A Method for Isolating Tumor Cells from Large Volume of Malignant Pleural Effusion and Its Efficacy Evaluation / 中国肺癌杂志

BACKGROUND@#Malignant plural effusion (MPE) is one of the most common specimen for liquid biopsy gene detection. This study aims to explore a method for isolating tumor cells from large volume of MPE and evaluate its efficacy and application prospect in gene detection.@*METHODS@#Pleural effusions (>500 mL) from 20 advanced lung cancer patients were obtained by effusion drainage and used to isolate tumor cells with cell separation media Percoll and Ficoll. Cell number and purity were calculated. DNA was extracted from the supernatant (etDNA), total cells and isolated tumor cells of pleural effusion (ETC-DNA) to detect the mutation of tumor-related genes by next-generation sequencing.@*RESULTS@#The median number of cells isolated from malignant pleural effusion was 8.50×10⁴ (interquel range: 9.25×10³-3.75×10⁵), 85.50%±5.80% of the cells were identified as tumor cells. The detection rates of epidermal growth factor receptor (EGFR) gene mutation of etDNA, total cell DNA and ETC-DNA were 70.00%, 50.00% and 70.00%, reseparately, while the median EGFR mutation abundance in 3 components was 16.05% (4.78%-43.06%), 1.09% (0.00%-2.39%), and 33.02% (18.50%-76.70%), respectively. ETC-DNA had good consistency with tissue DNA (P>0.999, kappa=1.000) and etDNA (P>0.999, kappa=1.000). ETC-DNA inclined to have higher EGFR mutation than etDNA, but the result was not statistically significant.@*CONCLUSIONS@#Our method can isolate large amount of tumor cells from a large volume of malignant pleural effusion with high purity. Using ETC-DNA as specimen improves the efficacy of gene detection, thus is worth further study.

Establishment of a novel TB-ARMS qPCR method for kras mutation detection / 生物工程学报

A simple, robust and highly sensitive TB-ARMS method based on qPCR technique was developed to detect kras mutations. The technique was evaluated, and its clinical application was investigated. Mutation specific primers for eight common kras mutations and wild type gene targeted blockers were designed and optimized. Moreover, a mutant-enriched condition was used in to improve the sensitivity and specificity of mutation detection. Constructed plasmids carrying mutant kras genes, as well as confirmed wild type genomic DNA, were used as standard samples for evaluation of the methodology. The performance of our new method was validated by comparing the results of our method with that of a commercial kras kit in testing 40 clinical samples. Preoperative plasma samples, as well as paired tissue samples, were tested in parallel for evaluation of its clinical application. We have developed a new TB-ARMS method for kras mutation detection that can detect minor mutant alleles with a frequency as low as 0.01% in a heterogeneous sample. We have successfully demonstrated its 0.01% detection sensitivity with highly specific mutant amplification in conjunction with selective wild type suppression by blocker under a mutant-enriched reaction condition. We also showed that our TB-ARMS method was more accurate than the commercial kras kit, which is widely used presently. Furthermore, we have validated our method as an efficient liquid biopsy method, and the results of the plasma DNA detection with our TB-ARMS method were in consistent with the sequencing results of paired tissue samples. In conclusion, our TB-ARMS qPCR method could be effectively applied in kras mutation test for clinical tissue samples, as well as for liquid biopsy samples such as plasma.

Use of uracil-DNA glycosylase enzyme to reduce DNA-related artifacts from formalin-fixed and paraffin-embedded tissues in diagnostic routine

