Amniotic fluid insulin-like growth factor binding protein 3 concentration as early indicator of fetal growth restriction.

OBJECTIVE: Insulin-like growth factor-I (IGF-I) is an important regulator of fetal growth and its bioavailability depends on insulin-like growth factor binding proteins (IGFBPs). Genes coding for IGF-I and IGFBP3 are polymorphic. We hypothesized that either amniotic fluid protein concentration at the beginning of the second trimester or genotype of one of these two genes could be predictive of abnormal fetal growth. STUDY DESIGN: Amniotic fluid samples (14-18 weeks of pregnancy) from 123 patients with appropriate for gestational age (AGA) fetuses, 39 patients with small for gestational age (SGA) fetuses and 34 patients with large for gestational age (LGA) were analyzed. Protein concentrations were evaluated by ELISA and gene polymorphisms by PCR. RESULTS: Amniotic fluid IGFBP3 concentrations were significantly higher in SGA compared to AGA group (P=0.030), and this was even more significant when adjusted to gestational age at the time of amniocentesis and other covariates (ANCOVA analysis: P=0.009). Genotypic distribution of IGF-I variable number of tandem repeats (VNTR) polymorphism was significantly different in SGA compared to AGA group (P=0.029). 19CA/20CA genotype frequency was threefold decreased in SGA compared to AGA group and the risk of SGA occurrence of this genotype was decreased accordingly: OR=0.289, 95%CI=0.1-0.9, P=0.032. Genotype distribution of IGFBP3(A-202C) polymorphism was similar in all three groups. CONCLUSIONS: High IGFBP3 concentrations in amniotic fluid at the beginning of the second trimester are associated with increased risks of SGA while 19CA/20CA genotype at IGF-I VNTR polymorphism is associated with reduced risks of SGA. Neither IGFBP3 concentrations, nor IGF-I/IGFBP3 polymorphisms are associated with modified risks of LGA.

Cloning and characterization of SmZF1, a gene encoding a Schistosoma mansoni zinc finger protein

The zinc finger motifs (Cys2His2) are found in several proteins playing a role in the regulation of transcripton. SmZF1, a Schistosoma mansoni gene encoding a zinc finger protein was initially isolated from an adult worm cDNA library, as a partial cDNA. The full sequence of the gene was obtained by subcloning and sequencing cDNA and genomic fragments. The collated gene sequence is 2181 nt and the complete cDNA sequence is 705 bp containing the full open reading frame of the gene. Analysis of the genome sequence revealed the presence of three introns interrupting the coding region. The open reading frame theoretically encodes a protein of 164 amino acids, with a calculated molecular mass of 18,667Da. The predicted protein contains three zinc finger motifs, usually present in transcription regulatory proteins. PCR amplification with specific primers for the gene allowed for the detection of the target in egg, cercariae, schistosomulum and adult worm cDNA libraries indicating the expression of the mRNA in these life cycle stages of S. mansoni. This pattern of expression suggests the gene plays a role in vital functions of different life cycle stages of the parasite. Future research will be directed to elucidate the functional role of SmZF1.

Molecular Modeling Approaches for Determining Gene Function: application to a Putative Poly-A Binding Protein from Leishmania amazonensis (LaPABP)

The great expansion in the number of genome sequencing projects has revealed the importance of computational methods to speed up the characterization of unknown genes. These studies have been improved by the use of three dimensional information from the predicted proteins generated by molecular modeling techniques. In this work, we disclose the structure-function relationship of a gene product from Leishmania amazonensis by applying molecular modeling and bioinformatics techniques. The analyzed sequence encodes a 159 aminoacids polypeptide (estimated 18 kDa) and was denoted LaPABP for its high homology with poly-A binding proteins from trypanosomatids. The domain structure, clustering analysis and a three dimensional model of LaPABP, basically obtained by homology modeling on the structure of the human poly-A binding protein, are described. Based on the analysis of the electrostatic potential mapped on the model's surface and conservation of intramolecular contacts responsible for folding stabilization we hypothesize that this protein may have less avidity to RNA than it's L. major counterpart but still account for a significant functional activity in the parasite. The model obtained will help in the design of mutagenesis experiments aimed to elucidate the mechanism of gene expression in trypanosomatids and serve as a starting point for its exploration as a potential source of targets for a rational chemotherapy.

