Autosomal Recessive Cone-rod Dystrophy Research Articles

Cone dystrophy with supernormal rod response – A rare case report

Cone dystrophy with supernormal rod response (CDSRR), also known as potassium channel subfamily V, member 2 (KCNV2) retinopathy, is a rare autosomal recessive cone rod dystrophy, which derives its name from its characteristic full-field electroretinography (ERG) features and the causative gene mutation (KCNV2 gene). It is one of the rare eye retinal dystrophies where ERG is pathognomonic and directly carries the genetic imprint of the condition. Here, we discuss the case of a 9-year-old Indian child with history of diminution of vision in both eyes. Full-field ERG showed a characteristic response of supranormal scotopic b-wave amplitude at high luminance intensities along with broadened trough of the a-wave with late negative component with reduced and delayed photopic response suggestive of CDSRR.

Variants in UBAP1L lead to autosomal recessive rod-cone and cone-rod dystrophy

PurposeProgressive inherited retinal degenerations (IRDs) affecting rods and cones are clinically and genetically heterogeneous and can lead to blindness with limited therapeutic options. The major gene defects have been identified in subjects of European and Asian descent with only few reports of North African descent. MethodsGenome, targeted next-generation, and Sanger sequencing was applied to cohort of ∼4000 IRDs cases. Expression analyses were performed including Chip-seq database analyses, on human-derived retinal organoids (ROs), retinal pigment epithelium cells, and zebrafish. Variants’ pathogenicity was accessed using 3D-modeling and/or ROs. ResultsHere, we identified a novel gene defect with three distinct pathogenic variants in UBAP1L in 4 independent autosomal recessive IRD cases from Tunisia. UBAP1L is expressed in the retinal pigment epithelium and retina, specifically in rods and cones, in line with the phenotype. It encodes Ubiquitin-associated protein 1-like, containing a solenoid of overlapping ubiquitin-associated domain, predicted to interact with ubiquitin. In silico and in vitro studies, including 3D-modeling and ROs revealed that the solenoid of overlapping ubiquitin-associated domain is truncated and thus ubiquitin binding most likely abolished secondary to all variants identified herein. ConclusionBiallelic UBAP1L variants are a novel cause of IRDs, most likely enriched in the North African population.

Gene therapy for RAB28: What can we learn from zebrafish?

The eye is particularly suited to gene therapy due to its accessibility, immunoprivileged state and compartmentalised structure. Indeed, many clinical trials are underway for therapeutic gene strategies for inherited retinal degenerations (IRDs). However, as there are currently 281 genes associated with IRD, there is still a large unmet need for effective therapies for the majority of IRD-causing genes. In humans, RAB28 null and hypomorphic alleles cause autosomal recessive cone-rod dystrophy (arCORD). Previous work demonstrated that restoring wild type zebrafish Rab28 via germline transgenesis, specifically in cone photoreceptors, is sufficient to rescue the defects in outer segment phagocytosis (OSP) observed in zebrafish rab28-/- knockouts (KO). This rescue suggests that gene therapy for RAB28-associated CORD may be successful by RAB28 gene restoration to cones. It also inspired us to critically consider the scenarios in which zebrafish can provide informative preclinical data for development of gene therapies. Thus, this review focuses on RAB28 biology and disease, and delves into both the opportunities and limitations of using zebrafish as a model for both gene therapy development and as a diagnostic tool for patient variants of unknown significance (VUS).

Association of ABCA4 Gene Variants in Patients with Autosomal Recessive Cone-Rod Dystrophy and Retinitis Pigmentosa Cohorts from South India

Association of ABCA4 Gene Variants in Patients with Autosomal Recessive Cone-Rod Dystrophy and Retinitis Pigmentosa Cohorts from South India

Dawn and dusk peaks of outer segment phagocytosis, and visual cycle function require Rab28.

RAB28 is a farnesylated, ciliary G‐protein. Patient variants in RAB28 are causative of autosomal recessive cone‐rod dystrophy (CRD), an inherited human blindness. In rodent and zebrafish models, the absence of Rab28 results in diminished dawn, photoreceptor, outer segment phagocytosis (OSP). Here, we demonstrate that Rab28 is also required for dusk peaks of OSP, but not for basal OSP levels. This study further elucidated the molecular mechanisms by which Rab28 controls OSP and inherited blindness. Proteomic profiling identified factors whose expression in the eye or whose expression at dawn and dusk peaks of OSP is dysregulated by loss of Rab28. Notably, transgenic overexpression of Rab28, solely in zebrafish cones, rescues the OSP defect in rab28 KO fish, suggesting rab28 gene replacement in cone photoreceptors is sufficient to regulate Rab28‐OSP. Rab28 loss also perturbs function of the visual cycle as retinoid levels of 11‐cRAL, 11cRP, and atRP are significantly reduced in larval and adult rab28 KO retinae (p < .05). These data give further understanding on the molecular mechanisms of RAB28‐associated CRD, highlighting roles of Rab28 in both peaks of OSP, in vitamin A metabolism and in retinoid recycling.

Long-Read Sequencing to Unravel Complex Structural Variants of CEP78 Leading to Cone-Rod Dystrophy and Hearing Loss.

