• Asian Pacific Journal of Cancer Prevention
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• v.14 no.11
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• pp.6653-6656
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• 2013

Background: The single most common proto-oncogene change in human neoplasms is a point mutation in RAS genes. A wide range of variation in frequency of KRAS mutations has been seen in hematologic malignancies. Despite this, RAS roles in leukemogenesis remain unclear. The frequency of KRAS mutations in CML has been reported to be between zero an 10%. Many attempts have been done to develop an anti-RAS drug as a therapeutic target. Materials and Methods: This cross sectional study was performed in Mashhad University of Medical Sciences, Mashhad, Iran from 2010-2012. In 78 CML patients (diagnosed according to WHO 2008 criteria) in chronic or accelerated phases, KRAS mutations in codons 12 and 13 were analyzed using a modified PCR-restriction fragment length polymorphism (RFLP) method. Results: We did not detect any KRAS mutations in this study. Conclusions: KRAS mutations are overall rare in early phase CML and might be secondary events happening late in leukemogenesis cooperating with initial genetic lesions.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.17
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• pp.7125-7128
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• 2014

Background: Acquired genetic alterations and presence of sensitizing mutations in the tyrosine kinase domain of EGFR and other signaling molecules have been found in different subsets of primary lung adenocarcinoma. The commonest EGFR mutations are small in frame deletions of exon 19 and a point mutation (L858R) in exon 21, having a combined occurrence of around 90%. The objective of this study was to determine the frequency and types of EGFR mutations in primary lung adenocarcinomas in Pakistan. Materials and Methods: EGFR mutations in tumor samples were screened by multiplex real time PCR. Briefly, DNA from formalin fixed paraffin-embedded tissue was amplified with primers and probes specific to 43 different EGFR mutations in a Cobas z 480 instrument. The assay detects mutations in four exons (18-21) of the EGFR gene. Results: Out of 94 patients, 65 were males and 29 females with a M:F ratio of 2.2: 1. The median age was 62 years (range, 28 - 85 years). In our biopsy samples 70 (74%) cases were of primary lung adenocarcinoma, whereas 24 (26%) were confirmed metastatic adenocarcinoma of primary lung origin. EGFR mutation was positive in 29% of the patients. The highest frequency of L858R was observed in 48% of these, followed by deletion in exon 19 (44%). In addition, other rare mutations such as compound G718X:S768I and insertions in exon 20 insertion were detected in approximately 4% of the patients. Conclusions: This study showed that Del 19 and L858R are the most frequent mutations in Pakistani lung adenocarcinoma patients and around 29% of the patients were found eligible for erlotinib therapy.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.12
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• pp.5147-5152
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• 2016

Background: Investigations of methods for detection of mutations have uncovered major weaknesses of direct sequencing and pyrosequencing, with their high costs and low sensitivity in screening for both known and unknown mutations. High resolution melting (HRM) analysis is an alternative tool for the rapid detection of mutations. Here we describe the accuracy of HRM in screening for KRAS and BRAF mutations in metastatic colorectal cancer (mCRCs) samples. Materials and Methods: A total of 1000 mCRC patients in Mehr Hospital, Tehran, Iran, from Feb 2008 to May 2012 were examined for KRAS mutations and 242 of them were selected for further assessment of BRAF mutations by HRM analysis. In order to calculate the sensitivity and specificity, HRM results were checked by pyrosequencing as the golden standard and Dxs Therascreen as a further method. Results: In the total of 1,000 participants, there were 664 (66.4%) with wild type and 336 (33.6%) with mutant codons 12 and/or 13 of the KRAS gene. Among 242 samples randomly checked for the BRAF gene, all were wild type by HRM. Pyrosequencing and Dxs Therascreen results were in line with those of the HRM. In this regard, the sensitivity and specificity of HRM were evaluated as 100%. Conclusion: The findings suggest that the HRM, in comparison with DNA sequencing, is a more appropriate method for precise scanning of KRAS and BRAF mutations. It is also possible to state that HRM may be an attractive technique for the detection of known or unknown somatic mutations in other genes.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.11
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• pp.4623-4627
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• 2014

Background: The epidermal growth factor receptor (EGFR) plays a vital role in the activation and inactivation of receptor tyrosine kinases. Mutations in exons 19 and 21 of EGFR are commonly found to be associated with non small cell lung carcinoma and triple negative breast cancer, enhancing sensitivity to EGFR targeting chemotherapeutic agents. Since amplification and prolonged activation of EGFR molecules have been identified in oral squamous cell carcinomas (OSCC), we investigated whether OSCCs carried mutations in exons 19 and 21 of EGFR to their incidence. Materials and Methods: Tumor chromosomal DNA isolated from forty surgically excised oral squamous cell carcinoma tissues was subjected to PCR amplification with intronic primers flanking exons 19 and 21 of the EGFR gene. The PCR amplicons were subsequently subjected to direct sequencing to elucidate the mutation status. Results: Data analysis of the EGFR exon 19 and 21 coding sequences did not show any mutations in the forty OSCC samples that were analyzed. Conclusions: To the best of our knowledge, this is the first study to have investigated the genetic status of exons 19 and 21 of EGFR in Indian OSCCs and identified that mutation in EGFR exon 19 and 21 may not contribute towards their genesis. The absence of mutations also indicates that oral cancerous lesions may not be as sensitive as other cancers to chemotherapeutic agents targeting EGFR.

