3.RESULITS
3.1 Pedigree and clinical characteristics
The proband included in our study (Fig. 1) was a 7-year-old boy from a Chinese family who was born at full term by spontaneous labor, and his binocular eyeball was large at birth. He was admitted to the hospital due to a further reduction in his visual acuity at 2 years of age and diagnosed with “congenital glaucoma”, and trabeculectomy for glaucoma in both eyes was performed. His best-corrected vision was 20/200 in the right eye and 20/133 in the left eye. IOP measured with the Goldmann tonometer was 34 mmHg in the right eye and 20 mmHg in the left eye. The operation history included ventricular septal defect repair in our hospital more than 4 years ago and sublingual cystectomy in our hospital 2 years ago. Figure 2 shows two photographs of the anterior segment of the proband’s eyes. The sclera of both eyes was pale blue, the cornea diameter was approximately 14 mm, and the axial depth of the anterior chamber was approximately 6 CT. Iris texture was not clear, with partial pigment loss. Extensive iridodialysis was observed, with the formation of a massive iris hiatus. The pupil had upward displacement, with ectropion uveae of the pupil margin and slight lens opacity (Fig. 2 A, B). The clinical features of the pedigree members are shown in Table 1.
3.2 Sanger sequencing and pathogenicity analysis of mutation sites
In our study, target capture high-throughput sequencing of 50 known candidate genes for iris disease using capture plates and gDNA samples from the proband was successfully carried out (Fig. 3). A heterozygous missense mutation (c.246C>A) was identified in this proband (nucleotide 246 in the coding region mutated from cytosine to adenine), leading to amino acid changes in p.S82R (amino acid no. 82 changed from serine to arginine), resulting in a missense mutation (Table 2). SIFT, PolyPhen 2, MutationTaster, GERP++ and REVEL were used to predict the pathogenic nature of the c.246C>A (p.S82R) mutation. The results showed that c.246C>A was rated as “deleterious” in SIFT, indicating that amino acid substitution at this conserved site was more likely to affect protein function. In PolyPhen-2, the mutation had a score of greater than 0.95, indicating that the mutation was likely to cause changes in protein structure or function. The mutation site was evaluated with Mutation Taster, with the assessment grade of “disease causing”. GERP++ had a score greater than 2, indicating that it was conserved, and the more conserved the site was, the greater the effect was on the protein. The REVEL score of a single missense variant ranged from 0 to 1, and the higher score reflected the higher possibility of disease caused by the variant. The score in REVEL was 0.968, indicating that the mutation might be pathogenic. Based on the above results, the mutation was predicted in the protein function prediction softwares SIFT, PolyPhen 2, MutationTaster, GERP++ and REVEL as deleterious, deleterious, deleterious, deleterious and deleterious respectively.According to the Standards and Guidelines for the Interpretation of Sequence Variants by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology in 2015, “likely pathogenic” was considered.
3.3 Mutation validation and cosegregation analysis
In our study, mutation identification and cosegregation analysis were completed using deoxy DNA sequencing (Fig. 3). The variant c.246C>A of FOXC1 was identified as a heterozygote in the proband (Table 2). The father and mother of the proband had wild-type genes and normal phenotypes, and the brother and grandmother also had wild-type genes and normal phenotypes. Thus, it was demonstrated that the c.246C>A variant of the FOXC1 gene co-segregated with the disease phenotype in this pedigree. This mutant was not present in the 100 normal racially matched controls (data not shown). In brief, these findings demonstrated complete segregation of this mutation in this family with Axenfeld-Rieger syndrome and determined its role in the pathogenesis of the disease.
3.4 Conservation analysis of the c.246C>A variant of FOXC1 and predictive analysis of the 3D model of the protein
By searching the Conserved Domain Database of the NCBI, gene conservation among different species was analyzed (Fig. 4). This study found that site p.S82 in the FOXC1 gene for serine(S) was highly conserved among species, such as human (NP_001444.2), house mouse (NP_032618.2), rat (NP_599165.1), zebrafish (NP_571803.1), and African clawed frog (NP_001007864.1), suggesting that 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 (Fig. 5).