scholarly journals DGGE Is More Sensitive for the Detection of Somatic Point Mutations than Direct Sequencing

BioTechniques ◽  
1999 ◽  
Vol 27 (2) ◽  
pp. 266-268 ◽  
Author(s):  
Barbara Trülzsch ◽  
Knut Krohn ◽  
Peter Wonerow ◽  
Ralf Paschke
Keyword(s):  
Direct Sequencing ◽  
Point Mutations

scholarly journals Prevalence of Gs alpha mutations in Korean patients with pituitary adenomas

2001 ◽  
Vol 168 (2) ◽  
pp. 221-226 ◽  
Author(s):  
HJ Kim ◽  
MS Kim ◽  
YJ Park ◽  
SW Kim ◽  
DJ Park ◽  
...  
Keyword(s):  
Tumor Size ◽  
Pituitary Adenomas ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Low Frequency ◽  
Oral Glucose ◽  
Fresh Tissue ◽  
Korean Patients ◽  
Gs Alpha ◽  
Non Functioning Pituitary Adenomas

The reported frequencies of Gs alpha mutations (gsp mutations) in growth hormone (GH)-secreting pituitary adenomas are variable (ranging from 4.4 to 43%), and the presence of these mutations in the other pituitary adenomas is still a matter of controversy. Previous clinical and biochemical analyses of patients with GH-secreting pituitary adenomas and gsp mutations produced conflicting results and did not demonstrate obvious characteristics. Therefore, we investigated the prevalence of gsp mutations in Korean patients with pituitary adenomas and elucidated the characteristics of these patients. Forty-four GH-secreting adenomas, 7 prolactin (PRL)-secreting adenomas and 32 clinically non-functioning adenomas were examined for the presence of point mutations in codon 201 and 227 of the Gs alpha gene using a nested PCR and direct sequencing of DNA extracted from fresh tissue or paraffin-embedded pituitary adenoma samples. Seven of the 44 GH-secreting pituitary adenomas had point mutations at codon 201 or 227; of these, five mutations were in codon 201 and two were in codon 227. In patients with gsp mutations, mean tumor size was significantly smaller than in patients without gsp mutations (15.9+/-8.7 mm vs. 24.9+/-14.9 mm, P<0.05). Age, sex, basal GH levels, GH response to oral glucose loading, GH response to octreotide and surgical outcome were not different in the two groups. One of the 32 clinically non-functioning pituitary adenomas had a point mutation at codon 201; none of the seven prolactinomas had these mutations. These results show that gsp mutations are not rare in Korean acromegalic patients and mean tumor size is significantly smaller in acromegalic patients with gsp mutations. Our results also confirm the low frequency of gsp mutations in clinically non-functioning pituitary adenomas and the absence of gsp mutations in prolactinoma.

scholarly journals Novel Point Mutations in the Dihydrofolate Reductase Gene of Plasmodium vivax: Evidence for Sequential Selection by Drug Pressure

2003 ◽  
Vol 47 (5) ◽  
pp. 1514-1521 ◽  
Author(s):  
Mallika Imwong ◽  
Sasithon Pukrittayakamee ◽  
Laurent Rénia ◽  
Franck Letourneur ◽  
Jean-Paul Charlieu ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Sequence Data ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Drug Pressure ◽  
Sequential Selection ◽  
Pcr Product ◽  
Reductase Gene ◽  
Antifolate Resistance ◽  
Type Sequence

