OBJECTIVESequences analysis of Ig variable regions from the peripheral blood mononuclear cells (PBMC) of patients with chronic B lymphocytic leukemia.

METHODSTotal RNA was isolated from PBMC of patients with chronic B lymphocytic leukemia, oligo-dT-primed cDNA was synthesized from RNA. The cDNA was amplified by Taq DNA polymerase with a set of specific 5' primers corresponding to Ig FR1 and 3' primers corresponding to CH1 (C micro /C) or CL (Ckappa/Clambda), the PCR products of variable regions of Ig heavy (IgH) and light (IgL) chains were sequenced by ABI PRISM Dye terminator cycle sequencing ready reaction kit and ABI PRISM 310 Genetic Analyzer. The gene homology of variable regions of IgH and IgL chains was compared by using DNA tools 5.1 system and "the international immunogenetics database".

RESULTSFour light chains and 3 heavy chains were amplified from 4 and 3 patients respectively. Homology analysis of the sequences of 4 light chains and 3 heavy chains were performed by DNA tools system. The sequences of light chains are high homologous. And the sequences of heavy chains are quite different. The homologous analysis of the sequences of variable region by using "the international immunogenetics database" showed that the sequences were higher homologous to idiotype gene of some B lymphocytic leukemia. Four VL genes belong to human Ig Vkappa subgroup I, 2 of 3 VH genes belong to VH3 family and 1 belongs to VH5 family.

CONCLUSIONIg genes have idiotype and same disease may have same idiotype.

Base Sequence ; Cloning, Molecular ; DNA, Complementary ; chemistry ; Gene Rearrangement ; Genes, Immunoglobulin ; Humans ; Immunoglobulin Fab Fragments ; genetics ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; immunology ; Molecular Sequence Data ; Polymerase Chain Reaction

No abstract available.
Genes, Immunoglobulin Heavy Chain* ; Immunoglobulin Heavy Chains* ; Immunoglobulins*

OBJECTIVETo evaluate the efficiency of the BIOMED-2 PCR assay and its implication in the diagnosis of mature B-cell non-Hodgkin's lymphomas.

METHODSClinical, morphological and immunohistochemical features of 72 cases of non-Hodgkin's lymphomas were studied, including 25 reactive lymphoid hyperplasia, 37 diffuse large B cell lymphomas (DLBCL) and 35 extranodal marginal zone lymphomas of mucosa associated lymphoid tissues (MALT lymphoma and in addition, 25 cases of reactive lymphoid hyperplasia were used as the controls). DNA was exacted from the paraffin embedded formalin fixed tissue blocks and the quality of DNA was assessed using the BIOMED-2 specimen control reaction. Adequate samples were then analyzed by BIOMED-2 for immunoglobulin heavy and kappa light chain rearrangements.

RESULTSAdequate DNA was obtained in 83 of 97 samples, including 60 mature B cell lymphomas and 23 reactive lymphoid hyperplasia. Clonal B-cell gene rearrangements were detected in 57 of 60 (95%) lymphomas. In contrast, clonal Ig gene rearrangements were not detected in any of the 23 cases of reactive lymphoid hyperplasia.

CONCLUSIONBIOMED-2 assay is highly sensitive and specific for the detection of clonal B cell gene rearrangement using routine paraffin embedded formalin fixed specimens.

Antigens, CD20 ; metabolism ; CD79 Antigens ; metabolism ; DNA, Neoplasm ; genetics ; Gene Rearrangement, B-Lymphocyte ; genetics ; Gene Rearrangement, B-Lymphocyte, Heavy Chain ; genetics ; Gene Rearrangement, B-Lymphocyte, Light Chain ; genetics ; Genes, Immunoglobulin ; Humans ; Immunophenotyping ; Lymphoma, B-Cell ; genetics ; immunology ; pathology ; Lymphoma, B-Cell, Marginal Zone ; genetics ; immunology ; pathology ; Lymphoma, Large B-Cell, Diffuse ; genetics ; immunology ; pathology ; Paraffin Embedding ; Pseudolymphoma ; genetics ; immunology ; pathology ; Sensitivity and Specificity

BACKGROUND: Immunoglobulin (Ig) gene rearrangement analysis is a useful additional tool to detect clonality of B-lymphoproliferative disease and the method to detect immunoglobulin gene rearrangement is required the high sensitivity and specificity. BIOMED-2 multiplex PCR was designed for the evaluation of molecular clonality of lymphoid lesions. We evaluated the usefulness of the BIOMED-2 multiplex PCR by comparing it with conventional nested PCR. METHODS: Sixteen patients with malignant lymphoma and 5 with reactive lymph nodes were examined to assess the sensitivity, specificity, and accuracy between conventional nested PCR and BIOMED-2. All 3 tests performed using the BIOMED-2 kit for immunoglobulin (Ig) heavy chain (IGH) gene, Igkappa light chain (IGK) gene, and Iglambda light chain (IGL) gene, were used to analyze clonality. RESULTS: Both the methods showed 100% specificity (95% confidence interval, 56.6-100.0). The combination of IGH and IGK BIOMED-2 tests with or without IGL revealed the highest sensitivity (87.5%; range, 64.0-96.5%) and accuracy (90%; range, 0.70-0.97). Compared to the conventional method, the BIOMED-2 test for IGH showed a higher sensitivity (62.5%; range, 38.6-81.5%) and accuracy (71%, 0.50-0.86). CONCLUSIONS: These results suggest that, compared to the conventional method, BIOMED-2 has higher sensitivity and allows for easier interpretation while evaluating the clonality of B-lymphoproliferative disease.
Gene Rearrangement ; Genes, Immunoglobulin ; Hematologic Neoplasms ; Humans ; Immunoglobulins ; Light ; Lymph Nodes ; Lymphoma ; Multiplex Polymerase Chain Reaction ; Polymerase Chain Reaction ; Sensitivity and Specificity

