Smith-Magenis syndrome (SMS) is a complex mental retardation syndrome caused by deletion of 17p11.2 region or mutation of the RAI1 gene (retinoic acid induced 1). Individuals with SMS typically exhibit speech and motor delays, mental retardation, characteristic craniofacial and skeletal anomalies, and a distinct neurobehavioral phenotype that includes sleep disturbances, stereotypes, and maladaptive and self-injurious behaviors. RAI1 is thought to be a transcription factor modulating the expression of genes involved in a variety of cellular functions. Previous studies have shown the RAI1 gene being induced by retinoic acid (RA), a derivative of vitamin A. RA plays a significant role in many processes such as immune function, neurogenesis and reproduction, and deprivation of RA causes craniofacial defects. We hypothesized that RA could be inducing RAI1 which then acts as a transcription factor in modulating the expression of multiple genes. To understand the consequences of clinical variation of RAI1 gene, we performed mutation screening and identified the first case of SMS without mental retardation. Using a zebrafish model, full-length rai1 gene was cloned and spatial and temporal expression of rai1 by in-situ hybridization was evaluated and the effect of RA on rai1 expression was subsequently analyzed. The data show rai1 expression in forebrain (diencephalon) and midbrain. A rai1 antisense morpholino will eventually be created to perform knockdown studies and rescue experiments. These studies will help in determining the significance of the rai1 gene, and its interacting molecular pathways responsible for growth, development, and behavior.

Previous studies showed haploinsufficiency of RAI1 is the main cause of Smith-Magenis syndrome (SMS). SMS is a developmental neurobehavioral syndrome characterized by intellectual disability, congenital anomalies, obesity, neurobehavioral abnormalities, and disrupted circadian sleep-wake pattern. SMS is caused by deletion or mutation of chromosomal region 17p11.2 that includes RAI1. Studies in the Elsea lab have shown that RAI1 is a dosage-sensitive gene. Haploinsufficiency of RAI1 leads to dysregulation of CLOCK, NR1D2, POMC, and BDNF, which are responsible for circadian rhythm, metabolism, and cognitive development. Based on the data from Elsea’s recent study on zebrafish, rai1 gene expression in zebrafish is regulated by retinoic acid. Treatment with retinoic acid increases the expression of rai1. In this study, we focused on the effect of retinoic acid on human RAI1 expression. We found the expression of RAI1 was enhanced by the treatment with retinoic acid. The different concentrations of the retinoic acid affect the levels of increase in expression, but we found little evidence that RAI1 expression was affected by the length of treatment time. In this study, we were unable to find consistency in the pattern of changes in the expression level in respect to the different treatment concentrations. We identified nine strong retinoic acid response element (RARE) candidate sites upstream of the start codon in human RAI1. Since there are possible RARE sites present in the upstream sequence of RAI1, it is more likely that RAI1 is regulated by retinoic acid. However, further experimental evidence will be needed to confirm those sites selected in silico are able to bind RAR/RXR heterodimers, to prove the selected sites are real RARE sites and were not identified by chance.

