Phenotypic and Genetic Alterations in Adult-Onset Cone and Cone-Rod Dystrophy.

INTRODUCTION: The objective of this study was to investigate the clinical characteristics and genetic spectrum of adult-onset cone/cone-rod dystrophy (AOCD/AOCRD) in Korean individuals. METHODS: This is a single-center, retrospective cross-sectional study. We analyzed 22 individuals with genetically confirmed cone dystrophy, with symptoms beginning after 30 years of age. All patients underwent comprehensive ophthalmic and electrophysiological examinations. Exome sequencing of 296 genes associated with inherited retinal disease was performed. The clinical features of patients with AOCD/AOCRD and the causative genes and variants detected by exome sequencing were analyzed. RESULTS: The median age at the first visit was 52 years (range, 31-76 years), and the most common initial symptom was reduced visual acuity. In most cases, fundus photography showed a bull's eye pattern with foveal sparing, consistent with perifoveal photoreceptor loss on optical coherence tomography. We identified disease-causing variants in six genes: RP1, CRX, CDHR1, PROM1, CRB1, and GUCY2D. Pathogenic variants in RP1, CRX, and CDHR1 were identified in 77% of the AOCD/AOCRD cases, including p.Cys1399LeufsTer5, p.Arg1933Ter, and p.Ile2061SerfsTer12 in RP1; p.Ter300GlnextTer118 in CRX; and p.Glu201Lys in CDHR1. No characteristic imaging differences were observed for any of the causative genes. Most of the RP1-related AOCD/AOCRD cases showed a decreased amplitude only in the photopic electroretinogram (ERG), whereas CRX-related AOCD/AOCRD cases showed a slightly decreased amplitude in both the scotopic and photopic ERGs. CONCLUSION: In case of visual impairment with bull's eye pattern of RPE atrophy recognized after the middle age, a comprehensive ophthalmic examination and genetic test should be considered, with the possibility of AOCD/AOCRD in East Asians.

RP2-Associated X-linked Retinopathy: Clinical Findings, Molecular Genetics, and Natural History.

PURPOSE: To review and describe in detail the clinical course, functional and anatomic characteristics of RP2-associated retinal degeneration. DESIGN: Retrospective case series. PARTICIPANTS: Male participants with disease-causing variants in the RP2 gene. METHODS: Review of all case notes and results of molecular genetic testing, retinal imaging (fundus autofluorescence [FAF] imaging, OCT), and electrophysiology assessment. MAIN OUTCOME MEASURES: Molecular genetic testing, clinical findings including best-corrected visual acuity (BCVA), qualitative and quantitative retinal imaging analysis, and electrophysiology parameters. RESULTS: Fifty-four molecularly confirmed patients were identified from 38 pedigrees. Twenty-eight disease-causing variants were identified, with 20 not previously clinically characterized. Fifty-three patients (98.1%) presented with retinitis pigmentosa. The mean age of onset (range ± standard deviation [SD]) was 9.6 years (1-57 ± 9.2 years). Forty-four patients (91.7%) had childhood-onset disease, with mean age of onset of 7.6 years. The most common first symptom was night blindness (68.8%). Mean BCVA (range ± SD) was 0.91 logarithm of the minimum angle of resolution (logMAR) (0-2.7 ± 0.80) and 0.94 logMAR (0-2.7 ± 0.78) for right and left eyes, respectively. On the basis of the World Health Organization visual impairment criteria, 18 patients (34%) had low vision. The majority (17/22) showed electroretinogram (ERG) evidence of a rod-cone dystrophy. Pattern ERG P50 was undetectable in all but 2 patients. A range of FAF findings was observed, from normal to advanced atrophy. There were no statistically significant differences between right and left eyes for ellipsoid zone width (EZW) and outer nuclear layer (ONL) thickness. The mean annual rate of EZW loss was 219 µm/year, and the mean annual decrease in ONL thickness was 4.93 µm/year. No patient with childhood-onset disease had an identifiable ellipsoid zone (EZ) after the age of 26 years at baseline or follow-up. Four patients had adulthood-onset disease and a less severe phenotype. CONCLUSIONS: This study details the clinical phenotype of RP2 retinopathy in a large cohort. The majority presented with early-onset severe retinal degeneration, with early macular involvement and complete loss of the foveal photoreceptor layer by the third decade of life. Full-field ERGs revealed rod-cone dystrophy in the vast majority, but with generalized (peripheral) cone system involvement of widely varying severity in the first 2 decades of life. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

A natural history study of autosomal dominant GUCY2D-associated cone-rod dystrophy.

