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 163 records · Page 9Linked to original sources

PubMed1992

Experimental analysis by site-directed mutagenesis of somatic mutation effects on affinity and fine specificity in antibodies specific for lysozyme.

To experimentally examine the functional roles of somatically derived structural variation in the lysozyme-binding mAb HyHEL-10, we have introduced three different point mutations and one insertion at two different sites in HyHEL-10 by site-directed mutagenesis and expression of the mutant antibodies. Mutation of Asp----Ala at position 101 of the H chain returns a somatically mutated residue to its germline sequence for HyHEL-10, and reduces affinity for chicken lysozyme by approximately 9000-fold. Lengthening the third H chain hypervariable region by two amino acids reduces affinity by about 2000-fold. Two mutations, Asp----Thr at position 101 in the H chain and Lys----Thr at position 49 in the L chain, model somatic differences found in another structurally related but functionally distinguishable mAb and minimally decrease affinity for chicken lysozyme. The H chain mutation Asp101VVH----Thr has little effect on affinity for other avian lysozymes but does alter relative fine specificity for these lysozymes. The L chain mutation Lys49VK----Thr increases affinity for duck lysozyme by approximately fivefold. Neither of the positions mutated, 101 in the H chain nor 49 in the L chain, nor the residues near the insertion contact lysozyme in the x-ray structure of the HyHEL-10 F(ab)-HEL complex. The results suggest that these mutations, which model observed somatic mutations, produce functional variation by indirect or long-range effects.

Amino Acid Sequence↗

PubMed1995

Analysis of VH genes used by neoplastic B cells in endemic Burkitt's lymphoma shows somatic hypermutation and intraclonal heterogeneity.

Tumor cell lines from six typical cases of endemic Epstein-Barr virus (EBV) genome-positive Burkitt's lymphoma (BL) have been investigated for usage and mutational pattern of Ig VH genes. The neoplastic cells all had a t(8;14) (q24;q32) translocation involving the c-myc protooncogene. The VH genes were derived from VH1, VH3 and VH4, and both the IgM-positive (four cases) and IgG-positive (two cases) were extensively mutated from germline sequence. In two cases, early and late passage tumor cells were available, and the VH nucleotide sequences were identical, indicating that mutations had not accumulated in vitro. In a further case, there was evidence of sequence heterogeneity, which appeared to have been generated in vivo, indicating that the tumor cell VH gene was able to undergo posttranslocation somatic hypermutation. Analysis of the relatively nonpolymorphic VH4 genes for the pattern of replacement or silent mutations did not show a role for antigen selection in the expressed sequences.

Amino Acid Sequence↗

PubMed1995

Splenic lymphoma with villous lymphocytes involves B cells with extensively mutated Ig heavy chain variable region genes.

Splenic lymphoma with villous lymphocytes (SLVL) is a recently defined subgroup of chronic B-cell lymphoproliferative diseases. The characteristic morphology of the tumor cells, together with phenotypic and cytogenetic findings, indicate that it is a distinct entity, but the nature of the cell or origin and its relationship to other low-grade lymphomas is unclear. For B-cell tumors, analysis of the variable region heavy chain (VH) genes used to encode the clonal Ig has shown marked differences between histologic categories, both in gene usage and extent of somatic mutation. An investigation of VH genes used in five typical cases of SLVL has shown somatic hypermutation from germline sequences in all cases, indicating that the cell of origin has been exposed to the hypermutation mechanism. However, no clonal heterogeneity was detectable, demonstrating that the tumor cell does not accumulate further mutations. These characteristics are similar to those found in mature postfollicular B cells, such as plasma cells. The distribution of mutations leading to replacement amino acids differed among the cases, with three of five cases showing clear evidence for antigen selection.

Aged↗

PubMed1997

Structural analysis of human antibodies to proteinase 3 from patients with Wegener granulomatosis.