Inactivating variants as well as a missense variant in the centrosomal CEP78 gene have been identified in autosomal recessive cone-rod dystrophy with hearing loss (CRDHL), a rare syndromic inherited retinal disease distinct from Usher syndrome. Apart from this, a complex structural variant (SV) implicating CEP78 has been reported in CRDHL. Here we aimed to expand the genetic architecture of typical CRDHL by the identification of complex SVs of the CEP78 region and characterization of their underlying mechanisms. Approaches used for the identification of the SVs are shallow whole-genome sequencing (sWGS) combined with quantitative polymerase chain reaction (PCR) and long-range PCR, or ExomeDepth analysis on whole-exome sequencing (WES) data. Targeted or whole-genome nanopore long-read sequencing (LRS) was used to delineate breakpoint junctions at the nucleotide level. For all SVs cases, the effect of the SVs on CEP78 expression was assessed using quantitative PCR on patient-derived RNA. Apart from two novel canonical CEP78 splice variants and a frameshifting single-nucleotide variant (SNV), two SVs affecting CEP78 were identified in three unrelated individuals with CRDHL: a heterozygous total gene deletion of 235 kb and a partial gene deletion of 15 kb in a heterozygous and homozygous state, respectively. Assessment of the molecular consequences of the SVs on patient’s materials displayed a loss-of-function effect. Delineation and characterization of the 15-kb deletion using targeted LRS revealed the previously described complex CEP78 SV, suggestive of a recurrent genomic rearrangement. A founder haplotype was demonstrated for the latter SV in cases of Belgian and British origin, respectively. The novel 235-kb deletion was delineated using whole-genome LRS. Breakpoint analysis showed microhomology and pointed to a replication-based underlying mechanism. Moreover, data mining of bulk and single-cell human and mouse transcriptional datasets, together with CEP78 immunostaining on human retina, linked the CEP78 expression domain with its phenotypic manifestations. Overall, this study supports that the CEP78 locus is prone to distinct SVs and that SV analysis should be considered in a genetic workup of CRDHL. Finally, it demonstrated the power of sWGS and both targeted and whole-genome LRS in identifying and characterizing complex SVs in patients with ocular diseases.

Expanding the Clinical and Genetic Spectrum of RAB28-Related Cone-Rod Dystrophy: Pathogenicity of Novel Variants in Italian Families

The small Ras-related GTPase Rab-28 is highly expressed in photoreceptor cells, where it possibly participates in membrane trafficking. To date, six alterations in the RAB28 gene have been associated with autosomal recessive cone-rod dystrophies. Confirmed variants include splicing variants, missense and nonsense mutations. Here, we present a thorough phenotypical and genotypical characterization of five individuals belonging to four Italian families, constituting the largest cohort of RAB28 patients reported in literature to date. All probands displayed similar clinical phenotype consisting of photophobia, decreased visual acuity, central outer retinal thinning, and impaired color vision. By sequencing the four probands, we identified: a novel homozygous splicing variant; two novel nonsense variants in homozygosis; a novel missense variant in compound heterozygous state with a previously reported nonsense variant. Exhaustive molecular dynamics simulations of the missense variant p.(Thr26Asn) in both its active and inactive states revealed an allosteric structural mechanism that impairs the binding of Mg2+, thus decreasing the affinity for GTP. The impaired GTP-GDP exchange ultimately locks Rab-28 in a GDP-bound inactive state. The loss-of-function mutation p.(Thr26Asn) was present in a compound heterozygosis with the nonsense variant p.(Arg137*), which does not cause mRNA-mediated decay, but is rather likely degraded due to its incomplete folding. The frameshift p.(Thr26Valfs4*) and nonsense p.(Leu13*) and p.(Trp107*) variants, if translated, would lack several key structural components necessary for the correct functioning of the encoded protein.

A New Cerkl Mouse Model Generated by CRISPR-Cas9 Shows Progressive Retinal Degeneration and Altered Morphological and Electrophysiological Phenotype.

PurposeClose to 100 genes cause retinitis pigmentosa, a Mendelian rare disease that affects 1 out of 4000 people worldwide. Mutations in the ceramide kinase-like gene (CERKL) are a prevalent cause of autosomal recessive cause retinitis pigmentosa and cone–rod dystrophy, but the functional role of this gene in the retina has yet to be fully determined. We aimed to generate a mouse model that resembles the phenotypic traits of patients carrying CERKL mutations to undertake functional studies and assay therapeutic approaches.MethodsThe Cerkl locus has been deleted (around 97 kb of genomic DNA) by gene editing using the CRISPR-Cas9 D10A nickase. Because the deletion of the Cerkl locus is lethal in mice in homozygosis, a double heterozygote mouse model with less than 10% residual Cerkl expression has been generated. The phenotypic alterations of the retina of this new model have been characterized at the morphological and electrophysiological levels.ResultsThis CerklKD/KO model shows retinal degeneration, with a decreased number of cones and progressive photoreceptor loss, poorly stacked photoreceptor outer segment membranes, defective retinal pigment epithelium phagocytosis, and altered electrophysiological recordings in aged retinas.ConclusionsTo our knowledge, this is the first Cerkl mouse model to mimic many of the phenotypic traits, including the slow but progressive retinal degeneration, shown by human patients carrying CERKL mutations. This useful model will provide unprecedented insights into the retinal molecular pathways altered in these patients and will contribute to the design of effective treatments.

