Modeling the long-range effect of an inversion downstream of EFNB1 concludes a 43-year molecular diagnostic odyssey for craniofrontonasal syndrome.

Craniofrontonasal syndrome (CFNS; MIM #304110) is a rare craniofacial disorder characterized by hypertelorism, a broad nasal root with a bifid nasal tip, orofacial clefting, and genital malformations caused by pathogenic variants in the X-linked gene EFNB1 (MIM *300035). CFNS exhibits sex-specific heterogeneity, with increased severity in females likely secondary to cellular interference related to random X-inactivation, resulting in mosaic EFNB1 expression. Previous studies have identified over 140 variants in EFNB1, but approximately 20% of CFNS have negative molecular testing, either due to a yet undiscovered causal gene or causal variants in regulatory regions not covered by traditional genetic testing methodologies. Here, we report a two-generation family with a clinical diagnosis of CFNS and negative clinical molecular testing. Research short-read genome testing identified a 2-Mb inversion together with two smaller deletions (13- and 7-bp), about 106-Kb downstream of EFNB1, which cosegregated with CFNS. Patient-derived fibroblasts reprogrammed into induced pluripotent stem cells (iPSCs) demonstrated two distinct iPSC populations in affected females, where one or other of the two X chromosomes was inactivated. In vitro assays further demonstrated that iPSCs with the active X chromosome bearing the inversion, exhibited a significant increase in EFNB1 expression, suggesting allelic imbalance contributes to mosaic EFNB1 expression. These findings nominate a novel causal variant type of CFNS, conclude a 43-year diagnostic odyssey for an affected family, and offer new hope for family planning for affected individuals.