Association of MDR-TB isolates with clinical characteristics of patients from Northern region of India.

Purpose: We sought to determine the characteristics and relative frequency of transmission of MDR‑TB in North India and their association with the clinical and epidemiological characteristics of TB‑patients. Materials and Methods: To achieve the objectives PCR‑SSCP, MAS‑PCR and direct DNA sequencing were used against 101 Mycobacterium tuberculosis isolates. Results: Multidrug‑resistant‑TB isolates were found to be significantly higher (P = 0.000) in previously treated patients in comparison to newly diagnosed patients. Further, significant differences (P = 0.003) were observed between different age groups (Mean ± SD, 28.6 ± 11.77) of the TB patients and multidrug resistance. Most frequent mutations were observed at codons 531 and 315 of rpoB and katG genes, respectively, in MDR‑TB isolates. Conclusion: Routine surveillance of resistance to anti‑TB drugs will improve timely recognition of MDR‑TB cases and help prevent further transmission in Northern India.

Mutation pattern of K-ras gene in colorectal cancer patients of Kashmir: A report.

Background and Aim: Colorectal cancer (CRC) is one of the leading malignancies worldwide. CRC has been reported to show geographical variation in its incidence, even within areas of ethnic homogeneity. The aim of this study is to identify K-ras gene mutations in CRC patients among the Kashmiri population, and to assess whether they are linked with the clinicopathological parameters. Materials and Methods: Paired tumor and normal tissue samples were collected from a consecutive series of 53 patients undergoing resective surgery for CRC. In addition blood was also collected from all the cases for ruling out germline mutation. Results: Colorectal patients, 22.64% (12 of 53), presented with mutations in K-ras constituting 13 missense mutations out of which 11 were G→A transition, one G→C transversion, and one G→T transversion. 61.5% percent of the mutations occurred in codon 12 and 38.5% in codon 13. One tumor contained missense mutations in both codons. K-ras mutations were significantly associated with advanced Dukes' stage (P < 0.05) and positive lymph node status (P < 0.05). Moreover Codon 12 K-ras mutations were associated with mucinous histotype (P < 0.05). Comparison of the mutation profile with other high-risk areas reflected both mucinous histotype differences and similarities indicating coexposure to a unique set of risk factors. Conclusion: Mutation of the K-ras gene is one of the commonest genetic changes in the development of human CRC, but it occurs in a rather low frequency in Kashmiri population.

Characterization of RPO B gene for detection of rifampicin drug resistance by SSCP and sequence analysis.

Purpose: Because of the emergence of multidrug-resistant tuberculosis in recent times, the rapid detection of resistance to the first-line anti-tuberculosis drug rifampicin was felt worldwide. Accordingly, this study was conducted to evaluate the diagnostic potential of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) for checking its utility as a rapid screening test for determination of rifampicin drug resistance. Materials and Methods: A total of 34 isolates of Mycobacterium tuberculosis ( M. tuberculosis ) (22 rifampicin resistant, 11 rifampicin sensitive and one control H37Rv) strains were analysed by PCR-SSCP and DNA sequencing within the 157-bp region of the rpo B gene (Ala 500 -Val 550 ). Results: Rifampicin resistance was detected successfully by PCR-SSCP in 20/22(90.90%) of rifampicin-resistant strains showing a total of nine different mutations in seven codon positions: codon 513 (CAA→CCA), 516 (GAC→GTC), 507 (GGC→GAC), 526 (CAC→GAC, TAC), 531 (TCG→TTG, TGG), 522 (TCG→TGG) and 533 (GTG→CCG). Two rifampicin-resistant strains showed an identical PCR-SSCP pattern with the wild type H37Rv; 77.27% rifampicin-resistant strains showed a single point mutation and 9.09% had no mutation. Three rifampicin-resistant strains showed characteristic double mutations at codon positions 526 and 531. Sensitivity and specificity were calculated as 90.90% and 100%. Conclusions: Rifampicin-resistant genotypes were mainly found in codon positions 516, 526 and 531. PCR-SSCP seems to be an efficacious method of predicting rifampicin resistance and substantially reduces the time required for susceptibility testing from 4 to 6 weeks to a few weeks.

