BRAF mutation detection and identification by cycling temperature capillary electrophoresis

Hinselwood, David C.; Abrahamsen, Torveig Weum; Ekstrøm, Per Olaf

doi:10.1002/elps.200410427

Cited by 24 publications

(29 citation statements)

References 56 publications

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“…Although some have argued that RAF1 inhibits BRAF activation in a kinase domaindependent fashion (19), other studies reported that human cancer-associated BRAF mutants with impaired kinase activity promote MEK activation by binding to and trans-activating RAF1 (12,51). Of note, these kinase-impaired mutations alter the activation segment of BRAF, and some (D594G and T599I) are analogous to NS-associated RAF1 mutants (16,18). Heidorn and colleagues recently reported that mice with a conditional kinase-dead Braf (Braf LSL-D594A ) allele, when combined with oncogenic Ras (Kras G12D ), induced melanomas in mice.…”

Section: Raf1mentioning

confidence: 99%

Increased BRAF Heterodimerization Is the Common Pathogenic Mechanism for Noonan Syndrome-Associated RAF1 Mutants

Yin

Simpson

et al. 2012

Molecular and Cellular Biology

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Noonan syndrome (NS) is a relatively common autosomal dominant disorder characterized by congenital heart defects, short stature, and facial dysmorphia. NS is caused by germ line mutations in several components of the RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway, including both kinase-activating and kinaseimpaired alleles of RAF1 (ϳ3 to 5%), which encodes a serine-threonine kinase for MEK1/2. To investigate how kinase-impaired RAF1 mutants cause NS, we generated knock-in mice expressing Raf1 D486N . Raf1 D486N/؉ (here D486N/؉) female mice exhibited a mild growth defect. Male and female D486N/D486N mice developed concentric cardiac hypertrophy and incompletely penetrant, but severe, growth defects. Remarkably, Mek/Erk activation was enhanced in Raf1 D486N -expressing cells compared with controls. RAF1 D486N , as well as other kinase-impaired RAF1 mutants, showed increased heterodimerization with BRAF, which was necessary and sufficient to promote increased MEK/ERK activation. Furthermore, kinase-activating RAF1 mutants also required heterodimerization to enhance MEK/ERK activation. Our results suggest that an increased heterodimerization ability is the common pathogenic mechanism for NS-associated RAF1 mutations. N oonan syndrome (NS) is a relatively common (1 in 1,000 to 2,500 live births) autosomal dominant disorder (28,30,45), characterized by short stature, craniofacial dysmorphia, a wide spectrum of congenital cardiac anomalies, and an increased risk of hematopoietic malignancy. Although NS is genetically heterogeneous (1, 4, 49), all known cases are caused by germ line mutations in conserved components of the canonical RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) (here RAS/ERK) cascade, a key regulator of cell proliferation, differentiation, and survival (13, 24). Mutations in PTPN11, which encodes the protein tyrosine phosphatase SHP2, account for approximately one-half of NS cases (46). Other known NS genes include SOS1 (ϳ10%) (38, 47), RAF1 (3 to 5%) (31, 36), KRAS (Ͻ2%) (41, 56), NRAS (9), and SHOC2 (10) (Ͻ1 to 2%). Mutations in some of these genes, as well as in genes encoding other RAS/ERK pathway components, also cause phenotypically related disorders, such as neurofibromatosis type 1 (NF1), Costello syndrome, cardiofacio-cutaneous (CFC) syndrome, and LEOPARD syndrome (named for the major features of this disorder, including multiple lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonic stenosis, abnormal genitalia, retardation of growth, and sensorneural deafness); together with NS, these syndromes are now termed "RASopathies" (49). How mutations in the same signaling pathway cause similar yet clearly distinct phenotypes remains unclear. Consequently, a detailed understanding of RASopathy pathogenesis should yield new insights into RAS/ERK pathway regulation.RAF family serine-threonine kinases (22,25,53) function as key RAS effectors, phosphorylating and a...

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Section: Raf1mentioning

confidence: 99%

Increased BRAF Heterodimerization Is the Common Pathogenic Mechanism for Noonan Syndrome-Associated RAF1 Mutants

Yin

Simpson

et al. 2012

Molecular and Cellular Biology

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“…The standard test currently employed to analyze patients' biopsies before initiation of vemurafenib treatment relies on real time PCR (the COBAS TM Test 10 ), whereby 5-10% of BRAFmutated melanoma cells can be detected in a background of normal cells. Alternative technologies to classic methods are under development, such as cycling temperature capillary electrophoresis 11 with sensitivity comparable to COBAS TM , silicon nanowire field-effect transistors 12 and a threedimensional gold nanowire platform 13 . The latter technologies have only been shown to work using synthetic oligonucleotide targets, larger gene fragments or still rely on an initial PCR amplification.…”

mentioning

confidence: 99%

Direct detection of a BRAF mutation in total RNA from melanoma cells using cantilever arrays

