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Polymorphic corneal amyloidosis
Polymorphic Corneal Amyloidosis A Disorder due to a Novel Mutation in the Transforming Growth Factor –Induced (BIGH3) Gene David E. Eifrig, Jr, MD, MBA,1 Natalie A. Afshari, MD,1 Harry W. Buchanan IV, MD,3 Brandy L. Bowling, MS,1 Gordon K. Klintworth, MD, PhD1,2 Purpose: To characterize the clinicopathologic phenotype as well as the molecular genetic basis of an autosomal dominant form of corneal amyloidosis. Design: Clinicopathologic and molecular genetic study of a family with a form of corneal amyloidosis. Participants: Forty-nine individuals from one family were studied. Methods: The medical records of affected family members were reviewed, and corneal tissue from those who had undergone penetrating keratoplasty (PK) was examined. Several family members were examined clinically, and corneas were photographed. Deoxyribonucleic acid from blood or buccal swabs was extracted from each consenting family member to determine the status of their transforming growth factor –induced (TGFBI) gene. The coding region of the TGFBI gene was analyzed for mutations in the proband’s DNA, and compared with the nucleotide sequences of normal individuals. This was performed by amplifying and sequencing all exons of the TGFBI gene. In all other family members, only exons 4, 8, 11, and 12 of the gene were amplified, sequenced, and analyzed for mutations. Main Outcome Measures: Clinicopathologic manifestations in relation to mutational status of the TGFBI gene. Results: Slit-lamp biomicroscopy revealed bilateral multiple polymorphic, polygonal, refractile, chipped ice– appearing gray and white opacities. There were also occasional deep filamentous lines that did not form a distinct lattice pattern. Corneal tissue of affected individuals who underwent PK contained widespread deposits of amyloid within the corneal stroma, particularly in the deep central stroma. Twelve members of the family were found to have a heterozygous single mutation in the TGFBI gene leading to a predicted amino acid substitution of aspartic acid for alanine (A546D). Nine of these individuals had ophthalmologist-documented corneal disease. The remaining 3, who were 11, 14, and 15 years old, were asymptomatic. In addition, 4 inconsequential polymorphisms with the nucleotide changes 387 G/A (R129R), 981 G/A (V327V), 1416 T/C (L472L), and 1620 C/T (F540F) were found. Conclusion: A distinct, progressive form of corneal amyloidosis with an autosomal dominant mode of inheritance is characterized clinically by the presence of refractile polymorphic corneal opacities. We have designated this entity, which is caused by an A546D mutation in the TGFBI gene, polymorphic corneal amyloidosis. Ophthalmology 2004;111:1108 –1114 © 2004 by the American Academy of Ophthalmology.
During the past few years, mutations in specific genes have been found to account for several inherited corneal disorOriginally received: June 20, 2003. Accepted: September 15, 2003. Manuscript no. 230398. 1 Department of Ophthalmology, Duke University Medical Center, Durham, North Carolina. 2 Department of Pathology, Duke University Medical Center, Durham, North Carolina. 3 Department of Surgery, Division of Ophthalmology, Sacred Heart Hospital, Allentown, Pennsylvania. Presented in part at: Association for Research in Vision and Ophthalmology Annual Meeting, May 7, 2003; Ft. Lauderdale, Florida. Research funding was provided by a research grant from the National Eye Institute, Bethesda, Maryland (grant no.: R01EY2712). Dr Afshari has a research career development award from Research to Prevent Blindness, New York, New York. Reprint requests to Gordon K. Klintworth, MD, PhD, 255 Medical Sciences Research Building, Box 3802 Duke University Medical Center, Durham, NC 27710.
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© 2004 by the American Academy of Ophthalmology Published by Elsevier Inc.
ders, and in many instances the clinicopathologic phenotypes correlate with the genetic mutation.1 Many corneal dystrophies are caused by a single nucleotide change, and some of these mutations occur in corneas with amyloid deposits. Amyloid deposition in the cornea occurs in numerous settings and is known to result from mutations in 3 different genes: GSN, M1S1, and transforming growth factor –induced (TGFBI; formerly called BIGH3).1,2 With regard to TGFBI alone, some 20 mutations are associated with 14 distinct phenotypes of corneal disorders.1 We report a form of inherited corneal amyloidosis characterized clinically by multiple polymorphic chipped ice–appearing foci within the stroma and occasional delicate linear opacities. Common complaints include decreased night vision, blurry vision, and dry eyes. The condition became symptomatic at a mean age of 33 years, and in many cases penetrating keratoplasty (PK) eventually became necessary. A novel mutation in TGFBI, A546D, cosegregates with the disorder. ISSN 0161-6420/04/$–see front matter doi:10.1016/j.ophtha.2003.09.043
Eifrig et al 䡠 Polymorphic Corneal Amyloidosis
Figure 1. Pedigree showing individuals with the A546D mutation in the transforming growth factor –induced (TGFBI) gene. Squares ⫽ males; circles ⫽ females.
After institutional review board approval was obtained, a 6-generation family was delineated (Fig 1). The ages ranged from 7 to 71 years. An ancestor of the proband, who suffered from a corneal disease, immigrated to the United States from Germany. The proband was discovered at Duke University Medical Center and was initially thought to be an example of an atypical lattice corneal dystrophy. Many other members of the family were examined clinically over 13 years by one of the authors. Seven affected members had undergone PK, and the corneal tissue was examined histopathologically. Six of these individuals underwent a total of 18 keratoplasties.
al (exons 1, 2, 8, 13, and 17)3 or Munier et al (exons 4 –7, 9 –12, and 14 –16).4,5 In exon 3 we designed and used the following forward and reverse primers: 5⬘-ACCTGTGAGGAACAGTGAAG-3⬘/5⬘-GCC TTTTATGTGGGTACTCC-3⬘. The resulting PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN, Valencia, CA) and sequenced on both strands using the BigDye Terminator Cycle Sequencer (Applied Biosystems, Foster City, CA) combined with an ABI PRISM 377 DNA Sequencer (Applied Biosystems). The resulting DNA sequencing gel was analyzed using ABI PRISM SeqScape Software (Applied Biosystems). The sequences of all exons were then aligned to the TGFBI cDNA using the web-based SeqWeb sequence analyzing software (Accelrys, San Diego, CA) to detect any nucleotide changes and resulting predicted amino acid alterations, if any, when compared with the TGFBI cDNA.
