Argininosuccinate lyase deficiency: evidence for heterogeneous structural gene mutations by immunoblotting.
(17/21)
Argininosuccinate lyase (AS lyase) deficiency is an inborn error of the urea cycle with extensive clinical and genetic heterogeneity. We investigated the biochemical basis of the enzyme defect and the genetic heterogeneity in this disorder using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting of fibroblast extracts. The AS lyase monomer in control fibroblasts was present in two bands of approximately 51 and approximately 49 Kd. Each of 28 mutant strains had some cross-reactive material (CRM) of the lower (approximately 49 Kd) MW, in quantities ranging from trace to substantial levels. The approximately 51 Kd band was found in only six mutants with near-normal amounts of AS lyase CRM or high residual enzyme activity. The residual AS lyase enzyme activity in a mutant did not necessarily reflect the amount of the 49-51 Kd monomer in that strain. In contrast, there was a strong general correlation between the quantity of 49-51 Kd CRM in a mutant and the frequency of complementation by that mutant. In addition to the CRM of normal molecular weight (MW) (49-51 Kd), the majority of mutants (but not controls) had significant CRM present in one to five bands of MW less than 49 Kd. The immunoprecipitation of at least one of these low MW bands was inhibited by purified human AS lyase. Mutants indistinguishable by clinical, enzymatic, or complementation analysis have been shown to be heterogeneous in their content of AS lyase CRM, greatly extending the number of distinct mutant alleles identified at this locus. These data demonstrate that multiple unique mutations in the structural gene coding for the monomer cause AS lyase deficiency and that the AS lyase monomers made by these mutants may be unstable. Integration of these findings with enzymatic and complementation data has indicated the functional domain of the AS lyase monomer likely to be altered in certain mutants. (+info)
Arginine, an indispensable amino acid for patients with inborn errors of urea synthesis.
(18/21)
The role of arginine as an essential amino was evaluated in four children with one of the deficiencies of carbamyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthetase, and argininosuccinase. Within 15-68 h after arginine deprivation nitrogen accumulated as ammonium or glutamine or both, but glutamine was quantitatively the largest nitrogen accumulation product. Concomitantly plasma and urinary urea levels decreased. Resumption of arginine intake (or citrulline in the case of ornithine transcarbamylase deficiency) promptly led to correction of the hyperammonemia, hyperglutaminemia and hypoargininemia. Ornithine was an unsatisfactory substitute for arginine. Arginine deprivation did not interfere with carbamyl phosphate synthesis as manifested by orotic aciduria. It is concluded that arginine is an indispensable amino acid for children with inborn errors of ureagenesis and its absence results in the rapid onset of symptomatic hyperammonemia. (+info)
Interallelic complementation in an inborn error of metabolism: genetic heterogeneity in argininosuccinate lyase deficiency.
(19/21)
We used complementation analysis as a probe for the detection of genetic heterogeneity within a single locus affected in a human disease, argininosuccinate lyase (L-argininosuccinate arginine-lyase, EC 4.3.2.1) deficiency. Fibroblasts cultured from 28 unrelated patients were fused in all possible pairwise combinations, and the argininosuccinate lyase activity in heterokaryons was assayed by measuring the incorporation of 14C from L-[ureido-14C]citrulline into acid-precipitable material. Partial complementation was observed in fusions involving 20 of the 28 strains, with the lyase activity increasing from 2- to 10-fold. Thirteen of the mutants were identified by the complementation analysis as being phenotypically unique. Of the 20 complementing strains, 3 were remarkable because they participated in all but 2 of the 32 positive complementation tests; 2 others constituted a unique subgroup that produced the highest increases in argininosuccinate lyase activity of all fusions. The 8 strains that did not complement any others consisted of two types: 3 mutants with the highest residual argininosuccinate lyase activity of all strains and 5 mutants with low residual activity. All of the mutants mapped to a single major complementation group. The data could be summarized as a circular complementation map with an attached linear tail, the mutants being distributed among 12 subgroups in a complex pattern. We conclude that all of these mutants are affected at a single locus, that extensive genetic heterogeneity is present in the mutant population, and that the affected locus in argininosuccinate lyase deficiency is likely to be the structural gene coding for that enzyme. (+info)
Intragenic complementation at the human argininosuccinate lyase locus. Identification of the major complementing alleles.
(20/21)
To determine the molecular and biochemical basis of intragenic complementation observed at the human argininosuccinate lyase (ASL) locus, we identified the ASL alleles in ASL-deficient cell strains with two unique complementation phenotypes: (i) frequent complementers, strains that participated in the majority of complementation events, and (ii) high activity complementers, strains in which complementation was associated with a relatively high level of restoration of ASL activity. Four mutations (Q286R, D87G, A398D, and a deletion of exon 13) were identified in the four strains examined. One of the two frequent complementers was homozygous, and the other heterozygous, for the Q286R allele. Similarly, one of the two high activity complementers was homozygous, and the other heterozygous, for the D87G allele. When the Q286R and D87G mutations were introduced by site-directed mutagenesis into wild-type ASL cDNA, each conferred loss of ASL activity in COS cell transfection assays. To test directly the hypothesis that intragenic complementation occurs at the ASL locus, one of the major complementation events observed previously, between strains carrying the Q286R and D87G alleles, was reconstructed in COS cell transfection assays. A partial restoration of ASL activity, comparable with the increase seen in the fibroblast complementation analysis, was observed on joint cotransfection of these two alleles. The results provide molecular confirmation of the major features of the ASL mutant complementation map, identify the Q286R and D87D alleles as the frequent and high activity complementing alleles, respectively, and provide direct proof of intragenic complementation at the ASL locus. (+info)
Peritoneal dialysis and exchange transfusion in a neonate with argininosuccinic aciduria.
(21/21)
Peritoneal dialysis rapidly reduced blood ammonia concentration in this child with arginino-succinic acid-lyase deficiency, whereas exchange transfusion did not. Yet this reduction in plasma ammonia level did not produce clinical improvement. We speculate that the effects of ammonia intoxication on the highly susceptible neonatal metabolism are due to an accumulation of toxic products and to an altered energy metabolism. Both aspects must be considered in any attempt to treat congenital hyperammonaemia. (+info)