DETECTION OF SOMATIC MUTATIONS IN THE BRAF GENE BY PYROSEQUENCING

Introduction. Detection of somatic mutations in the BRAF gene can be used in clinical oncology to clarify the diagnosis, select therapy and assess the prognosis of the disease. Pyrosequencing technology makes it possible to identify both already known and new mutations, as well as to determine the mutant allele ratio in the sample.The aim of the study was to develop the pyrosequencing-based method for detecting mutations in 592–601 codons of the BRAF gene.Material and Methods. The nucleotide sequences were obtained using «PyroMark Q24» instrument. The sensitivity and specificity of the method were estimated using dilutions of plasmid DNA samples containing the intact BRAF gene fragment mixed with sequence containing one of the mutations V600E, V600R, V600K, V600M, and K601E. The clinical testing was performed on 200 samples from thyroid nodules.Results. The developed method makes it possible to determine samples containing 2 % of the mutant allele for mutations V600K and V600R, 3 % for V600E and V600M, and 10 % for K601E. The pyrogram signal values for samples without mutations ranged from 0 to 19.5 % for different mutations. An analysis algorithm was developed to confirm the presence and differentiation of mutations in the 600 codon at a low proportion of the mutant allele based on the signals ratio on the pyrogram. The 47 clinical samples with mutations were found, 45 with V600E and 1 with V600_K601>E, for one sample, the type of mutation in the 600 codon could not be determined. The proportion of the mutant allele was 3.5–45 %. The concentration of extracted DNA less than 10 copies per mkl was obtained in 47 samples, of which 8 samples were found to have the mutations.Conclusion. The pyrosequencing-based method was developed for the detection of somatic mutations in 592–601 codons of the BRAF gene. The technique provided sufficient sensitivity to detect frequent mutations in the 600 codon and allowed the detection of rare mutations. Extraction of DNA from clinical samples obtained by fine-needle aspiration biopsy in most cases provided a sufficient concentration of DNA, which made it possible to use the technique in combination with cytological analysis without additional sampling. This approach can be applied to determine somatic mutations in DNA fragments of same length for other oncogenes.

Amplicon-Based Analysis of the Fungal Diversity Across Kenyan Soda Lakes

BackgroundMicroorganisms have been able colonize and thrive in environments characterized by low/high pH, temperature, salt or pressure. Examples of extreme environments are the soda lakes and soda deserts. The objective of this study was to explore the fungal diversity across soda lakes Magadi, Elmenteita, Sonachi and Bogoria in Kenya. A new set of primers was designed to amplify a fragment long enough for the 454-pyrosequencing technology. Results Analysis of the amplicons generated showed that the new primers amplified for eukaryotic groups. A total of 153,634 quality-filtered, non-chimeric sequences were used for community diversity analysis. The sequence reads were clustered into 502 operational taxonomic Units (OTUs) at 97% similarity using BLASTn analysis of which 432 were affiliated to known fungal phylotypes and the rest to other eukaryotes. Fungal OTUs were distributed across 107 genera affiliated to the phylum Ascomycota, Basidiomycota, Glomeromycotina and Incertae Sedis. The Phylum Ascomycota was the most abundant phylotype. Overall, fifteen (15) genera (Chaetomium, Monodictys, Arthrinium, Cladosporium, Fusarium, Myrothecium, Phyllosticta, Coniochaeta, Diatrype, Sarocladium, Sclerotinia, Aspergillus, Preussia and Eutypa) accounted for 65.3% of all the reads. The Genus Cladosporium was detected across all the samples at varying percentages with the highest being water from Lake Bogoria (51.4%). Good’s coverage estimator values ranged between 97 and 100%, an indication that the dominant phylotypes were represented in the data. ConclusionThese results provide useful insights that can guide cultivation dependent studies in order to understand the physiology and biochemistry of the as yet uncultured taxa.

