PDE6A-Associated Retinitis Pigmentosa, Clinical Characteristics, Genetics and Natural History.

PURPOSE: To analyze the genetics, clinical characteristics, and natural history of PDE6A-associated retinitis pigmentosa. DESIGN: Retrospective, longitudinal, observational cohort study. PARTICIPANTS: Patients with molecularly confirmed PDE6A-associated retinal dystrophy in a single tertiary referral center. METHODS: Review of medical records and retinal imaging, including fundus autofluorescence (FAF) imaging and spectral-domain optical coherence tomography (SD-OCT). Genetic results were reviewed, and the detected variants were assessed. RESULTS: Sixteen patients (32 eyes) were identified and evaluated longitudinally. Genetic analysis identified 14 variants in the PDE6A gene, including 8 novel variants. The mean age (±SD, range) was 34.8 years (± 17.4, 12 - 76) at baseline, with a mean follow-up time of 4.8 years. Best-corrected visual acuity (BCVA) was 0.45 ± 0.45 LogMAR (range 0.0 - 1.6) at baseline and 0.65 ± 0.7 LogMAR (range 0.0 - 2.3) at the last visit. BCVA was similar among eyes in 88% of patients. A hyperautofluorescent ring was observed on FAF in 50% and 44% of the eyes at baseline and follow up visit respectively, with a mean area of 9.7 ± 4.5mm2 at baseline and mean of 8.6 ± 4.8 mm2 at the follow-up visit. Mean horizontal ellipsoid zone width (EZW) at baseline was 1765 ± 1093 µm, which decreased to 1580 ± 1077 µm at follow up. Eighteen eyes exhibited cystoid macular oedema at baseline (56%), and 17 eyes (53%) at follow-up. There were statistically significant changes during the follow-up period in terms of BCVA, hyperautoflouroscent ring area and the EZW. CONCLUSIONS: This study highlights the natural history of PDE6A-retinopathy. The majority of the patients in this cohort had mild BCVA loss, and slowly progressive disease, based on FAF and OCT measurements.

A patient with albinism and retinitis pigmentosa, a case report.

Purpose: To present a case of molecularly confirmed oculocutaneous albinism (OCA) and retinitis pigmentosa (RP). Observations: A 46-year-old male with a lifelong established diagnosis of OCA and baseline best corrected visual acuity (BCVA) of 20/200, presented for worsening visual acuity over the last few years. BCVA was light perception and hand motion at face for the right and left eye, respectively. Fundus exam showed hypopigmented fundi with visible choroidal vessels and blunted foveal reflexes in both eyes. Optical coherence tomography showed foveal hypoplasia and outer retinal degenerative changes not typical of OCA. Fundus autofluorescence (FAF) imaging showed focal areas of decreased signal at the fovea, similar to areas of atrophy in an age matched patient with PDE6A-RP. Genetic testing identified a homozygous disease-causing variant in TYR c.1467dup, p. (Ala490Cysfs*20) causing OCA, and a homozygous pathogenic variant c.304C > A, p. (Arg102Ser) in PDE6A causing autosomal recessive RP. Conclusions and importance: This is the first report of a patient with OCA and RP. The lack of pigmentary changes can make the diagnosis of RP challenging in patients with albinism. FAF can show features suggestive of RP and genetic testing can establish the diagnosis. The findings described herein may help physicians diagnose an extremely rare phenotype.

Genetics, Clinical Characteristics, and Natural History of PDE6B-Associated Retinal Dystrophy.

