Analysis of RNA splicing defects in PITX2 mutants supports a gene dosage model of Axenfeld-Rieger syndrome

BACKGROUND: Axenfeld-Rieger syndrome (ARS) is associated with mutations in the PITX2 gene that encodes a homeobox transcription factor. Several intronic PITX2 mutations have been reported in Axenfeld-Rieger patients but their effects on gene expression have not been tested. METHODS: We present two new families with recurrent PITX2 intronic mutations and use PITX2c minigenes and transfected cells to address the hypothesis that intronic mutations effect RNA splicing. Three PITX2 mutations have been analyzed: a G>T mutation within the AG 3' splice site (ss) junction associated with exon 4 (IVS4-1G>T), a G>C mutation at position +5 of the 5' (ss) of exon 4 (IVS4+5G>C), and a previously reported A>G substitution at position -11 of 3'ss of exon 5 (IVS5-11A>G). RESULTS: Mutation IVS4+5G>C showed 71% retention of the intron between exons 4 and 5, and poorly expressed protein. Wild-type protein levels were proportionally expressed from correctly spliced mRNA. The G>T mutation within the exon 4 AG 3'ss junction shifted splicing exclusively to a new AG and resulted in a severely truncated, poorly expressed protein. Finally, the A>G substitution at position -11 of the 3'ss of exon 5 shifted splicing exclusively to a newly created upstream AG and resulted in generation of a protein with a truncated homeodomain. CONCLUSION: This is the first direct evidence to support aberrant RNA splicing as the mechanism underlying the disorder in some patients and suggests that the magnitude of the splicing defect may contribute to the variability of ARS phenotypes, in support of a gene dosage model of Axenfeld-Rieger syndrome

Lyst Mutation in Mice Recapitulates Iris Defects of Human Exfoliation Syndrome

PURPOSE. Human eyes with exfoliation syndrome (XFS) exhibit a distinctive pattern of iris transillumination defects that are recapitulated in Lyst mutant mice carrying the beige allele. The purpose of this study was to determine the anatomic basis for Lyst-mediated transillumination defects, test whether Lyst mutant mice develop other features of XFS, and describe the molecular basis of the beige mutation. METHODS. Lyst mutant mice and strain-matched controls were compared by clinical, histologic, immunohistochemical, and molecular genetic analyses. RESULTS. Slit-lamp examination showed that Lyst mutant mice uniformly exhibit XFS-like transillumination defects. Histologic analysis showed that these defects correlate with a sawtooth morphology of the iris pigment epithelium. Lyst mutant mice also produce an exfoliative-like material and exhibit pronounced pigment dispersion. Despite these insults, Lyst mutation does not cause increased intraocular pressure or optic nerve damage in the C57BL/6J genetic background. Sequence analysis identified that the beige mutation is predicted to delete a single isoleucine from the WD40 domain of the LYST protein, suggesting that this mutation is likely to disrupt a proteinprotein interaction. CONCLUSIONS. Lyst mutant eyes exhibit multiple features of XFS. Recent human genetic association studies have identified changes occurring in the LOXL1 gene as an important risk factor for XFS but also indicated that other factors contributing to risk likely exist. These results demonstrated that mutation of the Lyst gene can produce ocular features of human XFS and suggested that LYST or LYST-interacting genes may contribute to XFS. (Invest Ophthalmol Vis Sci. 2009;50:1205-1214) DOI: 10.1167/iovs.08-2791 E xfoliation syndrome (XFS) is a common age-related disorder primarily recognized by the pathologic accumulations of a fibrillar exfoliative material in the anterior chamber of the eye but also associated with several other ocular and systemic abnormalities. 7 In parallel to the clinical significance of exfoliative material in the diagnosis of XFS, much of the experimental work on XFS syndrome has focused on studies of exfoliative material. Such studies have shown that exfoliative material consists of an irregular conglomeration of randomly cross-banded fibrils approximately 30 nm in diameter surrounded by an amorphous matrix of glycoconjugates. 8 Exfoliative material also contains epitopes for a variety of proteins related to elastic microfibers, including fibrillin-1, 9 elastin, 10 latent TGF-␤ proteins, 11 lysyl oxidase, 4 and others. 3,4 These results and other experimental work on XFS have led to a hypothesis that XFS is a disease of elastosis. According to this theory, insults such as increased oxidative stress and elevated levels of TGF-␤1 likely trigger increased production of elastic microfibrils that are subsequently prone to aggregate and accumulate. A breakthrough in understanding XFS has been precipitated by genomewide association studies that have begun to unravel the genetic factors underlying XFS. XFS has long been appreciated to have strong hereditary contributions. 14 However, the influence of LOXL1 in XFS may not be as straightforward as is seemingly indicated by these impressive statistics. A multifactorial risk for XFS is suggested by the extremely high occurrence of high-risk LOXL1 alleles among the general population. Within the original Scandinavian populations studied, the high-risk haplotype of LOXL1 alleles was also detected at a frequency of approximately 50% in the general population, with approximately 25% of the general population homozygous for the haplotype. 14 Follow-up studies have also confirmed similar high-carrier frequencies. Here, we identify the Lyst gene as an additional gene potentially important to XFS. B6-Lyst bg-J mice homozygous for the beige-J (bg-J) allele recapitulate multiple ocular features of human XFS. Our initial consideration of B6-Lyst bg-J mice as a possible model of XFS was based on a resemblance of iris transillumination defects between these mice and human patients with XFS. In testing the anatomic basis for the Lyst

