The compound heterozygous mutations p.Arg124His and p.His174Asp in TGFBI
A 35-year-old woman (Proband 1; Family 1-Patient II-3) with confluent granular deposits, dense linear deposits, and extremely severe diffuse haze on her cornea visited our clinic. Her best-corrected visual acuity (BCVA) was 20/40 in both eyes (Fig. 1a). Genetic analysis revealed p.Arg124His and p.His174Asp missense mutations in TGFBI (Table 1). Her 38-year-old brother showed more diffuse haze in both eyes than in any usual GCD2 heterozygote of his age (Fig. 1a). The cornea of the proband’s father, who only harboured a heterozygous p.His174Asp mutation, was clear, whereas the mother, who was a GCD2 heterozygote, showed typical heterozygotic GCD2 phenotype, i.e., annular granular deposits and deep linear deposits in the cornea. The pedigree analysis showed both the proband and her brother to have inherited the mutations in trans-phase (i.e., p.His174Asp from the father and p.Arg124His from the mother) (Fig. 1b).
Table 1
Detected mutations increasing severity of granular corneal dystrophy 2 as a compound heterozygote with p.Arg124His in this study
Family
|
Nucleotide changea
|
Amino acid change
|
Amino acid sequence conservationb
|
Frequencies in the dbSNP databasec
|
Frequencies in the gnomAD databased
|
PP2e
|
MTf
|
PRO-VEANg
|
SIFTh
|
YUGCDF-1
YUGCDF-2
YUGCDF-3
|
c.520C > G
|
p.His174Asp
|
D. rerio
|
NA
|
0.000012
|
Dam
(0.999)
|
DC
(0.999)
|
Del
(-7.81)
|
Dam
(0.000)
|
YUGCDF-4
|
c.740T > A
|
p.Ile247Asn
|
D. rerio
|
rs376340498
C = 0.000008/1 (ExAC)
A = NA
|
C = 0.00001444
A = NA
|
Dam
(0.804)
|
DC
(0.999)
|
Del
(-5.14)
|
Dam
(0.000)
|
YUGCDF-5
|
c.263A > G
|
p.Tyr88Cys
|
D. rerio
|
NA
|
NA
|
Dam
(0.863)
|
DC
(0.999)
|
Del
(-6.93)
|
Dam
(0.000)
|
YUGCDF-6
|
c.770G > C
|
p.Arg257Pro
|
D. rerio
|
rs377709778
A = 0.00003/4 (ExAC)
C = NA
|
A = 0.00002890
C = NA
|
Dam
(0.977)
|
DC
(0.999)
|
Del
(-2.86)
|
Dam
(0.010)
|
YUGCDF-7
|
c.1631A > G
|
p.Asn544Ser
|
D. rerio
|
rs777288957
G = 0.00008/10 (ExAC)
|
0.000053
|
Benign
(0.281)
|
DC
(0.999)
|
Del
(-2.97)
|
Dam
(0.003)
|
YUGCDF-8
|
c.535C > T
|
p.Arg179*
|
D. rerio
|
rs886059924
(MAF = NA)
|
A = 0.000008133
T = NA
|
NA
|
DC
(1.000)
|
NA
|
NA
|
YUGCDF-9
|
c.1404T > A
|
p.Tyr468*
|
X. tropicalis
|
rs781643226
C = 0.000008/1 (ExAC)
A = NA
|
C = 0.000004064
A = NA
|
NA
|
DC
(1.000)
|
NA
|
NA
|
Abbreviations: Dam, damaging; DC, disease causing; Del, deleterious; MAF, minor allele frequency; MT, mutation taster; NA, not available; PP2, PolyPhen-2 prediction score Humvar; PROVEAN, Protein Variation Effect Analyzer; SIFT, Sorting Intolerant from Tolerant; SNP, single nucleotide polymorphism |
acDNA mutations are numbered according to the human cDNA reference sequence NM_004183; +1 corresponds to the A of ATG translation initiation codon. |
bAmino acid residue is continually conserved throughout evolution including the species as indicated. |
cdbSNP database (http://www.ncbi.nlm.nih.gov/SNP) |
dgnomAD browser (http://gnomad.broadinstitute.org/) |
ePolyPhen-2 prediction score HumVar ranges from 0 to 1.0; 0 = benign, 1.0 = probably damaging (http://genetics.bwh.harvard.edu/pph2/) |
fMutation taster (http://www.mutationtaster.org/). gPROVEAN (http://provean.jcvi.org/index.php). hSIFT (http://sift.jcvi.org/) |
A 49-year-old man (Proband 2; Family 2-Patient II-2), not related to Family 1, visited our clinic with visual disturbances complaint; he presented with very severe diffuse haze and stromal opacities compared to other patients (Fig. 1c). Genetic and segregation analyses of the patient’s family showed compound heterozygous p.Arg124His and p.His174Asp mutations in TGFBI (Supplementary Fig. S1a).