Positive control of swarming, rhamnolipid synthesis, and lipase production by the posttranscriptional RsmA/RsmZ system in Pseudomonas aeruginosa PAO1.

In Pseudomonas aeruginosa, the small RNA-binding, regulatory protein RsmA is a negative control element in the formation of several extracellular products (e.g., pyocyanin, hydrogen cyanide, PA-IL lectin) as well as in the production of N-acylhomoserine lactone quorum-sensing signal molecules. RsmA was found to control positively the ability to swarm and to produce extracellular rhamnolipids and lipase, i.e., functions contributing to niche colonization by P. aeruginosa. An rsmA null mutant was entirely devoid of swarming but produced detectable amounts of rhamnolipids, suggesting that factors in addition to rhamnolipids influence the swarming ability of P. aeruginosa. A small regulatory RNA, rsmZ, which antagonized the effects of RsmA, was identified in P. aeruginosa. Expression of the rsmZ gene was dependent on both the global regulator GacA and RsmA, increased with cell density, and was subject to negative autoregulation. Overexpression of rsmZ and a null mutation in rsmA resulted in quantitatively similar, negative or positive effects on target genes, in agreement with a model that postulates titration of RsmA protein by RsmZ RNA.

Promoter and transcription analysis of penicillin-binding protein genes in Streptococcus gordonii.

An optimally cross-linked peptidoglycan requires both transglycosylation and transpeptidation, provided by class A and class B penicillin-binding proteins (PBPs). Streptococcus gordonii possesses three class A PBPs (PBPs 1A, 1B, and 2A) and two class B PBPs (PBPs 2B and 2X) that are important for penicillin resistance. High-level resistance (MIC, > or =2 microg/ml) requires mutations in class B PBPs. However, although unmutated, class A PBPs are critical to facilitate resistance development (M. Haenni and P. Moreillon, Antimicrob. Agents Chemother. 50:4053-4061, 2006). Thus, their overexpression might be important to sustain the drug. Here, we determined the promoter regions of the S. gordonii PBPs and compared them to those of other streptococci. The extended -10 box was highly conserved and complied with a sigma(A)-type promoter consensus sequence. In contrast, the -35 box was poorly conserved, leaving the possibility of differential PBP regulation. Gene expression in a penicillin-susceptible parent (MIC, 0.008 microg/ml) and a high-level-resistant mutant (MIC, 2 microg/ml) was monitored using luciferase fusions. In the absence of penicillin, all PBPs were constitutively expressed, but their expression was globally increased (1.5 to 2 times) in the resistant mutant. In the presence of penicillin, class A PBPs were specifically overexpressed both in the parent (PBP 2A) and in the resistant mutant (PBPs 1A and 2A). By increasing transglycosylation, class A PBPs could promote peptidoglycan stability when transpeptidase is inhibited by penicillin. Since penicillin-related induction of class A PBPs occurred in both susceptible and resistant cells, such a mutation-independent facilitating mechanism could be operative at each step of resistance development.

Cryo-negative staining reveals conformational flexibility within yeast RNA polymerase I.

The structure of the yeast DNA-dependent RNA polymerase I (RNA Pol I), prepared by cryo-negative staining, was studied by electron microscopy. A structural model of the enzyme at a resolution of 1.8 nm was determined from the analysis of isolated molecules and showed an excellent fit with the atomic structure of the RNA Pol II Delta4/7. The high signal-to-noise ratio (SNR) of the stained molecular images revealed a conformational flexibility within the image data set that could be recovered in three-dimensions after implementation of a novel strategy to sort the "open" and "closed" conformations in our heterogeneous data set. This conformational change mapped in the "wall/flap" domain of the second largest subunit (beta-like) and allows a better accessibility of the DNA-binding groove. This displacement of the wall/flap domain could play an important role in the transition between initiation and elongation state of the enzyme. Moreover, a protrusion was apparent in the cryo-negatively stained model, which was absent in the atomic structure and was not detected in previous 3D models of RNA Pol I. This structure could, however, be detected in unstained views of the enzyme obtained from frozen hydrated 2D crystals, indicating that this novel feature is not induced by the staining process. Unexpectedly, negatively charged molybdenum compounds were found to accumulate within the DNA-binding groove, which is best explained by the highly positive electrostatic potential of this region of the molecule, thus, suggesting that the stain distribution reflects the overall surface charge of the molecule.

