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【Objective】 To investigate the distribution frequency and characteristics of Rh and Kell erythrocyte blood group antigens in Uygur population in Xinjiang, and to explore the molecular mechanism of K gene positive patients, so as to build a local rare blood group bank and improve the ability of clinical blood security. 【Methods】 From June 2018 to February 2020, blood samples of 4 000 unrelated Uygur healthy individuals from the Medical Examination Center of our hospital and other cooperative hospitals across the autonomous region were selected. Rh and Kell blood group antigens were detected using K/Rh antigen microcolumn gel cards. The exons of Kell gene were amplified by PCR and then subjected to electrophoresis and direct sequencing to investigate the molecular mechanism. 【Results】 In Xinjiang Uygur healthy population, 1) The RhD negative rate was 5.675% (227/4 000), including 5 phenotypes; RhD positive rate was 94.325% (3 773/4 000), including 9 phenotypes, which were in line with Hardy-Weinberg equilibrium distribution. The C/E antigen frequency in RhD negative and positive patients was 13.216%/4.185% vs 52.876%/25.788% (PC, g. 412A>G, exon 6, g. 133C>T, and g. 189T>C, respectively, two of which caused changes in amino acid sequence: alanine at position 193 to methionine (p.Ala193Met) and alanine 423 to valine (p.al423Val). The prediction of RNA secondary structure and protein conformation after mutation using relevant biological information software found that the mutation caused changes in RNA secondary structure, free energy, protein conformation and function. 【Conclusion】 The frequency of RhD antigen negative in Xinjiang Uygur population was higher than that in other ethnic groups, and the distribution of C/E antigen was different in D antigen negative/positive patients. The distribution of K antigen in Kell blood group system was higher than that in other ethnic groups (P<0.05). The primary and secondary structure changes of nascent peptide chain caused by a single point mutation in Kell gene may be one of the molecular mechanisms of K antigen positivity.
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Objective:To perform a prospective cohort study to identify individual susceptibility of glucocorticoid (GC) -associated osteonecrosis of the femoral head (GA-ONFH) and their clinical and genetic risk factors. Methods:The present prospective cohort study enrolled patients who received their first GC therapy between July 2015 and January 2018 at Zhongshan Hospital. All patients did not receive any GC treatment before enrollment. Further, they planned to start GC treatment with the dose (equivalent prednisone) of ≥30 mg/d, lasted ≥3 weeks, or pulse dose ≥200 mg/d, lasted ≥3 d. Blood samples were collected before GC treatment to evaluate bone metabolism and its released factors. Hip MRI was performed at the 1st, 3rd, 6th, 12th and 24th month to diagnose GA-ONFH. All patients were followed-up for ≥2 years. The endpoint was regarded as diagnosis of GA-ONFH or completion of 2 years follow-up. Lasso regression was performed to determine which clinical features were associated with GA-ONFH. A nested case-control sub-cohort (A, n=12) was established prospectively based on the main cohort by 1∶1 matching. Whole exome sequencing was performed to screen differential and functional candidate single nucleotide polymorphisms and insertion-deletions (SNP/InDels). Another sub-cohort (B, n=50) was constructed retrospectively in patients with GA-ONFH and non-ONFH patients received standard high dose GC treatment for more than two years. The candidate SNP/InDels were verified by Sanger sequencing based on the patients from sub-cohort B. Results:A total of 96 patients were enrolled of which 88 of them (32 