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ZINC FINGER C4H2 DOMAIN-CONTAINING PROTEIN; ZC4H2

ZINC FINGER C4H2 DOMAIN-CONTAINING PROTEIN; ZC4H2

Alternative titles; symbolsKIAA1166HGNC Approved Gene Symbol: ZC4H2Cytogenetic location: Xq11.2 Genomic coordinates (GRCh38): X:64,915,806-65,034,740 (from N...

Alternative titles; symbols

  • KIAA1166

HGNC Approved Gene Symbol: ZC4H2

Cytogenetic location: Xq11.2 Genomic coordinates (GRCh38): X:64,915,806-65,034,740 (from NCBI)

▼ Cloning and Expression
By sequencing clones obtained from a size-fractionated adult brain cDNA library, Hirosawa et al. (1999) cloned ZC4H2, which they designated KIAA1166. The transcript contains a number of repetitive elements in its 3-prime end. RT-PCR ELISA detected variable ZC4H2 expression in all adult and fetal tissues and specific adult brain regions examined. Highest expression was detected in adult liver, and lowest expression was detected in skeletal muscle, spleen, and testis.

By haplotype analysis, next-generation sequencing, array comparative genomic hybridization, and chromosome breakpoint mapping, Hirata et al. (2013) identified ZC4H2. The deduced protein contains 224 amino acids. In mouse, Zc4h2 was more highly expressed in brain and spinal cord during embryonic development and was downregulated postnatally and in the adult. Expression of Zc4h2 was also high in embryonic zebrafish brain and spinal cord. Immunofluorescence analysis revealed high Zc4h2 expression in nucleus, cytoplasm, and excitatory synapses of primary cultured hippocampal neurons. Database analysis revealed orthologs of ZC4H2 in mouse, rat, frog, and zebrafish.

In human fetal tissue, May et al. (2015) identified 2 ZC4H2 isoforms: a long 224-residue transcript and a shorter 201-residue transcript that lacked exon 1. Both transcripts are expressed in the brain, with the shorter isoform at a higher level. The short isoform was highly expressed in all regions of the brain and the spinal cord. The long isoform was weakly expressed in all regions with higher expression in the hypothalamus, pons, and medulla. It was not observed in the spinal cord or liver.

Frints et al. (2019) stated that there are 5 known ZC4H2 transcripts.

▼ Gene Function
Hirata et al. (2013) found that transient overexpression of ZC4H2 in mouse primary hippocampal neurons increased the number of dendritic spines. ZC4H2 localized to excitatory postsynaptic sites, as verified by colocalization with PSD95 (602887), but not to inhibitory synapses.

By RT-PCR analysis and knockdown experiments in Xenopus, Ma et al. (2017) found that Zc4h2 is expressed in the developing neural system and is involved in neural development. Knockdown or overexpression of Zc4h2 in Xenopus embryos and in the mammalian cell lines Hep3B and C2C12 showed that knockdown of Zc4h2 downregulated the expression level of bone morphogenetic protein (see BMP1, 112264) while its overexpression upregulated it. Immunoprecipitation in HEK293 cells demonstrated that Zc4h2 regulates BMP signaling by directly interacting with Smad1 (601595) and Smad5 (603110) through their MH2 domains, and in vitro ubiquitination assays further revealed that Zc4h2 binds and stabilizes those 2 proteins through reducing their association with the Smurf ubiquitin ligases (e.g., 605568) and thus their ubiquitination. Ma et al. (2017) also examined a group of ZC4H2 mutations which had been reported to cause Wieacker-Wolff syndrome (WRWF; 314580), and found that those ZC4H2 mutants weakened Smad-stabilizing activity compared to wildtype ZC4H2 proteins in HEK293 cells and Xenopus embryos.

▼ Mapping
By radiation hybrid analysis, Hirosawa et al. (1999) mapped the ZC4H2 gene to the X chromosome. Hirata et al. (2013) mapped the ZC4H2 gene to chromosome Xq11.2 by genomic sequence analysis.

