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.

In Vivo Selection of Fluoroquinolone-Resistant Escherichia coli Isolates Expressing Plasmid-Mediated Quinolone Resistance and Expanded-Spectrum β-Lactamase

A ciprofloxacin-resistant Escherichia coli isolate, isolate 1B, was obtained from a urinary specimen of a Canadian patient treated with norfloxacin for infection due to a ciprofloxacin-susceptible isolate, isolate 1A. Both isolates harbored a plasmid-encoded sul1-type integron with qnrA1 and blaVEB-1 genes. Isolate 1B had amino acid substitutions in gyrase and topoisomerase.

Functional Variants in NOS1 and NOS2A Are Not Associated with Progressive Hearing Loss in Ménière's Disease in a European Caucasian Population

Hearing loss in Meniere's disease (MD) is associated with loss of spiral ganglion neurons and hair cells. In a guinea pig model of endolymphatic hydrops, nitric oxide synthases (NOS) and oxidative stress mediate loss of spiral ganglion neurons. To test the hypothesis that functional variants of NOS1 and NOS2A are associated with MD, wed genotyped three functional variants of NOS1 (rs41279104,rs2682826, and a cytosine-adenosine microsatellite repeat in exon 1f) and the CCTTT repeat in the promoter of NOS2A gene (rs3833912) in two independent MD sets(273 patients in total) and 550 controls. A third cohort of American patients was genotyped as replication cohort for the CCTTT repeat. Neither allele nor genotype frequencies of rs41279104 and rs2682826 were associated with MD, although longer alleles of the cytosine-adenosine microsatellite repeat were marginally significant (corrected p = 0.05) in the Mediterranean cohort but not in a second Galicia cohort. Shorter numbers of the CCTTT repeat in NOS2A were significantly more frequent in Galicia controls (OR = 0.37 [CI, 0.18-0.76], corrected p =0.04), but this finding could not be replicated in Mediterranean or American case-control populations. Meta-analysis did not support an association between CCTTT repeats and risk for MD. Severe hearing loss (>75 dB) was also not associated with any functional variants studied. Functional variants of NOS1 and and NOS2A do not confer susceptibility for MD.

Granada Virus: a Natural Phlebovirus Reassortant of the Sandfly Fever Naples Serocomplex with Low Seroprevalence in Humans

A new member of the phlebovirus genus, tentatively named Granada virus, was detected in sandflies collected in Spain. By showing the presence of specific neutralizing antibodies in human serum collected in Granada, we show that Granada virus infects humans. The analysis of the complete genome of Granada virus revealed that this agent is likely to be a natural reassortant of the recently described Massilia virus (donor of the long and short segments) with ayet unidentified phlebovirus (donor of the medium segment)

Mutation analysis of genes that control the G1/S cell cycle in melanoma: TP53, CDKN1A, CDKN2A, and CDKN2B.

BACKGROUND The role of genes involved in the control of progression from the G1 to the S phase of the cell cycle in melanoma tumors in not fully known. The aim of our study was to analyse mutations in TP53, CDKN1A, CDKN2A, and CDKN2B genes in melanoma tumors and melanoma cell lines METHODS We analysed 39 primary and metastatic melanomas and 9 melanoma cell lines by single-stranded conformational polymorphism (SSCP). RESULTS The single-stranded technique showed heterozygous defects in the TP53 gene in 8 of 39 (20.5%) melanoma tumors: three new single point mutations in intronic sequences (introns 1 and 2) and exon 10, and three new single nucleotide polymorphisms located in introns 1 and 2 (C to T transition at position 11701 in intron 1; C insertion at position 11818 in intron 2; and C insertion at position 11875 in intron 2). One melanoma tumor exhibited two heterozygous alterations in the CDKN2A exon 1 one of which was novel (stop codon, and missense mutation). No defects were found in the remaining genes. CONCLUSION These results suggest that these genes are involved in melanoma tumorigenesis, although they may be not the major targets. Other suppressor genes that may be informative of the mechanism of tumorigenesis in skin melanomas should be studied.

An isoform of microtubule-associated protein 2 (MAP2) containing four repeats of the tubulin-binding motif.

