Genetic Diagnosis for 64 Patients with Inherited Retinal Disease.

The overlapping genetic and clinical spectrum in inherited retinal degeneration (IRD) creates challenges for accurate diagnoses. The goal of this work was to determine the genetic diagnosis and clinical features for patients diagnosed with an IRD. After signing informed consent, peripheral blood or saliva was collected from 64 patients diagnosed with an IRD. Genetic testing was performed on each patient in a Clinical Laboratory Improvement Amendments of 1988 (CLIA) certified laboratory. Mutations were verified with Sanger sequencing and segregation analysis when possible. Visual acuity was measured with a traditional Snellen chart and converted to a logarithm of minimal angle of resolution (logMAR). Fundus images of dilated eyes were acquired with the Optos® camera (Dunfermline, UK). Horizontal line scans were obtained with spectral-domain optical coherence tomography (SDOCT; Spectralis, Heidelberg, Germany). Genetic testing combined with segregation analysis resolved molecular and clinical diagnoses for 75% of patients. Ten novel mutations were found and unique genotype phenotype associations were made for the genes RP2 and CEP83. Collective knowledge is thereby expanded of the genetic basis and phenotypic correlation in IRD.

Delineating the Clinical Phenotype of Patients With the c.629C>G, p.Pro210Arg Mutation in Peripherin-2.

Purpose: More than 200 different mutations in peripherin-2 (PRPH2) are associated with multiple subtypes of inherited retinal diseases (IRDs), including retinitis pigmentosa and cone or macular diseases. Our goal was to understand how the poorly characterized PRPH2 mutation p.Pro210Arg (P210R) affects visual function and retinal structure as well as gain insight into the mechanism driving the clinical pathology. Methods: Eleven patients had clinical assessments including best-corrected visual acuity (BCVA), full field and multifocal electroretinography (ERG), static (spot size V) and kinetic perimetry (Octopus 900), and dark-adapted chromatic (DAC; Medmont; spot size V) perimetry. Images were acquired with the Optos ultra-wide field camera and spectral-domain optical coherence tomography (SD-OCT). Molecular characteristics of the P210R mutant protein were evaluated in vitro. Results: Patients with the P210R mutation had BCVA (Snellen) ranging from 20/15 to 20/80. Perimetry showed a reduction in sensitivity, while ERG findings suggested that cone function was more impaired than rod function. Scotomas were identified corresponding to atrophic retinal lesions. Imaging revealed heterogeneous outer retinal changes such as hyperfluorescent flecks, hypo-autofluorescence (AF) regions of atrophy, and thinning of the photoreceptor layer on SD-OCT. In vitro findings suggested that P210R-Prph2 retains the ability to interact with binding partner Rom1 but abnormally accumulates in the endoplasmic reticulum (ER), suggesting the protein does not fold properly. Conclusions: Rod and cone sensitivities were decreased in subjects with the P210R mutation in PRPH2. There was scotomatous vision loss that occurred within the macula, likely due to atrophy that occurs after drusen have formed and have begun to resolve. This suggests that although rod and cone photoreceptors are dependent on PRPH2, preventing blindness in this specific subgroup of patients could involve therapeutics that impede the formation or lifecycle of drusen.

Adherent but Not Suspension-Cultured Embryoid Bodies Develop into Laminated Retinal Organoids.

Human induced pluripotent stem cells (iPSCs) are differentiated into three-dimensional (3D) retinal organoids to study retinogenesis and diseases that would otherwise be impossible. The complexity and low yield in current protocols remain a technical challenge, particularly for inexperienced personnel. Differentiation protocols require labor-intensive and time-consuming dissection of optic vesicles (OVs). Here we compare this method with a suspension method of developing retinal organoids. iPSCs were differentiated with standard protocols but the suspension-grown method omitted the re-plating of embryoid bodies and dissection of OVs. All other media and treatments were identical between developmental methods. Developmental maturation was evaluated with RT-qPCR and immunocytochemistry. Dissection- and suspension-derived retinal organoids displayed temporal biogenesis of retinal cell types. Differences in retinal organoids generated by the two methods of differentiation included temporal developmental and the organization of neural retina layers. Retinal organoids grown in suspension showed delayed development and disorganized retinal layers compared to the dissected retinal organoids. We found that omitting the re-plating of EBs to form OVs resulted in numerous OVs that were easy to identify and matured along a retinal lineage. While more efficient, the suspension method led to retinal organoids with disorganized retinal layers compared to those obtained using conventional dissection protocols.

Functional Evaluation of Splicing for Variants of Uncertain Significance in Patients with Inherited Retinal Diseases.

