ATR-X mutations cause impaired nuclear location and altered DNA binding properties of the XNP/ATR-X protein

Mutations in the XNP/ATR-X gene, located in Xq13.3, are associated with sev eral X linked mental retardation syndromes, the best known being alpha thal assaemia with mental retardation (ATR-X). The XNP/ATR-X protein belongs to the family of SWI/SNF DNA helicases and contains three C2-C2 type zinc fing ers of unknown function. Previous studies have shown that 65% of mutations of XNP have been found within the zinc finger domain (encoded by exons 7, 8 , and the beginning of exon 9) while 35% of the mutations have been found i n the helicase domain extending over 3 kb at the C-terminus of the protein. Although different types of mutations have been identified, no specific ge notype-phenotype correlation has been found, suggesting that gene alteratio n leads to a loss of function irrespective of mutation type. Our aims were to understand the function of the XNP/ATR-X protein better, with specific a ttention to the functional consequences of mutations to the zinc finger dom ain. We used monoclonal antibodies directed against the XNP/ATR-X protein a nd performed immunocytochemical and western blot analyses, which showed alt ered or absent XNP/ATR-X expression in cells of affected patients. In addit ion, we used in vitro experiments to show that the zinc finger domain can m ediate double stranded DNA binding and found that the DNA binding capacity of mutant forms in ATR-X patients is severely reduced. These data provide i nsights into the understanding of the functional significance of XNP/ATR-X mutations.