Overview of variants in the ZIC3 gene. (a) Pedigrees of ZIC3 variant positive families. Squares, males; circles, females; diamonds with 'm', male stillborns/terminations; +, hemizygous mutation; +/−, heterozygous mutation; −/−, wild-type. Arrows indicate indexes. Reference sequences are based on Genbank accession numbers NM_003413.3, NP_003404.1 and NG_008115.1. Below pedigrees are representative Sanger sequences of the variants with the upper line being the reference sequence, the middle line a hemizygous male carrier and the bottom line a heterozygous female carrier. (b) Representation of the ZIC3 gene (NM_003413.3) and corresponding ZIC3 protein (NP_003404.1). On the right all disease associated variations known in literature. Black: pathogenic variants; blue: VUS. On the left variations detected in this study, in red, probably pathogenic mutations; blue, VUS. NES, nuclear export signal; NLS, nuclear localization signal; VUS, variants of unknown significance.