ZIC2 Mutations Research Articles

Heterotaxy and cardiac defect in a girl with chromosome translocation t(X;1)(q26;p13.1) and involvement of ZIC3

We report on a 2-year-old girl with situs ambiguus comprising right-sided stomach and spleen, left-sided liver and complex cardiac defect. Psychomotor development of this patient was normal, and no other major abnormalities were present. Chromosome analysis revealed a de novo balanced chromosome translocation t(X;1)(q26;p13.1). Molecular cytogenetic investigations identified a breakpoint spanning BAC clone on the X-chromosome containing the ZIC3 gene. Mutations in ZIC3 are associated with situs ambiguus and cardiac defects predominantly in males. This is the first report of a live born girl with an X-autosome translocation involving the ZIC3 region.

A novel heterozygous missense mutation 377T > C (V126A) of TGIF gene in a family segregated with holoprosencephaly and moyamoya disease

To identify whether any mutations of candidate genes including SHH, ZIC2, SIX3, and TGIF exist in a Taiwanese family segregated with holoprosencephaly (HPE) and moyamoya disease. Genotypes of the candidate genes SHH, ZIC2, SIX3, and TGIF were determined in the family members who were available for analysis by sequencing. In addition, genomic regions of another 50 unrelated Taiwanese (100 chromosomes) were studied to verify whether the nucleotide changes we found were mutations or polymorphisms. A novel missense mutation 377T > C and two polymorphisms (420A > G and 487C > T) in the TGIF gene were identified. No mutations in SHH, ZIC2 and SIX3 were found. The mother of the three HPE fetuses was found to be afflicted with moyamoya disease. A brief review of the mutations as well as polymorphisms reported in the TGIF gene up to 2005 is given. Molecular diagnosis can help genetic counseling in HPE, which is a heterogeneous disorder with its phenotypic and genotypic spectrum highly widened and variable. The possible association between TGIF mutation and moyamoya disease noted in our study also appeared to be novel.

In vitro analysis of partial loss-of-function ZIC2 mutations in holoprosencephaly: alanine tract expansion modulates DNA binding and transactivation

Heterozygous loss-of-function mutations in ZIC2 result in the severe brain malformation known as holoprosencephaly (HPE), indicating that forebrain development is exquisitely sensitive to the activity of this poorly understood transcription factor. To identify the regions of ZIC2 that are essential for activity, we have assessed the ability of a variety of ZIC2 mutant proteins to function in in vitro assays. Two sources of information were used to design relevant mutations. First, phenotype producing mutations in human and in mouse ZIC2 were mimicked and secondly, a comparative sequence analysis of the C-terminal was carried out. Analysis of these mutations suggests that either a decrease or an increase in ZIC2 mediated transcriptional activity can produce a forebrain phenotype. In addition, the analysis reveals that the C-terminal of ZIC2 contains both activation and repression domains. This region of ZIC2 contains an alanine-tract, and expansion of this domain is associated with HPE. In vitro analysis of proteins with alterations in alanine-tract length illustrates that the C-terminal alanine-tract of ZIC2 influences the strength of DNA binding and alters transcriptional activity in a promoter-specific manner. This finding provides a possible mechanism by which alanine-tract expansion mutations could alter the function of other transcription factors.

Phenotypic and molecular variability of the holoprosencephalic spectrum.

Since 1996, a European network has been organized from Rennes, France and holoprosencephalic files were collected for clinical and molecular study. Familial instances of typical and atypical holoprosencephaly (HPE) were found in 30% of cases. All affected children had psychomotor delay with microcephaly, often associated with endocrine, digestive, and respiratory abnormalities, and thermal dysregulation. Among 173 subjects in the molecular study, 28 heterozygous mutations were identified (16%): 15 SHH mutations, 6 ZIC2 mutations, 5 SIX3 mutations, and 2 TGIF mutations.

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

Mutations in the zinc finger transcription factor ZIC3 cause X-linked heterotaxy and have also been identified in patients with isolated congenital heart disease (CHD). To determine the relative contribution of ZIC3 mutations to both heterotaxy and isolated CHD, we screened the coding region of ZIC3 in 194 unrelated patients, including 61 patients with classic heterotaxy, 93 patients with heart defects characteristic of heterotaxy, and 11 patients with situs inversus totalis. Five novel ZIC3 mutations in three classic heterotaxy kindreds and two sporadic CHD cases were identified. None of these alleles was found in 97 ethnically matched control samples. On the basis of these analyses, we conclude that the phenotypic spectrum of ZIC3 mutations should be expanded to include affected females and CHD not typical for heterotaxy. This screening of a cohort of patients with sporadic heterotaxy indicates that ZIC3 mutations account for approximately 1% of affected individuals. Missense and nonsense mutations were found in the highly conserved zinc finger-binding domain and in the N-terminal protein domain. Functional analysis of all currently known ZIC3 point mutations indicates that mutations in the putative zinc finger DNA binding domain and in the N-terminal domain result in loss of reporter gene transactivation. It is surprising that transfection studies demonstrate aberrant cytoplasmic localization resulting from mutations between amino acids 253-323 of the ZIC3 protein, indicating that the pathogenesis of a subset of ZIC3 mutations results at least in part from failure of appropriate nuclear localization. These results further expand the phenotypic and genotypic spectrum of ZIC3 mutations and provide initial mechanistic insight into their functional consequences.

