Identification and Functional Analysis of ZIC3 Mutations in Heterotaxy and Related Congenital Heart Defects

Patients

For the purposes of the present study we categorized patients with laterality disorders into three subgroups: classic heterotaxy (familial and sporadic), CHD heterotaxy (familial and sporadic), and situs inversus (sporadic). Patients with classic heterotaxy have CCVM and evidence of disrupted left-right patterning in at least one other organ system, including any of the following anomalies: asplenia, polysplenia, evidence of splenic dysfunction, malrotation, omphalocele, abdominal situs inversus, bilateral bilobed lungs, bilateral trilobed lungs, abnormalities of the liver or gallbladder, or stomach situs. Patients were categorized as having CHD heterotaxy if they had normal situs in other organs or if insufficient data were available to make a determination. The types of CCVM included in this study were based on those described in the Baltimore-Washington Infant Study, which included laterality and looping defects. Patients categorized as affected by situs inversus had situs inversus totalis. The disorder was considered familial if a first-degree relative had a congenital heart defect of any type or if a second- or third-degree relative had evidence of a laterality disorder. Further details on enrollment criteria can be obtained at the Baylor College of Medicine Cardiovascular Genetics Web site. Controls were from the Baylor Polymorphism Resource, a set of lymphoblastoid cell lines developed from healthy adults in which ethnic information has been retained.

Phenotypic information was established via patient history and chart review, when available. Informed consent was obtained from all individuals in accordance with the protocol approved by the institutional review board of Baylor College of Medicine. Blood samples were collected from patients and parents, and Epstein-Barr virus–transformed cell lines were established. Genomic DNA was prepared from cell lines or fibroblast cultures (fetal samples) by standard procedures.

Mutation Detection

Exons 1–3 of the ZIC3 gene (GenBank accession number NM_008413) were amplified by PCR, using the following five primer sets: exon 1aF 5′ TCTTGCAGTGACGGAAAGTT-3′ and exon 1aR 5′-TTCATGTTCATGGGGCTGTA-3′; exon 1bF 5′-GCGCACGATCTATCTTCAGG-3′ and exon 1bR 5′-CCGCATATAACGGAAGAAGG-3′; exon 1cF 5′-TACAGCCCCATGAACATGAA-3′ and exon 1cR 5′-TCTCCCTCGCGCTCTAAGT-3′; exon 2F 5′-GCTGCTTGCCTCTGAGAAAC-3′ and exon 2R 5′-ACGTGGAAGACAGTGGGTTG-3′; and exon 3F 5′-GCTCTTGTTTTTGCTTGCAC-3′ and exon 3R 5′-CATTTCCATCTGATTGGTCTCA-3′. All PCRs were performed using 200 ng genomic DNA, 200 μM dNTPs, and 20 pmol of each primer. Samples were purified using Qiagen PCR purification and were sequenced using an ABI 3700.

Plasmids and Mutagenesis

A 1.4-kb Kpn1-DraI fragment of the human ZIC3 cDNA, encompassing the entire ORF, was subcloned in frame into the KpnI-NotI sites of a pHM6 expression vector (Roche) and into the eGFP vector (Clontech), to create an N-terminal hemagglutinin (HA)–tagged fusion and an N-terminal GFP fusion, respectively. ZIC3 mutations were introduced into the Kpn1-Dra1 fragment via Quik-Change site-directed mutagenesis (Stratagene), were sequenced in their entirety, and were subcloned into pHM6.

Transcriptional Assay

HeLa cells were maintained in α-modified Eagle medium with 2.5% fetal bovine serum and 7.5% newborn calf serum. Cells were transfected using lipofectamine (Roche) according to the manufacturer’s instructions. Cotransfections were performed using HA-ZIC3 constructs and an SV40 luciferase reporter plasmid (pGL3 promoter vector; Promega); results were normalized to a promoterless luciferase vector (pGL3 basic; Promega). A pRL-TK luciferase vector served as a control for transfection efficiency. Cells were harvested 48 h after transfection, and luciferase activities were determined using the Dual Luciferase Reporter Assay System (Promega). Firefly luciferase activities were normalized to Renilla luciferase activity. All results represent a minimum of four experiments.