▼ Molecular Genetics
Wieacker-Wolff syndrome

In affected members of 5 unrelated families with Wieacker-Wolff syndrome (WRWF; 314580), Hirata et al. (2013) identified 4 different missense mutations in the ZC4H2 gene (300897.0001-300897.0004). The mutations were found by exome sequencing in some of the families. The disorder showed X-linked inheritance and was characterized by onset of muscle weakness in utero. Affected boys were born with severe contractures and had delayed motor development, facial and bulbar weakness, characteristic dysmorphic facial features, skeletal abnormalities such as hip dislocation, scoliosis, and pes equinovarus, and later showed mental retardation. Carrier females had mild features of the disorder and demonstrated skewed X inactivation. Expression of 3 of the mutations in mouse primary neurons caused a significant decrease in synapse number and density, and none of the mutations was able to rescue the swimming defect of zebrafish morphants. The findings indicated that mutations in the ZC4H2 gene cause a clinically variable broad-spectrum neurodevelopmental disorder of the central and peripheral nervous systems.

In affected members of 4 unrelated families with WRWF, including the original family reported by Miles and Carpenter (1991) as having Miles-Carpenter syndrome, May et al. (2015) identified mutations in the ZC4H2 gene (300897.0004-300897.0007). The mutations were found by various methods, including whole-genome sequencing, X-chromosome exome sequencing, and direct sequencing of the ZC4H2 gene: all mutations were confirmed by Sanger sequencing and segregated with the disorder in the families. There were 3 missense and 1 splice site mutations. Molecular modeling of the mutant proteins suggested that all the missense mutations would destabilize the protein and result in a loss of function. Based on knockdown of the gene in zebrafish (see ANIMAL MODEL), May et al. (2015) suggested that ZC4H2 mutations adversely affect interneuron fate and connectivity throughout the brain and spinal cord, including a loss of GABAergic neurons. Clinical variability likely results from mutations affecting different isoforms as well as having different effects on the protein.

Frints et al. (2019) reported 11 males from 6 unrelated families (families 1, 4-6, 9, and 19) with hemizygous missense mutations in the ZC4H2 gene inherited from an unaffected or mildly affected mother (see, e.g., 300897.0002 and 300897.0011). The mutations, which were found by whole-exome or whole-genome sequencing, segregated with the disorder in the families. Two additional male patients (families 18 and 24) had de novo hemizygous missense variants (see, e.g., 300897.0002). All missense variants affected highly conserved residues, and none were found in the 1000 Genomes Project or gnomAD databases. Expression of 2 mutations, A200V and H70Q, failed to rescue swimming defects in zc4h2-null zebrafish, suggesting that missense mutations are pathogenic and likely result in decreased protein function. Frints et al. (2019) noted that complete loss-of-function ZC4H2 mutations are almost never observed in male patients (see GENOTYPE/PHENOTYPE CORRELATIONS).

Female-Restricted Wieacker-Wolff syndrome

In 14 unrelated females with female-restricted Wieacker-Wolff syndrome (WRWFFR; 301041), Frints et al. (2019) identified de novo heterozygous mutations or intragenic deletions in the ZC4H2 gene (see, e.g., 300897.0008, 300897.0010, 300897.0012-300897.0014). The mutations, which were found by whole-exome sequencing, whole-genome sequencing, or CNV analysis and confirmed by Sanger sequencing, were not found in the 1000 Genomes Project or gnomAD databases. In 1 family (family 3), a severely affected female fetus carried a heterozygous frameshift mutation (300897.0009) that was found in mosaic state (less than 10%) in the unaffected mother. A male infant with similar features in the same family had died 10 hours after birth, but DNA from that infant was not available. Most of the mutations were predicted to result in a loss of function and haploinsufficiency: there were 7 nonsense, splice site, or frameshift mutations, 5 intragenic deletions affecting only the ZC4H2 gene, and 2 missense variants affecting highly conserved residues. Functional studies of the variants and studies of patient cells besides X-inactivation studies were not performed.

