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At least 433 records · Page 24Linked to original sources
PubMed2025

Delivering effective genome sequencing in pediatric care: From research in the 100,000 Genomes Project to routine clinical practice.

PURPOSE: Genome sequencing (GS) is increasingly used to investigate rare conditions, primarily in children. The 100,000 Genomes Project (100KG) evaluated GS ahead of implementation in the English National Health Service. In 2020, the National Health Service Genomic Medicine Service (GMS) became the first public health care system to offer GS in routine clinical care. We investigate how learning from 100KG informed GMS service delivery. METHODS: We compare GS outcomes in children tested at a large pediatric hospital via GMS (n = 501) and 100KG research (n = 1759). RESULTS: GMS diagnostic yield (29%) was higher than that in 100KG (22%) (P < .0016). Median age at testing was 8 years in 100KG and 6 in the GMS (P < .05). In 100KG, the diagnostic yield was <10% for 15 indications, none of which are included in GMS testing. 100KG data showed little benefit to application of >3 panels. Use of fewer but larger GMS panels resulted in a significantly higher number of genes tested per patient: median 2801 vs 1373 in 100KG (P < .001). In 100KG, diagnostic yield was not significantly increased by testing more than 3 family members (n = 34/142, 24%). CONCLUSION: Learning from 100KG has informed GS clinical service delivery, resulting in higher diagnostic yields and earlier age at testing. Lessons are broadly applicable to all services providing GS, enabling earlier access to tailored management with fewer investigations.

Humans↗
PubMed1995

Improved detection of anti-HCV in post-transfusion hepatitis by a third-generation ELISA.

The sensitivity of ORTHO HCV 3.0 ELISA Test System (ELISA 3) for the detection of anti-HCV was compared with the second-generation ELISA, OR-THO HCV 2.0 ELISA Test System (ELISA 2). ELISA 3 differs from ELISA 2 in that it incorporates the HCV recombinant antigen NS5, in addition to recombinant antigens derived from the NS3, NS4 and core regions of the HCV genome. Specimens tested consisted of serial bleeds obtained from 21 individuals undergoing seroconversion following acquisition of post-transfusion HCV infection. ELISA 3 demonstrated significantly greater sensitivity than ELISA 2, detecting seroconversion earlier in 24% (5/21) of cases. Although one of these cases appeared to represent early seroconversion to NS5, most of the improved sensitivity of ELISA 3 appeared to derive from increased detectability of anti-c33c.

Enzyme-Linked Immunosorbent Assay↗
PubMed1999

Venezuelan equine encephalitis virus vectors expressing HIV-1 proteins: vector design strategies for improved vaccine efficacy.

A live virus vaccine vector has been constructed from a molecularly cloned attenuated strain of Venezuelan equine encephalitis virus (VEE). High levels of foreign protein expression are regulated by an additional copy of the 26 S viral subgenomic RNA promoter. The position of this additional promoter and foreign gene in the VEE genome was predicted to have a major influence on expression level of the heterologous protein. Two sites in the genome were tested to determine the optimal site for expression of the matrix/capsid (MA/CA) coding region of human immunodeficiency virus (HIV-1). One vector contained the additional promoter and the MA/CA genes immediately downstream of the VEE E1 gene at the 3' end of the genome. In the second vector, the additional promoter was introduced immediately upstream from the authentic 26 S subgenomic promoter. Significantly higher levels of MA/CA were expressed from the downstream vector compared to the upstream vector. However, the stability of expression for both vectors was similar following passage in baby hamster kidney cells (BHK) cells. In BALB/c mice, the two vectors elicited similar levels of cellular immune responses to MA/CA as determined by bulk cytotoxic T-lymphocyte assays and precursor frequency analysis, but the humoral response induced by the downstream vector was significantly stronger. At 11 months post boosting with the downstream vector, serum antibody levels against HIV MA/CA were undiminished, and MA/CA specific CTLp were detectable in all mice tested. These findings suggest that VEE vectors can be optimized to elicit strong, balanced and long-lived immune responses to foreign viral proteins.

AIDS Vaccines↗
PubMed1994

Elevated frequency of microsatellite mutations in TK6 human lymphoblast clones selected for mutations at the thymidine kinase locus.