A complex syndrome of left-right axis, central nervous system and axial skeleton defects in Zic3 mutant mice.

X-linked heterotaxy (HTX1) is a rare developmental disorder characterized by disturbances in embryonic laterality and other midline developmental field defects. HTX1 results from mutations in ZIC3, a member of the GLI transcription factor superfamily. A targeted deletion of the murine Zic3 locus has been created to investigate its function and interactions with other molecular components of the left-right axis pathway. Embryonic lethality is seen in approximately 50% of null mice with an additional 30% lethality in the perinatal period. Null embryos have defects in turning, cardiac development and neural tube closure. Malformations in live born null mice include complex congenital heart defects, pulmonary reversal or isomerism, CNS defects and vertebral/rib anomalies. Investigation of nodal expression in Zic3-deficient mice indicates that, although nodal is initially expressed symmetrically in the node, there is failure to maintain expression and to shift to asymmetric expression. Subsequent nodal and Pitx2 expression in the lateral plate mesoderm in these mice is randomized, indicating that Zic3 acts upstream of these genes in the determination of left-right asymmetry. The phenotype of these mice correctly models the defects found in human HTX1 and indicates an important role for Zic3 in both left-right and axial patterning.

Possible association of NTDs with a polyhistidine tract polymorphism in the ZIC2 gene

Neural tube defects (NTDs) and brain malformations represent a common finding in chromosome 13q deletion patients. Hemizygosity for ZIC2, which is located in the 13q32 critical deletion region, results in holoprosencephaly (HPE) in humans, and diminished expression of ZIC2 results in HPE as well as lumbosacral NTDs in mice. Taken together, these observations led us to hypothesize that ZIC2 mutations may be a cause of isolated NTD. To test this, we screened 192 NTD patients for mutations in ZIC2. While we did not find ZIC2 mutations in these patients, we did find some evidence of a possible association between a histidine tract polymorphism in ZIC2 and NTDs. Our sample was too small to reach definitive conclusions, but the evidence is sufficiently intriguing to encourage further research. If this association is confirmed, subtle alterations in ZIC2 activity may confer a risk of NTD.

Identification of novel mutations in SHH and ZIC2 in a South American (ECLAMC) population with holoprosencephaly

Holoprosencephaly (HPE) is genetically heterogeneous with four genes, SIX3, SHH, TGIF, and ZIC2 that have been identified to date and that are altered in 12% of patients. To analyze this prevalence in a South American population-based sample (57 HPE cases in 244,511 live and still births or 1 in 4300), we performed a mutational study of these genes in 30 unrelated children (26 newborns and 4 non-newborns) with HPE being ascertained by ECLAMC (Latin American Collaborative Study of Congenital Malformations). We identified three novel mutations: two were missense mutations of the SHH gene (Cys183-->Phe; His140-->Pro); the third mutation was a 2-bp deletion in the zinc-finger region of the ZIC2 gene. These molecular results explained 8% (2/26 newborn samples) of the HPE cases in this South American population-based sample, a proportion similar to our previously published data from a collection of cases.

Holoprosencephaly due to mutations in ZIC2: alanine tract expansion mutations may be caused by parental somatic recombination

We report on the prevalence of mutations in the zinc finger transcription factor gene, ZIC2, in a group of 509 unrelated individuals with isolated holoprosencephaly (HPE) and normal chromosomes. Overall, we encountered 16 HPE patients (from 15 unrelated families) with ZIC2 mutations. Thus, ZIC2 mutation was the apparent cause of HPE in 3-4% of cases. Seven mutations were frameshifts that were predicted to result in loss of function, further supporting the idea that ZIC2 haploinsufficiency can result in HPE. One mutation, an alanine tract expansion which is caused by the expansion of an imperfect trinucleotide repeat, occurred in seven patients from six different families. In three of those families, the father was found to be apparently mosaic for the mutation. We hypothesize that this mutation can arise through errors in somatic recombination, an extremely unusual mutation mechanism. In addition, one mutation resulted in a single amino acid change and one mutation was an in-frame deletion of 12 amino acids. The central nervous system malformations seen in patients with ZIC2 mutations ranged from alobar HPE (most common) to middle interhemispheric fusion defect (one case). Although severe facial anomalies are common in HPE, all of the patients with ZIC2 mutations had relatively normal faces, suggesting that ZIC2 mutations represent a large proportion of HPE cases without facial malformation.

