Rate Of Gene Deletion Research Articles

GST polymorphism as a predictive biomarker for modulating the susceptibility to chronic obstructive pulmonary disease: A North Indian study.

Chronic obstructive pulmonary disease (COPD) is commonly characterized by shortness of breath, coughing or expectoration. Smoking is the leading cause of COPD development, but only a small percentage of smokers develop symptoms, implying a genetic component. Glutathione S-transferase enzymes are responsible for detoxifying cigarette smoke components. The role of glutathione S-transferase T1 (GSTT1) and glutathione S-transferase M1 (GSTM1) gene polymorphism was assessed with COPD susceptibility and associated clinical parameters in the North Indian population. This was a cross-sectional study involving 200 COPD patients and 200 healthy individuals, with peripheral blood sampling and adequate questionnaires. Multiplex PCR was used for genotyping GSTT1 and GSTM1 gene polymorphism. Logistic regression was used to calculate the odds ratio and 95% confidence intervals to assess the COPD risk and GST polymorphisms. The GSTT1 gene deletion rate was higher in COPD cases (34.5%) than in healthy individuals (20.5%). A statistical relationship between the GSTT1(-) null genotype and COPD risk was observed (odds ratio=2.04, 95% CI=1.30-3.20, P=0.0019). After adjusting for covariates like age, sex and smoking status, a significant association was found for GSTT1(-) null genotype and COPD risk (adjusted odds ratio=2.90, 95% CI=1.43-5.87, P=0.003). The GSTT1(-) genotype was also significantly correlated with clinical parameters for COPD risk. Another primary observation was that females with the GSTT1(-) null genotype were more vulnerable to COPD than males with the same gene deletion. The GSTT1(-) null genotype strongly correlates with COPD development, while no association was observed in the GSTM1(-) null genotype in the North Indian population.

UroVysion™ fluorescence in situ hybridization (FISH) possibly has a high positive rate in carcinoma of non-urothelial lineages.

Background: Positive UroVysion™ fluorescence in situ hybridization (FISH) is generally considered as urothelial carcinoma (UC). We clarify if UroVysion™ FISH can be positive in carcinoma of non-urothelial lineages (CNUL), and verify the consistency of urine FISH and histological FISH in CNUL. Methods: All CNUL subjects detected by urine FISH assay due to haematuria from Tongji Hospital were screened. Meanwhile, 2 glandular cystitis and 2 urothelial carcinoma were served as negative or positive control. Paraffin-embedded tissue sections of all subjects were sent to the pathology department for histological FISH detection. Results: A total of 27 patients were included in this study, including 9 with adenocarcinomas, 11 with squamous cell carcinomas, and 7 with other tumour types. The overall positive rate in urine FISH was 64.00% (16/25) in patients with CNUL, 77.78% (7/9) in those with adenocarcinoma and 54.55% (6/11) in those with squamous carcinoma. There was a significant difference in the GLP p16 gene deletion rate between UC and CNUL (100% vs. 8.00%, p = 0.017). Histological FISH results showed that the histological results of 19 patients were consistent with their urine FISH results, and only one patient with stage Ⅲa urachal carcinoma had inconsistent histological FISH results (positive) and urine FISH (negative) results. Conclusion: We demonstrated for the first time the application value of FISH in CNUL on urine samples. Positive urine FISH tests indicate not only UC, but also CNUL. UroVysion™ FISH possibly has a high positive rate in CNUL. CNUL and UC have different genetic changes shown by FISH.

Evaluation of UroVysion for Urachal Carcinoma Detection.

