Genetic and Functional Analyses ofZIC3Variants in Congenital Heart Disease

Abstract

Mutations in zinc-finger in cerebellum 3 (ZIC3) result in heterotaxy or isolated congenital heart disease (CHD). The majority of reported mutations cluster in zinc-finger domains. We previously demonstrated that many of these lead to aberrant ZIC3 subcellular trafficking. A relative paucity of N- and C-terminal mutations has, however, prevented similar analyses in these regions. Notably, an N-terminal polyalanine expansion was recently identified in a patient with VACTERL, suggesting a potentially distinct function for this domain. Here, we report ZIC3 sequencing results from 440 unrelated patients with heterotaxy and CHD, the largest cohort yet examined. Variants were identified in 5.2% of sporadic male cases. This rate exceeds previous estimates of 1% and has important clinical implications for genetic testing and risk-based counseling. Eight of 11 were novel, including 5 N-terminal variants. Subsequent functional analyses included 4 additional reported but untested variants. Aberrant cytoplasmic localization and decreased luciferase transactivation were observed for all zinc-finger variants, but not for downstream or in-frame upstream variants, including both analyzed polyalanine expansions. Collectively, these results expand the ZIC3 mutational spectrum, support a higher than expected prevalence in sporadic cases, and suggest alternative functions for terminal mutations, highlighting a need for further study of these domains.

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

“…31,33,34 Additionally we did not identify any suspicious variants in patients with isolated TGA (30 patients with TGA, included in the subgroup cyanotic heart disease) comparable to earlier reports. 24,31,34 The final classification of variants in this study was based on a combination of in silico analysis as described in the Materials and Methods, together with functional data generated in this study as well as data from earlier publications. A summary of these data is presented in Supplementary Table 1 and Figure 1b.…”

“…However, since Wessels et al 35 detected a de novo expansion of 10-12 Ala residues and not all these variants have been functionally tested, the relevance of these variants remain to be determined. When assessing nuclear localization of mutated ZIC3 protein the two frame-shift variants p.(Val228Serfs*50) and p.(His281Argfs*62) are predicted to disrupt the previously mapped nuclear localization and export signals (NLS/NES) 36,37 and localization of these truncated proteins was divided over both nucleus and cytoplasm (Figure 2b and c) in line with Cowan et al 34 However, mutated p.(Asn371His) ZIC protein, predicted to disrupt one of the mapped NLS and located in a very conserved region in the ZF4 domain showed no different cell localization compared with WT protein and was completely localized to the nucleus in our study (Figure 3a-d). Bedard et al 36 showed that fragments in this region (350-380, 340-420 and 340-466) showed a mixture of cytoplasmic and nuclear localization and that a mutation in one of these regions might be sufficient to disrupt nuclear localization.…”

“…This single expansion Ala was also detected in in-house controls, in both male and female individuals. Together with the functional data of Cowan et al, 34 in which transactivation activity and nuclear localization are unaltered, it seems that this single expansion is at most a risk factor, but not solely causative. In addition, we also detected a contraction of the Ala repeat with three residues (10 to 7) in two patients.…”

“…We also detected several variations in the N-terminal alanine-tract (Figure 1b and Supplementary Table 1) as seen in other studies. 34,35 We detected the known single Ala expansion (10-11) three times. This single expansion Ala was also detected in in-house controls, in both male and female individuals.…”

Copy number variation as a genetic basis for heterotaxy and heterotaxy-spectrum congenital heart defects

“…75%) of familial X-linked pedigrees. Surprisingly, however, ZIC3 mutations underlie only a minority (3-5%) of sporadic heterotaxy cases [8,9]. Likewise, despite a conserved and central role for Nodal signalling in establishment of early molecular asymmetries, point mutations in Nodal pathway components are also not routinely identified and collectively explain only 5-10% of heterotaxy cases [10][11][12].…”