Analysis of individual immunoglobulin λ light chain genes amplified from single cells is inconsistent with variable region gene conversion in germinal-center B cell somatic mutation

Responding B cells in specific immune responses diversify their immunoglobulin genes and are selected on their variant antigen receptors in the microenviroment of the germinal center. The patterns of mutations previously reported for immunglobulin (Ig) genes have supported mechanistic hypotheses of either error-prone DNA synthesis or templated variable region gene conversion as the underlying mechanism in the generation of these mutations. To assess the role of gene conversion in germinal-center somatic mutation, we chose to examine nucleotide changes in mouse λ light chain genes which arose in response to a specific antigen. Laboratory mice possess three Vλ subexons, two of which differ from one another by only seven nucleotides, making these two subexons ideal for gene conversion. In the current study, we used six-parameter flow cytometry to isolate single λ light chain-expressing germinal-center B cells from two different time points in a primary immune response. We then individually amplified and sequenced individual V_λ1 genes from these single cells for mutational analysis. None of the 32 V_λ1 genes, containing a total of 40 mutations, showed evidence of gene conversion from either of the other Vλ subexons. Features such as the replacement to silent ratio of the mutations documented at the earlier time point indicate an absence of antigen-driven selection. These data indicate that V region gene conversion does not contribute to germinal-center somatic mutation and that gene conversion is not responsible for targeting mutation specifically to rearranged Ig genes. The biological implications are discussed.     

Rearrangement of upstream DH and VH genes to a rearranged immunoglobulin variable region gene inserted into the DQ52-JH region of the immunoglobulin heavy chain locus

We investigated gene rearrangements in the mutant IgH locus of a mouse strain generated by insertion of a rearranged heavy chain variable region gene (V_T15) into the DQ52-J_H region through gene targeting. In more than half of the B cells of heterozygous mutant mice, the mutant IgH locus was silenced by the rearrangement of an endogenous D_H or D_H and V_H gene to the inserted V_T15 gene. In these cases, a functional V_HD_HJ_H gene was present on the wild-type allele. The silencing rearrangement appeared to be mediated by recombination signal sequence (RSS)-like elements present in the “recipient” V_T15 gene. Among the many such elements on the inserted V_T15 gene, which apparently met the requirement for an RSS with respect to nucleotide sequence, only two were observed in the actual rearrangements. This indicates that targeting of the recombination machinery involves sequences in addition to the RSS motifs as they have been characterized so far. In homozygous mutant mice, most B cells appeared to carry the intact V_T15 gene on both mutant IgH alleles, although single-cell polymerase chain reaction revealed that silencing rearrangements occured frequently in B cell progenitors in the bone marrow. This observation indicates that once silencing rearrangements are initiated in a cell, they involve both V_T15 genes in most cases, reminiscent of normal D_H-J_H rearrangement. B cells which did not initiate such rearrangements develop to populate the peripheral B cell compartment.     

多囊卵巢综合征相关基因研究进展

多囊卵巢综合征是育龄妇女常见的生殖内分泌紊乱性疾病,以高雄激素血症、月经失调及高胰岛素血症为临床表现.迄今为止,其发病机制尚不清楚;但其生化特征提示其发病与高雄激素相关基因、胰岛素作用相关基因、促性腺激素功能失调相关基因及慢性炎症因子基因的异常相关.因此,对多卵巢综合征的遗传易感基因的研究将有助于进一步揭示其发病机制.     

Polymorphism of immunoglobulin lambda constant region genes in populations from France, Lebanon and Tunisia

Polymorphism of immunoglobulin lambda constant region (IGLC) genes has been studied in French, Lebanese and Tunisian people. The human IGLC polymorphisms appear as EcoRI restriction fragment length variations-8, 13, 18 or 23 kb-, these polymorphic fragments being related to a number of IGLC genes varying from six to nine per haploid genome. DNAs digested with the endonucleases EcoRI and HindIII were hybridized to a human IGLC probe and an immunoglobulin lambda intervening sequence region probe containing the J lambda 2 gene segment. Restriction fragments detected in Southern hybridizations were assigned to the IGLC locus map. Family studies allowed us to confirm the allelic nature of four of the different EcoRI restriction fragments observed. Frequencies of the corresponding alleles in French, Lebanese and Tunisian populations were determined and compared. The decrease of the 8-kb fragment (allele A1) frequency and, conversely, the increase of that of the 13-kb and 18-kb fragments (alleles A2 and A3) seemed to be correlated to a Negroid African contribution in the gene pool more important in Tunisia than in Lebanon.     

