Autosomal Recessive Retinal Dystrophy Research Articles

Generation of human induced pluripotent stem cell lines from a subject with UBAP1L-associated retinal dystrophy and CRISPR/cas9-corrected isogenic iPSC lines

A Human induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of a patient affected with an autosomal recessive retinal dystrophy carrying the homozygous c.910-7G>A variant in UBAP1L. Three isogenic control iPSC lines derived from this affected subject line were created using CRISPR/Cas9 engineering. All iPSC lines expressing the pluripotency markers, were able to differentiate into the three germ layers, and exhibit a normal karyotype. These cellular models will provide a powerful tool to study disease mechanisms associated with the recently reported UBAP1L- associated retinal dystrophy and better understand the role of the protein in retinal physiology.

Effective AAV-mediated gene replacement therapy in retinal organoids modeling AIPL1-associated LCA4

Biallelic variations in the aryl hydrocarbon receptor interacting protein-like 1 (AIPL1) gene cause Leber congenital amaurosis subtype 4 (LCA4), an autosomal recessive early-onset severe retinal dystrophy that leads to the rapid degeneration of retinal photoreceptors and the severe impairment of sight within the first few years of life. Currently, there is no treatment or cure for AIPL1-associated LCA4. In this study, we investigated the potential of adeno-associated virus-mediated AIPL1 gene replacement therapy in two previously validated human retinal organoid (RO) models of LCA4. We report here that photoreceptor-specific AIPL1 gene replacement therapy, currently being tested in a first-in-human application, effectively rescued molecular features of AIPL1-associated LCA4 in these models. Notably, the loss of retinal phosphodiesterase 6 was rescued and elevated cyclic guanosine monophosphate (cGMP) levels were reduced following treatment. Transcriptomic analysis of untreated and AAV-transduced ROs revealed transcriptomic changes in response to elevated cGMP levels and viral infection, respectively. Overall, this study supports AIPL1 gene therapy as a promising therapeutic intervention for LCA4.

Optical coherence tomography angiography in Bietti crystalline dystrophy.

Bietti crystalline dystrophy (BCD) is a rare autosomal recessive retinal dystrophy caused by pathogenic variants of CYP4V2 gene and characterized by shiny yellow deposits in the retina and progressive atrophy of the retinal pigment epithelium (RPE) and choriocapillaris. The main aim of the present study is to describe the optical coherence tomography angiography (OCTA) characteristics of a patient affected by BCD. A 59-years-old female with genetically confirmed BCD underwent an ophthalmological examination complete of OCTA performed in the atrophic retina, the junctional zone and the apparently normal retina. The area of choriocapillaris (CC) atrophy was compared to the area of RPE atrophy on fundus autofluorescence (FAF) imaging. A severe vessel density (VD) deficit at the level of superficial and deep capillary plexa as well as CC was registered in atrophic areas, which resulted deeper with respect to the junctional area, whereas the apparently preserved retina revealed VD values similar to that of control eyes. The area of RPE atrophy on FAF was larger (55.90 mm2 in right eye and 48.76 mm2 in left eye) than the area of CC atrophy on OCTA imaging (51.86 mm2 and 42.44 mm2 respectively in right and left eye). Our findings suggest that VD impairment of retinal plexa and CC follows the degeneration of RPE as demonstrated by the greater size of the area of RPE atrophy compared to CC atrophy. Further investigations based on OCTA imaging are necessary to enhance our knowledge of this rare disease.

The Clinical Spectrum and Disease Course of DRAM2 Retinopathy.