Clinical Evaluations
Histologic Evaluations
Slit-lamp biomicroscopy was performed on the eyes of affected individuals. Detailed clinical histories were obtained from participating members of the family, and particular attention was paid to the age of onset, initial presenting signs and symptoms, number of and dates of corneal grafts, and other ocular therapeutic procedures. Medical records were obtained from hospitals and physicians who had treated affected members of the family. Corneal thickness was measured by pachymetry (model DGH-550, DGH Technology Inc., Exton, PA) in 2 patients.
Corneal tissue obtained at PK was available from 4 affected individuals for study. Tissue sections of this material were examined by light microscopy after being stained with hematoxylin– eosin, Masson’s trichrome, periodic acid–Schiff, and Congo red.
Molecular Analysis
In this 6-generation family, 39 blood relatives were studied (Fig 1). Thirteen members of the family had a history of eye disease. Of these individuals, 4 are deceased, and brief ophthalmologic records were available for only 2. Eight of the living and affected subjects were available for study. The onset of clinical disease ranged from 28 to 40 years (mean, 33). Initial presenting symptoms and complaints for affected individuals included poor night vision with halos and glare (7/8), blurry vision (6/8), dry eyes (5/8), foreign body sensation (3/8), and photophobia (3/8) (Table 1). One af-
Materials and Methods Patients
Blood or buccal swab samples were obtained from 34 family members, and DNA was extracted using the Puregene Blood Kit (Gentra Systems, Minneapolis, MN) or the Puregene Buccal Cell Kit (Gentra Systems). All 17 exons of the TGFBI gene were analyzed in genomic DNA from the proband. This was done by amplifying the extracted DNA with the polymerase chain reaction (PCR) using the forward and reverse primers and the PCR conditions described by Afshari et
Results Clinical Findings
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Ophthalmology Volume 111, Number 6, June 2004 Table 1. Clinical Findings in Family Members with Corneal Opacities
Individual Case 1 2 3 4 5 6 7 8 9 10
Pedigree Identity V: 2 IV: 6 IV: 3 III: 7 IV: 16 IV: 7 IV: 8 III: 2 III: 4 III: 1
Age at Onset (yrs)
Gender
28 30 30 31 32 35 37 37 40 UA
Female Male Female Male Male Female Female Male Female Male
Symptoms at Onset PNV, FBS PNV, VA2, PNV, VA2, PNV, VA2, PNV, VA2, PNV, VA2, PNV, DE VA2 UA UA
DE, Ph DE FBS, Ph DE, FBS, Ph DE
Age at Corneal Graft None 43 None 35 33 53 None 47 59 38
Years of Symptoms before Initial Graft 7* 13 5* 4 2 18 6* 10 19 UA
DE ⫽ dry eye; FBS ⫽ foreign body sensation; Ph ⫽ photophobia; PNV ⫽ poor night vision; UA ⫽ unavailable data; VA2 ⫽ visual acuity decreased. *Years since onset but no corneal graft yet.
fected individual complained of rare, very mild ocular pain. The presence of corneal disease was discovered in 4 individuals by an ophthalmologist when they accompanied an affected relative to the clinic. Four affected individuals gave a history of ocular trauma before the onset of the corneal disorder. These 4 believed that the traumatic events were the cause of the corneal disease, despite the fact that they occurred 4, 7, 15, and 24 years before their formal diagnosis of corneal disease. Seven affected individuals underwent corneal transplantation due to a progressive corneal opacification. The age of PK ranged from 33 to 59 years (mean, 44). Five individuals underwent regrafts for various reasons (failed graft, herpes keratitis, or unknown) at 2, 11, 12, 14, and 20 years after the initial grafts. Slit-lamp biomicroscopy of the proband’s cornea revealed multiple bilateral polymorphic polygonal refractile gray and white opacities with occasional filamentous lines that did not form a distinct lattice pattern (Fig 2). Most corneas of affected family members contained multiple chipped ice–appearing opacities in the cornea. Occasional linear opacities overlapped and produced a focal small network. Other members of the family showed similar patterns. No guttae were noted on Descemet’s membrane. Corneal thickness was normal in the 2 patients in whom it was measured. The corneal disorder perplexed several ophthalmologists with expertise in corneal disease who examined affected members of the family at different institutions. The proband and 5 others were thought to have an atypical type of lattice corneal dystrophy.
Molecular Genetic Analysis
Histologic Analysis
Discussion
The corneal tissue that was available for review was notable for the presence of variably sized, irregularly shaped round amyloid deposits within the corneal stroma, which stained with Congo red and exhibited apple green dichroism when such preparations were examined under polarized light. In the milder cases, the corneal deposits were situated mostly in the posterior central cornea and were often adjacent to Descemet’s membrane, which, together with the corneal endothelium, was unremarkable (Fig 3A, B). In corneal tissue with more severe corneal opacifications, the amyloid deposits were larger and extended throughout the entire thickness of the cornea and also involved the peripheral cornea (Fig 3C, D). In one regrafted specimen, foci of amyloid were present along the margin of the original graft– host interface.
