fluorescent primers to identify point mutations such 
as sickle cell anemia and other hemoglobinopa- 
thies. Presently under investigation is the reverse 
dot-blot method, in which oligonucleotides are im- 
mobilized to filters and the patients' amplified 
DNAs are hybridized to them. Mutations in 35 differ- 
ent /3-thalassemia lesions as well as those in the he- 
moglobinopathies such as S, C, and E could be de- 
tected rapidly in this manner. 
Panels of filters are being designed for different 
regions of the world where /3-thalassemia is com- 
mon, including Asia, Africa, and the Mediterranean. 
Dr. Kan and his colleagues have collaborated with 
investigators in Sicily to initiate testing programs us- 
ing this approach. Also communications are being 
set up with 1 0 regional centers in the Guangdong 
province of China to institute testing programs in a 
region of 60 million population and a carrier rate 
of -3%. 
Control of Globin Gene Expression 
An important discovery made in the study of the 
control of globin gene expression is the identifica- 
tion of the functional importance of four hypersen- 
sitive sites (HS 1-4) upstream of the globin gene 
clusters. This region, called the locus control region 
(LCR), is believed to be responsible both for the 
high-level expression of globin genes in the ery- 
throid cells and for the developmental switches 
from embryonic to fetal to adult globin expression. 
The mechanism may be mediated through a series of 
DNA-protein interactions between the HS of the LCR 
and the globin gene promoters. To investigate these 
physiologically important interactions. Dr. Kan's 
laboratory has utilized the in vivo footprinting tech- 
nique to locate them in erythroid cells. 
Critical protein-binding motifs have been identi- 
fied in the hypersensitive sites, including NFE2/ 
API, GT/CAC, and GATA-1 sequences. Transfection 
experiments demonstrated that the NFE2/AP1 se- 
quences are critical for the action of the HS, as muta- 
tions or deletions abolish the function of the site. 
The GT and GATA sequences appear to be comple- 
mentary but not absolutely essential for the enhanc- 
ing activity. In cell lines that synthesize different 
types of globin, there was no discernible change in 
DNA-protein interactions in the different hypersen- 
sitive sites of the LCR, but the promoter region of 
the globin genes became footprinted in its TATA, 
CAAT, and CAC sequences when those genes be- 
came active. 
The utility of these protein-binding motifs is be- 
ing tested in gene transfer experiments. Because of 
the critical importance of the NFE2/AP1 sequences, 
they were inserted in a retrovirus vector that also 
carries the /3-globin gene. The NFE2/AP1 doubles 
retrovirally introduced /3-globin gene expression in 
erythroleukemia cells. Because the GT and GATA 
sequences were observed to be complementary in 
activity, they are being added to the vectors that 
carry the NFE2/AP1 site in order to seek further en- 
hancement of globin gene expression. 
Another way to control the diseases in sickle cell 
anemia and /3-thalassemia is to increase 7-globin 
gene expression. Dr. Kan's laboratory has studied 
the effect of butyrate on globin gene expression, in 
collaboration with Dr. Susan Perrine (Children's 
Hospital Oakland Research Institute). Dr. Perrine 
found that butyrate increased fetal hemoglobin pro- 
duction in tissue culture cells. In the fetal lamb 
model, the infusion of butyrate suppresses the 
switch from fetal to adult hemoglobin expression. 
Preliminary experiments showed that butyrates also 
enhance 7-globin gene expression in patients with 
thalassemia. The mechanism of induction of fetal 
hemoglobin expression by butyrate is being investi- 
gated. 
Red Cell Membrane Defects 
Dr. Kan's laboratory has investigated the molecu- 
lar basis of hemolytic anemias associated with ellip- 
tocytosis and spherocytosis. Previous studies had 
demonstrated that defects in protein 4.1, spectrin, 
and glycophorin could result in this group of dis- 
orders. Protein 4.1 is present not only in the ery- 
throid cell but in all tissues, and many isoforms are 
produced from a single gene by alternate splicing. A 
family with severe hemolytic anemia due to the 
complete absence of protein 4 . 1 unexpectedly re- 
vealed no other systemic defects. DNA analysis dis- 
closed a rearrangement of the gene in the region of 
the initiation codon of the red cell protein 4.1. How- 
ever, another upstream AUG is utilized for the syn- 
thesis of nonerythroid protein 4.1. Therefore the re- 
arrangement of the downstream AUG region does 
not affect the production of the nonerythroid pro- 
tein 4.1. The patients who have complete absence 
of erythroid protein 4.1 have no defects in other 
tissues, as the nonerythroid protein 4.1 continued 
to be produced normally. 
These projects were supported in part through a 
grant from the National Institute of Diabetes and Di- 
gestive and Kidney Diseases, National Institutes of 
Health. 
Dr. Kan is also Professor of Laboratory Medi- 
cine and Medicine and the Louis K. Diamond Pro- 
fessor of Hematology at the University of Califor- 
nia, San Francisco, and Honorary Professor at the 
University of Hong Kong. 
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