Pathogenic variants in DNA-damage regulated autophagy modulator 2 gene (DRAM2) cause a rare autosomal recessive retinal dystrophy and its disease course is not well understood. We present two Slovenian patients harboring a novel DRAM2 variant and a detailed review of all 23 other patients described to date. Whole exome and whole genome sequencing were performed in the two patients, and both underwent ophthalmological examination with a 2-year follow-up. PubMed was searched for papers with clinical descriptions of DRAM2 retinopathy. Patient 1 was homozygous for a novel variant, p.Met1?, and presented with the acute onset of photopsia and retina-wide retinopathy at the age of 35 years. The patient was first thought to have an autoimmune retinopathy and was treated with mycophenolate mofetil, which provided some symptomatic relief. Patient 2 was compound heterozygous for p.Met1? and p.Leu246Pro and presented with late-onset maculopathy at the age of 59 years. On review, patients with DRAM2 retinopathy usually present in the third decade with central visual loss, outer retinal layer loss on optical coherence tomography and a hyperautofluorescent ring on fundus autofluorescence. Either cone–rod or rod–cone dystrophy phenotype is observed on electroretinography, reflecting the importance of DRAM2 in both photoreceptor types. Non-null variants can result in milder disease.

Expanding the phenotype of TTLL5-associated retinal dystrophy: a case series

BackgroundInherited retinal dystrophies describe a heterogeneous group of retinal diseases that lead to the irreversible degeneration of rod and cone photoreceptors and eventual blindness. Recessive loss-of-function mutations in Tubulin Tyrosine Ligase Like 5 (TTLL5) represent a recently described cause of inherited cone–rod and cone dystrophy. This study describes the unusual phenotypes of three patients with autosomal recessive mutations in TTLL5. Examination of these patients included funduscopic evaluation, spectral-domain optical coherence tomography, short-wavelength autofluorescence, and full-field electroretinography (ffERG). Genetic diagnoses were confirmed using whole exome capture. Protein modeling of the identified variants was performed to explore potential genotype–phenotype correlations.ResultsGenetic testing revealed five novel variants in TTLL5 in three unrelated patients with retinal dystrophy. Clinical imaging demonstrated features of sectoral cone–rod dystrophy and cone dystrophy, with phenotypic variability seen across all three patients. One patient also developed high-frequency hearing loss during a similar time period as the onset of retinal disease, potentially suggestive of a syndromic disorder. Retinal structure findings were corroborated with functional measures including ffERG findings that supported these diagnoses. Modeling of the five variants suggest that they cause different effects on protein function, providing a potential reason for genotype–phenotype correlation in these patients.ConclusionsThe authors report retinal phenotypic findings in three unrelated patients with novel mutations causing autosomal recessive TTLL5-mediated retinal dystrophy. These findings broaden the understanding of the phenotypes associated with TTLL5-mediated retinal disease and suggest that mutations in TTLL5 should be considered as a potential cause of sectoral retinal dystrophy in addition to cone–rod and cone dystrophies.

An evaluation of pipelines for DNA variant detection can guide a reanalysis protocol to increase the diagnostic ratio of genetic diseases

Clinical exome (CE) sequencing has become a first-tier diagnostic test for hereditary diseases; however, its diagnostic rate is around 30–50%. In this study, we aimed to increase the diagnostic yield of CE using a custom reanalysis algorithm. Sequencing data were available for three cohorts using two commercial protocols applied as part of the diagnostic process. Using these cohorts, we compared the performance of general and clinically relevant variant calling and the efficacy of an in-house bioinformatic protocol (FJD-pipeline) in detecting causal variants as compared to commercial protocols. On the whole, the FJD-pipeline detected 99.74% of the causal variants identified by the commercial protocol in previously solved cases. In the unsolved cases, FJD-pipeline detects more INDELs and non-exonic variants, and is able to increase the diagnostic yield in 2.5% and 3.2% in the re-analysis of 78 cancer and 62 cardiovascular cases. These results were considered to design a reanalysis, filtering and prioritization algorithm that was tested by reassessing 68 inconclusive cases of monoallelic autosomal recessive retinal dystrophies increasing the diagnosis by 4.4%. In conclusion, a guided NGS reanalysis of unsolved cases increases the diagnostic yield in genetic disorders, making it a useful diagnostic tool in medical genetics.

