The reported germline repertoire of human immunoglobulin kappa chain genes is relatively complete and accurate

We describe a bioinformatic analysis of germline and rearranged immunoglobulin kappa chain (IGK) gene sequences, performed in order to assess the completeness and reliability of the reported IGK repertoire. In contrast to the reported heavy-chain gene repertoire, which includes many dubious sequences, only five IGK variable gene (IGKV) alleles appear to have been reported in error. There was, however, insufficient evidence to justify removing these IGKV genes from the germline repertoire. Bioinformatic analysis of apparent mismatches between reported germline genes and 1,863 expressed IGK sequences suggested the existence of two unreported IGKV polymorphisms. Genomic screening of 12 individuals led to the confirmation of both of these polymorphisms, IGKV1-16*02 and IGKV2-30*02. We also show that in contrast to the heavy chain, the IGK repertoire is dominated by sequences that use just a handful of kappa variable (IGKV) and junction (IGKJ) gene pairs. There is also little modification of IGKV and IGKJ genes by the processes of exonuclease removal and N nucleotide addition. The expressed IGK repertoire therefore lacks diversity and the junction region is particularly constrained. Remarkably, the analysis of a dataset of 435 relatively unmutated rearranged kappa genes showed that ten amino acid sequences account for almost 10% of the rearrangements, with identical sequences being derived from as many as seven independent sources. Such dominant sequences are likely to have important roles in the operation of the humoral immune response.     

Genomic screening by 454 pyrosequencing identifies a new human IGHV gene and sixteen other new IGHV allelic variants

Complete and accurate knowledge of the genes and allelic variants of the human immunoglobulin gene loci is critical for studies of B cell repertoire development and somatic point mutation, but evidence from studies of VDJ rearrangements suggests that our knowledge of the available immunoglobulin gene repertoire is far from complete. The reported repertoire has changed little over the last 15 years. This is, in part, a consequence of the inefficiencies involved in searching for new members of large, multigenic gene families by cloning and sequencing. The advent of high-throughput sequencing provides a new avenue by which the germline repertoire can be explored. In this report, we describe pyrosequencing studies of the heavy chain IGHV1, IGHV3 and IGHV4 gene subgroups in ten Papua New Guineans. Thousands of 454 reads aligned with complete identity to 51 previously reported functional IGHV genes and allelic variants. A new gene, IGHV3-NL1*01, was identified, which differs from the nearest previously reported gene by 15 nucleotides. Sixteen new IGHV alleles were also identified, 15 of which varied from previously reported functional IGHV genes by between one and four nucleotides, while one sequence appears to be a functional variant of the pseudogene IGHV3-25. BLAST searches suggest that at least six of these new genes are carried within the relatively well-studied populations of North America, Europe or Asia. This study substantially expands the known immunoglobulin gene repertoire and demonstrates that genetic variation of immunoglobulin genes can now be efficiently explored in different human populations using high-throughput pyrosequencing.     

Reconsidering the human immunoglobulin heavy-chain locus:

We have used a bioinformatics approach to evaluate the completeness and functionality of the reported human immunoglobulin heavy-chain IGHD gene repertoire. Using the hidden Markov-model-based iHMMune-align program, 1,080 relatively unmutated heavy-chain sequences were aligned against the reported repertoire. These alignments were compared with alignments to 1,639 more highly mutated sequences. Comparisons of the frequencies of gene utilization in the two databases, and analysis of features of aligned IGHD gene segments, including their length, the frequency with which they appear to mutate, and the frequency with which specific mutations were seen, were used to determine the reliability of alignments to the less commonly seen IGHD genes. Analysis demonstrates that IGHD4-23 and IGHD5-24, which have been reported to be open reading frames of uncertain functionality, are represented in the expressed gene repertoire; however, the functionality of IGHD6-25 must be questioned. Sequence similarities make the unequivocal identification of members of the IGHD1 gene family problematic, although all genes except IGHD1-14*01 appear to be functional. On the other hand, reported allelic variants of IGHD2-2 and of the IGHD3 gene family appear to be nonfunctional, very rare, or nonexistent. Analysis also suggests that the reported repertoire is relatively complete, although one new putative polymorphism (IGHD3-10*p03) was identified. This study therefore confirms a surprising lack of diversity in the available IGHD gene repertoire, and restriction of the germline sequence databases to the functional set described here will substantially improve the accuracy of IGHD gene alignments and therefore the accuracy of analysis of the V–D–J junction.Electronic Supplementary Material Supplementary material is available for this article at     

