ZIC2 Mutations Research Articles

A murineZic3transcript with a premature termination codon evades nonsense-mediated decay during axis formation

SUMMARYThe ZIC transcription factors are key mediators of embryonic development and ZIC3 is the gene most commonly associated with situs defects (heterotaxy) in humans. Half of patient ZIC3 mutations introduce a premature termination codon (PTC). In vivo, PTC-containing transcripts might be targeted for nonsense-mediated decay (NMD). NMD efficiency is known to vary greatly between transcripts, tissues and individuals and it is possible that differences in survival of PTC-containing transcripts partially explain the striking phenotypic variability that characterizes ZIC3-associated congenital defects. For example, the PTC-containing transcripts might encode a C-terminally truncated protein that retains partial function or that dominantly interferes with other ZIC family members. Here we describe the katun (Ka) mouse mutant, which harbours a mutation in the Zic3 gene that results in a PTC. At the time of axis formation there is no discernible decrease in this PTC-containing transcript in vivo, indicating that the mammalian Zic3 transcript is relatively insensitive to NMD, prompting the need to re-examine the molecular function of the truncated proteins predicted from human studies and to determine whether the N-terminal portion of ZIC3 possesses dominant-negative capabilities. A combination of in vitro studies and analysis of the Ka phenotype indicate that it is a null allele of Zic3 and that the N-terminal portion of ZIC3 does not encode a dominant-negative molecule. Heterotaxy in patients with PTC-containing ZIC3 transcripts probably arises due to loss of ZIC3 function alone.

Zic3 is required in the extra-cardiac perinodal region of the lateral plate mesoderm for left–right patterning and heart development

Mutations in ZIC3 cause human X-linked heterotaxy and isolated cardiovascular malformations. A mouse model with targeted deletion of Zic3 demonstrates an early role for Zic3 in gastrulation, CNS, cardiac and left-right axial development. The observation of multiple malformations in Zic3(null) mice and the relatively broad expression pattern of Zic3 suggest its important roles in multiple developmental processes. Here, we report that Zic3 is primarily required in epiblast derivatives to affect left-right patterning and its expression in epiblast is necessary for proper transcriptional control of embryonic cardiac development. However, cardiac malformations in Zic3 deficiency occur not because Zic3 is intrinsically required in the heart but rather because it functions early in the establishment of left-right body axis. In addition, we provide evidence supporting a role for Zic3 specifically in the perinodal region of the posterior lateral plate mesoderm for the establishment of laterality. These data delineate the spatial requirement of Zic3 during left-right patterning in the mammalian embryo, and provide basis for further understanding the molecular mechanisms underlying the complex interaction of Zic3 with signaling pathways involved in the early establishment of laterality.

An essential and highly conserved role for Zic3 in left–right patterning, gastrulation and convergent extension morphogenesis

Mutations in ZIC3 result in X-linked heterotaxy in humans, a syndrome consisting of left–right (L–R) patterning defects, midline abnormalities, and cardiac malformations. Similarly, loss of function of Zic3 in mouse results in abnormal L–R patterning and cardiac development. However, Zic3 null mice also exhibit defects in gastrulation, neural tube closure, and axial patterning, suggesting the hypothesis that Zic3 is necessary for proper convergent extension (C-E) morphogenesis. To further investigate the role of Zic3 in early embryonic development, we utilized two model systems, Xenopus laevis and zebrafish, and performed loss of function analysis using antisense morpholino-mediated gene knockdown. Both Xenopus and zebrafish demonstrated significant impairment of C-E in Zic3 morphants. L–R patterning was also disrupted, indicating that the role of Zic3 in L–R axis development is conserved across species. Correlation of L–R patterning and C-E defects in Xenopus suggests that early C-E defects may underlie L–R patterning defects at later stages, since Zic3 morphants with moderate to severe C-E defects exhibited an increase in laterality defects. Taken together, these results demonstrate a functional conservation of Zic3 in L–R patterning and uncover a previously unrecognized role for Zic3 in C-E morphogenesis during early vertebrate development.