Background: Patients with hematuria who are positive for urinary fluorescence in situ hybridization (FISH) are generally considered to have urothelial carcinoma. We determined whether UroVysion FISH could be used for the diagnosis of urachal carcinoma.Methods: Seven cases of urachal carcinoma with haematuria subjected to FISH analysis were retrospectively analyzed in our hospital from May 2012 to November 2019. Paraffin-embedded tissue sections from one FISH-positive and one FISH-negative urachal carcinoma were processed in strict accordance with the instructions of the UroVysion kit. Meanwhile, FISH data from the other 414 hematuria patients were collected as controls.Results: All 7 patients with urachal carcinoma were diagnosed with adenocarcinoma. According to Sheldon stage, six patients had stage IIIa and one patient had stage IVb. The sensitivity and specificity of urinary FISH for the diagnosis of urachal carcinoma were 71.43% (5/7) and 94.61% (281/297), respectively. The rates of polysomy for chromosomes 3 and 7 in positive patients were both 100% (5/5), whereas the rate of polysomy for chromosome 17 was 40% (2/5), and the chromosome 9p21 region (p16) gene deletion rate was 20% (1/5). Histological assessment and cytological FISH were consistent for urachal carcinoma. No significant difference was observed in the diagnostic efficacy between urachal carcinoma and urothelial carcinoma (71.43 vs. 87.18%, P = 0.245).Conclusions: Taken together, UroVysion FISH was found to be positive in a high proportion of pathologically confirmed urachal carcinoma of late stage with hematuria. Its chromosomal aberrations may be different from those of urothelial carcinoma, but more studies are needed to clarify their genetic background. Not all tumors showing abnormalities by FISH are urothelial carcinomas.

Association between TP53 gene deletion and protein expression in esophageal squamous cell carcinoma and its prognostic significance.

The aim of the present study was to investigate the association between tumor protein 53 (TP53) gene deletion and protein expression and clinical features in esophageal squamous cell carcinoma (ESCC), and to evaluate the predictive value of these two characteristics in the prognosis of ESCC. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were performed to detect the expression of p53 protein and gene deletion in ESCC tissue samples from different ethnic groups in Xinjiang, in order to analyze their association with clinicopathological characteristics and patient prognosis, as well as the sensitivity and specificity of the two methods. In addition, the results were further validated by tissue microarray from a different region. The positive rate of p53 protein expression was 54.5% (201/369) in the multi-ethnic group, and was significantly different between sex (P=0.026) and between tumor differentiation groups (P=0.032). FISH demonstrated that the TP53 gene deletion rate was 31.8% (68/214), which was significantly different between different tumor differentiation (P=0.002), lymph node metastasis (P=0.005) and vascular invasion (P<0.001) groups. The survival rate of patients with TP53 gene deletion was significantly lower than those without TP53 gene deletion (P<0.05). The positive rate of p53 protein expression in the tissue microarray was 58.1% (68/117), which was significantly different between the depth of invasion groups (P=0.011). The TP53 gene deletion rate was 47.9% (56/117), which significantly differed according to lymph node metastasis (P=0.003) and TNM stage (P=0.01). In addition, the total concordance rates of the two methods were 60.3 and 64.1%, respectively. There were also significant differences in the positive rate of TP53 gene deletion and protein expression in different stages of ESCC (P<0.05), which increased gradually with the progression of ESCC. The deletion of the TP53 gene in esophageal cancer was associated with poor prognosis and may be an important biomarker for evaluating the prognosis of patients with ESCC. The combination of FISH and IHC methods could significantly improve the detection rate of TP53 gene abnormalities and the accuracy of prognostic assessment of ESCC.

Correlation of methylthioadenosine phosphorylase (MTAP) loss with response to anti-folate therapy in urothelial bladder carcinoma (UBC).

