Cuevas EC, Bateman AC, Wilkins BS, et al. Microwave antigen ... mimicry by herpes simplex virus-type 1: autoimmune disease after viral infection. Science. ... specimens and four iris specimens were obtained from 16 patients without PEX.
IOVS, May 1999, Vol. 40, No. 6 9. Hagen EC, van de Vijver-Reenaldo H, de Keizer RJ, et al. Uveitis and anti-neutrophil cytoplasmic antibodies. Clin Exp Immunol. 1994;95:56-59. 10. Saxon A, Shanahan F, Landers C, Ganz T, Targan SR. A distinct subset of antineutrophil cytoplasmic antibodies is associated with inflammatory bowel disease. / Allergy Clin Immunol. 1990;86: 202-210. 11. Gordon LK, Eggena M, Hollanad GN, WeiszJM, Braun J. pANCA antibodies in patients with anterior uveitis: identification of a marker antibody usually associated with ulcerative colitis. / Clin Immunol. 1998;18:264-271. 12. Billing P, Tahir S, Calfin B, et al. Nuclear localization of the antigen detected by ulcerative colitis-associated perinuclear antineutrophil cytoplasmic antibodies. Am]Pathol. 1995;l47:979-987. 13. Vidrich A, Lee J, James E, Cobb L, Targan S. Segregation of pANCA antigenic recognition by DNase treatment of neutrophils: ulcerative colitis, type 1 autoimmune hepatitis, and primary sclerosing cholangitis. / Clin Immunol. 1995;15:293-299. 14. Sandborn WJ, Landers CJ, Steiner BL, Tremaine NJ, Targan SR. Association of antineutrophil cytoplasmic antibody is unexpectedly high in patients with treatment resistant, left-sided ulcerative colitis. Mayo Clin Proc. 1996;71:431-436. 15. Sandborn WJ, Landers CJ, Steiner BL, Tremaine NJ, Targan SR. Antineutrophil cytoplasmic antibody correlates with chronic pouchitis after ileal-anal pouch anastomosis. Am J Gastroenterol. 1996;90:740-747. 16. Shanahan F, Duerr R, Landers C, Yang H, Sutherland L, Rotter JT. Neutrophil autoantibodies in ulcerative colitis: a family study with evidence for genetic heterogeneity. Gastroenterology. 1992; 103: 456-461. 17. Seibold R, Slameschka D, Gregor M, Weber F. Neutrophil autoantibodies: a genetic marker in primary sclerosing cholangitis and ulcerative colitis. Gastroenterology. 1994;107:532-536. 18. Lyons JL, Rosenbaum JT. Uveitis associated with inflammatory' bowel disease compared with uveitis associated with spondyloarthropathy. Arch Ophthalmol. 1997;115:6l-64. 19- Soukiassian SH, Foster CS, Raiman MB. Treatment strategies for scleritis and uveitis associated with inflammatory bowel disease. AmJ Ophthalmol. 1994;118:601-6ll. 20. Banares AA, Jover JA, Fernandez-Gutierrez B, et al. Bowel inflammation in anterior uveitis and spondyloarthropathy. / Rheumatol. 1995;22:1112-1117. 21. Eggena M, Targan SR, Iwanczyk L, Vidrich A, Gordon LK, Braun J. Phage display cloning and characterization of an immunogenetic marker (perinuclear anti-neutrophil cytoplasmic antibody) in ulcerative colitis. / Immunol. 1996;156:4005-4011.
