Nature and frequency of mutations in the argininosuccinate gene that cause classical citrullinemia

Citrullinemia is an autosomal recessive disor- der caused by a genetic deficiency of argininosuccinate synthetase (ASS). So far 20 mutations in ASS mRNA have been identified in human classical citrullinemia, in- cluding 14 single base changes causing missense muta- tions in the coding sequence of the enzyme, 4 mutations associated with an absence of exons 5, 6, 7, or 13 in mRNA, 1 mutation with a deletion of the first 7 bases in exon 16 (which is caused by abnormal splicing), and 1 mutation with an insertion of 37 bases between the exon 15 and 16 regions in mRNA. In order to identify the ab- normality in the ASS gene causing the exon 7 and 13 deletion mutations and the 37-base insertion mutation be- tween exons 15 and 16 in mRNA, and to establish a DNA diagnostic test, we isolated and sequenced the genomic DNA surrounding each exon. The absence of exon 7 or 13 in ASS mRNA resulted from abnormal splicing caused by a single base change in the intron region: IVS-6 -2 (a tran- sition of A to G at the second nucleotide position within the 3" splice cleavage site of intron 6) and IVS-13 +5 (a transition of G to A at the fifth nucleotide position within the 5" splice cleavage site of intron 13), respectively. The IVS-6 2 mutation resulted in the creation of an MspI re- striction site. DNA diagnostic analysis of 33 Japanese al- leles with classical citrullinemia showed that 19 alleles had the IVS-6 -2 mutation (over 50% of the mutated alleles in Japanese patients). It was thus confirmed that one mu- tation is predominant in Japan. This differs from the situ- ation in the USA where there is far greater heterogeneity. The insertion mutation in mRNA on the other hand re- sulted from abnormal splicing caused by a 13-bp deletion