Neurodevelopmental disorders (NDDs) are a group of heterogeneous disease affecting the nervous system, including autism spectrum disorders (ASD), and present in about 1-3% of children. ASD is characterized by communicative impairments, difficulties in social interaction, behavior abnormalities and complex pathogenetic mechanisms. Increasing findings support an oligogenic model of inheritance with a combination of inherited and/or de novo variants, involved in different pathways and biological processes. The presence in the same individual of multiple deleterious variants in different genes could contribute, according to an additive model, to heterogeneity of clinical features observed in NDD patients. The genetic etiology of NDDs has not yet been fully clarified. Poland Syndrome (PS, OMIM 173800) is a rare congenital condition (incidence of 1-9/100.000) with variable phenotype characterized by pectoral muscle agenesis/hypoplasia that may include ipsilateral thoracic and upper limb anomalies. Most cases of PS are sporadic, familial recurrence has been observed (4%) with phenotypic heterogeneity. Different inheritance patterns have been reported and polygenic/multifactorial mechanisms have been hypothesized in some cases. The genetic etiology of PS remains unknown. Recent studies proved that the use of Whole Exome Sequencing (WES) together with the copy number variations (CNVs) analysis can help to the identification of the molecular bases of heterogeneous genetic disorders. In this study we aimed at investigating the genetic mechanisms underlying NDDs and PS. i) A retrospective Array-CGH analysis was performed on 526 NDD cases. The pathogenicity of CNVs was investigated through mining of literature and searching through databases reporting genes associated with NDD, particularly intellectual disability (ID) and ASD. (ii) Fifty-three ASD families were investigated by Whole exome sequencing, and potentially deleterious variants prioritized by custom filtering strategies including the use of Oligogenic Resource for Variant Analysis Platform (ORVAL) and enrichment analysis of candidate genes with GeneCodis4. (iii) A cohort of 30 PS patients were analysed by WES, and potentially deleterious variants prioritized by custom filtering strategies including oligogenic variant analysis. Functional analyses of identified variants included in vitro mutagenesis followed by cell imaging and gene reporter assays. Pathogenetic CNVs have been found in 42% of NDD cases, while 58% of the identified CNVs remain of uncertain significance. The study of arrays data led to the identification of new genes and "double-hit" mechanisms that could account for the NDDs. By using an oligogenic approach to the analysis of WES data, we identified inherited variants in different genes that globally could explain the complex phenotype in some cases. The study also revealed the involvement of different genes in pathways and biological processes common to several patients with NDD. In patients with PS, deleterious variants were found in two different genes, one involved in the pectoral muscle and cartilage development, and one involved in mechanisms of cell polarity and asymmetric cell divisions that could contribute synergistically to the PS phenotype, suggesting a digenic mechanism.
I disturbi del neurosviluppo (NDD) sono un gruppo eterogeneo di disordini che coinvolgono il sistema nervoso, tra cui i disordini dello spettro autistico (ASD), che colpiscono circa l'1-3% dei bambini. Sono state rilevate differenti varianti causative a carico di molti geni, a evidenziare una eterogeneità genetica, ma per molti pazienti l’origine genetica rimane ancora sconosciuta. Emergono sempre più evidenze a sostegno di un’origine più complessa, caratterizzata da meccanismi oligogenici che vedono coinvolti due o più geni, con diverso impatto (major gene o modifier). La presenza in uno stesso individuo di varianti deleterie in geni differenti potrebbe avere determinato con effetto additivo il quadro clinico diversificato osservato nei singoli pazienti. La Sindrome di Poland (PS. OMIM 173800), rara patologia congenita (prevalenza 1-9/100.000), è caratterizzata da agenesia/ipoplasia del muscolo pettorale, generalmente unilaterale, talvolta associata ad anomalie toraciche e/o dell’arto superiore. Di solito presente in forma sporadica, un 4% di casi può mostrare famigliarità con eterogeneità fenotipica anche all’interno della stessa famiglia. Per la maggior parte dei casi è stata ipotizzata un’origine poligenica/multifattoriale anche se sono stati riportati casi con ereditarietà autosomica dominante e autosomica recessiva. Al momento non sono noti geni associati a PS. i) È stata eseguita un’analisi retrospettiva dei risultati di Array-CGH su 526 casi NDD. È stata valutata la patogenicità dei CNV identificati attraverso lo studio della letteratura e la ricerca in database che riportano geni associati a NDD, in particolare a disabilità intellettiva (ID) e ASD. (ii) È stata effettuata l’analisi dell’intero esoma (WES) di 53 famiglie con ASD ed identificato alcuni geni/varianti candidati. Per indagare il meccanismo oligogenico sono stati utilizzati vari approcci. I dati WES sono stati analizzati utilizzando una nuova piattaforma