Exploration of the molecular genetics of exudative age-related macular degeneration.

Tam, Oi Sin Pancy.Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.Includes bibliographical references (leaves 101-128).Abstracts in English and Chinese.Table of ContentsTitle page --- p.iAbstract --- p.iii摘要 --- p.viAcknowledgements --- p.viiiTable of Contents --- p.ixList of Figures --- p.xiiiList of Tables --- p.xvAbbreviations --- p.xviiPublications related to the work of this thesis --- p.xxConference Presentations related to this thesis --- p.xxiChapter Chapter 1 --- IntroductionChapter 1.1 --- AMD --- p.1Chapter 1.2 --- Epidemiology --- p.4Chapter 1.3 --- Classification --- p.5Chapter 1.3.1 --- Dry AMD --- p.6Chapter 1.3.2 --- Wet/Exudative AMD --- p.9Chapter 1.4 --- Etiology and risk factors of AMD --- p.10Chapter 1.4.1 --- Gender and Ethnicity --- p.10Chapter 1.4.2 --- Smoking and vascular factors --- p.11Chapter 1.4.3 --- Genetic Factor --- p.11Chapter 1.5 --- Molecular Genetics of AMD --- p.12Chapter 1.5.1 --- Linkage studies --- p.12Chapter 1.5.2 --- Candidate genes search --- p.15Chapter 1.5.3 --- Genome-Wide Association --- p.18Chapter 1.5.3.1 --- Complement Factor H --- p.20Chapter 1.5.3.2 --- LOC387715 --- p.22Chapter 1.6 --- Statistical Analysis --- p.23Chapter 1.6.1 --- Genotyping --- p.23Chapter 1.6.2 --- Quality Assessment of Genetic Data --- p.24Chapter 1.6.3 --- Association Analysis --- p.26Chapter 1.6.4 --- Population Stratification --- p.26Chapter 1.6.5 --- Haplotype Analysis of Multiple SNPs --- p.26Chapter 1.6.6 --- Population Attributable Risk --- p.27Chapter 1.6.7 --- Interaction analysis --- p.28Chapter 1.7 --- Objectives --- p.28Chapter Chapter 2 --- Materials and Method --- p.30Chapter 2.1. --- Materials --- p.30Chapter 2.1.1. --- Proteins --- p.30Chapter 2.1.2. --- Chemicals --- p.30Chapter 2.1.3. --- Solutions and Buffers --- p.31Chapter 2.1.4. --- Reagents and Kits --- p.31Chapter 2.1.5. --- Apparatus --- p.32Chapter 2.1.6. --- Softwares --- p.32Chapter 2.2. --- Methods --- p.32Chapter 2.2.1. --- Study Subjects --- p.33Chapter 2.2.2. --- AMD atients --- p.33Chapter 2.2.3. --- Control Subjects --- p.34Chapter 2.2.4. --- DNA Extraction and Quantification --- p.34Chapter 2.2.5. --- Whole genome wide SNP scanning --- p.34Chapter 2.2.6. --- HTRA1 Genotyping --- p.38Chapter 2.2.6.1. --- Serial Polymerase Chain Reactions --- p.38Chapter 2.2.6.2. --- Cycle sequencing --- p.40Chapter 2.3. --- Statistical analysis --- p.40Chapter 2.3.1. --- Hardy-Weinberg Equilibrium Test --- p.40Chapter 2.3.2. --- Association Analysis: Linkage disequilibrium --- p.42Chapter 2.3.3. --- Haplotype Analysis --- p.43Chapter 2.3.4. --- Interaction Analysis --- p.43Chapter Chapter 3 --- Results --- p.46Chapter 3.1. --- Genome-wide Association Study of Exudative AMD --- p.46Chapter 3.1.1. --- Genotyping and Association Analysis --- p.46Chapter 3.1.2. --- Haplotype Analysis --- p.50Chapter 3.2. --- HTRA1 Genotyping --- p.57Chapter 3.2.1. --- Association Analysis --- p.57Chapter 3.2.2. --- Haplotype Analysis --- p.68Chapter 3.2.3. --- rsl 1200638 - Smoking Interaction --- p.68Chapter 3.2.4. --- rsl 1200638 - rs800292 Interaction --- p.74Chapter Chapter 4 --- Discussion --- p.79Chapter 4.1. --- Genome-wide Association Study of Exudative AMD --- p.79Chapter 4.1.1. --- Limitations and Concerns of Genome-Wide Association Study --- p.84Chapter 4.2. --- HTRA1 Genotyping --- p.85Chapter 4.2.1 --- Association and Haplotype Analysis --- p.85Chapter 4.2.2. --- HTRA1 --- p.87Chapter 4.2.3. --- Gene-Environment Interaction --- p.93Chapter 4.2.4. --- Gene-Gene Ineraction --- p.94Conclusions and Future Aspects --- p.97Electronic-Database Information --- p.100References --- p.10

