A unique Smith-Magenis patient with a de novo intragenic deletion on the maternally inherited overexpressed RAI1 allele

Abstract

RAI1 is a dosage-sensitive gene whose decreased or increased expression by recurrent and non-recurrent 17p11.2 deletions or duplications causes Smith-Magenis (SMS) or Potocki-Lupski syndromes (PTLS), respectively. Here we report on a 21-year-old female patient showing SMS phenotype who was found to carry a 3.4 kb de novo intragenic RAI1 deletion. Interestingly, a significant increase in RAI1 transcript levels was identified in the patient's, brother's and mother's peripheral blood cells. Allele-specific dosage analysis revealed that the patient's maternally inherited overexpressed RAI1 allele harbors the intragenic deletion, confirming the SMS diagnosis due to the presence of a single wild-type RAI1 functional allele. The mother and brother do not present any PTLS neurologic/behavioral clinical features. Extensive sequencing of RAI1 promoter and predicted regulatory regions showed no potential causative variants accounting for gene overexpression. However, the mother and both children share a novel private missense variant in RAI1 exon 3, currently classified as a VUS (uncertain significance), though predicted by two bioinformatic tools to disrupt the binding site of one specific transcription factor. The reported familial case, the second showing RAI1 overexpression in the absence of RAI1 duplication, may help to understand the regulation of RAI1 dosage sensitivity although its phenotypic effect remains to be determined.

Fig. 1. Facial features of the patient.

Front facial view of the patient at 15 (A) 17 (B) and 21 years (C), showing brachycephaly, broad face, midface hypoplasia, and thick eyebrows.

Fig. 2. Molecular characterization of RAI1 intragenic deletion and expression analyses.

A MLPA profile’s probes for RAI1 gene reveal in the proband a heterozygous deletion of RAI1 involving exon 5. Each black dot displays the final probe ratio for each locus analyzed in the patient compared to controls. Standard deviations were set up according to the Coffalyzer DB software v131211. B Graphic representation of RAI1 deletion characterized in the patient at genomic and transcript levels. Top panel: electropherogram showing the deletion breakpoints at genomic level. The 3.4 kb RAI1 deletion is represented by a red bar involving part of IVS4, exon 5, IVS5 and part of exon 6 (chr17:17710076-17713445, hg19). Black bars above RAI1 exons refer to MLPA probes. Bottom panel: electropherogram of the aberrant RAI1 transcript, derived from the activation of a new splice-site in exon 6. C Wild-type and mutated RAI1 mRNAs and corresponding proteins with the insertion of 64 new amino acids highlighted in red. D Scatter plots obtained using TaqMan probe on exon junction 3-4, showing global RAI1 expression in patient (circles), brother (squares), mother (triangles pointing upward), father (triangles pointing downward) relatively to normal controls (rhombuses). The horizontal black bars indicate the range between mean ±2 standard deviation values. Data were normalized against TBP housekeeping gene. Similar results were obtained after normalization against GAPDH housekeeping gene (data not shown) and by using the Taqman probe on exon junction 2–3 (data not shown). E Scatter plots using the Syber green probe designed across exon junction 4–5, involved in the deletion showing specific expression of wt RAI1 transcript in:patient (circles), brother (squares) and mother (triangles pointing upward), respectively. Data were normalized against TBP. Similar results were obtained after normalization against GAPDH housekeeping gene (data not shown). F Family tree summarizing the RAI1 genotype and transcripts.