 was more frequently discovered in the control group, and was statistically significantly associated with decreased caries susceptibility (p &#61; 0.001, OR &#61; 0.636, 95%CI &#61; 0.486-0.831). The G allele of rs2278163 was obviously correlated with elevated caries susceptibility (p &#61; 0.049, OR &#61; 1.314, 95%CI &#61; 1.000-1.725). DLX3 gene rs11656951 TT genotype was a protective factor for caries susceptibility in the subgroups gender, sweets intake, eating before sleep, brushing frequency, brushing with fluoride toothpaste, and dental visits. The GG genotype of rs2278163 was a risk factor for caries in subgroups eating before sleep, brushing without fluoride toothpaste, and regular dental. The TT genotype of rs11656951 was dramatically correlated with reduced caries risk in low (p &#61; 0.004, OR &#61; 0.387, 95%CI &#61; 0.202-0.742) and moderate/high (p &#61; 0.016, OR &#61; 0.360, 95%CI &#61; 0.154-0.840) groups. DLX3 gene rs11656951 TT genotype is a protective factor and rs2278163 GG genotype is a risk factor for caries susceptibility, especially in low and moderate/high subgroups.

A TBC1D24 gene variant coincides with STRC compound heterozygosity in a family with hearing loss: a case report

BackgroundHearing loss is a common inborn neurosensory condition. Hearing loss is very heterogeneous, and while screening programs exist for children, adolescents and adults with late-onset hearing loss often do not get referrals to geneticists.ObjectiveTo diagnose the cause of hearing impairment in two related late-onset hearing loss patients—father and son—on a molecular level. Both underwent audiological examinations, and both had moderate hearing loss.Case presentation.We used massive parallel sequencing, Sanger sequencing, MLPA, and standard audiological methods. We identified an inherited autosomal dominant likely causative variant in the TBC1D24 gene of both patients. They did not show any other TBC1D24 spectrum-related symptoms. Furthermore, the younger patient was found to be compound heterozygous for two variants in STRC gene.ConclusionsOnly a few dozen TBC1D24 hearing loss patients have been reported. On the contrary, STRC is a common hearing loss cause. We speculate that in the younger patient, the phenotype is caused by a combination of effects of both genes. The older patient’s phenotype is more likely caused only by the TBC1D24 variant. We believe that more attention should be paid to adolescent and adult-onset hearing loss patients, and more frequent referrals to geneticists are warranted.

Fluorescence Quantitative PCR Detection of ABL1 Kinase Region Mutations

To establish a highly sensitive and quantitative detection method for ABL1 kinase region mutations, provide strong support for the early diagnosis and treatment of chronic myeloid leukemia(CML). Sampele from 35 CML patients who were initially tested negative for ABL1 kinase region mutations by Sanger sequencing were collected. The ABL1 kinase region mutation was detected by the fluorescence quantitative detection kit of Shanghai Yuanqi Biopharmaceutical Technology Co., Ltd. The mutation rate was analyzed by △△Ct value method. The relative mutation rate of the final ABL1 kinase region was determined by dividing the mutation rate by the expression level of the fusion gene. Among the 35 CML patients initially tested negative for ABL1 mutations by the Sanger sequencing method, 7 cases of T315I mutation, 2 cases of T315A mutation, 2 cases of Y253H mutation, and 1 cases of E255K mutation after detection of the new method. The relative mutation rates range from 0.1% to 19.42%, which could not be detected by Sanger sequencing method. Subsequently, this method was used to detect the ABL1 mutation in 126 CML patients, and the positive rate exceeded that of the Sanger sequencing method. The BCR-ABL1 gene expression significantly reduced or negative after adjusting treatment strategy based on the mutation situation. Compared with Sanger sequencing, fluorescence quantitative PCR has higher sensitivity and can screen for low-frequency ABL1 kinase mutations in the early stage. Moreover, it can also perform relative quantitative analysis, so the method has good clinical application prospects for detecting ABL1 mutation.

