4.DISCUSSION
ARS is a rare genetic heterogeneous disorder characterized by a broad range of ocular and systemic abnormalities (Ito et al.,2014). In our current study, the heterozygous mutation c.246C>A(p.s82r) in the FOXC1 gene was found in the pedigree. ARS is often considered autosomal dominant, and some sporadic cases have also been reported. In this pedigree, the mutation in the FOXC1 gene was identified in the proband, and only the proband had typical signs and symptoms, while other members were normal, which indicated that the variant may be a spontaneous mutation. When inquiring about the family history, we knew that the maternal great grandmother and great grandmother of the proband were sisters, that is, the parents of the proband were consanguineous. Whether this may be the cause of spontaneous mutation of the gene remains to be further studied.
The specific function of the FOXC1 gene has not been fully elucidated. It is known to play an extremely important role in the regulation of embryonic and eye development, and FOXC1 mutations are frequently associated with Axenfeld-Rieger anomaly defined by anterior segment dysgenesis (ASD) with characteristic posterior embryotoxon, iris hypoplasia, and corectopia (Seifi et al.,2018). As an autosomal genetic dysplasia, ARS might manifest as a visual phenotype and/or nonvisual phenotype, and the visual phenotype includes underdeveloped iris stroma, underdeveloped microcornea, anterior synechiae, corneal opacity and juvenile glaucoma. The nonvisual phenotype includes mandibular hypoplasia, dental hypoplasia, abnormal periumbilical skin turnover, hearing impairment, congenital heart disease and renal dysplasia (Reis et al.,2012; Gauthier et al.,2020; Gokce et al.,2015). FOXC1 is also involved in the development of neural crest cells during embryonic development, mutation of which can cause the abnormal structure of aqueous outflow originating from neural crest cells, inducing increased intraocular pressure and blindness (Akula et al.,2019; Seo et al.,2017). In addition, its mutation can be accompanied by systemic abnormalities, such as impairment of facial organs derived from neural crest cells, hearing impairment, and cleft palate. Knockout of the FOXC1 gene in mice caused abnormal development of the eyes, heart, blood vessels, kidney, skeleton and other tissues or organs (Aldinger et al.,2009; Motojima et al.,2017; Motojima et al.,2017b). FOXC1-associated ARS is commonly reported without other systemic abnormalities (Zeynep et al.,2009; Strungaru et al.,2007). However, there are still patients with FOXC1 mutations demonstrating symptoms of systemic dysplasia, including congenital heart defects (CHDs), such as tetralogy of Fallot (Vande et al.,2018), sensorineural hearing loss (Souzeau et al.,2017) and brain abnormalities (Chrystal et al.,2019). The proband in the pedigree involved in our study presented with double eyeball enlargement at birth, was diagnosed with glaucoma, and underwent ventricular septal defect repair in our hospital when he was 3 years old, as well as sublingual cystectomy when he was 5. In addition, the child had a small tooth deformity and slight skin valgus around the umbilicus, indicating that the child had congenital ventricular septal defects and sublingual cysts. That is, this child had both visual and nonvisual phenotypes.
FOXC1 is expressed in the heart, kidney, eyes and brain; it is a member of the forkhead transcription factor family and plays an important role in embryogenesis, cell migration, cell differentiation, the expression of tissue-specific genes and tumorigenesis (Golson et al.,2016). FOXC1 expresses in heart, kidney, eyes and brain, it was a member of the forkhead transcription factor family and plays an important role in embryogenesis, cell migration, cell differentiation, the expression of tissue-specific genes and tumorigenesis. The forkhead (FH)structural domain of FOXC1 is very conserved and consists of 110 amino acids that recognize and bind to specific DNA sequences to activate target genes. Transcription and activation of downstream genes by the FOXC1 gene requires the involvement of two activation domains of AD-1 and AD-2, and inhibition of domain (ID) can attenuate the activity of these two domains (Lehmann et al.,2003). Mutations in the FOXC1 gene leading to ARS include missense mutations, nonsense mutations and insertion deletions and duplications of small fragments, most of these mutations are in the forkhead domain. There were few reports that the rearrangement of the chromosomal regions where the FOXC1 gene was located caused ARS (D’Haene et al.,2011; Chanda et al.,2008 ) and only one case of t(6,13) equilibrium translocation causing ARS was reported, in which FOXC1 was identified as the pathogenic gene(Nishimura et al.,1998) Patients with chromosome 6p25 deletion often show eye, cranial, facial, skeletal, heart and kidney deformities, hearing loss, or brain edema(D’Haene et al.,2011;Le Caignec et al.,2005; Descipio et al.,2005). The clinical symptoms of these patients varied, depending on the size of the missing chromosome fragment and the genes it contained. These patients also showed abnormalities in the anterior segment such as postembryonic ring and iris dysplasia, which was due to the absence of the FOXC1 gene or its regulatory elements(Lehmann et al.,2000; Nishimura et al.,2001; Lehmann et al.,2002).In this study, with the help of target capture high-throughput sequencing, a novel heterozygous missense mutation c.246C>A (nucleotide 246 in the coding region mutated from cytosine to adenine)was successfully identified in the proband of this family, resulting in amino acid changes p.S82R (amino acid 82 mutated from serine to arginine). Pathogenicity prediction results of this missense mutation of the FOXC1 gene c.246C>A (p.s82r) suggested that the mutation was identified as pathogenic. After filtering in the HGMD database, this mutation was identified as novel, thus extending its mutation spectrum.
ARS is often autosomal dominant, and some sporadic cases have been reported. In this ARS family, a new heterozygous mutation in the FOXC1 gene was identified only in the proband, while no FOXC1 gene mutation was identified in other family members, suggesting that the missense mutation c.246C>A might be sporadic, which may provide a strong basis for further study of this gene. By searching the Conserved Domain Database of NCBI, gene conservation among different species was analyzed. In our study, it was found that site p.S82 of the FOXC1 gene for serine S was highly conserved among species such as humans, house mice, rats, zebrafish, and African clawed frogs, suggesting that changes in this amino acid site might play an important role in the function of FOXC1. SWISS-MODEL was used to construct the three-dimensional structure of the FOXC1 region, and the results showed that the wild-type amino acid at position 82 was serine, which changed to arginine after mutation. The change from noncharged amino acids to positively charged amino acids might affect the function of proteins.
In summary, the heterozygous missense mutation c.246C>A in the FOXC1 gene was found in an ARS patient in this study. This site was highly conserved between the amino acid series of different species and located near the key protein domain. This mutation led to changes in the three-dimensional structure of the protein and was pathogenic. The discovery of this novel mutation further confirms the importance of FOXC1 in ARS, expands the mutation spectrum of the FOXC1 gene, and provides a new reference for further study of FOXC1 gene function.