Somatic DNA Rearrangements Research Articles

Childhood cancer mutagenesis caused by transposase-derived PGBD5.

Genomic rearrangements are a hallmark of most childhood tumors, including medulloblastoma, one of the most common brain tumors in children, but their causes remain largely unknown. Here, we show that PiggyBac transposable element derived 5 (Pgbd5) promotes tumor development in multiple developmentally accurate mouse models of Sonic Hedgehog (SHH) medulloblastoma. Most Pgbd5-deficient mice do not develop tumors, while maintaining normal cerebellar development. Ectopic activation of SHH signaling is sufficient to enforce cerebellar granule cell progenitor-like cell states, which exhibit Pgbd5-dependent expression of distinct DNA repair and neurodevelopmental factors. Mouse medulloblastomas expressing Pgbd5 have increased numbers of somatic structural DNA rearrangements, some of which carry PGBD5-specific sequences at their breakpoints. Similar sequence breakpoints recurrently affect somatic DNA rearrangements of known tumor suppressors and oncogenes in medulloblastomas in 329 children. This identifies PGBD5 as a medulloblastoma mutator and provides a genetic mechanism for the generation of oncogenic DNA rearrangements in childhood cancer.

Age-Related Clonal Hematopoiesis Is a Precursor for Adult Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) represents 20% of adult leukemias. Recent technologic advances have enabled detailed characterization of the genetic basis of leukemogenesis in ALL, including somatic structural DNA rearrangements and sequence mutations that disrupt lymphoid development, signaling, tumor suppression, and epigenetic modification. These studies also showed differences in the molecular profiles of pediatric vs adult ALL. However, adults with ALL, especially older adults (≥40 years), were underrepresented in these large series. Clinical outcomes of older adults with ALL are inferior to younger patients (<40 years) and the molecular basis for these differences is not completely understood.Hematopoietic stem cells accumulate DNA mutations with aging, and age-related clonal hematopoiesis (ARCH) has been linked to increased incidence of myeloid malignancies. The prevalence of ARCH increases logarithmically as the population ages, but its role in lymphoid leukemogenesis has not been fully established. We hypothesize that ARCH is a common precursor lesion for the development of ALL in older adults, and patients with ARCH-associated ALL have different clinical outcomes compared to patients whose disease do not harbor these mutations.We retrospectively studied adults with ALL treated at the University of Chicago and Moffitt Cancer Center between July 2014 and April 2021. Genetic profiling of tumor samples was performed by using Miseq Illumina next-generation sequencing (NGS) platform with a comprehensive sequencing panel covering commonly mutated myeloid and lymphoid genes. We classified pathogenicity using American College of Medical Genetics and Genomics guidelines.In total, 345 patients were studied: 286 (83%) had B-ALL, 49 (14%) had T-ALL and 10 (3%) had early T-precursor (ETP)-ALL. Overall, median age at diagnosis was 47 years (range, 18-88 years), and 211 (61%) were ≥40 years at diagnosis; 154 (45%) were women. Cytogenetic groups were as follows: 24% had Ph+ ALL, 13% had Ph-like ALL, and 3% had ALL with KMT2A rearrangement. The most frequent mutation in our adult ALL cohort was the loss of CDKN2A gene (32%), followed by mutations in TP53 (17%), IKZF1 (16%), NOTCH1 (9%), NRAS (9%), and JAK2 (6%) genes. Mutations involving the recurrently mutated genes in ARCH were seen in 110 of 345 patients (32%) with the following order of frequency: TP53 (17%), DNMT3A (5%), TET2 (4%), RUNX1 (3.5%), ASXL1 (3%), IDH1/2 (2%), BCORL1 (2%), EZH2 (1%), CUX1 (1%), and U2AF1 (1%) (Figure 1A). ARCH-associated mutations were more common in older adults (≥40 years) compared to young adults (41% vs 17%, p< 0.0001). Variant allelic frequencies (VAFs) for the ARCH-associated mutations were higher than the mutations involving signaling pathways, which suggests the ancestral nature of the former and secondary nature of the latter (Figure 1B). We further observed clonal dynamics in patients with serial diagnosis, remission and relapse samples available for sequencing. Founder ARCH clones re-emerged at the time of relapse (patient 92 and 100), and were also detectable at the time of complete remission with persistent measurable residual disease (patient 100) (Figure 1C). The overall survival (OS) for patients with ARCH-associated ALL was shorter than patients without ARCH, but the difference did not reach statistical significance (median OS, 39 months vs 84 months, p= 0.16) (Figure 1D).Our results indicate that ARCH is commonly identified as an ancestral event in older adults with ALL, with TP53 mutations being the most prevalent. Unlike patients with AML and TP53 mutations, patients with ALL and ARCH-associated mutations had comparable clinical outcomes to patients without ARCH. This may reflect the frequent use of antibody-based therapies (i.e. blinatumomab and inotuzumab) at diagnosis (on a clinical trial basis) or relapse in the two centers where these patients were treated. Collectively, these data suggest that ARCH may constitute a fertile soil for acute lymphoblastic leukemogenesis and further studies are warranted to interrogate the dynamic interplay between myeloid and lymphoid compartments of these patients. [Display omitted] DisclosuresStock: Pfizer: Consultancy, Honoraria, Research Funding; amgen: Honoraria; agios: Honoraria; jazz: Honoraria; kura: Honoraria; kite: Honoraria; morphosys: Honoraria; servier: Honoraria; syndax: Consultancy, Honoraria; Pluristeem: Consultancy, Honoraria. Shah: BeiGene: Consultancy, Honoraria; Incyte: Research Funding; Acrotech/Spectrum: Honoraria; Novartis: Consultancy, Other: Expenses; Pfizer: Consultancy, Other: Expenses; Amgen: Consultancy; Servier Genetics: Other; Jazz Pharmaceuticals: Research Funding; Precision Biosciences: Consultancy; Pharmacyclics/Janssen: Honoraria, Other: Expenses; Kite, a Gilead Company: Consultancy, Honoraria, Other: Expenses, Research Funding; Adaptive Biotechnologies: Consultancy; Bristol-Myers Squibb/Celgene: Consultancy, Other: Expenses.

