Molecular Vision 2003; 9:257-261 <>
Received 14 January 2003 | Accepted 13 June 2003 | Published 17 June 2003

Loss of heterozygosity in patients with pseudoexfoliation syndrome

Renata Zalewska,1 Witold Pepinski,2 Danuta Smolenska-Janica,1 Zofia Mariak,1 Ewa Proniewska-Skretek,1 Malgorzata Skawronska,2 Jerzy Janica2

Departments of 1Ophthalmology and 2Forensic Medicine, Medical University of Bialystok, Bialystok, Poland

Correspondence to: Witold Pepinski, Department of Forensic Medicine, Medical Academy of Bialystok, ul. Waszyngtona 13, 15-230 Bialystok, Poland; email:


Purpose: The purpose of the study was to evaluate the possible occurrence of loss of heterozygosity (LOH) in the anterior capsule, lens nucleus, iris, and trabeculum samples taken from patients with pseudoexfoliation syndrome (PEX). Loss of heterozygosity in a microsatellite marker locus indicates that the neighboring gene may be inactivated. Previous attempts to find a gene defect that might be responsible for pseudoexfoliation glaucoma have been unsuccessful.

Methods: Specimens of the anterior capsule, the lens nucleus, the iris, the trabeculum, and reference blood samples were collected from 19 PEX patients. Fluorescent multiplex PCR was used to amplify the microsatellite markers located on chromosomes 2, 4, 7, 12, 18, 19, and 21.

Results: LOH was found in 58% of the iris specimens and 50% of the anterior capsule specimens collected from PEX patients. The highest incidence of LOH was observed at the marker D7S820.

Conclusions: It is possible, that genetic factors may be involved in the etiology and pathogenesis of PEX.


Pseudoexfoliation syndrome (PEX) is a common and clinically significant systemic condition characterized by the pathological production and accumulation of an abnormal fibrillar extracellular material in the surface lining of the anterior and posterior chambers of the eye and is often associated with cataract and glaucoma. The characteristic fibrillar PEX material is composed of microfibrillar subunits surrounded by an amorphous matrix. The material has a complex glycoprotein/proteoglycan structure composed of a protein core surrounded by glycosaminoglycans [1,2]. It appears to be multifocally produced by various cell types, e.g. the nonpigmented cilliary epithelium, the iris pigment epithelium, the pre-equatorial lens epithelium, corneal endothelium, trabecular cells, vascular endothelia and smooth muscle cells of the iris. Immunohistochemical evidence of elastic fiber epitopes in PEX material and molecular biological data showing overexpression of elastic fiber components have led to the current pathogenetic concept, which explains PEX syndrome as a type of elastosis affecting elastic microfibrils in particular. Histochemically and ultrastructurally the material resembles that of the amyloid, but its fibers are four times thicker [3]. Degeneration also affects the connective tissue of the blood vessels, which increases the risk of vascular diseases. Myocardial ischemia, myocardial infarction, hypertension, stroke and aortal stenosis are common in PEX patients [4-7]. A genetic factor in PEX etiology was suggested by Aasved in 1975. Frequent familial glaucoma in the course of PEX has long indicated a possible genetic inheritance of the symptoms [8,9]. Genetic analyses of PEX patients has revealed genetic abnormalities within markers on chromosomes 1, 7, 9 and 13 [10]. A hereditary role in open angle glaucoma has been demonstrated for intraocular pressure, aqueous outflow, magnitude and rate of degenerative changes within the optic disc and response to treatment. A genetic influence is suggested in the regulation of apoptosis in ganglion cells and trabecular meshwork cells. The functional effects of gene products are likely to account for multiple lesions of the optic nerve in glaucoma patients [11-14]. A major risk for the development of glaucoma is pseudoexfoliation. It is found in 20-60% of patients in many regions of the world [15]. Previous attempts to find a gene defect that might be responsible for pseudoexfoliation glaucoma have been unsuccessful. In molecular diagnostics the importance of an indirect genetic analysis of natural genomic DNA polymorphism and genetic markers is gaining ground. One such method is the analysis of microsatellite polymorphism. Microsatellites or short tandem repeat polymorphic markers (STRs) are sequences of 2-7 base pairs (bp) in length which may be amplified via the polymerase chain reaction (PCR) using primers targeted at a specific STR sequence typically generating 50-500 bp sized fragments. The polymorphisms in STRs are due to the different number of copies of the repeat element that can occur in a population of individuals. Loss of heterozygosity in a microsatellite marker locus indicates that the neighboring gene may be inactivated [10]. The purpose of this paper was to evaluate the possible occurrence of loss of heterozygosity in the anterior capsule, lens nucleus, iris and trabeculum of PEX patients.