Ciliary Rab28 and the BBSome negatively regulate extracellular vesicle shedding.

Cilia both receive and send information, the latter in the form of extracellular vesicles (EVs). EVs are nano-communication devices that influence cell, tissue, and organism behavior. Mechanisms driving ciliary EV biogenesis are almost entirely unknown. Here, we show that the ciliary G-protein Rab28, associated with human autosomal recessive cone-rod dystrophy, negatively regulates EV levels in the sensory organs of Caenorhabditis elegans in a cilia specific manner. Sequential targeting of lipidated Rab28 to periciliary and ciliary membranes is highly dependent on the BBSome and the prenyl-binding protein phosphodiesterase 6 subunit delta (PDE6D), respectively, and BBSome loss causes excessive and ectopic EV production. We also find that EV defective mutants display abnormalities in sensory compartment morphogenesis. Together, these findings reveal that Rab28 and the BBSome are key in vivo regulators of EV production at the periciliary membrane and suggest that EVs may mediate signaling between cilia and glia to shape sensory organ compartments. Our data also suggest that defects in the biogenesis of cilia-related EVs may contribute to human ciliopathies.

A primer of deuterium in drug design

Future Medicinal ChemistryVol. 11, No. 16 CommentaryFree AccessA primer of deuterium in drug designSarah Cargnin, Marta Serafini & Tracey PiraliSarah CargninDepartment of Pharmaceutical Sciences, Università del Piemonte Orientale, Largo Donegani 2, 28100 Novara, ItalySearch for more papers by this author, Marta SerafiniDepartment of Pharmaceutical Sciences, Università del Piemonte Orientale, Largo Donegani 2, 28100 Novara, ItalySearch for more papers by this author & Tracey Pirali*Author for correspondence: E-mail Address: tracey.pirali@uniupo.itDepartment of Pharmaceutical Sciences, Università del Piemonte Orientale, Largo Donegani 2, 28100 Novara, ItalySearch for more papers by this authorPublished Online:20 Sep 2019https://doi.org/10.4155/fmc-2019-0183AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Deuterium in medicinal chemistryVery recently, deuterium (D), the heavy and nonradioactive isotope of hydrogen (H), has been a protagonist of cosmochemistry. Measurements of D/H isotopic ratio of water in minerals collected by a mission from the asteroid Itokawa have finally demonstrated that the composition of water is indistinguishable from that in terrestrial rocks, suggesting that asteroids could have been a potential source of water during the formation of Earth [1]. Cosmochemistry is not the only field where D has recently gained popularity. Indeed, this isotope is appealing also to medicinal chemists as a means of improving existing drugs or as a tool in the design of new lead compounds. This is particularly true since 2017, where the USFDA approved the first deuterated drug, deutetrabenazine [2]. A comprehensive review on medicinal chemistry of D has been recently published [3]. In the present commentary, we wish to critically highlight the key advantages of D and review the most recent advances in clinics with deuterated compounds. Indeed, this is a flourishing field, with a plethora of clinical trials being pursued.There is a sound reason for the success of this isotope in drug design: the D kinetic isotope effect. Due to the twofold higher mass of D compared with H, the C-D bond is much more resistant toward oxidative processes, for example catalyzed by CYP450 or by other enzymes involved in metabolism (for example; MAO, aldehyde oxidase), while retaining very similar steric properties. Such resistance must be proved nonetheless experimentally, case by case, as it is heavily dependent on the enzyme catalysis scenario and is more pronounced in those cases where the corresponding C-H bond cleavage is the rate-limiting step.Therefore, the H-D isosteric replacement in correspondence of an oxidizable soft-spot usually retains the pharmacodynamics, while improving the pharmacokinetics of a drug with a repercussion on half-life and/or of area under the curve values and, ultimately, on dose and/or dosing regimen. [4] The most striking example of this improvement is given by some inhibitors of aurora kinase reported in 2011 where the judicious incorporation of the two classical isosteres of H, D and fluorine, causes a significant increase of systemic drug exposure, together with a decrease of clearance, in four different animal species [5]. Although many other examples could be presented to show the ability of D to improve the pharmacokinetics of drugs, such amelioration does not always occur, and many factors that can mask or even revert the effects of deuteration come into play.D has implications that go beyond the mere increase of metabolic stability. For instance, deuteration can be capitalized upon to reduce toxicity, as demonstrated by many examples of deuteration that have followed over the years, including the recent report on the c-Met inhibitor JNJ38877605 [6]. Here, the soft-spot is the 2-position of quinoline ring which is extensively oxidized by aldehyde oxidase. While the classic H-F isosteric replacement [7] is not possible as it hampers the formation of a key H bond with a residue of the kinase, D incorporation on one hand retains the biological activity, and on the other improves the metabolic stability, thereby reducing the formation of a nephrotoxic metabolite and increases the oral exposure.D incorporation not only affects enzyme-catalyzed reactions but also pure chemical processes, such as epimerization. Many drugs are still sold as racemates due to chemically unstable stereocenters that make the two enantiomers interconverting. When incorporated in correspondence of chiral centres, D might decrease the rate of atom abstraction and stabilize the configuration in favor of the preferred enantiomer (the so-called D enabled chiral switching), as reported for CC-122, a thalidomide derivative in clinical trials for hematological cancers and solid tumors [8]. In