Immunofluorescence and Subcellular Localization

HeLa cells were seeded onto glass coverslips at 5 × 10⁵ cells/ml 12–18 h prior to transfection. The HA-ZIC3 expression constructs containing wild-type and mutant ZIC3 were transfected into HeLa cells by use of Lipofectamine (Roche), according to the manufacturer’s instructions. After 24 h, the cells were fixed in 4% paraformaldehyde/PBS on ice for 30 min, washed three times in PBS, and permeabilized in 0.3% Triton X/PBS for 2 min at room temperature. After a brief wash in 0.1% Triton X/PBS, the cells were blocked in 1% BSA/0.1% Triton X in PBS at room temperature for 1 h. The cells were incubated with a 1:250 dilution of an anti-HA rabbit polyclonal antibody (Novus Biologicals) for 2 h at room temperature, washed five times in PBS, incubated 1 h with a 1:500 dilution of Alexa Fluor 594 goat anti-rabbit IgG (Molecular Probes), washed three times in PBS then briefly in distilled water and mounted with DAPI. Cells were analyzed using fluorescence microscopy. At least 100 cells were counted for each construct. Cells were scored as “nuclear,” “nuclear and cytoplasmic,” or “cytoplasmic.”

Cohort Analysis

ZIC3 mutation analysis was performed by sequencing in a cohort of 20 familial and 145 sporadic cases of heterotaxy. Twenty-nine patients with atrial septal or ventricular septal defects without evidence of a laterality disorder were used as CHD controls. The 165 patients with heterotaxy included 59 females and 106 males, whereas the control CHD group consisted of 13 males and 16 females. The ethnicity of the patients with heterotaxy included 2% Asian (n=3), 3% African American (n=5), 42% white (n=69), 32% Hispanic (n=53), and 21% other or not determined (n=35).

Characteristics of the cohort are summarized in table 1. Patients with classic heterotaxy were subdivided on the basis of spleen position and number. Asplenia and polysplenia are thought to represent bilateral right-sidedness and bilateral left-sidedness, respectively; although they have overlapping spectra of anatomic defects, each has also been associated with characteristic cardiovascular malformations on the basis of autopsy series (Peoples et al. 1983; Van Praagh et al. ¹⁹⁹⁰; Phoon and Neill ¹⁹⁹⁴; Uemura et al. ¹⁹⁹⁵). In our cohort, the asplenia phenotype is more commonly associated with total anomalous pulmonary venous return and dextrocardia, whereas the polysplenia phenotype is more commonly associated with azygous continuation of the inferior vena cava and levocardia. These findings are consistent with previously published reports and indicate that this subpopulation of our cohort reflects the commonly identified anatomic abnormalities seen in heterotaxy. In general, the types of CCVM in the CHD heterotaxy group are similar, both in complexity and anatomy, to those found in the classic heterotaxy group. There is, however, an increased representation of L-TGA and a decreased representation of anomalous pulmonary venous return in the CHD heterotaxy group.

Identification of ZIC3 mutations

From a total of 194 patient samples, we identified 8 novel ZIC3 nucleotide changes: 2 nonsense mutations, 3 missense mutations, 2 silent nucleotide changes, and 1 nucleotide change in the 3′ untranslated region (table 2). The location of these novel point mutations, as well as those previously reported (Gebbia et al. 1997; Megarbane et al. ²⁰⁰⁰), and their predicted effects in the ZIC3 amino acid sequence are shown in figure 1.

We ascertained three pedigrees with transmission of heterotaxy consistent with X-linked inheritance. ZIC3 was sequenced in the probands (fig. 2), and two nonsense mutations and one missense mutation were identified. In each case, the mother of the proband was heterozygous for the mutation. In pedigree LAT 138, a C→A change results in a nonsense mutation and predicts protein truncation at amino acid 43. All previous mutations have clustered in the zinc finger–binding domains (Gebbia et al. 1997; Megarbane et al. ²⁰⁰⁰); therefore, this represents the most N-terminal mutation described to date. A second nonsense mutation, located in a conserved glutamine at amino acid 249, was found in pedigree LAT 129. This mutation results in truncation prior to the first zinc finger–binding domain.

The third familial mutation, C253S, is located at the first residue of the first zinc finger. Several factors suggest that this missense mutation is pathogenic. First, it is found in the highly conserved cysteine that initiates the first zinc finger domain and that forms one of the C2H2 zinc finger motifs. Second, this change was not found in 145 ethnically matched control chromosomes. Third, it functions abnormally in transactivation and subcellular-localization assays (see below). Figure 1 shows all currently identified ZIC3 mutations, including the five novel mutations identified in the present study, and illustrates the conservation of sequence across species found at the missense mutation sites.

