Somatic diversification of immunoglobulin heavy chain VDJ genes: evidence for somatic gene conversion in rabbits

Rabbits preferentially utilize only one of their multiple functional germline immunoglobulin VH genes. This preferential usage of one gene, VH1, raises the question of how rabbits generate antibody diversity. VDJ diversification was analyzed by cloning and sequencing VH1 gene rearrangements. Comparison of these sequences with that of germline VH1 identified clusters of nucleotide changes, including codon insertions and deletions. To investigate whether gene conversion was involved in this somatic diversification, we searched a data base of rabbit germline VH gene sequences for donor VH genes; potential donors were identified for five diversified regions. We conclude that somatic gene conversion has a major role in generating antibody diversity in rabbits. These studies provide clear evidence for somatic gene conversion of mammalian VDJ genes.     

基因测序技术的发展以及对个体化用药水平提高的影响

自基因测序技术发明之时起,就已开始运用在生命科学的研究中,对揭示生命本质的研究起到了关键作用。基因测序技术的运用推动了生命科学的发展,并由此引申了更多的科学问题;人们对未知领域的渴求又推动了基因测序技术的进步,发展出更高速、更低价的新技术。随着测序技术的逐步应用,临床个体化用药的水平有了极大的提高。基因测序技术目前已经成功应用于遗传基因多态性标志物的筛选中,使基因导向的合理用药成为可能;还成功应用于疾病组织突变位点标志物的筛查中,使肿瘤靶向用药成为可能;在病原体耐药基因突变检测中的应用,使基于细菌或病毒耐药突变的个体化用药成为可能。随着测序技术向更高通量、更高精度、更低成本的方向发展,基于基因检测的个体化健康时代将会到来。     

Selection and mutation in the “new” genetics: an emerging hypothesis

It has been anticipated that new, much more sensitive, next generation sequencing (NGS) techniques, using massively parallel sequencing, will likely provide radical insights into the genetics of multifactorial diseases. While NGS has been used initially to analyze individual human genomes, and has revealed considerable differences between healthy individuals, we have used NGS to examine genetic variation within individuals, by sequencing tissues “in depth”, i.e., oversequencing many thousands of times. Initial studies have revealed intra-tissue genetic heterogeneity, in the form of multiple variants of a single gene that exist as distinct “majority and “minority” variants. This highly specialized form of somatic mosaicism has been found within both cancer and normal tissues. If such genetic variation within individual tissues is widespread, it will need to be considered as a significant factor in the ontogeny of many multifactorial diseases, including cancer. The discovery of majority and minority gene variants and the resulting somatic cell heterogeneity in both normal and diseased tissues suggests that selection, as opposed to mutation, might be the critical event in disease ontogeny. We, therefore, are proposing a hypothesis to explain multifactorial disease ontogeny in which pre-existing multiple somatic gene variants, which may arise at a very early stage of tissue development, are eventually selected due to changes in tissue microenvironments.     

Detection of base substitution-type somatic mosaicism of the NLRP3 gene with >99.9% statistical confidence by massively parallel sequencing

Chronic infantile neurological cutaneous and articular syndrome (CINCA), also known as neonatal-onset multisystem inflammatory disease (NOMID), is a dominantly inherited systemic autoinflammatory disease and is caused by a heterozygous germline gain-of-function mutation in the NLRP3 gene. We recently found a high incidence of NLRP3 somatic mosaicism in apparently mutation-negative CINCA/NOMID patients using subcloning and subsequent capillary DNA sequencing. It is important to rapidly diagnose somatic NLRP3 mosaicism to ensure proper treatment. However, this approach requires large investments of time, cost, and labour that prevent routine genetic diagnosis of low-level somatic NLRP3 mosaicism. We developed a routine pipeline to detect even a low-level allele of NLRP3 with statistical significance using massively parallel DNA sequencing. To address the critical concern of discriminating a low-level allele from sequencing errors, we first constructed error rate maps of 14 polymerase chain reaction products covering the entire coding NLRP3 exons on a Roche 454 GS-FLX sequencer from 50 control samples without mosaicism. Based on these results, we formulated a statistical confidence value for each sequence variation in each strand to discriminate sequencing errors from real genetic variation even in a low-level allele, and thereby detected base substitutions at an allele frequency as low as 1% with 99.9% or higher confidence.     

