|Molecular Vision 2007;
Received 11 July 2007 | Accepted 15 October 2007 | Published 18 October 2007
TGFBI (BIGH3) gene mutations in Hungary - report of the novel F547S mutation associated with polymorphic corneal amyloidosis
(The first two authors contributed equally to this publication. György Vereb and András Berta are equally senior authors.)
Department of 1Ophthalmology, 2Biophysics and Cell Biology, 3Anatomy, Histology and Embryology, 4Clinical Biochemistry and Molecular Pathology, 7Clinical Research Center, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary, 5Department of Ophthalmology, Kenézy County Hospital Debrecen, Hungary and 6Department of Ophthalmology, City Hospital Kazincbarcika, Hungary
Correspondence to: L. Takács, Department of Ophthalmology, Medical and Health Science Center, University of Debrecen, Nagyerdei krt. 98. H4012-Debrecen, Hungary; Phone: +36 52 415816; FAX: +36 52 415816; email: email@example.com
Purpose: To identify mutations in the Transforming Growth Factor Beta Induced (TGFBI) gene in Hungarian patients with corneal dystrophy and to characterize histological features of their corneal buttons excised during penetrating keratoplasty.
Methods: Exons of TGFBI were sequenced in 38 members of 15 unrelated families with corneal dystrophy and exon 12 was also sequenced in 100 healthy controls from the same population. Immunohistological analysis of available corneal buttons excised during penetrating keratoplasty was also performed.
Results: Molecular genetic analysis revealed a heterozygous R124C mutation in 18 patients with lattice type I dystrophy. A R555W heterozygous mutation was detected in five patients with granular Groenouw type I corneal dystrophy and a R555Q heterozygous mutation was found in four patients clinically diagnosed with Reis-Bücklers (one patient) and Thiel-Behnke (three patients) dystrophy. Three patients with "atypical granular" dystrophy later diagnosed as Avellino dystrophy were heterozygous for the R124H mutation. A novel heterozygous mutation (T1640C) causing a F547S amino acid exchange was detected in a patient with polymorphic corneal amyloidosis. Immunohistochemistry showed the presence of BIGH3 protein deposits in all examined corneal buttons. Electron microscopy confirmed the presence of amyloid fibrils in the case of the novel mutation.
Conclusions: Our results indicate that molecular genetic analysis is required to confirm the diagnosis of corneal dystrophies. We report the first cases of Avellino dystrophy from Central-Eastern Europe. We conclude that the novel F547S mutation causes polymorphic corneal amyloidosis since no other mutations were detected in the TGFBI gene of this patient and the novel mutation could not be found in healthy controls.
5q31 linked corneal dystrophies are a clinically and histologically heterogeneous group of autosomal dominantly inherited corneal disorders. All these dystrophies result from mutations in the Transforming Growth Factor Beta Induced (TGFBI; BIGH3) gene, encoding for the BIGH3 protein, also called keratoepithelin . Corneal dystrophies caused by specific mutations of TGFBI include lattice corneal dystrophies type I (LCDI), type IIIA (LCDIIIA), type I/IIIA (LCDI/IIIA), and type IV(LCDIV), granular Groenouw type I corneal dystrophy (GCDI), Avellino corneal dystrophy (ACD), Thiel-Behnke dystrophy or corneal dystrophy of Bowman layer type II (CDB2), and Reis-Bücklers' dystrophy (RBCD). The most frequent types of these dystrophies result from mutations of two mutational hot spots in exons 4 and 12 of the TGFBI gene, representing codons 124 (LCDI, ACD, and RBCD) and 555 (GCD and CDB2), respectively. Mutations responsible for rare lattice dystrophies (LCD types III and IV) localize to other sites within exons 12, 13, and 14, encoding for the fourth fasciclin1 domain of the BIGH3 protein [2,3]. Histological examination of corneal specimens show amyloid deposits in lattice dystrophies and Avellino dystrophy, hyaline accumulation in granular dystrophy, and the presence of subepithelial fibrous material in Reis-Bücklers and Thiel-Behnke dystrophy . The two latter dystrophies, being relatively rare and somewhat similar in clinical appearance, were not always clearly distinguished in earlier studies [5,6]. However, the recent literature revealed distinct genetic, morphological, and clinical characteristics of these diseases. In Reis-Bücklers dystrophy, caused by the R124L mutation, subepithelial geographic corneal deposits can be seen on clinical examination, and rod shaped bodies can be observed by electron microscopy. Thiel-Behnke dystrophy, caused by the R555Q mutation, is characterized by honeycomb-shaped subepithelial deposits, which are composed of curly filaments as shown by electron microscopy [7,8].
