A novel denaturing high-performance liquid chromatography (D-HPLC) based method for kit mutation screening of patients (pts) with systemic mastocytosis (SM)

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 7085-7085
Author(s):  
S. Colarossi ◽  
S. Soverini ◽  
A. Gnani ◽  
M. Rondoni ◽  
S. Gatto ◽  
...  
Keyword(s):  
High Performance ◽  
Hplc Analysis ◽  
Systemic Mastocytosis ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Screening Method ◽  
Rflp Analysis ◽  
Rt Pcr ◽  
Kit Mutation ◽  
Pcr Product

7085 Background: SM is characterized by activating mutations of Kit tyrosine kinase. While the enzimatic site (ES) type mutation D816V renders Kit resistant to imatinib, regulatory type mutations are sensitive to inhibition. Kit mutations screening with sensitive methods is important for an appropriate therapeutic management of SM. Methods: Our aims were: to set up and optimize a D-HPLC-based screening method for mutations in critical regions of Kit; to assess the sensitivity and reliability of our D-HPLC assay as compared to RFLP analysis; to characterize additional mutations. The analysis was performed on 51 SM pts. Results: For each sample, a RT-PCR product spanning the catalytic and activation loops (ES) was screened in parallel by D-HPLC, followed by sequencing of D-HPLC-positive cases, and by RFLP according to an reported method for the D816V detection. By RFLP analysis, 34/51 pts were positive for the D816V. By D- HPLC analysis, an abnormal eluition profile was seen in 36/51 pts - all the 34 RFLP-positive cases as well as two additional pts. Direct sequencing confirmed the presence of the D816V in all the 34 RFLP-positive cases and showed that in two of these cases a I798I polymorphism was also present. The two pts scored positive by D-HPLC but negative by RFLP were found to have the I798I polymorphism. The 15 pts who did not harbour ES type mutations were further investigated by D-HPLC analysis of a RT-PCR product spanning the transmembrane (TM) and juxtamembrane (JM) domains. D-HPLC showed an abnormal elution profile in 5 pts. By direct sequencing one patient showed the K546K mutation and 4 pts showed the M541L polymorphism. Conclusions: Our D-HPLC-based assay proved a straightforward, reliable and sensitive method for Kit mutation analysis and highlighted the importance of screening for mutations other than the D816V. No significant financial relationships to disclose.

A Novel Denaturing-High Performance Liquid Chromatography (D-HPLC)-Based Method for Kit Mutation Screening of Patients (pts) with Systemic Mastocytosis (SM) Allows the Identification of Unreported Kit Variants.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4849-4849
Author(s):  
Sabrina Colarossi ◽  
Simona Soverini ◽  
Alessandra Gnani ◽  
Michela Rondoni ◽  
Simona Gatto ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Point Mutation ◽  
Hplc Analysis ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Rflp Analysis ◽  
Rt Pcr ◽  
Kit Mutation ◽  
Pcr Product ◽  
Tm Domain

Abstract SM is characterized by activating mutations of the Kit tyrosine kinase receptor. While the so-called ‘enzimatic site’ (ES) type mutation D816V renders Kit resistant to imatinib, ‘regulatory’ type mutations are sensitive to inhibition. Kit mutation screening with sensitive methods is important for appropriate therapeutic management of SM. Our aims were: to set up and optimize a D-HPLC-based screening method for mutations in different critical regions of the kit receptor; to assess the sensitivity and reliability of our D-HPLC assay as compared to RFLP analysis; to characterize additional mutations/polymorphisms. The analysis was performed on 37 SM pts. For each sample, a RT-PCR product spanning the catalytic and activation loops in the ES was screened in parallel by D-HPLC, followed by sequencing of D-HPLC-positive cases, and by RFLP according to an already reported method for the detection of the D816V. By RFLP analysis, 24/37 (65%) pts were positive for the D816V. By D-HPLC analysis, an abnormal eluition profile was seen in 26/37 (70%) pts - all the 24 pts scored as mutated by RFLP as well as two additional pts. Direct sequencing confirmed the presence of the D816V in all the 24 RFLP-positive cases, and showed that in two of these cases a I798I polymorphism was also present. The two pts scored positive by D-HPLC but negative by RFLP were found to have the same I798I polymorphism. The 11 pts who did not harbour ES type mutations were further investigated by D-HPLC analysis of a RT-PCR product corresponding to the transmembrane (TM) and juxtamembrane (JM) domains. D-HPLC showed an abnormal elution profile in 4 pts. Direct sequencing confirmed the presence of a point mutation in all cases. One patient showed a silent mutation at codon 546. Three pts showed a novel point mutation at codon 541 in the TM domain, resulting in a Met to Leu amino acid substitution. This is the second Kit TM domain mutation reported in a human disease and further supports the hypothesis of a role of the TM domain in regulating the enzymatic activity of an otherwise normal catalytic site. Further characterization of this novel mutant is ongoing. Morphologic and cytofluorimetric analyses of bone marrow biopsies and aspirates will be compared in order to assess whether the pts share any peculiar pathologic feature. Cos-7 cells are currently being transfected with M541L, D816V and wild-type kit in order to evaluate the effects of the M541L on kit enzymatic activity by western blot analysis of total and phosphorylated kit. Patient mast cells will be cultured in the presence or absence of kit ligand or imatinib, dasatinib and nilotinib in order to assess the sensitivity of the M541L to different kit inhibitors. Our novel D-HPLC-based assay proved a straightforward, reliable and sensitive method for kit mutation analysis. It also highlighted the importance of screening for mutations other than the D816V. D-HPLC analysis allowed us to find a novel M541L mutation which is now under characterization.

