A Rare De Novo RAI1 Gene Mutation Affecting BDNF-Enhancer-Driven Transcription Activity Associated with Autism and Atypical Smith-Magenis Syndrome Presentation

Abstract

Deletions and mutations involving the Retinoic Acid Induced 1 (RAI1) gene at 17p11.2 cause Smith-Magenis syndrome (SMS). Here we report a patient with autism as the main clinical presentation, with some SMS-like features and a rare de novo RAI1 gene mutation, c.3440G > A (p.R1147Q). We functionally characterized the RAI1 p.R1147Q mutant protein. The mutation, located near the nuclear localization signal, had no effect on the subcellular localization of the mutant protein. However, similar to previously reported RAI1 missense mutations in SMS patients, the RAI1 p.R1147Q mutant protein showed a significant deficiency in activating in vivo transcription of a reporter gene driven by a BDNF (brain-derived neurotrophic factor) intronic enhancer. In addition, expression of other genes associated with neurobehavioral abnormalities and/or neurodevelopmental disorders were found to be altered in this patient. These results suggest a likely contribution of RAI1, either alone or in combination of other factors, to social behavior and reinforce the RAI1 gene as a candidate gene in patients with autistic manifestations or social behavioral abnormalities.

Keywords: RAI1; Smith-Magenis syndrome; autism spectrum disorder; mutation; neurodevelopmental disorder.

Figure 1

Molecular analyses of a patient with autism. (A) A partial pedigree of a family with one child with autism. Nonsynonymous variants identified in the proband and his parents are shown. Grey filled symbol, affected autism; (B) Automated sequence chromatograms showing the RAI1 gene variation (arrow) in the proband. Triplet codon (underlined) and translated amino acids are shown; (C) An alignment of a region of human RAI1 showing the highly conserved 1147R residues (in bold type) altered in the proband; (D) Schematic representation of the domains (PolyQ, PolyS, nuclear localization signals (NLS1 and NLS2) and the PHD domain) contained in the RAI1 protein. The p.R1147Q alteration identified in this study is shown in bold. In addition, the previously described and functionally tested p.R1217Q, p.Q1389R, p.Q1562R and p.S1808N mutations are depicted.

Figure 2

In vitro evaluation of RAI1-p.R1147Q mutant. (A) Plasmid coding for the wild type or the mutant form of RAI1 was transfected in Neuro-2a cells and immunofluorescence was performed with anti-HA (green) antibody, while nuclei staining is shown with DAPI; (B) The subcellular localization (nuclear or cytoplasmic) of 200 positive cells for the immunodetection is summarized in the table; (C) The percentage of the reporter transcription activation is shown for RAI1-HA wild type (light grey column, N = 4) and p.R1147Q mutant (black column, N = 4); (D) Neuro-2a cells were co-transfected with a BDNF fused luciferase reporter plasmid, a β-galactosidase reporter plasmid, and either RAI1 wild type (white column, N = 3) or RAI1 p.R1147Q (dark grey column, N = 3). Twenty four hours post-transfection, the reporter proteins were measured from the cell lysates; (C,D) Activation of the reporter for the RAI1 wild type was considered 100% for normalization. Values represent mean ± SEM. (* = p < 0.05).