PREVALENCE OF THROMBOPHILIC GENETIC FACTORS AMONG PATIENTS WITH RETINITIS PIGMENTOSA ALMUTEZ M. GHARAIBEH, MD,* ABDALLA S. AWIDI, MD,† OSAMA H. ABABNEH, MD,* MOHAMMED A. ABU-AMEERH, MD,* MUHAMMAD A. AWIDI,† MOHANAD M. SALEH, MD,† ABDULBARI BENER, PHD,‡§ MOHAMMAD A. AL-KHATEEB, BSC,† MANAR R. DWEIK, MSC,† MUAWYAH D. ALBDOUR, MD* Purpose: To determine the prevalence of thrombophilic factors in patients with retinitis pigmentosa (RP). Methods: Fifty consecutive patients with RP and 50 controls matched by age and gender were tested for the presence of the following mutations: factor II (GA20210), factor V Leiden (GA1691), methylenetetrahydrofolate reductase (CT677), factor XIIIa (Val34/Leu), b-fibrinogen (GA455), tumor necrosis factor receptor (TNFRII) (M196R), plasminogen activator inhibitor-1 (PAI-1) (4 G/5 G), and plasminogen activator inhibitor-1 (PAI-1) (GA844). Results: The following heterozygous mutations were found in patients/controls: factor V Leiden (12/14), factor XIIIa (20/30), methylenetetrahydrofolate reductase 677 TT (48/52), b-fibrinogen GA455 (36/36), TNFRII (M196R) (40/42), PAI-1 4 G/5 G (40/48), and PAI-1 GA844 (50/52). The difference between patients with RP and the control group was not statistically significant for the prevalence of any of the studied factors (P . 0.05). Conclusion: In this study, thrombophilic mutations were not increased in patients with RP. Thrombophilic mutations do not seem to be risk factors for RP. Routine investigation of hereditary thrombophilia in these patients is not justified. RETINA 34:2147–2150, 2014

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II (GA20210), factor V Leiden (GA1691), methylenetetrahydrofolate reductase (MTHFR) (CT677), factor XIIIa (Val34/Leu), b-fibrinogen (GA455), TNFRII (M196R), plasminogen activator inhibitor-1 (PAI-1) (4 G/5 G), and plasminogen activator inhibitor-1 (PAI-1) (GA844) mutations. The association of these mutations with retinal artery occlusion and retinal vein occlusion was described in the literature.2–5 The rule of venous and arterial occlusion in RP secondary to thrombophilic factors has not been described. However, hemodynamic studies have demonstrated that RP is associated with a reduction in the retinal and choroidal blood flow.6,7 The association of RP and hereditary thrombophilic mutations has not been studied. The study of such mutations might shed some light on the prevention and treatment of RP. The aim of this study was to compare, for the first time, the prevalence of thrombophilic mutations in patients with RP with that in the normal control population without any clinical evidence of RP.

etinal dystrophies are a group of genetically and clinically heterogeneous disorders involving degeneration of the photoreceptors and resulting in partial or complete blindness. Retinitis pigmentosa (RP) is the most common clinical expression.1 Inherited thrombophilia may be defined as a group of disorders that increase the risk of developing venous or arterial thrombosis. Eight prevalent mutations have been described in association with thrombosis: factor

From the *Department of Ophthalmology, University of Jordan, Amman, Jordan; †Department of Medicine and Hematology, Faculty of Medicine, University of Jordan, Amman, Jordan; ‡Department of Medical Statistics and Epidemiology, Hamad Medical Corporation, Hamad General Hospital, Doha, Qatar; and §Department of Public Health and Medical Education, Weill Cornell Medical College, New York, New York. Supported by the Deanship of Scientific Research grant number 1024. None of the authors have any conflicting interests to disclose. Reprint requests: Abdalla S. Awidi, MD, Department of Medicine and Hematology, Faculty of Medicine, University of Jordan, Queen Rania Al Abdallah street, Amman, Jordan 11942; e-mail: [email protected]