PURPOSE: To analyze the clinical characteristics, natural history, and genetics of PDE6B-associated retinal dystrophy. DESIGN: Retrospective, observational cohort study. METHODS: Review of medical records and retinal imaging, including fundus autofluorescence (FAF) imaging and spectral-domain optical coherence tomography (SD-OCT) of patients with molecularly confirmed PDE6B-associated retinal dystrophy in a single tertiary referral center. Genetic results were reviewed, and the detected variants were assessed. RESULTS: Forty patients (80 eyes) were identified and evaluated longitudinally. The mean age (±SD, range) was 42.1 years (± 19.0, 10-86) at baseline, with a mean follow-up time of 5.2 years. Twenty-nine (72.5%) and 27 (67.5%) patients had no or mild visual acuity impairment at baseline and last visit, respectively. Best-corrected visual acuity (BCVA) was 0.56 ± 0.72 LogMAR (range -0.12 to 2.80) at baseline and 0.63 ± 0.73 LogMAR (range 0.0-2.80) at the last visit. BCVA was symmetrical in 87.5% of patients. A hyperautofluorescent ring was observed on FAF in 48 and 46 eyes at baseline and follow-up visit, respectively, with a mean area of 7.11 ± 4.13 mm2 at baseline and mean of 6.13 ± 3.62 mm2 at the follow-up visit. Mean horizontal ellipsoid zone width at baseline was 1946.1 ± 917.2 µm, which decreased to 1763.9 ± 827.9 µm at follow-up. Forty-four eyes had cystoid macular edema at baseline (55%), and 41 eyes (51.3%) at follow-up. There were statistically significant changes during the follow-up period in terms of BCVA and the ellipsoid zone width. Genetic analysis identified 43 variants in the PDE6B gene, including 16 novel variants. CONCLUSIONS: This study details the natural history of PDE6B-retinopathy in the largest cohort to date. Most patients had mild to no BCVA loss, with slowly progressive disease, based on FAF and OCT metrics. There is a high degree of disease symmetry and a wide window for intervention.

Best Vitelliform Macular Dystrophy Natural History Study Report 1: Clinical Features and Genetic Findings.

PURPOSE: To analyze the genetic findings, clinical spectrum, and natural history of Best vitelliform macular dystrophy (BVMD) in a cohort of 222 children and adults. DESIGN: Single-center retrospective, consecutive, observational study. PARTICIPANTS: Patients with a clinical diagnosis of BVMD from pedigrees with a likely disease-causing monoallelic sequence variant in the BEST1 gene. METHODS: Data were extracted from electronic and physical case notes. Electrophysiologic assessment and molecular genetic testing were analyzed. MAIN OUTCOME MEASURES: Molecular genetic test findings and clinical findings including best-corrected visual acuity (BCVA), choroidal neovascularization (CNV) rates, and electrophysiologic parameters. RESULTS: Two hundred twenty-two patients from 141 families were identified harboring 69 BEST1 variants. Mean age at presentation was 26.8 years (range, 1.3-84.8 years) and most patients (61.5%) demonstrated deterioration of central vision. Major funduscopic findings included 128 eyes (30.6%) with yellow vitelliform lesions, 78 eyes (18.7%) with atrophic changes, 49 eyes (11.7%) with fibrotic changes, 48 eyes (11.5%) with mild pigmentary changes, and 43 eyes (10.3%) showing a vitelliruptive appearance. Mean BCVA was 0.37 logarithm of the minimum angle of resolution (logMAR; Snellen equivalent, 20/47) for the right eye and 0.33 logMAR (Snellen equivalent, 20/43) for the left eye at presentation, with a mean annual loss rate of 0.013 logMAR and 0.009 logMAR, respectively, over a mean follow-up of 9.7 years. Thirty-seven patients (17.3%) received a diagnosis of CNV over a mean follow-up of 8.0 years. Eyes with CNV that received treatment with an anti-vascular endothelial growth factor (VEGF) agent showed better mean BCVA compared with eyes that were not treated with an anti-VEGF agent (0.28 logMAR [Snellen equivalent, 20/38] vs. 0.62 logMAR [Snellen equivalent, 20/83]). Most eyes exhibited a hyperopic refractive error (78.7%), and 13 patients (6.1%) received a diagnosis of amblyopia. Among the 3 most common variants, p.(Ala243Val) was associated with a later age of onset, better age-adjusted BCVA, and less advanced Gass stages compared with p.(Arg218Cys) and p.(Arg218His). CONCLUSIONS: BVMD shows a wide spectrum of phenotypic variability. The disease is very slowly progressive, and the observed phenotype-genotype correlations allow for more accurate prognostication and counselling. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Integrated Computational Study of the Light-Activated Structure of the AppA BLUF Domain and Its Spectral Signatures.

