[A Pipeline for the Error-free Identification of Somatic Alu Insertions in High-throughput Sequencing Data].

Retroelements are considered as one of the important sources of genomic variability in modern humans. It is known that transposition activity of retroelements in germline cells generates new insertions in various genomic loci and sometimes results in genetic diseases. Retroelements activity in somatic cells is restricted by different cellular mechanisms; however, there is an evidence for it in some tissue types. Somatic insertions can trigger tumorigenesis or participate in normal functioning such as generation of neurons' plasticity. In spite of the rapid development of high-throughput sequencing methods a confident detection of somatic insertions is still quite a challenging task. That, in part, is due to the absence of adequate bioinformatic tools for the analysis of sequencing data. Here, we propose an advanced computational pipeline for the identification of somatic insertions in datasets generated by selective amplification and high-throughput sequencing of genomic regions flanking insertions of AluYa5. Particular attention is paid for the identification of various artifacts arising in course of library preparation and the parameters for their filtration. Pipeline sensitivity is confirmed by in silico experiments with artificial datasets. Using the proposed pipeline we remove at least 80% of artifacts and preserve 75% of potentially somatic insertions. The approaches used in this work can be applied for the study of other mobile elements insertion variability.

Characterization of the T-cell Repertoire after Autologous HSCT in Patients with Ankylosing Spondylitis.

Autologous hematopoietic stem cell transplantation (HSCT), a safer type of HSCT than allogeneic HSCT, is a promising therapy for patients with severe autoimmune diseases (ADs). Despite the long history of medical practice, structural changes in the adaptive immune system as a result of autologous HSCT in patients with various types of ADs remain poorly understood. In this study, we used high-throughput sequencing to investigate the structural changes in the peripheral blood T-cell repertoire in adult patients with ankylosing spondylitis (AS) during two years after autologous HSCT. The implementation of unique molecular identifiers allowed us to substantially reduce the impact of the biases occurring during the preparation of libraries, to carry out a comparative analysis of the various properties of the T-cell repertoire between different time points, and to track the dynamics of both distinct T-cell clonotypes and T-cell subpopulations. In the first year of the reconstitution, clonal diversity of the T-cell repertoire remained lower than the initial one in both patients. During the second year after HSCT, clonal diversity continued to increase and reached a normal value in one of the patients. The increase in the diversity was associated with the emergence of a large number of low-frequency clonotypes, which were not identified before HSCT. Efficiency of clonotypes detection after HSCT was dependent on their abundance in the initial repertoire. Almost all of the 100 most abundant clonotypes observed before HSCT were detected 2 years after transplantation and remained highly abundant irrespective of their CD4+ or CD8+ phenotype. A total of up to 25% of peripheral blood T cells 2 years after HSCT were represented by clonotypes from the initial repertoire.

Tracking T-cell immune reconstitution after TCRαß/CD19-depleted hematopoietic cells transplantation in children.

αßT-cell-depleted allogeneic hematopoietic cell transplantation holds promise for the safe and accessible therapy of both malignant and non-malignant blood disorders. Here we employed molecular barcoding normalized T-cell receptor (TCR) profiling to quantitatively track T-cell immune reconstitution after TCRαß-/CD19-depleted transplantation in children. We demonstrate that seemingly early reconstitution of αßT-cell counts 2 months after transplantation is based on only several hundred rapidly expanded clones originating from non-depleted graft cells. In further months, frequency of these hyperexpanded clones declines, and after 1 year the observed T-cell counts and TCRß diversity are mostly provided by the newly produced T cells. We also demonstrate that high TCRß diversity at day 60 observed for some of the patients is determined by recipient T cells and intrathymic progenitors that survived conditioning regimen. Our results indicate that further efforts on optimization of TCRαß-/CD19-depleted transplantation protocols should be directed toward providing more efficient T-cell defense in the first months after transplantation.