A 41-year-old woman (Proband 3; Family 3), not related to either Family 1 or 2, presented with severe phenotypic variation (Fig. 1d) and showed both p.His174Asp and p.Arg124His mutations in TGFBI upon genetic analysis. We, however, could not trace her pedigree, since the patient refused clinical evaluation and genetic analysis of her family members.
To investigate the molecular mechanism of why the phenotype of GCD2 aggravate when p.His174Asp mutation is accompanied, the degree of aggregation of p.His174Asp, and p.Arg124His mutant proteins was examined. Mixture of the cultured media of p.His174Asp expressing cells and p.Arg124His corneal fibroblasts, showed increased TGFBIp oligomer and aggregate formation during in vitro experiments (Supplementary Fig. S2, see also Supplementary Note).
Compound heterozygous mutations, p.Arg124His and p.Ile247Asn, in TGFBI
A 35-year-old man (Proband 4; Family 4-Patient III-1) was referred to our hospital with severe diffuse corneal haze causing visual disturbance, which recurred after phototherapeutic keratectomy (PTK) at another clinic. We obtained photographs of the cornea, acquired at the other clinic at the age of 25 years; he had undergone PTK of the right cornea at the age of 24 years, while the left cornea had not been treated. The photograph of his left untreated cornea acquired at the age of 25 years (Fig. 2a left first) showed extremely severe diffuse haze and granular deposits. Both p.Ile247Asn and p.Arg124His missense mutations were detected in TGFBI (Table 1), and pedigree analysis confirmed the mutations to be located in different alleles (Fig. 2b). The proband’s father, aged 77 years, showed clear corneas (Fig. 2c) with only p.Ile247Asn mutation, whereas the proband’s mother harboured p.Arg124His mutation and her cornea showed typical GCD2 features. Moreover, the 62-year-old paternal uncle of the proband harboured only p.Ile247Asn mutation with a clear cornea. Since the proband experienced decreased visual acuity, additional PTK for each eye was performed separately in our clinic. Corneal opacity of the proband, however, recurred rapidly, becoming diffuse and dense even after several PTK ablations. After deep anterior lamellar keratoplasty (DALK) of his left eye, the cornea has remained clear since the past 3 years (Fig. 2a).
Similar to p.His174Asp, the p.Ile24yAsn mutation was also examined for the aggregation of TGFBIp. Mixture of the cultured media of p.Ile247Asn expressing cells and p.Arg124His corneal fibroblasts, showed increased TGFBIp oligomer and aggregate formation during in vitro experiments (Supplementary Fig. S2, see also Supplementary Note).
Heterozygous patients with GCD2 having additional p.Tyr88Cys, p.Arg257Pro, or p.Asn544Ser mutation in a different TGFBI allele
A 17-year-old woman (Proband 5; Family 5-Patient II-2) with numerous large granular deposits in the cornea visited our clinic. She experienced intermittent eye pain due to recurrent corneal erosion, although her BCVA was 20/25 in both eyes (Fig. 3a). Whole-TGFBI sequencing and pedigree analysis of the patient revealed that she harboured compound heterozygous p.Arg124His and p.Tyr88Cys mutations (Table 1, Supplementary Fig. S1b). No corneal deposit was detected in her 46-year-old father who harboured p.Tyr88Cys heterozygous mutation alone, whereas her 45-year-old mother harbouring p.Arg124His heterozygous mutation showed typical GCD2 heterozygous phenotype with discoid granular and star-shaped corneal deposits in both eyes (Fig. 3a). The proband’s sibling, with no mutation, showed no corneal opacity.