Inherited Retinal Diseases Due to RPE65 Variants: From Genetic Diagnostic Management to Therapy.

Inherited retinal diseases (IRDs) are a heterogeneous group of conditions that include retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) and early-onset severe retinal dystrophy (EO[S]RD), which differ in severity and age of onset. IRDs are caused by mutations in >250 genes. Variants in the RPE65 gene account for 0.6–6% of RP and 3–16% of LCA/EORD cases. Voretigene neparvovec is a gene therapy approved for the treatment of patients with an autosomal recessive retinal dystrophy due to confirmed biallelic RPE65 variants (RPE65-IRDs). Therefore, the accurate molecular diagnosis of RPE65-IRDs is crucial to identify ‘actionable’ genotypes—i.e., genotypes that may benefit from the treatment—and is an integral part of patient management. To date, hundreds of RPE65 variants have been identified, some of which are classified as pathogenic or likely pathogenic, while the significance of others is yet to be established. In this review, we provide an overview of the genetic diagnostic workup needed to select patients that could be eligible for voretigene neparvovec treatment. Careful clinical characterization of patients by multidisciplinary teams of experts, combined with the availability of next-generation sequencing approaches, can accelerate patients’ access to available therapeutic options.

Stargardt disease: Multimodal imaging: A review.

Stargardt disease (STGD1) is an autosomal recessive retinal dystrophy, characterised by bilateral progressive central vision loss and subretinal deposition of lipofuscin‐like substances. Recent advances in molecular diagnosis and therapeutic options are complemented by the increasing recognition of new multimodal imaging biomarkers that may predict genotype and disease progression. Unique non‐invasive imaging features of STDG1 are useful for gene variant interpretation and may even provide insight into the underlying molecular pathophysiology. In addition, pathognomonic imaging features of STGD1 have been used to train neural networks to improve time efficiency in lesion segmentation and disease progression measurements. This review will discuss the role of key imaging modalities, correlate imaging signs across varied STGD1 presentations and illustrate the use of multimodal imaging as an outcome measure in determining the efficacy of emerging STGD1 specific therapies.

Novel Heterozygous Deletion in Retinol Dehydrogenase 12 (RDH12) Causes Familial Autosomal Dominant Retinitis Pigmentosa.

Mutations in the retinol dehydrogenase 12 (RDH12) gene are primarily associated with Leber congenital amaurosis (LCA) type 13, a severe early onset autosomal recessive retinal dystrophy. Only one family with a heterozygous variant, associated with mild retinitis pigmentosa (RP), has been reported. We report a novel heterozygous variant [(c.759del; p.(Phe254Leufs∗24)], resulting in a frameshift and premature termination identified in two unrelated individuals with familial autosomal dominant RP. Both heterozygous variants are associated with a late onset RP phenotype, suggesting a possible genotype-phenotype correlation.

CRB2 Loss in Rod Photoreceptors Is Associated with Progressive Loss of Retinal Contrast Sensitivity.

Variations in the Crumbs homolog-1 (CRB1) gene are associated with a wide variety of autosomal recessive retinal dystrophies, including early onset retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). CRB1 belongs to the Crumbs family, which in mammals includes CRB2 and CRB3. Here, we studied the specific roles of CRB2 in rod photoreceptor cells and whether ablation of CRB2 in rods exacerbates the Crb1-disease. Therefore, we assessed the morphological, retinal, and visual functional consequences of specific ablation of CRB2 from rods with or without concomitant loss of CRB1. Our data demonstrated that loss of CRB2 in mature rods resulted in RP. The retina showed gliosis and disruption of the subapical region and adherens junctions at the outer limiting membrane. Rods were lost at the peripheral and central superior retina, while gross retinal lamination was preserved. Rod function as measured by electroretinography was impaired in adult mice. Additional loss of CRB1 exacerbated the retinal phenotype leading to an early reduction of the dark-adapted rod photoreceptor a-wave and reduced contrast sensitivity from 3-months-of-age, as measured by optokinetic tracking reflex (OKT) behavior testing. The data suggest that CRB2 present in rods is required to prevent photoreceptor degeneration and vision loss.