ClonoMatch: a tool for identifying homologous immunoglobulin and T-cell receptor sequences in large databases.

B-cell receptor (BCR) and T-cell receptor (TCR) repertoires are generated through somatic DNA rearrangements and are responsible for the molecular basis of antigen recognition in the immune system. Next-generation sequencing (NGS) of DNA and the falling cost of sequencing due to continued development of these technologies have made sequencing assays an affordable way to characterize the repertoire of adaptive immune receptors (sometimes termed the 'immunome'). Many new workflows have been developed to take advantage of NGS and have placed the resulting immunome datasets in the public domain. The scale of these NGS datasets has made it challenging to search through the Complementarity-determining region 3 (CDR3), which is responsible for imparting specific antibody-antigen interactions. Thus, there is an increasing demand for sequence analysis tools capable of searching through CDR3s from immunome data collections containing millions of sequences. To address this need, we created a software package called ClonoMatch that facilitates rapid searches in bulk immunome data for BCR or TCR sequences based on their CDR3 sequence or V3J clonotype. Documentation, software support and the codebase are all available at https://github.com/crowelab/clonomatch. This software is distributed under the GPL v3 license.

Distinct Classes of Complex Structural Variation Uncovered across Thousands of Cancer Genome Graphs

Cancer genomes often harbor hundreds of somatic DNA rearrangement junctions, many of which cannot be easily classified into simple (e.g., deletion) or complex (e.g., chromothripsis) structural variant classes. Applying a novel genome graph computational paradigm to analyze the topology of junction copy number (JCN) across 2,778 tumor whole-genome sequences, we uncovered three novel complex rearrangement phenomena: pyrgo, rigma, and tyfonas. Pyrgo are "towers" of low-JCN duplications associated with early-replicating regions, superenhancers, and breast or ovarian cancers. Rigma comprise "chasms" of low-JCN deletions enriched in late-replicating fragile sites and gastrointestinal carcinomas. Tyfonas are "typhoons" of high-JCN junctions and fold-back inversions associated with expressed protein-coding fusions, breakend hypermutation, and acral, but not cutaneous, melanomas. Clustering of tumors according to genome graph-derived features identified subgroups associated with DNA repair defects and poor prognosis.