Absolute quantification of somatic DNA alterations in human cancer

We describe a computational method that infers tumor purity and malignant cell ploidy directly from analysis of somatic DNA alterations. The method, named ABSOLUTE, can detect subclonal heterogeneity and somatic homozygosity, and it can calculate statistical sensitivity for detection of specific aberrations. We used ABSOLUTE to analyze exome sequencing data from 214 ovarian carcinoma tumor-normal pairs. This analysis identified both pervasive subclonal somatic point-mutations and a small subset of predominantly clonal and homozygous mutations, which were overrepresented in the tumor suppressor genes TP53 and NF1 and in a candidate tumor suppressor gene CDK12. We also used ABSOLUTE to infer absolute allelic copy-number profiles from 3,155 diverse cancer specimens, revealing that genome-doubling events are common in human cancer, likely occur in cells that are already aneuploid, and influence pathways of tumor progression (for example, with recessive inactivation of NF1 being less common after genome doubling). ABSOLUTE will facilitate the design of clinical sequencing studies and studies of cancer genome evolution and intra-tumor heterogeneity.     

I.29 lymphoma cells express a nonmutated VH gene before and after H chain switch

The I.29 B cell lymphoma consists of IgM+ and IgA+ cells which express the same germ-line VH gene. IgA+ cells of the I.29 lymphoma were derived from the IgM+ cells by a typical H chain switch recombination event. The IgM+ cells can be induced with LPS to undergo H chain switching in culture. It has been proposed that the somatic hypermutation process is activated during H chain switch, since V genes expressed in IgG+ and IgA+ cells have more frequently undergone mutation than those expressed in IgM+ cells. We have investigated this question by sequencing VH genes expressed before and after H chain switch in the I.29 lymphoma. We have also sequenced the germ-line VH gene corresponding to the gene expressed by I.29 cells to determine whether the VH gene expressed in the IgM+ cells had already undergone somatic mutation. Our results indicate that somatic mutation was not activated in the precursor cell for the I.29 lymphoma, nor during isotype switch in I.29 cells. It is possible that cells of the I.29 lymphoma, or their precursor, have not received the signal which induces somatic mutation, or that I.29 cells belong to a subset of B cells that cannot be induced to undergo any (or much) somatic mutation.     

A Method of Estimating the Numbers of Human and Mouse Immunoglobulin V-Genes

Mutations in immunoglobulin V-genes can be due to gene multiplication, allelic variations, mutations induced by antigens or somatic mutations, etc., and various combinations of these. Since the number of different mouse lambda light V-gene nucleotide sequences is relatively small, a pairwise comparison between these sequences can provide a rough idea as to the contributions of the above mechanisms to the number of nucleotide differences between sequences. A plot of occurrences against the number of differences suggests that differences between one to five can be attributed to somatic mutations. Six to 12 differences can be allelic. Thirteen to 17 may be due to allelic variations together with somatic mutations. Differences >17 appear to be derived from gene multiplication. Although these numbers are most likely somewhat different in humans, they can nevertheless provide a rough guide to sort out the effect of gene multiplication. Estimations of human heavy, kappa and lambda light chain immunoglobulin V-genes are in reasonably good agreement with recent experimental studies. For mouse kappa light and heavy chains, our estimations can provide some insight to future analyses by direct sequencing of these gene segments.     