Inherited retinal diseases (IRD) comprise a heterogeneous set of clinical and genetic disorders that lead to blindness. Given the emerging opportunities in precision medicine and gene therapy, it has become increasingly important to determine whether DNA variants with uncertain significance (VUS) are responsible for patients' IRD. This research was performed to assess the functional consequence of six VUS identified in patients with IRD. Clinical assessments included an ophthalmic examination, best-corrected visual acuity, and kinetic perimetry. Imaging was acquired with the Optos ultra-widefield camera and spectral domain optical coherence tomography (SD-OCT). Genetic testing was performed by Molecular Vision Laboratories. VUS that were predicted to alter splicing were analyzed with a minigene assay, which revealed that VUS in the genes OPA1, CNGB1, and CLUAP1 altered spicing mechanisms. Due to emerging gene and cell therapies, these results expand the genotype-phenotype correlations for patients diagnosed with an IRD.

Variable expressivity in patients with autosomal recessive retinitis pigmentosa associated with the gene CNGB1.

PURPOSE: In a cohort of eight families (11 patients) with autosomal recessive retinitis pigmentosa (arRP), we clinically characterized disease associated with mutations in CNGB1. METHODS: Visual function was determined by measuring the patients' visual acuity, dark- and light-adapted perimetry, and by full-field electroretinography. Retinal structure was evaluated with spectral-domain optical coherence tomography, fundus imaging, and autofluorescence imaging. RESULTS: Age of onset ranged from 4 to 49 years (mean [SD] 26 [17], median 27 years). The age at visit was 27-54 years, mean 37 (17). The range of visual acuity was logMAR -0.1 to 1.3 (Snellen 20/16 to 20/400) in the right eye and -0.1 to 0.9 (Snellen 20/16 to 20/160) in the left eye. Electrophysiological testing in five patients showed an absence of the rod response. Cone responses ranged from normal to severely reduced. The patients exhibited loss of rod vision more severe than cone vision. Funduscopic images showed widespread retinal degeneration with pigment clumping, optic disk pallor, arteriole attenuation, and a peri-foveal ring of hyper autofluorescence. Three families were tested for olfactory dysfunction and results indicated mild to complete anosmia in individuals with mutations in CNGB1. Genetic analysis revealed 6 novel variants, c.2127 C > G, p.Phe709Leu; c.1431 C > A, p.Cys477*; c.2034 G > A, p.Trp678*; c.2092 T > C, p.Cys698Arg; and c.583 + 2 T > C, c.2305-34 G > A and 3 variants that have been previously described, c.2957A>T, p.Asn986Ile; c.2544dup, p.Leu849Alafs*3; and c.2492 + 1 G > A. DISCUSSION: This is the first report for six novel CNGB1 variants associated with arRP. Two families had olfactory dysfunction in patients with arRP and family members who were heterozygous for a CNGB1 mutation. Additionally, findings demonstrated variable penetrance and expressivity of disease in these patients.

Disease Progression in Patients with Autosomal Dominant Retinitis Pigmentosa due to a Mutation in Inosine Monophosphate Dehydrogenase 1 (IMPDH1).

Purpose: Mutations in the inosine monophosphate dehydrogenase 1 (IMPDH1) gene are a common cause of inherited retinal degeneration (IRD). Due to species- and tissue-dependent expression of IMPDH1, there are no appropriate models of human IMPDH1 disease. Therefore, a limited understanding remains of disease expression and rates of progression for IMPDH1-related IRD. Methods: We evaluated semiautomated kinetic and chromatic static perimetry, spectral-domain optical coherence tomography (SD-OCT), and ultra-wide field fundus images with autofluorescence in a cohort of 12 patients (ages 11-58 at first visit). Ten patients had longitudinal data for which rates of progression were estimated. Results: Visual acuities were relatively stable over time and the photoreceptors within the central retina remained intact. Perifoveal photoreceptor loss measured over a period of years coincided with visual fields, which were constricted and progressed over time in all patients. Rod sensitivity showed a similar pattern of defect to that of the kinetic perimetry and the autofluorescence ultra-wide field imaging. Full-field electroretinograms were severely reduced and the dark-adapted rod and mixed responses were extinguished at earlier visits than the light-adapted cone responses. Conclusions: There was variability in disease severity at the first visit, but results show that the peripheral retina is more susceptible to the deleterious consequences of an IMPDH1 mutation. Given the pattern of degeneration and the alternatively spliced isoforms of IMPDH1, potential interventions may consider targeting the periphery early in disease, modulating transcript expression, and/or preserving central vision at late stages of the disease. Translational Relevance: These results inform clinical prognosis and offer evidence strategies toward therapeutic intervention.