et al. 2013

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Malignant melanoma, the deadliest form of skin cancer, is characterised by a predominant mutation in the BRAF gene. Drugs that target tumours carrying this mutation have recently entered the clinic. Therefore patients are routinely screened for mutations in this gene to determine whether they can benefit from this type of treatment. The current gold standard for mutation screening uses real time polymerase chain reaction (PCR) and sequencing methods. Here we show that an assay based on microcantilever arrays can detect the mutation nanomechanically without amplification in total RNA samples isolated from melanoma cells. The assay is based on a BRAF specific oligonucleotide probe. We detected mutant BRAF at a concentration of 500 pM in a 50-fold excess of the wild-type sequence. The method was able to distinguish melanoma cells carrying the mutation from wild type cells using as little as 20 ng/µl of RNA material, without prior PCR amplification and use of labels.The identification of alterations in specific signalling pathways and recurrent oncogenic mutations in particular types of cancers has led in the past decade to the explosion of targeted therapy approaches. In cutaneous melanoma, a significant improvement in overall survival has been achieved by vemurafenib and similar drugs that selectively inhibit tumours carrying a mutated BRAF 2 gene 1,2 . Additional drugs for combination therapies with higher efficacies and fewer side effects are in clinical trials 3 . BRAF is one of three RAF genes (rapidly accelerated fibrosarcoma A, B and C) encoding cytoplasmic protein serine/threonine kinases belonging to the mitogen-activated protein kinase (MAPK) signal transduction cascade, a pathway controlling various cellular processes such as proliferation, migration and survival 4,5 . BRAF somatic mutations are present in half of cutaneous melanomas. Over 90% of the mutations are a single T to A transversion at position 1799 in the BRAF coding sequence (cT1799A), which converts a valine amino acid residue at position 600 in the protein to a glutamic acid (V600E). This mutation renders the protein constitutively active, resulting in a deregulated MAPK pathway 6 and thus uncontrolled cell growth and cancer. BRAF mutations are also present in other neoplasms, including hairy cell leukemias, thyroid and colon carcinomas 7,8,9 . As the presence of the cT1799A/V600E BRAF (hereafter BRAF V600E ) mutation determines eligibility to BRAF inhibitor treatment, molecular screening of tumour biopsies is now carried out routinely. with sensitivity comparable to COBAS TM , silicon nanowire field-effect transistors 12 and a threedimensional gold nanowire platform 13 . The latter technologies have only been shown to work using synthetic oligonucleotide targets, larger gene fragments or still rely on an initial PCR amplification. In particular, they have not been applied to the direct identification of a mutated messenger RNA (mRNA) sequence in total RNA (constituted primarily by ribosomal RNA and containing all mRNAs transcribed from gen...

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“…Most recently we have illustrated how the Ekstrøm's laboratory development of cycling denaturing capillary electrophoresis (CyDCE) eliminated the labor-intensive optimization steps attendant on constant denaturing systems. 26,74,76,[81][82][83][84] .…”

Section: Applications In Human Dna Cells Tissues Tumors and Populamentioning

confidence: 99%

“…The data is then plotted as midpoint temperature versus the base sequence, creating a melting profile of the fragment 87 . The goal of melting profile analysis of DNA fragments with computer programs prior to DCE is to select and manipulate the target sequence, attach GC-clamp if necessary, so that the region of interest is in a low melting domain adjacent to a sequence with high temperature melting properties 28,53,82,87,[89][90][91] . The introduction of the thermally stable clamp increases the percentage of detectable DNA variants in the low melting domain by DGGE to close to 100% 92,93 .…”

Section: Design Of Dna Amplification Fragments With Appropriate Meltimentioning

confidence: 99%

“…The same method of allele separation was first demonstrated on a commercial capillary DNA sequencing in 2001 73,108 . DCE have been applied to 7 different capillary DNA sequencing instrument, ranging from single capillary up to 384 capillaries 38,56,73,74,78,81,82,84,89,105 . Due to the large variety of instrument and instrument protocols and setting, detailed running descriptions will not be given for each instrument.…”

Section: Denaturant Capillary Electrophoresis (Dce)mentioning

confidence: 99%

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Analysis of mutational spectra by denaturing capillary electrophoresis

et al. 2008

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Numbers and kinds of point mutant within DNA from cells, tissues and human population may be discovered for nearly any 75-250bp DNA sequence. High fidelity DNA amplification incorporating a thermally stable DNA "clamp" is followed by separation by denaturing capillary electrophoresis (DCE). DCE allows for peak collection and verification sequencing. DCE in a mode of cycling temperature, e.g.+/− 5°C, CyDCE, permits high resolution of mutant sequences using computer defined analytes without preliminary optimization experiments. DNA sequencers have been modified to permit higher throughput CyDCE and a massively parallel,~25,000 capillary system, has been designed for pangenomic scans in large human populations. DCE has been used to define quantitative point mutational spectra for study a wide variety of genetic phenomena: errors of DNA polymerases, mutations induced in human cells by chemicals and irradiation, testing of human genecommon disease associations and the discovery of origins of point mutations in human development and carcinogenesis.

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BRAF mutation detection and identification by cycling temperature capillary electrophoresis

Cited by 24 publications

References 56 publications

Increased BRAF Heterodimerization Is the Common Pathogenic Mechanism for Noonan Syndrome-Associated RAF1 Mutants

Increased BRAF Heterodimerization Is the Common Pathogenic Mechanism for Noonan Syndrome-Associated RAF1 Mutants

Direct detection of a BRAF mutation in total RNA from melanoma cells using cantilever arrays

Analysis of mutational spectra by denaturing capillary electrophoresis

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