Relationship among CFH and ARMS2 genotypes, macular pigment optical density, and neuroretinal function in persons without age-related macular degeneration

Purpose: To determine whether there is a difference in neuroretinal function and in macular pigment optical density between persons with high- and low-risk gene variants for age-related macular degeneration (AMD) and no ophthalmoscopic signs of AMD, and to compare the results on neuroretinal function to patients with manifest early AMD. Methods and Participants: Neuroretinal function was assessed with the multifocal electroretinogram (mfERG) for 32 participants (22 healthy persons with no AMD and 10 early AMD patients). The 22 healthy participants with no AMD had high- or low-risk genotypes for either CFH (rs380390) and/or ARMS2 (rs10490924). Trough-to-peak response densities and peak-implicit times were analyzed in 5 concentric rings. Macular pigment optical densitometry was assessed by customized heterochromatic flicker photometry. Results: Trough-to-peak response densities for concentric rings 1 to 3 were, on average, significantly greater in participants with high-risk genotypes than in participants with low-risk genotypes and in persons with early AMD after correction for age and smoking (p<0.05). The group peak- implicit times for ring 1 were, on average, delayed in the patients with early AMD compared with the participants with high- or low-risk genotypes, although these differences were not significant. There was no significant correlation between genotypes and macular pigment optical density. Conclusion: Increased neuroretinal activity in persons who carry high-risk AMD genotypes may be due to genetically determined subclinical inflammatory and/or histological changes in the retina. Neuroretinal function in healthy persons genetically susceptible to AMD may be a useful additional early biomarker (in combination with genetics) before there is clinical manifestation.

The relationship between CFH and ARMS2 genotypes and neuroretinal function in persons without age-related macular degeneration

Purpose: To determine whether neuroretinal function differs in healthy persons with and without common risk gene variants for age- related macular degeneration (AMD) and no ophthalmoscopic signs of AMD, and to compare those findings in persons with manifest early AMD. Methods and Participants: Neuroretinal function was assessed with the multifocal electroretinogram (mfERG) (VERIS, Redwood City, CA,) in 32 participants (22 healthy persons with no clinical signs of AMD and 10 early AMD patients). The 22 healthy participants with no AMD were risk genotypes for either the CFH (rs380390) and/or ARMS2 (rs10490920). We used a slow flash mfERG paradigm (3 inserted frames) and a 103 hexagon stimulus array. Recordings were made with DTL electrodes; fixation and eye movements were monitored online. Trough N1 to peak P1 (N1P1) response densities and P1-implicit times (IT) were analysed in 5 concentric rings. Results: N1P1 response densities (mean ± SD) for concentric rings 1-3 were on average significantly higher in at-risk genotypes (ring 1: 17.97 nV/deg2 ± 1.9, ring 2: 11.7 nV/deg2 ±1.3, ring 3: 8.7 nV/deg2 ± 0.7) compared to those without risk (ring 1: 13.7 nV/deg2 ± 1.9, ring 2: 9.2 nV/deg2 ±0.8, ring 3: 7.3 nV/deg2 ± 1.1) and compared to persons with early AMD (ring 1: 15.3 nV/deg2 ± 4.8, ring 2: 9.1 nV/deg2 ±2.3, ring 3 nV/deg2: 7.3± 1.3) (p<0.5). The group implicit times, P1-ITs for ring 1 were on average delayed in the early AMD patients (36.4 ms ± 1.0) compared to healthy participants with (35.1 ms ± 1.1) or without risk genotypes (34.8 ms ±1.3), although these differences were not significant. Conclusion: Neuroretinal function in persons with normal fundi can be differentiated into subgroups based on their genetics. Increased neuroretinal activity in persons who carry AMD risk genotypes may be due to genetically determined subclinical inflammatory and/or histological changes in the retina. Assessment of neuroretinal function in healthy persons genetically susceptible to AMD may be a useful early biomarker before there is clinical manifestation of AMD.

