Alternative titles; symbols
HGNC Approved Gene Symbol: SIX3
SNOMEDCT: 253159001, 38353004; ICD10CM: Q04.6;
Cytogenetic location: 2p21 Genomic coordinates (GRCh38): 2:44,941,702-44,946,071 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 2p21 | Holoprosencephaly 2 | 157170 | Autosomal dominant | 3 |
| Schizencephaly | 269160 | 3 |
The vertebrate SIX genes are homologs of the Drosophila 'sine oculis' (so) gene, which is expressed primarily in the developing visual system of the fly. Members of the SIX gene family encode proteins that are characterized by a divergent DNA-binding homeodomain and an upstream SIX domain, which may be involved both in determining DNA-binding specificity and in mediating protein-protein interactions. Genes in the SIX family have been shown to play roles in vertebrate and insect development or have been implicated in maintenance of the differentiated state of tissues (summary by Boucher et al., 2000).
Oliver et al. (1995) isolated a mouse cDNA encoding Six3. They analyzed the Six3 expression pattern using in situ hybridization to mouse embryo sections. In early development, Six3 mRNA demarcated the most anterior border of the developing mouse neural plate. The anterior early expression of Six3 in the neural plate was restricted to regions that later give rise to the most rostral nonneural and neural derivatives. Later in development, Six3 was expressed in the most anterior part of the neural tube. This expression pattern suggested that Six3 is involved in a process by which positional information is established at the anterior boundary of the developing mouse embryo. Six3 appears to be the mouse functional homolog of Drosophila 'so,' since the proteins share extensive sequence identity and Six3 is expressed during mouse eye development.
By searching an EST database for sequences related to Drosophila 'so' and murine Six3, Granadino et al. (1999) identified a cDNA encoding human SIX3. The predicted 332-amino acid protein contains a SIX domain followed by a homeobox. SIX3 shares 98% and 88% identity with the mouse and chicken Six3 proteins, respectively, and all 3 proteins have identical SIX and homeobox domains. Northern blot analysis revealed that the SIX3 gene was expressed as an approximately 3-kb mRNA only in the eye. In situ hybridization to fetal and adult human retinal tissues indicated that the SIX3 gene was expressed in the ganglion cells and in cells of the inner nuclear layer. This expression pattern was maintained in the fully differentiated eye.
Granadino et al. (1999) determined that the human SIX3 gene contains 2 exons and spans 4.4 kb.
By interspecific backcross analysis, Oliver et al. (1995) mapped the mouse Six3 gene to chromosome 17, in a region that shares homology of synteny with human chromosome 2p22-p16. Using FISH and radiation hybrid analysis, Granadino et al. (1999) confirmed the localization of the human SIX3 gene to chromosome 2p21-p16.
Loosli et al. (1999) found that injection of Six3 RNA into medaka fish embryos caused ectopic Pax6 (607108) and Rx2 (see 601881) expression in midbrain and cerebellum, resulting in the formation of ectopic retinal primordia. Injected mouse Six3 RNA initiated ectopic expression of endogenous medaka Six3, uncovering a feedback control of Six3 expression. Initiation of ectopic retina formation demonstrated a pivotal role for Six3 in vertebrate retina development and hinted at a conserved regulatory network underlying vertebrate and invertebrate eye development.
In a yeast 2-hybrid screen, Del Bene et al. (2004) identified the DNA replication-inhibitor geminin (602842) as a partner of Six3. Geminin inhibits cell cycle progression by sequestering Cdt1 (605525), the key component for the assembly of the prereplication complex. Del Bene et al. (2004) showed that Six3 efficiently competes with Cdt1 directly to bind to geminin, which reveals how Six3 can promote cell proliferation without transcription. In common with Six3 inactivation, overexpression of the geminin gene in medaka induces specific forebrain and eye defects that are rescued by Six3. Conversely, loss of Gem (in common with gain of Six3) promotes retinal precursor-cell proliferation and results in expanded optic vesicles, markedly potentiating Six3 gain-of-function phenotypes. Del Bene et al. (2004) concluded that the transcription factor Six3 and the replication-initiation inhibitor geminin act antagonistically to control the balance between proliferation and differentiation during early vertebrate eye development.
