Restriction Endonuclease Fragments Research Articles

Anaplasma platys-like strains in ruminants from Tunisia

Molecular diagnosis of Anaplasma platys and related strains (A. platys–like) in carnivores and ruminants is challenging due to co-infections with cross-reacting strains, and require post-amplification sequencing of the hemi-nested PCR products traditionally generated by targeting the groEL gene. In this study, a Restriction Enzyme Fragment Length Polymorphism (RFLP) assay coupled to hemi-nested groEL PCR was developed to discriminate among A. platys and genetically related strains. This novel approach was used for investigating A. platys-like infection in 963 domesticated ruminants (241 goats, 355 sheep, and 367 cattle) from 22 delegations located in North Tunisia. Overall prevalence rates of A. platys-like were 22.8, 11, and 3.5% in goats, sheep, and cattle, respectively. Alignment, identity comparison, and phylogenetic analysis of the groEL sequence variants obtained in this study confirmed RFLP data suggesting that Tunisian ruminants are infected by novel unclassified Anaplasma strains genetically related to A. platys. Compared to sequencing, RFLP assay allows fast detection of A. platys and A. platys-like pathogens in the same sample and has a potential value especially when screening ticks, cats and ruminants, which can be a common host for these two bacteria. This newly developed molecular technique would provide valuable molecular tool for epidemiological studies related to A. platys as well as remove concern over specificity of serological and molecular methods routinely used to identify diverse Anaplasma strains and species in wild and domestic ruminants.

Terminal Repeat Analysis of EBV Genomes.

Epstein-Barr virus (EBV) was the first human virus associated directly with human malignancies. During EBV infection of various host cells the double-stranded linear EBV DNA carried by the virions undergoes circularization. Since there are variable numbers of terminal repetitions (TRs) at the ends of the linear EBV genome, the resulting circular episomes enclose a variable number of TRs. Thus, in cells carrying viral episomes, the sizes of the terminal restriction enzyme fragments of EBV is affected by the number of TRs (Raab-Traub and Flynn Cell 47:883-889, 1986). Southern blot analysis revealed that in monoclonal proliferations, arising from a single cell, there was only a single band representing the joined EBV termini, whereas multiple terminal restriction enzyme fragments that differ in size were characteristic for oligoclonal or polyclonal proliferations. Using suitable probes, one can distinguish the episomal form from the linear EBV genomes that are formed during lytic EBV replication or during integration into the host genome. TR analysis is a useful tool for the determination of EBV clonality in different clinical samples and in cell lines carrying EBV genomes. A single terminal restriction enzyme fragment may indicate EBV infection at an early phase of clonal cell proliferation, whereas polyclonal EBV genomes may derive from multiple infections of proliferating cells.

Isolation and preliminary characterization of a new pathogenic iridovirus from redclaw crayfish Cherax quadricarinatus.

We report the preliminary characterization of a new iridovirus detected in diseased Cherax quadricarinatus collected from a farm in Fujian, China. Transmission electron microscopy identified numerous icosahedral particles (~150 nm in diameter) in the cytoplasm and budding from the plasma membrane of hematopoietic tissue cells. SDS-PAGE of virions semi-purified from the hemolymph of moribund C. quadricarinatus identified 24 proteins including a 50 kDa major capsid protein (MCP). By summing the sizes of DNA restriction endonuclease fragments, the viral genome was estimated to be ~150 kb in length. A 34 amino acid sequence deduced from a 103 bp MCP gene region amplified by PCR using degenerate primers targeted to MCP gene regions conserved among iridoviruses and chloriridoviruses was most similar (55% identity) to Sergestid iridovirus. Based on virion morphology, protein composition, DNA genome length, and MCP sequence relatedness, the virus identified has tentatively been named Cherax quadricarinatus iridovirus (CQIV). In addition, experimental infection of healthy C. quadricarinatus, Procambarus clarkii, and Litopenaeus vannamei with CQIV caused the same disease and high mortality, suggesting that CQIV poses a potential threat to cultured and wild crayfish and shrimp.

Role of Hyperhomocysteinemia and Methylene Tetrahydrofolate Reductase C677T Polymorphism in Idiopathic Portal Vein Thrombosis.

