Phenotypic spectrum of ARXopathies and functional analysis of a truncated human ARX gene product in neural cell cultures

Aim: Hemizygous mutations in the aristaless-related homeobox gene (ARX; Xp22.13), the human orthologue of the Drosophila gene aristaless, cause a wide spectrum of clinical phenotypes. Affected males present with mental retardation, which might be associated with dystonia, myoclonus epilepsy and in the most severe form of X-linked mental retardation with ambiguous genitalia (XLAG) with structural brain abnormalities including agenesis of the corpus callosum and lissencephaly. The aim of this study was to further characterize the genotype-phenotype correlation of ARXopathies and the function of ARX during neuronal differentiation.

Methods: Sequence analysis and QF-PCR to detect mutations and repeat expansions in a cohort of patients with lissencephaly and/or dystonia or myoclonus epilepsy with mental retardation. Establishment of fetal neural tissue cultures with the hemizygous ARX mutation c.1416_1417dupAC, resulting in a truncated protein with lack of the aristaless domain. Polyclonal antibodies were used to analyze these cells for the expression of nestin, GFAP, DCX, betaIIItubulin, calretinin, GAD67 and GABA known to be involved in cortical development and the differentiation of neuronal precursor cells.

Results: A causal ARX mutation could be identified in all 5 independent probands with characteristic features of XLAG. The four frameshift mutations resulted in either partial or complete loss of the aristaless domain, while the fifth proband was found to carry a missense mutation R332C in the highly conserved homeodomain. In one family with X-linked WEST syndrome the characteristic repeat expansion of the first polyalanine tract was identified. The established cell line in the absence of ARX is expressing markers characteristic for both cortical development and neuronal differentiation. Some cells were even found to express GAD67 and GABA, indicating the presence of mature GABAergic neurons.

Conclusion: XLAG is caused by hemizygous truncating mutations or missense mutations within highly conserved regions of the human ARX gene, while the milder ARXopathies result from distinct other ARX mutations which might conserve some residual protein function. In cultured fetal neural cells with the specific hemizygous ARX mutation c.1419_1420insAC and predicted loss of the aristaless domain no negative influence on neuronal differentiation and in particular no inhibitory effect on the development of GABAergic neurons were observed.