DISSERTATION SUBMITTED IN FULFILMENT OF THE

Tekspenuh

(1)CLINICAL, GENETIC AND CYTOKINE MARKERS OF RETINOPATHY OF PREMATURITY IN A COHORT OF. of. M. al. ay. a. MALAYSIAN INFANTS. ve r. si. ty. SYATIRAH ABU YAZIB. FACULTY OF MEDICINE. KUALA LUMPUR. U. ni. UNIVERSITY OF MALAYA. 2019.

(2) CLINICAL, GENETIC AND CYTOKINE MARKERS OF RETINOPATHY OF PREMATURITY IN A COHORT OF. M. al. ay. SYATIRAH ABU YAZIB. a. MALAYSIAN INFANTS. of. DISSERTATION SUBMITTED IN FULFILMENT OF THE. si. ty. REQUIREMENTS FOR THE DEGREE OF MEDICAL SCIENCES. ve r. FACULTY OF MEDICINE. UNIVERSITY OF MALAYA. U. ni. KUALA LUMPUR. 2019.

(3) UNIVERSITY OF MALAYA. ORIGINAL LITERARY WORK DECLARATION. Name of Candidate: Syatirah binti Abu Yazib Registration/Matric No: MGN140004 Name of Degree: Master of Medical Science Title of Project Paper/Research Report/Dissertation/Thesis (“this Work”):. ay. a. Clinical, Genetic and Cytokine markers of Retinopathy of prematurity in a cohort of Malaysian infant.. I do solemnly and sincerely declare that:. al. Field of Study: Ophthalmology. U. ni. ve r. si. ty. of. M. (1) I am the sole author/writer of this Work; (2) This Work is original; (3) Any use of any work in which copyright exists was done by way of fair dealing and for permitted purposes and any excerpt or extract from, or reference to or reproduction of any copyright work has been disclosed expressly and sufficiently and the title of the Work and its authorship have been acknowledged in this Work; (4) I do not have any actual knowledge nor do I ought reasonably to know that the making of this work constitutes an infringement of any copyright work; (5) I hereby assign all and every rights in the copyright to this Work to the University of Malaya (“UM”), who henceforth shall be owner of the copyright in this Work and that any reproduction or use in any form or by any means whatsoever is prohibited without the written consent of UM having been first had and obtained; (6) I am fully aware that if in the course of making this Work I have infringed any copyright whether intentionally or otherwise, I may be subject to legal action or any other action as may be determined by UM. Candidate’s Signature. Date:. Subscribed and solemnly declared before, Witness’s Signature Name: Designation:. Date:.

(4) ABSTRACT. Retinopathy of prematurity (ROP) is preventable childhood blindness happen to premature infants. Young gestational age, low birth weight, and high supplemental oxygen are known to be important clinical risk factors in ROP. Recent evidences suggested there are other factors either in the form of proteins or genetic alteration may. a. play a role in ROP pathogenesis. A total of 221 premature infants were included in the. ay. study consisting 80 infants diagnosed with ROP and 141 infants without ROP (Control). Study was investigated in three approaches; clinical risk factors, genetics and cytokine.. al. Statistical analysis were assessed according to three group; Control, ROP and Visually-. M. threatening ROP (VTROP), which is defined as eyes requiring laser treatment (laser or anti VEGF injections). Clinical data for each patient were collected using a standard pro. of. forma during ROP examinations in routine pediatric screening or retrieved from medical. ty. record unit, UMMC and comprehensively reviewed by medical officer. Non-biased. si. whole exome sequencing was done to first 20 consecutive DNA samples of patients. ve r. diagnosed with 10 of ROP patients and 10 control patients. Single variant was choose, SARDH polymorphism (rs582326) was identified in 5 out of 10 ROP samples and validated by Sanger sequencing validation. The remaining samples (n=201) were tested. ni. for the polymorphism using Taqman® Genotyping Assay, AssayID: C__2256321_20.. U. Serum levels cytokines VEGF-A, VEGF-C, VEGF-D, IGFBP1,IL-6, IL-8, IL-18, TGFalpha, TNF-alpha, HB-EGF, ANGPT2, EGF, Endoglin, PLGF, sCD40L, sFASL, PAI-1 and uPA were measured by multiplex protein array, BioPlex Pro™ Human Cancer Biomarker on 69 consecutive serum samples from similar cohort at 36-38 weeks of infant’s gestational ages. Data analysis on clinical risk factors showed significance differences in gestational age, (p<0.001), birth weight (p<0.001), duration of NICU stay (p<0.001), bronchopulmonary dysplasia (BPD), respiratory support (p<0.001) and. iii.

(5) invasive ventilation (p<0.001) between control and ROP/VTROP group. In addition, in multipe logistic regression, patients with younger gestational age (<29 weeks) the risk of having ROP is increase by 88% .As for invasive ventilation, it can be hypothesized as the longer duration of invasive ventilation, increase the odds of having ROP by 7 times and 3 times more likely to develop advance ROP which required a treatment (VTROP).In genetic analysis, there was a poor associations between SARDH polymorphism and ROP. a. in any of genetic models (dominant, recessive and additive), after adjusting for age, birth. ay. weight and gender. In addition, significance difference of VEGF-D (p=0.024) and IL8 (p=0.046) level was observed in VTROP compare to Non VTROP group. VEGF-D also. al. found significance (p=0.038) in VTROP versus Control group. Level of other cytokines. M. did not reveal any significance difference among other analyzed groups. All in all, clinical data risk factors analysis shown low gestational age was strongly associated in increased. of. risk of ROP. However, genotyping analysis shows poor association of ROP with SARDH. ty. gene (rs582326) in Malaysian population. Though, cytokine analysis shown higher. si. VEGF-D level may be further correlated with vision threatening ROP. All in all, these results were important as to determine potential factors that may contribute to the. U. ni. ve r. development of therapeutics and treatment for ROP.. iv.

(6) ABSTRAK. Retinopati pramatang (ROP) adalah penyakit kebutaan yang boleh terjadi kepada bayi pramatang. Secara amnya, bayi yang tidak cukup bulan, kurang berat lahir dan memerlukan bantuan oksigen yang tinggi berisiko tinggi untuk menghidap penyakit ROP. Faktor lain seperti perbezaan genetik dan kadar sitokin dalam darah juga mungkin memberi peranan penting dalam patogenesis ROP. Dalam penyelidikan ini, profil gen. a. disaring dari sampel darah untuk mengenalpasti mutasi polimorfisme tunggal yang. ay. terdapat dalam kohot kajian. Di samping itu, risiko klinikal pesakit dianalisa dan. al. perbezaan tahap sitokin dalam serum bayi pramatang dikaji. Saringan faktor klinikal serta. M. profil genetik (n=221) dan analisis perbandingan tahap sitokin (n=69) dijalankan ke atas tiga kumpulan utama, a. Pesakit ROP (ROP) (bayi pramatang yang menghidap penyakit. of. ROP b. Kumpulan kawalan (Control) (bayi pramatang yang tidak menghidap penyakit ROP) c. Pesakit ROP yang memerlukan rawatan (VTROP). Keputusan menunjukkan. ty. perbezaan yang signifikan (p<0.05) dalam beberapa faktor klinikal berisiko termasuk. si. umur kehamilan, berat lahir, tempoh pengudaraan invasif, tempoh berada di wad intensif. ve r. neonatal, kadar FiO2, PDA dan sepsis dalam ketiga-tiga analisis kumpulan. Di samping itu, dari aspek kajian genetik, saringan awal keseluruhan genom dijalankan kepada 20 terdiri daripada 10 pesakit ROP dan 10 NO ROP. Mutasi. ni. sampel DNA yang. U. polimorfisma tunggal yang unik dikenalpasti daripada analisis dan perubahan asid amino ini kemudian dibandingkan dengan pangkalan data gen (DisGeNet). Sepuluh gen telah dikenalpasti membawa mutasi unik yang berpotensi untuk membawa kesan terhadap penyakit ROP dikaji. Pencirian varian disaring sekali lagi menggunakan teknik saringan genetik yang berbeza iaitu penjujukan Sanger yang menumpukan kepada varian yang dipilih sahaja. Varian (rs582326) dari gen SARDH akhirnya dipilih untuk pengesahan akhir menggunakan Taqman® Genotyping Assay, AssayID: C__2256321_20. Model genetik dominan, resesif dan tambahan digunakan untuk menguji kaitan polimorfisme. v.