Microtubule-associated protein 2 (MAP2) exists in both high- and low-molecular mass isoforms, each of which has a tubulin-binding domain consisting of 3 imperfect tandem repeats of 31 amino acids containing a more highly conserved 18 amino acid 'core' sequence. We describe here a novel form of low molecular mass MAP2 (MAP2c) that contains an additional 4th repeat of this tubulin-binding motif. Like the 3 previously known repeat sequences, this 4th copy is highly conserved between MAP2 and the two other known members of the same gene family, tau and MAP4. In each of these three genes the additional 4th repeat is inserted between the 1st and 2nd repeats of the 3-repeat form of the molecule. Experiments with brain cell cultures, in which the relative proportions of neurons and glia had been manipulated by drug treatment, showed that 4-repeat MAP2c is associated with glial cells whereas 3-repeat MAP2c is expressed in neurons. Whereas 3-repeat MAP2c is expressed early in development and then declines, the level of 4-repeat MAP2c increases later in development, corresponding to the relatively late differentiation of glial cells compared to neurons. When transfected into non-neuronal cells, the 4-repeat version of MAP2c behaved indistinguishably from the 3-repeat form in stabilising and rearranging cellular microtubules. The presence of an additional 4th repeat of the tubulin-binding motif in all three members of the MAP2 gene family suggests that this variant arose prior to their differentiation from an ancestral gene.

Molecular epidemiology reveals long-term changes in HIV type 1 subtype B transmission in Switzerland.

BACKGROUND: Sequence data from resistance testing offer unique opportunities to characterize the structure of human immunodeficiency virus (HIV) infection epidemics. METHODS: We analyzed a representative set of HIV type 1 (HIV-1) subtype B pol sequences from 5700 patients enrolled in the Swiss HIV Cohort Study. We pooled these sequences with the same number of sequences from foreign epidemics, inferred a phylogeny, and identified Swiss transmission clusters as clades having a minimal size of 10 and containing >or=80% Swiss sequences. RESULTS: More than one-half of Swiss patients were included within 60 transmission clusters. Most transmission clusters were significantly dominated by specific transmission routes, which were used to identify the following patient groups: men having sex with men (MSM) (38 transmission clusters; average cluster size, 29 patients) or patients acquiring HIV through heterosexual contact (HETs) and injection drug users (IDUs) (12 transmission clusters; average cluster size, 144 patients). Interestingly, there were no transmission clusters dominated by sequences from HETs only. Although 44% of all HETs who were infected between 1983 and 1986 clustered with injection drug users, this percentage decreased to 18% for 2003-2006 (P<.001), indicating a diminishing role of injection drug users in transmission among HETs over time. CONCLUSIONS: Our analysis suggests (1) the absence of a self-sustaining epidemic of HIV-1 subtype B in HETs in Switzerland and (2) a temporally decreasing clustering of HIV infections in HETs and IDUs.

Structural analysis of TCR-ligand interactions studied on H-2Kd-restricted cloned CTL specific for a photoreactive peptide derivative.

To study the interaction of the TCR with its ligand, the complex of a MHC molecule and an antigenic peptide, we modified a TCR contact residue of a H-2Kd-restricted antigenic peptide with photoreactive 4-azidobenzoic acid. The photoreactive group was a critical component of the epitope recognized by CTL clones derived from mice immunized with such a peptide derivative. The majority of these clones expressed V beta 1-encoded beta chains that were paired with J alpha TA28-encoded alpha chains. For one of these TCR, the photoaffinity labeled sites were mapped on the alpha chain as a J alpha TA28-encoded tryptophan and on the beta chain as a residue of the C' strand of V beta 1. Molecular modeling of this TCR suggested the presence of a hydrophobic pocket that harbors this tryptophan as well as a tyrosine on the C' strand of V beta 1 between which the photoreactive side chain inserts. It is concluded that this avid binding principle may account for the preferential selection of V beta 1 and J alpha TA28-encoded TCR.

Orthologues of the Drosophila melanogaster E75 molting control gene in the filarial parasites Brugia malayi and Dirofilaria immitis.