Identification of genetic variants controlling RNA editing and their effect on RNA structure stabilization.

Post-transcriptional modification of RNA (RNA editing, RNAe) results in differences between the RNA transcript and the genomic DNA sequence (RDD). Enzymatic modification of adenosine to inosine (A2I) by ADAR is the most studied type of RNAe. However, few genetic association studies with A2I RNAe events have been conducted. Some studies have analyzed the inter-population RNAe-QTL diversity in humans, but the sample size of these studies was limited. Other types of RNA and DNA differences have been reported but are largely understudied. Here, we report a comprehensive analysis of all types of RDD, based on two independent datasets. We found that A2I was by far the most observed type of RDD. Moreover, manual curation suggests that A2I is likely the only enzymatically driven RNAe type observed in blood derived DNA, all other non-A2I RDD could either be attributed to sequencing and processing artifacts, or are a result of somatic DNA rearrangements. We then conducted an in-cis genetic association study and identified 472 genetic associations (RNAe-QTL), that were replicated in both datasets. We confirm the potential effect of the RNAe-QTL on RNA structure by showing that allele specific RNAe occurs in heterozygotes. Although the generally assumed function of RNAe is to destabilize double stranded RNA structure, we found clear evidence for the potential additional involvement of RNAe in maintaining RNA hairpin that has been altered by the RNAe-QTL. Our study confirms, in two independent datasets, the potential role of RNAe in maintaining RNA structure in the presence of genetic variation.

RNA sequencing identifies clonal structure of T-cell repertoires in patients with adult T-cell leukemia/lymphoma

The diversity of T-cell receptor (TCR) repertoires, as generated by somatic DNA rearrangements, is central to immune system function. High-throughput sequencing technologies now allow examination of antigen receptor repertoires at single-nucleotide and, more recently, single-cell resolution. The TCR repertoire can be altered in the context of infections, malignancies or immunological disorders. Here we examined the diversity of TCR clonality and its association with pathogenesis and prognosis in adult T-cell leukemia/lymphoma (ATL), a malignancy caused by infection with human T-cell leukemia virus type-1 (HTLV-1). We analyzed 62 sets of high-throughput RNA sequencing data from 59 samples of HTLV-1−infected individuals—asymptomatic carriers (ACs), smoldering, chronic, acute and lymphoma ATL subtypes—and three uninfected controls to evaluate TCR distribution. Based on these TCR profiles, CD4-positive cells and ACs showed polyclonal patterns, whereas ATL patients showed oligo- or monoclonal patterns (with 446 average clonotypes across samples). Expression of TCRα and TCRβ genes in the dominant clone differed among the samples. ACs, CD4-positive samples and smoldering patients showed significantly higher TCR diversity compared with chronic, acute and lymphoma subtypes. CDR3 sequence length distribution, amino acid conservation and gene usage variability for ATL patients resembled those of peripheral blood cells from ACs and healthy donors. Thus, determining monoclonal architecture and clonal diversity by RNA sequencing might be useful for prognostic purposes and for personalizing ATL diagnosis and assessment of treatments.

T cell receptor repertoires of mice and humans are clustered in similarity networks around conserved public CDR3 sequences.