One ZIC3 mutation was identified in the cohort of 145 patients with sporadic heterotaxy (table 2). The mutation, in exon 2, results in the substitution of glutamate for lysine at amino acid 405. This residue is highly conserved (fig. 1) and lies adjacent to a residue that participates in the C2H2 motif at amino acid 406. This change was not found in 145 ethnically matched control chromosomes. It is notable that this is the first female with CHD heterotaxy in whom a ZIC3 mutation has been identified.

The second mutation identified in a patient with nonheterotaxy CHD was from the group of 29 individuals with nonheterotaxy CHD. The patient has an atrial septal defect and pulmonic stenosis (table 3). No other malformation typically associated with heterotaxy was identified. The missense mutation at amino acid 217 results in a substitution of alanine for proline. It is located outside the zinc finger domain in a residue that is incompletely conserved among species (fig. 1). The results of mutation analysis in sporadic cases therefore indicate that ZIC3 mutations account for ∼1% (2/174; 95% CI 0.14%–4.0%) of this patient population. Furthermore, our data indicate that ZIC3 mutations do not underlie the male predominance seen in patients with heterotaxy.

Expansion of the Phenotypic Spectrum Associated with ZIC3 Mutations

Table 3 summarizes the phenotypes of the three familial cases (LAT 107, LAT 129, and LAT 138) and the two sporadic cases (LAT 818 and JT 15) in which ZIC3 mutations were identified. For each pedigree, the proband is listed first, followed by other affected family members for whom data were available. Although the familial cases demonstrate anatomic anomalies very consistent with findings in heterotaxy, they exhibit a number of abnormalities not previously described in association with ZIC3 mutations. Novel findings regarding cardiac lesions include two cases of hypoplastic left heart and two cases of total anomalous pulmonary venous return. Novel extracardiac anomalies include omphalocele. Midline defects are commonly associated with disorders of laterality, most frequently affecting genitourinary or musculoskeletal development (Gebbia et al. 1997; Ticho et al. ²⁰⁰⁰). Genitourinary abnormalities, particularly imperforate anus, have been consistently reported in families with ZIC3 mutations (Casey et al. 1993; Gebbia et al. ¹⁹⁹⁷). In concordance with this, two of the families identified in this study had affected members with imperforate anus. A variety of skeletal and renal defects were noted as well. In addition, a number of features not typically characteristic of heterotaxy syndromes, including dysmorphic facial features, cystic hygroma, and posterior embryotoxon were noted. It is interesting to note that patient LAT 138 IV-2 carried a tentative diagnosis of Alagille syndrome and patient LAT 129 III-6 was given a diagnosis of VATER association (MIM 192350). Because the clinical phenotype of patients with ZIC3 mutations is broad, it should be considered in the differential diagnosis of patients with multiple congenital anomalies, particularly when CCVMs are involved.

It is surprising that the ZIC3 mutations that were found in the subgroup of patients with sporadic CHD were not in males with classic heterotaxy. Both individuals had isolated heart defects without other manifestations of heterotaxy, reinforcing a previous observation that isolated congenital heart defects can result from ZIC3 deficiency (Megarbane et al. 2000). Furthermore, one of the patients was female, and the other patient had a constellation of heart malformations not typically associated with CHD heterotaxy. The finding of an affected female may reflect skewed X inactivation within the proband. However, Zic3-deficient mice, which closely mimic the anatomic abnormalities seen in humans, show left-right patterning abnormalities in a significant percentage (20%–40%) of heterozygous females (Purandare et al. 2002). Overall, these findings confirm previous speculation that ZIC3 mutations may be found both in females and in males and may be associated with isolated, nonheterotaxy CHD. They also reinforce the need for an understanding of the molecular determinants of looping cardiogenesis. Better understanding, in turn, may lead to a categorization of congenital heart defects more closely related to etiology and may aid the evaluation of potential candidate genes.

Mutations in ZIC3 Alter Reporter Gene Transactivation

To test the functional significance of mutations in ZIC3, site-directed mutagenesis was used to create previously published and newly identified mutations in the context of the ZIC3 ORF (Gebbia et al. 1997; Megarbane et al. ²⁰⁰⁰). ZIC3 has been shown to be a weak transcriptional activator and is hypothesized to act as a transcriptional coactivator (Mizugishi et al. 2001). The ZIC family physically and functionally interacts with GLI proteins via the zinc finger–binding domains, but direct transcriptional targets of ZIC3 have not yet been identified in vivo (Koyabu et al. 2001). In vitro, ZIC3 is able to activate a variety of target promoters, including SV40. In HeLa cells, transfection of wild-type HA-tagged ZIC3 demonstrated relatively strong transactivation of an SV40 luciferase reporter, with levels ∼200-fold higher than those of a promoterless control (data not shown). We subsequently tested mutant constructs containing nonsense, missense, or frameshift mutations described in the present study and in others (Gebbia et al. 1997; Megarbane et al. ²⁰⁰⁰). The results demonstrate that ZIC3 mutations produce aberrant reporter gene transactivation (fig. 3). Loss of activation occurs both with mutations in the putative DNA-binding domain of the zinc finger region as well as with mutations in the N-terminal domain. These results are consistent with previous work in Xenopus, which demonstrated that expression of the N-terminus of Zic3, in the absence of the zinc finger–binding domain, causes left-right patterning defects (Kitaguchi et al. 2000).