ABSTRACT Mutations in the dihydrofolate reductase (dhfr) genes of Plasmodium falciparum and P. vivax are associated with resistance to the antifolate antimalarial drugs. P. vivax dhfr sequences were obtained from 55 P. vivax isolates (isolates Belem and Sal 1, which are established lines originating from Latin America, and isolates from patient samples from Thailand [n = 44], India [n = 5], Iran [n = 2], and Madagascar [n = 2]) by direct sequencing of both strands of the purified PCR product and were compared to the P. vivax dhfr sequence from a P. vivax parasite isolated in Pakistan (isolate ARI/Pakistan), considered to represent the wild-type sequence. In total, 144 P. vivax dhfr mutations were found at only 12 positions, of which 4 have not been described previously. An F→L mutation at residue 57 had been observed previously, but a novel codon (TTA) resulted in a mutation in seven of the nine mutated variant sequences. A new mutation at residue 117 resulted in S→T (S→N has been described previously). These two variants are the same as those observed in the P. falciparum dhfr gene at residue 108, where they are associated with different levels of antifolate resistance. Two novel mutations, I→L at residue 13 and T→M at residue 61, appear to be unique to P. vivax. The clinical, epidemiological, and sequence data suggest a sequential pathway for the acquisition of the P. vivax dhfr mutations. Mutations at residues 117 and 58 arise first when drug pressure is applied. Highly mutated genes carry the S→T rather than the S→N mutation at residue 117. Mutations at residues 57 and 61 then occur, followed by a fifth mutation at residue 13.

scholarly journals Denaturing-HPLC-Based Assay for Detection of ABL Mutations in Chronic Myeloid Leukemia Patients Resistant to Imatinib

2004 ◽  
Vol 50 (7) ◽  
pp. 1205-1213 ◽  
Author(s):  
Simona Soverini ◽  
Giovanni Martinelli ◽  
Marilina Amabile ◽  
Angela Poerio ◽  
Michele Bianchini ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Large Scale ◽  
Kinase Inhibitor ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Kinase Domain ◽  
Imatinib Treatment ◽  
Abl Tyrosine Kinase ◽  
Reverse Transcription Pcr

Abstract Background: Despite the efficacy of the BCR-ABL tyrosine kinase inhibitor Imatinib mesylate for the treatment of chronic myeloid leukemia (CML), resistance has been observed in a proportion of cases, especially those with advanced stages of the disease. Point mutations within the ABL kinase domain are emerging as the most frequent mechanism for reactivation of kinase activity within the leukemic clone. Methods: We developed a denaturing-HPLC (D-HPLC)-based assay for screening for ABL point mutations. For each sample, two partially overlapping fragments of 393 and 482 bp corresponding to the kinase domain were amplified by nested reverse transcription-PCR and analyzed under selected temperature and acetonitrile gradient conditions. Fifty-one bone marrow and/or peripheral blood specimens from 27 CML patients who showed cytogenetic resistance to Imatinib were screened in parallel by D-HPLC and by direct sequencing. Results: In 12 of 27 (44%) patients, D-HPLC showed an abnormal elution profile suggesting the presence of a nucleotide change. Direct sequencing confirmed the presence of a point mutation in all cases. Conversely, all samples scored as wild type by D-HPLC showed no evidence of mutations by direct sequencing. In two cases, novel amino acid substitutions at codons already known for being hot-spots of mutation were identified (F311I and E355D). Conclusions: The proposed D-HPLC-based assay is highly specific and at least as sensitive as sequencing; with respect to the latter, it provides a much faster and less expensive semiautomated system for mutational screening. It may therefore potentially be a valuable tool for regular, large-scale testing of patients undergoing Imatinib treatment.

scholarly journals Genotypic Resistance inHelicobacter pyloriStrains Correlates with Susceptibility Test and Treatment Outcomes after Levofloxacin- and Clarithromycin-Based Therapies

2010 ◽  
Vol 55 (3) ◽  
pp. 1123-1129 ◽  
Author(s):  
Jyh-Ming Liou ◽  
Chi-Yang Chang ◽  
Wang-Huei Sheng ◽  
Yu-Chi Wang ◽  
Mei-Jyh Chen ◽  
...  
Keyword(s):  
Treatment Outcomes ◽  
Triple Therapy ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Kappa Coefficient ◽  
23S Rrna ◽  
Genotypic Resistance ◽  
Phenotypic Resistance ◽  
Therapeutic Outcomes ◽  
H Pylori

