Roberto Dell'omo
email: robertodellomo@hotmail.com

Kalliroi Konstantopoulou

Roger Wong

Carlos Pavesio

Mr Roberto

Roberto Dell

Presumed idiopathic outer lamellar defects of the fovea and chronic solar retinopathy. An OCT and Fundus Autofluorescence study

Keywords: Fundus autofluorescence, solar retinopathy, outer lamellar hole of the retina, optical coherence tomography

AIM:

To examine fundus autofluorescence (FAF) findings in eyes with presumed idiopathic outer lamellar defects (OLD) at the fovea and to discuss their pathogenesis.

METHODS: prospective observational case series of five eyes of four patients

presenting with OLD at the fovea defined as discrete lesions of 50-100μm in size located at the level of the outer retina on biomicroscopy and imaged on optical coherence tomography (OCT) as cylindric, well demarcated interruption of both hyperreflective bands corresponding to the inner/outer segments junction of photoreceptors and to the complex retinal pigment epithelium-choriocapillaris; none of the enrolled patients had history positive for direct sungazing, welding-arc or sunbed exposure, whiplash injury, ocular trauma, macular oedema/detachment or evidence of vitreo-macular traction. The corresponding FAF images were evaluated.

RESULTS: in eyes with OLD the neuroretina in the foveal region appeared to be thinner in comparison to fellow, unaffected eyes. FAF revealed well demarcated, hypoautofluorescent areas (corresponding in location to the OLD observed clinically and on OCT), surrounded by an irregular halo of relatively increased autofluorescence in the context of the greater hypoautofluorescent macular region.

CONCLUSION: biomicroscopy, OCT and FAF findings of presumed idiopathic OLD of the fovea strongly resembles those observed in association to chronic solar retinopathy. In association to OCT, FAF might represent an useful tecnique to detect subtle solar-induced injuries of the retina.

INTRODUCTION

Solar retinopathy [START_REF] Das | Solar chorioretinal burn[END_REF][START_REF] Agarwal | Solar retinitis[END_REF][START_REF] Gladstone | Solar retinitis after minimal exposure[END_REF][START_REF] Sadun | Solar retinopathy : a biophysical analysis[END_REF][START_REF] Rosen | Solar retinitis[END_REF] generally occurs in patients who have viewed an eclipse or have gazed directly at the sun, however it has also been described in patients who have minimally viewed the sun [START_REF] Gladstone | Solar retinitis after minimal exposure[END_REF] or who have been sunbathing, not sungazing . [START_REF] Gladstone | Solar retinitis after minimal exposure[END_REF][START_REF] Yannuzzi | Solar retinopathy. A Photobiologic and Geophysical Analysis[END_REF] Recently one of the largest series ever published on solar retinopathy [START_REF] Rai | Solar retinopathy. A study from Nepal and Germany Documenta[END_REF] reported that up to 49% of patients affected could not remember to have looked at the sun. A reddish, sharply demarcated foveal or juxtafoveal spot located at the level of outer retina, developing approximately two weeks after direct exposure to sunlight, is a well known sign of solar injury. This lesion is permanent and highly suggestive of solar retinopathy [START_REF] Gass | Stereoscopic Atlas of Macular Diseases[END_REF]. Similar biomicroscopic findings at the fovea have been reported in association to welding arc or sunbed exposure, ocular trauma, initial stages of idiopathic macular holes, persistent retinal defects following successful macular hole repair surgery, whiplash injuries, microaneurisms or hemorrages, idiopathic parafoveal telangiectasis or grouped under the generic terms of foveal spots [START_REF] Douglas | Foveal spot. A report of thirteen patients[END_REF] or microholes of the fovea centralis. [START_REF] Cairns | Microholes of the fovea centralis[END_REF][START_REF] Zambarakji | Macular microholes: pathogenesis and natural history[END_REF] Differential diagnosis is usually guided by history along with associated characteristic fluorescein angiography and OCT findings. Fundus autofluorescence (FAF), is a relatively novel, non invasive imaging technique based on the autofluorescent properties of natural occurring retinal fluorophores. [START_REF] Schmitz-Valckenberg | Fundus autofluorescence imaging. Review and perpectives[END_REF] It is generally accepted that the lipofuscin-laden cells of the retinal pigment epithelium (RPE) are the main source of FAF recorded with commercially available confocal scanning laser ophthalmoscopes. [START_REF] Schmitz-Valckenberg | Fundus autofluorescence imaging. Review and perpectives[END_REF][START_REF] Von Ruckmann | Distribution of fundus autofluorescence with a scanning laser ophthalmoscope[END_REF][START_REF] Trieschmann | Macular pigment: quantitative analysis on autofluorescence images[END_REF] The retinal pigment epithelium (RPE) with its high content in melanin is able to reduce photoreceptors exposure to potentially dangerous photons by absorping and scattering light forward into the choroid. The RPE is particularly exposed to damage when retina's defense mechanisms against toxic free radicals from light and oxygen are overwhelmed by light levels exceeding those typically encountered in a normal environment.

Subtle RPE abnormalities, overlooked on cursory ophthalmoscopy, can be efficiently imaged using FAF; reduction of the content of lipofuscin, the main fluorophore of the RPE, has been documented following light-induced loss of photoreceptors. [START_REF] Katz | Retinal light damage reduces autofluorescent pigment deposition in the retinal pigment epithelium[END_REF] In this study we report OCT and, for the best of our knowledge, the first description of FAF findings in eyes with presumed idiopathic outer lamellar defects (OLD) of the fovea and compare such findings with those reported in eyes with chronic solar retinopathy.

METHODS

A prospective observational case series aimed to investigate FAF findings in eyes with presumed idiopatic OLD at the fovea was designed and carried out at Moorfields Eye Hospital, NHS Trust, between January 2006 and June 2007. The study was performed in accordance to the tenets of the Declaration of Helsinky. Approval from local Institutional Review Board and informed consent from each enrolled subject were obtained.

The diagnosis was based on biomicroscopic identification of small, reddish, sharply demarcated outer lamellar defects at the fovea using a fundus contact lens (Goldmann three mirror lens, Ocular Instruments, Bellevue, Washington) and on third-generation OCT evaluation (Stratus Tomographer, Model 3000, Carl Zeiss Ophthalmic Systems Inc., Humphrey Division, Dublin, California). OCT examination was performed using six linear 6.00 mm scans oriented at intervals of 30 degrees and centered on the fixation point. To verify the accuracy of the OCT findings, the fundus area encompassing the lesions was additionally scanned using several vertical and horizontal scans of 5.00 mm in length. Retinal thickness was measured manually, using software callipers, at the foveal centre. OCT findings were considered consistent with diagnosis of OLD in presence of well demarcated interruption involving the hyperreflective bands corresponding to the inner/outer segments junction of photoreceptors and to the retinal pigment epithelium-choriocapillaris complex (Fig. 1,2); eyes with concomitant macular abnormalities detectable on OCT such as fine foveal epiretinal membrane or vitreomacular traction were excluded. Patients with evidence of other fundus disease, such as hereditary maculopathy, diabetes and other retinal vascular diseases, intraocular infection or inflammation, intraocular tumors, myopia, previous macular oedema, previous surgery for macular hole or retinal detachment, ocular trauma and whiplash injury were also excluded from the study.

Particular attention was paid in recording information about history of direct sungazing, welding arc and sunbed exposure, [START_REF] Mcginley | Sunbeds in current use in Scotland: A survey of their output and patterns of use[END_REF] or assumption in the past of compounds with photosensitizing properties. [START_REF] Kevin | Drug-induced photoallergic and phototoxic reactions[END_REF][START_REF] Costagliola | Retinal phototoxicity induced by hydrochlorothiazide after exposure to a UV tanning device[END_REF] All the participants received a detailed ophthalmologic examination including bestcorrected visual acuity on Snellen chart, slit-lamp biomicroscopy, indirect fundus examination, fundus photography, OCT 3 STRATUS and FAF evaluation at first visit and in occasion of follow-up appointments. For color fundus pictures, a high-resolution (1024 x 1024) fundus camera system (TRC-50IA/IMAGEnet 2000; Topcon, Tokyo, Japan) was used. FAF was recorded using a confocal scanning laser ophthalmoscope [Heidelberg Retina Angiograph, HRA, Heidelberg Engineering, Germany]. An argon blue laser (488 nm) was used for excitation, emitted light detected above 500 nm (barrier filter). Before recording FAF images, individual refraction was corrected by varying the optics of the instrument in the reflection mode. From each individual eye at least 16 FAF images were obtained by using the frame grabber (10.2 images per second). In order to amplify the FAF signal, the images were aligned and a mean image was calculated after automated detection and correction of eye movements using image analysis software. The digital images were saved on disk for further analysis and processing. OCT 3 STRATUS and FAF were performed for both eyes of all patients. In eyes diagnosed with outer lamellar defects at the fovea, OCT demonstrated discrete abnormalities in shape of focal interruption at the level of the hypereflective bands corresponding respectively to the junction between inner and outer photoreceptor segments and to the retinal pigment epithelium-choriocapillaris complex. In addition, in the affected eyes, the neuroretina at the fovea appeared to be substantially thinner in comparison to the fellow unaffected eyes (centrofoveal mean thickness: 127.2 μm and 183.7 μm in the affected and unaffected eyes respectively). A preserved foveal contour and a normal vitreoretinal interface with no evidence of vitreo-macular traction was noted in all cases. FAF revealed well demarcated, hypoautofluorescent areas (corresponding in location to the OLD observed clinically and to the defects of the outer retina/RPE layers detected by OCT) surrounded by an irregular halo of relatively increased autofluorescence in the context of the greater hypoautofluorescent macular region (Fig. 1,2). FAF images of the fellow, unaffected eyes were consistent with physiologic phenotypes previously described in the literature. [START_REF] Schmitz-Valckenberg | Fundus autofluorescence imaging. Review and perpectives[END_REF][START_REF] Trieschmann | Macular pigment: quantitative analysis on autofluorescence images[END_REF] Biomicroscopic, OCT and FAF features of the OLD didn't change over the course of the follow-up period.

RESULTS

Five eyes of four patients with diagnosis of presumed idiopathic

DISCUSSION

Solar retinopathy refers to maculopathy that occurs following exposure to solar radiation. [START_REF] Das | Solar chorioretinal burn[END_REF][START_REF] Agarwal | Solar retinitis[END_REF][START_REF] Gladstone | Solar retinitis after minimal exposure[END_REF][START_REF] Sadun | Solar retinopathy : a biophysical analysis[END_REF] There is a wide spectrum of solar-induced injuries and great individual variation in susceptibility for developing such lesions. [START_REF] Rosen | Solar retinitis[END_REF] Prolonged staring at the sun may cause only minimal abnormalities [START_REF] Rosen | Solar retinitis[END_REF] whereas minimal exposure (e.g. during sunbathing) may induce macular lesions. [START_REF] Gladstone | Solar retinitis after minimal exposure[END_REF] Many factors including characteristics of the light (wavelength, dose rate), ocular pigmentation, clarity of the ocular media and body temperature (influenced by climate, exercise or infection) along with unusual environmental conditions such as highly reflective surroundings and reduced atmospheric ozone [START_REF] Yannuzzi | Solar retinopathy. A Photobiologic and Geophysical Analysis[END_REF] are believed to influence the final injury on the retinal cells. Most cases are bilateral but often asymmetry between the two eyes is observed. Unilateral cases of solar retinopathy have been documented and are not uncommon.