Release of extracellular particles by recombinant vaccinia virus over-expressing the major envelope protein p37K.

During the replication cycle of vaccinia virus, four different forms of viral particles are produced. The two extracellular enveloped forms, cell-associated enveloped virus and extracellular enveloped virus, are responsible for cell-to-cell transmission and long-range spread of infection both in vivo and in vitro. Despite the biological importance of the enveloped forms, the mechanism of envelopment and the components involved in this process have been analysed only recently. Therefore the individual steps and the rate-limiting factors of the envelopment process are still unknown. The protein p37K, an unglycosylated but acylated envelope protein of molecular mass 37 kDa, has been shown to be essential for envelopment. However, this study shows that over-expression of p37K by vaccinia virus recombinants reduces rather than increases the yield of infectious enveloped virus which is mainly due to the enveloped virions exhibiting a strongly diminished specific infectivity.

Arterial properties in relation to genetic variations in the adducin subunits in a white population.

BACKGROUND: Adducin is a membrane skeleton protein, which consists of either alpha- and beta- or alpha- and gamma-subunits. We investigated whether arterial characteristics might be related to the genes encoding ADD1 (Gly460Trp-rs4961), ADD2 (C1797T-rs4984), and ADD3 (IVS11+386A>G-rs3731566). METHODS: We randomly recruited 1,126 Flemish subjects (mean age, 43.8 years; 50.3% women). Using a wall-tracking ultrasound system, we measured the properties of the carotid, femoral, and brachial arteries. We studied multivariate-adjusted phenotype-genotype associations, using a population- and family-based approach. RESULTS: In single-gene analyses, brachial diameter was 0.15 mm (P = 0.0022) larger, and brachial distensibility and cross-sectional compliance were 1.55 x 10(-3)/kPa (P = 0.013) and 0.017 mm(2)/kPa (P = 0.0029) lower in ADD3 AA than ADD3 GG homozygotes with an additive effect of the G allele. In multiple-gene analyses, the association of brachial diameter and distensibility with the ADD3 G allele occurred only in ADD1 GlyGly homozygotes. Otherwise, the associations between the arterial phenotypes in the three vascular beds and the ADD1 or ADD2 polymorphisms were not significant. In family-based analyses, the multivariate-adjusted heritability was 0.52, 0.38, and 0.30 for brachial diameter, distensibility, and cross-sectional compliance, respectively (P < 0.001). There was no evidence for population stratification (0.07 < or = P < or = 0.96). Transmission of the mutated ADD3 G allele was associated with smaller brachial diameter in 342 informative offspring (-0.12 +/- 0.04 mm; P = 0.0085) and in 209 offspring, who were ADD1 GlyGly homozygotes (-0.14 +/- 0.06 mm; P = 0.018). CONCLUSIONS: In ADD1 GlyGly homozygotes, the properties of the brachial artery are related to the ADD3 (A386G) polymorphism, but the underlying mechanism needs further clarification.

Production of recombinant TRAIL and TRAIL receptor: Fc chimeric proteins.

The tumor necrosis factor (TNF)/TNF receptor (TNFR) families of ligands and receptors are implicated in a variety of physiological and pathological processes and regulate cellular functions as diverse as proliferation, differentiation, and death. Recombinant forms of these ligands and receptors can act to agonize or antagonize these functions and are therefore useful for laboratory studies and may have clinical applications. A protocol is presented for the expression and purification of dimeric soluble receptors fused to the Fc portion of human IgG1 and of soluble, N-terminally Flag-tagged ligands. Soluble recombinant proteins are easier to handle than membrane-bound proteins and the use of tags greatly facilitates their detection and purification. In addition, some tags may provide enhanced biological activity to the recombinant proteins (mainly by oligomerization and stabilization effects) and facilitate their functional characterization. Expression in bacterial (for selected ligands) and eukaryotic expression systems (for ligands and receptors) was performed using M15 pREP4 bacteria and human embryonic kidney 293 cells, respectively. The yield of purified protein is about 1 mg/liter for the mammalian expression system and several milligrams per liter for the bacterial expression system. Protocols are given for a specific ligand-receptor pair, namely TRAIL (Apo-2L) and TRAIL receptor 2 (DR5), but can be applied to other ligands and receptors of the TNF family.