Unique among high passes: Insights into the genetic uniqueness among butterflies of Ladakh Trans-Himalaya through DNA barcoding.

The butterfly assemblage of Ladakh Trans-Himalaya demands a thorough analysis of their population genetic structure owing to their typical biogeographic affinity and their adaptability to extreme cold-desert climates. No such effort has been taken till date, and in this backdrop, we created a COI barcode reference library of 60 specimens representing 23 species. Barcodes were generated from freshly collected leg samples using the Sanger sequencing method, followed by phylogenetic clade analyses and divergence calculation. Our data represents 22% of Ladakh's Rhopaloceran fauna with the novel barcode submission for six species, including one Schedule II species, Paralasa mani. Contrary to the 3% threshold rule, the interspecific divergence between two species pairs of typical mountain genus Hyponephele and Karanasa was found to be 2.3% and 2.2%, respectively. The addition of conspecific global barcodes revealed that most species showed little increase in divergence value, while a two-fold increase was noted in a few species. Bayesian clade clustering outcomes largely aligned with current morphological classifications, forming monophyletic clades of conspecific barcodes, with only minor exceptions observed for the taxonomically complicated genus Polyommatus and misidentified records of Aulocera in the database. We also observed variations within the same phylogenetic clades forming nested lineages, which may be attributed to the taxonomic intricacies present at the subspecies level globally, mostly among Eurasian species. Overall, our effort not only substantiated the effectiveness of DNA Barcoding for the identification and conservation of this climatically vulnerable assemblage but also highlighted the significance of deciphering the unique genetic composition among this geographically isolated population of Ladakh butterflies.

Aspergillus aureoterreus, A. pseudonomiae, A. siamensis, and A. flocculosus-ochraceopetaliformis as Four New Pathogens of Eye Infection in DNA Barcoding Markers

Background: The use of a morphological method to determine Aspergillus species in routine laboratory investigation has always been challenged with low sensitivity and low specificity. Time-consuming specimen preparation and insufficient expertise are the crucial reasons. DNA barcoding markers have been introduced for identification as another gold standard method. Objective: To investigate DNA barcoding markers in identifying Aspergillus strains isolated from patients with eye infection. Methods: This study extracted 12 DNA samples of 12 Aspergillus strains. DNA barcoding genes, i.e., ITS, betatubulin (BT2), calmodulin (CAL), and actin (ACT) genes, were amplified and sequenced by polymerase chain reaction and Sanger's sequencing methods. All gene sequences were compared to those of the type or reference species collected from public databases. The species were identified with phylogenetic analyses in free applications from public websites. Results: ITS, BT2 and CAL, and ACT identified 5, 6, and 3 species and 3, 2, and 1 species complex of Aspergillus, respectively. DNA barcoding with multi-locus sequence typing revealed four new pathogens of human eye infection, including A. aureoterreus, A. pseudonomiae, A. siamensis, and A. flocculosus-ochraceopetaliformis. Conclusion: The DNA barcoding markers identified Aspergillus species with better specificity than the conventional method. The markers and techniques need to be used as tools for routine Aspergillus identification.

Molecular identification of lactic acid bacteria from traditional fermented foods and screening exopolysaccharide production by using food wastes.

In this study, lactic acid bacteria (LAB) isolation from fermented foods and molecular identification using magnetic bead technology were performed. And then exopolysaccharide (EPS) production possibility was tested in agar medium, and the positive ones were selected for the next step. The bacteria that could produce higher carbohydrate level were grown in MRS medium fortified with whey and pumpkin waste. In our study, 19 different LAB species were identified from fermented products collected from different places in Hatay(Türkiye) province. In molecular identification, universal primer pairs, p806R/p8FPL, and PEU7/DG74 were used for PCR amplification. After that, PCR products purified using paramagnetic bead technology were sequenced by the Sanger sequencing method. The dominant species, 23.8% of the isolates, were identified as Lactiplantibacillus plantarum. As a technological property of LAB, exopolysaccharide production capability of forty-two LAB isolate was tested in agar medium, and after eleven isolates were selected as positive. Two LAB (Latilactobacillus curvatus SHA2-3B and Loigolactobacillus coryniformis SHA6-3B) had higher EPS production capability when they were grown in MRS broth fortified with pumpkin waste and whey. The highest EPS content (1750mg/L glucose equivalent) was determined in Loigolactobacillus coryniformis SHA6-3B grown in MRS broth fortified with 10% pumpkin waste. Besides the produced EPS samples were validated with FTIR and SEM methods.