Abstract 17979: A Subset of Patients with Isolated Transposition of the Great Arteries and Double Outlet Right Ventricle Fall Within the Spectrum of Heterotaxy Syndrome

Background: Heterotaxy is a rare disorder of laterality. ZIC3 was the first gene in which human disease-causing mutations were identified. Mutations typically result in males with situs ambiguus and complex congenital heart disease; however affected females and one male with isolated transposition of the great arteries (TGA) have been reported. We hypothesized that a subset of patients with heart defects common to heterotaxy but without laterality defects would have ZIC3 mutations. We also sought to estimate the prevalence of ZIC3 mutations in sporadic heterotaxy. Methods and Results: Patients with TGA (n=169), double outlet right ventricle (DORV) (n=90), complete atrioventricular canal (n= 41) and heterotaxy (n=53) underwent sequencing of the ZIC3 open reading frame. We tested 90 patients with tetralogy of Fallot (TOF) to correlate genotype with phenotype. Three mutations were detected: 1 female with isolated DORV [c.49G>T (Gly17Cys)], 1 male with isolated TGA [c.98C>T (Ala33Val)], and 1 female with sporadic heterotaxy [841C>T (His280Tyr)] that were absent in 100 Caucasian and 100 African American control patients. We also identified a novel insertional mutation (CPFP333ins) in a family with heterotaxy. No mutations were found in cases with TOF. Transcriptional activation studies demonstrated significantly reduced transcription of the ZIC3 His280Tyr mutant construct. Conclusions: Isolated TGA and DORV, like heterotaxy, can arise from mutations in ZIC3, confirming a spectrum of disease that includes isolated cardiac malformations. ZIC3 mutations were found in 2% of patients with sporadic heterotaxy, therefore routine testing should be considered in patients with heterotaxy.

New findings for phenotype–genotype correlations in a large European series of holoprosencephaly cases

BackgroundHoloprosencephaly (HPE) is the most common forebrain defect in humans. It results from incomplete midline cleavage of the prosencephalon.MethodsA large European series of 645 HPE probands (and 699 relatives), consisting...

Mutations in ZIC3 and ACVR2B are a common cause of heterotaxy and associated cardiovascular anomalies

Heterotaxy syndrome is caused by left-right asymmetry disturbances and is associated with abnormal lateralisation of the abdominal and thoracic organs. The heart is frequently involved and the severity of the abnormality usually determines the outcome. We performed a direct sequence analysis of the coding sequence of genes including Zinc Finger Protein of the Cerebellum 3, Left-Right Determination Factor 2, Activin A Receptor Type IIB, and Cryptic in 47 patients with laterality defects and congenital cardiac disease. Of the 47 patients, 31 (66%) had atrioventricular septal defects, 34 (72%) had abnormal systemic venous return, 25 (53%) had transposed or malposed great arteries, and 20 (43%) had pulmonary venous abnormalities. We identified two novel genetic changes in Zinc Finger Protein of the Cerebellum 3, and these variants were not present in 100 ethnically matched control samples. One previously reported missense mutation in Activin A Receptor Type IIB was identified in two unrelated subjects. The genetic changes identified in this study are all located in conserved regions and are predicted to affect protein function in left-right axis formation and cardiovascular development. Mutations in Zinc Finger Protein of the Cerebellum 3 and Activin A Receptor Type IIB were identified in 4 of the 47 patients with heterotaxy syndrome for a yield of approximately 8.5%. Our results expand the mutation spectrum of monogenic heterotaxy syndrome with associated cardiac anomalies and suggest that there are other causes of heterotaxy yet to be identified.

Zic2 hypomorphic mutant mice as a schizophrenia model and ZIC2 mutations identified in schizophrenia patients

ZIC2 is a causal gene for holoprosencephaly and encodes a zinc-finger-type transcriptional regulator. We characterized Zic2(kd/+) mice with a moderate (40%) reduction in Zic2 expression. Zic2(kd/+) mice showed increased locomotor activity in novel environments, cognitive and sensorimotor gating dysfunctions, and social behavioral abnormalities. Zic2(kd/+) brain involved enlargement of the lateral ventricle, thinning of the cerebral cortex and corpus callosum, and decreased number of cholinergic neurons in the basal forebrain. Because these features are reminiscent of schizophrenia, we examined ZIC2 variant-carrying allele frequencies in schizophrenia patients and in controls in the Japanese population. Among three novel missense mutations in ZIC2, R409P was only found in schizophrenia patients, and was located in a strongly conserved position of the zinc finger domain. Mouse Zic2 with the corresponding mutation showed lowered transcription-activating capacity and had impaired target DNA-binding and co-factor-binding capacities. These results warrant further study of ZIC2 in the pathogenesis of schizophrenia.

