High-density Microarrays Research Articles

First report of two successive deletions on chromosome 15q13 cytogenetic bands in a boy and girl: additional data to 15q13.3 syndrome with a report of high IQ patient

15q13.3 syndrome is associated with a wide spectrum of neurological disorders. Among a cohort of 150 neurodevelopmental cases, we identified two patients with two close proximity interstitial hemizygous deletions on chromosome 15q13. Using high-density microarrays, we characterized these deletions and their approximate breakpoints. The second deletion in both patients overlaps in a small area containing CHRNA7 where the gene is partially deleted. The CHRNA7 is considered a strong candidate for the 15q13.3 deletion syndrome’s pathogenicity. Patient 1 has cognitive impairment, learning disabilities, hyperactivity and subtle dysmorphic features whereas patient 2 has mild language impairment with speech difficulty, mild dysmorphia, heart defect and interestingly a high IQ that has not been reported in 15q13.3 syndrome patients before. Our study presents first report of such two successive deletions in 15q13.3 syndrome patients and a high IQ in a 15q13.3 syndrome patient. Our study expands the breakpoints and phenotypic features related to 15q13.3 syndrome.

Aberrant expression of alternative splicing variants in multiple sclerosis - A systematic review.

Alternative splicing is an important form of RNA processing that affects nearly all human genes. The differential expression of specific transcript and protein isoforms holds the potential of novel biomarkers for complex diseases. In this systematic review, we compiled the existing literature on aberrant alternative splicing events in multiple sclerosis (MS). A systematic literature search in the PubMed database was carried out and supplemented by screening the reference lists of the identified articles. We selected only MS-related original research studies which compared the levels of different isoforms of human protein-coding genes. A narrative synthesis of the research findings was conducted. Additionally, we performed a case-control analysis using high-density transcriptome microarray data to reevaluate the genes that were examined in the reviewed studies. A total of 160 records were screened. Of those, 36 studies from the last two decades were included. Most commonly, peripheral blood samples were analyzed (32 studies), and PCR-based techniques were usually employed (27 studies) for measuring the expression of selected genes. Two studies used an exploratory genome-wide approach. Overall, 27 alternatively spliced genes were investigated. Nine of these genes appeared in at least two studies (CD40, CFLAR, FOXP3, IFNAR2, IL7R, MOG, PTPRC, SP140 and TNFRSF1A). The microarray data analysis confirmed differential alternative pre-mRNA splicing for 19 genes. An altered RNA processing of genes mediating immune signaling pathways has been repeatedly implicated in MS. The analysis of individual exon-level expression patterns is stimulated by the advancement of transcriptome profiling technologies. In particular, the examination of genes encoded in MS-associated genetic regions may provide important insights into the pathogenesis of the disease and help to identify new biomarkers.

Photoactivable Elimination of Tumorigenic Human Induced Pluripotent Stem Cells by Using a Lectin-Doxorubicin Prodrug Conjugate.

Human pluripotent stem cells (hPSCs) are attractive resources for regenerative medicine, but medical applications are hindered by their tumorigenic potential. Previously, a hPSC-specific lectin probe, rBC2LCN, was identified through comprehensive glycome analysis by using high-density lectin microarrays. Herein, a lectin-doxorubicin (DOX) prodrug conjugate, with controllable photolysis activation for the elimination of tumorigenic human induced pluripotent stem cells, has been developed. rBC2LCN was fused with a biotin-binding protein, tamavidin (BC2Tama), and the fusion protein was expressed in Escherichia coli and purified by means of affinity chromatography. BC2Tama was then conjugated with doxorubicin-photocleavable biotin (DOXPCB). The BC2Tama-DOXPCB conjugates were observed to bind to hPSCs followed by internalization. Upon exposure to ultraviolet light, DOX was released inside the cells, which allowed specific killing of the hPSCs. Thus, BC2Tama-DOXPCB should be useful for the targeted elimination of hPSCs contained in hPSC-derived cell therapy products. This is the first report of the generation of lectin-prodrug conjugates. BC2Tama should be applicable for the targeted delivery of various types of biotinylated compounds into hPSCs.

