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).