Body composition assessment by artificial intelligence can be a predictive tool for short-term postoperative complications in Hartmann's reversals.

BACKGROUND: Hartmann's reversal, a complex elective surgery, reverses and closes the colostomy in individuals who previously underwent a Hartmann's procedure due to colonic pathology like cancer or diverticulitis. It demands careful planning and patient optimisation to help reduce postoperative complications. Preoperative evaluation of body composition has been useful in identifying patients at high risk of short-term postoperative outcomes following colorectal cancer surgery. We sought to explore the use of our in-house derived Artificial Intelligence (AI) algorithm to measure body composition within patients undergoing Hartmann's reversal procedure in the prediction of short-term postoperative complications. METHODS: A retrospective study of all patients who underwent Hartmann's reversal within a single tertiary referral centre (Western) in Melbourne, Australia and who had a preoperative Computerised Tomography (CT) scan performed. Body composition was measured using our previously validated AI algorithm for body segmentation developed by the Department of Surgery, Western Precinct, University of Melbourne. Sarcopenia in our study was defined as a skeletal muscle index (SMI), calculated as Skeletal Muscle Area (SMA) /height2 < 38.5 cm2/m2 in women and < 52.4 cm2/m2 in men. RESULTS: Between 2010 and 2020, 47 patients (mean age 63.1 ± 12.3 years; male, n = 28 (59.6%) underwent body composition analysis. Twenty-one patients (44.7%) were sarcopenic, and 12 (25.5%) had evidence of sarcopenic obesity. The most common postoperative complication was surgical site infection (SSI) (n = 8, 17%). Sarcopenia (n = 7, 87.5%, p = 0.02) and sarcopenic obesity (n = 5, 62.5%, p = 0.02) were significantly associated with SSIs. The risks of developing an SSI were 8.7 times greater when sarcopenia was present. CONCLUSION: Sarcopenia and sarcopenic obesity were related to postoperative complications following Hartmann's reversal. Body composition measured by a validated AI algorithm may be a beneficial tool for predicting short-term surgical outcomes for these patients.

Comparison of CT derived body composition at the thoracic T4 and T12 with lumbar L3 vertebral levels and their utility in patients with rectal cancer.

BACKGROUND: Computed tomography (CT) derived body composition measurements of sarcopenia are an emerging form of prognostication in many disease processes. Although the L3 vertebral level is commonly used to measure skeletal muscle mass, other studies have suggested the utilisation of other segments. This study was performed to assess the variation and reproducibility of skeletal muscle mass at vertebral levels T4, T12 and L3 in pre-operative rectal cancer patients. If thoracic measurements were equivalent to those at L3, it will allow for body composition comparisons in a larger range of cancers where lumbar CT images are not routinely measured. RESEARCH METHODS: Patients with stage I - III rectal cancer, undergoing curative resection from 2010 - 2014, were assessed. CT based quantification of skeletal muscle was used to determine skeletal muscle cross sectional area (CSA) and skeletal muscle index (SMI). Systematic differences between the measurements at L3 with T4 and T12 vertebral levels were evaluated by percentile rank differences to assess distribution of differences and ordinary least product regression (OLP) to detect and distinguish fixed and proportional bias. RESULTS: Eighty eligible adult patients were included. Distribution of differences between T12 SMI and L3 SMI were more marked than differences between T4 SMI and L3 SMI. There was no fix or proportional bias with T4 SMI, but proportional bias was detected with T12 SMI measurements. T4 CSA duplicate measurements had higher test-retest reliability: coefficient of repeatability was 34.10 cm2 for T4 CSA vs 76.00 cm2 for T12 CSA. Annotation time (minutes) with L3 as reference, the median difference was 0.85 for T4 measurements and -0.03 for T12 measurements. Thirty-seven patients (46%) had evidence of sarcopenia at the L3 vertebral level, with males exhibiting higher rates of sarcopenia. However, there was no association between sarcopenia and post-operative complications, recurrence or hospital LOS (length of stay) in patients undergoing curative resection. CONCLUSIONS: Quantifying skeletal muscle mass at the T4 vertebral level is comparable to measures achieved at L3 in patients with rectal cancer, notwithstanding annotation time for T4 measurements are longer.