The keratopathy reported here differs in several respects from the other inherited corneal amyloidoses.1 The corneal opacities occur mainly in the posterior central corneal stroma, vary in size and shape, and mainly resemble chipped ice. Short filamentous opacities are also sometimes present, but in contrast to the lattice corneal dystrophies, these linear opacities rarely form a network, and when this occurs it is small and focal. Also, compared with the classic lattice corneal dystrophy, the age of onset is much later, and the deposits are deeper. Moreover, corneal erosions were not reported in our affected individuals. The literature contains a few somewhat similar cases, but
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The TGFBI gene was analyzed using DNA from 34 family members willing and able to give blood or buccal swabs. Twelve individuals (5 male and 7 female) exhibited a heterozygous nucleotide substitution of adenosine for cytosine at nucleotide 1637 in exon 12 of the TGFBI gene (Fig 4). This nucleotide change is predicted to change the amino acid alanine to aspartic acid at codon 546 (A546D). Most notably, this change cosegregated with the keratopathy, but only 9 of the 12 individuals with the nucleotide change had signs and symptoms of the corneal disease. The remaining 3 are asymptomatic adolescents, 11, 14, and 15 years old. In addition to the family’s unique heterozygous nucleotide change, leading to the amino acid change A546D, we found 4 heterozygous nucleotide substitutions in this family that neither cosegregated with the corneal abnormalities nor changed the encoded amino acid: guanine for adenosine at position 981 in exon 8 (V327V), thymine for cytosine at position 1416 in exon 11 (L472L), and cytosine for thymine at position 1620 in exon 12 (F540F). Furthermore, an analysis of the only other exon in TGFBI known to be associated with amyloid deposition, exon 4, was normal in all individuals except one. The exception was a 7-yearold male who had a heterozygous nucleotide change where adenosine was substituted for guanine at position 387, but which also left the amino acid unchanged (R129R). His other exons were normal.
Eifrig et al 䡠 Polymorphic Corneal Amyloidosis
Figure 2. Photographs of the cornea from 2 representative individuals taken by slit-lamp biomicroscopy. Right (A) and left (B) corneas of case 6, age 52 years (Table 1), illustrating variably shaped opacities in the central stroma. Note the linear forms in the left cornea. C, The left cornea is also illustrated 6 years later, just before penetrating keratoplasty, after further progression of the polymorphic opacities. D, The central and posterior deposits in the same individual are also shown at the same time. E, F, The second individual’s (case 7) corneas demonstrate the chipped ice–appearing corneal opacities at 43 years of age after 6 years of clinical disease.
with apparent differences. In 1940 Pillat6 reported a 3-generation family with an atypical corneal dystrophy characterized by the presence of bilateral fine punctate and short linear opacities in the axial and paraxial corneal stroma. Based on a histochemical study of affected tissue, Pillat concluded that the central changes were “amyloid or hyaline.” In 1975, Thomsitt and Bron7 documented similar flecklike stromal opacities in the cornea by retroillumination and reviewed 9 different clinical presentations of a condition they named polymorphic stromal dystrophy. The corneal opacities in their 9 patients differed in appearance, being flecklike, stellate, linear, and irregularly shaped, reminiscent of snowflakes. Furthermore, the distribution and
levels of stromal opacities varied, suggesting that their series included a heterogeneous collection of disorders. A few years later, Mannis et al8 documented a series of patients with polymorphic, punctate, and stromal amyloid deposits strikingly similar in appearance to ours. They called this entity polymorphic amyloid degeneration, and the authors concluded that “family studies failed to demonstrate hereditability,” despite the presence of 3 affected individuals in one family. However, in contrast to our family, the individuals in their series were largely asymptomatic. Moreover, the disorder was diagnosed during or after the fourth decade of life, which is at least 10 years older than in our family. In 1983, Krachmer et al9 described
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Eifrig et al 䡠 Polymorphic Corneal Amyloidosis Figure 3. Light microscopy of corneal tissue illustrating stromal deposits of amyloid. A, B, Mildly affected cornea of a 53-year-old female who had been symptomatic for 18 years. C, D, Severely affected cornea of a 49-year-old male who began to have corneal disease when 30 years old. A and C stain, Congo red; B and D stain, Congo red under polarized light. Figure 4. Nucleotide sequences of a portion of exon 12 of the transforming growth factor –induced gene from an unaffected family member (top) and the proband (bottom). The codon numbers are indicated at the top of the figure. The sequence in the proband shows a heterozygous single– base pair change at codon 546. The nucleotides cytosine (C) and adenosine (A) both appear at the second nucleotide position (arrow). The A is part of the mutant codon GAC, whereas the C is part of the normal GCC sequence. The unaffected sibling has both strands with the normal nucleotide and is thus homozygous for the normal GCC sequence.
