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At least 523 records · Page 29Linked to original sources

Functional genomic approaches using the nematode Caenorhabditis elegans as a model system.

Since the completion of the genome project of the nematode C. elegans in 1998, functional genomic approaches have been applied to elucidate the gene and protein networks in this model organism. The recent completion of the whole genome of C. briggsae, a close sister species of C. elegans, now makes it possible to employ the comparative genomic approaches for identifying regulatory mechanisms that are conserved in these species and to make more precise annotation of the predicted genes. RNA interference (RNAi) screenings in C. elegans have been performed to screen the whole genome for the genes whose mutations give rise to specific phenotypes of interest. RNAi screens can also be used to identify genes that act genetically together with a gene of interest. Microarray experiments have been very useful in identifying genes that exhibit co-regulated expression profiles in given genetic or environmental conditions. Proteomic approaches also can be applied to the nematode, just as in other species whose genomes are known. With all these functional genomic tools, genetics will still remain an important tool for gene function studies in the post genome era. New breakthroughs in C. elegans biology, such as establishing a feasible gene knockout method, immortalized cell lines, or identifying viruses that can be used as vectors for introducing exogenous gene constructs into the worms, will augment the usage of this small organism for genome-wide biology.

Animals↗

An update on the genetics of systemic lupus erythematosus.

The development of systemic lupus erythematosus has a strong genetic basis. The techniques to study the genetics of SLE have improved, and family-based studies have been gaining importance due to advances in the human genome project. Recently, complete genome scans with microsatellite markers and linkage analysis have been performed in human systemic lupus erythematosus in various populations. These studies represent the first step of a process towards the identification of susceptibility genes in SLE. We review here the results of the human genome scans performed until now and provide an update of the latest advances on the genetics of SLE in linkage studies and association analyses of candidate genes.

Female↗

Screening the genome to detect an association with hypertension.

We report tree-based association analysis as applied to the two Framingham cohorts and to the first replication of the simulated data obtained from the Genetic Analysis Workshop 13. For this analysis, familial association is ignored. The two endpoints examined are hypertension status at initial visit and time-to-hypertension, using a censored data approach. Although linkage association has previously been reported with hypertension, we found no association using the tree-based methodology.

Adult↗

Comparison of sib-pair and variance-components methods for genomic screening.

The statistical properties of sib-pair and variance-components linkage methods were compared using the nuclear family data from Problem 2. Overall, the power to detect linkage was not high for either method. The variance-components method had better power for detection of linkage, particularly when covariates were included in the model. Type I error rates were similar to nominal error rates for both methods.

Analysis of Variance↗

A genome wide linkage disequilibrium screen in Parkinson's disease.

Whole genome screening is increasingly used to identify genetic risk factors for complex diseases. In this study, a genome wide linkage disequilibrium (LD) screen was performed in a cohort of Parkinson's disease (PD) patients from the UK (n = 195) using pooled DNA to facilitate efficient genotyping of 5546 microsatellite markers. Allele frequencies were compared with those found in 2 previously typed disease free control populations, and the most interesting markers were selected for multiple repeat testing among the 3 pools. Markers were then individually genotyped in our original PD cohort and one of the original control groups, and independently in a second cohort of UK PD patients (n = 179), and additional controls. Using this 2-stage approach, we have been unable to find evidence for consistent association of any markers with sporadic PD. Subgroup analysis of the most promising marker shows some evidence that microsatellite marker D1S2886 is associated with familial forms of the disease.

Adult↗

Dynamics of mutation and recombination in a replicating population of complementing, defective viral genomes.