A major question in carcinogenesis is, How can a normal cell accumulate multiple mutations in different genes on different chromosomes, when the mutation rate of each gene is in the range of 10(-8) to 10(-5) per cell division? We hypothesize that many mutations may not be isolated events but rather are accompanied by concomitant mutations elsewhere in the genome. To test this hypothesis, 331 independent clones selected for new mutations at the thymidine kinase (TK) locus on chromosome 17q, and 243 nonselected control clones were examined for mutations in 12 random microsatellite loci dispersed throughout the genome. A total of 24 second-site mutations were identified in the TK mutant clones, compared with 3 in the control clones not selected for mutations at TK. The mutations include small deletions, insertions, and loss of heterozygosity. These results provide evidence that a global trans-acting mutagenic process exists in human cells. The activation of this process could be responsible for causing multiple essential mutations in tumor cells.

Base Sequence↗
PubMed2003

Genome-wide identification of chromosomal regions of increased tumor expression by transcriptome analysis.

Genes up-regulated in tumor cells provide attractive anticancer therapeutic targets. Although the general underlying mechanism for the increased expression in tumors is unknown, tumor-specific up-regulation of some genes can be attributed to aberrant DNA amplification, a phenomenon common to many tumors. Using a computational method, we constructed a general transcriptome map with the human genomic sequences and expressed sequence tags in the public database. The transcriptome map revealed nonrandom chromosomal regions (termed region of increased tumor expression) where clusters of genes exhibited increased expression in the 10 tumor tissue types tested. These genomic regions often correspond to experimentally verified tumor amplicons. Our large-scale transcriptome analysis led to identification of many additional chromosomal regions with increased tumor expression, regions that represent potential tumor amplicons.

Chromosomes, Human↗
PubMed1999

Genomic instability in silica- and cadmium chloride-transformed BALB/c-3T3 and tumor cell lines by random amplified polymorphic DNA analysis.

Our earlier studies using random amplified polymorphic DNA (RAPD) analysis have shown genetic instability in human lung cancer tissues. Here we have investigated the potential for genetic instability in silica- and cadmium chloride (CdCl2)-transformed BALB/c-3T3 cell lines. Non-transformed, transformed BALB/c-3T3 cells, and tumor cell lines (obtained by injecting nude mice with transformed cell lines) were analyzed for genomic changes. DNAs from 10 different transformed clones and their corresponding tumor cell lines were amplified individually by RAPD analysis using 10 arbitrary primers. DNA from non-transformed BALB/c-3T3 cells was used as a control to compare genetic alterations, if any, between non-transformed, transformed and tumor cell populations. PCR products from RAPD were electrophoretically separated on agarose gels and the banding profiles were visualized by ethidium bromide staining. Five of the 10 primers tested revealed genomic changes in silica-transformed cell lines when compared to non-transformed BALB/c-3T3 cells. Comparison of all 10 transformed and tumor cell lines showed varied degrees of genomic changes using all 10 primers. CdCl2-transformed cell lines displayed fewer genomic changes, only three of 10 primers showed a positive result. CdCl2-transformed cells and their corresponding tumor cell lines showed specific banding pattern differences in six of the 10 samples tested with six of the 10 primers. Changes in band intensity were the most commonly observed changes both in silica- and CdCl2-transformed and tumor cell lines. The results seem to indicate a progressive change in genomic rearrangements which may directly or indirectly be associated with progression of tumorigenesis.

3T3 Cells↗
PubMed1994

Advances in the molecular biology of gestational trophoblastic disease.

Gestational trophoblastic diseases are a heterogeneous pool of clinically and histopathologically defined entities with two clinically relevant features: reproductive failure and a high neoplastic potential. Here we review recent advances in understanding the biology and natural history of the most common form of trophoblastic disease, hydatidiform mole, with an emphasis on the clinical implications for patient management. There are no reliable genetic markers for predicting which subset of moles will behave aggressively, nor are there molecular diagnostic methods at present that offer advances over traditional histopathology. The predominant genetic finding in complete and partial hydatidiform moles is an imbalance of parental chromosomes, completely androgenetic (paternal) in the former and an extra paternal haploid set in the latter. This lack or imbalance of a maternal genomic contribution probably changes the gene expression since there is no evidence of gene mutation in these lesions.