(7) nukleotida tunggal SARDH rs582326 dengan ROP. Selain itu, dalam aspek sitokin, kadar sitokin VEGF-A, VEGF-C, VEGF-D, IGFBP1, IL-6, IL-8, IL-18, TGF-alpha, TNFalpha, HB -EGF, ANGPT2, EGF, Endoglin, PLGF, sCD40L, sFASL, PAI-1 dan uPA dalam serum pada sampel yang dikumpul dari Unit Rawatan Rapi Neonatal pada usia kehamilan 36-38 minggu dikaji. Asai sitokin digunakan dan setiap sampel dianalisis mengikut kumpulan kajian. Dapatan kajian menunjukkan perbezaan yang signifikan. a. (p<0.05) dalam sitokin VEGF-D (p=0.024) dan IL-8 (p=0.046) daripada analisis. ay. kumpulan VTROP dengan Non VTROP. Walaupun begitu, analisis bagi sitokin dalam kumpulan kajian yang lain tidak menunjukkan keputusan yang signifikan. Selain itu,. al. faktor VEGF D juga signifikan dalam analisis antara VTROP dan Non VTROP. M. (OR=9.309, p = 0.029). Kesimpulannya, kurang usia kehamilan dan kadar sitokin VEGFD yang tinggi berkait dengan peningkatan risiko dalam penyakit ROP yang lebih teruk.. of. Walaubagaimanapun, kajian genetik gen SARDH (rs582326) tidak menemukan perkaitan. ty. dengan ROP. Justeru itu, kajian yang lebih terperinci diperlukan untuk mengenalpasti gen. U. ni. ve r. si. yang berpotensi untuk mnyumbang kepada perawatan penyakit ROP.. vi.

(8) ACKNOWLEDGEMENTS. I would like to express my sincere thanks and utmost gratitude to: ALLAH S.W.T for HIS guidance, spiritual courage and giving me strength to face challenges and obstacles throughout this journey. Alhamdulillah. Associate Professor Tengku Ain Fathlun Tengku Kamalden for her unwavering guidance, advices, and most importantly, support my personal care and giving me opportunity to continue. a. my postgraduate studies under her close supervision in University of Malaya. Thank you for. ay. providing me with the motivation to develop a passion towards science and research and. al. expertise during the study.. M. Associate Professor Nurliza Khaliddin and Associate Prof Choo May May for their continuous support, clinical and motivational advices throughout the study. Thank you for. of. sharing your research experiences and expertise with me.. Dr Azanna Kamar and Dr Yao Man Choo for their continuous effort from peadiatric. si. progress smoothly.. ty. department to ensure patient recruitments, sample collection and follow up on schedule and. ve r. Prof Gopal Lingam for his guidance for his advices in understanding statistical analysis, and Dr Christine Peng Yee for managing and collecting patient’s clinical data for the results. ni. analysis.. U. Dr Rozieyatie Mohamad Saleh, for continuous support in sharing her research experiences,. expertise in laboratory work and kind guidance during writing process. Fatin Izzati, Fu Shun Wong, Nur Musfirah, Biobank Unit, and MBL unit for support, guidance and help in all laboratory works Thank you for your kind cooperation My parents and husband for financial support and my kids for their unconditional loves and concern. Their endless patience, gives me strength to proceed for my ambition and strive towards excellence. Lastly, Department of Ophthalmology and Faculty of Medicine for the master opportunity and facilities provided. Thank You!. 7.

(9) TABLE OF CONTENTS ABSTRACT. III. ABSTRAK. V. ACKNOWLEDGEMENTS. 7. TABLE OF CONTENTS. 8 13. LIST OF TABLES. 14. LIST OF SYMBOLS AND ABBREVIATION. 18. ay. CHAPTER 1: INTRODUCTION General Introduction. 20 20. al. 1.1. a. LIST OF FIGURES. Background..................................................................................................... 21. 1.1.2. Prevalence of ROP ......................................................................................... 22. of. The Retina. 23. ty. 1.2. M. 1.1.1. Embryology of retina ...................................................................................... 23. 1.2.2. Neurosensory retina ........................................................................................ 24. 1.2.3. Retinal pigment epithelium ............................................................................ 26. ve r. si. 1.2.1. Pathogenesis of Retinopathy of prematurity. ni. 1.3. General Classification of ROP. U. 1.4. 27. 28. 1.4.1. Severity and plus disease ................................................................................ 29. 1.4.2. Location .......................................................................................................... 31. 1.4.3. Extent .............................................................................................................. 32. 1.4.4. Regression and resolution............................................................................... 32. 1.5. Risk Factors. 1.5.1. 33. Gestational age ............................................................................................... 33. 8.

(10) 1.5.2. Birth weight .................................................................................................... 34. 1.5.3. Oxygen ........................................................................................................... 34. 1.5.4. Blood Transfusion .......................................................................................... 35. 1.5.5. Pharmacological factors ................................................................................. 36. 1.5.6. Co-morbidities of prematurity ........................................................................ 36. Management of ROP. Treatment of ROP........................................................................................... 37. Complication of ROP. 1.8. Studies on ROP. 40. 41. M. al. 1.7. a. 1.6.1. 37. ay. 1.6. Genetics study ................................................................................................ 41. 1.8.2. Cytokine study ................................................................................................ 71. Research Objectives. ty. 1.9. of. 1.8.1. Materials. 85 85. ve r. 2.1. si. CHAPTER 2: MATERIALS AND METHODOLOGY. 84. Consumables................................................................................................... 85. 2.1.2. Chemical reagents .......................................................................................... 85. ni. 2.1.1. U. 2.1.3 2.1.4. Kits ................................................................................................................. 86 Equipment....................................................................................................... 88. 2.2. Overall research workflow. 89. 2.3. Study design. 93. 2.4. Ethical aspects. 93. 2.5. Patient selection. 93. 9.

(11) 2.5.1 2.6. Inclusion and exclusion criteria ...................................................................... 93. Clinical risk factor study. 94. Proforma collection ........................................................................................ 94. 2.6.2. Fundus examination........................................................................................ 97. 2.6.3. Demographics and clinical risk factor data .................................................... 98. 2.6.4. Clinical Risk Factor Dataset ........................................................................... 98. 2.6.5. Statistical analysis .......................................................................................... 99. Genetics study. ay. 2.7. a. 2.6.1. 101. Samples Collection ....................................................................................... 102. 2.7.2. Collection and processing of blood and serum samples ............................... 102. 2.7.3. Collection and processing of saliva samples ................................................ 102. 2.7.4. DNA extraction ............................................................................................ 103. 2.7.5. DNA quantification and purity assessment .................................................. 104. 2.7.6. Whole exome sequencing (WES) ................................................................. 104. 2.7.7. Validation by Sanger sequencing ................................................................. 108. ve r. si. ty. of. M. al. 2.7.1. Genotyping by Real-Time PCR.................................................................... 109. 2.7.9. Allelic Discrimination plot analysis ............................................................. 111. ni. 2.7.8. Other experimental work done during SNPs screening process .................. 111. 2.7.11. Genetic analysis ............................................................................................ 114. U. 2.7.10. 2.8. Cytokine Study. 115. 2.8.1. Assays preparation........................................................................................ 116. 2.8.2. Running the assay ......................................................................................... 117. 2.8.3. Multiplex Elisa ............................................................................................. 118. 2.8.4. Cytokine analysis.......................................................................................... 118. CHAPTER 3: CLINICAL RISK FACTORS RESULTS. 119 10.

(12) 3.1. Overview of clinical risk factor results. 119. 3.2. Clinical risk factor Analysis 1: Control versus ROP. 121. 3.2.1. Multiple logistic regression: Analysis 1, Control Versus ROP .................... 123. 3.2.2. Final Multiple Logistic Regression model: Analysis 1 ................................ 123. 3.3. Clinical risk factor Analysis 2: Non VTROP versus VTROP. 124. Multiple logistic regression: Analysis 2, Non VTROP versus VTROP ....... 126. 3.3.2. Final Multiple Logistic Regression model: Analysis 2 ................................ 126. ay. Clinical risk factors Analysis 3: Control versus VTROP. 128. al. 3.4. a. 3.3.1. Multiple logistic regression: Analysis 3, Control versus VTROP ............... 130. 3.4.2. Final Multiple Logistic Regression model: Analysis 3 ................................ 130. 3.4.3. Summary of clinical risk factors finding ...................................................... 131. of. M. 3.4.1. 132. Whole Exome Sequencing (WES). 132. 4.2. Selection of candidate SNPs. 133. ve r. 4.1. si. ty. CHAPTER 4: GENETIC STUDY RESULTS. Sanger Sequencing results. ni. 4.3. Genotyping Results. U. 4.4. 138. 143. 4.4.1. GENETICS ANALYSIS 1: Control versus ROP ......................................... 146. 4.4.2. GENETICS ANALYSIS 2: Non VTROP versus VTROP........................... 148. 4.4.3. GENETICS ANALYSIS 3: Control versus VTROP ................................... 150. CHAPTER 5: CYTOKINE STUDY RESULTS. 153. 5.1. Patient Demographics and Characteristics. 153. 5.2. Cytokines Study Group Analysis. 154 11.