4521 Background: The MTAP gene encodes an essential enzyme for the salvage pathway of nucleotide synthesis and is frequently deleted in UBC. Anti-folate agents such as pemetrexed can effectively inhibit the de novo pathway of nucleotide synthesis and as a result, create a synthetic lethality in MTAP deficient UBC. We hypothesize that MTAP gene loss correlates with enhanced response to pemetrexed in UBC. Methods: We investigated MTAP gene deletion rates in the TCGA database and determined MTAP protein loss rates by immunohistochemistry (IHC) using a UBC tissue microarray (TMA) from 151 patients (pts). We then performed in vitro and in vivo studies using MTAP proficient and MTAP deficient bladder cancer cell lines. At the clinical level, we performed a retrospective analysis based on MTAP status of pts treated with pemetrexed as 2nd line at our institution between 2014 and 2018. We are now enrolling pts in a single-arm, open-label, phase II clinical trial (NCT02693717) with pemetrexed in pts with MTAP deficient UBC. Results: Per our TCGA and TMA IHC analyses, MTAP deficiency rate was 25.9% and 27.8%, respectively. MTAP deficient UBC cell lines were at least 40 times more sensitive to pemetrexed than MTAP proficient lines. Knockdown of the MTAP gene increased apoptosis rate by pemetrexed from approximately 20% to 60%. Additionally, pemetrexed significantly inhibited the growth of MTAP deficient or knockdown xenograft tumors but not MTAP proficient tumors. Retrospective analysis of 12 pts using RECIST criteria indicated that all 4 MTAP deficient UBC pts responded to pemetrexed whereas only 1 of 8 (12.5%) MTAP proficient UBC pts responded. Of the 6 pts enrolled on the clinical trial, 3 (50%) had complete or partial response, 1 had stable disease, 1 was not evaluable and 1 had disease progression. Combined analysis of the entire experience demonstrates a higher response rate in MTAP deficient UBC (70%) as compared to MTAP proficient UBC (12.5%). Conclusions: Our preclinical and clinical data demonstrate that MTAP loss in UBC leads to a state of synthetic lethality when treated with pemetrexed and should be further investigated as a novel biomarker to predict response to anti-folate agents.

Mutational and transcriptional landscape of spontaneous gene duplications and deletions in Caenorhabditis elegans

Gene duplication and deletion are pivotal processes shaping the structural and functional repertoire of genomes, with implications for disease, adaptation, and evolution. We employed a mutation accumulation (MA) framework partnered with high-throughput genomics to assess the molecular and transcriptional characteristics of newly arisen gene copy-number variants (CNVs) in Caenorhabditis elegans populations subjected to varying intensity of selection. Here, we report a direct spontaneous genome-wide rate of gene duplication of 2.9 × 10-5/gene per generation in C. elegans, the highest for any species to date. The rate of gene deletion is sixfold lower (5 × 10-6/gene per generation). Deletions of highly expressed genes are particularly deleterious, given their paucity in even the N = 1 lines with minimal efficacy of selection. The increase in average transcript abundance of new duplicates arising under minimal selection is significantly greater than twofold compared with single copies of the same gene, suggesting that genes in segmental duplications are frequently overactive at inception. The average increase in transcriptional activity of gene duplicates is greater in the N = 1 MA lines than in MA lines with larger population bottlenecks. There is an inverse relationship between the ancestral transcription levels of new gene duplicates and population size, with duplicate copies of highly expressed genes less likely to accumulate in larger populations. Our results demonstrate a fitness cost of increased transcription following duplication, which results in purifying selection against new gene duplicates. However, on average, duplications also provide a significant increase in gene expression that can facilitate adaptation to novel environmental challenges.

Effects of Glutathione S-Transferase Gene Polymorphisms and Antioxidant Capacity per Unit Albumin on the Pathogenesis of Chronic Obstructive Pulmonary Disease.

Objectives To study the effects of GSTM1, GSTT1 gene polymorphisms, and organism antioxidant capacity and related indicators such as antioxidant capacity per unit of albumin (AC/ALB) on chronic obstructive pulmonary disease (COPD). Methods Using polymerase chain reaction technology, GSTM1 and GSTT1 gene polymorphisms were detected in 33 COPD patients and 33 healthy people. The total antioxidant capacity (TAC) found in serum was determined using the I2/KI potentiometric, KMnO4 microtitration, and H2O2 potentiometric methods. The AC/ALB was defined as the TAC divided by the serum albumin concentration. Logistic regression analysis was carried out with biochemical screening indices, which was found to be closely related with the incidence of COPD. Results The GSTM1 and GSTT1 gene deletion rate in the COPD group was significantly higher than that in the control group (P < 0.05). The differences in serum TAC between the COPD and control groups, GSTM1 (+) and GSTM1 (−) groups, and GSTT1 (+) and GSTT1 (−) groups were statistically significant (P < 0.001). In addition, there was a significant difference in the AC/ALB between the COPD and control groups (P < 0.05). Logistic regression analysis showed that the incidence of COPD was closely related to the AC/ALB (P < 0.05). Conclusions GSTM1 and GSTT1 gene polymorphisms are closely correlated with the pathogenesis of COPD, while the AC/ALB plays a decisive role in the occurrence and development of COPD.

