Genetic and phenotypic heterogeneity in autosomal recessive retinal disease

Molecular genetics has transformed our understanding of disease and is gradually changing the way medicine is practiced. Genetic mapping provides a powerful approach to discover genes and biological processes underlying human disorders. Recent advances in DNA microarray and sequencing technology have significantly increased the power of genetic mapping studies and have ushered in a new era for biomedicine. In this thesis, linkage analysis (including homozygosity mapping), exome sequencing and candidate gene sequencing have been utilised to genetically dissect autosomal recessive retinal disease. Subsequently, clinical findings from patients found to be similar in terms of molecular pathology have been pooled. DNA and basic phenotypic data from over 500 unrelated individuals were available for the project. Disease-causing variants in three genes that have not been previously associated with human recessive disorders are reported: (a) biallelic mutations in TRPM1 abrogate ON bipolar cell function and cause complete congenital stationary night blindness; (b) biallelic mutations in KCNJ13, a gene encoding an inwardly rectifying potassium channel subunit cause Leber congenital amaurosis; (c) biallelic mutations in PLA2G5, a gene encoding group V phospholipase A2, cause benign fleck retina. The consequences of mutations in these and other disease-related genes (RDH5, GRM6, KCNV2, OAT and SAG) on retinal structure (spectral domain optical coherence tomography, fundus autofluorescence imaging) and visual function (electrophysiology, perimetry testing) have been studied; features that may have mechanistic relevance have been identified. Additionally, DNA sequence variation of a highly polymorphic gene (C2ORF71), recently associated with photoreceptor degeneration, has been studied and quantified in patient and control samples. Basic bioinformatics tools to analyse genomic data have been developed (bash, perl, python and R programming languages). Overall, results presented in this thesis contribute to an understanding of Mendelian retinal disease that is not only observational but also mechanistic

Dark-adapted red flash ERGs in healthy adults

Purpose: The x-wave of the dark-adapted (DA) ERG to a red flash reflects DA cone function. This exploratory study of healthy adults aimed to investigate changes in the DA red ERG with flash strength and during dark adaptation to optimise visualisation and therefore quantification of the x-wave. Methods: The effect of altering red flash strength was investigated in four subjects by recording ERGs after 20 minutes dark adaptation to red flashes (0.2–2.0 cd s m-2) using skin electrodes and natural pupils. The effect of dark adaptation duration was investigated in 16 subjects during 20 minutes in the dark, by recording DA 1.5 red ERGs at 1, 2, 3, 4, 5, 10, 15 and 20 minutes. Results: For a dark adaption period of 20 minutes, the x-wave was more clearly visualised to weaker (< 0.6 cd s m-2) red flash strengths: to stronger flashes it became obscured by the b-wave. For red flashes of 1.5 cd s m-2, the x-wave was most prominent in ERGs recorded after 1–5 minutes of dark adaptation: with longer dark-adaptation, it was subsumed into the b-wave’s rising edge. Conclusions: This small study suggests that x-wave visibility in healthy subjects after 20 minutes dark adaptation is improved by using flashes weaker than around 0.6 cd s m-2; for flash strengths of 1.5 cd s m-2, x-wave visibility is enhanced by recording after only around 5 minutes of dark adaptation. No evidence was found that interim red flash ERGs affecting the dark-adapted state of the normal retina

An ontological foundation for ocular phenotypes and rare eye diseases.

