Cells within tissues can be morphologically indistinguishable yet show molecular expression patterns that are remarkably heterogeneous. Here, we describe an approach for comprehensively identifying coregulated, heterogeneously expressed genes among cells that otherwise appear identical. The technique, called “stochastic profiling”, involves the repeated, random selection of very-small cell populations via laser-capture microdissection, followed by a customized single-cell amplification procedure and transcriptional profiling. Fluctuations in the resulting gene-expression measurements are then analyzed statistically to identify transcripts that are heterogeneously co-expressed. We stochastically profiled matrix-attached human epithelial cells in a three-dimensional culture model of mammary-acinar morphogenesis. Of 4,557 transcripts, we identified 547 genes with strong cell-to-cell expression differences. Clustering of this heterogeneous subset revealed several molecular “programs” implicated in protein biosynthesis, oxidative-stress responses, and nuclear factor-κB signaling, which were independently confirmed by RNA fluorescence in situ hybridization. Thus, stochastic profiling can reveal single-cell heterogeneities without measuring individual cells explicitly.

Mendelian and early-onset severe psychiatric phenotypes often involve genetic variants having a large effect, offering opportunities for genetic discoveries and early therapeutic interventions. Here, the index case is an 18-year-old boy, who at 14 years of age had a decline in cognitive functioning over the course of a year and subsequently presented with catatonia, auditory and visual hallucinations, paranoia, aggression, mood dysregulation, and disorganized thoughts. Exome sequencing revealed a stop-gain mutation in RCL1 (NM_005772.4:c.370 C > T, p.Gln124Ter), encoding an RNA 3′-terminal phosphate cyclase-like protein that is highly conserved across eukaryotic species. Subsequent investigations across two academic medical centers identified eleven additional cases of RCL1 copy number variations (CNVs) with varying neurodevelopmental or psychiatric phenotypes. These findings suggest that dosage variation of RCL1 contributes to a range of neurological and clinical phenotypes.

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