Advanced lymphoblastic clones detection in T-cell leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant neoplasm of the lymphocyte precursors that suffered malignant transformation arresting the lymphoid cell differentiation. Clinical studies revealed monoor, more rarely, oligoclonal nature of the disease. A precise identification of malignant clone markers is both the crucial stage of early diagnostics and the essential prognostic factor for therapeutic treatment. Here we present an improved system for unbiased detection of lymphoblastic clones in bone marrow aspirates of T-ALL patients. The system based on multiplex PCR of rearranged T-cell receptor locus (TRB) and straightforward sequencing of the resulted PCR fragments. Testing of the system on genomic DNA from Jurkat cell line and four clinical bone marrow aspirates revealed a set of unique TRB rearrangements that precisely characterize each of tested samples. Therefore, the outcome of the system produces highly informative molecular genetic markers for further monitoring of minimal residual disease in T-ALL patients.

Association of ERAP1 Allelic Variants with Risk of Ankylosing Spondylitis.

Ankylosing spondylitis (AS) belongs to a group of autoimmune diseases affecting the axial skeleton. Beside thehla-b*27allele, several other human genes that control the variety processes of immune homeostasis are considered to be associated with AS manifestation in different human populations. Among strong associated non-MHC geneserap1 encodingthe endoplasmic reticulum aminopeptidase 1 isoform was recently identified by single nucleotide polymorphisms (SNPs) meta analysis. In our study we inspected the genetic association of five non-synonymous coding SNPs fromerap1 withAS in Caucasians. We implemented the SSP-PCR system for precise genotyping of 87hla-b*27positive AS patients and 77hla-b*27healthy donors from the Russian population. Considerable differences in allele's frequencies within patients vs control cohort were shown for 3 of 5 SNPs under investigation. Using the EM-algorhitm we reconstructed 3-marker haplotypes that distinguish with high probability two cohorts due to differences in the haplotypes frequencies. In such a way both the sensitive, CCT, haplotype and the protective, TTC, one were predicted. To verify the calculation we determined genuine frequencies of 5-marker haplotypes in AS cohort by haplotyping of individual cDNA samples using improved SSP-PCR primer set. We demonstrated that the frequencies ofin silicareconstucted haplotypes and the frequencies of experimentally detected haplotypes are in a good agreement. Frequency of the risk haplotype CCT (rs17482078/10050860/2287987) detected within AS cohort reaches 88%, as well as the frequency calculated by EM-algorhitm.

Individual characterization of stably expanded T cell clones in ankylosing spondylitis patients.

Ankylosing spondylitis (AS) is commonly characterized by clonal expansions of T cells. However, these clonal populations are poorly studied and their role in disease initiation and progression remains unclear. Here, we performed mass sequencing of TCR V beta libraries to search for the expanded T cell clones for two AS patients. A number of clones comprising more than 5% of the corresponding TCR V beta family were identified in both patients. For the first time, expanded clones were shown to be stably abundant in blood samples of AS patients for the prolonged period (1.5 and 2.5 years for two patients, correspondingly). These clones were individually characterized in respect to their differentiation status using fluorescent cell sorting with CD27, CD28, and CD45RA markers followed by quantitative identification of each clone within corresponding fraction using real time PCR analysis. Stable clones differed in phenotype and several were shown to belong to the proinflammatory CD27 - /CD28 - population. Their potentially cytotoxic status was confirmed by staining with perforin-specific antibodies. Search for the TCR V beta CRD3 sequences homologous to the identified clones revealed close matches with the previously reported T cell clones from AS and reactive arthritis patients, thus supporting their role in the disease and proposing consensus TCR V beta CDR3 motifs for AS. Interestingly, these motifs were also found to have homology with earlier reported virus-specific CDR3 variants, indicating that viral infections could play role in development of AS.

From East to West: patterns of genetic diversity of populations living in four Eurasian regions.

