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PubMed2006

Contribution of germline BRCA1 and BRCA2 sequence alterations to breast cancer in Northern India.

BACKGROUND: A large number of distinct mutations in the BRCA1 and BRCA2 genes have been reported worldwide, but little is known regarding the role of these inherited susceptibility genes in breast cancer risk among Indian women. We investigated the distribution and the nature of BRCA1 and BRCA2 germline mutations and polymorphisms in a cohort of 204 Indian breast cancer patients and 140 age-matched controls. METHOD: Cases were selected with regard to early onset disease (< or =40 years) and family history of breast and ovarian cancer. Two hundred four breast cancer cases along with 140 age-matched controls were analyzed for mutations. All coding regions and exon-intron boundaries of the BRCA1 and BRCA2 genes were screened by heteroduplex analysis followed by direct sequencing of detected variants. RESULTS: In total, 18 genetic alterations were identified. Three deleterious frame-shift mutations (185delAG in exon 2; 4184del4 and 3596del4 in exon 11) were identified in BRCA1, along with one missense mutation (K1667R), one 5'UTR alteration (22C>G), three intronic variants (IVS10-12delG, IVS13+2T>C, IVS7+38T>C) and one silent substitution (5154C>T). Similarly three pathogenic protein-truncating mutations (6376insAA in exon 11, 8576insC in exon19, and 9999delA in exon 27) along with one missense mutation (A2951T), four intronic alterations (IVS2+90T>A, IVS7+75A>T, IVS8+56C>T, IVS25+58insG) and one silent substitution (1593A>G) were identified in BRCA2. Four previously reported polymorphisms (K1183R, S1613G, and M1652I in BRCA1, and 7470A>G in BRCA2) were detected in both controls and breast cancer patients. Rare BRCA1/2 sequence alterations were observed in 15 out of 105 (14.2%) early-onset cases without family history and 11.7% (4/34) breast cancer cases with family history. Of these, six were pathogenic protein truncating mutations. In addition, several variants of uncertain clinical significance were identified. Among these are two missense variants, one alteration of a consensus splice donor sequence, and a variant that potentially disrupts translational initiation. CONCLUSION: BRCA1 and BRCA2 mutations appear to account for a lower proportion of breast cancer patients at increased risk of harboring such mutations in Northern India (6/204, 2.9%) than has been reported in other populations. However, given the limited extent of reported family history among these patients, the observed mutation frequency is not dissimilar from that reported in other cohorts of early onset breast cancer patients. Several of the identified mutations are unique and novel to Indian patients.

Adolescent↗
PubMed1991

Three-dimensional structure of murine anti-p-azophenylarsonate Fab 36-71. 2. Structural basis of hapten binding and idiotypy.

Comparison between the structures and solvent-accessible surfaces of the antigen-binding fragments of two murine anti-p-azophenylarsonate monoclonal antibodies, one bearing a major cross-reactive idiotype of A/J strain mice (36-71) and one lacking the idiotype (R19.9; Lascombe et al., 1989), highlight the structural basis for the determination of hapten affinity and idiotypy. Since the sequence of R 19.9 is identical with the germline-encoded sequence at 16 positions in both heavy-chain and light-chain variable regions where somatic mutations and junctional differences have occurred to produce the 36-71 sequence, the structure of R 19.9 can be used to model the structure of the germline-encoded antibody (36-65) in the regions around these sites. These 16 sequence differences exclude the third heavy-chain complementarity-determining region because R 19.9 utilizes a D gene segment not associated with the predominant idiotype, which is 4 residues longer than the canonical D gene segment utilized in the sequences of 36-71 and 36-65. This difference between the structures of R 19.9 and 36-71 does not affect the validity of using the structure of R 19.9 to model the structure of 36-65 since the third heavy-chain complementarity-determining region is highly solvent-exposed in both 36-71 and R 19.9, and does not interact with any of these 16 sites. Comparing the structures of 36-71 and R 19.9 suggests that only three of the differences in the heavy-chain sequences, and three of the differences in the light-chain sequences of 36-71 and 36-65, increase the affinity for hapten.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Molecular characterization of the human immunoglobulin V lambda I germline gene repertoire.