Purpose:Portal vein thrombosis (PVT) is a rare and life-threatening vascular disorder characterized by obstruction or narrowing of the portal vein. Hyperhomocysteinemia and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism has been studied in PVT patients with conflicting results. In the present study the association of hyperhomocysteinemia and MTHFR C677T polymorphism with PVT risk was investigated in Iranians.Materials and Methods:Our study population consisted of 10 idiopathic PVT patients and 80 healthy control subjects matched for age and sex. MTHFR C677T polymorphism was genotyped by the polymerase chain reaction technique combined with restriction enzyme fragment length polymorphism (PCR-RFLP) technique and plasma total homocysteine (tHcy) levels were determined by enzyme immunoassay method.Results:Mean plasma tHcy levels were significantly higher in PVT patients (20.2±6.8) than control subjects (10.9±4.7) (P=0.001). Moreover, plasma tHcy levels were significantly higher in 677T allele carriers relative to 677C allele carriers in both PVT patients (P=0.01) and control subjects (P=0.03). Neither homozygote nor heterozygote genotypes of MTHFR C677T polymorphism correlated significantly with PVT risk (P>0.05). Moreover, MTHFR C677T polymorphism didn’t increase the risk of PVT under dominant (CT+TT vs. CC) or recessive (TT vs. CC+CT) genetic models analyzed (P>0.05). The difference in frequency of minor 677T allele between PVT patients and control subjects was not statistically significant (P>0.05).Conclusion:Based on the current study, we suggest that hyperhomocysteinemia constitutes a significant and common risk factor for PVT. Also, MTHFR C677T polymorphism is not a risk factor for PVT but is a contributing factor for elevated plasma tHcy levels.

Association of Methylenetetrahydrofolate Reductase C677T Polymorphism with Hyperhomocysteinemia and Deep Vein Thrombosis in the Iranian Population.

Purpose:Deep venous thrombosis (DVT) is a common but elusive condition characterized by a high morbidity and mortality rate. The aim of the present study was to investigate the correlation between methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism with plasma total homocysteine (tHcy) levels and DVT risk in an Iranian population.Materials and Methods:Our study population consisted of 67 patients with a diagnosis of DVT and 67 healthy subjects as controls. Genotyping of MTHFR C677T polymorphism was performed by the polymerase chain reaction technique combined with restriction enzyme fragment length polymorphism (PCR-RFLP) and measurement of tHcy levels was done by enzyme immunoassay method.Results:Plasma tHcy levels were significantly higher in DVT patients than controls (18.09±7.6 vs. 10.5±4.3, P=0.001). Also, plasma tHcy levels were significantly higher in MTHFR 677TT genotypes compared to 677CC genotypes in both DVT patients (P=0.016) and controls (P=0.03). Neither heterozygote nor homozygote genotypes of MTHFR C677T polymorphism was significantly correlated with DVT (P>0.05). The distribution of MTHFR C677T genotypes was similar between men and women in both DVT patients and controls (P>0.05). Moreover, the frequency of mutant 677T allele did not differ significantly between the two groups (28.3% vs. 21.6%, P=0.15).Conclusion:Based on this study, we propose that hyperhomocysteinemia but not homozygosity for MTHFR C677T polymorphism is a significant risk factor for DVT in the Iranian population. Also, MTHFR 677TT genotype is a determinant of elevated plasma tHcy levels.

Uncovering Human History: Origin through Preserved Names of Places!!!

When we are trying to uncover history and evolution of humanity we rely on Stonehenge evidence, paintings on rocks, writing on rocks, etc. With the advent of modern molecular biological techniques such as Human Leucocyte Antigen (HLA) typing, Polymerase Chain Reaction, Restriction enzyme Fragment Length Polymorphism (RFLP) analysis of DNA, whole genome sequencing we try to map out the origin of human evolution with reference to genetics and the associated cultural practices, migration patterns, beliefs and so on. With pros and cons of each methods one technique may not be superior/comprehensive over other. Instead, all the data and evidences can complement each other and come up with a comprehensive interpretation and conclusions.

Diversity of acidophilic prokaryotes at two acid mine drainage sites in Turkey.

