Systemic Evidence for Mitochondrial Dysfunction in Age-Related Macular Degeneration as Revealed by mtDNA Copy Number Measurements in Peripheral Blood.

Mitochondrial dysfunction is a common occurrence in the aging process and is observed in diseases such as age-related macular degeneration (AMD). Increased levels of reactive oxygen species lead to damaged mitochondrial DNA (mtDNA), resulting in dysfunctional mitochondria, and, consequently, mtDNA causes further harm in the retinal tissue. However, it is unclear whether the effects are locally restricted to the high-energy-demanding retinal pigment epithelium or are also systematically present. Therefore, we measured mtDNA copy number (mtDNA-CN) in peripheral blood using a qPCR approach with plasmid normalization in elderly participants with and without AMD from the AugUR study (n = 2262). We found significantly lower mtDNA-CN in the blood of participants with early (n = 453) and late (n = 170) AMD compared to AMD-free participants (n = 1630). In regression analyses, we found lower mtDNA-CN to be associated with late AMD when compared with AMD-free participants. Each reduction of mtDNA-CN by one standard deviation increased the risk for late AMD by 24%. This association was most pronounced in geographic atrophy (OR = 1.76, 95% CI 1.19-2.60, p = 0.004), which has limited treatment options. These findings provide new insights into the relationship between mtDNA-CN in blood and AMD, suggesting that it may serve as a more accessible biomarker than mtDNA-CN in the retina.

Genetic Risk Score Analysis Supports a Joint View of Two Classification Systems for Age-Related Macular Degeneration.

Purpose: The purpose of this study was to evaluate the utility of combining the Clinical Classification (CC) and the Three Continent age-related macular degeneration (AMD) Consortium Severity Scale (3CACSS) for classification of AMD. Methods: In two independent cross-sectional datasets of our population-based AugUR study (Altersbezogene Untersuchungen zur Gesundheit der Universität Regensburg), we graded AMD via color fundus images applying two established classification systems (CC and 3CACSS). We calculated the genetic risk score (GRS) across 50 previously identified variants for late AMD, its association via logistic regression, and area under the curve (AUC) for each AMD stage. Results: We analyzed 2188 persons aged 70 to 95 years. When comparing the two classification systems, we found a distinct pattern: CC "age-related changes" and CC "early AMD" distinguished individuals with 3CACSS "no AMD"; 3CACSS "mild/moderate/severe early AMD" stages, and distinguished CC "intermediate AMD". This suggested a 7-step scale combining the 2 systems: (i) "no AMD", (ii) "age-related changes", (iii) "very early AMD", (i.e. CC "early"), (iv) "mild early AMD", (v) "moderate early AMD", (vi) "severe early AMD", and (vii) "late AMD". GRS association and diagnostic accuracy increased stepwise by increased AMD severity in the 7-step scale and by increased restriction of controls (e.g. for CC "no AMD without age-related changes": AUC = 55.1%, 95% confidence interval [CI] = 51.6, 58.6, AUC = 62.3%, 95% CI = 59.1, 65.6, AUC = 63.8%, 95% CI = 59.3, 68.3, AUC = 78.1%, 95% CI = 73.6, 82.5, AUC = 82.2%, 95% CI = 78.4, 86.0, and AUC = 79.2%, 95% CI = 75.4, 83.0). A stepwise increase was also observed by increased drusen size and area. Conclusions: The utility of a 7-step scale is supported by our clinical and GRS data. This harmonization and full data integration provides an immediate simplification over using either CC or 3CACSS and helps to sharpen the control group.

Polygenic scores for estimated glomerular filtration rate in a population of general adults and elderly - comparative results from the KORA and AugUR study.