this case, the (-)-deuterated enantiomer is antitumorigenic, whereas the other enantiomer has no effect on tumor growth.Deuterated compounds in clinical development: an updateAs already mentioned, the first deuterated compound that has entered clinical practice is deutetrabenazine for the treatment of both choreas associated with Huntington's disease and tardive dyskinesia. This success has given industry confidence to invest in the development of deuterated drugs, and as a result, more than 20 deuterated drugs are currently in clinical development [3], with 6 of them (BMS-986165, AVP-786, RT001, ALK-001, donafenib, HC-1119) having reached Phase III clinical trials.There is ferment in the field, certainly, and relevant progress in the clinical testing of deuterated drugs has been made in the last few months. In December 2018, a new deuterated drug, HC-1119, publicly entered the panorama of deuterated medicines in clinical development. HC-1119 is the deuterated analog of enzalutamide, a second-generation competitive inhibitor of the androgen receptor which is effective for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Even if enzalutamide was shown to significantly prolong overall survival in mCRPC patients compared to placebo, a higher risk of seizure occurrence was detected in enzalutamide users [9]. Given that such toxicity was found to be dose dependent, a deuteration strategy was applied in order to improve enzalutamide's PK properties and potentially limit side effects. Since enzalutamide is metabolized to N-demethylenzalutamide mainly by CYP2C8 and CYP3A4/5, the N-CH3 moiety was substituted by N-CD3 in order to attenuate the N-demethylation pathway. As a result, HC-1119 was shown to have an ameliorated PK profile and a higher safety margin compared with its nondeuterated counterpart in different in vivo models [10,11]. Nowadays, five clinical trials for HC-1119 are registered on Clinicaltrials.gov. Three of these are Phase I, open-label, clinical trials aimed at evaluating the pharmacokinetic properties of HC-1119 in healthy volunteers (NCT03776968) or in patients affected by mCRPC (NCT03774056, NCT03778047). The remaining two studies are Phase III, multicenter, randomized, double-blind, clinical trials assessing the efficacy and safety of oral HC-1119 in mCRPC patients when compared with placebo (NCT03851640) or oral enzalutamide (NCT03850795). It should be noted that the use of enzalutamide as a direct comparator renders the study design deeply informative since it offers the chance of reliably assessing the value of the deuteration. The estimated dates of Phase III studies completions are reported to fall within 2021.Recent progresses in the field also concern ALK-001 and RT-001, the deuterated analogs of two nutritional compounds, vitamin A and linoleic acid, respectively. As we extensively described elsewhere [3], in these two examples D is key to the mechanism of action.In ALK-001 (d3-vitamin A), D at C20 position slows down dimerization and subsequent production of lipofuscin that is responsible for retinal degeneration [12]. For this reason, this compound reached a Phase II clinical trial in 2015 for the treatment of Stargardt's disease, a rare autosomal macular dystrophy commonly leading to blindness in young age (NCT02402660). In March 2019, a Phase II/III multicenter, randomized, double-masked, placebo-controlled study (NCT03845582) was initiated to test ALK-001 for the therapy of geographic atrophy (GA) secondary to age-related macular degeneration (AMD). GA, an advanced form of AMD, is characterized by atrophic lesions of the outer retina, in turn resulting in the progressive and irreversible loss of visual functions [13]. Even if GA and Stargardt's disease are recognized as distinct disorders, they share the accumulation of lipofuscin granules in the retinal pigment epithelium as one of the main pathogenic mechanisms. As for Strargardt's disease, there is no treatment authorized for GA and ALK-001 was hypothesized to represent a potential answer to unmet therapeutic needs in GA. The completion date of the clinical trial on GA is expected to be on 2022 and, as declared by the Sponsor, ALK001 will be tested also in other ophthalmological disorders, including intermediate dry-AMD, autosomal recessive retinitis pigmentosa and autosomal recessive cone-rod dystrophy [14].The incorporation of D in correspondence to the bis-allylic methylene group in linoleic acid has led to the discovery of RT001 (d2-linoleic acid ethyl ester) that is able to reduce the progress of lipid peroxidation [15]. Thanks to this peculiarity, it has entered the clinical development in 2015 for Friedrich's ataxia, a rare inherited neurodegenerative disorder associated with an increase in lipid peroxidation and characterized by areflexia, dysarthria and sensory deficits (NCT02445794). Three years later, a Phase II/III prospective open-label study was initiated to assess the efficacy and safety of RT001 in patients affected by infantile neuroaxonal dystrophy, a rare fatal neurodegenerative disorder characterized by a progressive loss of motor functions. On the basis of encouraging results seen in two Expanded Access patients, in February 2019 the Committee for Orphan Medical Products of the European Medicines Agency granted the Sponsor request for orphan medicinal product designation for the treatment of infantile neuroaxonal dystrophy with RT001 [16].Lastly, BMS-986165 and CTP-543 stand out as the most intensively studied deuterated compounds in 2019. BMS-986165 is a de novo designed deuterated compound that acts as an inhibitor of tyrosine kinase 2 and that is even more potent and selective compared to the already marketed ones [17]. BMS-986165 entered clinical development in 2015 for the treatment of autoimmune disorders, including psoriasis, systemic lupus erythematosus and inflammatory bowel diseases. Ten new clinical trials for BMS-986165 have started since the beginning of 2019, of which one is an open-label, multi-center, Phase III study aimed at