We apply an integrated approach combining microsecond MD simulations and (polarizable) QM/MM calculations of NMR, FTIR, and UV-vis spectra to validate the structure of the light-activated form of the AppA photoreceptor, an example of blue light using flavin (BLUF) protein domain. The latter photoactivate through a proton-coupled electron transfer (PCET) that results in a tautomerization of a conserved glutamine residue in the active site, but this mechanism has never been spectroscopically proven for AppA, which has been always considered as an exception. Our simulations instead confirm that the spectral features observed upon AppA photoactivation are indeed directly connected to the tautomer form of glutamine as predicted by the PCET mechanism. In addition, we observe small but significant changes in the AppA structure, which are transmitted from the flavin binding pocket to the surface of the protein.

Prognostication in Stargardt Disease Using Fundus Autofluorescence: Improving Patient Care.

PURPOSE: To explore fundus autofluorescence (FAF) imaging as an alternative to electroretinography as a noninvasive, quick, and readily interpretable method to predict disease progression in Stargardt disease (STGD). DESIGN: Retrospective case series of patients who attended Moorfields Eye Hospital (London, United Kingdom). PARTICIPANTS: Patients with STGD who met the following criteria were included: (1) biallelic disease-causing variants in ABCA4, (2) electroretinography testing performed in house with an unequivocal electroretinography group classification, and (3) ultrawidefield (UWF) FAF imaging performed up to 2 years before or after the electroretinography. METHODS: Patients were divided into 3 electroretinography groups based on retinal function and 3 FAF groups according to the extent of hypoautofluorescence and retinal background appearance. Fundus autofluorescence images of 30° and 55° were reviewed subsequently. MAIN OUTCOME MEASURES: Electroretinography and FAF concordance and its association with baseline visual acuity (VA) and genetics. RESULTS: Two hundred thirty-four patients were included in the cohort. One hundred seventy patients (73%) were in electroretinography and FAF groups of the same severity, 33 (14%) were in a milder FAF than electroretinography group, and 31 (13%) were in a more severe FAF than electroretinography group. Children < 10 years of age (n = 23) showed the lowest electroretinography and FAF concordance at 57% (9 of the 10 with discordant electroretinography and FAF showed milder FAF than electroretinography), and adults with adult onset showed the highest (80%). In 97% and 98% of patients, 30° and 55° FAF imaging, respectively, matched with the group defined by UWF FAF. CONCLUSIONS: We demonstrated that FAF imaging is an effective method to determine the extent of retinal involvement and thereby inform prognostication by comparing FAF with the current gold standard of electroretinography. In 80% of patients in our large molecularly proven cohort, we were able to predict if the disease was confined to the macula or also affected the peripheral retina. Children assessed at a young age, with at least 1 null variant, early disease onset, poor initial VA, or a combination thereof may have wider retinal involvement than predicted by FAF alone, may progress to a more severe FAF phenotype over time, or both. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

RPGR-Related Retinopathy: Clinical Features, Molecular Genetics, and Gene Replacement Therapy.

Retinitis pigmentosa GTPase regulator (RPGR) gene variants are the predominant cause of X-linked retinitis pigmentosa (XLRP) and a common cause of cone-rod dystrophy (CORD). XLRP presents as early as the first decade of life, with impaired night vision and constriction of peripheral visual field and rapid progression, eventually leading to blindness. In this review, we present RPGR gene structure and function, molecular genetics, animal models, RPGR-associated phenotypes and highlight emerging potential treatments such as gene-replacement therapy.

Fast Method for Excited-State Dynamics in Complex Systems and Its Application to the Photoactivation of a Blue Light Using Flavin Photoreceptor.

The excited-state dynamics of molecules embedded in complex (bio)matrices is still a challenging goal for quantum chemical models. Hybrid QM/MM models have proven to be an effective strategy, but an optimal combination of accuracy and computational cost still has to be found. Here, we present a method which combines the accuracy of a polarizable embedding QM/MM approach with the computational efficiency of an excited-state self-consistent field method. The newly implemented method is applied to the photoactivation of the blue-light-using flavin (BLUF) domain of the AppA protein. We show that the proton-coupled electron transfer (PCET) process suggested for other BLUF proteins is still valid also for AppA.

Functional evaluation in inherited retinal disease.