BACKGROUND: Polygenic scores (PGSs) combining genetic variants found to be associated with creatinine-based estimated glomerular filtration rate (eGFRcrea) have been applied in various study populations with different age ranges. This has shown that PGS explain less eGFRcrea variance in the elderly. Our aim was to understand how differences in eGFR variance and the percentage explained by PGS varies between population of general adults and elderly. RESULTS: We derived a PGS for cystatin-based eGFR (eGFRcys) from published genome-wide association studies. We used the 634 variants known for eGFRcrea and the 204 variants identified for eGFRcys to calculate the PGS in two comparable studies capturing a general adult and an elderly population, KORA S4 (n = 2,900; age 24-69 years) and AugUR (n = 2,272, age ≥ 70 years). To identify potential factors determining age-dependent differences on the PGS-explained variance, we evaluated the PGS variance, the eGFR variance, and the beta estimates of PGS association on eGFR. Specifically, we compared frequencies of eGFR-lowering alleles between general adult and elderly individuals and analyzed the influence of comorbidities and medication intake. The PGS for eGFRcrea explained almost twice as much (R2 = 9.6%) of age-/sex adjusted eGFR variance in the general adults compared to the elderly (4.6%). This difference was less pronounced for the PGS for eGFRcys (4.7% or 3.6%, respectively). The beta-estimate of the PGS on eGFRcrea was higher in the general adults compared to the elderly, but similar for the PGS on eGFRcys. The eGFR variance in the elderly was reduced by accounting for comorbidities and medication intake, but this did not explain the difference in R2-values. Allele frequencies between general adult and elderly individuals showed no significant differences except for one variant near APOE (rs429358). We found no enrichment of eGFR-protective alleles in the elderly compared to general adults. CONCLUSIONS: We concluded that the difference in explained variance by PGS was due to the higher age- and sex-adjusted eGFR variance in the elderly and, for eGFRcrea, also by a lower PGS association beta-estimate. Our results provide little evidence for survival or selection bias.

Cardiovascular Risk Factor Control in 70- to 95-Year-Old Individuals: Cross-Sectional Results from the Population-Based AugUR Study.

Cardiovascular risk factors such as high glucose, LDL-cholesterol, blood pressure, and impaired kidney function are particularly frequent in old-aged individuals. However, population-based data on the extent of cardiovascular risk factor control in the old-aged population is limited. AugUR is a cohort of the mobile "70+"-year-old population of/near Regensburg, recruited via population registries. We conducted cross-sectional analyses assessing the proportion of AugUR participants with LDL-cholesterol, HbA1c, or blood pressure beyond recommended levels and their association with impaired creatinine- and cystatin-based estimated glomerular filtration rate (eGFR, <60 mL/min/1.73 m2) or urine albumin-creatinine ratio (UACR, ≥30 mg/g). Among 2215 AugUR participants, 74.7% were taking lipid-, glucose-, blood-pressure-lowering, or diuretic medication. High LDL-cholesterol at ≥116 mg/dL was observed for 76.1% (51.1% among those with prior cardiovascular events). We found HbA1c ≥ 7.0% for 6.3%, and high or low systolic blood pressure for 6.8% or 26.5%, respectively (≥160, <120 mmHg). Logistic regression revealed (i) high HbA1c levels associated with increased risk for impaired kidney function among those untreated, (ii) high blood pressure with increased UACR, and (iii) low blood pressure with impaired eGFR, which was confined to individuals taking diuretics. Our results provide important insights into cardiovascular risk factor control in individuals aged 70-95 years, which are understudied in most population-based studies.

Photostress Recovery Time as a Potential Predictive Biomarker for Age-Related Macular Degeneration.

Purpose: The purpose of this study was to assess recovery time following photostress and its association with age-related macular degeneration (AMD) cross-sectionally and longitudinally in an elderly population-based cohort. Methods: We analyzed photostress recovery time (PRT) and AMD in >1800 AugUR study participants aged 70+ years. On color fundus images from baseline and 3-year follow-up, presence of AMD was graded manually (Three Continent AMD Consortium Severity Scale). Visual acuity (VA) was assessed via Early Treatment Diabetic Retinopathy Study (ETDRS) charts. After a 30-second bleaching of the macular region via direct ophthalmoscope, PRT was measured as the seconds to regain VA. Results: First, we analyzed 1208 AugUR participants cross-sectionally (288 with early AMD, and 78 with late AMD). Prolonged PRT was associated with early and late AMD versus no AMD (median PRT = 119.5, 198.0 versus 80.0 seconds, respectively; logistic regression odds ratio [OR] = 1.109-1.165 per 10 seconds, P values < 0.0001). Sensitivity analyses using alternative models or restricting to participants after cataract surgery revealed similar ORs. Second, the association was confirmed in an independent cross-sectional AugUR sample (n = 486). Third, in longitudinal analysis of 233 AugUR participants without AMD, prolonged PRT was associated with incident AMD ascertained 3 years later (follow-up time = 3.2 ± 0.2 years, OR = 1.112-1.162 per 10 seconds, P < 0.05). Overall, we demonstrate a significant association of prolonged PRT with AMD cross-sectionally and longitudinally in elderly individuals. Conclusions: Prolonged PRT might capture retinal function impairment after cell damage before early AMD is visible via color fundus imaging. Translational Relevance: Our results suggest PRT as quantitative predictive biomarker for incident AMD, making it potentially worthwhile also for clinical care.