BACKGROUND: The optical accessibility of the eye and technological advances in ophthalmic diagnostics have put ophthalmology at the forefront of data-driven medicine. The focus of this study is rare eye disorders, a group of conditions whose clinical heterogeneity and geographic dispersion make data-driven, evidence-based practice particularly challenging. Inter-institutional collaboration and information sharing is crucial but the lack of standardised terminology poses an important barrier. Ontologies are computational tools that include sets of vocabulary terms arranged in hierarchical structures. They can be used to provide robust terminology standards and to enhance data interoperability. Here, we discuss the development of the ophthalmology-related component of two well-established biomedical ontologies, the Human Phenotype Ontology (HPO; includes signs, symptoms and investigation findings) and the Orphanet Rare Disease Ontology (ORDO; includes rare disease nomenclature/nosology). METHODS: A variety of approaches were used including automated matching to existing resources and extensive manual curation. To achieve the latter, a study group including clinicians, patient representatives and ontology developers from 17 countries was formed. A broad range of terms was discussed and validated during a dedicated workshop attended by 60 members of the group. RESULTS: A comprehensive, structured and well-defined set of terms has been agreed on including 1106 terms relating to ocular phenotypes (HPO) and 1202 terms relating to rare eye disease nomenclature (ORDO). These terms and their relevant annotations can be accessed in http://www.human-phenotype-ontology.org/ and http://www.orpha.net/ ; comments, corrections, suggestions and requests for new terms can be made through these websites. This is an ongoing, community-driven endeavour and both HPO and ORDO are regularly updated. CONCLUSIONS: To our knowledge, this is the first effort of such scale to provide terminology standards for the rare eye disease community. We hope that this work will not only improve coding and standardise information exchange in clinical care and research, but also it will catalyse the transition to an evidence-based precision ophthalmology paradigm

An ontological foundation for ocular phenotypes and rare eye diseases.

BACKGROUND: The optical accessibility of the eye and technological advances in ophthalmic diagnostics have put ophthalmology at the forefront of data-driven medicine. The focus of this study is rare eye disorders, a group of conditions whose clinical heterogeneity and geographic dispersion make data-driven, evidence-based practice particularly challenging. Inter-institutional collaboration and information sharing is crucial but the lack of standardised terminology poses an important barrier. Ontologies are computational tools that include sets of vocabulary terms arranged in hierarchical structures. They can be used to provide robust terminology standards and to enhance data interoperability. Here, we discuss the development of the ophthalmology-related component of two well-established biomedical ontologies, the Human Phenotype Ontology (HPO; includes signs, symptoms and investigation findings) and the Orphanet Rare Disease Ontology (ORDO; includes rare disease nomenclature/nosology). METHODS: A variety of approaches were used including automated matching to existing resources and extensive manual curation. To achieve the latter, a study group including clinicians, patient representatives and ontology developers from 17 countries was formed. A broad range of terms was discussed and validated during a dedicated workshop attended by 60 members of the group. RESULTS: A comprehensive, structured and well-defined set of terms has been agreed on including 1106 terms relating to ocular phenotypes (HPO) and 1202 terms relating to rare eye disease nomenclature (ORDO). These terms and their relevant annotations can be accessed in http://www.human-phenotype-ontology.org/ and http://www.orpha.net/ ; comments, corrections, suggestions and requests for new terms can be made through these websites. This is an ongoing, community-driven endeavour and both HPO and ORDO are regularly updated. CONCLUSIONS: To our knowledge, this is the first effort of such scale to provide terminology standards for the rare eye disease community. We hope that this work will not only improve coding and standardise information exchange in clinical care and research, but also it will catalyse the transition to an evidence-based precision ophthalmology paradigm

A synonymous codon variant in two patients with autosomal recessive bestrophinopathy alters in vitro splicing of BEST1

Purpose: Autosomal recessive bestrophinopathy (ARB) is a newly defined retinal dystrophy caused by biallelic mutations in bestrophin-1 (BEST1) and is hypothesized to represent the null bestrophin-1 phenotype in humans. The aim was to determine whether a synonymous BEST1 variant, c.102C>T, identified in two unrelated ARB patients, alters pre-mRNA splicing of the gene. Additionally a detailed phenotypic characterization of this distinctive condition is presented for both patients.Methods: BEST1 was analyzed by direct sequencing. Patients underwent standard ophthalmic assessment. In silico and in vitro analysis using a minigene system was performed to assess whether a synonymous variant identified, c.102C>T p.Gly34Gly, alters pre-mRNA splicing of BEST1.Results: Both ARB patients harbored either proven (patient 1; c.102C>T p.Gly34Gly and c.572T>C p.Leu191Pro) or presumed (patient 2; c.102C>T p.Gly34Gly and c.1470_1471delCA, p.His490GlnfsX24) biallelic mutations in BEST1 and were found to have phenotypes consistent with ARB. In vitro analysis of the synonymous variant, c.102C>T p.Gly34Gly, demonstrated it to introduce a cryptic splice donor site 52 nucleotides upstream of the actual splice donor site.Conclusions: The novel BEST1 variant identified, c.102C>T p.Gly34Gly, alters pre-mRNA splicing in vitro and is potentially pathogenic. In vivo this splicing variant is predicted to lead to the production of an mRNA transcript with a premature termination codon (p.Glu35TrpfsX11) that is predicted to be degraded by NMD

