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Results for “positional cloning”

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PubMed2003

Positional cloning of Ncf1--a piece in the puzzle of arthritis genetics.

Positional cloning of susceptibility genes in complex diseases like rheumatoid arthritis in humans is hampered by aspects like genetic heterogeneity and environmental variations, while genetic studies in animal models contain several advantages. With animal models, the environment can be controlled, the genetic complexity of the disease is minimized and the disease onset can be predicted, which simplify diagnosis and characterization. We use pristane-induced arthritis in rats to investigate the inheritance of arthritis. Until now, we have identified 15 loci that significantly predispose rats to the development of arthritis. One of these arthritis loci has been isolated and confirmed to be caused by a polymorphism in the Ncf1 gene. In this review, we outline the methods used to identify Ncf1 as one single susceptibility gene in a complex puzzle of inherited factors that render susceptibility to a complex autoimmune disorder like arthritis.

Animals↗

PubMed1987

The effect of long-term marrow culture on the nonadherent Philadelphia chromosome-positive clone in chronic myelogenous leukemia: preliminary observations.

To evaluate the effect of long-term culture on the Philadelphia chromosome-positive nonadherent hemopoietic clone in chronic myelogenous leukemia, bone marrow aspirates from five patients were grown in culture for three to four weeks. In addition to control cultures, samples were incubated with recombinant alpha-interferon. Cytogenetic studies performed on the nonadherent hemopoietic cells demonstrated suppression of the Philadelphia chromosome-positive clones, and appearance of cells with normal diploid karyotype in two of the five patients studied; cells from the remaining three patients showed persistent Philadelphia chromosome-positive cells in 100% of the metaphases. Exposure to alpha-interferon did not enhance the suppression of the Philadelphia chromosome-positive clones. A good correlation was noted between in vitro results and in vivo cytogenetic response: the two patients whose in vitro studies showed diploid clones achieved complete hematologic remission and suppression of Philadelphia chromosome-positive metaphases with therapy. Our data suggest that long-term culture favors the growth of normal clones over Philadelphia chromosome-positive clones in the nonadherent hemopoietic cells of some patients with chronic myelogenous leukemia. The value of the procedure as an in vitro predictive test for residual normal stem cells and response to therapy and for autologous marrow in vitro purging needs further exploration.

Adult↗

PubMed1995

Comparison of the positional cloning methods used to isolate the BRCA1 gene.

A critical step in positional cloning is the identification of candidate genes from a large, genetically defined region. Candidate gene isolation by hybrid selection, genomic sequencing, and direct cDNA library screening identified 45 candidate gene fragments (CGFs) from a 600 kb genomic region that contains the BRCA1 gene. These CGFs define a minimum of 15 genes, six of which are newly localized to the BRCA1 region. We present an analysis of the efficiency and the sequences generated for each of these methods. We also compare our CGF set to those reported for the BRCA1 region by three other groups, revealing a surprising lack of overlap among the sets.

Breast Neoplasms↗

PubMed1995

Introduction to positional cloning.

The most common procedure for isolating genes that predispose to a certain disease is positional cloning. This process is initiated by mapping the responsible gene to its location on a chromosome linked to the disease. Successive cloning of the region results in the isolation of candidate genes. Finally, identification of mutations by mutational analysis points to the correct gene.

Chromosome Mapping↗

PubMedDate not supplied

Positional cloning of disease genes: advantages of genetic isolates.

Genetic isolates with a history of a small founder population, long-lasting isolation and population bottlenecks represent exceptional resources in the identification of disease genes. Specific rare, monogenic diseases become enriched, and families with multiple affected individuals occur frequently enough to be used in linkage analyses for locus identification. Further, the vast majority of cases are caused by the same mutation, and disease alleles reveal linkage disequilibrium (LD) with markers over significant genetic intervals; this facilitates disease locus identification by similarity search for a shared genotype or haplotype in small study samples consisting of few affected individuals. LD observed in disease alleles adds power to linkage analyses and helps to define the exact location of disease loci on the genetic map. Typically, based on the linkage disequilibrium and the ancient haplotype, the critical DNA region can be defined from the original 1- to 2-cM resolution obtained in linkage analysis to 50-200 kb, greatly facilitating the targeting of physical cloning and sequencing efforts. These advantages have been well demonstrated in the positional cloning of several rare monogenic diseases enriched in population isolates like the example of Finland used here. How useful genetic isolates will prove to be in the identification of complex disease genes is dependent on the genealogical history of the isolate, including the size of the founding population and the expansion rate during the history of the population.

