Alternative titles; symbols
HGNC Approved Gene Symbol: SIX5
Cytogenetic location: 19q13.32 Genomic coordinates (GRCh38) : 19:45,764,785-45,769,252 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 19q13.32 | Branchiootorenal syndrome 2 | 610896 | 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).
Boucher et al. (1995) identified SIX5 as a homeodomain protein gene downstream (centromeric) of the (CTG)n repeat in the DMPK gene (160900). RT-PCR analysis showed that the SIX5 gene, which they called DMAHP, is expressed in a number of human tissues, including skeletal muscle, heart, and brain.
Heath et al. (1997) used 2 different strategies to examine expression of the murine homolog of the DMAHP gene. The first approach, RT-PCR, detected spliced transcripts in a wide range of embryonic and adult tissues, in a pattern overlapping substantially with the expression of mouse DMPK. A second approach, the generation of transgenic mice expressing a lacZ reporter gene from a 4.3-kb DMAHP promoter fragment, also demonstrated expression in a range of tissues with potential links to the phenotype in myotonic dystrophy. They concluded that murine DMAHP has a similar pattern of expression to human DMAHP and that the mouse can serve as a useful model for functional studies of this gene, although species differences, such as the reduced CpG island (1.8 kb compared with 3.5 kb), must be kept in mind.
Myotonic dystrophy (160900) is a highly variable multisystem disease in which the classic adult-onset form displays progressive muscle wasting with myotonia, cataracts, heart block, gonadal atrophy, insulin resistance, and neuropsychiatric impairment. Its genetic basis is an expansion of CTG trinucleotide repeats in the DMPK protein kinase gene, though the pathophysiologic mechanism for multisystem degeneration in DM had yet to be defined. Among the triplet repeat expansion disorders, myotonic dystrophy is distinguished by the extended length of the repeat tract (5 to 13 kb in postmortem tissue) and its location in the 3-prime untranslated region of the DMPK gene. Thornton et al. (1997) noted that, in contrast to the profound muscle wasting that characterizes advanced myotonic dystrophy, only minor histopathologic abnormalities were found in DMPK knockout mice or in mice that overexpress a human DMPK transgene, making it unlikely that changes in DMPK activity provide a unitary explanation for the disease. Otten and Tapscott (1995) demonstrated that a DNase I (300081)-hypersensitive site located adjacent to the repeats on the wildtype allele is eliminated by repeat expansion, suggesting that large CTG-repeat arrays may be associated with a local chromatin environment that represses gene expression. Klesert et al. (1997) reported that the hypersensitive site contains an enhancer element that regulates transcription of the adjacent DMAHP homeobox gene. Analysis of DMAHP expression in cells of myotonic dystrophy patients with loss of the hypersensitive site revealed a 2- to 4-fold reduction in the steady-state DMAHP transcript levels relative to wildtype controls. Allele-specific analysis of DMAHP expression showed that steady-state transcript levels from the expanded allele were greatly reduced in comparison to those from the wildtype allele. Along the same line, Thornton et al. (1997) showed that DMAHP expression in myoblasts, muscle, and myocardium was reduced by the DM mutation in cis, and the magnitude of this effect depended on the extent of the CTG repeat expansion. These observations supported the hypothesis that DMAHP participates in the pathophysiology of DM.
Since the DM-associated (CTG)n repeat is located in the promoter region of SIX5, immediately downstream of DMPK, Winchester et al. (1999) hypothesized that dysfunction of this gene, which is homologous to the Drosophila eye development gene 'sine oculis,' is primarily responsible for the ophthalmic features of DM. The multicolored iridescent cataract is the most prominent feature of the eye pathology in DM. It is often the first and in some cases the only sign of the disease, occurring at a younger age than is expected for senile cataracts, and occurring in persons who show no muscle symptoms or who carry a premutation (CTG)n repeat allele. In an analysis of the expression of DMPK and SIX5 in normal human fetal and adult eyes, Winchester et al. (1999) found SIX5 transcripts in the adult corneal epithelium and endothelium, lens epithelium, ciliary body epithelia, cellular layers of the retina, and the sclera. SIX5 expression was not detected in fetal eyes. They also reported a restricted but partially overlapping expression pattern for DMPK transcripts and DMPK protein in normal fetal and adult eyes. Winchester et al. (1999) concluded that the expression of SIX5 and not DMPK in the adult lens indicated a role for SIX5 dysfunction in the development of adult-onset cataracts, the most frequently occurring eye phenotype in DM.
