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.

A novel ZIC3 gene mutation identified in patients with heterotaxy and congenital heart disease

“…A study in ZIC3 mutants overexpressing either wild-type ZIC3 mRNA or mutant ZIC3 mRNA showed that overexpression of wild-type ZIC3 mRNA resulted in cardiac laterality defects, posterior truncation or embryo lethality. However, the examined mutants (p.Val288Serfs*50, p.His281Argfs*62, p.Asn371His) exhibited less extensive phenotypes, indicating a partial loss of function 31 . To confirm the role of Zic3 in establishing LR asymmetry, we used an antisense MO to knockdown the expression of zebrafish zic3 .…”

“…The mutant ZIC3 construct was generated from the wild-type ZIC3 plasmid via site-directed mutagenesis and contained a C-terminal Myc-tag. The wild-type and mutant ZIC3 constructs were subsequently co-transfected into the NIH/3T3, H9C2, and 293T cell lines with an SV40 promoter-driven firefly luciferase reporter 22 , 31 . Our results showed that the ZIC3 mutation in the C2H2 domain significantly decreases reporter gene transactivation compared with the wild-type controls (Fig.…”

“…Moreover, ZIC3 is considered to act as a transcription factor, due to its ability to bind DNA and activate transcription 33 . Since the first gene was found to be associated with X-linked HTX through linkage analysis in five families in 1997, more than 36 pathogenic variants of the ZIC3 gene have been identified, providing substantial evidence that either loss or aberrant function of the ZIC3 protein is among the causes of heterotaxy 8 , 10 – 12 , 22 , 31 , 34 .…”

“…The wild-type and mutant mRNAs were synthesized with the mMESSAGE mMACHINE SP6 ULTRA Kit (Ambion, Inc). Then, 100 pg of mRNA was co-injected with 2.0 ng of zic3 TB-MO in 1-cell-stage embryos 31 . As a control, equal volumes of solution were injected.…”

Genetic Basis of Human Congenital Heart Disease

“…All types of inheritance including Xlinked, autosomal dominant, and autosomal recessive have been described with multiple implicated genes including LEFTYA, CRYPTIC, and ACVR2B (Belmont et al 2004). Pathogenic variants in ZIC3, a zinc-finger transcription factor involved in heart looping, are thought to contribute to ∼5% of HTX cases in males (Cowan et al 2014;Paulussen et al 2016).…”