The biodiversity of acidophilic prokaryotes in two acidic (pH 2.8-3.05) mine drainage (AMD) sites (Balya and Çan) in Turkey was examined using a combined cultivation-based and cultivation-independent approach. The latter included analyzing microbial diversity using fluorescent in situ hybridization (FISH), terminal restriction enzyme fragment length polymorphism (`T-RFLP), and quantitative PCR (qPCR). Numbers of cultivatable heterotrophic acidophilic bacteria were over an order of magnitude greater than those of chemolithotrophic acidophiles in both AMD ponds examined. Isolates identified as strains of Acidithiobacillus ferrivorans, Acidiphilium organovorum, and Ferrimicrobium acidiphilum were isolated from the Balya AMD pond, and others identified as strains of Leptospirillum ferriphilum, Acidicapsa ligni, and Acidiphilium rubrum from Çan AMD. Other isolates were too distantly related (from analysis of their 16S rRNA genes) to be identified at the species level. Archaeal diversity in the two ponds appeared to be far more limited. T-RFLP and qPCR confirmed the presence of Ferroplasma-like prokaryotes, but no archaea were isolated from the two sites. qPCR generated semiquantitative data for genera of some of the iron-oxidizing acidophiles isolated and/or detected, suggesting the order of abundance was Leptospirillum > Ferroplasma > Acidithiobacillus (Balya AMD) and Ferroplasma > Leptospirillum > Acidithiobacillus (Çan AMD).

SimRAD: an R package for simulation-based prediction of the number of loci expected in RADseq and similar genotyping by sequencing approaches.

Application of high-throughput sequencing platforms in the field of ecology and evolutionary biology is developing quickly with the introduction of efficient methods to reduce genome complexity. Numerous approaches for genome complexity reduction have been developed using different combinations of restriction enzymes, library construction strategies and fragment size selection. As a result, the choice of which techniques to use may become cumbersome, because it is difficult to anticipate the number of loci resulting from each method. We developed SimRAD, an R package that performs in silico restriction enzyme digests and fragment size selection as implemented in most restriction site associated DNA polymorphism and genotyping by sequencing methods. In silico digestion is performed on a reference genome or on a randomly generated DNA sequence when no reference genome sequence is available. SimRAD accurately predicts the number of loci under alternative protocols when a reference genome sequence is available for the targeted species (or a close relative) but may be unreliable when no reference genome is available. SimRAD is also useful for fine-tuning a given protocol to adjust the number of targeted loci. Here, we outline the functionality of SimRAD and provide an illustrative example of the use of the package (available on the CRAN at http://cran.r-project.org/web/packages/SimRAD).

Identifying and assessing infectious disease risks to the health of the Canadian population

Background: Considering the epitheliotropic properties of HPV due to their phylogeny, each region has its own distribution and prevalence of HPV infections, together with its specific genotipic distribution. Methods & Materials: In a retrospective analysis that was carriedon thedataod7411patients, several associationswereanalized that showthe connectionbetween thepresenceofHPVand the existance of cervical lesions. The data analised, as a representative sample of the female population in Macedonia, revealed a precise genotypic distribution and prevalence of the HPV infection among Macedonian women. The genptyping was done from cito-brush samples using PCR -RFLP technique with restriction endonuclease fragmentation of DNA. Results: The prevalence of HPV infection was established in 35,77% of the patients, highest being in the age group under 30 yrs of age, with 45,07% of the infected being in this age group. The genotypic distribution showed HPV 16 to be the most prevalent (23,39%), after which comes HPV 31 (10,68%) and HPV 53 (10,6%), and then comes HPV 18 (6,19%). Conclusion: Theprevalence of theHPV infection correlateswith theworld trend, yet the genotypic dsitribution is specific forMacedonia.

Rickettsiae and Ehrlichiae Within a City Park: Is the Urban Dweller at Risk?

Tick-borne diseases, such as spotted fever rickettsioses and ehrlichioses, are potentially severe and life-threatening infections. The incidences of these infections increase during warm weather months as ticks become active. Clinicians often consider outdoor activities in rural areas to be a risk factor for exposure to ticks and the pathogens they carry, but are those who live, work, and play within an urban environment excluded from this risk? In this study, we collected ticks from two urban parks in Little Rock, AR, to assess the presence of rickettsiae and ehrlichiae within an urban setting. A total of 273 ticks were collected during July, 2011. Amblyomma americanum was the predominant tick species, with 255 (93%) of those collected. The remaining 18 (7%) were Dermacentor variabilis. Ticks were separated and pooled into groups for further testing. Forty-two of the 43 (98%) A. americanum pools demonstrated molecular evidence for the presence of rickettsiae. None of the D. variabilis contained rickettsiae. Restriction enzyme fragment length polymorphism analysis and DNA sequencing revealed Rickettsia amblyommii to be the species present. One A. americanum pool from park A demonstrated the presence of Ehrlichia chaffeensis, the pathogen responsible for human monocytotropic ehrlichiosis. These results indicate that tick-borne pathogens are not limited to rural or suburban areas.