PURPOSE: To describe the natural history of autosomal dominant (AD) GUCY2D-associated cone-rod dystrophies (CRDs), and evaluate associated structural and functional biomarkers. METHODS: Retrospective analysis was conducted on 16 patients with AD GUCY2D-CRDs across two sites. Assessments included central macular thickness (CMT) and length of disruption to the ellipsoid zone (EZ) via optical coherence tomography (OCT), electroretinography (ERG) parameters, best corrected visual acuity (BCVA), and fundus autofluorescence (FAF). RESULTS: At first visit, with a mean age of 30 years (range 5-70 years), 12 patients had a BCVA below Australian driving standard (LogMAR ≥ 0.3 bilaterally), and 1 patient was legally blind (LogMAR ≥ 1). Longitudinal analysis demonstrated a deterioration of LogMAR by - 0.019 per year (p < 0.001). This accompanied a reduction in CMT of - 1.4 µm per year (p < 0.0001), lengthened EZ disruption by 42 µm per year (p = < 0.0001) and increased area of FAF by 0.05 mm2 per year (p = 0.027). Similarly, cone function decreased with increasing age, as demonstrated by decreasing b-wave amplitude of the light-adapted 30 Hz flicker and fused flicker (p = 0.005 and p = 0.018, respectively). Reduction in CMT and increased EZ disruption on OCT were associated with functional changes including poorer BCVA and decreased cone function on ERG. CONCLUSION: We have described the natural long-term decline in vision and cone function associated with mutations in GUCY2D and identified a set of functional and structural biomarkers that may be useful as outcome parameters for future therapeutic clinical trials.

Phenotypic variability in PRPH2 as demonstrated by a family with incomplete penetrance of autosomal dominant cone-rod dystrophy.

INTRODUCTION: Mutations in the peripherin-2 gene (PRPH2) are a common cause of inherited retinal dystrophies well known for their phenotypic diversity. We describe a novel presentation of the c.623G > A; p.(Gly208Asp) variant in association with cone-rod dystrophy and reduced penetrance. CASE DESCRIPTION: A 39-year-old man presents with a history of decreased visual acuity, photophobia, and dyschromatopsia. Fundus examination was largely unremarkable while spectral-domain optical coherence tomography (SD-OCT) demonstrated diffuse granularity at the ellipsoid zone. Full-field electroretinogram (ffERG) revealed a cone-rod dystrophy. Genetic testing revealed a heterozygous pathogenic variant, c.623G > A; p.(Gly208Asp), in the PRPH2 gene, also found in an unaffected brother. The 50-year-old brother had no visual symptoms and no findings on fundus examination. SD-OCT showed normal retinal architecture and ffERG was within normal limits bilaterally. CONCLUSION: This case report broadens the known phenotypic presentations of PRPH2-associated retinopathy and suggests that the PRPH2 variant c.623G > A; p.(Gly208Asp) may be associated with reduced penetrance.

Genetic characterization of 1210 Japanese pedigrees with inherited retinal diseases by whole-exome sequencing.

Inherited retinal diseases (IRDs) comprise a phenotypically and genetically heterogeneous group of ocular disorders that cause visual loss via progressive retinal degeneration. Here, we report the genetic characterization of 1210 IRD pedigrees enrolled through the Japan Eye Genetic Consortium and analyzed by whole exome sequencing. The most common phenotype was retinitis pigmentosa (RP, 43%), followed by macular dystrophy/cone- or cone-rod dystrophy (MD/CORD, 13%). In total, 67 causal genes were identified in 37% (448/1210) of the pedigrees. The first and second most frequently mutated genes were EYS and RP1, associated primarily with autosomal recessive (ar) RP, and RP and arMD/CORD, respectively. Examinations of variant frequency in total and by phenotype showed high accountability of a frequent EYS missense variant (c.2528G>A). In addition to the two known EYS founder mutations (c.4957dupA and c.8805C>G) of arRP, we observed a frequent RP1 variant (c.5797C>T) in patients with arMD/CORD.

Case Report: Multimodal Imaging Features of an ABCA4 Cone Dystrophy.

