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Мамедов Ильгар ЗияддиновичКандидат биологических наук Старший научный сотрудник (Лаборатория сравнительной и функциональной геномики) Тел.: Эл. почта: Imamedov@ibch.ru |
Избранные публикации
- (2016). Local fitness landscape of the green fluorescent protein. Nature 533 (7603), 397–401
[+]Fitness landscapes depict how genotypes manifest at the phenotypic level and form the basis of our understanding of many areas of biology, yet their properties remain elusive. Previous studies have analysed specific genes, often using their function as a proxy for fitness, experimentally assessing the effect on function of single mutations and their combinations in a specific sequence or in different sequences. However, systematic high-throughput studies of the local fitness landscape of an entire protein have not yet been reported. Here we visualize an extensive region of the local fitness landscape of the green fluorescent protein from Aequorea victoria (avGFP) by measuring the native function (fluorescence) of tens of thousands of derivative genotypes of avGFP. We show that the fitness landscape of avGFP is narrow, with 3/4 of the derivatives with a single mutation showing reduced fluorescence and half of the derivatives with four mutations being completely non-fluorescent. The narrowness is enhanced by epistasis, which was detected in up to 30% of genotypes with multiple mutations and mostly occurred through the cumulative effect of slightly deleterious mutations causing a threshold-like decrease in protein stability and a concomitant loss of fluorescence. A model of orthologous sequence divergence spanning hundreds of millions of years predicted the extent of epistasis in our data, indicating congruence between the fitness landscape properties at the local and global scales. The characterization of the local fitness landscape of avGFP has important implications for several fields including molecular evolution, population genetics and protein design.
ID:1529 - (2014). Distinctive properties of identical twins' TCR repertoires revealed by high-throughput sequencing. Proc. Natl. Acad. Sci. U.S.A. , [+]
Adaptive immunity in humans is provided by hypervariable Ig-like molecules on the surface of B and T cells. The final set of these molecules in each organism is formed under the influence of two forces: individual genetic traits and the environment, which includes the diverse spectra of alien and self-antigens. Here we assess the impact of individual genetic factors on the formation of the adaptive immunity by analyzing the T-cell receptor (TCR) repertoires of three pairs of monozygous twins by next-generation sequencing. Surprisingly, we found that an overlap between the TCR repertoires of monozygous twins is similar to an overlap between the TCR repertoires of nonrelated individuals. However, the number of identical complementary determining region 3 sequences in two individuals is significantly increased for twin pairs in the fraction of highly abundant TCR molecules, which is enriched by the antigen-experienced T cells. We found that the initial recruitment of particular TCR V genes for recombination and subsequent selection in the thymus is strictly determined by individual genetic factors. J genes of TCRs are selected randomly for recombination; however, the subsequent selection in the thymus gives preference to some α but not β J segments. These findings provide a deeper insight into the mechanism of TCR repertoire generation.
ID:1015 - (2014). Age-Related Decrease in TCR Repertoire Diversity Measured with Deep and Normalized Sequence Profiling. J. Immunol. 192 (6), 2689–98 [+]
The decrease of TCR diversity with aging has never been studied by direct methods. In this study, we combined high-throughput Illumina sequencing with unique cDNA molecular identifier technology to achieve deep and precisely normalized profiling of TCR β repertoires in 39 healthy donors aged 6-90 y. We demonstrate that TCR β diversity per 10(6) T cells decreases roughly linearly with age, with significant reduction already apparent by age 40. The percentage of naive T cells showed a strong correlation with measured TCR diversity and decreased linearly up to age 70. Remarkably, the oldest group (average age 82 y) was characterized by a higher percentage of naive CD4(+) T cells, lower abundance of expanded clones, and increased TCR diversity compared with the previous age group (average age 62 y), suggesting the influence of age selection and association of these three related parameters with longevity. Interestingly, cross-analysis of individual TCR β repertoires revealed a set >10,000 of the most representative public TCR β clonotypes, whose abundance among the top 100,000 clones correlated with TCR diversity and decreased with aging.
ID:1010 - (2013). Preparing unbiased T-cell receptor and antibody cDNA libraries for the deep next generation sequencing profiling. Front Immunol 4, 456 [+]
High-throughput sequencing has the power to reveal the nature of adaptive immunity as represented by the full complexity of T-cell receptor (TCR) and antibody (IG) repertoires, but is at present severely compromised by the quantitative bias, bottlenecks, and accumulated errors that inevitably occur in the course of library preparation and sequencing. Here we report an optimized protocol for the unbiased preparation of TCR and IG cDNA libraries for high-throughput sequencing, starting from thousands or millions of live cells in an investigated sample. Critical points to control are revealed, along with tips that allow researchers to minimize quantitative bias, accumulated errors, and cross-sample contamination at each stage, and to enhance the subsequent bioinformatic analysis. The protocol is simple, reliable, and can be performed in 1-2 days.