Expression of immunoglobulin genes in common variable immunodeficiency

Five common variable immunodeficiency (CVI) patients were analyzed for expression of immunoglobulin (Ig) genes. In the pokeweed mitogen (PWM)-induced Ig-production assay, the combination of T and B cells showed that all patients' T cells had normal helper functions and all patients' B cells had profound defects. The defective B-cell maturation stages based on their Ig gene expression patterns were variable. One of five patients showed normal -chain gene expression and nearly normal IgM production, but neither IgG nor IgA production, which suggested that this patient's B-cell defects might lie on a - to or - to class-switch stage. B cells in another patient showed low -chain gene expression and low IgM production, but an Ig enhancer region, which is an important region for expression of Ig genes, was intact. Thus, this patient might have a transacting factor defect which interacts with the Ig enhancer region. The other three patients showed no -chain gene expression and no IgM production. Thus, their B-cell defects lay on the B-cell maturation stage, similar to X-linked agammaglobulinemia. These results showed that primary B-cell defects in CVI occurred at several B-cell differentiation stages, which could be recognized by expression of Ig genes.     

Immunoglobulin gene conversion in chicken DT40 cells largely proceeds through an abasic site intermediate generated by excision of the uracil produced by AID-mediated deoxycytidine deamination

Diversification of the primary antibody repertoire in chickens is achieved by a gene conversion process that uses a set of immunoglobulin variable (IgV) pseudogenes as templates. Studies usingthe chicken DT40 B lymphoma cell line have shown that this gene conversion is dependent on activation-induced deaminase, which deaminates deoxycytidine to deoxyuridine in the IgV gene. The mechanism by which the resultant deoxyuridine/deoxyguanosine (dU/dG) mismatch acts to initiate the gene conversion process is unknown but likely involves either (i) recognition of the dU/dG pair by the mismatch repair complex or (ii) recognition of the dU itself by uracil-DNA glycosylase. To discriminate these possibilities, we have investigated the effects on IgV gene conversion of inhibiting uracil-DNA glycosylase. We find that such inhibition diminishes gene conversion, biasing instead towards point mutations. These results demonstrate that IgV gene conversion in DT40 cells is substantially dependent on uracil excision and implies that it proceeds by a pathway involving an abasic site, which could be acted upon by an apyrimidinic endonuclease to generate a DNA strand break facilitating the conversion process.     

Polymorphism in the promoter region of the CD14 gene and susceptibility to Brucellosis

A single-nucleotide polymorphism in the promoter region of the CD14 gene at position 159 has been implicated in susceptibility to infectious diseases. We sought to determine the association between CD14 C-159 T functional promoter polymorphism and brucellosis in Western Iranian population where the disease is endemic. The CD14 genotype was determined in 228 patients with brucellosis from a rural area and 129 healthy volunteers from the same area. The prevalence of genotype TT was significantly higher in the patients while the controls showed higher prevalence of genotype CC (34.5% vs 15.5%, 15.4% vs 25.6%, P = 0.009). Multiple logistic regression analysis after adjustment for gender demonstrated that the patients who were homozygous for allele T of promoter of CD14 gene had a significantly higher risk for developing brucellosis with odds ratio of 3.03 (95% CI, 5.2, 1.75 P = 0.0004). The existence of homozygous genotype of allele T of CD14 was an independent determinant for occurrence of arthritis among the patients with brucellosis (odds ratio of 3.92 (95% CI, 2.93, 5.88, P = 0.001).Our findings provide suggestive evidence of association of the CD14 gene polymorphism with susceptibility to development of brucellosis in Iranian populations.     