ABSTRACTThe accuracy of genotypic resistance to levofloxacin (gyrAmutations) and its agreement with treatment outcomes after levofloxacin-based therapy have not been reported. We aimed to assess the correlation.Helicobacter pyloristrains isolated from patients who received levofloxacin-based and clarithromycin-based triple therapies in a previous randomized trial were analyzed for point mutations ingyrAand 23S rRNA. PCR followed by direct sequencing was used to assess thegyrAand 23S rRNA mutations. An agar dilution test was used to determine the MICs of clarithromycin and levofloxacin. We found that the agreement between genotypic and phenotypic resistance to levofloxacin was best when the MIC breakpoint was >1 μg/ml (kappa coefficient, 0.754). The eradication rates in patients with and withoutgyrAmutations were 41.7% and 82.7%, respectively (P= 0.003). The agreement between genotypic and phenotypic resistance to clarithromycin was best when the MIC breakpoint was >2 μg/ml (kappa, 0.694). The eradication rates in patients with and without 23S rRNA mutations were 7.7% and 93.5%, respectively (P< 0.001). The agreements (kappa coefficient) between therapeutic outcomes after clarithromycin-based triple therapy and genotypic and phenotypic resistance were 0.671 and 0.356, respectively. The agreements (kappa coefficient) between therapeutic outcomes after levofloxacin-based triple therapy and genotypic and phenotypic resistance were 0.244 and 0.190, respectively. In conclusion,gyrAand 23S rRNA mutations inH. pyloristrains appeared to be better markers than phenotypic resistance in the prediction of treatment outcomes. The optimal breakpoints for levofloxacin and clarithromycin resistance appeared to be >1 μg/ml and >2 μg/ml, respectively.

Flow Cytometric FLT3 Expression in Acute Leukaemias Is of Diagnostic Value but Does Not Correlate with ITD / D835Y Mutation Status.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3014-3014
Author(s):  
Colin Ridler ◽  
Matthew Smith ◽  
Tim Milne ◽  
Fatema Ebrahim ◽  
Marion Macey ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mononuclear Cells ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Specific Marker ◽  
Flow Cytometric ◽  
Significant Difference ◽  
Flt3 Expression ◽  
Lineage Markers ◽  
B Lineage

Abstract High level expression of FLT3 in acute myeloid (AML) and lymphoid (ALL) leukaemias has been demonstrated by PCR techniques. This suggests that FLT3 and its ligand may play a significant role in the proliferation of leukaemic blast cells. Mutations of FLT3 are the most common genetic lesion in AML, occurring in 20–30% of cases, and confer an adverse prognosis. Flow cytometry was used as an alternative method to investigate FLT3 expression in acute leukaemia. Surface expression of FLT3 was measured in 111 bone marrow or peripheral blood samples of patients with AML or ALL using a phycoerythrin-labelled monoclonal IgG1 antibody (Coulter-Immunotech). Exons 14 – 15 and 20 were amplified from DNA extracted from mononuclear cells of 38 of the 111 samples. Internal tandem duplication (ITD) and mutation of the D835 position of the activation loop of FLT3 were identified by SSCP and direct sequencing. 82% (47/57) of AML and 81% (34/42) of B-lineage ALL samples showed ≥ 20% (range, 20 – 97%) positivity for FLT3 using flow cytometry. The Mean Fluorescence Intensity (MFI) was similar between the two groups and no significant difference was found between adult (82%) and paediatric (80%) samples. However, FLT3 expression in all 9 T-ALL samples (pro, intermediate and late subtypes) was negative with markedly reduced MFIs. Of the 25 AML cases tested for ITD/D835Y, 5 (20%) were positive (4 ITDs, 1 D835Y). Surface FLT3 expression in these 5 cases (% and MFI) was the same as in those with wild-type FLT3. No mutations of FLT3 were found in the ALL cases tested (9 B-ALL, 4 T-ALL). Of three biphenotypic leukaemias, one case with myeloid/B lineage positivity was FLT3+ by flow cytometry, while another with myeloid/T lineage markers, was FLT3−. A diagnostic conundrum was a patient with AML morphologically (cytogenetics failed): CD33+, MPO−, CD13− CD117−; T-lineage markers CD2+, CD5+, CD7+, as well as TdT+. The T-lineage specific marker cytoplasmic CD3 was negative. Only FLT3 positivity allowed a definitive diagnosis of AML to be made. In conclusion, surface FLT3 expression was present in > 80% of AML and B-lineage ALL, but in 0% (0/9) of T-ALL cases. Detection of this antigen should be included in the diagnostic immunophenotyping of acute leukaemias and is important where T-ALL needs to be excluded. Flow cytometric FLT3 expression cannot differentiate cases with or without ITD or point mutations of FLT3.