(13) 5.3. Serum Cytokine Profile Dataset. 155. Clinical risk factors cytokine dataset analysis 1 ........................................... 158. 5.3.2. CYTOKINE ANALYSIS 1: Control versus ROP........................................ 161. 5.3.3. Clinical risk factors cytokine dataset analysis 2 ........................................... 165. 5.3.4. CYTOKINE ANALYSIS 2: VTROP versus Non VTROP ........................ 168. 5.3.5. Clinical risk factors cytokine study dataset analysis 3 ................................. 172. 5.3.6. CYTOKINE ANALYSIS 3: Control versus VTROP group ........................ 175. a. 5.3.1. ay. CHAPTER 6: DISCUSSION. al. CHAPTER 7: CONCLUSION. M. CHAPTER 8: REFERENCES. 184 185 208. U. ni. ve r. si. ty. of. APPENDICES. 178. 12.

(14) LIST OF FIGURES Figure 1.1 :Retinal layer ............................................................................................................. 25 Figure 1.2 Pathogenesis of ROP ................................................................................................. 27 Figure 1.3 Stages of ROP ........................................................................................................... 29 Figure 1.4 Location of zones and extent of ROP ....................................................................... 31 Figure 1.5 ROP classification system by zones.......................................................................... 38. a. Figure 1.6 Schematic of Vascular Endothelial Growth Factor (VEGF) signalling. ................... 74. ay. Figure 2.1 Overall research workflow ........................................................................................ 91. al. Figure 2.2 :Patients recruitment samples collection flow .......................................................... 92. M. Figure 2.3 Retcam images indicates stage of ROP (a)immature peripheral retina, (b) stage 1, (c) stage 3 and (d) regressed ROP after laser treatment ............................................................. 97. of. Figure 2.4 :Workflow of statistical analysis used to analyze clinical risk factors ................... 100 Figure 4.1:Selection of candidate Single nucleotide polymorphisms (SNPs).......................... 134. ty. Figure 4.2 :Sanger Sequencing results (positive validation) .................................................... 139. si. Figure 4.3 :Sanger sequencing result (negative validation) ..................................................... 140. ve r. Figure 4.4 :Allelic discrimination plot of SARDH GENE Assay ID:C_2256321_20 .............. 144 Figure 4.5 :Genotype frequencies for SARDH SNP between Control and ROP ...................... 147. ni. Figure 4.6 :Genotype frequencies for SARDH SNP between Non VTROP and VTROP ........ 149. U. Figure 4.7 :Genotype frequencies for SARDH SNP between Control and VTROP ................ 151 Figure 5.1 :Relative serum cytokine levels comparison between ROP and NO ROP ............. 160 Figure 5.2 :Relative serum cytokine levels comparison between Non VTROP and VTROP . 167 Figure 5.3:Relative serum cytokine levels comparison between Control with VTROP .......... 174. 13.

(15) LIST OF TABLES Table 1.1: DisGeNet disease-gene database1 ............................................................................. 44 Table 1.2 :Summary of potential genes variant associated with ROP ....................................... 70 Table 1.3 :Summary of studied cytokine related to ROP ........................................................... 83 Table 2.1 Definition of clinical characteristics of risk factors ................................................... 95. a. Table 2.2:Sample collection details .......................................................................................... 102. ay. Table 2.3 :Real Time assay for one reaction volume ............................................................... 110 Table 2.4:Real-time PCR programme ...................................................................................... 111. al. Table 2.5 :Human Cancer Biomarker Assay ............................................................................ 116. M. Table 3.1 :Clinical characteristics of infants recruited in the study. ........................................ 120. of. Table 3.2 :Clinical risk factors between Control and ROP group ............................................ 122 Table 3.3 :Association of selected factors Control versus ROP ............................................... 124. ty. Table 3.4 :Clinical risk factors analysis between Non VTROP versus VTROP ...................... 125. si. Table 3.5 :Association of selected clinical factors, VTROP versus Non VTROP ................... 127. ve r. Table 3.6 :Clinical risk factors analysis between Control versus VTROP............................... 129 Table 3.7 :Association of clinical factors , Control versus VTROP ........................................ 130. ni. Table 4.1 : Summary of whole exome sequencing (WES) screening ...................................... 132. U. Table 4.2: Disease-gene association from DisGeNet database ................................................ 135 Table 4.3 :SNPs selection details ............................................................................................. 137 Table 4.4 :Sanger Sequencing validation results ...................................................................... 141 Table 4.5 :Allele frequency and percentage genotype in the study sample ............................. 145 Table 4.6 :Association of SARDH polymorphism in Control versus ROP .............................. 147 Table 4.7 :Association of SARDH polymorphism in Non VTROP versus VTROP ................ 149 Table 4.8 :Association of SARDH polymorphism in with VTROP versus Control ................. 151. 14.

(16) Table 5.1 :Demographics and Clinical Characteristics of the Studied Cohort ......................... 154 Table 5.2 :Gender frequency in the cytokines study sample .................................................... 155 Table 5.3 :Cytokines information for each panel and number of samples tested..................... 156 Table 5.4 :Clinical risk factors cytokine dataset (Analysis 1) .................................................. 158 Table 5.5 :Serum cytokine levels of analysis 1: Control versus ROP. ..................................... 159 Table 5.6 ANOVA test for analysis 1 (Control Versus ROP) .................................................. 162. a. Table 5.7 ANCOVA test; Effect of ROP to VEGF-A and IGFBP1 level ................................ 163. ay. Table 5.8 : Predicting risk of ROP in Group 1 cytokine analysis : VEGF A level ROP vs NO ROP .......................................................................................................................................... 164. al. Table 5.9: Clinical risk factors cytokine dataset (Analysis 2) .................................................. 165. M. Table 5.10 :Serum cytokine levels of analysis 2: VTROP versus Non VTROP group. ........... 166. of. Table 5.11 ANOVA test for analysis 2 .................................................................................... 169 Table 5.12 ANCOVA test; Effect of VTROP to VEGF-D and IL-6 level.............................. 170. si. ty. Table 5.13 Predicting risk in affecting VEGF-D level in Group 2 analysis VTROP versus Non VTROP group........................................................................................................................... 171. ve r. Table 5.14 :Clinical risk factors cytokine dataset (Analysis 3) ................................................ 172 Table 5.15 :Serum cytokine levels analysis 3: Control versus VTROP group ........................ 173. ni. Table 5.16 ANOVA test for analysis 3 .................................................................................... 176. U. Table 5.17 :ANCOVA- Group 3 analysis: GA affecting VEGF D in VTROP versus Control group ......................................................................................................................................... 177 Table 0.1: Result of LR test – Step 1 (Based on all 11 clinical risk variables) ........................ 210 Table 0.2: Result of LR test – Step 2 (Based on 10 clinical variables) .................................... 211 Table 0.3 :Result of LR test – Step 3 (Based on 9 clinical variables) ...................................... 211 Table 0.4: Result of LR test – Step 4 (Based on 8 clinical variables) ...................................... 212 Table 0.5: Result of LR test – Step 5 (Based on 7 clinical variables) ...................................... 212 Table 0.6: Result of LR test – Step 6 (Based on 6 clinical variables) ...................................... 212. 15.

(17) Table 0.7: Result of LR test – Step 7 (Based on 5 clinical variables) ...................................... 213 Table 0.8: Result of LR test – Step 8 (Based on 4 clinical variables) ...................................... 213 Table 0.9 :Result of LR test – Step 1 (Based on all 8 clinical risk variables) .......................... 213 Table 0.10 :Result of LR test – Step 2 (Based on 7 clinical risk variables) ............................. 214 Table 0.11 :Result of LR test – Step 3 (Based on 6 clinical risk variables) ............................. 215 Table 0.12 :Result of LR test – Step 4 (Based on 5 clinical risk variables) ............................. 215. a. Table 0.13 :Result of LR test – Step 5 (Based on 4 clinical risk variables) ............................. 215. ay. Table 0.14 :Result of LR test – Step 6 (Based on 3 clinical risk variables) ............................. 216. al. Table 0.15 :Result of LR test – Step 5 (Based on 2 clinical risk variables) ............................. 216. M. Table 0.16 :Result of LR test- Step 7 (based on 1 variable)..................................................... 216 Table 0.17 :Result of LR test – Step 1 (Based on all 11 clinical risk variables) ...................... 216. of. Table 0.18 :Result of LR test – Step 2 (Based on 10 clinical risk variables) ........................... 217. ty. Table 0.19 :Result of LR test – Step 3 (Based on 9 clinical risk variables) ............................. 217. si. Table 0.20 :Result of LR test – Step 4 (Based on 8 clinical risk variables) ............................. 218. ve r. Table 0.21 :Result of LR test – Step 5 (Based on 7 clinical risk variables) ............................. 219 Table 0.22 :Result of LR test – Step 6 (Based on 6 clinical risk variables) ............................. 219. ni. Table 0.23 :Result of LR test – Step 7 (Based on 5 clinical risk variables) ............................. 219. U. Table 0.24 :Result of LR test – Step 8 (Based on 4 clinical risk variables) ............................. 220 Table 0.25 :Result of LR test – Step 9 (Based on 3 clinical risk variables) ............................. 220 Table 0.26 :Result of LR test – Step 10 (Based on 2 clinical risk variables) ........................... 220. 16.