ITGA4 gene methylation status in chronic lymphocytic leukemia

Background: We aimed to investigate ITGA4 gene expression pattern and to explore its methylation heterogeneity in chronic lymphocytic leukemia (CLL). Patients & methods: Eighty one CLL patients and 75 healthy subjects were enrolled and prognostic evaluation of patients was assessed. ITGA4 q-realtime PCR was performed using Applied Biosystems, TaqMan gene expression assay. ITGA4 gene-specific CpG methylation was investigated in real time using pyrosequencing technology. Results: ITGA4 was differentially expressed in CLL patients. The CpG sites-1, 2 and 3 showed significantly higher mean levels than healthy controls (p = <0.001, 0.007 and 0.009). Significant association between CpG site-1 and CLL has been detected using age-adjusted logistic regression (p < 0.001). Conclusion: Hypermethylation at ITGA4 gene CpG sites (1,2,3) is a characteristic feature in CLL.

Catechol-O-methyltransferase gene promoter methylation as a peripheral biomarker in male schizophrenia

As an epigenetic modification, DNA methylation may reflect the interaction between genetic and environmental factors in the development of schizophrenia (SCZ). Catechol-O-methyltransferase (COMT) gene is a promising candidate gene of SCZ. In the present study, we investigate the association of COMT methylation with the risk of SCZ using bisulfite pyrosequencing technology. Significant association between DNA methylation of COMT and the risk of SCZ is identified (P = 1.618e−007). A breakdown analysis by gender shows that the significance is driven by males (P = 3.310e−009), but not by females. DNA methylation of COMT is not significantly associated with SCZ clinical phenotypes, including p300 and cysteine level. No interaction is found between COMT genotypes and the percent methylation of this gene. Receiver operating characteristic (ROC) curve shows that DNA methylation of COMT is able to predict the SCZ risk in males (area under curve [AUC] = 0.802, P = 1.91e−007). The current study indicates the clinical value of COMT methylation as a potential male-specific biomarker in SCZ diagnosis.

Microsatellite loci in the tiger shark and cross-species amplification using pyrosequencing technology

The tiger shark (Galeocerdo cuvier) has a global distribution in tropical and warm temperate seas, and it is caught in numerous fisheries worldwide, mainly as bycatch. It is currently assessed as near threatened by the International Union for Conservation of Nature (IUCN) Red List. In this study, we identified nine microsatellite loci through next generation sequencing (454 pyrosequencing) using 29 samples from the western Atlantic. The genetic diversity of these loci were assessed and revealed a total of 48 alleles ranging from 3 to 7 alleles per locus (average of 5.3 alleles). Cross-species amplification was successful at most loci for other species such asCarcharhinus longimanus, C. acronotusandAlopias superciliosus. Given the potential applicability of genetic markers for biological conservation, these data may contribute to the population assessment of this and other species of sharks worldwide.

Microsatellite loci in the tiger shark and cross-species amplification using pyrosequencing technology

The tiger shark (Galeocerdo cuvier) has a global distribution in tropical and warm temperate seas, and is caught in numerous fisheries worldwide, mainly as bycatch. It is currently assessed as near threatened by the International Union for Conservation of Nature (IUCN) Red List. In this study we identified 9 microsatellite loci through next generation sequencing (454 pyrosequencing) using 29 samples from the western Atlantic. The genetic diversity of these loci was assessed and revealed a total of 48 alleles ranging from 3 to 7 alleles per locus (average of 5.3 alleles). Cross-species amplification was assessed in three other species: Carcharhinus longimanus, C. acronotus and Alopias superciliosus. Given the potential applicability of genetic markers for biological conservation, these data may contribute to the population assessment of this and other species of sharks worldwide.

Microsatellite loci in the tiger shark and cross-species amplification using pyrosequencing technology

The tiger shark (Galeocerdo cuvier) has a global distribution in tropical and warm temperate seas, and is caught in numerous fisheries worldwide, mainly as bycatch. It is currently assessed as near threatened by the International Union for Conservation of Nature (IUCN) Red List. In this study we identified 9 microsatellite loci through next generation sequencing (454 pyrosequencing) using 29 samples from the western Atlantic. The genetic diversity of these loci was assessed and revealed a total of 48 alleles ranging from 3 to 7 alleles per locus (average of 5.3 alleles). Cross-species amplification was assessed in three other species: Carcharhinus longimanus, C. acronotus and Alopias superciliosus. Given the potential applicability of genetic markers for biological conservation, these data may contribute to the population assessment of this and other species of sharks worldwide.