1. Severe Phenotypes of Granular Corneal Dystrophy Type 2 and Genetic Analysis Results Yong Woo Ji, MD, Eung Kweon Kim, MD, PhD, Seung-Il Choi, PhD, Bong-Yoon Kim, PhD, Tae-im Kim, MD, PhD, and Hyung Keun Lee, MD, PhD Corneal Dystrophy Research Institute, Department of Ophthalmology, Severance Hospital, Yonsei University College of Medicine, Seoul, Korea The authors have no financial or proprietary interest in any of the products or techniques mentioned in this presentation.

2. Granular corneal dystrophy Type 2 (GCD2) Also called Avellino Corneal Dystrophy GCD2 is an autosomal dominant disorder caused by an Arg124His (R124H) mutation in the transforming growth factor- β-induced (TGFBI) gene on chromosome 5q31. GCD2 have prevalence of about 11.5 affected patients per 10,000 individuals in Korea. Among corneal dystrophies related to TGFBI gene mutation, GCD2 shows the highest prevalence. Progressive accumulation and aggregation of hyaline and amyloid in the corneal stroma with aging is a characteristic clinical feature of GCD2.

3. Heterozygous GCD2 In the early stage of expression in GCD2 heterozygotes, granular deposits are detected in the superficial stromal layer, while lattice deposits are in the deeper stroma later, resulting in diffuse stromal haze with visual impairment. Corneal opacities worsen slowly and most patients maintain vision and may experience severe visual impairment only late in life time. Each Patient In one’s Teens In Twenties In Sixties

4. Heterozygous GCD2 However, distinctive heterozygous GCD2 can express diverse phenotypes and unpredictable rapid progression threatening vision even within family or young age. Molecular Vision 2012;18:1755-1762 Cornea 2013;32:296–300 Fig 1. Minimal phenotype of heterozygous GCD2 in 34 year-old male patient Fig 2. Severe phenotype of heterozygous GCD2 in 35 year-old female patient

5. Purpose GCD2 heterozygotes showed extremely varied phenotypes between individual patients. However, the genetic etiology of this variation has not been discussed previously. We investigated second TGFBI factors related to more severe expressivity of heterozygous GCD2 for patient’s age.

6. Methods (This study was performed in accordance with the Declaration of Helsinki and was approved by the Severance Hospital Institutional Review Board) Patients and Clinical evaluation 2 proband cases and their families with heterozygous GCD2 : Jan 2007 - Dec 2014 at Yonsei University Medical Center, Seoul, Korea Slit lamp examination and photography Genomic DNA Preparation and Mutation Analyses Full sequencing of DNA from peripheral blood : Polymerase chain reaction and Sanger sequencing for all exons of TGFBI gene

7. Results Case 1 (F/35) Heterozygous GCD2 Patient with Second R179X Mutation on TGFBI gene Clinical evaluation A 35 year-old woman visited our clinic for extensive severe multiple discoid granular corneal deposits on both eyes resembling homozygous GCD2. She had best corrected visual acuity (BCVA) 20/50 on both eyes and no other ocular history. Molecular genetic analysis A heterozygous C→T transition at nucleotide 535 of exon 5 at the first position of codon 179 (Arg179Stop, R179X) with was detected on the TGFBI gene in GCD2 patient.

8. Results Case 1 (F/35) Heterozygous GCD2 Patient with Second R179X Mutation on TGFBI gene Pedigree of family members with genetic analyses And clinical evaluation R179X mutation of TGFBI gene is silent by itself. However, when it accompanied with heterozygous R124H, compound heterozygous patient had severe phenotype even in younger age (35 year-old).

9. Clinical evaluation A 25 year-old man was referred with severe diffuse corneal haze with multiple granular and lattice deposits causing visual disturbance, even after phototherapeutic keratectomy (PTK) for both eyes at other clinic. His BCVA was 20/50 on right and 20/60 on left eye. Additional PTK for each eye was performed separately 5 years ago at our clinic. Therefore he had still favorable vision, BCVA 20/25 on both eyes. Molecular genetic analysis A heterozygous T→A transition at nucleotide 740 of exon 6 at the second position of codon 247 (Ile247Asn, I247N) with was detected on the TGFBI gene in GCD2 patient. Results Case 2 (M/25) Heterozygous GCD2 Patient with Second I247N Mutation on TGFBI gene

10. adfas Pedigree of family members with genetic analyses And clinical evaluation I247N mutation of TGFBI gene is silent by itself. However, when it accompanied with heterozygous R124H, compound GCD2 phenotypes become severe and recur fast after PTK ablation, even with heterozygote. Results Case 2 (M/25) Heterozygous GCD2 Patient with Second I247N Mutation on TGFBI gene

11. Conclusions Heterozygous GCD2 (Arg124His mutation of TGFBI gene) Silent second mutations of TGFBI gene on the other chromosome by itself Compound heterozygosity manifests more severe phenotype of GCD2 than individual age’s  Second silent mutations on TGFBI gene is worthy of detection as a genetic marker of severe heterozygous GCD2 Arg179Stop and Ile247Asn mutation, etc….