Deacetylation of mycothiol-derived 'waste product' triggers the last biosynthetic steps of lincosamide antibiotics.

The immediate post-condensation steps in lincomycin biosynthesis are reminiscent of the mycothiol-dependent detoxification system of actinomycetes. This machinery provides the last proven lincomycin intermediate, a mercapturic acid derivative, which formally represents the 'waste product' of the detoxification process. We identified and purified new lincomycin intermediates from the culture broth of deletion mutant strains of Streptomyces lincolnensis and tested these compounds as substrates for proteins putatively involved in lincomycin biosynthesis. The results, based on LC-MS, in-source collision-induced dissociation mass spectrometry and NMR analysis, revealed the final steps of lincomycin biosynthesis, i.e. conversion of the mercapturic acid derivative to lincomycin. Most importantly, we show that deacetylation of the N'-acetyl-S-cysteine residue of the mercapturic acid derivative is required to 'escape' the detoxification-like system and proceed towards completion of the biosynthetic pathway. Additionally, our results, supported by l-cysteine-13C3, 15N incorporation experiments, give evidence that a different type of reaction catalysed by the homologous pair of pyridoxal-5'-phosphate-dependent enzymes, LmbF and CcbF, forms the branch point in the biosynthesis of lincomycin and celesticetin, two related lincosamides.

Lincosamide synthetase--a unique condensation system combining elements of nonribosomal peptide synthetase and mycothiol metabolism.

In the biosynthesis of lincosamide antibiotics lincomycin and celesticetin, the amino acid and amino sugar units are linked by an amide bond. The respective condensing enzyme lincosamide synthetase (LS) is expected to be an unusual system combining nonribosomal peptide synthetase (NRPS) components with so far unknown amino sugar related activities. The biosynthetic gene cluster of celesticetin was sequenced and compared to the lincomycin one revealing putative LS coding ORFs shared in both clusters. Based on a bioassay and production profiles of S. lincolnensis strains with individually deleted putative LS coding genes, the proteins LmbC, D, E, F and V were assigned to LS function. Moreover, the newly recognized N-terminal domain of LmbN (LmbN-CP) was also assigned to LS as a NRPS carrier protein (CP). Surprisingly, the homologous CP coding sequence in celesticetin cluster is part of ccbZ gene adjacent to ccbN, the counterpart of lmbN, suggesting the gene rearrangement, evident also from still active internal translation start in lmbN, and indicating the direction of lincosamide biosynthesis evolution. The in vitro test with LmbN-CP, LmbC and the newly identified S. lincolnensis phosphopantetheinyl transferase Slp, confirmed the cooperation of the previously characterized NRPS A-domain LmbC with a holo-LmbN-CP in activation of a 4-propyl-L-proline precursor of lincomycin. This result completed the functional characterization of LS subunits resembling NRPS initiation module. Two of the four remaining putative LS subunits, LmbE/CcbE and LmbV/CcbV, exhibit low but significant homology to enzymes from the metabolism of mycothiol, the NRPS-independent system processing the amino sugar and amino acid units. The functions of particular LS subunits as well as cooperation of both NRPS-based and NRPS-independent LS blocks are discussed. The described condensing enzyme represents a unique hybrid system with overall composition quite dissimilar to any other known enzyme system.

Sequence analysis of porothramycin biosynthetic gene cluster.

The biosynthetic gene cluster of porothramycin, a sequence-selective DNA alkylating compound, was identified in the genome of producing strain Streptomyces albus subsp. albus (ATCC 39897) and sequentially characterized. A 39.7 kb long DNA region contains 27 putative genes, 18 of them revealing high similarity with homologous genes from biosynthetic gene cluster of closely related pyrrolobenzodiazepine (PBD) compound anthramycin. However, considering the structures of both compounds, the number of differences in the gene composition of compared biosynthetic gene clusters was unexpectedly high, indicating participation of alternative enzymes in biosynthesis of both porothramycin precursors, anthranilate, and branched L-proline derivative. Based on the sequence analysis of putative NRPS modules Por20 and Por21, we suppose that in porothramycin biosynthesis, the methylation of anthranilate unit occurs prior to the condensation reaction, while modifications of branched proline derivative, oxidation, and dimethylation of the side chain occur on already condensed PBD core. Corresponding two specific methyltransferase encoding genes por26 and por25 were identified in the porothramycin gene cluster. Surprisingly, also methyltransferase gene por18 homologous to orf19 from anthramycin biosynthesis was detected in porothramycin gene cluster even though the appropriate biosynthetic step is missing, as suggested by ultra high-performance liquid chromatography-diode array detection-mass spectrometry (UHPLC-DAD-MS) analysis of the product in the S. albus culture broth.