Abstract

It is estimated that ∼1% of the world's population has intellectual disability, with males affected more often than females. OGT is an X-linked gene encoding for the enzyme O-GlcNAc transferase (OGT), which carries out the reversible addition of N-acetylglucosamine (GlcNAc) to Ser/Thr residues of its intracellular substrates. Three missense mutations in the tetratricopeptide (TPR) repeats of OGT have recently been reported to cause X-linked intellectual disability (XLID). Here, we report the discovery of two additional novel missense mutations (c.775 G>A, p.A259T, and c.1016 A>G, p.E339G) in the TPR domain of OGT that segregate with XLID in affected families. Characterization of all five of these XLID missense variants of OGT demonstrates modest declines in thermodynamic stability and/or activities of the variants. We engineered each of the mutations into a male human embryonic stem cell line using CRISPR/Cas9. Investigation of the global O-GlcNAc profile as well as OGT and O-GlcNAc hydrolase levels by Western blotting showed no gross changes in steady-state levels in the engineered lines. However, analyses of the differential transcriptomes of the OGT variant-expressing stem cells revealed shared deregulation of genes involved in cell fate determination and liver X receptor/retinoid X receptor signaling, which has been implicated in neuronal development. Thus, here we reveal two additional mutations encoding residues in the TPR regions of OGT that appear causal for XLID and provide evidence that the relatively stable and active TPR variants may share a common, unelucidated mechanism of altering gene expression profiles in human embryonic stem cells.

Highlights

  • It is estimated that ϳ1% of the world’s population has intellectual disability, with males affected more often than females

  • Using exome sequencing to determine the etiology of the disease, we discover two separate novel missense mutations in the tetratricopeptide repeats (TPRs) domain of O-GlcNAc transferase (OGT) that segregate with X-linked Intellectual disability (ID) (XLID) in these patients

  • It was unsurprising that we found no significant changes (CTD 110.6, p Ͼ 0.4; RL2, p Ͼ 0.7; MannWhitney U test, data not shown) in the levels of O-GlcNAc in the X-linked intellectual disability (XLID) OGT-TPR mutant-expressing lines compared with WT (Fig. 5, a and b)

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Summary

ARTICLE cro

Three missense mutations in the tetratricopeptide (TPR) repeats of OGT have recently been reported to cause X-linked intellectual disability (XLID). Two additional mutations encoding residues in the TPR regions of OGT that appear causal for XLID and provide evidence that the relatively stable and active TPR variants may share a common, unelucidated mechanism of altering gene expression profiles in human embryonic stem cells. Using exome sequencing to determine the etiology of the disease, we discover two separate novel missense mutations in the TPR domain of OGT that segregate with X-linked ID (XLID) in these patients. To use as an ex vivo human isogenic model to study the effects of the XLID mutations on early development and disease pathogenesis, we engineered a human male embryonic stem cell line using CRISPR/Cas to express these OGT variants. Analysis of the transcriptome of these cells, reveals shared dysregulation of genes and pathways involved in early development, providing a starting point for dissecting the impact of OGT variants on common phenotypes in the XLID-affected individuals

Case reports of three patients with intellectual disability
This study
OGT variant WT
Discussion
Study samples
Protein expression and purification
Thermal stability assay
Enzyme kinetics
Cell lines and culture maintenance
Cell lysis and Western blotting

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