Boucher et al. (1995) identified the SIX5 gene in chromosome 19q13.3, centromeric to the DMPK gene (605377).
Branchiootorenal syndrome (BOR2; 610896) is an autosomal dominant developmental disorder characterized by the association of branchial arch defects, hearing loss, and renal anomalies. Mutations in the EYA1 gene (601653) were identified as a cause of the BOR syndrome. A member of the SIX family of proteins, unc-39 (SIX5), was found to interact directly with eya-1 in Caenorhabditis elegans as identified by high-throughput, yeast 2-hybrid analysis (Li et al., 2004). Hoskins et al. (2007) hypothesized that this interaction would be conserved in humans and that interactors of EYA1 represent good candidate genes for BOR. They therefore screened a cohort of 95 patients with BOR for mutations in SIX5. Four different heterozygous missense mutations were identified in 5 individuals with BOR2. Functional analyses of these mutations demonstrated that 2 mutations (600963.0001, 600963.0004) affected EYA1-SIX5 binding and the ability of SIX5 or the EYA1-SIX5 complex to activate gene transcription.
In 1 of the patients reported by Hoskins et al. (2007) as carrying a mutation in the SIX5 gene (T552M; 600963.0004), Krug et al. (2011) identified a mutation in the EYA1 gene, a deletion removing exons 3, 4, and 5. This patient's DNA had been tested for EYA1 mutations by direct sequencing, but not for abnormal copy number. This observation, in addition to the extreme rarity of SIX5 mutations, caused Krug et al. (2011) to reconsider the role of SIX5 in branchiootorenal syndrome etiology.
Klesert et al. (2000) replaced the first exon of Six5 with a beta-galactosidase reporter gene. Histochemical detection of beta-galactosidase activity demonstrated expression as early as 8.5 days postcoitum in the anterior neural folds. Between 10.5 and 15.5 days postcoitum, there was low expression in smooth muscle of stomach, esophagus, and urogenital sinus, and in skeletal muscle of the tongue, as well as scattered expression in the myotome and developing limb muscle. A higher level of expression was seen in the sclerotome, meninges, adrenal gland, and cartilaginous areas of developing bones and trachea. In the eye, expression was evident in the retina, the cornea, the vasculature on the posterior surface of the lens, and faintly in the lens-fiber layer. There was no reliable expression detected in the adult mouse. Disruption of Six5 function did not affect viability or fertility. Homozygous mutant mice had no apparent abnormalities of skeletal muscle function, but developed lenticular opacities at a higher rate than controls. Klesert et al. (2000) concluded that Six5 deficiency contributes to the cataract phenotype in myotonic dystrophy, and that myotonic dystrophy represents a multigenic disorder.
Sarkar et al. (2000) also studied Six5 expression in mouse. By in situ hybridization, they detected Six5 expression in the adult corneal epithelium and endothelium, inner and outer epithelium of the ciliary body, anterior lens epithelium, ganglion cells, cells of the inner nuclear layer, and photoreceptor cells of the retina. Sarkar et al. (2000) deleted the entire Six5 gene and replaced it with a neo cassette. They found that the rate and severity of cataract formation was inversely related to Six5 dosage and was temporally progressive. Six5 +/- and Six5 -/- mice showed increased steady-state levels of the sodium-potassium-ATPase alpha-1 subunit (182310) and decreased Dm15 mRNA levels. Sarkar et al. (2000) suggested that altered ion homeostasis within the lens may contribute to cataract formation. As ocular cataracts are a characteristic feature of DM, these results demonstrated that decreased Six5 transcription is important in the etiology of DM. The authors concluded that their data supported the hypothesis that DM is a contiguous gene syndrome associated with the partial loss of both DMPK and SIX5.
Sato et al. (2002) overexpressed a constitutively active form of Six5 in murine P19 embryonal carcinoma cells. Using expression profiling in cDNA arrays, they identified 21 potential target genes whose expression level increased by the treatment. Genes expressed in the somites, skeletal muscles, brain, and meninges comprised the majority, suggesting a role for Six5 in the development and function of mesodermal tissues and brain. One of these genes, Igfbp5 (146734), was also decreased in Six5-deficient mouse fibroblasts, and the response of human IGFBP5 to MyoD (159970)-induced muscle conversion was altered in cells of DM patients. The authors concluded that Six5 is an activator that directs Igfbp5 expression, and hypothesized that reduced SIX5 expression in DM contributes to the DM phenotype.