Quality control usage in high-density microarrays reveals differential gene expression profiles in ovarian cancer

There are several existing reports of microarray chip use for assessment of altered gene expression in different diseases. In fact, there have been over 1.5 million assays of this kind performed over the last twenty years, which have influenced clinical and translational research studies. The most commonly used DNA microarray platforms are Affymetrix GeneChip and Quality Control Software along with their GeneChip Probe Arrays. These chips are created using several quality controls to confirm the success of each assay, but their actual impact on gene expression profiles had not been previously analyzed until the appearance of several bioinformatics tools for this purpose. We here performed a data mining analysis, in this case specifically focused on ovarian cancer, as well as healthy ovarian tissue and ovarian cell lines, in order to confirm quality control results and associated variation in gene expression profiles. The microarray data used in our research were downloaded from ArrayExpress and Gene Expression Omnibus (GEO) and analyzed with Expression Console Software using RMA, MAS5 and Plier algorithms. The gene expression profiles were obtained using Partek Genomics Suite v6.6 and data were visualized using principal component analysis, heat map, and Venn diagrams. Microarray quality control analysis showed that roughly 40% of the microarray files were false negative, demonstrating over- and under-estimation of expressed genes. Additionally, we confirmed the results performing second analysis using independent samples. About 70% of the significant expressed genes were correlated in both analyses. These results demonstrate the importance of appropriate microarray processing to obtain a reliable gene expression profile.

Regional methylome profiling reveals dynamic epigenetic heterogeneity and convergent hypomethylation of stem cell quiescence-associated genes in breast cancer following neoadjuvant chemotherapy

BackgroundNeoadjuvant chemotherapy (NAC) induces a pathological complete response (pCR) in ~ 30% of patients with breast cancer. However, aberrant DNA methylation alterations are frequent events during breast cancer progression and acquisition of chemoresistance. We aimed to characterize the inter- and intra-tumor methylation heterogeneity (MH) in breast cancer following NAC.MethodsDNA methylation profiles of spatially separated regions of breast tumors before and after NAC treatment were investigated using high-density methylation microarray. Methylation levels of genes of interest were further examined using multiplexed MethyLight droplet digital PCR (ddPCR).ResultsWe have discovered different levels of intra-tumor MH in breast cancer patients. Moreover, NAC dramatically altered the methylation profiles and such changes were highly heterogeneous between the patients. Despite the high inter-patient heterogeneity, we identified that stem cell quiescence-associated genes ALDH1L1, HOPX, WNT5A and SOX9 were convergently hypomethylated across all the samples after NAC treatment. Furthermore, by using MethyLight ddPCR, we verified that the methylation levels of these 4 genes were significantly lower in breast tumor samples after NAC than those before NAC.ConclusionsOur study has revealed that NAC dramatically alters epigenetic heterogeneity in breast cancer and induces convergent hypomethylation of stem cell quiescence-associated genes, ALDH1L1, HOPX, WNT5A and SOX9, which can potentially be developed as therapeutic targets or biomarkers for chemoresistance.

Genetic Imbalance Is Associated With Functional Outcome After Ischemic Stroke.

Background and Purpose- We sought to explore the effect of genetic imbalance on functional outcome after ischemic stroke (IS). Methods- Copy number variation was identified in high-density single-nucleotide polymorphism microarray data of IS patients from the CADISP (Cervical Artery Dissection and Ischemic Stroke Patients) and SiGN (Stroke Genetics Network)/GISCOME (Genetics of Ischaemic Stroke Functional Outcome) networks. Genetic imbalance, defined as total number of protein-coding genes affected by copy number variations in an individual, was compared between patients with favorable (modified Rankin Scale score of 0-2) and unfavorable (modified Rankin Scale score of ≥3) outcome after 3 months. Subgroup analyses were confined to patients with imbalance affecting ohnologs-a class of dose-sensitive genes, or to those with imbalance not affecting ohnologs. The association of imbalance with outcome was analyzed by logistic regression analysis, adjusted for age, sex, stroke subtype, stroke severity, and ancestry. Results- The study sample comprised 816 CADISP patients (age 44.2±10.3 years) and 2498 SiGN/GISCOME patients (age 67.7±14.2 years). Outcome was unfavorable in 122 CADISP and 889 SiGN/GISCOME patients. Multivariate logistic regression analysis revealed that increased genetic imbalance was associated with less favorable outcome in both samples (CADISP: P=0.0007; odds ratio=0.89; 95% CI, 0.82-0.95 and SiGN/GISCOME: P=0.0036; odds ratio=0.94; 95% CI, 0.91-0.98). The association was independent of age, sex, stroke severity on admission, stroke subtype, and ancestry. On subgroup analysis, imbalance affecting ohnologs was associated with outcome (CADISP: odds ratio=0.88; 95% CI, 0.80-0.95 and SiGN/GISCOME: odds ratio=0.93; 95% CI, 0.89-0.98) whereas imbalance without ohnologs lacked such an association. Conclusions- Increased genetic imbalance was associated with poorer functional outcome after IS in both study populations. Subgroup analysis revealed that this association was driven by presence of ohnologs in the respective copy number variations, suggesting a causal role of the deleterious effects of genetic imbalance.