Generation of two iPS cell lines (FRIMOi003-A and FRIMOi004-A) derived from Stargardt patients carrying ABCA4 compound heterozygous mutations

Recessive Stargardt disease (STGD1) is an autosomal recessive retinal dystrophy, caused by mutations in the retina-specific ATP-binding cassette transporter (ABCA4) gene, which plays a role as a retinaldehyde flippase in the photoreceptor outer segments. In this work, two human induced pluripotent stem cell (iPSC) lines were generated from STGD1 patients carrying compound heterozygous mutations in ABCA4. Skin fibroblasts were reprogrammed with the Yamanaka factors using a non-integrating, Sendai virus-based approach. Both iPSC lines displayed typical embryonic stem cell morphology, had normal karyotype, expressed several pluripotency markers and were able to differentiate into all three germ layers.Resource tableUnlabelled TableUnique stem cell lines identifierFRIMOi003-A, FRIMOi004-AAlternative names of stem cell linesSTGD1_FiPS4F1.5, STGD2_FiPS4F1.7InstitutionFundació de Recerca de l'Institut de Microcirurgia Ocular, Barcelona, SpainContact information of distributorMarina Riera, genetica.riera@imo.esEsther Pomares, pomares@imo.esType of cell linesiPSCOriginHumanCell SourceDermal fibroblastsClonalityClonalMethod of reprogrammingSendai virusMultiline rationaleSame disease non-isogenic cell linesGene modificationYesType of modificationHereditaryAssociated diseaseStargardt diseaseGene/locusABCA4 on chromosome 1p22.1Method of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock dateNovember 2018Cell line repository/bankhttp://www.isciii.es/ISCIII/es/contenidos/fd-el-instituto/fd-organizacion/fd-estructura-directiva/fd-subdireccion-general-investigacion-terapia-celular-medicina-regenerativa/fd-centros-unidades/fd-banco-nacional-lineas-celulares/fd-lineas-celulares-disponibles/lineas-de-celulas-iPS.shtmlEthical approvalPatients informed consent was obtainedEthics approval was received from the Ethics Committee of Institut de Microcirurgia Ocular (170505_117)

Novel clinical findings in autosomal recessive NR2E3-related retinal dystrophy.

To evaluate the clinical phenotype of autosomal recessive NR2E3-related retinal dystrophy. We retrospectively studied 11 patients carrying out at least 2 NR2E3 mutations; they had undergone comprehensive ophthalmological examination, fundus photography, optical coherence tomography, electrophysiological testing, and visual field at the Regional Reference Center for Hereditary Retinal Degenerations of the Eye Clinic in Florence. Five females and six males with a diagnosis of NR2E3-related retinal dystrophy were included in the study. All patients complained of nyctalopia. Visual acuity ranged from 0.00 logMAR to hand motion. Two patients presented bull's eye maculopathy, and one of these was characterized by a triple hyper-autofluorescent ring at the fundus autofluorescence examination. Three patients showed small yellowish dots and spots at the mid-periphery. One patient was characterized by widespread subretinal drusenoid deposits (SDD) at the posterior pole. Four patients showed vitreous abnormalities. Optical coherence tomography (OCT) examinations detected variable degrees of abnormal retinal lamination and schitic changes. Seven patients were compound heterozygous and four were homozygous for mutations in NR2E3. Our study confirmed high variable phenotype in autosomal recessive NR2E3-related retinal dystrophy. Bull's eye maculopathy, subretinal drusenoid deposits, and foveal hypoplasia represent novel clinical findings in NR2E3-related retinal dystrophy. Macular involvement was detectable in all the patients, and the abnormal foveal avascular zone (FAZ) supports the role of NR2E3 in retinal development.