Ophthalmoscopic examination in acute phases may reveal a small yellowish spot in the centre of the foveal region surrounded by an area of faint grey, granular pigmentation.

[8] This spot usually fades gradually within few weeks and assumes a reddish, sharply circumscribed appearance at the fovea, which Gass [START_REF] Gass | Stereoscopic Atlas of Macular Diseases[END_REF] described as pathognomonic of solar retinopathy.

Recently, N. Rai et al. [START_REF] Rai | Solar retinopathy. A study from Nepal and Germany Documenta[END_REF] have described clinical findings of 319 patients presenting with retinal lesions in the fovea highly suggestive of solar retinopathy, underscoring that only 51% of the examined patients could report history of sun-gazing in the past.

Therefore, although diagnosis may be straightforward in patients with acute loss of vision following prolonged sun exposure, patients who present months to years later may present more of a diagnostic challenge.

Histopathological studies [START_REF] Tso | The human fovea after sun gazing[END_REF][START_REF] Hope-Ross | Ultrastructural findings in solar retinopathy[END_REF] have demonstrated that the RPE and outer segments of the photoreceptor layer are most susceptible to solar damage and that the outer lamellar defects visible a few weeks after the insult are probably caused by focal loss of the photoreceptors, which occurs maximally few days after exposure. [START_REF] Thanos | Exposure to a solar eclipse causes neuronal death in the retina[END_REF] OCT findings of chronic solar retinopathy seem to correlate with histopathology observations; Garg et al. [START_REF] Garg | Optical Coherence Tomography of Chronic Solar Retinopathy[END_REF] described small, hyporeflective space in the outer retinal and retinal pigment epithelium (RPE) layers and thinning of the overlying fovea in a series of four patients with documented history of chronic solar retinopathy. Similar lesions involving both the outer retina and the inner retinal pigment epithelium had been previously described by Huang et al. [START_REF] Huang | Optical coherence tomography findings in photic maculopathy[END_REF] in three cases of photic maculopathy.

In our study, eyes with biomicroscopic and OCT findings consistent with diagnosis of OLD, strongly resembling chronic solar retinopathy, were further analyzed using FAF.

FAF imaging of the ocular fundus depends both on stimulated emission of light from molecules, chiefly lipofuscin, in the RPE cells and on light attenuating factors such as macular pigment, cone and RPE pigment in the fovea. [START_REF] Schmitz-Valckenberg | Fundus autofluorescence imaging. Review and perpectives[END_REF][START_REF] Von Ruckmann | Distribution of fundus autofluorescence with a scanning laser ophthalmoscope[END_REF][START_REF] Trieschmann | Macular pigment: quantitative analysis on autofluorescence images[END_REF] In the eyes with OLD at the fovea we observed a characteristic autofluorescence pattern consisting of small areas of decreased autofluorescence, corresponding in location to the OLD, surrounded by a ring of faint increased autofluorescence in the context of the relatively hypoautofluorescent macular area.

To the best of our knowledge this pattern has not been associated with other maculopathies and doesn't fit any of the physiologic FAF phenotypes previously described in the literature. [START_REF] Schmitz-Valckenberg | Fundus autofluorescence imaging. Review and perpectives[END_REF][START_REF] Von Ruckmann | Distribution of fundus autofluorescence with a scanning laser ophthalmoscope[END_REF][START_REF] Trieschmann | Macular pigment: quantitative analysis on autofluorescence images[END_REF] Interestingly, similar FAF findings (i.e. small focal areas of decreased autofluorescence corresponding to the OLD visible on biomicroscopy) have been reported in association to documented cases of solar retinopathy ( Dr. L.A. Yannuzzi, lecture on Fundus Autofluorescence, 14 April 2005, Fermo, Italy and Prof A. Bird, personal communication, 2007).

The well demarcated hypoautofluorescent areas corresponding to the OLD visible on funduscopy and on OCT could be secondary to lack of RPE cells or more likely to dead photoreceptors with consequent reduced accumulation of lipofuscin in the underlying RPE cells as it has been demonstrated in previous studies evaluating the effect on FAF of light-induced damage to the retina. [START_REF] Katz | Retinal light damage reduces autofluorescent pigment deposition in the retinal pigment epithelium[END_REF] Conversely, the halo of relatively increased signal in the context of macular hypofuorescence might be due to lipofuscin accumulation secondary to disturbances occurring at the level of photoreceptors-RPE interface. We didn't observe any obvious alterations of the layers corresponding to the junction between outer and inner photoreceptor segments and to the RPE-choriocapillaris complex in the juxtafoveal areas on OCT 3 images, but it is possible that new generation Spectral-domain OCTs, with their improved image resolution and sensitivity, may reveal subtle changes undiscovered by time-domain devices.

Another hypothesis could be that the increased signal is secondary to alterations in the distribution or to reduction of the luteal pigment in the perifoveal region. It has been demonstrated that any defect involving the foveal tissue, including lamellar macular holes and macular pseudoholes [START_REF] Bottoni | Diagnosis of macular pseudoholes and lamellar macular holes: is optical coherence tomography the "gold standard"?[END_REF] , may alter the degree of foveal FAF by decreasing the amount of masking macular pigment, and therefore increasing the foveal FAF.

Studies revealing alterations affecting optical density measurement and lateral distribution of macular pigment in eyes with features similar to those reported in this paper, might validate such hypothesis.

In our series a substantial thinning of the foveal region was observed in eyes with foveal OLD in comparison to the fellow, unaffected eyes with a resulting area of relatively increased autofluorescent signal. It remains uncertain if such retinal thinning is secondary to the damage inflicted to the retina by the light or if it represents just an anatomical peculiarity making these eyes more susceptible than contralateral to develop light-induced injuries.

Depletion of macular pigment leading to an increased FAF signal has been described in eyes with idiopathic macular telangiectasis [START_REF] Issa | Idiopathic macular teleangiectasia[END_REF], where an impaired trafficking and/or storage of lutein and zeaxanthin has been proposed to play a role; a similar deficiency could in theory make some eyes more prone to develop solar retinopathy.

We acknowledge that the present study presents several limitations: the sample of patients analysed is small; all the patients are females whereas solar maculopathy equally affects both genders; furthermore, although the series presented shares clinical and OCT features commonly seen in association to solar retinopathy, no patient reported history of solar exposure. Consequently, we cannot exclude that the macular changes herein described, are actually secondary to undefined forms of macular distrophies or to resolved, subtle chorioretinal inflammatory disease (for example acute retinal pigment epitheliitis or acute macular neuroretinitis). Self-limiting forms of central serous chorioretinopathy might be considered in the differential diagnosis too. Another possibility is that the OLD were caused by a vitreous-retinal interface syndrome; however, none of the eyes presented in this series had evidence of vitreous-macular traction; the stability of symptoms and funduscopic appearance of the lesions during the follow-up, makes, in our opinion, an interface syndrome for the pathogenesis of these OLD, at least unlikely.

In conclusion our findings suggest that presumed idiopathic OLD at the fovea might be secondary to chronic solar retinopathy. Further studies evaluating larger number of patients affected by this relatively uncommon maculopathy are warranted.

  photosensitizing properties.Mean follow-up (±SD) was 10.2 (±2.2) months. All four patients were females. Mean (±SD) age was 52,5 (±4,4) years.Characteristics of the examined eyes are summarized in the Table.

Figure 1 .

 1 Figure 1. (A and B) Case 1, color fundus photograph showing small, reddish, sharply demarcated spots at the fovea in both eyes (BE). (C and D) Optical coherence tomography reveals interruption of the thin hyperreflective band corresponding to the junction between inner and outer segment of photoreceptors and of the inner portion of the hyperreflective band corresponding to the retinal pigment epithelium/choriocapillaris complex (arrows); note the partial posterior vitreous detachment not involving the fovea in the right eye. (E and F) Fundus autofluorescence shows well demarcated, hypoautofluorescent areas corresponding in location to the outer lamellar defect observed clinically surrounded by an irregular halo of hyperautofluorescence in the context of the greater hypofluorescent area of the macular region of BE.

Figure 2 .

 2 Figure 2. (A) Case 3, color fundus photograph showing a small red lesion at the fovea and surrounding drusen-like deposits in the right macula. (B) Funduscopy in the left eye (LE) is unremarkable; (C) optical coherence tomography (OCT) in the right eye reveals interruption of the thin hyperreflective band corresponding to the junction between inner and outer segment of photoreceptors and of the inner portion of the hyperreflective band corresponding to the retinal pigment epithelium/choriocapillaris complex (arrow). (D) OCT shows a normal retinal structure in the LE; (E) fundus autofluorescence (FAF) of the RE shows a well-demarcated, hypoautofluorescent lesion corresponding in location to the outer lamellar defect observed clinically; the drusen-like lesions seen ophthalmoscopically are hyperautofluorescent. (F) FAF findings in the LE appear

Table .

 . Summary of the characteristics of the sample.Mild or moderate nuclear sclerosis was present in all but one eye, which had previously undergone uncomplicated phacoemulsification with posterior chamber intraocular lens implantation. One patient presented foveal outer lamellar defects in BE, the other three had unilateral lesions. In the five eyes with outer lamellar defects, best-corrected visual acuity was 6/12 in one, 6/9 in two and 6/6 in two eyes; visual acuity in the three fellow eyes of the patients with unilateral defect was 6/6. On biomicroscopic examination none

	pt/age/	eye affected ( OLD	VA	central foveal thickness
	gender	at the fovea on		on OCT
		biomicroscopy and
		OCT )
	1. 47/F BE	6/6 RE	RE: 118 μm
			6/6 LE	LE: 127 μm
	2. 51/F RE	6/9 RE	RE: 125 μm
			6/6 LE	LE: 182 μm
	3. 55/F RE	6/9 RE	RE: 112 μm
			6/6 LE	LE: 194 μm
	4. 57/F LE	6/9 RE	RE: 175 μm
			6/12 LE
	Abbreviations: pt: patient; F: female; OLD: outer lamellar defect; OCT: optical
	coherence tomography; BE: both eyes; VA: visual acuity; RE: right eye; LE: left eye.

LE: 154 μm of the patients had a complete posterior vitreous detachment (PVD). None of the patients demonstrated any retinal haemorrhage, lipid or exudate, full thickness macular hole, or operculum. In one patient drusen-like deposits were present at the macula in the eye with outer lamellar defect (Fig 2).

ACKNOWLEDGMENTS

The authors are indebted to Professor Alan C. Bird for his scholarly contribution to the field.

