HEMPAS (Hereditary dyserythroblastic multinuclearity associated with positive acidified serum lysis test) is a human genetic disease characterized by defective glycosylation of polylactosaminoglycan proteins in erythroid cells. Previously obtained data are implicating a defect in each patient of one of three enzymes, N-acetylglucosaminyltransferase II (GnT II), galactosyltransferase (GT) or alpha-mannosidase II (alpha-MII). Over the next five years, the gene defect in each HEMPAS will be determined to establish molecular genetics of HEMPAS. First, HEMPAS variant G.K.'s GT gene will be determined whether his GT is defective. To accomplish this, nucleotide sequence of the GT from variant G.K. will be amplified by employing a polymerase chain reaction (PCR). The G.K.'s GT sequence will be compared to the normal GT sequence which has been determined previously by cDNA cloning. If a mutation of GT is identified, an expression vector having GT cDNA with the same mutation as in the G.K. variant will be constructed. The trafficking of the mutated GT in COS-1 cells transfected by the vector will be examined as to whether mutated GT is secreted from the cells as observed in G.K. cells. Second, gene mutation of alpha-MII found in HEMPAS G.C. will be analyzed further. G.C. cells express a low level of alpha-MII poly(A)+mRNA. Regulatory regions such as the promoter region and the transcription initiation site of alpha-MII gene will be investigated and genetic mutation leading to the low alpha-MII mRNA production will be determined. In order to correlate low alpha-MII activity and morphological abnormality of HEMPAS erythroid cells, the morphology of erythroblasts cultured in vitro in the presence of swainsonine (alpha-MII inhibitor) will be investigated. The third project will be to determine whether a majority of HEMPAS patients are defective in GnT II. A human placenta cDNA library will be screened and cDNA for GnT II will be isolated to determine the sequence of normal GnT II. Then the HEMPAS patients' DNA and mRNA will be examined by Southern and Northern analysis, respectively. The GnT II cDNA sequence in HEMPAS patients will be determined through mRNA-based PCR. Determination of the primary defect of HEMPAS will provide a molecular basis for future diagnosis and genetic therapy of this disease.
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