Rare novel variants in the ZIC3 gene cause X-linked heterotaxy

Abstract

Variants in the ZIC3 gene are rare, but have demonstrated their profound clinical significance in X-linked heterotaxy, affecting in particular male patients with abnormal arrangement of thoracic and visceral organs. Several reports have shown relevance of ZIC3 gene variants in both familial and sporadic cases and with a predominance of mutations detected in zinc-finger domains. No studies so far have assessed the functional consequences of ZIC3 variants in an in vivo model organism. A study population of 348 patients collected over more than 10 years with a large variety of congenital heart disease including heterotaxy was screened for variants in the ZIC3 gene. Functional effects of three variants were assessed both in vitro and in vivo in the zebrafish. We identified six novel pathogenic variants (1,7%), all in either male patients with heterotaxy (n=5) or a female patient with multiple male deaths due to heterotaxy in the family (n=1). All variants were located within the zinc-finger domains or leading to a truncation before these domains. Truncating variants showed abnormal trafficking of mutated ZIC3 proteins, whereas the missense variant showed normal trafficking. Overexpression of wild-type and mutated ZIC protein in zebrafish showed full non-functionality of the two frame-shift variants and partial activity of the missense variant compared with wild-type, further underscoring the pathogenic character of these variants. Concluding, we greatly expanded the number of causative variants in ZIC3 and delineated the functional effects of three variants using in vitro and in vivo model systems.

Figure 1

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.

Figure 1

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.

Figure 2

(a,b) Autopsy report photographs of the male index fetus of family 23 217. (a) Opened thorax and abdomen with situs inversus. (b) Graphic representation of situs inversus. (c–h) Autopsy report photographs of male index fetus of family 21 753 (c) heart from the back, (d) heart from the left side, (e) opened left ventricle, (f) organs seen from the backside, (g) opened thorax and abdomen and (h) liver from the backside. A detailed clinical description of the fetus is described in the result section.

Figure 3

Representative subcellular localization of WT and mutated ZIC3 proteins. (a–d) First column represent WT or mutant GFP-tagged ZIC3 proteins transfected in HeLa cells. Second column is the nuclear DAPI staining and the third column is both columns merged. A minimum of 50 cells were scored for each construct. Scoring was based on three localization classifications: nuclear, cytoplasmic or both (e).

Figure 4

Functional assessment of variants in zebrafish. (a) Representative micrographs of cmlc2:GFP zebrafish embryo's displaying cardiac laterality defects. The picture on the left shows a normally located left-sided heart (left-sided heart), while the picture in the middle indicates an intermediately positioned heart (midline heart). The picture on the right illustrates an aberrantly located right-sided heart (right-sided heart). (b) Quantification of phenotypes upon expression of wild-type human ZIC3 protein and of the indicated variants. A minimum of 80 embryos was analyzed. uic, uninjected control.