LICENCE FOR PUBLICATION

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in BJO and any other BMJPGL products and sublicences such use and exploit all subsidiary rights.

COMPETING INTEREST

None declared.


MD Eugenio Triay
email: eugenio.triay@sankterik.se

Louise Bergman

Bo Nilsson

Charlotta All-Ericsson

Stefan Seregard

Time trends in the incidence of conjunctival melanoma in Sweden

PURPOSE. To study time trends in the incidence of conjunctival melanoma in Sweden.

METHODS. All patients with conjunctival melanoma 1960 to 2005 in Sweden were identified through the Swedish Cancer Registry, cross-checked against hospital files and validated by histopathologic review (97.5%) or detailed hospital records (2.5%). The crude and age-standardized incidences were estimated separately for each gender and the annual change in incidence over time was estimated using a regression model with logarithmic incidence numbers. Time trends of the largest diameter, thickness and location of the tumor when diagnosed were analyzed.

RESULTS. The age-standardized incidence of conjunctival melanoma increased significantly in males (n = 89) from 0.10 cases/million to 0.74 cases/million (P = 0.001) and in females (n = 81) from 0.06 cases/million to 0.45 cases/million (P = 0.007). The annual relative change in age-standardized incidence was 16.9 % (95 % CI, 12.2 % -21.6 %) in males and 19.5 % (95% CI, 9.3 % -29.7 %) in females. The age-specific incidence was higher in men and women 65 years and older (1.48 and 1.39 cases/million, respectively) than in younger men and women (0.3 and 0.2 cases/million, respectively). During the period of study, tumors became smaller (P = 0.005) and thinner (P = 0.002) at the time of diagnosis and increasingly arose from parts of the conjunctiva exposed to ultraviolet radiation (P = 0.001).

CONCLUSIONS. The incidence of conjunctival melanoma increased in Sweden during the period 1960 through 2005.

Conjunctival melanoma arises from melanocytes lodged in the basilar layer of the conjunctival epithelium and causes death in 30 % or more of the patients. [START_REF] Seregard | Conjunctival Melanoma[END_REF] Conjunctival melanoma is distinctly uncommon and though 95 % of all melanoma cases are cutaneous and 3-4 % of tumors occur in the eye, only about 5 % of melanoma arising in the ocular region has a conjunctival origin. [START_REF] Seregard | New aspects on the pathogenesis of the conjunctival melanoma[END_REF] Population based studies indicate that the incidence of conjunctival melanoma is probably within the range 0.024 -0.08 per 100,000. [START_REF] Seregard | Conjunctival Melanoma[END_REF][START_REF] Seregard | New aspects on the pathogenesis of the conjunctival melanoma[END_REF][START_REF] Yu | Conjunctival melanoma: is it increasing in the United States?[END_REF][START_REF] Tuomaala | Population-based assessment of clinical characteristics predicting outcome of conjunctival melanoma in whites[END_REF][START_REF] Missotten | Conjunctival melanoma in the Netherlands: A nationwide study[END_REF][START_REF] Isager | Uveal and conjunctival malignant melanoma in Denmark, 1943-97: Incidence and validation study[END_REF] Given recent advances in the epidemiology and molecular biology of conjunctival melanoma, it now seems likely that the molecular mechanisms driving conjunctival melanoma tumorigenesis are more akin to those of cutaneous and mucous membrane melanoma than those of melanoma arising in the uveal tract. [START_REF] Seregard | Conjunctival Melanoma[END_REF][START_REF] Li | Uveal melanocytes do not respond to or express receptors for alpha-melanocyte stimulating hormone[END_REF][START_REF] Henkind | External ocular pigmentation[END_REF][START_REF] Yu | Conjunctival melanoma: is it increasing in the United States?[END_REF][START_REF] Tuomaala | Population-based assessment of clinical characteristics predicting outcome of conjunctival melanoma in whites[END_REF] Specifically, the incidence of cutaneous melanoma is increasing with 4 -5 % annually in the Western world and this rise is largely being attributed to increased exposure to ultraviolet radiation (UVR). [START_REF] Isager | Uveal and conjunctival malignant melanoma in Denmark, 1943-97: Incidence and validation study[END_REF][START_REF] Inskip | Trends in the incidence of ocular melanoma in the United States, 1974-1998[END_REF] The uvea is largely protected from UVR exposure and uveal melanoma incidence rates appear stable or even declining. [START_REF] Bergman | Incidence of uveal melanoma in Sweden from 1960 to 1998[END_REF] Interestingly, large parts of the conjunctiva are exposed to UVR and recent studies suggest that conjunctival melanoma incidence rates may be rising and parallel those of cutaneous melanoma. [START_REF] Yu | Conjunctival melanoma: is it increasing in the United States?[END_REF][START_REF] Tuomaala | Population-based assessment of clinical characteristics predicting outcome of conjunctival melanoma in whites[END_REF]

MATERIALS AND METHODS

The criteria for inclusion were patients with primary conjunctival melanoma, diagnosed in Sweden 1960 through 2005 with the diagnosis validated by histopathologic review by a trained ophthalmic pathologist or by detailed hospital records including clinical photographs or drawings. The Swedish National Cancer Registry, founded in 1958, receives reports when a malignant disease is diagnosed. Compulsory dual registration from a clinician and a histopathologist or cytopathologist ensures a high inclusion rate. Each patient is identified through a unique national registration number, and the registry is estimated to include more than 95% of all cases of cancer in the country. [START_REF] Mattson | Completeness of the Swedish Cancer Register: non-notified cancer cases recorded on death certificates in 1978[END_REF] These data were combined with those of the Swedish Ocular Oncology Service and Eye Pathology Laboratory based at Karolinska University Hospital until 1990, and thereafter at St Eriks Eye Hospital in Stockholm. This tertiary referral centre manages nearly all cases of conjunctival and uveal melanoma in Sweden.

The research protocol for the present study was approved by the Human Ethics Committee at the Karolinska Institute, in accordance with the statutes of the World Medical Association's Declaration of Helsinki. Files from the Swedish National Cancer Registry during the period 1960 through 2005 [START_REF]The Cancer Registry: Cancer incidence in Sweden[END_REF] were searched for patients with conjunctival melanoma, using both the International Classifications of Diseases, and the Seventh, Eight, Ninth and Tenth Revision (ICD-7, ICD-8, ICD-9 and ICD-10) code and the Systematized Nomenclature of Medicine (SNOMED) code. All the patients reported from Cancer Registry were cross-checked against the files of the Ocular Oncology Service and Eye Pathology Laboratory based in Stockholm. Data on the Swedish population were collected from the Statistics Sweden. 13 During the study period, 170 cases of conjunctival melanoma were registered with the Swedish National Cancer Registry. A histopathologic review of archival tissue indicated that 21 cases were erroneously classified as conjunctival melanoma, as follows: 10 cases were choroidal melanoma, 5 iris melanoma, 3 ciliary body melanoma, 2 cutaneous melanoma and 1 case was a breast cancer. In addition, 14 patients were classified as conjunctival melanoma with the Swedish National Cancer Registry but as there no hospital records or histopathologic samples to validate this, all 35 cases were excluded from this study, leaving 135 patients with conjunctival melanoma filed with the Swedish National Cancer Registry 1960 -2005.

When searching our local pathology and hospital files, a total of 151 patients with conjunctival melanoma were found; 116 of these matched the files of the Swedish National Cancer Registry but 35 cases not previously registered were disclosed and formally notified.

Hence, the Swedish National Cancer Registry's non-inclusion rate of conjunctival melanoma was 20.6 %. The 35 cases not previously registered were added to the 135 validated cases from the Swedish Cancer Registry leaving 170 patients with conjunctival melanoma to be included in this study; 166 (97.5 %) of these were confirmed by histopathologic examination and 4 (2.5 %) by detailed hospital records only. The size of each tumor was assessed from clinical photographs by two independent observers. The pathological grading and histological grade were assessed (tables 1-2). Most tumors were bulbar and multifocal (table 3). No evidence of primary tumor pT(is)

Melanoma of the conjunctiva confined to the epithelium pT1a Melanoma of the bulbar conjunctiva not more than 0.5 mm in thickness with invasion of the substantia propria pT1b

Melanoma of the bulbar conjunctiva more than 0.5 mm but not more than 1.5 mm in thickness with invasion of the substantia propria pT1c

Melanoma of the bulbar conjunctiva greater than 1.5 mm in thickness with invasion of the substantia propria pT2a

Melanoma of the palpebral, forniceal or caruncular conjunctiva not more than 0.5 mm in thickness with invasion of the substantia propria pT2b

Melanoma more than 0.5 but not greater than 1.5 mm in thickness with invasion of the substantia propria. pT2c

Melanoma of the palpebral, forniceal or caruncular conjunctiva greater than 1.5 mm in thickness with invasion of the substantia propria. pT3

Melanoma invades the eye, eyelid, nasolacrimal system, sinuses or orbit pT4

Melanoma invades the central nervous system *Note: pT(is) Melanoma in situ (includes the term primary acquired melanosis with atypia) replacing greater than 75 % of the normal epithelial thickness, with cytologic features of epithelioid cells, including abdundant cytoplasm, vesicular nuclei or prominent nucleoli, and/or presence of intraepithelial nests of atypical cells. During the period 1960 through 2005, the overall age-standardized incidence of conjunctival melanoma in the Swedish population increased 7-fold, from 0.08 cases/million to 0.56 cases/million (P = 0.001). In the male population, the age-standardized incidence increased from 0.10 to 0.74 cases/million (P < 0.001) and this was paralleled by a similar increase among women from 0.06 to 0.45 cases/million (P = 0.007) during the period of study (Fig 3).

By applying a log regression model, we determined the annual change in incidence rate. The male incidence increased by 16.9 % (95 % CI, 12.2 % -21.6 %) yearly and the female incidence increased by 19.5 % (95 % CI, 9. Model-based fitted values with linear regression shown no significant increase in conjunctival melanoma arising from conjunctiva protected from ultraviolet radiation (P for slope 0.275), but tumors arising from conjunctiva exposed to ultraviolet radiation increased (P for slope < 0.001) during the period of study. The difference between slopes was statistically significant (P = 0.001). The total number of cases of UV-exposed conjunctival melanoma was 134. The number of UV-nonexposed conjunctival melanoma was 33. In three cases it was not possible to completely identify the location of the tumor.

