State, Matthew W.

INTRODUCTION The human cerebral cortex has undergone an extraordinary increase in size and complexity during mammalian evolution. Cortical cell lineages are specified in the embryo, and genetic and epidemiological evidence implicates early cortical development in the etiology of neuropsychiatric disorders such as autism spectrum disorder (ASD), intellectual disabilities, and schizophrenia. Most of the disease-implicated genomic variants are located outside of genes, and the interpretation of noncoding mutations is lagging behind owing to limited annotation of functional elements in the noncoding genome. RATIONALE We set out to discover gene-regulatory elements and chart their dynamic activity during prenatal human cortical development, focusing on enhancers, which carry most of the weight upon regulation of gene expression. We longitudinally modeled human brain development using human induced pluripotent stem cell (hiPSC)–derived cortical organoids and compared organoids to isogenic fetal brain tissue. RESULTS Fetal fibroblast–derived hiPSC lines were used to generate cortically patterned organoids and to compare oganoids’ epigenome and transcriptome to that of isogenic fetal brains and external datasets. Organoids model cortical development between 5 and 16 postconception weeks, thus enabling us to study transitions from cortical stem cells to progenitors to early neurons. The greatest changes occur at the transition from stem cells to progenitors. The regulatory landscape encompasses a total set of 96,375 enhancers linked to target genes, with 49,640 enhancers being active in organoids but not in mid-fetal brain, suggesting major roles in cortical neuron specification. Enhancers that gained activity in the human lineage are active in the earliest stages of organoid development, when they target genes that regulate the growth of radial glial cells. Parallel weighted gene coexpression network analysis (WGCNA) of transcriptome and enhancer activities defined a number of modules of coexpressed genes and coactive enhancers, following just six and four global temporal patterns that we refer to as supermodules, likely reflecting fundamental programs in embryonic and fetal brain. Correlations between gene expression and enhancer activity allowed stratifying enhancers into two categories: activating regulators (A-regs) and repressive regulators (R-regs). Several enhancer modules converged with gene modules, suggesting that coexpressed genes are regulated by enhancers with correlated patterns of activity. Furthermore, enhancers active in organoids and fetal brains were enriched for ASD de novo variants that disrupt binding sites of homeodomain, Hes1, NR4A2, Sox3, and NFIX transcription factors. CONCLUSION We validated hiPSC-derived cortical organoids as a suitable model system for studying gene regulation in human embryonic brain development, evolution, and disease. Our results suggest that organoids may reveal how noncoding mutations contribute to ASD etiology. Summary of the study, analyses, and main results. Data were generated for iPSC-derived human telencephalic organoids and isogenic fetal cortex. Organoids modeled embryonic and early fetal cortex and show a larger repertoire of enhancers. Enhancers could be divided into activators and repressors of gene expression. We derived networks of modules and supermodules with correlated gene and enhancer activities, some of which were implicated in autism spectrum disorders (ASD).

A Fine Balance Intellectual and neurological disabilities can arise from diverse developmental aberrations. Novarino et al. (p. 394 , published online 6 September; see the Perspective by Beaudet ) have now determined the genetic basis for one such disorder for a small group of patients. Exome sequencing led to identification of mutations in a kinase BCKDK ( Branched Chain Ketoacid Dehydrogenase Kinase ) that regulates metabolism of branched-chain amino acids such as valine, leucine, and isoleucine. Mice with homozygous mutations in the BCKDK gene showed developmental and neurological abnormalities resembling those in certain mouse autism models. Analysis of transport mechanisms responsible for carrying amino acids across the blood-brain barrier revealed competition between the branched-chain amino acids and large neutral amino acids. Nutritional supplementation with extra branched-chain amino acids in the diet of mice carrying homozygous mutations in the BCKDK gene normalized their phenotype.

Patent ductus arteriosus (PDA) is a common congenital heart disease that results when the ductus arteriosus, a muscular artery, fails to remodel and close after birth. A syndromic form of this disorder, Char syndrome, is caused by mutation in TFAP2B , the gene encoding a neural crest-derived transcription factor. Established features of the syndrome are PDA, facial dysmorphology, and fifth-finger clinodactyly. Disease-causing mutations are missense and are proposed to be dominant negative. Because only a small number of families have been reported, there is limited information on the spectrum of mutations and resulting phenotypes. We report the characterization of two kindreds (K144 and K145) with Char syndrome containing 22 and 5 affected members, respectively. Genotyping revealed linkage to TFAP2B in both families. Sequencing of TFAP2B demonstrated mutations in both kindreds that were not found among control chromosomes. Both mutations altered highly conserved bases in introns required for normal splicing as demonstrated by biochemical studies in mammalian cells. The abnormal splicing results in mRNAs containing frameshift mutations that are expected to be degraded by nonsense-mediated mRNA decay, resulting in haploinsufficiency; even if produced, the protein in K144 would lack DNA binding and dimerization motifs and would likely result in haploinsufficiency. Examination of these two kindreds for phenotypes that segregate with TFAP2B mutations identified several phenotypes not previously linked to Char syndrome. These include parasomnia and dental and occipital-bone abnormalities. The striking sleep disorder in these kindreds implicates TFAP2B-dependent functions in the normal regulation of sleep.