Detection of the V617F Mutation of JAK2 Using a Novel Fully Automated SNP Super-Rapid Detector.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4640-4640
Author(s):  
Ruriko Tanaka ◽  
Junya Kuroda ◽  
Eishi Ashihara ◽  
Takayuki Ishikawa ◽  
Tomohiko Taki ◽  
...  
Keyword(s):  
Cell Line ◽  
Residual Disease ◽  
Detection System ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Wild Type ◽  
Snp Detection

Abstract The JAK2 V617F substitution mutation (JAK2V617F) is one of the genetic hallmarks of chronic myeloproliferative diseases (CMPDs), such as polycythemia vera (PV), essential thrombocythemia (ET), or chronic idiopathic myelofibrosis (CIMF). Accurate and rapid detection of this mutation is essential for diagnosing and treating CMPDs today. We have developed a novel, rapid, sensitive and fully-automated single nucleotide polymorphism (SNP) detection system, termed ARKRAY SNP Detection System (ASDS), and used it to detect JAK2V617F in patients with CMPDs. With ASDS, diagnosis requires only 100ml of whole blood (or DNA), and the system automatically performs DNA extraction and PCR. The detection of both wild type and mutant jak2 alleles from PCR amplicons was measured by the increase in fluorescence produced by the dissociation of a JAK2V617F-specific guanine-quenching probe, and was completed within 75 minutes. In dilution assays of HEL cells (a JAK2V617F-positive leukemia cell line) using MYL cells (a chronic myelogenous leukemia (CML)-derived cell line with wild type JAK2), the system reliably quantified the mutation in a cell population containing as few as 1.0% mutant cells (Figure). We tested 44 samples from CMPDs patients using ASDS and direct sequencing (DS) (13 PV, 23 ET, 3 CIMF, 1 chronic myelomonocytic leukemia (CMMoL), 1 chronic neutrophilic leukemia (CNL), 3 unclassifiable CMPD (uCMPD)), which included samples from 3 post-allogenic bone marrow transplantation (BMT) patients with CIMF or uCMPD. Using ASDS, we detected JAK2V617F in 12/13 PV, 13/23 ET, 0/1 CIMF without BMT, 1/1 CMMoL, 0/1 CNL and 1/3 uCMPD. Overall, these results were comparable to previous results using relatively sensitive detection strategies, such as allele-specific PCR (AS-PCR). One of the 3 post-BMT CIMF samples was positive for JAK2V617F, which indicated that there were residual disease clones after BMT. ASDS detected JAK2V617F in one PV and eight ET patients, while DS failed to detect the mutation in these same samples, which clearly indicated that ASDS has a higher sensitivity than DS. JAK2V617F was absent in all samples from secondary erythrocytemia and healthy volunteers. Collectively, these results demonstrate that ASDS is a powerful and convenient tool for detecting JAK2V617F. With its associated high sensitivity, convenience and rapidity, this system will enable “Point-of-care” testing in clinical laboratories and “Patient-oriented” therapy for CMPDs. ASDS could also be applied to the detection of other point mutations relevant to cancer treatment, such as mutations in the BCR-ABL kinase domain that are associated with CML. Figure Figure

