Disruption of RFX family transcription factors causes autism, attention-deficit/hyperactivity disorder, intellectual disability, and dysregulated behavior

Abstract

Purpose: We describe a novel neurobehavioral phenotype of autism spectrum disorder (ASD), intellectual disability, and/or attention-deficit/hyperactivity disorder (ADHD) associated with de novo or inherited deleterious variants in members of the RFX family of genes. RFX genes are evolutionarily conserved transcription factors that act as master regulators of central nervous system development and ciliogenesis.

Methods: We assembled a cohort of 38 individuals (from 33 unrelated families) with de novo variants in RFX3, RFX4, and RFX7. We describe their common clinical phenotypes and present bioinformatic analyses of expression patterns and downstream targets of these genes as they relate to other neurodevelopmental risk genes.

Results: These individuals share neurobehavioral features including ASD, intellectual disability, and/or ADHD; other frequent features include hypersensitivity to sensory stimuli and sleep problems. RFX3, RFX4, and RFX7 are strongly expressed in developing and adult human brain, and X-box binding motifs as well as RFX ChIP-seq peaks are enriched in the cis-regulatory regions of known ASD risk genes.

Conclusion: These results establish a likely role of deleterious variation in RFX3, RFX4, and RFX7 in cases of monogenic intellectual disability, ADHD and ASD, and position these genes as potentially critical transcriptional regulators of neurobiological pathways associated with neurodevelopmental disease pathogenesis.

Figure 1.. Pedigrees of reported individuals with RFX3, RFX4, and RFX7 variants.

Pedigrees and clinical photographs of individuals with variants in RFX3, RFX4, and RFX7. (A) RFX3, RFX4, and RFX7 case pedigrees. All pedigrees show de novo origin of variants except for RFX3-8a-d: a 33 year-old affected mother carrying the variant p.(Leu496Alafs*7) with transmission to three children, and pedigree RFX4-3a-c: three affected children homozygous for p.(Thr247Met). (B) Individuals with RFX3 variants. (C) Individuals with RFX4 variants. (D) Individuals with RFX7 variants.

Figure 2.. Distribution and predicted deleteriousness of RFX variants.

(A) Mapping of selected RFX variants to domains. Whole gene deletion and intronic variants are not illustrated. RFX3 (NP_602304.1), RFX4 (NP_998759.1), RFX7 (NP_073752.5). (C) Missense variant deleteriousness scores for the currently reported variants (current) and prior reported variants (prior) in RFX3, 4, and 7. The distribution of MPC scores for missense variants reported in this study is significantly different from that of prior reported missense variants, Kolmogorov-Smirnov (K-S) test p-value <0.05 (p-value=0.015). MPC, Missense badness, PolyPhen-2, and Constraint. NsynD, Nonsynonymous Damaging score. CADD, Combined Annotation Dependent Depletion.

Figure 3.. RFX3, RFX4, and RFX7 expression patterns in human cortex and haploinsufficiency gene dosage model.

(A) Transcriptomic cell types in the prenatal human cortex identified by single-cell RNA-sequencing. (B) RFX3, 4, and 7 expression patterns in single cells of the prenatal human cortex. (C) Heatmap of RFX3, 4, and 7 expression levels among cell types in the prenatal human cortex. (D) Transcriptomic cell types in the postnatal human cortex identified by single-cell RNA-sequencing. (E) RFX3, 4, and 7 expression patterns in single cells of the postnatal human cortex. (F) Heatmap of RFX3, 4, and 7 expression levels among cell types in the postnatal human cortex. (G) The enrichment of KEGG pathways, ciliary genes, ASD risk gene sets, and ASD differentially expressed genes (DEGs) among RFX3 ChIP-seq binding targets. Pathways and ASD gene sets are ranked by their statistical significance (p.adjust values, Benjamini-Hochberg’s correction). Red arrows indicate ASD risk gene sets and ASD DEGs. X-axis shows the number of genes bound by RFX in their promoter regions. (H) Binding of RFX family transcription factors bind to X-box motif in promoter regions of ciliary and immunologic genes. Target gene lists obtained from Piasecki, Durand, Reith, Sugiaman-Trapman.^,– Model of RFX gene dose-dependent regulation of genes. In tissues with higher expression of RFX genes, ASD genes are activated. Lower levels of RFX genes are sufficient to activate ciliary genes. vRG, ventricular radial glia. oRG, outer radial glia. PgG2M, cycling progenitors G2/M phase. PgS, cycling progenitors S phase. IP, intermediate progenitors. ExN, migrating excitatory. ExM, maturing excitatory. ExM-U, maturing excitatory upper enriched. ExDp1, excitatory deep layer 1. ExDp2, excitatory deep layer 2. InMGE, interneuron MGE. InCGE, interneuron CGE. OPC, oligodendrocyte precursor cells. End, endothelial. Per, pericyte. Mic, microglia. Neu-mat, immature neurons. Neu-NRGN, NRGN expressing neurons. L5/6, layer 5/6 excitatory neurons. L5/6-CC, layer 5/6 excitatory cortico-cortical projection neurons. L4, layer 4 excitatory neurons. L2/3, layer 2/3 excitatory neurons. IN-SST, somatostatin interneurons. IN-PV, parvalbumin interneurons. IN-SV2C, SVC2 expressing interneurons. IN-VIP, VIP interneurons. AST-FB, fibrous astrocytes. AST-PP, protoplasmic astrocytes. OPC, oligodendrocyte precursor cells.