We determined the structure of five IgM autoAbs to proteinase-3 (PR3). These Abs are highly specific for Wegener's granulomatosis (WG) and may be involved in the pathogenesis of vasculitis in WG. Five clonal lymphoblastoid cell lines secreting Abs to PR3 were derived from four patients' B cells. From 3 to 5% of supernatants from wells contained detectable anti-PR3 Abs, indicating that anti-neutrophil cytoplasmic Ab specificity represents a sizable part of the IgM B cell repertoire in patients with WG. Mu heavy chains of WG1, WG4-1, and WG4-2 clones belonged to the VH3 subgroup. WG4-1 and WG4-2 heavy chains were identical, indicating an oligoclonal expansion of autoreactive B cells in this patient. WG4-1 (and WG4-2) used the VH3-23 V(H) gene, the product of which was shown to directly bind PR3. Heavy chains of WG2 and WG3 derived from VH4-59 and VH1-2 genes, respectively. Comparison with germline sequences showed that three of the five V(H) genes from clonal lines were somatically mutated with a R:S ratio in complementarity-determining regions of 3:0, 5:1, and 5:1, respectively. Three kappa light chains derived from the Vkappa4 gene, and one derived from a Vkappa1 gene. In these four Vkappa genes, there were overall R:S ratios of mutation of 8:1 and 0:7 in complementarity-determining regions and framework regions, respectively. These data suggest that the production of these autoantibodies, which are increasingly important in the diagnosis and management of WG, are influenced by an Ag-driven process.

Amino Acid Sequence↗

PubMed1998

CD4+ T cells mature in the absence of MHC class I and class II expression in Ly-6A.2 transgenic mice.

The TCRs expressed on T lymphocytes recognize foreign peptides bound to MHC molecules. This reactivity is the basis of specific immune response to the foreign Ag. How such specificities are generated in the thymus is still being debated. Signals generated through TCR upon interaction with self MHC-peptide complexes are critical for maturation of the CD4+ helper and CD8+ cytotoxic subsets. We have observed maturation of CD4+ but not CD8+ T cells in Ly-6A.2 transgenic MHC null mice. Since there can be no interactions with MHC molecules in these mice, these CD4+ cells must express the T cell repertoire that exists before positive and negative selection. Interestingly, despite an absence of selection by MHC molecules, the CD4+ cells that mature recognize MHC molecules at a frequency as high as in CD4+ cells in normal mice. These results demonstrate that: 1) the germline sequences encoding TCRs are biased toward reactivity to MHC molecules; and 2) CD4+ cells as opposed to CD8+ cells have distinct lineage commitment signals. These results also suggest that signals originating from Ly-6 can promote or substitute for signals generated from TCR that are required for positive selection. Moreover, this animal model offers a system to study T cell development in the thymus that can provide insights into mechanisms of lineage commitment in developing T cells.

Animals↗

PubMed2026

Integrating germline and tumor sequencing to improve hereditary cancer diagnosis and care.

A subset of cancers arises due to inherited germline pathogenic variants in specific genes, known as hereditary cancers. These genes typically include tumor suppressors, DNA repair and replication fidelity genes, and occasionally oncogenes. In most hereditary cancer syndromes, Knudson's two-hit hypothesis applies, where a second somatic event inactivates the remaining allele of a tumor suppressor or DNA repair gene, leading to tumorigenesis. Advancements in genome-wide sequencing have significantly enhanced our understanding of the mutational processes involved in hereditary cancers. In particular, the assessment of microsatellite instability (MSI), tumor mutational burden (TMB), and mutational signatures has emerged as a powerful tool for the identification of hereditary tumors. Tumors with high or ultra-high TMB often reflect underlying DNA repair deficiencies, while specific mutational signatures can pinpoint the defective pathway. These tumor mutational features are especially informative in syndromes involving mismatch repair (MMR), homologous recombination (HR), base excision repair (BER), nucleotide excision repair (NER), and polymerase proofreading. Moreover, tumor sequencing aids in the interpretation of germline variants, identifies somatic mosaicism, and helps differentiate hereditary from sporadic cancers. Additionally, tumor molecular features associated with DNA repair deficiencies offer insights into personalized therapies, such as the use of PARP inhibitors for BRCA1/2-deficient tumors and immune checkpoint inhibitors for MMR- and polymerase proofreading-deficient cancers. Tumor profiling also uncovers actionable mutations in oncogenes like RET and VHL, which can be targeted with specific therapies. This review explores the integration of tumor molecular features with germline genetic data to refine diagnosis, risk assessment, and therapeutic strategies in hereditary cancer.

