ONGOING PROJECTS
Our multidisciplinary team is our strength. Since 2015 we are working on neurodevelopmental disorders. Read below to know something more about our ongoing projects.
VALIDATION OF A PRECLINICAL MODEL FOR THE EVALUATION AND DEVELOPMENT OF NEW THERAPEUTICAL APPROACHES IN DUP15Q DISEASE
Funding: Nonsolo15 Foundation
Dup15q syndrome is a neurodevelopmental disorder determined by the presence of one or more supernumerary copies of the q11.2-q13.1 region of chromosome 15 of maternal origin. The incidence of the syndrome is estimated to be about 1 in every 30000 live births.
Typical manifestations of the syndrome include central hypotonia, global psychomotor developmental delay, moderate to severe intellectual disability, autism, and epilepsy.
Epilepsy affects more than 50 percent of affected individuals, most frequently begins in childhood, and is generally drug resistant.
The frequency and severity of dup15q-associated epilepsy denote the need to deepen our knowledge of the mechanisms of epileptogenesis related to the syndrome and develop specific therapeutic strategies. In this perspective, it is important to have preclinical models that are representative of the main pathophysiological mechanisms underlying the disorder and significantly correlated with clinically observable variables.
The present project proposes an innovative approach to study mechanisms of epileptogenesis and to optimize treatment prediction.
At the end of August 2022, 4 fibroblast lines from 4 different dup15q patients were obtained. Between September and October these lines were expanded and frozen at Prof. Alfredo Brusco's laboratory, ready for the next step.
In mid-December 2022, these fibroblasts were shipped to the Austria-based company, Neurolentech GmbH, to be reprogrammed into induced pluripotent stem cells (hiPSCs). After about 3 months, once ready, these patient’s derived stem cells, together with healthy control derived pluripotent stem cells, will be sent to the University of Twente in Enschede, the Netherlands, at the laboratory of Dr. Monica Frega.
There, they will be differentiated into cortical glutamatergic neurons and, together with those derived from control subjects, will be cultured, and studied on micro-electrode arrays (MEAs), which are cell culture dishes with embedded micro-electrodes that allow non-invasive measurement of neuronal network activity.
We expect that the networks created by hiPSCs of patients with epilepsy will show different types of activity compared to healthy people, leading to the identification of a signature, or "electrical signature," characteristic of affected patients.
Next, both groups of neurons, will be treated with possible drugs that could counteract epilepsy and the effect in vitro will be evaluated.
In this way, it is possible to evaluate the possibility of reversing this "electrical signature," returning it to the physiological levels observed in unaffected controls.
This study will allow in vitro testing of the efficacy of drug treatments directly on patient-derived cells, enabling the development of personalized therapies.
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MULTIOMIC STRATEGIES TO IMPLEMENT THE DIAGNOSTIC WORKFLOW OF RARE DISEASES
Funding: PNRR2022 Ministero della Salute
Project code: PNRR-MR1-2022-12376067
Exome sequencing (ES), used to diagnose rare genetic disorders, has a limited yield (30-40%), and a major
challenge is a need for additional functional tests to classify uncertain variants. To fulfil these clinical
requests, we will provide a multiomic approach to improve current diagnostic pipelines in the National
Health System.
Starting from a survey of ES-negative cases, we will develop bioinformatics tools to perform a routine re-
analysis of ES data. The computational pipeline will be based on a state-of-the-art method specifically
tailored to neurodevelopmental disorders by training it on known causative variants. The sole reanalysis is
expected to solve a fraction of cases - thanks to the improved bioinformatic pipeline and updated
biomedical knowledge - which represent a very efficient way to use already produced genomic data. After
the reanalysis step, most patients will remain unsolved with either no candidate variants or Variants with
Unknown clinical Significance (VUS) and will be subject to triage to address them toward one of the
predefined procedures for functional validation: (1) those with likely splicing-altering VUS in phenotypically-
compatible genes will be addressed to a standardized experimental workflow (minigene assay and RT-PCR);
(2) those with VUS in genes that have been associated to known and reliable episignatures will be
addressed to the methylation assay; (3) all the rest, including those without identified VUS will be
addressed to Whole Transcriptome Sequencing (WTS). The combination of triage and predefined functional
tests is designed to achieve the best results in a rational and efficient way.