Functional assessments are a fundamental part of the clinical evaluation of patients with inherited retinal diseases (IRDs). Their importance and impact have become increasingly notable, given the significant breadth and number of clinical trials and studies investigating multiple avenues of intervention across a wide range of IRDs, including gene, pharmacological and cellular therapies. Moreover, the fact that many clinical trials are reporting improvements in vision, rather than the previously anticipated structural stability/slowing of degeneration, makes functional evaluation of primary relevance. In this review, we will describe a range of methods employed to characterise retinal function and functional vision, beginning with tests variably included in the clinic, such as visual acuity, electrophysiological assessment and colour discrimination, and then discussing assessments often reserved for clinical trials/research studies such as photoaversion testing, full-field static perimetry and microperimetry, and vision-guided mobility testing; addressing perimetry in greatest detail, given it is commonly a primary outcome metric. We will focus on how these tests can help diagnose and monitor particular genotypes, also noting their limitations/challenges and exploring analytical methodologies for better exploiting functional measurements, as well as how they facilitate patient inclusion and stratification in clinical trials and serve as outcome measures.

From crystallographic data to the solution structure of photoreceptors: the case of the AppA BLUF domain.

Photoreceptor proteins bind a chromophore, which, upon light absorption, modifies its geometry or its interactions with the protein, finally inducing the structural change needed to switch the protein from an inactive to an active or signaling state. In the Blue Light-Using Flavin (BLUF) family of photoreceptors, the chromophore is a flavin and the changes have been connected with a rearrangement of the hydrogen bond network around it on the basis of spectroscopic changes measured for the dark-to-light conversion. However, the exact conformational change triggered by the photoexcitation is still elusive mainly because a clear consensus on the identity not only of the light activated state but also of the dark one has not been achieved. Here, we present an integrated investigation that combines microsecond MD simulations starting from the two conflicting crystal structures available for the AppA BLUF domain with calculations of NMR, IR and UV-Vis spectra using a polarizable QM/MM approach. Thanks to such a combined analysis of the three different spectroscopic responses, a robust characterization of the structure of the dark state in solution is given together with the uncovering of important flaws of the most popular molecular mechanisms present in the literature for the dark-to-light activation.

Ultrafast Transient Infrared Spectroscopy of Photoreceptors with Polarizable QM/MM Dynamics.

Ultrafast transient infrared (TRIR) spectroscopy is widely used to measure the excitation-induced structural changes of protein-bound chromophores. Here, we design a novel and general strategy to compute TRIR spectra of photoreceptors by combining µs-long MM molecular dynamics with ps-long QM/AMOEBA Born-Oppenheimer molecular dynamics (BOMD) trajectories for both ground and excited electronic states. As a proof of concept, the strategy is here applied to AppA, a blue-light-utilizing flavin (BLUF) protein, found in bacteria. We first analyzed the short-time evolution of the embedded flavin upon excitation revealing that its dynamic Stokes shift is ultrafast and mainly driven by the internal reorganization of the chromophore. A different normal-mode representation was needed to describe ground- and excited-state IR spectra. In this way, we could assign all of the bands observed in the measured transient spectrum. In particular, we could characterize the flavin isoalloxazine-ring region of the spectrum, for which a full and clear description was missing.

Structural evaluation in inherited retinal diseases.

Ophthalmic genetics is a field that has been rapidly evolving over the last decade, mainly due to the flourishing of translational medicine for inherited retinal diseases (IRD). In this review, we will address the different methods by which retinal structure can be objectively and accurately assessed in IRD. We review standard-of-care imaging for these patients: colour fundus photography, fundus autofluorescence imaging and optical coherence tomography (OCT), as well as higher-resolution and/or newer technologies including OCT angiography, adaptive optics imaging, fundus imaging using a range of wavelengths, magnetic resonance imaging, laser speckle flowgraphy and retinal oximetry, illustrating their utility using paradigm genotypes with on-going therapeutic efforts/trials.

Heart Failure Drug Modifies the Intrinsic Dynamics of the Pre-Power Stroke State of Cardiac Myosin.