Detection of BCR-ABL Kinase Domain Mutations in Chronic Myeloid Leukemia Patients Treated with Tirosin-Kinase Inhibitors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4261-4261
Author(s):  
Gonzalo Manrique ◽  
Roberta Bittencout ◽  
Verónica Pérez ◽  
Vanesa Sholl ◽  
Monica Cappetta ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Direct Sequencing ◽  
Low Cost ◽  
Mutation Screening ◽  
Point Mutations ◽  
Kinase Domain ◽  
Rapid Screening ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Allele Specific

Abstract Background. Point mutations in the kinase domain (KD) of the BCR-ABL are the most frequent mechanism of drug resistance in CML patients treated with kinase inhibitors (TKI). More than 80 mutations with different frequency and clinical significance have been reported. One of them, the T315I confers resistance to all TKIs available. The detection of mutations in KD allows early identification of high-risk patients and therefore guides clinical therapy decisions. Aim. To assess the mutation status of a group of CML pts resistant to TKI from Uruguay (n=35) and Brazil (n=30). Methods. KD mutation screening was performed by RT-PCR and direct sequencing according to Branford et al. (2002). Additionally, we developed a rapid, specific, sensitive and low cost allele specific (AS)-RT-PCR assay to identify T315I, using Branford’s KD amplification primers in combination with an allele specific primer for the T315I point mutation detection. BCR-ABL transcript levels were also measured by RQ-PCR according to international recommendations. Results and Discussion. RT-PCR and direct sequencing analyses performed in all pts showed the presence of T315l mutation in 3/65 cases. Other 11 showed the alternative mutations Y253H (n=2), E450A, G250E (n=2), E459K (n=2), E450G, F317L (n=2) and E255K; and the remaining 55 showed no mutations in the ABL KD. All 65 samples together with cDNA from 15 non-resistant CML pts and 10 cDNA from non-CML were analyzed by AS-RT-PCR assay for T315l mutation in order to validate the method. T315l was identified in the 3 samples in which the mutation was previously detected by direct sequencing and in 1 pt that had been classified as KD mutation negative. This result was then confirmed by direct sequencing of the AS-PCR product. T315 was neither detected in samples positive for other mutations nor in samples of non-resistant CML and non-CML patients, supporting the specificity of the method. Assessment of the sensitivity of the AS-RT-PCR was performed on serial dilutions experiments using RNA from T315 positive pt into RNA from CML-T315l negative pt, showing that the T315I mutation was detectable to a level of 0.01 % by AS-PCR, while through direct sequencing method the sensitivity was 10–20%. The prevalence of mutations in our study was 15/65 (23%). Conclusions. Our results showed that the AS-RT-PCR described here is a convenient and easy tool to be used in a clinical routine laboratory for rapid screening for BCR-ABL T315. This, together with direct sequencing, constitutes a suitable approach for CML resistance monitoring and therapeutic choice.

