NATURE AND FREQUENCY OF MUTATIONS IN THE ARGININOSUCCINATE SYNTHETASEGENE THAT CAUSE CLASSICAL CITRULLINEMIA

Citrullinemia is an autosomal recessive disorder caused by a genetic d eficiency of argininosuccinate synthetase (ASS). So far 20 mutations i n ASS mRNA have been identified in human classical citrullinemia, incl uding 14 single base changes causing missense mutations in the coding sequence of the enzyme, 4 mutations associated with an absence of exon s 5, 6, 7, or 13 in mRNA, 1 mutation with a deletion of the first 7 ba ses 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 mR NA. In order to identify the abnormality in the ASS gene causing the e xon 7 and 13 deletion mutations and the 37-base insertion mutation bet ween exons 15 and 16 in mRNA, and to establish a DNA diagnostic test, we isolated and sequenced the genomic DNA surrounding each exon. The a bsence of exon 7 or 13 in ASS mRNA resulted from abnormal splicing cau sed by a single base change in the intron region: IVS-6(-2) (a transit ion of A to G at the second nucleotide position within the 3' splice c leavage site of intron 6) and IVS-13(+5) (a transition of G to A at th e fifth nucleotide position within the 5' splice cleavage site of intr on 13), respectively. The IVS-6(-2) mutation resulted in the creation of an MspI restriction 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 mutation is predominant in Japan. This differs from the situation in the USA where there is far greater hete rogeneity. The insertion mutation in mRNA on the other hand resulted f rom abnormal splicing caused by a 13-bp deletion at the splice-junctio n between exon 15 and intron 15. The deletion had a short direct repea t (CTCAGG) at the breakpoint junction and presumably resulted from sli pped mispairing.