Identification of a Novel ZIC3 Isoform and Mutation Screening in Patients with Heterotaxy and Congenital Heart Disease

Patients with heterotaxy have characteristic cardiovascular malformations, abnormal arrangement of their visceral organs, and midline patterning defects that result from abnormal left-right patterning during embryogenesis. Loss of function of the transcription factor ZIC3 causes X-linked heterotaxy and isolated congenital heart malformations and represents one of the few known monogenic causes of congenital heart disease. The birth incidence of heterotaxy-spectrum malformations is significantly higher in males, but our previous work indicated that mutations within ZIC3 did not account for the male over-representation. Therefore, cross species comparative sequence alignment was used to identify a putative novel fourth exon, and the existence of a novel alternatively spliced transcript was confirmed by amplification from murine embryonic RNA and subsequent sequencing. This transcript, termed Zic3-B, encompasses exons 1, 2, and 4 whereas Zic3-A encompasses exons 1, 2, and 3. The resulting protein isoforms are 466 and 456 amino acid residues respectively, sharing the first 407 residues. Importantly, the last two amino acids in the fifth zinc finger DNA binding domain are altered in the Zic3-B isoform, indicating a potential functional difference that was further evaluated by expression, subcellular localization, and transactivation analyses. The temporo-spatial expression pattern of Zic3-B overlaps with Zic3-A in vivo, and both isoforms are localized to the nucleus in vitro. Both isoforms can transcriptionally activate a Gli binding site reporter, but only ZIC3-A synergistically activates upon co-transfection with Gli3, suggesting that the isoforms are functionally distinct. Screening 109 familial and sporadic male heterotaxy cases did not identify pathogenic mutations in the newly identified fourth exon and larger studies are necessary to establish the importance of the novel isoform in human disease.

Mutations in CDON, Encoding a Hedgehog Receptor, Result in Holoprosencephaly and Defective Interactions with Other Hedgehog Receptors

Holoprosencephaly (HPE), a common human congenital anomaly defined by a failure to delineate the midline of the forebrain and/or midface, is associated with diminished Sonic hedgehog (SHH)-pathway activity in development of these structures. SHH signaling is regulated by a network of ligand-binding factors, including the primary receptor PTCH1 and the putative coreceptors, CDON (also called CDO), BOC, and GAS1. Although binding of SHH to these receptors promotes pathway activity, it is not known whether interactions between these receptors are important. We report here identification of missense CDON mutations in human HPE. These mutations diminish CDON's ability to support SHH-dependent gene expression in cell-based signaling assays. The mutations occur outside the SHH-binding domain of CDON, and the encoded variant CDON proteins do not display defects in binding to SHH. In contrast, wild-type CDON associates with PTCH1 and GAS1, but the variants do so inefficiently, in a manner that parallels their activity in cell-based assays. Our findings argue that CDON must associate with both ligand and other hedgehog-receptor components, particularly PTCH1, for signaling to occur and that disruption of the latter interactions is a mechanism of HPE.

Zic2 hypomorphic mutant mice as a schizophrenia model and ZIC2 mutations identified in schizophrenia patients

ZIC2 is a causal gene for holoprosencephaly and encodes a zinc-finger-type transcriptional regulator. We characterized Zic2kd/+ mice with a moderate (40%) reduction in Zic2 expression. Zic2kd/+ mice showed increased locomotor activity in novel environments, cognitive and sensorimotor gating dysfunctions, and social behavioral abnormalities. Zic2kd/+ brain involved enlargement of the lateral ventricle, thinning of the cerebral cortex and corpus callosum, and decreased number of cholinergic neurons in the basal forebrain. Because these features are reminiscent of schizophrenia, we examined ZIC2 variant-carrying allele frequencies in schizophrenia patients and in controls in the Japanese population. Among three novel missense mutations in ZIC2, R409P was only found in schizophrenia patients, and was located in a strongly conserved position of the zinc finger domain. Mouse Zic2 with the corresponding mutation showed lowered transcription-activating capacity and had impaired target DNA-binding and co-factor-binding capacities. These results warrant further study of ZIC2 in the pathogenesis of schizophrenia.