Decreased expression of peroxisome proliferator-activated receptor alpha and liver fatty acid binding protein after partial hepatectomy of rats and mice.

BACKGROUND AIMS: Marked changes in metabolism, including liver steatosis and hypoglycemia, occur after partial hepatectomy. Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a nuclear hormone receptor that is activated by fatty acids and involved in hepatic fatty acid metabolism and regeneration. Liver fatty acid binding protein (LFABP) is an abundant protein in liver cytosol whose expression is regulated by PPAR alpha. It is involved in fatty acid uptake and diffusion and in PPAR alpha signaling. The aim of this study was to investigate the expression of PPAR alpha and LFABP during liver regeneration. METHODS: Male Sprague-Dawley rats and male C57 Bl/6 mice were subjected to 2/3 hepatectomy and LFABP and PPAR alpha mRNA and protein levels were measured at different time points after surgery. The effect of partial hepatectomy was followed during 48 h in rats and 72 h in mice. RESULTS: PPAR alpha mRNA and protein levels were decreased 26 h after hepatectomy of rats. The LFABP mRNA and protein levels paralleled those of PPAR alpha and were also decreased 26 h after hepatectomy. In mice, the mRNA level was decreased after 36 and 72 h after hepatectomy. In this case, LFABP mRNA levels decreased more slowly after partial hepatectomy than in rats. CONCLUSIONS: A marked decrease in PPAR alpha expression may be important for changed gene expression, e.g. LFABP, and metabolic changes, such as hypoglycemia, during liver regeneration.