A 28-year-old man (Proband 6; Family 6-Patient III-1) was referred to our clinic owing to visual disturbance due to corneal opacities in both eyes. BCVA of the patient was 20/40 for the right eye and 20/35 for the left eye, with both eyes showing confluent granular deposits and dense lattice deposits (Fig. 3b). Genetic analysis of the proband showed p.Arg124His and p.Arg257Pro missense mutations in TGFBI (Table 1, Supplementary Fig. S1c). His 54-year-old father, who only harboured heterozygous p.Arg257Pro mutation, did not show any deposit (Fig. 3b), whereas his 51-year-old mother and 49-year-old maternal uncle, both GCD2 heterozygotes, showed typical annular granular deposits with deep linear deposits (Fig. 3b), confirming trans-phase mutations in the proband.
A 32-year-old woman (Proband 7; Family 7-Patient III-11), who had undergone laser-assisted sub-epithelial keratomileusis (LASEK) in both her eyes at another clinic, visited our clinic with complains of visual disturbance, with severe diffuse stromal opacities. She underwent PTK in our clinic to remove the opacity in her right eye; however, her corneal opacity recurred rapidly after PTK (Fig. 3c). Genetic and pedigree analyses revealed that she carried both p.Arg124His and p.Asn544Ser mutations in a different allele of TGFBI (Table 1, Supplementary Fig. S1d). We could not find older living family members with p.Asn544Ser mutation alone in her family, and the younger members (aged 15 and 4 years) who harboured p.Asn544Ser mutation alone did not show any opacity.
We happened to identify a 22-year-old woman with p.Asn544Ser mutation alone in a different family during routine genetic screening before refractive surgery, following which we analysed the whole family of the patient for p.Asn544Ser mutation (Supplementary Fig. S1g). Two family members, one 82-year-old woman and the other 74-year-old woman, who were p.Asn544Ser heterozygotes, did not show any corneal opacity. A 56-year-old woman with p.Asn544Ser mutation alone (Family 10-Patient III-5), who had undergone LASEK 4 years ago on her left cornea only, to correct myopia of 2.5 D, showed single, small, white opacity, 1.4 mm away from the pupil centre (Supplementary Fig. S1h).
Heterozygous patients with GCD2 and additional nonsense mutations (p.Arg179* or p.Tyr468*) in the opposite allele of TGFBI
A 35-year-old woman (Proband 8; Family 8-Patient III-2) visited our clinic for treating extensive severe multiple discoid granular corneal deposits, resembling those observed in homozygous GCD2, and diffuse haze in both eyes. She had BCVA of 20/50 in both eyes and no other ocular history (Fig. 4a). After whole-TGFBI sequencing, she was found to carry compound heterozygous p.Arg124His and p.Arg179* [CGA (Arg) → TGA (stop codon)] mutations (Table 1). The proband’s 76-year-old father and 36-year-old elder sister were found to be p.Arg124His heterozygotes with their corneas expressing the typical GCD2 phenotypes (father: superficial breadcrumb-like deposits with deep spiny deposits) (Fig. 4a). The younger sister, without mutation, had no corneal abnormality. The proband’s mother and two maternal aunts, who were 70-, 64-, and 56-year-old, respectively, showed clear corneas despite having a missense mutation (p.Arg179*) in TGFBI (Fig. 4a, and Supplementary Fig. S1e). The findings for this family have been previously reported,21 except for the results of the genetic tests of the proband’s mother and maternal aunts.
A 21-year-old man (Proband 9; Family 9-Patient II-1) with numerous large granular deposits in both eyes visited our clinic (Fig. 4b). Both p.Arg124His and p.Tyr468* [TAT (Tyr) → TAA (stop codon)] mutations were detected in TGFBI (Table 1). The proband’s 44-year-old mother harbouring p.Arg124His mutation showed only mild typical corneal opacities compared to the proband (Fig. 4b). Although we could not test whether the his father harboured p.Tyr468* mutation, the proband was likely compound heterozygous for these mutations, assuming that p.Tyr468* mutation did not occur de novo (Supplementary Fig. S1f).