In a previous study, we documented that serial passage of a biological clone of foot-and-mouth disease virus (FMDV) at high multiplicity of infection (moi) in cell culture resulted in viral populations dominated by defective genomes that included internal in-frame deletions, affecting the L and capsid-coding regions, and were infectious by complementation. In the present study, analyses of the defective genomes present in individual viral plaques, and of consensus nucleotide sequences determined for the entire genomes of sequential samples, have revealed a continuous dynamics of mutation and recombination. At some points of high genetic instability, multiple minority genomes with different internal deletions co-existed in the population. At later passages, a new defective RNA arose and displaced a related, previously dominant RNA. Nucleotide sequences of the different genomic forms found in sequential isolates have revealed an accumulation of mutations at an average rate of 0.12 substitutions per genome per passage. At the regions around the deletion sites, substantial, minor or no nucleotide sequence identity is found, suggesting relaxed sequence requirements for the occurrence of internal deletions. Competition experiments indicate a selective advantage of late phase defective genomes over their precursor forms. The defective genome-based FMDV retained an expansion of host cell tropism, undergone by the standard virus at a previous stage of the same evolutionary lineage. Thus, despite a complex dynamics of mutation and recombination, and phases of high genetic instability, a biologically relevant phenotypic trait was stably maintained after the evolutionary transition towards a primitive genome segmentation. The results extend the concept of a complex spectrum of mutant genomes to a complex spectrum of defective genomes in some evolutionary transitions of RNA viruses.

Animals↗

Rat model of familial combined hyperlipidemia as a result of comparative mapping.

Total genome scan was carried out in 266 F2 intercrosses from the Prague hypertriglyceridemic (HTG) rat that shares several clinical characteristics with human metabolic syndrome. Two loci for plasma triglycerides (TG) were localized on chromosome 2 (Chr 2) (LOD 4.4, 3.2). The first locus overlapped with the rat syntenic region of the human locus for the metabolic syndrome and for small, dense LDL, while the second overlapped with the syntenic region of another locus for small, dense LDL in humans by the comparative mapping approach. Loci for TG on rat Chr 13 (LOD 3.3) and Chr 1 (LOD 2.7) overlapped with the syntenic region of loci for human familial combined hyperlipidemia (FCHL) in Finnish and Dutch populations, respectively. The concordances of loci for TG localized in this study with previously reported loci for FCHL and its related phenotypes are underlying the generalized importance of these loci in dyslipidemia. These data suggest the close relationship between dyslipidemia in HTG rats and human FCHL, establishing a novel animal model for exploration of pathophysiology and therapy based on genomic determinants.

Animals↗

Evidence for multiple hybrid groups in Trypanosoma cruzi.

A role for parasite genetic variability in the spectrum of Chagas disease is emerging but not yet evident, in part due to an incomplete understanding of the population structure of Trypanosoma cruzi. To investigate further the observed genotypic variation at the sequence and chromosomal levels in strains of standard and field-isolated T. cruzi we have undertaken a comparative analysis of 10 regions of the genome from two isolates representing T. cruzi I (Dm28c and Silvio X10) and two from T. cruzi II (CL Brener and Esmeraldo). Amplified regions contained intergenic (non-coding) sequences from tandemly repeated genes. Multiple nucleotide polymorphisms correlated with the T. cruzi I/T. cruzi II classification. Two intergenic regions had useful polymorphisms for the design of classification probes to test on genomic DNA from other known isolates. Two adjacent nucleotide polymorphisms in HSP 60 correlated with the T. cruzi I and T. cruzi II distinction. 1F8 nucleotide polymorphisms revealed multiple subdivisions of T. cruzi II: subgroups IIa and IIc displayed the T. cruzi I pattern; subgroups IId and IIe possessed both the I and II patterns. Furthermore, isolates from subgroups IId and IIe contained the 1F8 polymorphic markers on different chromosome bands supporting a genetic exchange event that resulted in chromosomes V and IX of T. cruzi strain CL Brener. Based on these analyses, T. cruzi I and subgroup IIb appear to be pure lines, while subgroups IIa/IIc and IId/IIe are hybrid lines. These data demonstrate for the first time that IIa/IIc are hybrid, consistent with the hypothesis that genetic recombination has occurred more than once within the T. cruzi lines.

Animals↗

Using PAC nested deletions to order contigs and microsatellite markers at the high repetitive sequence containing Npr3 gene locus.