Genetic instability of the sFRP1 gene in hepatocellular carcinoma in Chinese people / 解剖学报

Objective To examine loss of heterozygosity (LOH) and microsatellite instability (MSI) of locus D8S532 on chromosome 8 and their influence on the expression of sFRP1 in the hepatocellular carcinoma (HCCs), which may provide an experimental evidence for clarifying the mechanism of sFRP1 gene and tumor development. Methods DNA was extracted from formalin-fixed paraffin-embedded tissues. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and ordinary silver stain were used to study LOH and MSI of locus D8S532. Envision immunohistochemistry, Leica-Qwin computerized imaging system and Image-Pro PluS (IPP) version 4.5 professional imaging analysis software were used to assess the expression of sFRP1. Results The detection rates of LOH and MSI of locus D8S532 in the 36 specimens of HCC were 11.11% and 8.33% respectively. The down-regulation of sFRP1 was observed in 31 of 36 HCCs (86.11%) compared with non-carcinoma liver tissues, and the positive rate of sFRP1 protein of the HCCs was 52.78%( 19/36 ). The frequency of LOH was lower in the cases with positive expression of sFRP1 protein than those negative (0 vs 23.53%, P <0.05). Conclusion It was a common phenomenon that expression of sFRP1 protein is negative or low in Chinese with HCCs. The genetic instability of sFRP1 gene was one of causes, which lead to HCCs. LOH may play a major role in negative expression of sFRP1.

p53 Mutation and p53 Protein Expression in Gastric Cancer Tissues

PURPOSE: Variable changes occur in the progression from normal gastric epithelium to cancer, including many tumor, tumor suppressor and DNA repair genes, as well as growth factor and its receptors. The mutation and protein expression of the p53 gene may be useful prognostic factors, but their significance is still uncertain. METHODS: Specimens from 296 gastric cancer patients, treated by a curative gastrectomy, between March 1999 and April 2001, at Kyungpook National University Hospital, were used. The p53 gene mutation was assessed using a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, and the overexpression of tumor p53 protein using immunohistochemistry. The correlation between the results and clinicopathological parameters were then analyzed. RESULTS: The mutation and protein overexpression of the p53 gene were shown in 61 (20.6%) and 124 (41.9%) tumors, respectively. Of the 61 cases with a p53 mutation, 43 (70.5%) showed overexpression of the p53 protein, and of the 235 without mutation of the p53 gene, 81 (34.5%) had no overexpression of the p53 protein, and also showed statistical significance (P< 0.001). The mutation and protein overexpression of the p53 gene showed no significant differences according to age, gender, stage, location and gross type, but of the 138 intestinal and 128 of the diffuse types, 33 (23.9%) and 18 (14.1%) cases, respectively, showed p53 mutation (P=0.027); whereas, of the 150 well differentiated and 142 poorly differentiated tumors, 75 (50%) and 18 (33.8%), respectively, showed overexpression of the p53 protein. Also, of the 138 intestinal and 128 diffuse types, 71 (51.4%) and 43 (33.6%) showed overexpression of the p53 protein. There were no significant differences in the 5 year survival according to the mutation and protein overexpression of the p53 gene. CONCLUSION: The mutation and protein overexpression of the p53 gene, as assessed by PCR-SSCP and immunohistochemistry, respectively, showed a statistically significant correlation, but had little value as prognostic factors following a curative gastrectomy.

p53 Gene Mutation in Gastric Cancer Tissue

PURPOSE: p53 is one of the most commonly mutated genes in human tumors. The aim of this study was to analyze p53 mutation in gastric cancer and its correlations with the clinicopathologic variables to clarify the usefulness of p53 mutation as a prognostic factor. MATERIALS AND METHODS: Specimens from 331 patients with gastric cancer who underwent a gastrectomy between March 1999 and April 2001 at the Kyungpook National University Hospital were used. p53 gene mutations were assessed by using a polymerase chain-reaction single-strand conformation polymorphism (PCR-SSCP) analysis. The correlations between p53 gene mutation and clinocopathologic parameters were analyzed. RESULTS: p53 mutations were found in 66 (19.9%) tumors. Among those 66 cases, mutations were seen in 23 tumors at exon 5, in 8 at exon 6, in 21 at exon 7, and in 17 at exon 8. Two mutations were shown in 3 tumors. Thirty-six (23.1%) of 156 intestinal-type tumors and 19 (13.1%) of 145 diffuse-type tumors showed p53 gene mutation (P=0.007). The frequency of p53 gene mutation didn't show any significant differences according to age, sex, stage, location, or gross type. Exon 5 mutations showed more frequently in intestinal-type tumors than in diffuse-type tumors (9.7% vs. 2.8%, P=0.024), and p53 mutation were more frequent in lymph nodes metastasis group than lymph nodes non-metastasis group with statistical significance (25.0% vs 15.6%, P=0.034). The five-year survival rate showed no statistically significant difference with p53 mutation (P=0.704). CONCLUSION: p53 mutations assessed by PCR-SSCP had little value as a prognostic factor after gastrectomy in patients with gastric cancer.