SA18 - ANALYSIS OF GENETIC VARIANTS IN MEXICAN CHILDREN WITH AUTISM SPECTRUM DISORDER: AN IMMUNGENOMIC APPROACH

Background Autism spectrum disorders encompass a large number of symptoms such as difficulty in social interaction, poor communication skills and repetitive behaviors. Recently, new hypotheses have emerged about the relationship between psychiatric diseases and abnormalities in the immune system, where evidence suggests that a part of the etiology can be explained by immunological alterations. Genomic wide association studies in Latino and Mexican populations are scarce, so in this study we used a sample of pediatric patients of Mexican mestizo origin with ASD diagnosis. Methods As a first approach to the study of genetic variants in 120 mexican children with ASD, using a high density microarray (Psycharray, Illumina) and 45 controls. This array contains variants related to common psychiatric conditions and immune system. For the analysis, the Plink and Eigensoft softwares were used. Genotypic values 1×10-4 were taken. A logistic regression was carried out and adjusted for gender and ancestry. Results As we expected, immune system genes are involved in ASD. Further, we found some genes associated with mental diseases. Some SNPs are close to immune genes as TNFRSF19 (P=4×10-4), ATG16L1 (P=2×10-4) within genes as MLIP (P=4×10-5), NUBP1 (P=4×10-4) and ATP9A (P=3×10-4). SNPs located at intergenic regions were found statistically significant either. In chromosome 4 and 7 were found a signal of several intergenic SNPs with a significant P. Discussion This is the first study in children with ASD performed in Mexican population. These results give us an idea of the SNPs contained in genes that can explain part of the etiopathogenesis of the disorder. Pathway analyses are necessary to elucidate gene interaction. Intergenic SNPs with significant P need to minucious analyses. In addition, it lays the foundation for future functional studies on the genes involved.

Copy number alterations and copy-neutral loss of heterozygosity in Ukrainian patients with primary myelofibrosis

To examine frequencies and spectrum of genomic alterations in Ukrainian patients diagnosed with primary myelofibrosis(PMF). We enrolled 30 Ukrainian patients diagnosed with PMF who were previously tested for usual mutations in mye-loproliferative neoplasms driver genes (JAK2, MPL and CALR). Genomic DNA samples were obtained from peripheral blood leukocytes of these patients. Copy number alterations and copy-neutral loss of heterozygosity (cnLOH) were assessed using a high-density CytoScanHD microarray platform. Statistical significance was evaluated by the Fisher exact test. We identified frequent genomic alterations, but no significant difference in the rates of copy-number loss, copy-number gain, cnLOH, or multiple genomic alterations were found in the groups of PMF patients that were positive for one of the usual mutations in driver genes or negative for such mutations (33.3% and 55.6%, p = 0.4181, 19.0% and 11.1%, p = 1.0000, 61.9% and 44.4%, p = 0.4434, 33.3% and 55.6%, p=0.4181, respectively). The most frequent alterations were cnLOH at 1p36-1p22, 9p24.3-9p13.3 and 11q12.3-11q25; copy number loss at 7q21-7q36.3 and 13q12.3-13q14.3. Copy number alterations and cnLOH commonly affected the EZH2, LAMB4, CBL, CUX1, ATM, RB1 and TP53 genes, in addition to JAK2, MPL and CALR. We demonstrated the spectrum of genomic alterations in the groups of the Ukrainian PMF patients with or without the usual mutations in the specific driver genes. We identified several potential genes, which may be involved in the myeloproliferative neoplasms development and their phenotype modification (EZH2, LAMB4, CBL, CUX1, ATM, RB1 and TP53).

