Downloaded from http://bjo.bmj.com/ on August 18, 2017 - Published by group.bmj.com Clinical science Variant lattice corneal dystrophy associated with compound heterozygous mutations in the TGFBI gene Lydia Bai-Tsin Ann,1 Alessandro Abbouda,2 Ricardo F Frausto,1 Samira Huseynli,2 Kishan Gupta,1 Jorge L Alió,2,3 Anthony J Aldave1

1Stein Eye Institute, David ABSTRACT which TGFBI mutations are relatively common, Geffen School of Medicine, Background/Aims To report the clinical, such as Korea where 1 in every 875 individuals University of California, Los histopathological and genetic features of a variant of carries the mutation associated with GCD type II, Angeles, California, USA 2Vissum, Instituto Oftalmológico lattice corneal dystrophy (LCD) associated with two individuals who are homozygous for the mutant de Alicante, Universidad Miguel pathogenic mutations in the transforming growth allele demonstrate an earlier onset and more severe Hernández, Alicante, Spain factor-B-induced (TGFBI) gene. manifestation of the affected phenotype.7 We are 3 Division of Ophthalmology, Methods Clinical characterisation was performed by aware of only seven reports of six unrelated fam- Universidad Miguel Hernández, Alicante, Spain slit lamp examination and in vivo confocal microscopic ilies who are heterozygous for two different TGFBI imaging (IVCM). Histopathological characterisation was mutations (table 1). A determination of whether fi Correspondence to performed with light microscopic examination of an the identi ed mutations were on the same or Anthony J Aldave, The Stein Eye excised corneal button and a peripheral blood samples opposite alleles was made in four of the seven Institute, 100 Stein Plaza, UCLA, were collected for TGFBI screening. reports, with the identified mutations being present Los Angeles, CA 90095-7003, Results A 42-year-old woman presented with on the opposite allele in three of the four reports. USA; ​aldave@​jsei.​ucla.​edu progressive photophobia and decreased visual acuity in We describe the clinical and histopathological fea- LB-TA and AA contributed both eyes. Slit lamp examination demonstrated punctate tures of a novel variant LCD associated with the p. equally. and linear branching opacities in the mid and posterior (Val113Ile) and p.(Leu558Pro) mutations in the corneal stroma, corresponding to hyper-reflective TGFBI gene. As p.(Val113Ile) has been previously Received 27 August 2015 opacities noted on IVCM and amyloid deposition noted described in association with a GCD phenotype, Revised 16 April 2016 Accepted 19 June 2016 on histopathological examination of an excised corneal the p.(Leu558Pro) mutation appears to be the Published Online First button. TGFBI screening revealed two previously reported major determinant of the resultant phenotype, a 11 July 2016 heterozygous missense mutations: c.337G>A novel variant of LCD.89 (p.(Val113Ile)) in exon 4 and c.1673T>C (p. (Leu558Pro)) in exon 12. Screening of an affected sibling MATERIALS AND METHODS with a similar phenotype revealed that she was also In this study, we followed the tenets of the heterozygous for both mutations, while screening of Declaration of Helsinki in the treatment of the parti- another sibling with punctate but not linear stromal cipants reported. Study approval was obtained from opacities revealed that she was heterozygous for only the the Institutional Review Board at the University of p.(Leu558Pro) mutation. California, Los Angeles (IRB#11000020). Conclusions The p.(Val113Ile) mutation results in an alteration of the atypical LCD phenotype associated with Clinical evaluation the p.(Leu558Pro) mutation. This represents only the After informed consent was obtained, a detailed second report of the alteration of the phenotype of a clinical examination, including corneal pachymetry TGFBI dystrophy by a second, non-homozygous (Visante OCT; Carl Zeiss Meditec, Dublin, CA, pathogenic mutation, and thus provides insight into the USA), non-contact specular microscopy (SP-3000P; phenotype-genotype correlation of the TGFBI dystrophies. Topcon Oakland, CA, USA) and in vivo confocal microscopy (IVCM) (Confoscan; Nidek Technologies, Fremont, CA, USA), was performed INTRODUCTION for two members of a family with multiple Mutations in the transforming growth members affected by an apparently dominantly factor-B-induced gene (TGFBI; MIM 601692), inherited corneal disorder to determine their located on chromosome 5q31, have been associated affected status. with a variety of corneal dystrophies, including: lattice corneal dystrophy (LCD) associated with Histopathological analysis stromal amyloid deposition; granular corneal dys- Corneal tissue excised at the time of a penetrating trophy (GCD) type I and II, characterised by keratoplasty (PK) was fixed and analysed using light amorphous stromal aggregates and the Bowman microscopy following staining with H&E, Congo layer dystrophies, also known as Thiel-Behnke and red and periodic acid-Schiff (PAS). – Reis Buckler (RBCD) corneal dystrophies.1 5 To date, more than 50 different autosomal dominant Genetic analysis fi To cite: Ann LB-T, coding region mutations have been identi ed in Genomic DNA was extracted from saliva samples Abbouda A, Frausto RF, TGFBI, with the vast majority of the affected indi- (Oragene saliva collection kits; DNA Genotek, et al. Br J Ophthalmol viduals demonstrating a single pathogenic coding Ottawa, ON, Canada) collected from study sub- 2017;101:509–513. region mutation.26However, in populations in jects. All 17 coding exons of TGFBI were amplified

