IDENTIFICATION OF NOVEL MECHANISMS FOR NEUROLOGICAL CONDITIONS OVERLAPPING SMITH-MAGENIS SYNDROME
Creators: name: SCIARRILLO, MARIA
Publisher: name: Universit� degli Studi di Milano
Year: 2018

description: Smith Magenis Syndrome (SMS, OMIM#182290) is a sporadic dominant disorder, with an estimated prevalence of 1:15000-25000 and results from RAI1 gene haploinsufficiency due to either 17p11.2 deletion or RAI1 mutation. SMS has a clinically recognizable phenotype characterized by distinct physical features, neurodevelopmental delay, cognitive impairment and behavioral problems which are reported in 75-100% of SMS cases, and include remarkable sleep disturbance (primarily due to circadian rhythms impairment), stereotypies, maladaptive, self-injurious behavior and sensory processing issues. SMS diagnosis is challenging due to the lack of a striking early childhood facial phenotype, maladaptive behavior that escalates with age, and overlapping syndromes that share with SMS most of the clinical signs and might switch to a differential diagnosis, i.e. 2q23.1 deletion syndrome (OMIM#156200) and Brachydactly Mental Retardation syndrome (BDMR, OMIM#600430). Despite RAI1 is recognized as the disease-causing gene, 50% of patients with a clinical suspicion of SMS do not have the classical genetic defects, thus it is likely that at genomic level other loci different from RAI1, if disrupted, eventually explain SMS similar phenotypes (SMS-like). Hence, even if several animal models support RAI1 crucial contribution to brain development and plasticity, by interacting at chromatin promoter and enhancer regions, compelling evidences on its function, regulators, interactors, and targets are still missing. In order to unveil the molecular basis of SMS-like syndrome and to clarify RAI1 molecular function, the main aim of this project will be a genetic and functional investigation of RAI1 and candidate genes possibly implicated in SMS-like clinical manifestation. A previously selected cohort of 40 SMS-like patients with a clinical suspicion of SMS but without the classical microdeletion at 17p11.2 or RAI1 mutation was available in Medical Cytogenetics and Molecular Genetics Laboratory. High resolution array CGH screening of whole cohort was used to identify Copy Number Variants (CNVs) potentially containing dosage-sensitive genes eventually involved in neurological integrity maintenance, cognition and development, thus putatively implicated in SMS and ?SMS-like? clinical condition. Among 40 SMS-like patients cohort the whole genome analysis pinpointed the attention on a CNV, specifically a 54 kb maternal deletion on Xq13.3 (chrX:74772380-74826319, hg19) in one male patient (SMS1). The Xq13.3 deletion does not involve any gene but contains highly conserved region, a predicted insulator and maps 29 kb far from 5? end of the ZDHHC15 (Zinc Finger DHHC domain-containing protein 15) gene which encodes for palmitoyl-transferase 15 ubiquitously expressed, but highly expressed in the brain. ZDHHC15 was considered an interesting gene possibly implicated in patient phenotype onset due to its function and because was previously associated to a nonsyndromic X-linked intellectual disability. RT-qPCR and digital PCR analyses performed on SMS1 cDNA from peripheral blood revealed a significant downregulation of the ZDHHC15 transcript, supporting that the CNV involving a predicted insulator element results in gene expression alteration by a position effect. Consistent with a possible involvement of ZDHHC15 in SMS-like phenotypes, subsequent Sanger sequencing of all male patients within the cohort was performed and identified a maternally inherited transversion, c.*182A&gt;C, on ZDHHC15 3?UTR in a second male patient (SMS2). In order to clarify any transcriptional effect on ZDHHC15 regulation caused by transversion, both relative RT-qPCR and digital PCR were carried out and allowed to show a slight but not significant ZDHHC15 downregulation in SMS2 cDNA from peripheral blood. Since 3?UTR can be target of several miRNAs playing a role in mRNA regulation, was investigated if in SMS2 the c.*182A&gt;C variant might have altered the normal target region of any miRNAs. Bioinformatic tools enable to select two miRNAs predicted to interact specifically with wild type ZDHHC15 3?UTR (miR-142-5p and miR-5590-3p) and three specifically with mutated ZDHHC15 3?UTR (miR-922; miR-191-5p and miR-4797-5p). Luciferase assay on HEK293T validated a specific and significant effect on wild type ZDHHC15 3?UTR sequence for miR-5590-3p and on mutated ZDHHC15 3?UTR sequence for miR-4797-3p, supporting the initial hypothesis of a possible transcriptional alterations due to A&gt;C transversion. The identification of two different alterations on ZDHHC15 regulatory regions in two unrelated cases in such small cohort of SMS-like patients further supported the possible direct or indirect involvement of ZDHHC15 in RAI1 pathway. To test in vitro whether a transient knockdown of RAI1 and ZDHHC15 would lead to change in expression of genes associated to the regulation of circadian rhythms we used silencing experiments on human BE(2)-M17 neuroblastoma cell line. Both RAI1 and ZDHHC15 silenced cells displayed significant deregulation of expression in up to half of the circadian genes. Moreover, nine out of main sixteen circadian gene proteins tested were predicted to be palmitoylated supporting an eventual role of ZDHHC15 in circadian rhythms control. In silico palmitoylation predictions and silencing experiments corroborate the idea of interconnection among RAI1, ZDHHC15 and circadian rhythms, but further analysis are needed to get a mechanistic insight. In conclusion the combined genomic and functional approach used, highlight ZDHHC15 as a promising candidate gene involved in SMS/SMS-like phenotypes.