Translational Read-Through Drugs (TRIDs) Are Able to Restore Protein Expression and Ciliogenesis in Fibroblasts of Patients with Retinitis Pigmentosa Caused by a Premature Termination Codon in <i>FAM161A</i>

Ataluren and Gentamicin are translational readthrough drugs (TRIDs) that induce premature termination codon (PTC) readthrough, resulting in the production of full-length proteins that usually harbor a single missense substitution. FAM161A is a ciliary protein which is expressed in photoreceptors, and pathogenic variants in this gene cause retinitis pigmentosa (RP). Applying TRIDs on fibroblasts from RP patients due to PTC in the FAM161A (p.Arg523*) gene may uncover whether TRIDs can restore expression, localization and function of this protein. Fibroblasts from six patients and five age-matched controls were starved prior to treatment with ataluren or gentamicin, and later FAM161A expression, ciliogenesis and cilia length were analyzed. In contrast to control cells, fibroblasts of patients did not express the FAM161A protein, showed a lower percentage of ciliated cells and grew shorter cilia after starvation. Ataluren and Gentamicin treatment were able to restore FAM161A expression, localization and co-localization with α-tubulin. Ciliogenesis and cilia length were restored following Ataluren treatment almost up to a level which was observed in control cells. Gentamicin was less efficient in ciliogenesis compared to Ataluren. Our results provide a proof-of-concept that PTCs in FAM161A can be effectively suppressed by Ataluren or Gentamicin, resulting in a full-length functional protein

Unique combination of clinical features in a large cohort of 100 patients with retinitis pigmentosa caused by FAM161A mutations

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Retinal Structure and Function in a Knock-in Mouse Model for the FAM161A-p.Arg523∗ Human Nonsense Pathogenic Variant

Purpose: Pathogenic variants in FAM161A are the most common cause of retinitis pigmentosa in Israel. Two founder pathogenic variants explain the vast majority of cases of Jewish origin, 1 being a nonsense variant (p.Arg523∗). The aim of this study was to generate a knock-in (KI) mouse model harboring the corresponding p.Arg512∗ pathogenic variant and characterize the course of retinal disease. Design: Experimental study of a mouse animal model. Subjects/Participants/Controls: A total of 106 Fam161a knock-in mice and 29 wild-type mice with C57BL/6J background particiapted in this study. Methods: Homozygous Fam161a p.Arg512∗ KI mice were generated by Cyagen Biosciences. Visual acuity (VA) was evaluated using optomotor tracking response and retinal function was assessed by electroretinography (ERG). Retinal structure was examined in vivo using OCT and fundus autofluorescence imaging. Retinal morphometry was evaluated by histologic and immunohistochemical (IHC) analyses. Main Outcome Measures: Visual and retinal function assessments, clinical imaging examinations, quantitative histology, and IHC studies of KI as compared with wild-type (WT) mice retinas. Results: The KI model was generated by replacing 3 bp, resulting in p.Arg512∗. Homozygous KI mice that had progressive loss of VA and ERG responses until the age of 18 months, with no detectable response at 21 months. OCT showed complete loss of the outer nuclear layer at 21 months. Fundus autofluorescence imaging revealed progressive narrowing of blood vessels and formation of patchy hyper-autofluorescent and hypo-autofluorescent spots. Histologic analysis showed progressive loss of photoreceptor nuclei. Immunohistochemistry staining showed Fam161a expression mainly in photoreceptors cilia and the outer plexiform layer (OPL) in WT mice retinas, whereas faint expression was evident mainly in the cilia and OPL of KI mice. Conclusions: The Fam161a - p.Arg512∗ KI mouse model is characterized by widespread retinal degeneration with relatively slow progression. Surprisingly, disease onset is delayed and progression is slower compared with the previously reported knock-out model. The common human null mutation in the KI mouse model is potentially amenable for correction by translational read-through-inducing drugs and by gene augmentation therapy and RNA editing, and can serve to test these treatments as a first step toward possible application in patients. Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article

Nonsyndromic Retinitis Pigmentosa in the Ashkenazi Jewish Population: Genetic and Clinical Aspects

To analyze the genetic and clinical findings in retinitis pigmentosa (RP) patients of Ashkenazi Jewish (AJ) descent, aiming to identify genotype-phenotype correlations. Cohort study. Retinitis pigmentosa patients from 230 families of AJ origin. Sanger sequencing was performed to detect specific founder mutations known to be prevalent in the AJ population. Ophthalmologic analysis included a comprehensive clinical examination, visual acuity (VA), visual fields, electroretinography, color vision testing, and retinal imaging by OCT, pseudocolor, and autofluorescence fundus photography. Inheritance pattern and causative mutation; retinal function as assessed by VA, visual fields, and electroretinography results; and retinal structural changes observed on clinical funduscopy as well as by pseudocolor, autofluorescence, and OCT imaging. The causative mutation was identified in 37% of families. The most prevalent RP-causing mutations are the Alu insertion (c.1297_8ins353, p.K433Rins31*) in the male germ cell-associated kinase (MAK) gene (39% of families with a known genetic cause for RP) and c.124A>G, p.K42E in dehydrodolichol diphosphate synthase (DHDDS) (33%). Additionally, disease-causing mutations were identified in 11 other genes. Analysis of clinical parameters of patients with mutations in the 2 most common RP-causing genes revealed that MAK patients had better VA and visual fields at relatively older ages in comparison with DHDDS patients. Funduscopic findings of DHDDS patients matched those of MAK patients who were 20 to 30 years older. Patients with DHDDS mutations were referred for electrophysiologic evaluation at earlier ages, and their cone responses became nondetectable at a much younger age than MAK patients. Our AJ cohort of RP patients is the largest reported to date and showed a substantial difference in the genetic causes of RP compared with cohorts of other populations, mainly a high rate of autosomal recessive inheritance and a unique composition of causative genes. The most common RP-causing genes in our cohort, MAK and DHDDS, were not described as major causative genes in other populations. The clinical data show that in general, patients with biallelic MAK mutations had a later age of onset and a milder retinal phenotype compared with patients with biallelic DHDDS mutations