Ann LB-T, et al. Br J Ophthalmol 2017;101:509–513. doi:10.1136/bjophthalmol-2015-307602 509 Downloaded from http://bjo.bmj.com/ on August 18, 2017 - Published by group.bmj.com Clinical science

Table 1 Previous reports of compound heterozygous mutations in transforming growth factor-B-induced (TGFBI) gene Author Year Mutation 1 Mutation 2 Location† Clinical description

Dighiero et al10 2000 c.371G>T c.373_378delACGGAG Cis ▸ Round or snow-flake-like deposits appearing as dense, rod-shaped p.(Arg124Leu) p.(Thr125_Glu126del) structures in the central, anterior stroma Sakimoto et al11 2003 c.370C>T c.1408C>T Trans ▸ Lattice-like stromal opacities p.(Arg124Cys) p.(Gly470*) Klintworth et al12‡ 2004 c.1637C>A c.1652C>A Not ▸ Refractile lattice-like corneal stromal deposits characterised by branching p.(Ala546Asp) p.(Pro551Gln) determined and non-branching lattice figures ▸ Delicate filamentous and discrete, short, irregularly shaped stromal deposits and corneal haze Aldave et al13‡ 2004 c.1637C>A c.1652C>A Not ▸ Symmetric, radially arranged, branching refractile lines within and p.(Ala546Asp) p.(Pro551Gln) determined surrounding an area of central anterior stromal haze ▸ Multiple polymorphic, refractile deposits in the mid and posterior stroma Yamada et al14 2009 c.371G>A c.1631A>G Trans ▸ Granular and stellate anterior stromal opacities p.(Arg124His) p.(Asn544Ser) ▸ Lattice lines in 2/3 affected individuals Zhong et al15 2010 c.1541G>C c.1545T>A Not ▸ Anterior stromal refractile deposits characterised by branching and p.(Arg514Pro) p.(Phe515Leu) determined non-branching lattice lines ▸ Delicate, filamentous, discrete, short and irregularly shaped stromal deposits and corneal haze Yam et al16 2012 c.307-308delCT c.371G>A Trans ▸ Multiple granular, refractile, ice flake-like grey-whitish opacities p.(Ser104Lysfs*26) p.(Arg124His) Current study 2016 c.384G>A c.1673T>C Trans ▸ Discrete and confluent punctate stromal opacities p.(Val113Ile) p.(Leu558Pro) ▸ Fine linear and branching stromal opacities involving the posterior central and paracentral cornea All mutation descriptions have been updated from their original published format for adherence to Human Genome Variation Society nomenclature (http://www.hgvs.org/mutnomen/disc.html). †This column refers to whether the pair of mutations were identified on the same (cis) or opposite (trans) alleles. ‡The unique pedigrees for each of these reports were confirmed to have a common founder by haplotype analysis.