The mouse antibody heavy chain repertoire is germline-focused and highly variable between inbred strains

The human and mouse antibody repertoires are formed by identical processes, but like all small animals, mice only have sufficient lymphocytes to express a small part of the potential antibody repertoire. In this study, we determined how the heavy chain repertoires of two mouse strains are generated. Analysis of IgM- and IgG-associated VDJ rearrangements generated by high-throughput sequencing confirmed the presence of 99 functional immunoglobulin heavy chain variable (IGHV) genes in the C57BL/6 genome, and inferred the presence of 164 IGHV genes in the BALB/c genome. Remarkably, only five IGHV sequences were common to both strains. Compared with humans, little N nucleotide addition was seen in the junctions of mouse VDJ genes. Germline human IgG-associated IGHV genes are rare, but many murine IgG-associated IGHV genes were unmutated. Together these results suggest that the expressed mouse repertoire is more germline-focused than the human repertoire. The apparently divergent germline repertoires of the mouse strains are discussed with reference to reports that inbred mouse strains carry blocks of genes derived from each of the three subspecies of the house mouse. We hypothesize that the germline genes of BALB/c and C57BL/6 mice may originally have evolved to generate distinct germline-focused antibody repertoires in the different mouse subspecies.     

Partial versus productive immunoglobulin heavy locus rearrangements in chronic lymphocytic leukemia: implications for B-cell receptor stereotypy

The frequent occurrence of stereotyped heavy complementarity-determining region 3 (VH CDR3) sequences among unrelated cases with chronic lymphocytic leukemia (CLL) is widely taken as evidence for antigen selection. Stereotyped VH CDR3 sequences are often defined by the selective association of certain immunoglobulin heavy diversity (IGHD) genes in specific reading frames with certain immunoglobulin heavy joining (IGHJ ) genes. To gain insight into the mechanisms underlying VH CDR3 restrictions and also determine the developmental stage when restrictions in VH CDR3 are imposed, we analyzed partial IGHD-IGHJ rearrangements (D-J) in 829 CLL cases and compared the productively rearranged D-J joints (that is, in-frame junctions without junctional stop codons) to (a) the productive immunoglobulin heavy variable (IGHV )-IGHD-IGHJ rearrangements (V-D-J) from the same cases and (b) 174 D-J rearrangements from 160 precursor B-cell acute lymphoblastic leukemia cases (pre-B acute lymphoblastic leukemia [ALL]). Partial D-J rearrangements were detected in 272/829 CLL cases (32.8%). Sequence analysis was feasible in 238 of 272 D-J rearrangements; 198 of 238 (83.2%) were productively rearranged. The D-J joints in CLL did not differ significantly from those in pre-B ALL, except for higher frequency of the IGHD7-27 and IGHJ6 genes in the latter. Among CLL carrying productively rearranged D-J, comparison of the IGHD gene repertoire in productive V-D-J versus D-J revealed the following: (a) overuse of IGHD reading frames encoding hydrophilic peptides among V-D-J and (b) selection of the IGHD3-3 and IGHD6-19 genes in V-D-J junctions. These results document that the IGHD and IGHJ gene biases in the CLL expressed VH CDR3 repertoire are not stochastic but are directed by selection operating at the immunoglobulin protein level.     

<Emphasis Type="Italic">IGHV1</Emphasis>,<Emphasis Type="Italic"> IGHV5</Emphasis> and<Emphasis Type="Italic"> IGHV7</Emphasis> subgroup genes in the Rhesus macaque

