Prevalence Estimates of Predicted Pathogenic COL4A3-COL4A5 Variants in a Population Sequencing Database and Their Implications for Alport Syndrome.

BACKGROUND: The reported prevalence of Alport syndrome varies from one in 5000 to one in 53,000 individuals. This study estimated the frequencies of predicted pathogenic COL4A3-COL4A5 variants in sequencing databases of populations without known kidney disease. METHODS: Predicted pathogenic variants were identified using filtering steps based on the ACMG/AMP criteria, which considered collagen IV α3-α5 position 1 Gly to be critical domains. The population frequencies of predicted pathogenic COL4A3-COL4A5 variants were then determined per mean number of sequenced alleles. Population frequencies for compound heterozygous and digenic combinations were calculated from the results for heterozygous variants. RESULTS: COL4A3-COL4A5 variants resulting in position 1 Gly substitutions were confirmed to be associated with hematuria (for each, P<0.001). Predicted pathogenic COL4A5 variants were found in at least one in 2320 individuals. p.(Gly624Asp) represented nearly half (16 of 33, 48%) of the variants in Europeans. Most COL4A5 variants (54 of 59, 92%) had a biochemical feature that potentially mitigated the clinical effect. The predicted pathogenic heterozygous COL4A3 and COL4A4 variants affected one in 106 of the population, consistent with the finding of thin basement membrane nephropathy in normal donor kidney biopsy specimens. Predicted pathogenic compound heterozygous variants occurred in one in 88,866 individuals, and digenic variants in at least one in 44,793. CONCLUSIONS: The population frequencies for Alport syndrome are suggested by the frequencies of predicted pathogenic COL4A3-COL4A5 variants, but must be adjusted for the disease penetrance of individual variants and for the likelihood of already diagnosed disease and non-Gly substitutions. Disease penetrance may depend on other genetic and environmental factors.

Simultaneous assessment of the macrobiome and microbiome in a bulk sample of tropical arthropods through DNA metasystematics.

Conventional assessments of ecosystem sample composition are based on morphology-based or DNA barcode identification of individuals. Both approaches are costly and time-consuming, especially when applied to the large number of specimens and taxa commonly included in ecological investigations. Next-generation sequencing approaches can overcome the bottleneck of individual specimen isolation and identification by simultaneously sequencing specimens of all taxa in a bulk mixture. Here we apply multiple parallel amplification primers, multiple DNA barcode markers, 454-pyrosequencing, and Illumina MiSeq sequencing to the same sample to maximize recovery of the arthropod macrobiome and the bacterial and other microbial microbiome of a bulk arthropod sample. We validate this method with a complex sample containing 1,066 morphologically distinguishable arthropods from a tropical terrestrial ecosystem with high taxonomic diversity. Multiamplicon next-generation DNA barcoding was able to recover sequences corresponding to 91% of the distinguishable individuals in a bulk environmental sample, as well as many species present as undistinguishable tissue. 454-pyrosequencing was able to recover 10 more families of arthropods and 30 more species than did conventional Sanger sequencing of each individual specimen. The use of other loci (16S and 18S ribosomal DNA gene regions) also added the detection of species of microbes associated with these terrestrial arthropods. This method greatly decreases the time and money necessary to perform DNA-based comparisons of biodiversity among ecosystem samples. This methodology opens the door to much cheaper and increased capacity for ecological and evolutionary studies applicable to a wide range of socio-economic issues, as well as a basic understanding of how the world works.

Mitochondrial and nuclear phylogenetic analysis with Sanger and next-generation sequencing shows that, in Área de Conservación Guanacaste, northwestern Costa Rica, the skipper butterfly named Urbanus belli (family Hesperiidae) comprises three morphologically cryptic species.