The retinoic acid induced 1 (RAI1) gene maps within the Smith-Magenis syndrome (SMS) region on chromosome 17p11.2. Interstitial deletion of 17p11.2 including RAI1 or mutation of RAI1 results in SMS, while duplication of 17p11.2, including RAI1, results in the dup(17)(p11.2) syndrome. Smith-Magenis syndrome is a complex disorder characterized by a constellation of ~30 features that includes mental retardation, sleep disturbance, craniofacial defects, neurological and behavioral anomalies, and variable systemic features. Dup(17)(p11.2) syndrome is characterized by mental retardation, craniofacial defects, developmental delay, failure to thrive, and hyperactivity. We hypothesized that RAI1 is a dosage-sensitive gene with specific roles in SMS and dup(17)(p11.2) syndrome. To understand the clinical consequences of haploinsufficiency of RAI1 in humans, 60 SMS patients were evaluated by fluorescent in situ hybridization and/or sequencing of RAI1 to identify 17p11.2 deletions or intragenic mutations. Phenotypic comparison between patients with deletions and those with RAI1 mutations show that 21 of 30 SMS features are the result of haploinsufficiency of RAI1. Other features such as cardiac and renal anomalies, speech and motor delay, chronic respiratory and ear infections, hypotonia, short stature, and ear and eye anomalies are associated with 17p11.2 deletions rather than RAI1 mutations (P <0.05). Mouse models were evaluated using both qualitative and quantitative methodologies for phenotypic consequences due to altered Rai1 dosage. To this extent, BAC transgenic mice overexpressing Rai1 1.5-fold (hemizygous) or 2-fold (homozygous) and Rai1-targeted heterozygous (Rai1+/-) mice with 0.5-fold dosage were utilized. Compared to wild type littermates, Rai1 overexpressing mice have growth retardation, increased locomotor activity, gait abnormalities, and abnormal anxiety-related behavior, while Rai1+/- mice are obese and hypoactive. Analyses of homozygous BAC transgenic mice revealed a dosage-dependent exacerbation of the phenotype. Both the Rai1-overexpressors and Rai1-haploinsufficient mice showed neurological deficits. To identify target genes altered due to haploinsufficiency of RAI1, RNA-interference-based knockdown (~50%) of RAI1 was achieved in HEK293T cell lines. Genome-wide gene expression profiling showed that ~60 genes were upregulated and ~200 genes were downregulated due to RAI1 haploinsufficiency. Real-time qPCR not only confirmed the gene expression profile in HEK293 cells but also in lymphoblastoid cell lines obtained from SMS patients with 17p11.2 deletion. Further, our analysis has identified several RAI1 downstream genes, implicated in circadian activity, growth regulation, lipid biosynthesis, and neuronal regulation, which are potential candidate genes for non-deletion/non-RAI1-mutation cases of SMS and SMS-like phenotypes. These results show that Rai1 dosage has major consequences on molecular processes involved in growth, development, circadian rhythm, and neurological and behavioral functions, thus providing evidence for several dosage-thresholds for phenotypic manifestations causing dup(17p11.2) syndrome or Smith-Magenis syndrome in humans.

Smith-Magenis Syndrome (SMS) [OMIM, #182290] is a congenital anomaly and mental retardation (MCA/MR) syndrome associated with deletion of chromosome17p11.2 [1]. The clinical phenotype has been well described and includes minor craniofacial anomalies, self-injurious behaviors as well as sleep disturbances, speech delays, and obesity [1,2,3]. The incidence of SMS is estimated to be ~ 1:15,000 - 25,000 births [2,6]. Among SMS patients, ~90% are comprised of 17p11.2 deletions, while ~10% have RAI1 mutations [8]. All 17p11.2 deletions associated with SMS include RAI1 deletion [10]. RAI1 is thought to function as a transcriptional factor although its cellular role is still unclear. First, in order to better understand the role of RAI1 as a transcriptional factor and its relation to SMS, we confirmed that RAI1 regulates BDNF within an intronic region. This sequence was further narrowed down by utilizing the luciferase reporter assay. This test confirmed what was previously found using ChIP-chip assay and microarray analysis of Rai1+/- mice hypothalami. Next, in order to evaluate the role of Bdnf, an ampakine drug was administered to the Rai1+/- mouse model. A mouse model is a powerful tool for studying a specific gene. Rai1+/- mice exhibit the SMS phenotypes of obesity, craniofacial abnormalities, reduced pain sensitivities, seizures and others. Many physical, neurological, and behavioral tests were performed on the mice to see if any of the phenotypes can be rescued. Interestingly, twice-daily injections of ampakine CX1837 restored the pain sensitivities in Rai1+/- mice. The hot plate data suggest that BDNF potentially has a role in regulating the SMS phenotype of decreased pain sensitivity. In order to evaluate other genes that are altered as a result of the CX1837 ampakine drug, the whole brain's global gene expression was evaluated via microarray analysis. Two potential pain-related genes were identified to be upregulated due to drug administration, which could account for the pain phenotypes observed. One of the genes upregulated in treated mice was Osm, which is interesting because Osm is responsible for pain sensitivity. Further analysis is needed to confirm that an ampakine drug can potentially be used to treat SMS patients.