Gene Deletion by Synthesis in Yeast.

Targeted gene deletion is a useful tool for understanding the function of a gene and its protein product. We have developed an efficient and robust gene deletion approach in yeast that employs oligonucleotide-based gene synthesis. This approach requires a deletion cassette composed of three modules: a central 1397-bp KanMX4 selection marker module and two 366-bp gene-specific flanking modules. The invariable KanMX4 module can be used in combination with different pairs of flanking modules targeting different genes. The two flanking modules consist of both sequences unique to each cassette (chromosomal homologous regions and barcodes) and those common to all deletion constructs (artificial linkers and restriction enzyme sites). Oligonucleotides for each module and junction regions are designed using the BatchBlock2Oligo program and are synthesized on a 96-well basis. The oligonucleotides are ligated into a single deletion cassette by ligase chain reaction, which is then amplified through two rounds of nested PCR to obtain sufficient quantities for yeast transformation. After removal of the artificial linkers, the deletion cassettes are transformed into wild-type diploid fission yeast SP286 cells. Verification of correct clone and gene deletion is achieved by performing check PCR and tetrad analysis. This method with proven effectiveness, as evidenced by a high success rate of gene deletion, can be potentially applicable to create systematic gene deletion libraries in a variety of yeast species.

Chemical Inhibitors of Non-Homologous End Joining Increase Targeted Construct Integration in Cryptococcus neoformans.

The development of a biolistic transformation protocol for Cryptococcus neoformans over 25 years ago ushered in a new era of molecular characterization of virulence in this previously intractable fungal pathogen. However, due to the low rate of homologous recombination in this species, the process of creating targeted gene deletions using biolistic transformation remains inefficient. To overcome the corresponding difficulty achieving molecular genetic modifications, members of the Cryptococcus community have investigated the use of specific genetic backgrounds or construct design strategies aimed at reducing ectopic construct integration via non-homologous end joining (NHEJ). One such approach involves deletion of components of the NHEJ-associated Ku heterodimer. While this strategy increases homologous recombination to nearly 100%, it also restricts strain generation to a ku80Δ genetic background and requires subsequent complex mating procedures to reestablish wild-type DNA repair. In this study, we have investigated the ability of known inhibitors of mammalian NHEJ to transiently phenocopy the C. neoformans Ku deletion strains. Testing of eight candidate inhibitors revealed a range of efficacies in C. neoformans, with the most promising compound (W7) routinely increasing the rate of gene deletion to over 50%. We have successfully employed multiple inhibitors to reproducibly enhance the deletion rate at multiple loci, demonstrating a new, easily applied methodology to expedite acquisition of precise genetic alterations in C. neoformans. Based on this success, we anticipate that the use of these inhibitors will not only become widespread in the Cryptococcus community, but may also find use in other fungal species as well.

TP53 gene deletion in esophageal cancer tissues of patients and its clinical significance

The aim of this study was to examine TP53 gene deletion in esophageal cancer (EC) tissues obtained from patients and to evaluate its clinical significance. Forty surgical specimens from patients with esophageal squamous cell carcinoma were examined for TP53 gene deletion using the fluorescence insitu hybridization (FISH) technique. Thirty-two male and 8female patients were enrolled, with an average age of 56years. TP53 gene deletion was significantly higher in poorly-differentiated EC cases compared to well-differentiated cases (P=0.028). The TP53 gene deletion rate was also significantly higher in the group with lymph node metastasis compared to the group without lymph node metastasis (P=0.0313). The TP53 gene deletion rate was shown to be correlated with the level of differentiation and lymph node metastasis in EC; it may therefore be an important molecular marker for evaluating the condition of EC in patients.