To advance our understanding of the human immunoglobulin V lambda germline gene contribution to normal as well as autoimmune responses, we have isolated and sequenced six germline genes of the V lambda I subgroup. These genes can be divided into three sub-subgroups on the basis of greater than or equal to 93% nucleotide sequence homology and greater than or equal to 88% deduced amino acid sequence similarity. Examination of all cDNA and protein sequences available for expressed V lambda I genes supports the assignment of these three sub-subgroups. Sequence comparisons also suggest that germline gene members of two of these sub-subgroups, I-a and I-b, are preferentially utilized in the expressed V lambda I repertoire. This finding may be at least partially attributable to regulatory sequence abnormalities apparent in two of the other V lambda I germline genes (Humlv101 and Humlv104) which may interfere with their expression.

Amino Acid Sequence↗
PubMed1983

Allelic immunoglobulin VH genes in two mouse strains: possible germline gene recombination.

The nucleotide sequence of two germline immunoglobulin heavy chain variable region (VH) genes of mouse BALB/c origin was determined. These two genes are highly homologous to each other. They both have the unusual codon CCT for proline at position 7, which so far has been found only in a specific set of VH genes, called the NPb family. We show that the two VH genes belong to this set. One of our BALB/c genes, VH124, is more homologous to a C57BL/6 NPb VH gene than to any BALB/c VH gene, and we propose that these two genes are alleles. A comparison of the substitutions between these two genes with published sequences of all other BALB/c and C57BL/6 NPb VH genes reveals evidence for past homologous recombination events between related germline VH genes Homologous recombination may play an important role in the diversification of germline immunoglobulin VH genes.

Alleles↗
PubMed1999

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 Vlambda 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.

Alleles↗
PubMed1990

Molecular cloning of a human immunoglobulin heavy chain variable (VH) region with anti-myelin-associated glycoprotein activity.

A cDNA clone that encodes the heavy chain variable region (VH) of an IgM M-protein with anti-myelin-associated glycoprotein (MAG) activity secreted by chronic lymphocytic leukemia cells (B-C11) from a patient with peripheral neuropathy was cloned and sequenced. The JH region was identical to the germline JH4 sequence except for deletion of a thymidine residue at the site of D-JH recombination, and the D region showed greatest homology to DM2. Sequence analysis of the VH region revealed greatest homology to VH26, a member of the VH3 gene family, but homology was only 83.7% over 326 bases, suggesting that it was derived from as yet an unidentified member of the VH3 gene family.

Amino Acid Sequence↗
PubMed2001

Radiation and germline mutation at repeat sequences: Are we in the middle of a paradigm shift?

Two assumptions are commonly made in the estimation of genetic risk: (1) that the seven specific loci in the mouse constitute a suitable basis for extrapolation to genetic disease in humans, and (2) that mutations are induced by radiation damage (energy-loss events leading to double-stranded damage) occurring within the gene and are induced linearly with dose, at least at low doses. Recent evidence on the mutability of repeat sequences is reviewed that suggests that neither of these assumptions is as well founded as we like to think. Repeat sequences are common in the human genome, and alterations in them may have health consequences. Many of them are unstable, both spontaneously and after irradiation. The fact that changes in DNA repeat sequences can clearly arise as a result of radiation damage outside the sequence concerned and the likely involvement of some sort of signal transduction process mean that the nature of the radiation dose response cannot be assumed. While the time has not come to abandon the current paradigms, it would seem sensible to invest more effort in exploring the induction of changes in repeat sequences after irradiation and the consequences of such changes for health.

Genetic Diseases, Inborn↗
PubMed2005

piggyBac internal sequences are necessary for efficient transformation of target genomes.

