Final Report Summary - CBCD (Understanding the basis of cerebellar and brainstem congenital defects: from clinical and molecular characterisation to the development of a novel neuroembryonic in vitro model)
Cerebellar and brainstem congenital defects (CBCDs) represent a heterogeneous group of congenital or early-onset disorders characterized by extreme phenotypic variability, that yield major consequences in terms of morbidity and mortality and represent a common cause of termination of pregnancy when diagnosed prenatally. Along with typical cerebellar signs such as ataxia and nystagmus, patients often present intellectual disability and other cognitive and behavioral dysfunctions, visual problems and variable multiorgan involvement including the kidneys, skeleton and liver. When this project started, the genetic basis of many CBCDs was still undetermined.
This project, articulated in four workpackages, addressed the following aims: i) to perform a detailed clinical, neuroradiological (WP1) and genetic characterization (WP2-3) of large cohorts of CBCD patients; ii) to improve knowledge on the genetic basis of mendelian and sporadic CBCDs (WP2-3); iii) to make use of in vitro models based on mouse embryonic stem cells (mESCs) and then patient-derived induced pluripotent stem cells (iPSCs) differentiated towards the neuronal lineage to assess in vitro the developmental defects underlying some CBCDs (WP4). The overall goal was to improve pre- and post-natal diagnosis, genetic counseling, patients' management and prognosis, and to help the development of rehabilitation strategies targeted to specific problems in CBCD patients.
The results generated in the course of the project are briefly summarized here:
WP1: Over 1.200 patients with various forms of CBCDs, including about half with Joubert syndrome (JS) and related ciliopathies, have been recruited from Italian and international centres. For each patient, a detailed clinical questionnaire, brain MRI and blood/DNA samples were collected, and neuroimaging have been evaluated by expert neuroradiologists to reach a consensus diagnosis. A careful assessment of clinical and instrumental data allowed defining some novel clinical features associated to CBCDs and reporting specific laboratory findings as sensitive biomarkers of organ involvement, improving management and prognosis. Detailed assessment of the brain malformation and/or DTI-tractography have been performed in a subset of patients (e.g. with tubulinopathies, Poretti-Boltshauser syndrome and ponto-cerebellar hypoplasias) allowing a fine characterization of the defect. Long-term follow-up of patients also allowed to delineate the natural history of some common CBCDs such as JS, describing the outcome and quality of life of these patients when reaching adult age.
WP2: Next-generation-sequencing of known genes has been performed in about 600 patients with various mendelian CBCDs (JS, ponto-cerebellar hypoplasias, Poretti-Boltshauser syndrome, tubulinopathies), reaching a genetic diagnosis in about two-thirds of cases, and allowing the definition of relevant gene-phenotype correlates, with important implications for counselling and management. Whole exome sequencing in mutation-negative families led to the identification and functional characterization of several novel CBCD genes causative of either JS (TMEM216, CEP41, PDE6D, SUFU, KIAA0586) or other CBCDs (GSX2, TTL, TOE1, FSD1L).
WP3: over 100 sporadic CBCD patients underwent high resolution array, allowing the detection of chromosomal imbalances in about 10%. We excluded a major role for chromosomal defects in common sporadic CBCDs such as isolated cerebellar hypoplasia, with relevant implications for the diagnostic path. A high-resolution custom array has been designed to search for heterozygous copy number variants in 26 JS patients in whom only one pathogenic mutation was previously identified by NGS analysis, increasing the diagnostic yield of about 10%.
WP4: We developed and characterized mESCs and patient-derived iPSCs to study the embryonic neural specification in vitro. We showed that these cells possess cilia and that two JS-related proteins, Meckelin and Jouberin, are necessary for neuronal development. This optimized model is currently being used in the lab as a main tool to compare the neurodevelopmental defects among iPSCs from JS patients carrying mutations in distinct genes.
This project, articulated in four workpackages, addressed the following aims: i) to perform a detailed clinical, neuroradiological (WP1) and genetic characterization (WP2-3) of large cohorts of CBCD patients; ii) to improve knowledge on the genetic basis of mendelian and sporadic CBCDs (WP2-3); iii) to make use of in vitro models based on mouse embryonic stem cells (mESCs) and then patient-derived induced pluripotent stem cells (iPSCs) differentiated towards the neuronal lineage to assess in vitro the developmental defects underlying some CBCDs (WP4). The overall goal was to improve pre- and post-natal diagnosis, genetic counseling, patients' management and prognosis, and to help the development of rehabilitation strategies targeted to specific problems in CBCD patients.
The results generated in the course of the project are briefly summarized here:
WP1: Over 1.200 patients with various forms of CBCDs, including about half with Joubert syndrome (JS) and related ciliopathies, have been recruited from Italian and international centres. For each patient, a detailed clinical questionnaire, brain MRI and blood/DNA samples were collected, and neuroimaging have been evaluated by expert neuroradiologists to reach a consensus diagnosis. A careful assessment of clinical and instrumental data allowed defining some novel clinical features associated to CBCDs and reporting specific laboratory findings as sensitive biomarkers of organ involvement, improving management and prognosis. Detailed assessment of the brain malformation and/or DTI-tractography have been performed in a subset of patients (e.g. with tubulinopathies, Poretti-Boltshauser syndrome and ponto-cerebellar hypoplasias) allowing a fine characterization of the defect. Long-term follow-up of patients also allowed to delineate the natural history of some common CBCDs such as JS, describing the outcome and quality of life of these patients when reaching adult age.
WP2: Next-generation-sequencing of known genes has been performed in about 600 patients with various mendelian CBCDs (JS, ponto-cerebellar hypoplasias, Poretti-Boltshauser syndrome, tubulinopathies), reaching a genetic diagnosis in about two-thirds of cases, and allowing the definition of relevant gene-phenotype correlates, with important implications for counselling and management. Whole exome sequencing in mutation-negative families led to the identification and functional characterization of several novel CBCD genes causative of either JS (TMEM216, CEP41, PDE6D, SUFU, KIAA0586) or other CBCDs (GSX2, TTL, TOE1, FSD1L).
WP3: over 100 sporadic CBCD patients underwent high resolution array, allowing the detection of chromosomal imbalances in about 10%. We excluded a major role for chromosomal defects in common sporadic CBCDs such as isolated cerebellar hypoplasia, with relevant implications for the diagnostic path. A high-resolution custom array has been designed to search for heterozygous copy number variants in 26 JS patients in whom only one pathogenic mutation was previously identified by NGS analysis, increasing the diagnostic yield of about 10%.
WP4: We developed and characterized mESCs and patient-derived iPSCs to study the embryonic neural specification in vitro. We showed that these cells possess cilia and that two JS-related proteins, Meckelin and Jouberin, are necessary for neuronal development. This optimized model is currently being used in the lab as a main tool to compare the neurodevelopmental defects among iPSCs from JS patients carrying mutations in distinct genes.