Keratoconus International Consortium (KIC)- advancing keratoconus research.

CLINICAL RELEVANCE: The Keratoconus International Consortium (KIC) will allow better understanding of keratoconus. BACKGROUND: Keratoconus is a disorder characterised by corneal elevation and thinning, leading to reduced vision. The current gaps in understanding of this disease will be discussed and the need for a multi-pronged and multi-centre engagement to enhance our understanding of keratoconus will be highlighted. DESIGN: KIC has been established to address the gaps in our understanding of keratoconus with the aim of collecting baseline as well as longitudinal data on several fields. PARTICIPANTS: Keratoconus and control (no corneal condition) subjects from different sites globally will be recruited in the study. METHODS: KIC collects data using an online, secure database, which enables standardised data collection at member sites. Data fields collected include medical history, clinical features, quality of life and economic burden questionnaires and possible genetic sample collection from patients of different ethnicities across different geographical locations. RESULTS: There are currently 40 Australian and international clinics or hospital departments who have joined the KIC. Baseline data has so far been collected on 1130 keratoconus patients and indicates a median age of 29.70 years with 61% being male. A total of 15.3% report a positive family history of keratoconus and 57.7% self-report a history of frequent eye rubbing. CONCLUSION: The strength of this consortium is its international, collaborative design and use of a common data collection tool. Inclusion and analyses of cross-sectional and longitudinal data will help answer many questions that remain in keratoconus, including factors affecting progression and treatment outcomes.

Binding of Gtf2i-ß/δ transcription factors to the ARMS2 gene leads to increased circulating HTRA1 in AMD patients and in vitro.

The disease-initiating molecular events for age-related macular degeneration (AMD), a multifactorial retinal disease affecting many millions of elderly individuals worldwide, are still unknown. Of the over 30 risk and protective loci so far associated with AMD through whole genome-wide association studies (GWAS), the Age-Related Maculopathy Susceptibility 2 (ARMS2) gene locus represents one of the most highly associated risk regions for AMD. A unique insertion/deletion (in/del) sequence located immediately upstream of the High Temperature Requirement A1 (HTRA1) gene in this region confers high risk for AMD. Using electrophoretic mobility shift assay (EMSA), we identified that two Gtf2i-ß/δ transcription factor isoforms bind to the cis-element 5'- ATTAATAACC-3' contained in this in/del sequence. The binding of these transcription factors leads to enhanced upregulation of transcription of the secretory serine protease HTRA1 in transfected cells and AMD patient-derived induced pluripotent stem cells (iPSCs). Overexpression of Htra1 in mice using a CAG-promoter demonstrated increased blood concentration of Htra1 protein, caused upregulation of vascular endothelial growth factor (VEGF), and produced a choroidal neovascularization (CNV)-like phenotype. Finally, a comparison of 478 AMD patients to 481 healthy, age-matched controls from Japan, India, Australia, and the USA showed a statistically increased level of secreted HTRA1 blood concentration in AMD patients compared with age-matched controls. Taken together, these results suggest a common mechanism across ethnicities whereby increased systemic blood circulation of secreted serine protease HTRA1 leads to subsequent degradation of Bruch's membrane and eventual CNV in AMD.

Eye rubbing in the aetiology of keratoconus: a systematic review and meta-analysis.

PURPOSE: Keratoconus is a potentially blinding condition that slowly deforms the cornea in young people. Despite the increasing prevalence of keratoconus, the exact aetiology of the condition is unknown. This first systematic review examines the evidence of eye rubbing and its association with keratoconus and presents the findings of the meta-analysis. METHODS: Two independent reviewers searched the electronic databases for all potential articles published from 1st of January 1900 to 31st of July 2020 on eye rubbing and keratoconus. The researchers assessed the methodological quality of the studies using the Newcastle-Ottawa scale for observational studies. The assessment for statistical heterogeneity was estimated using chi-square and I-square (I2) tests. A p value of < 0.05 was considered as statistically significant and I2 < 30% as homogenous. Begg funnel plot was used to interpret the asymmetry or small study effects. RESULTS: Eight case-control studies were included in this systematic review. Two studies assessed eye rubbing without odds ratios and thus were excluded. The pooled odds ratios for the six remaining studies included in the meta-analysis was 6.46 (95% CI 4.12-10.1). The study results were heterogenous (I2 = 71.69 [95% CI 35.14-87.88]). All the studies scored moderate quality methodology on the Newcastle-Ottawa scale. Begg funnel plot showed asymmetry supporting heterogeneity. CONCLUSION: Eye rubbing showed consistent association with keratoconus. However, the current evidence is limited to only a small number of case-control studies which present as heterogeneous and of sub-optimal methodological quality. Additionally, the cause-effect temporal relationship cannot be determined. Further studies are needed to address this intricate relationship of eye rubbing and its induction, ongoing progression, and severity of keratoconus.