DISCUSSION

This study demonstrates a dramatic and statistically significant increase in the incidence of conjunctival melanoma in Sweden 1960 through 2005. Are these results correct? There have been two recent studies on time trends in conjunctival melanoma incidence based on closely similar but not identical datasets from the National Cancer Institute's Surveillance Epidemiology, and End Results (SEER) program. The SEER database covered in 1992 approximately 14 % of the population of the United States. [START_REF] Inskip | Trends in the incidence of ocular melanoma in the United States, 1974-1998[END_REF] One of the previous studies based on the SEER database detected 153 cases of conjunctival melanoma during the period 1974 -1998; [START_REF] Inskip | Trends in the incidence of ocular melanoma in the United States, 1974-1998[END_REF] the other found 206 cases when including only 2 more years, from 1973 to 1999. [START_REF] Yu | Conjunctival melanoma: is it increasing in the United States?[END_REF] Whilst the first study failed to detect a statistically significant increase in conjunctival melanoma incidence, [START_REF] Inskip | Trends in the incidence of ocular melanoma in the United States, 1974-1998[END_REF] the second study detected a biennial increase of 11.2 % (95 % CI; 6.3 -16.3) in White men. [START_REF] Yu | Conjunctival melanoma: is it increasing in the United States?[END_REF] Remarkably this rise was not seen in women. In contrast, the study reported herein is the first to demonstrate a statistically significant increase in the incidence of conjunctival melanoma for both males and females with no apparent gender difference. Similarly, a previous study found no difference between genders suggesting that in Finland, the rise in conjunctival melanoma incidence largely parallels the increase seen in cutaneous melanoma incidence. [START_REF] Tuomaala | Population-based assessment of clinical characteristics predicting outcome of conjunctival melanoma in whites[END_REF] A rise in cutaneous melanoma incidence is apparent in many Western countries and in Sweden a 5 % annual increase has been reported during 1976 through 1994. [START_REF] Månsson-Brahme | Trends in incidence of cutaneous malignant melanoma in a Swedish population 1976-1994[END_REF] Notably, the 17.1 % (95 % CI; 11.2 % -23.0 %) annual increase of the incidence we report is more than 3 times the rise in cutaneous melanoma incidence in a near identical population. [START_REF] Månsson-Brahme | Trends in incidence of cutaneous malignant melanoma in a Swedish population 1976-1994[END_REF] There are many potential flaws to consider before accepting this sharp rise in incidence. Firstly, the completeness of registration may have been incomplete during the early years of the study, giving rise to ascertainment bias. However, the proportion of cases validated by histopathologic examination remained high throughout the period of study reported herein. Also, data derived from both types of sources (National Registry and Ocular Oncology Centre) included in this study showed a similar trend with incidence rising from 1970 and reaching a plateau after 1990. Interestingly, this plateau parallels the levelling off noticed for cutaneous melanoma incidence in Northern Europe since the mid 1990s. [START_REF] De Vries | Changing epidemiology of malignant cutaneous melanoma in Europe 1953-1997: Rising trends in incidence and mortality but recent stabilizations in Western Europe and decrease in Scandinavia[END_REF] Secondly, the incidence of conjunctival melanoma in Sweden during the first period of the study (from 1960 to the mid 1970s) was lower (0.08 to 0.20 per million) than the crude annual incidence reported from neighbouring countries like Finland with a mean of 0.4 per million [START_REF] Tuomaala | Population-based assessment of clinical characteristics predicting outcome of conjunctival melanoma in whites[END_REF] , or Denmark with 0.45 cases per million. [START_REF] Isager | Uveal and conjunctival malignant melanoma in Denmark, 1943-97: Incidence and validation study[END_REF] The low incidence seen in Sweden until the mid 1970s would somewhat accentuate the rise in incidence seen in later years.

Thirdly, because the incidence of conjunctival melanoma differs markedly between ethnically different populations, [START_REF] Hu | Population-based incidence of conjunctival melanoma in various races and ethnic groups and comparison with other melanoma[END_REF] a rising incidence occurring through a long period of time could be (partially) explained by an ethnicity shift of that population. The SEER population includes 14 % of the United States population and is more much more ethnically heterogenous (including higher proportions of Blacks and Hispanics with a lower incidence rate) than the Swedish population, which like the Finnish population is largely made up of Whites. [START_REF] Hu | Population-based incidence of conjunctival melanoma in various races and ethnic groups and comparison with other melanoma[END_REF] Notably, there has been a small but growing immigration from both European and non-European countries to Sweden in the past few decades. [START_REF] Månsson-Brahme | Trends in incidence of cutaneous malignant melanoma in a Swedish population 1976-1994[END_REF] However, the net immigration rate remains comparatively low: the Swedish rate is estimated at 0.86/1000 population compared with 3.5/1000 population for the United States during 2000. [START_REF]The World Fact Book 2000[END_REF] Data from the Swedish National Bureau of Statistics indicate that the proportion of the population of non-European origin in 1960 was 0.2 % and in 1998 had increased to 4.0 %. 18 Fourthly, diverse factors might explain the increase of reported cases to the Swedish National Cancer Registry during the recent decades as, for instance, health awareness of the population or a higher accessibility to ophthalmologic examination through the expansion of cataract surgery and screening programs for diabetic retinopathy that has been introduced in the last twenty five years. However, only very small subsets of the Swedish population have annual ophthalmological follow-up. The statistically significant decrease of the largest tumor diameter and tumor thickness we noted is consistent with lesions being diagnosed at an earlier stage in recent years, but even so this is insufficient to fully account for the increase in overall incidence observed throughout the 46-years period of the study.

Increased intermittent UVR exposure through ambient sunlight has been directly implicated in the significant rise in cutaneous melanoma incidence apparent in fair skinned societies. [START_REF] De Vries | Changing epidemiology of malignant cutaneous melanoma in Europe 1953-1997: Rising trends in incidence and mortality but recent stabilizations in Western Europe and decrease in Scandinavia[END_REF] Not much evidence is available on the pathogenesis of conjunctival melanoma, but as most of the conjunctiva is UVR exposed, it is conceivable that any rise in conjunctival melanoma incidence could be attributed to similar molecular mechanisms as in the pathogenesis of cutaneous melanoma. [START_REF] Seregard | New aspects on the pathogenesis of the conjunctival melanoma[END_REF] Interestingly, the rise in conjunctival melanoma incidence we have shown in this study could largely be attributed to conjunctival melanoma occurring more frequently in conjunctiva exposed to UVR. Similarly, there was no detectable increase in conjunctival melanoma arising from the tarsal conjunctiva (outlining the inner surface of the eyelids) with minimal sunlight exposure. Taken together with the time trend, this suggests that UVR exposure may play a major part in the pathogenesis of conjunctival melanoma.

RESULTSAFigure 1

 1 Figure 1The age distribution of conjunctival melanoma in the Swedish population during the period 1960 through 2005.

Figure 2 Age

 2 Figure 2 Age-specific incidence rates of conjunctival melanoma in the population of Sweden during the period 1960 through 2005.

3

 3 Figure 3 Annual age-standardized incidence compared to the observed incidence in the Swedish population for (A) males, (B) females and (C) both genders combined during the period 1960 through 2005. Solid line: observed incidence; broken line: age-standardized rates when standardized against the Swedish population of 1970 to 1974.

Figure 4

 4 Figure 4

Table 1

 1 Pathological staging of primary tumour according to American Joint Committee on Cancer (AJCC) Staging Manual, Seventh Edition (2009). Springer Science and Business Media (www.springerlink.com). Reproduced with permission.

	Primary Tumor (pT)	Number of Cases
	______________________________________________________
	pTX	14
	pT1a	29
	pT1b	48
	pT1c	37
	pT2a	4
	pT2b	10
	pT2c	19
	pT3	9
	______________________________________________________
	Total	170

Table 2 Histological

 2 conjunctiva) or as conjunctiva with minimal UVR exposure (tarsal and forniceal conjunctiva). The level for statistical significance was a set at P < 0.01. Data were processed with statistical-analysis software (SPSS, ver. 15.0; SPSS Inc., Chicago, IL).

	Histological Grade (G)	Number of Cases
	______________________________________________________
	GX		18
	G0		0
	G1		34
	G2		0
	G3		94
	G4		24
	______________________________________________________
	Total		170
	The histologic grade represents the origin of the primary tumor. Because this study only included invasive
	melanoma of the conjunctiva there is no case of G0 or G2.
	GX	Origin cannot be assessed
	G0	Primary acquired melanosis without cellular atypia
	G1	Conjunctival nevus
	G2	Primary acquired melanosis with cellular atypia (epithelial disease only)
	G3	Primary acquired melanosis with epithelial cellular atypia and invasive melanoma
	G4	De novo malignant melanoma

grade of primary tumour according to American Joint Committee on Cancer (AJCC) Staging Manual, Seventh Edition (2009). Springer Science and Business Media (www.springerlink.com). Reproduced with permission.

Each gender was analyzed separately, and the patients were divided into eight age groups, each spanning 10 years, except the youngest (0 -24 years) and the oldest (> 85 years) age groups. The analysis over time was performed for 5-year periods from 1960 except the period 2000 to 2005.

An overall crude and age-standardized incidence rate in the total Swedish population was calculated, and the genders were further analyzed separately. Age-standardization on incidence numbers over the study period was performed by a direct method, with the Swedish location of the tumor when at diagnosed. Tumor location was coded as conjunctiva chronically exposed to ambient solar radiation and hence UVR (most of the bulbar and limbal and all caruncular

Table 4 Stratum

 4

Weights of the Swedish Population during the period 1970 to 1974

Supported by grants from Karin Sandqvist's Foundation for the Visually Impaired, St. Eriks Eye Research Foundation and from the Swedish Research Council. right to grant on behalf of all authors and does grant on behalf of all authors,


E Van Aken

E A De Letter

M Veckeneer

L Derycke

T Van Enschot

I Geers

S Delanghe

J R Delanghe

correspondence. E Van Aken

Transthyretin levels in the vitreous correlate with change in visual acuity after vitrectomy

Keywords: vitreous, transthyretin, biochemical marker, vitrectomy

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in BJO and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence (http://bjo.bmj.com/ifora/licence.pdf).

Competing interests: none declared INTRODUCTION

TTR is a 55 kDa homotetrameric protein with a dimer configuration that is synthesized in the liver, choroid plexus, visceral yolk sac and retinal pigment epithelium. Each monomer is a 127 residue polypeptide rich in beta sheet structure.

Association of two monomers forms an extended beta sandwich. Further association of another identical set of monomers produces the homotetrameric structure. In serum, it functions as a carrier for thyroxin and retinol-binding protein (RBP). It has two thyroxin binding sites per tetramer at the interface between the latter set of dimers, and two binding sites for RBP on the outer surface. As a carrier protein for thyroxin in serum, it acts in concert with thyroxin binding globulin and albumin.