Loss of Heterozygosity in Pseudoexfoliation Syndrome Vassilios P. Kozobolis,1'2 Efstathios T. Detorakis,1'2 George Sourvinos,5 Ioannis G. Pallikaris,1'2 and Demetrios A. Spandidos5
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22. Barbas CF, Kang AS, Lerner R, Benkovic SJ. Assembly of combinatorial antibody libraries on phage surfaces: the gene 111 site. Proc NatlAcadSci USA. 1991 ;88:7978-7982. 23. Cuevas EC, Bateman AC, Wilkins BS, et al. Microwave antigen retrieval in immunocytochemistry: a study of 80 antibodies./ Clin Pathol. 1994;47:448-452. 24. Taylor CR, Shi SR, Chaiwun B, Young L, Iman SA, Cote RJ. Strategies for improving the immunohistochemical staining of various intranuclear prognostic markers in formalin-paraffin sections: androgen receptor, estrogen receptor, progesterone receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 antigen revealed by antigen retrieval techniques. Hum Pathol. 1994;25:263270. 25. Sobajima J, Ozaki S, Uesugi H, et al. Prevalence and characterization of perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) directed against HMG1 and HMG2 in ulcerative colitis. Clin Exp Immunol. 1998;111:402- 407. 26. Parseghian MH, Henschen AH, Krieglstein KG, Hamkalo BA. A proposal for a coherent mammalian histone HI nomenclature correlated with amino acid sequences. Protein Sci. 1994;3:575587. 27. Liao L, Singhwani R, Rojkind M, Factor S, Leinwand L, Diamond B. Antibody-mediated autoimmune myocarditis depends on genetically determined target organ sensitivity. / Exp Med. 1995;18l: 1123-1131. 28. Zhao Z-S, Granucci F, Yeh L, Schaffer PA, Cantor H. Molecular mimicry by herpes simplex virus-type 1: autoimmune disease after viral infection. Science. 1998;279:1344 -1347. 29- Forrester JV, Liversidge J, Dick A, et al. What determines the site of inflammation in uveitis and chorioretinitis. Eye. 1997;ll:l62-l66. 30. McMenamin PG. The distribution of immune cells in the uveal tract of the normal eye. Eye. 1997;11:183-193. 31. Broekhuyse RM, Kuhlmann ED, Winkens HJ, Van Vugt AHM. Experimental autoimmune anterior uveitis (EAAU), a new form of experimental uveitis, I: induction by a detergent-insoluble, intrinsic protein fraction of the retinal pigment epithelium. Exp Eye Res. 1991;52:465-474. 32. Prendergast RA, Iliff CE, Coskuncan NM, et al. T-cell traffic and the inflammatory response in experimental autoimmune uveoretinitis. Invest Ophthalmol Vis Sci. 1998;39:752-762. 33. Butler TL, McMenamin PG. Resident and infiltrating immune cells in the uveal tract in the early and late stages of experimental autoimmune uveoretinitis. Invest Ophthalmol Vis Sci. 1996;37: 2195-2210.
METHODS. Twelve iris specimens, 12 anterior capsule spec-
imens, and respective blood samples were obtained from 17 patients with PEX (13 men), who were undergoing glaucoma and cataract surgery. Sixteen anterior capsule specimens and four iris specimens were obtained from 16 patients without PEX. Polymerase chain reaction was used to amplify 10 highly polymorphic microsatellite markers located on chromosomes 1, 7, 9, and 13. RESULTS. Overall, 83-3% (20/24) of PEX specimens and
PURPOSE. Pseudoexfoliation (PEX) syndrome is character-
ized by the accumulation of a material of unknown origin in the anterior structures of the eye. Loss of heterozygosity (LOH) in a genetic locus indicates the presence of a gene located in the same region that could be implicated in the development or the progression of a disease. In this study, the occurrence of LOH in tissues involved in PEX and the possible correlation of LOH incidence with clinical parameters were evaluated.
