Functional analysis of Sonic Hedgehog variants associated with holoprosencephaly in humans using a CRISPR/Cas9 zebrafish model.

Genetic variation in the highly conserved Sonic Hedgehog (SHH) gene is one of the most common genetic causes for the malformations of the brain and face in humans described as the holoprosencephaly clinical spectrum. However, only a minor fraction of known SHH variants have been experimentally proven to lead to abnormal function. Employing a phenotypic rescue assay with synthetic human messenger RNA variant constructs in shha-/- knockout zebrafish, we evaluated 104 clinically reported in-frame and missense SHH variants. Our data helped us to classify them into loss of function variants (31), hypomorphic variants (33), and nonpathogenic variants (40). We discuss the strengths and weaknesses of currently accepted predictors of variant deleteriousness and the American College of Medical Genetics and Genomics guidelines for variant interpretation in the context of this functional model; furthermore, we demonstrate the robustness of model systems such as zebrafish as a rapid method to resolve variants of uncertain significance.