4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ a patient with both posterior crocodile shagreen and polymorphic amyloid degeneration having punctate polymorphic stromal opacities that appeared white with direct illumination and refractile with indirect illumination. In considering what to designate the keratopathy found in our family, we have chosen the descriptive term polymorphic corneal amyloidosis, because the opacities within the cornea possess many forms (polymorphic) and result from amyloid deposits (amyloidosis). In addition to the clinicopathologic features, the genetic findings of this family are also unique. Several mutations in the TGFBI gene are responsible for a variety of inherited corneal stromal diseases,1 and a nucleotide change in the TGFBI gene that alters the encoded amino acid probably affects the tertiary or quaternary structure of the encoded protein, but why this leads to amyloid deposition remains to be determined. From studies on corneal disorders caused by mutations in the TGFBI gene, the mutant encoded TGFBI protein or fragments of it probably accumulate within the corneal stroma.10 –12 In this family the heterozygous nucleotide change cosegregates with the keratopathy, and is predicted to replace alanine in residue 546, in the Fascilin 4 domain of the TGFBI protein, with a very different one, aspartic acid. Although the function of the Fascilin 4 domain remains unknown, several mutations in this region lead to specific corneal phenotypes.3,5,13 It is noteworthy that mutations have been detected in this same codon, 546, in other families with corneal amyloidosis. We have observed a combination of A546D and P551Q in a family with lattice corneal dystrophy type I (Invest Ophthalmol Vis Sci 39[suppl]:S513, 1998). Dighiero et al reported a different mutation in the same residue, 546 (or A546T), which resulted in a phenotype very different from ours and which is designated French lattice corneal dystrophy type IIIA by the authors.13 Their patients had “ropy and thick lattice lines located predominantly in the central corneas, some nodular opacities, a diffuse haziness between the lines,” and a history of recurrent epithelial erosions. Individuals in our family did not suffer from epithelial erosions. Yet, the ages of onset of corneal disease in persons with the A546T and A456D mutations were somewhat similar: 35 to 40 years and 33 to 34 years, respectively. Hopefully, differences and similarities in phenotypes produced by specific amino acid changes in the TGFBI protein will provide clues to the molecular milieu that leads to the strikingly different features of these different forms of corneal amyloidosis. Moreover, localized physical differences in the coding regions of the TGFBI gene may further provide clues to help in understanding these diseases. Normally, residue 546 in TGFBI protein is alanine, a
hydrophobic aliphatic amino acid with a methyl side group. In individuals with the A546D-mutated TGFBI gene, the predicted aspartic acid (D) at residue 546 is hydrophilic, with a CH2COO side chain, and thus changed from a hydrophobic amino acid to a hydrophilic one that is negatively charged at physiologic pH. In the A546T mutation, threonine is substituted, and is an uncharged molecule with a polar hydroxyl group that, due to the carboxylate side chain, increases bonding to hydrogen, which increases water solubility and thus increases bonding with other polar molecules. Moreover, these 3 amino acids at residue 546 have very different preferences for forming shapes within the protein complex. Alanine, the normal amino acid, prefers ␣ helices, whereas aspartic acid prefers turns that “tend to disrupt ␣ helices,” and threonine prefers to form  sheets.14 Presumably these altered charges, solubilities, and shape preferences in the mutated TGFBI protein lead to a tertiary or quaternary structure different than that of the naturally occurring protein, and this then leads to differently sized degradation products after proteolysis. Further studies are needed to determine why these and other specific mutations in the TGFBI gene, such as R124C and R124H, lead to amyloid deposition. In addition to nucleotide changes responsible for the corneal amyloidosis in our family, several other heterozygous nucleotide changes that coded for inconsequential single nucleotide polymorphisms were detected. These changes neither cosegregate with this corneal disorder nor alter the encoded TGFBI protein (the changes are 1620 C/T [F540F], 981 G/A [V327V], 1416 T/C [L472L], and 387 G/A [R129R]). With the exception of R129R, which has not been documented, to the best of our knowledge, all of these single nucleotide polymorphisms have been previously reported: F540F,15–17 V327V,15,17 and L472L.15,17,18 Several other polymorphisms have also been discovered since investigators started to sequence the TGFBI gene. These have included 294 A/T (P98P),17 405 C/T (P135P),17 852 C/A (A284A),17 651 C/G (L217L),15 1028G/A (E328E),19 1439 G/A (A480V),18 1632 T/C (N544N),15 C to T substitution within intron 4,17 an insertion within intron 12 (IVS12⫹23A/G),17 an insertion within intron 5 (IVS5⫹7insA),15 and a C to T substitution in intron 10.18 We have described a unique phenotype with a novel mutation in the TGFBI gene, which can be compared with other mutations in the same domain and even the same codon. Further study of the protein structures and interactions that lead directly to these differences should shed light on how amyloid is formed, and ultimately lead to therapies to prevent its deposition. We believe that, to advance therapy, linking clinical manifestations with the molecular pro-
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Ophthalmology Volume 111, Number 6, June 2004 cesses must remain the mutual goal of clinicians and researchers working together to uncover the clues provided by these inborn errors of nature related to diseases of the eye.
References 1. Klintworth GK. The molecular genetics of the corneal dystrophies— current status. Front Biosci 2003;8:d687–713. 2. Klintworth GK. Proteins in ocular disease. In: Garner A, Klintworth GK, eds. Pathobiology of Ocular Disease: A Dynamic Approach. Part B. 2nd ed. New York: Marcel Dekker; 1994:973–1032. 3. Afshari NA, Mullally JE, Afshari MA, et al. Survey of patients with granular, lattice, Avellino, and Reis-Bu¨ cklers corneal dystrophies for mutations in the BIGH3 and gelsolin genes. Arch Ophthalmol 2001;119:16 –22. 4. Munier FL, Korvatska E, Djemai A, et al. Kerato-epithelin mutations in four 5q31-linked corneal dystrophies. Nat Genet 1997;15:247–51. 5. Munier FL, Frueh BE, Othenin-Girard P, et al. BIGH3 mutation spectrum in corneal dystrophies. Invest Ophthalmol Vis Sci 2002;43:949 –54. 6. Pillat A. Zur Frage der familia¨ ren Hornhautentartung: Ueber eine eigenartige tiefe schollige und periphere gitterfo¨ rmige famila¨ re Hornhautdystrophie. Klin Monatsbl Augenheilkd 1940;104:571– 88. 7. Thomsitt J, Bron AJ. Polymorphic stromal dystrophy. Br J Ophthalmol 1975;59:125–32. 8. Mannis MJ, Krachmer JH, Rodrigues MM, Pardos GJ. Polymorphic amyloid degeneration of the cornea. A clinical and histopathologic study. Arch Ophthalmol 1981;99:1217–23. 9. Krachmer JH, Dubord PJ, Rodrigues MM, Mannis MJ. Cor-
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10. 11. 12.
13. 14. 15. 16. 17. 18. 19.