A previously reported piggyBac minimal sequence cartridge, which is capable of efficient transposition in embryo interplasmid transposition assays, failed to produce transformants at a significant frequency in Drosophila melanogaster compared with full-length or less extensive internal deletion constructs. We have re-examined the importance of these internal domain (ID) sequences for germline transformation using a PCR strategy that effectively adds increasing lengths of ID sequences to each terminus. A series of these piggyBac ID synthetic deletion plasmids containing the 3xP3-ECFP marker gene are compared for germline transformation of D. melanogaster. Our analyses identify a minimal sequence configuration that is sufficient for movement of piggyBac vectored sequences from plasmids into the insect genome. Southern hybridizations confirm the presence of the piggyBac transposon sequences, and insertion site analyses confirm these integrations target TTAA sites. The results verify that ID sequences adjacent to the 5' and 3' terminal repeat domains are crucial for effective germline transformation with piggyBac even though they are not required for excision or interplasmid transposition. Using this information we reconstructed an inverted repeat cartridge, ITR1.1k, and a minimal piggyBac transposon vector, pXL-BacII-ECFP, each of which contains these identified ID sequences in addition to the terminal repeat configuration previously described as essential for mobility. We confirm in independent experiments that these new minimal constructs yield transformation frequencies similar to the control piggyBac vector. Sequencing analyses of our constructs verify the position and the source of a point mutation within the 3' internal repeat sequence of our vectors that has no apparent effect on transformation efficiency.

Animals↗
PubMed1981

Antibody diversity: somatic hypermutation of rearranged VH genes.

The immune response to phosphorylcholine in BALB/c mice has been well characterized. Amino acid sequence analyses of heavy-chain variable (VH) regions from 19 myeloma and hybridoma immunoglobulins binding phosphorylcholine show that 10 are identical (the prototype T15 VH sequence) and 9 are distinct variants differing by one to eight residues. A T15 VH DNA probe was used to isolate four closely related members of the T15 VH gene family, including one encoding the T15 VH sequence, from a sperm genomic library. A comparison of the protein and germline VH sequences suggested that most of the immune response to phosphorylcholine is derived from the T15 germline VH gene segment. The variant heavy chains from the M167 and M603 alpha immunoglobulins differ in their VH protein sequences from T15 by eight and three residues, respectively. We analyzed the somatic variability in and around the coding regions of these two variant VH genes by comparing them with the corresponding regions of the appropriate germline gene segments. The somatic variation has three properties: it is extensive and is found in flanking as well as coding sequences (for example, at least 44 substitutions for the M167 sequence and 10 substitutions for the M603 sequence); in the coding regions, it includes many silent as well as replacement substitutions; and it is focal in nature and centered around the rearranged VH genes. Although the mutations extend into the neighboring upstream and downstream flanking sequences, sequences approximately 5 kb upstream and downstream from the VH genes show no substitutions. Moreover, the associated heavy-chain constant genes (C alpha) from both variant alpha genes are unaltered, indicating that a closely linked and coexpressed gene is unmutated. We conclude that this somatic variation is generated by a special hypermutational mechanism highly localized in its site of execution and highly restricted in its time of operation during B-cell development.

Animals↗
PubMed2004

Rapid identification of germline mutations in retinoblastoma by protein truncation testing.