• Tuberculosis and Respiratory Diseases
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• v.82 no.2
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• pp.143-150
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• 2019

Background: The purpose of this study was to analyze the relationship between the gene mutation patterns by the GenoType MTBDRplus (MTBDRplus) assay and the phenotypic drug susceptibility test (pDST) results of isoniazid (INH) and prothionamide (Pto). Methods: A total of 206 patients whose MTBDRplus assay results revealed katG or inhA mutations were enrolled in the study. The pDST results were compared to mutation patterns on the MTBDRplus assay. Results: The katG and inhA mutations were identified in 68.0% and 35.0% of patients, respectively. Among the 134 isolated katG mutations, three (2.2%), 127 (94.8%) and 11 (8.2%) were phenotypically resistant to low-level INH, high-level INH, and Pto, respectively. Among the 66 isolated inhA mutations, 34 (51.5%), 18 (27.3%) and 21 (31.8%) were phenotypically resistant to low-level INH, high-level INH, and Pto, respectively. Of the 34 phenotypic Pto resistant isolates, 21 (61.8%), 11 (32.4%), and two (5.9%) had inhA, katG, and both gene mutations. Conclusion: It is noted that Pto may still be selected as one of the appropriate multidrug-resistant tuberculosis regimen, although inhA mutation is detected by the MTBDRplus assay until pDST confirms a Pto resistance. The reporting of detailed mutation patterns of the MTBDRplus assay may be important for clinical practice, rather than simply presenting resistance or susceptibility test results.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.6
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• pp.775-783
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• 2018

Objective: The purpose of this study was to investigate the genetic effects of six keratin (KRT) genes on the wool traits of 418 Chinese Merino (Xinjiang type) (CMXT) individuals. Methods: To explore the effects and association of six KRT genes on sheep wool traits, The polymerase chain reaction-based single-strand conformation polymorphism (PCR-SSCP), DNA sequencing, and the gene pyramiding effect methods were used. Results: We report 20 mutation sites (single-nucleotide polymorphisms) within the six KRT genes, in which twelve induced silent mutations; five induced missense mutations and resulted in $Ile{\rightarrow}Thr$, $Glu{\rightarrow}Asp$, $Gly{\rightarrow}Ala$, $Ala{\rightarrow}Ser$, $Se{\rightarrow}His$; two were nonsense mutations and one was a same-sense mutation. Association analysis showed that two genotypes of the KRT31 gene were significantly associated with fiber diameter (p<0.05); three genotypes of the KRT36 gene were significantly associated with wool fineness score and fiber diameter (p<0.05), three genotypes of the KRT38 gene were significantly associated with the number of crimps (p<0.05); and three genotypes of the KRT85 gene were significantly associated with wool crimps score, body size, and fiber diameter (p<0.05). Analysis of the gene pyramiding effect between the different genotypes of the gene loci KRT36, KRT38, and KRT85, each genotype in a gene locus was combined with all the genotypes of another two gene loci and formed the different three loci combinations, indicated a total of 26 types of possible combined genotypes in the analyzed population. Compared with the other combined genotypes, the combinations CC-GG-II, CC-HH-IJ, CC-HH-JJ, DD-HH-JJ, CC-GH-IJ, and CC-GH-JJ at gene loci KRT36, KRT38, and KRT85, respectively, had a greater effect on wool traits (p<0.05). Conclusion: Our results indicate that the mutation loci of KRT31, KRT36, KRT38, and KRT85 genes, as well as the combinations at gene loci KRT36, KRT38, and KRT85 in CMXT have significant effects on wool traits, suggesting that these genes are important candidate genes for wool traits, which will contribute to sheep breeding and provide a molecular basis for improved wool quality in sheep.