Background: Detection of somatic mutations is a mandatory practice for therapeutic definition in precision oncology. However, somatic mutation detection protocols use DNA from formalin-fixed and paraffin-embedded (FFPE) tumor tissues, which can result in detection of nonreproducible sequence artifacts, especially C:G > T:A transitions, in DNA. In recent studies, DNA pretreatment with uracil DNA glycosylase (UDG), an enzyme involved in base excision repair, significantly reduced the number of DNA artifacts after mutation detection by next-generation sequencing (NGS) and other methods, without affecting the capacity to detect real mutations. This study aimed to evaluate the effects of UDG enzymatic pretreatment in reducing the number of DNA sequencing artifacts from FFPE tumor samples, to improve the accuracy of genetic testing in the molecular diagnostic routine. Methods: We selected 12 FFPE tumor samples (10 melanoma, 1 lung, and 1 colorectal tumor sample) with different storage times. We compared sequencing results of a 16-hotspot gene panel of NGS libraries prepared with UDG-treated and untreated samples. Results: All UDG-treated samples showed large reductions in the total number of transitions (medium reduction of 80%) and the transition/transversion ratio (medium reduction of 75%). In addition, most sequence artifacts presented a low variant allele frequency (VAF < 10%) which are eliminated with UDG treatment. Conclusion: Including UDG enzymatic treatment before multiplex amplification in the NGS workflow significantly decreased the number of artifactual variants detected in FFPE samples. Thus, including this additional step in the current methodology should improve the rate of true mutation detection in the molecular diagnostic routine.

Applicable Value of AMSS-PCR in Lung Cancer Gene Mutation Detection / 中国肺癌杂志

BACKGROUND@#The detection of driver oncogenes of lung cancer is of great importance. There are various gene detection techniques nowadays which are different from each other. We carried out this study to investigate the specificity and sensitivity of assay panels based on an Amplification Refractory Mutation System-polymerase chain reaction (ARMS-PCR) technique of Amplification Mutation Specific System (AMSS) in detection of lung cancer gene mutation. To estimate the applicable value of assay panels in clinical settings.@*METHODS@#We collected cancer tissue specimens or fluid specimens from 309 patients. Mutation results were presented for those samples previously detected by ARMS-PCR. In comparison, we carried out AMSS-PCR using (epidermal growth factor receptor, EGFR) assay panel and Six-Alliance assay panel as well as Sanger sequencing. Software SPSS 22.0 (SPSS IBM) was used for statistical analysis.@*RESULTS@#The rates of consistency between the results by assay panels and Sanger sequencing or ARMS-PCR were 97.41% and 97.73%, respectively. Besides, EGFR assay panel had higher consistency rates with other detection methods than Six-Alliance assay panel. As for consistency test, the Kappa values of assay panels with Sanger sequencing, assay panels with ARMS-PCR, and ARMS-PCR with Sanger sequencing were 0.946, 0.953, and 0.913, respectively. The receiver operating characteristic curve (ROC) area under curve (AUC) of assay panels was 0.976 referring to Sanger sequencing, and 0.975 as ARMS-PCR was referred to.@*CONCLUSIONS@#AMSS-PCR can make an optimal cancer gene mutation detection method for clinical settings.

Progress in digital PCR technology and application / 生物工程学报

Digital PCR is an emerging analysis technology for absolute quantification after realtime-PCR. Through digital PCR, single DNA molecules are distributed into isolated reactions, and the product with fluorescence signal can be detected and analyzed after amplification. With the advantages of higher sensitivity and accuracy, digital PCR, independent of a standard curve, is developing rapidly and applied widely to the next generation sequencing and detection fields, such as gene mutation, copy number variation, microorganism, and genetically modified food. In this article, we reviewed the quantitative method and research progress of digital PCR technology in the main application fields.

Molecular analysis of exons 6 and 7 of phenylalanine hydroxylase gene mutations in Phenylketonuria patients in Western Iran.

Background: Phenylketonuria (PKU) is an inborn error of amino acid metabolism that results from a deficiency of phenylalanine hydroxylase (PAH). According to PAH database, exons 6 and 7 and their flanking introns of PAH gene contain the greatest number of mutant alleles. Therefore, as a preliminary study, nucleotide sequence analysis of exons 6 and 7 of the PAH gene has been performed in 25 PKU patients whose ancestors lived in Kermanshah province of Iran. To date, there has been no mutation data describing the genotypes of the PKU disease in this Kurdish ethnic region background. Materials and Methods: Twenty-five patients (aged between 2 and 23 years) participated in this study. The DNA fragments containing two exons of the PAH gene [6 and 7] and their exon-flanking intronic sequences were amplified and sequenced. Results: The total of detected mutations were R261X (8%), R176X (4%), R243Q (4%), R243X (2%) and R261Q (2%), as they accounted for 20% of all mutant alleles in this study. The identified polymorphisms are: IVS5 -54 G > A (22%), Q232Q (8%) and V245V (4%). All of the detected mutations in this study are related to CpG dinucleotides in the PAH gene sequence. Conclusion: The frequency of R261X, the most common mutation in our study, in Iranian population is <5%. Furthermore, there is no report of detection of R176X and R243Q in Isfahan and Azeri Turkish populations. These findings confirm the common Mediterranean mutations in this local population, although with more or lower frequencies than those reported in other related studies in Iran. Therefore, it may be necessary to study the PAH gene mutations in other provinces of Iran separately.

