The role of Aristaless related homeobox (ARX) gene mutations in intellectual disability.

Abstract

Intellectual disability (ID) affects ~1-3% of the population, profoundly impacting the lives of affected individuals and their families. An approximate 30% excess of males with ID implicates X-chromosome genes. The most common inherited form of ID is fragile-X syndrome, affecting ~1/5,000 live male births. Another X-linked gene, the aristaless related homeobox (ARX) gene, is also frequently mutated causing X-linked ID (XLID). At least 50 pathogenic mutations spanning the ARX open reading frame (ORF) have been reported in 110 families. These mutations cause at least 10 clinically distinct pathologies, all of which include ID. These clinical entities range in severity from X-linked lissencephaly with ambiguous genitalia (XLAG) to mild ID with no other consistent clinical features. Of the known ARX mutations 60% occur in the section of the ORF that encodes for the first two tracts of uninterrupted alanine, ie polyalanine (pA) tracts. This is likely due to the extraordinarily high GC content of these regions of the gene (>97%). Two recurrent mutations (c.304ins(GCG)₇ – pA1 and c.429_452dup – pA2) arise from expansion of their respective pA tracts. The c.429_452dup mutation alone accounts for ~40% of all reported ARX mutations. To assess the frequency of ARX mutations among the intellectually disabled, genomic DNA from 613 individuals were screened for the most frequent ARX mutations. Of these, 500/613 samples were screened for mutations in the entire ARX ORF by either SSCP, dHPLC or direct Sanger sequencing. A subset of 94/500 patients were also screened for sequence variations in ultraconserved (uc) elements flanking the ARX gene, which likely act as ARX enhancers. Subsequently, using transient transfection studies we assessed the subcellular localisation of selected mutations and wildtype ARX proteins. Six different ARX mutations were detected in eight individuals (8/613; 1.3%) and potentially pathogenic sequence variations were found in uc elements in three more individuals. A total of five duplication mutations were discovered in pA2, two larger than the recurrent c.429_452dup, confirming exon 2 of ARX as a mutation ‘hot spot’. Increased aggregation was observed as a function of pA1 and pA2 length, aligning with the patient’s phenotypic severity. Three missense mutations were detected. A familial c.81G>C mutation caused a premature termination codon in exon 1, leading to Ohtahara syndrome (OS) and West syndrome (WS) in two male cousins. Although the c.81G>C mutation should truncate the ARX protein, reinitiation of translation at a down-stream methionine codon (c.121_123) likely occurs, ‘rescuing’ these patients from the otherwise severe XLAG phenotype. Two point mutations (c.1074G>T/p.R358S; c.1136G>T/ p.R379L) that alter key residues within the homeodomain were found in two individuals with brain/genital malformations and led to increased ARX protein mislocalisation. These mutations impair vital properties of ARX’s transcription factor function by perturbing its localisation into the nucleus (p.R379L) or DNA binding (p.R358S). This study confirms that ARX mutations contribute significantly to XLID and that the majority of mutations occur within exon 2, specifically within the region of pA2. Moreover, there is a correlation between the subcellular localization of the mutant protein and the clinical severity in the patients.