• Clinical and Experimental Pediatrics
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• v.53 no.3
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• pp.397-407
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• 2010

Purpose : The F9 gene is known to be the causative gene for hemophilia B, but unfortunately the detection rate for restriction fragment length polymorphism-based linkage analysis is only 55.6%. Direct DNA sequencing can detect 98% of mutations, but this alternative procedure is very costly. Here, we conducted multiplex polymerase chain reactions (PCRs) and conformation sensitive gel electrophoresis (CSGE) to perform a screened DNA sequencing for the F9 gene, and we compared the results with direct sequencing in terms of accuracy, cost, simplicity, and time consumption. Methods : A total of 27 unrelated hemophilia B patients were enrolled. Direct DNA sequencing was performed for 27 patients by a separate institute, and multiplex PCR-CSGE screened sequencing was done in our laboratory. Results of the direct DNA sequencing were used as a reference, to which the results of the multiplex PCR-CSGE screened sequencing were compared. For the patients whose mutation was not detected by the 2 methods, multiplex ligation-dependent probe amplification (MLPA) was conducted. Results : With direct sequencing, the mutations could be identified from 26 patients (96.3%), whereas for multiplex PCRCSGE screened sequencing, the mutations could be detected in 23 (85.2%). One patient's mutation was identified by MLPA. A total of 21 different mutations were found among the 27 patients. Conclusion : Multiplex PCR-CSGE screened DNA sequencing detected 88.9% of mutations and reduced costs by 55.7% compared with direct DNA sequencing. However, it was more labor-intensive and time-consuming.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.11
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• pp.5659-5663
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• 2012

To investigate the expression of hPOT1 in the HeLa cell line and screen point mutations of hpot1 in different tumor tissues a two step osmotic method was used to extract nuclear proteins. EMSA was performed to determine the expression of hPOT1 in the HeLa cell line. PCR was also employed to amplify the exon14 sequence of the hpot1 gene in various of cancer tissues. A SV gel and PCR clean-up system was performed to enrich PCR products. DNAStar was used to analyse the exon14 sequence of the hpot1 gene. hPOT1 was expressed in the HeLa cell line and the signal was gradually enhanced as the amount of extracted nuclear proteins increased. The DNA fragment of exon14 of hpot1 was successfully amplified in the HeLa cell line and all cancer tissues, point mutations being observed in 2 out of 3 cases of endometrial cancer (66.7%) despite the hpot1 sequence being highly conserved. However, the sequence of hpot1 exon14 do not demonstrate point mutations in most cancer tissues. Since hPOT1 was expressed in HeLa cell and the probability of gene point variants was obviously higher in endometrial cancer than other cancers, it may be involved in the pathogenesis of gynecological cancers, especially in cervix and endometrium.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.2
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• pp.154-159
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• 2017

Objective: This study was designed to characterize the DNA polymorphisms of the melanocortin-1 receptor (MC1R) gene in indigenous Saudi Arabian sheep breeds exhibiting different color coats, along with individuals of the Sawaknee breed, an exotic sheep imported from Sudan. Methods: The complete coding region of MC1R gene including parts of 3' and 5' untranslated regions was amplified and sequenced from three the indigenous Saudi sheep; Najdi (generally black, n = 41), Naeimi (generally white with brown faces, n = 36) and Herri (generally white, n = 18), in addition to 13 Sawaknee sheep. Results: Five single nucleotide polymorphisms (SNPs) were detected in the MC1R gene: two led to nonsynonymous mutations (c.218 T>A, p.73 Met>Lys and c.361 G>A, p.121 Asp>Asn) and three led to synonymous mutations (c.429 C>T, p.143 Tyr>Tyr; c.600 T>G, p.200 Leu>Leu, and c.735 C>T, p.245 Ile>Ile). Based on these five SNPs, eight haplotypes representing MC1R $E^d$ and $E^+$ alleles were identified among the studied sheep breeds. The most common haplotype (H3) of the dominant $E^d$ allele was associated with either black or brown coat color in Najdi and Sawaknee sheep, respectively. Two other haplotypes (H6 and H7) of $E^d$ allele, with only the nonsynonymous mutation A218T, were detected for the first time in Saudi indigenous sheep. Conclusion: In addition to investigating the MC1R allelic variation in Saudi indigenous sheep populations, the present study supports the assumption that the two independent nonsynonymous Met73Lys and Asp121Asn mutations in MC1R gene are associated with black or red coat colors in sheep breeds.

• Neonatal Medicine
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• v.18 no.2
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• pp.370-373
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• 2011

Citrin deficiency caused by the SLC25A13 gene mutations is associated with both neonatal-onset type II citrullinemia (CTLN2), also known as neonatal intrahepatic cholestasis caused by citrin deficiency and adult-onset CTLN2. Neonatal-onset CTLN2 is an autosomal recessive disorder characterized by poor growth, intrahepatic cholestasis, and increased serum citrulline. A 16-days old infant with hyperammonemia was referred for evaluation of increased plasma citrulline diagnosed using tandem mass spectrometry. Blood amino acid analysis showed significant elevation of citrulline. Mild elevation in serum galactose levels had been found. DNA analysis of the SLC25A13 gene in this patient showed two novel compound heterozygous mutations, c.221C>T in exon4 and c.1645C in exon16 (p.[Ser74Phe]+[Gln549X]). We suggest that infants with a high serum citrulline level on a tandem mass screening test are candidates for gene analysis and blood amino acid analysis for neonatal-onset CTLN2.