Molecular Genetics of Age-related Macular Degeneration

Age-related macular degeneration (AMD; OMIM # 603075) is an eye disease of the elderly, signs of which appear after the age of 50. In the Western world it is a leading cause of permanent visual loss with a prevalence of 8.5% in persons under 54 years of age and of 37% in persons over 75 years of age. Early forms of AMD may be asymptomatic, but in the late forms usually a central scotoma in the visual field follows severely complicating daily tasks. Smoking, age, and genetic predisposition are known risk factors for AMD. Until recently no true susceptibility genes had been identified though the composition of drusen deposits, the hallmarks of AMD, has suggested that the complement system might play a role in the pathogenesis of AMD. When four groups reported in March 2005, that, on chromosome 1q32, a Y402H variant in the complement factor H (CFH) gene confers risk for AMD in independent Caucasian samples, a new period in the field of genetic research of AMD started. CFH is a key regulator of the complement system. Thus, it is logical to speculate, that it plays a role in the pathogenesis of AMD. We performed a case-control association study to analyse whether the CFH Y402H variant contain a risk for AMD in the Finnish population. Although the population of Finland represents a genetic isolate, the CFH Y402H polymorphism was associated with AMD also in our patient sample with similar risk allele frequencies as in the other Caucasian populations. We further evaluated the effects of this variant, but no association between lesion subtype (predominantly classic, minimally classic or occult lesion) or lesion size of neovascular AMD and the CFH Y402H variant was detected. Neither did the variant have an effect on the photodynamic therapy (PDT) outcome. The patients that respond to PDT carried the risk genotype as frequently as those who did not respond, and no difference was found in the number of PDT sessions needed in patients with or without the risk genotypes of CFH Y402H. Functional analyses, however, showed that the binding of C-reactive protein (CRP) to CFH was significantly reduced in patients with the risk genotype of Y402H. In the past two years, the LOC387715/ high-temperature requirement factor A1 (HTRA1) locus on 10q26 has also been repeatedly associated with AMD in several populations. The recent discovery of the LOC387715 protein on the mitochondrial outer membrane suggests that the LOC387715 gene, not HTRA1, is the true predisposing gene in this region, although its biological function is still unknown. In our Finnish patient material, patients with AMD carried the A69S risk genotype of LOC387715 more frequently than the controls. Also, for the first time, an interaction between the CFH Y402H and the LOC387715 A69S variants was found. The most recently detected susceptibilty gene of AMD, the complement component 3 (C3) gene, encodes the central component of the complement system, C3. In our Finnish sample, an additive gene effect for the C3 locus was detected, though weaker than the effects for the two main loci, CFH and LOC387715. Instead, the hemicentin-1 or the elongation of very long chain fatty acids-like 4 genes that have also been suggested as candidate genes for AMD did not carry a risk for AMD in the Finnish population. This was the first series of molecular genetic study of AMD in Finland. We showed that two common risk variants, CFH Y402H and LOC387715 A69S, represent a high risk of AMD also in the isolated Finnish population, and furthermore, that they had a statistical interaction. It was demonstrated that the CFH Y402H risk genotype affects the binding of CFH to CRP thus suggesting that complement indeed plays an important role in the pathogenesis of AMD.Silmänpohjan ikärappeuma on yleisin näkövammaisuutta aiheuttava sairaus länsimaissa. Suomessa sitä esiintyy noin 40%.lla yli 70-vuotiaista. Se on silmän verkkokalvon tarkan näkemisen alueen sairaus, joka voi aiheuttaa näkökentän keskeiseen osaan laajan puutosalueen, ja voi siten heikentää näkökykyä oleellisesti. Silmänpohjan ikärappeuman kiistattomia riskitekijöitä ovat ikä, perintötekijät ja tupakointi. Molekyyligeneettisten tutkimusten avulla on voitu viime vuosina osoittaa, että silmänpohjan ikärappeumalla on vahva immunologinen tausta. Kaksi ikärappeumaan liittyvistä alttiusgeeneistä komplementtifaktori H ja C3 vaikuttavat ihmisen immuunipuolustukseen. HTRA1 ja LOC387715-geenit ovat uusia geenejä, joiden toiminnasta on vielä vähän tietoa. Näistä LOC3817715 on uusi mitokondrion ulkokalvon proteiini, joka ilmentyy verkkokalvon soluissa ja vaikuttaa todennäköiseltä alttiusgeeniltä. Tässä väitöskirjatyössä on tutkittu edellä mainittuja silmän ikärappeuman alttiusgeenejä 300 suomalaisen potilaan aineistossa. Kyseisten alttiusgeenien variantit assosioituvat ikärappeumaan myös suomalaisilla potilailla ja riskialleelien frekvenssit olivat samaa tasoa kuin muissa kaukasialaisissa populaatioissa. Väitöskirjassa selvitettiin tarkemmin CFH Y402H polymorfismin merkitystä ikärappeuman synnyssä. CFH Y402H genotyypin ei todettu vaikuttavan siihen, minkälainen pitkälle edenneen ikärappeuman muoto (predominantly classic, minimally classic tai occult) potilaalla oli tai siihen, miten ikärappeumapotilas reagoi fotodynamiseen hoitoon, joka on viime vuosiin asti ollut pääasiallinen kostean ikärappeuman hoitomuoto. Väitöskirjassa osoitettiin myös, että tekijä H:n Y402H:n genotyyppi vaikutti tulehdusta ilmentävän C-reaktiivisen proteiinin sitoutumiseen. Silmän ikärappeumaa voidaan pitää oligogeenisenä tautina eli sen syntyyn vaikuttavat vain harvat suuren riskin sisältämät geenit. Niiden löytyminen auttaa osaltaan uusien hoitomuotojen kehittämisessä