dHPLC as a Rapid and Reliable Screening Method for A-Thalassemia Screening.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3774-3774
Author(s):  
Luca Bernasconi ◽  
Roberto Herklotz ◽  
Martin Hergersberg ◽  
Andreas Huber
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Restriction Analysis ◽  
Direct Sequencing ◽  
Single Gene ◽  
Globin Gene ◽  
Mutation Screening ◽  
Screening Method ◽  
Screening Methods ◽  
Intrauterine Death ◽  
Globin Genes

Abstract Background: a-thalassemia, the most prevalent of all thalassemias, is an inherited single gene disorder of the hemoglobin synthesis characterized by the absence or the reduced expression of a-globin genes resulting in an imbalanced ratio between a- and b-gobin chain synthesis. While some DNA alterations lead to microcytic anemia, the most others cause severe anemia and intrauterine death if co-inherited in a homozygous constellation. The most common genetic defects leading to the a-thalassemia phenotype are deletions of one or more of the two highly homologous a-globin genes. Less frequently, a-thalassemia derives from nondeletional mutations located in parts of the a-globin gene that are critical for its normal expression. So far the identification of the genetic background of a-thalassemic patients has included specific amplification of each a-globin gene followed by laborious and expensive mutation analysis methods such as denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism (SSCP) or direct sequencing of the entire a-genes. Method: We attempted to evaluate the performance of denaturing high performance liquid chromatography (dHPLC) technique to identify nondeletional mutations located in the a1- or a2-globin gene. Due to the high sequence homology of a1- and a2-globin genes, separate amplification of the two genes followed by nested PCR were performed. This generated four overlapping amplicons (a-dHPLC1 to 4) covering the whole a1/a2-globin locus including the 3′- and 5′-untranslated regions. The formation of DNA hetero-/homoduplexes was ensured by denaturing and slow reannealing of crude PCR products. Through a DNA separation column under partial-denaturing conditions, DNA homoduplexes were retained longer than their corresponding heteroduplexes generating distinguishable chromatographic profiles and hence allowing for the differentiation between wildtype and mutant DNA. Samples which differed from the wildtype in their elution profile were sequenced in both directions. Results: 50 patients showing a presumptive a-thalassemic hematological profile not due to deletional mutations (excluded by gap PCR technique) were tested. 10 carriers of a-thalassemia nondeletional alleles (previously identified by endonuclease restriction analysis) and 10 healthy patients were also included in the evaluation. Conclusion: Our results show that dHPLC is a reliable, sensitive, and specific screening method to detect pointmutations and small deletions located in the a-globin genes. Furthermore, the low costs of analysis and the rapidity of the screening (about 6 min/sample) make the dHPLC technique an appropriate alternative to technically demanding and time consuming mutation screening methods such as DGGE or SSCP analysis.

Frequency of JAK2 Exon 12 Mutations in JAK2 Exon 14 V617F-Negative Patients: High Frequency in ET Patients.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2539-2539
Author(s):  
Veronique M. Duke ◽  
Nicole Gurunlian ◽  
Bi Yogashangari ◽  
Tom Colley ◽  
Christopher McNamara ◽  
...  
Keyword(s):  
High Performance ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Correct Diagnosis ◽  
Myeloproliferative Disorders ◽  
Janus Kinase ◽  
Polycythaemia Vera ◽  
Chiari Syndrome ◽  
Gene Scanning ◽  
V617f Mutation