Animals↗
PubMed2004

An efficient Monte Carlo approach to assessing statistical significance in genomic studies.

MOTIVATION: Multiple hypothesis testing is a common problem in genome research, particularly in microarray experiments and genomewide association studies. Failure to account for the effects of multiple comparisons would result in an abundance of false positive results. The Bonferroni correction and Holm's step-down procedure are overly conservative, whereas the permutation test is time-consuming and is restricted to simple problems. RESULTS: We developed an efficient Monte Carlo approach to approximating the joint distribution of the test statistics along the genome. We then used the Monte Carlo distribution to evaluate the commonly used criteria for error control, such as familywise error rates and positive false discovery rates. This approach is applicable to any data structures and test statistics. Applications to simulated and real data demonstrate that the proposed approach provides accurate error control, and can be substantially more powerful than the Bonferroni and Holm methods, especially when the test statistics are highly correlated.

Algorithms↗
PubMed2006

A genome wide linkage search for breast cancer susceptibility genes.

Mutations in known breast cancer susceptibility genes account for a minority of the familial aggregation of the disease. To search for further breast cancer susceptibility genes, we performed a combined analysis of four genome-wide linkage screens, which included a total of 149 multiple case breast cancer families. All families included at least three cases of breast cancer diagnosed below age 60 years, at least one of whom had been tested and found not to carry a BRCA1 or BRCA2 mutation. Evidence for linkage was assessed using parametric linkage analysis, assuming both a dominant and a recessive mode of inheritance, and using nonparametric methods. The highest LOD score obtained in any analysis of the combined data was 1.80 under the dominant model, in a region on chromosome 4 close to marker D4S392. Three further LOD scores over 1 were identified in the parametric analyses and two in the nonparametric analyses. A maximum LOD score of 2.40 was found on chromosome arm 2p in families with four or more cases of breast cancer diagnosed below age 50 years. The number of linkage peaks did not differ from the number expected by chance. These results suggest regions that may harbor novel breast cancer susceptibility genes. They also indicate that no single gene is likely to account for a large fraction of the familial aggregation of breast cancer that is not due to mutations in BRCA1 or BRCA2.

Breast Neoplasms↗
PubMed1999

Application of an ordered subset analysis approach to the genetics of alcoholism.

For complex diseases, underlying etiologic heterogeneity may reduce power to detect linkage. Thus, methods to identify more homogeneous subgroups within a given sample in a linkage study may improve detection of putative susceptibility loci. In this study we describe an ordered subsetting approach that utilizes disease-related quantitative trait data to complement traditional linkage analysis. This approach uses family-based lod scores derived from the initial genome screen and a family-based descriptor of the trait of interest. The goal of the approach is to identify more homogeneous subgroups of the data by ranking families based on their quantitative trait data. Permutation testing is used to assess statistical significance. This approach can be adapted to a variety of linkage methods and may provide a means to dissect some of the underlying heterogeneity in complex disease genetics.

Alcoholism↗
PubMed2006

Foot differentiation and genomic plasticity in Hydra: lessons from the PPOD gene family.

In Hydra, developmental processes are permanently active to maintain a simple body plan consisting of a two-layered, radially symmetrical tube with two differentiated structures, head and foot. Foot formation is a dynamic process and includes terminal differentiation of gastric epithelial cells into mucous secreting basal disc cells. A well-established marker for this highly specialized cell type is a locally expressed peroxidase (Hoffmeister et al. 1985). Based on the foot-specific peroxidase activity, the gene PPOD1 has been identified (Hoffmeister-Ullerich et al. 2002). Unexpectedly, this approach led to the identification of a second gene, PPOD2, with high sequence similarity to PPOD1 but a strikingly different expression pattern. Here, we characterize PPOD2 in more detail and show that both genes, PPOD1 and PPOD2, are members of a gene family with differential complexity and expression patterns in different Hydra species. At the genomic level, differences in gene number and structure within the PPOD gene family, even among closely related species, support a recently proposed phylogeny of the genus Hydra and point to unexpected genomic plasticity within closely related species of this ancient metazoan taxon.

Animals↗
PubMedDate not supplied

Identification of duck plague virus by polymerase chain reaction.

