There are three overall goals for this proposal. First, although a large number of mutant alleles which produce reduced expression of the beta-globin gene have been characterized, a number of suspected new and uncharacterized alleles still require analysis. Those already available in our lab comprise 8-10 in number and are from Chinese, Blacks, and Kurdish ethnic origins. In addition, we have two Greek non-deletion delta beta-thalassemia patients who will analyzed and may carry mutations which provide important information about the nature of hemoglobin switching. In this work we will develop for routine use a new method of genomic DNA sequencing which relies on prior amplification of desired regions of DNA. These techniques will allow us to quickly characterize beta-gene mutations that lead to mildly abnormal phenotypes. Second, we will continue studies of the evolution of the beta- globin gene cluster specifically determining whether variation 3' to the beta gene in the L1 region is due to simple point mutation or gene conversion of L1 sequences. We will also study the region between the delta- and beta-globin genes 1) to determine the nature of sequence randomization within the region 1 kb 5' to the beta- globin gene and 2) to delimit the region responsible for the recombination hotspot. Third, we will improve methods of direct detection of known mutant alleles to devise simplified direct detection schemes for gene diagnosis. The development of improved schemes for the detection of beta-globin gene defects will serve as a model which can then be transferred, when appropriate, to the genetic screening for carriers of other common autosomal recessive traits, such as cystic fibrosis and PKU.