Dislocated Lens Nuclei Simulating Choroidal Melanomas

• Three patients underwent echographic examinations for lesions suspected of being choroidal malignant melanomas. All patients were pseudophakic, and two had extensive optic nerve damage from inflammatory glaucoma. In all three patients echography demonstrated an intravitreal lens nucleus that had been lost during cataract extraction. To our knowledge, dislocated lens nuclei have not been reported previously in the differential diagnosis of choroidal melanoma. This diagnosis should be considered in the aphakic or pseudophakic patient with a fundus mass lesion. Echography is especially helpful in establishing the correct diagnosis

Variance Owing to Observer, Repeat Imaging, and Fundus Camera Type on Cup-to-disc Ratio Estimates by Stereo Planimetry

Objective: To determine and compare variance components in linear cup-to-disc ratio (LCDR) estimates by computer-assisted planimetry by human experts, and automated machine algorithm (digital automated planimetry). Design: Prospective case series for evaluation of planimetry. Participants: Forty-four eyes of 44 consecutive patients from the outpatient Glaucoma Service at University of Iowa with diagnosis of glaucoma or glaucoma suspect were studied. Methods: Six stereo pairs of optic nerve photographs were taken per eye: 3 repeat stereo pairs using simultaneous fixed-stereo base fundus camera (Nidek 3Dx) and another 3 repeat stereo pairs using sequential variable-stereo base fundus camera (Zeiss). Each optic disc stereo pair was digitized and segmented into cup and rim by 3 glaucoma specialists (computer-assisted planimetry) and using a computer algorithm (digital automated planimetry), and LCDR was calculated for each segmentation (either specialist or algorithm). A linear mixed model was used to estimate mean, SD, and variance components of measurements. Main Outcome Measures: Average LCDR, interobserver, interrepeat, intercamera coefficients of variation (CV) of LCDR and their 95% tolerance limits. Results: There was a significant difference in LCDR estimates among the 3 glaucoma specialists. The interobserver CV of 10.65% was larger than interrepeat (6.7%) or intercamera CV (7.6%). For the algorithm, the LCDR estimate was significantly higher for simultaneous stereo fundus images (Nidek, mean: 0.66) than for sequential stereo fundus images (Zeiss, mean: 0.64), whereas interrepeat CV for Nidek (4.4%) was lower than Zeiss (6.36%); the algorithm's interrepeat and intercamera CV were 5.47% and 7.26%, respectively. Conclusions: Interobserver variability was the largest source of variation for glaucoma specialists, whereas their interrepeat and intercamera variability is comparable with that of the algorithm. DAP reduces variability on LCDR estimates from simultaneous stereo images, such as the Nidek 3Dx

A Vector Force Model of Upper Eyelid Position in the Setting of a Trabeculectomy Bleb