Swept source optical coherence tomography and optical coherence tomography angiography in pediatric enhanced S-cone syndrome: a case report

BackgroundEnhanced S-cone syndrome is an autosomal recessive retinal dystrophy related to a defect in a nuclear receptor gene (NR2E3) that leads to alteration in cells development from rod to S-cone. This retinal dystrophy may be associated with retinal schisis. The aim of this report is to describe structural optical coherence tomography and optical coherence tomography angiography features in a case of enhanced S-cone syndrome associated with macular schisis.Case presentationA Caucasian 13-year-old girl underwent measurement of best corrected visual acuity, ophthalmoscopic evaluation, and fundus autofluorescence examination. Photopic and scotopic electroretinography were carried out as well. Enhanced S-cone syndrome was suspected on the basis of clinical and electrophysiological findings. Structural optical coherence tomography and optical coherence tomography angiography allowed the further characterization of the associated macular schisis.Genetic analysis not only confirmed the diagnosis but increased the clinical novelty of this case report by showing two variations in the NR2E3 gene probably related to the phenotype: a missense variation c.1118T>C which leads to the substitution of leucine with proline in amino acid position 373, and c.349+5G>C, which involves a gene sequence near a splicing site.ConclusionsSwept source structural optical coherence tomography (B scans and “en face” images) and optical coherence tomography angiography allowed the observation of retinal structural details and the involvement of each retinal layer and capillary plexus in enhanced S-cone syndrome. Of interest, neither of the two NR2E3 gene variants found in this case report have been linked to any form of retinopathy.

Mutations in known disease genes account for the majority of autosomal recessive retinal dystrophies.

Retinal dystrophies (RDs) are hereditary blinding eye conditions that are highly variable in their clinical presentation. The remarkable genetic heterogeneity that characterizes RD was a major challenge in establishing the molecular diagnosis in these patients until the recent advent of next-generation sequencing. It remains unclear, however, what percentage of autosomal recessive RD remain undiagnosed when all established RD genes are sequenced. We enrolled 75 families in which RD segregates in an apparently autosomal recessive manner. We show that the yield of a multigene panel that contains known RD genes is 67.5%. The higher yield (82.3%) when whole exome sequencing was implemented instead was often due to hits in genes that were not included in the original design of the panel. We also show the value of homozygosity mapping even during the era of exome sequencing in uncovering cryptic mutations. In total, we describe 45 unique likely deleterious variants (of which 18 are novel including one deep intronic and one genomic deletion mutation). Our study suggests that the genetic heterogeneity of autosomal recessive RD is approaching saturation and that any new RD genes will probably account for only a minor role in the mutation burden.

A novel, homozygous nonsense variant of the CDHR1 gene in a Chinese family causes autosomal recessive retinal dystrophy by NGS‐based genetic diagnosis

Retinal dystrophy is an inherited, heterogeneous, chronic and progressive disorder of visual functions. The mutations of patients with autosomal recessive retinal retinopathy cone‐and‐rod dysfunction and macular dystrophy have not been well described in the Chinese population. In this study, a three‐generation Chinese retinal dystrophy family was recruited. Ophthalmic examinations were performed. Targeted next generation sequencing (TGS) was used to identify causative genes, and Sanger sequencing was conducted to verify candidate mutations and co‐segregation. Reverse transcription (RT)‐PCR was applied to investigate the spatial and temporal expression patterns of cdhr1 gene in mouse. A novel, homozygous, deleterious and nonsense variant (c.T1641A; p.Y547*) in the CDHR1 gene was identified in the family with autosomal recessive retinal dystrophy, which was co‐segregated with the clinical phenotypes in this family. RT‐PCR analysis revealed that cdhr1 is ubiquitously expressed in eye, particularly very high expression in retina; high expression in lens, sclera, and cornea; and high expression in brain. In conclusion, our study is the first to indicate that the novel homozygous variant c.T1641A (p.Y547*) in the CHDR1 gene might be the disease‐causing mutation for retinal dystrophy in our patient, extending its mutation spectrums. These findings further the understanding of the molecular pathogenesis of this disease and provide new insights for diagnosis as well as new implications for genetic counselling.