ID:995 - (2013). High-throughput identification of antigen-specific TCRs by TCR gene capture. Nat. Med. , [+]
The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.
ID:885 - (2013). MiTCR: software for T-cell receptor sequencing data analysis. Nat. Methods 10 (9), 813–4 ID:886
- (2013). Pairing of T-cell receptor chains via emulsion PCR. European journal of immunology , [+]
Our ability to analyze adaptive immunity and engineer its activity has long been constrained by our limited ability to identify native pairs of heavy-light antibody chains and alpha-beta T-cell receptor (TCR) chains - both of which comprise coupled "halves of a key", collectively capable of recognizing specific antigens. Here we report a cell-based emulsion RT-PCR approach that allows the selective fusion of the native pairs of amplified TCR alpha and beta chain genes for complex samples. A new type of PCR suppression technique was developed that makes it possible to amplify the fused library with minimal noise for subsequent analysis by high-throughput paired-end Illumina sequencing. With this technique, single analysis of a complex blood sample allows identification of multiple native TCR chain pairs. This approach may be extended to identify native antibody chain pairs and, more generally, pairs of mRNA molecules that are co-expressed in the same living cells. This article is protected by copyright. All rights reserved.
ID:849 - (2012). Next generation sequencing for TCR repertoire profiling: platform-specific features and correction algorithms. European journal of immunology , [+]
The T-cell receptor (TCR) repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next generation sequencing (NGS) is becoming a powerful tool for deep TCR profiling; yet, questions abound regarding the methodological approaches for sample preparation and correct data interpretation. Accumulated PCR and sequencing errors along with library preparation bottlenecks and uneven PCR efficiencies lead to information loss, biased quantification, and generation of huge artificial TCR diversity. Here, we compare Illumina, 454, and Ion Torrent platforms for individual TCR profiling, evaluate the rate and character of errors, and propose advanced platform-specific algorithms to correct massive sequencing data. These developments are applicable to a wide variety of NGS applications. We demonstrate that advanced correction allows the removal of the majority of artificial TCR diversity with concomitant rescue of most of the sequencing information. Thus, this correction enhances the accuracy of clonotype identification and quantification as well as overall TCR diversity measurements.
ID:733 - (2012). First autologous hematopoietic SCT for ankylosing spondylitis: a case report and clues to understanding the therapy. Bone marrow transplantation , ID:732
- (2011). Quantitative tracking of T cell clones after haematopoietic stem cell transplantation. EMBO Mol Med 3 (4), 201–7 [+]
Autologous haematopoietic stem cell transplantation is highly efficient for the treatment of systemic autoimmune diseases, but its consequences for the immune system remain poorly understood. Here, we describe an optimized RNA-based technology for unbiased amplification of T cell receptor beta-chain libraries and use it to perform the first detailed, quantitative tracking of T cell clones during 10 months after transplantation. We show that multiple clones survive the procedure, contribute to the immune response to activated infections, and form a new skewed and stable T cell receptor repertoire.
ID:453 - (2010). Contribution of functional KIR3DL1 to ankylosing spondylitis. Cellular & molecular immunology , [+]
Increasing evidence points to a role for killer immunoglobulin-like receptors (KIRs) in the development of autoimmune diseases. In particular, a positive association of KIR3DS1 (activating receptor) and a negative association of KIR3DL1 (inhibitory receptor) alleles with ankylosing spondylitis (AS) have been reported by several groups. However, none of the studies analyzed these associations in the context of functionality of polymorphic KIR3DL1. To better understand how the KIR3DL1/3DS1 genes determine susceptibility to AS, we analyzed the frequencies of alleles and genotypes encoding functional (KIR3DL1*F) and non-functional (KIR3DL1*004) receptors. We genotyped 83 AS patients and 107 human leukocyte antigen (HLA)-B27-positive healthy controls from the Russian Caucasian population using a two-stage sequence-specific primer PCR, which distinguishes KIR3DS1, KIR3DL1*F and KIR3DL1*004 alleles. For the patients carrying two functional KIR3DL1 alleles, those alleles were additionally genotyped to identify KIR3DL1*005 and KIR3DL1*007 alleles, which are functional but are expressed at low levels. KIR3DL1 was negatively associated with AS at the expense of KIR3DL1*F but not of KIR3DL1*004. This finding indicates that the inhibitory KIR3DL1 receptor protects against the development of AS and is not simply a passive counterpart of the segregating KIR3DS1 allele encoding the activating receptor. However, analysis of genotype frequencies indicates that the presence of KIR3DS1 is a more important factor for AS susceptibility than the absence of KIR3DL1*F. The activation of either natural killer (NK) or T cells via the KIR3DS1 receptor can be one of the critical events in AS development, while the presence of the functional KIR3DL1 receptor has a protective effect. Nevertheless, even individuals with a genotype that carried two inhibitory KIR3DL1 alleles expressed at high levels could develop AS.Cellular & Molecular Immunology advance online publication, 6 September 2010; doi:10.1038/cmi.2010.42.