for direct sequencing using previously described primers and also denied a history of corneal erosions, demonstrated similar conditions.13 17 18 The minor allele frequencies (MAF) of iden- punctate and linear mid and deep stromal opacities to those tified variants were assigned using the Database of Single seen in the proband’s corneas. A PK was performed in each eye, Nucleotide Polymorphisms (National Center for Biotechnology with evidence of recurrent dystrophic deposits noted 3 years Information, National Library of Medicine; Bethesda, MD, USA after transplantation. The proband’s 43-year-old and (dbSNP Build ID: 138)). 38-year-old affected sisters (figure 1; II-3 and II-5) demonstrated similar-appearing PAD-like discrete stromal opacities as well as branching mid and posterior stromal opacities in each cornea. RESULTS fi Clinical evaluation However, the 50-year-old elder sister of the proband ( gure 1; The proband (figure 1; II-4) is a 42-year-old woman who pre- II-1) demonstrated only the PAD-like mid and posterior stromal fi sented to one of the authors ( JLA), with a 5-year history of opacities, without evidence of the ne linear and branching photophobia and loss of visual acuity in each eye. She denied a stromal opacities observed in her affected siblings. The pro- ’ history of recurrent corneal erosions. Corrected distance visual band s father was not available for examination due to advanced acuity (CDVA) measured 20/60 in each eye. Slit lamp examin- age and limited mobility. However, based on previous ocular ation demonstrated discrete and confluent punctate opacities examinations, he developed corneal stromal deposits when he ’ involving the mid and posterior central corneal stroma of each was approximately 40 years old. The proband s mother was not eye (figure 2). The opacities, which resembled those described in available for examination or enrolment in this study due to polymorphic amyloid degeneration (PAD), appeared grey-white advanced age and senility, but the family denied a known on direct illumination and translucent on retroillumination. In history of her having a corneal dystrophy. addition, fine linear and branching stromal opacities involving the posterior central and paracentral cornea but sparing the Histopathological analysis corneal periphery were observed in each eye (figure 2). Central Histopathology of the proband’s corneal button demonstrated a corneal thickness was 573 mm oculus dexter and 571 mm oculus normal epithelium and Bowman’s layer. Located within the sinister. Specular microscopic imaging demonstrated a normal corneal stroma, elongated fusiform structures were observed endothelial cell morphology and density, ranging between 2700 oriented parallel to the stromal lamellae that demonstrated posi- and 2900 cells/mm2. IVCM imaging demonstrated white opaci- tive staining with PAS and Congo red stains (figure 3B). ties of varying sizes and shapes primarily located in the mid and Histopathological analysis of the proband’s brother’s cornea was posterior stroma (figure 3A). The patient underwent an unevent- not performed. ful femtosecond laser-assisted PK in the left eye. One year after the keratoplasty procedure, CDVA in the left eye had improved Genetic analysis to 20/25 and no evidence of recurrent dystrophic deposition was Screening of TGFBI exons 1–17 in the proband demonstrated two noted in the graft. previously described heterozygous missense mutations, c.384G>A Four of the proband’s five siblings were also examined by one (p.(Val113Ile)) in exon 4 and c.1673T>C (p.(Leu558Pro)) in of the authors ( JLA). Slit lamp biomicroscopic examination of a exon 12 (figure 1), and a common synonymous substitution, 46-year-old male sibling of the proband (figure 1; II-2), who c.651G>C (p.(Leu217Leu)), in exon 6 (MAF=0.318).89

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Figure 1 Pedigree of family with a variant form of lattice corneal dystrophy. Filled symbols represent affected individuals; open symbols represent unaffected individuals; question mark (?) indicates individuals of undetermined affected status; arrowhead indicates the proband; asterisks indicate individuals in whom transforming growth factor-B-induced (TGFBI) screening was performed. Chromatograms demonstrate the TGFBI mutations c.337G>A (p.(Val113Ile)), identified in individuals II-3 and II-4, and c.1673T>C (p.(Leu558Pro)), identified in individuals II-1, II-3 and II-4.

