Harris et al reported a total of 15 distinct variants in RFX3: two frameshift variants, two canonical splice donor variants, eight missense variants, one in-frame deletion, one 42 kb deletion removing the last two exons of RFX3, and one 227 kb deletion involving only RFX3. In one family, an affected parent transmitted a frameshift variant to three affected children; all other variants were de novoand novel. All variants were absent from gnomAD.
RFX3exhibits intolerance to loss-of-function variation in human population databases (gnomAD, pLI scores = 1.00). This combined with the fact that the majority of variants identified are predicted to cause outright protein truncation or gene deletion (20 out of 33) strongly supports a loss-of-function / haploinsufficiency model. All missense variants affect highly conserved amino acids (PhastCons vertebrate, mammalian, and primate scores ranging from 0.99–1.00).
RFX transcription factors are defined by a conserved, specialized winged-helix type DNA binding domain (DBD) that recognize the X-box motif. In addition to the DBD, RFX3 has three known domains that are associated with dimerization (DD). All of the non-truncating (missense or in-frame deletion) variants identified in RFX3were found to be located in the DBD or one of the dimerization domains.
RFX3has been reported to have relatively high expression in human cortex. Single-cell transcriptomes from developing and adult human cortex revealed that in developing human cortex, RFX3is most highly expressed in maturing excitatory upper enriched neurons, and in adult cortex RFX3 was most highly expressed in glutamatergic layer 2/3 neurons. These expression profiles suggest that RFXdeleterious variants may lead to our observed neurodevelopmental phenotypes by altering early developmental cell fates or by impacting the function of upper-layer cortical neurons.
Enriched expression of RFX3in upper cortical layer neurons places this gene in cells that are involved in communication between regions of the cortex important for higher cognition and social behavior, raising the possibility that haploinsufficiency may disrupt either the developmental specification, synaptic connectivity, or electrophysiological function of this set of neurons. Projection neurons in this layer have been implicated in ASD by analyses of co-expression networks of autism genes, and superficial cortical neurons exhibit the strongest amount of differential gene expression in ASD brains compared to controls.
RFX3