OBJECTIVE: To demonstrate the utility of protein truncation testing (PTT) for rapid detection and sequencing of germline mutations in the retinoblastoma tumor suppressor gene (RB1). METHODS: We performed PTT, a technique based on the in vitro synthesis of protein from amplified RNA, on 27 probands from 27 kindreds with hereditary retinoblastoma. In 4 kindreds, PTT was also performed on 1 additional affected relative. Ten unrelated patients without retinoblastoma were included as negative control subjects. All PTT-detected mutations were further analyzed by focused sequencing of genomic DNA. When no mutation was detected by PTT, we performed exon-by-exon sequencing, as well as cytogenetic analysis by Giemsa-trypsin-Giemsa banding and by fluorescent in situ hybridization for RB1. The results of proband testing were used for direct genetic testing by polymerase chain reaction and sequencing in 11 relatives from 7 of the 27 kindreds. RESULTS: Of the probands tested, 19 (70%) of 27 tested positive for germline mutations by PTT. In 1 kindred, the proband had negative PTT results but an additional affected relative had positive PTT results. Focused DNA sequencing of 1 patient with positive PTT results from each of the 20 kindreds with positive PTT results revealed truncating mutations in 19 kindreds. Four demonstrated frameshift deletions, 6 had splice site mutations, and 9 showed nonsense mutations. Further analysis by genomic exon-by-exon sequencing and karyotype analysis of the 8 probands who tested negative for germline mutations by PTT revealed 1 splice site mutation, 2 missense mutations, and 1 chromosomal deletion. Focused sequencing based on positive PTT results was successfully used to confirm shared truncating mutations in additional affected family members in 2 kindreds. Using a multitiered approach to genetic testing, 23 (85%) of 27 kindreds had mutations identified and those detected by PTT received a positive result in as few as 7 days. In control subjects, PTT produced no false-positive results. CONCLUSIONS: Protein truncation testing is an effective, rapid single-modality screen for germline mutations in patients with retinoblastoma. When used as an initial screen, PTT can increase the yield of additional testing modalities, such as sequencing and chromosomal analysis, providing a timely and cost-effective approach for the diagnosis of heritable germline mutations in patients with retinoblastoma.Clinical Relevance The clinical application of PTT in retinoblastoma will improve detection of germline retinoblastoma mutations, which will supply critical information for prognosis, treatment planning, follow-up care, and genetic counseling.

DNA Mutational Analysis↗
PubMed1993

Variable region genes of anti-HIV human monoclonal antibodies: non-restricted use of the V gene repertoire and extensive somatic mutation.

The extent of the expressed human V gene repertoire for the most part has been derived from fetal cDNA libraries, autoantibodies, and myeloma proteins. In order to continue to explore the utilization of the VH and VL gene repertoire in response to exogenous viral antigens, the heavy and light chain cDNAs from four human anti-HIV monoclonal antibodies were PCR amplified from human-mouse heterohybridomas, cloned, and nucleotide sequence analysis performed. Of the monoclonals analyzed, three were directed against gp120 and one reacted with gp41. Three of the antibodies were of the IgG1 lambda isotype and one was an IgG1 kappa. Three of the four heavy chains were derived from VHI gene segments and one VHII was observed. D segments showed evidence of D-D joining and three JH4 and one JH5 gene were utilized. Two V lambda II lambda chains and one from the V lambda III gene family were observed and the single kappa chain sequenced was from the V kappa III family. DNA sequence comparison with known germline gene segments identified putative precursor V gene segments for one of the heavy chains and two light chains. Comparison of the expressed amino acid sequences with the predicted germline sequences indicated that changes were clustered in the CDRs and FR3 regions of the V gene segments. We reported previously the nucleotide sequences of five human monoclonal antibodies from HIV-infected individuals, three of which utilized VHIV, one VHV and one a VHI gene segment and also found extensive evidence of somatic mutation. Collectively, our results indicate that an antigen driven response is functioning following HIV infection and, surprisingly, to date we have not encountered a VHIII gene segment. Since VHIII is the largest human VH gene family, it may well be that this under-representation has both functional and clinical implications.

Amino Acid Sequence↗
PubMed2026

T-rex: standardized analysis of germline variants in whole-exome sequencing trios.

Whole-exome sequencing (WES) enables the identification of rare germline variants contributing to pediatric diseases. Trio-based sequencing, comparing affected children with their parents, is particularly effective for rare disease genetics. However, WES data analysis requires bioinformatics expertise, varies across institutions, and is often incompatible with clinical workflows. We developed T-Rex (Trio Rare variant analysis of EXomes), a cross-platform desktop application that enables the standardized and local analysis of WES germline Trio data without the need for programming knowledge. T-Rex integrates state-of-the-art tools for alignment, dual-variant calling (GATK HaplotypeCaller&#x2009;+&#x2009;VarScan2), annotation (SNPEff/SNPSift), rare-variant filtering based on population frequencies (gnomAD), and family-based statistical testing, including the Transmission Disequilibrium Test with multiple-testing correction. Benchmarking of the dual-caller strategy on the Genome in a Bottle Ashkenazim Trio demonstrates high precision (99.2%) while maintaining robust sensitivity (91.1%). User testing (n&#x2009;=&#x2009;13) confirmed quick learning across clinicians and researchers. Application to a cohort of n&#x2009;=&#x2009;121 pediatric cancer Trio datasets, filtering for rare protein-coding variants (MAF&#x2009;&#x2264;&#x2009;0.1% in gnomAD v4.1), validated all assessable previously reported pathogenic variants. Overall, T-Rex enables clinicians to robustly analyze WES Trio data in compliance with data protection regulations without requiring additional software licenses. As one of the first platforms for comprehensive WES Trio analysis that requires no programming expertise while providing reproducible, end-to-end workflows for clinical genomics, T-Rex facilitates collaborative research between clinics and reduces reliance on external providers.

