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. 2013:19:333-47.
Epub 2013 Feb 7.

Contribution of the latent transforming growth factor-β binding protein 2 gene to etiology of primary open angle glaucoma and pseudoexfoliation syndrome

Sahar Jelodari-Mamaghani  1 , Ramona Haji-Seyed-Javadi, Fatemeh Suri, Naveed Nilforushan, Shahin Yazdani, Kambiz Kamyab, Elahe Elahi
Affiliations

Contribution of the latent transforming growth factor-β binding protein 2 gene to etiology of primary open angle glaucoma and pseudoexfoliation syndrome

Sahar Jelodari-Mamaghani et al. Mol Vis. 2013.

Abstract

Purpose: To assess for the first time the possible contribution of latent transforming growth factor (TGF)-beta binding protein 2 (LTBP2), an extracellular matrix (ECM) protein that associates with fibrillin-1-containing microfibrils, to the etiology of primary open angle glaucoma (POAG) and pseudoexfoliation (PEX) syndrome. Mutations in LTBP2 have previously been shown to be the cause of primary congenital glaucoma (PCG) and other disorders that often manifest as secondary glaucoma.

Methods: All exons of LTBP2 were sequenced in the DNA of 42 unrelated patients with POAG and 48 unrelated patients with PEX syndrome. Contribution of candidate variations to disease was assessed by screening in control individuals and use of biochemical, bioinformatics, and evolutionary criteria, and in one case by segregation analysis within the family of a proband with POAG. Microscopy was performed on the skin of a patient with PEX syndrome whose condition developed into PEX glaucoma during the course of the study and on the skin of her son previously identified with PCG who harbored the same LTBP2 mutation.

Results: Among the 30 sequence variations observed in LTBP2, five found in five patients with POAG and two found in two patients with PEX glaucoma syndrome may contribute to their diseases. One of the mutations was observed in a patient with POAG and in a patient with PEX glaucoma syndrome. Light, fluorescent, and electron microscopy showed that a mutation present in one of the individuals affected with PEX glaucoma syndrome and in her son affected with PCG causes disruptions in the ECM.

Conclusions: Some LTBP2 sequence variations can contribute to the etiology of POAG and PEX glaucoma syndrome. It is not expected that in these diseases LTBP2 mutations behave in a strictly Mendelian fashion with complete penetrance. In conjunction with recent findings, the results suggest that anomalies in the ECM are among the factors that can contribute to POAG and PEX glaucoma syndrome. LTBP2 and other related ECM protein coding genes should be screened in larger cohorts with these diseases, which are common disorders and important to the public health.