Генетически-фенотипический профиль детей с атипичным гемолитико-уремическим синдромом

Atypical hemolytic uremic syndrome (aHUS) is a life-threatening disease caused by dysregulation of the alternative complement pathway as a result of mutations in complement genes or the formation of antibodies (AB) to some of the complement factors. Identification of the gene mutations incidence and the corresponding phenotypic features in pediatric patients with aHUS in Russia is aimed at improving the diagnosis and prognosis of the disease as well as treatment tactics planning for patient management. The purpose of this research was to determine the patterns of progression, prognosis and outcomes of aHUS in children depending on their genetic profile. Materials and methods used: the Article represents data from a retro- and prospective analyses of the characteristics of the course of aHUS in 172 children depending on their genetic profile performed in Jan. 2012-Feb. 2022. The sporadic form of aHUS was diagnosed after exclusion of the other forms of thrombotic microangiopathy (TMA), the familial form (in cases with the familial records), the acquired (CFH-Ab HUS) in cases with AB increase to factor H. AutoAb against factor H was determined by enzyme-linked immunosorbent assay (ELISA-VIDITEST anti-complement factor H). The search for mutations (n=167) was carried out using Next-generation Sequencing (NGS) and Sanger sequencing methods. Results: the sporadic form of aHUS was diagnosed in 96.9% of cases, familial in 3.1% and CFH-Ab HUS in 23.8%. The genetic nature of aHUS was confirmed in 53.4% of cases. Pathogenic mutations were detected in 8.6% of cases, probably pathogenic in 42.8% and those with unclear clinical significance in 46.8%. The complement genes mutations were detected in 88.5% of cases and mutations of genes not related to the complement system (ADAMTS13, DGKE, INF2) in 11.5%. The most frequently identified mutations were in the CFH (17.1%), C3 (12.9%) and CD46 (12.9%) genes and less frequently in CFI (7.1%), THBD (5.7%), CFHR5 (5.7%) and CFB (1.4%). In children with CFH-Ab HUS, the CFHR1/CFHR3 deletion was detected in 87.8% of cases, including in 19.4% in combination with mutations of unknown clinical significance. Multiple organ failure syndrome had developed in 77.8% of cases in children with pathogenic/probably pathogenic mutations, in 85.3% in patients with mutations of unknown clinical significance, in 91.8% in patients without mutations and in 83.3% in patients with CFHR1/CFHR3 deletion. Chronic kidney disease (CKD) stages 3 to 5 in sporadic/familial aHUS, regardless of genetic profile, had counted for 14.5% and 16.7% in CFH-Ab HUS. Lethal outcome occurred in 4.3% of cases in the presence of mutations, in 4.9% in the absence of mutations and in 2.8% in cases of CFHR1/CFHR3 deletion. Relapses of aHUS prior to the Eculizumab treatment had developed more often in the presence of CFHR1/CFHR3 mutations and deletion in comparison with children without mutations (31.4% v. 13.1%, p=0.0313 and 44.4% v. 13.1%, p= 0.004). Relapse of aHUS after discontinuation of the Eculizumab therapy had developed in 16.4% of cases. Risk factors for the relapse of aHUS were as follows: patient’s age above 12 months old at the time of the development of aHUS, severe arterial hypertension in the acute period, the use of Eculizumab at the fourth week after the onset of the disease and/or later, recurrent course of aHUS and the presence of CKD prior to the start of the Eculizumab therapy. Conclusion: The genetic profile of patients does not determine the severity and outcome of aHUS though it does affect the relapse rate. The prognosis and survival of patients with aHUS are determined by the severity of the acute period and the timeliness of the complement blocking therapy management.