Diversity of T cell receptor (TCR) repertoires, generated by somatic DNA rearrangements, is central to immune system function. However, the level of sequence similarity of TCR repertoires within and between species has not been characterized. Using network analysis of high-throughput TCR sequencing data, we found that abundant CDR3-TCRβ sequences were clustered within networks generated by sequence similarity. We discovered a substantial number of public CDR3-TCRβ segments that were identical in mice and humans. These conserved public sequences were central within TCR sequence-similarity networks. Annotated TCR sequences, previously associated with self-specificities such as autoimmunity and cancer, were linked to network clusters. Mechanistically, CDR3 networks were promoted by MHC-mediated selection, and were reduced following immunization, immune checkpoint blockade or aging. Our findings provide a new view of T cell repertoire organization and physiology, and suggest that the immune system distributes its TCR sequences unevenly, attending to specific foci of reactivity.

Abstract 4888: Human PGBD5 DNA transposase promotes site-specific oncogenic mutations in rhabdoid tumors

Abstract Genomic rearrangements are a hallmark of childhood solid tumors, but their mutational causes remain poorly understood. Here, we identify the piggyBac transposable element derived 5 (PGBD5) gene as an enzymatically active human DNA transposase expressed in the majority of rhabdoid tumors, a lethal childhood cancer. Using assembly-based whole-genome DNA sequencing, we observed previously unknown somatic genomic rearrangements in primary human rhabdoid tumors. These rearrangements were characterized by deletions and inversions involving PGBD5-specific signal (PSS) sequences at their breakpoints, with some recurrently targeting tumor suppressor genes, leading to their inactivation. PGBD5 was found to be physically associated with human genomic PSS sequences that were also sufficient to mediate PGBD5-induced DNA rearrangements in rhabdoid tumor cells. We found that ectopic expression of PGBD5 in primary human cells was sufficient to promote penetrant cell transformation in vitro and in immunodeficient mice in vivo. This activity required specific catalytic residues in the PGBD5 transposase domain, as well as end-joining DNA repair, and induced distinct structural rearrangements, involving PSS-associated breakpoints, similar to those found in primary human rhabdoid tumors. Thus, PGBD5 defines a distinct class of oncogenic mutators and induces site-specific somatic DNA rearrangements in human cancer. Citation Format: Anton G. Henssen, Richard Koche, Jiali Zhuang, Eileen Jiang, Casie Reed, Amy Eisenberg, Eric Still, Elias Rodríguez-Fos, Santiago Gonzalez, Montserrat Puiggròs, Andrew N. Blackford, Christopher E. Mason, Elisa de Stanchina, Mithat Gönen, Anne-Katrin Emde, Minita Shah, Kanika Arora, Catherine Reeves, Nicholas D. Socci, Elizabeth Perlman, Cristina R. Antonescu, Charles W. Roberts, Hanno Steen, Elizabeth Mullen, Stephen P. Jackson, David Torrents, Zhiping Weng, Scott A. Armstrong, Alex Kentsis. Human PGBD5 DNA transposase promotes site-specific oncogenic mutations in rhabdoid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4888. doi:10.1158/1538-7445.AM2017-4888

Genetic Basis of Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, and despite cure rates exceeding 90% in children, it remains an important cause of morbidity and mortality in children and adults. The past decade has been marked by extraordinary advances into the genetic basis of leukemogenesis and treatment responsiveness in ALL. Both B-cell and T-cell ALL comprise multiple subtypes harboring distinct constellations of somatic structural DNA rearrangements and sequence mutations that commonly perturb lymphoid development, cytokine receptors, kinase and Ras signaling, tumor suppression, and chromatin modification. Recent studies have helped to understand the genetic basis of clonal evolution and relapse and the role of inherited genetic variants in leukemogenesis. Many of these findings are of clinical importance, and ongoing studies implementing clinical sequencing in the management of leukemia are expected to improve diagnosis, monitoring of residual disease, and early detection of relapse and to guide precise therapies. Here, we provide a concise review of genomic studies in ALL and discuss the role of genomic testing in clinical management.

The piggyBac transposon-derived genes TPB1 and TPB6 mediate essential transposon-like excision during the developmental rearrangement of key genes in Tetrahymena thermophila.