DNA extraction from clotted blood in genotyping quality.

DNA extraction from frozen blood clots is challenging. Here, the authors applied QIAGEN Clotspin Baskets and the Gentra Puregene Blood Kit for DNA extraction to cellular fraction of 5.5 ml whole blood without anticoagulating additives. The amount and quality of extracted DNA were assessed via spectrophotometer and gel electrophoresis. Results from array-based genotyping were analyzed. All steps were compared with DNA isolated from anticoagulated blood samples from a separate study. The quality and concentration of DNA extracted from clotted blood were comparable to those of DNA extracted from anticoagulated blood. DNA yield was on average 27 µg per ml clotted blood, with an average purity of 1.87 (A260/A280). Genotyping quality was similar for both DNA sources (call rate: 99.56% from clotted vs 99.49% from anticoagulated blood).

Self-report of chronic diseases in old-aged individuals: extent of agreement with general practitioner medical records in the German AugUR study.

BACKGROUND: To estimate prevalence and incidence of diseases through self-reports in observational studies, it is important to understand the accuracy of participant reports. We aimed to quantify the agreement of self-reported and general practitioner-reported diseases in an old-aged population and to identify socio-demographic determinants of agreement. METHODS: This analysis was conducted as part of the AugUR study (n=2449), a prospective population-based cohort study in individuals aged 70-95 years, including 2321 participants with consent to contact physicians. Self-reported chronic diseases of participants were compared with medical data provided by their respective general practitioners (n=589, response rate=25.4%). We derived overall agreement, over-reporting/under-reporting, and Cohen's kappa and used logistic regression to evaluate the dependency of agreement on participants' sociodemographic characteristics. RESULTS: Among the 589 participants (53.1% women), 96.9% reported at least one of the evaluated chronic diseases. Overall agreement was >80% for hypertension, diabetes, myocardial infarction, stroke, cancer, asthma, bronchitis/chronic obstructive pulmonary disease and rheumatoid arthritis, but lower for heart failure, kidney disease and arthrosis. Cohen's kappa was highest for diabetes and cancer and lowest for heart failure, musculoskeletal, kidney and lung diseases. Sex was the primary determinant of agreement on stroke, kidney disease, cancer and rheumatoid arthritis. Agreement for myocardial infarction and stroke was most compromised by older age and for cancer by lower educational level. CONCLUSION: Self-reports may be an effective tool to assess diabetes and cancer in observational studies in the old and very old aged. In contrast, self-reports on heart failure, musculoskeletal, kidney or lung diseases may be substantially imprecise.

Relative Telomere Length Is Associated With Age-Related Macular Degeneration in Women.

Purpose: Relative telomere length (RTL) is a biomarker for physiological aging. Premature shortening of telomeres is associated with oxidative stress, which is one possible pathway that might contribute to age-related macular degeneration (AMD). We therefore aimed to investigate the association between RTL and AMD in a well-characterized group of elderly individuals. Methods: We measured RTL in participants of the AugUR study using a multiplex quantitative PCR-based assay determining the ratio between the telomere product and a single-copy gene product (T/S ratio). AMD was assessed by manual grading of color fundus images using the Three Continent AMD Consortium Severity Scale. Results: Among the 2262 individuals 70 to 95 years old (627 with AMD and 1635 without AMD), RTL was significantly shorter in individuals with AMD compared to AMD-free participants. In age- and sex-adjusted logistic regression analyses, we observed an 8% higher odds for AMD per 0.1 unit shorter RTL (odds ratio [OR] = 1.08; 95% confidence interval [CI], 1.02-1.14; P = 0.005). The estimates remained stable when adjusted for smoking, high-density lipoprotein cholesterol, cardiovascular disease, diabetes, and hypertension. Interestingly, this association was only present in women (OR = 1.14; 95% CI, 1.06-1.23; P < 0.001), but not in men (OR = 1.01; 95% CI, 0.93-1.10; P = 0.76). A significant sex-by-RTL interaction on AMD was detected (P = 0.043). Conclusions: Our results show an association of RTL with AMD that was restricted to women. This is in line with altered reactive oxygen species levels and higher telomerase activity in women and provides an indication for a sex-differential pathway for oxidative stress and AMD.