males and 56 females, mean age 42.30 years) completed follow-up. Eight cases (9.1%) were diagnosed with GA-ONFH. The median time from the start of GC therapy to the diagnosis of ONFH was 53.00(34.00,13.50) days. The baseline characteristics, such as age, sex and body mass index, indicated no significant difference between the ONFH group and the non-ONFH group. The cumulative GC dose of the ONFH patients in the first month was higher than that of non-ONFH [32.74(29.55, 47.05) mg/kg vs. 24.00(21.10, 29.45) mg/kg, Z=-2.410, P=0.016]. However, there was no significant difference of patients who underwent pulse therapy (37.5% vs. 10.0%, adjusted χ 2=2.829, P=0.093). The ratio of serum apolipoprotein B/apolipoprotein A1 (ApoB/ApoA1) in patients with ONFH was higher than that in non-ONFH group before GC use [0.95(0.80, 1.50) vs. 0.70(0.60, 0.80), Z=-2.875, P=0.000]. Due to the multicollinearity, Lasso regression model was performed to reduce overfitting. All variables were included in the model. The results suggested that higher ApoB/ApoA1 ratio, lower serum β-c-terminal telopeptide (β-CTX) and higher cumulative GC dose in the first month were the top three risk factors of GA-ONFH. This model had an accuracy of 0.982 in internal validation. Seven differential candidate SNP/InDels were found by whole exome sequencing of sub-cohort A. We further verified these SNP/InDels in sub-cohort B. The patients with COLEC12 mutation (rs2305027, G1816A) were at risk of GA-ONFH ( OR=6.00, 95% CI: 1.17, 30.73). Conclusion:Higher first-month GC dose, lower serum β-CTX level before treatment, higher ApoB/ApoA1 ratio and COLEC12 mutation (rs2305027, G1816A) could increase the risk of GA-ONFH.
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RESUMEN Objetivos: el objetivo principal del estudio fue evaluar la supervivencia global en pacientes con carcinoma colorrectal metastásico (CCRm) cuyos tumores tuvieran el gen KRAS mutado frente al no mutado. Materiales y métodos: se analizaron los datos de las historias clínicas de pacientes con CCRm (enero 2010 - diciembre 2013) de diferentes hospitales de Lima Metropolitana, cuyos tumores tuvieron evaluación del estado mutacional del exón 2, gen KRAS. Se usaron la curva de supervivencia de Kaplan-Meier y la prueba de long rank o Breslow para las comparaciones de las curvas de supervivencia. Resultados: de los 320 casos analizados, hubo 227 pacientes (70,93%) con KRAS no mutado y 93 (29,07%) con KRAS mutado. La supervivencia global de pacientes con CCRm y KRAS mutado fue mayor que los pacientes con KRAS no mutado (hazart ratio: 0,73; IC 95% 0,55 - 0,98; p=0,037). Conclusión: la población con CCRm y KRAS mutado estudiada en centros médicos de Lima Metropolitana tuvo una supervivencia mayor, comparada con la no mutada, comportamiento diferente a lo encontrado en la literatura mundial.
ABSTRACT Objectives : The main goal for this study was to evaluate overall survival in mCRC patients with mutated vs. wild type KRAS gene (exon 2) status. Materials and Methods : Between January 2010 and December 2013, data from clinical records of mCRC patients from different hospitals in Lima, stating an assessment of the KRAS gene mutation status (exon 2), were analyzed. Kaplan-Meier survival estimates and Log-Rank or Breslow Test were used to compare the survival curves. Results : Three-hundred and twenty cases were analyzed. There were 227 patients (70.93%) with wild type KRAS and 93 (29.07%) with mutated KRAS. The overall survival of mCRC patients and mutated KRAS was higher than that of patients with wild type KRAS (HR: 0.73; 95% CI: 0.55-0.98; P= 0.037). Conclusion : Patients with mCRC and mutated KRAS who were studied in Lima have higher survival rates compared to wild type patients, being this different from what is found in the world literature.