MERTK mutation update in inherited retinal diseases.

MER tyrosine kinase (MERTK) encodes a surface receptor localized at the apical membrane of the retinal pigment epithelium. It plays a critical role in photoreceptor outer segment internalization prior to phagocytosis. Mutations in MERTK have been associated with severe autosomal recessive retinal dystrophies in the RCS rat and in humans. We present here a comprehensive review of all reported MERTK disease causing variants with the associated phenotype. In addition, we provide further data and insights of a large cohort of 1,195 inherited retinal dystrophies (IRD) index cases applying state-of-the-art genotyping techniques and summarize current knowledge. A total of 79 variants have now been identified underlying rod-cone dystrophy and cone-rod dystrophy including 11 novel variants reported here. The mutation spectrum in MERTK includes 33 missense, 12 nonsense, 12 splice defects, 12 small deletions, two small insertion-deletions, three small duplications, and two exonic and three gross deletions. Altogether, mutations in MERTK account for ∼2% of IRD cases with a severe retinal phenotype. These data are important for current and future therapeutic trials including gene replacement therapy or cell-based therapy.

A founder mutation in CERKL is a major cause of retinal dystrophy in Finland.

To study the genetic aetiology of retinal dystrophies (RD) in Finnish patients. A targeted next-generation sequencing (NGS) panel of 105 retinal dystrophy genes was used in a cohort of 55 RD patients. The overall diagnostic yield was 60% demonstrating the power of this approach. Interestingly, a missense mutation c.375C>G p.(Cys125Trp) in the CERKL gene was found in 18% of the patients in either a homozygous or compound heterozygous state. Data from Exome Aggregation Consortium (ExAC) Browser show that the CERKL c.375C>G p.(Cys125Trp) allele is enriched in the Finnish population and thus is a founder mutation. Furthermore, we report the clinical picture of 18 patients with mutations in the CERKL gene. CERKL mutations cause a macular-onset disease, in which symptoms first become apparent at the second decade. We also detected other novel founder mutations in the CERKL, EYS, RP1, ABCA4 and GUCY2D genes. Our report indicates that the first diagnostic test for Finnish patients with sporadic or autosomal recessive RD should be a targeted test for founder mutations in the CERKL, EYS, RP1, ABCA4 and GUCY2D genes. These results confirm the utility of NGS-based gene panels as a powerful method for mutation identification in RD, thus enabling improved genetic counselling for these families.

Mutation detection of Pakistani families with autosomal recessive retinal dystrophies

PurposeHereditary retinal dystrophies (RD) are a group of heterogeneous disorders caused by mutations in over 200 genes. RDs can be subdivided into different groups based on the primary degeneration of rod or cone photoreceptor cells. This study was conducted to investigate the underlying RD genes and mutation in consanguineous families from Pakistan.MethodsFamilies were recruited after informed consent. Peripheral blood was collected and genomic DNA was extracted according to standard procedures. Homozygosity mapping was performed using Affymetrix Gene Chip Human Mapping 250 K‐NspI arrays. The data were analyzed using Homozygosity Mapper software. Primers for amplifying all exons and intron boundaries of all genes were designed with Primer3plus software followed by PCR and Sanger sequencing. Minigene splicing assay and DNA walking were performed on respective samples.ResultsHomozygosity mapping identified a novel locus in family A, one novel gene (C8ORF37) in family B and a large novel genomic deletion of the (LCA5) gene in family C.ConclusionsOur study indicates the heterogeneous nature of retinal dystrophies in Pakistan. Although earlier studies have explored a number of RD families, but underlying genes are still unknown for significant proportion of Pakistani families. Theoretically the traditional screening methods (homozygosity mapping based candidate gene sequencing) were successful, however SNP based genome wide scan were further implemented to improve the detection of underlying variations responsible for Retinal Dystrophies. Therefore the amalgamation of traditional and modern molecular techniques is required for accurate identification of mutations. It is anticipated that these findings will contribute to future genetic testing in Pakistani families to minimize the risk of recessive disorders.