Coding Region of IRF6 Gene May Not Be Causal for Van Der Woude Syndrome in Cases from India

2009 ◽  
Vol 46 (5) ◽  
pp. 541-544 ◽  
Author(s):  
Akhtar Ali ◽  
Subodh Kumar Singh ◽  
Rajiva Raman
Keyword(s):  
Present Report ◽  
Direct Sequencing ◽  
Geographical Area ◽  
Point Mutations ◽  
Coding Region ◽  
Van Der Woude Syndrome ◽  
Coding Regions ◽  
Irf6 Gene ◽  
Novel Variants ◽  
History Of

Objective: Evaluation of the IRF6 gene in Van der Woude syndrome cases from an Indian population. Subjects: Nine affected and four unaffected individuals from seven families with Van der Woude syndrome as well as five normal controls (with no history of Van der Woude or any other congenital malformation and belonging to the same geographical area as the families with Van der Woude syndrome). Method: Direct sequencing of all coding regions and exon-intron boundaries of the IRF6 gene. Results: Five novel variants: IVS1+3900 A>G, 191 T>C, IVS4+775 C>T, IVS8+218 C>T, 1511 T>A (Ser 416 Arg) and two known variants: IVS6+27 C>G, 1083 G>A (V274I) were detected. Except for one, all were in noncoding regions either in 3′UTR or in introns. There was only one mutation in the coding region, detected in a normal control. Conclusion: The present report indicates that point mutations in the coding region of the IRF6 gene may not be a major cause of Van der Woude syndrome in Indian populations.

scholarly journals Evidence of Selective Sweeps in Genes Conferring Resistance to Chloroquine and Pyrimethamine in Plasmodium falciparum Isolates in India

2009 ◽  
Vol 54 (3) ◽  
pp. 997-1006 ◽  
Author(s):  
Tonya Mixson-Hayden ◽  
Vidhan Jain ◽  
Andrea M. McCollum ◽  
Amanda Poe ◽  
Avinash C. Nagpal ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Cerebral Malaria ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Drug Resistant ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Mild Malaria ◽  
Resistant Genotypes

ABSTRACT Treatment of Plasmodium falciparum is complicated by the emergence and spread of parasite resistance to many of the first-line drugs used to treat malaria. Antimalarial drug resistance has been associated with specific point mutations in several genes, suggesting that these single nucleotide polymorphisms can be useful in tracking the emergence of drug resistance. In India, P. falciparum infection can manifest itself as asymptomatic, mild, or severe malaria, with or without cerebral involvement. We tested whether chloroquine- and antifolate drug-resistant genotypes would be more commonly associated with cases of cerebral malaria than with cases of mild malaria in the province of Jabalpur, India, by genotyping the dhps, dhfr, pfmdr-1, and pfcrt genes using pyrosequencing, direct sequencing, and real-time PCR. Further, we used microsatellites surrounding the genes to determine the origins and spread of the drug-resistant genotypes in this area. Resistance to chloroquine was essentially fixed, with 95% of the isolates harboring the pfcrt K76T mutation. Resistant genotypes of dhfr, dhps, and pfmdr-1 were found in 94%, 17%, and 77% of the isolates, respectively. Drug-resistant genotypes were equally likely to be associated with cerebral malaria as with mild malaria. We found evidence of a selective sweep in pfcrt and, to a lesser degree, in dhfr, indicating high levels of resistance to chloroquine and evolving resistance to pyrimethamine. Microsatellites surrounding pfcrt indicate that the resistant genotypes (SVMNT) were most similar to those found in Papua New Guinea.