Ciliated protozoans perform extreme forms of programmed somatic DNA rearrangement during development. The model ciliate Tetrahymena thermophila removes 34% of its germline micronuclear genome from somatic macronuclei by excising thousands of internal eliminated sequences (IESs), a process that shares features with transposon excision. Indeed, piggyBac transposon-derived genes are necessary for genome-wide IES excision in both Tetrahymena (TPB2 [Tetrahymena piggyBac-like 2] and LIA5) and Paramecium tetraurelia (PiggyMac). T. thermophila has at least three other piggyBac-derived genes: TPB1, TPB6, and TPB7 Here, we show that TPB1 and TPB6 excise a small, distinct set of 12 unusual IESs that disrupt exons. TPB1-deficient cells complete mating, but their progeny exhibit slow growth, giant vacuoles, and osmotic shock sensitivity due to retention of an IES in the vacuolar gene DOP1 (Dopey domain-containing protein). Unlike most IESs, TPB1-dependent IESs have piggyBac-like terminal inverted motifs that are necessary for excision. Transposon-like excision mediated by TPB1 and TPB6 provides direct evidence for a transposon origin of not only IES excision machinery but also IESs themselves. Our study highlights a division of labor among ciliate piggyBac-derived genes, which carry out mutually exclusive categories of excision events mediated by either transposon-like features or RNA-directed heterochromatin.

Chapter Four - The Role of BCR Isotype in B-Cell Development and Activation

The development and function of B lymphocytes critically depend on the non-germline B-cell antigen receptor (BCR). In addition to the diverse antigen-recognition regions, whose coding sequences are generated by the somatic DNA rearrangement, the variety of the constant domains of the Heavy Chain (HC) portion contributes to the multiplicity of the BCR types. The functions of particular classes of the HC, particularly in the context of the membrane BCR, are not completely understood. The expression of the various classes of the HC correlates with the distinct stages of B-cell development, types of B-cell subsets, and their effector functions. In this chapter, we summarize and discuss the accumulated knowledge on the role of the μ, δ, and γ HC isotypes of the conventional and precursor BCR in B-cell differentiation, selection, and engagement with (auto)antigens.

Evaluation of IGHV Ultra-Deep Sequences for Activation-Induced Deaminase Characteristics in CLL Cells after T Cell Stimulation