Recurrent partial rhombencephalosynapsis and holoprosencephaly in siblings with a mutation of ZIC2

Rhombencephalosynapsis (RES) is a rare congenital brain malformation typically identified by magnetic resonance imaging and characterized by fusion of the cerebellar hemispheres and dentate nuclei and vermian agenesis or hypogenesis. Although RES is frequently found in conjunction with other brain malformations and/or congenital anomalies, no specific molecular etiology has been discovered to date and no animal models exist. We identified two half sisters with alobar or semi-lobar holoprosencephaly (HPE) and partial RES, suggesting that genes linked to HPE may also contribute to RES. A deletion of seven base pairs in exon one of the ZIC2 gene (c.392_98del7) was identified in each of the two half sisters with HPE and partial RES. To identify genetic causes of RES and to assess whether genes identified in HPE have a role in RES, we tested 11 additional individuals with RES by high-resolution chromosome analysis, chromosomal microarray analysis, and sequencing of four HPE genes. No mutations in ZIC2 or in other genes that cause HPE were identified, suggesting that mutation of ZIC2 is a rare cause of, or contributor to, RES associated with HPE. In addition, an individual with a complex rearrangement of chromosome 22q13.3 and RES was identified, suggesting the presence of a dosage-sensitive gene that may contribute to RES in this region.

The unfolding clinical spectrum of holoprosencephaly due to mutations in SHH, ZIC2, SIX3 and TGIF genes.

Holoprosencephaly is a severe malformation of the brain characterized by abnormal formation and separation of the developing central nervous system. The prevalence is 1:250 during early embryogenesis, the live-born prevalence is 1:16 000. The etiology of HPE is extremely heterogeneous and can be teratogenic or genetic. We screened four known HPE genes in a Dutch cohort of 86 non-syndromic HPE index cases, including 53 family members. We detected 21 mutations (24.4%), 3 in SHH, 9 in ZIC2 and 9 in SIX3. Eight mutations involved amino-acid substitutions, 7 ins/del mutations, 1 frame-shift, 3 identical poly-alanine tract expansions and 2 gene deletions. Pathogenicity of mutations was presumed based on de novo character, predicted non-functionality of mutated proteins, segregation of mutations with affected family-members or combinations of these features. Two mutations were reported previously. SNP array confirmed detected deletions; one spanning the ZIC2/ZIC5 genes (approx. 100 kb) the other a 1.45 Mb deletion including SIX2/SIX3 genes. The mutation percentage (24%) is comparable with previous reports, but we detected significantly less mutations in SHH: 3.5 vs 10.7% (P=0.043) and significantly more in SIX3: 10.5 vs 4.3% (P=0.018). For TGIF1 and ZIC2 mutation the rate was in conformity with earlier reports. About half of the mutations were de novo, one was a germ line mosaic. The familial mutations displayed extensive heterogeneity in clinical manifestation. Of seven familial index patients only two parental carriers showed minor HPE signs, five were completely asymptomatic. Therefore, each novel mutation should be considered as a risk factor for clinically manifest HPE, with the caveat of reduced clinical penetrance.

Mutations in ZIC2 in human holoprosencephaly: description of a Novel ZIC2 specific phenotype and comprehensive analysis of 157 individuals

BackgroundHoloprosencephaly (HPE), the most common malformation of the human forebrain, may be due to mutations in genes associated with non-syndromic HPE. Mutations in ZIC2, located on chromosome 13q32, are a...

DNA hypomethylation, transient neonatal diabetes, and prune belly sequence in one of two identical twins

One known genetic mechanism for transient neonatal diabetes is loss of methylation at 6q24. The etiology of prune belly sequence is unknown but a genetic defect, affecting the mesoderm from which the triad abdominal muscle hypoplasia, urinary tract abnormalities, and cryptorchidism develop, has been suggested. We investigated a family, including one twin, with transient neonatal diabetes and prune belly sequence. Autoantibody tests excluded type 1 diabetes. Microsatellite marker analysis confirmed the twins being monozygotic. We identified no mutations in ZFP57, KCNJ11, ABCC8, GCK, HNF1A, HNF1B, HNF3B, IPF1, PAX4, or ZIC3. The proband had loss of methylation at the 6q24 locus TNDM and also at the loci IGF2R, DIRAS3, and PEG1, while the other family members, including the healthy monozygotic twin, had normal findings. The loss of methylation on chromosome 6q24 and elsewhere may indicate a generalized maternal hypomethylation syndrome, which accounts for both transient neonatal diabetes and prune belly sequence.