Visual function and CFH/ARMS2 risk genotypes in macular dystrophy due to maternally inherited diabetes and deafness

Maternally inherited diabetes and deafness (MIDD) is an autosomal dominant inherited syndrome caused by the mitochondrial DNA (mtDNA) nucleotide mutation A3243G. It affects various organs including the eye with external ophthalmoparesis, ptosis, and bilateral macular pattern dystrophy.1, 2 The prevalence of retinal involvement in MIDD is high, with 50% to 85% of patients exhibiting some macular changes.1 Those changes, however, can vary between patients and within families dramatically based on the percentage of retinal mtDNA mutations, making it difficult to give predictions on an individual’s visual prognosis...

Neovascular age-related macular degeneration risk based on CFH, LOC387715/HTRA1, and smoking

Age-related macular degeneration (AMD) is the major cause of blindness in the elderly. Those with the neovascular end-stage of disease have irreversible loss of central vision. AMD is a complex disorder in which genetic and environmental factors play a role. Polymorphisms in the complement factor H (CFH) gene, LOC387715, and the HTRA1 promoter are strongly associated with AMD. Smoking also contributes to the etiology. We aimed to provide a model of disease risk based on these factors.

The association between macular pigment optical density and CFH, ARMS2, C2/BF, and C3 genotype.

Age-related macular degeneration (AMD) is the most common cause of blindness in older people in developed countries, and risk for this condition may be classified as genetic or environmental, with an interaction between such factors predisposing to this disease. This study investigated the relationship between AMD risk genes, macular pigment optical density (MPOD), which may protect against AMD, and serum concentrations of the macular carotenoids, lutein (L) and zeaxanthin (Z). This was a cross-sectional study of 302 healthy adult subjects. Dietary intake of L and Z was assessed by food frequency questionnaire, and MPOD was measured by customized heterochromatic flicker photometry. We also calculated MPOD Area as the area of MP under the spatial profile curve, to reflect MP across the macula. Serum L and Z were measured by HPLC. Genotyping of tag SNPs in the genes CFH, ARMS2, C3, C2 and BF was undertaken with multiplex polymerase chain reaction (PCR) and primer extension methodology (ABI Snapshot, ABI Warrington UK) on DNA extracted from peripheral blood. The mean ± SD (range) age of the subjects in this study was 48 ± 11 (21-66) years. There was a statistically significant association between CFH genotype and family history of AMD, with subjects having two non-risk CFH haplotypes (n =35), or one non-risk and one protective CFH haplotype (n = 33), being significantly more likely to have a negative family history of AMD (Pearson Chi square: p = 0.001). There was no significant association between the AMD risk genes investigated and either MPOD (One way ANOVA: p > 0.05) or serum concentrations of L or Z (One way ANOVA: p > 0.05, for both). Subjects who were homozygous for risk alleles of both CFH and ARMS2 (n = 4) had significantly lower MPOD at 0.5_ and 1_ retinal eccentricity (Independent samples t test: p

Persons with age-related maculopathy risk genotypes and clinically normal eyes have reduced mesopic vision

PURPOSE: To determine if participants with normal visual acuity, no ophthalmoscopically signs of age-related maculopathy (ARM) in both eyes and who are carriers of the CFH, LOC387715 and HRTA1 high-risk genotypes (“gene-positive”) have impaired rod- and cone-mediated mesopic visual function compared to persons who do not carry the risk genotypes (“gene-negative”).---------- METHODS: Fifty-three Caucasian study participants (mean 55.8 ± 6.1) were genotyped for CFH, LOC387715/ARMS2 and HRTA1 polymorphisms. We genotyped single nucleotide polymorphisms (SNPs) in the CFH (rs380390), LOC387715/ARMS2 (rs10490924) and HTRA1 (rs11200638) genes using Applied Biosystems optimised TaqMan assays. We determined the critical fusion frequency (CFF) mediated by cones alone (Long, Middle and Short wavelength sensitive cones; LMS) and by the combined activities of cones and rods (LMSR). The stimuli were generated using a 4-primary photostimulator that provides independent control of the photoreceptor excitation under mesopic light levels. Visual function was further assessed using standard clinical tests, flicker perimetry and microperimetry.---------- RESULTS: The mesopic CFF mediated by rods and cones (LMSR) was significantly reduced in gene-positive compared to gene-negative participants after correction for age (p=0.03). Cone-mediated CFF (LMS) was not significantly different between gene-positive and -negative participants. There were no significant associations between flicker perimetry and microperimetry and genotype.---------- CONCLUSIONS: This is the first study to relate ARM risk genotypes with mesopic visual function in clinically normal persons. These preliminary results could become of clinical importance as mesopic vision may be used to document sub-clinical retinal changes in persons with risk genotypes and to determine whether those persons progress into manifest disease.

