Basi genetiche e dello sviluppo della Sindrome di Opitz G/BBB
  • Responsabile: dott.ssa Germana Meroni

L’obiettivo di questo progetto è di identificare i meccanismi patogenetici implicati nella Sindrome di Opitz (OS), una forma genetica di disabilità intellettiva con difetti congeniti dello sviluppo. I processi mediante i quali mutazioni nel gene MID1 causano i difetti in OS sono ancora sconosciuti e la completa comprensione è ulteriormente complicata dall’eterogeneità clinica e genetica di questa sindrome. Nell’ambito del progetto utilizzeremo risorse uniche combinando genetica umana e murina in un approccio robusto per l’identificazione dei componenti molecolari implicati nello sviluppo della linea mediana. L’utilizzo di modelli murini che riproduce i difetti neurologici di OS permetterà di identificare i meccanismi coinvolti nell’ipoplasia cerebellare. La parallela caratterizzazione molecolare di una numerosa coorte di pazienti affronterà il problema dell’eterogeneità genetica in OS. L’integrazione dei due approcci definirà in meccanismi patogenetici e l’eterogeneità genetica con un forte impatto sia nelle conoscenze di base sia nella diagnosi di disabilità intellettive.

Background and Significance

Opitz Syndrome (OS) is a genetic form of intellectual disability with congenital midline defects caused by mutations in the MID1 gene, which encodes an E3 ubiquitin ligase of still unidentified developmental role. Full understanding of the pathogenesis is further complicated by OS clinical heterogeneity. In addition, our large cohort of OS DNA samples fully screened for MID1 mutations shows an elevated percentage of patients (more than 60%) with no detectable mutations in the MID1 gene pointing to genetic heterogeneity underlying this defect. To start addressing these issues, we generated a mouse model that recapitulates the neurological abnormalities observed in OS patients: anterior cerebellar vermis hypoplasia and behavioral impairments. Our approach integrating the OS mouse model study with the molecular characterization of the collection of OS samples points to comprehensively unravel OS pathogenetic mechanisms. Besides the strong impact on OS pathogenesis and diagnosis, this project has wider significance: a) abnormalities of the cerebellum are common to several disorders and the developmental processes governing its complex morphology and function are still unknown, yet crucial to understand human cerebellar pathologies; b) several mental retardation and congenital malformation syndromes share some of the OS midline defects complicating the diagnosis, which will be aided by the proposed comprehensive molecular characterization.

Specific aims

Aim 1: The proposed project is articulated into three integrated objectives that fully exploit two unique resources: an OS mouse model and a large collection of OS DNA samples. Aim1. Unravel the molecular events triggered by the lack of Mid1 in the cerebellum. We will exploit the Mid1 knock-out mouse line to dissect the mechanisms that contribute to the embryonic formation of the anterior vermis, through a cerebellum-specific transcriptomic and proteomic approach.

Aim 2: Investigate the genetic heterogeneity of OS. We will perform exome-sequencing of a group of patients showing the typical OS synopsis without carrying MID1 mutations to identify OS pathogenetic variants in novel genes.

Aim 3: Establish a pipeline for the molecular diagnosis of OS. We will integrate the list of genes/proteins altered in the mouse model (Aim 1) and those with candidate pathogenetic variants (Aim 2) to select genes to be validated in a large cohort (200 DNA sample) of non-MID1-mutated OS and OS-like samples.

Hypothesis

The mechanisms through which MID1 alterations cause the OS defects are largely unknown and, to further complicate our understanding of this disorder, the genetics underlying this disease appears to be more heterogeneous than thought. We hypothesize that the pathways altered in the OS mouse model we have generated reflect the pathogenetic mechanisms implicated in the human condition. The genes within these networks may underscore other genetic forms of OS and/or of OS-like conditions. The proposed combined approach exploiting both the animal model and a large cohort of OS patients will improve our understanding of the disease developmental mechanisms and allow the identification of additional OS (and OS-like) causative genes thus impacting on: i) the knowledge of cerebellar and other midline structures development; ii) the pipelines for patients' molecular screening; and iii) the re-definition, through molecular data, of the diagnostic criteria for a class of syndromic forms of intellectual disabilities.

Preliminary data

We performed transcriptome analysis upon laser capture microdissection of the developing cerebellum of Mid1 KO and WT mid-gestation embryos. We obtained differentially expressed genes (cut-off p<0.05; fold >1) that are significantly enriched in defined KEGG pathways and GO terms (cut off p<0.05). We have started the process of validation in new sets of embryos by real-time RT-PCRand RNA in situ hybridization. In parallel, we selected the first 12 genomic DNAs of OS male patients with typical clinical signs (Mental retardation, cerebellar hypoplasia, Hypertelorism, Hypospadias, laryngotracheoesophageal abnormalities) and performed Illumina exome-sequencing. Sequences were aligned to NCBI build 37 human genome reference using BWA. Upon mapping and variant calling (GATK), SNVs that are >1% in ESP6500SI, 1000Genome, and in our internal database were filtered out. We obtained variants in approximately 400 genes per patient with approximately 50 genes shared by at least 4/5 samples.

Materials and Methods

Mouse cerebellar studies. The mice used are Mid1 knock-out males generated in our laboratory and control wild-type littermates. Differential quantitative label-free proteomic analysis will be performed at the FingerPrints Proteomics Facility, University of Dundee, UK. RNA in situ hybridization, immunohistochemistry, and immunoblot will be employed to study thegenes and peptides differentially expressed in the knock-out embryos. Exome sequencing. In collaboration with Danilo Licastro (CBM, Trieste) we will perform exome sequencing using the

Illumina platform and reagents. Sequence data will be analyzed following the pipeline already employed in the first group of patients following the guidelines in the "GATK best practices v4". Targeted sequencing. We will design a oligonucleotide probe set for the targeted enrichment of the OS candidate genes. The design of the probes will be performed using the Illumina DesignStudio tool and sequencing performed as above.

Impact and Translational Implications

The accomplished experiments will: a) improve the counselling and management of OS and other midline intellectual disabilities with novel diagnostic molecular criteria; b) open the possibility to explore the effect of inhibitory/agonist compounds in the mouse model investigating possible treatments for some of OS clinical signs; c) add knowledge on the complex cerebellar developmental processes and on pathogenetic mechanisms that may be implicated in other disorders with cerebellar hypoplasia.

Periodo: 
da 01/12/2014 a 30/11/2017

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