Association of fucosyltransferase 2 gene polymorphism with the susceptibility to norovirus infection in Han Chinese population.

Fucosyltransferase 2 (FUT2) gene, which regulates the formation of Histoblood group antigens, could determine the human susceptibility to norovirus. This study aimed to investigate the correlation between FUT2 gene polymorphism and susceptibility to norovirus gastroenteritis in Han Chinese population. A total of 212 children patients with acute gastroenteritis were enrolled. The stool and serum samples were collected respectively. We used the qPCR method to detect the norovirus infection status from the stool samples, and we used serum samples to detect the FUT2 polymorphism. A case-control study was conducted to investigate the three common SNPs polymorphisms (rs281377, rs1047781, and rs601338) of FUT2 gene with sanger sequencing method. The results indicated that the homozygous genotypes and mutant allele of rs1047781 (A385T) would downgrade the risk of norovirus gastroenteritis in Chinese Han population (AA vs. TT, odds ratio [OR] = 0.098, 95% confidence interval [CI] = 0.026-0.370, p = 0.001; AA + AT vs. TT, OR = 0.118. 95% CI = 0.033-0.424, p = 0.001; A vs. T, OR = 0.528, 95% CI = 0.351-0.974, p = 0.002). There were no significant difference of rs281377 (C357T) and rs601338 (G428A) polymorphisms between norovirus positive and norovirus negative groups (p > 0.05). The haplotype T-T-G was less susceptible (OR = 0.49, 95% CI = 0.31-0.79, p = 0.0034) to norovirus infection compared to other haplotypes. Our results investigated the relationship between the FUT2 gene polymorphisms and norovirus susceptibility in Han Chinese population, and firstly revealed that children with homozygous genotypes and mutant alleles of FUT2 rs1047781 (A385T) were less susceptible to norovirus gastroenteritis.

Exploring oral bacterial compositional network in two oral disease groups using a convergent approach of NGS-molecular diagnostics.

Since most of the commonly known oral diseases are explained in link with balance of microbial community, an accurate bacterial taxonomy profiling for determining bacterial compositional network is essential. However, compared to intestinal microbiome, research data pool related to oral microbiome is small, and general 16S rRNA screening method has a taxonomy misclassification issue in confirming complex bacterial composition at the species level. Present study aimed to explore bacterial compositional networks at the species level within saliva of 39 oral disease patients (Dental Caries group: n = 26 and Periodontitis group: n = 13) through comparison with public Korean-specific healthy oral microbiome data. Here, we applied comprehensive molecular diagnostics based on qRT-PCR and Sanger sequencing methods to complement the technical limitations of NGS-based 16S V3-V4 amplicon sequencing technology. As a result of microbiome profiling at the genus level, relative frequencies of many nitrate-reducing bacteria within each oral disease group were found to be significantly low compared to the healthy group. In addition, the molecular diagnostics-based bacterial identification method allowed the determination of the correct taxonomy of screened primary colonizers (Streptococcus and Actinomyces unclassification clusters) for each oral disease. Finally, as with the results of microbiome profiling at the genus level, many core-species classified within the saliva of each oral disease group were also related to nitrate-reduction, and it was estimated that various pathogens associated with each disease formed a bacterial network with the core-species. Our study introduced a novel approach that can compensate for the difficulty of identifying an accurate bacterial compositional network at the species level due to unclear taxonomy classification by using the convergent approach of NGS-molecular diagnostics. Ultimately, we suggest that our experimental approach and results could be potential reference materials for researchers who intend to prevent oral disease by determining the correlation between oral health and bacterial compositional network according to the changes in the relative frequency for nitrate-reducing species.

Mutation Analysis of Exon 1 in the Hemoglobin Subunit Beta (HBB) Gene in Beta-Thalassemia.

