Alternative titles; symbols
HGNC Approved Gene Symbol: STX4
Cytogenetic location: 16p11.2 Genomic coordinates (GRCh38): 16:31,033,095-31,040,168 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 16p11.2 | ?Deafness, autosomal recessive 123 | 620745 | Autosomal recessive | 3 |
Li et al. (1994) described the nucleotide sequence of STX4A, a syntaxin gene isolated from a placenta library. (The gene was previously symbolized STX2.) It encodes a predicted 297-amino acid protein that is 89% identical to the amino acid sequence of rat Stx4a.
By immunohistochemical analysis of rat brain sections, Kennedy et al. (2010) showed that Stx4 was expressed throughout brain. In rat hippocampal neurons, Stx4 showed a punctate distribution in the somatodendritic compartment, and Stx4 clusters often localized to dendritic spines. Immunogold electron microscopy of CA1 rat hippocampus revealed frequent Stx4 labeling near spine membranes at sites lateral to postsynaptic densities, with occasional labeling of presynaptic membranes. Biochemical fractionation of mouse brain revealed Stx4 in synaptosome fractions, but not in synaptic vesicle fractions.
Schrauwen et al. (2023) performed in silico analysis of mouse RNA expression datasets for the murine ortholog of human STX4, Stx4a, and observed that Stx4a is highly expressed during inner ear development, with widespread expression in sensory epithelium. Stx4a was upregulated during later developmental stages, including in the inner and outer hair cells and in the spiral and vestibular ganglion cells. Stx4a was also expressed widely during early craniofacial development, such as in the paraxial mesoderm at embryonic day (E) 8.5, and maxillary arch epidermal ectoderm (E9.5). Whole-mount immunostaining of Stx4a in cochlear tissue of 12-day-old wildtype mice revealed Stx4a immunoreactivity in stereocilia as well as throughout the outer and inner hair cell bodies and in the plasma membrane.
Gross (2014) mapped the STX4 gene to chromosome 16p11.2 based on an alignment of the STX4 sequence (GenBank AF026007) with the genomic sequence (GRCh37).
Mandon et al. (1996) stated that vesicle-associated membrane proteins (VAMPs) or synaptobrevins (see 185880) are proposed to bind to cognate vesicle-targeting receptors in target membranes, which include several members of the syntaxin family. The molecular mechanisms responsible for targeting of vesicles containing aquaporin-2 (AQP2; 107777) to the apical plasma membrane of renal collecting duct cells may involve the vesicle-targeting protein STX4. Among the known syntaxin isoforms, only STX1 (STX1A; 186590) and STX4 bind VAMP2 (185881) with high affinity. Thus, STX1 and STX4 could be considered candidates for a role in targeting of the AQP2/VAMP2-containing vesicles to the apical plasma membrane in collecting duct cells. In contrast to STX1, which is expressed predominantly in the central nervous system, STX4 is expressed in a variety of tissues including the kidney. Mandon et al. (1996) used sequence data for rat Stx1a, Stx1b (601485), and Stx4 from Bennett et al. (1993) to design PCR primers for RT-PCR analysis of rat tissues. They detected Stx4 mRNA in the apical plasma membrane of collecting duct principal cells. They also demonstrated in the rat kidney the presence of a protein with the characteristics of Stx4 in the apical plasma membrane of inner medullary-collecting duct cells.
To clarify the physiologic function of STXBP3 (608339) in insulin-stimulated GLUT4 (SLC2A4; 138190) exocytosis, Kanda et al. (2005) generated mouse embryos deficient in the Stx4-binding protein Stxbp3 and developed Stxbp3 -/- adipocytes from their mesenchymal fibroblasts. The insulin-induced appearance of Glut4 at the cell surface was enhanced in Stxbp3 -/- adipocytes compared to +/+ cells. Wortmannin, an inhibitor of PI3K, inhibited insulin-stimulated Glut4 externalization in +/+ but not -/- adipocytes. Kanda et al. (2005) suggested that disruption of the interaction between STX4 and STXBP3 in adipocytes might result in enhancement of insulin-stimulated GLUT4 externalization.