A polymerase chain reaction (PCR) assay was developed for detecting duck plague virus. A 765-bp EcoRI fragment cloned from the genome of the duck plague vaccine (DP-VAC) virus was sequenced for PCR primer development. The fragment sequence was found by GenBank alignment searches to be similar to the 3' ends of an undefined open reading frame and the gene for DNA polymerase protein in other herpesviruses. Three of four primers sets were found to be specific for the DP-VAC virus and 100% (7/7) of field isolates but did not amplify DNA from inclusion body disease of cranes virus. The specificity of one primer set was tested with genome templates from other avian herpesviruses, including those from a golden eagle, bald eagle, great horned owl, snowy owl, peregrine falcon, prairie falcon, pigeon, psittacine, and chicken (infectious laryngotracheitis), but amplicons were not produced. Hence, this PCR test is highly specific for duck plague virus DNA. Two primer sets were able to detect 1 fg of DNA from the duck plague vaccine strain, equivalent to five genome copies. In addition, the ratio of tissue culture infectious doses to genome copies of duck plague vaccine virus from infected duck embryo cells was determined to be 1:100, making the PCR assay 20 times more sensitive than tissue culture for detecting duck plague virus. The speed, sensitivity, and specificity of this PCR provide a greatly improved diagnostic and research tool for studying the epizootiology of duck plague.

Animals↗
PubMed2003

Mapping loci influencing blood pressure in the Framingham pedigrees using model-free LOD score analysis of a quantitative trait.

This paper presents a method of performing model-free LOD-score based linkage analysis on quantitative traits. It is implemented in the QMFLINK program. The method is used to perform a genome screen on the Framingham Heart Study data. A number of markers that show some support for linkage in our study coincide substantially with those implicated in other linkage studies of hypertension. Although the new method needs further testing on additional real and simulated data sets we can already say that it is straightforward to apply and may offer a useful complementary approach to previously available methods for the linkage analysis of quantitative traits.

Adult Children↗
PubMed1993

Simple repetitive (GAA)n loci in the human genome.

In order to investigate the organization (and inheritance) of simple tandem (GAA)n repeats in the human genome, different restriction enzymes were employed for DNA digestion followed by separation of the resulting fragments by agarose gel electrophoresis. Frequently cutting enzymes (4 bp recognition sites) revealed highly complex multilocus banding patterns after conventional horizontal submarine gels. Fragments larger than 25 kb, resulting from digestions with rarely cutting enzymes (6 bp recognition sites), were separated by pulsed-field gel electrophoresis (PFGE). Hybridizations were carried out directly in the gel matrix. Nearly all of the enzymes produce at least one predominant signal band after hybridization with (GAA)6. The frequency of (GAA)n stretches was estimated on human chromosome 4 by probing a respective cosmid library. In comparison with other simple di-, tri- and tetranucleotide repeats, (GAA)n stretches appear underrepresented. Hybridization of a fetal human brain cDNA library indicated very few expressed (GAA)n repeats. These data are discussed with particular reference to genomic organization and other simple repetitive trinucleotide stretches.

Chromosome Mapping↗
PubMed1993

Confidence in evolutionary trees from biological sequence data.

The reliable construction of evolutionary trees from nucleotide sequences often depends on randomization tests such as the bootstrap and PTP (cladistic permutation tail probability) tests. The genomes of bacteria, viruses, animals and plants, however, vary widely in their nucleotide frequencies. Where genomes have independently acquired similar G+C base compositions, signals in the data arise that cause methods of evolutionary tree reconstruction to estimate the wrong tree by grouping together sequences with similar G+C content. Under these conditions randomization tests can lead to both the rejection of the correct evolutionary hypothesis and acceptance of an incorrect hypothesis (such as with the contradictory inferences from the photosynthetic rbcS and rbcL sequences). We have proposed one approach to testing for G+C content problem. Here we present a formalization of this method, a frequency-dependent significance test, which has general application.

Base Sequence↗
PubMed2000

Characterization of a highly glycopeptide-resistant Enterococcus gallinarum isolate.