Highly polymorphic di- and tetranucleotide repeats in and around Npr3, a potential candidate gene for hypertension, have been identified using a novel approach. Because this chromosomal site is rich in repetitive DNA and difficult to sequence, P1 artificial chromosomes were retrofitted with a loxP transposon to map the gene sequence within a clone using a series of nested deletions. Sequences from ends of deletions 1-3 kb apart identified a (CA)(20) and a (TA)(18)-(CA)(8) repeat 8 kb upstream and within an intron of Npr3, respectively. DNA from 17 individuals was analyzed for length polymorphisms in these and eight additional repeats identified in 200 kb of working draft sequence from this region in GenBank. The sequence contigs and microsatellite repeats from GenBank were ordered using the P1-derived artificial chromosome deletion series. Several of these repeats were found to vary considerably in length in the set of genomic DNA tested. Since this site in chromosome 5p has recently been implicated in disease in studies with genetically hypertensive rats, the microsatellite markers reported here will be useful for genetic analysis and may even be implicated in the disease process in humans. We discuss how these types of data are useful for interpreting draft DNA sequence coming out of the genome projects, and the utility of deletion clones as a resource for ordering contigs and gap filling.

Alleles↗

False positive rates in a genomic screen for complex quantitative traits.

We conducted a genomic screen for genes associated with Q1, Q2, and Q3 in 239 nuclear pedigrees from replicate 115, Problem Set 2A. We compared false positive (FP) and true positive (TP) rates for three significance levels and two map densities. Using the 2 cM genetic map and alpha = 0.05 produced the most FP but detected the greatest number of major genes. Following up only 31 plateaus (two or more adjacent markers with significant results) from the 2 cM screen eliminated some FP, but failed to detect MG3 for Q3. Multipoint analysis reduced the number of priority regions from 31 to seven; only two of these regions were TP. Replication of the two-point analysis of plateau markers in replicate 80 detected all of the genes associated with Q1 and Q2, but not Q3. Multipoint analysis in replicate 80 failed to replicate any genes associated with Q1, Q2, or Q3, but "replicated" two FP regions. While FP may be reduced by decreasing map density, considering only plateaus for follow up and decreasing significance levels, such adjustments may also fail to detect weak TP. Multipoint analysis and replication in independent data sets may not be reliable methods of distinguishing FP from TP.

Chromosome Mapping↗

A method for meta-analysis of genome searches: application to simulated data.

Genome searches have been performed for many complex traits, and in some cases several searches have been performed in a single disease. Replication of significant results is rare, and a systematic method of reviewing results from a number of searches is needed. A method for meta-analysis is presented which provides a systematic descriptive overview of the separate analyses while dealing with some of the problems specific to meta-analysis of genome searches. The results of two separate meta-analyses correctly indicate the presence of susceptibility loci on chromosomes 1, 3, and 5 in the GAW11 Problem 2 data.

Genetic Linkage↗

Screening candidate genes for mutations in patients with hypogonadotropic hypogonadism using custom genome resequencing microarrays.

OBJECTIVE: The purpose of this study was to determine the consistency of calling single nucleotide polymorphisms (SNPs) by custom genome resequencing microarrays compared with capillary DNA sequencing. STUDY DESIGN: Amplified genomic DNA from 23 patients with hypogonadotropic hypogonadism was hybridized to microarrays containing 30 kilobases of sequence from 6 different candidate genes. Capillary DNA sequencing was performed in 10 patients. RESULTS: For 10 patients with > or =90% of bases called, 49 SNPs in 5 of 6 genes were identified. Of the 490 bases, 75 were ambiguous (read as "N"), and 415 were able to be called an A, C, G, or T. Of 415 called, 401 (96.6%) sequences were confirmed by DNA sequencing. All homozygotes (285/285) were called identically, while sequence from 89.2% (116/130) of heterozygotes agreed by both methods. The level of agreement between microarray calls and capillary DNA sequencing demonstrated substantial accuracy. CONCLUSION: Custom genome resequencing microarrays are highly consistent with capillary sequencing in calling individual bases in genomic DNA from patients with human disease.

Adolescent↗

[Study of a comparative statistical analytical model for nucleotide sequences in the genome of viruses of the Papovaviridae family].