Humans↗

PubMed1991

Spontaneous mutation at the hypervariable mouse minisatellite locus Ms6-hm: flanking DNA sequence and analysis of germline and early somatic mutation events.

Hypervariability at minisatellite loci is maintained by spontaneous mutation to new-length alleles. At the most variable loci, mutation rate is directly measurable by pedigree analysis. The mouse minisatellite locus Ms6-hm has a germline mutation rate of 2.5% per gamete and is therefore one of the most unstable loci yet identified in the mouse genome. Mutation events at this locus also occur during early mouse development, resulting in mice mosaic for cells carrying a common non-parental allele in different somatic tissues and the germline. The DNA sequence flanking Ms6-hm is rich in dispersed repetitive elements; the minisatellite array has expanded from within a member of the Mouse Transcript family which is flanked by two additional Mouse Transcript elements, and a B2 element lies further 3' to the minisatellite. To define the characteristics of mutation events at Ms6-hm we have analysed 19 germline and 13 somatic length-change events. Germline mutation events at Ms6-hm are not accompanied by the exchange of flanking markers in three informative mutant alleles analysed.

Alleles↗

PubMed2004

Despite extensive similarity in germline DH and JH sequence, the adult Rhesus macaque CDR-H3 repertoire differs from human.

Rhesus macaques (Macaca mulatta) and chimpanzees (Pan troglodytes) are frequently used models for the study and treatment of human infectious diseases, including AIDS. Confidence in the equivalence to human of the humoral immune responses of these higher primates has grown as studies of immunoglobulin germline sequences have documented average identities of 90% or greater to human counterparts. The most variable component of the immunoglobulin heavy chain, complementarity determining region 3 (CDR-H3) is the product of somatic (junctional) as well as germline (combinatorial) mechanisms of diversity. Located at the center of the antigen binding site, CDR-H3 often exerts a dominant role in antibody specificity and affinity. To test whether similarity in germline DH and JH sequence would yield similarity in CDR-H3 composition in the expressed repertoire, we compared IgM CDR-H3 transcripts from Rhesus and chimpanzee blood to human. In fetal Rhesus, the range and mean of CDR-H3 lengths was similar to that observed in fetal human. However, the Rhesus repertoire of adult muCDR-H3 transcripts did not contain the longer hypervariable intervals that humans begin to express late in the second trimester of fetal life. Conversely, the adult chimpanzee repertoire included more long CDR-H3 structures than human. The differences between these adult repertoires reflected fine changes in N addition and terminal nucleotide loss. We conclude that the same mechanisms that refine and shape CDR-H3 diversity during ontogeny can also be used to fine tune and individualize species-specific antibody repertoires despite germline immunoglobulin sequence similarity.

Age Factors↗

PubMed1992

Immunoglobulin variable region gene expression in response to Haemophilus influenzae type b polysaccharide.

The mechanism(s) responsible for the ontogenic patterns of acquisition of the antibody repertoire is unknown. The immune response to Haemophilus influenzae type b (Hib) capsular polysaccharide provides an excellent model system in which to examine the ontogeny of immunoglobulin variable region expression. A panel of hybridomas secreting human antibodies specific for Hib capsular polysaccharide was developed using peripheral blood lymphocytes from donors immunized with Hib vaccines. Nucleotide sequence analysis of the heavy chain V regions expressed by four of these hybridomas suggests selective use of members of the VHIII gene family in combination with different D and J segments. The nucleotide sequences were highly homologous to two candidate germline gene sequences. Others have reported that these particular germline sequences are expressed in fetal liver, suggesting that the inability of young children to produce antibody to the Hib capsular polysaccharide is not due to failure to express these VH regions early in ontogeny.