Characterization of the Replicon Region of the <i>Enterococcus faecium</i> Plasmid pEV105 Potentially Used in the Construction of Cloning Vector

A replication probe vector (pUB380) was constructed for detecting the replicon of LAB plasmid. A cryptic plasmid, pEV105, was isolated fromEnterococcus faeciumKLDS 6.0718. Multiple restriction endonuclease fragments of pEV105 were separately ligased to pUB380. After a series of subcloning and electrotransformation, the minimal replicon of pEV105 was isolated on a 2.5 kbPstI-XbalI fragment. Replicon based on this region followed a theta-type mechanism of replication inEnterococcus faeciumKLDS 6.0718. The minimal replicon DNA fragment was sequenced and the result shows that the gene sequence homology has 99.8 % compared with partial sequence of pCIZ2 fromEnterococcus faeciumL50. Five putative ORFs were concluded by software. The result of alignments indicated two ORFs, repAl05 and repBl05, encoding two putative proteins RepAl05 and RepBl05 of 245 and 178 amino acids respectively. The 2.5 kb fragment of minimal replicon as a stable replicon is feasible for constructing food-grade cloning and expressing vector of lactic acid bacteria.

Use of restriction enzyme fragment length polymorphism (RFLP) of the 16S–23S rRNA internal transcribed spacer region (ITS) for identification of fish mycobacteria

PCR targeting the 16S–23S rRNA gene internally transcribed spacer (ITS) region has been proposed as a rapid and reliable method for the detection of Mycobacterium species in human clinical specimens. Because of variation in ITS sequences amongst Mycobacterium species, a single PCR amplification can be used to differentiate slowly growing and rapidly growing species within this genus. In the present study, analysis by ITS-PCR and ITS-restriction fragment length polymorphism (RFLP) was found to be a useful, simple and rapid method compared to current molecular and phenotypic techniques. The ITS was amplified from 13 reference strains and 59 fish isolated mycobacteria using a set of published PCR primers. After PCR, the banding patterns generated allowed slowly growing mycobacteria to be differentiated from all other rapidly growing species, with the exception of Mycobacterium conceptionense. HaeIII was selected as one of two restriction enzymes that, together with the knowledge about amplicon sizes, would produce an acceptable level of discrimination in the resulting RLFP patterns, especially in the rapidly growing group of mycobacteria. After digestion with Sau96I, the amplified products of most isolates of Mycobacterium fortuitum, including subtypes II and V and those 2 isolates with new patterns (220, 100bp), presented identical or very similar patterns as obtained by HaeIII digestion. All isolates of Mycobacterium marinum, Mycobacterium chelonae and Mycobacterium gordonae, whose PCR products were not digested with HaeIII, produced two well-defined fragments with the Sau96I restriction enzyme.

Detection of avian reoviruses causing tenosynovitis in breeder flocks in Iran by reverse transcription-polymerase chain reaction (RT-PCR) and restriction enzyme fragment length polymorphism (RFLP)

BACKGROUND:Avian reoviruses (ARVs) are members of the Orthoreovirus genus; one of the 12 genera of the Reoviridae family. The ARVs are the cause of some important diseases in poultry such as reovirus-induced arthritis, tenosynovitis, chronic respiratory disease, and mal-absorption syndrome. OBJECTIVES: In this study, the presence of ARVs in the Iranian breeder flocks was investigated through reverse transcription- polymerase chain reaction (RT-PCR) and restriction enzyme fragment length polymorphism (RFLP). METHODS: A total of 800 fecal swab samples were initially collected from breeder flocks (older than 45 weeks of age). They were then sent to the laboratory in containers with PBS, and after that they were pooled and finally to 120 samples were obtained. The total RNA extracted from the pooled fecal samples were used to amplify the selected parts of the S1 (1023 bp) and S4 (437 bp) genes from the ARV field isolates using RT-PCR. The positive RT-PCR amplified products were further analyzed by RFLP using five restriction enzymes. RESULTS: Based on the findings, 5 samples were positive with the S1 primer and 6 samples were with the S4 one. The patterns observed after the digestion of PCR products revealed that the isolates of this study were identical to both the S1133 vaccine and standard strains. CONCLUSIONS: The findings suggested that the RT-PCR/RFLP analysis might be considered as a simple and rapid approach for the differentiation of ARV isolates. This study was the first molecular detection of the ARVs presence in the Iranian breeder flocks using the RT- PCR amplification of the S1 and S4 genes and RFLP analysis.

