Identification and characterization of a missense mutation in O-GlcNAc transferase that segregates with disease in a family with X-linked intellectual disability
MetadataShow full item record
O-GlcNAc transferase (OGT) is responsible for the addition of the β-N-acetylglucosamine post-translational modification to serine/threonine residues of hundreds of nuclear and cytoplasmic proteins. In a focused X chromosome exome next generation sequencing of 30 probands with X-linked Intellectual disability (ID), a novel missense mutation in the OGT gene (Xq13.1) has been identified in a family with three affected males. The mutation occurs in the tetratricopeptide(TPR) region [762G>T (p.L254F)]of the transferase. The clinical phenotypes of these patients include hypospadia, clinodactyly, short stature, microcephaly, and- ID. To study the physiological role of this mutation, lymphoblastoid cell lines from two affected males, one mother and three unaffected related males were isolated. Steady-state OGT protein levels are decreased in the patient samples compared to the carrier and normal control in agreement with molecular modeling that predicts the mutation to be destabilizing. This was further validated by half-life studies that demonstrate a faster turnover of the L254F-OGT. We have generated a recombinant L254F-OGT that has allowed us to perform activity studies and L254F-OGT is active in vivo against protein substrates and in vitro against a synthetic peptide. Surprisingly, steady-state global O-GlcNAc levels remain grossly unaffected in XLID. The same samples, however, show a decrease in steady-state O-GlcNAcase (OGA, the enzyme that removes O-GlcNAc from proteins) levels. These findings imply a compensation mechanism exists, although imperfect, given the phenotype of the patients, for maintaining global O-GlcNAc levels. L254F-OGT patients also show a decrease in OGA steady state mRNA levels and luciferase reporter expression. OGT has been previously shown to exist in a co-repressor complex to down regulate gene expression. We have observed that there is enrichment of OGT at the proximal promoter region of OGA leading us to hypothesize that OGT regulates OGA transcription in the patient lymphoblastoids. In parallel, global transcriptome analysis by performing RNA deep sequencing has revealed that there are disease specific changes in gene expression. Currently we are determining the mechanism of regulation of OGA gene repression by OGT as well as validating targets obtained by RNA deep sequencing analysis. Finally, we will generate induced pluripotent stem (iPS) cells from the fibroblast of affected patients and carriers in order to explore the imperfect compensatory mechanism in derived neural lineages due to the specific phenotypes observed. For the first time, we have identified and partially characterized a missense mutation in OGT that causes a disease, XLID.