Immunoglobulin VH genes in thymic MALT lymphoma are biased toward a restricted repertoire and are frequently unmutated

Thymic MALT lymphoma shows certain distinctive features among MALT lymphomas, such as expression of IgA isotype, consistent lack of API2-MALT1 gene fusion, and very strong association with autoimmune disease, especially Sjogren's syndrome. To help clarify the nature of the clonal lymphoid infiltrates, we analysed the usage and somatic hypermutation of the Ig heavy chain variable region (V(H)) genes in nine different cases. The V(H) rearrangement was potentially functional in all cases and was restricted to the V(H)3 family. V(H) usage was biased toward V(H)3-30 (five cases) and V(H)3-23 (three cases) segments, which have both been frequently expressed by autoimmune B cells. Somatic hypermutation was absent in five cases. Fewer than the expected replacement mutations were found in the framework regions in two cases, indicating a negative antigen selection pressure. Ongoing mutation was absent in all cases. D segment usage was varied, whereas J(H) segment usage was restricted to J(H)4. The observed patterns of V(H) usage and mutations suggested that specific antigens may play a pathologically relevant role in the genesis or progression of thymic MALT lymphoma.     

Immunoglobulin VH genes of the goldfish, Carassius auratus: a re-examination

Five VH-related genomic sequences from the goldfish, Carassius auratus, have been characterized. One of these sequences appeared to be a functional gene, and four to be pseudogenes. The main conclusions drawn from this study were that: (1) With minor exceptions, goldfish VH genes conform to the typical pattern of vertebrate VH genes in terms of their structure (they encode an intron-split hydrophobic leader, 3 framework and 2 complementarity-determining regions, and possess a typical 3' recombination signal sequence for VH to D joining) and regulatory sequences (possession of a typical upstream octameric promoter). (2) The sequences indicate that goldfish possess multiple families of VH sequences (at least three). Two of these families contain approximately 6 and 10 members, as judged from Southern blot hybridization experiments. (3) Goldfish VH gene families are distributed throughout the members of the species in the manner typical of that of VH families in other vertebrate species. Thus, this observation corrects the previous conclusion (Wilson et al., Proc. natn. Acad. Sci. U.S.A. 85, 1566-1570, 1988) that VH genes are discontinuously distributed in the goldfish population.     

A map of VH genes located next to the DH region in the Igh locus of two congenic Igh-recombinant mouse strains

A new congenic mouse strain (C57BL/6-Igh-Vb-Ca) with a recombinant chromosome 12 is described. It carries the Igh-1a allele, but shows the serological characteristics of C57BL/6 when analyzed for idiotype expression with respect to the antigens dextran and (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP). We analyzed liver DNA from one animal for restriction fragment length polymorphism by hybridization to probes detecting members of nine VH gene families and DH segments, and compared it to DNA from animals carrying the nonrecombinant haplotypes Igha and Ighb, respectively. The breakpoint of recombination maps to the region carrying members of VH gene families VGAM3.8, PC7183 and Q52. The CB8KN strain which according to the serological analysis carries a recombinant Igh locus (Igh-Va-Cb) on BALB/c background was also analyzed. In this strain the breakpoint of recombination again maps to the region carrying members of VH gene families VGAM3.8, PC7183 and Q52. Our results show that the VH genes of families PC7183 and Q52 are interspersed and map to the region next to the DH locus. At least one gene from the VGAM3.8 family also maps to this region in the Igha and the Ighb haplotype.     

Properties of overlapping genes are conserved across microbial genomes

There are numerous examples from the genomes of viruses, mitochondria, and chromosomes that adjacent genes can overlap, sharing at least one nucleotide. Overlaps have been hypothesized to be involved in genome size minimization and as a regulatory mechanism of gene expression. Here we show that overlapping genes are a consistent feature (approximately one-third of all genes) across all microbial genomes sequenced to date, have homologs in more microbes than do non-overlapping genes, and are therefore likely more conserved. In addition, the size, phase (reading frame offset), and distribution, among other characteristics, of overlapping genes are most consistent with the hypothesis that overlaps function in the regulation of gene expression. The upstream sequences and conservation of overlapping orthologs of two model organisms from the genus Prochlorococcus that have significantly different GC-content, and therefore different nucleotide sequences for orthologs, are also consistent with small overlapping sequence regions and programmed shifts in reading frame as a common mechanism in the regulation of microbial gene expression.     