Copy number variation analysis of twin pairs discordant for cleft lip with or without cleft palate

Non-syndromic cleft lip with or without cleft palate (nsCL/P) is a frequent orofacial malformation. The comparison of concordance rate observed in monozygotic and dizygotic twins supports high level of heritability and a strong genetic component. However, phenotype concordance for orofacial cleft in monozygotic twins is about 50%. The aim of the present investigation was to detect postzygotic events that may account for discordance in monozygotic twins. High-density SNP microarrays hybridization was used to genotype two pairs of monozygotic twins discordant for nsCL/P. Discordant SNP genotypes and copy number variants were analyzed to identify genetic differences responsible of phenotype discrepancy. A number of differences were observed, none involving known nsCL/P candidate genes or genomic regions. Considering the limitation of the study, related to the small sample size and to the large-scale investigation method, the results suggest that the detection of discordant events in other monozygotic twin pairs would be remarkable and warrant further investigations.

Chip-SIP: Stable Isotope Probing Analyzed with rRNA-Targeted Microarrays and NanoSIMS.

Chip-SIP is a stable isotope probing (SIP) method for linking microbial identity and function in mixed communities and is capable of analyzing multiple isotopes (13C, 15N, and 18O) simultaneously. This method uses a high-density microarray to separate taxon-specific 16S (or 18S) rRNA genes and a high sensitivity magnetic sector secondary ion mass spectrometer (SIMS) to determine the relative isotope incorporation of the rRNA at each probe location. Using a maskless array synthesizer (MAS), we synthesize multiple unique sequences to target hundreds of taxa at the ribosomal operational taxonomic unit (OTU) level on an array surface, and then analyze it with a NanoSIMS 50, using its high-spatial resolution imaging capability to generate isotope ratios for individual probes. The Chip-SIP method has been used in diverse systems, including surface marine and estuarine water, rhizosphere, and peat soils, to quantify taxon-specific relative incorporation of different substrates in complex microbial communities. Depending on the hypothesis and experimental design, Chip-SIP allows the user to compare the same community incorporating different substrates, different communities incorporating the same substrate(s), or quantify how a community responds to treatment effects, such as temperature or nutrient concentrations.

Model-based Evaluation of Gene Expression Changes in Response to Leishmania Infection.

Since the development of high-density microarray technology in the late 1990s, global host gene expression changes in response to various stimuli have been extensively studied. More than a dozen peer-reviewed publications have investigated the effect of Leishmania infection in various models since 2001. This review covers the transcriptional changes in macrophage models induced by various Leishmania species and summarizes the resulting impact these studies have on our understanding of the host response to leishmaniasis in vitro. Characterization of the similarities and differences between various model systems will not only further our understanding of Leishmania-induced changes to macrophage gene expression but also identify potential therapeutic targets in the future.

The long noncoding RNA KIAA0125 is upregulated in ameloblastomas

Ameloblastoma and adenomatoid odontogenic tumor (AOT) are jaw tumors derived from the teeth forming apparatus. While ameloblastoma is a destructive, debilitating lesion, with conventional surgical treatment leading to facial deformity and morbodities, AOT shows indolent clinical behavior. The underlying molecular mechanisms associated with their biological behavior are unknown. The use of high-density whole-genome microarray analysis in ameloblastomas and AOT revealed high frequency of genomic gain at 14q32.33, which encompasses the long noncoding RNA (lncRNA) gene KIAA0125. In the present study, we aimed to investigate the expression profile of KIAA0125 in these tumors. Thirteen samples were included (five solid/multicystic ameloblastomas, four AOT, and four dental follicles). The relative quantification of KIAA0125 expression was obtained by qPCR and interactions of KIAA0125 were in silico predicted. We detected higher levels of KIAA0125 transcripts in the ameloblastoma group compared to dental follicles (p = 0.042). The expression levels of KIAA0125 in AOT were not different from that of dental follicles. KIAA0125 was predicted to interact with 41 miRNA families. Four miRNAs of these families have been previously reported differentially expressed in ameloblastoma, being miR-135a-5p, miR-204-5p and miR-205-5p upregulated, and miR-150-5p downregulated. The lncRNA KIAA0125 is likely involved in the ameloblastoma pathobiology. LncRNAs hold strong promise as therapeutic targets and experimental validation of this lncRNA functions may lead to tailored therapies targeting KIAA0125 in extensive and recurrent ameloblastoma cases.