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Objective@#To establish patient derived xenograft (PDX) model of malignant peritoneal mesothelioma (MPM), and to identify the key characteristics of tumor biology of the model, so as to provide an experiment platform for studying the pathologic mechanisms and new therapeutic strategies for MPM.@*Methods@#Surgically excised MPM tumor tissues were inoculated subcutaneously in BALB/c-nu/nu mice for 3 stable passages. In the 4th passage, the subcutaneous tumors were harvested under aseptic conditions, cleaned and made into MPM tumor cell homogenate. Four nude mice (two males and two females) were selected and one male and one female nude mouse were inoculated in the abdominal cavity at the dose of 100 μL, others were inoculated at a dose of 200 μL. The PDX model of MPM was established. The changes of body mass in nude mice were measured regularly, the extent of abdominal and pelvic tumors was judged by experimental peritoneal cancer index (ePCI) score, and the pathologic characteristics of tumors were analyzed.@*Results@#The subcutaneous and abdominal animal models of MPM were successfully established. The subcutaneous tumor model grew into tumor on the 20th day, followed by a slow growth stage between the 20th and 29th day, then a rapid growth stage between the 30th and 57th day. According to the dose of tumor cells (100, 200 μL) and timing (14th and 69th days after grafting), the abdominal tumor model successfully simulated the early and late clinical stages of MPM. The HE staining results of the MPM nude mice model showed that the tumor was epithelial mesothelioma and invaded most of the organs, including liver, spleen, pancreas, mesentery. Immunohistochemical staining for calretinin, cytokeratin 5/6, WT1 and Ki-67 were positive. Whole-genome exon sequencing identified 26 and 36 high frequency gene mutations in tumors derived from the PDX model and clinical sample from patients, including 21 common gene mutations.@*Conclusions@#The PDX model of MPM is established. The model is characterized by highly malignant tumor with rapid growth and high invasiveness.
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To establish patient derived xenograft (PDX) model of malignant peritoneal mesothelioma (MPM), and to identify the key characteristics of tumor biology of the model, so as to provide an experiment platform for studying the pathologic mechanisms and new therapeutic strategies for MPM. Surgically excised MPM tumor tissues were inoculated subcutaneously in BALB/c-nu/nu mice for 3 stable passages. In the 4th passage, the subcutaneous tumors were harvested under aseptic conditions, cleaned and made into MPM tumor cell homogenate. Four nude mice (two males and two females) were selected and one male and one female nude mouse were inoculated in the abdominal cavity at the dose of 100 μL, others were inoculated at a dose of 200 μL. The PDX model of MPM was established. The changes of body mass in nude mice were measured regularly, the extent of abdominal and pelvic tumors was judged by experimental peritoneal cancer index (ePCI) score, and the pathologic characteristics of tumors were analyzed. The subcutaneous and abdominal animal models of MPM were successfully established. The subcutaneous tumor model grew into tumor on the 20th day, followed by a slow growth stage between the 20th and 29th day, then a rapid growth stage between the 30th and 57th day. According to the dose of tumor cells (100, 200 μL) and timing (14th and 69th days after grafting), the abdominal tumor model successfully simulated the early and late clinical stages of MPM. The HE staining results of the MPM nude mice model showed that the tumor was epithelial mesothelioma and invaded most of the organs, including liver, spleen, pancreas, mesentery. Immunohistochemical staining for calretinin, cytokeratin 5/6, WT1 and Ki-67 were positive. Whole-genome exon sequencing identified 26 and 36 high frequency gene mutations in tumors derived from the PDX model and clinical sample from patients, including 21 common gene mutations. The PDX model of MPM is established. The model is characterized by highly malignant tumor with rapid growth and high invasiveness.