The nonsense mutations all show significant loss of activation, including a 1477–1478insTT frameshift mutation that results in a premature stop codon at amino acid 408. All but one of the missense mutations also shows a loss of transactivation. The exception is the N-terminal mutation, P217A. In this case, a significant and reproducible increase in transcriptional activation is noted. This mutation was not found in 145 ethnicity-matched control chromosomes or in 412 chromosomes within the CHD cohort. It is of interest that the phenotype of this patient, which consisted of an atrial septal defect and pulmonic stenosis, is not characteristic of heterotaxy (table 3). It is therefore interesting to speculate that activation of ZIC3 may contribute to additional phenotypic abnormalities separable from classic heterotaxy. Zic1 has previously been shown to play a role in the subcellular localization, and thus posttranslational control, of Gli1 and Gli3 in a cell-type–specific manner. Given the known physical interaction between ZIC and GLI proteins, it is possible that increases or decreases in ZIC3 expression alter the stoichiometry of the GLI proteins at the cellular level. Further mutation and functional analyses, both in vitro and in vivo, will be required to establish whether activating mutations or overexpression of the ZIC3 transcription factor plays a significant role in CHD.

Subcellular Localization

To further assess the functional significance of ZIC3 mutations, transient transfections were performed using ZIC3-mutant constructs to determine subcellular localization by use of immunofluorescence. Initially, the appropriate expression pattern of ZIC3 and the effect of epitope tagging were assessed using two constructs, an N-terminal HA tagged ZIC3 and an N-terminal eGFP-ZIC3 fusion. These constructs demonstrated nuclear localization of ZIC3 in both HeLa and P19 teratocarcinoma cells (fig. 4 and data not shown), and HA-tagged constructs were used for the remainder of the experiments. It is interesting that all of the missense mutations tested, with the exception of P217A, showed abnormal subcellular localization (fig. 4 and data not shown). HA-ZIC3 was detected in the cytoplasm or in both cytoplasm and nucleus. In some cells, localization within the cytoplasm was in a stippled distribution. To compare the relative distribution of ZIC3 by construct, 100–200 cells were counted for each construct and were scored for nuclear, nuclear and cytoplasmic, or cytoplasmic localization. The results are shown in figure 5 and indicate that loss of nuclear localization occurs for mutations between amino acids 253 and 323. Nuclear-localization–prediction programs fail to identify a nuclear localization sequence (NLS) for ZIC3. The lack of a classical NLS suggests that ZIC3 may enter the nucleus as a complex associated with other factors. Alternatively, ZIC3 may contain a novel NLS or may enter the nucleus directly via an unconventional pathway. The results obtained using these known patient mutations delimit a critical protein domain(s) required for nuclear localization. Furthermore, these results indicate that the pathogenesis of a subset of ZIC3 mutations results from failure of the mutant protein to localize to the nucleus.

In addition to abnormalities in subcellular localization, mutations also affected protein stability. Two truncating mutations, S43X and Q249X, resulted in absent or nearly absent protein (data not shown). Furthermore, on the basis of the number of HA-expressing cells in this assay, protein stability also appeared to be qualitatively diminished with several of the missense mutations. A summary of the mutations with their associated clinical information and results of functional analyses is shown in table 4.

In summary, our data demonstrate that ZIC3 mutations account for ∼1% of sporadic CHD heterotaxy. The additional mutations identified in the present study indicate that mutations outside the zinc finger–binding domain can cause heterotaxy and further broaden the genotypic and phenotypic spectrum of defects caused by mutations in this gene. In addition, our data provide methods for functional evaluation of ZIC3 mutations and indicate that the pathogenesis of a subset of mutations results from aberrant subcellular localization. Future studies directed toward identifying the interactions required for proper cellular localization should provide valuable insight into the mechanisms by which ZIC3 functions in left-right patterning.