Antibody responses to Zika virus proteins in pregnant and non-pregnant macaques.

The specificity of the antibody response against Zika virus (ZIKV) is not well-characterized. This is due, in part, to the antigenic similarity between ZIKV and closely related dengue virus (DENV) serotypes. Since these and other similar viruses co-circulate, are spread by the same mosquito species, and can cause similar acute clinical syndromes, it is difficult to disentangle ZIKV-specific antibody responses from responses to closely-related arboviruses in humans. Here we use high-density peptide microarrays to profile anti-ZIKV antibody reactivity in pregnant and non-pregnant macaque monkeys with known exposure histories and compare these results to reactivity following DENV infection. We also compare cross-reactive binding of ZIKV-immune sera to the full proteomes of 28 arboviruses. We independently confirm a purported ZIKV-specific IgG antibody response targeting ZIKV nonstructural protein 2B (NS2B) that was recently reported in ZIKV-infected people and we show that antibody reactivity in pregnant animals can be detected as late as 127 days post-infection (dpi). However, we also show that these responses wane over time, sometimes rapidly, and in one case the response was elicited following DENV infection in a previously ZIKV-exposed animal. These results suggest epidemiologic studies assessing seroprevalence of ZIKV immunity using linear epitope-based strategies will remain challenging to interpret due to susceptibility to false positive results. However, the method used here demonstrates the potential for rapid profiling of proteome-wide antibody responses to a myriad of neglected diseases simultaneously and may be especially useful for distinguishing antibody reactivity among closely related pathogens.

Association of Circulating Tumor Cells (CTCs) and Genomic Signatures in Prostate Cancer Patients

Intermediate and high-risk prostate cancer patients who are candidates for radiotherapy (RT) are very heterogeneous. Genomic classifiers add to clinical-pathologic risk factors and have the potential to become integral to risk stratification. Since circulating tumor cells (CTCs) are determinate of metastasis and genomic predictors of CTCs are not yet understood in this population, we investigated the associations of CTC counts with several genomic classifiers in patients enrolled in a randomized Phase II radiotherapy clinical trial (BLaStM, NCT02307058). CTCs were enumerated in blood samples from 31 patients using a microfilter system. The number of single CTCs, CTC clusters and their sum were recorded. Several gene signatures were investigated. Decipher® (metastatic risk), PORTOS (predictor of RT response) and PAM50 (predictor hormone response, which includes gene expression-based "intrinsic" subtypes: luminal A, luminal B, HER2-enriched, and basal-like), were quantified in tissue obtained by MRI-Ultrasound fused biopsies. A high-density gene expression microarray processed at a biosciences laboratory was used. A Poisson regression model with demographic and disease characteristics variables (PSA, Gleason Score (GS), T stage, %tumor in biopsies, age, race/ethnicity) were used in the univariable analysis (UVA). Multivariable analysis (MVA) included the statistically significant variables, where each biomarker was examined, along with chosen covariates for association with CTC counts in a one-at-a-time manner. CTCs were evident in over 70% of patients tested. The single cell and cluster median values in 8 mL of blood were 33 (range 0-343) and 4 (range 0-26). In MVA, several of the signatures were associated with total CTC number (single cells and clusters). When the biopsy core containing the highest Gleason score (GS) and %tumor were analyzed, the significant relationships included PORTOS (RR 6.00; p<0.001), PAM50_basal (RR 2.21; p<0.001), and Decipher (RR 1.16; p=0.048). Since multiple biopsies were analyzed for gene expression, when the cores with the highest signature value were selected, PORTOS surged (RR=47.10; p<0.0001) in the MVA. Despite the relatively small sample size, the results indicate a strong signal related to radiation sensitivity. NCCN risk categories were not significantly related to CTC counts or gene signatures. Determinants of prostate cancer lethality in intermediate to high-risk prostate cancer are poorly understood. Our results show an association between tumor gene expression alterations and CTC number, indicating that genes linked to radiation sensitivity have a role in early metastatic risk.