Recurrent structural variation, clustered sites of selection, and disease risk for the complement factor H (CFH) gene family.

Structural variation and single-nucleotide variation of the complement factor H (CFH) gene family underlie several complex genetic diseases, including age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome (AHUS). To understand its diversity and evolution, we performed high-quality sequencing of this ∼360-kbp locus in six primate lineages, including multiple human haplotypes. Comparative sequence analyses reveal two distinct periods of gene duplication leading to the emergence of four CFH-related (CFHR) gene paralogs (CFHR2 and CFHR4 ∼25-35 Mya and CFHR1 and CFHR3 ∼7-13 Mya). Remarkably, all evolutionary breakpoints share a common ∼4.8-kbp segment corresponding to an ancestral CFHR gene promoter that has expanded independently throughout primate evolution. This segment is recurrently reused and juxtaposed with a donor duplication containing exons 8 and 9 from ancestral CFH, creating four CFHR fusion genes that include lineage-specific members of the gene family. Combined analysis of >5,000 AMD cases and controls identifies a significant burden of a rare missense mutation that clusters at the N terminus of CFH [P = 5.81 × 10-8, odds ratio (OR) = 9.8 (3.67-Infinity)]. A bipolar clustering pattern of rare nonsynonymous mutations in patients with AMD (P < 10-3) and AHUS (P = 0.0079) maps to functional domains that show evidence of positive selection during primate evolution. Our structural variation analysis in >2,400 individuals reveals five recurrent rearrangement breakpoints that show variable frequency among AMD cases and controls. These data suggest a dynamic and recurrent pattern of mutation critical to the emergence of new CFHR genes but also in the predisposition to complex human genetic disease phenotypes.

Corneal supply and the use of technology to reduce its demand: A review.

Recovery and access to end-of-life corneal tissue for corneal transplantation, training and research is globally maldistributed. The reasons for the maldistribution are complex and multifaceted, and not well defined or understood. Currently there are few solutions available to effectively address these issues. This review provides an overview of the system, key issues impacting recovery and allocation and emphasises how end-user ophthalmologists and researchers, with support from administrators and the wider sector, can assist in increasing access long-term through sustaining eye banks nationally and globally. We posit that prevention measures and improved surgical techniques, together with the development of novel therapies will play a significant role in reducing demand and enhance the equitable allocation of corneas.

Risk factors and association with severity of keratoconus: the Australian study of Keratoconus.

SIGNIFICANCE: Our results show that asthmatic patients tend to have more severe KC and thus close monitoring for disease progression would be advised, and appropriate treatment strategies may be actioned stabilise the condition that may reduce the need for future corneal transplantation. PURPOSE: To explore a wide range of risk factors associated with the severity of keratoconus (KC). METHODS: A cross-sectional study of KC patients was undertaken in Melbourne, Australia. A questionnaire addressing age, gender, educational background, ocular and medical history, smoking and alcohol consumption, and physical examination comprising anthropometric measurements was collected; eye examination was undertaken. The associations between a range of risk factors and the severity of KC were determined using univariate and multivariable linear regression analyses. RESULTS: A total of 260 KC subjects were included in this study. Mean age of subject was 35.5 (SD = 14.8) years and the majority of the subjects were European 171 (68.2%). Initial univariate regression analysis identified the following risk factors at the p < 0.1 level with KC: higher body mass index, smoking cigarettes, diabetes, rheumatoid arthritis and asthma were associated with increased severity of KC, whereas eczema was associated with less severe KC. Following multivariable regression analysis, only asthma remained as a significant risk factor associated with 2.2 diopters (D) steeper average mean keratometry compared to KC subjects having no asthma [p = 0.03; ß = 2.18; 95% confidence intervals: 1.22, 4.14]. CONCLUSION: Our study describes the comprehensive assessment of all the known risk factors in a large KC cohort recruited in Australia. Our study has reported asthma as the only risk factor found to be significantly associated with the severity of KC. The results of this study allow us to better understand the aetiology of KC and such knowledge could be useful in instigate systemic management of patients to slow or prevent KC.