(18) 17. ve r. ni. U ty. si of ay. al. M. a.

(19) >. more than. °C. degree Celsius. µg. Microgram. µl. Microliter. µM. Micromolar. AGTR1. Angiotensin II receptor type 1. BDNF. Brain derived neurophatic factor. bp. base pair. CFH. Complement factor H. COL18A1. collagen, type XVIII, alpha 1. COL5A1. collagen, type V, alpha 1. CRYBA4. Crystallin,beta A4. DNA. Deoxyrbonucleic acid. dNTP. Deoxynucleotide. M. of. ty. si. ve r. DR. ay. less than. al. <. a. LIST OF SYMBOLS AND ABBREVIATION. Endothelial nitric oxide synthase. ni. eNOS3. Diabetic Retinopathy. U. FGD6. FYVE, RhoGEF and PH domain containing 6. FZD4. Frizzled 4. hr. Hour. INVS. Inversin. LRP5. Low density lipoprotein related protein 5. LRP5. Low density lipoprotein receptor-related protein 5. ND. Norrie Disease. 18.

(20) PCR. Polymerase chain rection. PI3K. Phosphoinositide 3. RLF. Retrolental Fibroplasia. ROP. Retinopathy of prematurity. RPE. Retinal pigment epithelium. rpm. Rotation per minute. rpm. rotation per minute. SARDH. Sarcosine dehydrogenase. sec. Second. a. Next Generation Sequencing. ay. NGS. al. Nanogram. M. ng. of. SERPINF1 Serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1 SFSWAP Splicing factor,suppressor of white-apricot family Single Nucleotide polymorphism. TBE. Tris Borate EDTA. TGFB1. Transforming growth beta-1 gene. ve r. si. ty. SNP. TNF receptor-associated factor 2. UN-HDI. United Nation Human Development Index. UV. Ultraviolet. ni. TRAF2. Volt. VEGF. Vascular Endothelial Growth Factor. VLBW. Very low birth weight infant. U. V. 19.

(21) CHAPTER 1: INTRODUCTION 1.1 General Introduction Retinopathy of prematurity (ROP) is an avoidable blinding ocular disease and a major problem affecting premature babies. Rate of blindness in ROP varies significantly between countries, and it depends on the respective neonatal care services, efficiency in ROP screening. a. and accessibility to treatment. According to WHO (World Health Organization), the number of. ay. blind children in Asian continent is estimated to be 1 million every year. In Malaysia, the. al. prevalence of avoidable childhood blindness is up to 50.5% and more than 10% are caused by. M. ROP (K. M. Chang, Patel, Tajunisah, & Subrayan, 2015).. Known major risk factors of ROP are low birth weight (Kinsey, Jacobus, & Hemphill, 1956;. of. Palmer et al., 1991), high supplemental oxygen (Kinsey et al., 1956) and low gestational age. ty. (Choo, Martin, Theam, & Chan, 2009). Neonatal care services in Malaysia has advanced. si. significantly (Blencowe et al., 2013) with increased survival rates in babies as premature as 26 weeks and with birth weight as low as 500 gram birth weight. This increases the risk of ROP. ve r. (Mittal, Dhanireddy, & Higgins, 1998). Malaysia adhere to the Ministry of Health, National Clinical Practice Guidelines (2005) of ROP. Screening of ROP is systematically carried out. ni. according to the clinical risk factors. Early detection intervention will prevent progression of the. U. disease.. However, ROP may not always be predicted from the clinical risk factors alone. In some cases, babies with low risk factors show aggressive form of disease while some babies with very high risk show mild disease and resolves spontaneously (Agarwal, Agarwal, Buratto, Apple, & Ali, 2002; Wheatley, Dickinson, Mackey, Craig, & Sale, 2002). This suggest that other factors are involved in the development of ROP.. 20.

(22) These factors can be genetic variants, which may influence the abnormal development of the premature retina there re evidences that show a strong number of variants that increases the risk of ROP (Bizzarro et al., 2006). This study was conducted with the aim to identify genetic polymorphism and to comprehensively analyze and compare the cytokine levels in ROP patient in Malaysian population, as to provide further understanding on disease factors and mechanism. The outcome of this study may suggest other potential therapeutic option such as. a. pharmacokinetic or gene therapy for future treatment preference in ROP.. ay. 1.1.1 Background. al. Early in 1940’s, ROP, also known as retrolental fibroplasia (RLF) is first described by. M. Terry(1942), when he noted the development of membranes on the retina in a visually impaired infant. Henceforth, more cases were described and identified by other neonatologist throughout. of. the world. Through serial examinations, physicians discovered that the condition is not congenital but developed after birth, either because of environmental influences or clinical. si. ty. factors that led to the development of the disorder (Phelps, 2001).. ve r. Other than oxygen, other factors influencing ROP development are light exposure, bacteremia, anemia, vitamin defiencies, electrolyte disturbance and hypercapnia (Pierce E, 2000). Oxygen supplement became the focus of research in ROP risk factors as it became the. ni. leading cause of peadiatric blindness by the early 1950s (Silverman, 1980). Limitation of oxygen. U. use with more cautious monitoring resulted in decreased ROP incidence and disappeared in early 1960s. Thus, the disease was rarely introduced to the medical student and the clinical practice was not actively discussed (Phelps, 1992).. In the early 1980s, re-emerging era of ROP began with the advancement of neonatal intensive care at an astounding rate. At this point of time, there was no general classification of the disease; no agreement on identifying the ROP severity or progression and no specific intervention and guidelines were available. (Phelps, 2001).. 21.

(23) In 1981, ophthalmologist and neonatologist gathered in an international meeting and developed a system, known as International Classification of ROP (ICROP). It allows more accurate determination and diagnosis of ROP across time and space. It also provides a more accurate documentation for clinical trials. In 1983, RLF term is change to ROP and this medical terminology is used henceforth.. Now, ROP screening is a routine examination carried out in all high-risk infants.. a. Development of prevention and treatment method of ROP disease is important as it will affect. ay. the visual acuity of the child.. al. 1.1.2 Prevalence of ROP. M. ROP prevalence around the world varies depending on the advancement of the neonatal care services. In the developed countries, where the financial resources are unlimited with economy. of. stability, the neonatal units are equipped with technology and trained qualified personnel to. ty. support and provide optimum care for extremely premature newborns. Based on United Nation. si. Development Program- Human Development Index (UN-HDI), the rate of ROP prevalence. ve r. among these countries reach to 5-8%(B. Darlow, Horwood, & Clement, 1993). However, this is not so in the medium and less developed country.. ni. ROP prevalence in very low birth weight infants were studied in many populations throughout. U. the world, including Egypt (19.2%)(Mohamed et al., 2009)India (24.0%)(Murthy, Babu, Benakappa, & Murthy, 2006), Brazil (25.5%)(Fortes Filho, Eckert, Procianoy, Barros, & Procianoy, 2009), Pakistan(32.4%).These does not differ in southeast asian countries, like Singapore (29.2%), China (10.8%) (J. Chen & Smith, 2007) and Malaysia (17.4%). Low birth weight and gestational age were known as significant risk in ROP in these populations (B. A. Darlow, Horwood, & Clemett, 1992). 22.

(24) A survey conducted in schools with visual impairments (schools with visual impairments) showed that more than 10% of the children with visual impairments were affected by ROP. ROP is believed to be one of the most contributing factors for the higher number of blind children reported in Asian countries (Koay, Patel, Tajunisah, Subrayan, & Lansingh, 2015).The impact of blindness due to ROP is significant to the society. To date, gestational age and birthweight are the most significant contributing factors. However, it is important to look for other factors that. a. may contribute to the management and hopefully reduce the incidence of ROP particularly in the. ay. Malaysian population.. al. 1.2 The Retina. M. 1.2.1 Embryology of retina. Retina is a photosensitive layer that converts light images into nerve impulses. It is divided. of. into two main components, neurosensory retina and retinal pigment epithelium (RPE). In normal infant, retinal development begins at 16th week gestation and vascularization process completed. ty. after reach 40 weeks of gestational age (Asano & Dray, 2014). Formation of retinal vasculature. si. starts from the center of the optic nerve where an adequate level of growth factors (ie; Vascular. ve r. Endothelial Growth Factor (VEGF) and Insulin Growth Factor (IGF)) secreted (Heidary, Vanderveen, & Smith, 2009).. ni. This will then stimulate the migration and development of retinal vascular network. These. U. factors, together with other component are normally alleviated in the uterus before birth. In premature infant, this development interrupted and forced to progress in the extrauterine environment. Its either vascular growth in the retina slows or abnormally grows which lead to retinal detachment.. 23.