94.11% (16/17) of patients with PEX had LOH. The highest incidence of LOH was observed in marker D13S175 (41.6%) followed by D7S478 and D7S479 (37.5%). Only three non-PEX specimens displayed LOH. The number of loci lost was directly related to the altitude of the patients' present residence, but the number lost did not differ significantly between the iris and capsule samples. CONCLUSIONS. The occurrence of LOH in tissues involved in
PEX implies a genetic role in PEX pathogenesis at a cellu-
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lar level. The correlation of LOH incidence with the altitude of the patient's residence, could indicate an increased susceptibility to UV radiation of the chromosomal regions examined. (Invest Ophthalmol Vis Set. 1999;40: 1255-1260) seudoexfoliation (PEX) syndrome is a degenerative condition affecting people more than 40 years of age, characterized by deposits of material of unknown origin and nature in the anterior structures of the eye, such as the anterior capsule, trabecular meshwork, ciliary body, pupillary margin, and iris epithelium.1'2 The existence of proteoglycans in pseudoexfoliative material and the chemical similarity to elastic tissue has lead to the concept that PEX is a disturbance of basal membrane synthesis.3'4 Pseudoexfoliative material has been said to be produced by metabolically disturbed epithelial cells of the capsule,5'6 pigment epithelial cells of the iris and ciliary body,7'8 or pericytes of the iris and ciliary body. 910 There is evidence that PEX is systemic, apparently involving aberrant connective-tissue metabolism throughout the body.'' A pattern of autosomal dominant heredity has been described.12 Loss of heterozygosity (LOH) in highly polymorphic microsatellite markers is a somatic cellular event characterizing a clone of cells.13 The detection of LOH is commonly used for the identification of sites in the genome with high probability for the presence of candidate dominant genes.l4 The finding of LOH at a microsatellite marker locus implies that a nearby gene could be affected by the genetic lesion.1516 The highly polymorphic microsatellite DNA, composed of repetitive sequences of dinucleotides (CA)n and, rarely, trinucleotides, has been used for genetic linkage analysis in various neoplastic and nonneoplastic conditions.16 The purpose of this study was to evaluate the existence of LOH in tissues possibly implicated in the synthesis of pseudoexfoliative material, and to determine the correlation of this genetic alteration with clinical and epidemiologic parameters.
P
METHODS Tissue specimens and blood samples were obtained from patients treated at the Ophthalmological Clinic of the University Hospital of Heraklion, Crete, who had pseudoexfoliative features apparent in the anterior segment after pupil dilation. All patients with PEX who underwent cataract or glaucoma surgery during the study were included. The research followed the tenets of the Declaration of Helsinki. Specimens were also obtained from patients without PEX who were of comparable age and gender. Epidemiologic and clinical information such as age, gender, place of residence, and visual acuity and intraocular pressure were taken from the patients' files for use in statistical analysis. In the patients undergoing cataract surgery the anterior capsule was obtained,
From the Departments of 'Ophthalmology and 'Virology, University of Crete; and the 2Vardinoyannion Eye Institute of Crete, Greece. Submitted for publication April 15, 1998; revised November 19, 1998; accepted December 21, 1998. Proprietary interest category: N. Reprint requests: Demetrios A. Spandidos, School of Medicine, University of Crete, Department of Virology, POB 1393, Heraklion, Crete, 71110 Greece.
IOVS, May 1999, Vol. 40, No. 6 whereas in those undergoing glaucoma surgery, a segment of peripheral iris was obtained from the peripheral iridectomy site. In the patients undergoing combined cataract-glaucoma surgery, the anterior capsule and the segment of peripheral iris were both obtained. The iris and capsule specimens were thoroughly rinsed with normal saline after excision, to remove the remaining blood. Blood samples were obtained from each patient by venipuncture. Immediately after surgery, tissue specimens and blood samples were stored at -70°C and 4°C, respectively. DNA extraction was performed by means of a standard protocol using organic detergents. A 25-juJ polymerase chain reaction (PCR) analysis was performed, containing 200 ng genomic DNA, 1 fiM of each primer, 250 JU,M deoxyribonucleoside triphosphate, 2.5 jui 10X buffer (670 mM Tris-HCl [pH 8.5], 166 mM ammonium sulfate, 67 mM magnesium chloride, 1.7 mg/ml bovine serum albumin, 100 