SEARCH · PubMed Health

Results for “germline sequencing”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 145 records · Page 8Linked to original sources

PubMed2004

Analysis of familial male breast cancer for germline mutations in CHEK2.

We have previously shown that the1100delC variant of the cell-cycle-checkpoint kinase gene CHEK2, which is carried by approximately 1% of the population confers a two-fold increase in female breast cancer and a 10-fold increase in male breast cancer. To extend our knowledge on the role of CHEK2 in susceptibility to male breast cancer we have screened a series of 26 breast cancer cases with male representation for germline sequence variation in the CHEK2 gene. One individual was found to harbour the 1100delC variant. No other mutations were identified. Variants other than 1100delC are rare in male breast cancer.

Breast Neoplasms, Male↗

PubMed2005

Jalpha-gene segment usage and the CDR3 diversity of porcine TCRalpha-chain cDNA clones from the PBL of a five-month-old pig and the thymus of a one-month-old pig.

Porcine T-cell receptor alpha (TCRalpha)-chain cDNA clones were isolated from libraries made from two different sources, the thymus of a 1-month-old LW strain pig and the peripheral blood lymphocytes (PBL) of a 5-month-old Clawn strain pig. Among 109 cDNA clones with the Jalpha-gene segment, 44 different Jalpha-gene segments were found out of the 61 Jalpha-gene segments previously identified in the porcine germline sequence. Among the 103 complete TCRalpha-chain cDNA clones with the rearranged Valpha- and Jalpha-gene segments, 33 different Valpha-gene segments were identified, which randomly rearranged to Jalpha-gene segments indicating lack of any specific combinations between Valpha- and Jalpha-gene segments with only one exception of the same set of Jalpha-gene segments in duplicate clones. Among the cDNA clones from PBL of an individual 5-month-old Clawn strain pig, a broad distribution of the Jalpha-gene segment usage was observed over the entire Jalpha-gene cluster. The Jalpha-gene segment usage in an individual 1-month-old thymus from a LW strain pig also gave a pattern consistent with the 5-month-old pig. These distributions of the Jalpha-gene segment usage were similar to the previously reported patterns for human T-cells and those of adult murine T-cells. Among the porcine cDNA clones isolated, TCRalpha-chain CDR3 length ranged from 4 to 14 amino acids with the average being 9.35 amino acids. Present report provides groundwork for further studies on porcine TCRalpha-chain expression.

Amino Acid Sequence↗

PubMed1998

Insertions and deletions in hypervariable loops of antibody heavy chains contribute to molecular diversity.

Antibody diversity, a molecular feature which allows these proteins to specifically interact with a diverse set of targets, is created at the genetic level by a variety of means. These include germline gene segment recombination, junctional diversity and single basepair (bp) substitution. We here demonstrate that a human high affinity antibody specific for an exogenous protein antigen carry three amino acid residues immediately adjacent to the first hypervariable loop of the heavy chain. These additional residues are shown not to be encoded by the germline repertoire. We also describe the characteristics of insertions and deletions, not found in any known germline sequence, within the first and second hypervariable loops of other previously described antibody-encoding genes. These findings demonstrate that insertions or deletions of entire codons provide yet another approach by which the human antibody repertoire is diversified in vivo. Since these major genetic modifications occur within or immediately adjacent to loops contributing to the antigen-binding site, they are likely to affect the binding properties of the mutated antibodies.

Animals↗

PubMed2002

Novel insight into antibody diversification from cattle.

The bovine preimmune repertoire develops in the absence of maternal antibodies due to the placental barrier formed by syndesmochorial type of placenta. The limited germline sequence diversity, both at the heavy and light chain loci, imposes constraints on generation of combinatorial diversity in cattle. The cattle, thus, must employ other strategies for antibody diversification. Analysis of VDJ rearrangements in adult cattle have led identification of generation of large IgM antibody molecules that may have an exceptionally long CDR3H region (up to 61 amino acids). The IgM antibodies with an exceptionally long CDR3H are indeed functional as some of these recognize structurally dissimilar antigens. The antibody diversification in cattle involves generation of an exceptionally long CDR3H in addition to point somatic mutations.

