Dissecting the cellular and genetic disruptions in autism spectrum disorder comorbidities

Abstract

Around 80% of children with an autism spectrum disorder (ASD) have at least one other neuropsychiatric co-morbidity. The most common include attention-deficit/hyperactivity disorder, epilepsy, and depression, with co-occurrence rates at up to 70%. The causes of such disorders are known to have a high degree of inherited risk and have recently been shown to involve disruptions to gene expression programmes in important fetal cortical cell-types. However, there remains a gap in understanding the mechanisms surrounding these disorders and in the identification of key genetic and cellular players at risk during cortical development.

Therefore, we here analysed a publically available single-nuclei RNA sequencing dataset from ASD prefrontal cortex samples including each co-morbidity of interest listed above for differentially expressed genes and cellular disruptions. Here it was found that the cell-type with the most interesting and neurodevelopment-related disruptions were excitatory neurons, uncovered through a range of proportion and interaction computational analyses. The top most significant genes in excitatory neurons were then explored in a single-cell RNA sequencing fetal prefrontal cortex dataset to uncover which of these disrupted genes had relevant expression trajectories in developing excitatory neurons. Candidates were shortlisted for novelty and reliable expression patterns across multiple datasets, and modelled in vitro for analysis of functional disruptions to neurogenesis.

Four candidate genes, HNRNPC, CXXC4, C1QTNF4, and GNAL, were shortlisted after in silico analysis as they have no known role in neurodevelopment or the disorders and matched the above criteria. One gene – CXXC4 - appeared most interesting in the end, showing brain-specificity, disruption in excitatory neurons in the disorder sequencing dataset and promising interactions with neurodevelopmental regulators such as MEF2C. The candidates were then knocked down in an in vitro iPSC to neuron system modelled to reflect neurodevelopment to validate their potential role. The in vitro validation interestingly revealed a potential role of CXXC4 in neuronal proliferation and maturation as knockdown seen a reduction in the growth of cells and at day 10, had significantly increased the expression of neuronal markers TUBB3 and MAP2.

The overall goal of the study was to unveil novel gene expression signatures involved in important neurodevelopmental processes that, when disrupted, lead to each of the ASD co-morbidities of interest. This would help to provide a better understanding of co-occurring conditions at a transcriptomic and cell-type level and thereby aid in expanding research for earlier diagnosis, care, and intervention. We here identified a potential novel role of CXXC4 in neuronal proliferation and maturation that when upregulated, may lead to the development of ASD.

Date of Award	Jul 2024
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Northern Ireland Department for the Economy
Supervisor	Vijay Tiwari (Supervisor) & Mei Chen (Supervisor)