体细胞变异对神经系统常见肿瘤和发育异常类疾病的致病性

在生物体发育过程中各种内源性及外源性因素均可造成DNA损伤,引起体细胞变异.研究表明体细胞变异对肿瘤具有致病性作用,而体细胞变异对神经系统发育异常类疾病的致病性鲜有报道.新一代测序技术的发展,尤其是全外显子测序,靶向深度测序的应用大大提高了低频体细胞变异检出的敏感性,使科研人员重新认识了体细胞变异在神经系统肿瘤和发育异常类疾病发生中的致病性.本文综述了体细胞变异在神经系统肿瘤和发育异常类疾病致病性方面的研究进展,旨在为今后研究该类疾病的遗传病因提供新的思路,同时也为新药开发提供理论依据.     

Global, comparative analysis of tissue-specific promoter CpG methylation

Understanding cell-type-specific epigenetic codes on a global level is a major challenge after the sequencing of the human genome has been completed. Here we applied methyl-CpG immunoprecipitation (MCIp) to obtain comparative methylation profiles of coding and noncoding genes in three human tissues, testis, brain, and monocytes. Forty-four mainly testis-specific promoters were independently validated using bisulfite sequencing or single-gene MCIp, confirming the results obtained by the MCIp microarray approach. We demonstrate the previously unknown somatic hypermethylation at many CpG-rich, testis-specific gene promoters, in particular in ampliconic areas of the Y chromosome. We also identify a number of miRNA genes showing tissue-specific methylation patterns. The comparison of the obtained tissue methylation profiles with corresponding gene expression data indicates a significant association between tissue-specific promoter methylation and gene expression, not only in CpG-rich promoters. In summary, our study highlights the exceptional epigenetic status of germ-line cells in testis and provides a global insight into tissue-specific DNA methylation patterns.     

Molecular cloning of an immunoglobulin kappa constant gene from NZB mouse

An EcoRI fragment carrying the immunoglobulin C kappa gene and multiple J gene segments from the DNA of the NZB strain of mouse was cloned into lambda Ch 4A DNA. Subsequent characterization of the clone by heteroduplex analysis, restriction-enzyme mapping and DNA sequencing demonstrated that the organization of the J gene segments and the C kappa gene of NZB mouse was similar, if not identical, to that of DNA from the Balb/c strain of mouse. Since the amino acid sequence of the light chain of a plasmacytoma of NZB mouse shows a J region sequence different from that of Balb/c mouse, our results indicate that the new J sequence arose by somatic mutation.     

Expressed antibody repertoires in human cord blood cells: 454 sequencing and IMGT/HighV-QUEST analysis of germline gene usage, junctional diversity, and somatic mutations

Human cord blood cell-derived IgM antibodies are important for the neonate immune responses and construction of germline-based immunoglobulin libraries. Several previous studies of a relatively small number of sequences found that they exhibit restrictions in the usage of germline genes and in the diversity of the variable heavy chain complementarity determining region 3 compared to adults. To further characterize such restrictions on a larger scale and to compare the early B-cell diversity to adult IgM repertoires, we performed 454 sequencing and IMGT/HighV-QUEST analysis of cord blood IG libraries from two babies and determined germline gene usage, V-D-J rearrangement, VHCDR3 diversity, and somatic mutations to characterize human neonate repertoire. Most of the germline subgroups were identified with frequencies comparable to those present in the adult IgM repertoire except for the IGHV1-2 gene that was preferentially expressed in the cord blood cells. The gene usage diversity contributed to 1,430 unique IGH V-D-J rearrangement patterns while the exonuclease trimming and N region addition at the V-D-J junctions along with gene diversity created a wide range of VHCDR3 with different lengths and sequence variability. We observed a lower degree of somatic mutations in the CDR and framework regions of antibodies from cord blood cells compared to adults. These results provide insights into the characteristics of human cord blood antibody repertoires, which have gene usage diversity and VHCDR3 lengths similar to that of the adult IgM repertoire but differ significantly in some of the gene usages, V-D-J rearrangements, junctional diversity, and somatic mutations.     