Economic impact of keratoconus using a health expenditure questionnaire: A patient perspective.

IMPORTANCE: This is the first study to estimate the lifetime costs associated with keratoconus based on a questionnaire completed by patients and highlights the significant economic burden of the disease. As keratoconus affects individuals from a young age, the study highlights keratoconus as a public health concern. BACKGROUND: Keratoconus is a disorder characterized by corneal steepening and thinning, leading to reduced visual acuity. To date, there have been no studies evaluating the economic costs of keratoconus from a patient's perspective. DESIGN: A randomized cross-sectional study undertaken in Australia where keratoconus subjects were recruited from public and private ophthalmology and optometry clinics. PARTICIPANTS: A total of 100 participants completed the questionnaire: median age was 31 years and 57% were males. METHODS: A keratoconus health expenditure questionnaire was designed to assess direct and indirect expenditures for each individual. MAIN OUTCOME MEASURES: Total direct and indirect costs associated with the condition were calculated along with the estimated lifetime per capita costs. RESULTS: The total cost related to direct and indirect care was estimated to be AUD 3365. By applying our cost data to keratoconus prevalence data for the Australian population, the total cost is estimated to be approximately AUD 44.7 million per year in Australia. CONCLUSIONS AND RELEVANCE: Our results show that the costs associated with the diagnosis and management of keratoconus represent a significant cost to patients. An understanding of this is important not only to individuals and their families, but also health care providers, health insurers and the wider health system.

Twin Registries Moving Forward and Meeting the Future: A Review.

Twin registries have developed as a valuable resource for the study of many aspects of disease and society over the years in many different countries. A number of these registries include large numbers of twins with data collected at varying information levels for twin cohorts over the past several decades. More recent expansion of twin datasets has allowed for the collection of genetic data, together with many other levels of 'omic' information along with multiple demographic, physiological, health outcomes and other measures typically used in epidemiologic research. Other twin data sources outside these registries reflect research interests in particular aspects of disease or specific phenotypic assessment. Twin registries have the potential to play a key role in many aspects of the artificial intelligence/machine learning-driven projects of the future and will continue to keep adapting to the changing research landscape.

Loss of Function of P2X7 Receptor Scavenger Activity in Aging Mice: A Novel Model for Investigating the Early Pathogenesis of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a leading cause of irreversible, severe vision loss in Western countries. Recently, we identified a novel pathway involving P2X7 receptor scavenger function expressed on ocular immune cells as a risk factor for advanced AMD. In this study, we investigate the effect of loss of P2X7 receptor function on retinal structure and function during aging. P2X7-null and wild-type C57bl6J mice were investigated at 4, 12, and 18 months of age for macrophage phagocytosis activity, ocular histological changes, and retinal function. Phagocytosis activity of blood-borne macrophages decreased with age at 18 months in the wild-type mouse. Lack of P2X7 receptor function reduced phagocytosis at all ages compared to wild-type mice. At 12 months of age, P2X7-null mice had thickening of Bruchs membrane and retinal pigment epithelium dysfunction. By 18 months of age, P2X7-null mice displayed phenotypic characteristics consistent with early AMD, including Bruchs membrane thickening, retinal pigment epithelium cell loss, retinal functional deficits, and signs of subretinal inflammation. Our present study shows that loss of function of the P2X7 receptor in mice induces retinal changes representing characteristics of early AMD, providing a valuable model for investigating the role of scavenger receptor function and the immune system in the development of this age-related disease.

A genome-wide association study of corneal astigmatism: The CREAM Consortium.