The role of CTCFL/BORIS in Meiosis

The complexity of mammalian genome organization demands a complex interplay of DNA and proteins to orchestrate proper gene regulation. CTCF, a highly conserved, ubiquitously expressed protein has been postulated as a primary organizer of genome architecture because of its roles in transcriptional activation/repression, insulation and imprinting. Diverse regulatory functions are exerted through genome wide binding via a central eleven zinc finger DNA binding domain and an array of diverse protein-protein interactions through N- and C- terminal domains. CTCFL has been identified as a paralog of CTCF expressed only in spermatogenic cells of the testis. CTCF and CTCFL have a highly homologous DNA-binding domain, while the flanking amino acid sequences exhibit no significant similarity. Genome- wide mapping of CTCF binding sites has been carried out in many cell types, but no data exist for CTCFL apart from a few identified loci. The lack of high quality antibodies prompted us to generate an endogenously flag-tagged CTCFL mouse model using BAC recombination. IHC staining using anti-flag antibodies confirmed CTCFL localization to type Β spermatogonia and preleptotene spermatocytes and a mutually exclusive pattern of expression with CTCF. ChIP followed by high-throughput sequencing identified 10,382 binding sites showing 70% overlap but representing only 20% of CTCF sites. Consensus sequence analysis identified a significantly longer binding motif with prominently less ambiguity of base calling at every position. The significant difference between CTCF and CTCFL genomic binding patterns proposes that their binding to DNA is differentially regulated. Analysis of CTCFL binding to methylated regions on a genome wide scale identified approximately 1,000 loci. Methylation-independent binding of CTCFL might be at least one of the mechanisms that ensures distinct binding patterns of CTCF and CTCFL since CTCF binding is methylation- sensitive. Co-localization of CTCF with cohesin has been well established and analysis of CTCFL and SMC3 overlap identified around 3,300 binding sites from which two related but distinct consensus sequence motifs were derived. Because virtually all data for cohesin binding originate from mitotically proliferating cells, the anticipated overlap is expected to be considerably higher in meiotic cells. Meiosis-specific cohesin subunit Rec8 is specific for spermatocytes and 6 out of the 12 identified binding sites are also bound by CTCFL. In conclusion, this was the first genome-wide mapping of CTCFL binding sites in spermatocytes, the only cell type where CTCF is not expressed. CTCFL has a unique binding site repertoire distinct from CTCF, binds to methylated sequences and shows a significant overlap with cohesin binding sites. Future efforts will be oriented towards deciphering the role CTCFL plays in conversion of chromatin structure and function from mitotic to meiotic chromosomes. - La complexité de l'organisation du génome des mammifères exige une interaction particulière entre ADN et protéines pour orchestrer une régulation appropriée de l'expression des gènes. CTCFL, une protéine ubiquitaire très conservée, serait le principal organisateur de l'architecture du génome de par son rôle dans l'activation / la répression de la transcription, la protection et la localisation des gènes. Diverses régulations sont opérées, d'une part au travers d'interactions à différents endroits du génome par le biais d'un domaine protéique central de liaison à l'ADN à onze doigts de zinc, et d'autre part par des interactions protéine-protéine variées au niveau de leur domaine N- et C-terminal. CTCFL a été identifié comme un paralogue de CTCF exprimé uniquement dans les cellules spermatiques du testicule. CTCFL et CTCF ont un domaine de liaison à l'ADN très homologue, tandis que les séquences d'acides aminés situées de part et d'autre de ce domaine ne présentent aucune similitude. Une cartographie générale des sites de liaison au CTCF a été réalisée pour de nombreux types cellulaires, mais il n'existe aucune donnée pour CTCFL à l'exception de l'identification de quelques loci. L'absence d'anticorps de bonne qualité nous a conduit à générer un modèle murin portant un CTCFL endogène taggué grâce à un procédé de recombinaison BAC. Une coloration IHC à l'aide d'anticorps anti-FLAG a confirmé la présence de CTCFL au niveau des spermatogonies de type Β et des spermatocytes au stade préleptotène, et une distribution mutuellement exclusive avec CTCF. Une méthode de Chromatine Immunoprecipitation (ChIP) suivie d'un séquençage à haut débit a permis d'identifier 10.382 sites de liaison montrant 70% d'homologie mais ne représentant que 20% des sites CTCF. L'analyse de la séquence consensus révèle un motif de fixation à l'ADN nettement plus long et qui comporte bien moins de bases aléatoires à chaque position nucléotidique. La différence significative entre les séquences génomiques des sites de liaison au CTCF et CTCFL suggère que leur fixation à l'ADN est régulée différemment. Appliquée à l'échelle du génome, l'étude de l'interaction de CTCFL avec des régions méthylées de l'ADN a permis d'identifier environ 1.000 loci. Contrairement à CTCFL, la liaison de CTCF dépend de l'état de méthylation de l'ADN ; cette modification épigénétique constitue donc au moins un des mécanismes de régulation expliquant une localisation de CTCF et CTCFL à des sites distincts du génome. La co- localisation de CTCF avec la cohésine étant établie, l'analyse de la superposition des séquences de CTCFL avec la sous-unité SMC3 identifie environ 3.300 sites de liaison parmi lesquels deux mêmes motifs consensus distincts par leur séquence sont mis en évidence. La presque quasi-totalité des données sur la cohésine ayant été établie à partir de cellules en prolifération mitotique, il est probable que la similitude au sein des séquences consensus soit encore plus grande dans le cas des cellules en méiose. La sous-unité Rec8 de la cohésine propre à l'état de méiose est spécifiquement exprimée dans les spermatocytes. Or 6 des 12 sites de liaison identifiés sont également utilisés par CTCFL. Pour conclure, ce travail constitue la première cartographie à l'échelle du génome des sites de liaison de CTCFL dans les spermatocytes, seul type cellulaire où CTCFL n'est pas exprimé. CTCFL possède un répertoire unique de sites de fixation à l'ADN distinct de CTCF, se lie à des séquences méthylées et présente un nombre important de sites de liaison communs avec la cohésine. Les perspectives futures sont d'élucider le rôle de CTCFL dans le remodelage de la structure de la chromatine et de définir sa fonction dans le processus de méiose.