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.

四氟乙烯等离子体聚合反应的基础研究

[I] 等离子体聚四氟乙烯的ESCA表征 本研究采用外部电容耦合式聚合装置，频率为13.56MHz，合成了等离子体聚四氟乙烯（PPTFE）。应用ESCA表征了辉光压和非辉光压产物的结构，同时应用CNDO进到了理论计算。结果表明，在辉光压上非辉光压中制得的PPTFE结构差别很大。非辉光内淀积物结构亦现差异。辉光压内的PPTFE是高度支化交联的，在本实验的典型反应条件下，PPTFE的F/C比为1.42，Gs区五个峰确定归属后，各主要对应基团的相对组成约为：CF_3－21%、CF_2－32%、CF－22%、－C from | to | of — 20%，其余为少量的CFH、CH_2（II）、CH_2（I）。在非辉光压内可得到近於线线型的PPTFE，其F/C为2.08，端基CF_3约占15%，CF_2占78%，代表支化交联的基团CF、－C from | to | of －皆为零，另外应用X射线衍射法证实，在非辉光压中形成的PPTFE具有类似于聚四氟乙烯的（PTFE）结晶，这一实验结果至今未见文献报导。深入分析这一结果，并运用ESR、MS、~(19)F－NMR等表征手段，进一步提出四氟乙烯等离子体聚合反应机理。这方面在[II]中论述。本工作提出五类十三种模型化合物，并运用Siegbahn的电荷电位模型同时结合使用CNDO/2电子计算机程序；还运用Pauling价键模型，分别应用这两种理论方法，计算了PPTFE中ClS结合能位移（ΔEi），所得结果相近，从而为ClS区五个峰的归属提供理论依据。五个峰归属为：(1) 284.7(ev) － CH_2(I)，(2) 287.0(ev) － －C from | to | of －、CH_2(II)，(3) 289.0(ev) －CF、CFH，(4) 291.5(ev) － CF_2，(5) 293.6(ev) － CF_3。通过对模型化合物中ClS的ΔEi计算结果，推导出F作为α、β、γ位碳上的取代基对α位碳上IS电子结合能位移影响所产生的效应值，并初步总结出表达这种效应的经验式：ΔEi ＝ aα ＋ bβ ＋ cγ式中α、β、γ分别为2.23 ± 0.08(ev)、0.27 ± 0.02(ev)、0.20 ± 0.02(ev)，a、b、c为取代基F的个数。实验结果还证明，淀积位置、功率、压力、等离子气体如Ar、He、N_2等反应条件对PPTFE膜结构有影响，并应用能量梯度解释这种影响；另外发现，同功率、同压力但不同淀积位置上以及同淀积位置但不同功率或不同压力时的PPTFE膜结构所受能量梯度的影响。本工作根据等离子体聚四氟工烯结构的表征结果，并结合ESR、MS等有关分析数据，提出了PPTFE的结构模型。[II] 等郭子体聚四氟乙烯的反应机理 鉴于等离子体聚合反应的复杂性，目前对等离子体聚合反应机理争论较大，其焦点是：反应的活性中心是离子还是自由基；反应地点是在气相还是在反应体系的固体表面。本工作通过对等离子体聚四氟乙烯气相产物的研究证实：等离子体聚合反就是通过自由基历程，引发和初级链增长主要在气相，脱F和支化交联反应在表面上进行。聚合反应装置同[I]中所述，聚合条件是功率60瓦，压力1 * 10~(-1)乇。通过液氟冷阱收集气相生成物，然后制备成有机溶液和本体溶液。气相生成物的ESR分析结果表明，PPTFE膜中存在自由基浓度约在10~(18)自旋数／克，峰形为一条反对称吸收线，宽为175G，g值是2.005。气相生成物的丙酮、苯、环已烷、已烷等有机溶液的ESR谱相同，皆为20条精细结构分裂谱线。另外，通过一系列实验，检测了桔黄色本体溶液的ESR谱，终于得到具有160条超精细结构的分裂谱线，自由基浓度高达10~(20)自旋数／克。初步认为是几种自由基的混合物，固谱图十分复杂，目前解谱尚有困难，需进一步研究。等高子体气相生成物中有大量自由基并且得到其精细和超精细分裂谱线，这一实验结果至今未见文献报导。这个结果进一步证实了聚合反应的活性中心主要是自由基。考察了PPTFE膜的自由基在90 ℃时随时间变化的ESR谱，发现一开始衰减很快；研究了PPTFE膜的自由基在不同气氛下常温衰减情况，结果指出：辉光压和非辉光压的PPTFE膜中自由基在空气中衰减快，辉光压的PPTFE膜中自由基在真空中及单体气氛中衰减缓慢。气相生成物的有机溶液在90 ℃时随时间变化的ESR谱表明，各峰衰减速率不同，证实并非一种自由基，溶液的自由基能与吡啶反应生成棕红色物质，可能是吡啶盐。本实验用GC－MS联用对气相冷凝物本体溶液进行了分析，比文献上只用MS与聚合反应体系联用分析气相混合物的方法，对分析反应机理提供更为有说服力的实验结果。结果表明，四氟乙烯等离子体聚合的气相生成物是直链的全氟烷烃（分子中碳原子数为C_3－C_8），还有含碳原子数为C_4、C_5、C_6、C_8全氟环烷烃，也可能是括分子链两端是自由基的直链全氟烷烃，还有二氟卡宾。从TFE等离子体聚合的气相中产生齐聚物及其结构特征，可以推断气相中发生了链引发反应和初级增长反应，引发反应的历程首先是TFE单体分子中π键断裂，其次是C~σ-C的σ键均裂，然后通过自由基的复合或诱导反应进行链的初级增长反应。还进行了气相生成物有机溶液的~(19)F－NMR分析，其结果初步看来与GC－MS的结果相一致。还探索了不同条件下在非辉光压内得到的PPTFE膜，进一步表明，在顺着气流方向的淀积位置上的膜结构是接近线型的。这个结果与用质谱、核磁的表征结果相符：从不用方面证实了上述反应历程。此外，通过对PPTFE及PTFE在Ar等离子体系中的刻蚀实验，表明了聚合膜上发生六量脱F反应。由这个结果并结合PPTFE膜及气相产物的结构表征，可以推测到PPTFE膜的支化，交联是通过表面反应进行的。综合四氟乙烯等离子体聚合反应机理的讨论，本论文建议了TFE的等离子体聚合反应历程模型。