Objective: To investigate the mutational features of the immunoglobulin heavy chain variable region (IgHV) gene in patients with chronic lymphocytic leukemia (CLL) using immunophenotypic and molecular genetic methods. Methods: The laboratory results of 266 CLL patients who underwent IgHV gene examination at Sino-US diagnostics laboratory from February 2020 to February 2021 were analyzed for the IgVH mutational status and presence of specific IgVH fragments. In addition, their immunophenotypic, molecular, chromosomal karyotypic, and FISH profiles were investigated and correlated with the IgVH mutational status. Results: Among 266 patients, 172 were male and 94 were female, with a media age of 67 years (20-82 years).There were more patients with mutated IgHV (m-IgHV) than unmutated IgHV (un-IgHV) (69.2%∶30.8%). There was association of VH family and the presence of gene fragments: the overall incidence of VH families including VH3 family (142/266, 53.4%), VH4 family (75/266, 28.2%), and VH1 family (34/266, 12.8%) was about 95%, among which the proportion of VH4-34 (26/266, 9.8%), VH3-23 (25/266, 9.4%), VH3-7 (24/266, 9.0%), and VH4-39 (16/266, 6.0%) was about 35%. VH3-20 and VH3-49 only occurred in un-IgHV (P<0.05). In addition, the expression rates of CD38 (26.3% vs. 3.0%), CD79b (71.1%∶45.5%) and 11q deletion (25.5%∶5.3%) were higher in un-IgHV, and single trisomy 12 (37.9%∶5.6%) were more commonly found in m-IgHV (P<0.05). MYD88 was one of the major mutation genes in m-IgHV, while ATM had the highest mutation rate in un-IgHV. Conclusion: CLL patients have differential expression in terms of IgHV gene mutations, correlating to their immunophenotype and genetics characteristics.
Male ; Female ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/genetics* ; Immunoglobulin Variable Region/genetics* ; Genes, Immunoglobulin Heavy Chain ; Mutation ; Immunoglobulin Heavy Chains/genetics* ; Prognosis

Almost all patients with multiple myeloma (MM) have chromosomal translocation which can result in genetic variation. There are mainly five types of chromosomal translocations, involving the IGH gene translocation to 11q13 (CCND1), 4p16 (FGFR/MMSET), 16q23 (MAF), 6p21 (CCND3) and 20q11 (MAFB). It is possible that all IGH translocations converge on a common cell cycle signal pathway. Some MM develops through a multistep transformation from monoclonal gammopathy of undetermined significance (MGUS) to smoldering MM (SMM) and eventually to MM and plasma cell leukemia (PCL). Similarly to what Darwin proposed in the mid-19th century-random genetic variation and natural selection in the context of limited resources, MM clonal evolution follow branching and nonlinear mode. The failure of MM treatment is usually related with the minimal subclone which is hardly found at newlydiagnosed.
Clonal Evolution ; Cyclin D1 ; Genes, Immunoglobulin Heavy Chain ; Humans ; Multiple Myeloma ; genetics ; Translocation, Genetic

The aim of this study was to investigate the bcl-2/IgH expression levels in patients with follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) and its clinical significance. The bcl-2/IgH expression levels in bone marrow (BM) and/or peripheral blood (PB) of 20 patients were detected by using SYBR Green I real-time polymerase chain reaction, and the dynamic monitoring for bcl-2/IgH expression level in 4 of these patients was performed. The results showed that in patients with bcl-2/IgH-positive there was no statistically significant difference in the relative copy-numbers of bcl-2/IgH fusion gene in BM and PB (4.23 and 2.73 respectively, p = 0.107), but the difference was significant before and after treatment (3.61 and 2.69 respectively, p = 0.000), the expression level of bcl-2/IgH fusion gene in newly diagnosed and relapsed group was remarkably higher than that in remission group (p = 0.008). The bcl-2/IgH expression level in PB increased evidently 3 months prior to the clinical relapse in one case out of dynamically monitored 4 cases. It is concluded that the bcl-2/IgH expression level is associated with the disease status, the expression level is high in newly diagnosed and relapsed patients and low in those who achieved remission, the bcl-2/IgH fusion gene expression level decreased evidently after therapy, this change may be related to the clinical disease progression, the results suggest that peripheral blood can be regarded as the resource for detection of bcl-2/IgH fusion gene.
Adult ; Aged ; Female ; Gene Expression ; Genes, Immunoglobulin Heavy Chain ; Genes, bcl-2 ; Humans ; Lymphoma ; genetics ; Male ; Middle Aged ; Oncogene Proteins, Fusion ; genetics ; Polymerase Chain Reaction ; methods ; Translocation, Genetic

OBJECTIVETo study the relationship between immunoglobulin variable heavy chain (IgVH) gene mutation status and clinical features, pathologic findings and biologic behavior of mantle cell lymphoma (MCL).