Humans↗
PubMed2002

The Rhesus monkey immunoglobulin IGHD and IGHJ germline repertoire.

In order to facilitate molecular analysis of antibody responses in Rhesus monkeys ( Macaca mulatta), we used PCR techniques to clone and sequence the germline IGHD gene repertoire and the IGHD7- IGHJ6 locus in its entirety. We identified 30 distinct Rhesus DH genes belonging to seven subgroups and their recombination signal sequences that together share an average of 91% identity with their human counterparts, six potentially functional IGHJ genes and their recombination signal sequences that together share 93% identity with their human counterparts, as well as a novel IGHJ gene, IGHJ5 beta, which is a duplicated variant of IGHJ5. The presence, on average, of one additional IGHD gene in Rhesus IGHD subgroups when compared with human and one additional IGHJ gene suggests Rhesus has undergone at least two independent duplications beyond those that mark the human IGHD/IGHJ locus. Amino acid sequence composition is highly conserved between Rhesus and human, with IGHD insertions and deletions limited to three-nucleotide multiples, which serve to preserve enrichment for tyrosine, glycine, and serine residues in IGHD reading frame 1. The high degree of conservation between human and Rhesus IGHD and IGHJ genes supports the hypothesis that the germline repertoire encodes evolutionarily preferred antibody sequence as a result of selection for function.

Animals↗
PubMed1995

A subgroup of human VH3 germline genes that encode a high-avidity synovial rheumatoid factor.

We have previously derived and identified a highly avid monoclonal IgM rheumatoid factor (mRF), C6, from unstimulated rheumatoid synovial cells (RSC). At the time, the closet VH germline gene, VH26, demonstrated only 88% homology with C6. To identify the germline counterpart of C6, genomic DNA from the same rheumatoid arthritis (RA) patient from whom C6 was derived was used in the polymerase chain reaction (PCR). Four of the six closely related germline genes that we sequenced had exonic regions that were identical with the VH region of C6 cDNA. These six germline sequences differed in their intronic regions, suggesting that they were distinct, but closely related genomic sequences. To further evaluate the extent of these related genes we identified nine additional germline genes having VH-encoding exons that were 86-97% identical to the C6 cDNA sequence. Furthermore, we examined the polymorphic nature of the C6 VH gene using single strand conformation polymorphism (SSCP), and identified two peaks, confirming the existence of highly homologous genes. The sequence and polymorphism data suggest that: (1) the VH region of the high avidity mRF C6 was derived from an unmutated germline gene; (2) C6 was encoded by a VH gene belonging to a set of homologous genes within the larger VH3 family; and (3) in addition to somatic rearrangements of B-cell genes and antigen-driven somatic mutation, gene duplication and conversion events of germline genes could be important in generating diversity and polyclonality among high-affinity pathogenic autoantibodies.

Amino Acid Sequence↗
PubMed1990

Detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin hypervariable region specific oligonucleotide probes.