BACKGROUND: Skipper butterflies (Hesperiidae) are a relatively well-studied family of Lepidoptera. However, a combination of DNA barcodes, morphology, and natural history data has revealed several cryptic species complexes within them. Here, we investigate three DNA barcode lineages of what has been identified as Urbanus belli (Hesperiidae, Eudaminae) in Área de Conservación Guanacaste (ACG), northwestern Costa Rica. RESULTS: Although no morphological traits appear to distinguish among the three, congruent nuclear and mitochondrial lineage patterns show that "Urbanus belli" in ACG is a complex of three sympatric species. A single strain of Wolbachia present in two of the three cryptic species indicates that Urbanus segnestami Burns (formerly Urbanus belliDHJ01), Urbanus bernikerni Burns (formerly Urbanus belliDHJ02), and Urbanus ehakernae Burns (formerly Urbanus belliDHJ03) may be biologically separated by Wolbachia, as well as by their genetics. Use of parallel sequencing through 454-pyrosequencing improved the utility of ITS2 as a phylogenetic marker and permitted examination of the intra- and interlineage relationships of ITS2 variants within the species complex. Interlineage, intralineage and intragenomic compensatory base pair changes were discovered in the secondary structure of ITS2. CONCLUSION: These findings corroborate the existence of three cryptic species. Our confirmation of a novel cryptic species complex, initially suggested by DNA barcode lineages, argues for using a multi-marker approach coupled with next-generation sequencing for exploration of other suspected species complexes.

Polygenic risk scores in atrial fibrillation: Associations and clinical utility in disease prediction.

Atrial fibrillation (AF) is a common heart arrhythmia and a major cause of cardioembolic stroke. Therefore, accurate prediction is desirable to allow high-risk individuals to be identified early and their risk lowered before complications arise. Polygenic risk scores (PRSs) have become a popular method of quantifying aggregated genetic risk from common variants, but their clinical value in AF remains uncertain. This literature review summarizes the associations between PRS and AF risk and discusses the evidence for the clinical utility of PRS for AF prediction. Stroke risk in patients with AF is also considered. Despite consistent associations between PRS and AF risk, the performance of PRS as a stand-alone tool for AF prediction was poor. However, addition of PRS to the existing AF prediction models commonly improved the predictive performance above that of the clinical models alone, including in cohorts with comorbid cardiovascular disease. Associations between PRS and cardioembolic stroke risk in patients with AF have also been reported, but improvements in stroke prediction models from PRS have been minimal. PRS are likely to add value to the existing clinical AF prediction models; however, standardization of PRS across studies and populations will likely be required before they can be meaningfully adopted into routine clinical practice.

The Polygenic Score Catalog: new functionality and tools to enable FAIR research.

Polygenic scores (PGS) have transformed human genetic research and have multiple potential clinical applications, including risk stratification for disease prevention and prediction of treatment response. Here, we present a series of recent enhancements to the PGS Catalog (www.PGSCatalog.org), the largest findable, accessible, interoperable, and reusable (FAIR) repository of PGS. These include expansions in data content and ancestral diversity as well as the addition of new features. We further present the PGS Catalog Calculator (pgsc_calc, https://github.com/PGScatalog/pgsc_calc), an open-source, scalable and portable pipeline to reproducibly calculate PGS that securely democratizes equitable PGS applications by implementing genetic ancestry estimation and score normalization using reference data. With the PGS Catalog & calculator users can now quantify an individual's genetic predisposition for hundreds of common diseases and clinically relevant traits. Taken together, these updates and tools facilitate the next generation of PGS, thus lowering barriers to the clinical studies necessary to identify where PGS may be integrated into clinical practice.

Integrated clinical risk prediction of type 2 diabetes with a multifactorial polygenic risk score.