Animals↗

PubMed2002

Variation in the manganese superoxide dismutase gene (SOD2) is not a major cause of radiotherapy complications in breast cancer patients.

BACKGROUND AND PURPOSE: Small proportions of patients receiving radiotherapy develop marked long-term radiation damage. It is thought that this is due, at least in part, to intrinsic differences in cellular radiosensitivity, but the underlying mechanism is unknown. Reactive oxygen species are involved in cellular radiation damage, hence inter-individual differences in free radical detoxification may be related to radiosensitivity. Within mitochondria manganese superoxide dismutase (MnSOD) provides a major defence against oxidative damage by reactive oxygen species. MnSOD has been linked to expression of malignant phenotype and apoptosis and polymorphic variation in the gene, SOD2 to risk of breast cancer. MATERIALS AND METHODS: Forty-one breast cancer patients developing marked changes in breast appearance after radiotherapy and 39 patients who showed no clinically detectable reaction after radiotherapy were analyzed for germline sequence variation in SOD2. RESULTS: The Ala-9Val polymorphism was detected, but no other sequence variants were detected in SOD2. Both alleles of the Ala-9Val polymorphism were equally distributed between the two patient groups. CONCLUSIONS: Sequence variation in SOD2 is not the major cause of radiotherapy complications in women with breast cancer.

Breast↗

PubMed1997

Somatic mutation of the MEN1 gene in parathyroid tumours.

Primary hyperparathyroidism is a common disorder with an annual incidence of approximately 0.5 in 1,000 (ref. 1). In more than 95% of cases, the disease is caused by sporadic parathyroid adenoma or sporadic hyperplasia. Some cases are caused by inherited syndromes, such as multiple endocrine neoplasia type 1 (MEN1; ref. 2). In most cases, the molecular basis of parathyroid neoplasia is unknown. Parathyroid adenomas are usually monoclonal, suggesting that one important step in tumour development is a mutation in a progenitor cell. Approximately 30% of sporadic parathyroid tumours show loss of heterozygosity (LOH) for polymorphic markers on 11q13, the site of the MEN1 tumour suppressor gene. This raises the question of whether such sporadic parathyroid tumours are caused by sequential inactivation of both alleles of the MEN1 gene. We recently cloned the MEN1 gene and identified MEN1 germline mutations in fourteen of fifteen kindreds with familial MEN1 (ref. 10). We have studied parathyroid tumours not associated with MEN1 to determine whether somatic mutations in the MEN1 gene are present. Among 33 tumours we found somatic MEN1 gene mutation in 7, while the corresponding MEN1 germline sequence was normal in each patient. All tumours with MEN1 gene mutation showed LOH on 11q13, making the tumour cells hemi- or homozygous for the mutant allele. Thus, somatic MEN1 gene mutation for the mutant allele. Thus, somatic MEN1 gene mutation contributes to tumorigenesis in a substantial number of parathyroid tumours not associated with the MEN1 syndrome.

Chromosomes, Human, Pair 11↗

PubMed2004

Chronic lymphocytic leukemia with 6q- shows distinct hematological features and intermediate prognosis.