Ultra-deep sequencing has revolutionized our ability to acquire large amounts of genetic data. We have applied this technology towards understanding the mutational process in B-cell chronic lymphocytic leukemia (CLL), which may be a key to understanding CLL pathogenesis. Acquisition of new cytogenetic aberrations and gene mutations in the CLL clone is associated with worse patient outcome. CLL is not unique in this aspect, as new somatic mutations and DNA rearrangements are also found during the evolution of other solid and liquid tumors. In many of these, activation-induced deaminase (AID), an enzyme normally expressed in germinal center B lymphocytes to induce IGHV-D-J mutations and isotype class switch recombination, is abnormally expressed. Its mutational activity, acting outside of the Ig loci, is implicated in the evolution to more aggressive disease.In CLL, the detection of leukemic cells expressing AID ex vivo correlates with significantly shorter patient survival. To test if AID mutational activity is functional in CLL cells and therefore could contribute to CLL evolution, we analyzed mutations in IGHV-D-J, the preferred substrate for AID. Because the rate of AID-induced mutation is low and only a small percentage of CLL cells express AID ex vivo, we used ultra-deep sequencing to analyze CLL cells that were activated under conditions that simulate the CLL microenvironment. Specifically, CLL cells were activated (1) in vitro by simulating the provision of T-cell help or (2) in vivo after adoptive transfer into alymphoid recipient mice, which requires the presence of T-cells for CLL cell growth. Each of these conditions induce AID in a large fraction of CLL cells.To analyze IGHV-D-J mutations, the specific CLL clone IGHV was amplified from cDNA obtained on day 0 or from the activated CLL samples using IGHV family-specific and IGHM primers to enable subsequent comparison of IGHV-D-J with IGHM mutation frequencies. Three unmutated IGHV CLL (U-CLL) and 3 mutated IGHV (>2% compared to germline) CLL (M-CLL) samples were sequenced with the Roche 454 FLX system, resulting in a total of 1,367,522 sequence reads.After using the Roche 454 algorithm to trim sequence reads, they were prepared using custom R scripts that separated 5'IGHV and 5'IGHM primer sequences, aligned sequences to the CLL clone IGHV-D-J rearrangement, and removed poor quality (<20) sequences, insertions, and deletions. Beginning at the 5' end, the script also extracted blocks of sequences of the same length for day 0 and activated samples, which are required for subsequent analyses. After these preparations, the resulting 724,855 sequence blocks were subjected to clonal analyses with custom R scripts. The dominant CLL clone accounted for 94.5% (684,691) of the sequences. Subclone sequences occurring more than once were extracted. After comparison to day 0, new subclones could be identified in all samples after activation (3.22 – 28.70 new subclones / read bp *106).To evaluate AID mutational characteristics in new subclones, SHMTool (http://scb.aecom.yu.edu/shmtool) was employed to calculate mutation frequencies in IGHV-D-J relative to the IGHM constant region, at AID mutation hotspot sites (GYW or WRC), at AID mutation coldspot sites (SYC or GRS), at C/G base pairs, and at error-prone DNA polymerase eta repair hotspot sites (WA or TW). To calculate statistical significance, we utilized a custom R script that used a bootstrap method to account for the large sample sizes provided by ultra-deep sequencing as well as to correct for differences in sequencing sample size.All samples showed an increase in IGHV-D-J versus IGHM mutations after T cell activation. Five of 6 cases showed an increase in AID hostpot mutation frequency. AID coldspot mutation frequency decreased in 3/6 CLL cases. Percent transition mutation at C/G sites was higher than random in 2/6 CLL cases, which correlated with low frequencies of DNA polymerase eta hotspot mutation. In the other 4/6 CLL cases, the lower percent transitions at C/G sites may reflect the contribution of error-prone DNA repair.In summary, we developed a method to analyze ultra-deep IGHV-D-J sequences that revealed AID mutational characteristics in both U-CLL and M-CLL cells after activation with T-cell help in vitro or in vivo. These data are consistent with the hypothesis that AID, perhaps along with error-prone DNA repair, creates new mutations leading to the evolution of aggressive CLL. Disclosures:Rai:Sanofi: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

A common copy-number breakpoint of ERBB2 amplification in breast cancer colocalizes with a complex block of segmental duplications

IntroductionSegmental duplications (low-copy repeats) are the recently duplicated genomic segments in the human genome that display nearly identical (> 90%) sequences and account for about 5% of euchromatic regions. In germline, duplicated segments mediate nonallelic homologous recombination and thus cause both non-disease-causing copy-number variants and genomic disorders. To what extent duplicated segments play a role in somatic DNA rearrangements in cancer remains elusive. Duplicated segments often cluster and form genomic blocks enriched with both direct and inverted repeats (complex genomic regions). Such complex regions could be fragile and play a mechanistic role in the amplification of the ERBB2 gene in breast tumors, because repeated sequences are known to initiate gene amplification in model systems.MethodsWe conducted polymerase chain reaction (PCR)-based assays for primary breast tumors and analyzed publically available array-comparative genomic hybridization data to map a common copy-number breakpoint in ERBB2-amplified primary breast tumors. We further used molecular, bioinformatics, and population-genetics approaches to define duplication contents, structural variants, and haplotypes within the common breakpoint.ResultsWe found a large (> 300-kb) block of duplicated segments that was colocalized with a common-copy number breakpoint for ERBB2 amplification. The breakpoint that potentially initiated ERBB2 amplification localized in a region 1.5 megabases (Mb) on the telomeric side of ERBB2. The region is very complex, with extensive duplications of KRTAP genes, structural variants, and, as a result, a paucity of single-nucleotide polymorphism (SNP) markers. Duplicated segments are varied in size and degree of sequence homology, indicating that duplications have occurred recurrently during genome evolution.ConclusionsAmplification of the ERBB2 gene in breast tumors is potentially initiated by a complex region that has unusual genomic features and thus requires rigorous, labor-intensive investigation. The haplotypes we provide could be useful to identify the potential association between the complex region and ERBB2 amplification.

