Human adenylosuccinate lyase (ADSL), cloning and characterization of full-length cDNA and its isoform, gene structure and molecular basis for ADSL deficiency in six patients

Abstract

Adenylosuccinate lyase (ADSL) is a bifunctional enzyme acting in de novo purine synthesis and purine nucleotide recycling. ADSL deficiency is a selectively neuronopathic disorder with psychomotor retardation and epilepsy as leading traits. Both dephosphorylated enzyme substrates, succinylaminoimidazole-carboxamide riboside (SAICAr) and succinyladenosine (S-Ado), accumulate in the cerebrospinal fluid (CSF) of affected individuals with S-Ado/SAICAr concentration ratios proportional to the phenotype severity. We studied the disorder at various levels in a group of six patients with ADSL deficiency. We identified the complete ADSL cDNA and its alternatively spliced isoform resulting from exon 12 skipping. Both mRNA isoforms were expressed in all the tissues studied with the non-spliced form 10-fold more abundant. Both cDNAs were expressed in Escherichia coli and functionally characterized at the protein level. The results showed only the unspliced ADSL to be active. The gene consists of 13 exons spanning 23 kb. The promotor region shows typical features of the housekeeping gene. Eight mutations were identified in a group of six patients. The expression studies of the mutant proteins carried out in an attempt to study genotype-phenotype correlation showed that the level of residual enzyme activity correlates with the severity of the clinical phenotype. All the mutant enzymes studied in vitro displayed a proportional decrease in activity against both of their substrates. However, this was not concordant with strikingly different concentration ratios in the CSF of individual patients. This suggests either different in vivo enzyme activities against each of the substrates and/or their different turnover across the CSF-blood barrier, which may be decisive in determining disease severity.

Testing for Ancient Selection Using Cross-population Allele Frequency Differentiation

“…The nonsynonymous mutation (A429V) is in the C-terminal domain of the protein ( Figure 6B) and lies in a highly conserved position [primate PhastCons = 0.953; GERP score = 5.67 (Siepel et al 2005;Cooper et al 2010;Kircher et al 2014)]. The ancestral amino acid is conserved across the tetrapod phylogeny, and the mutation is only three residues away from the most common causative SNP for severe adenylosuccinase deficiency (Maaswinkel-Mooij et al 1997;Marie et al 1999;Kmoch et al 2000;Race et al 2000;Edery et al 2003). The change has the highest probability of being disruptive to protein function, of all the nonsynonymous modern-human-specific changes that lie in the top-scoring regions (C score = 17.69).…”

Familial juvenile hyperuricaemic nephropathy (FJHN): linkage analysis in 15 families, physical and transcriptional characterisation of the FJHN critical region on chromosome 16p11.2 and the analysis of seven candidate genes

“…Genomic fragments covering promotor region (about 500 bp upstream from the most cDNA 5' end) and all of the exons and intron-exon boundaries were PCR amplified from genomic DNA and sequenced as previously described. 37 All candidate genes were analysed in one proband and one unaffected family member in each family. Genetic variations were screened against SNP and UNIGENE databases.…”

Biochemical and structural analysis of 14 mutant adsl enzyme complexes and correlation to phenotypic heterogeneity of adenylosuccinate lyase deficiency

“…The ADSL subunits are encoded in humans by a single ADSL gene (MIM# 608222), which spans 23 kb on chromosome 22q13.1-13.2. The gene consists of 13 exons, and it is ubiquitously expressed as two alternatively spliced mRNA isoforms [Fon et al, 1993;Kmoch et al, 2000;Van Keuren et al, 1987].…”