Use of the chicken lysozyme 5' matrix attachment region to generate high producer CHO cell lines.

Scaffold or matrix attachment region (S/MAR) genetic elements have previously been proposed to insulate transgenes from repressive effects linked to their site of integration within the host cell genome. We have evaluated their use in various stable transfection settings to increase the production of recombinant proteins such as monoclonal antibodies from Chinese hamster ovary (CHO) cell lines. Using the green fluorescent protein coding sequence, we show that S/MAR elements mediate a dual effect on the population of transfected cells. First, S/MAR elements almost fully abolish the occurrence of cell clones that express little transgene that may result from transgene integration in an unfavorable chromosomal environment. Second, they increase the overall expression of the transgene over the whole range of expression levels, allowing the detection of cells with significantly higher levels of transgene expression. An optimal setting was identified as the addition of a S/MAR element both in cis (on the transgene expression vector) and in trans (co-transfected on a separate plasmid). When used to express immunoglobulins, the S/MAR element enabled cell clones with high and stable levels of expression to be isolated following the analysis of a few cell lines generated without transgene amplification procedures.

Epigenetic features of human mesenchymal stem cells determine their permissiveness for induction of relevant transcriptional changes by SYT-SSX1.

BACKGROUND: A characteristic SYT-SSX fusion gene resulting from the chromosomal translocation t(X;18)(p11;q11) is detectable in almost all synovial sarcomas, a malignant soft tissue tumor widely believed to originate from as yet unidentified pluripotent stem cells. The resulting fusion protein has no DNA binding motifs but possesses protein-protein interaction domains that are believed to mediate association with chromatin remodeling complexes. Despite recent advances in the identification of molecules that interact with SYT-SSX and with the corresponding wild type SYT and SSX proteins, the mechanisms whereby the SYT-SSX might contribute to neoplastic transformation remain unclear. Epigenetic deregulation has been suggested to be one possible mechanism. METHODOLOGY/PRINCIPAL FINDINGS: We addressed the effect of SYT/SSX expression on the transcriptome of four independent isolates of primary human bone marrow mesenchymal stem cells (hMSC). We observed transcriptional changes similar to the gene expression signature of synovial sarcoma, principally involving genes whose regulation is linked to epigenetic factors, including imprinted genes, genes with transcription start sites within a CpG island and chromatin related genes. Single population analysis revealed hMSC isolate-specific transcriptional changes involving genes that are important for biological functions of stem cells as well as genes that are considered to be molecular markers of synovial sarcoma including IGF2, EPHRINS, and BCL2. Methylation status analysis of sequences at the H19/IGF2 imprinted locus indicated that distinct epigenetic features characterize hMSC populations and condition the transcriptional effects of SYT-SSX expression. CONCLUSIONS/SIGNIFICANCE: Our observations suggest that epigenetic features may define the cellular microenvironment in which SYT-SSX displays its functional effects.

The redox potential interferes with the expression of laminin binding molecules in Bacteroides fragilis

The Bacteroides fragilis ATCC strain was grown in a synthetic media with contrasting redox potential (Eh) levels [reduced (-60 mV) or oxidised (+100mV)] and their adhesion capacity to extracellular matrix components was evaluated. The strain was capable of adhering to laminin, fibronectin, fibronectin + heparan sulphate and heparan sulphate. A stronger adherence to laminin after growing the strain under oxidising conditions was verified. Electron microscopy using ruthenium red showed a heterogeneous population under this condition. Dot-blotting analyses confirmed stronger laminin recognition by outer membrane proteins of cells cultured at a higher Eh. Using a laminin affinity column, several putative laminin binding proteins obtained from the cultures kept under oxidising (60 kDa, 36 kDa, 25 kDa and 15 kDa) and reducing (60 kDa) conditions could be detected. Our results show that the expression of B. fragilis surface components that recognise laminin are influenced by Eh variations.

Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing

BACKGROUND: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. METHODS: We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform. RESULTS: We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations. CONCLUSIONS: HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.