The diversity of the antibody response is achieved, in part, by rearrangement of different immunoglobulin (Ig) genes. The Ig heavy chain is made up of a variable region (IGHV), a diversity region (IGHD) and a joining region (IGHJ). Human germline IGHV genes have been grouped into seven multigene subgroups. Size and usage of these subgroups is not equal, the IGHV3 subgroup is the most commonly used (36%), followed by IGHV1/7 (26%), then IGHV4, IGHV5, IGHV2, IGHV6 (15%, 12%, 4%, 3% respectively). The rhesus macaque (Macaca mulatta) is a useful non-human primate model for studies of infection and the database of germline Ig genes for the macaque is gradually growing to become a useful tool in the study of B-cell responses. The proportions of IGHV subgroup usage in the macaque are similar to those in man. Representatives from IGHV3 and IGHV4 subgroups for the macaque have been published, as have germline sequences of the IGHD and IGHJ genes. However, to date there have been no sequences published from the second largest IGHV subgroup, IGHV1. We report the isolation and sequencing of a genomic fragment containing an IGHV1 gene from the macaque. Polymerase chain reaction (PCR) primers designed from this sequence enabled us to amplify and sequence 25 new IGHV1 germline genes. We also isolated two IGHV7 genes, using the same primers, and two IGHV5 genes, using human IGHV5 primers.     

Uneven Distribution of Repetitive Trinucleotide Motifs in Human Immunoglobulin Heavy Variable Genes

Insertions and deletions of entire codons have recently been discovered as a mechanism by which B cells, in addition to conventional base substitution, evolve the antibodies produced by their immunoglobulin genes. These events frequently seem to involve repetitive sequence motifs in the antibody-encoding genes, and it has been suggested that they occur through polymerase slippage. In order to better understand the process of codon deletion, we have analyzed the human immunoglobulin heavy variable (IGHV) germline gene repertoire for the presence of trinucleotide repeats. Such repeats would ensure that the reading frame is maintained in the case of a deletional event, as slippage over multiples of three bases would be favored. We demonstrate here that IGHV genes specifically carry repetitive trinucleotide motifs in the complementarity-determining regions (CDR) 1 and 2, thus making these parts of the genes that encode highly flexible structures particularly prone to functional deletions. We propose that the human IGHV repertoire carries inherent motifs that allow an antibody response to develop efficiently by targeting codon deletion events to the parts of the molecule that are likely to be able to harbor such modifications. Received: 10 April 2001 / Accepted: 27 August 2001     

Immunoglobulin heavy- and light-chain repertoire in splenic marginal zone lymphoma

The considerable heterogeneity in morphology, immunophenotype, genotype, and clinical behavior of splenic marginal zone lymphoma (SMZL) hinders firm conclusions on the origin and differentiation stage of the neoplastic cells. Immunoglobulin (IG) gene usage and somatic mutation patterns were studied in a series of 43 SMZL cases. Clonal IGHV-D-J rearrangements were amplified in 42/43 cases (4 cases carried double rearrangements). Among IGHV-D-J rearrangements, IGHV3 and IGHV4 subgroup genes were used with the highest frequency. Nineteen IGHV genes were unmutated (> 98% homology to the closest germline IGHV gene), whereas 27/46 were mutated. Clonal IGKV-J and IGLV-J gene rearrangements were amplified in 36/43 cases, including 31 IGKV-J (8/31 in lambda light-chain expressing cases) and 12 IGLV-J rearrangements; 9/31 IGKV and 6/12 IGLV sequences were mutated. IGKV-J and IGLV-J rearrangements used 14 IGKV and 9 IGLV different germline genes. Significant evidence for positive selection by classical T-dependent antigen was found in only 5/27 IGHV and 6/15 IGKV+IGLV mutated genes. These results provide evidence for the diverse B-cell subpopulations residing in the SMZ, which could represent physiologic equivalents of distinct SMZL subtypes. Furthermore, they indicate that in SMZL, as in other B cell malignancies, a complementarity imprint of antigen selection might be witnessed either by IGHV, IGKV, or IGLV rearranged sequences.     

Expression of cloned immunoglobulin genes introduced into mouse L cells.

Functionally rearranged immunoglobulin heavy-chain (gamma 2b) and light-chain (lambda 1 and kappa) genes were introduced into mouse L tk- cells by co-transformation with the Herpes virus tk gene. Cloned cell lines were selected in HAT medium and tested for the presence of transfected immunoglobulin gene sequences by Southern blotting analysis. It was found that the gamma 2b gene was accurately transcribed at a low level in transfected mouse L cells and cytoplasmic gamma 2b, heavy-chain protein was detected by immunoprecipitation of cell extracts. Light-chain genes, on the other hand, were not accurately transcribed. Instead, lambda 1 or kappa RNA species were detected which were approximately 200 to 300 bases longer than the authentic mRNAs. These results suggest that the expression of rearranged heavy-chain and light-chain genes are controlled differently and that these differences can be seen in transfected, non-lymphoid cells.     