open source di analisi: ORVAL. È stata eseguita analisi di Gene Enrichment in pathways e processi biologici associati a NDD per i geni candidati ottenuti sia dall’analisi CNV che da WES utilizzando gli strumenti bionformatici GeneCodis4 e Cytoscape. (iii) Sono stati analizzati mediante WES 30 pazienti con PS che avevano caratteristiche fenotipiche simili a un caso indice in cui erano stati identificati 2 geni candidati per lo sviluppo della patologia. Sono stati effettuati studi funzionali delle varianti identificate per verificarne l’effetto su localizzazione cellulare e livelli di espressione delle proteine codificate dai geni mutati. Per questo obbiettivo, sono stati effettuati mutagenesi in vitro ed analisi di localizzazione cellulare (western blot di lisati cellulari frazionati, ImageStream e Opera Phenix), e test della luciferasi. Sono state trovate CNVs patogenetiche in circa il 42% dei casi NDD, mentre il 58% delle CNVs identificate rimangono di significato incerto. Lo studio dei dati array ha portato all’identificazione di nuovi geni e nuovi meccanismi (“double-hit” mechanisms) che potrebbero essere alla base di NDD. Meccanismi oligogenici sono stati anche riscontrati in alcuni casi della coorte di pazienti ASD analizzati con WES. Dallo studio effettuato è inoltre emerso il coinvolgimento di geni diversi in pathway e processi biologici comuni a più pazienti con NDD. Nei pazienti con PS sono state riscontrate varianti deleterie in due geni implicati nella formazione di muscolo pettorale e cartilagine e in meccanismi di polarità cellulare e divisioni cellulari asimmetriche che potrebbero contribuire sinergicamente al fenotipo PS facendo ipotizzare un meccanismo digenico.
Studio di meccanismi oligogenici alla base di patologie complesse dello sviluppo: disturbi del neurosviluppo e sindrome di Poland.
CERMINARA, MARIA
2022-05-13
Abstract
Neurodevelopmental disorders (NDDs) are a group of heterogeneous disease affecting the nervous system, including autism spectrum disorders (ASD), and present in about 1-3% of children. ASD is characterized by communicative impairments, difficulties in social interaction, behavior abnormalities and complex pathogenetic mechanisms. Increasing findings support an oligogenic model of inheritance with a combination of inherited and/or de novo variants, involved in different pathways and biological processes. The presence in the same individual of multiple deleterious variants in different genes could contribute, according to an additive model, to heterogeneity of clinical features observed in NDD patients. The genetic etiology of NDDs has not yet been fully clarified. Poland Syndrome (PS, OMIM 173800) is a rare congenital condition (incidence of 1-9/100.000) with variable phenotype characterized by pectoral muscle agenesis/hypoplasia that may include ipsilateral thoracic and upper limb anomalies. Most cases of PS are sporadic, familial recurrence has been observed (4%) with phenotypic heterogeneity. Different inheritance patterns have been reported and polygenic/multifactorial mechanisms have been hypothesized in some cases. The genetic etiology of PS remains unknown. Recent studies proved that the use of Whole Exome Sequencing (WES) together with the copy number variations (CNVs) analysis can help to the identification of the molecular bases of heterogeneous genetic disorders. In this study we aimed at investigating the genetic mechanisms underlying NDDs and PS. i) A retrospective Array-CGH analysis was performed on 526 NDD cases. The pathogenicity of CNVs was investigated through mining of literature and searching through databases reporting genes associated with NDD, particularly intellectual disability (ID) and ASD. (ii) Fifty-three ASD families were investigated by Whole exome sequencing, and potentially deleterious variants prioritized by custom filtering strategies including the use of Oligogenic Resource for Variant Analysis Platform (ORVAL) and enrichment analysis of candidate genes with GeneCodis4. (iii) A cohort of 30 PS patients were analysed by WES, and potentially deleterious variants prioritized by custom filtering strategies including oligogenic variant analysis. Functional analyses of identified variants included in vitro mutagenesis followed by cell imaging and gene reporter assays. Pathogenetic CNVs have been found in 42% of NDD cases, while 58% of the identified CNVs remain of uncertain significance. The study of arrays data led to the identification of new genes and "double-hit" mechanisms that could account for the NDDs. By using an oligogenic approach to the analysis of WES data, we identified inherited variants in different genes that globally could explain the complex phenotype in some cases. The study also revealed the involvement of different genes in pathways and biological processes common to several patients with NDD. In patients with PS, deleterious variants were found in two different genes, one involved in the pectoral muscle and cartilage development, and one involved in mechanisms of cell polarity and asymmetric cell divisions that could contribute synergistically to the PS phenotype, suggesting a digenic mechanism.File | Dimensione | Formato | |
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