scholarly journals Clinical description and mutational profile of a Moroccan series of patients with Rubinstein Taybi syndrome

2021 ◽  
Vol 21 (2) ◽  
pp. 960-967
Author(s):  
Siham Chafai Elalaoui ◽  
Wiam Smaili ◽  
Julien Van-Gils ◽  
Patricia Fergelot ◽  
Ilham Ratbi ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Major Gene ◽  
Point Mutations ◽  
Molecular Study ◽  
Molecular Data ◽  
Clinical Expertise ◽  
Cgh Array ◽  
Clinical Description ◽  
Moroccan Patients ◽  
Crebbp Gene

Background: Rubinstein-Taybi syndrome (RSTS; OMIM 180849) is a rare autosomal dominant developmental disorder with an estimated prevalence of one case per 125,000 live births. RSTS is characterized by typical face, broad thumbs and halluces, short stature, and intellectual disability. Facial dysmorphy is characteristic with microcephaly, low frontal hairline, arched eyebrows, long eyelashes, convex profile of nose, narrow palate, and micrognathia. RSTS is mainly due to mutations or microdeletions of the CREBBP gene (about 60%) and more rarely of the EP300 gene (8%). Objective: Clinical description and identification of mutations of patients with Rubinstein Taybi syndrome. Methods: PCR and direct sequencing of CREBBP gene. Results: We report here, the clinical and molecular data of a series of six Moroccan patients with a phenotype of RSTS. The molecular study of the major gene CREBBP (by Sanger Sequencing followed by CGH array, if sequence normal) revealed point mutations in five patients. For the sixth patient, CGH array revealed a microdeletion carrying the CREBBP gene. Through this work, we emphasize the importance of clinical expertise in the diagnosis, management and genetic counseling in Rubinstein Taybi syndrome. Keywords: Rubinstein Taybi syndrome; CREBBP gene; mutation; Moroccan.

Genetic Basis and Carrier Detection of Hemophilia B of Chinese Origin

1993 ◽  
Vol 69 (03) ◽  
pp. 247-252 ◽  
Author(s):  
Shu-Wha Lin ◽  
Ming-Ching Shen
Keyword(s):  
Amino Acid ◽  
Gene Deletion ◽  
Direct Sequencing ◽  
Screening Method ◽  
Point Mutations ◽  
Factor Ix ◽  
Hemophilia B ◽  
Sscp Analysis ◽  
Amino Acid Residues ◽  
Chinese Origin

SummaryWe have characterized the genetic defects of 17 hemophilia B patients of Chinese origin by means of the polymerase chain reaction (PCR) and direct sequencing. The single-strand conformation polymorphism (SSCP) was used as an initial screening method to analyze the entire coding region and the flanking introns of each individual’s factor IX gene. The abnormal exons were subsequently amplified and the nucleotide sequence determined. Of the 17 patients studied, 16 had single point mutations and one had a gross gene deletion of exons VII and VIII of factor IX. Among these 16 factor IX variants with point mutations 13 were missense and two were nonsense mutations. The remaining one had a nucleotide deleted, resulting in frame shifting at amino acid residue 97. A total of ten novel mutations, including the one with gross gene deletion, are reported in this study which have not been described previously. Five of the remaining seven variants were missense mutations with novel amino acids substituted for residues 127, 132, 180, 207, and 215, respectively. Mutations containing different amino acid residues at those positions have been reported. The last two are variants that have already been described to contain mutations at amino acid residues 333 and 365, respectively. To evaluate the efficiency of SSCP analysis in assessing the mutated exons and to further confirm our results we sequenced the entire exons of all 17 factor IX genes. The mutations detected by SSCP method were indeed the only mutation identified in each factor IX variant. The SSCP analysis and direct sequencing have also allowed us to circumvent the difficulties of carrier determination for Chinese by direct detection of the abnormal factor IX alleles inherited by the females.

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