SPECIFIC DIAGNOSIS OF HEMOGLOBINOPATHIES

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The cause of sickle cell disease (SCD), the most common mutation in the beta-globin gene, is the substitution of valine for glutamic acid at the sixth residue of the beta chain. The incidence of the disorder among African-Americans is approximately 1 in 500 births, with about 8% of African-Americans being heterozygous for Hb S. Since SCD is a significant cause of morbidity and mortality, we propose to devise an accurate, automated and cost-effective methods capable of increasing the confidence level of neonatal diagnosis of frequently-encountered, clinically-significant sickling hemoglobinopathies. Related to these goals this project aims to establish an allele-specific fluorescence-tagged gene amplification protocol to assay for the A to T mutation in the sequence encoding codon 6 of the human beta-globin gene. The rapid nonradioactive approach will allow direct detection of the normal or the beta-s-globin allele in genomic DNA without the additional steps of probe hybridization or restriction enzyme cleavage. Advanced techniques will be developed and utilize for screening for variants of SCD by developing a strategy for rapidly detecting additional known mutations within the beta-globin gene in compound heterozygotes for variants of SCD. Samples from compound heterozygotes for beta-s and other known beta-gene mutations will be analyzed for 4-6 mutations per reaction by multiplex or a competitive allele-specific fluorescence-tagged gene amplification protocol. Samples with uncharacterized mutations will be diagnosed by automated fluorescence-based DNA sequence analysis. Automated electrophoresis combined with real time multicolor fluorescence detection of unique labeled primers will provide the means to detect PCR-generated fragments in a single lane. Finally, to be investigated is the feasibility of a non-invasive method of prenatal diagnosis by analysis of fetal trophoblast cells obtained from maternal peripheral blood. Fetal trophoblast cells from maternal peripheral blood during pregnancy will be identified and isolated by means of monoclonal antibodies of unique specificity and high affinity against trophoblast membrane proteins. It is anticipated that fetal trophoblast cells can be isolated during the first trimester of pregnancy. Although the yield of such cells is low, a sufficient number can be isolated to allow amplification by PCR, thus enabling identification of the sickle cell gene and ultimately allowing widespread noninvasive prenatal screening.