Using a large panel of highly selective markers for specific cell subtypes and a subset of autism candidate genes, we detected discrete pathological patches of abnormal laminar cytoarchitecture and disorganization in the majority of analyzed samples of prefrontal and temporal cortexes, but not occipital cortex, obtained from the boys and girls with autism who were included in our study. Serial analysis and three-dimensional reconstruction of multiple cellular markers revealed these regions to be focal patches of abnormal gene expression measuring 5 to 7 mm in length and spanning multiple contiguous neocortical layers. These patches were characterized by a decrease in the number of cells expressing layer- or cell-type–specific markers that are normally present in fully differentiated cortical neurons, as well as decreased expression of certain autism candidate genes.

The presentation of the patches was consistent within case samples but varied across cases. No cortical layer was uniformly spared, and the clearest evidence of abnormal expression was found in layers 4 and 5. Reduced marker expression was not due to a reduced number of neurons; the identity of the unlabeled neurons in the patches remains to be determined. These neurons may be layer-appropriate neurons that failed to express the marker, neurons in an immature or perturbed developmental state, or layer-inappropriate neurons. Our data are consistent with an early prenatal origin of autism or at least prenatal processes that may confer a predisposition to autism.

Although our data suggest a novel pathological mechanism in autism, they do not identify the mechanism. The identified laminar disorganization could result from migration defects resulting in the failure of cells to reach their targeted destination and the accumulation of such cells in nearby regions, as has been seen in mouse models.28 Alternatively, patches could reflect de novo changes early in neurodevelopmental processes, potentially in gene sequence or epigenetic state, which yield patch regions of affected progenitor cells adjacent to regions of unaffected progenitor cells. To test either model, a targeted analysis across large regions of neocortical tissue obtained from children with autism would be required, with detailed comparisons of gene sequence, methylation state, and expression profiles in identified regions with cortical patches, as compared with regions without such patches.

Even though we did not preselect for specific clinical endophenotypes, we identified pathological cortical patches in 10 of 11 case samples (91%) and in 1 of 11 control samples (9%). Because we sampled only small portions of cortex yet observed focal patches in nearly every case sample, the most parsimonious explanation is that pathological patches are widespread across prefrontal and temporal cortex in children with autism. Given the well-described phenotypic heterogeneity in autism, the presence of a relatively similar pathological feature across cases was unexpected. However, the features that we describe here may explain some of the heterogeneity of autism: disorganized patches in different locations could disrupt disparate functional systems in the prefrontal and temporal cortexes and potentially influence symptom expression, response to treatment, and clinical outcome. Within this model, the observation of an apparent patch in one control sample also raises the possibility of a subclinical patch phenotype.

We did not observe in the patches obvious abnormalities of marker expression specific to either microglia or astroglia, a finding that shows that the lack of in situ signal was not a nonspecific-tissue or processing artifact affecting messenger RNA integrity in general. The post hoc RT-PCR experiment guided by in situ hybridization further confirmed our original finding that patch regions represent areas of quantitative decrease in signal rather than artifacts from processing.

The strength of the standardized high-throughput colorimetric in situ hybridization used here is the reproducibility of labeling across large gene panels and the sensitivity of the method to label the soma of expressing cells in serial thin-tissue sections. This platform has been used extensively for genomewide mapping of brainwide transcript distributions in mouse brain25 and for targeted analysis in brain tissue obtained from nonhuman primates and from humans (www.brain-map.org). Our study design was informed by the accumulated knowledge of cell-type–specific gene expression and was tested to show similar specificity in a subset of genes that, when expressed, selectively label tissues in children's brains. Although in situ hybridization is semiquantitative, we were able to identify focal differences in cellular (laminar) distributions and decreased expression levels across cortical regions. We advise caution, however, in defining any nonpatch region as “normative” autism cortex, because previous studies have shown widespread pathological features (e.g., overabundance of neurons) in the prefrontal cortex of children with autism.3

Although our sample size was small in comparison with postmortem studies of adult diseases, it is as large as or larger than that in most previous postmortem studies of autism. The study was not limited by tissue quality, since frozen blocks obtained from each patient were evaluated and selected for high RNA integrity numbers before being sectioned and stained on in situ hybridization. Interpretation was not confounded by other variables of interest: pathological cortical patches were present in boys and girls, in high- and low-functioning children, and regardless of the cause of death or postmortem interval. The only two children with autism who had a history of medical complications were those with the least severe patch defects: Patient 21, the only child with autism in whom we did not detect patches, was the only child in our study with a history of severe seizures, and Patient 16, who had in utero exposure to cocaine and heroin, had the mildest pathological features with respect to patches (Table S2 in the Supplementary Appendix). Otherwise, prenatal and perinatal developmental histories were unremarkable and did not involve prematurity.

In conclusion, we identified discrete patches of disorganized cortex in the majority of postmortem samples obtained from young autistic children that we examined. These patches occurred in regions mediating the functions that are disturbed in autism: social, emotional, communication, and language functions. Such abnormalities may represent a common set of developmental neuropathological features that underlie autism and probably result from dysregulation of layer formation and layer-specific neuronal differentiation at prenatal developmental stages.