Abstract The identification of the G to T point (V617F) mutation in exon 14 of the JH2 domain of the Janus kinase 2 gene (JAK2) has recently simplified the diagnosis of many patients affected with a variety of myeloproliferative disorders (MPDs). More recently JAK2 exon 12 mutations have been described in these disorders; although the role of these mutations is as yet unclear. There have been only two investigations which have identified the presence of exon 12 mutations in clinical diagnosis among polycythaemia vera (PV) (Scott et al NEJM 2007, Pardanani et al Leukaemia 2007). A total of 725 patients diagnosed clinically with a variety of MPDs were investigated for both the frequently occurring exon 14 V617F and for the rarer exon 12 mutations. Methods: Peripheral blood samples from all patients were screened for the V617F mutation by gene-scaning on an ABI 3130 fragment analyser. 478 of these patients were further screened for the exon 12 mutant allele by denaturing high performance liquid chromatography (Transgenomic WAVE®) and direct sequencing. Results: Of 725 patients screened for the V617F mutation 204 (28%) were found to be heterozygous (HET) and 30 (4%) homozygously (HM) mutated while 491 (68%) were wildtype (WT). 478 were further tested for the exon 12 mutation and eight were found to carry mutations. Two of the patients had previously described mutations while three novel mutations were described in the remaining six patients. Only one of these was in the PV group while the majority of exon 12 mutations clustered in the essential thrombocythemia (ET) (4/8) and Budd Chiari patients (2/8). All exon 12 mutations were identified at very low level. Conclusions: Combined rapid gene-scanning and WAVE analysis allows for the characterisation of a large cohort of patients diagnosed clinically to have a variety of MPDs. This helps with the correct diagnosis of a frequently difficult group of patients. Detection of exon 12 mutations should be used in patients who are WT for V617F, including patients in the ET and the Budd Chiari syndrome groups. Both V617F and exon 12 mutation analysis tests should be included in primary screening in the routine management of MPD patients. Diagnosis Total WT for V617F (mutant exon 12) HET for V617F (mutant exon 12) HM for V617F (mutant exon 12) Results of Exon 14 V617F and exon 12 mutation screening Budd Chiari/PVT 60 42 (2) 16 (0) 2 (0) Polycythemia Vera (PV) 187 112 (1) 63 (0) 12 (0) Reactive PV 28 28 (0) 0 (none tested) 0 (none tested) ET 198 123 (4) 71 (none tested) 4 (none tested) MPD 123 87 (1) 29 (none tested) 7 (none tested) Inspecified diagnosis 129 99 (none tested) 25 (none tested) 5 (none tested) TOTAL 725 491 (8) 204 (0) 30 (0)

scholarly journals A Genetic Shift in the Virus Strains that Cause Mosaic in Louisiana Sugarcane

Plant Disease ◽  
2007 ◽  
Vol 91 (4) ◽  
pp. 453-458 ◽  
Author(s):  
M. P. Grisham ◽  
Y.-B. Pan
Keyword(s):  
Mosaic Virus ◽  
Rflp Analysis ◽  
Diseased Plant ◽  
Rt Pcr ◽  
Pcr Product ◽  
Polymerase Chain ◽  
Genetic Shift ◽  
Virus Isolates ◽  
Virus Strains ◽  
Sugarcane Industry

Leaf samples from 693 sugarcane plants showing mosaic symptoms were collected in 2001, 2002, and 2003 at 12 locations within the Louisiana sugarcane industry. Virus isolates associated with the diseased plants were identified using reverse-transcriptase polymerase chain reaction (RT-PCR) to distinguish between Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV). No SCMV strain was associated with any diseased plant collected during the survey. RT-PCR-based restriction fragment length polymorphism (RFLP) analysis showed that SrMV strains I, H, and M were associated with 67, 10, and 2% of the plants with mosaic symptoms, respectively. In previous surveys conducted between 1978 and 1995, over 90% of the plants sampled were infected with SrMV strain H. The remaining plants mostly were infected with SrMV strain I, except for an occasional sample with SrMV strain M. RT-PCR showed that approximately 13% of the samples collected between 2001 and 2003 were infected with SrMV, but the RFLP banding pattern did not match any described strain. Twelve plants were co-infected by two SrMV strains and two plants by three SrMV strains. No RT-PCR product was produced by either the SCMV- or the SrMV-specific RT-PCR primer set for 8% of the plants showing mosaic symptoms, suggesting that another virus may cause sugarcane mosaic in Louisiana.

PDGFRA Mutations in Gastrointestinal Stromal Tumors: Frequency, Spectrum and In Vitro Sensitivity to Imatinib

2005 ◽  
Vol 23 (23) ◽  
pp. 5357-5364 ◽  
Author(s):  
Christopher L. Corless ◽  
Arin Schroeder ◽  
Diana Griffith ◽  
Ajia Town ◽  
Laura McGreevey ◽  
...  
Keyword(s):  
Gastrointestinal Stromal Tumors ◽  
High Performance ◽  
Kinase Inhibitor ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Growth Factor Receptor ◽  
Stromal Tumors ◽  
Pdgfra Mutation ◽  
Factor Receptor Alpha