To develop a sensitive and specific assay for minimal residual disease in acute lymphoblastic leukemia (ALL), we exploited the enormous diversity of genomic sequences created by immune receptor gene rearrangements. To isolate clone-specific sequences, we first synthesized oligonucleotides that match conserved variable (VH) and joining (JH) sequences flanking the third hypervariable region (HVR3) in the rearranged immunoglobulin heavy chain (IgH) locus. In polymerase chain reactions (PCR), these primers were then used to amplify the intervening HVR3 segments from leukemic DNA samples. Of 12 B-lineage ALLs studied, ten generated one or more fragments of the size expected for HVR3 gene segments. Thus, this single pair of amplimers was sufficient to isolate HVR3 sequences from a majority of acute lymphoblastic leukemias. To verify that the amplified fragments originated from HVR3 alleles and to assess their diversity, we sequenced 7 PCR products derived from 6 leukemias. In addition to elements of recognized D segments, each of the 7 fragments contained novel VH-D and D-JH junctional sequences, including N nucleotides, not known to be present in the germline. Each sequence was unique, and allele-specific oligonucleotide probes hybridized only to HVR3 segments from which the probes were derived. Therefore, as anticipated, these HVR3 segments appeared to possess the diversity required to serve as clonal markers for leukemic populations. To demonstrate that these amplified HVR3 alleles could serve as the basis for a sensitive and specific assay to detect rare leukemic cells, we analyzed in detail one pre-B leukemia that had rearranged 2 IgH alleles. The HVR3 sequences were shown to be linked to rearranged JH-containing restriction fragments in digests of genomic DNA, establishing their origin in the leukemic cells. We synthesized oligonucleotides corresponding to the unique junctional sequences in the HVR3 segments. Using these novel amplimers in an allele-specific amplification and hybridization procedure, we showed that this assay can detect 10 leukemic cells in a background of 10(6) normal blood mononuclear cells. In contrast, the leukemic HVR3 sequences were not detected in extracts of normal or unrelated remission leukemic leukocytes. We conclude that the assay for specific IgH HVR3 sequences is a realistic strategy for detection of minimal residual disease in B-lineage ALL.

Alleles↗
PubMed1996

Characterization of anti-sperm antibodies and their coding cDNA sequences by Epstein-Barr virus transformed B cell lines from lymphocytes of infertile women possessing anti-sperm antibodies.

Epstein-Barr virus (EBV)-transformed B cell lines that produce human antisperm antibodies were established using peripheral blood lymphocytes (PBLs) from infertile women with sperm immobilizing antibodies in their sera. We obtained three stable cell populations (designated B1, B2, D5) of transformed PBLs originating from three different patients. They produced IgM sperm-reacting antibodies directed against the tail of live, methanol-fixed and NaIO4-treated human spermatozoa. The established antisperm antibodies recognized noncarbohydrate sperm membrane antigens with different specificity and distribution in the male reproductive system. Antisperm antibody-B2 corresponding antigen appears to be specific for the male reproductive system. This antigen is excreted from the epithelial cells of the ductus epididymidis and bound to the spermatozoa in the lumen of the ductus. Antisperm antibodies B1 and D5 corresponding antigens were expressed on the spermatozoa in the seminiferous tubules and were common to the secretions of the ductus epididymidis, prostate and some other somatic organs. The cDNA of the immunoglobulin heavy chain genes were analyzed by reverse transcription polymerase chain reaction (RT-PCR) using RNA extracted from these clones. The immunoglobulin heavy chain cDNA sequences of these antisperm antibodies showed extremely high homology to previously reported immunoglobulin germline DNA sequences, implying that these antisperm antibodies might be natural autoantibodies rather than antibodies stimulated by external antigen.

Antibodies, Monoclonal↗
PubMed1992

Immunoglobulin heavy and light chain gene sequences of a human CD5 positive immunocytoma and sequences of four novel VHIII germline genes.

To analyse the V genes expressed by an IgM lambda CD5-positive immunocytoma heavy and light chain V region genes were cloned and sequenced. The heavy chain is composed of a previously undescribed VHIII gene joined to an unknown D gene and to JH4. The light chain V region is composed of a V lambda II gene rearranged to J lambda 1. In an attempt to clone the germline counterpart of the VHIII gene expressed in the immunocytoma PCR amplifications of genomic DNA were carried out and four previously unknown VHIII genes were identified. As several independent clones for the heavy and light chain V region genes were sequenced the rate of somatic mutation of the V genes was calculated to be below 2 x 10(-5)/bp/cell division.

Amino Acid Sequence↗