Functional Predictors of Causative Cis-Regulatory Mutations in Mendelian Disease

Undiagnosed neurodevelopmental disease is significantly associated with rare variants in cis-regulatory elements (CRE) but demonstrating causality is challenging as target gene consequences may differ from a causative variant affecting the coding region. Here, we address this challenge by applying a procedure to discriminate likely diagnostic regulatory variants from those of neutral/low-penetrant effect. We identified six rare CRE variants using targeted and whole genome sequencing in 48 unrelated males with apparent X-linked intellectual disability (XLID) but without detectable coding region variants. These variants segregated appropriately in families and altered conserved bases in predicted CRE targeting known XLID genes. Three were unique and three were rare but too common to be plausibly causative for XLID. We compared the cis-regulatory activity of wild-type and mutant alleles in zebrafish embryos using dual-color fluorescent reporters. Two variants showed striking changes: one plausibly causative (FMR1CRE) and the other likely neutral/low-penetrant (TENM1CRE). These variants were “knocked-in” to mice and both altered embryonic neural expression of their target gene. Only Fmr1CRE mice showed disease-relevant behavioral defects. FMR1CRE is plausibly disease-associated resulting in complex misregulation of Fmr1/FMRP rather than loss-of-function. This is consistent both with absence of Fragile X syndrome in the probands and the observed electrophysiological anomalies in the FMR1CRE mouse brain. Although disruption of in vivo patterns of endogenous gene expression in disease-relevant tissues by CRE variants cannot be used as strong evidence for Mendelian disease association, in conjunction with extreme rarity in human populations and with relevant knock-in mouse phenotypes, such variants can become likely pathogenic.