neal posterior crocodile shagreen and polymorphic amyloid degeneration. Arch Ophthalmol 1983;101:54 –9. Klintworth GK, Valnickova Z, Enghild JJ. Accumulation of beta ig-h3 gene product in corneas with granular dystrophy. Am J Pathol 1998;152:743– 8. Streeten BW, Qi Y, Klintworth GK, et al. Localization of beta ig-h3 protein in 5q31-linked corneal dystrophies and normal corneas. Arch Ophthalmol 1999;117:67–75. Hedegaard CJ, Thøgersen ID, Enghild JJ, et al. Transforming growth factor beta induced protein accumulation in granular corneal dystrophy type III (Reis-Bu¨ cklers dystrophy). Identification by mass spectrometry in 15 year old two-dimensional protein gels. Mol Vis 2003;9:355–9. Dighiero P, Drunat S, Ellies P, et al. A new mutation (A546T) of the beta ig-h3 gene responsible for a French lattice corneal dystrophy type IIIA. Am J Ophthalmol 2000;129:248 –51. Stryer L. Protein structure and function. In: Berg JM, Tymoczko JL, Stryer L, eds. Biochemistry. 5th ed. San Francisco: W.H. Freeman; 2002:41–74. Schmitt-Bernard CF, Guittard C, Arnaud B, et al. BIGH3 exon 14 mutations lead to intermediate type I/IIIA of lattice corneal dystrophies. Invest Ophthalmol Vis Sci 2000;41:1302– 8. Kim HS, Yoon SK, Cho BJ, et al. BIGH3 gene mutations and rapid detection in Korean patients with corneal dystrophy. Cornea 2001;20:844 –9. Korvatska E, Munier FL, Djemai A, et al. Mutation hot spots in 5q31-linked corneal dystrophies. Am J Hum Genet 1998; 62:320 – 4. Tsujikawa M, Shimomura Y, Okada M, et al. Novel polymorphisms in the beta ig-h3 gene. J Hum Genet 1998;43:214 –5. Tsujikawa K, Tsujikawa M, Yamamoto S, et al. Allelic homogeneity due to a founder mutation in Japanese patients with lattice corneal dystrophy type IIIA. Am J Med Genet 2002; 113:20 –2.
During the past few years, mutations in specific genes have been found to account for several inherited corneal disorOriginally received: June 20, 2003. Accepted: September 15, 2003. Manuscript no. 230398. 1 Department of Ophthalmology, Duke University Medical Center, Durham, North Carolina. 2 Department of Pathology, Duke University Medical Center, Durham, North Carolina. 3 Department of Surgery, Division of Ophthalmology, Sacred Heart Hospital, Allentown, Pennsylvania. Presented in part at: Association for Research in Vision and Ophthalmology Annual Meeting, May 7, 2003; Ft. Lauderdale, Florida. Research funding was provided by a research grant from the National Eye Institute, Bethesda, Maryland (grant no.: R01EY2712). Dr Afshari has a research career development award from Research to Prevent Blindness, New York, New York. Reprint requests to Gordon K. Klintworth, MD, PhD, 255 Medical Sciences Research Building, Box 3802 Duke University Medical Center, Durham, NC 27710.
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© 2004 by the American Academy of Ophthalmology Published by Elsevier Inc.
ders, and in many instances the clinicopathologic phenotypes correlate with the genetic mutation.1 Many corneal dystrophies are caused by a single nucleotide change, and some of these mutations occur in corneas with amyloid deposits. Amyloid deposition in the cornea occurs in numerous settings and is known to result from mutations in 3 different genes: GSN, M1S1, and transforming growth factor –induced (TGFBI; formerly called BIGH3).1,2 With regard to TGFBI alone, some 20 mutations are associated with 14 distinct phenotypes of corneal disorders.1 We report a form of inherited corneal amyloidosis characterized clinically by multiple polymorphic chipped ice–appearing foci within the stroma and occasional delicate linear opacities. Common complaints include decreased night vision, blurry vision, and dry eyes. The condition became symptomatic at a mean age of 33 years, and in many cases penetrating keratoplasty (PK) eventually became necessary. A novel mutation in TGFBI, A546D, cosegregates with the disorder. ISSN 0161-6420/04/$–see front matter doi:10.1016/j.ophtha.2003.09.043
Eifrig et al 䡠 Polymorphic Corneal Amyloidosis
Figure 1. Pedigree showing individuals with the A546D mutation in the transforming growth factor –induced (TGFBI) gene. Squares ⫽ males; circles ⫽ females.
After institutional review board approval was obtained, a 6-generation family was delineated (Fig 1). The ages ranged from 7 to 71 years. An ancestor of the proband, who suffered from a corneal disease, immigrated to the United States from Germany. The proband was discovered at Duke University Medical Center and was initially thought to be an example of an atypical lattice corneal dystrophy. Many other members of the family were examined clinically over 13 years by one of the authors. Seven affected members had undergone PK, and the corneal tissue was examined histopathologically. Six of these individuals underwent a total of 18 keratoplasties.
al (exons 1, 2, 8, 13, and 17)3 or Munier et al (exons 4 –7, 9 –12, and 14 –16).4,5 In exon 3 we designed and used the following forward and reverse primers: 5⬘-ACCTGTGAGGAACAGTGAAG-3⬘/5⬘-GCC TTTTATGTGGGTACTCC-3⬘. The resulting PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN, Valencia, CA) and sequenced on both strands using the BigDye Terminator Cycle Sequencer (Applied Biosystems, Foster City, CA) combined with an ABI PRISM 377 DNA Sequencer (Applied Biosystems). The resulting DNA sequencing gel was analyzed using ABI PRISM SeqScape Software (Applied Biosystems). The sequences of all exons were then aligned to the TGFBI cDNA using the web-based SeqWeb sequence analyzing software (Accelrys, San Diego, CA) to detect any nucleotide changes and resulting predicted amino acid alterations, if any, when compared with the TGFBI cDNA.
Clinical Evaluations
Histologic Evaluations
Slit-lamp biomicroscopy was performed on the eyes of affected individuals. Detailed clinical histories were obtained from participating members of the family, and particular attention was paid to the age of onset, initial presenting signs and symptoms, number of and dates of corneal grafts, and other ocular therapeutic procedures. Medical records were obtained from hospitals and physicians who had treated affected members of the family. Corneal thickness was measured by pachymetry (model DGH-550, DGH Technology Inc., Exton, PA) in 2 patients.
Corneal tissue obtained at PK was available from 4 affected individuals for study. Tissue sections of this material were examined by light microscopy after being stained with hematoxylin– eosin, Masson’s trichrome, periodic acid–Schiff, and Congo red.