Filarial parasites cause debilitating diseases in humans and domesticated animals. Brugia malayi and Dirofilaria immitis are transmitted by mosquitoes and infect humans and dogs, respectively. Their life cycle is punctuated by a series of cuticular molts as they move between different hosts and tissues. An understanding of the genetic basis for these developmental transitions may suggest potential targets for vaccines or chemotherapeutics. Nuclear receptor (NR) proteins have been implicated in molting in the free-living nematode Caenorhabditis elegans and have well characterized roles in molting during larval development of Drosophila melanogaster. For example, the D. melanogaster E75 (NR1D3) NR gene is required for molting and metamorphosis, as well as egg chamber development in adult females. We have identified Bm-nhr-11and Di-nhr-6, B. malayi and D. immitis orthologues of E75. Both genes encode canonical nuclear receptor proteins, are developmentally regulated, and are expressed in a sex-specific manner in adults.

Quantitation of endogenous mouse mammary tumor virus superantigen expression by lymphocyte subsets.

Superantigens (SAg) encoded by endogenous mouse mammary tumor viruses (Mtv) interact with the V beta domain of the T cell receptor (TcR-V beta). Presentation of Mtv SAg can lead to stimulation and/or deletion of the reactive T cells, but little is known about the quantitative aspects of SAg presentation. Although monoclonal antibodies have been raised against Mtv SAg, they have not been useful in quantitating SAg protein, which is present in very low amounts in normal cells. Alternative attempts to quantitate Mtv SAg mRNA expression are complicated by the fact that Mtv transcription occurs from multiple loci and in different overlapping reading frames. In this report we describe a novel competitive polymerase chain reaction assay which allows the locus-specific quantitation of SAg expression at the mRNA level in lymphocyte subsets from mouse strains with multiple endogenous Mtv loci. In B cells as well as T cells (CD4+ or CD8+), Mtv-6 SAg is expressed at the highest levels, followed by Mtv-7 SAg and (to a much lesser extent) Mtv-8,9. Consistent with functional Mtv-7 SAg presentation studies, we find that Mtv-7 SAg expression is higher in B cells than in CD8+ T cells and very low in the CD4+ subset. The overall hierarchy in Mtv SAg expression (i.e. Mtv-6 &gt; Mtv-7 &gt; Mtv 8,9) was also observed for mRNA isolated from neonatal thymus. Furthermore, the kinetics of intrathymic deletion of the corresponding TcR-V beta domains during ontogeny correlated with the levels of Mtv SAg expression. Collectively our data suggest that T cell responses to Mtv SAg are largely controlled by SAg expression levels on presenting cells.

Prevalent role of TCR alpha-chain in the selection of the preimmune repertoire specific for a human tumor-associated self-antigen.

The specificity of recognition of pMHC complexes by T lymphocytes is determined by the V regions of the TCR alpha- and beta-chains. Recent experimental evidence has suggested that Ag-specific TCR repertoires may exhibit a more V alpha- than V beta-restricted usage. Whether V alpha usage is narrowed during immune responses to Ag or if, on the contrary, restricted V alpha usage is already defined at the early stages of TCR repertoire selection, however, has remained unexplored. Here, we analyzed V and CDR3 TCR regions of single circulating naive T cells specifically detected ex vivo and isolated with HLA-A2/melan-A peptide multimers. Similarly to what was previously observed for melan-A-specific Ag-experienced T cells, we found a relatively wide V beta usage, but a preferential V alpha 2.1 usage. Restricted V alpha 2.1 usage was also found among single CD8(+) A2/melan-A multimer(+) thymocytes, indicating that V alpha-restricted selection takes place in the thymus. V alpha 2.1 usage, however, was independent from functional avidity of Ag recognition. Thus, interaction of the pMHC complex with selected V alpha-chains contributes to set the broad Ag specificity, as underlined by preferential binding of A2/melan-A multimers to V alpha 2.1-bearing TCRs, whereas functional outcomes result from the sum of these with other interactions between pMHC complex and TCR.