Computational Mass Spectrometry–Based Proteomics

Proteomics is defined as the system-widecharacterization of all the proteins in anorganism in terms of their sequence,localization, abundance, post-translationalmodifications, and biomolecular interac-tions. Modern proteomic investigationsare increasingly quantitative and compre-hensive [1]. Examples include the relativequantification of over 4,000 proteins inhaploid and diploid yeast, which identifiedthe pheromone signaling pathway asenriched in differential abundance [2];determination of site- and time-specificdynamics of more than 6,000 phosphory-lation sites of HeLa cells stimulated withepidermal growth factor [3]; and charac-terization of 232 multiprotein complexesin Saccharomyces cerevisiae, which proposednew cellular roles for 344 proteins [4].Such investigations are now successfullyutilized in functional biology [5,6], geno-mics [7,8], and biomedical research [9].Challenges of proteomic studies stemfrom the complexity of the proteome andto its broad dynamic range. For example,the human genome contains around20,000 protein coding genes. Their trans-lation, combined with splicing or proteol-ysis, yields an estimated 50,000–500,000proteins, and over 10 million differentprotein forms can be derived by somaticDNA rearrangements and post-transla-tional modifications [10]. The abundanceof protein species in human plasma spansmore than 10 orders of magnitude [11].Unlike oligonucleotides, proteins cannotbe amplified, and therefore the objectivesof proteomics are achieved by sensitiveand scalable technologies identifying andquantifying proteins [12]. The overallmass spectrometry–based proteomic work-flow is summarized in Figure 1.

Natural Killer Cells: From Basic Research to Treatments

SPECIALTY GRAND CHALLENGE article Front. Immunol., 03 June 2011Sec. NK and Innate Lymphoid Cell Biology volume 2 - 2011 | https://doi.org/10.3389/fimmu.2011.00018

Invariant natural killer T cells: bridging innate and adaptive immunity

Cells of the innate immune system interact with pathogens via conserved pattern-recognition receptors, whereas cells of the adaptive immune system recognize pathogens through diverse, antigen-specific receptors that are generated by somatic DNA rearrangement. Invariant natural killer T (iNKT) cells are a subset of lymphocytes that bridge the innate and adaptive immune systems. Although iNKT cells express T cell receptors that are generated by somatic DNA rearrangement, these receptors are semi-invariant and interact with a limited set of lipid and glycolipid antigens, thus resembling the pattern-recognition receptors of the innate immune system. Functionally, iNKT cells most closely resemble cells of the innate immune system, as they rapidly elicit their effector functions following activation, and fail to develop immunological memory. iNKT cells can become activated in response to a variety of stimuli and participate in the regulation of various immune responses. Activated iNKT cells produce several cytokines with the capacity to jump-start and modulate an adaptive immune response. A variety of glycolipid antigens that can differentially elicit distinct effector functions in iNKT cells have been identified. These reagents have been employed to test the hypothesis that iNKT cells can be harnessed for therapeutic purposes in human diseases. Here, we review the innate-like properties and functions of iNKT cells and discuss their interactions with other cell types of the immune system.

RAG: a recombinase diversified

During B cell and T cell development, the lymphoid-specific proteins RAG-1 and RAG-2 act together to initiate the assembly of antigen receptor genes through a series of site-specific somatic DNA rearrangements that are collectively called variable-diversity-joining (V(D)J) recombination. In the past 20 years, a great deal has been learned about the enzymatic activities of the RAG-1-RAG-2 complex. Recent studies have identified several new and exciting regulatory functions of the RAG-1-RAG-2 complex. Here we discuss some of these functions and suggest that the RAG-1-RAG-2 complex nucleates a specialized subnuclear compartment that we call the 'V(D)J recombination factory'.