Immunoglobulin VH gene expression in human aging.

Immune responses change in aging humans, but it is not known whether there is an age-associated change in the expressed B cell repertoire. We compared Ig VH cDNA libraries from circulating B cells of five elderly and three young human adults. As in young persons, nearly two-thirds of the cDNA clones from older subjects had zero to three V(H) mutations, although there was more individual variation among the elderly. V(H)4 family expression increased in older subjects, both in unmutated and in mutated cDNA clones, whereas V(H)3 family expression predominated in young adults. To test for bias toward activated cells in the cDNA libraries, we studied two older persons by both cDNA library analysis and single-cell RT-PCR. In one subject, more than 85% of VH segments were unmutated by either analysis. In the second, mutated Ig segments were much more frequent in cDNA clones than in consecutive single cells; however, V(H) family usage and high representation of particular genes were similar in both analyses. While aging humans continue to produce naive B cells with unmutated Ig genes, a shift to greater use of the V(H)4 family members and expression of particular genes may reflect changes in selection of developing B cells before affinity maturation toward reactivity with foreign antigen.     

Immunoglobulin VH gene analysis in gastric MALT lymphomas.

The majority of gastric mucosa-associated lymphoid tissue (MALT) lymphomas are successfully treated with Helicobacter pylori eradication alone. However, certain subsets of these tumors are resistant to the eradication treatment. As API2-MALT1 fusion is a feature of one of these subsets, we divided gastric MALT lymphomas into three groups: eradication-responsive and API2-MALT1 fusion-negative (Group A), eradication-resistant and fusion-negative (Group B), and eradication-resistant and fusion-positive (Group C). To characterize further gastric MALT lymphomas, we analyzed VH genes, which do not change in the course of tumor progression, by extensive subcloning of the monoclonal PCR products of 45 cases. VH3-23 and VH3-30 were preferentially used in Group A tumors (14/23 cases, 61%) as compared with Group B (1/10 cases, 10%, P=0.0094) and Group C (2/12 cases, 17%, P=0.017). Tumors of Groups B and C used variegated VH fragments, and no dominant VH fragments were noted. Somatic mutation was detected in most of the cases. Ongoing mutation was detected in 3/45 cases (7%), when assessed according to strict criteria for a confirmed mutation. These findings suggest that inflammation-dependent tumors (Group A) may be derived from a highly restricted, probably H. pylori-associated, B cell subset and may not often progress to those that are inflammation-independent (Groups B and C). Although considered to be common in this tumor, ongoing mutation may be infrequent when assessed by strict criteria.     

Immunoglobulin heavy chain genes in humans are located on chromosome 14

The human immunoglobulin heavy chain gene complex has been assigned to chromosome 14 by filter hybridization of restriction digests of mouse-human somatic cell hybrids. Cloned DNA probes for both variable and constant regions were used.     

Genetics of mouse antibodies. II. Recombination between VH genes and allotype

The BAB/14 (BALB/c.C57BL/Ka-Ig-1^b/Hz) congenic mouse strain was found to respond to (4-hydroxy-3-nitrophenyl)acetyl (NP)-chicken globulin immunization with nonheteroclitic anti-NP antibodies as does the BALB/c inbred partner strain. This is in contrast to the heteroclitic anti-NP response of the C57BL/Ka allotype donor strain and indicates that the allotype linked V_H-NP gene was inherited with the V_H-DEX gene from BALB/c rather than with the allotype genes from C57BL/Ka. This verifies the interpretation of BAB/14 as an immunoglobulin gene recombinant. Two recombinants between V_H-DEX and Ig-1 allotype genes have been found among 554 chromosomes tested yielding a recombination frequency of 0.4%. Analysis of the BAB/14 recombinant showed that this crossover occurred between V_H genes implying that V_H genes and C_H genes are not organized in separate clusters separated by a large spacer region but are contiguous. Analysis of the recombination frequencies indicates that the total number of V_H genes must be at least 50.     

