Attenuation of an amino-terminal premature stop codon mutation in the ATRX gene by an alternative mode of translational initiation.

N onsense mutations located in the 59 end of the coding sequence of a gene are commonly considered to be null alleles. Not only do such mutations result in the production of a truncated and usually inactive protein product, but premature stop codon mutations that occur upstream of the last exon–exon junction are also known to activate nonsense mediated decay (NMD) which results in the specific degradation of the affected mRNA. 2 However, several mechanisms may allow either fully or partially functional protein to be produced from alleles containing a premature stop codon mutation and this phenomenon may lead to considerable amelioration of the resulting phenotype. First, translational decoding (miscoding) of the stop codon as a sense codon, which can occur naturally due to the context of the stop codon, may result in low level ‘‘leaky’’ expression of full length protein. Although no examples of stop codon readthrough leading to disease amelioration have been clearly documented in humans, this mechanism has been implied for several premature stop codons, most notably for a premature stop codon mutation in the cystic fibrosis transmembrane conductance regulator gene (MIM 602421) leading to mild pulmonary presentation. 5 In this case, the equivalent mutation in a yeast gene, Ste6, has been shown to be suppressed at levels as high as 10%. Second, an internally truncated form of the protein may be expressed due to altered processing of the mRNA in which the stop codon containing exon (and sometimes adjacent exons) are removed from the mature mRNA. A correlation between exon skipping and suppression of disease symptoms has been documented in several cases of Becker muscular dystrophy (MIM 300376), a milder form of Duchenne muscular dystrophy. 8 Third, protein expression in some genes can be rescued by translational initiation at internal start codons downstream from a premature stop codon or frameshift mutation. This might occur by leaky scanning, internal ribosome entry, or through reinitiation following termination of translation. This kind of mechanism has been implicated for mild disease presentation in several diseases, including adrenal hypoplasia congenita (AHC; MIM 300200), Nijmegen breakage syndrome (NBS; MIM 251260), attenuated adenomatous polyposis coli (APC; MIM 175100), and one form of peroxisome-biogenesis disorder (PBD; MIM 601758). We examined the possibility that one of these mechanisms might be responsible for the unusually mild phenotype of patients carrying a premature stop mutation in the ATRX gene. ATRX is an X encoded member of the SNF2 protein family which exhibits ATPase and chromatin remodelling activities. Constitutional mutations give rise to a form of syndromal mental retardation (ATR-X syndrome; MIM 301040) associated with severe to profound learning difficulties, facial dysmorphism, genital abnormalities, and alpha thalassaemia. Somatic mutations have recently been identified in the clonal haematological disorder alpha thalassaemia myelodysplasia syndrome (ATMDS; MIM 300448). In ATR-X syndrome, the majority of mutations are missense and lie within two highly conserved regions of the gene, a PHD-like zinc finger domain and a helicase domain. True null mutations appear to have severe consequences: a mouse ATRX knockout is embryonic lethal (unpublished data). It was intriguing, therefore, to find that a milder variant of ATR-X syndrome was associated with a 109CRT mutation which gives rise to a premature stop codon, R37X. In the family reported by Guerrini et al, of the four affected male cousins, one had profound mental retardation which is typical of the condition, but a second had moderate mental retardation and two had mild mental retardation and were able to live partially independent lives. Subsequently, another unrelated male with moderate mental retardation has been identified with the same mutation. In this report we demonstrate, through analysis of the endogenous protein, that cell lines from individuals with the R37X mutation express approximately 20% of the wildtype levels of ATRX protein. Alternate spicing is excluded as the mechanism for this level of protein expression. We show that it is not due to stop codon readthrough, but instead it is Key points