Missense Mutation R338W in ARHGEF9 in a Family with X-linked Intellectual Disability with Variable Macrocephaly and Macro-Orchidism.

Philip Long,Charles E Schwartz,Kirsten Harvey,Robert J Harvey,Simone Grannò,Victoria M James,Roger E Stevenson,Patrick Tarpey,John P Johnson,Melanie M May

doi:10.3389/fnmol.2015.00083

Abstract

Non-syndromal X-linked intellectual disability (NS-XLID) represents a broad group of clinical disorders in which ID is the only clinically consistent manifestation. Although in many cases either chromosomal linkage data or knowledge of the >100 existing XLID genes has assisted mutation discovery, the underlying cause of disease remains unresolved in many families. We report the resolution of a large family (K8010) with NS-XLID, with variable macrocephaly and macro-orchidism. Although a previous linkage study had mapped the locus to Xq12-q21, this region contained too many candidate genes to be analyzed using conventional approaches. However, X-chromosome exome sequencing, bioinformatics analysis and segregation analysis revealed a novel missense mutation (c.1012C>T; p.R338W) in ARHGEF9. This gene encodes collybistin (CB), a neuronal GDP-GTP exchange factor previously implicated in several cases of XLID, as well as clustering of gephyrin and GABAA receptors at inhibitory synapses. Molecular modeling of the CB R338W substitution revealed that this change results in the substitution of a long electropositive side-chain with a large non-charged hydrophobic side-chain. The R338W change is predicted to result in clashes with adjacent amino acids (K363 and N335) and disruption of electrostatic potential and local folding of the PH domain, which is known to bind phosphatidylinositol-3-phosphate (PI3P/PtdIns-3-P). Consistent with this finding, functional assays revealed that recombinant CB CB2SH3−R338W was deficient in PI3P binding and was not able to translocate EGFP-gephyrin to submembrane microaggregates in an in vitro clustering assay. Taken together, these results suggest that the R338W mutation in ARHGEF9 is the underlying cause of NS-XLID in this family.

Highlights

Intellectual disability (ID) is characterized by significantly impaired intellectual and adaptive function, and is often defined by an IQ score below 70 in addition to deficits in two or more adaptive behaviors that affect everyday life
Positively-charged residues are thought to be critical for interaction of the pleckstrin homology (PH) domain with the membrane (Xiang et al, 2006) and R338W clearly changes the electrostatic potential of the PH domain, as visualized using Adaptive PoissonBoltzmann Solver (APBS) (Figures 2A–C)
Given the key role of CB in mediating gephyrin clustering at inhibitory synapses, we investigated whether the R338W substitution affected the ability of CB to translocate gephyrin

Summary

Introduction

Intellectual disability (ID) is characterized by significantly impaired intellectual and adaptive function, and is often defined by an IQ score below 70 in addition to deficits in two or more adaptive behaviors (e.g., social skills, problem solving) that affect everyday life. ID is subdivided into syndromal ID, where ID is associated with other clinical, morphological, or behavioral symptoms or non-syndromal ID, where intellectual deficits appear without other associated defects (Stevenson et al, 2012). Despite screening for mutations in selected known XLID genes by conventional linkage/candidate gene analysis or array CGH for examining copy number variants (CNVs), large number of families mapping to the X-chromosome remained unresolved (Lubs et al, 2012). These cases either represent undiscovered disease-relevant mutations in known genes, or causal mutations in novel XLID loci that remain to be identified

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Conclusion