Argininosuccinic acid lyase (
ASAL) deficiency is a clinically heterogeneous autosomal recessive
urea cycle disorder. We previously established by complementation analysis that 28
ASAL-deficient patients have heterogeneous mutations in a single gene. To prove that the
ASAL structural gene is the affected locus, we sequenced polymerase chain reaction-amplified
ASAL cDNA of a representative mutant from the single complementation group. Fibroblast strain 944 (approximately 1% of residual
ASAL activity), from a late-onset patient who was the product of a consanguineous mating, had only a single base-pair change in the coding region, a C-283----T transition at a CpG dinucleotide in exon 3. This substitution converts Arg-95 to Cys (R95C), occurs in a stretch of 13 residues that is identical in yeast and human
ASAL, and was present in both of the patient's alleles but not in 14 other mutant or 10 normal alleles. Expression in COS cells demonstrated that the R95C mutation produces normal amounts of
ASAL mRNA but little
protein and less than 1%
ASAL activity. We observed that amplified
cDNA from mutant 944 and normal cells (liver, keratinocytes, lymphoblasts, and fibroblasts) contained, in addition to the expected 5' 513-base-pair band, a prominent 318-base-pair
ASAL band formed by the splicing of exon 2 from the transcript. The short transcript maintains the
ASAL reading frame but removes Lys-51, a residue that may be essential for catalysis, since it binds the argininosuccinate substrate. We conclude (i) that the identification of the R95C mutation in strain 944 demonstrates that virtually all
ASAL deficiency results from defects in the
ASAL structural gene and (ii) that minor alternative splicing of the coding region occurs at the
ASAL locus.