(25) 1.2.2 Neurosensory retina. The neurosensory retina is a thin transparent layer of neural tissue. Retinal cell layers are arranged in a highly organized manner and in histological sections appear eight distinct layers as shown in Figure 1.5.. The most sensitive and most important structure for vision is called macula, in which comprises of high concentrations of cones which responsible for a fine vision (acuity) and high. a. quality color vision. The specialized area, fovea which is for a high quality vision and the rest of. ay. the retina is for a peripheral vision.. al. Light strike the retina and passes through photoreceptors, rodes and cones. It is then converts. M. into the electrical energy and modified the electrical signals to the ganglion cells (axons) by connector neurons. These axons then track along the retina surface and enter the optic nerve for. of. translations(Batterbury, Bowling, & Kanski, 1999).Rods are used for detection of movement and. ty. for vision in a low light level. Rods and cones are distributed to the entire retina for their. si. functional requirements (Jogi, 2009). External to the neurosensory retina lays the RPE, a single. U. ni. ve r. layer of pigmented cells which are essential to the photoreceptor physiology.. 24.

(26) a ay al M of ty. Figure 1.1 :Retinal layer. U. ni. ve r. si. Figure 1.1 image was from Saidha et al.,(2011). Retrieved January 8 2018 from http://brain.oxfordjournals.org/content/134/2/518. 25.

(27) 1.2.3 Retinal pigment epithelium. Retinal pigment epithelium (RPE) is a monolayer pigmented cell forming part of the retinal barrier. It is derived from the neural tube tissue which undergoes differentiation into transporting epithelium (Bok, 1993). The main function of RPE is to absorb light which focused by the lens on the retina and provide the maintenance to the photoreceptor. The voltagedependent ion conductance principles of the RPE enable the stabilization of ions composition in. a. the sub-retinal spaces which is essential in photoreceptor excitation(Marmor & Wolfensberger,. ay. 1998). Besides, RPE also metabolically insulate and support the overlying neural retina by transporting water and metabolic end products from the sub-retinal space to the blood.. al. Furthermore, it is also responsible for the nutrients level as it is also take out nutrients such as. M. retinol and fatty acids.. of. RPE is a part of complex system which control the permeability, vascular supply, growth, repair and other processes vital to the function of retina. The surrounding tissues behaviors are. ty. affected by the several growth factors secreted by RPE which essential for the maintenance of. si. the structural integrity of the retina and choriocapillaries. RPE cells recycle vitamin A for the. ve r. formation of photopigments, transport water and metabolites, renew photoreceptors and help to reduce damage by scattered light. Impairment of RPE function, in effect of aging can lead to loss. ni. of retinal function (Agarwal et al., 2002).. U. Factors produced by the RPE include platelet-derived growth factor (PDGF), pigment. epithelium-derived factor (PEDF), vascular endothelial growth factor(VEGF), fibroblast growth factor (FGF), and transforming growth factor (TGF)(Adamis et al., 1993). PDGF carries specific roles in cell growth and healing process which modulates the maintenance of the cell. PEDF serves as neuroprotectant and structural maintenance and vascular inhibitor for the endothelial cell proliferation. VEGF stimulate the normal growth of vascular tissues and prevents cell apoptosis(Spilsbury, Garrett, Shen, Constable, & Rakoczy, 2000). As for. 26.

(28) inflammation and regeneration , FGF and TGF will be responsible to insist for moderate stable condition (Park & Hollenberg, 1989).. 1.3 Pathogenesis of Retinopathy of prematurity Retinopathies is group of diseases that happen when retina is damaged which may cause vision impairment. There are several types of retinopathy including diabetic retinopathy, hypertensive retinopathy, central serous retinopathy and retinopathy of prematurity (ROP).. a. Pathogenesis of ROP was described in two phase hypotheses in pathogenesis of ROP (M. U. ni. ve r. si. ty. of. M. al. ay. Elizabeth Hartnett & Penn, 2012).. Figure 1.2 Pathogenesis of ROP Figure 1.2 image was from Smith. L (2003). Retrieved August 10 2019 from https://www.sciencedirect.com/science/article/pii/S1084275603001192. 27.

(29) In phase 1 ROP, where there is avasocessation period, and phase 2 where there is a vasoproliferation process. In phase 1, there is hyperoxia-induced vasocessation where the retinal vasculature is exposed to excessive oxygen levels. Insulin growth factor (IGF) was reduce from normal level and this also suppressed vascular growth factors (VEGF) and results in arrested. a. development of retinal blood vessels.. ay. In Phase 2, a hypoxia-induced vasoproliferation stage where IGF level was increase, VEGF is also elevated which results in abnormal growth of retinal blood vessels. The dynamic. al. relationship between oxygen supplementation is uncertain, as both hyperoxia and hypoxia both. M. plays a role in ROP pathogenesis (M Elizabeth Hartnett & Penn, 2012; McInnes, 2012). The effect growth factor and cytokines level in blood also suggested to play a role in mechanism of. of. ROP.. ty. Comprehensive study about the ROP mechanism will also enhanced understanding of. si. pathogenesis of adult ischemic retinal vasculopathies like diabetic retinopathy (diabetis mellitus. ve r. complication) and age related macular degeneration (visual impairment in erderly) which potent an adequately similar pattern. Mechanism of ROP pathogenesis were described involve ocular. ni. circulation, vasoobliteration and neovascularization.. U. 1.4 General Classification of ROP General classification of ROP (International Classification of Retinopathy of Prematurity,. ICROP) described levels of severity of ROP based on several parameters: zone, stage, extent of stage and presence of plus disease. In previous classification by Reese et al., the International classification of acute ROP involved retinal detachment and ROP sequelae. There is a expanded version of classification to ensure consistency between centers and countries.. 28.

(30) Two concepts in the expanding revision published were pre-plus disease and aggressive posterior ROP (AP-ROP). There are four parameters described in ICROP classification including severity, location, extent and plus disease. Progression of surgical intervention were also reported.. 1.4.1 Severity and plus disease. ROP severity is define according to stages (stage 0-5) which based on clinical presentation. Figure 1.3 Stages of ROP. ni. ve r. si. ty. of. M. al. ay. a. between the vascularized and avascular retina.. U. Figure 1.3 image was retrieved on August,10 2019 from https://nei.nih.gov/health/rop/rop. Clinical presentation of stages of ROP as presented in figure 1.3 were each described as below;. Stage 1; there is presence of distinct demarcation lines between perfused and non-perfused (immature) retina. Spontaneous regression usually occurs when the retinal vessels eventually extend peripherally and perfuse the immature retina (Fletcher & Corrêa, 2011). This is the. 29.

(31) mildest form of ROP, and requires no treatment. These babies will usually grow up with normal vision. Stage 2; a ridge develops at the demarcation line. Like in stage 1, these eyes may improve without treatment. After few weeks the blood vessels start to grow and the retina become fully vascularized. Stage 3; extensive development of new vessels grows on the posterior surface of the ridge or. a. anterior towards the vitreous cavity. It known as severe state of abnormal blood vessel growth.. ay. Patient who is diagnosed with stage 3 with a plus disease require treatment immediately to prevent retinal detachment.. al. Stage 4; fibrovascular tissue will start to pull the retina and will cause a partial retinal. M. detachment. These tissues will cause bleeding and fibrovascuar tissues may pulls the retina and blood vessels away from the wall of the eye and even causes bleeding.. of. Stage 5; The avascularized retina will fully detach and this will causes severe visual impairment. ty. and even blindness (Gole et al., 2005; Pediatrics, 1984).. si. Plus disease; Described more florid stage of ROP. The initial sign of plus disease is tortuosity. ve r. of the retinal arterioles and congestion of the retinal veins close to optic disc. Other signs include iris vascular engorgement, pupillary rigidity and hazy vitreous which indicate progressive. ni. vascular incompetence. It may appear at any stages of ROP and often use as indicator early. U. severe ROP. Plus disease requires treatment immediately and increased like hood of an unfavorable outcome(Albert & Jakobiec, 2000).. Pre-plus disease;. The severe form is plus disease while the inadequate characteristics like posterior vascular abnormalities with insufficient arteriolar tortuosity does not classified to plus but consider as pre-plus, as it still demonstrate more arterial tortuosity and more venous dilation than normal. It also increase the possibility of peripheral retinopathy to happen. In time, the. 30.

(32) abnormalities of the vessel may progress to plus disease as the vessels dilate and become more tortuous. Aggressive posterior ROP (APROP); Define as the severe form of ROP, rapidly progressing and ill-defined nature of retinopathy. Characterized by distinctive appearance of active \ plus disease at posterior location either zone 1 or zone II of the vascular abnormality at the junction of vascular and avascular. It is difficult to differentiate arterioles and venules in APROP. Stage. a. 1 to stage 2 progression may not been observed, and the flat network of vessels is easily be. al. ay. missed. (Gole et al., 2005)(ROP thesis). M. 1.4.2 Location. By anatomical, retina is divided according to zones, zone 1, 2 and 3. The divided zones. U. ni. ve r. si. ty. of. centered on the optic disk. Details of the measurement of zones briefly described as below;. Figure 1.4 Location of zones and extent of ROP Figure 1.4 image was retrieved August 10 2019 from https://www.rnib.org.uk/eye-health/eye-conditions/retinopathy-of-prematurity. 31.