Cytogenetic and fluorescence in situ hybridization studies were successfully performed in 217 chronic lymphocytic leukemia (CLL). In all, 13 patients with 6q21 deletion were identified and characterized in comparison with 92 patients with 'favourable' karyotype (normal or 13q-), 69 cases with 'intermediate risk' (1-2 anomalies) and 43 cases with 'unfavourable' karyotype (complex, 11q- or 17p-). Six out of 13 cases with 6q- showed an excess of atypical lymphocytes, a finding confirmed at the histologic level; >20% CD38+ cells were seen in 5/6 cases. IGVH mutational status revealed >98% homology to the germline sequence in 4/10 cases. When compared with the 'favourable' group, patients with 6q- showed a higher white blood cell (WBC) count, frequent splenomegaly, atypical morphology, CD38+ and short time from diagnosis to first treatment and short survival. A higher median WBC count was found in the 6q- group vs the intermediate-risk group; survival was shorter in the unfavourable group. To ascertain if the 6q- anomaly was an independent factor predicting for an inferior outcome among those patients with 'favourable' cytogenetics, we performed an analysis of prognostic factors in 105 patients (92 'favourable' plus 13 with 6q-), showing that the 6q- chromosome maintained its prognostic significance at multivariate analysis (P=0.02) along with stage (P=0.01). We conclude that CLL with 6q- is characterized by a high incidence of atypical morphology, classical immunophenotype with CD38 positivity and intermediate incidence of IGVH somatic hypermutation. Clinicobiological features and outcome show that this cytogenetic subset of CLL should be allocated in an intermediate-risk category.

ADP-ribosyl Cyclase↗

PubMed2002

Extensive CDR3H length heterogeneity exists in bovine foetal VDJ rearrangements.

Analysis of seven variable-diversity-joining (VDJ) gene rearrangements in B splenocytes from a 125-day-old bovine foetus revealed an extensive heavy-chain complementarity-determining region 3 (CDR3H) length variation (9-56 codons). Indeed, the global CDR3H size spectratyping of foetal VDJ rearrangements substantiated such an extensive heterogeneity and was comparable with that noted in peripheral B lymphocytes of adult cattle. These observations are in contrast to species such as humans with extensive germline combinatorial capability where shorter CDR3H length is noted early during B-cell development. Exceptionally long CDR3H (as in adult cattle) was noted in two foetal VDJ rearrangements encoded by a single germline VH gene. Further, two VH genes (gl.110.20 and BF2B5) were preferentially expressed in the foetal VDJ rearrangements. The DH gene-encoded CDR3H region of foetal VDJ rearrangements is remarkable for repetitive GGT (glycine) and TAT (tyrosine) codons that favour the recruitment of somatic hypermutations. It appears that closely related germline DH genes, preferentially used in the hydrophilic reading frame, encode varying CDR3H lengths early during B-cell ontogeny in cattle. A comparison of germline and expressed VH genes, especially in the CDR1 and CDR2, confirms that somatic hypermutations contribute to immunoglobulin (Ig)M antibody diversification in cattle. The biased nucleotide base use and high occurrence of 'hot-spot' triplet (AGPy; AG pyrimidine base) in the CDRs predisposes to somatic hypermutations. Overall, these observations suggest that extensive CDR3H length heterogeneity, including the generation of exceptionally long CDR3H (up to 56 amino acids), and somatic hypermutations contribute to IgM antibody diversification in cattle. The extensive CDR3H length heterogeneity early during the B-cell development may compensate for constraints imposed on antibody diversification owing to the limited germline sequence diversity of genetic elements in cattle.

Amino Acid Sequence↗

PubMed1997

H-ras oncogene activation in invasive UVR-induced corneal sarcomas of the opossum Monodelphis domestica.

Chronic exposure to ultraviolet radiation (UVR) induces corneal sarcomas in the South American opossum Monodelphis domestica. Cell lines are readily established from these tumors. Northern blotting of mRNA from six such cell lines revealed high expression of the H-ras oncogene. H-ras cDNA from an eye tumor cell line was cloned and characterized; the germline sequence of codons 12, 13, and 61 was confirmed by examination of H-ras sequences amplified from liver DNA by the polymerase chain reaction. The Monodelphis H-ras coding sequence is 84-89% identical to that of other vertebrates at the nucleotide level, and the predicted 189-amino-acid sequence differs by 2-12 amino acids from that of other vertebrates. Analysis of 12 primary invasive corneal sarcomas induced by chronic UVR exposure revealed no evidence of H-ras gene amplification or rearrangement. One tumor was heterozygous for an activating point mutation in codon 61 of the H-ras gene; the tumor was also homozygous for a point mutation at an adjacent site in codon 62. These results provide additional evidence for the functional importance and consequent evolutionary conservation of the ras oncogenes.