Purpose: To identify genes and genetic markers associated with corneal astigmatism. Methods: A meta-analysis of genome-wide association studies (GWASs) of corneal astigmatism undertaken for 14 European ancestry (n=22,250) and 8 Asian ancestry (n=9,120) cohorts was performed by the Consortium for Refractive Error and Myopia. Cases were defined as having >0.75 diopters of corneal astigmatism. Subsequent gene-based and gene-set analyses of the meta-analyzed results of European ancestry cohorts were performed using VEGAS2 and MAGMA software. Additionally, estimates of single nucleotide polymorphism (SNP)-based heritability for corneal and refractive astigmatism and the spherical equivalent were calculated for Europeans using LD score regression. Results: The meta-analysis of all cohorts identified a genome-wide significant locus near the platelet-derived growth factor receptor alpha (PDGFRA) gene: top SNP: rs7673984, odds ratio=1.12 (95% CI:1.08-1.16), p=5.55×10-9. No other genome-wide significant loci were identified in the combined analysis or European/Asian ancestry-specific analyses. Gene-based analysis identified three novel candidate genes for corneal astigmatism in Europeans-claudin-7 (CLDN7), acid phosphatase 2, lysosomal (ACP2), and TNF alpha-induced protein 8 like 3 (TNFAIP8L3). Conclusions: In addition to replicating a previously identified genome-wide significant locus for corneal astigmatism near the PDGFRA gene, gene-based analysis identified three novel candidate genes, CLDN7, ACP2, and TNFAIP8L3, that warrant further investigation to understand their role in the pathogenesis of corneal astigmatism. The much lower number of genetic variants and genes demonstrating an association with corneal astigmatism compared to published spherical equivalent GWAS analyses suggest a greater influence of rare genetic variants, non-additive genetic effects, or environmental factors in the development of astigmatism.

Pooled genome wide association detects association upstream of FCRL3 with Graves' disease.

BACKGROUND: Graves' disease is an autoimmune thyroid disease of complex inheritance. Multiple genetic susceptibility loci are thought to be involved in Graves' disease and it is therefore likely that these can be identified by genome wide association studies. This study aimed to determine if a genome wide association study, using a pooling methodology, could detect genomic loci associated with Graves' disease. RESULTS: Nineteen of the top ranking single nucleotide polymorphisms including HLA-DQA1 and C6orf10, were clustered within the Major Histo-compatibility Complex region on chromosome 6p21, with rs1613056 reaching genome wide significance (p = 5 × 10-8). Technical validation of top ranking non-Major Histo-compatablity complex single nucleotide polymorphisms with individual genotyping in the discovery cohort revealed four single nucleotide polymorphisms with p ≤ 10-4. Rs17676303 on chromosome 1q23.1, located upstream of FCRL3, showed evidence of association with Graves' disease across the discovery, replication and combined cohorts. A second single nucleotide polymorphism rs9644119 downstream of DPYSL2 showed some evidence of association supported by finding in the replication cohort that warrants further study. CONCLUSIONS: Pooled genome wide association study identified a genetic variant upstream of FCRL3 as a susceptibility locus for Graves' disease in addition to those identified in the Major Histo-compatibility Complex. A second locus downstream of DPYSL2 is potentially a novel genetic variant in Graves' disease that requires further confirmation.

Multiallelic copy number variation in the complement component 4A (C4A) gene is associated with late-stage age-related macular degeneration (AMD).

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of vision loss in Western societies with a strong genetic component. Candidate gene studies as well as genome-wide association studies strongly implicated genetic variations in complement genes to be involved in disease risk. So far, no association of AMD with complement component 4 (C4) was reported probably due to the complex nature of the C4 locus on chromosome 6. METHODS: We used multiplex ligation-dependent probe amplification (MLPA) to determine the copy number of the C4 gene as well as of both relevant isoforms, C4A and C4B, and assessed their association with AMD using logistic regression models. RESULTS: Here, we report on the analysis of 2645 individuals (1536 probands and 1109 unaffected controls), across three different centers, for multiallelic copy number variation (CNV) at the C4 locus. We find strong statistical significance for association of increased copy number of C4A (OR 0.81 (0.73; 0.89);P = 4.4 × 10(-5)), with the effect most pronounced in individuals over 78 years (OR 0.67 (0.55; 0.81)) and females (OR 0.77 (0.68; 0.87)). Furthermore, this association is independent of known AMD-associated risk variants in the nearby CFB/C2 locus, particularly in females and in individuals over 78 years. CONCLUSIONS: Our data strengthen the notion that complement dysregulation plays a crucial role in AMD etiology, an important finding for early intervention strategies and future therapeutics. In addition, for the first time, we provide evidence that multiallelic CNVs are associated with AMD pathology.