Purpose Gastrointestinal stromal tumors (GISTs) commonly harbor oncogenic mutations of the KIT tyrosine kinase, which is a target for the kinase inhibitor imatinib. A subset of GISTs, however, contains mutations in the homologous kinase platelet derived growth factor receptor alpha (PDGFRA), and the most common of these mutations is resistant to imatinib in vitro. Little is known of the other types of PDGFRA mutations that occur in GISTs. Materials and Methods We determined the KIT and PDGFRA mutation status of 1,105 unique GISTs using a combination of denaturing high-performance liquid chromatography and direct sequencing. Results There were 80 tumors (7.2%) with a PDGFRA mutation: 66 in exon 18, 11 in exon 12, and three in exon 14. Transient expression of representative PDGFRA isoforms in CHO cells revealed imatinib sensitivity of exon 12 mutations (SPDHE566-571R and insertion ER561-562) and an exon 14 substitution (N659K). However, most isoforms with a substitution involving codon D842 in exon 18 (D842V, RD841-842KI, DI842-843IM) were resistant to the drug, with the exception of D842Y. Interestingly, other mutations in exon 18 (D846Y, N848K, Y849K and HDSN845-848P) were all imatinib sensitive. Proliferation studies with BA/F3 cell lines stably expressing selected PDGFRA mutant isoforms supported these findings. Conclusion Including our cases, there are 289 reported PDGFRA-mutant GISTs, of which 181 (62.6%) had the imatinib-resistant substitution D842V. However, our findings suggest that more than one third of GISTs with PDGFRA mutations may respond to imatinib and that mutation screening may be helpful in the management of these tumors.

scholarly journals Denaturing High-Performance Liquid Chromatography Detection of Ribosomal Mutations Conferring Macrolide Resistance in Gram-Positive Cocci

2004 ◽  
Vol 48 (1) ◽  
pp. 297-304 ◽  
Author(s):  
Annie Canu ◽  
Ahmed Abbas ◽  
Brigitte Malbruny ◽  
François Sichel ◽  
Roland Leclercq
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
High Performance ◽  
Mutation Screening ◽  
Screening Method ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Gram Positive ◽  
Automated Method ◽  
Gram Positive Cocci

ABSTRACT Mutations in genes coding for L4 (rplD) or L22 (rplV) ribosomal proteins or in 23S rRNA (rrl gene) are reported as a cause of macrolide resistance in streptococci and staphylococci. This study was aimed at evaluating a denaturing high-performance liquid chromatography (DHPLC) technique as a rapid mutation screening method. Portions of these genes were amplified by PCR from total DNA of 48 strains of Streptococcus pneumoniae (n = 22), Staphylococcus aureus (n = 16), Streptococcus pyogenes (n = 6), Streptococcus oralis (n = 2), and group G streptococcus (n = 2). Thirty-seven of these strains were resistant to macrolides and harbored one or several mutations in one or two of the target genes, and 11 were susceptible. PCR products were analyzed by DHPLC. All mutations were detected, except a point mutation in a pneumococcal rplD gene. The method detected one mutated rrl copy out of six in S. aureus. This automated method is promising for screening of mutations involved in macrolide resistance in gram-positive cocci.

scholarly journals A novel detection method for heteroduplex DNA using carbodiimide-induced interrupted primer extension

2010 ◽  
Vol 1 (1) ◽  
pp. 5
Author(s):  
Tania Tabone ◽  
Richard Cotton ◽  
Ninan Mathew ◽  
J. Des Parkin ◽  
Judy Savige
Keyword(s):  
Novel Mutation ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Screening Method ◽  
Primer Extension ◽  
Cystic Kidney Disease ◽  
Primer Binding Site ◽  
Cystic Kidney ◽  
Renal Diseases ◽  
Inherited Disease

Direct sequencing may be problematic in demonstrating mutations where inherited disease results from multiple different heterozygous variants in large genes. We describe here a novel mutation screening method based on the ability of carbodiimide to bind mismatched DNA and interrupt primer extension thereby identifying both a heterozygous variant and its location. This assay detected all four classes of DNA mismatch in 550 bp engineered plasmid fragments and in two dominantly inherited renal diseases. In patients with thin basement membrane nephropathy, the method demonstrated multiple variants within a single amplicon including some close to the primer binding site. This method also detected a complex mutation in medullary cystic kidney disease type 2 (c.278-289 del/insCCGGCTCCT) as multiple termination events and, furthermore, correctly identified five affected and 28 unaffected family members. Carbodiimide-induced interrupted primer extension identifies heterozygous variants in large or multiexonic genes, where the variants differ in each family, their locations are unknown, and even if there are multiple known non-pathogenic variants within the same amplicon. This assay incorporates a “universal” protocol that detects all types of mutations without the need for further optimization, and potentially detects mutations where the proportion of heteroduplex is less than 50%.

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