evaluating the efficacy and safety of BMS-986165 in patients affected by moderate-to-severe psoriasis (NCT03924427). Conversely, CTP-543 is the deuterated analog of ruxolitinib (d8.ruxolitinib), a JAK1 and JAK2 inhibitor, for which four clinical trials have started since March 2019 (Phase I: NCT03880136; Phase II: NCT03898479, NCT03941548, NCT03811912). These studies are expected to be concluded between 2019 and 2021.Overall, the growing swarm of clinical trials on deuterated compounds clearly reflects the efforts made by the industrial world in unravelling the therapeutic potential of deuterated drugs. Yet, it must be acknowledged that for a medicinal chemist point of view clinical research is overly slow in feeding back information, with efficacy and safety data that are eagerly awaited to understand whether to pursue this strategy more often. Trials that will compare deuterated and non-deuterated compounds, in particular, will be highly informative.Concluding remarksD substitution is the smallest possible structural change that can be made to a molecule and it follows that its incorporation appears to be particularly useful for those soft positions that do not tolerate changes in steric hindrance or electronic properties. Despite the minimal change produced, it is becoming clear that D can provide significant advantages of different natures, according to each specific molecule, that go further beyond the simple prolongation of the half-life. It must be stressed that developing a deuterated drug is seemingly trivial, but is actually challenging and to be successful cannot disregard some concerns.First of all, D incorporation does not solve issues for every compound and in every position. A judicious choice of both the molecule and the site of deuteration must be undertaken, exploring the metabolic fate and the enzymology of the given compound. This is necessary to enable an informed decision regarding the suitability of the approach. In this regard, it has been estimated that less than 10% of all FDA-approved drugs are amenable to deuteration because of their chemical structure, or because they are metabolized in a way that D kinetic isotope effect would not be significant. Furthermore, potential metabolic switches (the deuteration of one position increases the metabolism at another site) can occur after deuteration, diverting the compound through other biotransformation routes. It follows that, if more than one oxidizable soft-spot is present, an initial per-deuteration of these positions is suggested, followed by progressive reversion to H until disappearance of the positive outcome given by D.A high attrition rate in the translation from in vitro to in vivo systems must be considered, especially in relation to the multiple factors that are not easy to be predicted and might hamper the correlation between in vitro and in vivo D kinetic isotope effect, including competing elimination mechanisms (for example Phase II reactions or urinary/biliary excretion of unchanged drug). Interspecies variability is an additional element of vulnerability that, conversely, can be foreseen at an early stage of development by careful evaluation of microsomal clearance values in microsomes of different species. In this regard, we would like to highlight a very recent paper that reports a MS/MS method that allows to simultaneously assess the metabolic stability toward liver microsomes of a 1:1 mixture of deuterated and nondeuterated compounds, with the advantage of being a rapid screening tool able to minimize experimental bias [18].Finally, the synthesis of deuterated compounds is far from trivial: on one hand the deuterated pool strategy is limited by a narrow scope and relatively high cost of deuterated substrates, while on the other the exchange approach might suffer from low selectivity and D content, together with harsh and not always green experimental conditions [19]. It is therefore important to carefully evaluate from the very beginning the degree of D enrichment and selectivity that can be obtained, and maintained, during the process.On these bases, the effects of deuteration incorporation are often unpredictable and the lack of obviousness for successfully patenting deuterated drugs comes from the unpredictability nature of such approaches. Many compounds when deuterated will not show a useful and significant difference compared with the precursor, and hence the demonstration of a positive outcome should be enough to satisfy this requirement. Nevertheless, patentability concerns are currently arising, with a variegate picture of allowed, rejected and allowed after restriction applications. This is somehow counterbalanced by the fact that, in economic and regulatory terms, an unquestionable edge of D switch is the possibility of faster and less costly clinical trials, that can partly capitalize on the extensive data previously collected on the nondeuterated version. Therefore, due to intellectual property issues, the era of D switching seems to be near to an ending, but D incorporation is currently establishing itself as a useful drug design strategy during de novo drug discovery, as demonstrated by the advent of BMS-98616. Besides that, as an important barrier to competition, claiming deuterated versions of new molecules in patent applications right from the start has already emerged as a trend for the large pharma companies, in some cases just as an elusive way to broaden the Markush structures, in others as a concrete result substantiated by examples and data.In a very recent report on FDA drug approvals of 2018, fluorine has deserved nomination as the ‘Atom of the Year’ [20]. Thanks to its high complementarity with this halogen in reducing drug attrition rate, we believe that D will soon gain the attention it deserves. There are already several deuterated drugs in the pipeline, and it is foreseen that a number of them will follow the example of deutetrabenazine and hit the market in years to come.Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.