▼ Genotype/Phenotype Correlations
Among a large cohort of 23 families, including male and female patients, with WRWF or WRWFFR, respectively, Frints et al. (2019) observed that no males, except possibly a patient from family 3 who died at age 10 hours, carried hemizygous nonsense, frameshift, or splice site mutations or intragenic deletions affecting the ZC4H2 gene. In contrast, these types of putative complete loss-of-function mutations were only observed in females affected with WRWFFR, and occurred de novo. The findings suggested that mutations predicted to result in complete loss of ZC4H2 protein are very rare in males; moreover, the pathologic mechanism in females is most likely haploinsufficiency.

▼ Cytogenetics
In a boy with sporadic occurrence of WRWF, Hirata et al. (2013) identified a de novo paracentric inversion on the X chromosome with breakpoints at Xq11.2, involving ZC4H2, and Xq28, which did not contain any known genes. RT-PCR on patient cells showed no detectable ZC4H2 transcripts, indicating that the rearrangement abolished ZC4H2 expression. In addition, whole-genome microarray analysis identified small heterozygous deletions at Xq11.2 in 2 mildly affected girls. The girls had distal muscle weakness, camptodactyly, equinovarus foot deformity or contracture of the Achilles tendon, language deficits, and intellectual disability. The deletions were 826 and 321 kb, respectively, and included ZC4H2 but no other adjacent genes. These findings indicated that heterozygous deletions of ZC4H2 can result in a clinical phenotype, even in females.

Using array-CGH analysis, Zanzottera et al. (2017) identified a de novo heterozygous 429-kb Xq11.2 deletion, including ZC4H2 as the only known gene, in a female patient with features of Wieacker-Wolff syndrome similar to the severity seen in males with the condition. X-inactivation was found to be random (not skewed). Zanzottera et al. (2017) suggested that female carriers can be more severely affected than previously observed. In a female patient with features of a severe form of Wieacker-Wolff syndrome, Okubo et al. (2018) identified a de novo heterozygous 395-kb deletion at Xq11.2 that included ZC4H2 as the only known gene. The phenotype in this patient was more severe than that in the patient reported by Zanzottera et al. (2017) and included arthrogryposis multiplex congenita, severe intellectual disability, spastic quadriplegia, and progressive brain atrophy.

▼ Animal Model
Hirata et al. (2013) found that morpholino-mediated knockdown of Zc4h2 in zebrafish resulted in no gross abnormalities, but morphants showed impaired swimming capability and impaired alpha-motoneuron development. The number of neuromuscular endplates was reduced in morphants, and endplates appeared disorganized.

May et al. (2015) found expression of the zc4h2 zebrafish ortholog in the developing central nervous system, mainly in differentiating progenitors and mature neurons and/or glia. Expression was localized mainly in the nucleus. Zc4h2-null zebrafish showed abnormal flexion of the pectoral fins and active movements of the pectoral fins, continuous swimming movements, and balance problems. They also had abnormally positioned eyes, an open mouth, and continuous jaw movements. These abnormalities were associated with a loss of markers of the V2a and V2b interneurons in the hindbrain and spinal cord, as well as a significant reduction in the number of GABAergic interneurons in the midbrain tegmentum, as demonstrated by decreased gad1 expression (605363). Wildtype human ZC4H2 was able to restore the behavioral abnormalities of mutant fish as well as gad1 expression.

▼ ALLELIC VARIANTS ( 14 Selected Examples):

.0001 WIEACKER-WOLFF SYNDROME
ZC4H2, VAL63LEU
In affected members of a family with Wieacker-Wolff syndrome (WRWF; 314580) originally reported by Wieacker et al. (1985), Hirata et al. (2013) identified a c.187G-C transversion in exon 3b of the ZC4H2 gene, resulting in a val63-to-leu (V63L) substitution at a highly conserved residue in the coiled-coil domain. The mutation, which was identified by exome sequencing of the X chromosome and confirmed by Sanger sequencing, segregated with the disorder and was not found in several large control exome databases.

