Resumo

The aim of this study was the application of a phenotype-driven N-ethyl-N-nitrosourea (ENU) mutagenesis screen in mice for the identification of recessive mutations affecting mouse central nervous system development. Mouse ENU mutagenesis program was set up at CEDEME/UNIFESP, in São Paulo, Brazil. Two months-old BALB/c male mice were treated with three weekly doses of ENU (80-95mg/kg b.w.). After a sterile period of about four months, those males were crossed to untreated BALB/c females and a protocol of 3 generations was followed to recover recessive mutations. Four recessive mutant mice bearing varied phenotypes were isolated. One of the mice that gave rise to a lineage is a mutant mouse we named BALB/c atxrec1. The phenotype includes ataxia, tremor, disbasia, involuntary head movement, absence of Moro reflex, absence of free-fall righting reflex, in addition to a discrete delay in palpebral aperture and male infertility in 95% of the animals. Based on these phenotypic characteristics and due to the lack of cytoarchitectural defects in the brain, our hypothesis is that the mutant mice BALB/c atxrec1 could present a metabolic hereditary disease. To verify our hypothesis of a form of inborn error of metabolism, the profile of amino-acid excretion was determined in urine samples from BALB/c and BALB/c atxrec1. Paper chromatography revealed an increase in threonine, glycine and arginine, and a decrease in leucine, isoleucine and acid gamma aminobutyric, in BALB/c atxrec1 urine compared to the urine of wild-type animals. This metabolic profile suggested deficiency in guanidinoacetate N-methyltransferase (GAMT). Mapping of BALB/c atxrec1 by microsatellite analysis showed linkage of the mutation to a region of chromosome 10, in a chromosomal subregion between the markers D10Mit194 and D10Mit95 (LOD score of 4,016 close to D10Mit194 and LOD score of 1,761 close to D10Mit95). The gene coding GAMT is localized in chromosome 10, at 43cM, which is included in the region defined by the microsatellite mapping. Our next step is to sequence GAMT to determine if the mutation causing BALB/c atxrec1 phenotype is in that gene. If this is correct, as suggested by the phenotypical analysis performed so far, BALB/c atxrec1 mice could be a new animal model for the study of GAMT deficiency syndrome.