GENETIC HEMATOLOGY 
Stuart H. Ormn, M.D., Investigator 
The central focus of Dr. Orkin's laboratory is the 
study of the structure, expression, and function of 
specific genes that are relevant to the normal bi- 
ology and pathology of hematopoietic cells. The 
ultimate goals are to understand the molecular 
basis for cellular commitment and differentiation, 
determine the mechanisms for regulation and coor- 
dination of gene expression in developing blood 
cells, and apply basic knowledge to the manage- 
ment of inherited human disorders of hemato- 
poietic cells. 
I. Globin Gene Expression. 
The genes of the (3-globin family are subject to 
precise regulation, both with respect to cell lineage 
and the temporal expression of individual mem- 
bers. To approach molecular mechanisms of gene 
regulation, Dr. Orkin initially directed attention to 
the identification of nuclear DNA-binding proteins 
that might participate in control of fetal (7)-globin 
gene transcription. An erythroid cell-specific fac- 
tor (GF-1) was detected that recognizes a short- 
sequence motif present in the 7-globin promoter in 
the vicinity of a single-base change (at position 
-175) associated with hereditary persistence of fetal 
hemoglobin (HPFH) into adult life. By expression 
of site-specific mutagenesis it was established that 
the overexpression of this mutant promoter is ery- 
throid-specific and is dependent on the availability 
of binding sites for the erythroid factor. Indepen- 
dent work by others showed that the sequence 
motif recognized by this erythroid factor is not lim- 
ited to the 7-globin promoter but is found in the 
promoters and enhancers of the majority of ery- 
throid-expressed genes of human, mouse, and 
avian origin. 
The human protein was purified to homogeneity 
and subjected to direct peptide sequencing to de- 
termine the nature of the erythroid factor. In addi- 
tion, the cDNA encoding the mouse protein was 
isolated and characterized by expression of recom- 
binant clones in mammalian cells. GF-1 of both 
human and mouse origin is a 413-amino acid poly- 
peptide that contains two novel cysteine-cysteine 
zinc finger-like DNA-binding domains. This DNA- 
binding region is extraordinarily conserved across 
species. GF-1 is expressed not only in developing 
erythroid cells but also in cells of the mega- 
karyocytic (platelet) lineage. This indicates that this 
regulatory factor is expressed in a bipotential hema- 
topoietic progenitor that subsequently further com- 
mits to a single-cell lineage. GF-1 is encoded by a 
single gene on the X chromosome. Current efforts 
are devoted to understanding the role of GF-1 in 
transcriptional activation of genes, the regulation of 
the GF-1 gene in developing hematopoietic cells, 
and the involvement of GF-1 in cellular commit- 
ment and hemoglobin switching in development. 
II. Molecular Genetics of Superoxide Generation by 
Phagocytes. 
Phagocytic cells produce superoxide via an 
NADPH-oxidase complex as part of a major bacteri- 
cidal host defense system. Deficiency in this system 
leads to an immune disorder, chronic granuloma- 
tous disease (CGD), the major variety of which is 
X-linked (X-CGD). By reverse genetics it was pre- 
viously established that X-CGD is due to mutation 
of a gene encoding the glycoprotein subunit of an 
unusual, neutrophil-specific heterodimeric cytopro- 
tein b. Subsequent research has proceeded in three 
directions. 1) Mutations leading to X-CGD and rarer 
autosomal forms of CGD have been characterized 
in an effort to delineate critical portions of the rele- 
vant proteins. These studies have led to the identifi- 
cation of a single-base change in the X-CGD gene 
that results in a nonfunctional cytochrome b mole- 
cule in white blood cells and to the discovery of 
mutations in the other subunit of the cytochrome 
in a rare form of autosomal CGD. 2) The molecular 
basis for regulation of the X-CGD gene in white 
blood cells has been pursued by expression of gene 
constructs and study of nuclear DNA-binding pro- 
teins that interact with promoter elements. 3) The 
mechanism(s) by which the cytokine interferon-7 
augments transcription of the X-CGD gene and 
ameliorates the cytochrome b deficit in some 
X-CGD patients has been investigated. These stud- 
ies have revealed that interferon-7 can reprogram 
white cell progenitors in vivo to express increased 
levels of the neutrophil cytochrome. Assessment of 
the efficacy of interferon-7 in management of CGD 
is being performed as part of an international con- 
trolled clinical trial. 
III. Gene Transfer and Modification in Hematopoi- 
etic Cells. 
In collaboration with Dr. David Williams (HHMI, 
Children's Hospital, Boston), Dr. Orkin has contin- 
ued to develop strategies for the efficient transfer 
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