BACKGROUND AND PURPOSE: Adequate treatment of emergency infection involving antibiotic-resistant bacteria such as vancomycin-resistant Enterococcus requires a convergence of clinical and bacteriologic techniques. An isolate of Enterococcus gallinarum, designated as TSGH63, is known to be uncommonly vancomycin-resistant. This study investigated the genetic determinant for this unique characteristic. METHODS: After completing the conventional identification and sensitivity tests, the genomic content of E. gallinarum TSGH63 was extracted and analyzed by pulse-field electrophoresis. A set of specific primers for vanA, vanB, vanC1, and vanC2/C3 genes was then applied in a multiplex polymerase chain reaction (PCR) to differentiate its genetic content. To locate the determinant for high vancomycin resistance, the electrophoresis profile was further analyzed by Southern blot using the digoxigenin (DIG)-labeled vanA gene probe. Finally, interspecies transfer of the vancomycin-resistance determinant of E. gallinarum TSGH63 was tested by a conjugation experiment in vitro. RESULTS: A 50-kb plasmid was identified in the analysis of the genomic extract of E. gallinarum TSGH63 by pulse field electrophoresis. Using multiplex PCR, we demonstrated that E. gallinarum TSGH63 harbors a vanA gene in addition to a vanC1 gene. The DIG-labeled vanA gene-specific probe bound to the plasmid exclusively on the Southern blot. The plasmid-carried vanA gene, but not the vanC1 gene, was found to be transferable from TSGH63 to E. faecalis JH2-2 by conjugation in vitro. CONCLUSIONS: This is the first report of isolation of E. gallinarum with a high level of resistance to glycopeptides in Taiwan. The demonstrated interspecies transfer of the vancomycin-resistance gene highlights the importance of stringent control of the use of vancomycin.

Conjugation, Genetic↗
PubMed1997

Identification of a sequence within U5 required for human immunodeficiency virus type 1 to stably maintain a primer binding site complementary to tRNA(Met).

Initiation of reverse transcription of human immunodeficiency virus type 1 (HIV-1) occurs by extension from the 3' end of a cellular tRNA complexed to the primer binding site (PBS) located near the 5' end of the viral RNA genome. Although the PBSs for all naturally occurring HIV-1 viruses are complementary to the 3'-terminal 18 nucleotides of tRNA(Lys)3, we identified an HIV-1 virus which contained a PBS complementary to the 3' nucleotides of tRNA(Met); the PBS of this virus was not stable upon extended culture and reverted back to the wild type (S.-M. Kang, J. K. Wakefield, and C. D. Morrow, Virology 222:401-414, 1996). To further characterize the virus with a PBS complementary to tRNA(Met), a DNA fragment encompassing the PBS and U5 region from this proviral genome was substituted for the same region in the infectious HIV-1 proviral clone [named pHXB2(AC-Met)]. Three additional proviral genomes were also created: pHXB2(Met), which is isogenic with pHXB2 except for the PBS complementary to tRNA(Met); pHXB2(Met-AC-Met), which contains the PBS sequence complementary to the 3'-terminal nucleotides and the sequence upstream of this PBS in U5 complementary to the anticodon region of tRNA(Met); and pHXB2(Met-C-Met), which contains two G-to-C changes predicted to disrupt complementarity within the tRNA(Met) anticodon region. Viruses derived from the transfection of these proviral genomes were infectious, although the appearance of the viruses was delayed compared to that of the wild-type virus. PCR amplification and DNA sequence analysis of the PBS regions from proviral genomes revealed that the PBSs from viruses derived from pHXB2(Met) and pHXB2(AC-Met) reverted back to the wild type by days 16 and 44 postcoculture, respectively. Two new, novel mutant viruses were identified among viruses derived from pHXB2(Met-C-Met) at day 35 postcoculture: one contained a PBS complementary to tRNA(Lys)1,2, while the second maintained a PBS complementary to tRNA(Met) but contained a 26-nucleotide deletion in U5 upstream of the anticodon-complementary region. By day 125 postcoculture, the PBS in the virus from this culture had reverted back to the wild type, complementary to tRNA(Lys)3. In contrast, the viruses derived from pHXB2(Met-AC-Met) stably maintained a PBS complementary to tRNA(Met) during the 125-day culture period examined. The results of these studies support the idea that HIV-1 can maintain a PBS complementary to alternative tRNAs provided that the appropriate complementarity exists between the U5-PBS region of the viral RNA genome and the tRNA molecule used to initiate reverse transcription.

Animals↗