Human IgA- and IgM-secreting intestinal plasma cells carry heavily mutated VH region genes

Single IgA- or IgM-secreting plasma cells were isolated from histological sections of human jejunum and terminal ileum, and Ig heavy chain variable (V_H ) region genes were amplified and sequenced. Taken together, 62 of 63 cells analyzed harbored somatically mutated V_H region genes, indicating that the vast majority of both IgA- and IgM-secreting intestinal plasma cells derive from germinal center B cells. On average, rearranged V_H genes of IgA- and IgM-secreting plasma cells showed a mutation frequency of 9.0 % and 8.5 %, respectively, which exceeds the level of somatic mutation of V region genes carried by human memory B cells. Moreover, we detected deletions or insertions in the complementarity-determining regions of 5 of the 58 functional V_H region genes analyzed, suggesting that these alterations may contribute to the diversification of the human antibody repertoire in the course of an immune reaction.     

The preparation of mouse VH fragments and the characterization of heterologous anti-mouse VH antibodies

VH fragments were prepared from several mouse IgM molecules by cyanylation. In all cases VH fragments were purified to homogeneity by using Ig light chain affinity columns. Several different anti-VH antisera were prepared in rabbits and the specificities of these antibodies were studied. Two patterns of cross-reactivities were observed: (a) some anti-VH antibodies reacted only with closely related VH molecules, e.g., anti-VH HPC52 anti bodies reacted only with VH of phosphorylcholine-binding myeloma or hybridoma proteins, and concordantly, stained about 4% of mouse spleen B cells; (b) on the other hand, antisera-like anti-VH 104E and 8916 antibodies were very cross-reactive. Binding assays showed that both of these anti-VH antibodies reacted with 50-60% of mouse immunoglobulins. However, they recognized mainly nonoverlapping populations of mouse immunoglobulins, and thus the pool of these antibodies reacted with about 95% of mouse VH regions. Concordantly, anti-VH 104E antibodies stained in the fluorescence-activated cell sorter (FACS) analysis more than 50% of mouse spleen B cells. Cross-reactive anti-VH antibodies ("anti-framework") did not stain T cells nor did they immunoprecipitate VH-like molecules which were synthesized by T cells.     

A VH gene is located within 95 Kb of the human immunoglobulin heavy chain constant region genes

Using cosmids covering about 117 Kb upstream of the human immunoglobulin chain C mu gene, we have identified a potentially functional VH gene, belonging to the VHVI subgroup. This VHVI gene is only about 95 Kb from the C mu gene and is probably the first functional VH segment of the Igh locus. These results illustrate the proximity of the human VH, DH and JH segments involved in creation of the complete heavy chain genes.     

Five skeletal myosin heavy chain genes are organized as a multigene complex in the human genome

Myosin heavy chain (MyHC) isoforms are encoded by a multigenefamily in vertebrates. We used genomic DNA mapping by pulsefield gel electrophoresis to demortstrate that, in humans, theembryonic, fetal, fast IIB and IIX MyHC genes and a gene codingfor a non-identified striated muscle MyHC fast-type isoform(NI), are contained within a 320 kb SalI genomic fragment. Thelocus is flanked by two CpG islands, separated by 580 kb. Inorder to further characterize the MyHC genes, a human genomiclibrary constructed in yeast artificial chromosomes (YAC) wasscreened and five independent clones were Isolated. Characterizationof these YACs revealed that one of them contains at least fiveMyHC genes, based on partial sequencing of their conserved thirdcoding exons. Three of these genes correspond to those encodingthe embryonic, fetal and fast IIB MyHC isoforms. Moreover, inthis YAC done the embryonic and fetal genes, on the one hand,and the adult fast (IIB, IIX and NI) genes, on the other hand,are contained within two different ClaI fragments. This resultsuggests that the genes encoding the two developmental formsare adjacent In the human genome and that temporal regulationof the MyHC genes might be related to their organization withinthe locus. These data represent the first direct evidence forthe existence in the human genome of a MyHC multigene locusthat contains at least five genes.     