Evolution of microbial "streamer" growths in an acidic, metal-contaminated stream draining an abandoned underground copper mine.

A nine year study was carried out on the evolution of macroscopic “acid streamer” growths in acidic, metal-rich mine water from the point of construction of a new channel to drain an abandoned underground copper mine. The new channel became rapidly colonized by acidophilic bacteria: two species of autotrophic iron-oxidizers (Acidithiobacillus ferrivorans and “Ferrovum myxofaciens”) and a heterotrophic iron-oxidizer (a novel genus/species with the proposed name “Acidithrix ferrooxidans”). The same bacteria dominated the acid streamer communities for the entire nine year period, with the autotrophic species accounting for ~80% of the micro-organisms in the streamer growths (as determined by terminal restriction enzyme fragment length polymorphism (T-RFLP) analysis). Biodiversity of the acid streamers became somewhat greater in time, and included species of heterotrophic acidophiles that reduce ferric iron (Acidiphilium, Acidobacterium, Acidocella and gammaproteobacterium WJ2) and other autotrophic iron-oxidizers (Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans). The diversity of archaea in the acid streamers was far more limited; relatively few clones were obtained, all of which were very distantly related to known species of euryarchaeotes. Some differences were apparent between the acid streamer community and planktonic-phase bacteria. This study has provided unique insights into the evolution of an extremophilic microbial community, and identified several novel species of acidophilic prokaryotes.

TGF-β1 T869C Polymorphism May Affect Susceptibility to Idiopathic Pulmonary Fibrosis and Disease Severity

Transforming growth factor-β1 (TGF-β1) is a key cytokine that plays a critical role in idiopathic pulmonary fibrosis (IPF). The genotypes of T869C polymorphism may be associated with the susceptibility to fibrotic lung disease. We investigated a single-nucleotide polymorphism at exon 1 nucleotide position 29 (T→C) of the TGF-β1 gene. Eighty-five healthy controls and 85 subjects with surgically confirmed IPF were investigated using polymerase chain reaction and restriction enzyme fragment length polymorphism techniques. The IPF patients consisted of 55 men and 30 women. The mean age was 61±8years. Fifty-one (60%) of the 85 IPF patients were smokers and 34 were nonsmokers. The distribution of genotypes between IPF patients and controls was significantly different (IPF: TT 43.5% and TC or CC 56.5%; controls: TT 27.1% and TC or CC 72.9%, p=0.037). TT genotype was significantly associated with decreased PaO2 and increased D(A-a)O2 upon initial diagnosis (p=0.006 and 0.009, respectively). There was a positive association between TT genotype and IPF development (odds ratio [OR]=2.1, 95% confidence interval [CI]=1.1-4.0, p=0.028). This study suggests that the TGF-β1 gene T869C polymorphism may affect susceptibility to IPF in Koreans. Larger studies are required to confirm the genetic association of TGF-β1 gene polymorphism and IPF.

The 8-hydroxydeoxyguanosine concentrations according to hormone therapy and S326C polymorphism of OGG1 gene in postmenopausal women