The CTG expansion causing DM results in transcriptional silencing of the flanking SIX5 allele. Sarkar et al. (2004) generated Six5 knockout and heterozygous mice by targeted disruption and demonstrated a strict requirement of Six5 for both spermatogenic cell survival and spermiogenesis. Leydig cell hyperproliferation and increased intratesticular testosterone levels were observed in the Six5 -/- mice. Although increased FSH (see 136530) levels were observed in the Six5 +/- and Six5 -/- mice, serum testosterone levels and intratesticular inhibin alpha (INHA; 147380) and inhibin beta-B (INHBB; 147390) levels were not altered in the Six5 mutant animals when compared with controls. Steady-state c-Kit (164920) levels were reduced in the Six5 -/- testis. The authors concluded that decreased c-Kit levels could contribute to the elevated spermatogenic cell apoptosis and Leydig cell hyperproliferation in the Six5 -/- mice. They hypothesized that the reduced SIX5 levels may contribute to the male reproductive defects in DM1.
In a patient with branchiootorenal syndrome (BOR2; 610896) manifesting bilateral dysplastic kidneys and preauricular tag on the right but no hearing loss, Hoskins et al. (2007) identified a heterozygous 472G-A transition in the SIX5 gene that resulted in an ala158-to-thr (A158T) amino acid substitution. Using yeast 2-hybrid analysis and luciferase assays, Hoskins et al. (2007) showed that this mutation reduced EYA1-SIX5 binding and the ability of the EYA1-SIX5 complex to activate gene transcription.
In a patient with a clinical diagnosis of branchiootorenal syndrome (BOR2; 610896) who had bilateral dysplastic kidneys and reduced renal function, bilateral hearing loss and cervical fistulae, and right-sided hemifacial microsomia, preauricular sinus, and pinna malformation, Hoskins et al. (2007) identified a heterozygous 886G-A transition in the SIX5 gene that resulted in an ala296-to-thr (A296T) amino acid substitution.
In a patient with a clinical diagnosis of branchiootorenal syndrome (BOR2; 610896), Hoskins et al. (2007) found a heterozygous 1093G-A transition in the SIX5 gene that resulted in a gly365-to-arg (G365R) amino acid substitution.
This variant, formerly titled BRANCHIOOTORENAL SYNDROME 2, has been reclassified based on the findings of Krug et al. (2011).
In 2 unrelated patients with the BOR syndrome (BOR2; 610896), Hoskins et al. (2007) found heterozygosity for the same missense mutation in the SIX5 gene, a 1655C-T transition that resulted in a thr552-to-met (T552M) substitution. One patient had no hearing loss but had bilateral hypoplastic kidneys and cervical fistulae; the second patient had agenesis of the left kidney and hypoplasia of the right kidney, bilateral cervical fistulae, and bilateral hearing loss. Using yeast 2-hybrid analysis and luciferase assays, Hoskins et al. (2007) showed that this mutation reduced EYA1-SIX5 binding and the ability of the EYA1-SIX5 complex to activate gene transcription.
Krug et al. (2011) demonstrated that one of the patients carrying the T552M mutation had a 3-exon deletion in the EYA1 gene (601653). This patient's DNA had been tested for EYA1 mutations by direct sequencing, but not for abnormal copy number. This patient had an affected twin brother and an affected father; both the T552M SIX5 and the 3-exon deletion in EYA1 were found in these family members.
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Sarkar, P. S., Appukuttan, B., Han, J., Ito, Y., Ai, C., Tsai, W., Chai, Y., Stout, J. T., Reddy, S. Heterozygous loss of Six5 in mice is sufficient to cause ocular cataracts. Nature Genet. 25: 110-114, 2000. [PubMed: 10802668] [Full Text: https://doi.org/10.1038/75500]
Sarkar, P. S., Paul, S., Han, J., Reddy, S. Six5 is required for spermatogenic cell survival and spermiogenesis. Hum. Molec. Genet. 13: 1421-1431, 2004. [PubMed: 15163633] [Full Text: https://doi.org/10.1093/hmg/ddh161]
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Winchester, C. L., Ferrier, R. K., Sermoni, A., Clark, B. J., Johnson, K. J. Characterization of the expression of DMPK and SIX5 in the human eye and implications for pathogenesis in myotonic dystrophy. Hum. Molec. Genet. 8: 481-492, 1999. [PubMed: 9949207] [Full Text: https://doi.org/10.1093/hmg/8.3.481]