(33) Zone I: a circle that has a radius twice the disk-foveal distance approximately about 30º.. Zone II: extends from the edge of zone I to the ora serrata on nasal side and encircles the anatomic equator. Zone III: includes all part temporally, superiorly and inferiorly anterior to zone II. No anatomic landmarks identifying zone III, when the directly nasal retina is fully vascularized indicate zone. a. III has been entered. ay. 1.4.3 Extent. al. The extent of the affected sites refers to the circumferential involvement recorded by clock hour. The clock hours is also known as 30º sectors observation. The starting point of the sectors. M. 3 o’clock position is located on the right of both eyes, meaning the right eye would be on the. ty. 1.4.4 Regression and resolution. of. nasal side and in the temporal side of the left eyes during general examination by practitioner.. si. Regression and resolution of ROP is not well studied because once the need of surgical. ve r. intervention passed, active surveillance does not really a necessity. However, it shown evidence of clinical pathology in diagnosed patient an also a sign that the severe patient responded to. ni. treatment.. U. Rate of ROP regression reported to reflects by respective postmenstrual age (PMA), described. when sign of lessening plus disease and the failure development of ROP. Ridges become thins and breaks up. Then, vessels grow through the ridge into the peripheral avascular retina thus ROP was resolved. The mean time of onset of signs of resolution in children with birth weight less than 1251g was 38.6 weeks (Wright, Spiegel, & Hengst, 2013).. Normally, all stage 1 and stage 2 ROP will undergo up to 80% resolution (Pierce E, 2000). Nevertheless, resolution varies in stage 3 cases, which it may or may not resolve depending on. 32.

(34) severity. One of the signs of incomplete resolution is retinal dragging. There is 74% of all ROP shown sign of resolution by 40 weeks and 90% by 44 weeks of PMA. (Repka, Palmer, & Tung, 2000).. 1.5 Risk Factors ROP is a complex disease that involved many known risk factors and related closely with prematurity complication. Major risk factor include low gestational age and low birth weight are. a. the most powerful predictor (Choo et al., 2009). There are also several other clinical factors had. ay. been implicated to the pathogenesis of ROP such as high oxygen supplemental (Kinsey et al.,. al. 1956), mechanical ventilation (Hussain, Clive, & Bhandari, 1999), blood transfusion (Hay &. M. Bell, 2000, pharmacological factor (B. A. Darlow et al., 1992) and other co-morbidities (Lin, Lin, Tsai, Lin, & Su, 2003).. of. 1.5.1 Gestational age. ty. Averagely, 15 million preterm babies were born each year and preterm birth happens at ratio. si. of 1 to 10 in every delivery (H. Chang et al., 2014). Premature babies define as babies which. ve r. born alive before 37 weeks of pregnancy. Babies are considered very premature when they are born less than 32 weeks and extremely premature for less than 28 weeks.. ni. These group of very and extreme premature babies carried higher risk to ROP disease. Each. U. year, it was an estimated 32,000 premature babies developed severe visual impairment and blindness (Gilbert, 2008). It is somehow inevitable to prevent preterm birth related to certain pregnancy complication (Newnham et al., 2014). This is because pregnancy complications due to many factors and may difference amongst each individual maternal morbididities.. 33.

(35) 1.5.2 Birth weight. Low birth weight has been established as a major risk factor for ROP. Strong evidences shown the development of retinopathy of prematurity was inversely proportional to weight and gestational age at birth.. ROP frequency remains elevated among very low birth weight infants associated with low gestational age as major risk factors. A study in Singapore found that the incidence of ROP in. a. any stages in infants weighing less than 1000g, was as high as 55.4%, with 13.7% having. ay. threshold disease(Shah, Yeo, Ling, & Ho, 2005). Many other studies showed that low birth. al. weight is associated with ROP(Chye, Lim, Leong, & Wong, 1999; Fortes Filho et al., 2009;. M. Inder, Clemett, Austin, Graham, & Darlow, 1997; Lin et al., 2003).. Apart of that, research in Taiwan with very low birth weight infants (<1250 gram) also had. of. been related with threshold disease in ROP supported the theory(Lin et al., 2003).However,. ty. reported data still consider on the overall health condition as an independent factor of ROP.(Ng,. ve r. 1.5.3 Oxygen. si. Shaw, Fielder, & Levene, 1988). Excessive supplemental oxygen during the early postnatal period has been linked to ROP. It. ni. was believed to release of angiogenic factors which is associated with abnormal retinal. U. angiogenesis (Cavallaro et al., 2014; J. Chen & Smith, 2007; Stahl et al., 2010).. Clinical studies supported the association of oxygen and ROP. Transcutaneous PO2 events. over 80mmHg affect in ROP severity and occurrence (Campbell, 1951; Flynn et al., 1992; Kinsey et al., 1977). In the first week of birth, oxygen levels usually crucial for ROP development (Cunningham, Mclntosh, Fleck, & Elton, 1995). This is because oxygen levels affect retinal development of infant and the excess amount of oxygen level may impact the outcome of abnormal retinal vasculature development of infant’s eye.. 34.

(36) High saturation levels of oxygen (94-98%) had shown a higher chance of ROP requiring treatment and as compare to (70-90%) incidence with no additional shift in neurologic morbidity in the latter(M. L. Chen, Guo, Smith, Dammann, & Dammann, 2010). The ideal levels of oxygen use for clinical practice is still ongoing to be specified (Sola et al., 2014; Tin & Gupta, 2007) . In addition, repeated cycles of hypoxia and hyperoxia (and hence repeated oxygen fluctuations) may also contribute to the rapid progression of ROP to more advance stage (Hay & Bell, 2000).. a. Though, risk of oxygen in ROP is often depends on infant health (Chow, Wright, & Sola,. ay. 2003). Decreased oxygen levels will reduce the incidence of blindness in ROP case, increase the. al. survival rate of infants and higher morbidity (D. Bolton & Cross, 1974; Cross, 1973).. M. 1.5.4 Blood Transfusion. Blood transfusion is often needed preterm infants especially those very low birth weight. of. (VLBW) infants. Over 50% of preterm infants require multiple blood transfusions during. ty. hospitalization and at least one blood transfusion during their stay at neonatal intensive care unit. si. (NICU) (Strauss, 1997). Multiple blood transfusions may induce hyperoxia in premature babies.. ve r. This is because adult donor blood’s hemoglobin (HbA) level of affinity to oxygen is lower compared with fetal hemoglobin (HbF). Thus, blood transfusions leads to excessive of oxygen. ni. released to the tissue, ultimately become risk in ROP progression too (Hay & Bell, 2000).. U. Blood transfusion on VLBW infants is important to improve their weight gain, improved. oxygenation and lower the lactic acidosis (Mikaniki, Shirzadian, & Mikaniki, 2012). However, there are few potential risks, including infection, cardiovascular and metabolic complications, elevated oxidative stress. These risk factors associate are believe to be related with chronic lung disease and ROP (R. Cooke, Drury, Yoxall, & James, 1996; Saugstad, 2003; Wheatley et al., 2002). By several reported evidences, a more restrictive transfusion guideline were adopted many neonatal units to reduce the transfusion frequency (Alagappan, Shattuck, & Malloy, 1997;. 35.

(37) Franz & Pohlandt, 2001; Maier et al., 2000; Widness et al., 1996). Nevertheless, researcher still identify potentials and safety benefits of restricting blood transfusion.. 1.5.5 Pharmacological factors. There are several pharmacologic intervention studies to prevent the progression of ROP (Beharry, Valencia, Lazzaro, & Aranda, 2016) and some have also shown a promising effect to the patient of ROP, despite the oxygen therapy control alone. As we know, oxygen has been one. a. of the important risk factor of ROP since excessive usage of oxygen may lead to damaging effect. ay. on the retina.. al. In respiration process, reactive oxygen species (ROS) were also produced as a byproduct. M. which may trigger lipid peroxidation reactions and DNA damage. Inadequate or excessive ROS in antioxidant system will lead to various pathologies such as hypoxia, vascular dysfunction and. of. ROP development. Several studies had shown a positive effect of antioxidants for treatment and. ty. prevention of ROP(Spinnato et al., 2007).. si. Study by Darlow et., al (1992) found that preterm infants treated with indomethacin were. ve r. 1.5 times more likely to develop ROP than untreated infants. Up to 68% of premature babies who received dopamine at first week of life had higher ROP incidence (Allegaert et al.,. ni. 2004).Xanthine had also been identified as predictor in acute ROP (Hammer et al.,. U. 1986).Prenatal steroids are routinely administered to all mothers with preterm labour to prevent respiratory distress syndrome. However, prenatal steroids treatment was also linked to ROP development and progression (Aggarwal, Agarwal, Deorari, & Paul, 2002; C.-S. Yang, Chen, Lee, Hsu, & Liu, 2001). 1.5.6 Co-morbidities of prematurity. Systemic co-morbidities of prematurity was recently found to play a significant role in the development of ROP (Lin et al., 2003) such as bronchopulmonary disease, intraventricular. 36.