Identification of a novel role of ZIC3 in regulating cardiac development

Mutations in ZIC3 cause X-linked heterotaxy, a disorder characterized by abnormal lateralization of normally asymmetric thoracic and abdominal organs. Animal models demonstrate an early role for ZIC3 in embryonic left-right (LR) patterning. ZIC3 mutations have also been described in patients with isolated cardiovascular malformations. We wished to address the hypothesis that ZIC3 has plieotropic effects in development and may regulate cardiac development independent of its role in LR patterning. We observed significantly reduced expression of several markers of cardiac lineage commitment in Zic3(null/y) embryonic stem cells including atrial natriuretic factor (ANF), Nkx2.5 and Tbx5. Likewise, ANF expression-a molecular marker of trabecular myocardium and a direct target of multiple cardiac-specific transcription factors-was severely reduced in E9.5 Zic3 null hearts. Trabecular myocardium was reduced in these embryos. This finding was similar to that observed in embryos with cardiac-specific ablation of serum response factor (SRF), a direct transcriptional regulator of ANF expression. While ZIC3 by itself had no effect on the ANF promoter, it could bind to and inhibit a cardiac alpha-actin promoter through its zinc finger domains. We observed that ZIC3 could function as a coactivator of SRF on both cardiac alpha-actin and ANF promoters. The zinc fingers of ZIC3 and the mcm1, agamous deficiens SRF (MADS) box motif of SRF were found to mediate their physical and functional interactions. These findings reveal a novel role of ZIC3 in regulating cardiac gene expression and may explain, in part, the association of ZIC3 mutation with cardiovascular malformations.

ZIC2-dependent Transcriptional Regulation Is Mediated by DNA-dependent Protein Kinase, Poly(ADP-ribose) Polymerase, and RNA Helicase A

The Zic family of zinc finger proteins is essential for animal development, as demonstrated by the holoprosencephaly caused by mammalian Zic2 mutation. To determine the molecular mechanism of Zic-mediated developmental control, we characterized two types of high molecular weight complexes, including Zic2. Complex I was composed of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku70/80, and poly(ADP-ribose) polymerase; complex II contained Ku70/80 and RNA helicase A; all the components interacted directly with Zic2 protein. Immunoprecipitation, subnuclear localization, and in vitro phosphorylation analyses revealed that the DNA-PKcs in complex I played an essential role in the assembly of complex II. Stepwise exchange from complex I to complex II depended on phosphorylation of Zic2 by DNA-PK and poly-(ADP-ribose) polymerase. Phosphorylated Zic2 protein made a stable complex with RNA helicase A, and complex II could interact with RNA polymerase II. Phosphorylation-dependent transformation of Zic2-containing molecular complexes may occur in transcriptional regulation.

Characterization of the interactions of human ZIC3 mutants with GLI3

ZIC3, a GLI superfamily transcription factor, is involved in establishing normal embryonic left-right patterning. Multiple abnormalities in the central nervous system (CNS) and axial skeleton have also been observed in mice bearing a Zic3 null allele, mice with a Zic3 overexpression allele, and the majority of patients carrying ZIC3 mutations. Previous studies indicate that ZIC3 protein can bind to the GLI consensus binding site (GLIBS) and physically interact with GLI3, a transcription factor involved in multiple aspects of neural and skeletal development. We investigated in vitro interactions of ZIC3 with GLI3 and the effect of ZIC3 mutations identified in patients with either heterotaxy or isolated cardiovascular malformations. Electrophoresis mobility shift assay (EMSA) revealed that all five intact zinc finger (ZF) domains were necessary for binding of ZIC3 to GLIBS. Inclusion of GLIBS upstream of a basal TK promoter had no effect on the activation of the promoter by ZIC3 alone, but it enhanced the synergistic activation of ZIC3 and GLI3. Wild-type (WT) ZIC3 showed specific binding to GLI3 in GST-pull-down assays. Nonsense and frameshift ZIC3 mutants lacking one or more of the zinc finger domains did not physically interact with GST-GLI3; however, two missense mutants c.1213A>G (p.K405E, fifth ZF domain), and c.649C>G (p.P217A, conserved N-terminal domain) retained binding. Luciferase reporter assays indicated that both p.P217A and p.K405E mutants also retained coactivation with GLI3 of reporter gene expression activity, while all the GLI3-nonbinding ZIC3 mutants lacked this activity. Interestingly, no CNS or skeletal abnormalities were observed in patients bearing the p.P217A or p.K405E mutations.

