X-linked sideroblastic anemia: identification of the mutation in the erythroid-specific delta-aminolevulinate synthase gene (ALAS2) in the original family described by Cooley

摘要: In 1945, Thomas Cooley described the first cases of X-linked sideroblastic anemia (XLSA) in two brothers from a large family which inheritance disease was documented through six generations. Almost 40 years later enzymatic defect XLSA identified as deficient activity erythroid-specific form delta-aminolevulinate synthase (ALAS2), enzyme heme biosynthetic pathway. To determine nature mutation ALAS2 gene causing Cooley's original family, genomic DNAs were isolated affected hemizygotes, and each exon PCR amplified sequenced. A single transversion (A to C) 5. The predicted substitution leucine for phenylalanine at residue 165 (F165L) highly conserved domain catalytic core shared by all species. No other nucleotide changes found sequencing 11 exons, including intron/exon boundaries, 1 kb 5'-flanking 350 nucleotides 3'-flanking sequence. introduced an Mse I site restriction analysis PCR-amplified DNA confirmed presence lesion three obligate heterozygotes generations this family. Carrier diagnosis additional members one proband's sisters. After prokaryotic expression affinity purification both mutant normal fusion proteins, specific F165L about 26% normal. cofactor, pyridoxal 5'-phosphate, activated and/or stabilized purified recombinant vitro, consistent with pyridoxine-responsive hemizygotes