A detailed phenotypic assessment of individuals affected by MFRP-related oculopathy

Purpose: To determine the spectrum of mutations and phenotypic variability within patients with mutations in membrane-type frizzled related protein gene (MFRP).Methods: Individuals were initially ascertained based on a phenotype similar to that previously published in association with MFRP mutations. Affected patients underwent a full ophthalmic examination (best-corrected visual acuity, slit-lamp examination, applanation tonometry, and fundoscopy), color fundus photography, optical coherence tomography, autofluorescence imaging, and electrophysiology. MFRP was identified by a genome-wide scan in the fourth-largest autozygous region in one consanguineous family. Sanger sequencing of all the exons and intron-exon boundaries of MFRP was undertaken in the affected individuals.Results: Seven affected individuals from four families were identified as having mutations in MFRP. Patients from two families were homozygous for mutations already previously described (c. 1143_1144 insC and c. 492 delC), while those from the other two were compound heterozygous for mutations (c. 201G>A and c. 491_492 insT, and c. 492 delC, and c. 1622_1625 delTCTG), three of which were novel. There was considerable phenotypic variability within and among families. Autofluorescence imaging revealed the central macula to be relatively well preserved. Foveal cysts and optic nerve head drusen were present in two of the four families. Electrophysiology results showed rod-cone dystrophy with mild to moderate reduction in macular function in all affected members.Conclusions: We report three novel MFRP mutations and expand the phenotypic data available on patients with MFRP mutations

Assessment of the incorporation of CNV surveillance into gene panel next-generation sequencing testing for inherited retinal diseases.

BACKGROUND: Diagnostic use of gene panel next-generation sequencing (NGS) techniques is commonplace for individuals with inherited retinal dystrophies (IRDs), a highly genetically heterogeneous group of disorders. However, these techniques have often failed to capture the complete spectrum of genomic variation causing IRD, including CNVs. This study assessed the applicability of introducing CNV surveillance into first-tier diagnostic gene panel NGS services for IRD. METHODS: Three read-depth algorithms were applied to gene panel NGS data sets for 550 referred individuals, and informatics strategies used for quality assurance and CNV filtering. CNV events were confirmed and reported to referring clinicians through an accredited diagnostic laboratory. RESULTS: We confirmed the presence of 33 deletions and 11 duplications, determining these findings to contribute to the confirmed or provisional molecular diagnosis of IRD for 25 individuals. We show that at least 7% of individuals referred for diagnostic testing for IRD have a CNV within genes relevant to their clinical diagnosis, and determined a positive predictive value of 79% for the employed CNV filtering techniques. CONCLUSION: Incorporation of CNV analysis increases diagnostic yield of gene panel NGS diagnostic tests for IRD, increases clarity in diagnostic reporting and expands the spectrum of known disease-causing mutations