The RNA-binding protein HuR regulates DNA methylation through stabilization of DNMT3b mRNA

The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3'UTR prompted studies to investigate if this transcript associated with HuR. The interaction between HuR and DNMT3b mRNA was studied by immunoprecipitation of endogenous HuR ribonucleoprotein complexes followed by RT-qPCR detection of DNMT3b mRNA, and by in vitro pulldown of biotinylated DNMT3b RNAs followed by western blotting detection of HuR. These studies revealed that binding of HuR stabilized the DNMT3b mRNA and increased DNMT3b expression. Unexpectedly, cisplatin treatment triggered the dissociation of the [HuR-DNMT3b mRNA] complex, in turn promoting DNMT3b mRNA decay, decreasing DNMT3b abundance, and lowering the methylation of repeated sequences and global DNA methylation. In summary, our data identify DNMT3b mRNA as a novel HuR target, present evidence that HuR affects DNMT3b expression levels post-transcriptionally, and reveal the functional consequences of the HuR-regulated DNMT3b upon DNA methylation patterns.

Improved efficiency of a Salmonella-based vaccine against human papillomavirus type 16 virus-like particles achieved by using a codon-optimized version of L1.

Cervical cancer results from cervical infection by human papillomaviruses (HPVs), especially HPV16. An effective vaccine against these HPVs is expected to have a dramatic impact on the incidence of this cancer and its precursor lesions. The leading candidate, a subunit prophylactic HPV virus-like particle (VLP) vaccine, can protect women from HPV infection. An alternative improved vaccine that avoids parenteral injection, that is efficient with a single dose, and that induces mucosal immunity might greatly facilitate vaccine implementation in different settings. In this study, we have constructed a new generation of recombinant Salmonella organisms that assemble HPV16 VLPs and induce high titers of neutralizing antibodies in mice after a single nasal or oral immunization with live bacteria. This was achieved through the expression of a HPV16 L1 capsid gene whose codon usage was optimized to fit with the most frequently used codons in Salmonella. Interestingly, the high immunogenicity of the new recombinant bacteria did not correlate with an increased expression of L1 VLPs but with a greater stability of the L1-expressing plasmid in vitro and in vivo in absence of antibiotic selection. Anti-HPV16 humoral and neutralizing responses were also observed with different Salmonella enterica serovar Typhimurium strains whose attenuating deletions have already been shown to be safe after oral vaccination of humans. Thus, our findings are a promising improvement toward a vaccine strain that could be tested in human volunteers.

Hepatitis B genotype G and high frequency of lamivudine-resistance mutations among human immunodeficiencyvirus/hepatitis B virus co-infected patients in Brazil

In this study, we evaluated the hepatitis B virus (HBV) genotype distribution and HBV genomic mutations among a group of human immunodeficiency virus-HBV co-infected patients from an AIDS outpatient clinic in São Paulo. HBV serological markers were detected by commercially available enzyme immunoassay kits. HBV DNA was detected using in-house nested polymerase chain reaction and quantified by Cobas Amplicor. HBV genotypes and mutations in the basal core promoter (BCP)/pre-core/core regions and surface/polymerase genes were determined by sequencing. Among the 59 patients included in this study, 55 reported prior use of lamivudine (LAM) or tenofovir. HBV DNA was detected in 16/22 patients, with a genotype distribution of A (n = 12,75%), G (n = 2,13%), D (n = 1,6%) and F (n = 1,6%). The sequence data of the two patients infected with genotype G strongly suggested co-infection with genotype A. In 10 patients with viremia, LAM-resistance mutations in the polymerase gene (rtL180M + rtM204V and rtV173L + rtL180M + rtM204V) were found, accompanied by changes in the envelope gene (sI195M, sW196L and sI195M/sE164D). Mutations in the BCP and pre-core regions were identified in four patients. In conclusion, genotype G, which is rarely seen in Brazil, was observed in the group of patients included in our study. A high prevalence of mutations associated with LAM-resistance and mutations associated with anti-HBs resistance were also found among these patients.

Recommender System Based on Consumer Product Reviews

Consumer reviews, opinions and shared experiences in the use of a product is a powerful source of information about consumer preferences that can be used in recommender systems. Despite the importance and value of such information, there is no comprehensive mechanism that formalizes the opinions selection and retrieval process and the utilization of retrieved opinions due to the difficulty of extracting information from text data. In this paper, a new recommender system that is built on consumer product reviews is proposed. A prioritizing mechanism is developed for the system. The proposed approach is illustrated using the case study of a recommender system for digital cameras