Gene Mutation Analysis of A Child with Familial Hypercholesterolemia and His Family / 实用儿科临床杂志

Objective To screen the mutation of certain gene of a 10-years-old boy with multiple xanthomas and very high level of cholesterol who could be diagnosed as homozygous familial hypercholesterolemia (FH),to explore the relationship between the genotype and phenotype,and to discuss the molecular pathologic mechanism.Methods The basic information of life styles were asked from the boy and his familial members.The blood was drown to examine the lipid and genes.The boy was examined with electrocardiogram examination,ultrasonography and coronary CT angiography (CTA) to evaluate the degree of atherosclerosis.Peripheral blood DNA of the boy and his parents were extracted by phenol-chloroform method and investigated for mutations of promoter and all 18 exons of low density lipoprotein receptor(LDLR) gene.Screening was carried out by using Touch-down polymerase chain reaction (PCR) and single strand conformation polymorphism(PCR-SSCP),combined with DNA sequence analysis.In addition,the apolipoprotein B100 gene(apoB100) for known mutations (R3500Q) which caused familial defective apoB100 was screened by PCR-DNA sequence analysis.Results 1.The level of cholesterol of his parents were higher than the normal.2.Several clinical manifestations of atherosclerosis were detected from that boy.Increased intima-media thickness and plaques were detected in the common carotid artery.Mitral valve regurgitation was found by echocardiography.Coronary stenosis was confirmed by CTA.3.No mutations R3500Q of apoB100 was observed.4.A homozygous mutation in exon13 of the LDLR gene (D601Y) were identified in the boy and his parents harbour D601Y heterozygous mutation due to a single base pair substitution of G for T in the codon for residue 1864.Conclusions The final diagnosis of the boy with multiple xanthomas was homozygous FH.His disease was caused by D601Y homozygous mutation in exon13 of the LDLR gene inherited from his heterozygous parents.

Compound Heterozygosis Mutation of Low Density Lipoprotein Receptor Gene in Familial Hypercholestero-lemia Family / 实用儿科临床杂志

Objective To identify mutations site and clinical characteristics of a familial hypercholesterolemia(FH) proband diagnosed clinically through DNA sequencing and family analysis in the proband and his family members of 3 generations.Methods Blood samples and clinical data of the kindred of total 29 from 3 generations members were collected.Proband had a physical examination electrocar-diogrom and vascular ultrasound.The proband and his family members took routine clinical exams,and genomic DNA was isolated.The promoter region and the 18 exons of low density liporotein receptor(LDLR) gene were screened by Touch down polymerase chain reaction -single strand conformation polymorphism(PCR-SSCP) and DNA sequencing.The result of sequencing were matched gene sequence published in the BLAST database.Results 1.Increased intima-media thickness and plaque were detected in the common carotid artery,right subclavian artery of the proband.Aortic valve regurgitation was found by echocardiography.2.No mutation R3500Q of ApoB100 was observed.3.Two heterozygous mutations in exon 10 and 13 of LDLR gene (W462X and A606T) were identified.The proband and 5 members of paternal relatives showed W462X heterozygosis mutation in exon 10 of LDLR gene which introduced the change from tryptophone to a new stop codon.The proband's mother and grandmother harboured A606T heterozygous mutation in exon 13 of LDLR gene due to a single base pair substitution of G for A in the codon for residue 1 879.Conclusions Disease causing mutations of proband are W462X and A606T compound heterozygosis mutation in exon 10 and 13 of LDLR gene inherited from mother and father.Proband shows homozyous phenotype though the genotype analysis indicates heterozygous mutations.

STUDY ON GENETIC INSTABILITY OF nm23H1 GENE IN CHINESE WITH SQUAMOUS CELL LUNG CARCINOMA / 解剖学报

0.05).Conclusion The increase in the amount of nm23H1 protein expression can play an important role in restraining metastasis of squamous cell lung carcinoma.The heredity instability(MSI and LOH) of nm23H1 gene may not be implicated in expression of the gene and pathogenesis and progression of squamous cell lung carcinoma.