Using RT-PCR, immunoblot analysis, and immunofluorescence microscopy, Sander et al. (2008) demonstrated that human intestinal mast cells (MCs) expressed SNAP23 (602534), STX1B, STX2 (132350), STX3 (STX3A; 600876), STX4, and STX6 (603944), but not SNAP25 (600322). MCs also expressed VAMP3 (603657), VAMP7 (300053), and VAMP8 (603177), but, in contrast with rodent MCs, they expressed only low levels of VAMP2 (185881). VAMP7 and VAMP8 translocated to the plasma membrane and interacted with SNAP23 and STX4 upon activation. Inhibition of STX4, SNAP23, VAMP7, or VAMP8, but not VAMP2 or VAMP3, resulted in markedly reduced high-affinity IgE receptor-mediated histamine release. Sander et al. (2008) concluded that human MCs express a specific pattern of SNAREs and that VAMP7 and VAMP8, but not VAMP2, are required for rapid degranulation.
Using rat hippocampal neurons, Kennedy et al. (2010) showed that exocytosis of recycling endosomes at dendritic spines occurred at Stx4-enriched sites immediately lateral to postsynaptic densities. Disruption of Stx4 either acutely or chronically blocked membrane fusion and cargo delivery triggered by synaptic activation, and acute inhibition of Stx4 abolished long-term potentiation. Kennedy et al. (2010) concluded that STX4 is a central component of the SNARE machinery that mediates rapid, activity-dependent fusion of recycling endosomes in dendritic spines.
Autosomal Recessive Deafness 123
In 8 affected members from 2 sibships of a large consanguineous Pakistani family (pedigree 4768) segregating autosomal recessive nonsyndromic hearing loss (DFNB123; 620745), Schrauwen et al. (2023) identified homozygosity for a splice site mutation in the STX4 gene (186591.0002) that segregated with disease in the pedigree. The variant was present at low minor allele frequency in the gnomAD database-v2, and was not found in other public variant databases, including gnomAD-v3.
Associations Pending Confirmation
For discussion of a possible association between variation in the STX4 gene and early-onset dilated cardiomyopathy (CMD), see 186591.0001.
After ascertaining a 9-year-old boy (patient 1) with CMD and a homozygous missense mutation in the STX4 gene (see 186591.0001), Perl et al. (2022) used GeneMatcher to identify another individual with biallelic STX4 mutations: a male infant (patient 2) with multiple anomalies noted prenatally, including frontal edema, persistent ductus arteriosus, oligo/anhydramnios, hypoplastic kidneys, duodenal atresia with severely dilated echogenic bowel loops, and overlapping fingers. Postnatal evaluation after delivery at 30 weeks' gestation showed multiple malformations, including pulmonary hypoplasia, hepatomegaly, duodenal atresia, renal hypoplasia, small urinary bladder, scoliosis, clubfoot, and musculoskeletal contractures of the right hand, elbow, and foot. Echocardiography showed normal cardiac arrangement with moderately reduced left ventricular function. Facial dysmorphisms included small dysplastic low-set and posteriorly rotated ears, retrognathia, and tent-shaped mouth. He had massive ubiquitous edema and required ventilatory support; he died 5 days after birth due to multiorgan failure. The infant was compound heterozygous for a 2-bp deletion (c.89_90delGC), causing a frameshift predicted to result in a premature termination codon (Gly30AspfsTer28), and a splice site mutation (c.232+4A-C). His unaffected parents were each heterozygous for 1 of the mutations. The authors suggested that there is a requirement for STX4 during normal human development, and that the gene plays a role in cardiac physiology and neuromuscular function.
