Glycerol kinase deficiency is a human genetic disorder characterized by considerable clinical heterogeneity. This enzyme deficiency maps to the midportion of the short arm of the X chromosome, the Xp21 region, and is closely linked genetically to the loci for a number of other disorders, including Duchenne muscular dystrophy, retinitis pigmentosa, ornithine transcarbamylase deficiency, congenital adrenal hypoplasia, and chronic granulomatous disease. The purpose of the proposed research is to clarify the clinical heterogeneity of glycerol kinase deficiency and to refine the genetic relationships of these linked disorders at the molecular level.
The specific aims of this proposal are: to clone the full length cDNA and corresponding genomic sequence for the human hepatic form of glycerol kinase in order to investigate molecular heterogeneity at the DNA and mRNA levels, to improve diagnosis and carrier detection, and to establish by linkage analysis the relative position of the glycerol kinase locus to other markers in the Xp21 region. The methodology proposed involves three cloning strategies: screening a cDNA library with synthetic oligonucleotide probes constructed after purification and sequencing of human liver glycerol kinase; using the technique of subtractive hybridization between labeled cDNA prepared from normal poly (A)+RNA and unlabeled mRNA from a patient with glycerol kinase deficiency and a documented Xp21 deletion; or screening a lambda gtll library with antiglycerol kinase antibody. The glycerol kinase protein has been purified to homogeneity in this investigator's laboratory. Although the N-terminus is blocked, successful CNBr fragmentation has been carried out, and amino acid sequence data is anticipated in the near future. Therefore, this strategy should provide a successful approach, but the others offer available alternatives. These investigations will significantly improve our understanding of the clinical and molecular heterogeneity among patients with glycerol kinase deficiency, and will also enhance our knowledge of the linkage relationships between loci in this region of the human genome. This work will serve as the foundation for evaluation of the biology of the glycerol kinase and porin which form a regulable and reversible enzyme-receptor system.
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