In several corneal dystrophies, the deposits contain the BIGH3 protein as demonstrated by immunohistological examination [9,10].
In this study, we present the results of a molecular genetic analysis of 15 families referred to our department between 1997 and 2006 because of presumed BIGH3-related corneal dystrophies. Immunohistochemical studies of available dystrophic corneal buttons excised during penetrating keratoplasty were performed using a polyclonal antibody detecting the BIGH3 protein. The clinical and histological characteristics of a corneal dystrophy similar to polymorphic corneal amyloidosis, caused by the novel F547S mutation, are described.
Thirty-eight members from 15 families that were referred to the participating ophthalmology services between 1997 and 2006 were examined. One hundred unrelated, healthy volunteers from the same population who showed no sign of corneal disease on clinical examination were also recruited. All examinations were performed according to the tenets of the Declaration of Helsinki. All patients and controls were enrolled in the study after informed consent.
Clinical examinations included assessment of best-corrected visual acuity (BCVA), slit lamp examination, and slit lamp photography. On clinical examination, 18 patients showed the characteristics of lattice corneal dystrophy type I, five patients had granular dystrophy Groenouw type I, one patient was diagnosed with Reis-Bücklers, and three with Thiel-Behnke dystrophy. Three patients had "atypical granular" dystrophy and one patient was diagnosed with "atypical lattice" corneal dystrophy. Seven family members did not show any clinical sign of corneal dystrophy.
Molecular genetic analysis
Peripheral blood (9 ml) was taken from patients, unaffected family members, and 100 healthy volunteers. Genomic DNA was isolated from buffy coat using QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany). Exons of TGFBI were amplified using polymerase chain reaction (PCR) primers encompassing entire exons and short segments of flanking introns. Exon 17 was sequenced in three overlapping segments. Primers that we have optimized for amplification are listed in Table 1. For exon 4, they are identical to those previously published . Primer annealing temperature was adjusted separately for each PCR reaction and ranged from 50 °C-60 °C. An initial step of denaturation at 95 °C was followed by 35-45 cycles of annealing, elongation at 72 °C, and denaturation at 95 °C. PCR products purified by ultrafiltration were sequenced with an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA).
Histology and immunohistochemistry
Corneal buttons of 15 patients affected by lattice corneal dystrophy type I (LCDI; 10 cases from five families), corneal dystrophy of Bowman layer type II (CDB2; two cases, one case initially diagnosed as Reis-Bücklers dystrophy), Groenouw corneal dystrophy type I (GCDI; two cases), and polymorphic corneal amyloidosis (one case) were either fixed in 98% ethanol-2% acetic acid solution and embedded in paraffin or frozen in TissueTek OCT (Bayer, Pittsburgh, PA) and stored in liquid nitrogen until use. One of the two CDB2 and GCDI corneal buttons were recurrences 15 and 11 years after the first transplantations, respectively. The patients with Avellino dystrophy had best corrected visual acuities between 20/30 and 20/20, therefore keratoplasty was not performed and corneal buttons with Avellino dystrophy could not be subjected to histological analysis. Tissue sections were stained with hematoxylin-eosin, congo red, and Masson's trichrome in each case. Congo red stained sections were examined under polarized light as well. Polyclonal anti-BIGH3 antibody against an 18 amino-acid peptide identical with the NH2-terminal portion of the protein was prepared in chickens as described . Sections of corneal buttons were placed on β-methacrylopropyl-trimetoxy-silane-coated glass slides (Sigma, Schnelldorf, Germany), blocked with 5% horse serum, and then incubated with 1:100 diluted polyclonal antisera. Slides were developed with Sigma Fast Red tablets (Sigma) according to the manufacturer's instructions. Slides were examined with a Nikon Eclipse TS100 microscope (Nikon Instruments Europe B.V., Badhoevedorp, Netherlands) with 20X and 40X objectives.