Incidence, progression and risk factors of age-related macular degeneration in 35-95-year-old individuals from three jointly designed German cohort studies.

OBJECTIVE: To estimate age-related macular degeneration (AMD) incidence/progression across a wide age range. METHODS AND ANALYSIS: AMD at baseline and follow-up (colour fundus imaging, Three Continent AMD Consortium Severity Scale, 3CACSS, clinical classification, CC) was assessed for 1513 individuals aged 35-95 years at baseline from three jointly designed population-based cohorts in Germany: Kooperative Gesundheitsforschung in der Region Augsburg (KORA-Fit, KORA-FF4) and Altersbezogene Untersuchungen zur Gesundheit der Universität Regensburg (AugUR) with 18-year, 14-year or 3-year follow-up, respectively. Baseline assessment included lifestyle, metabolic and genetic markers. We derived cumulative estimates, rates and risk factor association for: (1) incident early AMD, (2) incident late AMD among no AMD at baseline (definition 1), (3) incident late AMD among no/early AMD at baseline (definition 2), (4) progression from early to late AMD. RESULTS: Incidence/progression increased by age, except progression in 70+-year old. We observed 35-55-year-old with 3CACSS-based early AMD who progressed to late AMD. Predominant risk factor for incident late AMD definition 2 was early AMD followed by genetics and smoking. When separating incident late AMD definition 1 from progression (instead of combined as incident late AMD definition 2), estimates help judge an individual's risk based on age and (3CACSS) early AMD status: for example, for a 65-year old, 3-year late AMD risk with no or early AMD is 0.5% or 7%, 3-year early AMD risk is 3%; for an 85-year old, these numbers are 0.5%, 21%, 12%, respectively. For CC-based 'early/intermediate' AMD, incidence was higher, but progression was lower. CONCLUSION: We provide a practical guide for AMD risk for ophthalmology practice and healthcare management and document a late AMD risk for individuals aged <55 years.

Changes in healthcare seeking and lifestyle in old aged individuals during COVID-19 lockdown in Germany: the population-based AugUR study.

BACKGROUND: Containment measures in the COVID-19 pandemic protected individuals at high risk, particularly individuals at old age, but little is known about how these measures affected health-related behavior of old aged individuals. We aimed to investigate the impact of the spring 2020 lockdown in Germany on healthcare-seeking and health-related lifestyle in the old aged and to identify susceptible subgroups. METHODS: We conducted a follow-up survey among the pre-pandemically well-characterized participants of our AugUR cohort study, residents in/around Regensburg aged 70+ years and relatively mobile. A self-completion questionnaire on current behavior, perceived changes, and SARS-Cov-2 infection was mailed in May 2020, shortly before contact restrictions ended. Pre-pandemic lifestyle and medical conditions were derived from previous study center visits. RESULTS: Among 1850 survey participants (73-98 years; net-response 89%), 74% were at increased risk for severe COVID-19 according to medical conditions; four participants reported SARS-CoV-2 infection (0.2%). Participants reported changes in behavior: 29% refrained from medical appointments, 14% increased TV consumption, 26% reported less physical activity, but no systematic increase of smoking or alcohol consumption. When comparing during- and pre-lockdown reports of lifestyle within participant, we found the same pattern as for the reported perceived changes. Women and the more educated were more susceptible to changes. Worse QOL was perceived by 38%. CONCLUSIONS: Our data suggest that the spring 2020 lockdown did not affect the lifestyle of a majority of the mobile old aged individuals, but the substantial proportions with decreased physical activity and healthcare-seeking are markers of collateral damage.

Distribution and specificity of high-sensitivity cardiac troponin T in older adults without acute cardiac conditions: cross-sectional results from the population-based AugUR study.