In studies in Xenopus laevis, Masse et al. (2007) demonstrated that overexpression of ectonucleoside triphosphate diphosphohydrolase-2 (ENTPD2; 602012), an ectoenzyme that converts ATP to ADP, resulted in increased expression of Pax6, Rx1, and Six3 and caused ectopic eye-like structures, with occasional complete duplication of the eye. In contrast, downregulation of endogenous Entpd2 decreased Rx1 and Pax6 expression. Masse et al. (2007) concluded that ENTPD2 therefore acts upstream of these eye field transcription factors (EFTFs). To test whether ADP, the product of ENTPD2, might act to trigger eye development through P2Y1 receptors, selective in Xenopus for ADP, Masse et al. (2007) simultaneously knocked down expression of the genes encoding Entpd2 and the P2y1 receptor (601167). This prevented the expression of Rx1 and Pax6 and eye formation completely.
Jeong et al. (2008) provided evidence that SIX3 acts as a regulator of SHH (600725) transcription via binding to a highly conserved enhancer of SHH, SBE2 (SHH brain enhancer-2), located 460 kb upstream of the SHH coding sequence. SBE2 is a 10-nucleotide sequence conserved in human, mouse, chicken, and frog for over 350 million years. Pull-down assays using mouse brain extracts and electrophoretic mobility shift assays (EMSAs) demonstrated that SIX3 and SBE2 bind directly. Jeong et al. (2008) identified a 444C-T transition within the SBE2 enhancer sequence in 1 of 474 patients with holoprosencephaly (HPE); this patient had no other mutations in HPE-associated genes. The variant was not observed in DNA samples from 450 unrelated control individuals, but the father was an unaffected carrier, consistent with reduced penetrance. In vitro studies showed that SIX3 bound the 444C variant of SBE2 with higher affinity than the 444T variant. Jeong et al. (2008) postulated that this variant resulted in decreased SHH expression, which contributed to the development of HPE. The data suggested that SIX3 is a direct regulator of SHH expression in the anterior diencephalon.
Schell et al. (1996) mapped a locus for holoprosencephaly (HPE2; 157170) to a 1-Mb interval on 2p21 and defined a minimal critical region by a set of 6 overlapping deletions and 3 clustered translocations in HPE patients. Wallis et al. (1999) described the isolation and characterization of the homeobox-containing SIX3 gene from the HPE2 minimal critical region. They showed that at least 2 of the HPE-associated translocation breakpoints in 2p21 are less than 200 kb from the 5-prime end of SIX3. Mutational analysis identified 4 different mutations in the homeodomain of SIX3 that were predicted to interfere with transcriptional activation and were associated with HPE. Wallis et al. (1999) proposed that SIX3 is the HPE2 gene, essential for the development of the anterior neural plate and eyes in humans.
Laflamme et al. (2004) investigated the functional consequence of 3 holoprosencephaly-related missense mutations of the SIX3 gene with respect to the ability of the protein to interact with and stimulate the transcriptional activity of the nuclear receptor NOR1 (NR4A3; 600542). Using glutathione S-transferase fusion protein pull-down assays and transient cotransfections of Neuro-2a cells with expression and reporter vectors, they found that 1 mutation, L226V (603714.0001), did not alter the properties of SIX3 toward NOR1. Another mutation, V250A (603714.0003), resulted in the production of a highly unstable protein in Neuro-2a cells. The third mutation, R257P (603714.0002), resulted in a mutant SIX3 protein that no longer interacted with NOR1 in vivo. These observations suggested that different SIX3 mutations in HPE2 may affect different signaling pathways, and that 1 of these pathways may involve the nuclear receptor NOR1.
In 6 Brazilian patients with HPE2, Ribeiro et al. (2006) identified 5 missense mutations and 2 frameshift mutations in the SIX3 gene (see, e.g., 603714.0005) Comparison of patients with missense versus frameshift mutations showed essentially no difference. Experience with these patients suggested that SIX3 mutations result in a more severe phenotype than other gene mutations for holoprosencephaly. Three mutations were paternally transmitted, 2 were maternal, and 1 was a de novo event. The 5 parental mutation carriers appeared normal.