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Materials and Methods Study Sample and Study Design A total of 50 patients with RP were enrolled during a study period of 1 year. We studied 50 unrelated age-/gender-matched control group subjects. All control subjects had negative personal and family history of RP and had no signs of RP or other major eye diseases. The results in both groups were defined by determining the prevalence of different thrombophilic mutations. Inclusion Criteria and Exclusion Criteria To be defined to have RP, the patients underwent complete ophthalmic examinations. All patients had progressive decline of visual acuity and night blindness; all had the classical clinical triad of RP retinal findings, including diffusely scattered bone spicule pigmentation, attenuated blood vessels, and waxy pallor of the optic nerve. During diagnosis, routine visual field testing was not performed for those patients. Patients with atypical patterns of RP (sectoreal RP, pericentric RP, RP albescens, RP with exudative vasculopathy, or pigmented paravenous chorioretinal atrophy) were excluded. Any patient who lacked the classical triad was excluded. All patients were recruited from the ophthalmology clinic at the University Hospital, University of Jordan (UJ); laboratory tests were performed at the Homeostasis and Thrombosis laboratory, Faculty of Medicine, UJ. The study was approved by the institutional review board. Written informed consent was obtained from all study participants in accordance with the Declaration of Helsinki. Blood Samples Blood samples were obtained from patients and controls on the day of enrollment and were drawn in 4.5 mL, 0.5 M EDTA. DNA analysis for the following mutations was performed: factor II (GA20210), factor V Leiden (GA1691), methylenetetrahydrofolate reductase (CT677), factor XIIIa (Val34/Leu), b-fibrinogen (GA455), TNFRII (M196R), PAI-1 (4 G/5 G), and PAI-1 (GA844).

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Genomic DNA was tested for the polymorphism by polymerase chain reaction–restriction fragment length polymorphism method, where the polymerase chain reaction products were digested with restriction endonucleases, and the restriction fragments were separated by agarose gel electrophoresis as described in the literature: FII (GA20210),8 FV Leiden (GA1691),8 methylenetetrahydrofolate reductase (CT677),9 factor XIIIa (Val34/Leu),10 b-fibrinogen (GA455),11 TNFRII (M196R),11 PAI-1 (4 G/5 G),12 and PAI-1 (GA844).12 Controls on each gel included a known heterozygote, homozygous, and a normal control known not to possess any of the mutations. Statistical Analysis The results of statistical analysis are presented as mean and percentages. The data were analyzed using the Statistical Packages for Social Sciences (SPSS Inc, Chicago, IL). The level P , 0.05 was considered as the cutoff value for significance.

Results The mean age of the 30 male and 20 female patients with RP was 29.94 years, whereas that of the control group was 24.4 years. The SD for the patients was 19.18, whereas the SD for controls was 16.3. No statistical difference was observed between the study and control groups regarding gender (P = 0.641) (Table 1). Table 2 shows the prevalence of thrombophilic factors among the RP and control groups. The most frequent thrombophilia in both groups was PAI-1 (GA844) mutation, which was similar in patients with RP and the control group (P = 0.593). None of the patients with RP or controls had factor II (GA20210) mutation, b-fibrinogen (GA455)/homozygous mutation, or factor V Leiden (GA1691)/homozygous mutation. The incidence of mutations was almost equally frequent in both groups. The statistical difference between patients with RP and the control group was not significant for the prevalence of any of the studied thrombophilic mutations. Table 1. Characteristics of Patients

Thrombophilic Mutations Screening The genotype of the mutations was detected by genomic DNA isolation from 300 mL of the buffy coat using the Wizard Genomic DNA Purification kit (Promega, Madison, WI) according to manufactures instructions.



Gender

RP N = 50, n (%)

Controls N = 50, n (%)

Male Female

30 (60) 20 (40)

26 (56) 24 (44)

*Two-sided P values were calculated.