In the present study a model for comparative analysis of nucleotide sequences has been developed, in order to evaluate statistical features of nucleotide distribution in DNA strands without any genetic relationship. Every DNA strand has been considered as a finite Markov chain; a matrix, whose elements represent the number of couplings between a nucleotide and the following one in 5'-3' direction, has been used for every DNA strand, and the statistical relationship has been detected by using Kendall's test. The genomes of Polyomavirus (strain A2) and DPV have been analysed by the proposed model; a substantial likeness between the behaviour of nucleotide distribution on all four DNA strands analysed has been shown; the strongest likeness concerned the complementary strands of Polyomavirus as well as the homologous sense strands of both viruses.

Base Sequence↗

Empirical evaluation of genome scans for linkage of a quantitative trait associated with a complex disorder.

Nonparametric sib-pair analysis (Haseman-Elston) was used to search for evidence of linkage between a putative locus for a complex quantitative trait Q1 and genome-wide markers (367 markers from 10 chromosomes) for the first 100 replicates of nuclear family data. The characteristics of the statistically positive linkage results [the magnitude of p-values (p), the number of supporting flanking markers, and the percentage of positive replicates] were compared for true linkage (major and minor genes) and false positive evidence for linkage. Discriminant analysis was used to evaluate which characteristics of these statistically positive linkage results are good indicators to discriminate true linkage from false positive evidence for linkage. Sensitivity and false positive rates of several proposed criteria for linkage, as well as the criteria based on our results were evaluated. The relationship between the map location of the marker with the lowest p-value and the map location of the true underlying gene was also evaluated, which provided useful information for fine mapping and replication studies.

Age Distribution↗

Cloning of a sapB homologue (sapB2) encoding a putative 112-kDa Campylobacter fetus S-layer protein and its use for identification and molecular genotyping.

A sap gene encoding a surface layer protein was isolated from a Campylobacter fetus ssp. fetus CIP 53.96T cosmid library. This sap gene, which shows significant homology with the sapB conserved region, was named sapB2. The complete ORF of 3339 nucleotides encodes a 1112-amino acid polypeptide with a calculated molecular mass of 112 kDa. High homology with the sapB gene was found in a region beginning 67 bp before the ORF and proceeding 546 bp into the ORF. Similarly, 98% homology with the sapA2 gene was observed in a 2038-bp region beginning 540 bp after the initiation codon. In the present study, we show that this sapB2 gene has two main interesting features: the 5' end of the region which presents high homology with the sapA2 homologue was found to be present in every C. fetus strain, and the fragment (IG01) comprising the region which presents homology with the sapB conserved region and the 5' end of the sapA2 homologue region, when used as a probe, can reveal genomic polymorphism among C. fetus strains. We exploited these features to develop a PCR assay for the specific detection of C. fetus and to set up a method for typing C. fetus isolates. The PCR assay was found to be species-specific. Oligonucleotide primers derived from the 5' end of sapA2 homologue region were used in a polymerase chain reaction test on genomic DNA extracted from 101 Campylobacter fetus, 18 Campylobacter non-fetus and seven non-Campylobacter strains. A 220-bp fragment was amplified only when C. fetus DNA was used as a target. In Southern blot analysis, the IG01 probe was found to hybridize only with DNA extracted from C. fetus strains. Moreover, IG01 hybridized with several fragments of HindIII-digested DNA, giving a specific pattern for each strain.

Amino Acid Sequence↗

The $1000 genome: ethical and legal issues in whole genome sequencing of individuals.

Progress in gene sequencing could make rapid whole genome sequencing of individuals affordable to millions of persons and useful for many purposes in a future era of genomic medicine. Using the idea of $1000 genome as a focus, this article reviews the main technical, ethical, and legal issues that must be resolved to make mass genotyping of individuals cost-effective and ethically effective. It presents the case for individual ownership of a person's genome and its formation, and shows the implications of that position for rights to informed consent and privacy over sequencing, testing, and disclosing genomic information about identifiable individuals. Legal recognition of a person's right to control his or her genome and the information that it contains is essential for further progress in applying genomic discoveries to human lives.

Adult↗