OBJECTIVE: European guidelines recommended a uniform upper reference limit of high-sensitivity cardiac troponin T (hsTnT) to rule out non-ST segment elevation myocardial infarction. Our study aimed to provide a hsTnT reference distribution and to assess the specificity of the 14 ng/L cut-off value in the mobile population ≥70 years of age. DESIGN: A cross-sectional analysis was performed in the German AugUR study (Altersbezogene Untersuchungen zur Gesundheit der University of Regensburg). SETTING: Study population was the mobile population aged 70+ years living in the city and county of Regensburg, Germany. PARTICIPANTS: A random sample was derived from the local population registries of residence. Of the 5644 individuals invited, 1133 participated (response ratio=20.1%). All participants came to the study centre and were mentally and physically mobile to conduct the protocol (face-to-face interview, blood draw and standardised transthoracic echocardiography). None of the participants was in an acute state of myocardial infarction. RESULTS: Among the 1129 individuals with hsTnT measurements (overall median=10.0 ng/L(25th, 75th percentile)=(7.0, 15.0 ng/L)), hsTnT was higher among the older individuals and higher among men (men 70-74 years median=9.6 ng/L (7.2, 13.1 ng/L); men 90-95 years median=21.2 ng/L (14.6, 26.0 ng/L); women 70-74 years median=6.3 ng/L (4.7, 8.7 ng/L); and women 90-95 years median=18.0 ng/L (11.0, 21.0 ng/L)). In participants with impaired kidney function (eGFRcrea <60 mL/min/1.73 m2), hsTnT was elevated (median=13.6 ng/L (9.4, 20.6 ng/L)).Specificity of recommended upper reference limit, 14 ng/L, is 68%. Most false positives were among men aged >79 years (specificity=34%). In a healthy subgroup (n=96, none of the following: overt heart disease, impaired renal function, blood pressure >160/100 mm Hg, left ventricular hypertrophy and diastolic/systolic dysfunction), specificity was 90%. CONCLUSION: In the elderly population without acute myocardial infarction, hsTnT further increases with age showing different levels for men and women. The specificity of the 14 ng/L cut-off is considerably lower than 99%, even in healthy subjects.

Frequency of hand eczema in the elderly: Cross-sectional findings from the German AugUR study.

BACKGROUND: Hand eczema (HE) is a chronic inflammatory skin disease caused by a genetic predisposition and environmental exposures. There is a lack of population-based studies on skin diseases in the elderly. OBJECTIVES: Our aim was to estimate the frequency of HE in the elderly to determine its burden of disease in this particular population. METHODS: We analyzed data from the research platform AugUR, a study on chronic diseases in the elderly (n = 1133, ages 70-95 years, mean age 77.6, 45.1% women). Raw frequencies were estimated using self-reports on physician-diagnosed HE from a standardized personal interview. Frequencies were standardized to the Bavarian population weighted by gender and 5-year age-groups. RESULTS: In our sample 2.7% (95% confidence interval [CI] 1.6-4.3) of the paticipants reported to ever have been diagnosed with HE. Among those 57% were male. After standardization, the frequency was estimated at 2.8% (95% CI 1.9-3.9). There were no differences between male and female participants. CONCLUSIONS: Compared to other studies on lifetime frequency of HE, our estimates seem to be remarkably lower. More in-depth studies with validated diagnoses are warranted to precisely estimate the burden of HE in the elderly.

Genome-wide association meta-analysis for early age-related macular degeneration highlights novel loci and insights for advanced disease.