Adult↗

PubMed2006

Assessing radiation-associated mutational risk to the germline: repetitive DNA sequences as mutational targets and biomarkers.

This review assesses recent data on mutational risk to the germline after radiation exposure obtained by molecular analysis of tandemly repeated DNA loci (TRDLs): minisatellites in humans and expanded simple tandem repeats in mice. Some studies, particularly those including exposure to internal emitters, indicate that TRDL mutation can be used as a marker of human radiation exposure; most human studies, however, are negative. Although mouse studies have suggested that TRDL mutation analysis may be more widely applicable in biomonitoring, there are important differences between the structure of mouse and human TRDLs. Mutational mechanisms probably differ between the two species, and so care should be taken in predicting effects in humans from mouse data. In mice and humans, TRDL mutations are largely untargeted with only limited evidence of dose dependence. Transgenerational mutation has been observed in mice but not in humans, but the mechanisms driving such mutation transmission are unknown. Some minisatellite variants are associated with human diseases and may affect gene transcription, but causal relationships have not yet been established. It is concluded that at present the TRDL mutation data do not warrant a dramatic revision of germline or cancer risk estimates for radiation.

Animals↗

PubMed2026

Precision medicine and parental experience: a longitudinal study of psychosocial responses to germline genomic results in pediatric oncology.

INTRODUCTION: Precision medicine has become central to pediatric oncology, with germline genomic sequencing commonly integrated into routine care. Families must interpret complex genomic findings during emotionally vulnerable periods, generating mixed reactions ranging from clarity and relief to anxiety and uncertainty. Palliative care clinicians, genetic counselors, psychologists, social workers, and oncology providers may each contribute to supporting families as they interpret and integrate these findings over the course of a child's cancer care and beyond. Little is known about the trajectory of parental emotional and cognitive responses after receiving germline sequencing results, limiting clinicians' ability to anticipate support needs across the cancer care continuum. This study quantitatively examines parental emotional and cognitive responses across time following disclosure of germline sequencing results in a pediatric oncology setting. METHODS: Parents (n = 218) self-reported sequencing-related distress, positive feelings, intrusive thoughts, certainty, and self-efficacy using validated measures at two longitudinal follow-up points after disclosure of their child's germline test results. Outcomes were compared across germline test result types (pathogenic/likely pathogenic [P/LP], n = 31 [14%]; variants of uncertain significance [VUS], n = 86 [39%]; and negative, n = 101[46%]). RESULTS: Parents of children receiving P/LP or P/LP+VUS results reported significantly higher distress yet greater positive feelings than parents receiving negative results. Notably, certainty and self-efficacy increased from Timepoint 1 (median 254 days following return of results) to Timepoint 2 (median 537 days). Intrusive thoughts did not significantly differ by genetic result type or change over time; however, the factors contributing to intrusive thoughts could not be determined from the current study. DISCUSSION: These findings provide insight into how families adapt to germline genomic information following a pediatric cancer diagnosis. As precision medicine becomes increasingly embedded in pediatric oncology, structured follow-up and communication that address families' evolving informational and psychosocial needs are essential to ensure care that is scientifically precise, emotionally attuned, and centered on the family experience.

family-centered care↗

PubMed2026

Diverse mediators of cancer predisposition uncovered by germline whole genome sequencing of unexplained familial cancers.