Retinol is transported bound to RBP, which itself is bound to TTR. [START_REF] Monaco | Structure of a complex of two plasma proteins: transthyretin and retinol-binding protein[END_REF] RBP has higher affinity for TTR when it is bound to a retinol ester. [START_REF] Noy | Interactions of retinol with binding proteins: studies with retinolbinding protein and with transthyretin[END_REF] The transport of protein-bound ligands across lipid-water interfaces, such as cell membrane surfaces, is thought to involve obligatory dissociation of protein-bound ligand into the free state before transmembrane transport. [START_REF] Mendel | The free hormone hypothesis: a physiologically based mathematical model[END_REF] The retinol-RBP-TTR complex cannot freely pass through cell membranes. STRA6 is a multitransmembrane domain protein functioning as the high-affinity tissue receptor for plasma RBP and mediating cellular uptake of vitamin A from the vitamin A-RBP complex. [START_REF] Kawaguchi | A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A[END_REF] It has been suggested that formation of the complex between TTR and single, or multiple, RBP molecules, prevents renal clearance of the retinol/RBP complex. [START_REF] Kanai | Retinolbinding protein: the transport protein for vitamin A in human plasma[END_REF] In the eye, TTR is bound to retinol and RBP in various ocular tissues. In contrast to retinol, which is derived solely from the serum, TTR is synthesized locally. Plasma TTR does not cross Bruch's membrane into the retina. Ocular TTR is synthesized in the retinal pigment epithelium (RPE) and in the pigmented epithelium of the ciliary body, the corneal endothelium and the optic nerve fibre layer of the retina and is transported to specific locations within the eye. [START_REF] Getz | Transthyretin localization in cultured and native human retinal pigment epithelium[END_REF][START_REF] Dwork | Distribution of transthyretin in the rat eye[END_REF][START_REF] Pfeffer | Expression of transthyretin and retinol binding protein mRNAs and secretion of transthyretin by cultured monkey retinal pigment epithelium[END_REF] Although the physiological role of TTR in the eye is unknown, it is possible that it participates in retinol cycling.

TTR is able to deposit as amyloid fibrils in senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP). At least 20 TTR mutations are associated with amyloidosis. [START_REF] Sandgren | Ocular amyloidosis, with special reference to the hereditary forms with vitreous involvement[END_REF] Vitreous amyloid fibrils are the result of local synthesis of mutated TTR.

Nuclear cataract is associated with low protein intake and low serum levels of TTR. [START_REF] Delcourt | Pathologies Oculaires Liées à l'Age study group. Albumin and transthyretin as risk factors for cataract: the POLA study[END_REF] Immunohistochemical analysis of drusen in patients with age-related macular degeneration showed the presence of TTR. [START_REF] Mullins | Drusen associated with aging and agerelated macular degeneration contain proteins common to extracellular deposits associated with atherosclerosis, elastosis, amyloidosis, and dense deposit disease[END_REF] Comparison of aqueous humour between myopic and non-myopic patients showed a higher TTR level in the former. [START_REF] Duan | Proteomic analysis of aqueous humor from patients with myopia[END_REF] There exist no biochemical markers to differentiate between patients with excellent and poorer visual acuity after vitrectomy. Little is known about the TTR concentrations in normal and pathological human vitreous fluid. As there are no known reports of humans lacking TTR, the importance of TTR to vision has not been described. In view of the presumed key role of TTR in the vitamin A cycle, we wanted to explore the use of TTR in human vitreous fluid as a biochemical marker protein for retinal function.

MATERIALS AND METHODS

Subjects

Vitreous samples obtained from human cadavers (n=73), obtained through vitreous tap 4 mm from the limbus in the temporal upper or lower quadrant, were used for establishing references values for TTR. In table 1, the age, sex, post-mortem interval, and possible pre-existing diseases are provided. The median post mortem interval was 1.5 days (IQR 0.9-2.4 days). In addition, the cause, mechanism and manner of death of each individual are specified. Furthermore, we analyzed 77 freshly collected vitreous fluid samples submitted to our laboratory. Samples were collected by two vitreoretinal surgeons (MV in the Rotterdam Eye Hospital, the Netherlands, and EVA in Ghent University Hospital, Belgium). A vitreous tap of 0.5 ml was performed at the start of every vitrectomy procedure via a syringe connected to the collecting canula of the vitreous cutter. All samples were immediately stored at 4°C. Informed consent was obtained from each patient. According to diagnosis, the patient could be divided in longstanding retinal detachment (n=29), fresh retinal detachment (n=17), macular holes (n = 20), or advanced proliferative diabetic retinopathy (n=11). As definitions we used: "longstanding retinal detachment" for retinal detachments of patients with typical symptoms for a retinal detachment for at least 1 week before surgical intervention, "fresh retinal detachment" for retinal detachments of patients with typical symptoms for a retinal detachment for less than 1 week before surgical intervention, "macular hole" for stage 3 (>400 μ) macular holes characterized by OCT existing more than 6 months, and "advanced proliferative diabetic retinopathy" for patients with a fundus partially obscured by vitreous haemorrhage according to ETDRS standards. [START_REF] Davis | Risk factors for high-risk proliferative diabetic retinopathy and severe visual loss: Early Treatment Diabetic Retinopathy Study Report #18[END_REF] Pre-and postoperative visual acuity are measured at Snellen charts at 6 meters. Material 917a as a standard and commercially available reagents (Instruchemie, Delfzijl, The Netherlands) on a Modular P system (Roche). [START_REF] Orsonneau | An improved pyrogallol red-molybdate method for determining total urinary protein[END_REF] The 2.5 and 97.5 percentiles of the human cadaver specimens were used for calculating the reference interval. All samples were kept frozen at -20°C until analysis. Between run coefficient of variation was 5.86 % and the detection limit was 3.35 mg/l.

Biochemical investigations

TTR

Clinical investigations

In the patient group, best corrected visual acuity (Snellen) was evaluated before and 3-4 months after vitrectomy. The diagnosis of all preoperative conditions was made by fundoscopy by a trained vitreoretinal surgeon. Additionally, an Optical Coherence Tomography (OCT) was performed for patients presenting with macular hole.

Patients that had yet undergone cataract surgery before were defined "pseudophakic". Patients that had not yet undergone cataract surgery before or had cataract surgery simultaneously with the vitrectomy were defined "phakic".

Characteristics of patients are found in Table 2.

Statistics

P values <0.05 were considered significant. Agreement between data was evaluated by Spearman rank analysis. The lower limit of detection was calculated as the mean value -3 SD for a blank sample. Diagnostic accuracy was assessed by ROC analysis using commercially available statistical software (MedCalc ® , SPSS). The variance of TTR was studied using a stepwise backward elimination logistic regression model, starting with all covariates in the model followed by a stepwise removal of the term associated with the highest p-value.

RESULTS

Reference values

In the reference group (n = 73, median age: 46 years; IQR 35-58 years), median total protein concentration was 538 mg/l (IQR 269-987 mg/l). Considering the causes of death and the pre-existing pathologies, our control cases were a varied group and thus represent the normal population, including a few diabetics (n = 10; see Table 1).

Distribution of total protein was skewed. Median TTR concentration was 18 mg/l (IQR 4-24 mg/l) (Table 2). The concentration of TTR was correlated with total protein concentration of the vitreous fluid (correlation coefficient: 0.459; p<0.01). TTR concentrations were not significantly affected by the post-mortem interval.

Values in various pathological conditions

Preoperative and postoperative visual acuity are given in Table 3. Total protein levels in patients with fresh or longstanding retinal detachment and in patients with proliferative diabetic retinopathy were higher than total protein reference levels.

Among the patient groups, highest TTR values were recorded in the patients presenting with proliferative diabetic retinopathy and lowest in patients presenting with macular hole stage 3 (Table 4). Median TTR values in any pathological condition did not differ statistically with reference TTR values. TTR values in patients who were phakic prior to the vitrectomy procedure (median 22.5 mg/l) were higher than in pseudophakic patients (median 12 mg/l) (fig 1). TTR/total protein ratios were calculated, as this ratio corrects for the vascular permeability changes. TTR/total protein ratios in any disease did not differ with reference TTR/total protein values. Reference TTR/total protein ratios in the vitreous (2.2%) largely exceed the TTR/total protein levels in serum (0.4%-0.5%). The reference TTR values correlated with total protein levels in the vitreous. The origin of vitreous proteins includes the tissues surrounding the vitreous, the hyalocytes of the vitreous, or the surrounding vasculature. The much higher TTR/total protein ratios found in the vitreous as compared to serum values implicates that simple diffusion from the serum cannot be the source of vitreous TTR. We conclude that the physiological level of TTR found in the vitreous of cadaver eyes is derived from surrounding tissues as RPE cells.

We found a significant correlation between postoperative change in visual acuity and high TTR concentrations in the vitreous preoperatively. Ramakrishnan et al. found low TTR levels in the vitreous of diabetic patients. [START_REF] Ramakrishnan | Transthyretin (prealbumin) in eye structures and variation of vitreous-transthyretin in diseases[END_REF] TTR levels in the former study however were not measured by immunonephelometry but calculated semi quantitatively by densitometry after electrophoresis. Our results show that TTR levels in the vitreous are not related to diagnosis but to RPE function. TTR expression is a marker of differentiation of RPE cells in vitro. [START_REF] Pfeffer | Expression of transthyretin and retinol binding protein mRNAs and secretion of transthyretin by cultured monkey retinal pigment epithelium[END_REF] We presume a higher metabolic state of RPE cells in patients with high TTR expression, and thus a better regeneration after a vitrectomy procedure.

The function of TTR in the vitreous remains to be elucidated. RPE of the eye and choroid plexus of the brain are developmental and functional homologues, sharing many of the characteristics of transporting epithelia: the cells are joined by tight apical membrane junctions and they regulate the transport of fluids and serum proteins into their respective humours. The secretion of TTR by the choroid plexus of the brain provides the mechanism for the regulation of T4 (thyroxin) levels in the brain. TTR in the CSF is involved in the delivery of T4 to stem cells and progenitor cells within the brain, which require T4 for regulation of the cell cycle. [START_REF] Richardson | Cell and molecular biology of transthyretin and thyroid hormones[END_REF][START_REF] Lemkine | Adult neural stem cell cycling in vivo requires thyroid hormone and its alpha receptor[END_REF] The abundance of TTR mRNA in RPE is, save for the choroid plexus in the brain, unmatched by any other tissue of the body. [START_REF] Pfeffer | Expression of transthyretin and retinol binding protein mRNAs and secretion of transthyretin by cultured monkey retinal pigment epithelium[END_REF] TTR protein is secreted predominantly in an apical direction by RPE cells in vitro. [START_REF] Ong | Synthesis and secretion of retinol-binding protein and transthyretin by cultured retinal pigment epithelium[END_REF] The fact that the retinol metabolism is altered in TTR-null mice suggests that TTR plays a major role in the retinol metabolism in the interphotoreceptor matrix (IPM). [START_REF] Palha | Thyroid hormone distribution in the mouse brain: the role of transthyretin[END_REF] Interphotoreceptor retinoidbinding protein (IRBP), a non-selective scavenger and stabilizer of retinoids released from photoreceptors and pigment epithelial cells, is the most abundant soluble protein component of the IPM. IRBP plays a direct role in the release of all-trans retinol from the rods. [START_REF] Qtaishat | Interphotoreceptor retinoid-binding protein (IRBP) promotes the release of all-trans retinol from the isolated retina following rhodopsin bleaching illumination[END_REF] All-trans retinol generated in rod photoreceptors upon the bleaching of rhodopsin is known to move from the rods to the RPE, where it is enzymatically converted to 11-cis retinal in the retinoid visual cycle. Experiments conducted on toad RPE-eyecups, mammalian RPE cells and isolated RPE membranes indicate that IRBP specifically promotes the release of 11-cis retinal from the RPE apical surface, a step necessary for the movement of 11-cis retinal to the rods. [START_REF] Edwards | IRBP enhances removal of 11-cis -retinaldehyde from isolated RPE membranes[END_REF] IRBP thereby directly supports the regeneration of rhodopsin in the visual cycle. [START_REF] Pepperberg | Interphotoreceptor retinoid-binding protein (IRBP). Molecular biology and physiological role in the visual cycle of rhodopsin[END_REF] An emerging concept is that the central role of retinoid-binding proteins in the visual cycle is to achieve tight control of retinoid concentration while protecting the retinal cells from the toxicity of free retinoids. In analogy with the CSF where thyroxin concentrations are titrated by binding to TTR, a spatial gradient of free IRBP molecules and IRBP/retinoid complexes might as well be regulated by TTR in the IPM.