BACKGROUND: Advanced age-related macular degeneration (AMD) is a leading cause of blindness. While around half of the genetic contribution to advanced AMD has been uncovered, little is known about the genetic architecture of early AMD. METHODS: To identify genetic factors for early AMD, we conducted a genome-wide association study (GWAS) meta-analysis (14,034 cases, 91,214 controls, 11 sources of data including the International AMD Genomics Consortium, IAMDGC, and UK Biobank, UKBB). We ascertained early AMD via color fundus photographs by manual grading for 10 sources and via an automated machine learning approach for > 170,000 photographs from UKBB. We searched for early AMD loci via GWAS and via a candidate approach based on 14 previously suggested early AMD variants. RESULTS: Altogether, we identified 10 independent loci with statistical significance for early AMD: (i) 8 from our GWAS with genome-wide significance (P < 5 × 10- 8), (ii) one previously suggested locus with experiment-wise significance (P < 0.05/14) in our non-overlapping data and with genome-wide significance when combining the reported and our non-overlapping data (together 17,539 cases, 105,395 controls), and (iii) one further previously suggested locus with experiment-wise significance in our non-overlapping data. Of these 10 identified loci, 8 were novel and 2 known for early AMD. Most of the 10 loci overlapped with known advanced AMD loci (near ARMS2/HTRA1, CFH, C2, C3, CETP, TNFRSF10A, VEGFA, APOE), except two that have not yet been identified with statistical significance for any AMD. Among the 17 genes within these two loci, in-silico functional annotation suggested CD46 and TYR as the most likely responsible genes. Presence or absence of an early AMD effect distinguished the known pathways of advanced AMD genetics (complement/lipid pathways versus extracellular matrix metabolism). CONCLUSIONS: Our GWAS on early AMD identified novel loci, highlighted shared and distinct genetics between early and advanced AMD and provides insights into AMD etiology. Our data provide a resource comparable in size to the existing IAMDGC data on advanced AMD genetics enabling a joint view. The biological relevance of this joint view is underscored by the ability of early AMD effects to differentiate the major pathways for advanced AMD.

Retinal Layer Thicknesses in Early Age-Related Macular Degeneration: Results From the German AugUR Study.

Purpose: To systematically analyze thicknesses of retinal layers in an older population and their link to early age-related macular degeneration (AMD). Methods: In the AugUR baseline survey from a population aged ≥70 years, we conducted multimodal retinal imaging, including spectral-domain optical coherence tomography. Autosegmentation of eight distinct retinal layers was followed by manual correction of segmentation errors. AMD status was graded on color fundus images according to the Three Continent AMD Consortium Severity Scale. We tested the association of early AMD on retinal layer thicknesses by using linear mixed models and replicated significant results in independent data also from the AugUR platform. Results: When comparing layer thicknesses between early AMD and no AMD (822 eyes, 449 participants), the retinal pigment epithelium/Bruch's membrane complex demonstrated a statistically significant thickening (e.g., P = 6.41 × 10-92 for severe early versus no AMD) and photoreceptor layers showed a significant thinning. Autosegmented retinal layer thicknesses revealed similar associations as manually corrected values but underestimated some effects. Independent replication analysis in 1026 eyes (546 participants) confirmed associations (e.g., P = 9.38 × 10-36 for retinal pigment epithelium/Bruch's membrane complex, severe early versus no AMD). Conclusions: This first population-based study on spectral-domain optical coherence tomography-derived retinal layer thicknesses in a total of ∼1000 individuals provides insights into the reliability of autosegmentation and layer-specific reference values for an older population. Our findings show a difference in thicknesses between early AMD and no AMD for some retinal layers, suggesting these as potential imaging biomarkers. The thinning of photoreceptor layers substantiates a photoreceptor cell loss/damage already occurring in early AMD.

On the impact of different approaches to classify age-related macular degeneration: Results from the German AugUR study.

While age-related macular degeneration (AMD) poses an important personal and public health burden, comparing epidemiological studies on AMD is hampered by differing approaches to classify AMD. In our AugUR study survey, recruiting residents from in/around Regensburg, Germany, aged 70+, we analyzed the AMD status derived from color fundus images applying two different classification systems. Based on 1,040 participants with gradable fundus images for at least one eye, we show that including individuals with only one gradable eye (n = 155) underestimates AMD prevalence and we provide a correction procedure. Bias-corrected and standardized to the Bavarian population, late AMD prevalence is 7.3% (95% confidence interval = [5.4; 9.4]). We find substantially different prevalence estimates for "early/intermediate AMD" depending on the classification system: 45.3% (95%-CI = [41.8; 48.7]) applying the Clinical Classification (early/intermediate AMD) or 17.1% (95%-CI = [14.6; 19.7]) applying the Three Continent AMD Consortium Severity Scale (mild/moderate/severe early AMD). We thus provide a first effort to grade AMD in a complete study with different classification systems, a first approach for bias-correction from individuals with only one gradable eye, and the first AMD prevalence estimates from a German elderly population. Our results underscore substantial differences for early/intermediate AMD prevalence estimates between classification systems and an urgent need for harmonization.