ju-M /3-mercaptoethanol, and 1% [wt/vol] Triton X-100), and 1 U Tag DNA polymerase. The reactions were denatured for 3 minutes at 94°C, and DNA was amplified for 30 cycles at 94°C, then 50°C, 52°C, 55°C (annealing temperature depending on the primer used each time), and 72°C, each step. Ten microliters of the PCR product was electrophoresed in a 10% polyacrylamide gel and silver stained. Gels were scanned, and the intensity of the bands corresponding to the microsatellite alleles was quantitated by an image analysis system (Adobe Photoshop, ver. 5.0; Adobe, San Jose, CA). The analyses were performed twice, and the results were highly reproducible. A comparison of the electrophoretic patterns of the amplified marker segments in the normal and pathologic tissues was performed. LOH was defined as a decrease (at least 50%) in intensity of one allele compared with that of the other, determined after comparison of pathologic and normal DNA. In few cases, although LOH occurred, the PCR amplification of polymorphic microsatellite alleles resulted in an allelic imbalance due to the presence, in decreased levels, of a contaminating band. This band corresponded to the deleted allele that is present in the normal tissue. Generally, this "contamination" is attributed to the presence of normal DNA derived by peripheral blood or adjacent normal tissue.17 Traces of blood could have remained on the specimens even after thorough rinsing with normal saline, especially in the case of iris samples, because of hemorrhage during iridectomy. However, because of excision under the surgical microscope and subsequent rinsing, we think the bulk of DNA obtained was derived from the tissues that were targeted (i.e., anterior capsule and iris). In heterogeneous specimens, a load of 30% of affected cells is considered necessary to identify LOH (some reports claim an LOH identification with only 10% of affected cells per specimen).'3 Ten microsatellite markers were examined (Table 1). PEX has been reported to be related to exposure to solar light.18 Given that investigators in studies of pathologic conditions of the skin and eyes relating to UV radiation19'20 have reported a high incidence of LOH in markers on chromosomes 9 and 13, markers located on these chromosomes were examined. Additionally, markers on chromosome 7 were evaluated, because a number of candidate genes for connective tissue proteins such as collagen 1A2, laminin Bl, and elastin have been localized to chromosomal region 7q.21'22 Because PEX is strongly associated with glaucoma,1'2 and there are reports of a glaucoma-
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TABLE 1. Incidence of LOH
LOH per Chromosome
LOH per Marker %of
%of
Marker
Location
Specimens*
Patientsf
Chromosome
D1S80 D1S416 D9S109 D9S112 D9S50 D7S478 D7S479 D13S155 D13S168 D13S175
Iq42
20.80 (5/24) 16.66 (4/24) 8.3 (2/24) 12.5 (3/24) 20.8 (5/24) 37.50 (9/24) 37.50 (9/24) 16.66 (4/24) 16.66 (4/24) 41.66(10/24)
29.41 (5/17) 23.52 (4/17) 11.76(2/17) 17.64 (3/17) 23.52 (4/17) 41.17 (7/17) 35.29 (6/17) 23.52 (4/17) 23.52 (4/17) 52.94 (9/17)
1 1 9 9 9 7 7 13 13 13
1
9q31 9q31-q34 9p21 7pl5-q22 7q21-q22 13ql4.3-q21.2 13ql4.3 13qll
%of
% of Specimens*
Patientsf
37.50 (9/24)
52.94 (9/17)
54.16(13/24)
58.82 (10/17)
33.33 (8/24)
41.17(7/17)
54.16(13/24)
70.58 (12/17)
Data in parentheses represent the number of 'specimens or fpatients. related gene located on chromosome I,23'24 markers on chromosome 1 were also examined. Results were analyzed using a statistical analysis program commonly used in biomedical assays (SPSS 6.0 for Windows; SPSS, Chicago, IL). Statistical significance was set at P < 0.05. Twenty-four tissue specimens were obtained from patients with PEX, 12 anterior capsule and 12 iris portions. The specimens were collected from 17 patients (13 men; 76.5%). PEX was bilateral in 10 (58.8%) of 17 patients included in the study. The average age of the patients was 76.6 ±1.8 years (mean ± SE; range, 60-88 years), 77.7 ± 2.6 years (range, 60-88 years) in the men and 71.4 ± 0.7 years (range, 69-74 years) in the women. The average altitude of the patients' present residences was 186.4 ± 38.5 m (range, 0-560 m). Sixteen anterior capsule and four iris specimens from patients without PEX, were also obtained. The specimens were collected from 16 patients (10 men; 62.5%). The average age of the patients was 78.5 ±1.9 years (range, 62-85 years), 76.6 ± 2.1 years (range, 63-84 years) in men and 72.5 ± 0.8 years (range, 62-85 years) in women. The average altitude of the patients' present residences was 167.4 ± 47.4 m (range, 0-480 m) The differences in age, gender, and altitude of residence between patients with and without PEX was statistically insignificant.
