MOLECULAR STUDIES OF HUMAN GENETIC DISEASES 
ARrauR L. Beaudet, M.D., Investigator 
Research in Dr. Beaudet's laboratory is focused 
on molecular approaches to a number of human 
genetic diseases, including spinocerebellar ataxia, 
cystic fibrosis, deficiencies in urea cycle en2ymes, 
and disorders of cell adhesion molecules. Much of 
this research is conducted in collaboration with Dr. 
William E. O'Brien (HHMI Senior Associate). 
I. Argininosuccinate Synthetase Locus and 
Citrullinemia. 
Genetic deficiency of argininosuccinate syn- 
thetase (AS) causes neonatal citrullinemia, which is 
characterized by increased blood ammonia, mental 
retardation, and early death without treatment. 
Seven single-base missense mutations causing 
citrullinemia were identified by sequencing clones 
obtained by amplification of cDNA from cultured fi- 
broblasts. Four additional alleles are associated 
with the absence of a single exon sequence in the 
cDNA, and another mutation is a single-base splic- 
ing abnormality resulting in the absence of seven 
base pairs in the cDNA sequence. The characteriza- 
tion of these 12 mutant alleles indicates that the 
mutations causing citrullinemia are extremely het- 
erogeneous, and all nonconsanguineous patients 
studied to date are compound heterozygotes. The 
mutation causing citrullinemia in Friesian cattle was 
identified as a nonsense mutation, at codon 86, and 
a heterozygote detection method using the poly- 
merase chain reaction for analysis of genomic DNA 
was developed. 
DNA polymorphisms were identified within the 
human AS gene and were used to map the gene 
more precisely through the CEPH (Centre d'Etude 
du Polymorphisme Humain) collaborative linkage 
study. The human gene maps to chromosome 9q34 
close to the ABO blood group with a recombina- 
tion fraction of 0.04. These polymorphisms also 
provide improved methods for prenatal diagnosis 
of citrullinemia. Similar mapping studies were per- 
formed in the mouse, using recombinant inbred 
strains. The mouse gene maps to chromosome 2 
with close linkage to the fifth component of com- 
plement, placing the gene in a conserved linkage 
group between mouse and human, including the 
fifth component of complement, the AS gene, the 
Abelson oncogene, and adenylate kinase- 1. 
Studies aimed at developing somatic gene ther- 
apy for various human diseases are being per- 
formed, using the AS gene and citrullinemia as a 
model system. Although this gene is usually ex- 
pressed at high levels in hepatocytes, it is possible 
that expression in bone marrow-derived cells 
would provide an adequate correction. The human 
cDNA sequence was introduced into a modified N2 
vector for expression from the long terminal repeat 
(LTR) with the viral structure LTR-cDNA-LTR. Retro- 
viral titers of 3-5 x 10*^ were obtained using the 
GP+E-86 (ecotropic) and G^-\-envMA12 (ampho- 
tropic) packaging cell lines. Both packaging cell 
lines have been used for infection of mouse bone 
marrow cells, followed by reimplantation of bone 
marrow into lethally irradiated recipient mice. All 
mice express human AS activity in mouse periph- 
eral blood. Most mice experience decreased levels 
of expression 8-15 wk post-transplantation, but 
many mice continue to express at significant levels 
beyond 20 wk. Retroviral infection of human and 
baboon bone marrow cells is being studied in vitro 
in preparation for in vivo experiments in baboons. 
Work is under way with a goal of developing a 
mouse mutant for citrullinemia. The mouse gene 
has been extensively characterized, and recombi- 
nant DNA constructions suitable for homologous 
recombination in embryonic stem (ES) cells are 
being prepared. 
II. Gene Cloning for Spinocerebellar Ataxia. 
Spinocerebellar ataxia (SCA 1) is a dominantly in- 
herited neurodegenerative disorder that is mapped 
to the short arm of chromosome 6. Dr. Huda 
Zoghbi has performed extensive studies to map 
regionally the SCA 1 locus on chromosome 6p. A 
detailed somatic cell hybrid panel for regional map- 
ping on chromosome 6p was developed. A genomic 
DNA library was prepared from a somatic cell hy- 
brid containing chromosome 6p as the only identi- 
fiable human chromosomal material. Over 100 
clones from this library were isolated and regionally 
mapped. Subsequently a set of radiation-induced 
hybrid cells was isolated containing small portions 
of chromosome 6p. Current data indicate that the 
SCA 1 locus maps centromeric to the HLA region 
and that flanking DNA markers are available. Efforts 
continue to map the SCA 1 locus precisely, with the 
goal of cloning the gene. 
III. Molecular Genetic Studies of Cystic Fibrosis. 
Cystic fibrosis (CF) is the most frequent lethal au- 
tosomal recessive disease in Caucasians, and the 
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