Cancer frequently clusters in families due to shared environment and genetics. However, many familial cancer cases lack a clinically recognized pathogenic germline variant (PGV). We analyzed germline genomes and family history from 2,726 individuals without a PGV in the All of Us Research Program, including 1,496 cases across 18 cancer types with extensive family history and 1,230 family history-negative, cancer-free controls. We identified allelic series of rare structural variants inactivating MSH2 in individuals with phenotypes consistent with Lynch syndrome and BRCA1 in breast cancer. Cancer polygenic risk scores were enriched in cases and correlated with patterns of cancer diagnoses within families. Exome-wide rare variant analyses nominated six candidate predisposition genes, including TSTD2 and BRAT1 in thyroid and breast cancer, respectively. Overall, polygenic risk and rare variants impacting known genes explained a median of 5% of unexplained familial cancers, increasing to 11% when including newly nominated risk factors.

Journal Article↗

PubMed2000

Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries.

We constructed a single-chain Fv antibody library that permits human complementarity-determining region (CDR) gene fragments of any germline to be incorporated combinatorially into the appropriate positions of the variable-region frameworks VH-DP47 and VL-DPL3. A library of 2 x 109 independent transformants was screened against haptens, peptides, carbohydrates, and proteins, and the selected antibody fragments exhibited dissociation constants in the subnanomolar range. The antibody genes in this library were built on a single master framework into which diverse CDRs were allowed to recombine. These CDRs were sampled from in vivo-processed gene sequences, thus potentially optimizing the levels of correctly folded and functional molecules, and resulting in a molecule exhibiting a lower computed immunogenicity compared to naive immunoglobulins. Using the modularized assembly process to incorporate foreign sequences into an immunoglobulin scaffold, it is possible to vary as many as six CDRs at the same time, creating genetic and functional variation in antibody molecules.

Germ-Line Mutation↗

PubMed2005

Chromatin and RNAi factors protect the C. elegans germline against repetitive sequences.

Protection of genomes against invasion by repetitive sequences, such as transposons, viruses, and repetitive transgenes, involves strong and selective silencing of these sequences. During silencing of repetitive transgenes, a trans effect ("cosuppression") occurs that results in silencing of cognate endogenous genes. Here we report RNA interference (RNAi) screens performed to catalog genes required for cosuppression in the Caenorhabditis elegans germline. We find factors with a putative role in chromatin remodeling and factors involved in RNAi. Together with molecular data also presented in this study, these results suggest that in C. elegans repetitive sequences trigger transcriptional gene silencing using RNAi and chromatin factors.

Animals↗

PubMedDate not supplied

Invention and mystery in hypotrich DNA.

Hypotrichs have evolved extraordinary ways of organizing, manipulating, and replicating the DNA in their micronuclear and macronuclear genomes. Short macronuclear DNA molecules containing single genes are created by excision from chromosomes, accompanied by massive elimination of the germline DNA sequences between genes. Germline genes themselves are interrupted by multiple noncoding segments called internal eliminated segments, or IESs, that divide genes into multiple macronuclear-destined segments, or MDSs. The functional significance of this organization is unknown. Over evolutionary time IESs accumulate mutations rapidly, are inserted into or excised from genes, and shift position along DNA molecules. MDSs are ligated to create functional genes when IESs are spliced out of micronuclear DNA during macronuclear development. MDSs in some germline genes are in scrambled disorder and become unscrambled in association with IES elimination. Replication of DNA in the macronucleus is accomplished by organization of replication enzymes and factors into a structure that sweeps through the macronucleus to replicate the many millions of gene-sized DNA molecules. The significance of many of the bizarre DNA phenomena in the evolutionary/functional success of hypotrichs is still unclear.

Animals↗

PubMed2006

Identification of somatic and germline mitochondrial DNA sequence variants in prostate cancer patients.