Characteristic Ocular Features in Cases of Autosomal Recessive PROM1 Cone-Rod Dystrophy

PurposeTo define characteristic ocular features in a group of patients with autosomal recessive (AR) PROM1 cone-rod dystrophy (CRD).MethodsThree males and one female from three unrelated families were first seen at the ages of 15 to 22 years and diagnosed with CRD. Clinical testing available for review included full-field electroretinogram (ERG) in three patients, as well as near-infrared autofluorescence (NIR-AF), spectral-domain optical coherence tomography (SD-OCT), and color fundus photography in all four patients. Whole exome sequencing (WES) was performed on all cases, and whole genome sequencing (WGS) was performed in two families.ResultsWES found compound heterozygous PROM1 variants in one isolated male, plus heterozygous variants in the remaining patients. WGS uncovered deleterious PROM1 variants in these two families. ERG showed markedly reduced cone-isolated amplitudes and variably reduced rod-isolated amplitudes. The dark-adapted combined rod and cone responses demonstrated notably reduced a-wave amplitudes and moderately reduced b-waves, and the resultant waveform resembled the normal rod-isolated response. On fundus examination, oval-shaped macular lesions were observed, as were several small, circular hypoautofluorescent lesions within the posterior pole on NIR-AF. Three patients showed extramacular circular atrophic lesions.ConclusionsThe autofluorescence changes, peripheral retinal abnormalities, and ERG findings have not been emphasized in previous reports of AR PROM1, but they became a recognizable phenotype in this cohort of patients. A similar constellation of findings may be observed in CRD due to CDHR1, a functionally related gene. The pattern of abnormalities reported herein may help to focus genetic screening in patients with these findings.

Macular Dystrophy and Cone-Rod Dystrophy Caused by Mutations in the RP1 Gene: Extending the RP1 Disease Spectrum.

To describe the clinical and genetic spectrum of RP1-associated retinal dystrophies. In this multicenter case series, we included 22 patients with RP1-associated retinal dystrophies from 19 families from The Netherlands and Japan. Data on clinical characteristics, visual acuity, visual field, ERG, and retinal imaging were extracted from medical records over a mean follow-up of 8.1 years. Eleven patients were diagnosed with autosomal recessive macular dystrophy (arMD) or autosomal recessive cone-rod dystrophy (arCRD), five with autosomal recessive retinitis pigmentosa (arRP), and six with autosomal dominant RP (adRP). The mean age of onset was 40.3 years (range 14-56) in the patients with arMD/arCRD, 26.2 years (range 18-40) in adRP, and 8.8 years (range 5-12) in arRP patients. All patients with arMD/arCRD carried either the hypomorphic p.Arg1933* variant positioned close to the C-terminus (8 of 11 patients) or a missense variant in exon 2 (3 of 11 patients), compound heterozygous with a likely deleterious frameshift or nonsense mutation, or the p.Gln1916* variant. In contrast, all mutations identified in adRP and arRP patients were frameshift and/or nonsense variants located far from the C-terminus. Mutations in the RP1 gene are associated with a broad spectrum of progressive retinal dystrophies. In addition to adRP and arRP, our study provides further evidence that arCRD and arMD are RP1-associated phenotypes as well. The macular involvement in patients with the hypomorphic RP1 variant suggests that macular function may remain compromised if expression levels of RP1 do not reach adequate levels after gene augmentation therapy.

A pilot investigation of audiovisual processing and multisensory integration in patients with inherited retinal dystrophies

BackgroundIn this study, we examined audiovisual (AV) processing in normal and visually impaired individuals who exhibit partial loss of vision due to inherited retinal dystrophies (IRDs).MethodsTwo groups were analyzed for this pilot study: Group 1 was composed of IRD participants: two with autosomal dominant retinitis pigmentosa (RP), two with autosomal recessive cone-rod dystrophy (CORD), and two with the related complex disorder, Bardet-Biedl syndrome (BBS); Group 2 was composed of 15 non-IRD participants (controls). Audiovisual looming and receding stimuli (conveying perceptual motion) were used to assess the cortical processing and integration of unimodal (A or V) and multimodal (AV) sensory cues. Electroencephalography (EEG) was used to simultaneously resolve the temporal and spatial characteristics of AV processing and assess differences in neural responses between groups. Measurement of AV integration was accomplished via quantification of the EEG’s spectral power and event-related brain potentials (ERPs).ResultsResults show that IRD individuals exhibit reduced AV integration for concurrent audio and visual (AV) stimuli but increased brain activity during the unimodal A (but not V) presentation. This was corroborated in behavioral responses, where IRD patients showed slower and less accurate judgments of AV and V stimuli but more accurate responses in the A-alone condition.ConclusionsCollectively, our findings imply a neural compensation from auditory sensory brain areas due to visual deprivation.