Molecular Analysis
In this 6-generation family, 39 blood relatives were studied (Fig 1). Thirteen members of the family had a history of eye disease. Of these individuals, 4 are deceased, and brief ophthalmologic records were available for only 2. Eight of the living and affected subjects were available for study. The onset of clinical disease ranged from 28 to 40 years (mean, 33). Initial presenting symptoms and complaints for affected individuals included poor night vision with halos and glare (7/8), blurry vision (6/8), dry eyes (5/8), foreign body sensation (3/8), and photophobia (3/8) (Table 1). One af-
Materials and Methods Patients
Blood or buccal swab samples were obtained from 34 family members, and DNA was extracted using the Puregene Blood Kit (Gentra Systems, Minneapolis, MN) or the Puregene Buccal Cell Kit (Gentra Systems). All 17 exons of the TGFBI gene were analyzed in genomic DNA from the proband. This was done by amplifying the extracted DNA with the polymerase chain reaction (PCR) using the forward and reverse primers and the PCR conditions described by Afshari et
Results Clinical Findings
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Ophthalmology Volume 111, Number 6, June 2004 Table 1. Clinical Findings in Family Members with Corneal Opacities
Individual Case 1 2 3 4 5 6 7 8 9 10
Pedigree Identity V: 2 IV: 6 IV: 3 III: 7 IV: 16 IV: 7 IV: 8 III: 2 III: 4 III: 1
Age at Onset (yrs)
Gender
28 30 30 31 32 35 37 37 40 UA
Female Male Female Male Male Female Female Male Female Male
Symptoms at Onset PNV, FBS PNV, VA2, PNV, VA2, PNV, VA2, PNV, VA2, PNV, VA2, PNV, DE VA2 UA UA
DE, Ph DE FBS, Ph DE, FBS, Ph DE
Age at Corneal Graft None 43 None 35 33 53 None 47 59 38
Years of Symptoms before Initial Graft 7* 13 5* 4 2 18 6* 10 19 UA
DE ⫽ dry eye; FBS ⫽ foreign body sensation; Ph ⫽ photophobia; PNV ⫽ poor night vision; UA ⫽ unavailable data; VA2 ⫽ visual acuity decreased. *Years since onset but no corneal graft yet.
fected individual complained of rare, very mild ocular pain. The presence of corneal disease was discovered in 4 individuals by an ophthalmologist when they accompanied an affected relative to the clinic. Four affected individuals gave a history of ocular trauma before the onset of the corneal disorder. These 4 believed that the traumatic events were the cause of the corneal disease, despite the fact that they occurred 4, 7, 15, and 24 years before their formal diagnosis of corneal disease. Seven affected individuals underwent corneal transplantation due to a progressive corneal opacification. The age of PK ranged from 33 to 59 years (mean, 44). Five individuals underwent regrafts for various reasons (failed graft, herpes keratitis, or unknown) at 2, 11, 12, 14, and 20 years after the initial grafts. Slit-lamp biomicroscopy of the proband’s cornea revealed multiple bilateral polymorphic polygonal refractile gray and white opacities with occasional filamentous lines that did not form a distinct lattice pattern (Fig 2). Most corneas of affected family members contained multiple chipped ice–appearing opacities in the cornea. Occasional linear opacities overlapped and produced a focal small network. Other members of the family showed similar patterns. No guttae were noted on Descemet’s membrane. Corneal thickness was normal in the 2 patients in whom it was measured. The corneal disorder perplexed several ophthalmologists with expertise in corneal disease who examined affected members of the family at different institutions. The proband and 5 others were thought to have an atypical type of lattice corneal dystrophy.
Molecular Genetic Analysis
Histologic Analysis
Discussion
The corneal tissue that was available for review was notable for the presence of variably sized, irregularly shaped round amyloid deposits within the corneal stroma, which stained with Congo red and exhibited apple green dichroism when such preparations were examined under polarized light. In the milder cases, the corneal deposits were situated mostly in the posterior central cornea and were often adjacent to Descemet’s membrane, which, together with the corneal endothelium, was unremarkable (Fig 3A, B). In corneal tissue with more severe corneal opacifications, the amyloid deposits were larger and extended throughout the entire thickness of the cornea and also involved the peripheral cornea (Fig 3C, D). In one regrafted specimen, foci of amyloid were present along the margin of the original graft– host interface.
The keratopathy reported here differs in several respects from the other inherited corneal amyloidoses.1 The corneal opacities occur mainly in the posterior central corneal stroma, vary in size and shape, and mainly resemble chipped ice. Short filamentous opacities are also sometimes present, but in contrast to the lattice corneal dystrophies, these linear opacities rarely form a network, and when this occurs it is small and focal. Also, compared with the classic lattice corneal dystrophy, the age of onset is much later, and the deposits are deeper. Moreover, corneal erosions were not reported in our affected individuals. The literature contains a few somewhat similar cases, but
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The TGFBI gene was analyzed using DNA from 34 family members willing and able to give blood or buccal swabs. Twelve individuals (5 male and 7 female) exhibited a heterozygous nucleotide substitution of adenosine for cytosine at nucleotide 1637 in exon 12 of the TGFBI gene (Fig 4). This nucleotide change is predicted to change the amino acid alanine to aspartic acid at codon 546 (A546D). Most notably, this change cosegregated with the keratopathy, but only 9 of the 12 individuals with the nucleotide change had signs and symptoms of the corneal disease. The remaining 3 are asymptomatic adolescents, 11, 14, and 15 years old. In addition to the family’s unique heterozygous nucleotide change, leading to the amino acid change A546D, we found 4 heterozygous nucleotide substitutions in this family that neither cosegregated with the corneal abnormalities nor changed the encoded amino acid: guanine for adenosine at position 981 in exon 8 (V327V), thymine for cytosine at position 1416 in exon 11 (L472L), and cytosine for thymine at position 1620 in exon 12 (F540F). Furthermore, an analysis of the only other exon in TGFBI known to be associated with amyloid deposition, exon 4, was normal in all individuals except one. The exception was a 7-yearold male who had a heterozygous nucleotide change where adenosine was substituted for guanine at position 387, but which also left the amino acid unchanged (R129R). His other exons were normal.