SIGNIFICANCE: Cone dystrophies and cone-rod dystrophies are a group of rare inherited pathologies characterized by degeneration of cone photoreceptors and subsequent rod involvement. The identification of causative genes is essential for diagnosis, and advanced imaging is acquiring great value in the characterization of the different phenotypic expressions. PURPOSE: We describe genotype-phenotype associations of an autosomal recessive ABCA4-associated cone dystrophy using multimodal imaging. CASE REPORT: A 34-year-old woman presented with progressive visual acuity decay. Visual acuity was 20/32 for her right eye and 20/25 for her left eye. A central scotoma was detected on a 10-2 Humphrey visual field in both eyes. Funduscopy revealed perifoveal retinal pigment epithelial changes, and fundus autofluorescence using blue excitation light showed decreased autofluorescence in the central fovea of both eyes with surrounding annular ring of increased autofluorescence in the perifoveal zone; green excitation light fundus autofluorescence was more accurate in the characterization of the size, perimeter, and circularity of central hypofluorescent lesions. Optical coherence tomography revealed an incomplete focal cavitation in both foveas, and optical coherence tomography angiography images showed a reduction in the superficial and deep capillary plexus density, an increased foveal avascular area, and subtle voids in choriocapillaris blood flow. Electroretinography was consistent with cone dystrophy, and molecular testing revealed the alteration of the ABCA4 gene. CONCLUSIONS: The identification of an incomplete focal cavitation could alert the clinician to consider early ABCA4 central cone dystrophy. The patient in this case also exhibited reduced vessel density in the foveal area. Both of these characteristics could be important features related to the underlying genetic mutation.

Clinical characterization and the improved molecular diagnosis of autosomal dominant cone-rod dystrophy in patients with SCA7.

Purpose: To evaluate the retinal phenotype and genetic features of Chinese patients with spinocerebellar ataxia type 7 (SCA7). Methods: Detailed ophthalmic examinations, including electroretinograms, fundus photography, fundus autofluorescence and optical coherence tomography, were performed to analyse the retinal lesions of patients with SCA7. A molecular genetic analysis was completed to confirm the number of CAG repeats in ATXN7 gene on the patients and their family members. Results: Eight patients from three families with SCA7 were included in this study. Trinucleotide repeat was expanded from 43 to 113 in the affected patients. The affected patients were characterized by different degrees of cone-rod dystrophy, which is positively related to the number of CAG repeats and age. All patients complained of progressive bilateral visual loss, and most cases reported visual disturbance earlier than gait movement or dysarthria. A coarse granular appearance of the macular region on scanning laser ophthalmoscopy, hypofluorescence in the macula on autofluorescence, retinal atrophy on optic coherence tomography, depression of multifocal electroretinograms and prominent abnormalities in cone-mediated responses on electrograms are the general features of SCA7-related retinopathy. Hyperreflective dots in the outer retinal layers and choroidal vessel layers are a common sign in optic coherence tomography in the advanced stage. Conclusions: SCA7 shows a cone-rod dystrophy phenotype. The multimodal imaging of the retina is beneficial to detect the early lesions of cone-rod dystrophy related to SCA7.

ISOLATED MACULOPATHY AND MODERATE ROD-CONE DYSTROPHY REPRESENT THE MILDER END OF THE RDH12-RELATED RETINAL DYSTROPHY SPECTRUM.

PURPOSE: To describe an isolated maculopathy and an intermediate rod-cone dystrophy phenotype as the milder end of the RDH12-related retinal dystrophy spectrum. METHODS: Seven patients (17-34 years of age) underwent an extensive ophthalmic workup including psychophysical and electrophysiological testing and multimodal imaging. RESULTS: Three patients have isolated macular disease. Best-corrected visual acuity (BCVA) ranges from 20/125 to 20/40 with normal visual fields or only limited central, relative scotomata, and normal full-field ERGs. Both optical coherence tomography scans and autofluorescent imaging hint at relatively better-preserved foveal quality initially. An intermediate rod-cone phenotype in four patients is characterized by a central retinal dystrophy extending just beyond the vascular arcades, characteristic peripapillary sparing, and additional scattered atrophic patches. Again, foveal quality is initially better on optical coherence tomography scans. Best-corrected visual acuity ranges from counting fingers to 20/32. Goldmann visual fields vary from central scotomata to severe generalized abnormalities. ERGs range between mild and severe rod-cone dysfunction. Nine distinct RDH12 pathogenic variants, two of which are novel, are identified. CONCLUSION: The classic phenotype of RDH12-related early-onset retinal dystrophy is expanded to include an isolated maculopathy and intermediate dystrophy phenotype, characterized by its later onset and milder course with a fair visual potential until much later in life, emphasizing the phenotypic heterogeneity of RDH12-related retinopathy.