RESULTS Overall, 83.3% (20/24) of PEX specimens and 94.1% (16/17) of patients with PEX, showed LOH at one or more loci. Eight specimens showed loss in one locus, two in two loci, three in two loci, four in four loci, three in five loci, and one in six loci, whereas in four specimens no loss was found. The incidence of LOH in the microsatellite markers and chromosomes examined is presented in Table 1. In Figure 1, representative cases of LOH are presented. In Table 2, the loci lost in the examined markers for each anterior capsule and iris specimen are presented, together with the age, gender, altitude of residence, and intraocular pressure of patients from whom the specimens were obtained. Only three occurrences of LOH (two in anterior capsule specimens and one in iris specimens) were observed in the group of patients without PEX, in markers D9S50 (9p21),
D13S168 (13ql4.3), and D13S175 (13qll). The difference between the percentage of specimens displaying LOH in PEXaffected and non-PEX-affected patients was statistically significant (Yate's corrected, X2 = 13.85; P *** 0). The total number of loci lost was directly correlated with the altitude of the patients' present residences and was statistically significant (bivariate correlation coefficient = 0.42; P = 0.04). The respective correlations with visual acuity and intraocular pressure were statistically insignificant (bivariate correlation coefficients = 0.22 and 0.06; P = 0.32 and 0.76, respectively), whereas a statistically significant inverse correlation with patients' ages was observed (bivariate correlation coefficient = -0.43; P = 0.04). Seventy-five percent (9/12) of the anterior capsule specimens and 91.70% (11/12) of the iris specimens displayed LOH in at least one locus (/-test value = 0.32; P = 0.75). Eighty percent (12/15) of specimens from male patients and 85.7% (6/7) from female patients displayed LOH at one or more loci (t-test value = 0.02; P = 0.975). LOH in at least one locus was displayed by 88.9% (8/9) of specimens from patients with unilateral PEX and 71.4% (10/14) of specimens from patients with bilateral PEX (one-tailed Fisher's exact test, P = 0.52; two-tailed Fisher's exact-test, P = 1.00). In some cases, LOH incidence in individual markers or chromosome arms showed a significant or nearly significant correlation with clinical and epidemiologic parameters, such as age, altitude of present residence, and intraocular pressure. The average age of patients displaying LOH in one or both examined markers in chromosome 7 (D7S478 and D7S479) was 72.4 years, whereas the respective average age of patients without LOH in these markers was 82 years; the difference is statistically significant (/-test value = 3-2; two-tailed significance «= 0). A similar, almost statistically significant difference, was observed in marker D9S50. The average age of patients with LOH in this marker was 70.4 years, whereas the average age of those without LOH in D9S50 was 78.1 years (/-test value = 2.00; two-tailed significance = 0.06). The incidence of LOH in D9S50 showed an almost statistically significant correlation with the altitude of the patients' present residences. The average altitude of residences of patients who had LOH in D9S5O was 327.5 m, whereas the respective altitude of residences of those without LOH was 155 m (f-test value = 1.8; two-tailed significance = 0.08). The
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7 D73479 B N
7 D9SS0 B N
I
mm
7 D9S112
6 D1S80 N
I
N
I
I
B
2.1
FIGURE 1. Representative examples of LOH detected in PEX syndrome. Arrows indicate the position of a deleted allele. Faint bands in the position of a deleted allele are interpreted as contamination by adjacent normal DNA. N, normal (DNA derived from leukocytes, used as control); I, iris; B, capsular bag. intraocular pressure was significantly correlated with LOH incidence in marker D9S109. The average intraocular pressure in patients who had LOH in D9S109, was 29 nim Hg, whereas the respective value in those without LOH was 17 mm Hg (/-test value = 2.1; two-tailed significance = 0.04). The Mest value and two-tailed statistical significance of the difference in age, altitude of residence, and intraocular pressure between pa-