Mitochondrial DNA polymorphism A4917G is independently associated with age-related macular degeneration.

The objective of this study was to determine if MTND2*LHON4917G (4917G), a specific non-synonymous polymorphism in the mitochondrial genome previously associated with neurodegenerative phenotypes, is associated with increased risk for age-related macular degeneration (AMD). A preliminary study of 393 individuals (293 cases and 100 controls) ascertained at Vanderbilt revealed an increased occurrence of 4917G in cases compared to controls (15.4% vs.9.0%, p = 0.11). Since there was a significant age difference between cases and controls in this initial analysis, we extended the study by selecting Caucasian pairs matched at the exact age at examination. From the 1547 individuals in the Vanderbilt/Duke AMD population association study (including 157 in the preliminary study), we were able to match 560 (280 cases and 280 unaffected) on exact age at examination. This study population was genotyped for 4917G plus specific AMD-associated nuclear genome polymorphisms in CFH, LOC387715 and ApoE. Following adjustment for the listed nuclear genome polymorphisms, 4917G independently predicts the presence of AMD (OR = 2.16, 95%CI 1.20-3.91, p = 0.01). In conclusion, a specific mitochondrial polymorphism previously implicated in other neurodegenerative phenotypes (4917G) appears to convey risk for AMD independent of recently discovered nuclear DNA polymorphisms.