Eifrig et al 䡠 Polymorphic Corneal Amyloidosis
Figure 2. Photographs of the cornea from 2 representative individuals taken by slit-lamp biomicroscopy. Right (A) and left (B) corneas of case 6, age 52 years (Table 1), illustrating variably shaped opacities in the central stroma. Note the linear forms in the left cornea. C, The left cornea is also illustrated 6 years later, just before penetrating keratoplasty, after further progression of the polymorphic opacities. D, The central and posterior deposits in the same individual are also shown at the same time. E, F, The second individual’s (case 7) corneas demonstrate the chipped ice–appearing corneal opacities at 43 years of age after 6 years of clinical disease.
with apparent differences. In 1940 Pillat6 reported a 3-generation family with an atypical corneal dystrophy characterized by the presence of bilateral fine punctate and short linear opacities in the axial and paraxial corneal stroma. Based on a histochemical study of affected tissue, Pillat concluded that the central changes were “amyloid or hyaline.” In 1975, Thomsitt and Bron7 documented similar flecklike stromal opacities in the cornea by retroillumination and reviewed 9 different clinical presentations of a condition they named polymorphic stromal dystrophy. The corneal opacities in their 9 patients differed in appearance, being flecklike, stellate, linear, and irregularly shaped, reminiscent of snowflakes. Furthermore, the distribution and
levels of stromal opacities varied, suggesting that their series included a heterogeneous collection of disorders. A few years later, Mannis et al8 documented a series of patients with polymorphic, punctate, and stromal amyloid deposits strikingly similar in appearance to ours. They called this entity polymorphic amyloid degeneration, and the authors concluded that “family studies failed to demonstrate hereditability,” despite the presence of 3 affected individuals in one family. However, in contrast to our family, the individuals in their series were largely asymptomatic. Moreover, the disorder was diagnosed during or after the fourth decade of life, which is at least 10 years older than in our family. In 1983, Krachmer et al9 described
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Eifrig et al 䡠 Polymorphic Corneal Amyloidosis Figure 3. Light microscopy of corneal tissue illustrating stromal deposits of amyloid. A, B, Mildly affected cornea of a 53-year-old female who had been symptomatic for 18 years. C, D, Severely affected cornea of a 49-year-old male who began to have corneal disease when 30 years old. A and C stain, Congo red; B and D stain, Congo red under polarized light. Figure 4. Nucleotide sequences of a portion of exon 12 of the transforming growth factor –induced gene from an unaffected family member (top) and the proband (bottom). The codon numbers are indicated at the top of the figure. The sequence in the proband shows a heterozygous single– base pair change at codon 546. The nucleotides cytosine (C) and adenosine (A) both appear at the second nucleotide position (arrow). The A is part of the mutant codon GAC, whereas the C is part of the normal GCC sequence. The unaffected sibling has both strands with the normal nucleotide and is thus homozygous for the normal GCC sequence.
4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ a patient with both posterior crocodile shagreen and polymorphic amyloid degeneration having punctate polymorphic stromal opacities that appeared white with direct illumination and refractile with indirect illumination. In considering what to designate the keratopathy found in our family, we have chosen the descriptive term polymorphic corneal amyloidosis, because the opacities within the cornea possess many forms (polymorphic) and result from amyloid deposits (amyloidosis). In addition to the clinicopathologic features, the genetic findings of this family are also unique. Several mutations in the TGFBI gene are responsible for a variety of inherited corneal stromal diseases,1 and a nucleotide change in the TGFBI gene that alters the encoded amino acid probably affects the tertiary or quaternary structure of the encoded protein, but why this leads to amyloid deposition remains to be determined. From studies on corneal disorders caused by mutations in the TGFBI gene, the mutant encoded TGFBI protein or fragments of it probably accumulate within the corneal stroma.10 –12 In this family the heterozygous nucleotide change cosegregates with the keratopathy, and is predicted to replace alanine in residue 546, in the Fascilin 4 domain of the TGFBI protein, with a very different one, aspartic acid. Although the function of the Fascilin 4 domain remains unknown, several mutations in this region lead to specific corneal phenotypes.3,5,13 It is noteworthy that mutations have been detected in this same codon, 546, in other families with corneal amyloidosis. We have observed a combination of A546D and P551Q in a family with lattice corneal dystrophy type I (Invest Ophthalmol Vis Sci 39[suppl]:S513, 1998). Dighiero et al reported a different mutation in the same residue, 546 (or A546T), which resulted in a phenotype very different from ours and which is designated French lattice corneal dystrophy type IIIA by the authors.13 Their patients had “ropy and thick lattice lines located predominantly in the central corneas, some nodular opacities, a diffuse haziness between the lines,” and a history of recurrent epithelial erosions. Individuals in our family did not suffer from epithelial erosions. Yet, the ages of onset of corneal disease in persons with the A546T and A456D mutations were somewhat similar: 35 to 40 years and 33 to 34 years, respectively. Hopefully, differences and similarities in phenotypes produced by specific amino acid changes in the TGFBI protein will provide clues to the molecular milieu that leads to the strikingly different features of these different forms of corneal amyloidosis. Moreover, localized physical differences in the coding regions of the TGFBI gene may further provide clues to help in understanding these diseases. Normally, residue 546 in TGFBI protein is alanine, a