A vector force model for the determination of upper eyelid position in the setting of a trabeculectomy bleb is presented. The model is used to explain the clinical courses of 5 patients with bleb-induced upper eyelid malposition and the efficacy of modalities previously described for the treatment of bleb-induced upper eyelid retraction. The novel use of botulinum toxin in the treatment of bleb-induced eyelid retraction and unique surgical considerations in patients with trabeculectomy blebs undergoing upper eyelid surgery are discussed. A vector force analysis was conducted and a force diagram constructed. The clinical and surgical courses of 5 patients with trabeculectomy blebs and upper eyelid malposition were reviewed. The vector force model was applied to these cases and the previously described treatment modalities for bleb-induced upper eyelid retraction. Vector force analysis demonstrates that in the case of trabeculectomy bleb-induced upper eyelid retraction, the net force vector, which represents the sum of all the individual forces acting on the eyelid, has a positive vertical component resulting in superior displacement of the eyelid. In contrast, bleb-induced ptosis results when the net force vector has a negative vertical component. In 3 patients, alterations in the bleb resulted in resolution of upper eyelid malposition. Botulinum toxin was used to achieve a normal upper eyelid position in 1 patient with lateral canthal tendon disinsertion and unilateral eyelid retraction and 1 patient with bilateral eyelid retraction. One patient developed unilateral ptosis in concert with the emergence of a large Tenon cyst that resolved with the treatment of the cyst via eyelid massage. One patient with unilateral ptosis and an ipsilateral bleb underwent external levator advancement but was unable to achieve the desired upper eyelid height as retraction over the bleb occurred with any attempt to elevate the eyelid above a marginal reflex distance of 1.5 mm. The efficacy of previously reported modalities for the treatment of trabeculectomy bleb-induced upper eyelid retraction can be explained by either a reduction in the positive vertical component of the net force vector or augmentation of the negative vertical component. A vector force model systematically accounts for the multiple determinants of upper eyelid position in the setting of a trabeculectomy bleb. This model provides a framework for the evaluation of bleb-induced upper eyelid malposition and offers a logical, mathematical explanation for the occurrence of bleb-induced upper eyelid retraction and the usefulness of previously reported treatment modalities for this clinical entity

Automated segmentation of the optic disc from stereo color photographs using physiologically plausible features. Investigative ophthalmology & visual science

PURPOSE. To evaluate a novel automated segmentation algorithm for cup-to-disc segmentation from stereo color photographs of patients with glaucoma for the measurement of glaucoma progression. METHODS. Stereo color photographs of the optic disc were obtained by using a fixed stereo-base fundus camera in 58 eyes of 58 patients with suspected or open-angle glaucoma. Manual planimetry was performed by three glaucoma faculty members to delineate a reference standard rim and cup segmentation of all stereo pairs and by three glaucoma fellows as well. Pixel feature classification was evaluated on the stereo pairs and corresponding reference standard, by using feature computation based on simulation of photoreceptor color opponency and visual cortex simple and complex cells. An optimal subset of 12 features was used to segment all pixels in all stereo pairs, and the percentage of pixels assigned the correct class and linear cup-to-disc ratio (LCDR) estimates of the glaucoma fellows and the algorithm were compared to the reference standard. RESULTS. The algorithm was able to assign cup, rim, and background correctly to 88% of all pixels. Correlations of the LCDR estimates of glaucoma fellows with those of the reference standard were 0.73 (95% CI, 0.58 -0.83), 0.81 (95% CI, 0.70 -0.89), and 0.86 (95% CI, 0.78 -0.91), respectively, whereas the correlation of the algorithm with the reference standard was 0.93 (95% CI, 0.89 -0.96; n ϭ 58). CONCLUSIONS. The pixel feature classification algorithm allows objective segmentation of the optic disc from conventional color stereo photographs automatically without human input. The performance of the disc segmentation and LCDR calculation of the algorithm was comparable to that of glaucoma fellows in training and is promising for objective evaluation of optic disc cupping. (Invest Ophthalmol Vis Sci

Linkage of Rieger syndrome to the region of the epidermal growth factor gene on chromosome 4

Rieger syndrome is an autosomal dominant disorder of morphogenesis in which previous cytogenetic arrangements have suggested chromosome 4 as a candidate chromosome. Using a group of highly polymorphic short tandem repeat polymorphisms (STRP), including a new tetranucleotide repeat for epidermal growth factor (EGF), significant linkage of Rieger syndrome to 4q markers has been identified. Tight linkage to EGF supports its role as a candidate gene, although a recombinant in an unaffected individual has been identified. This study demonstrates the utility of using polymorphic STRP markers when only a limited number of small families are available for study

Evaluation of optinueurin sequence variations in 1,048 patients with open-angle glaucoma

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