Age-related Macular Degeneration: from risk profiles towards prediction models

Age-related macular degeneration (AMD) is a chronic disease and the leading cause of blindness in elderly in the Western World. Due to the aging population the number of affected persons is expected to increase, which will almost include 17 million affected persons in Europe in 2040. Newly associated genes and environmental factors have helped to elucidate a large part of the pathogenesis of AMD. These newly identified genes are involved in the complement cascade, lipid metabolism, extracellular matrix remodulation and angiogenesis. Thyroid hormone has been identified as a newly potential risk factor. An important modifiable risk factor is dietary intake. A diet of 200 grams of vegetables a day, 2 large pieces of fruit a day and 2 times fish per week has been associated with a 42% lower risk of AMD. This beneficial effect is most likely due to carotenoids, like lutein and zeaxanthin, and omega-3-fatty acids. A prediction model including demographic, genetic and environmental data could distinguish with an accuracy of 87% between those who will develop end stage AMD and those who will not. These findings can help future studies to further unravel the pathogenesis of AMD and ultimately develop preventative measures for this blinding disease

Abstracts of 51st EASD Annual Meeting

Background and aims: Presence and frequency of beta cell (BC) dysfunction(BCD) and insulin resistance (IR) in patients with newly diagnosedtype 2 diabetes mellitus (NDT2D) are imperfectly known, becauseprevious studies used small cohorts and/or only surrogate indexes of BCfunction and IR.We sought to assess BC function and IR with state-of-artmethods in the VNDS.Materials and methods: In 712 GADA-negative, drug naïve, consecutiveItalian NDT2D patients we assessed: 1. standard parameters; 2. insulinsensitivity (IS) by the euglycaemic insulin clamp); 3. BC functionby state-of-art modeling of prolonged (5 hours) OGTT-derived glucose/C-peptide curves. Thresholds for BCD and IR were the 25th percentilesof BC function and IS assessed with the same methods of the VNDS inItalian subjects with normal glucose regulation of the GENFIEV (n=340)and GISIR (n=386) studies, respectively.Results: In the VNDS, 89.8% [95% C.I.: 87.6 - 92.0%] and87.8% [85.4 - 90.2] patients had BCD and IR, respectively. Patientswith only one defect were 19.7% [16.8 - 22.6]. IsolatedBCD and isolated IR were present in 10.9% [8.6 - 13.2] and8.9% [6.8 - 11.0] patients, respectively. Coexistence of BCDand IR was observed in 78.9% [75.9 - 81.9] of the patients.1.4% [0.5 - 2.3] of the patients had no detectable alterations inBC function and IS. Patients (19.7%) with only one metabolicdefect had lower BMI, fasting glucose, HbA1c, triglycerides andBC function, and higher HDL-cholesterol and IS than patientswith both BCD and IR (p<0.01 or less after Bonferroni’scorrection).Conclusion: In conclusion, in NDT2DM patients: 1. at least 75.9% haveboth BCD and IR; 2. At least 87.6% and 85.4% have BCD and IR,respectively; 3. At least 16.8% have only one defect and a significantlydifferent (milder) metabolic phenotype compared to patients with bothdefects. These findings may be relevant to therapeutic strategies centeredon the metabolic phenotype of the patient.Clinical Trial Registration Number: NCT00879801; NCT01526720Supported by: University of Veron