Novel mutations in MERTK associated with childhood onset rod-cone dystrophy

PurposeTo report the clinical phenotype in patients with a retinal dystrophy associated with novel mutations in the MER tyrosine kinase (MERTK) gene.MethodsA consanguineous family of Middle Eastern origin was identified, and affected members underwent a full clinical evaluation. Linkage analysis was performed using the Affymetrix 50K chip. Regions of homozygosity were identified. The positional candidate genes protocadherin 21 (PCDH21), retinal G protein-coupled receptor (RGR), and MERTK were polymerase chain reaction (PCR) amplified and sequenced. Long-range PCR was performed to characterize the deletion. Two hundred and ninety-two probands with autosomal recessive, childhood onset, retinal dystrophies were analyzed using the Asper Ophthalmics Leber congenital amaurosis chip to screen for known MERTK mutations.ResultsAnalysis of a 50K-Affymetrix whole genome scan identified three regions of homozygosity on chromosomes 2 and 10. Screening of the candidate gene MERTK showed a possible deletion of exon 8. Long-range PCR identified a ~9 kb deletion within MERTK that removes exon 8. Screening of DNA from a panel of Saudi Arabian patients with autosomal recessive retinitis pigmentosa identified a second consanguineous family with the same mutation. One patient with a known MERTK mutation (p.R651X) was identified using the Asper Ophthalmics Leber congenital amaurosis chip. Further screening of the gene identified a second novel splice site mutation in intron 1. The phenotype associated with these identified MERTK mutations is of a childhood onset rod-cone dystrophy with early macular atrophy. The optical coherence tomography (OCT) appearance is distinctive with evidence of debris beneath the sensory retina.ConclusionsMutations in MERTK are a rare cause of retinal dystrophy. Non homologous recombination between Alu Y repeats near or within disease genes may be an important cause of retinal dystrophies

Vision in observers with enhanced S-cone syndrome: an excess of S-cones connected mainly to conventional S-cone pathways but also a faster pathway

Purpose: The effect of increased numbers of S-cone photoreceptors in enhanced S-cone syndrome (ESCS) was investigated psychophysically in six ESCS observers to understand more about the relative cone sensitivities and postreceptoral organization. Methods: Measures of temporal sensitivity or delay were made: S- and L-cone temporal acuity (critical flicker fusion or cff), S-cone temporal contrast sensitivity, and S-cone delay. Results: ESCS observers showed uniform enhancements of S-cone cff of between 0.85 and 6.25 Hz, but reductions in L-cone cff. They also showed higher S-cone temporal-contrast-sensitivities at medium and high S-cone adaptation levels with sensitivity functions that peaked near 7.5 Hz but fell off at lower and higher frequencies; in contrast, the mean normal function was flat at low frequencies and fell-off only at high frequencies. The S-cone signal, as in the normal, is subject to large phase delays. Conclusions: We interpret the enhancements in cff as increases in S-cone number in ESCS of between 1.39 and 11.32 times normal density (with a mean of 3.48). The peaked ESCS contrast-sensitivity functions are consistent with S-cone signal interactions that increase sensitivity at intermediate frequencies through constructive interference but decrease it at lower and higher frequencies through destructive interference. Measures of S-cone delays relative to L- and M-cone signals show that the predominant S-cone signals in ESCS are negative and delayed as in normal observers, but reveal another faster, positive S-cone signal. This signal is also likely to be the cause of constructive and destructive interference in the contrast-sensitivity data of ESCS observers

Predicting risk of cardiovascular disease using retinal OCT imaging

We investigated the potential of optical coherence tomography (OCT) as an additional imaging technique to predict future cardiovascular disease (CVD). We utilised a self-supervised deep learning approach based on Variational Autoencoders (VAE) to learn low-dimensional representations of high-dimensional 3D OCT images and to capture distinct characteristics of different retinal layers within the OCT image. A Random Forest (RF) classifier was subsequently trained using the learned latent features and participant demographic and clinical data, to differentiate between patients at risk of CVD events (MI or stroke) and non-CVD cases. Our predictive model, trained on multimodal data, was assessed based on its ability to correctly identify individuals likely to suffer from a CVD event(MI or stroke), within a 5-year interval after image acquisition. Our self-supervised VAE feature selection and multimodal Random Forest classifier differentiate between patients at risk of future CVD events and the control group with an AUC of 0.75, outperforming the clinically established QRISK3 score (AUC= 0.597). The choroidal layer visible in OCT images was identified as an important predictor of future CVD events using a novel approach to model explanability. Retinal OCT imaging provides a cost-effective and non-invasive alternative to predict the risk of cardiovascular disease and is readily accessible in optometry practices and hospitals