Prostate cancer (PCa) is the second most frequent cancer among men in the European Union and the most common in the United States. Older age and a positive family history of PCa are important risk factors, but little is known about the disease aetiology. Mitochondria are involved in essential cellular pathways, some of which have been associated with tumorigenesis. We analysed the presence of sequence variants, depletion and rearrangements in the mitochondrial DNA (mtDNA) of PCa patients. Sequencing of the D-loop and genes RNR1 and 2, ND3, ND4L and ND4, and long-range and real-time PCR techniques were carried out on 51 samples (41 from patients and 10 from controls). Normal, hyperplastic or tumour samples were obtained from 17 patients. Six patients' seminal vesicles were also investigated as an additional patient's control tissue (these structures seldom develop tumours). Neither depletion nor mtDNA rearrangements were detected. In contrast, 94 mtDNA sequence variants were identified, 9 previously unreported. The regions presenting more sequence variants were MT-DLOOP (52%), MT-RNR2 (14%) and MT-ND4 (13%). The patients' seminal vesicles studied showed the same set of variants as the corresponding prostate, suggesting either that the pathogenic role of these particular variants is minor or that they participate in the prostatic carcinogenesis in combination with other factors absent in seminal vesicles. Five patients (29.4%) harboured eight somatic changes in the mtDNA. One affects a conserved residue and three have not been previously described. The analysis of other genes in the mtDNA molecule might demonstrate an even higher incidence of mtDNA somatic variants in these PCa patients.

Aged↗

PubMed2002

Systemic lupus erythematosus patients under immunosuppressive treatment express high levels of the immunoglobulin lambda variable IGLV8S1 gene with silent somatic mutations.

Systemic lupus erythematosus (SLE) patients express high titers of somatically mutated serum autoantibodies against nuclear structures including double-stranded DNA. These somatic mutations accumulate codons for basic amino acids in the immunoglobulin variable regions of both, heavy and light chains, facilitating binding to nucleic acids. The variable (V) immunoglobulin lambda 8 (IGLV8S1) gene contributes to autoreactive B-cell repertoire of auto-immune patients. Accumulation of immune complexes of these anti-DNA autoantibodies causes severe systemic inflammation in SLE. The current treatment of lupus disease is based on immunosuppressive drugs, but the precise role for this therapy remains to be defined. To evaluate the in vivo effect of combined immunosuppressive treatment on B-lymphocytes repertoire of SLE patients, we have developed an approach using the IGLV8S1 gene as a marker. The transcription of this gene in treated SLE patients was increased. However, we observed a trend, in these patients, to conserve complementarity determining regions (CDRs) and framework regions (FRs) of Vlambda8 polypeptide light chain deduced sequence, from its germline counterpart. Sequencing IGLV8S1 cDNA of untreated SLE patients, taken as a control for treatment effect, displayed a decreased frequency of silent somatic mutations (consequently high frequency of replacement mutations) in the Vlambda8 polypeptide chain deduced sequence. These data suggest that the immunosuppressive drug treatment modulates the positive selection of somatically mutated Vlambda8 light chain.

Adult↗

PubMed2002

Germline mutations and sequence variants of the macrophage scavenger receptor 1 gene are associated with prostate cancer risk.

Deletions on human chromosome 8p22-23 in prostate cancer cells and linkage studies in families affected with hereditary prostate cancer (HPC) have implicated this region in the development of prostate cancer. The macrophage scavenger receptor 1 gene (MSR1, also known as SR-A) is located at 8p22 and functions in several processes proposed to be relevant to prostate carcinogenesis. Here we report the results of genetic analyses that indicate that mutations in MSR1 may be associated with risk of prostate cancer. Among families affected with HPC, we identified six rare missense mutations and one nonsense mutation in MSR1. A family-based linkage and association test indicated that these mutations co-segregate with prostate cancer (P = 0.0007). In addition, among men of European descent, MSR1 mutations were detected in 4.4% of individuals affected with non-HPC as compared with 0.8% of unaffected men (P = 0.009). Among African American men, these values were 12.5% and 1.8%, respectively (P = 0.01). These results show that MSR1 may be important in susceptibility to prostate cancer in men of both African American and European descent.

Aged↗