The X-linked mouse mutation Bent tail is associated with a deletion of the Zic3 locus.

Bent tail (BN:) is a spontaneous, semi-dominant mutation on the mouse X chromosome that produces tail deformities and, rarely, open neural tube defects. Analysis of 292 normal male and affected male and female progeny from an intraspecific back-cross involving BN: supports a gene order of cen-DXMit89-18.5 +/- 2.3 cM-DXMit166-1.4 +/- 0.7 cM-BN:-1.0 +/- 0.6 cM-DXMit140 -4.8 +/- 1.3 cM-DXBay6-tel. A high frequency of sex chromosomal non-disjunction, unrelated to the BN: mutation, was also identified in the background strain. Refined genetic and physical mapping of the BN: critical region demonstrate that the mutation is associated with a <170 kb submicroscopic deletion that includes the anonymous microsatellite marker DXMit208 as well as the entire Zic3 locus. Human mutations in ZIC3 are associated with left-right axis malformations (MIM 306955, 208530, 207100). Abnormalities of abdominal and thoracic situs were also detected in viable BN: males and females. The presence of anal and spinal abnormalities in some of the human patients and the deletion of Zic3 in BN: mice support a key role for this gene in neural tube development and closure.

A deletion encompassing Zic3 in bent tail, a mouse model for X-linked neural tube defects.

Bent tail is a mouse model for human neural tube defects. Bent tail mice are characterized by a shortened, kinked tail. We have observed numerous aberrations in Bent tail embryos including exencephaly, rotation defects and occasionally omphalocele, orofacial schisis and situs abnormalities. Exencephaly was seen in >10% of all embryos and resulted from a closure defect of the hindbrain. Bent tail maps to the proximal part of the X chromosome. By haplotype analysis we have appointed the Bent tail locus to a 1.1 cM interval between markers DXMit159 and DXMit143. Subsequent analysis has revealed the presence of a deletion in all affected animals. The deletion is approximately 1 Mb in size and encompasses the gene for ZIC:3, a zinc finger transcription factor expressed in murine neuroectoderm and dorsal axial mesoderm during neurulation. ZIC:3 is a homolog of the Drosophila segmentation gene odd-paired. Although the Bent tail phenotype probably is the result of the deletion of several genes, combining data on ZIC:3 expression and function of ZIC: genes in the mouse shows that deletion of Zic3 alone is compatible with a major role of this gene in the congenital malformations of the Bent tail mouse. In man, mutations in ZIC3 are associated with situs abnormalities. These patients occasionally also show spina bifida, indicating that genetic variation in human ZIC3 may contribute to other congenital malformations, including neural tube defects.

The structure and function of genes causing human holoprosencephaly

Defective patterning of the ventral forebrain combined with incomplete separation of the eye field during the human embryonic gastrulation and early neurulation stages leads to holoprosencephaly (HPE), the most common malformation of the brain in humans. Both genetic and environmental perturbations can cause this extremely heterogeneous condition. Recently, several genes have been shown to cause HPE in humans including mutations in Sonic Hedgehog, SIX3,ZIC2, and TGIF. Here we summarize the known functions of these factors and discuss the potential roles of additional factors associated with cyclopia in animal models.

Holoprosencephaly due to mutations in ZIC2, a homologue of Drosophila odd-paired.

Holoprosencephaly (HPE) is the most common structural anomaly of the human brain and is one of the anomalies seen in patients with deletions and duplications of chromosome 13. On the basis of molecular analysis of a series of patients with hemizygous deletions of the long arm of chromosome 13, we have defined a discrete region in band 13q32 where deletion leads to major developmental anomalies (the 13q32 deletion syndrome). This approximately 1-Mb region lies between markers D135136 and D13S147. Patients in which this region is deleted usually have major congenital malformations, including brain anomalies such as HPE or exencephaly, and digital anomalies such as absent thumbs. We now report that human ZIC2 maps to this critical deletion region and that heterozygous mutations in ZIC2 are associated with HPE. Haploinsufficiency for ZIC2 is likely to cause the brain malformations seen in 13q deletion patients.

The compulsion of chirality: toward an understanding of left-right asymmetry.

