Single-Neuron Sequencing to Explore Somatic Genetic Variants in Normal and Pathological Human Brain Development

The human brain is one of the most exquisite structures in nature, featuring extreme functional complexity and capacities that allow for advanced cognitive abilities. During the development of the human brain, neural progenitors undergo massive proliferation, which is known to inevitably result in spontaneous mutations; yet the degree of somatic mosaicism within the human brain is unexplored. Several hypotheses have been proposed that various types of somatic mosaicism may serve as an adaptive mechanism to diversify neurons and thereby promote the functional complexity of human brains. Previously proposed mechanisms to increase somatic mosaicism within the brain include elevated somatic LINE-1 element retrotransposition, and the creation of somatic aneuploidy during neurogenesis. On the other hand, genomic diversity needs to be balanced by genomic stability, in order to protect against deleterious mutations that reduce the fitness of the cells, or oncogenic mutations that might promote cancers. In fact, brain-specific somatic mutations have also been proposed to contribute to the unexplained burden of neurological diseases. To directly study genomic variability from cell-to-cell within the human brain, we developed a method to isolate and amplify single neuronal genomes from postmortem and surgically resected human brain tissues. We quantified the frequency of somatic LINE-1 retrotransposition events and aneuploidy in human cortical neurons, and found that the frequencies of both are low, with no sign of brain-specific elevation, arguing against the hypotheses that these two mutational sources are obligate generators of neuronal diversity. Additionally, aneuploidy analysis was performed on bulk and single cortical cells from a hemimegalencephaly brain. Hemimegalencephaly is an asymmetrical brain overgrowth syndrome caused by somatic mutations in brain. Single-cell analysis identified an unexpected mosaic tetrasomy of chromosome 1q, affecting both neuronal and glial populations, as a genetic cause of hemimegalencephaly. These results demonstrate that single-neuron sequencing allows systematic assessment of genomic diversity in the human brain and the identification and characterization of pathogenic somatic mutations underlying neurological disorders.