Common N-acetylgalactosamine-6-sulfate sulfatase (GALNS) exon mutations in Brazilian patients with mucopolysaccharidosis IVA (MPS IVA)

Morquio A Syndrome (mucopolysaccharidosis IVA - MPS IVA, OMIM# 253000) is an autosomal recessive inborn error of metabolism caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). We investigated five unrelated Brazilian MPS IVA families for mutations in exons 4, 5, 9 and 10 of the GALNS gene. Six out of the 10 mutant alleles were identified. Taken together with a previous study, which included six unrelated families, common mutations among Brazilian patients were p.N164T, p.G116S and p.G301C. Among one hundred control subjects three novel silent mutations were found (p.A107A; GCC -> GCT, p.Y108Y; TAC -> TAT, p.P357P; CCG -> CCA). Screening starting with exons 4, 5, 9, 10 and 11 may be a good strategy for genotyping of Brazilian patients since these exons include 73 percent of all mutations identified in the current and previous studies.

Gene chip and its use in the detection of mutations / 医学研究生学报

Gene chip is an important biological technology, which is developing quickly recently. In this article, we reviewed the origin, development, basic operation of gene chip. Several kinds of gene chips including tiled array, direct allele specific fluorescence targeting (DAFT) array, labeled auxiliary oligonucleotides array, ligase detection reaction array, and peptide nucleic acid array were introduced as examples for their use in mutation detection. The prospect of the use of gene chip in optimizing drug therapy was also discussed.

Detection of Gene Mutation and Relationship between Phenotype and Genotype in Four Chinese Families with Osteogenesis Imperfecta / 实用儿科临床杂志

T mutation was detected in the 4th propositus at the 9th intron,but any COL1A1 or COL1A2 gene mutation was detected in the third propositus and the other members in the former families.Conclusions The genetic mutation of COL1A1 may result in OI in China,but other mutations may also exist.Moreover,the phenotype was influenced not only by OI genotype,but also by the genetic background,environment and other factors.

Connexin 26 and autosomal recessive non-syndromic hearing loss.

Prelingual deafness occurs with a frequency of 1 in 1000 live births and is divided into syndromic and non-syndromic forms contributing 40 and 60% respectively. Autosomal recessive non-syndromic hearing loss (ARNSHL) is responsible for 80% cases of childhood deafness. Nearly all genes localized for ARNSHL cause prelingual, severe to profound, sensorineural hearing impairment. ARNSHL is genetically heterogeneous and at least 39 loci have been identified. The most significant finding to date has been the discovery of mutations in GJB2 gene at the DFNB1 locus on chromosome 13q12 as the major cause of profound prelingual deafness. This was first reported in a Tunisian family in 1994 and thereafter in many different countries. GJB2 gene encodes the gap-junction protein, connexin 26 (Cx26), mutations in which have become the first genetic marker of inherited hearing loss. Allele-specific polymerase chain reaction (AS-PCR), single stranded conformation polymorphism (SSCP) and sequencing methods have been developed for the detection of mutations in Cx26 gene. In India as well, the Cx26 mutations are being screened in families with hearing impaired children using these molecular methods. Therefore, in order to create awareness among the clinicians and the affected families; we have attempted to review the Cx26 gene mutations responsible for autosomal recessive type of non-syndromic hearing loss. The efficacy and utility of Cx26 gene analysis might open the path to proper counseling of families for carrier detection and prenatal diagnosis. It may even facilitate the development of strategies in future for the treatment of this common genetic disorder.