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Aims: To establish the common rules of exon combinatorics during RNA splicing. Study Design: Inferring a plausible statistical model of exon combinatorics from the annotated models of human genes during RNA splicing. Place and Duration of Study: Department of Genetics (Belarusian State University), Proteome and Genome Research Unit (Luxembourg Institute of Health), Department of Genetics (Lomonosov Moscow State University) and Moscow Center of Experimental Embryology and Reproductive Biotechnologies, between January 2017 and July 2019. Methodology: We used human mRNA and EST sequences from GenBank (1093522 unique records in total) and linear models of the human genes from Ensembl (58051 genes), AceView (72384 genes), ECgene (57172 genes), NCBI RefSeq (54262 genes), UCSC Genome Browser (58037 genes) and VEGA (54950 genes) to calculate a combinatorial index of human exons. We inferred the most plausible statistical model describing the distribution of combinatorial index of human exons using Clauset’s mathematical formalism. Predictors of the combinatorial index values and functional outcomes of the predefined behavior of exons during splicing were also determined. Results: Power-law is the most plausible statistical model describing the combinatorics of exons during RNA splicing. The combinatorial index of human exons is defined by more than 90% by the 138 features that have different importance. The most important of these features are the abundance of exon in transcripts, the strength of splice sites, the rank of exon in transcripts and the type of exon. Analysis of the marginal effects shows that different values of the same feature have unequal influence on the combinatorial index of human exons. Power-law behavior of exons during RNA splicing pre-determines structural diversity of transcripts, low sensitivity of splicing process to random perturbations and its high vulnerability to manipulation with highly combinative exons. Conclusion: Exons widely involved in alternative splicing are a part of the common power-law phenomenon in human cells. The power-law behavior of exons during RNA splicing gives the unique characteristics to human genes.
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The epidermal growth factor receptor (EGFR) exon 19 deletion (19del) and the L858R point mutation of exon 21 are the most common types of EGFR mutations in non-small cell lung cancer (NSCLC).Treatment with EGFR tyrosine kinase inhibitors (TKI) can provide better survival benefit for some patients with advanced NSCLC.Clinical studies have shown that patients with these two types of mutations have different benefits in EGFR-TKI therapy.However,most patients treated with EGFR-TKI develop resistance after 12 months of treatment,the most common of which is the EGFR gene T790M mutation.In order to study the mechanism of resistance to TKI in NSCLC patients,and to develop new therapeutic methods and rational treatment strategies,further research on tumor characteristics in the whole disease progression and treatment process is indispensable.Exploring and comparing the differences between 19del and L858R and T790M in obtaining resistance to TKI is of great clinical significance.
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The epidermal growth factor receptor (EGFR) exon 19 deletion (19del) and the L858R point mutation of exon 21 are the most common types of EGFR mutations in non-small cell lung cancer (NSCLC). Treatment with EGFR tyrosine kinase inhibitors (TKI) can provide better survival benefit for some patients with advanced NSCLC. Clinical studies have shown that patients with these two types of mutations have different benefits in EGFR-TKI therapy. However, most patients treated with EGFR-TKI develop resistance after 12 months of treatment, the most common of which is the EGFR gene T790M mutation. In order to study the mechanism of resistance to TKI in NSCLC patients, and to develop new therapeutic methods and rational treatment strategies, further research on tumor characteristics in the whole disease progression and treatment process is indispensable. Exploring and comparing the differences between 19del and L858R and T790M in obtaining resistance to TKI is of great clinical significance.
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@# Objective： ：To detect the gene mutation in cholangiocarcinoma patients using the next generation sequencing (NGS) technology, and to analyze its correlation to the prognosis of the patients. Methods: From June 2016 to June 2018, 40 patients diagnosed with cholangiocarcinoma received NGS examination to screen the possible mutations (single base mutation, structural variation, copy number variation and gene fusion, etc.). The disease control rates (DCR), progression-free survival (PFS) and overall survival (OS) of the patients, who received the first line therapy, were retrospectively reviewed to analyze the relationship between signaling pathway as well as its genetic variation and the prognosis of cholangiocarcinoma patients. Results: The median PFS of patients with and without TP53 mutation was 11.0 and 8.3 months, respectively (P=0.332), while OS was 14.3 and 32.9 months, respectively (P=0.041). The median PFS of patients with and without PI3K mutations was 8.3 and 11.0 months, respectively (P=0.285), while OS was 14.3 and 37.0 months, respectively (P=0.020). The median PFS of patients with and without mTOR pathway mutations was 6.3 and 10.3 months, respectively (P=0.020), while OS was 15.6 and 19.6 months, respectively (P=0.892). There was no significant effect of pathway-related gene mutations on patients’survival. Conclusion: The prognosis of cholangiocarcinoma patients with TP53 and PI3K pathway activation had obviously poor prognosis than those without. No significant difference was observed between the patients with and without mTOR pathway activation and IDH mutation.