High-Density RNA Microarrays Synthesized In Situ by Photolithography.

While high‐density DNA microarrays have been available for over three decades, the synthesis of equivalent RNA microarrays has proven intractable until now. Herein we describe the first in situ synthesis of mixed‐based, high‐density RNA microarrays using photolithography and light‐sensitive RNA phosphoramidites. With coupling efficiencies comparable to those of DNA monomers, RNA oligonucleotides at least 30 nucleotides long can now efficiently be prepared using modified phosphoramidite chemistry. A two‐step deprotection route unmasks the phosphodiester, the exocyclic amines and the 2′ hydroxyl. Hybridization and enzymatic assays validate the quality and the identity of the surface‐bound RNA. We show that high‐density is feasible by synthesizing a complex RNA permutation library with 262144 unique sequences. We also introduce DNA/RNA chimeric microarrays and explore their applications by mapping the sequence specificity of RNase HII.

A Systematic Analysis Workflow for High-Density Customized Protein Microarrays in Biomarker Screening.

High-density protein microarrays constitute a promising high-throughput platform for the characterization of protein expression patterns, biomarker discovery, and validation. Different types of protein microarrays have been described according to several features (such as content, format, and detection system) presenting advantages and disadvantages which are relevant for the specific application and purposes. Therefore, an experimental design is key for any screening based on protein microarrays assays; in fact, the data analysis strategy is directly related to the experimental design, type of protein microarray and consequently the final outcome, the data and results interpretation, is also directly linked. Here, it is proposed a systematic workflow for biomarker discovery based on tailor-made protein microarrays platforms which obtain comprehensively info for the functional protein characterization in high-throughput format.

Antibody Cross-Reactivity in Antivenom Research.

Antivenom cross-reactivity has been investigated for decades to determine which antivenoms can be used to treat snakebite envenomings from different snake species. Traditionally, the methods used for analyzing cross-reactivity have been immunodiffusion, immunoblotting, enzyme-linked immunosorbent assay (ELISA), enzymatic assays, and in vivo neutralization studies. In recent years, new methods for determination of cross-reactivity have emerged, including surface plasmon resonance, antivenomics, and high-density peptide microarray technology. Antivenomics involves a top-down assessment of the toxin-binding capacities of antivenoms, whereas high-density peptide microarray technology may be harnessed to provide in-depth knowledge on which toxin epitopes are recognized by antivenoms. This review provides an overview of both the classical and new methods used to investigate antivenom cross-reactivity, the advantages and disadvantages of each method, and examples of studies using the methods. A special focus is given to antivenomics and high-density peptide microarray technology as these high-throughput methods have recently been introduced in this field and may enable more detailed assessments of antivenom cross-reactivity.

Runs of homozygosity in a selected cattle population with extremely inbred bulls: Descriptive and functional analyses revealed highly variable patterns.