ID:370 - (2010). Normalization of genomic DNA using duplex-specific nuclease. BioTechniques 48 (6), 351–355
[+]An application of duplex-specific nuclease (DSN) normalization technology to whole-genome shotgun sequencing of genomes with a large proportion of repetitive DNA is described. The method uses a thermostable DSN from the Kamchatka crab that specifically hydrolyzes dsDNA. In model experiments on human genomic DNA, we demonstrated that DSN normalization of double-stranded DNA formed during C0t analysis is effective against abundant repetitive sequences with high sequence identity, while retaining highly divergent repeats and coding regions at baseline levels. Thus, DSN normalization applied to C0t analysis can be used to eliminate evolutionarily young repetitive elements from genomic DNA before sequencing, and should prove invaluable in studies of large eukaryotic genomes, such as those of higher plants.
ID:339 - (2010). A new set of markers for human identification based on 32 polymorphic Alu insertions. EJHG , [+]
A number of genetic systems for human genetic identification based on short tandem repeats or single nucleotide polymorphisms are widely used for crime detection, kinship studies and in analysis of victims of mass disasters. Here, we have developed a new set of 32 molecular genetic markers for human genetic identification based on polymorphic retroelement insertions. Allele frequencies were determined in a group of 90 unrelated individuals from four genetically distant populations of the Russian Federation. The mean match probability and probability of paternal exclusion, calculated based on population data, were 5.53 x 10(-14) and 99.784%, respectively. The developed system is cheap and easy to use as compared to all previously published methods. The application of fluorescence-based methods for allele discrimination allows to use the human genetic identification set in automatic and high-throughput formats.European Journal of Human Genetics advance online publication, 24 February 2010; doi:10.1038/ejhg.2010.22.
ID:338 - (2010). Optogenetic in vivo cell manipulation in KillerRed-expressing zebrafish transgenics. BMC Dev. Biol. 10, 110 [+]
KillerRed (KR) is a novel photosensitizer that efficiently generates reactive oxygen species (ROS) in KR-expressing cells upon intense green or white light illumination in vitro, resulting in damage to their plasma membrane and cell death.
ID:508 - (2009). Individual characterization of stably expanded T cell clones in ankylosing spondylitis patients. Autoimmunity 42 (6), 525–36 [+]
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.
ID:276 - (2009). [The oligoclonal expansion of T cells: study of its stability over time]. Bioorg. Khim. 36 (2), 206–14 [+]
A novel experimental approach to the investigation of the repertoire of peripheral T lymphocytes of patients suffering from ankylosing spondylitis (AS) is proposed. This approach is based on the wide-range sequencing of cDNA of the beta-chain of the T-cellular receptor (TcR). The results of the analysis of the diversity of sequences of the TcR antigen-binding domain (CDR3) inside the total pool of one patient with AS are presented by the example of the second V family (BV2) of TcR. The expansion of six independent TcR-expressing clones of T cells with a similar amino acid sequence of the CDR3 domains was proposed based on the results of the comparative structural analysis of the clone libraries of the cDNA of TcR BV2. The long-time stable expansion of these T clones was demonstrated during the development of the disease by specific monitoring.
ID:405 - (2009). [Retroposons in modern human genome evolution]. Bioorg. Khim. 35 (6), 779–88 [+]
The ascertainment of the rates and driving forces of human genome evolution along with the genetic diversity of populations or separate population groups remains a topical problem of fundamental and applied genomics. According to the results of comparative analysis, the most numerous human genome structure peculiarities are connected with the distribution of mobile genetic retroelements - LTR, LINE1, SVA, and Alu repeats. Due to the wide distribution in different genome loci, conversed retropositional activity, and the retroelements regulatory potential, let us regard them as one of the significant evolutionary driving forces and the source of human genome variability. In the current review, we summarize published data and recent results of our research aimed at the analysis of the evolutionary impact of the young retroelements group on the function and variability of the human genome. We examine modern approaches of the polygenomic identification of polymorphic retroelements inserts. Using an original Internet resource, we analyze special features of the genomic polymorphic inserts of Alu repeats. We thoroughly characterize the strategy of large-scale functional analysis of polymorphic retroelement inserts. The presented results confirm the hypothesis of the roles of retroelements as active cis regulatory elements that are able to modulate surrounding genes.