BackgroundThe 8-hydroxydeoxyguanosine (8-OHdG) is widely used for determination of DNA damage since it is excised from oxidative damaged DNA with endonuclease repair enzymes coded by 8-oxoguanine DNA N-glycosylase gene (OGG1). The present study aimed at investigating whether hormone therapy (HT) may influence on the blood/urinary 8-OHdG levels and whether the level of 8-OHdG is different according to OGG1 S326C polymorphism in postmenopausal women receiving HT. MethodsIn 102 postmenopausal women receiving HT, the 8-OHdG levels were measured in the blood and urine using high performance liquid chromatography (HPLC) before HT and 3months after HT. The genotyping of the S326C polymorphism of the OGG1 was performed by polymerase chain reaction (PCR) and restriction enzyme fragment length polymorphism (RFLP) analysis. ResultsAfter HT, mean blood 8-OHdG level significantly decreased compared to those before HT (P=0.003), while urinary 8-OHdG level did not show any difference according to HT. Pre-HT level of 8-OHdG was not different according to OGG1 genotypes and similar finding was demonstrated in post-HT 8-OHdG concentration. ConclusionsThese findings imply that hormone therapy can reduce blood 8-OHdG concentration, one of the markers of oxidative damage. Further study is needed to confirm this association in larger population.

A mutation in the type II hair keratin KRT86 gene in a Han family with monilethrix

Monilethrix, a congenital disease of hair, is usually associated with mutations in keratin genes, like KRT81, KRT83 and KRT86. We conducted this study to investigate the mutation of type II human basic hair keratin hHb/KRT gene in a Han family with monilethrix and obtain information for potential pathogenic mechanism study of monilethrix. Peripheral blood samples were drawn for genomic DNA detection. Exon 1 and exon 7 of the KRT81, KRT83 and KRT86 genes were amplified by PCR. All PCR products were sequenced directly using an ABI 310 DNA sequencer. These sequences were aligned with the standard sequences in GenBank using the BLAST software. PCR products were digested with restriction endonuclease and restriction fragment length polymorphism (RFLP) analysis was performed. In this study, we identified one novel mutation, which is a heterozygous transitional mutation of G→A at position 1,289 in exon 7 of the KRT86 gene [R430Q (KRT86)]. RFLP assays for the novel mutation excluded the possibility of polymorphism. The R430Q mutation of the KRT86 gene may be pathogenic for monilethrix. Meanwhile, we did not find any novel mutation or recurrent mutation in exons 1 and 7 of KRT81 and KRT83 and exon 1 of KRT86. There is a potential pathogenic gene in the subjects and our results expand the spectrum of mutations in the hHb6 gene.

Biochemical and genetic risk factors for atherosclerosis in systemic lupus erythematosus

IntroductionSystemic lupus erythematosus (SLE) is associated with an increase in the risk of premature cardiovascular complications caused by accelerated atherosclerosis which significantly contributes to morbidity and mortality. Carotid ultrasonography is a very sensitive imaging tool to detect premature atherosclerosis and measurements of carotid intima–media thickness (IMT) assess the extent and the severity of systemic atherosclerosis. The pathogenesis of accelerated atherosclerosis in SLE is not clear; inflammation and endothelial dysfunction in addition to genetic risk factors represent important factors in the onset of atherosclerosis. Aim of the workTo evaluate the relation between asymmetric dimethylarginine (ADMA), high sensitive C-reactive protein (hs-CRP), monocyte chemoattractant protein-1 (MCP-1) (both serum levels and the genotypes of the MCP-1 A−2518G polymorphism) with the development of carotid atherosclerosis in patients with SLE and their relation to disease activity. Patients and methodsIn the present study, 30 non-menopause SLE female patients and 20 healthy age-matched females were included. Both patients and controls were subjected to evaluation of body mass index (BMI), IMT, serum glucose, serum lipids, hs-CRP, ADMA, MCP-1 (both serum level and gene polymorphism). Serum ADMA, hs-CRP, and MCP-1, levels were measured by enzyme-linked immunosorbent assay. MCP-1 genomic variants were detected by polymerase chain reaction followed by restriction enzyme–fragment analysis. ResultsValues for IMT, hs-CRP, ADMA and MCP-1 were significantly higher in patients with SLE than in healthy controls with more significant increase in SLE patients with IMT ⩾1mm than in those with IMT <1mm. Carotid IMT was significantly positively correlated with all the studied variables except for age, BMI and FBS, but significantly negatively correlated with HDL-C in all SLE patients. G/G genotype of MCP-1 A−2518G gene was more frequent in SLE patients than controls. IMT, hs-CRP, ADMA and MCP-1 from patients with G/G phenotypes were markedly higher than those from patients with the A/A genotype.In multiple regression analysis, ADMA and MCP-1 were the strongest independent determinants of IMT in SLE patients. ConclusionsAssessment of high levels of ADMA, hs-CRP, MCP-1, in addition to the MCP-1 A−2518G polymorphism may play a role in the pathogenesis of accelerated atherosclerosis in SLE patients and would be useful in identifying the risk of developing cardiovascular disease. Kindly suggestDevelopment of a novel therapy targeting ADMA and MCP-1 may have a potential role in preventing the progression of increased IMT in SLE patients.