Screening of TGFBI in the proband’s 43-year-old affected sister following amino acids: 134–236 (FAS1–1), 242–372 (FAS1–2), (figure 1; II-3) demonstrated the same missense and silent muta- 373–501 (FAS1–3) and 502–632 (FAS1–4).819All TGFBI tions present in the heterozygous state. However, screening of mutations associated with dystrophic corneal deposition, includ- TGFBI in the proband’s 50-year-old elder sister (figure 1; II-1) did ing the p.(Leu558Pro) mutation that we report, are located in not demonstrate the c.384G>A mutation in exon 4 or the the fourth FAS1 domain, except for those affecting amino acid c.651G>C variant in exon 6, but did demonstrate heterozygosity residues 113 and 123–126 in the first FAS1 domain and amino for the c.1673T>C mutation in exon 12 (figure 1). acid 501 in the third FAS1 domain.15818The valine at pos- ition 113 is located in the α1 helix of the first FAS1 domain and DISCUSSION is strictly conserved in various species, indicating its importance TGFBI-associated corneal dystrophies are associated with extra- in normal protein function.820Indirect in vitro evidence sug- cellular depositions of insoluble protein aggregates, which can gests that the presence of valine-valine at positions 112–113 be amyloid (as in LCD), granular/non-amyloid (GCD type I and may play an important role in amyloid fibril formation, yet the RBCD), or a mixture of both (GCD type II), in the cornea.8 exact role of Val113 in TGFBI folding remains to be eluci- The protein encoded by TGFBI is 683 amino acids long and dated.21 Missense mutations in the fourth FAS1 domain lead to contains four tandemly repeated fasciclin 1 (FAS1) domains of dystrophic corneal deposition by altering TGFBI structure, sta- approximately 130 residues each, so named due to their bility and subsequent protein processing or by affecting TGFBI sequence similarity to Drosophila melanogaster axonal guidance turnover and fibrillation rates, causing increased aggregation.2 molecule FAS1.19 These four FAS1 domains correspond to the Specifically, a proteolytic cleavage site has been shown to be

Figure 2 Slit lamp photomicrographs of individual (figure 1; II-4) with variant lattice corneal dystrophy. Discrete opacities are seen in the central posterior stroma while fine branching linear opacities are observed in the paracentral corneal stroma (arrows).

