Aberrant Proliferative and Organizational Pathways with Disrupted Cortical Lamination In Young Autistic Males

Background: Autism is a highly heritable neurodevelopmental disorder with abnormal brain enlargement and dysfunction in early years, but the genetic mechanisms remain unknown.  Aberrant brain overgrowth, then premature growth arrest and decline is a well-documented observation, but genetic and expression studies have failed to explain this abnormal growth pattern. Due to the scarcity of well-preserved brain tissue, few studies investigated genome-wide expression patterns in the autism brain, especially in regions that display the most flagrant growth abnormality.  Using small sample size and subsets of the genome, previous studies have suggested possible alterations in expression levels of neurodevelopmental and immune-related genes.  To confirm and extend these findings, we used tissue from key brain regions and tested the hypothesis that genotypic variation may account for the gene dysregulation of these pathways.  Furthermore, cortical phenotypic correlates of aberrant genetic expression must be examined to identify possible morphological consequences of these expression abnormalities and genotypic variation associated with autism.

Objectives: We aimed to identify genome-wide expression signatures, copy number variation (CNV), and cortical microstructure in autistic and control tissue from prefrontal cortex where growth abnormality is pronounced. We then sought to expand upon the genetic analyses using publicly available genotypic datasets.

Methods: Genome-wide expression analyses were performed in 33 autistic and control postmortem frozen tissue cases (ages 2-56 years). mRNA was processed with the DASL protocol and hybridized on Illumina microarray.  Differential expression was assessed using 2-way ANOVA with diagnosis and categorical age as variables of interest and validated by RT-PCR. Enrichment analyses were performed using Metacore. Nissl staining and in situ hybridization (ISH) of laminar specific genes were performed to identify microstructural correlates of aberrant expression in a subset of young autistic males.  Nissl sections were examined by a blinded neuropathologist and through blinded stereological measurement of neuron density. Subjective impressions of ISH abnormalities in autistic cases were systematically recorded.

Results:   Pathways and processes mediating cell cycle, DNA damage, apoptotic, cellular specification and neural patterning functions were aberrantly expressed in the young autistic prefrontal cortex. CNVs were highly enriched in cell cycle and cytoskeleton processes. In AGRE/BROAD/JHI datasets, genes mediating cell cycle processes were significantly associated with autism, but not genes regulating inflammation, apoptosis, or synaptogenesis. Histological examination of sections from young cases correctly identified 6/7 autistic cases as having areas of pathologically disorganized cortex. Regions neighboring or overlapping with these disorganized areas showed deficits of ISH laminar marker labeling.  Examination of neuron density showed a 17% increase in regions showing disorganization or ISH deficits compared to controls. 

Conclusions:  Dysregulation of proliferative processes, particularly cell cycle and DNA-damage pathways during neurodevelopment, could give rise to increased cell density and susceptibility to incorporation of DNA-damaged cells in the autistic cortex. Aberrant expression of neural patterning genes may lead to gross morphological and functional lateralization and anterior/posterior abnormality patterns identified by MRI and fMRI in individuals with autism. Finally, these expression deficits may be related to common and rare genotypic variation, playing a mechanistic role in the pathogenesis of autism.  Other implications will be discussed.