One-quarter of the corneal button of the patient having the F547S mutation was fixed in 1% glutaraldehyde, dehydrated in methanol, and embedded in Epon. Ultrathin sections were placed on coated 300 mesh copper grids and post-contrasted in uranyl acetate-lead citrate. Sections were examined with a Jeol 1010 transmission electron microscope at 80 kV.
Results of genetic and immunohistochemical examinations are summarized in Table 2. In all patients diagnosed with LCDI dystrophy, the TGFBI R124C mutation was found. No mutation in TGFBI was detected in seven clinically unaffected LCDI family members. In five patients showing the clinical signs of GCDI, the heterozygous R555W mutation was present. In one patient initially diagnosed as having Reis-Bücklers dystrophy and in three patients with CDB2 (Thiel-Behnke) dystrophy, the heterozygous R555Q mutation was detected. Thus, the diagnosis of Reis-Bücklers dystrophy was modified to CDB2 (Thiel-Behnke) dystrophy based on the results of the molecular genetic analysis.
Immunohistological examination of 10 LCDI corneal buttons with a polyclonal antibody against the NH2-terminal portion of the BIGH3 protein revealed strong staining of the intraepithelial and subepithelial deposits and weaker staining around deep stromal deposits while no labeling within the deep deposits was observed (Figure 1A,B). Intra- or interfamilial differences were not noted among LCDI patients. In GCDI corneas, strong staining of the superficial and weaker staining of the deep granular deposits was observed (Figure 1C,D). In the CDB2 corneal buttons, strong labeling of the subepithelial fibrous deposit could be detected (Figure 1E,F).
In three members of a family, clinical examinations revealed breadcrumb-like deposits in the central anterior cornea, which had linear extensions toward the deeper stroma. Between the deposits, the cornea was clear. Based on the clinical appearance, the initial diagnosis was "atypical granular" dystrophy. Molecular genetic examination revealed the previously reported R124H mutation and established the diagnosis of Avellino dystrophy.
In both corneas of a 46-year-old patient, slit lamp examination revealed central, snowflake-like deposits mixed with fine linear and ice-chipped appearing deposits, which were more abundant in the central than in the peripheral cornea. Deposits were seen throughout the corneal stroma and numerous pre-Descemet deposits could be observed, however, no corneal guttae were present. A diffuse haze was seen between the central corneal deposits (Figure 2A,B). The visual acuity of the patient was 20/200 (right eye) and 30/200 (left eye). A novel heterozygous mutation, T1640C, causing the F547S amino acid exchange was detected in exon 12 of TGFBI in this patient (Figure 2F). Histological examination of her corneal button showed abundant congo red positive deposits mainly in the deep layers of the corneal stroma (Figure 2C), yielding green birefringence when examined under polarized light (Figure 2D). Immunohistochemistry showed strong staining of the deposits after labeling with a polyclonal anti-BIGH3 antibody (Figure 2E). Electron microscopic examination detected electron-dense deposits consisting of 8-10 nm thick, straight, nonbranching fibrils among collagen lamellae of the corneal stroma (Figure 3). All these findings indicate the presence of BIGH3 containing amyloid deposits in the cornea of this patient. The novel T1640C mutation was not detected in the 100 unrelated healthy controls from the same population. Therefore, we conclude that the T1640C mutation causing the F547S amino acid exchange in the BIGH3 protein is responsible for the corneal dystrophy of this patient.
In accordance with many previous reports [12-15], we have detected most of the mutations at codons 124 and 555 of TGFBI, indicating that these sites represent mutational hot spots in Hungarian corneal dystrophy patients as well. In four cases, the initial clinical diagnosis had to be corrected based on the results of the molecular genetic examinations. In one case, initially diagnosed as Reis-Bücklers dystrophy, honeycomb-shaped subepithelial deposits as well as characteristic histological features of CDB2 were seen, thus the incorrect diagnosis could have resulted from confusing earlier literature data [1,5,6], rather than an unusual clinical picture.
Different staining patterns of normal corneas and BIGH3 containing deposits were published with various antibodies reacting with the NH2-terminal, COOH-terminal, or middle portions of the BIGH3 protein [10,11,16]. Regarding LCDI and GCDI, our findings corroborate earlier reports on the absence of NH2-terminal immuno-reactive BIGH3 in intrastromal deposits. In CDB2, staining of the deposits has been reported with an antibody to the middle portion of BIGH3 . Our results indicate that CDB2 deposits also contain the NH2-terminal segment of BIGH3.
