Molecular genetics of X-linked sideroblastic anemia and erythroid-specific delta-aminolevulinate synthase.
Item
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Title
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Molecular genetics of X-linked sideroblastic anemia and erythroid-specific delta-aminolevulinate synthase.
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Identifier
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AAI9510650
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identifier
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9510650
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Creator
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Cotter, Philip David.
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Contributor
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Adviser: David F. Bishop
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Date
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1994
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Genetics
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Abstract
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X-linked sideroblastic anemia is a hematologic disorder characterized by hypochromic, microcytic anemia, elevated serum iron and the presence of ringed sideroblasts in the bone marrow. The erythroid-specific {dollar}\delta{dollar}-aminolevulinate synthase (ALAS2) gene, the first enzyme in heme biosynthesis, has been considered a candidate gene for XLSA. Each of the eleven exons including the intron/exon boundaries, 1 kb 5{dollar}\prime{dollar} and 350 bp 3{dollar}\prime{dollar} of the ALAS2 gene were PCR-amplified, subcloned and sequenced from patients with XLSA.;The first mutation in the ALAS2 gene was identified in exon 9 (I476N) from a patient with pyridoxine-responsive XLSA. Characterization of the normal and mutant recombinant enzymes showed that the mutant enzyme was responsive in vitro to pyridoxal 5{dollar}\prime{dollar}-phosphate (PLP), consistent with the patient's clinical response to pyridoxine. This result demonstrated for the first time that XLSA was due to a molecular defect in the ALAS2 gene. Subsequently, the causative ALAS2 mutation (F165L) was identified from the family in which XLSA was originally described in 1945 by Cooley. Similarly, recombinant mutant enzyme was shown to be responsive in vitro to PLP.;PCR analysis of somatic cell and radiation hybrids was used to refine the chromosomal localization of the housekeeping (ALAS1) and erythroid (ALAS2) genes. ALAS1 was localized to chromosome band 3p21.1 and ALAS2 to the distal subregion of Xp11.21. This localization of ALAS2 conclusively demonstrated separate loci for XLSA (Xp11.21) and XLSA with ataxia (linked to Xq13).;An additional four ALAS2 mutations were identified in unrelated families with pyridoxine-responsive XLSA. All four, R411C, R448Q, R452C and R452H, were in exon 9 the region encoding the lysine (K386) involved in PLP binding. Whereas the majority of ALAS2 mutations to date have been private, the R448Q and R452H mutations were identified as common mutations and together account for over one third of XLSA families. Two other mutations, K299Q and A172T, were identified in elderly patients who presented after the seventh decade and showed a remarkable response to pyridoxine, delineating a late onset form of XLSA. Recombinant mutant enzymes were markedly unstable, but could be stabilized in vitro by PLP.;A polymorphism was identified in the 3{dollar}\prime{dollar} region of the ALAS2 gene that in combination with sequence analysis allowed the exclusion of ALAS2 in the etiology of sideroblastic anmeia in one family and provided evidence for autosomal inheritance.
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Type
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dissertation
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Source
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PQT Legacy Restricted.xlsx
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degree
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Ph.D.