We found higher TTR concentration in phakic patients than in pseudophakic patients.

The vitreous proteome is found to be different in pseudophakic patients. [START_REF] Neal | Alterations in human vitreous humour following cataract extraction[END_REF] Lens cortical fibre debris for example is leaking into the vitreous during cataract extraction.

Cataract surgery might thus account for the easier passage of TTR from the posterior chamber to the anterior chamber, resulting in a higher outflow via the trabecular meshwork.

Total protein levels in the vitreous of diabetic patients were 4.3 x higher than reference total protein levels. Electrophoresis of vitreous fluid proteins in diabetics revealed a plasma-like protein pattern suggesting the increased protein concentration is due to a major diffusion from the pathological vessels. Increased vascular permeability and pathological neovascularization are major aspects of diabetic retinopathy. Reduced integrity of adherens junctions and tight junctions of human diabetic retina have been reported. [START_REF] Davidson | Reduced expression of the adherens junction protein cadherin-5 in a diabetic retina[END_REF][START_REF] Peters | Angiopoietin modulation of vascular endothelial growth factor: Effects on retinal endothelial cell permeability[END_REF] TTR values in the vitreous of diabetic patients were fairly similar to reference TTR values, as can be deduced from the following example. A vitreous cavity with a 4 ml content is filled with blood corresponds to an influx of only 12 μg TTR from the serum into the vitreous cavity (3 mg/l TTR x 4 ml).

The amount of TTR that has originated from the serum into the vitreous cavity cannot cause a significant rise in the TTR concentration already present in the vitreous.

In conclusion, vitreous TTR concentrations strongly exceed the values obtained in serum. Therefore, their concentrations in vitreous fluid reflect local production by the RPE. The present study suggests that TTR can be used as a biochemical marker for assessing retinal function. . Agreement was fair between the TTR concentration (x) and the sight measurements (y). The following regression equation was obtained: y (Snellen) = 0.0462 + 0.0042 log X (mg/L); rho Spearman = 0.408; p =0.012; 95% confidence interval 0.106-0.641.

Legends of figures.

  was measured immunonephelometrically on undiluted vitreous fluid samples with use of commercially available Behring antibodies on a Behring Nephelometer II analyzer (Dade Behring) standardized against the widely accepted WHO/College of American Pathologists Certified Reference Material 470. Total protein in vitreous fluid was measured by a pyrogallol red method (n = 129) with Standard Reference

  Peroperative vitreous TTR values correlated well with the visual outcome following treatment (fig2). In a logistic regression model, both change in visual acuity and lens status were the only variables which proved to explain the variance of TTR (multiple correlation coefficient: 0.494; phakic status: t= 2.767; p=0.0084; and change in visual acuity t=2.924: p= 0.0056)DISCUSSIONThis is the first report to the authors' knowledge about a biochemical marker related to change in visual acuity after vitrectomy and for measuring TTR in vitreous samples by immunonephelometry. In the present study, high concentrations of TTR (mean: 19 mg/l) were found in vitreous fluid of cadaver eyes. The observed values are comparable to the ones observed in cerebrospinal fluid (mean: 17 mg/l). Median serum TTR levels are 300 mg/l (IQR 250-350 mg/l). Total protein levels in vitreous fluid are 538 mg/l and are comparable with total protein levels in serum and in CSF (150-500 mg/l). Total protein levels in serum are much higher (64 10 3 -83 10 3 mg/l).

Figure 1

 1 Figure 1 Box-Whisker plots for TTR values in vitreous samples of patients.

Figure 2

 2 Figure 2 Correlation between vitreous TTR concentration in patients and

Table 2 Characteristics of patients.

 2

		Retinal	Retinal	Advanced	Macular hole
				proliferative
		detachment	detachment		Stage 3
				diabetic
		> 1 week	< 1 week
				retinopathy
	Number	29	17	11	20
	Age (range)	71 (61-79)	57 (51-69)	56 (50-59)	71 (61-74)
	Phakic	15 (52%)	6 (35%)	6 (55%)	14 (70%)
	Men	13 (45%)	8 (47%)	7 (64%)	7 (35%)

Table 3 . Preoperative visual acuity, postoperative visual acuity and change in visual acuity for all patients individually in Snellen (6 meters). Visual acuity of 0.017 (1/60) equals fingers counting at 1 metre, visual acuity of 0.003 (1/300) equals hand movement at 1 metre. Mean values +/-SD for each patient group are given below. The Kruskal-Wallis one-way analysis of variance by ranks did not show a statistically significant difference between the patient groups (p>0.05).

 3

		Visual acuity	Visual	acuity	Change	in
		preoperatively	postoperatively	visual acuity
	Retinal detachment>1week	0.1	0.9		0.8
	(n=29)	0.7	1.0		0.3
		0.2	0.7		0.5
		0.003	0.017		0.014
		0.16	0.3		0.14
		0.1	0.1		0
		0.05	0.9		0.85
		0.05	0.7		0.65
		0.003	0.4		0.397
		0.02	0.6		0.58
		0.003	0.02		0.017
		0.003	0.2		0.197
		0.7	0.7		0
		0.003	0.2		0.197
		0.1	0.16		0.06
		1.0	0.7		-0.3
		0.05	0.2		0.15
		0.003	0.017		0.004
		0.017	0.3		0.283
		0.9	0.8		-0.1
		0.1	0.16		0.06
		0.4	0.4		0
		0.2	0.16		-0.04
		0.02	0.02		0
		0.003	0.2		0.197
		0.003	0.15		0.147
		0.003	0.6		0.597
		0.017	0.16		0.143
		0.2	0.16		-0.04
	mean±SD	0.2±0.28	0.4±0.3		0.2±0.3
	Retinal detachment<1week	0.003	0.4		0.397
	(n=17)	0.8	1		0.2
		1.0	0.7		-0.3
		0.003	0.6		0.597
		0.003	0.9		0.897
		0.017	0.9		0.883
		0.003	0.5		0.497

Table 4 Reference values and values for total protein and TTR in vitreous fluid of the various patient groups. The within-and between-run values for total protein and TTR are summarized.

 4

	Retinal condition Total protein (mg/l)	TTR (mg/l)	TTR/total protein (%)
		median IQR	median IQR	median IQR
	reference	538	269-987	18	4-24	2.2	0.9-3.9
	RDD>1 week old 781*	460-1881	19	12-24 1.6	0.6-3.3
	RDD<1 week old 816*	427-1527	14	7-25	1.8	1.7-3.1
	Diabetes mellitus 2326*	1880-2818 20	10-24 1.3	1.2-1.4
	Macular hole	701	241-1456	13	9-25	1.5	1.1-3.2

*: P<0.05 vs reference values. Mann-Whitney U-test.

ABSTRACT

Background/aim: Little is known about biochemical markers related to change in visual acuity after vitrectomy. We investigated the potential use of transthyretin (TTR), a carrier of the retinol/retinol-binding protein, as a biochemical marker protein.

Methods:

We measured TTR using immunonephelometry in a group of patients (n=77) in longstanding (> 1 week) retinal detachment (n=29), fresh (< 1 week) retinal detachment (n=17), macular holes (n=20), or diabetic retinopathy (n=11). Vitreous samples were taken at the start of every vitrectomy procedure. For reference values, cadaver specimens (n=73) were used.

Results: Reference values for vitreous TTR (median 18 mg/l; IQR 4-24 mg/l) comprised 2.2% of reference values for vitreous protein levels (median 538 mg/l; IQR 269-987 mg/l). Vitreous TTR values of patients were comparable in all disorders.

Vitreous TTR values were higher in phakic (median 22.5 mg/l; IQR 10-27 mg/l) than in pseudophakic patients (median 12 mg/l; IQR 8-19 mg/l)(p=0.06). Postoperative change in visual acuity correlated well with vitreous TTR values found peroperatively (r s =0.408; p=0.012). Both change in visual acuity and lens status were the only variables which proved to explain the variance of TTR (multiple correlation coefficient: 0.494; phakic status: t=2.767; p=0.0084; and change in visual acuity t=2.924: p=0.0056).

Conclusion:

Vitreous fluid concentrations of TTR can be regarded as a biochemical marker for retinal function.


Tiia Kangas-Kontio

Satu Vavuli

Sakari Kakko

Jaana Penna

Eeva-Riitta Savolainen

Markku Savolainen

Dr Johanna Liinamaa
email: johanna.liinamaa@oulu.fi

Kangas-Kontio

Kakko Sj

Savolainen E-R

Savolainen Mj

Liinamaa Mj

Polymorphism of the Manganese Superoxide Dismutase Gene But not of Vascular Endothelial Growth Factor Gene Is a Risk Factor for Diabetic Retinopathy

Keywords: Diabetes, diabetic complication, oxidative stress, retinopathy

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Diabetic retinopathy is a microvascular complication of diabetes in which vessel endothelium is damaged due to oxidative stress, leukocyte adhesion, altered coagulation and inflammation [START_REF] Camera | Diabetic microangiopathy: physiopathological, clinical and therapeutic aspects[END_REF]. Vascular alterations due to hypoxia and biochemical changes in the eye in diabetic retinopathy lead to accumulation of blood and fluid in the retinal tissue.

These angiogenic stimuli and the subsequent increased production of vascular endothelial growth factor (VEGF) may lead to retinal neovascularisation. These fragile neovascular vessels can leak blood into the vitreous cavity compromising vision. Fibrous tissue growth may also lead to retinal detachment and blindness if left untreated [START_REF] Morello | Etiology and natural history of diabetic retinopathy: an overview[END_REF].

Diabetic retinopathy is associated with diabetes duration and poor glycemic control, but several studies have indicated that variations in the onset and severity of diabetic retinopathy are not totally explained by known risk factors [START_REF] Uhlmann | Genetics of diabetic retinopathy[END_REF]. Furthermore, familial aggregation of diabetic retinopathy has been found [START_REF] Hanis | Genetics of diabetic retinopathy[END_REF]. Associations between diabetic retinopathy and several candidate genes have been reported and VEGF, among others, has been suggested to be one of the most promising genetic loci involved in diabetic retinopathy [START_REF] Uhlmann | Genetics of diabetic retinopathy[END_REF].