Domene et al. (2008) developed 2 biosensor assays in zebrafish to test the activity of mutant SIX3 alleles. One assay measured the ability of SIX3 alleles to rescue the zebrafish headless phenotype, and the other assay measured the ability of SIX3 alleles to induce dorsalized phenotypes, known to arise from overexpression and shifting of balance in the BMP (112264) pathway. The study showed that 89% of putative deleterious mutations causing HPE are significant loss-of-function alleles. The majority of mutations affect the BMP repressor function. Point mutations in the N-terminal Groucho-binding eh1-like motif decreased SIX3 function in all assays, indicating that this corepressor activity is essential. However, some truncated mutant proteins lacking the homeodomain still retained residual activity, as long as the first 39 amino acids of the SIX3 domain were intact.
Hehr et al. (2010) provided evidence that SIX3 mutations may contribute to schizencephaly (269160), which may be considered part of the larger phenotypic spectrum of HPE. Heterozygous SIX3 mutations (603714.0008-603714.0010) were identified in 2 unrelated patients and a fetus with schizencephaly, respectively, only 1 of whom also had HPE. Two of the mutations had previously been found in patients with variable manifestations of HPE.
Lens regeneration in adult newts is a classic example of how cells can faithfully regenerate a complete organ through the process of transdifferentiation. After lens removal, the pigment epithelial cells of the dorsal, but not the ventral, iris dedifferentiate and then differentiate to form a new lens. The genes SIX3 and PAX6 (607108) induce ectopic lenses during embryogenesis. Grogg et al. (2005) tested these genes, as well as members of the BMP (see 112264) pathway that regulate establishment of the dorsal-ventral axis in embryos, for their ability to induce lens regeneration. They showed that the lens can be regenerated from the ventral iris when the Bmp pathway is inhibited and when the iris is transfected with Six3 and treated with retinoic acid. In intact irides, Six3 is expressed at higher levels in the ventral than in the dorsal iris. During regeneration, however, only expression in the dorsal iris is significantly increased. Such an increase is seen in ventral irides only when they are induced to transdifferentiate by Six3 and retinoic acid or by Bmp inhibitors. Grogg et al. (2005) concluded that lens regeneration can be achieved in noncompetent adult tissues and that this regeneration occurs through a gene regulatory mechanism that is more complex that the dorsal expression of lens regeneration-specific genes.
Geng et al. (2008) found that deletion of 1 Six3 allele or replacement of wildtype Six3 with a Six3 allele containing the HPE-associated V250A mutation recapitulated most features of human HPE in mice. Reduced amounts of functional Six3 protein failed to activate Shh expression in the rostral diencephalon ventral midline of mutant mice, ultimately causing HPE.
Wallis et al. (1999) identified a de novo leu226-to-val (L226V) in the SIX3 gene in a patient with holoprosencephaly (HPE2; 157170).
Laflamme et al. (2004) demonstrated that this mutation did not alter the properties of SIX3 toward NOR1 in vitro.
Wallis et al. (1999) identified an arg257-to-pro (R257P) missense mutation in the SIX3 gene in a patient with a sporadic holoprosencephaly (HPE2; 157170). The patient presented with semilobar HPE, microphthalmia, and iris coloboma. The eye findings were considered consistent with the role described for SIX3 in eye development.
Laflamme et al. (2004) demonstrated that the SIX3 protein carrying this mutation did not interact with NOR1 (600542) in vivo.
Wallis et al. (1999) were able to study DNA from 3 of 4 affected fetuses with semilobar holoprosencephaly (HPE2; 157170); all carried a val250-to-ala (V250A) mutation. The proband in this family, who had HPE microsigns, also had the V250A mutation. Similar variability of penetrance in HPE families can be seen with other HPE genes such as SHH (600725), the site of mutations causing HPE3 (142945).
Laflamme et al. (2004) demonstrated that this mutation resulted in the production of a highly unstable protein in Neuro-2a cells.