Although it has long been clear that correct development of left–right (LR) asymmetry requires that tissues in the early embryo know whether they lie to the left or right of the midline, the molecular mechanisms that invariantly orient the LR axis have remained obscure. The recent demonstration that the iv (inverted viscerum) mutation in the mouse may be caused by a mutation in a gene encoding an axonemal dynein heavy chain has been much anticipated (Afzelius 1976; Brown et al. 1991; Levin and Nascone 1997) and sheds light on the earliest steps in the determination of LR asymmetry (Supp et al. 1997). However, many questions are also raised, such as what the roles of axonemal versus cytoplasmic dynein are, and how dynein action is transmitted across fields of cells, a prerequisite to the large-scale asymmetric gene expression known to be involved in determination of body asymmetry (Fujinaga 1996; Levin et al. 1997). In this review we discuss the nature of the information flow from molecular chirality to morphological and behavioral asymmetry as well as some possible molecular candidates for these processes. We also address the timing of initial LR decisions during embryogenesis, and evolutionary aspects of asymmetry. Most internal organs in the chest and abdomen of all vertebrates lie asymmetrically along the LR body axis despite external bilateral symmetry of the organism itself. In all normal individuals, the LR axis is invariantly oriented such that the apex of the heart points to the left, the aorta loops to the right and the inferior vena cava runs to the left of the spinal column. Similarly, the right lung is divided into three lobes whereas the left has only two. Beneath the diaphragm, the stomach and spleen are on the left and the intestine runs from right to left. Deviation from this normal pattern of asymmetry (situs solitus) can lead to complete mirror-image reversals of internal organ placement and anatomy (situs inversus) or randomization of organ situs (heterotaxy) as well as some loss of asymmetry (isomerism) (Burn 1991; WinerMuram 1995). Complete situs inversus does not appear to confer any adverse effects on the individual, yet, nonetheless, is estimated to occur in only 1/20,000 humans (although this is commonly thought to under-represent the actual number). Heterotaxia, in contrast, usually results in multiple abnormalities many of which, such as complex heart or vascular defects, are fatal without surgical intervention. Similarly, isomerisms such as Ivemark’s sequence (right isomerism, characterized by asplenia) as well as left isomerism (characterized by polysplenia) frequently compromise viability but, in less severe cases, may escape clinical detection (Burn 1991). A hallmark of most sporadic, familial, and experimentally-induced cases of laterality defects is that the organism does not lose its asymmetry; rather, individual organs (separately or together) can exhibit mirror-image asymmetry (Levin et al. 1995; Fujinaga 1996). This has led to the idea that asymmetric development or placement of an individual organ is distinct from the mechanism that orients the LR axis during development. In the absence of LR cues, therefore, the individual organs often become unbiased and develop with either normal or inverted asymmetry. The search for genes that control the overall pattern of asymmetry has provided some insight into early events leading to LR specification. The first demonstrations of asymmetric gene expression preceding organogenesis was made in chick embryos (Levin et al. 1995). Subsequent studies using mouse, Xenopus, and zebrafish embryos suggests that details of the left and right cascades of gene activation may not be conserved (Matzuk et al. 1995; Chiang et al. 1996; Collignon et al. 1996; Lowe et al. 1996); however, in all species examined, it appears that the left-sided gene cascade culminates in expression of nodal, which encodes a TGFb family member (Fig. 1, 2). An important aspect of the these studies is that misexpression of either leftor right-sided genes unbiases organ situs and leads to heterotaxia (Levin et al. 1997; Sampath et al. 1997). This, combined with the expression of the cascades prior to organogenesis, suggests that nodal and other downstream genes, such as lefty-1 and lefty-2 [also encoding TGFb family members (Meno et al. 1996)], provide LR cues to the developing organs. An important implication of this work is that mutations in these genes are likely to underlie both familial and sporadic cases of laterality defects in humans. This is likely to be the case for a familial X-linked situs abnormality that results from mutations in Zic3 (Gebbia et al. 1997), a gene encoding a zinc finger transcription factor. Interestingly, Zic3 shares structural similarity with the product of the Drosophila pair–rule gene odd paired (opa) and Corresponding author. E-MAIL mmercola@warren.med.harvard.edu; FAX (617) 432-1144.

X-linked situs abnormalities result from mutations in ZIC3.

Vertebrates position unpaired organs of the chest and abdomen asymmetrically along the left-right (LR) body axis. Each structure comes to lie non-randomly with respect to the midline in an overall position designated situs solitus, exemplified in humans by placement of the heart, stomach and spleen consistently to the left. Aberrant LR axis development can lead to randomization of individual organ position (situs ambiguus) or to mirror-image reversal of all lateralized structures (situs inversus). Previously we mapped a locus for situs abnormalities in humans, HTX1, to Xq26.2 by linkage analysis in a single family (LR1) and by detection of a deletion in an unrelated situs ambiguus male (Family LR2; refs 2,3). From this chromosomal region we have positionally cloned ZIC3, a gene encoding a putative zinc-finger transcription factor. One frameshift, two missense and two nonsense mutations have been identified in familial and sporadic situs ambiguus. The frameshift allele is also associated with situs inversus among some heterozygous females, suggesting that ZIC3 functions in the earliest stages of LR-axis formation. ZIC3, which has not been previously implicated in vertebrate LR-axis development, is the first gene unequivocally associated with human situs abnormalities.