Omecamtiv mecarbil (OM), currently investigated for the treatment of heart failure, is the first example of a new class of drugs (cardiac myotropes) that can modify muscle contractility by directly targeting sarcomeric proteins. Using atomistic molecular dynamics simulations, we show that the binding of OM to the pre-power stroke state of cardiac myosin inhibits the functional motions of the protein and potentially affects Pi release from the nucleotide binding site. We also show that the changes in myosin ATPase activity induced by a set of OM analogues can be predicted from their relative affinity to the pre-power stroke state compared to the near rigor one, indicating that conformational selectivity plays an important role in determining the activity of these compounds.

An Activity-Based Probe Targeting Non-Catalytic, Highly Conserved Amino Acid Residues within Bromodomains.

Bromodomain-containing proteins are epigenetic modulators involved in a wide range of cellular processes, from recruitment of transcription factors to pathological disruption of gene regulation and cancer development. Since the druggability of these acetyl-lysine reader domains was established, efforts were made to develop potent and selective inhibitors across the entire family. Here we report the development of a small molecule-based approach to covalently modify recombinant and endogenous bromodomain-containing proteins by targeting a conserved lysine and a tyrosine residue in the variable ZA or BC loops. Moreover, the addition of a reporter tag allowed in-gel visualization and pull-down of the desired bromodomains.

Allosteric modulation of cardiac myosin dynamics by omecamtiv mecarbil.

New promising avenues for the pharmacological treatment of skeletal and heart muscle diseases rely on direct sarcomeric modulators, which are molecules that can directly bind to sarcomeric proteins and either inhibit or enhance their activity. A recent breakthrough has been the discovery of the myosin activator omecamtiv mecarbil (OM), which has been shown to increase the power output of the cardiac muscle and is currently in clinical trials for the treatment of heart failure. While the overall effect of OM on the mechano-chemical cycle of myosin is to increase the fraction of myosin molecules in the sarcomere that are strongly bound to actin, the molecular basis of its action is still not completely clear. We present here a Molecular Dynamics study of the motor domain of human cardiac myosin bound to OM, where the effects of the drug on the dynamical properties of the protein are investigated for the first time with atomistic resolution. We found that OM has a double effect on myosin dynamics, inducing a) an increased coupling of the motions of the converter and lever arm subdomains to the rest of the protein and b) a rewiring of the network of dynamic correlations, which produces preferential communication pathways between the OM binding site and distant functional regions. The location of the residues responsible for these effects suggests possible strategies for the future development of improved drugs and the targeting of specific cardiomyopathy-related mutations.

In silico analysis of the binding of anthelmintics to Caenorhabditis elegansP-glycoprotein 1.

Macrocyclic lactones (ML) are important anthelmintics used in animals and humans against parasite nematodes, but their therapeutic success is compromised by the spread of ML resistance. Some ABC transporters, such as p-glycoproteins (Pgps), are selected and overexpressed in ML-resistant nematodes, supporting a role for some drug efflux proteins in ML resistance. However, the role of such proteins in ML transport remains to be clarified at the molecular level. Recently, Caenorhabditis elegans Pgp-1 (Cel-Pgp-1) has been crystallized, and its drug-modulated ATPase function characterized in vitro revealed Cel-Pgp-1 as a multidrug transporter. Using this crystal structure, we have developed an in silico drug docking model in order to study the binding of ML and other anthelmintic drugs to Cel-Pgp-1. All tested ML bound with high affinity in a unique site, within the inner chamber of the protein, supporting that ML may be transported by Cel-Pgp-1. Interestingly, interacting residues delineate a ML specific fingerprint involving H-bonds, including T1028. In particular, benzofurane and spiroketal moieties bound to specific sub-sites. When compared with the aglycone ML, such as moxidectin and ivermectin aglycone, avermectin anthelmintics have significant higher affinity for Cel-Pgp-1, likely due to the sugar substituent(s) that bind to a specific area involving H-bonds at Y771. Triclabendazole, closantel and emodepside bound with good affinities to different sub-sites in the inner chamber, partially overlapping with the ML binding site, suggesting that they could compete for Cel-Pgp-1-mediated ML transport. In conclusion, this work provides novel information on the role of nematode Pgps in transporting anthelmintics, and a valuable tool to predict drug-drug interactions and to rationally design new competitive inhibitors of clinically-relevant nematode Pgps, to improve anthelmintic therapeutics.