CDHR1 mutations in retinal dystrophies

We report ophthalmic and genetic findings in patients with autosomal recessive retinitis pigmentosa (RP), cone-rod dystrophy (CRD) or cone dystrophy (CD) harboring potential pathogenic variants in the CDHR1 gene. Detailed ophthalmic examination was performed in seven sporadic and six familial subjects. Mutation screening was done using a customized next generation sequencing panel targeting 105 genes implicated in inherited retinal disorders. In one family, homozygosity mapping with subsequent candidate gene analysis was performed. Stringent filtering for rare and potentially disease causing variants following a model of autosomal recessive inheritance led to the identification of eleven different CDHR1 variants in nine index cases. All variants were novel at the time of their identification. In silico analyses confirmed their pathogenic potential. Minigene assays were performed for two non-canonical splice site variants and revealed missplicing for the mutant alleles. Mutations in CDHR1 are a rare cause of retinal dystrophy. Our study further expands the mutational spectrum of this gene and the associated clinical presentation.

Ablation of EYS in zebrafish causes mislocalisation of outer segment proteins, F-actin disruption and cone-rod dystrophy

Mutations in EYS are associated with autosomal recessive retinitis pigmentosa (arRP) and autosomal recessive cone-rod dystrophy (arCRD) however, the function of EYS and the molecular mechanisms of how these mutations cause retinal degeneration are still unclear. Because EYS is absent in mouse and rat, and the structure of the retina differs substantially between humans and Drosophila, we utilised zebrafish as a model organism to study the function of EYS in the retina. We constructed an EYS-knockout zebrafish-line by TALEN technology which showed visual impairment at an early age, while the histological and immunofluorescence assays indicated the presence of progressive retinal degeneration with a cone predominately affected pattern. These phenotypes recapitulate the clinical manifestations of arCRD patients. Furthermore, the EYS−/− zebrafish also showed mislocalisation of certain outer segment proteins (rhodopsin, opn1lw, opn1sw1, GNB3 and PRPH2), and disruption of actin filaments in photoreceptors. Protein mislocalisation may, therefore, disrupt the function of cones and rods in these zebrafish and cause photoreceptor death. Collectively, these results point to a novel role for EYS in maintaining the morphological structure of F-actin and in protein transport, loss of this function might be the trigger for the resultant cellular events that ultimately lead to photoreceptor death.

Whole-Organism Developmental Expression Profiling Identifies RAB-28 as a Novel Ciliary GTPase Associated with the BBSome and Intraflagellar Transport.

Primary cilia are specialised sensory and developmental signalling devices extending from the surface of most eukaryotic cells. Defects in these organelles cause inherited human disorders (ciliopathies) such as retinitis pigmentosa and Bardet-Biedl syndrome (BBS), frequently affecting many physiological and developmental processes across multiple organs. Cilium formation, maintenance and function depend on intracellular transport systems such as intraflagellar transport (IFT), which is driven by kinesin-2 and IFT-dynein motors and regulated by the Bardet-Biedl syndrome (BBS) cargo-adaptor protein complex, or BBSome. To identify new cilium-associated genes, we employed the nematode C. elegans, where ciliogenesis occurs within a short timespan during late embryogenesis when most sensory neurons differentiate. Using whole-organism RNA-Seq libraries, we discovered a signature expression profile highly enriched for transcripts of known ciliary proteins, including FAM-161 (FAM161A orthologue), CCDC-104 (CCDC104), and RPI-1 (RP1/RP1L1), which we confirm are cilium-localised in worms. From a list of 185 candidate ciliary genes, we uncover orthologues of human MAP9, YAP, CCDC149, and RAB28 as conserved cilium-associated components. Further analyses of C. elegans RAB-28, recently associated with autosomal-recessive cone-rod dystrophy, reveal that this small GTPase is exclusively expressed in ciliated neurons where it dynamically associates with IFT trains. Whereas inactive GDP-bound RAB-28 displays no IFT movement and diffuse localisation, GTP-bound (activated) RAB-28 concentrates at the periciliary membrane in a BBSome-dependent manner and undergoes bidirectional IFT. Functional analyses reveal that whilst cilium structure, sensory function and IFT are seemingly normal in a rab-28 null allele, overexpression of predicted GDP or GTP locked variants of RAB-28 perturbs cilium and sensory pore morphogenesis and function. Collectively, our findings present a new approach for identifying ciliary proteins, and unveil RAB28, a GTPase most closely related to the BBS protein RABL4/IFT27, as an IFT-associated cargo with BBSome-dependent cell autonomous and non-autonomous functions at the ciliary base.

Identification of Novel Mutations in the LRR-Cap Domain of C21orf2 in Japanese Patients With Retinitis Pigmentosa and Cone-Rod Dystrophy.

C21orf2 encodes a ciliary protein related to syndromic and nonsyndromic retinal degeneration. The purpose of this study was to identify novel mutations of C21orf2 associated with syndromic autosomal recessive retinitis pigmentosa (arRP) and autosomal recessive cone-rod dystrophy (arCRD) by using whole exome sequencing of a Japanese cohort. Whole exome sequencing was performed on DNA from affected and healthy members from 147 families with retinal degenerations. Identified nonsense and missense mutations were further restricted by using the reported single nucleotide variation frequencies and inherited patterns. The effect of the mutations was examined by in vitro assays. Novel mutations in C21orf2 were found in Japanese patients with arRP with skeletal defects or arCRD. Compound heterozygous mutations, from one family (p.V111M and p.Y107H), and a homozygous mutation, from another family (p.Y107C), were all located in the leucine-rich repeat C-terminal domain required for protein stabilization. C21orf2 was expressed in the retina through the developing to the mature stage, and the protein localized to the photoreceptor cilia in the adult retina. In vitro expression showed reduced levels and affected localizations of mutated protein products compared to the wild type. The identified C21orf2 mutations decreased protein stability and affected cytoplasmic localization of C21orf2. Since C21orf2 was required for ciliogenesis, our data suggested that reduced levels of functional C21orf2 induced photoreceptor degradation through abnormal cilia formation, leading to arRP or arCRD in the retina.