Smith Magenis Syndrome (SMS) is a multiple congenital anomalies/ mental retardation disorder caused by deletion or mutation of the RAI1 gene on chromosome 17p11.2. The majority of patients with SMS phenotypes have a deletion or mutation of RAI1. However, some patients have been observed with SMS-like phenotypes and yet have no deletions or mutations in the RAI1 gene. One possible explanation could be aberrant methylation of RAI1 since RAI1 is present and yet may be silenced. In order to study this possibility, patient cell lines were treated with 5-Aza-2’-deoxycytidine. RNA was extracted and real-time PCR was used to check the RAI1 expression status on the cells. RAI1 is thought to be a transcription factor, but the DNA binding sequence is still unknown. Sequences from ChIP-chip data were compared to identify a consensus sequence. One gene which contained this consensus sequence was the chemokine-like receptor-1 gene (CMKLR1), which was investigated by luciferase assay. CMKLR1 showed upregulation when co-transfected with RAI1.

Smith-Magenis Syndrome (SMS; OMIM #182290) is a multiple congenital abnormality and intellectual disability (ID) disorder caused by either an interstitial deletion of the 17p11.2 region containing the retinoic acid induced-1 (RAI1) gene or a mutation of the RAI1 gene. Individuals diagnosed with SMS typically present characteristics such as ID, self-injurious behavior, sleep disturbance, ocular and otolaryngological abnormalities, craniofacial and skeletal abnormalities, neurological and behavioral abnormalities, as well as other systemic defects and manifestations. Previous work by Vyas in 2009 showed temporal expression of rai1 in zebrafish embryos as early as 9 hpf. We hypothesize that there is maternal rai1 expression as early as zero hours post fertilization in wild type embryos. Using end-point PCR, we found that in fact there is maternal rai1 expression is detectable as early as 2 hours post fertilization (hpf) in wild type zebrafish embryos. Furthermore, we quantified rai1 expression using qPCR and found that rai1 expression declines significantly after 6 hpf. We hypothesize that a down regulation of rai1 or loss of rai1 will lead to morphological phenotypes, especially if that loss of rai1 function occurs during the earliest stages of zebrafish embryogenesis. Using a rai1morpholino oligonucleotide (MO), we found a loss of rai1 expression did not induce a morphological phenotype in in wild type embryos; furthermore, we also found that a loss of maternal rai1 expression did not induce a morphological phenotype as well. Utilizing a mutant rai1 zebrafish line, we found that both rai1 +/fh370 progeny nor rai1 fh370/fh370 progeny exhibited a morphological phenotype and that downstream targets such as bdnf were not affected by a reduction or complete loss of rai1. Prior research has shown that retinoic acid (RA) can induce rai1 expression. We hypothesize that RA can induce expression of rai1 during zebrafish embryogenesis. Using wild type fish and a rai1 in situ hybridization probe, we found that RA treatment at 25 hpf induced expression of rai1. The construction of a rai1 overexpression vector used for overexpression studies was started. Further development of GFP expression vector and zebrafish rai1 antibody are needed to determine if the morpholino is reducing rai1 protein expression.