Intraclonal cell expansion and selection driven by B cell receptor in chronic lymphocytic leukemia

The mutational status of the immunoglobulin heavy-chain variable region (IGHV) genes utilized by chronic lymphocytic leukemia (CLL) clones defines two disease subgroups. Patients with unmutated IGHV have a more aggressive disease and a worse outcome than patients with cells having somatic IGHV gene mutations. Moreover, up to 30% of the unmutated CLL clones exhibit very similar or identical B cell receptors (BcR), often encoded by the same IG genes. These "stereotyped" BcRs have been classified into defined subsets. The presence of an IGHV gene somatic mutation and the utilization of a skewed gene repertoire compared with normal B cells together with the expression of stereotyped receptors by unmutated CLL clones may indicate stimulation/selection by antigenic epitopes. This antigenic stimulation may occur prior to or during neoplastic transformation, but it is unknown whether this stimulation/selection continues after leukemogenesis has ceased. In this study, we focused on seven CLL cases with stereotyped BcR Subset #8 found among a cohort of 700 patients; in six, the cells expressed IgG and utilized IGHV4-39 and IGKV1-39/IGKV1D-39 genes, as reported for Subset #8 BcR. One case exhibited special features, including expression of IgM or IgG by different subclones consequent to an isotype switch, allelic inclusion at the IGH locus in the IgM-expressing cells and a particular pattern of cytogenetic lesions. Collectively, the data indicate a process of antigenic stimulation/selection of the fully transformed CLL cells leading to the expansion of the Subset #8 IgG-bearing subclone.     

iHMMune-align: hidden Markov model-based alignment and identification of germline genes in rearranged immunoglobulin gene sequences

MOTIVATION: Immunoglobulin heavy chain (IGH) genes in mature B lymphocytes are the result of recombination of IGHV, IGHD and IGHJ germline genes, followed by somatic mutation. The correct identification of the germline genes that make up a variable VH domain is essential to our understanding of the process of antibody diversity generation as well as to clinical investigations of some leukaemias and lymphomas. RESULTS: We have developed iHMMune-align, an alignment program that uses a hidden Markov model (HMM) to model the processes involved in human IGH gene rearrangement and maturation. The performance of iHMMune-align was compared to that of other immunoglobulin gene alignment utilities using both clonally related and randomly selected IGH sequences. This evaluation suggests that iHMMune-align provides a more accurate identification of component germline genes than other currently available IGH gene characterization programs. AVAILABILITY: iHMMune-align cross-platform Java executable and web interface are freely available to academic users and can be accessed at http://www.emi.unsw.edu.au/~ihmmune/.     

Identification of two new alleles, IGHV3-23*04 and IGHJ6*04, and the complete sequence of the IGHV3-h pseudogene in the human immunoglobulin locus and their prevalences in Danish Caucasians

More than 100 variable (V), 27 diversity (D), and six joining (J) genes are encoded in the human heavy chain locus, and many of these genes exists in different allelic forms. The number of genes and the allelic differences help to create diversity in the immunoglobulin receptors, a key feature of the adaptive immune system. We here report the identification of two novel and seemingly functional alleles of human heavy chain genes. The variable IGHV3-23*04 allele is found with an allele frequency of 0.21 amongst Danish Caucasians, whereas the novel joining IGHJ6*04 allele is rare (allele frequency 0.02). We also report the full sequence of IGHV3-h. The gene exists in two allelic forms but is only found in 58% of the Danish Caucasians studied. The methionine translation initiation codon is mutated, ATG→AAG, and we therefore propose that the gene is a pseudogene incapable of being translated.     

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.     

HAPPY mapping of a YAC reveals alternative haplotypes in the human immunoglobulin VH locus.

We have identified and sequenced 14 human immunoglobulin VH segments cloned in a yeast artificial chromosome, and have used a rapid PCR-based technique (HAPPY mapping, 12) to derive the order and approximate distances between them. The sequences mapped comprise thirteen germline VH segments and one rearranged VH3 gene. Comparison of our map with other data suggests the existence of at least two distinct haplotypes, differing in the presence or absence of the consecutive genes DP-78, DP-46 and DP-64, and in the duplication of segments DP-49 and DP-65. Screening of ten individuals confirms the existence of both haplotypes, and indicates that both are common amongst the population.     