Bias in somatic hypermutation of human VH genes

Translationally silent mutations, which are not antigen selected,of human V_H6 Ig gene rearrangements isolated from human spleenwere analyzed for bias to gain insight into intrinsic featuresof the mutation process. Sixty-three clones representing 38V_H6DJ rearrangements had an overall mutation frequency of 4.5%,a replacement/silent (R/S) mutation ratio of 2.1 and 167 uniquesilent mutations. The silent mutations showed bias in: (I) targetingto CDR1 and CDR2, (II) an increased frequency of mutations ofA compared to T nucleotide bases on the coding strand, and (III)an increased frequency of transitions versus transvereions.Bias of CG over CA, of GC over GT and of AC over AT transvereionswas also present. Hot spots of mutation were observed, somewhich corresponded to potential sites of stem - loop formation.The results suggest that the somatic mutation process in manmay be targeted to the complementarity determining region forsome V genes, exhibits specific base substitutions favoringtransitions and specific types of transversions, and may beoccurring on only one DNA strand.     

Immunoglobulin VH gene expression in Ly-1+ and conventional B lymphocytes

Lymphocyte populations in which Ly-1 B cells are differentially represented were studied for the expression of ten VH gene families, either by an RNA colony blot assay or by in situ hybridization of single cells, in BALB/c and C57BL/6 mice. The comparisons of cells from lymph nodes, Peyer's patches and adult spleen (poor in Ly-1 B cells) with cells from peritoneal cavity and neonatal spleen (rich in Ly-1 B cells) were confirmed by the analysis of adult peritoneal Ly-1- and Ly-1+ B cells sorted on the fluorescence-activated cell sorter. The results indicate that the peritoneal Ly-1+ B subset uses the whole spectrum of known VH gene families, and shows a preferential utilization of CP12 VH genes, most likely as a result of a selective process during life.     

All VH11 genes expressed in peritoneal lymphocytes encode anti-bromelain-treated mouse red blood cell autoantibodies but other VH gene families contribute to this specificity

We have studied the relationship between B cell reactivity to bromelain-treated autologous mouse erythrocytes (BrMRBC) and expression of the VH11 gene family in splenic, peritoneal and pleuropericardial cell populations from normal C57BL/6 mice. B lymphocytes producing antibodies to BrMRBC were selectively enriched or depleted from normal populations by rosette formation with BrMRBC, followed by centrifugation over density gradients. This selection method, based on the presence of functional receptors (membrane IgM), is harmless for the cells and allowed subsequent cloning in agar (colony-forming unit-B). The utilization of the 10 VH gene families was then scored in mRNA colony blot assays. The analysis of greater than 650 anti-BrMRBC clones and greater than 350 VH11-expressing colonies indicates that about half of those antibody reactivities are encoded by VH11 genes. Furthermore, it appears that all VH11-expressing B cells in the peritoneal cavity produce anti-BrMRBC antibodies.     

Immunoglobulin VH gene expression in human B cell lines and tumors: biased VH gene expression in chronic lymphocytic leukemia

We have studied frequencies of VH gene utilization in a panel of monoclonal Epstein-Barr virus (EBV)-transformed B cell lines derived from human adult and fetal tissues as well as in monoclonal B cells obtained from fresh chronic lymphocytic leukemia (CLL) samples. The results show that IgM-secreting EBV cell lines from both fetal and adult tissues utilize VH genes from particular families roughly in proportion to estimated family size, suggesting that the repertoire of sigM-positive B cells in both fetal and adult organs is 'normalized' with respect to the V(H) gene family. In contrast, we find a highly biased pattern of VH gene expression in CLLs. The significance of these findings is discussed in the context of mechanisms that could be involved in normal B cell repertoire development and in the process of malignant transformation of precursors of CLL.