Smith-Magenis syndrome (SMS) is a complex disorder caused by haploinsufficiency of RAI1 and characterized by sleep disturbances, behavioral abnormalities, mental retardation, and obesity in teens and adults. Rai1+/- mice are obese after 20 weeks. Dup(17)(p11.2) syndrome is a complex disorder associated with overexpression of RAI1. A transgenic mouse model of dup(17)(p11.2) syndrome overexpresses Rai1 and results in a mouse that is growth delayed. In order to characterize the obese phenotypes of mouse models of SMS and the role of RAI1 in obesity, daily food intake and serum levels of insulin, glucose, PPY, and leptin were measured; adiposity was studied by characterizing fat deposition; and gene expression was studied in the hypothalamus. These studies show that Rai1+/- mice are hyperphagic, consume more during the inactive light phase, and have altered satiety genes in the hypothalamus. Adiposity studies have shown WT females have a higher body fat content and visceral fat proportion than males, but Rai1-Tg and Rai1+/- females have similar fat deposition patterns as WT males. Hypothalamic gene expression studies show that many genes and pathways are affected by Rai1 and Rai1 dosage, including many genes associated with obesity and satiety.

Smith-Magenis syndrome (SMS) is a multiple congenital abnormalities intellectual disability syndrome that results from a deletion of chromosome 17p11.2 or mutation of the retinoic acid inducted one gene (RAI1). SMS is characterized by a multitude of phenotypic features including craniofacial defects, short stature, obesity, intellectual disability, self-abusive behavior, sleep disturbance and behavioral abnormalities. Interestingly, although SMS is a clearly defined syndrome with a known molecular change at its foundation, ~40% of all candidate cases sent to the Elsea lab for evaluation do not have a mutation or deletion of RAI1. We hypothesize that at least one other locus must be responsible for this Smith-Magenis-like (SMS-like) phenotype. To address this hypothesis, we first compiled a cohort of 52 subjects who had been referred to the Elsea lab for a clinical diagnosis of SMS. Once these individuals were confirmed to not have an RAI1 mutation or deletion, their phenotypes were compiled and statically analyzed to distinguish whether SMS and SMS-like cohorts are different in the prevalence of the core phenotypes of SMS such as, but not limited to, sleep disturbance, self-abusive behavior and developmental delay. SMS-like and SMS cohorts are not different in prevalence for these core features. Next, all SMS-like subjects were sent for whole genome array comparative genomic hybridization (aCGH) to identify duplications or deletions of each individual’s genome which contribute to the phenotype observed. We identified 6 pathogenic copy number variants (CNVs) in six individuals which contribute directly to the clinical phenotype, including two del(2)(q37). This study enabled us to draw relationships between SMS and other syndromes that had never been appreciated before and helped to identify pathways in which RAI1 may function. Using the data from our SMS-like study we were able to further characterize two known syndromes; Deletion 2q37 syndrome (brachydactyly mental retardation syndrome) and deletion 2q23 syndrome. With regard to deletion 2q37, syndrome we used genomic data from known and new deletion 2q37 subjects to refine the critical region to one gene: the histone deacetylase 4 gene (HDAC4). Using both clinical and molecular clues, we were able to identify one subject from our SMS-like cohort who has an insertion in HDAC4 which results in a premature stop codon. We conclude from this study that mutation of HDAC4 results in brachydactyly mental retardation syndrome. With regard to deletion 2q23 syndrome there were only five known cases in the published literature to which we were able to add two more. Using as similar approach to our del2q37 study we refined the critical region for this syndrome to one gene, the methyl binding domain 5 gene (MBD5). Using a molecular and clinical approach we were able to conclude that haploinsufficiency of MBD5 results in the core phenotypes seen in del2q23 syndrome including microcephaly, intellectual disabilities, severe speech impairment, and seizures. Using all the data generated from the three previous studies we set out to characterize the molecular function of RAI1. We hypothesize that RAI1 is a transcription factor that regulates gene expression of core genes involved in development, neurological function, and circadian rhythm. Using a ChIP-chip based approach we identified 257 transcripts we believe RAI1 regulates. Following up on these transcripts, using in vitro and in vivo methods, we have been able to conclude that RAI1 is a positive regulator of CLOCK, the master regulator of the central circadian cycle. Taken together, these studies have given us insight into the specific molecular changes that contribute to SMS and SMS-like syndromes. We have unveiled pathways and genes which are important to normal human development and behavior and identified novel functions of RAI1. These studies will provide the foundation for the future discovery of the pathways affected.