TABLE
2. Loci Deleted in the Examined Markers
Code
Age
Sex
Altitude
Anterior capsule specimens F 0 1 69 2 0 F 72 M 60 70 5 M 4 0 78 72 F 360 5 88 M 350 6 M 7 250 63 72 8 340 M M 320 9 85 10 M 320 85 11 74 0 F 12 72 M 0 Iris specimens 1 0 F 69 2 72 F 0 60 70 M 3 4 78 M 0 72 F 360 5 6 M 350 88 M 7 0 87 8 M 86 340 560 M 70 9 M 10 86 90 11 M 380 79 12 M 81 340
tients with and without LOH in selected markers (D7S478, D7S479, D9S50, and D9S1O9) is shown in Table 3DISCUSSION Tumor allelotyping, the genotypic analysis of all 23 chromosomes to determine regions of interstitial deletions, was intro-
t
D13S155
D13S168
D13S175
D9S5O
D9S112
14 24 21 10 12
H H H H H
H LOH H H
H
H
H
LOH H H H LOH H
H
LOH
H H H H LOH
10
19
LOH LOH H H
H H H H H H H LOH H H H H
H LOH LOH H
LOH O H H
LOH
H
H
H
H H
H LOH
H H
LOH H LOH H H
H H
H
H H H H H
H H H H LOH H H LOH O H H H
H H H H H H H H H H H H
H H H H H H H H LOH LOH H H
H H H LOH LOH H H H H LOH H H
11 14 14 24 20 14 24 21 10 12 10 13
41 34 24 20 22
H H H H H H H H H H H
LOH H LOH
H H LOH H H H H H LOH H H LOH
LOH H H H H LOH H LOH LOH H LOH LOH
H
D9S1O9 D1S80
H H H H H
H H H H H H
D1S416
D7S479
D7S478
H
H H
LOH H H H LOH H H LOH H H H LOH
H LOH H H LOH H LOH H H H H LOH
H H H H H LOH LOH H LOH H H H
LOH H LOH H LOH H LOH H LOH H H H
LOH H H H H
H
H
LOH H H LOH H
LOH H LOH LOH H H
In the first four cases, both the anterior capsule specimen and an iris specimen were obtained from the same patient, t, intraocular pressure; H, heterozygosity; LOH, loss of heterozygosity.
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TABLE 3. £-Test Value and Two-Tailed Statistical Significance of the Difference D7S478
Age Altitude Intraocular pressure
D9S50
D7S479
D9S109
Mest
P
f-Test
P
Mest
P
t-Test
P
1.85 0.31
0.07 0.76
2.19 0.31
0.04 0.76
1.96 1.8
0.06 0.08
0.28 1.14
0.77 0.26
0.92
0.36
1.35
0.19
0.7
0.44
2.11
0.04
Data represent difference in age, altitude of residence, and intraocular pressure between patients with and without LOH in the presence of selected markers (D7S478, D7S479, D9S5O, and D9S109). duced by Vogelstein et al.25 It is based on Knudson's paradigm of deleted anti-oncogenes.25 LOH, the loss of an allelic band in tumor DNA that is present in constitutional DNA, is recognized as indicative of putative tumor-suppressor genes.26 The systematic analysis of LOH in multiple loci in tumor specimens can provide the basis for preliminary models of molecular multistep progression in some tumor types.26 LOH may arise from different mechanisms.27 The locus may become hemizygous, through deletion or chromosome loss without reduplication, or homozygous, through mitotic recombination, gene conversion, or chromosome loss followed by reduplication. In the latter case, the resultant two alleles could be normal (if the first retained allele was normal) or mutated (if the first mutated allele was mutated). Microsatellites have been used extensively for tumor allelotyping and allelic imbalance analysis.13 Their usefulness as genetic markers is enhanced by their ubiquity, PCR typeability, Mendelian codominant inheritance, and extreme polymorphism.13 In addition to its presence in neoplasias, LOH has been found in benign conditions, such as actinic keratosis of the skin,19 ocular pterygium,20 and atherosclerotic plaques.28 The fact that tissues implicated in PEX pathogenesis, such as anterior capsule and iris, display LOH indicates a possible genetic role in the pathogenesis of the condition. In this case, genetic damage successively affecting both alleles of the gene or genes involved (Knudson's two-hit hypothesis), could lead to the expression of the PEX phenotype. Apart from pathologic conditions, neoplastic or nonneoplastic, LOH has also been reported in phenotypically normal tissue, as in normal bladder mucosa in the presence of cancer of the urinary bladder29 and in normal tissue