A Missense Mutation in DHDDS, Encoding Dehydrodolichyl Diphosphate Synthase, Is Associated with Autosomal-Recessive Retinitis Pigmentosa in Ashkenazi Jews

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal degenerations caused by mutations in at least 50 genes. Using homozygosity mapping in Ashkenazi Jewish (AJ) patients with autosomal-recessive RP (arRP), we identified a shared 1.7 Mb homozygous region on chromosome 1p36.11. Sequence analysis revealed a founder homozygous missense mutation, c.124A>G (p.Lys42Glu), in the dehydrodolichyl diphosphate synthase gene (DHDDS) in 20 AJ patients with RP of 15 unrelated families. The mutation was not identified in an additional set of 109 AJ patients with RP, in 20 AJ patients with other inherited retinal diseases, or in 70 patients with retinal degeneration of other ethnic origins. The mutation was found heterozygously in 1 out of 322 ethnically matched normal control individuals. RT-PCR analysis in 21 human tissues revealed ubiquitous expression of DHDDS. Immunohistochemical analysis of the human retina with anti-DHDDS antibodies revealed intense labeling of the cone and rod photoreceptor inner segments. Clinical manifestations of patients who are homozygous for the c.124A>G mutation were within the spectrum associated with arRP. Most patients had symptoms of night and peripheral vision loss, nondetectable electroretinographic responses, constriction of visual fields, and funduscopic hallmarks of retinal degeneration. DHDDS is a key enzyme in the pathway of dolichol, which plays an important role in N-glycosylation of many glycoproteins, including rhodopsin. Our results support a pivotal role of DHDDS in retinal function and may allow for new therapeutic interventions for RP

Exome sequencing and cis-regulatory mapping identify mutations in MAK, a gene encoding a regulator of ciliary length, as a cause of retinitis pigmentosa

Contains fulltext : 96905.pdf (publisher's version ) (Closed access)A fundamental challenge in analyzing exome-sequence data is distinguishing pathogenic mutations from background polymorphisms. To address this problem in the context of a genetically heterogeneous disease, retinitis pigmentosa (RP), we devised a candidate-gene prioritization strategy called cis-regulatory mapping that utilizes ChIP-seq data for the photoreceptor transcription factor CRX to rank candidate genes. Exome sequencing combined with this approach identified a homozygous nonsense mutation in male germ cell-associated kinase (MAK) in the single affected member of a consanguineous Turkish family with RP. MAK encodes a cilium-associated mitogen-activated protein kinase whose function is conserved from the ciliated alga, Chlamydomonas reinhardtii, to humans. Mutations in MAK orthologs in mice and other model organisms result in abnormally long cilia and, in mice, rapid photoreceptor degeneration. Subsequent sequence analyses of additional individuals with RP identified five probands with missense mutations in MAK. Two of these mutations alter amino acids that are conserved in all known kinases, and an in vitro kinase assay indicates that these mutations result in a loss of kinase activity. Thus, kinase activity appears to be critical for MAK function in humans. This study highlights a previously underappreciated role for CRX as a direct transcriptional regulator of ciliary genes in photoreceptors. In addition, it demonstrates the effectiveness of CRX-based cis-regulatory mapping in prioritizing candidate genes from exome data and suggests that this strategy should be generally applicable to a range of retinal diseases

Mutations in RAB28, encoding a farnesylated small GTPase, are associated with autosomal-recessive cone-rod dystrophy

Item does not contain fulltextThe majority of the genetic causes of autosomal-recessive (ar) cone-rod dystrophy (CRD) are currently unknown. A combined approach of homozygosity mapping and exome sequencing revealed a homozygous nonsense mutation (c.565C>T [p.Glu189*]) in RAB28 in a German family with three siblings with arCRD. Another homozygous nonsense mutation (c.409C>T [p.Arg137*]) was identified in a family of Moroccan Jewish descent with two siblings affected by arCRD. All five affected individuals presented with hyperpigmentation in the macula, progressive loss of the visual acuity, atrophy of the retinal pigment epithelium, and severely reduced cone and rod responses on the electroretinogram. RAB28 encodes a member of the Rab subfamily of the RAS-related small GTPases. Alternative RNA splicing yields three predicted protein isoforms with alternative C-termini, which are all truncated by the nonsense mutations identified in the arCRD families in this report. Opposed to other Rab GTPases that are generally geranylgeranylated, RAB28 is predicted to be farnesylated. Staining of rat retina showed localization of RAB28 to the basal body and the ciliary rootlet of the photoreceptors. Analogous to the function of other RAB family members, RAB28 might be involved in ciliary transport in photoreceptor cells. This study reveals a crucial role for RAB28 in photoreceptor function and suggests that mutations in other Rab proteins may also be associated with retinal dystrophies