Using Western blot analysis, Ma et al. (2017) showed that V63L mutant ZC4H2 had weaker SMAD-stabilizing activity compared to wildtype ZC4H2 in HEK293 cells.

.0002 WIEACKER-WOLFF SYNDROME
ZC4H2, ARG198GLN
In affected members of a family with Wieacker-Wolff syndrome (WRWF; 314580), Hirata et al. (2013) identified a c.593G-A transition in exon 6 of the ZC4H2 gene, resulting in an arg198-to gln (R198Q) substitution at a highly conserved residue in the zinc finger domain. The mutation, which was identified by linkage analysis and deep sequencing of the linked interval, segregated with the disorder and was not found in several large control exome databases.

Using Western blot analysis, Ma et al. (2017) showed that R198Q mutant ZC4H2 had weaker SMAD-stabilizing activity compared to wildtype ZC4H2 in HEK293 cells.

In 2 unrelated males (family 4 and 24) with WRWF, Frints et al. (2019) identified a hemizygous R198Q mutation in the ZC4H2 gene. One mutation was inherited from an unaffected mother, whereas the other occurred de novo. Functional studies of the variant and studies of patient cells were not performed. The mutations, which were found by exome sequencing and confirmed by Sanger sequencing, were not present in the gnomAD database.

.0003 WIEACKER-WOLFF SYNDROME
ZC4H2, PRO201SER
In affected members of a family with Wieacker-Wolff syndrome (WRWF; 314580) originally reported by Hennekam et al. (1991), Hirata et al. (2013) identified a c.601C-T transition in exon 6 of the ZC4H2 gene, resulting in a pro201-to-ser (P201S) substitution at a highly conserved residue in the zinc finger domain. The mutation segregated with the disorder and was not found in several large control exome databases.

Using Western blot analysis, Ma et al. (2017) showed that P201S mutant ZC4H2 had weaker SMAD-stabilizing activity compared to wildtype ZC4H2 in HEK293 cells.

.0004 WIEACKER-WOLFF SYNDROME
ZC4H2, ARG213TRP
In affected members of 2 unrelated families with Wieacker-Wolff syndrome (WRWF; 314580), Hirata et al. (2013) identified a c.637C-T transition in exon 6 of the ZC4H2 gene, resulting in an arg213-to-trp (R213W) substitution at a highly conserved residue. The mutation, which was identified by exome sequencing and confirmed by Sanger sequencing, segregated with the disorder and was not found in several large control exome databases.

May et al. (2015) identified an R213W mutation in the ZC4H2 gene in affected members of a family (K8615) with WRWF. There were 3 affected males who were hemizygous for the mutation, and 3 female carriers, only 1 of whom had mild intellectual disability. Molecular modeling predicted that the mutation would result in destabilization of the protein. Transfection of the mutation into COS7 cells showed that the majority of the mutant R213W protein localized outside of the nucleus, whereas the wildtype protein resided in the nucleus. These findings suggested that the mutation disturbs the nuclear localization domain. The R213W mutation was able only to partially restore gad1 (605363) expression in zc4h2-null zebrafish, suggesting that the mutation results in a loss of function.

Using Western blot analysis, Ma et al. (2017) showed that R213W mutant ZC4H2 had weaker SMAD-stabilizing activity compared to wildtype ZC4H2 in HEK293 cells.