We have analyzed the distribution and patterns of the genetic diversity of eight Alu loci (ACE, ApoA1, PV92, TPA25, NBC27, NBC102, NBC148, and NBC182) in 1,049 individuals representing 16 populations of the Volga-Ural region (Bashkirs, Tatars, Komis, Maris, Mordvins, and Udmurts), Central Asia (Kazakhs, Uzbeks, and Uighurs), the North Caucasus (Karachays, Kumyks, Kuban Nogays, and Karanogays), and Central South Siberia (Yakuts, Kalmyks and Evenks). Geographic divide between Europe and Asia, e.g. the Ural Mountains and the Caspian Sea, can also be considered as a genetic boundary. The data indicates that the populations of the two boundary regions between Europe and Asia, the Volga-Ural region of Russia, and populations of the North Caucasus are more similar to European than to Asian populations. Finally, Siberian and Central Asian populations are genetically closely related to each other.

Genome-wide experimental identification and functional analysis of human specific retroelements.

Retroelements (REs) actively reshape genomes through genomic rearrangements, creation of new genes and modulation of the regulatory machinery of existing genes, thus introducing genomic novelties which potentially may be subject to natural selection. Thousands of RE integrations, presumably distinguishing the human and chimpanzee genomes, might well be involved in modern human speciation. In this self-review we describe our recent results on genome-wide identification of human specific RE integrations and their transcriptional activity obtained with three new experimental techniques (TGDA, DiffIR and SDDIR) developed by us for such studies. A new mechanism of the formation of retroelements involving template switches during L1-mediated mRNA reverse transcription, revealed in this research, will also be described in the review.

A rare event of insertion polymorphism of a HERV-K LTR in the human genome.

Human endogenous retroviruses (HERVs), which constitute a significant part of the human genome, might have a serious impact on primate evolution. Over a hundred insertions of HERV-K(HML-2) family members distinguish the human genome from other primate genomes. However, only three cases of insertion polymorphisms have been reported so far, all for endogenous HERV-K proviruses. This suggests that some retroviral integrations occurred rather recently in human genome evolution. In this report, we describe a very rare case of true insertion polymorphism of a solitary HERV-K LTR in the human genome. Distribution of the LTR-containing allele was tested in 5 Africans and 83 individuals from three Russian populations. The allele frequency appeared to be relatively high in populations of both European and Asian origin. The detected polymorphic LTR could be a useful molecular genetic marker of the corresponding genomic region.

Identification of paralogous HERV-K LTRs on human chromosomes 3, 4, 7 and 11 in regions containing clusters of olfactory receptor genes.

A locus harboring a human endogenous retroviral LTR (long terminal repeat) was mapped on the short arm of human chromosome 7 (7p22), and its evolutionary history was investigated. Sequences of two human genome fragments that were homologous to the LTR-flanking sequences were found in human genome databases: (1) an LTR-containing DNA fragment from region 3p13 of the human genome, which includes clusters of olfactory receptor genes and pseudogenes; and (2) a fragment of region 21q22.1 lacking LTR sequences. PCR analysis demonstrated that LTRs with highly homologous flanking sequences could be found in the genomes of human, chimp, gorilla, and orangutan, but were absent from the genomes of gibbon and New World monkeys. A PCR assay with a primer set corresponding to the sequence from human Chr 3 allowed us to detect LTR-containing paralogous sequences on human chromosomes 3, 4, 7, and 11. The divergence times for the LTR-flanking sequences on chromosomes 3 and 7, and the paralogous sequence on chromosome 21, were evaluated and used to reconstruct the order of duplication events and retroviral insertions. (1) An initial duplication event that occurred 14-17 Mya and before LTR insertion - produced two loci, one corresponding to that located on Chr 21, while the second was the ancestor of the loci on chromosomes 3 and 7. (2) Insertion of the LTR (most probably as a provirus) into this ancestral locus took place 13 Mya. (3) Duplication of the LTR-containing ancestral locus occurred 11 Mya, forming the paralogous modern loci on Chr 3 and 7.