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Objective To investigate the impact of sample typeon the detection of c-KIT exon 17 mutation in acute myeloid leukemia (AML) patients. Methods A retrospective study was conducted on 51 bone marrow samples collected from 37 AML patients [17 maleand 20 female, with a median age of 33 (range from 1 to 82)] at diagnosis or after treatment from June 2016 to August 2018. Of the 37 cases of AML, 24 were t(8; 21) AML, 11 were inv(16)/t(16;16) AML and 2 were non-CBF-AML. RNA and DNA were simultaneously extracted from every sample. PCR followed by Sanger sequencing were used to screen c-KIT exon 17 mutation, and the comparisons were made between paired cDNA and DNAsamples. Results (1) Of the 51 paired samples, 14 pairs were simultaneously detected positive for c-KITmutation in both of cDNA and DNA samples, but 17 pairs were detected negative in both, and the remaining 20 pairswere only detected positive for the mutation in cDNA but not in DNA, with an inconsistency rate of 39.2％. The positive rate of detecting c-KITmutation was significantly higher in cDNA than in DNA samples (66.7％vs 27.5％,P=0.000073). (2)Inconsistent mutation results between paired cDNA and DNA samples occurred in t(8;21)AML, inv(16)AML and non-CBF-AML patients with the inconsistency rate of 36.4％(12/33), 27.2％(3/11) and 71.4％ (5/7), respectively. (3)The inconsistency rate was significantly higher in samples collected after treatment compared with those collected at diagnosis (72.7％vs 13.8％, P=0.00003). (4) All 5 serially monitored patients with c-KITmutation had inconsistency in mutation detection between cDNA and DNA samples during follow up. Conclusion cDNA improves the detection of c-KIT exon 17 mutation in AML patients compared with DNA, which is especially common after treatment.
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Objective@#To study the clinicopathologic characteristics and immunophenotype of lymphomatoid papulosis(LyP), followed by exon mutation analysis with focus on gene mutations involved in apoptosis pathway and other possible pathogenic genes.@*Methods@#Clinical data analysis and immunohistochemical staining were carried out in 20 cases of LyP. Whole exome sequencing technology was employed in 2 cases of type C of LyP.@*Results@#Of the 20 cases, there were 9 males and 11 females with a median age of 28.6 years. Nineteen patients presented with multiple papules and nodules, and one case presented with only one tumor nodule. Of the fifteen cases with available followed-up data, all were alive (20-155 months). Histologically, the tumors primarily involved the dermis and subcutaneous layer, in which 6 were type A, 3 were type B, 10 were type C and 1 was type D. Main infiltration patterns included wedge-shaped, band-like, sheets and large nodular. Immunohistochemistry showed that most cases expressed CD30 in the large tumor cells. Sixteen cases expressed CD3, 17 cases expressed CD4 and 8 cases expressed CD8. Sixteen cases expressed TIA1. Ten cases expressed GrB and 1 case expressed CD15. All but one case did not expressed CD20. All cases did not express ALK1.A total of 101 common non-synonymous mutations were detected in 2 cases of LyP type C by whole exome sequencing, including 87 missense mutations, 6 missense mutation/frame-shift deletions, 2 missense mutation/nonframe-shift deletions, 5 frame-shift deletions, 1 missense mutations/synonymous mutation. Syndecan-1(SDC1), COL4A1, Laminin-5 were involved in the extracellular matrix receptor pathway.@*Conclusions@#Clinical presentations are crucial for the diagnosis of LyP. LyP has a favorable prognosis. SDC1, COL4A1 and Laminin-5 gene mutations may be associated with tumor recurrence or progression into a higher gradelymphoma.