Reticular Pseudodrusen and Their Association with Age-Related Macular Degeneration: The Melbourne Collaborative Cohort Study.

PURPOSE: To determine the prevalence of reticular pseudodrusen (RPD) and its association with age-related macular degeneration (AMD) and AMD risk factors in a large sample. DESIGN: Community-based cohort study in Melbourne, Victoria, Australia. PARTICIPANTS: A total of 21,130 participants 48 to 86 years of age available for ophthalmic assessment at follow-up from 2003 through 2007. METHODS: Lifestyle, diet, and anthropometric measurements were obtained at baseline and follow-up. At follow-up, digital macular color photographs were graded for early, intermediate, and late AMD as well as the presence of RPD. Data were analyzed using multinomial logistic regression controlling for age, gender, smoking, country of birth, and diet. MAIN OUTCOME MEASURES: Detection of RPD based on color fundus photographs. RESULTS: Prevalence of RPD was 0.41% (87 of 21,130 participants), with 51% having bilateral RPD. Patients with RPD were older compared with patients with large drusen (>125 µm; 76±4 vs. 68±9 years; P < 0.001). Increasing age, female gender, being a current smoker, as well as focal pigmentary abnormalities and large drusen (>125 µm) were associated with a higher prevalence of RPD. Presence of geographic atrophy (GA) was associated with the highest odds of having RPD (odds ratio [OR], 153; 95% confidence interval [CI], 53-442), followed by choroidal neovascularization (CNV; OR, 90; 95% CI, 26-310), intermediate AMD (OR, 33; 95% CI, 14-77), and early AMD (OR, 12; 95% CI, 5-31) compared with those with no AMD. The ARMS2 single nucleotide polymorphism (SNP) rs10490924, HTRA1 SNPs rs11200638 and rs3793917, and CFH SNPs rs393955, rs1061170, and rs2274700 were associated with increased prevalence of RPD (all P < 0.05). CONCLUSIONS: Reticular pseudodrusen are highly concurrent with AMD and have similar associations with known AMD risk factors such as age, gender, smoking, and genetic risk factors. Reticular pseudodrusen are associated more strongly with GA than with CNV. Although RPD are not specific to AMD, they are likely to be a strong risk factor for progression to late-stage AMD, similar to focal pigmentary abnormalities and large drusen.

Meta-analysis of genome-wide association studies in five cohorts reveals common variants in RBFOX1, a regulator of tissue-specific splicing, associated with refractive error.

Visual refractive errors (REs) are complex genetic traits with a largely unknown etiology. To date, genome-wide association studies (GWASs) of moderate size have identified several novel risk markers for RE, measured here as mean spherical equivalent (MSE). We performed a GWAS using a total of 7280 samples from five cohorts: the Age-Related Eye Disease Study (AREDS); the KORA study ('Cooperative Health Research in the Region of Augsburg'); the Framingham Eye Study (FES); the Ogliastra Genetic Park-Talana (OGP-Talana) Study and the Multiethnic Study of Atherosclerosis (MESA). Genotyping was performed on Illumina and Affymetrix platforms with additional markers imputed to the HapMap II reference panel. We identified a new genome-wide significant locus on chromosome 16 (rs10500355, P = 3.9 × 10(-9)) in a combined discovery and replication set (26 953 samples). This single nucleotide polymorphism (SNP) is located within the RBFOX1 gene which is a neuron-specific splicing factor regulating a wide range of alternative splicing events implicated in neuronal development and maturation, including transcription factors, other splicing factors and synaptic proteins.

Age-related macular degeneration phenotypes associated with mutually exclusive homozygous risk variants in CFH and HTRA1 genes.