The study material comprised 19 patients, 12 females and 7 males, with pseudoexfoliation syndrome referred to the Ophthalmology Department, Medical University of Bialystok. The mean age was 75.5 (SD 4.7), range 64-86. Of 16 patients with a history of cataract, 9 were also treated for pseudoexfoliative glaucoma. The intraocular pressure was abnormal in 5 patients. All patients underwent extracapsular removal of the cataract with a posterior chamber intraocular implant. In 5 patients, additional trabeculectomy with peripheral iridectomy was performed. In 3 patients with no significant lens opacities an increased intraocular pressure which was resistant to local treatment or laser therapy, trabeculectomy with peripheral iridectomy was performed. The study material included specimens of the anterior capsules (16 samples), the lens nuclei (16 samples), iris specimens (12 samples) and trabeculum specimens (8 samples). Twenty individuals, 12 females and 8 males, aged 62-89, mean age 74 (SD 5.2) served as a control group. Twelve patients underwent cataract surgery, 5 patients underwent glaucoma surgery and 3 patients had cataract and glaucoma surgery. No signs of exfoliation were observed in the control group. Reference blood samples were collected from all the patients. The material was stored in a refrigerator. Standard organic methods were used for DNA isolation. Additional microcolumn purification was performed where necessary. Fluorescent multiplex PCR was used to amplify microsatellite loci included in commercially available human identification kits: AmpFlSTR Profiler and AmpFlSTR SGM Plus (Applied Biosystems, Foster City, CA) according to the manufacturer's recommendations. Genotyping was performed in a 310 ABI Prism Genetic Analyzer (Applied Biosystems) using the GeneScan Analysis v3.1 and Genotyper v2.5 software. The following microsatellite markers were examined: D2S1338 - 2q35-37.1, FGA - 4q28, D7S820 - 7q21, VWA - 12p12pter, D18S51 - 18q21.3, D19S433 - 19q12-13.1, D21S11 - 21q11.2-q21. Glaucomatous optic neuropathy is a disease characterized by remodeling of the extracellular matrix and loss of retinal ganglion cell axons at the level of the optic nerve head. Astrocytes, the major cell type in the optic nerve head, may participate in this process by production of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). The marker on chromosome 7 was included, because the presence of specific MMPs and TIMPs in the optic nerve head may participate in homeostasis and remodeling of the extracellular matrix in glaucoma [16]. Alterations of iridal vessels in PEX were noted using iris fluorescein angiography demonstrating fluorescein leakage and microneovascularization. Electron microscopic studies of iris vessels in PEX revealed deposition of PEX material in the vessel walls with consecutive disorganization of the normal vessel structure, loss of endothelial and adventitial cells and thinning and fenestration of the endothelial lining [17]. The authors decided to examine the marker VWA - 12p12pter; intron 40 of the human von Willebrand factor gene, associated with the endothelial structure [18]. It has been reported that in the aqueous humor of glaucoma patients endothelin levels were significantly higher than those in control subjects. The endothelin converting enzyme (ECE) cleaves the big endothelin to the biologically active 21 amino acid peptide endothelin [19]. The ECE gene is mapped to 2q36-q37, therefore the D2S1338 (2q35-q37.1) marker was used in the study. It has been proposed that the death of ganglion cells by apoptosis represents a common pathway that leads to glaucomatous vision loss, however the mechanism of ganglion cell apoptosis is not well understood [20]. Tatton proposed the model of neuronal apoptosis [21]. Many authors described a number of proteins that play role in apoptosis including Bcl-2 [22]. In our study the marker D18S51 - 18q21.3 with Bcl-2 localization was examined. Additionally, a marker on chromosome 19 (D19S433 - 19q12-13.1) was investigated, because candidate genes for cytochrome c reductase involved in apoptosis were localized in the chromosomal region 19q12 [21]. Because PEX material is histochemically and ultrastructurally related to amyloid we decided to examine two markers associated with amyloidosis: FGA - 4q28 and D21S11 - 21q11.2-q21 [3]. The analyses were performed twice, so the results were highly reproducible. Loss of heterozygosity (LOH) was defined as a decrease (at least 50%) in peak height of an allele compared with that of the other determined after comparison of normal and pathologic DNA. Frequency of LOH was calculated for respective samples. Clinical information including age, gender, and intraocular pressure were taken from the patients records for use in statistical analysis. The Student's t-test for paired samples was used in statistical analyses, employing the SPSS software package v.8.0 for Windows (SPSS Inc., Chicago, IL). Frequency of LOH in the iris and the anterior capsule samples were statistically tested using Fisher's exact test.