It is expected that most of the patients will be addressed to WTS which has been proved to be very
effective in identifying regulatory variants. To overcome the inaccessibility of the affected tissue (i.e.,
central nervous system), our approach will be based on the sampling of peripheral blood and exfoliated
epithelial cells. This alternative is well supported by recent data and makes the overall sampling procedure
a lessened burden for clinicians and patients. Episignatures analysis is especially effective if the studied
subject is compared with a large dataset of already known cases. Therefore, methylation will be shared
with the group of prof Sadikovic from London (Canada) who is already a long-standing collaborator of one
of our units.
Our project leverages a multidisciplinary team with a synergistic and complementary integration that
includes expertise in clinical genomics, molecular genomics, computational biology, and molecular
diagnostics. Another important aspect of the project will be the detailed collection of procedures, results,
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and expenses in a structured and quarriable database that will be implied to assess the diagnostic
sensitivity and cost-effectiveness performances.
NEUDIG
Unveiling the hidden side of NEUrodevelopmental DIsorder Genetics
Funding: PRIN 2020
Code: 20203P8C3X
Role: Participant laboratory
Neurodevelopmental disorders (NDDs) are a group of disorders caused by the disruption of essential neurodevelopmental processes. NDDs include autism spectrum disorder, intellectual disability, attention deficit hyperactivity disorder, and epilepsy. Familial NDDs have been instrumental for identifying the contribution of genetic factors to the pathogenesis of NDDs. It has emerged that the phenotypic outcome of NDDs depends upon highly penetrant rare/de novo monogenic variants or common low risk variants leading to multifactorial/polygenic disease. Focusing on the former category, we have been collecting a large survey of 1,100 NDD families analysed by a-CGH and trio-WES. Despite the implementation of sequencing technologies and the numerous novel NDD-causative genes identified, the percentage of patients who remain undiagnosed at the molecular level is still high (70%).
Multiple reasons can account for this: lack of information which leads to missed pathogenic variants (gene unknown at the time of the analysis; scanty information on the variants found); technical restriction of screening methods (low covered regions; missed structural variants); incomplete bioinformatic analyses. Furthermore, many novel genes are still to be annotated and uncommon disease patterns are easily missed (e.g., novel imprinting disorders, TAR-like phenotype, TADopathies).
We aim to further clarify the complex genetic bases of NDDs exploiting an integrated multidisciplinary team. We will start from the harmonization and re-analysis of our trio whole-exome sequencing dataset. We will combine several variant filtering options and evaluate incomplete penetrance/variable expressivity and missed CNVs. A selected group of 50 undiagnosed families (quad) will constitute the core of our project: we will perform whole genome sequencing and prepare patient-derived cortical neuronal cell lines generated from induced pluripotent stem (iPS) cells. These cells will be used for tissue-specific transcriptomic profiling neuron-derived and integrated transcriptomic/genomic studies. We will perform network and pathway analyses exploiting up-to-date machine learning models for variant interpretation. The final task of our project will involve functional characterization of selected variants by genetic, biochemical, cellular and epigenomic assays.
We expect to identify new genes and genomic mechanisms involved in NDDs. In addition, the present project will produce significant deliverables: a unique collection with genomic and phenotypic information for NDDs, standardized procedures to extract maximal information from genomic data, allowing iteration and sharing among different centers; a valuable set of iPS cell lines from patients with NDDs that will be made available to the scientific community, a comprehensive and expandable functional map of molecular pathways involved in NDD, protocols and materials for a functional diagnostic pipeline to interpret unconventional genomic variants.
