MOLECULAR STUDIES OF HUMAN GENETIC DISEASES 
Arthur L. Beaudet, M.D., Investigator 
Leukocyte and Endothelial Cell 
Adhesion Molecules 
Inflammatory cell adhesion molecules (CAMs) on 
the surface of leukocytes and endothelial cells play 
a major role in inflammatory responses by mediat- 
ing leukocyte-endothelial interactions, leukocyte- 
leukocyte interactions, leukocyte emigration from 
the vasculature, and other cellular processes. These 
molecules include leukocyte integrins (GDI 8, 
GDI la, GDI lb, GDllc, VLA-4), members of the 
immunoglobulin superfamily (ICAM-1, IGAM-2, 
VGAM), and the selectins (P-, E-, and L-selectin). Dr. 
Beaudet's laboratory is examining the hypothesis 
that reduced levels of expression of the inflamma- 
tory GAMs will be correlated with reduced suscepti- 
bility to common human diseases that have an in- 
flammatory component, such as atherosclerosis, 
inflammatory bowel disease, rheumatoid arthritis, 
insulin-dependent diabetes mellitus, and autoim- 
mune diseases. Mutations in inflammatory GAMs are 
being introduced and tested in mice through gene 
targeting, to determine whether genetic variation in 
these loci might be an important variable in disease 
susceptibility. If the hypothesis is proved correct, 
pharmacologic methods to reduce expression or 
function of inflammatory GAMs should reduce or 
prevent disease processes that are strongly in- 
fluenced by inflammatory GAM expression. 
The GDIS gene encodes the subunit, which 
can combine with any of three different a subunits 
to form leukocyte integrins. GDIS deficiency in hu- 
mans causes granulocytosis, impaired leukocyte em- 
igration, serious susceptibility to bacterial infec- 
tions, and early death in the severe form of the 
disorder. Human GDIS deficiency is an excellent 
candidate for somatic gene therapy using ex vivo 
infection of bone marrow cells. Toward this goal, 
human GD 1 S borne by a retrovirus vector has been 
used for expression in granulocytes in transplanted 
mice, for correction of the adhesion defect in defi- 
cient lymphoblasts, and for expression in deficient 
human bone marrow cultured in vivo. The rele- 
vance of more-modest variations in GDIS expres- 
sion in common human diseases is unexplored. 
To obtain a GDIS mutant mouse, the laboratory 
used an insertion vector for gene targeting. Homo- 
zygous mutant animals are healthy and fertile when 
raised in a specific pathogen-free (SPF) environ- 
ment. Mutant animals have a moderate leukocytosis 
similar to that seen in mild forms of human GDIS 
deficiency. Immunostaining and fluorescence- 
activated cell sorting (FAGS) analysis of mutant ani- 
mals reveal a 70-90% reduction of surface expres- 
sion of GDIS on lymphocytes and granulocytes. 
Residual expression of GDIS was proved to be 
due to a cryptic promoter that lies in the plasmid 
backbone of the insertion construct just upstream of 
the exon containing the ATG initiator codon. This 
mutation provides a hypoinflammatory mouse 
model, and the effect of the mutation on various 
inflammatory diseases is being studied. There is a 
modest but significant delay in transplantation re- 
jection in mutant animals used as recipients in neo- 
natal heart transplants. 
IGAM-1 is a transmembrane adhesion protein that, 
while relatively widely expressed, is particularly 
important on the endothelial surface, where it medi- 
ates leukocyte emigration through interaction with 
some of the ^2 integrins. No human deficient pa- 
tients are known. A null mutation was introduced 
into the mouse IGAM-1 gene, using gene targeting 
with a replacement construct. Homozygous mutant 
mice are healthy and fertile in an SPF environment. 
Immunostaining of lung, where IGAM-1 is maxi- 
mally expressed, reveals complete absence of ex- 
pression in mutant animals, and immunostaining 
and FAGS analysis of splenocytes from mutant ani- 
mals similarly reveals a complete absence of expres- 
sion. Examination of mutant animals in a chemical 
(thioglycoUate) peritonitis model indicates re- 
duced emigration of leukocytes to the peritoneal 
cavity — as reflected in elevated numbers of gran- 
ulocytes in the peripheral blood and reduced granu- 
locytes in the peritoneal fluid — although leukocyte 
emigration by IGAM-1 -independent mechanisms is 
substantial. 
The project to obtain GDIS and IGAM-1 mutant 
mice was supported by a grant from the National 
Institutes of Health. 
To test the hypothesis that genetic variation in 
inflammatory GAMs might correlate with common 
disease susceptibilities, a series of DNA polymor- 
phisms have been identified in some of these genes, 
with particular focus on IGAM-1 and the selectin 
gene cluster. All three known selectin genes are lo- 
cated within a 200-kb region on human chromo- 
some 1 . These polymorphisms, including a common 
amino acid polymorphism in IGAM-1, have been 
used for linkage analysis in humans and will be 
tested soon in appropriate populations to search for 
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