The genetic maps for most of the human chromosomes have been generated using restriction fragment length polymorphisms (RFLPs). Of the 5,000 known RFLPs that have been described, there are only 1,500 polymorphisms in coding genes. This represents a small fraction of the 50-100,000 genes that are believed to be encoded in the entire genome. In order to characterize polymorphisms in biologically important human genes, we have employed both standard approaches, as well as new methods to identify genetic markers. These methods include identification of RFLPs by DNA hybridization, characterization of single-stranded conformation polymorphisms (SSCPs) in non-coding portions of genes, and the use of microsatel-lite loci in and near known genes. Polymorphisms have been described in the tryptophan hydroxylase gene (TPH), the gamma 2 subunit of the GABA receptor (GABARG2), a transcription factor gene that regulates apolipoproteins, a tyrosine kinase gene (RLK), transketolase, a serotonin receptor (HTR1A), and the vacuolar H+-ATPase gene. Many of these genetic markers have been typed in the Centre D'Etude du Polymorphisme Humain collection of three generation families. These families are employed in developing a genetic map of the human genome. In addition, we have begun to establish a collection of highly informative markers that can be typed by the polymerase chain reaction. These markers are microsatellites, regions of the genome that contain repetitive sequences 2-5 base pairs in length, that are highly polymorphic. To date, we have synthesized primers for 65 microsatellite loci and have tested them to confirm their polymorphism. These markers are extremely valuable for linkage mapping projects, as well as analysis of allele sharing amongst siblings. One particular marker that we are extensively studying is in the tumor necrosis factor (TNF) gene cluster. The TNF genes are located within the HLA locus and play a crucial role in immune function. We have also established a microsatellite repeat in the CD4 gene. Both the CD4 and TNF polymorphisms are being typed in groups of patients exposed to HIV to look for associations with disease in these individuals.