CAPRIN1-RELATED NEURODEVELOPMENTAL DISORDERS: SEARCHING FOR AFFECTED PATHWAYS AND FOR THERAPEUTIC APPROACHES
Funding: Fondazione Emma ed Ernesto Rulfo 2022
Background and preliminary data
The Cell cycle Associated PRoteIN 1 (CAPRIN1) (MIM *601178) is an haploinsufficient gene (GnomAD pLi=0.97) highly expressed in brain, whose product is and involved in the transport of mRNA in neurons. This gene is a strong candidate for neurodevelopmental disorders; a Caprin1+/- mouse model is characterized by a reduction in social interactions, lower response to novelty1 and impaired synaptic plasticity2.
In the past years, we identified nine cases carrying Loss-of-function (LoF) variants in CAPRIN1, characterized by language impairment (100% of cases), speech delay (89%), intellectual disability (88%), ADHD (86%), ASD (68%), developmental delay (33%) and seizures (22%) (manuscript in preparation). The characterization of patients-derived cells (fibroblasts, PBMC or EBV-transformed lymphoblastoid B-cell lines), showed us a half dose of CAPRIN1 mRNA and protein, and the exclusive expression of the wt allele, supporting CAPRIN1 haploinsufficiency.
We characterized the effect of CAPRIN1 loss, using cortical neurons derived from hiPSCs engineered with CRISPR/Cas9 technology. Our results, showed a complete disruption of neuronal organization, reduced processes length and increased neuronal death in CAPRIN1+/- neurons, likely mediated by an increased oxidative stress and calcium influx. We also observed a reduced electrical neuronal activity, using multi-electrode arrays (MEA), and an increase in the global translation rate in the CAPRIN1+/- neurons.
Rationale and aim of the work
CAPRIN1 plays a central role in synaptic plasticity, and it is expected to regulate many of its actors. For this reason, we think that studying CAPRIN1 we will obtain a wider knowledge on the genetic basis of neurodevelopmental disorders.
Furthermore, we want to understand the pathways impaired by CAPRIN1 haploinsufficiency, with the long-term purpose of finding therapeutic approaches that could be used to treat patients affected by CAPRIN1-related neurodevelopmental disorder.
UNDERSTANDING TANGO2 PATHOGENIC MECHANISMS IN HIPSCS-DERIVED NEURONS
Funding: TANGO2Research Foundation 2022
TANGO2 gene is associated with an autosomal recessive disorder characterized by recurrent metabolic encephalomyopathic crises with rhabdomyolysis, cardiac arrhythmias, and neurodegeneration (MIM #616878). Despite the scientific efforts, pathogenic mechanisms underlying TANGO2-related disease are still largely unknown, as well as the reasons behind the pleiotropic nature of the syndrome and the significant clinical variability among patients.
Because an important lack of knowledge relates to the impairment of the neurological compartment, our work will focus on investigating the role of TANGO2 in neurons and provide initial insights into the genetic bases of the disease clinical variability.
We will generate human induced pluripotent stem cells (hiPSC) from fibroblasts of two siblings with the same TANGO2 genotype, but clinically discordant (one affected by a severe, the other by a mild/inapparent form). Patients-derived hiPSC will be used to obtain neuronal progenitor cells (NPC) and mature neurons in vitro. Using these cells, we will study cellular morphology and vitality, and perform a transcriptome analysis, comparing the two siblings with healthy control subjects. In addition, we will perform exome sequencing of patients’ DNA, to better investigate the potential impact of variants in genes for TANGO2-related proteins.
With our project, we will combine next generation sequencing technologies (exome sequencing and transcriptome) and innovative in vitro models (hiPSCs-derived NPC and neurons), to shed light on altered pathways in neurons and the mechanism behind the different severity degrees observed in patients, potentially identifying genetic modifiers of the disease.