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Figures

Figure 1
Figure 1
Screening of c.1999A>C mutation by an allele-specific polymerase chain reaction. Polymerase chain reaction (PCR) was performed in presence of two pairs of primers, one for amplification of a 456 bp fragment in FBN1 and the other for amplification of a 259 bp fragment in LTBP2. The former served as control for efficacy of the PCR reaction. The 3′ terminus of the forward primer for the LTBP2 fragment was designed to amplify only the mutated allele (c.1999C) and not the wild-type allele. M, size markers; patient with PEX, template was from patient with PEX carrying a mutation; template in all other lanes is from control individuals. The arrow on the left shows the migration position of the LTBP2 product, and the arrow on the right shows migration position of FBN1 product. Comparable results were obtained in all 100 controls screened.
Figure 2
Figure 2
Screening of c.4912G>A mutation by restriction fragment length polymorphism. Polymerase chain reaction products of exon 34 were digested with HpyCH4IV. Three bands are evident in the electrophoretic pattern of mutation carrier (POAG patient), and only two bands in the electrophoretic pattern of the control individual. M, size markers.
Figure 3
Figure 3
Positions of putative disease-associated mutations in latent transforming growth factor-beta binding protein 2. Positions of disease-associated mutations observed here are shown above the schematic representation of the latent transforming growth factor-beta binding protein 2 (LTBP2) structure, and the positions of the mutations previously reported are presented below the diagram. Diseases associated with the mutations are also given. Symbols used for the various LTBP2 domains are indicated; horizontal lines represent protein regions not known to be specific domains.
Figure 4
Figure 4
LTBP2 mutation p.Arg495Gln in a family with primary open angle glaucoma. A: Pedigree of the family. 216T, 211L, and 216L are the ID numbers of living members of the family. Filled circles and squares: affected with glaucoma; open circles and squares: unaffected phenotype; wt=wild-type allele, m=mutated allele. B: Chromatograms showing homozygous wild-type LTBP2 genotype c.1484G (top) and heterozygous mutated genotype c.1484G>A (bottom). C: Fundus image of the proband’s left eye. Diffuse optic disc atrophy, enlarged cup-to-disc ratio with notching of the neuroretinal rim in the inferior pole of the disc, and severe nerve fiber layer loss especially in inferior area are evident.
Figure 5
Figure 5
Visual field defects in the left eye of patient with POAG (211L). The single field analysis printout shows severe visual field loss with dense superior arcuate scotoma that threatens the fixation and inferior nasal field defect.
Figure 6
Figure 6
Light microscope images of skin tissue sections from patients with p.Tyr1792fsX55-causing mutations and age-matched control individuals. A–D: Orcein Geimsa–stained elastic fibers of a homozygous carrier with primary congenital glaucoma (PCG; A) and control individual (C), a heterozygous carrier with pseudoexfoliation (PEX) syndrome (B) and a control individual (D) visualized with a light microscope. Elastic fibers were sparser and fragmented in each patient compared to the respective control. Examples of fragmented fibers and sparse regions are shown, respectively, with arrows and * symbol. E–H: Trichrome stained collagen fibers of individual with PCG (E) and control individual (G), individual with PEX syndrome (F) and control individual (H) visualized with a light microscope. Collagen fibers, stained with blue, were sparser in each patient compared to the respective control. Arrector pili muscles appear red in G.
Figure 7
Figure 7
Transmission electron microscope images of skin tissue sections from patients with p.Tyr1792fsX55-causing mutations and age matched control individuals. Representative electron micrographs of a homozygous carrier with primary congenital glaucoma (PCG; A) and the control individual (C), and a heterozygous carrier with pseudoexfoliation (PEX) syndrome (B) and the control individual (D). More areas that appear devoid of extra cellular matrix (ECM) structures are evident in the image taken from the patients’ samples (A, B), and the ECM of the patient with PCG (A) is sparser than that of his older mother with PEX syndrome (B). Examples of sparse regions are shown with a star (∗). f, fibroblast; cb, collagen bundles.
Figure 8
Figure 8
Fluorescent microscope images of skin tissue sections from patients with p.Tyr1792fsX55-causing mutations and age-matched control individuals stained for latent transforming growth factor-beta binding protein 2. Representative immunofluorescent cryosections from a homozygous carrier with primary congenital glaucoma (PCG; A) and the control individual (C), and from a heterozygous carrier with pseudoexfoliation (PEX) syndrome (B) and the control individual (D). Fibers stained for latent transforming growth factor-beta binding protein 2 (LTBP2) in the patient with PCG are thinner and noticeably fewer (A) compared with the control individual (C). Fibers in the patient with PEX syndrome are also thinner, but dense and convoluted (B) compared to the control individual (D). Examples of longer thicker fibers in the control sections are shown with arrows; these were not seen in the sections of patients’ tissues. Negative control is shown in Figure 9.
Figure 9
Figure 9
Fluorescent microscope images of skin tissue sections from patients with p.Tyr1792fsX55-causing mutations and age-matched control individuals stained for fibrillin-1. Representative immunofluorescent cryosections from a homozygous carrier with primary congenital glaucoma (PCG; A) and the control individual (C), and a heterozygous carrier with pseudoexfoliation (PEX) syndrome (B) and the control individual (D). The thick and long fibers that stained for fibrillin-1 in the control individuals (C, D; arrows) were not observed in multiple sections derived from the patient tissues (A, B). Fibers in the patient with PEX syndrome are dense and convoluted (B) compared to the control individuals (D). Negative control is shown in E.
Figure 10
Figure 10
Images of eyes of a patient with pseudoexfoliation glaucoma syndrome and a patient with primary congenital glaucoma with heterozygous and homozygous p.Tyr1792fsX55-causing mutation in LTBP2, respectively. A and B: Images showing deposition of pseudoexfoliation material (arrows) on the endothelial surface of the cornea (arrow in A) and at the pupillary border of the iris (arrow in B) in the eye of an individual diagnosed with pseudoexfoliation glaucoma (PEXG) syndrome. C: Fundus photograph of the same PEXG individual exhibiting characteristic features of glaucomatous optic neuropathy. Diffuse neuroretinal rim thinning with more involvement of the inferior rim, notching and peripapillary atrophy all around the disc are evident. D: Image of an eye of an individual with PCG showing diffuse corneal opacity and calcific band keratopathy.
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