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associated with the p.(Asn544Ser) mutation. Allele-specific cloning revealed that the two mutations were on different chro- mosomes, thus explaining the combined phenotype. We also performed strand-specific sequencing to determine whether the two mutations were on the same allele. Unfortunately, the large size of the amplicon prevented successful molecular cloning of the 11 kb region, and neither of the proband’s parents were available for DNA collection. However, as the proband’s eldest sister was heterozygous for only the p.(Leu558Pro) mutation, it can be reasonably presumed that the p.(Val113Ile) and (p. (Leu558Pro) mutations identified in the proband and another sister are present on separate chromosomal strands. As such, it is likely that each parent harboured one of the two mutations. Rather than producing a compound phenotype, as reported for individuals with the Arg124His and Asn544Ser mutations affecting separate alleles, the mutations that we report, which also are located in the first and fourth FAS1 domains, produce a phenotype distinct from that associated with the mutation in the first FAS1 domain but consistent with that associated with the mutation in the fourth FAS1 domain. This suggests that the fourth FAS1 domain mutation, p.(Leu558Pro) is the major determinant of phenotype. However, the difference in pheno- type between the proband and her sister who demonstrated punctate mid and posterior stromal opacities as well as linear and branching mid and deep stromal opacities, and the pro- band’s eldest sister, who demonstrated only the punctate mid Figure 3 In vitro confocal microscopic imaging of the central posterior stroma of individual (figure 1; II-4) with variant lattice corneal and posterior stromal opacities, indicates that the p.(Val113Ile) dystrophy. Hyper-reflective extracellular structures are seen, mutation does alter the phenotype associated with the p. corresponding to the dystrophic deposits noted clinically (A). (Leu558Pro) mutation. The identification of other individuals Histopathological examination of the same cornea following with compound heterozygous mutations in the first and fourth penetrating keratoplasty demonstrates panstromal extracellular deposits FAS1 domains will determine whether this is a consistent that displayed positive staining with the Congo red stain (B). finding, and thus provide further insight into the mechanisms via which the identified missense mutations determine the morphology of the dystrophic deposits that characterise the TGFBI corneal dystrophies. between the wild-type Arg557 and Leu558 residues in the core 22 Contributors AA, JLA and AJA conceived and designed the study. LB-TA, RF, AA, of the fourth FAS1 domain. Thus, the p.(Leu558Pro) muta- fi fi KG and JLA were involved in data acquisition, data analysis and creation of gures tion may result in a mutant TGFBI protein that is signi cantly and tables. LB-TA, RF and AJA were responsible for interpretation of results. SH less susceptible to proteolysis, disrupting the normal degradation and AA wrote the original version of the manuscript, which was rewritten by LB-TA and turnover of corneal TGFBI, as demonstrated for the p.- and extensively revised by AJA. All authors contributed to the critical revision of (Arg555Trp) mutation, leading to dystrophic corneal depos- successive drafts of the manuscript and gave final approval for the submitted manuscript. ition.22 The fact that the individual reported herein does not present with a phenotype characteristic of GCD previously asso- Funding Support for the conduct of this study was provided by the National Eye fi Institute (grant R01EY022082 to AJA and core grant P30EY000331) and by an ciated with the identi ed missense mutation involving Val113 unrestricted grant from Research to Prevent Blindness (AJA) and by a grant from the suggests that the disruption of the proteolytic cleavage site is of Spanish Ministry of Economy and Competitiveness, Instituto Carlos III, Red Temática primary importance in terms of its effect on TGFBI function. de Investigación Cooperativa en Salud “Patología ocular del envejecimiento, calidad We report the clinical, histopathological and molecular visual y calidad de vida”, Subproyecto de Calidad Visual (RD07/0062) (JLA). genetic features of a novel variant of LCD in a Spanish family. Competing interests None declared. The phenotype in the family that we report differs considerably Patient consent Obtained. from the phenotype previously reported in association with the Provenance and peer review Not commissioned; externally peer reviewed. p.(Val113Ile) mutation, resembling more the clinical phenotype of ‘polymorphic corneal amyloidosis’ that has been used to describe the appearance of individuals with atypical LCD asso- 9 ciated with the p.(Leu558Pro) mutation. To the best of our REFERENCES knowledge, only six families have been reported with affected 1 Weiss JS, Møller HU, Aldave AJ, et al. IC3D classification of corneal dystrophies— individuals who are heterozygous for two different TGFBI edition 2. Cornea 2015;34:117–59. – mutations (table 1).21016 23 However, in five of the six fam- 2 Poulsen ET, Runager K, Risør MW, et al. 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Variant lattice corneal dystrophy associated with compound heterozygous mutations in the TGFBI gene Lydia Bai-Tsin Ann, Alessandro Abbouda, Ricardo F Frausto, Samira Huseynli, Kishan Gupta, Jorge L Alió and Anthony J Aldave

Br J Ophthalmol 2017 101: 509-513 originally published online July 11, 2016 doi: 10.1136/bjophthalmol-2015-307602

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