Genomic screening of ABCA4 and array CGH analysis underline the genetic variability of Greek patients with inherited retinal diseases

BackgroundRetinal dystrophies are a clinically and genetically heterogeneous group of disorders which affect more than two million people worldwide. The present study focused on the role of the ABCA4 gene in the pathogenesis of hereditary retinal dystrophies (autosomal recessive Stargardt disease, autosomal recessive cone-rod dystrophy, and autosomal recessive retinitis pigmentosa) in patients of Greek origin. Materials and methodsOur cohort included 26 unrelated patients and their first degree healthy relatives. The ABCA4 mutation screening involved Sanger sequencing of all exons and flanking regions. Evaluation of novel variants included sequencing of control samples, family segregation analysis and characterization by in silico prediction tools. Twenty five patients were also screened for copy number variations by array-comparative genomic hybridization. ResultsExcluding known disease-causing mutations and polymorphisms, two novel variants were identified in coding and non-coding regions of ABCA4. Array-CGH analysis revealed two partial deletions of USH2A and MYO3A in two patients with nonsyndromic autosomal recessive retinitis pigmentosa. ConclusionsThe ABCA4 mutation spectrum in Greek patients differs from other populations. Bioinformatic tools, segregation analysis along with clinical data from the patients seemed to be crucial for the evaluation of genetic variants and particularly for the discrimination between causative and non-causative variants.

Screening of ABCA4 Gene in a Chinese Cohort With Stargardt Disease or Cone-Rod Dystrophy With a Report on 85 Novel Mutations.

Mutations in the ABCA4 gene are heterogeneous and somewhat ethnic specific and can result in autosomal recessive Stargardt disease (STGD1), cone or cone-rod dystrophy (CRD), and retinitis pigmentosa. The objective of this study was to determine the ABCA4 mutation detection rate and mutation spectrum in a cohort of Chinese patients with STGD1 or CRD and describe the clinical features of the patients with ABCA4 mutations. A total of 161 probands were recruited for genetic analysis; these included 96 patients diagnosed with STGD1 and 65 individuals with CRD. All probands underwent ophthalmic examinations. All coding exons and exon-intron boundaries of the ABCA4 gene were screened for mutations by PCR-based DNA sequencing, followed by analyses for pathogenicity by in silico programs. We found at least two disease-causing ABCA4 alleles in 102 unrelated patients (63.4%), one disease-causing allele in 16 patients (9.9%), and no disease-causing allele in 43 affected individuals (26.7%), giving an overall mutation detection rate of 73.3% (118/161). In total, 136 disease-causing variants of the ABCA4 gene, including 85 novel ones, were identified. The identified mutations included 77 (57.0%) missense, 19 (14.1%) nonsense, 23 (17.0%) splicing effect, and 16 (11.9%) frameshift small insertion or deletion mutations. The most frequent mutation in this cohort was c.2424C>G p.Y808X, representing 4.7% of all screened alleles (15/322). The mutation spectrum of the ABCA4 gene in Chinese patients is quite different from that for Caucasian patients. The establishment of the mutation profile will facilitate ABCA4 screening and risk evaluation for Chinese patients with STGD1.

Clinical Utility Gene Card for: autosomal recessive cone-rod dystrophy.

ABCA4 ATP-binding cassette, sub-family A (ABC1), member 4 1p21-23 ADAM9 A disintegrin and metalloproteinase domain 9 8p11.23 C8orf37 Chromosome 8 open reading frame 37 8q22.1 CDHR1 Cadherin-related family, member 1 10q23.1 CERKL Ceramide kinase-like 2q31.3 CNGB3 Cyclic nucleotide-gated channel, beta-3 18q21.3 CORD1 Locus 18q21.1-q21.3 CORD8 Locus 1q12-q24 CORD17 Locus 10q26.1 CRX Cone-rod homeobox-containing gene 19q13.33 EYS Eyes shuthomolog (Drosophila) 6q12 FSCN2 Fascin homolog 2, actin-bundling protein, retinal 17q25.3 GUCY2D Guanylatecyclase 2D, membrane 17p13.1 KCNV2 Potassium channel, voltage-gated, subfamily v, member 2 9p24.2 PDE6C Phosphodiesterase 6C, cGMP-specific, cone, alpha prime 10q24 POC1B POC1 centriolar protein homolog B 12q21.33 PROM1 Prominin 1 4p15.32 RAB28 RAS-associated protein 28 4p15.33 RPE65 Retinal pigment epithelium-specific protein 65kDa 1p31 RPGRIP1 Retinitis pigmentosa GTPase regulator-interacting protein 14q11.2 TULP1 Tubby-like protein 1 6p21.31