.0005 WIEACKER-WOLFF SYNDROME
ZC4H2, LEU66HIS
In affected members of a family (K8070) with X-linked mental retardation originally described by Miles and Carpenter (1991) (WRWF; 314580), May et al. (2015) identified a c.197T-A transversion in the ZC4H2 gene, resulting in a leu66-to-his (L66H) substitution at a highly conserved residue. The mutation, which was found by next-generation resequencing of 718 genes on the X chromosome and confirmed by Sanger sequencing, segregated with the disorder in the family. It was not found in the dbSNP or 1000 Genomes Project databases, or in 1,302 control X chromosomes. Molecular modeling predicted that the mutation would destabilize the protein. The L66H mutation was able only to partially restore gad1 (605363) expression in zc4h2-null zebrafish, suggesting that the mutation results in a loss of function.

.0006 WIEACKER-WOLFF SYNDROME
ZC4H2, IVS2DS, G-A, +5
In 2 males from a family (K9333) with Wieacker-Wolff syndrome (WRWF; 314580), May et al. (2015) identified a hemizygous G-to-A transition (c.225+5G-A) in intron 2 of the ZC4H2 gene, resulting in a splicing defect with an in-frame insertion of 15 residues (Val75ins15aa). The wildtype transcript was not found by RT-PCR of patient cells. The mutation, which was found by next-generation sequencing of the X chromosome and confirmed by Sanger sequencing, segregated with the disorder in the family. Two carrier females were unaffected, whereas 1 was reported to have speech impediment, seizures, and abnormal gait.

.0007 WIEACKER-WOLFF SYNDROME
ZC4H2, ARG18LYS
In 3 males from an Italian family (K9611) with Wieacker-Wolff syndrome (WRWF; 314580), May et al. (2015) identified a c.53G-A transition in exon 1 of the ZC4H2 gene, resulting in an arg18-to-lys (R18K) substitution at a highly conserved residue. The mutation, which was found by whole-genome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family. The mutation was not found in the dbSNP or 1000 Genomes Project database. Molecular modeling predicted that the mutation would destabilize the protein. May et al. (2015) found that the R18K mutation would affect only the long isoform of ZC4H2, since the short isoform lacks exon 1. The short isoform was highly expressed in all brain regions and spinal cord of human fetal tissue, whereas the long form was not expressed in spinal cord. Four carrier females had variable degrees of intellectual disability. The R18K mutation was able to restore gad1 expression (605363) in zc4h2-null zebrafish. May et al. (2015) noted that the phenotype in this family was milder compared that of patients with other mutations.

.0008 WIEACKER-WOLFF SYNDROME, FEMALE-RESTRICTED
ZC4H2, IVS2DS, T-C, +2
In a 3-year-old girl (family 2) with female-restricted Wieacker-Wolff syndrome (WRWFFR; 301041), Frints et al. (2019) identified a de novo heterozygous T-to-C transition in intron 2 of the ZC4H2 gene (c.225+2T-C, NM_018684.3), predicted to result in a splicing defect. The mutation, which was found by exome sequencing and confirmed by Sanger sequencing, was not found in the 1000 Genomes Project or gnomAD databases. Functional studies or studies of the variant in patient cells were not performed, but the variant was predicted to result in a loss of function and haploinsufficiency. Patient leukocytes showed completely skewed X-inactivation (100:0%).

.0009 WIEACKER-WOLFF SYNDROME, FEMALE-RESTRICTED
ZC4H2, 2-BP DEL, 275AG
In a female fetus (family 3) with female-restricted Wieacker-Wolff syndrome (WRWFFR; 301041), Frints et al. (2019) identified a heterozygous 2-bp deletion (c.274delAG, NM_018684.3) in exon 5 of the ZC4H2 gene, predicted to result in a frameshift and premature termination (Glu92ValfsTer7) in the coiled-coil domain. The mutation, which was found by exome sequencing and confirmed by Sanger sequencing, was not found in the 1000 Genomes Project or gnomAD databases. The variant was inherited from the unaffected mother who was mosaic (less than 10%) for the mutation. Functional studies or studies of the variant in patient cells were not performed, but the variant was predicted to result in a loss of function and haploinsufficiency. X-inactivation studies of leukocytes and fibroblasts from the mother and daughter were interpreted as a random pattern (78:22 in the mother, 61:39 in the daughter). An affected male infant was also born in this family, but he died at 10 hours of age and DNA was not available for study.

