Isolation Of Disease Genes Research Articles

CIS--cloning of identical sequences between two complex genomes.

Development of the methods permitting cloning of identical sequences between two sources of DNA can be very useful for many purposes, including isolation of disease genes. Here we describe a new method called CIS (cloning of identical sequences). A combination of digestion with MvnI, treatment with mung bean nuclease, UDG (uracil-DNA glycosylase) and PCR with 5'-methyl-dCTP and dUTP was used to isolate identical sequences between two micro-cell hybrid lines (MCH). In a control experiment, mouse MCH903.1 and MCH939.2 containing human chromosome 3 from different individuals, were compared using the CIS procedure. Only background fluorescence in-situ hybridization (FISH) was achieved. In another experiment, mouse MCH903.1, containing complete human chromosome 3, and rat MCH429.11, containing a part of human 3q from the same chromosome were compared. The experiment showed that the original MCH429.11 and the DNA purified using the CIS procedure had identical FISH patterns to human metaphase chromosomes, thus demonstrating the efficiency of CIS.

Construction of a 2.5-Mb Integrated Physical and Gene Map of Distal 21q22.3

The gene-rich telomeric region of 21q harbors several loci relevant to human diseases including autoimmune polyglandular disease type I, nonsyndromic deafness, Knobloch syndrome, holoprosencephaly, and bipolar affective disorder. A contig of genomic clones in this region would facilitate the isolation of these genes. However, distal 21q22.3 has yet been poorly mapped, presumably due to the presence of sequences that are underrepresented in yeast artificial chromosome (YAC) libraries. We generated a framework of YACs and used these clones as starting points for the isolation of a combination of bacterial artificial chromosome clones, P1-derived artificial chromosome clones, and cosmid clones by chromosome walking procedures. These studies resulted in the construction of a high-resolution contig map spanning the 2.5-Mb region from PFKL to the telomere, ∼2 Mb of which are covered by ready-to-sequence contigs. Within this map we determined the location and relative distance of 21 markers. These include 9 established genetic markers, the order of which is cen–PFKL–D21S154–D21S170– D21S171–D21S1903–D21S1897–D21S112–D21S1446–D21S1575–tel. Moreover, we established the precise map position of 13 genes and 4 ESTs including the recently isolated genes C21ORF2, SMT3H1, RNA editing deaminase 1 (ADARB1), folate transporter (SLC19A1), COL18A1, lanosterol synthase (LSS-PEN), pericentrin (PCNT), and arginine methyltransferase (HRMT1L1). This integrated map provides a useful resource for the mapping and isolation of disease genes and for the construction of a complete transcription map of distal 21q as well as for large-scale sequencing efforts.

Integrated Map of the Chromosome 8p12–p21 Region, a Region Involved in Human Cancers and Werner Syndrome

Detailed physical maps of the human genome are important resources for the identification and isolation of disease genes and for studying the structure and function of the genome. To improve the definition of the 8p12–p21 chromosomal region, an integrated physical and genetic map was constructed extending from the genesNEFLtoFGFR1.The map comprises a series of contigs (the larger of these being around 9 Mb) of yeast artificial chromosomes (YACs) spanning the proximal region of deletion involved in a broad range of human cancers, including breast carcinomas, and in the Werner syndrome. In addition, losses of heterozygosity at 8p markers and linkage analysis of breast cancer families were also detailed. Finally, several genes potentially involved in 8p-associated diseases, namelyGTF2E2, PPP2CB,andHGL,were precisely mapped within the YAC contigs. The reported map and contigs of YACs should facilitate the search for putative genes involved in sporadic and familial breast cancer as well as in the Werner syndrome.

Integration of physical, breakpoint and genetic maps of chromosome 22. Localization of 587 yeast artificial chromosomes with 238 mapped markers.

Detailed physical maps of the human genome are important resources for the identification and isolation of disease genes and for studying the structure and function of the genome. We used data from STS content mapping of YACs and natural and induced chromosomal breakpoints to anchor contigs of overlapping yeast artificial chromosome (YAC) clones spanning extensive regions of human chromosome 22. The STSs were assigned to specific regions (bins) on the chromosome using cell lines from a somatic hybrid mapping panel defining a maximum of 25 intervals. YAC libraries were screened by PCR amplification of hierarchical pools of yeast DNA with 238 markers, and a total of 587 YAC clones were identified. These YACs were assembled into contigs based upon their shared STS content using a simulated annealing algorithm. Fifteen contigs, containing between 2 and 74 STSs were assembled, and ordered along the chromosome based upon the cytogenetic breakpoint, meiotic and PFG maps. Additional singleton YACs were assigned to unique chromosomal bins. These ordered YAC contigs will be useful for identifying disease genes and chromosomal breakpoints by positional cloning and will provide the foundation for higher resolution physical maps for large scale sequencing of the chromosome.

The isolation of disease genes by positional cloning.

The isolation of disease genes by positional cloning.