In three members of one family, molecular genetic examination revealed the R124H mutation and established the diagnosis of Avellino dystrophy, which has not been reported from Hungary or the Eastern-European region so far. This type of dystrophy was first described in an American-Italian family  but was since found in many populations and seem to be especially frequent in Asia [18-21]. Our study confirms that the R124H mutation may occur in any population.
In a patient initially diagnosed as "atypical lattice" corneal dystrophy, we detected the novel T1640C mutation causing the F547S amino acid exchange in BIGH3. No other mutation in TGFBI could be detected in this patient. The novel T1640C mutation was not found in 100 unrelated healthy subjects from the same population, indicating that the mutation does not represent a common polymorphism in Hungary. Histological and electronmicroscopic examination showed amyloid deposits in the corneal stroma, and immunohistochemistry showed the presence of BIGH3 in the patient's corneal deposits. These results indicate that T1640C is a novel mutation of TGFBI causing corneal amyloidosis. This mutation seems to be one of the rare mutations in the fourth fasciclin1 (FAS1) domain of BIGH3, which cause various forms of corneal amyloidosis with characteristic lattice lines described as LCDI, LCDI/IIIA, LCDIIIA, LCDIV, and polymorphic corneal amyloidosis [3,22-28]. In 2003, Clout and Hohenester  established a structural model of the fourth FAS1 domain of BIGH3, based on the crystal structure of drosophila fasciclin1. Using this model, they concluded that with the exception of R124 and R555, all amino acids implicated so far in dystrophy-related mutation sites in BIGH3 are highly conserved and probably have a role in protein folding and maintenance of protein structure. Phenylalanine 547 is located in a 10 amino acid long conserved region, which extends from Y537 to F547 . According to the model, F547 would interact with L518 and other side chains in the formation of a buried hydrophobic protein core. The exchange of the hydrophobic phenylalanine in position 547 to serine, a hydrophilic and much smaller molecule, could disrupt this core and cause severe structural instability and protein misfolding. Interestingly, five mutations have been described in the conserved region between Y537 and F547, three of them (T538R, F540S, and A546T) causing LCDIIIA [3,31,32], one (N544S) causing LCDIV , and another (A546D) causing polymorphic corneal amyliodosis . In the latter dystrophy, central deposits with chipped-ice appearance as well as fine, deep stromal linear deposits were described, and histology showed the presence of amyloid deposits in the corneal stroma. Because of its comparable clinical and histological characteristics, we have classified the dystrophy caused by the F457S mutation as polymorphic corneal amyloidosis.
Collectively, our results demonstrate that molecular genetic analysis is necessary to establish the appropriate diagnosis of corneal dystrophies. Detailed genetic and histological characterization helps our understanding of the pathomechanisms of TGFBI-related corneal dystrophies.
This work was supported by the Hungarian National Research Fund grants OTKA F046321 and K68616. G.V. is the recipient of an Öveges grant from the Hungarian National Office for Research and Technology.
1. Munier FL, Korvatska E, Djemai A, Le Paslier D, Zografos L, Pescia G, Schorderet DF. Kerato-epithelin mutations in four 5q31-linked corneal dystrophies. Nat Genet 1997; 15:247-51.
2. Korvatska E, Munier FL, Djemai A, Wang MX, Frueh B, Chiou AG, Uffer S, Ballestrazzi E, Braunstein RE, Forster RK, Culbertson WW, Boman H, Zografos L, Schorderet DF. Mutation hot spots in 5q31-linked corneal dystrophies. Am J Hum Genet 1998; 62:320-4.
3. El-Ashry MF, Abd El-Aziz MM, Larkin DF, Clarke B, Cree IA, Hardcastle AJ, Bhattacharya SS, Ebenezer ND. A clinical, histopathological, and genetic study of Avellino corneal dystrophy in British families. Br J Ophthalmol 2003; 87:839-42.