hydrophobic aliphatic amino acid with a methyl side group. In individuals with the A546D-mutated TGFBI gene, the predicted aspartic acid (D) at residue 546 is hydrophilic, with a CH2COO side chain, and thus changed from a hydrophobic amino acid to a hydrophilic one that is negatively charged at physiologic pH. In the A546T mutation, threonine is substituted, and is an uncharged molecule with a polar hydroxyl group that, due to the carboxylate side chain, increases bonding to hydrogen, which increases water solubility and thus increases bonding with other polar molecules. Moreover, these 3 amino acids at residue 546 have very different preferences for forming shapes within the protein complex. Alanine, the normal amino acid, prefers ␣ helices, whereas aspartic acid prefers turns that “tend to disrupt ␣ helices,” and threonine prefers to form  sheets.14 Presumably these altered charges, solubilities, and shape preferences in the mutated TGFBI protein lead to a tertiary or quaternary structure different than that of the naturally occurring protein, and this then leads to differently sized degradation products after proteolysis. Further studies are needed to determine why these and other specific mutations in the TGFBI gene, such as R124C and R124H, lead to amyloid deposition. In addition to nucleotide changes responsible for the corneal amyloidosis in our family, several other heterozygous nucleotide changes that coded for inconsequential single nucleotide polymorphisms were detected. These changes neither cosegregate with this corneal disorder nor alter the encoded TGFBI protein (the changes are 1620 C/T [F540F], 981 G/A [V327V], 1416 T/C [L472L], and 387 G/A [R129R]). With the exception of R129R, which has not been documented, to the best of our knowledge, all of these single nucleotide polymorphisms have been previously reported: F540F,15–17 V327V,15,17 and L472L.15,17,18 Several other polymorphisms have also been discovered since investigators started to sequence the TGFBI gene. These have included 294 A/T (P98P),17 405 C/T (P135P),17 852 C/A (A284A),17 651 C/G (L217L),15 1028G/A (E328E),19 1439 G/A (A480V),18 1632 T/C (N544N),15 C to T substitution within intron 4,17 an insertion within intron 12 (IVS12⫹23A/G),17 an insertion within intron 5 (IVS5⫹7insA),15 and a C to T substitution in intron 10.18 We have described a unique phenotype with a novel mutation in the TGFBI gene, which can be compared with other mutations in the same domain and even the same codon. Further study of the protein structures and interactions that lead directly to these differences should shed light on how amyloid is formed, and ultimately lead to therapies to prevent its deposition. We believe that, to advance therapy, linking clinical manifestations with the molecular pro-
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Ophthalmology Volume 111, Number 6, June 2004 cesses must remain the mutual goal of clinicians and researchers working together to uncover the clues provided by these inborn errors of nature related to diseases of the eye.
References 1. Klintworth GK. The molecular genetics of the corneal dystrophies— current status. Front Biosci 2003;8:d687–713. 2. Klintworth GK. Proteins in ocular disease. In: Garner A, Klintworth GK, eds. Pathobiology of Ocular Disease: A Dynamic Approach. Part B. 2nd ed. New York: Marcel Dekker; 1994:973–1032. 3. Afshari NA, Mullally JE, Afshari MA, et al. Survey of patients with granular, lattice, Avellino, and Reis-Bu¨ cklers corneal dystrophies for mutations in the BIGH3 and gelsolin genes. Arch Ophthalmol 2001;119:16 –22. 4. Munier FL, Korvatska E, Djemai A, et al. Kerato-epithelin mutations in four 5q31-linked corneal dystrophies. Nat Genet 1997;15:247–51. 5. Munier FL, Frueh BE, Othenin-Girard P, et al. BIGH3 mutation spectrum in corneal dystrophies. Invest Ophthalmol Vis Sci 2002;43:949 –54. 6. Pillat A. Zur Frage der familia¨ ren Hornhautentartung: Ueber eine eigenartige tiefe schollige und periphere gitterfo¨ rmige famila¨ re Hornhautdystrophie. Klin Monatsbl Augenheilkd 1940;104:571– 88. 7. Thomsitt J, Bron AJ. Polymorphic stromal dystrophy. Br J Ophthalmol 1975;59:125–32. 8. Mannis MJ, Krachmer JH, Rodrigues MM, Pardos GJ. Polymorphic amyloid degeneration of the cornea. A clinical and histopathologic study. Arch Ophthalmol 1981;99:1217–23. 9. Krachmer JH, Dubord PJ, Rodrigues MM, Mannis MJ. Cor-
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10. 11. 12.
13. 14. 15. 16. 17. 18. 19.
neal posterior crocodile shagreen and polymorphic amyloid degeneration. Arch Ophthalmol 1983;101:54 –9. Klintworth GK, Valnickova Z, Enghild JJ. Accumulation of beta ig-h3 gene product in corneas with granular dystrophy. Am J Pathol 1998;152:743– 8. Streeten BW, Qi Y, Klintworth GK, et al. Localization of beta ig-h3 protein in 5q31-linked corneal dystrophies and normal corneas. Arch Ophthalmol 1999;117:67–75. Hedegaard CJ, Thøgersen ID, Enghild JJ, et al. Transforming growth factor beta induced protein accumulation in granular corneal dystrophy type III (Reis-Bu¨ cklers dystrophy). Identification by mass spectrometry in 15 year old two-dimensional protein gels. Mol Vis 2003;9:355–9. Dighiero P, Drunat S, Ellies P, et al. A new mutation (A546T) of the beta ig-h3 gene responsible for a French lattice corneal dystrophy type IIIA. Am J Ophthalmol 2000;129:248 –51. Stryer L. Protein structure and function. In: Berg JM, Tymoczko JL, Stryer L, eds. Biochemistry. 5th ed. San Francisco: W.H. Freeman; 2002:41–74. Schmitt-Bernard CF, Guittard C, Arnaud B, et al. BIGH3 exon 14 mutations lead to intermediate type I/IIIA of lattice corneal dystrophies. Invest Ophthalmol Vis Sci 2000;41:1302– 8. Kim HS, Yoon SK, Cho BJ, et al. BIGH3 gene mutations and rapid detection in Korean patients with corneal dystrophy. Cornea 2001;20:844 –9. Korvatska E, Munier FL, Djemai A, et al. Mutation hot spots in 5q31-linked corneal dystrophies. Am J Hum Genet 1998; 62:320 – 4. Tsujikawa M, Shimomura Y, Okada M, et al. Novel polymorphisms in the beta ig-h3 gene. J Hum Genet 1998;43:214 –5. Tsujikawa K, Tsujikawa M, Yamamoto S, et al. Allelic homogeneity due to a founder mutation in Japanese patients with lattice corneal dystrophy type IIIA. Am J Med Genet 2002; 113:20 –2.