Association of mutation types and distribution characteristics of dystrophin gene with clinical symptoms in Chinese population

Duchenne muscular dystrophy (DMD) is X-linked disorder caused by mutations in the dystrophin gene. To investigate mutation types and distribution characteristics of dystrophin gene in Chinese DMD patients, we used Multiplex Ligation-Dependent Probe Amplification (MLPA) to analyze the dystrophin gene in 720 DMD patients, their mothers, and 20 normal adult males. Results showed that detection rate was 64.9% (467/720) in all the patients, gene deletion rate was 54.3% (391/720), and gene duplication rate was 10.6% (76/720). The rate of deletion mutant occurred in Exon 45-54 was 71.9% (281/391) in all gene deletion patients; meanwhile, the rate of gene duplication occurred in Exon 1-40 was 82.9% (63/76) in all gene duplication ones. In all the patients with gene deletion and duplication, the rate of DMD and IMD was 90.6% (423/467), and BMD, 9.4% (44/467). This indicates that the main reason of duchenne muscular dystrophy is dystrophin gene deletion mutation, which would occur in any gene unevenly with hot spots of mutation. The location and fragment length of gene deletion and duplication cannot decide the severity of clinical symptoms directly.

Gene Space Dynamics During the Evolution of Aegilops tauschii, Brachypodium distachyon, Oryza sativa, and Sorghum bicolor Genomes

Nine different regions totaling 9.7 Mb of the 4.02 Gb Aegilops tauschii genome were sequenced using the Sanger sequencing technology and compared with orthologous Brachypodium distachyon, Oryza sativa (rice), and Sorghum bicolor (sorghum) genomic sequences. The ancestral gene content in these regions was inferred and used to estimate gene deletion and gene duplication rates along each branch of the phylogenetic tree relating the four species. The total gene number in the extant Ae. tauschii genome was estimated to be 36,371. The gene deletion and gene duplication rates and total gene numbers in the four genomes were used to estimate the total gene number in each node of the phylogenetic tree. The common ancestor of the Brachypodieae and Triticeae lineages was estimated to have had 28,558 genes, and the common ancestor of the Panicoideae, Ehrhartoideae, and Pooideae subfamilies was estimated to have had 27,152 or 28,350 genes, depending on the ancestral gene scenario. Relative to the Brachypodieae and Triticeae common ancestor, the gene number was reduced in B. distachyon by 3,026 genes and increased in Ae. tauschii by 7,813 genes. The sum of gene deletion and gene duplication rates, which reflects the rate of gene synteny loss, was correlated with the rate of structural chromosome rearrangements and was highest in the Ae. tauschii lineage and lowest in the rice lineage. The high rate of gene space evolution in the Ae. tauschii lineage accounts for the fact that, contrary to the expectations, the level of synteny between the phylogenetically more related Ae. tauschii and B. distachyon genomes is similar to the level of synteny between the Ae. tauschii genome and the genomes of the less related rice and sorghum. The ratio of gene duplication to gene deletion rates in these four grass species closely parallels both the total number of genes in a species and the overall genome size. Because the overall genome size is to a large extent a function of the repeated sequence content in a genome, we suggest that the amount and activity of repeated sequences are important factors determining the number of genes in a genome.

Prognostic Significance of Molecular Cytogenetics in Chinese Patients with Chronic Lymphocytic Leukemia: A Prospective Unicentric Analysis.