4. Yanoff M, Fine BS. Ocular Pathology: A Text and Atlas. 2nd ed. Philadelphia: Harper & Row; 1982.
5. Mashima Y, Nakamura Y, Noda K, Konishi M, Yamada M, Kudoh J, Shimizu N. A novel mutation at codon 124 (R124L) in the BIGH3 gene is associated with a superficial variant of granular corneal dystrophy. Arch Ophthalmol 1999; 117:90-3.
6. Okada M, Yamamoto S, Tsujikawa M, Watanabe H, Inoue Y, Maeda N, Shimomura Y, Nishida K, Quantock AJ, Kinoshita S, Tano Y. Two distinct kerato-epithelin mutations in Reis-Bucklers corneal dystrophy. Am J Ophthalmol 1998; 126:535-42.
7. Dighiero P, Niel F, Ellies P, D'Hermies F, Savoldelli M, Renard G, Delpech M, Valleix S. Histologic phenotype-genotype correlation of corneal dystrophies associated with eight distinct mutations in the TGFBI gene. Ophthalmology 2001; 108:818-23.
8. Ridgway AE, Akhtar S, Munier FL, Schorderet DF, Stewart H, Perveen R, Bonshek RE, Odenthal MT, Dixon M, Barraquer R, Escoto R, Black GC. Ultrastructural and molecular analysis of Bowman's layer corneal dystrophies: an epithelial origin? Invest Ophthalmol Vis Sci 2000; 41:3286-92.
9. Streeten BW, Qi Y, Klintworth GK, Eagle RC Jr, Strauss JA, Bennett K. Immunolocalization of beta ig-h3 protein in 5q31-linked corneal dystrophies and normal corneas. Arch Ophthalmol 1999; 117:67-75.
10. Korvatska E, Munier FL, Chaubert P, Wang MX, Mashima Y, Yamada M, Uffer S, Zografos L, Schorderet DF. On the role of kerato-epithelin in the pathogenesis of 5q31-linked corneal dystrophies. Invest Ophthalmol Vis Sci 1999; 40:2213-9.
11. Takacs L, Boross P, Tozser J, Modis L Jr, Toth G, Berta A. Transforming growth factor-beta induced protein, betaIG-H3, is present in degraded form and altered localization in lattice corneal dystrophy type I. Exp Eye Res 1998; 66:739-45.
12. Takahashi K, Murakami A, Okisaka S. Kerato-epithelin mutation (R 555 Q) in a case of reis-Bucklers corneal dystrophy. Jpn J Ophthalmol 2000; 44:191.
13. El-Ashry MF, Abd El-Aziz MM, Hardcastle AJ, Bhattacharya SS, Ebenezer ND. A clinical and molecular genetic study of autosomal-dominant stromal corneal dystrophy in British population. Ophthalmic Res 2005; 37:310-7.
14. Konishi M, Mashima Y, Yamada M, Kudoh J, Shimizu N. The classic form of granular corneal dystrophy associated with R555W mutation in the BIGH3 gene is rare in Japanese patients. Am J Ophthalmol 1998; 126:450-2.
15. Pampukha VM, Drozhyna GI, Livshits LA. TGFBI gene mutation analysis in families with hereditary corneal dystrophies from Ukraine. Ophthalmologica 2004; 218:411-4.
16. Escribano J, Hernando N, Ghosh S, Crabb J, Coca-Prados M. cDNA from human ocular ciliary epithelium homologous to beta ig-h3 is preferentially expressed as an extracellular protein in the corneal epithelium. J Cell Physiol 1994; 160:511-21.
17. Folberg R, Alfonso E, Croxatto JO, Driezen NG, Panjwani N, Laibson PR, Boruchoff SA, Baum J, Malbran ES, Fernandez-Meijide R. Clinically atypical granular corneal dystrophy with pathologic features of lattice-like amyloid deposits. A study of these families. Ophthalmology 1988; 95:46-51.
18. Dighiero P, Ellies P, Valleix S, D'hermies F, Savoldelli M, Pouliquen Y, Legeais JM, Renard G. [Avellino dystrophy. Current diagnostic criteria]. J Fr Ophtalmol 2000; 23:735-7.
19. Francis PJ, Johnson S, Edmunds B, Kelsell RE, Sheridan E, Garrett C, Holder GE, Hunt DM, Moore AT. Genetic linkage analysis of a novel syndrome comprising North Carolina-like macular dystrophy and progressive sensorineural hearing loss. Br J Ophthalmol 2003; 87:893-8.
