Allele-biased expression in differentiating human neurons: implications for neuropsychiatric disorders

Abstract

Stochastic processes and imprinting, along with genetic factors, lead to monoallelic or allele-biased gene expression. Stochastic monoallelic expression fine-tunes information processing in immune cells and the olfactory system, and imprinting plays an important role in development. Recent studies suggest that both stochastic events and imprinting may be more widespread than previously considered. We are interested in allele-biased gene expression occurring in the brain because parent-of-origin effects suggestive of imprinting appear to play a role in the transmission of schizophrenia (SZ) and autism spectrum disorders (ASD) in some families. In addition, allele-biased expression could help explain monozygotic (MZ) twin discordance and reduced penetrance. The ability to study allele-biased expression in human neurons has been transformed with the advent of induced pluripotent stem cell (iPSC) technology and next generation sequencing. Using transcriptome sequencing (RNA-Seq) we identified 801 genes in differentiating neurons that were expressed in an allele-biased manner. These included a number of putative SZ and ASD candidates, such as A2BP1 (RBFOX1), ERBB4, NLGN4X, NRG1, NRG3, NRXN1, and NLGN1. Overall, there was a modest enrichment for SZ and ASD candidate genes among those that showed evidence for allele-biased expression (chi-square, p = 0.02). In addition to helping explain MZ twin discordance and reduced penetrance, the capacity to group many candidate genes affecting a variety of molecular and cellular pathways under a common regulatory process - allele-biased expression - could have therapeutic implications.

Figure 1. Sanger sequencing of selected X-linked genes.

Top panel in each strip is sequence of genomic DNA (gDNA) for iPSC-1, a control subject, confirming heterozygosity of each SNP. Other strips are sequences of cDNA derived from undifferentiated (undiff) and neurons cultivated under growth and differentiation conditions A and B (see Methods S1 for details). The RNA samples used to generate the Sanger sequencing data for iPSC-1 protocol A (undifferentiated iPSCs and neurons) were the same samples used for RNA-Seq.

Figure 2. Imprinted genes.

SNPs in KCNQ1 and CTNNA3 were validated by Sanger sequencing. See legend Figure 1 for details. iPSC-1 (II) is a biological replicate of iPSC-1.

Figure 3. Validation of A2BP1.

SZ39, SZ97 and iPSC-15 are iPSC lines developed using fibroblasts from patients with SZ.

Figure 4. Validation of NRG1 and ERBB4.

The NRG1 SNPs rs4602844 and rs1481757 map near the promoter of the NRG1 isoform HRG-β1C, and an intron in the GGF2/HRG-β1D isoforms, respectively.

Figure 5. Reference vs alternative RNA-Seq reads at heterozygous SNPs.

Y-axis, number (Nb) of reads with the alternative allele (211 in iPSCs, 619 in neurons); X-axis, number of reads with the reference allele (332 in iPSCs, 923 in neurons). Biallelic (grey); allele-biased expression (black).