Detection of classical 17p11.2 deletions, an atypical deletion and RAI1 alterations in patients with features suggestive of Smith-Magenis syndrome

Abstract

Smith-Magenis syndrome (SMS) is a complex disorder whose clinical features include mild to severe intellectual disability with speech delay, growth failure, brachycephaly, flat midface, short broad hands, and behavioral problems. SMS is typically caused by a large deletion on 17p11.2 that encompasses multiple genes including the retinoic acid induced 1, RAI1, gene or a mutation in the RAI1 gene. Here we have evaluated 30 patients with suspected SMS and identified SMS-associated classical 17p11.2 deletions in six patients, an atypical deletion of ~139 kb that partially deletes the RAI1 gene in one patient, and RAI1 gene nonsynonymous alterations of unknown significance in two unrelated patients. The RAI1 mutant proteins showed no significant alterations in molecular weight, subcellular localization and transcriptional activity. Clinical features of patients with or without 17p11.2 deletions and mutations involving the RAI1 gene were compared to identify phenotypes that may be useful in diagnosing patients with SMS.

Figure 1

(a) Array CGH profiles of chromosome 17 in six patients with the classical deletion of SMS and schematic representation of chromosome 17p11.2. Representative gene content of the 17p11.2 deletion is shown. The RAI1 gene is indicated by a red arrow. Other deletions involving the 17p11.2 region described by Elsea and Girirajan are shown below. (b) Array CGH profile of chromosome 17 in SAG7567 and schematic representation of the RAI1 gene transcript. Black bars indicate qPCR amplicons used for confirmation of deletions found by arrayCGH. (c) Analysis by qPCR, which confirmed the occurrence of deletion of exon 3 of the RAI1 gene in six patients of SMS: PT1 (SAG6339) PT2 (SAG7131), PT3 (SAG7132), PT4 (SAG7571), PT5 (SAG5120) and PT6 (SAG6052), along with normal controls (NL1 and NL2). (d) Analysis by quantitative real-time PCR, which confirmed the occurrence of deletion of exon 2 of the RAI1 gene in one patient of SMS. The parental samples do not carry the deletion, shown with two normal controls (NL1 and NL2).

Figure 2

Automated sequence chromatograms showing RAI1 variants in patients. Triplet codon (underlined) and translated amino acids are shown. The two RAI1 gene heterozygous variations, (a) a c.3650G>A (p.R1217Q) in patient SAG4739 and (b) a c.4166A>G (p.Q1389R) in patient SAG6888 are shown. (c) Alignment of selected region of human RAI1 showing residues (in bold types) altered in patients that are conserved in other mammals.

Figure 3

In vitro evaluation of two point mutations associated with SMS. (a) Schematic representation of RAI1-HA, RAI1-HA R1217Q and RAI1-HA Q1389R. In blue is represented the poly-Gln domain, in yellow: poly-Ser domains, the in silico described nuclear localization signals are depicted in black, in slanted line: the PHD domain. The coding sequence for HA epitope is represented in red. Both generated mutants were confirmed by DNA sequencing (data not shown). (b) The molecular weight of mutated proteins was calculated by western blot analysis with anti-HA antibody in Neuro-2a cells extracts. The obtained molecular weight is depicted and also the controls for the immunoreactivity are shown (e/v, extracts transfected only with the empty vector and u/t represents untransfected cells control). Anti-β-tubulin was used as a loading control. (c) The percentage of the reporter transcription activation is shown for RAI1-HA R1217Q (white) and RAI1-HA Q1389R (gray) compared with RAI1-HA wild-type protein (black). Values represent mean±SEM. (d) Each plasmid was transfected in Neuro-2a cells and immunofluorescence was performed with anti-HA (green) antibody, whereas nuclei staining is shown with DAPI. (e) The subcellular localization of 200 positive cells for the immunodetection is summarized. α, Antibody against.

Figure 4

Facial features of patients with (a) a classical deletion (SAG6052), (b) an atypical deletion (SAG7567), (c) a nonsynonymous variant, p.R1217Q (SAG4739) and (d) a patient from the SMS-like group, with no known alteration.