Using CRISPR/Cas9, Perl et al. (2022) generated a stx4 mutant zebrafish allele. Heterozygous carriers of the stx allele were equivalent to wildtype zebrafish. However, by 72 hours postfertilization (hpf), all homozygous stx4 mutants exhibited a complex of defects, including myocardial dysfunction with linearized hearts, pericardial edema, bradycardia, microcephaly, loss of the midbrain/hindbrain boundary, otic vesicle dysgenesis, neuronal atrophy and cell death, and touch insensitivity. A small proportion (less than 10%) of stx4 mutants had hearts that ceased to beat entirely by 72 hpf, and approximately 20% of stx4 mutants exhibited hemorrhage of the intersegmental and cranial vasculature. None of the stx4 mutants survived past 5 days postfertilization, presumably due to defects in multiple organs. Ca(2+) imaging in 72-hpf explanted hearts of stx4 mutants and their wildtype clutchmates revealed that the Ca(2+)-transient amplitude in stx4 mutant atria was significantly diminished compared with wildtype atria, and the Ca(2+)-transient duration in paced stx4 mutant ventricles was significantly shorter than in wildtype ventricles. The authors concluded that cardiac dysfunction observed in stx4 mutants is at least partially due to defects in Ca(2+) handling in cardiomyocytes. Further experiments with L-type Ca(2+) channel (LTCC) agonists and antagonists suggested that stx4 mutants have reduced sarcolemmal LTCC function, which likely contributes to the cardiac dysfunction observed in the setting of stx4 loss of function.
Schrauwen et al. (2023) generated zebrafish with morpholino-based knockdown of the STX4 ortholog by targeting the start codon site (ATG) and the zebrafish 5-prime donor splice site (SS) corresponding to the human c.232+6T-C variant (186591.0002). Both the ATG and SS morpholinos showed a significant difference in response to stimuli (startle response) compared to controls. The injected larvae also showed severe morphologic developmental defects with edema, and had a greater head size compared to control larvae. In addition, neuromast cells of the ATG and SS morpholinos did not show uptake of dye, suggesting that mechanotransduction function was disrupted in those cells. The authors concluded that morpholino-based knockdown of stx4 in zebrafish results in loss of hearing and mechanotransduction function along with severe morphologic defects.
This variant is classified as a variant of unknown significance because its contribution to cardiomyopathy (see 115200) has not been confirmed.
In a 9-year-old boy (patient 1) with congenital sensorineural hearing loss, hypotonia, and global developmental delay, who presented with severe biventricular dilated cardiomyopathy at age 3 years and underwent cardiac transplantation within 2 months, Perl et al. (2022) identified homozygosity for a c.718C-T transition (c.718C-T, NM_004604.4) in exon 9 of the STX4 gene, resulting in an arg240-to-trp (R240W) substitution within the highly conserved coiled-coil SNARE homology domain. His unaffected parents were heterozygous for the mutation and his unaffected brother did not carry the mutation. Three heterozygous alleles of the same 240 residue were present in the gnomAD database, yielding a global minor allele frequency of 0.00000796. The patient had progressive muscular weakness posttransplant, remained ventilator-dependent, and experienced poor somatic growth on enteral feeds. The authors generated zebrafish with a stx4 R241W variant, the analogous zebrafish mutation to R240W, and observed profound myocardial dysfunction and bradycardia. In addition, unlike wildtype stx4, the R241W mutant could not rescue the pleiotropic defects of stx4-null mutants. R241W mutant zebrafish showed a high degree of variability in their bradycardia, suggesting that the variant likely produces a hypomorphic protein.