Combining information from multiple GWASs for a disease and its risk factors has proven a powerful approach for development of polygenic risk scores (PRSs). This may be particularly useful for type 2 diabetes (T2D), a highly polygenic and heterogeneous disease where the additional predictive value of a PRS is unclear. Here, we use a meta-scoring approach to develop a metaPRS for T2D that incorporated genome-wide associations from both European and non-European genetic ancestries and T2D risk factors. We evaluated the performance of this metaPRS and benchmarked it against existing genome-wide PRS in 620,059 participants and 50,572 T2D cases amongst six diverse genetic ancestries from UK Biobank, INTERVAL, the All of Us Research Program, and the Singapore Multi-Ethnic Cohort. We show that our metaPRS was the most powerful PRS for predicting T2D in European population-based cohorts and had comparable performance to the top ancestry-specific PRS, highlighting its transferability. In UK Biobank, we show the metaPRS had stronger predictive power for 10-year risk than all individual risk factors apart from BMI and biomarkers of dysglycemia. The metaPRS modestly improved T2D risk stratification of QDiabetes risk scores for 10-year risk prediction, particularly when prioritising individuals for blood tests of dysglycemia. Overall, we present a highly predictive and transferrable PRS for T2D and demonstrate that the potential for PRS to incrementally improve T2D risk prediction when incorporated into UK guideline-recommended screening and risk prediction with a clinical risk score.

A phylogenetic analysis of relationships among genera of Conopidae (Diptera) based on molecular and morphological data.

Members of the family Conopidae (Diptera) have been the focus of little targeted phylogenetic research. The most comprehensive test of phylogenetic support for the present subfamily classification of Conopidae is presented here using 66 specimens, including 59 species of Conopidae and seven outgroup taxa. Relationships among subfamily clades are also explored. A total of 6824 bp of DNA sequence data from five gene regions (12S ribosomal DNA, cytochrome c oxidase subunit I, cytochrome b, 28S ribosomal DNA and alanyl-tRNA synthetase) are combined with 111 morphological characters in a combined analysis using both parsimony and Bayesian methods. Parsimony analysis recovers three shortest trees. Bayesian analysis recovers a nearly identical tree. Five monophyletic subfamilies of Conopidae are recovered. The rarely acknowledged Zodioninae is restored, including the genera Zodion and Parazodion. The genus Sicus is removed from Myopinae. Morphological synapomorphies are discussed for each subfamily and inter-subfamily clade, including a comprehensive review of the character interpretaions of previous authors. Included are detailed comparative illustrations of male and female genitalia of representatives of all five subfamilies with new morphological interpretation.

Next-generation sequencing technologies for environmental DNA research.

Since 2005, advances in next-generation sequencing technologies have revolutionized biological science. The analysis of environmental DNA through the use of specific gene markers such as species-specific DNA barcodes has been a key application of next-generation sequencing technologies in ecological and environmental research. Access to parallel, massive amounts of sequencing data, as well as subsequent improvements in read length and throughput of different sequencing platforms, is leading to a better representation of sample diversity at a reasonable cost. New technologies are being developed rapidly and have the potential to dramatically accelerate ecological and environmental research. The fast pace of development and improvements in next-generation sequencing technologies can reflect on broader and more robust applications in environmental DNA research. Here, we review the advantages and limitations of current next-generation sequencing technologies in regard to their application for environmental DNA analysis.

A Systematic Review of Pathogenic COL4A5 Variants and Proteinuria in Women and Girls With X-linked Alport Syndrome.

Introduction: Women and girls with X-linked Alport syndrome have a risk of disease progression that is difficult to predict. This systematic review examined whether proteinuria correlated with genotype and disease severity in this population. Methods: PubMed and Scopus were searched for manuscripts from the past 20 years with "COL4A5," "female," "proteinuria" and related terms. Genotypes and clinical data for women and girls with pathogenic heterozygous COL4A5 variants were extracted. Features were then compared between females with proteinuria or without proteinuria; and genotype-phenotype correlations for age at proteinuria and kidney failure determined. Results: Three-hundred sixty-six women and girls with COL4A5 variants and a median age of 29 years (interquartile range 15-46) were identified. Eighty-eight (24%) had large rearrangements or truncating variants, 63 (17%) had splicing variants, and 215 (59%) had missense changes. In all, 236 (64%) had proteinuria, 56 (16%) had kidney failure, 40 (16%) had a hearing loss, and 15 (7%) had ocular abnormalities. Women and girls with proteinuria were more likely to have large rearrangements or truncating variants (P = 0.005), and less likely to have missense changes (P = 0.0002). Those with proteinuria were also more likely to develop kidney failure (P < 0.0001). Women and girls with truncating, large or splicing variants developed proteinuria earlier than those with missense changes (P = 0.001, P < 0.0001 respectively). Those whose proteinuria was detected before the age of 15 progressed to kidney failure sooner (P < 0.0001). Conclusion: Proteinuria correlates with a more severe genotype in women and girls with X-linked Alport syndrome and is an indicator of disease severity and likely progression.