Pasquier et al. (2000) reported a new HPE (HPE2; 157170) family, presenting a wide spectrum of clinical features ranging from cyclopia to hypotelorism, in which a mutation was found for the first time in the SIX domain of the SIX3 gene: a GG insertion created a frameshift leading to a nonsense mutation downstream in the homeodomain region, where the 4 previously reported mutations had been found.
Ribeiro et al. (2006) described holoprosencephaly (HPE2; 157170) in an infant heterozygous for 2 mutations in cis in the SIX3 gene: a 2-bp insertion (406_407dupGC), and a 206G-A transition resulting in a gly69-to-asp (G69D) substitution. The father had no microform of HPE but had the same mutations, as did the paternal grandmother. The proposita had microcephaly, hypotelorism, upslanting palpebral fissures, prominent eyes, absent nasal septum, flat nasal bridge, malar hypoplasia, premaxillary agenesis, and wide median agenesis of the philtral area. CT scan of the brain showed alobar HPE.
In affected members of a large kindred with variable manifestations of holoprosencephaly (HPE2; 157170), Solomon et al. (2009) identified a heterozygous 339G-T transversion in the SIX3 gene, resulting in a trp113-to-cys (W113C) substitution in the SIX domain and complete loss of protein function. The proband was ascertained at birth because of alobar HPE, macrocephaly, severe hypotelorism, short nose with upturned nares, hypoplastic philtrum, and low-set ears. In a family review, 2 deceased individuals had full HPE as observed in the proposita, 5 had died in early infancy from unknown causes, and at least 9 had a subtle facial microform with short angular nose with hypotelorism or narrow nasal bridge. The authors commented that the studies of this family spanned 15 years and that the analysis was complicated by reduced penetrance, variable expressivity, and phenocopies.
Holoprosencephaly 2
In a father and his 2 children with variable manifestations of holoprosencephaly (HPE2; 157170), Lacbawan et al. (2009) identified a heterozygous 385G-T transversion in the SIX3 gene, resulting in a glu129-to-ter (E129X) substitution. The father had microform HPE, the proband had lobar HPE, and the daughter had alobar HPE. This same mutation had been identified by Domene et al. (2008) in a patient with HPE, and was demonstrated by those authors to result in complete loss of function in a zebrafish biosensor assay.
Schizencephaly
Hehr et al. (2010) identified a de novo heterozygous E129X mutation in the SIX3 gene in a fetus with alobar HPE and evidence of schizencephaly (269160). Complex cerebral malformations were identified by ultrasound at 27 weeks' gestation, including microbrachycephaly, and a large unilateral schizencephalic cleft of the left posterior cerebral hemisphere. The findings suggested that schizencephaly may in some cases be part of the broad HPE spectrum.
Holoprosencephaly 2
In 3 unrelated probands with variable manifestations of holoprosencephaly (HPE2; 157170), Lacbawan et al. (2009) identified a heterozygous 109G-T transversion in the SIX3 gene, resulting in a gly37-to-cys (G37C) substitution. One patient had unspecified HPE, 1 had lobar, and 1 had the middle interhemispheric variant; the unaffected mother of this last patient also carried the mutation. Functional studies in zebrafish by Domene et al. (2008) indicated that the G37C substitution was a hypomorphic allele.
Holoprosencephaly 2 and Schizencephaly
Hehr et al. (2010) identified a heterozygous G37C mutation in the SIX3 gene an 8-year-old girl with unilateral schizencephaly (269160) of the left hemisphere without evidence of HPE. She had normal intelligence, but slight spastic paresis of the right leg. Her father, paternal grandfather, and aunt also carried the mutation, and clinical examination of the father showed no evidence of HPE.
In a German female infant with schizencephaly (269160), Hehr et al. (2010) identified a heterozygous 499G-T transversion in the SIX3 gene, resulting in an ala167-to-ser (A167S) substitution immediately adjacent to an eh1-like motif in a highly conserved domain. The pregnancy was complicated by diabetes mellitus, and postnatal imaging showed a complex brain malformation with extended occipitotemporal open-lip schizencephaly on the left side and a cystic formation within the left hemisphere. The mutation was not found in 200 controls.
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