(38) haemorrhage (Liu et al., 2005), sepsis (Liu et al., 2005; Shah et al., 2005) respiratory distress syndrome and intra-ventricular haemorrhage (Watts, Adams, Thomas, & Bunce, 2000). Other risk factors that has been associated with ROP are candidemia (Karlowicz, Giannone, Pestian, Morrow, & Shults, 2000), carbon dioxide tension (Liao, Lai, & Kuo, 2000), raised in serum bilirubin levels (Yeo, Perlman, Hao, & Mullaney, 1998) and assisted conception(Watts & Adams, 2000) and maternal preeclampsia (Hussain et al., 1999). Researcher suggest that some. a. of these co-morbidities might be related to the pathway of retinopathy development.. ay. 1.6 Management of ROP. al. The ROP management guideline in Malaysia requires that screening of premature babies be. M. performed from as early as 4-6 weeks after birth. Premature babies eligible for ROP screening include those with birth weight less than 1500 g, or gestational age less than 32 weeks, or any. of. other infants with unstable condition (high oxygen levels, mechanical ventilation, respiratory distress syndrome, intraventricular haemorrhage, anemia, chronic hypoxia, bradycardia and. ty. seizures) that increased the risk of ROP. Screening of all premature infants is conducted at. si. regular intervals weekly or fortnightly until retina is fully vascularized, which usually occur in. ve r. 36-40 weeks of gestational age.. Examination of normal fundus of extremely premature infants might be tricky since it is. ni. difficult to visualize the detail condition of blood vessels in the retina since it is thin and straight.. U. Moreover, timing of examinations is important so it can be treated early before becoming more severe.. 1.6.1 Treatment of ROP. Nowadays, good neonatal care practices and advance technology in developed countries has improved the survival rate more premature infants up to 6-8%. This leads to more cases of ROP(Domanico, Davis, Coleman, & Davis, 2011). Although ROP cases increase over the time, it is still in constant phase around the world (Martin, Fanaroff, & Walsh, 2011). Early detection. 37.

(39) through timely screening guidelines, effective follow up appointment and relatively good timing of treatment is important in preventing blindness in infants diagnosed with ROP (Martin et al., 2011).. 1.6.1.1 Indication of treatment. In severe cases of ROP, laser photocoagulation was recommended as treatment option.. ve r. si. ty. of. M. al. ay. close observation for progression of ROP (Gole et al., 2005).. a. Type 1 ROP is treated with laser photocoagulation treatment to the eyes within 48-72 hours and. Figure 1.5 ROP classification system by zones. ni. Figure 1.5 image was retrieved on August,10 2019 from http://www.adhb.govt.nz/newborn/guidelines/developmental/rop.htm. Type 1 of ROP. U. i. 1. Zone 1, any stage of ROP with plus disease 2. Zone 1, stage 3 with or without plus disease 3. Zone 2, stage 2 or 3 with plus disease These eyes have highly active ROP and should be considered for early treatments.. 38.

(40) ii. Type 2 of ROP. 1.Zone 1, stage 1 or 2 with no plus disease 2. Zone 2 stage 3 with no plus disease. 1.6.1.2 Treatment options. (a) Cryotheraphy (CRYO-ROP). a. Cryotheraphy is a technique used to destroy a variety type of abnormal cell by using an. ay. extremely cold liquid or instrument while preserving the surrounding cells from injury. It is applied trans-sclerally to the avascular zone anterior to the acute ROP lesion. Using a retinal. al. probe, the cryotherapy lesions are applied confluently. The endpoint of cryotherapy is the. M. appearance of whitening of the retina due to freezing. Ocular complications of cryotherapy include eyelid edema, lacerations and haemorrhage of the conjunctiva and preretinal and vitreous. of. haemorrhage(Hoyt & Taylor, 2012).. ty. The CRYO-ROP study (C. f. R. o. P. C. Group, 1988, 1990a, 1990b, 2001; Quinn et al., 1996). si. showed that cryotherapy for threshold ROP produced a significant benefit for structural status of. ve r. the eye and for vision. However, retinal ablation in eyes with severe ROP did not necessarily result in normal retinal structure or the development of visual acuity in the normal range.. ni. (b) Laser treatment. U. Introduction of portable diode and argon lasers delivered through indirect ophthalmoscope. encourage the usage of laser for treatments of acute ROP. Laser therapy is one of the standard treatments for advance ROP and it became a procedure of choice in all over the world. This is because it is effective, fast and affordable in terms of operational cost. Laser can be accurately placed, is simpler than cryotherapy to administer in experienced hands, and may be easier to deliver and more effective in zone 1 disease. It causes less tissues destruction and fewer. 39.

(41) complications. Complications of laser include corneal, iris, and lens burns and also cataract. Retinal or vitreous hemorrhage may occur but is probably not laser specific(Good, 2004).. (c) Anti-VEGF treatment The BEAT-ROP (bevacizumab eliminates the angiogenic threat of retinopathy of prematurity) study provides evidence that anti-VEGF treatment may result in outcomes at least as good as or better than, those currently achieved with laser photocoagulation (Régnier,. a. Malcolm, Allen, Wright, & Bezlyak, 2014) Randomized clinical trials need to be undertaken to. ay. determine the ocular and systemic adverse effect. These studies must have sufficient follow-up. al. to determine the visual function and neurodevelopmental outcomes (Vishak J John, 2012). It will require series of clinical trials and testing must be done to prove effectiveness of study drug used. (d) Retinal detachment surgery. of. M. for treatment and to protect patient’s safety.. ty. Retinal detachment happens when the retina is pulled away from the blood vessels which. si. essentially supply oxygen and nutrients. Sometimes, vitreous intact with the retina might move. ve r. the retina away and tear the retina. Fluid will fill through the part and lifting the retina off from eye. Retina will not be functioning when it is detached, and vision will automatically become blurry. If detachment of retina is not treated promptly, serious problem may occur and causes. U. ni. blindness.. 1.7. Complication of ROP Infant who are diagnosed with ROP are at risk of developing other ocular complications. such as myopia, squint (resulting from the dragging of the optic disc), amblyopia, retinal detachment, decreased visual acuity, macular ectopia, nystagmus, cataract, microcornea and angle-closure glaucoma (Drenser, Trese, & Capone Jr, 2010; Kaiser & Trese, 2001; Kaiser, Trese, Williams, & Cox, 2001; R. Robinson & O'Keefe, 1993; Tufail et al., 2004; Ward & Beachy, 2003). Studies have demonstrated a strong correlation between degree of ROP with. 40.

(42) presence of high myopia (nearsightedness), and as much 80 % of infants with ROP reported to develop (Garcia-Valenzuela & Kaufman, 2005; B. T. Smith & Tasman, 2005). The occurrence of squints is reported to be 23%-47% in infants who have ROP and 10%-20% in premature infants without ROP(Kushner, 1982).. 1.8 Studies on ROP Studies in ROP were divided into fundamental and clinical studies. Most of it including. a. aspects of genetics and cytokine association to the disease mechanism. Generally, studies were. ay. performed by collecting demographics data and clinical observation data (PRO-forma) as to. al. obtain related information that will facilitate in the research. The demographic data of patients,. M. medical history including description of procedures, ailments or diseases and assessment of various clinical parameters were collected and recorded in the PRO-forma form. This data was. Genetics study. ty. 1.8.1. of. then converted into excel data and cleaned before used as dataset in SPSS.. si. For decades, variation in genes had been studied by geneticist on its contribution to various. ve r. disease variation. It is either identifying the individual genes with variations on mendelian patterns of inheritance basis in single gene disease (autosomal dominant, autosomal recessive. ni. and X-linked) (Kondo et al., 2013; Toomes et al., 2004) or finding the association of genetic. U. susceptibility to disease as sequence of event and interrelated with effects of many genes (Z. Chen et al., 1993). Great concerns have been made recently to the association between particular genetic polymorphism and mutation to a specific disease. Previously, several mutation and polymorphism of norrie disease (ND) gene, vascular endothelial growth factor (VEGF) gene, frizzled 4(FZD4) and low density lipoprotein related protein 5 (LRP5) ,endothelial nitric oxide synthase (NOS3) gene, transforming growth beta-1 gene (TGFB1),complement factor H (CFH),angiotensin II receptor type 1 (AGTR1) gene, and brain derived neurophatic factor 41.