METHODSIgVH gene was amplified in 60 cases of MCL with FR1-JH and FR2-JH primers in BIOMED-2. The sequence was determined by cloning. The IgVH somatic mutational status was analyzed using NCBI's Ig-Blast tool. The relationship between IgVH gene mutation status and clinicopathologic features was also analyzed.

RESULTSForty percent (24 cases, 28 functional Ig genes) of the MCL cases displayed somatically mutated VH genes (defined as > 2% mutated), whereas 60.0% (36 cases, 40 functional Ig genes) showed unmutated VH genes. The most widely used genes were VH3-21 (27.9%) and VH4-34 (19.1%). The former were mainly used by unmutated cases, while the later mainly by mutated cases.Intraclonal heterogeneity was noted in 19 cases. There was no correlation of VH mutation status and specific VH gene with survival (P > 0.05).

CONCLUSIONSMCL comprises at least two subsets that do not correlate with morphology: one with unmutated VH genes and one with mutated VH genes. The biased use of VH3-21 and VH4-34 is noted. The nonrandom usage of IgVH segments suggests specific antigens may play a role in the pathogenesis and progression of MCL subsets. There is no correlation of IgVH mutation status and specific VH gene with survival.

DNA Primers ; Female ; Genes, Immunoglobulin Heavy Chain ; genetics ; Humans ; Immunoglobulin Variable Region ; genetics ; Lymphoma, Mantle-Cell ; genetics ; mortality ; pathology ; Male ; Mutation ; Prognosis

OBJECTIVETo study the rearrangement of immunoglobulin (Ig) heavy chain variable region (V(H)) genes in human neonates with different gestational ages (GA).

METHODSPeripheral blood from the neonates with GA of 27 weeks (4 cases), 28-32 weeks (9 cases), 33-36 weeks (12 cases), and 37-42 weeks (13 cases) was collected. RT-PCR was used to amplify the Ig V(H) gene, and the PCR products were separated by electrophoresis and analyzed using 6% denaturing PAGE gel.

RESULTSAll Ig V(H) family genes had several rearranged genes in each GA group, and the neonates with different GA showed no significant difference in the median molecular weight for each rearranged Ig V(H) family gene.

CONCLUSIONThe neonates with GA of 27-42 weeks exhibit diversity in Ig V(H) gene rearrangement, and for the same Ig V(H) family, the median length of the arranged Ig V(H) genes is independent of the gestational age.

Gene Rearrangement ; Genes, Immunoglobulin Heavy Chain ; Gestational Age ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Infant, Newborn ; Multigene Family ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

OBJECTIVETo investigate the overrepresentation of specific gene segments of immunoglobulin heavy chain variable region (IgVH) among unmutated and mutated chronic lymphocytic leukemia (CLL) patients and its prognostic implication.

METHODSMultiplex PCR was used to identify the expression of IgVH segment and its mutation status in CLL.

RESULTSAnalyses were successfully performed in 80 of 85 samples. Marked skewed IgVH families were disclosed. The most commonly used VH was VH3 (40.0%), followed by VH4 (30.0%), VHI (13.8%), VH2 (10.0%) and VH5, VH7 (2.5%). Fifty-six patients (70.0%) had mutated VH, 24 (30.0%) unmutated VH. Nine cases (11.3%) were with 100% germline sequence. Fifteen cases (15/24, 62.5%) in VH4, 29 (29/32, 90.7%) in VH3, and 4 (4/11, 36.3%) in VH1 had mutated VH. The most frequently used IgVH gene was VH4-39 (13.8%), and VH4-34 (8.8%). J4 (36/66, 54.5%) and D3 (25/66, 37.8%) were the most frequently used in J and D genes. The progression-free survival (PFS) was 82 and 17 months (P = 0.000), and the overall survival (OS) was 90 and 41 months (P = 0.009), respectively, for mutated and unmutated cases. Recurrent CDR3 sequences were found in our patients and 2 patients with VH1-69 had CDR3 sequences highly similar to those reported in literature.

CONCLUSIONThere is difference in IgVH gene segment usage and mutational status in different area CLL patients. Recurrent CDR3 sequences were found in specific IgVH gene segments, which highlights the importance of immunoglobulin mediated stimulation in the development of CLL.

Adult ; Aged ; Aged, 80 and over ; DNA Mutational Analysis ; Female ; Gene Rearrangement, B-Lymphocyte, Heavy Chain ; Genes, Immunoglobulin ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; immunology ; pathology ; Male ; Middle Aged ; Mutation