Compound heterozygous mutations in TGFBI cause a severe phenotype of granular corneal dystrophy type 2

We investigated the clinical and genetic features of patients with severe phenotype of granular corneal dystrophy type 2 (GCD2) associated with compound heterozygosity in the transforming growth factor-β-induced (TGFBI) gene. Patients with severe GCD2 underwent ophthalmic examination (best-corrected visual acuity test, intraocular pressure measurement, slit-lamp examination, and slit-lamp photograph analysis) and direct Sanger sequencing of whole-TGFBI. The patient’s family was tested to determine the pedigrees. Five novel mutations (p.(His174Asp), p.(Ile247Asn), p.(Tyr88Cys), p.(Arg257Pro), and p.(Tyr468*)) and two known mutations (p.(Asn544Ser) and p.(Arg179*)) in TGFBI were identified, along with p.(Arg124His), in the patients. Trans-phase of TGFBI second mutations was confirmed by pedigree analysis. Multiple, extensive discoid granular, and increased linear deposits were observed in the probands carrying p.(Arg124His) and other nonsense mutations. Some patients who had undergone phototherapeutic keratectomy experienced rapid recurrence (p.(Ile247Asn) and p.(Asn544Ser)); however, the cornea was well-maintained in a patient who underwent deep anterior lamellar keratoplasty (p.(Ile247Asn)). Thus, compound heterozygosity of TGFBI is associated with the phenotypic variability of TGFBI corneal dystrophies, suggesting that identifying TGFBI second mutations may be vital in patients with extraordinarily severe phenotypes. Our findings indicate the necessity for a more precise observation of genotype–phenotype correlation and additional care when treating TGFBI corneal dystrophies.

www.nature.com/scientificreports/ 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.  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) www.nature.com/scientificreports/ 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. www.nature.com/scientificreports/  www.nature.com/scientificreports/ 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.

Compound heterozygous mutations, p.(Arg124His) and p.(Ile247Asn), in TGFBI.
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-yearold 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.

Discussion
The current study demonstrated that simultaneous presence of mutations, such as p.  24,25 . Further, intracellular TGFBIp is reportedly cleared out via lysosomes, and this function is impaired in cultured GCD2 corneal fibroblasts 26 . We suspected the double production of mutated TGFBIps from p.(Arg124His) allele and heterozygous mutation in opposite alleles to result in an intracellular TGFBIp status closer to that seen in GCD2 homozygotes than in heterozygotes. Elucidating the mechanism underlying the formation of differential phenotypes for each different second mutation requires further studies.
Interestingly, all the heterozygotes of p.  Fig. S1h). The 56-year-old woman with p.(Asn544Ser) mutation alone, who had undergone LASEK 4 years ago, showed only a small opacity. We could not determine whether this opacity was a result of scarring after LASEK or the exacerbation of silent www.nature.com/scientificreports/ cornea having p.(Asn544Ser)-only mutation. A study conducted in Japan reported lattice corneal dystrophy in a 68-year-old patient with a p.(Asn544Ser) mutation only 27 , although the underlying cause could not be precisely explained. However, the data suggested that more reports of cases with silent second mutations would be required to verify the safety of refractive surgery. There are abundant reports of exacerbation of TGFBI-related corneal dystrophies, including GCD2 or GCD1 with deposits, following corneal trauma such as laser-assisted in situ keratomileusis 28,29 , LASEK, photorefractive keratectomy, and refractive keratotomy 4,30,31 .  32 reported that the macroscopic, microscopic, and ultrastructural appearance of TGFBI-null mouse cornea remain unaffected, suggesting that partial or complete knockdown of TGFBI could be a potential therapy against TGFBI-linked (Arg124His) mutation showed milder phenotypes than the proband, and the mother carrying heterozygous TGFBI p.(Arg179*) showed no corneal opacity. (b) Slit-lamp photographs of Proband 9 and his mother. Proband 9 harboured compound heterozygous TGFBI p.(Tyr468*) and p.(Arg124His) mutations and showed more severe discoid granular deposits than his mother who harboured heterozygous TGFBI p.(Arg124His) mutation. www.nature.com/scientificreports/ corneal dystrophies. Currently, correction of p.(Arg124His) mutation in cornea by knocking out the mutated allele is being attempted for the treatment of GCD2 33,34 . Our present data indicate that the knockout of the allele should be very precise during gene therapy, in case of the heterozygote, such that only the allele containing p.(Arg124His) mutation is treated, leaving the normal-sequence allele intact. If the wild-type allele is altered to stop codon while the p.(Arg124His)-mutant allele is kept unchanged, it may result in the final corneal lesion being similar to that observed in compound heterozygotes with stop codon in the present study. Family 1 in this study was previously reported to have extremely varied GCD2 heterozygote phenotypes 10 . However, the exact reason for severe phenotypic variability in the family was not known then; here, we showed the compound heterozygous mutation to be a causative factor among multiple genetic and environmental factors. Moreover, we showed that the patients with compound heterozygous TGFBI mutations experienced rapid recurrence after PTK. Identifying the reason underlying why the cornea remaining clear for an extended period after DALK in Proband 4 of this study would require further investigation.
In conclusion, this study reported five novel and two known genetic mutations that can predict the occurrence of the severe phenotype of heterozygous GCD2. Since phenotypic variations may indicate the presence of a new genetic change, whole-TGFBI sequencing would be necessary when a heterozygous patient with severe GCD2 is identified. Identification of cases with second mutations could more thoroughly explain the pathophysiology of GCD2.

Clinical investigation. This study was approved by the Institutional Review Board of Yonsei University
College of Medicine (IRB No. 4-2012-0209) and followed the tenets of the Declaration of Helsinki. Written informed consent was obtained from all participants. All participants agreed to have their photos published. The patients who visited Severance Hospital from January 2007 to October 2020 underwent a detailed ophthalmological examination, including BCVA test, intraocular pressure measurement, slit-lamp examination, and slit-lamp photograph analysis. Among the patients with GCD2, the ones who presented severe phenotypes and had no other history of ocular or systemic diseases were selected as candidates for whole-TGFBI sequencing. After identifying other mutations, in addition to p.(Arg124His), family members were enrolled in the study to perform segregation analysis.
Genomic DNA preparation and mutation analysis. TGFBI was analysed as described previously, after receiving informed consent from the participants 29 . Briefly, 2 ml of blood was drawn from each subject and genomic DNA was extracted from their peripheral leukocytes using a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Primers were designed to amplify all 17 exons of TGFBI (Supplementary Table S1). Each polymerase chain reaction (PCR) was performed using a 20-μl reaction mixture (Maxime PCR Premix kit; iNtRON Biotechnology, Seongnam-si, Gyeonggi-do, Korea) containing 100 ng of genomic DNA, 10 pmol each of forward and reverse primers, and distilled water. Samples were amplified through 35 PCR cycles; each cycle consisted of denaturation for 20 s at 94 °C, annealing for 15 s at 58 °C, and extension for 50 s at 72 °C. PCR was performed using a 96-well thermal cycler (Applied Biosystems, Foster City, CA, USA). Subsequently, Sanger sequencing was performed for all exons; to screen for mutations, we compared the DNA sequence of patients with the complementary DNA sequence of TGFBI obtained from GenBank (NC_000005.10).
TGFBIp oligomer formation, aggregate analysis, and immunoblotting. To determine oligomer formation and aggregation ability, both wild-type and p.Arg124His-mutant TGFBIp were incubated with either p.(His174Asp)-or p.(Ile247Asn)-mutant TGFBIp for 1 h at 37 °C, following which immunoblotting was performed 35  www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.