A Deep Learning Algorithm for Prediction of Age-Related Eye Disease Study Severity Scale for Age-Related Macular Degeneration from Color Fundus Photography.

PURPOSE: Age-related macular degeneration (AMD) is a common threat to vision. While classification of disease stages is critical to understanding disease risk and progression, several systems based on color fundus photographs are known. Most of these require in-depth and time-consuming analysis of fundus images. Herein, we present an automated computer-based classification algorithm. DESIGN: Algorithm development for AMD classification based on a large collection of color fundus images. Validation is performed on a cross-sectional, population-based study. PARTICIPANTS: We included 120 656 manually graded color fundus images from 3654 Age-Related Eye Disease Study (AREDS) participants. AREDS participants were >55 years of age, and non-AMD sight-threatening diseases were excluded at recruitment. In addition, performance of our algorithm was evaluated in 5555 fundus images from the population-based Kooperative Gesundheitsforschung in der Region Augsburg (KORA; Cooperative Health Research in the Region of Augsburg) study. METHODS: We defined 13 classes (9 AREDS steps, 3 late AMD stages, and 1 for ungradable images) and trained several convolution deep learning architectures. An ensemble of network architectures improved prediction accuracy. An independent dataset was used to evaluate the performance of our algorithm in a population-based study. MAIN OUTCOME MEASURES: κ Statistics and accuracy to evaluate the concordance between predicted and expert human grader classification. RESULTS: A network ensemble of 6 different neural net architectures predicted the 13 classes in the AREDS test set with a quadratic weighted κ of 92% (95% confidence interval, 89%-92%) and an overall accuracy of 63.3%. In the independent KORA dataset, images wrongly classified as AMD were mainly the result of a macular reflex observed in young individuals. By restricting the KORA analysis to individuals >55 years of age and prior exclusion of other retinopathies, the weighted and unweighted κ increased to 50% and 63%, respectively. Importantly, the algorithm detected 84.2% of all fundus images with definite signs of early or late AMD. Overall, 94.3% of healthy fundus images were classified correctly. CONCLUSIONS: Our deep learning algoritm revealed a weighted κ outperforming human graders in the AREDS study and is suitable to classify AMD fundus images in other datasets using individuals >55 years of age.

Genetic pleiotropy between age-related macular degeneration and 16 complex diseases and traits.

BACKGROUND: Age-related macular degeneration (AMD) is a common condition of vision loss with disease development strongly influenced by environmental and genetic factors. Recently, 34 loci were associated with AMD at genome-wide significance. So far, little is known about a genetic overlap between AMD and other complex diseases or disease-relevant traits. METHODS: For each of 60 complex diseases/traits with publicly available genome-wide significant association data, the lead genetic variant per independent locus was extracted and a genetic score was calculated for each disease/trait as the weighted sum of risk alleles. The association with AMD was estimated based on 16,144 AMD cases and 17,832 controls using logistic regression. RESULTS: Of the respective disease/trait variance, the 60 genetic scores explained on average 4.8% (0.27-20.69%) and 16 of them were found to be significantly associated with AMD (Q-values < 0.01, p values from < 1.0 × 10-16 to 1.9 × 10-3). Notably, an increased risk for AMD was associated with reduced risk for cardiovascular diseases, increased risk for autoimmune diseases, higher HDL and lower LDL levels in serum, lower bone-mineral density as well as an increased risk for skin cancer. By restricting the analysis to 1824 variants initially used to compute the 60 genetic scores, we identified 28 novel AMD risk variants (Q-values < 0.01, p values from 1.1 × 10-7 to 3.0 × 10-4), known to be involved in cardiovascular disorders, lipid metabolism, autoimmune diseases, anthropomorphic traits, ocular disorders, and neurological diseases. The latter variants represent 20 novel AMD-associated, pleiotropic loci. Genes in the novel loci reinforce previous findings strongly implicating the complement system in AMD pathogenesis. CONCLUSIONS: We demonstrate a substantial overlap of the genetics of several complex diseases/traits with AMD and provide statistically significant evidence for an additional 20 loci associated with AMD. This highlights the possibility that so far unrelated pathologies may have disease pathways in common.