Assignment of Heregulin (HGL) to Human Chromosome 8p22-p11 by PCR Analysis of Somatic Cell Hybrid DNA

Detailed physical maps of the human genome are important resources for the identification and isolation of disease genes and for studying the structure and function of the genome. To improve the definition of the 8p12–p21 chromosomal region, an integrated physical and genetic map was constructed extending from the genesNEFLtoFGFR1.The map comprises a series of contigs (the larger of these being around 9 Mb) of yeast artificial chromosomes (YACs) spanning the proximal region of deletion involved in a broad range of human cancers, including breast carcinomas, and in the Werner syndrome. In addition, losses of heterozygosity at 8p markers and linkage analysis of breast cancer families were also detailed. Finally, several genes potentially involved in 8p-associated diseases, namelyGTF2E2, PPP2CB,andHGL,were precisely mapped within the YAC contigs. The reported map and contigs of YACs should facilitate the search for putative genes involved in sporadic and familial breast cancer as well as in the Werner syndrome.

Isolation and characterization of a yeast artificial chromosome (YAC) contig around the human steroid sulfatase gene

The region surrounding the steroid sulfatase (STS) locus on Xp22.3 is of particular interest since it represents a deletion hot spot, shares homology with the proximal long arm of the Y chromosome (Yq11.2), and contains genes for several well-described X-linked disorders. Here we describe yeast artificial chromosomes (YACs) covering 450 kb around the STS gene. Eight YAC clones were isolated from a human YAC library. Their STS exon content was determined and the overlap of the clones characterized. Two of the YAC clones were found to contain the entire STS gene. The most proximal and the most distal ends of the YAC contig were cloned but neither of them crossed the breakpoints in any of the previously described patients with entire STS gene deletions. This is consistent with deletions larger than 500 kb in all these patients. One of the YAC clones was found to contain sequences from the STS pseudogene on Yq11.2. Two anonymous DNA sequences, GMGXY19 and GMGXY3, previously mapped in the vicinity of the STS locus, were found within the YAC contig and their assignment with respect to the STS locus was thus possible. This contig is useful for the overlap cloning of the Xp22.3 region and for reverse genetic strategies for the isolation of disease genes in the region. Furthermore, it may provide insight into the molecular mechanisms of deletion and translocation events on Xp22.3 and in the evolution of sex chromosomes.

The molecular basis of genetic disease.

The pace of localization and characterization of genes affected in human genetic disorders is quickening. Many important genes were localized or characterized recently: genes for in cystic fibrosis, NF-2, Marfan's syndrome and xeroderma pigmentosum, to name a few. Also, in the past 15 months, the CFTR gene affected in cystic fibrosis has been isolated, the first disease gene to be isolated without use of previous cytogenetic clues, such as deletions or translocations in sporadic cases. Other examples should follow, although we have been disappointed to date by the difficulties encountered in the isolation of Huntington's disease gene which was localized a number of years ago to distal chromosome 4p. It is still very difficult to isolate a disease gene without critical cytogenetic information. New improved techniques for finding the desired expressed sequences in a large cloned segment of human DNA are needed. Our ability to find mutant alleles of a given sequence has expanded greatly with the recent technical advances in denaturing gradient gel electrophoresis, chemical cleavage, and single-stranded conformational electrophoresis. One would predict that information derived from the human genome project will have a major impact upon the isolation of further disease genes. As whole regions of human chromosomes or indeed entire chromosomes are physically mapped and cloned as continuous, overlapping YACs (yeast artificial chromosomes), isolation of disease genes will become easier and easier.(ABSTRACT TRUNCATED AT 250 WORDS)

Til 1--a human lymphoblastoid cell line with minimal DNA methylation.

Methylation has been shown to be correlated with several fundamental cellular processes, including changes in gene expression, alterations in chromatin structure and inactivation of the mammalian X chromosome. It is possible, therefore, that the methylation status of a particular sequence may reflect involvement in a number of processes. Given this potentially confused situation, it is clear that many studies would be facilitated if unmethylated or minimally methylated DNA from a mammalian source were available. A major use of such DNA would be in the construction of long-range physical maps. In many cases, long-range physical maps are a prerequisite for the eventual isolation of disease genes that have been localised to a particular chromosomal region by other means (e.g. genetic linkage studies). Many of the enzymes used in such long-range mapping experiments are methylation sensitive, which makes it difficult to determine how many sites for a particular enzyme are present in any DNA sequence. Here we report the finding of a minimally methylated DNA in the human lymphoblastoid cell line, Til 1. The methylation level of Til 1 DNA was analysed in several ways and compared with that in human lymphocytes, placental tissue and other lymphoblastoid cell lines. The results showed clear and reproducible differences in methylation among the cell types, both at a global level and in the vicinities of specific DNA sequences. Lymphocyte DNA had the highest level of methylation while placental DNA and cell line DNA had lower but similar levels. Til 1 had abnormally low levels of 5-methylcytosine when measured directly, and no detectable methylation at any of the restriction sites examined.