Chromosome Mapping↗

PubMed2000

Positional cloning and functional analysis of the gene responsible for Nijmegen breakage syndrome, NBS1.

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, combined immunodeficiency, and a high incidence of lymphoid tumor. Cells from NBS patients show chromosomal instability, hypersensitivity to ionizing radiation and abnormal p53-mediated cell cycle regulation. We cloned the underlying gene for NBS, designated NBS1, by complementation-assisted positional cloning from the candidate region 8q21. Large genomic sequencing, as well as a search using computer programs, provides a powerful approach for identifying the underlying gene for a disease. The NBS1 gene encodes a protein of 754 amino acids that has FHA and BRCT domains which often are conserved in cell-cycle checkpoint proteins. The gene has weak homology to the yeast (Saccharomyces cerevisiae) Xrs2 protein in the N-terminus region. Like yeast Xrs2, the NBS1 protein forms a complex with hRAD50/hMRE11, and the complex is condensed as foci in the nucleus after irradiation, indicative that this triple-complex is a crucial factor in DNA repair. Functional analysis of the NBS1 protein is in progress and it should provide further clues to understanding the repair mechanism of radiation-induced DNA double-strand breaks.

Amino Acid Sequence↗

PubMed2000

Fine mapping of quantitative trait loci using advanced intercross lines of mice and positional cloning of the corresponding genes.

High-resolution mapping of quantitative trait loci (QTLs) is an essential step towards positional cloning and identification of the corresponding genes. Most QTL detection and mapping studies in mice have been carried out using F2 intercross and backcross populations. As a consequence of the limited number of recombination events in small chromosomal regions, this has generally permitted mapping to only relatively large confidence intervals of 20 to 40 cM. A number of population designs have been proposed to increase recombination level in crosses. This includes advanced intercross lines (AIL) described by Darvasi and Soller [Genomics. 1995; 141: 1199-1207]. In this report demonstration of the utility of the AIL approach for fine mapping of QTL, which previously had been mapped with 95% confidence interval to 20 to 40 cM in a F2 intercross, will be presented. The methodological approaches to go from the fine-mapped QTL to the identification of the actual genes and mutations are discussed.

Animals↗

PubMed1993

Neurofibromatosis type 1. Beyond positional cloning.

Recent advances in molecular genetics have enabled researchers to more rapidly identify human disease genes. The identification of these genes by positional cloning has opened the door to a better understanding of such diseases through a more complete appreciation of the molecular biologic processes that underlie them. In this review, the approaches taken to dissect the function of the gene for von Recklinghausen neurofibromatosis are presented. These general approaches involve identification of the protein product, determination of its relation to other known proteins, analysis of its distribution in tissues, within cells, and over development, and dissection of its role in producing the disease phenotype. Last, the insights gained from studying the molecular biology of the neurofibromatosis type 1 gene have direct impact on other biologic processes, such as neoplasia, cellular differentiation, and growth factor-mediated signal transduction as well as potential application to improved treatments for neurofibromatosis type 1 and cancer.

Cloning, Molecular↗

PubMed2006

Positional cloning: complex cardiovascular traits.

Cardiovascular traits represent the quintessential complex trait derived from the confluence of numerous genetic and environmental risk factors. Therefore, positional cloning of cardiovascular genes contributing to the common forms of cardiovascular disease is much more challenging than the mapping and isolation of genes contributing to rare Mendelian phenocopies of cardiovascular disease. Success requires careful and systematic phenotyping, large numbers of families that contain multiple patients, and high-quality genotyping covering for both genome scan and the follow-up case-control association study testing the genes and LD blocks within the linkage peaks.

Cardiovascular Diseases↗

PubMed1996

Positional cloning of a novel potassium channel gene: KVLQT1 mutations cause cardiac arrhythmias.