NT-proBNP Predicts Cardiovascular Death in the General Population Independent of Left Ventricular Mass and Function: Insights from a Large Population-Based Study with Long-Term Follow-Up.

AIMS: B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) predict cardiovascular endpoints in patients and all-cause death in the general population. This was assigned to their association with clinical cardiac remodelling defined as changes in size, shape and function of the heart. The aim of this study was to evaluate whether NT-proBNP and BNP were associated with cardiovascular and overall death independent of clinical cardiac remodelling measured by echocardiography as left ventricular hypertrophy (LVH), diastolic dysfunction and left ventricular ejection fraction (EF). METHODS AND RESULTS: In a general population-based cohort study from Germany (KORA-S3) with subjects' baseline age ranging from 25 to 74 years, cardiac morphology and function were assessed as left ventricular mass (LVM), diastolic dysfunction and EF by echocardiography and circulating NT-proBNP and BNP were measured at baseline. In 1,223 subjects with mortality follow-up information, we examined the association of baseline NT-proBNP and BNP with cardiovascular mortality (number of deaths = 52, median follow-up time = 12.9years) using Cox regression without and with adjustment for cardiovascular risk factors, LVM, diastolic dysfunction and EF. The risk of cardiovascular mortality increased with higher NT-proBNP levels measured at baseline (hazard ratio HR = 1.67 per unit increment in logNT-proBNP, p = 2.78*10-4, adjusted for age and sex). This increased risk persisted after adjustment for cardiovascular risk factors, LVM, diastolic dysfunction and EF (HR = 1.73; p = 0.047). When excluding subjects with relevant LVH (LVM to body surface area > 149g/m2 in men / 122g/m2 in women), the NT-proBNP association with mortality was still significant (n = 1,138; number of deaths = 35; HR = 1.48; p = 0.04). We found similar results for BNP. CONCLUSION: Our data confirms NT-proBNP and BNP as predictor of cardiovascular mortality in a large general population-based study with long-term follow-up. Our study extends previously published population-based studies to younger and potentially healthier individuals without relevant LVH, diastolic dysfunction or LVD.

The German AugUR study: study protocol of a prospective study to investigate chronic diseases in the elderly.

BACKGROUND: The majority of patients suffering from chronic health disabilities is beyond 70 years of age. Typical late-onset chronic diseases include those affecting the heart, the kidney, cancer, and conditions of the eye such as age-related macular degeneration. These diseases disable patients for many years and largely compromise autonomy in daily life. Due to challenges in recruiting the elderly, the collection of population-based epidemiological data as a prerequisite to understand associated risk factors and mechanisms is commonly done in the general population within an age-range of 20 to 70 years. METHODS/DESIGN: We establish the German AugUR study (Age-related diseases: understanding genetic and non-genetic influences - a study at the University of Regensburg), a prospective study in the mobile elderly general population in and around Regensburg in eastern Bavaria. In the long term, we aim to recruit 3,000 persons of Caucasian ethnicity with at least 70 years of age via residents' registration offices and conduct 3-year follow-ups. The study protocol includes a standardized interview regarding social and life-style factors, medication history, quality-of-life, and existing diagnoses of common diseases. The participants undergo medical examinations for ophthalmological, cardiovascular or diabetes-related conditions, and general measurements of body shape and fitness. The program is particularly tailored for the elderly. Biobanking of whole blood, serum, plasma, and urine is conducted and standard laboratory measurements are performed in fresh samples. DISCUSSION: AugUR is specifically designed as a research platform to host studies of late onset diseases. Consequently, this platform will help (1) to unravel the genetic and non-genetic etiology of disease development and progression, (2) to serve as control group of elderly individuals for comparisons with various patient groups, (3) to derive prevalence and incidence data on chronic diseases, and (4) to provide clinical reference parameters for the elderly mobile general population. This data will foster our understanding of disease mechanisms, which may ultimately help to improve prevention, diagnosis, and therapy for frequent chronic diseases. Here we present the baseline study protocol of AugUR.

Generation of highly purified human cardiomyocytes from peripheral blood mononuclear cell-derived induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T, α-actinin and myosin light chain 2 (MLC2V). In a subsequent metabolic selection with lactate, the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%, whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular, atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified, functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling, drug discovery, and regenerative medicine.