Natural Killer T Cells and Autoimmune Disease

Natural killer T (NKT) cells are an unusual subset of innate immune cells that express a surface receptor generated by somatic DNA rearrangement, a hallmark of cells of the adaptive immune system. NKT cells express a highly restricted repertoire of T cell receptors that recognize glycolipid antigens bound with the antigen-presenting molecule CD1d. A hallmark of NKT cells is their capacity to produce copious amounts of immunomodulatory cytokines upon antigenic stimulation, which endows these cells with potent immunoregulatory properties. Consequently, NKT cells have been implicated in regulating a wide variety of immune responses, including immune responses against autoantigens. In patients and mice with a variety of autoimmune diseases, numbers and functions of NKT cells are disturbed, but the relevance of these findings to the etiology of autoimmunity remains to be fully established. Nevertheless, in some mouse models of autoimmunity, NKT cell-deficiency exacerbates disease, suggesting that NKT cells play a role in suppressing autoimmunity. Conversely, specific activation of NKT cells with glycolipid antigens generally protects mice against the development of autoimmunity. Most of these studies have employed the potent sponge-derived NKT cell antigen alpha-galactosylceramide (alpha-GalCer). However, alpha-GalCer treatment in mice was associated with detrimental side effects and treatment efficacy was influenced by a variety of parameters, resulting sometimes in disease exacerbation rather than protection. Recent efforts have focused on developing NKT cell agonists with superior treatment efficacy than alpha-GalCer. Collectively, these studies have identified NKT cells as attractive targets for treatment of human autoimmune diseases.

Mitochondrial DNA as a Cancer Biomarker

As part of a national effort to identify biomarkers for the early detection of cancer, we developed a rapid and high-throughput sequencing protocol for the detection of sequence variants in mitochondrial DNA. Here, we describe the development and implementation of this protocol for clinical samples. Heteroplasmic and homoplasmic sequence variants occur in the mitochondrial genome in patient tumors. We identified these changes by sequencing mitochondrial DNA obtained from tumors and blood from the same individual. We confirmed previously identified primary lung tumor changes and extended these findings in a small patient cohort. Eight sequence variants were identified in stage I to stage IV tumor samples. Two of the sequence variants identified (22%) were found in the D-loop region, which accounts for 6.8% of the mitochondrial genome. The other sequence variants were distributed throughout the coding region. In the forensic community, the sequence variations used for identification are localized to the D-loop region because this region appears to have a higher rate of mutation. However, in lung tumors the majority of sequence variation occurred in the coding region. Hence, incomplete mitochondrial genome sequencing, designed to scan discrete portions of the genome, misses potentially important sequence variants associated with cancer or other diseases.

Genomic rearrangements on VCAM1, SELE, APEG1 and AIF1 loci in atherosclerosis

The inflammatory nature of atherosclerosis has been well established. However, the initial steps that trigger this response in the arterial intima remain obscure. Previous studies reported a significant rate of genomic alterations in human atheromas. The accumulation of genomic rearrangements in vascular endothelium and smooth muscle cells may be important for disease development. To address this issue, 78 post-mortem obtained aortic atheromas were screened for microsatellite DNA alterations versus correspondent venous blood. To evaluate the significance of these observations, 33 additional histologically normal aortic specimens from age and sex-matched cases were examined. Loss of heterozygosity (LOH) was found in 47,4% of the cases and in 18,2% of controls in at least one locus. The LOH occurrence in aortic tissue is associated to atherosclerosis risk (OR 4,06, 95% CI 1,50 to 10,93). Significant genomic alterations were found on 1p32-p31, 1q22-q25, 2q35 and 6p21.3 where VCAM1, SELE, APEG1 and AIF1 genes have been mapped respectively. Our data implicate somatic DNA rearrangements, on loci associated to leukocyte adhesion, vascular smooth muscle cells growth, differentiation and migration, to atherosclerosis development as an inflammatory condition.