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AIM:To explore the association of the mutation in PPP2R3A exons and retinoblastoma.METHODS:Hospital-based case control study was taken.Retinoblastoma patients (15 cases, as case group) and matched controls (30 controls, as control group) were recruited in this study.Genomic DNA obtained from formalin fixed paraffin embedded (FFPE) and peripheral blood were used as template.PPP2R3A gene exon sequences were detected by PCR-sequencing.Homology analysis was performed using blastn in GenBank.RESULTS:Analyzing PPP2R3A DNA sequences (1001bp) from 15 cases, two reported SNPs had been detected, including rs34629706 and rs144802055.Rs34629706 also occurred in the control group.Rs144802055 appeared only in the case group.CONCLUSION:PPP2R3A gene SNPs of rs34629706 is unrelated to the incidence of retinoblastoma.Relations between rs144802055 and RB needs to be further explored.
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Background Congenital cataract is an important cause of blindness and amblyopia in children,and about 50％ of congenital cataract is hereditary.Objective The aim of this study was to determine the diseasecausing gene of one Hui congenital cataract pedigree by using exon combined target region capture sequencing chip of eye diseases.Methods This study was approved by Ethic Committee of Ningxia People's Hospital and followed Declaration of Helsinki.One Hui congenital cataract pedigree was recruited in Ningxia Eye Hospital in 2011.All the disease history of the members in this family were collected and recorded,and the eye examinations were performed.The peripheral blood specimens were collected from family members and 300 healthy individuals for the extraction of DNA.Exon combined target region capture sequencing chip of eye diseases was used to screen the candidate diseasecausing mutations,then PCR and direct sequencing were used to confirm the disease-causing mutations.Results This H ui family included 61 members of 6 generations,and 18 patients were diagnosed in serial 5 passages,conforming to autosomal dominant inheritance pattern.Among 18 cataract patients,7 individuals were associated with nystagmus and strabismus,and 4 patients had high myopia.Eight candidate pathogenetic mutations were detected by exon combined target region capture sequencing chip of eye diseases and bioinformatics method,with 5 mutations in noncoding regions and 3 in coding regions.The mutation P24T of CYRGD gene was confirmed as pathogenic mutation of this pedigree by using PCR and direct sequencing methods.These mutations co-segregated with affected members of the family,and the mutations were not found in the unaffected family members and 300 unrelated controls.Conclusions P24T of CYRGD gene mutation is confirmed as pathogenic mutation of this pedigree.Exon combined target region capture sequencing chip provides a new approach to detect disease-causing mutations of congenital cataract with diversity clinical phenotypes.
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Background: The classical forms of severe Spinal Muscular Atrophy type is well recognized by pediatricians. Case Characteristics: A hypotonic neonate with severe respiratory distress at birth. Observation: Homozygous absence of exons 7 of the Survival Motor Neuron I gene. Outcome: Died 108 days after admission when respiratory support was withdrawn at the request of the parents. Message: Spinal Muscular Atrophy should be kept in mind in the differential diagnosis for unexplained severe generalized hypotonia and severe respiratory distress immediately after birth in the neonates.
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Objective: To sequence the ATP7B gene in patients with Wilson's disease (WD) and to analyze the relationship between the mutations and WD. Methods: The genomic DNA was obtained from the oral mucosal cells of 67 clinically diagnosed WD patients; PCR was used to amplify all the exons 5' end→ 3' end of ATP7B gene. And the PCR products were subjected to DNA direct sequencing for mutations. Results: We found that the ATP7B gene mutation rate was 77.61% (52/67) in WD patients. Of these patients, 16 had homozygote mutations (including 12 patients with Arg778Leu and 4 with Arg919Gly), 5 had complex mutations, and 31 had simple hetrozygote mutations. Five types of the ATP7B gene complex mutations were rarely reported in China. Conclusion: We have identified 5 complex mutations of ATP7Bgene, which might be related to the development and progression of WD and deserves further study.