.0010 WIEACKER-WOLFF SYNDROME, FEMALE-RESTRICTED
ZC4H2, CYS206PHE
In a 23-year-old woman (family 8) with female-restricted Wieacker-Wolff syndrome (WRWFFR; 301041), Frints et al. (2019) identified a de novo heterozygous c.617G-T transversion (c.617G-T, NM_018684.3) in exon 5 of the ZC4H2 gene, predicted to result in a cys206-to-phe (C206F) substitution at a highly conserved residue in the zinc finger domain. The mutation, which was found by exome sequencing and confirmed by Sanger sequencing, was not found in the 1000 Genomes Project or gnomAD databases. Functional studies or studies of the variant in patient cells were not performed, but the variant was predicted to disrupt the protein function and cause haploinsufficiency.

.0011 WIEACKER-WOLFF SYNDROME
ZC4H2, LYS217ARG
In 4 affected males in a multigenerational Serbian family (family 9) with Wieacker-Wolff syndrome (WRWF; 314580), Frints et al. (2019) identified a hemizygous c.650A-G transition (c.650A-G, NM_018684.3) in exon 5 of the ZC4H2 gene, predicted to result in a lys217-to-arg (K217R) substitution at a highly conserved residue in the C terminus. The mutation, which was found by exome sequencing and confirmed by Sanger sequencing, was not found in the 1000 Genomes Project or gnomAD databases. Functional studies of the variant and studies of patient cells were not performed. There were 5 female carriers in the family, 2 of whom were over the age of 60 and had mild features.

.0012 WIEACKER-WOLFF SYNDROME, FEMALE-RESTRICTED
ZC4H2, 195-KB DEL
In an 18-year-old Dutch woman (family 11) with female-restricted Wieacker-Wolff syndrome (WRWFFR; 301041), Frints et al. (2019) identified a de novo heterozygous 195-kb deletion in the ZC4H2 gene that affected the first exons of all ZC4H2 transcripts, but no other genes. The mutation, which was found by CGH-array analysis, was not found in the 1000 Genomes Project or gnomAD databases. Functional studies of the variant and studies of patient cells were not performed, but the variant was predicted to result in a loss of function and haploinsufficiency.

.0013 WIEACKER-WOLFF SYNDROME, FEMALE-RESTRICTED
ZC4H2, TRP142TER
In a 5-year-old girl of Scottish descent (family 14) with female-restricted Wieacker-Wolff syndrome (WRWFFR; 301041), Frints et al. (2019) identified a de novo heterozygous c.426G-A transition (c.426G-A, NM_018684.3) in exon 4 of the ZC4H2 gene, predicted to result in a trp142-to-ter (W142X) substitution. The mutation, which was found by exome sequencing and confirmed by Sanger sequencing, was not found in the 1000 Genomes Project or gnomAD databases. Functional studies of the variant and studies of patient cells were not performed, but the variant was predicted to result in a loss of function and haploinsufficiency.

.0014 WIEACKER-WOLFF SYNDROME, FEMALE-RESTRICTED
ZC4H2, GLN23TER
In a 10-year-old girl (family 16) with female-restricted Wieacker-Wolff syndrome (WRWFFR; 301041), Frints et al. (2019) identified a de novo heterozygous c.67C-T transition (c.67C-T, NM_018684.3) in exon 2 of the ZC4H2 gene, predicted to result in a gln23-to-ter (Q23X) substitution in the coiled-coil domain. The mutation, which was found by exome sequencing and confirmed by Sanger sequencing, was not found in the 1000 Genomes Project or gnomAD databases. Functional studies of the variant and studies of patient cells were not performed, but the variant was predicted to result in a loss of function and haploinsufficiency.

Tags: Xq11.2