Gastric cancer: basic aspects

The worldwide incidence and mortality of gastric cancer (GC) remain high, and new concepts for diagnosis and treatment are needed. In this review, we summarize recent studies that applied next-generation sequencing approaches and also report the latest development in microRNA research. Two recently published studies identified somatic mutations in ARID1A gene in GC using exome sequencing. On the other hand, dysregulation of microRNA expression can alter processes such as proliferation, apoptosis, invasion, and metastasis. These novel markers may prove to be useful in earlier diagnosis and as prognostic or predictive markers in patients with GC .     

Absence of somatic mosaicism in 17 families with hemophilia B: an analysis with a sensitivity 10- to 1000-fold greater than that of sequencing gels

Most estimates of germ-line mosaicism have been derived from families in which there has been transmission of a mutated allele to two or more children by an unaffected individual. Previously, analyses for somatic mosaicism detected five such individuals by PCR-based sequencing and haplotype analysis at a sensitivity of approximately 1 mutant per 10 wild-type alleles. To determine whether mutations that occur later in embryogenesis also give rise to somatic mosaicism, we analyzed leukocyte DNA from 17 individuals in whom a mutation in the factor IX gene was known to have originated. Methods capable of detecting 1 mutant allele in 100–10 000 were utilized, and no further examples of somatic mosaicism were detected. If confirmed by future studies, the paucity of somatic mosaicism with mutant:wild-type allele frequencies ranging from 1:10 to 1:1000 (relative to the 11% of somatic mosaicism detected with mutant:wild-type allele frequencies of 1:1 to 1:10) may reflect a higher mutation rate and/or germ-line lineage allocation very early in embryogenesis. Received: 14 July 1995 / Revised: 1 April 1996     

Multiplex DNA deletion detection and exon sequencing of the hypoxanthine phosphoribosyltransferase gene in Lesch-Nyhan families

The Lesch-Nyhan (LN) syndrome is a genetically lethal human neurological disease that results from mutations that inactivate the hypoxanthine phosphoribosyltransferase (HPRT) gene. The elucidation of the complete DNA sequence of the human HPRT gene locus has enabled the construction of multiple oligonucleotide primer sets for the simultaneous in vitro amplification of all nine HPRT exons. The multiplex polymerase chain reaction provides a facile assay for the detection of HPRT exon deletions and the reaction products can be analyzed by direct automated fluorescent DNA sequencing to identify subtle alterations in the gene. Alterations have been identified in the HPRT genes from 15 independent LN cases, and 10 LN family studies were performed. The sequencing method uses solid supports and is sufficiently simple and sensitive to be a favored approach for LN diagnosis. LN heterozygotes can be diagnosed without reference to the affected male. In addition, these procedures will be useful for somatic mutagenesis studies.     

Targeted deep resequencing of the human cancer genome using next-generation technologies

Next-generation sequencing technologies have revolutionized our ability to identify genetic variants, either germline or somatic point mutations, that occur in cancer. Parallelization and miniaturization of DNA sequencing enables massive data throughput and for the first time, large-scale, nucleotide resolution views of cancer genomes can be achieved. Systematic, large-scale sequencing surveys have revealed that the genetic spectrum of mutations in cancers appears to be highly complex with numerous low frequency bystander somatic variations, and a limited number of common, frequently mutated genes. Large sample sizes and deeper resequencing are much needed in resolving clinical and biological relevance of the mutations as well as in detecting somatic variants in heterogeneous samples and cancer cell sub-populations. However, even with the next-generation sequencing technologies, the overwhelming size of the human genome and need for very high fold coverage represents a major challenge for up-scaling cancer genome sequencing projects. Assays to target, capture, enrich or partition disease-specific regions of the genome offer immediate solutions for reducing the complexity of the sequencing libraries. Integration of targeted DNA capture assays and next-generation deep resequencing improves the ability to identify clinically and biologically relevant mutations.