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Objective To establish a single-tube detecting system for the simultaneous identification of JAK2 V617F and JAK2 exon12 mutations.Methods Genomic DNA of cell line PC-3 was utilized as the wild type control,while genomic DNA of cell line HEL and plasmids with diverse JAK2 exon 12 mutations were used as the positive controls for JAK2 V617F and exon12 mutations.Multiplex PCR was performed to amplify the different amplicons combined with high-resolution melting (HRM) analysis,which established the multiplex detecting system for JAK2 V617F and exon12 mutations.Meanwhile 42 cases of polycythemia vera patients were collected to detect 2 kinds of JAK2 mutations by the above system and routine methods.Results The multiplex JAK2 mutations detecting system was successfully established by multiplex PCR combined with high-resolution melting curve analysis,which could simultaneously detect JAK2 V617F and JAK2 exon12 mutations.The analytical sensitivities of 2 mutations in this system were both up to 5％ and the precision (coefficient of variation) of intra-and inter-assay of the melting temperature (Tm) of 2 amplicons were separately less than 0.01％.37 cases were identified JAK2 V617F mutations from 42 polycythemia vera patients,while 2 JAK2 exon12 mutations cases were found from 5 JAK2 V617F negative patients.Compared with routine methods,the results matched the rate of 100％.Two cases of JAK2 exon 12 mutations were confirmed to the mutation types of H538K539delinsL and F537-I546dul10 + F547L by cloning and sequencing.Conclusions This method can simultaneously detect two kinds of JAK2 mutations in the peripheral blood and will contribute to the molecular diagnosis of myeloproliferative neoplasms,especially polycythemia vera.
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Objective To evaluate the role of μ opioid receptor exon 7 in the analgesic efficacy of endomorphin-2 in rats.Methods Twenty-four male Sprague-Dawley rats in which IT catheters were successfully implanted,weighing 220-260 g,were randomly divided into 3 groups (n =8 each) using a random number table:normal saline control group (group C),negative siRNA control group (group N-siRNA) andμ opioid receptor exon 7 siRNA group (group E7-siRNA).In C,N-siRNA and E7-siRNA groups,30μl saline solution,negative siRNA plasmid 20 μl + lipofectamine 2000 (10 μl),and μ opioid receptor siRNA plasmid 20μ1 + lipofectamine 2000 (10 μl) were intrathecally injected once a day for 3 consecutive days.The mechanical pain threshold was measured on 4th day (baseline).Endomorphin-2 10 μg was injected intrathecally at 1 h after measurement of the pain threshold.The mechanical pain threshold was measured at 5,20,40 and 60 min after endomorphin-2 injection,and the analgesic efficacy was calculated.Results There was no significant difference in the baseline pain threshold among the three groups.Compared with group C,no significant difference was found in the analgesic efficacy at each time point after endomorphin-2 injection in group N-siRNA,and the analgesic efficacy was significantly decreased at 5 and 20 min after endomorphin-2 injection in group E7-siRNA.Conclusion μ opioid receptor exon 7 is involved in the analgesic efficacy of endomorphin-2 in rats.
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Objective To investigate the aberrant methylation and expression of growth arrest and DNA-damage-in-ducible 45 gamma (GADD45G) gene in gastric cardia adenocarcinoma (GCA). Methods Bisulfite conversion-methylation specific polymerase chain reaction method (BS-MSP) and immunohistochemistry method were used respectively to detect the methylation status and protein expression of GADD45G in 138 GCA tumor tissues and corresponding normal tissues. Re-sults The methylation status of GADD45G distal promoter (region 1) was not detected in GCA tumor tissues and corre-sponding normal tissues. For GADD45G region 2 and region 3, the BS-MSP results of region 3 were identical to that of re-gion 2. The methylation frequency of proximal promoter and exon 1 in GADD45G island 2 (region 2 and region 3) in GCA tu-mor tissues (49.3%, 68/138) was significantly increased compared to that in corresponding normal tissues (0, P＜0.01). The methylation status of this two sites in tumor tissues was associated with TNM stage of tumors (P＜0.05). The protein expres-sion of GADD45G in tumor tissues was significantly decreased than that in corresponding normal tissues (P ＜ 0.05),and threre was a significant negative correlation with methylation status of GADD45G proximal promoter and exon 1 (rs=-0.398). Conclusion The decreased expression of GADD45G by hypermethylation of proximal promoter exon 1 of the gene may play an important role in gastric cardia adenocarcinoma.
