R. F. Rieder

Cerebrovascular accidents in children with sickle-cell disease and alpha-thalassemia.

Alpha-thalassemia occurs commonly in black Americans, ~ with approximately 30% lacking one a-glob{n gene (athai-2 genotype: aa/a-) and 4% lacking two genes (athai- 1 genotype: a-/a- or, rarely, aa/--). In individuals with sickle-cell disease, a-thalassemia attenuates some of the hematologic manifestations, 2-4 although the extent of clinical benefit remains controversial?. 5 Before routine implementation of long-term transfusion programs for children with sickle-cell disease and cerebrovascular accident, 6, 7 CVA was ~i significant cause of death in the young sickle-cell disease population? Because the favorable effects of a-thalassemia on hematologic manifestations are apparent by the age of 1 year, 9 it is possible that a reduction in incidence of CVA, which occurs at a mean age of 7 years, s might contribute to prolonged survivaP in patients with sickle cell-a-thalassemia. In fact, a reduced incidence of a-thalassemia has been reported in a small group of patients with sickle-cell disease who have had a CVA? ~ The a-globin gene status of 20 of our sickle-cell disease patients with a history of CVA has been ascertained and is the basis for this report. METHODS

Transcriptional upregulation of γ-globin by phenylbutyrate and analogous aromatic fatty acids

Abstract Phenylbutyrate has been shown recently to induce fetal hemoglobin (HbF) production in patients with sickle cell anemia and β thalassemia. We have now examined related aromatic fatty acids in order to define the range of active structures and identify plausible mechanisms of action. Structure-function analysis revealed that for effective stimulation of HbF in erythroid precursors: (1) the ideal length for the aliphatic side chain is four carbons; (2) oxygen or sulfur substitutions in the carboxylic chain are allowed, as evidenced by the equal or increased activity of phenoxypropionate, benzylthioglycolate, and benzyloxyacetate compared with phenylbutyrate; and (3) blocking the carboxylate group by conversion to the amide form greatly reduces potency. Molecular analysis indicated that the prototype agent, phenylbutyrate, increases HbF production through transcriptional activation of the γ-globin gene. The latter contains a butyrate responsive promoter known to up-regulate transcription in the presence of short-chain fatty acids of three to five carbons. To determine whether stimulation of an element in this promoter by phenylbutyrate and its analogues might contribute to their mechanism of action, we used a transient expression system involving K562 erythroleukemia cells transfected with a luciferase reporter gene driven by the minimum γ-globin promoter. Transcriptional activation in this experimental system correlated well with the capacity of an aromatic fatty acid to increase HbF production in erythroid precursors ( r = 0.94). Our studies identify potent analogues of phenylbutyrate for the treatment of β-chain hemoglobinopathies, and suggest that stimulation of a butyrate responsive promoter may be responsible for their activity.

Deletion of a Repeated AG Dinucleotide at the Exon 1–Intron 1 Junction Causes α-Gene Dysfunction without an mRNA Splicing Defect

In a-thalassemia, the deficiency in a-globin synthesis is most frequently due to deletion of one or more of the normal complement of four a-globin genes. Hemoglobin H disease usually results from the absence of three a genes (genotype --/-a). This disorder has a low incidence in black subjccts, due to a rarity of the -/ haplotype. We have previously reported that the deletion of one of two tandemly repeated AG dinucleotides at the 3’ end of exon 1 near the junction with intron 1 is responsible for a dysfunctional a-globin gene in a black family with a-thalassemia and Hh H disease.’ The loss of the two nucleotidcs results in a reading frameshift with the generation of a downstream stop codon in exon 2 at codon 55. We have now found the same genetic lesion in a second, unrelated black family, in which five mcmbcrs have Hb H disease.’ Because of uncertainty of the effect of nonsense codons on the expression of mRNA, the mutant gene was studied in COS-7 cells, using plasmid-based vcctors.’ A 1.5-kb Psi I fragment containing the abnormal gene (aT) with 5‘ and 3’ sequences was ligated to plasmid pLTNlha after removal of the normal a gene. The aT and normal a plasmids (together with plasmid pLTN3BhP as an internal control) were transfected into COS-7 cells. When production of the normal a and the aT mRNA was compared to that of the f3 mRNA, the a/p and aT/p ratios were 1.6 and 1.8, respectively. Thc 5’ end of the aT mRNA was analyzed by primer extension analysis; the extension products mapped to the correct initiation site. The effect of the dinucleotide dclction on the removal o f the first intron was studied by S, nuclease protection analyses. Correct removal of the intron at both the 5’ donor and 3’ acceptor sites was noted. The 3’ end of the aT mRNA from transfected COS-7 cells also was analysed by S, digestion; the results indicated correct processing. Our studies showed that the a-thalassemic globin gene is stably expressed at a level comparable to that of a normal a-globin gene when introduced into COS cells, using a plasmid vector. The resultant a-thalassemia mRNA also appeared to havc correct 5‘ and 3’ ends; and removal of intron 1 proceeded normally at the 5’ and 3’ splice sites, despite the alteration in the consensus sequence near the 5’ donor site. Because the AG dinucleotide is tandernly rcpeated at the 3‘ end of exon 1 of the a-globin gene in the sequence GAGAGgt, the presently described deletion does not destroy the sequence immediately 5’ to the invariant gt, which is common to all known introns. This seems a likely explanation for the lack of interference with splicing noted in the present study. This work also suggests that the decrease in the