The analysis of runs of homozygosity (ROH), using high throughput genomic data, has become a valuable and frequently used methodology to characterize the genomic and inbreeding variation of livestock and wildlife animal populations. However, this methodology has been scarcely used in highly inbred domestic animals. Here, we analyzed and characterized the occurrence of ROH fragments in highly inbred (HI; average pedigree-based inbreeding coefficient FPED = 0.164; 0.103 to 0.306) and outbred Retinta bulls (LI; average FPED = 0.008; 0 to 0.025). We studied the length of the fragments, their abundance, and genome distribution using high-density microarray data. The number of ROH was significantly higher in the HI group, especially for long fragments (>8Mb). In the LI group, the number of ROH continuously decreased with fragment length. Genome-wide distribution of ROH was highly variable between samples. Some chromosomes presented a larger number of fragments (BTA1, BTA19, BTA29), others had longer fragments (BTA4, BTA12, BTA17), while other ones showed an increased ROH accumulation over specific loci (BTA2, BTA7, BTA23, BTA29). Similar differences were observed in the analysis of 12 individuals produced by a similar inbred event (FPED3 = 0.125). The correlation between the fraction of the genome covered by ROH (FROH) and FPED was high (0.79), suggesting that ROH-based estimations are indicative of inbreeding levels. On the other hand, the correlation between FPED and the microsatellite-based inbreeding coefficient (FMIC) was only moderate (r = 0.44), suggesting that STR-based inbreeding estimations should be avoided. Similarly, we found a very low correlation (r = -0.0132) between recombination rate and ROH abundance across the genome. Finally, we performed functional annotation analyses of genome regions with significantly enriched ROH abundance. Results revealed gene clusters related to pregnancy-associated proteins and immune reaction. The same analysis performed for regions enriched with recently formed ROH (> 8 Mb) showed gene clusters related to flagellum assembly. In both cases, the processes were related to male and female reproductive functions, which may partially explain the reduced fertility associated with inbred populations.

LTR-retrotransposon transcriptome modulation in response to endotoxin-induced stress in PBMCs

BackgroundHuman Endogenous Retroviruses (HERVs) and Mammalian apparent LTR-retrotransposons (MaLRs) represent the 8% of our genome and are distributed among our 46 chromosomes. These LTR-retrotransposons are thought to be essentially silent except in cancer, autoimmunity and placental development. Their Long Terminal Repeats (LTRs) constitute putative promoter or polyA regulatory sequences. In this study, we used a recently described high-density microarray which can be used to study HERV/MaLR transcriptome including 353,994 HERV/MaLR loci and 1559 immunity-related genes.ResultsWe described, for the first time, the HERV transcriptome in peripheral blood mononuclear cells (PBMCs) using a cellular model mimicking inflammatory response and monocyte anergy observed after septic shock. About 5.6% of the HERV/MaLR repertoire is transcribed in PBMCs. Roughly one-tenth [5.7–13.1%] of LTRs exhibit a putative constitutive promoter or polyA function while one-quarter [19.5–27.6%] may shift from silent to active. Evidence was given that some HERVs/MaLRs and genes may share similar regulation control under lipopolysaccharide (LPS) stimulation conditions. Stimulus-dependent response confirms that HERV expression is tightly regulated in PBMCs. Altogether, these observations make it possible to integrate 62 HERVs/MaLRs and 26 genes in 11 canonical pathways and suggest a link between HERV expression and immune response. The transcriptional modulation of HERVs located close to genes such as OAS2/3 and IFI44/IFI44L or at a great distance from genes was discussed.ConclusionThis microarray-based approach revealed the expression of about 47,466 distinct HERV loci and identified 951 putative promoter LTRs and 744 putative polyA LTRs in PBMCs. HERV/MaLR expression was shown to be tightly modulated under several stimuli including high-dose and low-dose LPS and Interferon-γ (IFN-γ). HERV incorporation at the crossroads of immune response pathways paves the way for further functional studies and analyses of the HERV transcriptome in altered immune responses in vivo such as in sepsis.

Investigation of Yersinia pestis and Yersinia pseudotuberculosis strains from Georgia and neighboring countries in the Caucasus by high-density SNP microarray.

Yersinia pestis, the causative agent of plague, is a recently evolved clone of the enteropathogenic bacterium Yersinia pseudotuberculosis. Y. pestis has been extensively studied for decades; however, there are insufficient data about the intra-species diversity of this microorganism in certain parts of the world, including the Caucasus region. Using a high-density single-nucleotide polymorphism (SNP) microarray, we genotyped a total of 46 Y. pestis isolates from two plague foci in Georgia and neighboring Caucasus countries together with 12 Y. pseudotuberculosis isolates from Georgia. The genotyping microarray captured a total of 13,525 SNP positions across the Y. pestis and Y. pseudotuberculosis genomes and plasmids with high-throughput capability and superior reproducibility. From this analysis, we confirmed the presence of two independent and relatively distant phylogenetic groups of Y. pestis in the Caucasus region. The signature SNP patterns identified from this study will allow assay development for plague surveillance and pseudotuberculosis diagnostics.