ID:509 - (2009). [A new database on polymorphic retroelements in human genome (PRED)]. Mol. Biol. (Mosk.) 42 (4), 721–7 [+]
Comparison of primate genomes sequences has confirmed the evidence that substantial part of intra- and interspecies differences is provided by retroelements. Human genome contains thousands of polymorphic retroelement copies considered to be perspective molecular genetic markers of new generation. However utilization of polymorphic retroelements as molecular genetic markers is limited due to lack of systematic data on their number, genomic context and distribution among human populations. We have created first bilingual (Russian/English) internet-resource devoted to known polymorphic retroelements discovered in human genome by our group as well as by other researchers worldwide. The database contains information about each retroelement copy location, position relative to known and predicted genes, frequency of alleles in human populations and others. Our internet portal allows to perform a search in database using multiple search conditions and available on http://labcfg.ibch.ru/home.html. The database provides an opportunity to investigate distribution of polymorphic retroelements in human genome and to design new genetic markers for various population and medical studies.
ID:510 - (2007). Most recent AluY insertions in human gene introns reduce the content of the primary transcripts in a cell type specific manner. Gene 390 (1-2), 122–9 [+]
Предложена общая стратегия широко масштабного анализа влияния ретроэлементов на функционирование генома приматов, использующая явление инсерционного полиморфизма Alu элементов генома человека. Предложен метод сравнения транскрипционной активности аллельных вариантов генов человека, отличающихся по интронным инсерциям AluY элементов. Открыт ингибиторный эффект диморфных AluY инсерций на экспрессию пораженного аллеля и показан тканеспецифический характер проявления обнаруженного эффекта.
ID:100 - (2005). Whole-genome experimental identification of insertion/deletion polymorphisms of interspersed repeats by a new general approach. Nucleic Acids Res. 33 (2), e16 [+]
Разработана уникальная технология экспериментального сравнительно-структурного анализа смесевых образцов геномной ДНК, направленная на полногеномную идентификацию полиморфных инсерций отдельных классов ретроэлементов. Эффективность нового экспериментального подхода к изучению инсерционного полиморфизма ретроэлементов генома человека доказана открытием обширной группы диморфных инсерций AluY элементов, до 40% представителей которой не могли быть идентифицированы биоинформатическими методами анализа.
ID:99 - (2004). A rare event of insertion polymorphism of a HERV-K LTR in the human genome. Genomics 84 (3), 596–9 [+]
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.
ID:511 - (2004). Unusually long target site duplications flanking some of the long terminal repeats of human endogenous retrovirus K in the human genome. J. Gen. Virol. 85 (Pt 6), 1485–8 [+]
Human endogenous retroviruses (HERVs) make up a substantial part of the human genome. HERVs and solitary long terminal repeats (solo LTRs) are usually flanked by 4-6 nt short direct repeats through the well-known mechanism of their integration. A number of solo LTRs flanked by unusually long direct repeats were detected in the human genome. These unusual structures might be a product of an alternative virus insertion mechanism.
ID:512 - (2003). Human-specific subfamilies of HERV-K (HML-2) long terminal repeats: three master genes were active simultaneously during branching of hominoid lineages. Genomics 81 (2), 149–56 [+]
Полный структурный анализ специфичных для генома человека LTR-элементов. Впервые доказана множественная ретропозиция эндогенных ретровирусов в ходе эволюции генома человека.
ID:96 - (2002). Genome-wide comparison of differences in the integration sites of interspersed repeats between closely related genomes. Nucleic Acids Res. 30 (14), e71 [+]
Представлен окончательный вариант экспериментального подхода к полногеномному сравнительному анализу распределения участков интеграции LTR-элементов в геномах близкородственных видов. Впервые идентифицировано и предварительно охарактеризовано 11 инсерций LTR-элементов, отличающих геном человека от генома шимпанзе.
ID:18 - (2002). A technique for genome-wide identification of differences in the interspersed repeats integrations between closely related genomes and its application to detection of human-specific integrations of HERV-K LTRs. Genomics 79 (3), 413–22 [+]
В статье опубликован новый метод, позволяющий сравнивать распределение геномных повторов между близкородственными геномами при помощи метода вычитающей гибридизации
ID:17