Allelic polymorphisms and RFLP in the human immunoglobulin lambda light chain locus

The organization of the human immunoglobulin lambda light chain locus (IGL) was recently described. This locus has been entirely sequenced. To evaluate the extent of the genomic variability existing inside that locus, we compiled all the available sequences of germline IGLV genes to find variants of Vλ sequences. We also looked for RFLP polymorphisms in a reputedly highly polymorphic human population from eastern Senegal, and compiled all RFLP data previously published. Analysis of these data indicates that IGLV alleles are frequent and increase the diversity of the lambda light chain repertoire in the human population. In contrast, RFLP and polymorphism by insertion and/or deletion are limited in that locus. This observation reinforces our hypothesis that the human IGL locus has undergone less evolutionary shuffling than the human kappa or heavy-chain loci. Received: 23 December 1998 / Accepted: 1 March 1999     

双峰驼重链抗体组库的基本特征研究

目的:利用二代高通量测序技术,了解双峰骆驼循环B细胞重链抗体(HCAbs)组库的组成和基本特征。方法:通过分离骆驼外周血单核细胞(PBMC),提取m RNA,利用多重PCR和Illumina Mi-seq高通量测序技术对三头双峰骆驼的重链抗体可变区进行深度测序,分析了重链抗体组库V、J基因组成、重排时末端基因删除数和V-J基因配对率,以及CDR3的长度、香农多样性指数(Shannon index)、氨基酸组成分布等基本特征。结果:鉴定出平均每头骆驼130000条有效数据和67561条独特CDR3序列,HCAbs含量较高的V基因为IGHV1S45、IGHV1S50和IGHV1S52,J基因为IGHJ4和IGHJ6,所对应的V-J基因配对含量大于40%;CDR3的长度主要分布在10-30个氨基酸之间,含量较高的氨基酸为丙氨酸、甘氨酸和半胱氨酸;CDR3区域70%以上的平均长度为20个氨基酸长度,其中V基因长度为3 bp,J基因长度分布在1-18 bp。结论:双峰骆驼B细胞重链抗体组库由巨大的、不均匀分布(以少数VJ基因克隆占大多数)的和具有高度多样性的多克隆抗体构成,较长CDR3和富含丙氨酸、甘氨酸和半胱氨酸是HCAbs的重要特征。     

Camel heavy-chain antibodies: diverse germline V(H)H and specific mechanisms enlarge the antigen-binding repertoire

The antigen-binding site of the camel heavy-chain antibodies devoid of light chain consists of a single variable domain (V(H)H) that obviously lacks the V(H)-V(L) combinatorial diversity. To evaluate the extent of the V(H)H antigen-binding repertoire, a germline database was constructed from PCR-amplified V(H)H/V(H) segments of a single specimen of Camelus dromedarius. A total of 33 V(H)H and 39 V()H unique sequences were identified, encoded by 42 and 50 different genes, respectively. Sequence comparison indicates that the V(H)Hs evolved within the V(H) subgroup III. Nevertheless, the V(H)H germline segments are highly diverse, leading to a broad structural repertoire of the antigen-binding loops. Seven V(H)H subfamilies were recognized, of which five were confirmed to be expressed in vivo. Comparison of germline and cDNA sequences demonstrates that the rearranged V(H)Hs are extensively diversified by somatic mutation processes, leading to an additional hypervariable region and a high incidence of nucleotide insertions or deletions. These diversification processes are driven by hypermutation and recombination hotspots embedded in the V(H)H germline genes at the regions affecting the structure of the antigen-binding loops.     

Direct cDNA cloning of the rearranged immunoglobulin variable region

A major problem in the study of multigene families is the effort required to clone and sequence these genes. We describe a method to rapidly clone and sequence immunoglobulin variable region gene sequences without constructing cDNA libraries. Because immunoglobulin variable-region genes are flanked by conserved sequences, we have been able to apply the polymerase chain reaction (PCR) to clone and sequence both the light- and heavy-chain rearranged immunoglobulin genes from small numbers of hybridoma cells. This method will greatly facilitate the construction of chimeric mouse/human monoclonal antibodies for immunoglobulin structural studies as well as for therapeutic use.