Several clinical studies have reported increased VEGF levels in aqueous and vitreous fluids in patients with diabetic retinopathy [START_REF] Caldwell | Vascular endothelial growth factor and diabetic retinopathy: pathophysiological mechanisms and treatment perspectives[END_REF]. VEGF is a multifunctional cytokine which promotes angiogenesis and vascular permeability in highly vascular tissues such as kidneys and retina. It induces endothelial cell proliferation, promotes endothelial cell migration and functions both as a vasodilator and as an antiapoptotic factor [START_REF] Alon | Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity[END_REF]. The VEGF gene located on chromosome 6 is highly polymorphic containing eight exons.

The retinal damage caused by hyperglycaemia has been linked to increased oxidative stress. The overproduction of reactive oxygen species, especially superoxide, by the mitochondrial electron-transport chain is prevented by overexpression of the enzyme manganese superoxide dismutase (MnSOD), the mitochondrial form of superoxide dismutase. MnSOD catalyses the breakdown of superoxide into hydrogen peroxide and thus protects the endothelial cells from oxidative stress [START_REF] Kowluru | Role of mitochondrial superoxide dismutase in the development of diabetic retinopathy[END_REF]. The most common polymorphism of MnSOD (a missense polymorphism changing alanine to valine at position 9 from the first amino acid of the mature protein or the 16th amino acid from the beginning of the signal sequence, Ala16Val), evokes functional consequences and this mutation has been identified as a risk factor for diabetic nephropathy [START_REF] Möllsten | A functional polymorphism in the manganese superoxide dismutase gene and diabetic nephropathy[END_REF].

The purpose of this study is to investigate the role of VEGF and MnSOD polymorphisms in diabetic retinopathy. The identification of the genes associated with diabetic retinopathy would forms a basis for understanding the biochemical mechanisms of the disease and provide new tools for identification of patients at risk.

MATERIALS AND METHODS

Study subjects

This study is a case-control study. Recruited subjects were diabetic patients (type 1 or type 2) attending fundus imaging for screening of diabetic retinopathy in Oulu City Health Centres or patients with diabetic retinopathy from the Department of Ophthalmology, Oulu University Hospital. The diagnosis and grading of diabetic retinopathy was conducted by an expert ophthalmologist from 60 degree fundus photographs through dilated pupils and by clinical examination. In order to avoid diabetics being misclassified as free of diabetic retinopathy because of a brief diabetes duration, we included only patients with a diabetes duration of at least 15 years. All subjects were interviewed about their health status (e.g. type of diabetes, duration of diabetes, medications). We performed basic blood tests (e.g. fasting blood glucose level, glycated haemoglobin, creatinine, CRP) as well as determinations of plasma lipids and lipid metabolism.

A total of 313 patients were recruited and 229 diabetics completed the questionnaire and provided blood samples. They were divided into two study groups: The diabetic retinopathy (DR) group consisted of 131 diabetics with diabetic retinopathy and the diabetic control (DC) group of 98 diabetic controls without retinopathy or with very mild background retinopathy (only a few microaneurysms). The control group (C) consisted of the control cohort of 526 inhabitants of the city of Oulu (Opera Control Cohort). These samples have been collected earlier and the group has been characterised [START_REF] Rantala | Prevalence of the metabolic syndrome in drugtreated hypertensive patients and control subjects[END_REF].

The vitrous samples were collected from 39 diabetic patients with proliferative diabetic retinopathy who underwent vitreous surgery in Oulu University Hospital, Oulu, Finland.

None of these patients had had anti-VEGF treatment and all gave their informed consent.

Vitreous collection was performed at the beginning of classic three-port pars plana vitrectomy before starting intravitreal infusion with balanced salt solution. Undiluted vitreous samples (200-500 μl) were aspirated into a tuberculin syringe attached into a vitreous cutter and immediately refrigerated after collection. Vitreous was aliquoted, transferred into sterile tubes and frozen at -70 ºC.

Participation in the study was voluntary and all subjects had the right to revoke their permission without affecting their future treatment. Patient data were collected from the medical records of the Oulu University Hospital and Oulu City Health Centres. This study was approved by the Ethical Committee of the Oulu University Hospital and all the study subjects provived an informed consent.

Methods

Genomic DNA was isolated from leukocytes using the salting-out technique. The genotyping for VEGF SNPs was done using TaqMan® SNP Genotyping Assays and Applied Biosystems 7000 Real-Time PCR System (Applied Biosystems, Foster City, CA).

The current study focuses on five identified polymorphisms of the VEGF gene: rs699947 (-2578A>C, numbering from the beginning of translation, located at the promoter), rs2010963 (-634C>G, located at 5' UTR), rs2146323 (A>C, intron 2), rs3025033 (A>G, intron 7) and rs3025039 (+936C>T, located at 3' UTR). A further three SNPs rs699947, rs2010963 and rs3025039 were selected based on previous positive findings that these SNPs can affect VEGF expression [START_REF] Shahbazi | Vascular endothelial growth factor gene polymorphisms are associated with acute renal allograft rejection[END_REF][START_REF] Koukourakis | VEGF gene sequence variation defines VEGF gene expression status and angiogenic activity in non-small cell lung cancer[END_REF] and rs2146323 and rs3025033 were selected (with selection criteria of minor allele frequency over 10 % and r 2 threshold of 0.8) from

HapMap to cover all the haploblocks of the gene. The MnSOD polymorphism was selected based on its previous association with diabetic nephropathy [START_REF] Möllsten | A functional polymorphism in the manganese superoxide dismutase gene and diabetic nephropathy[END_REF]. MnSOD genotyping was conducted as previously described [START_REF] Kakko | The signal sequence polymorphism of the MnSOD gene is associated with the degree of carotid atherosclerosis[END_REF].

Plasma and vitreous VEGF concentrations were assessed using a QuantiGlo Chemiluminescent human VEGF immunoassay (cat. no QVE00B, R&D Systems, Minneapolis, MN, USA). The assay was performed according to the manufacturer's instructions. The detection limit of the assay is 6.4 pg/ml and the assay is linear up to 20 000 pg/ml. According to the manufacturer, the intra-assay coefficient of variation was 4.0 % with an inter-assay coefficient of variation 7.9 %. The laboratory analyses (glucose control, lipids etc.) were performed as routine samples in the Oulu University Hospital.

Statistical analysis

Statistical analysis was done using the SPSS program version 14.0 (SPSS Inc., Chicago, IL, USA). The differences in the distribution of genotypes and alleles between the study groups were analysed by Fisher's exact test for 2 × 2 contingency table. The differences between the VEGF levels were analysed by Student's t-test. Haplotypes were constructed and haplotype-based association analysis was performed with the THESIAS ("Testing Haplotype Effects In Association Studies") program based on the maximum likelihood model and linked to the SEM algorithm [START_REF] Tregouet | Cox proportional hazards survival regression in haplotype-based association analysis using the Stochastic-EM algorithm[END_REF]. With THESIAS it is possible to estimate haplotype frequencies and their effects on the phenotype of interest simultaneously with covariates. Covariates (sex, diabetes type and glycated haemoglobin) were included in the analysis when indicated.

The power of the study was estimated by the PAWE program (Power for the Association With Errors) for the comparison of the allele frequencies between the study groups. In the power analysis, the power was assumed to be 80 % and the level for the statistical significance 0.05. The rare allele frequency of the studied SNPs in the control group varied between 0.16 and 0.48 (Table 3 and4). If the rare allele frequency is 0.16 in the control group, it should be 0.075 or 0.27 in the case group for the difference in the allele frequencies to be detected as being statistically significant. If the rare allele frequency is 0.48 in the control group, it should be 0.35 or 0.62 in the case group to be detected as statistically significant. Our power to detect the observed difference in the MnSOD allele frequencies (0.46 vs. 0.56, Table 4) between the study groups to be statistically significant (with the statistical significance level of 0.05) was estimated to be 55%.

RESULTS

The characteristics of the study groups are presented in Table 1. The mean ages of the patients at the DR, DC and C groups were 59.6, 56.1 and 51.4 years, respectively. The mean duration of diabetes was 24.3 years in the DR group and 24.7 years in the DC group. The prevalence of diabetic complications and hypertension was significantly higher in the DR group than in the DC group. Smoking habits and alcohol consumption did not differ significantly between the study groups. The levels of glycated haemoglobin and plasma lipids for the study groups are shown in Table 2.

VEGF SNPs

The genotype and allele frequencies are presented in Table 3. There were no significant differences in allele and genotype frequencies between the DR and DC groups. We also analyzed VEGF alleles and genotype frequencies separately for type I and type II diabetes, but we did not find a significant difference (please see Supplemental Tables). A total of 10 haplotypes were reconstructed from the five VEGF SNPs using the THESIAS program but the haplotype frequencies did not differ significantly between study groups (data not shown). The inclusion of covariates (age, diabetes type and glycated haemoglobin) did not change the results of the haplotype analysis.

MnSOD SNP

The genotype and allele frequencies for the MnSOD Ala16Val polymorphism are presented in Table 4. We found a higher frequency of the AlaAla genotype (p=0.03) and the Ala allele (p=0.04) in the DR group when compared to the DC group. No such difference was found when the DC group was compared with the control group or when all diabetics were compared with the control group. We also analyzed MnSOD alleles and genotype frequencies separately for type I and type II diabetes, but we did not find a significant difference (please see Supplemental Tables).

Plasma and vitreous VEGF levels in VEGF and MnSOD genotypes

The plasma and vitreous fluid VEGF levels in different VEGF and MnSOD genotypes are presented in Table 5. The number of subjects in some of the groups was small, and no significant difference between plasma and vitreous VEGF and VEGF genotypes was found, exept for higher plasma VEGF in the VEGF rs2010963 GG genotype than CG genotype, but there were no subjects in CC genotype at all. However, plasma and vitreous VEGF concentrations were associated with MnSOD gene, as there was a trend that the ValVal genotype was associated with a higher plasma and vitreous VEGF concentration (p=0.0330 and p=0.0170, respectively).

DISCUSSION

Diabetic retinopathy is a leading cause of new-onset blindness in the working-age population in the developed countries [START_REF] Caldwell | Vascular endothelial growth factor and diabetic retinopathy: pathophysiological mechanisms and treatment perspectives[END_REF]. It is considered as a microvascular complication of diabetes. In diabetic retinopathy the endothelium is damaged due to oxidative stress, leukocyte adhesion, altered coagulation and inflammation [START_REF] Camera | Diabetic microangiopathy: physiopathological, clinical and therapeutic aspects[END_REF], and this leads to leakage of vessels and neovascularization. The current study has examined the role of genetic polymorphisms of the VEGF and MnSOD genes in diabetic retinopathy. We found an association between MnSOD gene polymorphism and diabetic retinopathy. In this study, we selected VEGF SNPs covering all the haploblocks of the gene, and therefore, the negative results of the VEGF polymorphisms should predict the association of the whole VEGF gene due to the strong linkage disequilibrium inside the haploblocks.