B-cell chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia in the Western countries, however, infrequent in the Eastern. It is characterized by a highly variable clinical course; some patients survive more than 20 years, whereas others die within a few months of diagnosis. The characteristics of Chinese patients with CLL compared with the Western countries have not yet been clarified. The aim of this study was to prospectively explore characteristics and prognostic significance of molecular cytogenetic aberrations in Chinese patients with CLL. Interphase fluorescence in situ hybridization (FISH) and a panel of probes (LSI D13S319,LSI p53,LSI ATM,CEP 12,LSI MYB,LSI IGHC/IGHV) were used to detect cytogenetics abnormalities in 95 patients with CLL. Cytogenetics aberrations and their association with some other prognostic factors were analyzed. Kaplan-Meier was used for survival time. Out of the 95 CLL patients, molecular cytogenetic aberrations were found in 69 (72.6%) cases and 25 (26.3%) patients showed more than two kinds of abnormalities. The most frequent abnormalities detected were del(13q14) in 46 cases (48.4%), followed by trisomy of chromosome 12 in 22 patients (23.2%), 14q32 rearrangement in 21 patients (22.1%), del(17p13) in 16 patients (16.8%), del(11q22) in 9 patients (9.5%) and del(6q23) in 5 patients (5.3%). There were no significant differences of molecular cytogenetic aberrations in sex, age, Binet stages, peripheral lymphocyte count, and the levels of lactate dehydrogenase (LDH), β2-microglobulin (β2-MG), and ZAP-70. The TP53 and ATM gene deletion rates were higher in the group of CD38 high expression than that in the group of low expression (P=0.047 and P=0.001). No patient with TP53 and ATM gene deletion achieved complete response (CR) among 41 patients received treatment with fludarabine. The survival time was shorter in patients with high levels of LDH (P=0.028), β2-MG (P=0.012), and CD38 (P=0.000), and with TP53 gene deletion (P=0.000). Patients with sole del(13q14) had longer survival time than those with other abnormalities (P=0.044). It was showed that panel FISH has greatly increased the sensitivity of cytogenetic analyses and del(13q14) was the most frequent abnormality in CLL. Detection of molecular cytogenetic aberrations with FISH had important prognostic significance in CLL. The patients with sole del(13q14) had favorable outcome, and with del(17p13) had poor outcome.

Chromosome 9 and 17 aberrations and p53 gene deletion detected by fluorescence in situ hybridization in renal-cell carcinoma.

Nuclear grade and tumor stage have been reported as important prognostic factors for renal-cell carcinoma (RCC), but tumors of similar stage and grade can still exhibit wide variations in biologic behavior and clinical outcome. Fluorescence in situ hybridization (FISH) has recently been applied to RCC. This study was designed to investigate whether aberrations of some chromosomes or genes detected by FISH are related to the progression of RCC. We examined 52 patients with RCC, including 31 patients without metastasis (control group) and 21 patients with either concurrent or subsequent metastasis (metastatic group). Paraffin-embedded specimens of the primary tumors were analyzed by FISH for aberrations of chromosomes 9 and 17, as well as for p53 gene alterations. The incidence of aberrations of chromosome 9 was higher in the metastatic group than in the control group. The p53 gene deletion rate was significantly higher in the metastatic group than in the control group. When the metastatic group was separated into concurrent and subsequent metastasis subgroups, chromosome 17 aberrations as well as p53 gene deletion were significantly more common in the subsequent metastasis group than in the control group. Numerical aberrations of chromosome 17 as well as p53 gene deletion detected by FISH may be markers of chromosomal instability in RCC and are probably associated with an increased propensity to metastasize.

Spinal muscular atrophy.

Spinal muscular atrophy is a common cause of disability in childhood and is characterized by weakness and wasting of voluntary muscle. It is frequently fatal. The gene for this disorder has been identified as the SMN gene and is part of a highly complex duplicated region of chromosome 5 that is subject to a high rate of gene deletion and gene conversion. The severity of muscle weakness correlates with the amount of full-length SMN protein produced. Molecular genetic studies support a model in which patients are compound heterozygotes of deleted and converted alleles that predicts a progressively decreasing amount of protein product with severity of muscle weakness. The function of SMN is beginning to be understood and it appears to be involved in ribonucleoprotein biogenesis and thus indirectly in post-transcriptional processing of mRNA. There are theoretical grounds for motor neurons having a cell-specific vulnerability to disturbances of mRNA processing and transport and these are briefly reviewed.