PURPOSE: To determine age-related macular degeneration (AMD) phenotypes associated with mutually exclusive homozygotic risk variants in rs1061170 (CFH) and rs11200638 (HTRA1). METHODS: Nested case-control study of 2,982 eyes (2,129 control, 809 drusen ≥125 µm, 44 advanced AMD) homozygous for CFH [TT or CC] and HTRA1 [GG or AA] were analyzed using logistic regression and generalized estimating equations specifically regards to homozygous risk variants in one but homozygous no-risk in the other gene. RESULTS: In early AMD, [CFH HTRA1] and [CFH HTRA1] were associated with central drusen (odds ratio [95% confidence interval] = 4.13 [2.97-5.73] and 3.65 [1.88-7.09], respectively). However, only [CFH HTRA1] was associated with central drusen occupying ≥50% area (13.9 [2.97-64.7]). In advanced AMD, [CFH HTRA1] was associated with geographic atrophy (4.04 [1.57-10.4]), whereas [CFH HTRA1] was associated with neovascular AMD (36.5 [8.3-160.9]). In doubly homozygous risk groups [CFH HTRA1], odds ratios were multiplicative. CONCLUSION: Central but not peripheral drusen location was strongly associated with both [CFH HTRA1] and [CFH HTRA1]. Only [CFH HTRA1] was significantly associated with increased central drusen area.

Technical considerations for genotyping multi-allelic copy number variation (CNV), in regions of segmental duplication.

BACKGROUND: Intrachromosomal segmental duplications provide the substrate for non-allelic homologous recombination, facilitating extensive copy number variation in the human genome. Many multi-copy gene families are embedded within genomic regions with high levels of sequence identity (>95%) and therefore pose considerable analytical challenges. In some cases, the complexity involved in analyzing such regions is largely underestimated. Rapid, cost effective analysis of multi-copy gene regions have typically implemented quantitative approaches, however quantitative data are not an absolute means of certainty. Therefore any technique prone to degrees of measurement error can produce ambiguous results that may lead to spurious associations with complex disease. RESULTS: In this study we have focused on testing the accuracy and reproducibility of quantitative analysis techniques. With reference to the C-C Chemokine Ligand-3-like-1 (CCL3L1) gene, we performed analysis using real-time Quantitative PCR (QPCR), Multiplex Ligation-dependent Probe Amplification (MLPA) and Paralogue Ratio Test (PRT). After controlling for potential outside variables on assay performance, including DNA concentration, quality, preparation and storage conditions, we find that real-time QPCR produces data that does not cluster tightly around copy number integer values, with variation substantially greater than that of the MLPA or PRT systems. We find that the method of rounding real-time QPCR measurements can potentially lead to mis-scoring of copy number genotypes and suggest caution should be exercised in interpreting QPCR data. CONCLUSIONS: We conclude that real-time QPCR is inherently prone to measurement error, even under conditions that would seem favorable for association studies. Our results indicate that potential variability in the physicochemical properties of the DNA samples cannot solely explain the poor performance exhibited by the real-time QPCR systems. We recommend that more robust approaches such as PRT or MLPA should be used to genotype multi-allelic copy number variation in disease association studies and suggest several approaches which can be implemented to ensure the quality of the copy number typing using quantitative methods.

Can genetic associations change with age? CFH and age-related macular degeneration.

Genetic variation in the gene encoding complement factor H (CFH) on chromosome 1q31 has repeatedly been associated with an increased risk of age-related macular degeneration (AMD); however, previous studies have had inadequate numbers of participants across a sufficiently wide age range to determine whether the association varies by age. We conducted a genetic case-control study using data from 2294 cases and 2294 controls selected from the Melbourne Collaborative Cohort Study, matched on age, sex and region of origin. Four consistently replicated CFH single-nucleotide polymorphisms (SNPs) were genotyped: rs1061170 (Y402H), rs2274700, rs393955 and rs800292; their relationship with AMD prevalence was determined across the age range 48-86. A difference in genotype frequencies was seen across age groups, where the low-risk homozygote prevalence rose with each increasing age group. Associations with early AMD were strongly modified by age for three of the four SNPs (interaction P-value: 0.01-0.00003). An inverse association between the high-risk homozygote for each SNP and early AMD was observed in the younger age groups [odds ratios (OR) range 0.37-0.48 for age <55], reversing to a positive association with increasing age (OR 1.87-2.8 for age >75). The direction of associations for this gene change was from inverse to risk with increasing age. These findings have important implications for predictive models for AMD and potentially other age-related diseases which extrapolate risks from older cohorts, as they assume homogeneity of association by age, which might not exist.