Insight into the origin and clonal history of B-cell tumors as revealed by analysis of immunoglobulin variable region genes

Summary: Recombination of V_H D_H and J_H genes is a unique first step in normal B-cell development. Subsequent differentiation to a mature plasma cell is accompanied by further events in the Ig genes, including V_I-J_t joining, somatic hypermutation and isotype switching. Chromosomal changes leading to B-cell tumors can occur at many points in this sequence, and may be partly a consequence of the genetic mobility and mutability permitted in order to generate a diverse antibody repertoire, V genes of neo-plastic B cells may reflect the point of maturation reached by the B cell of origin, prior to transformation, Analysis of tumors therefore provides useful information on V-gene patterns in normal B cells, and may add another dimension to classification of B-cell tumors. Transformation ma)' also preserve cell populations normally destined to die by apoptosis. Tumor cells arrested in the sire where somatic hypermutation and isotype switch are occurring can still be subject to these processes, and could be influenced by persisting antigen. However, mutation is silenced at the point of exit lo the periphery, leading lo fixed mutational patterns in tumors of mature B cells, V-gene analysis provides an invaluable tool for understanding the genesis of neoplastic change. It also has a clear clinical relevance in tracking tumor cells, measuring residual disease, and finally in offering the opportunity of developing vaccines for treatment.     

Inactivation of rabbit immunoglobulin lambda chain variable region genes by the insertion of short interspersed elements of the C family

Two rabbit germ-line genes encoding immunoglobulin lambda light chain V regions were cloned from a rabbit genomic liver DNA library and characterized. One, V lambda 1, is separated by at least 8 kb from any other V lambda gene. The second, V lamdba 4, forms part of a three-gene cluster with two functional V lambda genes recently reported. Both V lambda 1 and V lambda 4 have structural features rendering them pseudogenes. The coding regions have frame-shift mutations which would yield defective protein products; both genes are also interrupted by the insertions of short, interspersed repetitive elements of the C family. In the V lambda 1 gene, the 369-bp insert is located upstream of the gene between the putative TATA box and the leader exon, whereas in gene V lambda 4, the 360-bp insert interrupts the FR2 at codon 48c. In addition, the sequence of the complement-determining region 3 of gene V lambda 1 is very similar to the mouse DSP2.6 sequence.     

Sequence analysis of immunoglobulin variable region genes that encode autoantibodies expressed by lymphomas of mucosa associated lymphoid tissue

Aim—To determine whether the immunoglobulin genes used by three gastric mucosa associated lymphoid tissue type lymphomas with known autoreactivity are mutated from germline as mutation from germline is an indicator of exposure to a mutational mechanism which characteristically acts on B cells as they undergo a follicle centre response.     

抗人红细胞H抗原单链抗体基因克隆和表达

目的 克隆抗人红细胞H抗原单克隆抗体轻、重链可变区(VH、VL)基因并构建单链抗体(ScFv)基因及其表达载体,实现其在原核细胞中的表达.方法 从分泌抗人红细胞H抗原单克隆抗体的杂交瘤细胞株2FA中提取总RNA,采用RT-PCR法获得抗人红细胞H抗原单克隆抗体的VH、VL基因;利用重叠引物延伸法(splicing by overlap extension,SOE)将轻重链可变区基因连接,并引入连接肽(Linker)编码序列,构建VH-Linker-VL结构的单链抗体(ScFv)基因,将其克隆到原核表达载体pET-his中,转化BL21(DE3)plysS细胞,IPTG诱导表达;获得的目的蛋白经纯化后,用SDS-PAGE与Westem blotting对目的蛋白进行鉴定,间接EHISA、竞争ELISA和免疫荧光法检测目的蛋白的活性.结果 克隆的VH基因长度为351 bp,属于鼠抗体可变区重链基因家族I(B)亚群;VL基因长度为339 bp,属于鼠抗体可变区轻链基因家族I亚群;SOE法克隆的单链抗体基因为750 bp;构建的含原核表达载体的菌株经IPTG诱导后,SDS-PAGE及Western blotting法检测到Mr 32 000的目的蛋白(表达的ScFv);ScFv纯化后,经免疫荧光法、间接与竞争HLISA法检测到该ScFv蛋白具有生物结合活性.结论 成功地克隆了抗人红细胞H抗原单克隆抗体VH与VL基因和ScFv基因,构建ScFv基因的表达载体,实现了ScFv在大肠杆菌BL21(DE3)plysS细胞中的活性表达,为基于红细胞H抗原的免疫检测技术建立奠定了基础.     