P* 0.641

THROMBOPHILIC FACTORS AND PIGMENTOSA  GHARAIBEH ET AL Table 2. Prevalence of Thrombophilic Mutations Among Patients With RP and Controls Mutation Factor II (GA20210) Normal Homozygous Heterozygous FV Leiden (GA1691) Normal Homozygous Heterozygous MTHFR (C . T677) Normal Homozygous Heterozygous Factor XIIIa (Val34/Leu) Normal Homozygous Heterozygous b Fibrinogen (GA455) Normal Homozygous Heterozygous TNFRII (M196R) Normal Homozygous Heterozygous PAI-1 (4 G/5 G) Normal Homozygous Heterozygous PAI-1 (GA844) Normal Homozygous Heterozygous

RP N = 50, Controls N = 50, n (%) n (%)

P*

50 (100)

50 (100)

1

43 (86) 1 (2) 6 (12)

43 (86) 0 7 (14)

0.584

22 (44) 4 (8) 24 (48)

17 (34) 7 (14) 26 (52)

37 (74) 3 (6) 10 (20)

33 (66) 2 (4) 15 (30)

32 (64) — 18 (36)

32 (64) — 18 (36)

26 (52) 4 (8) 20 (40)

25 (50) 4 (8) 21 (42)

16 (32) 14 (28) 20 (40)

19 (38) 7 (14) 24 (48)

11 (22) 14 (28) 25 (50)

14 (28) 10 (20) 26 (52)

0.463

0.490

0.582 0.978

0.228 0.593

*Two-sided P values based on Pearson chi-square or fisher’s exact test.

Discussion Retinal perfusion is known to be reduced in RP.13 In advanced stages of RP, not only the retinal but also the choroidal circulation is affected.14 This might contribute to the retinal pigment epithelium degeneration. These findings may suggest a role of retinal ischemia as a contributing factor in photoreceptors apoptosis. The observation that breathing carbon dioxide, a strong vasodilator, improved occular hemodynamics and had a beneficial effect on the remaining visual field area in patients with RP,15 may support this as well. To the best of our knowledge, this is the first work in the literature that studied the relation between thrombophilic mutations and RP.

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Prothrombin (factor II) gene mutation (G20210A) is an established risk factor for venous thrombosis,16 although it was not related to either type of central retinal vein occlusion.17 Parc et al18 suggested that prothrombin G20210A mutation may be associated with central retinal artery occlusion, and thus affecting the retinal blood flow. None of our patients with RP or controls had FII (GA20210) mutation. Factor V Leiden mutation was not found to be related to any type of central retinal vein occlusion,19 although it enhanced the risk of developing neovascular complications in central retinal vein occlusion.20 Heterozygous mutations of factor V Leiden were found in 12% of our patients with RP and 14% of controls with no statistically significant P value (0.584). Similar statistically insignificant P values were found between patients with RP and controls for methylenetetrahydrofolate reductase CT677 heterozygous, b-fibrinogen GA455/heterozygous, and TNFRII (M196R) heterozygous mutations. Factor XIIIa Val34Leu mutation has been reported to be associated with an increased risk for spontaneous episodes of subconjunctival hemorrhage.21 In our study, no difference was found between patients with RP and controls in terms of heterozygousity of this mutation with (P = 0.490). Syndromic forms of RP are not uncommon. The existence of such syndromic forms of RP puts RP at the crossroad of several medical specialties.22 This interaction between different pathologies and genetically determined programmed cell death might help in treating such diseases, if we were able to control the triggering factors. In this study, multiple thrombophilic mutations had no significant role as risk factors for patients with RP. Routine investigation of hereditary thrombophilia in these patients is not justified. Key words: retinitis pigmentosa, thrombophilia, risk factors, inheritance. Acknowledgments The authors thank the patients who participated, the staff of Ophthalmology Clinic at Jordan University Hospital, and Homeostasis and Thrombosis Laboratory staff at the Faculty of Medicine for technical assistance. This work was supported by the deanship of scientific research, UJ Grant no 1234/2009. References 1. Singh HP, Jalali S, Narayanan R, Kannabiran C. Genetic analysis of Indian families with autosomal recessive retinitis pigmentosa by homozygosity screening. Invest Ophthalmol Vis Sci 2009;50:4056–4071. 2. Ben-Ami R, Zeltser D, Leibowitz I, Berliner SA. Retinal artery occlusion in a patient with factor V Leiden and prothrombin