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Objective To set up a new diagnostic platform based on microarray exon-capture and next-generation sequencing for detecting small mutations in dystrophin gene.The sensitivity and specificity of the method were assessed in clinical settings and the distribution of small mutations in Chinese Duchenne muscular dystrophy/Becker muscular dystrophy (DMD/BMD) patients were also analyzed.Methods Forty-one DMD/BMD patients diagnosed by the clinical criteria without large deletion or duplication (≥ 1exon) were recruited from Peking Union Medical College Hospital consecutively.Genomic DNA was extracted from blood samples.The libraries were prepared.Then exon and intron-exon flanking sequences of DMD gene were captured by custom microarray.Targeted next-generation sequencing and Sanger Sequencing were conducted.The patients who were not detected any disease-causing mutation were performed muscle biopsy.Results Thirty-eight subjects were detected small mutations in DMD gene.All single nucleotide variants (SNVs) and insertion & deletions (INDELs) were validated by Sanger sequencing.Twenty-one novel mutations were reported.The distribution of SNVs and INDELs was similar to other international DMD databases.Upon immunohistochemistry staining of dystrophin protein,1 of 3 mutation-undetected patients was diagnosed as DMD,2 of them were excluded.The specificity of the method was 100％,while the sensitivity was 97.4％.Conclusions Our microarray-captured next-generation sequencing assay could detect SNVs and INDELs with high sensitivity and specificity.Its advantages are economic,time-saving and stable.The platform is suitable for clinical gene diagnosis.
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ObjectiveTo investigate the clinical significance of pyrosequencing assay for determining K-ras mutations in exon 2 codons 12 and 13 in clinical colorectal cancer tissues.Methods Genomic DNA,extracted from K-ras mutant cell lines SW480 (homozygous,c.35G ＞ T), DLD-1 (heterozygous,c.38G ＞ A) and wild-type HT-29,was first used as the sequencing template respectively to test the accuracy of pyrosequencing methodology.The SW480 and DLD-1 DNA was separately mixed with wild-type HT-29 DNA in proportions of 2％,3％,5％,10％,20％,30％ and 50％,the sensitivity for mutation detection was measured separately by pyrosequencing assay and directed Sanger DNA sequencing in the serial DNA mixture samples.The pyrosequencing assay results were compared with the corresponding Sanger sequencing and the datas were analysized by Fisher exact test.Pyrosequencing analysis was then performed for screening K-ras exon 2 mutations at codons 12 and 13 on DNA isolated from a panel of 30 colorectal cancer samples derived fromclinicalformalin-fixed andparaffinembedded(FFPE)tissues.ResultsCancer cell lines with known K-ras mutations ( SW480 and DLD-1 ) were readily detectable by pyrosequencing-based analysis.When the proportions of mutant colorectal cancer cell line DNA were 5％ and 10％ content,the mutation rates of K-ras gene detected by conventional Sanger DNA sequencing were 33.3％ (4/12) and 58.3％ (7/12) respectively,whereas the mutation rates detected by pyrosequencingbased assay were 91.7％ (11/12) and 100％(12/12) respectively,there were significant differences between those two sequencing methodology ( P ＜0.05).Furthermore,we found 10 patients with K-ras exon 2 point mutations at codons 12 and 13 by pyrosequencing-based assay from 30 colorectal cancer FFPE tissues,the point mutation rate was 33.3％ (10/30) and all of the mutations determined were heterozygous.The codon 12 was most frequently affected [30％ (9/30)].Mutations with the highest frequency were G ＞ A transitions [ 50％ ( 5/10 ) ],followed by G ＞ T transversions [ 30％ ( 3/10 ) ].Conclusion The pyrosequencing assay provides an accurate and sensitive method for mutation screening of K-ras exon 2 codons 12 and 13 in routine diagnostic specimens,thereby allowing the selection of the cancer treatment in clinical individualized practice.