(43) (BDNF) gene were found to be associated with ROP (refer table 1.2) (M. E. Hartnett et al., 2014; Poggi et al., 2015; Shastry, Hejtmancik, Plager, Hartzer, & Trese, 1995). However, this data is inconsistent and varies among researcher throughout the world. To date, genetic compositions which is specific to the development of ROP are still unknown because of complexity of disease and various interrelated factors in mechanism of the disease.. Some researcher focuses on targeted gene candidates, though there may be other unidentified. a. variants that are left untested but important to ROP. However, identification of variants and. ay. affected genes that contribute to the disease are often complicated (Balasubramanian, Brown, &. al. Reed, 2002). Currently, with the advance technology, it is now possible to simultaneously cross-. M. examine million sites in any individual’s genomic DNA with the purpose of finding associations between the diseases and carried genetic variation.. of. Therefore, screening of samples by non-biased sequencing approach might give a better point. ty. of view on potential genetic polymorphisms and its association with ROP. Polymorphism is. si. different with mutation. A mutation is defined as changes in DNA sequence away from normal.. ve r. This implies there is a normal allele that is prevalent in the population and that the mutation changes this to a rare and abnormal variant. In contrast, a polymorphism a DNA sequence variation that is common in the population. In a sequencing project, Single Nucleotide. ni. Polymorphisms (SNPs) and DNA mutations are defined as DNA variants detectable in >1 % or. U. <1 % of the population, respectively. (Karki, Pandya, Elston, & Ferlini, 2015).. 1.8.1.1. Single Nucleotide Polymorphism. Single Nucleotide Polymorphism (SNP) is a single base pair in genomic DNA which is difference in sequence alternative found in exons, introns, intergenic regions in promoters and enhancers. SNPs are more likely to yield, upon collection a functional or physiologically relevant allele than another polymorphism. It carried a tremendous effect on molecular biology and. 42.

(44) genetics. This includes genetic data of various types and resolution, including DNA sequences, genotype, haplotype, allele sharing, gene expression and protein expression.. SNP is single base pair position in genomic DNA at which different sequence alternatives (alleles) exist in normal individuals in some population, where in the least frequent allele has abundance of 1% or greater. The information provided by the SNP databases is a very important and valuable resource for research, Nevertheless, their usefulness is determined by quality and. ay. databases, issues of quality are particularly important.. a. coverage, Since there are literally hundreds of sources that deposit SNP information into. al. Previous genetic studies in ROP had been done to the targeted gene, either it is screened on. M. individuals with severe ROP disease or it is compared with normal individuals (Table 1.2) (Kondo et al., 2013; Poggi et al., 2015). Targeted genes studied in ROP often involved several. of. selected candidates polymorphisms by researchers which studies the association with genetic. ty. eye disease, systemic disease such as inflammation, angiogenesis and many others (Amano et. si. al., 2003; Kondo et al., 2013) These factors triggered unbalanced regulation of these targeted. ve r. genes which may influences the development of abnormal vascular activity in the eyes mainly in ROP.. ni. In other diseases, SNP was often use to investigate potential gene marker associated to disease. U. mechanisms. Studies in ROP often start with selection of potential genes, therefore they might be some other marker that left untested. Method of non-biased wide and deep screening of potential SNP marker such as whole exome sequencing can detect variant in sequence of several genes, that may affect the regulation of protein or metabolic condition in the body, that may leads to ROP mechanism. Several SNPs in genes were chosen in this study based on reported studies in disease-gene database (DisGeNET) (Table 1.1).. 43.

(45) Table 1.1: DisGeNet disease-gene database1 Associated disease. Liver mitochondrial matrix flavoenzyme that catalyzes the oxidative demethylation of sarcosine. study. Diabetic Retinopathy (DR). Shared disease genes: SDS,SDHB,TP53,VEGFA,CDKN2A,MTO R,INS,TNF,HIF1A,IGF1R. 1. M. ve. rs i. Shared disease genes : VEGFA,IL6,TNF,TP53,IGF1,MMP9,PTGS 2,TGFB1,ICAM1,IL1B. Uveitis, Diabetic Retinopathy, Retinopathy of prematurity. -Expression of vascular endothelial growth factor and pigment epithelial-derived factor in a rat model of retinopathy of prematurity.. of. Neurotrophic protein; induces extensive neuronal differentiation in retinoblastoma cells. Potent inhibitor of angiogenesis.. -Polymorphisms of sorbitol dehydrogenase (SDH) gene and susceptibility to diabetic retinopathy. -Association between sorbitol dehydrogenase gene polymorphisms and type 2 diabetic retinopathy.. ty. SERPINF1. Related studies. Review. a. SARDH. Gene function. Gene polymorphism association in DR, related to angiogenesis mechanism. Shared disease genes including VEGFA and IGF1R which likely to associated with the protein involve in progression of ROP disease.. al ay. Gene. -Genetic contributions to the development of retinopathy of prematurity.. Gene is also known as PEDF , which regulate angiogenesis through multiple mechanisms, including downregulation of VEGF expression, decreased levels of PEDF have been associated with proliferative diabetic retinopathy and agerelated macular degeneration (AMD). A potential treatment of ROP, neuroprotective and anti-inflammatory , potentially may regulate VEGF-induced pathologic angiogenesis, and has been safely tolerated in a human clinical trial.. U. ni. information and data in the table were adapted and retrieved from http://www.disgenet.org. 44.

(46) Required for normal renal development and establishment of left-right axis. Probably acts as a molecular switch between different Wnt signaling pathways. Inhibits the canonical Wnt pathway by targeting cytoplasmic disheveled (DVL1) for degradation by the ubiquitinproteasome. Involved in the organization of apical junctions in kidney cells together with NPHP1, NPHP4 and RPGRIP1L/NPHP8 (By similarity).. Nephronophthisis 2, renal dysplasia and retinal aplasia (disorder), retinitis pigmentosa. ty. Age related macular degeneration.. Reported presence of RP in a patient with NPHP type 2 and INVS mutations . Present in chronic renal disease that may also associated with eye problem.. -Lack of NPHP2 mutations in a newborn infant with Joubert syndrome-related disorder presenting as end-stage renal disease. -New loci and coding variants confer risk for age-related macular degeneration in East Asians.(rs10507047). Identified significant important different in genome-wide and exome-wide study of AMD provides new insights into the genetic mechanisms of AMD in East Asians.. ve. rs i. May activate CDC42, a member of the Ras-like family of Rho- and Rac proteins, by exchanging bound GDP for free GTP. May play a role in regulating the actin cytoskeleton and cell shape.. -Retinitis pigmentosa and renal failure in a patient with mutations in INVS.. Review. -Active transport and diffusion barriers restrict Joubert Syndromeassociated ARL13B/ARL-13 to an Inv-like ciliary membrane subdomain.. Shared disease genes: NPHP3,INS,NPHP1,UMOD,ACE,MUC1,IFNG ,TNF,IL6,MMP2. FGD6. Related studies. al ay. Associated study disease. M. INVS. Gene function. of. Gene. a. Table 1.1:DisGeNet disease-gene database1 (continue-1). 1. ni. Shared disease genes: ABCA4,ANGPT2,CX3CR1,SERPINF1,RORA ,RAC1,ANXA2,PLEK,FLT1. U. information and data in the table were adapted and retrieved from http://www.disgenet.org. 45.

(47) Table 1.1:DisGeNet disease-gene database1 (continue-2) Gene. Gene function. Associated study. Related studies. Review. Knobloch synrome. rs i. Corneal Diseases, Keratoconus. ve. Shared disease genes;. ty. VEGFA,TNF,TP53,IL6,EGFR,MMP9,CD KN2A,BCL2,PTGS2,IL1B. Minor connective tissue component of nearly ubiquitous distribution. Type V collagen binds to DNA, heparan sulfate, thrombospondin, heparin, and insulin. ni. COL1A1,IL6,TGFB1,IGF1,MMP9,COL1A 2, VEGFA,ILIB,MMP3,COL3A1. U. Gene is related to Knobloch Syndrome which is associated with degeneration of eye. Potential role as anti-angiogenic factor. of. Shared disease genes:. COL5A1. -Inhibition of retinal neovascularization by intraocular viral-mediated delivery of anti-angiogenic agents.. al ay. COLA18A probably plays a major role in determining the retinal structure as well as in the closure of the neural tube. Endostatin potently inhibits endothelial cell proliferation and angiogenesis. May inhibit angiogenesis by binding to the heparin sulfate proteoglycans involved in growth factor signaling. M. COL18A1. a. disease. -New loci associated with central cornea thickness include COL5A1, AKAP13 and AVGR8.. A potential important variant in ROP.. -Genetic association of COL5A1 variants in keratoconus patients suggests a complex connection between corneal thinning and keratoconus. -Evaluating the association between keratoconus and the corneal thickness genes. 1. information and data in the table were adapted and retrieved from http://www.disgenet.org 46.