Introduction Thalassemia is a widely prevalent monogenic hematological disorder found worldwide. It exists in two forms: alpha- and beta-thalassemia. Alterations in the hemoglobin subunit beta (HBB) gene cause beta-thalassemia, with missense and point mutations affecting beta-globin synthesis. Consequently, genetic screening for beta-thalassemia is essential for genetic counseling, carrier screening, and prenatal diagnosis. Aim and objective This study aims to examine and identify mutations in the exon 1 region of the HBB gene in beta-thalassemia patients from the Vijayapura region. Methods This study involved 47 clinically diagnosed children with beta-thalassemia from a hospital in Vijayapura, India. Detailed clinical histories of all patients were recorded. Genomic DNA was extracted from the blood samples of these patients and subjected to polymerase chain reaction (PCR) using exon-specific primers for the HBB gene. The PCR products were then sequenced using the capillary-based Sanger sequencing method to identify mutations in the HBB gene. Results A total of 47 clinically diagnosed beta-thalassemia patients were included in the study, comprising 30 males and 17 females, aged between one and 20 years. Sequencing analysis of exon 1 in the beta-globin gene identified 17 beta-thalassemia variants. The most common mutation observed was T>G, G>C, C>A, and C>T in the exon 1 region of the HBB gene. Conclusion This study identifies the pattern of beta-thalassemia mutations, aiding in the prevention of the disorder through prenatal diagnosis and genetic counseling. Mutations can alter codon sequences, affecting protein production. Research highlights the importance of a primary prevention program to analyze mutations and sequence variations at the molecular level, thereby helping to address numerous genetic disorders.

Evidence of Trangency and Humanization in Mice Obtained at the SCBMT of FMBA of Russia by Sanger Sequencing Method

Evidence of Trangency and Humanization in Mice Obtained at the SCBMT of FMBA of Russia by Sanger Sequencing Method

Evaluation of the FPMC respiratory panel for detection of respiratory tract pathogens in nasopharyngeal swab and sputum specimens

ObjectivesThe performance of the new Respiratory Pathogen panel (fluorescent probe melting curve, FPMC) for the qualitative detection of 12 organisms (chlamydia pneumoniae, mycoplasma pneumoniae, adenovirus, influenza A virus, influenza B virus, parainfluenza virus, rhinovirus, etc.) was assessed.MethodsProspectively collected nasopharyngeal swab (NPS) and sputum specimens (n = 635) were detected by using the FPMC panel, with the Sanger sequencing method as the comparative method.ResultsThe overall percent concordance between the FPMC analysis method and the Sanger sequencing method was 100% and 99.66% for NPS and sputum specimens, respectively. The FPMC testified an overall positive percent concordance of 100% for both NPS and sputum specimens. The FPMC analysis method also testified an overall negative percent concordance of 100% and 99.38% for NPS and sputum specimens, respectively.ConclusionsThe FPMC analysis method is a stable and accurate assay for rapid, comprehensive detecting for respiratory pathogens.

Validation of a novel NGS based BCR::ABL1 kinase domain mutation detection assay in Indian cohort

The efficacy and treatment outcome of a CML patient are heavily dependent on BCR::ABL1 kinase domain (KD) mutation status. Next-generation sequencing technology is a bright alternative to the previously used sanger sequencing method due to its global presence in diagnostic setups, massive parallel sequencing ability, and far better sensitivity. In the present study, we have demonstrated a new protocol for kinase domain mutation analysis using the next-generation sequencing (NGS) method using the ion torrent sequencing platform. This protocol uses RNA as the starting material, followed by nested PCR to amplify the fusion transcript, which is subsequently used as a template for NGS. Initial validation and comparison of this assay with the sanger sequencing (SS) method yielded 95.23% agreement. CML samples (n = 121) with a failure to TKI response were subjected to this newly developed NGS-based assay to detect KD mutations, from which samples were found to have mutations with a sensitivity ranging from 2.32 to 93.41%. A total of 34.71% of samples (n = 42) were found to be positive for one or more KD mutations, whereas 65.29% of samples (n = 81) were found to be negative. Nine samples out of 42 positive samples, i.e., 21.42%, were found to have compound mutations. This is one of the first studies from India, which includes more than 160 samples and is analyzed by the NGS approach for KD mutation analysis.