Genetic factors contribute to the risk of sudden death from cardiac arrhythmias. Here, positional cloning methods establish KVLQT1 as the chromosome 11-linked LQT1 gene responsible for the most common inherited cardiac arrhythmia. KVLQT1 is strongly expressed in the heart and encodes a protein with structural features of a voltage-gated potassium channel. KVLQT1 mutations are present in affected members of 16 arrhythmia families, including one intragenic deletion and ten different missense mutations. These data define KVLQT1 as a novel cardiac potassium channel gene and show that mutations in this gene cause susceptibility to ventricular tachyarrhythmias and sudden death.

Amino Acid Sequence↗

PubMed1992

Isolation of the Arabidopsis ABI3 gene by positional cloning.

Arabidopsis abi3 mutants are altered in various aspects of seed development and germination that reflect a decreased responsiveness to the hormone abscisic acid. The ABI3 gene has been isolated by positional cloning. A detailed restriction fragment length polymorphism (RFLP) map of the abi3 region was constructed. An RFLP marker closely linked to the abi3 locus was identified, and by analyzing an overlapping set of cosmid clones containing this marker, the abi3 locus was localized within a 35-kb region. An 11-kb subfragment was then shown to complement the mutant phenotype in transgenic plants, thereby further delimiting the position of the locus. A candidate ABI3 gene was identified within this fragment as being expressed in developing fruits. The primary structure of the encoded protein was deduced from sequence analysis of a corresponding cDNA clone. In the most severe abi3-4 allele, the size of this predicted protein was reduced by 40% due to the presence of a point mutation that introduced a premature stop codon. The predicted ABI3 protein displays discrete regions of high similarity to the maize viviparous-1 protein.

Amino Acid Sequence↗

PubMed2001

Positional cloning of a novel Fanconi anemia gene, FANCD2.

Fanconi anemia (FA) is a genetic disease with birth defects, bone marrow failure, and cancer susceptibility. To date, genes for five of the seven known complementation groups have been cloned. Complementation group D is heterogeneous, consisting of two distinct genes, FANCD1 and FANCD2. Here we report the positional cloning of FANCD2. The gene consists of 44 exons, encodes a novel 1451 amino acid nuclear protein, and has two protein isoforms. Similar to other FA proteins, the FANCD2 protein has no known functional domains, but unlike other known FA genes, FANCD2 is highly conserved in A. thaliana, C. elegans, and Drosophila. Retroviral transduction of the cloned FANCD2 cDNA into FA-D2 cells resulted in functional complementation of MMC sensitivity.

Alleles↗

PubMed1995

Positional cloning and sequence analysis of the Drosophila clock gene, timeless.

The Drosophila genes timeless (tim) and period (per) interact, and both are required for production of circadian rhythms. Here the positional cloning and sequencing of tim are reported. The tim gene encodes a previously uncharacterized protein of 1389 amino acids, and possibly another protein of 1122 amino acids. The arrhythmic mutation tim01 is a 64-base pair deletion that truncates TIM to 749 amino acids. Absence of sequence similarity to the PER dimerization motif (PAS) indicates that direct interaction between PER and TIM would require a heterotypic protein association.

Amino Acid Sequence↗

PubMed2005

Family studies and positional cloning of genes for asthma and related phenotypes.

Although it is not yet known how many genes may contribute to the susceptibility or the severity of asthma and related phenotypes, genome-wide screens and positional cloning techniques have been successful in identifying contributing genes in multiple populations. The results of these studies provide additional insight into the molecular mechanisms responsible for the development of a variety of phenotypes. Replication with additional populations--particularly in large-scale studies--has been used to distinguish between false positive results or population-specific effects or to further quantify the conferred risk. Even when individual markers do not replicate in multiple population, association of the same region or gene has been useful in directing future studies. As further understanding of linkage disequilibrium patterns within the genome has allowed greater efficiency for genetic studies, advances in high-throughput genotyping technology, genetic analysis methodologies, and a more in-depth understanding of clinical phenotypes has made genome-wide studies more accessible and cost-effective. In the future, identification of function variants with clinical relevance may be used to influence the diagnosis and treatment of asthma.