adjacent to breast carcinoma.30 LOH found in anterior capsule and iris specimens without clinical signs of PEX could represent a silent genotypic alteration, without phenotypic consequences for the tissue (perhaps caused by the exposure of such tissues to solar light, because chromosomal regions 9p and 13q, in which LOH has been observed, are particularly susceptible to damage from UV radiation19'20). In contrast, LOH in non-PEX specimens could be an indication that the tissues examined possess a potential for pathologic evolution, because transformation of non-PEX-affected eyes to PEX-affected eyes over time has been reported.31 Another possibility is the subclinical existence of pseudoexfoliative material in eyes in which PEX is not diagnosed. Histologic examination of eyes without clinically evident PEX features has revealed pseudoexfoliative material.32 In the present study, taking into account that patients with and without clinically evident PEX did not differ significantly in the epidemiologic parameters recorded, the fact
that LOH was significantly more common in the PEX-affected group supports the concept of genetic involvement in the pathogenesis of PEX. The high incidence of LOH in chromosomal regions 13q, 9q, and 9p has been reported in conditions strongly related to exposure to UV light, such as actinic keratosis19 and basal cell carcinoma.33 PEX has also been reported to be associated with increased exposure to solar light.18 The common incidence of LOH in the present study in microsatellite markers located in the same regions could reflect an increased susceptibility of these chromosomal regions to UV light. The significant correlation of cumulative incidence of LOH with the altitude of the patients' present residence, which is known to be directly correlated with exposure to UV radiation,34 supports the concept of increased genetic material susceptibility to UV light in the chromosomal areas examined. Pseudoexfoliative material is thought to result from abnormal basement membrane production by degenerated epithelial cells.35 An immunologic relationship of PEX material with elastic tissue was also shown, suggesting that an abnormal stimulus or defective regulation of matrix synthesis exists in the disease.3637 In the present study, a relatively high incidence of LOH was displayed in region 7q, where genes for various connective tissue proteins, such as elastin, laminin Bl, and collagen 1A2 are potentially located,2122 possibly implying that genes in this area play a role in PEX pathogenesis. PEX is generally an age-related condition.1'2 No correlation has been reported between patients' age and the incidence of LOH in microsatellite markers.38'39 However, in the case of sporadic invasive ductal breast carcinoma, LOH incidence in markers flanking the ATM gene (chromosome 11) has been reported to be correlated with early age of tumor diagnosis in a set of tumors.40 The inverse correlation of age, in the present study, with LOH incidence in markers D7S479 and D7S478 (and, at a lower level of statistical significance, in marker D9S50) may reflect genetic damage that accelerates the sequence of events that lead to the PEX phenotype. Taking into account that LOH in D9S109 was not observed among patients without glaucoma, the significant association between LOH incidence in this marker and intraocular pressure may imply a genetic lesion that predisposes to PEX and glaucoma. It has been suggested that a single locus disturbance may cause PEX, an increase in intraocular pressure, and a degeneration of the iris pigment epithelium.41 The LOH incidence did not differ significantly between the anterior capsule and the iris specimens, suggesting that both tissues could be equally involved in PEX pathogenesis.
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Reports
Further research is needed to examine whether LOH reflects a genetic disturbance leading to the production of pseudoexfoliative material in the tissues evaluated.
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