In 8 affected members from 2 sibships of a large consanguineous Pakistani family (pedigree 4768) segregating autosomal recessive nonsyndromic deafness (DFNB123; 620745), Schrauwen et al. (2023) identified a splice site mutation in intron 3 of the STX4 gene (c.232+6T-C, NM_004604.5). Minigene assay, confirmed by Sanger sequencing, demonstrated skipping of exon 3, causing a frameshift predicted to result in a transcript targeted by nonsense-mediated decay. The unaffected parents and an unaffected sib were heterozygous for the mutation, which was present at low minor allele frequency (7.98 x 10(-6)) in the gnomAD-v2 database, but was not found in several other population databases, including TOPMed Bravo, Greater Middle East (GME), and gnomAD-v3. Zebrafish with morpholino-based knockdown of stx4 at the 5-prime donor site corresponding to the human c.232+6T-C variant showed loss of hearing and mechanotransduction function, along with severe morphologic defects.
Bennett, M. K., Garcia-Arraras, J. E., Elferink, L. A., Peterson, K., Fleming, A. M., Hazuka, C. D., Scheller, R. H. The syntaxin family of vesicular transport receptors. Cell 74: 863-873, 1993. [PubMed: 7690687] [Full Text: https://doi.org/10.1016/0092-8674(93)90466-4]
Gross, M. B. Personal Communication. Baltimore, Md. 6/24/2014.
Kanda, H., Tamori, Y., Shinoda, H., Yoshikawa, M., Sakaue, M., Udagawa, J., Otani, H., Tashiro, F., Miyazaki, J., Kasuga, M. Adipocytes from Munc18c-null mice show increased sensitivity to insulin-stimulated GLUT4 externalization. J. Clin. Invest. 115: 291-301, 2005. [PubMed: 15690082] [Full Text: https://doi.org/10.1172/JCI22681]
Kennedy, M. J., Davison, I. G., Robinson, C. G., Ehlers, M. D. Syntaxin-4 defines a domain for activity-dependent exocytosis in dendritic spines. Cell 141: 524-535, 2010. [PubMed: 20434989] [Full Text: https://doi.org/10.1016/j.cell.2010.02.042]
Li, H., Hodge, D. R., Pei, G. K., Seth, A. Isolation and sequence analysis of the human syntaxin-encoding gene. Gene 143: 303-304, 1994. [PubMed: 8206394] [Full Text: https://doi.org/10.1016/0378-1119(94)90117-1]
Mandon, B., Chou, C.-L., Nielsen, S., Knepper, M. A. Syntaxin-4 is localized to the apical plasma membrane of rat renal collecting duct cells: possible role in aquaporin-2 trafficking. J. Clin. Invest. 98: 906-913, 1996. [PubMed: 8770861] [Full Text: https://doi.org/10.1172/JCI118873]
Perl, E., Ravisankar, P., Beerens, M. E., Mulahasanovic, L., Smallwood, K., Sasso, M. B., Wenzel, C., Ryan, T. D., Komar, M., Bove, K. E., MacRae, C. A., Weaver, K. N., Prada, C. E., Waxman, J. S. Stx4 is required to regulate cardiomyocyte Ca2+ handling during vertebrate cardiac development. Hum. Genet. Genomics Adv. 3: 100115, 2022. [PubMed: 35599850] [Full Text: https://doi.org/10.1016/j.xhgg.2022.100115]
Sander, L. E., Frank, S. P. C., Bolat, S., Blank, U., Galli, T., Bigalke, H., Bischoff, S. C., Lorentz, A. Vesicle associated membrane protein (VAMP)-7 and VAMP-8, but not VAMP-2 or VAMP-3, are required for activation-induced degranulation of mature human mast cells. Europ. J. Immun. 38: 855-863, 2008. [PubMed: 18253931] [Full Text: https://doi.org/10.1002/eji.200737634]
Schrauwen, I., Ghaffar, A., Bharadwaj, T., Shah, K., Rehman, S., Acharya, A., Liaqat, K., Lin, N. S., Everard, J. L., Khan, A., Ahmed, Z. M., Ahmad, W., Riazuddin, S., Leal, S. M. Syntaxin 4 is essential for hearing in human and zebrafish. Hum. Molec. Genet. 32: 1184-1192, 2023. [PubMed: 36355422] [Full Text: https://doi.org/10.1093/hmg/ddac257]