Amino Acid Sequence↗

PubMed2003

Molecular structural and functional characterization of tumor suppressive anti-ErbB-2 monoclonal antibody by phage display system.

To investigate the molecular structural and functional characteristics of tumor-suppressive anti-ErbB-2 monoclonal antibody (mAb) SER4, we performed mAb-gene cloning and epitope mapping by a phage display system. Structural analysis demonstrated that both the heavy chain (HC) and light chain variable regions are highly homologous with the derived germline sequences, while the HC complementarity determining region (HCDR) 3 has a relatively short length and biased amino acid usage. A cloned gene-derived recombinant Fab (rFab) fragment showed antigen binding activity and specificity comparable to the parent mAb. Cross-linking of the rFab fragment with the anti-Fab antibody elicited cell growth inhibition in vitro. These results imply that the cloned genes actually encode the Fab part of SER4. The epitope mimetic peptide (mimotope) isolated by panning a phage-displayed random peptide library against SER4 showed no cross-reactivity with mAbs other than SER4. The mimotope was found to be homologous with (87)AHNQVRQVPLQR(98) in the extracellular domain of ErbB-2 by means of a clustalw search. Since SER4 causes the growth inhibition of ErbB-2 positive cells, the predicted epitope sequence may constitute the putative functional domain of ErbB-2.

Amino Acid Sequence↗

PubMed1986

A joining-diversity-joining complex generated by inversion mechanism and a variable-diversity complex in the beta-chain gene of the human T-cell receptor.

We have analysed an inactive allele of the beta-chain gene of the T-cell receptor in a human T-cell line HPB-ALL. Comparison with germline sequences showed that HPB-ALL has a joining (J)-diversity (D)-J complex recombined in head-to-head configuration and a variable (V)-D complex in tail-to-tail configuration. These results demonstrate that the inversion mechanism functions in the beta-chain gene of the T-cell receptor. The presence of the V-D complex suggests that V-D recombination could occur prior to D-J recombination although there is no definite proof that the V-D complex is an intermediate to form the V-D-J complex.

Base Sequence↗

PubMed2000

Design and synthesis of germline-based hemi-humanized single-chain Fv against the CD18 surface antigen.

The 6.7 murine monoclonal antibody (mAb) recognizes the human CD18 antigen and is therefore of interest as an anti-inflammatory agent. The 6.7 heavy variable chain (VH) was humanized using the closest human germline sequence as the template on to which to graft the murine complementary determining regions (CDRs). Two versions were proposed, one in which the residue proline 45 of the murine form was maintained and another in which this framework residue was changed to the leucine found in the human sequence. These VH humanized versions were expressed in the yeast Pichia pastoris as hemi-humanized single-chain Fv (scFvs), with the VL from the murine antibody. The scFv from the murine antibody was also expressed. The binding activities of the murine and both hemi-humanized scFvs were determined by flow cytometry analysis. All the constructions were able to recognize human lymphocytes harboring CD18, indicating successful humanization with transfer of the original binding capability. Some differences between the two hemi-humanized versions were observed. The method used was simple and straightforward, with no need for refined structural analyses and could be used for the humanization of other antibodies.

Amino Acid Sequence↗

PubMed2002

Immunoglobulin gene rearrangement analysis in composite hodgkin disease and large B-cell lymphoma: evidence for receptor revision of immunoglobulin heavy chain variable region genes in Hodgkin-Reed-Sternberg cells?