20. Holland EJ, Daya SM, Stone EM, Folberg R, Dobler AA, Cameron JD, Doughman DJ. Avellino corneal dystrophy. Clinical manifestations and natural history. Ophthalmology 1992; 99:1564-8.
21. Konishi M, Mashima Y, Nakamura Y, Yamada M, Sugiura H. Granular-lattice (Avellino) corneal dystrophy in Japanese patients. Cornea 1997; 16:635-8.
22. Pieramici SF, Afshari NA. Genetics of corneal dystrophies: the evolving landscape. Curr Opin Ophthalmol 2006; 17:361-6.
23. Mashima Y, Yamamoto S, Inoue Y, Yamada M, Konishi M, Watanabe H, Maeda N, Shimomura Y, Kinoshita S. Association of autosomal dominantly inherited corneal dystrophies with BIGH3 gene mutations in Japan. Am J Ophthalmol 2000; 130:516-7.
24. Endo S, Nguyen TH, Fujiki K, Hotta Y, Nakayasu K, Yamaguchi T, Ishida N, Kanai A. Leu518Pro mutation of the beta ig-h3 gene causes lattice corneal dystrophy type I. Am J Ophthalmol 1999; 128:104-6.
25. Klintworth GK. The molecular genetics of the corneal dystrophies--current status. Front Biosci 2003; 8:d687-713.
26. Chau HM, Ha NT, Cung LX, Thanh TK, Fujiki K, Murakami A, Kanai A. H626R and R124C mutations of the TGFBI (BIGH3) gene caused lattice corneal dystrophy in Vietnamese people. Br J Ophthalmol 2003; 87:686-9.
27. Stewart H, Black GC, Donnai D, Bonshek RE, McCarthy J, Morgan S, Dixon MJ, Ridgway AA. A mutation within exon 14 of the TGFBI (BIGH3) gene on chromosome 5q31 causes an asymmetric, late-onset form of lattice corneal dystrophy. Ophthalmology 1999; 106:964-70.
28. Warren JF, Abbott RL, Yoon MK, Crawford JB, Spencer WH, Margolis TP. A new mutation (Leu569Arg) within exon 13 of the TGFBI (BIGH3) gene causes lattice corneal dystrophy type I. Am J Ophthalmol 2003; 136:872-8.
29. Clout NJ, Hohenester E. A model of FAS1 domain 4 of the corneal protein beta(ig)-h3 gives a clearer view on corneal dystrophies. Mol Vis 2003; 9:440-8 <http://www.molvis.org/molvis/v9/a56/>.
30. Skonier J, Neubauer M, Madisen L, Bennett K, Plowman GD, Purchio AF. cDNA cloning and sequence analysis of beta ig-h3, a novel gene induced in a human adenocarcinoma cell line after treatment with transforming growth factor-beta. DNA Cell Biol 1992; 11:511-22.
31. Dighiero P, Drunat S, Ellies P, D'Hermies F, Savoldelli M, Legeais JM, Renard G, Delpech M, Grateau G, Valleix S. A new mutation (A546T) of the betaig-h3 gene responsible for a French lattice corneal dystrophy type IIIA. Am J Ophthalmol 2000; 129:248-51.
32. Stix B, Leber M, Bingemer P, Gross C, Ruschoff J, Fandrich M, Schorderet DF, Vorwerk CK, Zacharias M, Roessner A, Rocken C. Hereditary lattice corneal dystrophy is associated with corneal amyloid deposits enclosing C-terminal fragments of keratoepithelin. Invest Ophthalmol Vis Sci 2005; 46:1133-9.
33. Nakagawa Asahina S, Fujiki K, Enomoto Y, Murakami A, Kanai A. [Case of late onset and isolated lattice corneal dystrophy with Asn544Ser (N544S) mutation of transforming growth factor beta-induced (TGFBI, BIGH3) gene]. Nippon Ganka Gakkai Zasshi 2004; 108:618-20.
34. Eifrig DE Jr, Afshari NA, Buchanan HW 4th, Bowling BL, Klintworth GK. Polymorphic corneal amyloidosis: a disorder due to a novel mutation in the transforming growth factor beta-induced (BIGH3) gene. Ophthalmology 2004; 111:1108-14.