Genotype-Phenotype Correlations for Pathogenic COL4A3-COL4A5 Variants in X-Linked, Autosomal Recessive, and Autosomal Dominant Alport Syndrome.

Alport syndrome is inherited as an X-linked (XL), autosomal recessive (AR), or autosomal dominant (AD) disease, where pathogenic COL4A3 - COL4A5 variants affect the basement membrane collagen IV α3α4α5 network. About 50% of pathogenic variants in each gene (major rearrangements and large deletions in 15%, truncating variants in 20%, splicing changes in 15%) are associated with "severe" disease with earlier onset kidney failure, and hearing loss and ocular abnormalities in males with XL inheritance and in males and females with AR disease. Severe variants are also associated with early proteinuria which is itself a risk factor for kidney failure. The other half of pathogenic variants are missense changes which are mainly Gly substitutions. These are generally associated with later onset kidney failure, hearing loss, and less often with major ocular abnormalities. Further determinants of severity for missense variants for XL disease in males, and in AD disease, include Gly versus non-Gly substitutions; increased distance from a non-collagenous interruption or terminus; and Gly substitutions with a more (Arg, Glu, Asp, Val, and Trp) or less disruptive (Ala, Ser, and Cys) residue. Understanding genotype-phenotype correlations in Alport syndrome is important because they help predict the likely age at kidney failure, and the need for early and aggressive management with renin-angiotensin system blockade and other therapies. Genotype-phenotype correlations also help standardize patients with Alport syndrome undergoing trials of clinical treatment. It is unclear whether severe variants predispose more often to kidney cysts or coincidental IgA glomerulonephritis which are recognized increasingly in COL4A3-, COL4A4 - and COL4A5-associated disease.

Pathogenicity of missense variants affecting the collagen IV α5 carboxy non-collagenous domain in X-linked Alport syndrome.

X-linked Alport syndrome is a genetic kidney disease caused by pathogenic COL4A5 variants, but little is known of the consequences of missense variants affecting the NC1 domain of the corresponding collagen IV α5 chain. This study examined these variants in a normal (gnomAD) and other databases (LOVD, Clin Var and 100,000 Genomes Project) to determine their pathogenicity and clinical significance. Males with Cys substitutions in the collagen IV α5 NC1 domain reported in LOVD (n = 25) were examined for typical Alport features, including age at kidney failure. All NC1 variants in LOVD (n = 86) were then assessed for structural damage using an online computational tool, Missense3D. Variants in the ClinVar, gnomAD and 100,000 Genomes Project databases were also examined for structural effects. Predicted damage associated with NC1 substitutions was then correlated with the level of conservation of the affected residues. Cys substitutions in males were associated with the typical features of X-linked Alport syndrome, with a median age at kidney failure of 31 years. NC1 substitutions predicted to cause structural damage were overrepresented in LOVD (p < 0.001), and those affecting Cys residues or 'buried' Gly residues were more common than expected (both p < 0.001). Most NC1 substitutions in gnomAD (88%) were predicted to be structurally-neutral. Substitutions affecting conserved residues resulted in more structural damage than those affecting non-conserved residues (p < 0.001). Many pathogenic missense variants affecting the collagen IV α5 NC1 domain have their effect through molecular structural damage and 3D modelling is a useful tool in their assessment.

