Cystathionine beta-synthase (CBS) catalyzes the condensation of homocysteine (Hcy) and serine to cystathionine, which is then hydrolyzed to cysteine by cystathionine gamma-lyase. Inactivation of CBS results in CBS-deficient homocystinuria more commonly referred to as classical homocystinuria, which, if untreated, results in mental retardation, thromboembolic complications, and a range of connective tissue disorders. The molecular mechanisms that underlie the pathology of this disease are poorly understood. We report here the generation of a new mouse model of classical homocystinuria in which the mouse cbs gene is inactivated and that exhibits low-level expression of the human CBS transgene under the control of the human CBS promoter. This mouse model, designated “human only” (HO), exhibits severe elevations in both plasma and tissue levels of Hcy, methionine, S-adenosylmethionine, and S-adenosylhomocysteine and a concomitant decrease in plasma and hepatic levels of cysteine. HO mice exhibit mild hepatopathy but, in contrast to previous models of classical homocystinuria, do not incur hepatic steatosis, fibrosis, or neonatal death with approximately 90% of HO mice living for at least 6 months. Tail bleeding determinations indicate that HO mice are in a hypercoagulative state that is significantly ameliorated by betaine treatment in a manner that recapitulates the disease as it occurs in humans. Our findings indicate that this mouse model will be a valuable tool in the study of pathogenesis in classical homocystinuria and the rational design of novel treatments.

Cystathionine beta-synthase (CBS) deficient homocystinuria is an inherited metabolic defect that if untreated typically results in mental retardation, thromboembolism and a range of connective tissue disturbances. A knockout mouse model has previously been used to investigate pathogenic mechanisms in classical homocystinuria (Watanabe et al., PNAS 92 (1995) 1585–1589). This mouse model exhibits a semi-lethal phenotype and the majority of mice do not survive the early neonatal period. We report here that the birth incidence of cbs (−/−) mice produced from heterozygous crosses is non-Mendelian and not significantly improved by treatment with either the Hcy lowering compound betaine or the cysteine donor N-acetylcysteine. Betaine treatment did improve survival of cbs (−/−) mice and restored fertility to female cbs (−/−) mice but did so without significantly lowering Hcy levels. Surviving cbs (−/−) mice failed to show any alteration in coagulation parameters compared to wild-type controls. Moribund cbs (−/−) mice exhibited severe liver injury and hepatic fibrosis while surviving cbs (−/−) mice although less severely affected, still exhibited a level of severe liver injury that is not found in the human disease. The hepatopathy observed in this model may offer an explanation for the failure of cbs (−/−) mice to respond to betaine or exhibit a hypercoagulative phenotype. We conclude that although this model provides useful data on the biochemical sequelae of classical homocystinuria, it does not successfully recapitulate a number of important features of the human disease and its use for studying mechanisms in homocystinuria should be treated with caution as the hepatopathy produces changes which could influence the results.

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.

The CDO(-/-) mouse clearly demonstrates that H2S/HS(-) production in tissues can exceed the capacity of the animal to oxidize sulfide to sulfate and demonstrates that pancreas and lung are more susceptible to toxicity from endogenous H2S/HS(-)production than are liver and kidney.

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