Disease Expression in Autosomal Recessive Retinal Dystrophy Associated With Mutations in the DRAM2 Gene.

To determine the disease course of retinal dystrophy caused by recessive variants in the DRAM2 (damage-regulated autophagy modulator 2) gene. Sixteen individuals with DRAM2-retinopathy were examined (six families; age range, 19-56 years, includes one pre-symptomatic case). The change in visual acuity over time was studied, and electrophysiology (n = 6), retina-tracking perimetry (n = 1), fundus autofluorescence (FAF) imaging (n = 6), and optical coherence tomography (OCT; n = 12) were performed. All symptomatic patients presented with central visual loss (15/15) unaccompanied either by nyctalopia or light-hypersensitivity; most (11/15) developed symptoms in the third decade of life. A granular macular appearance, often with associated white/yellow dots, was an early fundoscopic feature. There was an ill-defined ring of hyperautofluorescence on FAF. Optical coherence tomography revealed loss of the ellipsoid zone perifoveally in a 19-year-old pre-symptomatic individual. The central atrophic area enlarged over time and fundoscopy showed peripheral degeneration in seven of the nine individuals that were examined ≥ 10 years after becoming symptomatic; some of these subjects developed nyctalopia and light hypersensitivity. Electrophysiology revealed generalized retinal dysfunction in three of the five individuals that were tested ≥ 10 years after becoming symptomatic. Patients with DRAM2-retinopathy are typically asymptomatic in the first two decades of life and present with central visual loss and a maculopathy. A faint hyperautofluorescent ring on FAF can be a suggestive feature. The retinal periphery is frequently affected later in the disease process. Photoreceptor degeneration is likely to be the primary event and future studies on DRAM2-retinopathy are expected to provide important insights into retinal autophagy.

Hidden Genetic Variation in LCA9-Associated Congenital Blindness Explained by 5'UTR Mutations and Copy-Number Variations of NMNAT1.

ABSTRACTLeber congenital amaurosis (LCA) is a severe autosomal‐recessive retinal dystrophy leading to congenital blindness. A recently identified LCA gene is NMNAT1, located in the LCA9 locus. Although most mutations in blindness genes are coding variations, there is accumulating evidence for hidden noncoding defects or structural variations (SVs). The starting point of this study was an LCA9‐associated consanguineous family in which no coding mutations were found in the LCA9 region. Exploring the untranslated regions of NMNAT1 revealed a novel homozygous 5′UTR variant, c.‐70A>T. Moreover, an adjacent 5′UTR variant, c.‐69C>T, was identified in a second consanguineous family displaying a similar phenotype. Both 5′UTR variants resulted in decreased NMNAT1 mRNA abundance in patients’ lymphocytes, and caused decreased luciferase activity in human retinal pigment epithelial RPE‐1 cells. Second, we unraveled pseudohomozygosity of a coding NMNAT1 mutation in two unrelated LCA patients by the identification of two distinct heterozygous partial NMNAT1 deletions. Molecular characterization of the breakpoint junctions revealed a complex Alu‐rich genomic architecture. Our study uncovered hidden genetic variation in NMNAT1‐associated LCA and emphasized a shift from coding to noncoding regulatory mutations and repeat‐mediated SVs in the molecular pathogenesis of heterogeneous recessive disorders such as hereditary blindness.