Digenic Alport Syndrome.

Digenic Alport syndrome refers to the inheritance of pathogenic variants in COL4A5 plus COL4A3 or COL4A4 or in COL4A3 plus COL4A4 Where digenic Alport syndrome includes a pathogenic COL4A5 variant, the consequences depend on the sex of the affected individual, COL4A5 variant "severity," and the nature of the COL4A3 or COL4A4 change. A man with a pathogenic COL4A5 variant has all his collagen IV α3α4α5-heterotrimers affected, and an additional COL4A3 or COL4A4 variant may not worsen disease. A woman with a pathogenic COL4A5 variant has on average 50% of her heterotrimers affected, which is increased to 75% with a further COL4A3 or COL4A4 variant and associated with a higher risk of proteinuria. In digenic Alport syndrome with pathogenic COL4A3 and COL4A4 variants, 75% of the heterotrimers are affected. The COL4A3 and COL4A4 genes occur head-to-head on chromosome 2, and inheritance is autosomal dominant when both variants affect the same chromosome (in cis) or recessive when they affect different chromosomes (in trans). This form of digenic disease results in increased proteinuria and a median age of kidney failure intermediate between autosomal dominant and autosomal recessive Alport syndrome. Previous guidelines have suggested that all pathogenic or likely pathogenic digenic variants should be identified and reported. Affected family members should be identified, treated, and discouraged from kidney donation. Inheritance within a family is easier to predict if the two variants are considered independently and if COL4A3 and COL4A4 variants are known to be inherited on the same or different chromosomes.

Genotype-phenotype correlations for COL4A3-COL4A5 variants resulting in Gly substitutions in Alport syndrome.

Alport syndrome is the commonest inherited kidney disease and nearly half the pathogenic variants in the COL4A3-COL4A5 genes that cause Alport syndrome result in Gly substitutions. This study examined the molecular characteristics of Gly substitutions that determine the severity of clinical features. Pathogenic COL4A5 variants affecting Gly in the Leiden Open Variation Database in males with X-linked Alport syndrome were correlated with age at kidney failure (n = 157) and hearing loss diagnosis (n = 80). Heterozygous pathogenic COL4A3 and COL4A4 variants affecting Gly (n = 304) in autosomal dominant Alport syndrome were correlated with the risk of haematuria in the UK 100,000 Genomes Project. Gly substitutions were stratified by exon location (1 to 20 or 21 to carboxyl terminus), being adjacent to a non-collagenous region (interruption or terminus), and the degree of instability caused by the replacement residue. Pathogenic COL4A5 variants that resulted in a Gly substitution with a highly destabilising residue reduced the median age at kidney failure by 7 years (p = 0.002), and age at hearing loss diagnosis by 21 years (p = 0.004). Substitutions adjacent to a non-collagenous region delayed kidney failure by 19 years (p = 0.014). Heterozygous pathogenic COL4A3 and COL4A4 variants that resulted in a Gly substitution with a highly destabilising residue (Arg, Val, Glu, Asp, Trp) were associated with an increased risk of haematuria (p = 0.018), and those adjacent to a non-collagenous region were associated with a reduced risk (p = 0.046). Exon location had no effect. In addition, COL4A5 variants adjacent to non-collagenous regions were over-represented in the normal population in gnomAD (p < 0.001). The nature of the substitution and of nearby residues determine the risk of haematuria, early onset kidney failure and hearing loss for Gly substitutions in X-linked and autosomal dominant Alport syndrome.

The evolution of marine dwelling in Diptera.