Asthma↗

PubMed2004

Visualization of the S-locus region in Ipomoea trifida: toward positional cloning of self-incompatibility genes.

Self-incompatibility (SI) in Ipomoea trifida is regulated by a single S locus with multiple alleles. Identification of SI genes in the S -locus region by positional cloning is one of the most important goals for understanding sexual reproduction in this species. Despite our intensive efforts to construct bacterial artificial chromosome (BAC) contigs covering the S -locus region, a gap was observed in the core region of the potential S locus. In order to confirm the physical linkage of two non-overlapping BAC contigs in the S -locus region and to determine the size of the gap between them, fluorescence in-situ hybridization (FISH) was performed on mitotic chromosomes and extended DNA fibres using previously isolated S -linked BAC clones as probes. The information obtained from this work would be useful for molecular cloning of the SI genes by a chromosome walking approach. In addition, we showed that strong suppression of recombination in the S locus was not related to the centromere because the S locus was mapped to one end of a chromosome.

Chromosomes, Artificial, Bacterial↗

PubMed2002

Positional cloning identifies a novel cyclophilin as a candidate amplified oncogene in 1q21.

Gains of 1q21-q23 have been associated with metastasis and chemotherapy response, particularly in bladder cancer, hepatocellular carcinomas and sarcomas. By positional cloning of amplified genes by yeast artificial chromosome-mediated cDNA capture using magnetic beads, we have identified three candidate genes (COAS1, -2 and -3) in the amplified region in sarcomas. COAS1 and -2 showed higher amplification levels than COAS3. Most notably, amplification was very common in osteosarcomas, where in particular COAS2 was highly expressed. COAS1 has multiple repeats and shows no homology to previously described genes, whereas COAS2 is a novel member of the cyclosporin-binding peptidyl-prolyl isomerase family, very similar to cyclophilin A. COAS2 was overexpressed almost exclusively in aggressive metastatic or chemotherapy resistant tumours. Although COAS2 was generally more amplified than COAS1, it was not expressed in well-differentiated liposarcomas, where amplification of this region is very common. All three genes were found to be amplified and over-expressed also in breast carcinomas. The complex nature of the 1q21-23 amplicons and close proximity of the genes make unequivocal determination of the gene responsible difficult. Quite likely, the different genes may give selective advantages to different subsets of tumours.

Amino Acid Sequence↗

PubMed2002

Positional cloning of rice semidwarfing gene, sd-1: rice "green revolution gene" encodes a mutant enzyme involved in gibberellin synthesis.

A rice semidwarfing gene, sd-1, known as the "green revolution gene," was isolated by positional cloning and revealed to encode gibberellin 20-oxidase, the key enzyme in the gibberellin biosynthesis pathway. Analysis of 3477 segregants using several PCR-based marker technologies, including cleaved amplified polymorphic sequence, derived-CAPS, and single nucleotide polymorphisms revealed 1 ORF in a 6-kb candidate interval. Normal-type rice cultivars have an identical sequence in this region, consisting of 3 exons (558, 318, and 291 bp) and 2 introns (105 and 1471 bp). Dee-Geo-Woo-Gen-type sd-1 mutants have a 383-bp deletion from the genome (278-bp deletion from the expressed sequence), from the middle of exon 1 to upstream of exon 2, including a 105-bp intron, resulting in a frame-shift that produces a termination codon after the deletion site. The radiation-induced sd-1 mutant Calrose 76 has a 1-bp substitution in exon 2, causing an amino acid substitution (Leu [CTC] to Phe [TTC]). Expression analysis suggests the existence of at least one more locus of gibberellin 20-oxidase which may prevent severe dwarfism from developing in sd-1 mutants.

Amino Acid Sequence↗

PubMed1994

[From positional cloning to gene identification and its function in genetic diseases].

The huge development of genetic tools, together with the availability of powerful computers have enabled the mapping of several loci responsible for genetic diseases by the positional cloning technique, also called reverse genetics. The step consisting of identifying the gene itself remains a difficult step and the function of the gene identified may remain difficult to establish. In this chapter, we will examine the various steps allowing the identification of the gene, techniques allowing the creation of animal models of human diseases and the recent progresses of these techniques.

Animals↗