A ROD-CONE DYSTROPHY IS SYSTEMATICALLY ASSOCIATED TO THE RTN4IP1 RECESSIVE OPTIC ATROPHY.

PURPOSE: RTN4IP1 biallelic mutations cause a recessive optic atrophy, sometimes associated to more severe neurological syndromes, but so far, no retinal phenotype has been reported in RTN4IP1 patients, justifying their reappraisal. METHODS: Seven patients from four families carrying biallelic RTN4IP1 variants were retrospectively reviewed, with emphasis on their age of onset, visual acuity, multimodal imaging including color and autofluorescence frames, spectral-domain optical coherence tomography with RNFL and macular analyses. RESULTS: Seven patients from four RTN4IP1 families developed in their first decade of life a bilateral recessive optic atrophy with severe central visual loss, and primary nystagmus developed in 5 of 7 patients. Six patients were legally blind. In a second stage, the seven individuals developed a rod-cone dystrophy, sparing the macular zone and the far periphery. This retinal damage was identified by 55° field fundus autofluorescence frames and also by spectral-domain optical coherence tomography scans of the temporal part of the macular zone in five of the seven patients. Full-field electroretinography measurements disclosed reduced b-wave amplitude of the rod responses in all patients but two. Family 4 with the p.R103H and c.601A > T (p.K201*) truncating mutation had further combined neurological signs with cerebellar ataxia, seizures, and intellectual disability. CONCLUSION: RTN4IP1 recessive optic atrophy is systematically associated to a rod-cone dystrophy, which suggests that both the retinal ganglion cells and the rods are affected as a result of a deficit in the mitochondrial respiratory chain. Thus, systematic widefield autofluorescence frames and temporal macular scans are recommended for the evaluation of patients with optic neuropathies.

Functional characterization of the first missense variant in CEP78, a founder allele associated with cone-rod dystrophy, hearing loss, and reduced male fertility.

Inactivating variants in the centrosomal CEP78 gene have been found in cone-rod dystrophy with hearing loss (CRDHL), a particular phenotype distinct from Usher syndrome. Here, we identified and functionally characterized the first CEP78 missense variant c.449T>C, p.(Leu150Ser) in three CRDHL families. The variant was found in a biallelic state in two Belgian families and in a compound heterozygous state-in trans with c.1462-1G>T-in a third German family. Haplotype reconstruction showed a founder effect. Homology modeling revealed a detrimental effect of p.(Leu150Ser) on protein stability, which was corroborated in patients' fibroblasts. Elongated primary cilia without clear ultrastructural abnormalities in sperm or nasal brushes suggest impaired cilia assembly. Two affected males from different families displayed sperm abnormalities causing infertility. One of these is a heterozygous carrier of a complex allele in SPAG17, a ciliary gene previously associated with autosomal recessive male infertility. Taken together, our data indicate that a missense founder allele in CEP78 underlies the same sensorineural CRDHL phenotype previously associated with inactivating variants. Interestingly, the CEP78 phenotype has been possibly expanded with male infertility. Finally, CEP78 loss-of-function variants may have an underestimated role in misdiagnosed Usher syndrome, with or without sperm abnormalities.

Expanding the genotypic spectrum of Jalili syndrome: Novel CNNM4 variants and uniparental isodisomy in a north American patient cohort.

Jalili syndrome is a rare multisystem disorder with the most prominent features consisting of cone-rod dystrophy and amelogenesis imperfecta. Few cases have been reported in the Americas. Here we describe a case series of patients with Jalili syndrome examined at the National Eye Institute's Ophthalmic Genetics clinic between 2016 and 2018. Three unrelated sporadic cases were systematically evaluated for ocular phenotype and determined to have cone-rod dystrophy with bull's eye maculopathy, photophobia, and nystagmus. All patients had amelogenesis imperfecta. Two of these patients had Guatemalan ancestry and the same novel homozygous CNNM4 variant (p.Arg236Trp c.706C > T) without evidence of consanguinity. This variant met likely pathogenic criteria by the American College of Medical Genetics guidelines. An additional patient had a homozygous deleterious variant in CNNM4 (c.279delC p.Phe93Leufs*31), which resulted from paternal uniparental isodisomy for chromosome 2p22-2q37. This individual had additional syndromic features including developmental delay and spastic diplegia, likely related to mutations at other loci. Our work highlights the genotypic variability of Jalili syndrome and expands the genotypic spectrum of this condition by describing the first series of patients seen in the United States.