Full-sized HERV-K (HML-2) human endogenous retroviral LTR sequences on human chromosome 21: map locations and evolutionary history.

One of the evolutionary mechanisms for acquisition of novel functional sequences can be domestication of exogenous retroviruses that have been integrated into the germ line. The whole genome mapping of such elements in various species could reveal differences in positions of the retroviral integration and suggest possible roles of these differences in speciation. Here, we describe the number, locations and sequence features of the human endogenous retrovirus HERV-K (HML-2) long terminal repeat (LTR) sequences on human chromosome 21. We show that their distribution along the chromosome is not only non-random but also roughly correlated with the gene density. Amplification of orthologous LTR sites from a number of primate genomes produced patterns of presence and absence for each LTR sequence and allowed determination of the phylogenetic ages and evolutionary order of appearance of individual LTRs. The identity level and phylogenetic age of the LTRs did not correlate with their map locations. Thus, despite the non-random distribution of LTRs, they have apparently been inserted randomly into the chromosome relative to each other. As evidenced in previous studies of chromosomes 19 and 22, this is a characteristic of HERV-K integration.

Solitary HERV-K LTRs possess bi-directional promoter activity and contain a negative regulatory element in the U5 region.

Reporter gene analysis of HERV-K solitary long terminal repeats (LTRs) showed that they retain detectable activity in human teratocarcinoma cells, and can direct the transcription in both orientations relative to the reporter gene. Deletion analysis demonstrated the possible existence of alternative promoters within the LTR as well as a silencer-like element in the U5 region. Our results indicate also that all-trans-retinoic acid is capable of modulating expression of the reporter gene directed by a HERV-K LTR in NT2/D1 cells.

Differences in HERV-K LTR insertions in orthologous loci of humans and great apes.

The classification of the long terminal repeats (LTRs) of the human endogenous retrovirus HERV-K (HML-2) family was refined according to diagnostic differences between the LTR sequences. The mutation rate was estimated to be approximately equal for LTRs belonging to different families and branches of human endogenous retroviruses (HERVs). An average mutation rate value was calculated based on differences between LTRs of the same HERV and was found to be 0.13% per million years (Myr). Using this value, the ages of different LTR groups belonging to the LTR HML-2 subfamily were found to vary from 3 to 50Myr. Orthologous potential LTR-containing loci from different primate species were PCR amplified using primers corresponding to the genomic sequences flanking LTR integration sites. This allowed us to calculate the phylogenetic times of LTR integrations in primate lineages in the course of the evolution and to demonstrate that they are in good agreement with the LTR ages calculated from the mutation rates. Human-specific integrations for some very young LTRs were demonstrated. The possibility of LTRs and HERVs involvement in the evolution of primates is discussed.

Fine mapping of a polymorphic CA repeat marker on human chromosome 19 and its use in population studies.

A highly polymorphic microsatellite (CA)n-marker (CAct685) previously isolated from human chromosome 19 cosmid library was localized near GPI in 19q13.1. For the fine localization of this marker, the hybridization with chromosome 19-specific cosmid libraries assembled in contigs was used. Polymorphism analysis of the marker in 12 populations of Russia and neighboring countries showed 14 alleles containing from 16 to 30 repeat units. Populations belonging to Indo-European, Uralic and Altaic linguistic families demonstrated a great similarity in allele frequency profiles. Differences between these populations were lower for CAct685 than for classical markers. Allele distribution of CAct685 in a Chukchi population belonging to the Chukchi-Kamchatkan linguistic family differs from those in all other populations, that may be typical for Mongoloid population or reflect an ethnic history of Chukchi as a small population. Thus use of the CAct685 marker seems to be effective for analysis of distant peoples.

A human endogenous retrovirus-like (HERV) LTR formed more than 10 million years ago due to an insertion of HERV-H LTR into the 5' LTR of HERV-K is situated on human chromosomes 10, 19 and Y.