Marine dwelling in Diptera has been relatively unexplored and the frequency of transitions to the marine environment and the evolutionary history remain poorly understood. By reviewing records from the World Register of Marine Species and using ancestral state reconstruction methods, we build on the fly tree of life phylogeny and ecological descriptions of marine life history. Our ancestral state reconstruction analyses suggest marine dwelling is lacking as an ancestral trait for the most recent common ancestor to Diptera. While many transitions in Empidoidea, Sciomyzoidea, Tipulomorpha, and Culicomorpha seem to have been gradual, other transitions in Tephritoidea and Tabanomorpha were found likely to have been stochastic occurrences. From the collection of 532 marine species, we reveal several independent transitions to the marine environment throughout the fly tree of life. Considering the results from our analysis, we outline potential adaptations for marine flies and discuss the barriers of colonizing the marine environment and the implications to the mechanisms for salt tolerance.

Placement of Conopidae (Diptera) within Schizophora based on mtDNA and nrDNA gene regions.

The first attempt to phylogenetically place Conopidae using molecular characters, as well as the largest molecular analysis of relationships within Schizophora (Diptera) to date, is presented. Twenty-eight taxa from 11 acalyptrate families and seven acalyptrate superfamilies are represented. Nearly 12,800 bp of sequence data from 10 genes representing both mitochondrial (cytochrome oxidase I (COI), cytochrome b (cytB), and 12S) and nuclear genes (28S, the carbamoyl phosphate synthetase region of CAD (CAD), elongation factor-1alpha (EF-1alpha), white, alanyl-tRNA synthetase (AATS), triose phosphate isomerase (TPI), and phosphogluconate dehydrogenase (PGD)) are analysed. Parsimony and Bayesian analyses strongly support the monophyly of both Conopidae and Schizophora. While in the parsimony analysis, Conopidae are placed as sister to the remaining Schizophora, the Bayesian analysis recovers a Conopidae+Lauxaniidae clade. The value of nuclear, mitochondrial, ribosomal, and protein-coding gene sequence data for answering phylogenetic questions at different levels of divergence is evaluated.

Band-cutting no more: A method for the isolation and purification of target PCR bands from multiplex PCR products using new technology.

A procedure for the isolation of target PCR bands from multiplex PCR products using the E-Gel iBase Power System, an E-Gel Safe Imager Real-Time Transilluminator, and E-Gel CloneWell 0.8% SYBR Safe agarose cassettes is presented. The collected isolates are suitable for direct use in sequencing reactions without need of further purification. Reductions in time spent per sample and cost per sequence produced compared to traditional "band-cutting" methods are demonstrated. An added benefit is that the new procedure does not require exposure to ethidium bromide or ultraviolet radiation.

Observations on hilltopping in thick-headed flies (Diptera: Conopidae).

Direct observations of hilltopping behaviour in the thick-headed flies (Diptera: Conopidae) have only been mentioned once in the literature. Hilltop collecting, however, may be an effective way to survey these endparasitoids. The first evidence of hilltopping in species belonging to the subfamilies Myopinae and Dalmanniinae is presented and discussed. Field observations were conducted on Colle Vescovo, Italy and Mount Rigaud, Canada, and museum specimens were examined. Observations and records indicate that four species in the genera Dalmannia, Myopa, and Zodion are hilltoppers on Colle Vescovo, while three species in the genera Myopa and Physocephala are hilltoppers on three hilltops near Ottawa, Canada. Fifteen additional species of conopids have been collected on hilltops and could possibly utilize hilltops in some years as a part of their mating strategy. Detailed phenologies and observations of mating and perching behaviours are given for species in the genera Dalmannia, Myopa, Physocephala, and Zodion. The importance of hilltop habitat preservation is stressed.

Is periodontitis a risk factor for ischaemic stroke, coronary artery disease and subclinical atherosclerosis? A Mendelian randomization study.