The iris and the anterior capsule specimens from 10/19 patients with PEX (53%) displayed LOH. No LOH was found in trabeculum and lens nucleus specimens nor in the control group samples. The differences in mean age and gender between patients with and without PEX were statistically insignificant. The frequency of LOH in the respective microsatellite markers for respective anterior capsule and iris specimens is presented in Table 1. Seven patients (36%) displayed LOH at 1 locus and three patients (11%) at 3 loci. Overall, 7/12 iris specimens and 8/16 anterior capsule specimens (58% and 50%, respectively) displayed LOH. At D7S820 LOH was found in 11 specimens from 8 patients (42%), at FGA in 6 specimens from 4 patients (19%) and at VWA in 3 specimens from 3 patients (15%). Figure 1, Figure 2, and Figure 3 present LOH patterns in the analyzed specimens as compared with normal DNA isolated from corresponding blood samples. Figure 1 shows electrophoregrams (Genotyper pattern) of LOH at locus D2S1338. The anterior capsule DNA showed complete deletion of allele 18 (sample 5, lower panel) when compared with the paired reference blood DNA (sample 3, middle panel). Figure 2 shows electrophoregrams (Genotyper pattern) of LOH at locus FGA. The anterior capsule DNA showed complete deletion of allele 22 (sample 18, middle panel) when compared with the paired reference blood DNA (sample 19, lower panel). Figure 3 shows electrophoregrams (Genotyper pattern) of LOH at locus FGA. The iris DNA showed complete deletion of allele 18 (sample 18, middle panel) when compared with the paired reference blood DNA (sample 30, lower panel). The incidence of LOH in the iris and the anterior capsule specimens is presented in Table 2. The locus D7S820 was significantly subject to LOH in the anterior capsule specimens (p=0.0034), whereas the respective value in the iris specimens was insignificant (p=0.1580), based on the Fisher exact test. The incidence of LOH at D2S1338, FGA, and VWA was statistically insignificant (p=0.8452, p=0.3895, and p=0.8062, respectively). No LOH was found at the other microsatellite markers. The average intraocular pressure in patients with LOH was 25.8 mm Hg, whereas the respective value in those without LOH was 17.1 mm Hg. The differences in mean intraocular pressure between patients with and without PEX were statistically insignificant (p=0.1378).


Microsatellite markers are commonly used in the analysis of allelic imbalance. Presence of LOH at numerous loci in tumor specimens may contribute to the development of a model of cancerogenous progression [23]. Application of microsatellite loci as genetic markers is common, due to their abundance in the genome, extreme polymorphism and amplification by the PCR reaction. The issue of possible genetic lesions in pseudoexfoliation syndrome has hitherto gained little attention. Until recently only a single paper reporting LOH incidence in PEX patients has been published [10]. In our study a panel of 7 microsatellite markers, different from those used in the study mentioned above, was applied. LOH was found in 58% of the iris specimens and in 50% of the anterior capsule specimens collected from PEX patients. Kozobolis et al. [10] reported the LOH incidence in the iris and the anterior capsule as 91.7% and 75.0%, respectively. In our material, the highest LOH incidence was found at D7S820 (42% of patients), the locus located on chromosome 7. This chromosome also carries gene sequences encoding elastin, collagen 1A2 and laminin B1 [1,5,10]. Kozobolis et al. [10] found LOH at D7S478 (7p15-q22) and at D7S479 (7q21-q22) for 41.17% and 35.30% of patients, respectively. Considering the fact that pseudoexfoliation syndrome tends to be commonly recognized as an elastosis, the high incidence of genetic lesions on chromosome 7 would appear to be of a great importance. Our findings suggest, that glaucomatous optic neuropathy and PEX may result from alterations in a candidate gene for metalloproteinase participating in homeostasis and remodeling of the extracellular matrix. The Fisher exact test revealed, that D7S820 was significantly subject to LOH in the anterior capsule specimens, but not in the iris specimens, which may be due to the smaller sample size (16 versus 12). Although being statistically insignificant, the occurrence of LOH at D2S1338, FGA, and VWA loci is noteworthy, suggesting possible association of the syndrome with amyloidosis and involvement of endothelium in PEX pathogenesis. However, no reports on LOH at these loci in PEX patients have been found in the available literature. No LOH was found at the other microsatellite markers. Further studies with larger numbers of patients are needed to elucidate the contribution of genetic and non-genetic factors to the development of pseudoexfoliation syndrome and pseudoexfoliation glaucoma.

In conclusion, we have determined that the highest frequency of LOH in PEX patients was found at locus D7S820 and that it is possible that genetic factors are involved in the etiology and pathogenesis of PEX.


This work was supported by the Medical University of Bialystok and the State Committee for Scientific Research grant 3-57691.


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