CAPRIN1: A NEW GENE INVOLVED IN AUTISM SPECTRUM DISORDER
Funding: Fondazione Emma ed Ernesto Rulfo 2021
Introduction
Autism Spectrum Disorders (ASDs) are a group of neurodevelopmental diseases with a significant - and extremely heterogeneous - genetic component. Novel scientific developments in the field of molecular genetics have allowed to greatly increase our knowledge of their genetic determinants. Approximately 5-15% of ASD cases result from copy number variants (CNVs), i.e., deletions/duplications of specific chromosomal regions, the most common being at 7q11, 15q11–13, and 22q11.2. Nowadays, the screening for pathogenic CNVs by microarrays is so important to be considered a first-level approach in the molecular diagnostic of ASD. More recently, high throughput DNA sequencing technologies have allowed to screen for the entire subset of human genes (exome) in trios which allowed identifying more than a hundred of different single-gene diseases associated with ASD. At least 1,000-1,500 genes are estimated to be involved in ASD. All genetic modes of inheritance are possible, but noteworthy, de novo mutations are frequent and explain severe ASD cases, accounting for the reduced number of familiar cases. The list of candidate genes involved in ASD is continuously increasing as the complexity of data supporting their pathogenicity (see SFARI database). ASD-causing genes have started providing clues on functional pathways involved in their pathogenesis. We are uncovering that three main pathways seem critically destroyed in ASD: synapse development and function; growth, transcription regulation and protein synthesis; serotonin signaling and neuropeptides. We are at the dawn of ASD genetics, which is starting to unravel the pathogenic mechanisms associated with these so far undecipherable disorders (Brusco and Ferrero, Genomic Architecture of ASD, 2019).
Preliminary data
We have identified Cell cycle-Associated PRoteIN 1 (CAPRIN1, MIM601168) as a possible novel gene associated with autism spectrum disorders. Caprin-1 is a ubiquitously expressed RNA-binding protein, whose gene maps on 11p13. Caprin-1 protein localizes in neuronal RNA granules of dendrites, where it binds key mRNAs involved in neuronal activity and synaptic plasticity, such as CamKIIα, BDNF, CREB, MAP2 and TrkB. Through its N-terminal domain, Caprin-1 is also able to repress mRNA local translation when it is released from neuronal granules, following synaptic stimulations. The translational repression is limited to specific targets since absence of Caprin-1 does not affect global rates of protein synthesis. Through its HR1 and HR2, Caprin-1 is able to interact with FMRP and G3BP1, which are known to be components of ribonucleoprotein (RNP) complexes such as RNA granules, forming a Caprin-1/G3BP1/FMRP complex. Caprin-1 is suggested to be a scaffold protein able to mediate the formation of distinct RNP complexes. Caprin1+/- mice show alteration in social behaviour, lower response to novelty and difficulties in reversal learning compared to controls. Heterozygous Caprin1 deficiency in animal models cause alteration in the structure of dendritic spines, that further cause a reduction in the postsynaptic response to stimulation, impairment of long-term memory formation and degeneration of neural networks. Moreover, Caprin1 loss in neurons causes differential expression of a subset of mRNAs that encode proteins involved in AMPAR localization, membrane potential control and actin reorganization.
Rationale and aim of the work
We have collected eight cases with de novo or inherited heterozygous loss-of-function (LoF) variants in CAPRIN1, whose phenotype includes language delay and autism spectrum disorder (ASD), often associated with attention deficit hyperactivity disorder (ADHD), intellectual disability (ID) and seizures. CAPRIN1 is an haploinsufficient gene (pLI=0.97, GnomAD) highly expressed in brain [PMID:27525107;31367017;28135719]. It is a strong candidate for ND, because it encodes a protein hub which regulates several synaptic proteins (e.g., FMRP, G3BP1). In animal models, Caprin1 is crucial in the development of dendrites and dendritic spines [PMID:20861386;26865403]; behavioural studies on Caprin1+/- mice have shown a reduction in social interactions, lower response to novelty and glutamatergic receptor-mediated alterations in long-term potentiation (LTP) mechanisms [PMID:26865403;29157358].
Despite the above strong evidences, a clinical and molecular description of patients carrying pathogenic variants in CAPRIN1 has not yet been published. We aim at proving CAPRIN1 causes a novel synaptopathy, providing a functional characterization in human cells.