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G20210A mutations. Blood Coagul Fibrinolysis 2002;13: 57–59. Marcucci R, Sodi A, Giambene B, et al. Cardiovascular and thrombophilic risk factors in patients with retinal artery occlusion. Blood Coagul Fibrinolysis 2007;18:321–326. Biancardi AL, Gadelha T, Borges WI, et al. Thrombophilic mutations and risk of retinal vein occlusion. Arq Bras Oftalmol 2007;70:971–974. Greiner1 K, Peetz D, Winkgen1 A, et al. Genetic thrombophilia in patients with retinal vascular occlusion. Int Ophthalmol 1999;23:155–160. Cellini M, Strobbe E, Gizzi C, et al. ET-1 plasma levels ocular blood flow retinitis pigmentosa. Can J Physiol Pharmacol 2010;88:630–635. Falsini B, Anselmi GM, Marangoni D, Subfoveal choroidal blood flow and central retinal function in retinitis pigmentosa. Invest Ophthalmol Vis Sci 2011;52:1064–1069. Huber S, Mcmasteter KJ, Voelkerding KV. Analytical evaluation of primer engineered multiplex polymerase chain reactionrestriction fragment length polymorphism for detection of factor V leiden and prothrombin G20210A. J Mol Diagn 2000;2: 153–157. Yi P, Pogribny I, James SL. Multiplex PCR for simultaneous detection of 677 C–T and 1296 A–C polymorphisms in methylenetetrahydrofolate reductase gene for population studies of cancer risk. Cancer Lett 2002;181:209–213. Hancer VS, Diz-Kucukkaya R, Bilge AK, et al. The association between factor XIII Val34Leu polymorphism and early myocardial infraction. Circ J 2006;70:239–242. Mannucci PM, Mari D, Merati G, et al. Gene polymorphisms predicting high plasma levels of coagulation and fibrinolysis proteins. Arterioscler Thromb Vasc Biol 1997; 17:755–759.



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12. Al-Anasari AS, Ollier WER, Villarreal J, et al. Tumor necrosis factor receptor II (TNFRII) exon 6 polymorphism in systemic lupus erythematosus. Tissue Antigens 2000;55:97–99. 13. Grunwald JE, Maguire AM. Retinal hemodynamics in retinitis pigmentosa. Am J Ophthalmol 1996;122:502–508. 14. Schmidt KG, Pillunat LE, Kohler K, Flammer J. Ocular pulse amplitude is reduced in patients with advanced retinitis pigmentosa. Br J Ophthalmol 2001;85:678–682. 15. Tacke CM, Pillunat LE, Lang GK. Ocular carbon dioxide reactivity in retinitis pigmentosa. Perimetry results. Ophthalmologe 1993;90:510–514. 16. Marjot T, Yadav S, Hasan N, et al. Genes associated with adult cerebral venous thrombosis. Stroke 2011;42:913–918. 17. Kalayci D, Gürgey A, Güven D, et al. Factor V Leiden and prothrombin 20210 A mutations in patients with central andbranch retinal vein occlusion. Acta Ophthalmol Scand 1999; 77:622–624. 18. Parc C, Tiberghien E, Pierre-Kahn V. Ocular artery thrombosis as an initial presentation of a prothrombin G20210A mutation. J Fr Ophtalmol 2010;33:380–382. 19. Aras S, Yilmaz G, Alpas I, et al. Retinal vein occlusion, factor V Leiden and prothrombin 20210 G: A mutations. Eur J Ophthalmol 2001;11:351–355. 20. Hvarfner C, Hillarp A, Larsson J. Influence of factor V Leiden on the development of neovascularisation secondary to central retinal vein occlusion. Br J Ophthalmol 2003;87: 305–306. 21. Incorvaia C, Costagliola C, Parmeggiani F, et al. Recurrent episodes of spontaneous subconjunctival hemorrhage in patients with factor XIII Val34Leu mutation. Am J Ophthalmol 2002;134:927–929. 22. Dufier JL. Early therapeutic trials for retinitis pigmentosa. Bull Acad Natl Med 2003;187:1685–1692.

Prevalence of thrombophilic genetic factors among patients with retinitis pigmentosa.

To determine the prevalence of thrombophilic factors in patients with retinitis pigmentosa (RP)...
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