Mapping Of Candidate Disease Genes

The Human Genome Project was initiated in the United States on October 1,1990, with the goal of elucidating the genetic architecture of the human genome. In February 2001, a working draft of the human genome was published (6), and fine sequencing of the genome continues. The sequencing of the human genome impacts greatly on the study of human disease. The identification, isolation, and characterization of the genes involved in normal biological processes as well as disease states should greatly increase as a result of a known gene sequence. The completion of the HGP will result in the accumulation of vast amounts of sequencing data to be used for the discovery of fundamental biological processes, isolation of candidate disease genes, and the diagnosis, treatment and prevention of human disease.

The classical approaches to identifying genes involved in human disease include functional and positional cloning. In functional cloning, the disease gene is usually isolated only after the underlying physiological defect has been elucidated. The study of the gene function precedes gene identification and chromosomal mapping. Examples of disease genes isolated through functional cloning include phenylalanine hydroxylase in phenylketonuria (8) and the P-hemoglobin gene in P-thalassemia (9). In positional cloning, gene mapping precedes the identification of the disease gene (10). This approach is classically used when there is little or no understanding about the function of a defective gene. Generally, blood samples from families in which the genetic disorder is segregating are collected. Genetic mapping then limits the region of the genome to a size that allows molecular cloning of the interval, analysis of encoded genes, and identification of mutations. Finally, functional analysis can be applied to the gene. The mutated gene that gives rise to Duchenne muscular dystrophy was one of the many disease genes isolated via its chromosomal location by positional cloning (11).

Our laboratory employs the products of the HGP and the technique of positional cloning to identify human liver tumor suppressor genes. The 11p11.2-p12 chromosomal region was demonstrated to harbor a candidate disease gene [in hepatocellular carcinoma (HCC)], not through pedigree analysis, as in classical positional cloning, but through the use of human/rat microcell hybrid cell lines. Analysis of chromosome 11 microsatellite markers indicated that the locus conferring tumor suppression in our model system was located on human 11p11.2-p12. More refined mapping was performed using EST markers that were in the candidate region (12). All ESTs and genes identified within the delimited region of DNA (11p11.2-p12) became candidates for the disease (HCC). Through deletion mapping of suppressed MCH cell lines in our laboratory with EST markers, we were able to identify candidate liver tumor suppressor genes. EST sequence can then be compared to other sequence data for identification of homology to any other DNA sequence. The gene corresponding to the EST can then be identified through further mapping and sequence analysis. Characterization of candidate genes is then necessary to demonstrate that the disease is associated with a mutation in a particular candidate gene.

The HGP is evolving from mapped chromosomes to the determination of complete DNA sequence through the incorporation of partial genomic sequence tracts into the underlying architecture of previously mapped regions. Although the HGP has nearly finished the goal of completely sequencing the human genome, there are still gaps and long tracts of unfinished sequencing data. The influx of new sequence information is dramatic; the position of genes and markers in the draft sequence changes daily. It is important to note, however, that until this information is complete, researchers need to incorporate the new sequence data with mapped data they have generated in their own laboratories.

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