Identification of single nucleotide variations in the AGPAT6 gene and its association with milk production traits in Murrah buffalo

The present study investigated the association of genetic variations within the 5′UTR exons 1-1511, 3′UTR exons 1512-1723 of the sn-1-acylglycerol-3-phosphate-O-acyltransferase 6 (AGPAT6) gene of buffalo with milk production traits, using the PCR-SSCP and Sanger sequencing method. The study revealed two unique conformational patterns: ‘1’ and ‘2’ in 100 Murrah buffaloes. Ten primer pairs were used for amplification of the AGPAT6 gene and in total nine single nucleotide polymorphisms (SNPs) were identified: viz., two in Exon 4 and seven in Exon8. The frequency of SSCP pattern 1 was found to be consistently high in the buffalo population under study. The preliminary results showed substantial variations in the distribution of SSCP variant frequencies of pattern 1 and 2 within Murrah buffaloes. However, these variants of exon4 and exon 8 had non-significant association with milk yield, whereas the same variants in terms of SNPs in the coding region were found to have a significant (P≤0.01) association with fat percentage. This suggests that AGPAT6 affects the milk fat percentage, which is an economically important trait, and that further study of this gene is warranted in Murrah buffaloes. The polymorphisms observed can be used as molecular markers to assist selection.

Мутации в генах KRAS/NRAS и эффективность индукционной терапии по трехкомпонентным схемам на основе бортезомиба у пациентов с впервые диагностированной множественной миеломой

AIM. To identify the KRAS and NRAS gene mutations in patients with newly diagnosed multiple myeloma (ММ) and to classify them according to the depth of antitumor response to bortezomib-based triplet induction therapy. MATERIALS &amp; METHODS. The trial enrolled 89 patients with newly diagnosed MM prior to chemotherapy. Among them, there were 45 women and 44 men aged 30–82 years (median 58.5 years). ММ was diagnosed according to IMWG criteria (2014). Bone marrow (BM) plasma cells were isolated from the aspirate using gradient method with subsequent immunomagnetic CD138 marker selection. The KRAS and NRAS gene mutations in BM CD138+ cells were identified with Sanger sequencing method. The proteomic programs MutationTaster, Polyphen2, and FATHMM-XF were used for mutation analysis in the KRAS and NRAS genes. All patients received bortezomib-based triplet chemotherapy as first-line treatment. The response depth was assessed after completing 6 cycles of PAD and VCD regimens. Antitumor response was evaluated according to IMWG (2016) criteria. RESULTS. The mutation rate in the gene family RAS was 42 % (37/89). The analysis focused on the data from 33 patients with mutations detected and response identified after 6 cycles of treatment. In 22 out of 33 patients, deep response was not achieved, whereas 11 patients showed complete remission (CR) + very good partial remission (VGPR). In the group of patients without mutations in the gene family RAS, the response to therapy meeting the CR + VGPR criteria was 64 % (27/42). The differences appeared to be significant (p = 0.008). The clinical data and the evaluation of primary treatment outcomes provided the basis for distinguishing a group of 9 prognostically unfavorable mutations: NRAS Gly13Asp, Gln61His; KRAS Gly12Ala, Gly12Asp, Gly12Val, Gly13Asp, Gln61Arg, Gln61His, and Ala146Val. CONCLUSION. The mutations in KRAS and NRAS belonging to the gene family RAS had a negative effect on the efficacy of the bortezomib-based triplet induction therapy. Mutation variants in the RAS family genes differed in prognostic significance. The analysis results helped to identify the mutation variants associated with the worse response to therapy: NRAS Gly13Asp, Gln61His; KRAS Gly12Ala, Gly12Asp, Gly12Val, Gly13Asp, Gln61Arg, Gln61His, and Ala146Val.