Previous studies on VEGF polymorphisms have suggested that the C allele of the -634C>G (rs2010963) polymorphism is a genetic risk factor for diabetic retinopathy [START_REF] Awata | A common polymorphism in the 5'-untranslated region of the VEGF gene is associated with diabetic retinopathy in type 2 diabetes[END_REF] and diabetic macular oedema at least in a Japanese population of type 2 diabetics [START_REF] Awata | Functional VEGF C-634G polymorphism is associated with development of diabetic macular edema and correlated with macular retinal thickness in type 2 diabetes[END_REF].

The CC genotype of the -634C>G SNP has been associated with higher serum and vitreous levels of VEGF ( 16), ( 17) but it was not correlated with proliferative retinopathy in the Slovene population or in a British population [START_REF] Ray | Association of the VEGF gene with proliferative diabetic retinopathy but not proteinuria in diabetes[END_REF]. A study of Polish type 2 diabetics detected an association of C allele of -634C>G polymorphism with the development of nonproliferative diabetic retinopathy [START_REF] Szaflik | An association between vascular endothelial growth factor gene promoter polymorphisms and diabetic retinopathy[END_REF]. However, the difference in study populations may account for the difference between these previous studies and the current study, as we did not find this SNP associating with diabetic retinopathy. Although the VEGF gene itself might not be associated with diabetic retinopathy, there might be mutations in other genes regulating VEGF expression, e.g. transcription factors such as hypoxia-inducible factor 1 α (20).

Diabetic retinopathy is associated with oxidative stress and hyperglycaemia-induced mitochondrial reactive oxygen-species overproduction is a key event in the development of diabetic complications. Overexpression of MnSOD in transgenic mice has inhibited retinal VEGF expression, protected the retina from diabetes-induced abnormalities in the mitochondria and prevented vascular histopathology [START_REF] Kanwar | Oxidative damage in the retinal mitochondria of diabetic mice: possible protection by superoxide dismutase[END_REF]. Recently, in a Slovenian population, a significantly higher frequency of the ValVal genotype of the Ala16Val polymorphism of the MnSOD was found in patients with diabetic retinopathy compared to those without diabetic retinopathy [START_REF] Petrovic | Manganese superoxide dismutase gene polymorphism (V16A) is associated with diabetic retinopathy in Slovene (Caucasians) type 2 diabetes patients[END_REF]. In a Korean population of type 2 diabetics, Ala16Val polymorphism of the Mn-SOD gene was not related to the progression of diabetic retinopathy, but was associated with diabetic macular oedema in patients with a mean age 53 years [START_REF] Lee | Association of manganese superoxide dismutase gene polymorphism (V16A) with diabetic macular edema in Korean type 2 diabetic patients[END_REF]. However, our results could not confirm these reports as we found a higher frequency of AlaAla genotype in diabetic retinopathy. This discrepancy could be due to the study population age, since previously a discrepancy between polymorphism of the MnSOD gene has been described in patients with breast cancer, in the younger group (median age 34) there was an association to ValVal [START_REF] Bergman | Polymorphism in the manganese superoxide dismutase (MnSOD) gene and risk of breast cancer in young women[END_REF] but in older individuals to AlaAla [START_REF] Mitrunen | Association between manganese superoxide dismutase (MnSOD) gene polymorphism and breast cancer risk[END_REF].

The strength of our study is a homogenous and well-characterized study population. The VEGF gene was also analysed thoroughly utilizing haplotypic information, in addition to using only genotypes of single SNPs. The limitation of the current study is a rather small study population. However, with the current sample size a major locus for diabetic retinopathy should be detected as specified by the power analysis. It is possible that a minor effect of VEGF gene on the risk of diabetic retinopathy is missed.

In conclusion, in the current study we found a significant association between MnSOD Ala16Val polymorphism and diabetic retinopathy. However, the studied VEGF SNPs studied were not associated with the risk of diabetic retinopathy and therefore it is unlikely that VEGF gene is a major locus determining the risk of diabetic retinopathy The data are expressed as number of genotypes and alleles (percentages). DR, diabetic retinopathy, DC, diabetic control group, and C, controls. The data are expressed as number of genotypes (percentage) or mean (SD).

Table 1 .

 1 Clinical characteristics of the study groups.

	DR	DC	C

Table 2 .

 2 Glycemic control and plasma lipids, creatinine and plasma VEGF in the study

	groups.
		DR	DC	C
		n=131	n=98	n=526
	Plasma cholesterol (mmol/l)	4.6 (1.1)	4.6 (0.9)	5.7 (1.1)
	HDL cholesterol (mmol/l)	1.3 (0.4)	1.5 (0.5)	1.4 (0.4)
	LDL cholesterol (mmol/l)	2.8 (0.9)	2.6 (0.7)	3.5 (1.0)
	Triglycerides (mmol/l)	1.7 (1.4)	1.1 (0.7)	1.4 (0.8)
	Fasting plasma glucose (mmol/l) 9.0 (3.9)	8.5 (2.9)
	Glycated haemoglobin (%)	9.2 (1.9)	8.3 (1.3)
	Creatinine (μmol/l)	106.4 (91.5)	72.6 (20.4)
	Plasma VEGF (pg/ml)	145.0 (104.5)
	Vitreous VEGF (pg/ml)	1040.3 (1276.5)
	The data are expressed as mean (SD). DR, diabetic retinopathy, DC, diabetic control
	group, and C, controls.

Table 3 .

 3 Allele and genotype frequencies of the five VEGF SNPs studied.

		DR	DC	C	P-values (DR vs. DC)
	rs699947
	Genotype frequencies
	AA	38 (33) 27 (28) 173 (30)	AA and AC vs. CC 0.1744
	AC	58 (48) 38 (40) 250 (46)
	CC	30 (19) 31 (32) 101 (24)
	Allele frequencies
	A	134 (57) 92 (48) 596 (53)	A vs. C	0.2926
	C	118 (43) 100 (52) 452 (47)
	rs2010963
	Genotype frequencies
	CC	10 (6)	5 (5)	32 (8)	CC and CG vs. GG 0.5846
	CG	42 (32) 39 (41) 170 (33)
	GG	74 (61) 52 (54) 322 (59)
	Allele frequencies
	C	62 (22) 49 (26) 234 (25)	C vs. G	0.8259
	G	190 (78) 143 (74) 814 (75)
	rs2146323
	Genotype frequencies
	AA	19 (15) 13 (13) 106 (21)	AA vs. AC and CC 0.8479
	AC	61 (48) 44 (45) 223 (44)
	CC	47 (37) 40 (41) 178 (35)
	Allele frequencies
	A	99 (39) 70 (36) 435 (43)	A vs. C	0.5560
	C	155 (61) 124 (64) 579 (57)
	rs3025033	n (%)	n (%)	n (%)
	Genotype frequencies
	AA	88 (69) 71 (73) 363 (71)	AA vs. AG and GG 0.5550
	AG	34 (27) 22 (23) 135 (26)
	GG	5 (4)	4 (4)	15 (3)
	Allele frequencies
	A	210 (83) 164 (85) 861 (84)	A vs. G	0.6107
	G	44 (17) 30 (15) 165 (16)
	rs3025039
	Genotype frequencies
	CC	88 (70) 72 (75) 368 (70)	CC vs. CT and TT 0.4514
	CT	35 (27) 21 (22) 140 (28)
	TT	3 (3)	3 (3)	16 (2)
	Allele frequencies
	C	211 (84) 165 (86) 876 (84)	C vs. T	0.5952
	T	41 (16) 27 (14) 172 (16)

Table 4 .

 4 Allele and genotype frequencies of the MnSOD Val16Ala polymorphism.The data are expressed as number of genotypes (percentages). DR, diabetic retinopathy, DC, diabetic control group, and C, controls.

		DR	DC	C	P-values (DR vs. DC)
	Genotype frequencies
	AlaAla	40 (32)	18 (19)	115 (23)	AlaAla vs. AlaVal	0.0316
	AlaVal	56 (44)	49 (51)	246 (50)	and ValVal
	ValVal	30 (24)	29 (30)	132 (27)
	Allele frequencies
	Ala	136 (54) 85 (44)	476 (48)	Ala vs. Val	0.0449
	Val	116 (46) 107 (56) 510 (52)

Table 5 .

 5 VEGF levels of plasma and vitreous fluid in different genotype groups shown for all the genotyped SNPs.

		n (%) Plasma VEGF (pg/ml) Vitreous VEGF (pg/ml)
	VEGF rs699947
	AA	15 (38) 193.4 (139.0)	1388.3 (1802.3)
	AC	19 (49) 109.3 (63.6)	825.7 (842.9)
	CC	5 (13)	128.2 (35.8)	811.4 (419.4)
	AA and AC vs. CC		p = 0.9812	p = 0.4427
	VEGF rs2010963
	CC	0 (0)
	CG	15 (39) 100.0 (46.4)	690.1 (397.8)
	GG	24 (61) 171.2 (120.0)	1259.1 (1570.4)
	CC and CG vs. GG		p < 0.0001	p = 0.0799
	VEGF rs2146323
	AA	5 (13)	148.7 (167.8)	925.4 (934.0)
	AC	22 (56) 152.3 (90.2)	1288.2 (1602.7)
	CC	12 (31) 128.7 (107.8)	633.6 (314.8)
	AA vs. AC and CC		p = 0.9878	p = 0.6126
	VEGF rs3025033
	AA	27 (69) 159.4 (101.4)	1118.3 (1480.1)
	AG	11 (28) 115.2 (113.8)	890.7 (658.8)
	GG	1 (3)	97.9 ( )	577.5 ( )
	AA vs. AG and GG		p = 0.9419	p = 0.3512
	VEGF rs3025039
	CC	27 (69) 159.4 (101.4)	1118.3 (1480.1)
	CT	11 (28) 115.2 (113.8)	890.7 (658.8)
	TT	1 (3)	97.9 ( )	577.5 ( )
	CC vs. CT and TT		p = 0.9419	p = 0.3512
	MnSOD
	AlaAla	12 (31) 122.3 (76.3)	457.4 (253.3)
	AlaVal	21 (54) 147.3 (105.6)	1158.1 (928.1)
	ValVal	6 (15)	178.5 (148.4)	1793.5 (2691.4)
	AlaAla vs. AlaVal and ValVal p = 0.0330	p = 0.0170

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the skillful technical assistance of Kirsi Kvist-Mäkelä, Marja-Leena Kytökangas, Saara Korhonen and Sari Pyrhönen. Dr. Liinamaa was supported by a grant from the Eye Foundation, Helsinki, Finland and Prof. Savolainen, Dr.

Kakko and Kangas-Kontio M.Sc. by Academy of Finland and Sigrid Juselius Foundation.