Immunoglobulin heavy chain gene (IgH) rearrangement was studied in a patient showing the occurrence of classical Hodgkin disease and large B-cell lymphoma (LBCL) in the same lymph node. The VHDHJH region was amplified by polymerase chain reaction, the template being the DNA extracted from single Hodgkin and Reed-Sternberg and LBCL cells, microdissected on hematoxylin-eosin-stained sections by laser capture. A repeated VH4DH3JH4 segment was found in Reed-Sternberg cells, whereas a repeated VH3DH3JH4 segment was observed in LBCL cells. Rearranged VH genes carried somatic mutations in both populations, indicating a common germinal center cell origin. The IgH rearrangement found in clonally related Reed-Sternberg cells differed from the one of LBCL cells in the VH region but showed the same JH and DH segments with no variation from the respective germline sequence. The DH-JH junction is the first immunoglobulin gene segment rearranged in precursor B cells. Because the possibility of secondary Ig gene rearrangement in peripheral lymphoid organs has recently been reported, in the patient described here Reed-Sternberg and LBCL cells might originate from a common precursor in which secondary VH replacement took place during the germinal center reaction, giving rise to two different clonally related lymphomas.

Adult↗

PubMed1995

Idiotypic oligonucleotide probes to detect myeloma cells by mRNA in situ hybridization.

An mRNA in situ hybridization (ISH) method which used non-radioactive idiotypic oligonucleotide probes has been used to detect malignant cells in the bone marrow and peripheral blood of patients with multiple myeloma. For each of two patients with multiple myeloma a pair of biotinylated antisense oligonucleotide probes (18-22mer) was prepared from non-germline sequences of the rear-ranged immunoglobulin heavy chain (IgH) gene. These oligonucleotide sequences were not homologous with any previously published sequence. The probes from each patient were specific as shown by a failure to hybridize to any cells from six other myeloma patients and four normal individuals. Specific staining of IgH gene mRNA occurred only when the myeloma cells and the sequence of the probe used were from the same patient. Using simultaneous fluorescent immunocytochemistry it was shown that more than 95% of the ISH-positive cells expressed the malignant light chain in their cytoplasm. ISH positive cells were found in 1-4% of the peripheral blood mononuclear fraction of these two patients. These studies show that idiotypic oligonucleotide IgH gene probes can be used to identify individual cells belonging to the malignant clone and offer the possibility of developing innovative tumour-specific therapeutic procedures using antisense technology for patients with myeloma.

Base Sequence↗

PubMed2001

Somatic mutation and germline variants of MINPP1, a phosphatase gene located in proximity to PTEN on 10q23.3, in follicular thyroid carcinomas.

Various genes have been identified to play a role in the pathogenesis of follicular thyroid tumors. Cowden syndrome is the only known familial syndrome with an increased risk of both follicular thyroid adenoma (FA) and carcinoma (FTC). Germline mutations in the tumor suppressor gene PTEN, which encodes a dual-specificity phosphatase, have been found in up to 80% of patients with Cowden syndrome suggesting a role of PTEN in the pathogenesis of follicular thyroid tumors. Although somatic intragenic mutations in PTEN, which maps to 10q23.3, are rarely found in follicular tumors, loss of heterozygosity (LOH) of markers within 10q22-24 occurs in about 25%. Recently, another phosphatase gene, MINPP1, has been localized to 10q23.3. MINPP1 has the ability to remove 3-phosphate from inositol phosphate substrates, a function that overlaps that of PTEN. Because of this overlapping function with PTEN and the physical location of MINPP1 to a region with frequent LOH in follicular thyroid tumors, we considered it to be an excellent candidate gene that could contribute to the pathogenesis of follicular thyroid tumors. We analyzed DNA from tumor and corresponding normal tissue from 23 patients with FA and 15 patients with FTC for LOH and mutations at the MINPP1 locus. LOH was identified in four malignant and three benign tumors. One of these FTCs with LOH was found to harbor a somatic c.122C > T or S41L mutation. We also found two germline sequence variants, c.809A > G (Q270R) and IVS3 + 34T > A. The c.809A > G variant was found in only one patient with FA but not in patients with FTC or normal controls. More interestingly, IVS3 + 34T > A was found in about 15% of FA cases and normal controls but not in patients with FTC. These results suggest a role for MINPP1 in the pathogenesis of at least a subset of malignant follicular thyroid tumors, and MINPP1 might act as a low penetrance predisposition allele for FTC.