A chimeric long terminal repeat (LTR) containing the whole LTR of a human endogenous retrovirus-like element of the H family (HERV-H) inserted downstream of the core enhancer region of the 5' LTR of a HERV-K retroelement was detected and sequenced in the human 19p12 locus, known to be enriched with genes encoding zinc finger proteins. Similar chimeras were also detected in human chromosomes 10 and Y in human-hamster hybrid cells containing individual human chromosomes. This finding was interpreted as evidence of transpositions of the chimera in the genome. PCR analyses detected the chimera in the genomes of chimpanzee and gorilla, but not in that of orangutan. These data demonstrate that the chimera appeared in the primate germ cells more than 10 million years ago, before divergence of the human/chimpanzee and the gorilla lineages. The combination of the two LTRs forms a new regulatory system that can be involved in nearby gene expression.

High polymorphism level of genomic sequences flanking insertion sites of human endogenous retroviral long terminal repeats.

The polymorphism at the multitude of loci adjacent to human endogenous retrovirus long terminal repeats (LTRs) was analyzed by a technique for whole genome differential display based on the PCR suppression effect that provides selective amplification and display of genomic sequences flanking interspersed repeated elements. This strategy is simple, target-specific, requires a small amount of DNA and provides reproducible and highly informative data. The average frequency of polymorphism observed in the vicinity of the LTR insertion sites was found to be about 12%. The high incidence of polymorphism within the LTR flanks together with the frequent location of LTRs near genes makes the LTR loci a useful source of polymorphic markers for gene mapping.

Identification and localization of differences between Escherichia coli and Salmonella typhimurium genomes by suppressive subtractive hybridization.

The availability of bacterial genome sequences raises an important new problem - how can one move from completely sequenced microorganisms as a reference to the hundreds and thousands of other strains or isolates of the same or related species that will not be sequenced in the near future? An efficient way to approach this task is the comparison of genomes by subtractive hybridization. Recently we developed a sensitive and reproducible subtraction procedure for comparison of bacterial genomes, based on the method of suppression subtractive hybridization (SSH). In this work we demonstrate the applicability of subtractive hybridization to the comparison of the related but markedly divergent bacterial species Escherichia coli and Salmonella typhimurium. Clone libraries representing sequence differences were obtained and, in the case of completely sequenced E. coli genome, the differences were directly placed in the genome map. About 60% of the differential clones identified by SSH were present in one of the genomes under comparison and absent from the other. Additional differences in most cases represent sequences that have diverged considerably in the course of evolution. Such an approach to comparative bacterial genomics can be applied both to studies of interspecies evolution - to elucidate the "strategies" that enable different genomes to fit their ecological niches - and to development of diagnostic probes for the rapid identification of pathogenic bacterial species.

High resolution mapping DNAs by R-loop atomic force microscopy.

R-loops formed by short RNA transcripts have been imaged by atomic force microscopy (AFM) at a constant force in the height mode. The technique was applied to mapping the human endogenous retrovirus K10 family (HERV-K10) long terminal repeats (LTR) within individual plasmids and cosmids. RNA probes specific for the U3 (384 nt) and U5 (375 nt) LTR regions separated by a span of 200 bp were used for R-loop formation with LTRs located within plasmid (3.8 kb) or cosmid ( approximately 40 kb) DNAs. R-loops stabilized by glyoxal treatment and adsorbed onto the mica surface in the presence of magnesium ions looked like looped out segments of RNA:DNA hybrids. The total yield of R-loops was usually approximately 95%. The RNA:DNA hybrids were found to be 12-15% shorter than the corresponding DNA:DNA duplex. The two regions of the LTR could be easily discerned in the AFM images as clearly separated loops. R-loop positions determined on cosmids by AFM were accurate to approximately 0.5% of the cosmid length. This technique might be easily adapted for mapping various sequences such as gene exons or regulatory regions and for detecting insertions, deletions and rearrangements that cause human genetic diseases.