Craniofacial, skeletal, and cardiac defects associated with altered embryonic murine Zic3 expression following targeted insertion of a PGK-NEO cassette

Mutation in ZIC3 (OMIM #306955), a zinc finger transcription factor, causes heterotaxy (situs ambiguus) or isolated congenital heart defects in humans. Mice bearing a null mutation in Zic3 have left-right patterning defects with associated cardiovascular, vertebra/rib, and central nervous system malformations. Although XZic3 is thought to play a critical role in Xenopus neural crest development, no defects in tissues derived from neural crest are apparent in adult Zic3(null) mice. In this study we have characterized the effect of a PGK-neo cassette insertion 5' of the Zic3 locus. The Zic3 transcript in this new allele is up-regulated in ES cells and in E9.0 embryos, but no ectopic expression was detected. Unlike the Zic3(null) mutation in which only 20% of mutant animals survive to adulthood, there was no evidence of excess fetal death caused by the Zic3(neo) allele. Zic3(neo) mutant mice exhibited hemifacial microsomia, asymmetric low set ears, axial skeletal defects, kyphosis and scoliosis; a combination of defects which mimics Goldenhar Syndrome. Some Zic3(neo) mice had evidence of left-right axis patterning defects, but cardiac malformation was much less common than in the Zic3(null) mutants. A six-week old hemizygous mouse was found to have thoraco-cervical ectopia cordis, an extremely rare congenital malformation in humans and for which there is no precedent in a mouse model.

Nuclear import and export signals are essential for proper cellular trafficking and function of ZIC3

Missense, frameshift and nonsense mutations in the zinc finger transcription factor ZIC3 cause heterotaxy as well as isolated congenital heart disease. Previously, we developed transactivation and subcellular localization assays to test the function of ZIC3 point mutations. Aberrant cytoplasmic localization suggested that the pathogenesis of ZIC3 mutations results, at least in part, from failure of appropriate cellular trafficking. To further investigate this hypothesis, the nucleocytoplasmic shuttling properties of ZIC3 have been examined. Subcellular localization assays designed to span the entire open-reading frame of wild-type and mutant ZIC3 proteins identified the presence of nucleocytoplasmic transport signals. ZIC3 domain mapping indicates that a relatively large region containing the zinc finger binding sites and a known GLI interacting domain is required for transport to the nucleus. Site-directed mutagenesis of critical residues within two putative nuclear localization signals (NLSs) leads to loss of nuclear localization. No further decrease was observed when both NLS sites were mutated, suggesting that mutation of either NLS site is sufficient for loss of importin-mediated nuclear localization. Additionally, we identify a cryptic CRM-1-dependent nuclear export signal (NES) within ZIC3, and identify a mutation within this region in a patient with heterotaxy. These results provide the first evidence that control of cellular trafficking of ZIC3 is critical for function and suggest a possible mechanism for transcriptional control during left-right patterning. Identification of mutations in mapped NLS or NES domains in heterotaxy patients demonstrates the functional importance of these domains in cardiac morphogenesis and allows for integration of structural analysis with developmental function.

Holoprosencephaly: Clinical, anatomic, and molecular dimensions

Holoprosencephaly is addressed under the following headings: alobar, semilobar, and lobar holoprosencephaly; arrhinencephaly; agenesis of the corpus callosum; pituitary abnormalities; hindbrain abnormalities; syntelencephaly; aprosencephaly/atelencephaly; neural tube defects; facial anomalies; median cleft lip; minor facial anomalies; single maxillary central incisor; holoprosencephaly-like phenotype; epidemiology; genetic causes of holoprosencephaly; teratogenic causes of holoprosencephaly; SHH mutations; ZIC2 mutations; SIX3 mutations; TGIF mutations; PTCH mutations; GLI2 mutations; FAST1 mutations; TDGF1 mutations; and DHCR7 mutations.