Immunohistochemical study of immunoglobulin light chain amyloidosis with antibodies to the immunoglobulin light chain variable region

To detect immunoglobulin (Ig) light chain amyloidosis (AL amyloidosis) in formalin-fixed, paraffin-embedded tissue sections by immunohistochemistry, polyclonal antibodies were generated against synthetic peptides corresponding to amino acids 1-19 of the Ig lambda light chain V lambda VI subgroup (anti-V lambda VI (1-19)) and the Ig kappa light chain Vkappa I subgroup (anti-Vkappa I (1-19)). Anti-V lambda VI (1-19) antibody reacted with amyloid deposits in 21 of 22 Alambda amyloidosis cases, and anti-Vkappa I (1-19) antibody reacted with amyloid deposits in 10 of 11 Akappa amyloidosis cases. Immunoreactivity varied in intensity by case and within specimens. Surprisingly, amyloid deposits were positive for anti-V kappa I (1-19) staining in one case of Alambda amyloidosis. Analysis of anti-V lambda VI (1-19) and anti-Vkappa I (1-19) antibody reactivity by ELISA showed some cross-reactivity with peptides other than antigen peptides. The antibodies were not reactive in all cases of AL amyloidosis examined but may be useful, together with anti-Ig constant region antibodies, for immunohistochemical diagnosis of AL amyloidosis.     

TET3 dioxygenase modulates gene conversion at the avian immunoglobulin variable region via demethylation of non-CpG sites in pseudogene templates

Diversification of the avian primary immunoglobulin (Ig) repertoire is achieved in developing B cells by somatic hypermutation (SHM) and gene conversion (GCV). GCV is a type of homologous recombination that unidirectionally transfers segments of Ig pseudogenes to Ig variable domains. It is regulated by epigenetic mechanisms like histone modifications, but the role of DNA methylation remains unclear. Here, we demonstrate that the chicken B-cell line DT40 lacking TET3, a member of the TET (Ten-eleven translocation) family dioxygenases that facilitate DNA demethylation, exhibited a marked reduction in GCV activity in Ig variable regions. This was accompanied by a drop in the bulk levels of 5-hydroxymethylcytosine, an oxidized derivative of 5-methylcytosine, whereas TET1-deficient or TET2-deficient DT40 strains did not exhibit such effects. Deletion of TET3 caused little effects on the expression of proteins required for SHM and GCV, but induced hypermethylation in some Ig pseudogene templates. Notably, the enhanced methylation occurred preferably on non-CpG cytosines. Disruption of both TET1 and TET3 significantly inhibited the expression of activation-induced cytidine deaminase (AID), an essential player in Ig diversification. These results uncover unique roles of TET proteins in avian Ig diversification, highlighting the potential importance of TET3 in maintaining hypomethylation In Ig pseudogenes.     

Remarkable selective glycosylation of the immunoglobulin variable region in follicular lymphoma

Follicular lymphoma (FL) generally expresses immunoglobulin (Ig) with somatically mutated variable (V) region genes. Surprisingly, these almost always carry introduced motifs available for N-glycosylation (Asn-X-Ser/Thr). Introduced motifs are uncommon on normal B cells, but are on other germinal center (GC)-associated B-cell malignancies suggesting a site-specific role. They are not evident in mutated chronic lymphocytic leukemia (CLL) or myeloma. Recently, we found that the glycosylation sites are unusual in containing oligomannose glycans, which are apparently displayed on tumor cell surface IgM. This suggests a potential interaction with a mannose receptor in the GC. However, natural N-glycosylation sites exist in germline (GL) V region genes, particularly the V4-34 gene expressed by normal B cells and by some malignancies, including CLL, potentially undermining the selective importance for FL. To compare oligosaccharide addition at the introduced and natural sites, we expressed V region genes as single chain Fv (scFv) and analyzed the added glycans. In contrast to introduced sites, which were oligomannosylated, the natural GL motif in the V4-34 sequence had no added sugars. The remarkable selective glycosylation within the heavy chain V region gene of FL apparently permits only limited processing to oligomannose at somatically mutated motifs, creating a feature exploitable by GC lymphomas.     

Molecular analysis of a patient with hydrops fetalis caused by β-glucuronidase deficiency,and evidence for additional pseudogenes

A patient with hydrops fetalis caused by β-glucuronidase deficiency was found to be homozygous for a C to T transition at nucleotide position 672 in his cDNA. Genomic analysis showed the presence of pseudogenes for the β-glucuronidase gene. After separation of PCR products of the gene and the pseudogenes it was shown that the patient and his father were heterozygous for the C-T 672 transition and the mother did not carry the mutation. © 1993 Wiley-Liss, Inc.