BACKGROUND AND AIMS: Observational studies have reported an association between periodontitis and cardiovascular disease but whether this association is causal is uncertain. We therefore used Mendelian randomization to test whether periodontitis is causally associated with stroke, coronary artery disease, or subclinical atherosclerosis. METHODS: A two-sample Mendelian randomization analysis was carried out using five single nucleotide polymorphisms previously associated with periodontitis in genome-wide association studies. Summary data were drawn from MEGASTROKE and combined with de novo analyses of UK Biobank for stroke and its major subtypes (up to 44,221 cases, 739,957 controls) and CARDIoGRAMplusC4D and UK Biobank for coronary artery disease (122,733 cases, 424,528 controls). We used existing data on carotid intima-media thickness in UK Biobank as a marker of subclinical atherosclerosis (N = 22,179). Causal estimates were obtained using inverse-variance weighted Mendelian randomization. Sensitivity analyses were performed using weighted median and MR-Egger approaches. RESULTS: No association was found between periodontitis and any stroke (odds ratio [OR] per doubling in the odds of periodontitis 0.99, 95% confidence interval [CI] 0.97 to 1.02), ischaemic stroke (OR 1.00, 95% CI 0.97 to 1.03) or its major subtypes (p > 0.4), or coronary artery disease (OR 1.01, 95% CI 0.99 to 1.03). Similarly, we found no association for periodontitis and subclinical atherosclerosis (ß -0.002, 95% CI -0.004 to 0.001). These results were consistent across a series of sensitivity analyses. CONCLUSIONS: These findings provide no robust evidence for a causal relationship between periodontitis and stroke or coronary artery disease. This suggests that associations reported in observational studies may represent confounding.

Diptera of Canada.

The Canadian Diptera fauna is updated. Numbers of species currently known from Canada, total Barcode Index Numbers (BINs), and estimated numbers of undescribed or unrecorded species are provided for each family. An overview of recent changes in the systematics and Canadian faunistics of major groups is provided as well as some general information on biology and life history. A total of 116 families and 9620 described species of Canadian Diptera are reported, representing more than a 36% increase in species numbers since the last comparable assessment by JF McAlpine et al. (1979). Almost 30,000 BINs have so far been obtained from flies in Canada. Estimates of additional number of species remaining to be documented in the country range from 5200 to 20,400.

Microvascular retinopathy and angiographically-demonstrated coronary artery disease: A cross-sectional, observational study.

Epidemiological studies suggest retinal microvascular abnormalities predict cardiac events. This study examined microvascular features associated with coronary artery abnormalities. This was a single-centre, cross-sectional, observational study of 144 consecutive subjects undergoing coronary angiography for clinical indications. Their angiograms were deidentified and graded for disease (Leaman score, LAD stenosis ≥ 70%, number of vessels stenosed ≥ 70%), and Thrombolysis in Myocardial Infarction (TIMI) blush score. Subjects also underwent retinal photography (KOWA non-mydriatic camera, Japan), and their deidentified retinal images were graded for hypertensive microvascular retinopathy (Wong and Mitchell classification), vessel calibre using a computer-assisted method (IVAN, U Wisconsin), and diabetic retinopathy (modified Airlie House scheme) independently by a trained grader and an ophthalmologist. Retinal abnormalities were compared between subjects with high and low angiography scores using one way ANOVA, Chi squared and logistic regression analysis (StataCorp, Texas). Subjects had a mean age of 61 years (range 32-88), and included 101 males (70%). Seventeen (12%) had Leaman scores > 10.5, 46 (32%) had LAD stenosis, 13 (9%) had ≥ 3 arteries stenosed, and 20 (14%) had TIMI blush scores < 1. Twenty-six subjects (18%) had a retinal hemorrhage, and 115 (74%) a mild or moderate hypertensive retinopathy. Fifty-five (38%) had diabetes, and 24 (17%) a background (n = 20) or proliferative (n = 4) diabetic retinopathy. A retinal hemorrhage (p = 0.046), moderate microvascular retinopathy (p = 0.08) and proliferative diabetic retinopathy (p = 0.04) were all associated with a higher Leaman score. Venular calibre was increased with triple vessel disease (205.7 ± 21.6 µm, and 193.7 ± 22.3 µm in normals, p = 0.03). Diabetic retinopathy correlated with an increased TIMI blush score (p = 0.01). Retinal microvascular imaging warrants further evaluation in identifying the presence, extent and nature of coronary artery disease.