Adenoma↗

PubMed2000

Relative roles of somatic and Darwinian evolution in shaping the antibody response.

The need for a highly specific system of recognition in immunity has resulted in the evolution of several somatic mechanisms such as V(D)J recombination, to diversify the repertoire of B cells. Therefore, repertoire diversification is the driving force for the cells that constitute the bulk of the response to unpredictable pathogens, the B2 naive B cells. Predictability of antigen, on the other hand, has played a major role in shaping the neonatal repertoire, in which evolution to recognize commonly encountered pathogens has driven the germline sequence of several VH segments that are used frequently in the neonatal repertoire. A third population, the memory B cell population, is generated to respond to a known pathogen, but predictability of the pathogen is not acquired until after a first exposure. Therefore, it is somatic evolution in germinal centers that drives the generation of high-affinity memory B cells.

Animals↗

PubMed2003

The in vitro and in vivo effects of anti-galactose antibodies on endothelial cell activation and xenograft rejection.

We have previously produced a series of antigalactose (anti-Gal) hybridomas and characterized their heavy chain gene usage. Here we have quantified the affinity of these Abs for the alpha-Gal epitope and characterized their in vitro effects on endothelial cell activation and apoptosis. We report that anti-Gal mAbs derived from Gal(-/-) mice show a range of affinity for the alpha-Gal epitope, and that affinity was generally increased as the V(H) gene usage transitioned from germline sequences to sequences exhibiting somatic maturation. Despite an 85-fold range in affinity, all the anti-Gal mAbs examined induced alpha-Gal-specific endothelial cell activation, and after prolonged exposure induced endothelial cell apoptosis in a complement-independent manner. Only murine anti-Gal mAbs of the IgM or IgG3 subclass, but not IgG1, were effective at initiating complement-dependent cell lysis. Using a novel rat to mouse xenograft model, we examined the in vivo ability of these mAbs to induce xenograft rejection and characterized the rejection using histology and immunohistochemistry. Infusion of complement-fixing IgG3 mAbs resulted in either hyperacute rejection or acute vascular rejection of the xenograft. Surprisingly, infusion of an equal amount of a high affinity anti-Gal IgG1 mAb, that fixed complement poorly also induced a rapid xenograft rejection, which we have labeled very acute rejection. These studies emphasize the importance of in vivo assays, in addition to in vitro assays, in understanding the role of anti-Gal IgG-mediated tissue injury and xenograft rejection.

Animals↗

PubMed2002

Multivariate analysis of prognostic factors in CLL: clinical stage, IGVH gene mutational status, and loss or mutation of the p53 gene are independent prognostic factors.

This study evaluates the prognostic significance of genetic abnormalities (detected at or shortly after presentation), clinical stage, lymphocyte morphology, CD38 expression, and IGVH gene status in 205 patients with chronic lymphocytic leukemia (B-CLL). Deletion of chromosome 11q23, absence of a deletion of chromosome 13q14, atypical lymphocyte morphology, and more than 30% CD38 expression are significantly associated with the presence of unmutated IGVH genes. Advanced stage, male sex, atypical morphology, more than 30% CD38 expression, trisomy 12, deletion of chromosome 11q23, loss or mutation of the p53 gene, and unmutated IGVH genes are all poor prognostic factors in a univariate analysis. However, only 98% or more homology of IGVH genes to the germline sequence, loss or mutation of the p53 gene, and clinical stage retain prognostic significance in a multivariate analysis. The median survival of patients with mutated IGVH genes, unmutated IGVH genes, and loss or mutation of the p53 gene regardless of IGVH gene status is 310, 119, and 47 months, respectively. These data should facilitate the design of new trials for the management of patients presenting with advanced disease or poor prognosis early stage disease.

ADP-ribosyl Cyclase↗