VEGFA amplification/deletion in human breast tumors.

e21017 Background: The anti-VEGF antibody, bevacizumab, has been FDA approved for the treatment of breast cancer. While germline variability (i.e. single nucleotide polymorphisms) may serve as a predictive marker for anti-VEGF therapy, to date tumor-specific variability has not. More specifically, amplification or deletion of the VEGFA gene has not been studied or evaluated as a predictive or prognostic biomarker for breast cancer. Methods: A VEGFA/centromere enumerization-6 (CEN-6) probe was created and validated using DNA clones and restriction enzyme fragment measurements. The final product contained a RP11-710-L16 probe covering 183 KB including the VEGFA gene and flanking regions. The CEN-6 probe was developed with fluorescein isothiocyanate labeled peptide nucleic acid oligonucleotides. VEGFA and CEN-6 probes were tested on metaphase spreads to exclude cross-hybridization to other chromosomes. A tissue microarray containing 93 breast cancers was analyzed for the presence of VEGFA gene amplification and/or deletion using a FISH protocol similar to the TOP2A FISH pharmDx Kit. VEGFA/CEN-6 signal ratio in cancer cells was recorded. Normal cells in the tissue sections served as an internal positive control of pretreatment and hybridization efficiency. A ratio of <0.8 was considered deleted, ≥ 1.5-1.99 was considered borderline amplified, whereas a ratio≥ 2.0 was considered amplified. Results: Of the 93 core tissue specimens, FISH analysis was successful in 80 cases (86%) of the cases. Of these, 11% were found to demonstrate VEGFA deletions, 9% were borderline amplified and 5% were amplified. Thus, 25% in total had genetic aberrations of this gene. The aberrations did not correlate with ER or HER2 expression, tumor size, nodal status or grade. Conclusions: VEGFA gene aberrations are relatively common in primary breast cancers. Their role as predictors of response to anti-VEGF therapies remains to be studied. Result n = 80 % Deletion (<0.8) 9 11 Normal (0.8-1.49) 60 75 Borderline (1.5-1.99) 7 9 Amplified (≥2.0) 4 5 Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Dako Denmark A/S Molecular Pathology

Abstract 2208: High-resolution analysis of insertional mutagenesis screens to study genetic interactions in heterogeneous tumors

Abstract Insertional mutagenesis (IM) screens in genetically engineered mouse models of cancer are a powerful tool to identify genes or regions in the genome contributing to tumorigenesis. Current methods to identify the integration sites are not quantitative and do not give information about the clonality of a given insertion in a tumor. Consequently, the results of IM screens are hard to interpret since the obtained data are incomplete and highly biased due to technical variation. We have developed and optimized a next-generation sequencing-based method for ultra high-throughput analysis of insertional mutations in a less biased and quantitative manner. Integration sites of retroviral or transposon based insertional mutagens are amplified using Splinkerette-mediated PCR and subsequent 454 sequencing. In contrast to current protocols, based on restriction enzyme (RE) fragmentation, we randomly shear gDNA, which allows a random ligation of the Splinkerette. This enables us to generate randomly sized amplicons per integration, which reduces amplification- and sequencing bias compared to amplicons with a fixed length per integration using RE based methods. Since Splinkerette ligation can occur at every base pair, we can determine the amount of cells containing the given insertion by counting unique ligation points, each representing a unique chromosome, and thereby a cell, in the tumor. This latter feature, addressing clonality of an insertional mutagen, will allow the identification of both clonal and sub-clonal mutations in genetically heterogeneous tumors. More importantly, we are now able to study co-occurring mutations, e.g. underlying synthetic lethality, which cannot be performed reliably using the current method. The insertional mutagenesis data obtained with the improved method will represent the biological situation of a given integration within a tumor, thereby facilitating subsequent validation experiments. Additionally, data obtained from our retroviral- and transposon-based screens in various mouse models of cancer will serve as a catalogue of cancer-relevant mutations that can be used for cross-species comparative analysis of the large numbers of mutations found in human breast cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2208.