Features, genetics and their correlation in Jalili syndrome: a systematic review.

Jalili syndrome is a rare genetic disorder first identified by Jalili in Gaza. Amelogenesis imperfecta and cone-rode dystrophy are simultaneously seen in Jalili syndrome patients as the main and primary manifestations. Molecular analysis has revealed that the CNNM4 gene is responsible for this rare syndrome. Jalili syndrome has been observed in many countries around the world, especially in the Middle East and North Africa. In the current scoping systematic review we searched electronic databases to find studies related to Jalili syndrome. In this review we summarise the reported clinical symptoms, CNNM4 gene and protein structure, CNNM4 mutations, attempts to reach a genotype-phenotype correlation, the functional role of CNNM4 mutations, and epidemiological aspects of Jalili syndrome. In addition, we have analysed the reported mutations in mutation effect prediction databases in order to gain a better understanding of the mutation's outcomes.

PHENOTYPIC CHARACTERISTICS OF ROD-CONE DYSTROPHY ASSOCIATED WITH MYO7A MUTATIONS IN A LARGE FRENCH COHORT.

PURPOSE: To document the rod-cone dystrophy phenotype of patients with Usher syndrome type 1 (USH1) harboring MYO7A mutations. METHODS: Retrospective cohort study of 53 patients (42 families) with biallelic MYO7A mutations who underwent comprehensive examination, including functional visual tests and multimodal retinal imaging. Genetic analysis was performed either using a multiplex amplicon panel or through direct sequencing. Data were analyzed with IBM SPSS Statistics software v. 21.0. RESULTS: Fifty different genetic variations including 4 novel were identified. Most patients showed a typical rod-cone dystrophy phenotype, with best-corrected visual acuity and central visual field deteriorating linearly with age. At age 29, binocular visual field demonstrated an average preservation of 50 central degrees, constricting by 50% within 5 years. Structural changes based on spectral domain optical coherence tomography, short wavelength autofluorescence, and near-infrared autofluorescence measurements did not however correlate with age. Our study revealed a higher percentage of epiretinal membranes and cystoid macular edema in patients with MYO7A mutations compared with rod-cone dystrophy patients with other mutations. Subgroup analyses did not reveal substantial genotype-phenotype correlations. CONCLUSION: To the best of our knowledge, this is the largest French cohort of patients with MYO7A mutations reported to date. Functional visual characteristics of this subset of patients followed a linear decline as in other typical rod-cone dystrophy, but structural changes were variable indicating the need for a case-by-case evaluation for prognostic prediction and choice of potential therapies.

RPGR-Associated Dystrophies: Clinical, Genetic, and Histopathological Features.

This study describes the clinical, genetic, and histopathological features in patients with RPGR-associated retinal dystrophies. Nine male patients from eight unrelated families underwent a comprehensive ophthalmic examination. Additionally, the histopathology of the right eye from a patient with an end-stage cone-rod-dystrophy (CRD)/sector retinitis pigmentosa (RP) phenotype was examined. All RPGR mutations causing a CRD phenotype were situated in exon ORF15. The mean best-corrected visual acuity (BCVA, decimals) was 0.58 (standard deviation (SD)): 0.34; range: 0.05-1.13); and the mean spherical refractive error was -4.1 D (SD: 2.11; range: -1.38 to -8.19). Hyperautofluorescent rings were observed in six patients. Full-field electroretinography responses were absent in all patients. The visual field defects ranged from peripheral constriction to central islands. The mean macular sensitivity on microperimetry was 11.6 dB (SD: 7.8; range: 1.6-24.4) and correlated significantly with BCVA (r = 0.907; p = 0.001). A histological examination of the donor eye showed disruption of retinal topology and stratification, with a more severe loss found in the peripheral regions. Reactive gliosis was seen in the inner layers of all regions. Our study demonstrates the highly variable phenotype found in RPGR-associated retinal dystrophies. Therapies should be applied at the earliest signs of photoreceptor degeneration, prior to the remodeling of the inner retina.

Novel mutations in c2orf71 causing an early onset form of cone-rod dystrophy: A molecular diagnosis after 20 years of clinical follow-up.

Purpose: Cone rod-dystrophies (CRDs) are pigmentary retinopathies mainly involving cones. CRDs typically present with decreased visual acuity and loss of sensitivity in the central visual field, reflecting the primary dysfunction of cones associated with night blindness and concentric visual field loss due to rod dysfunction. We describe the phenotype, natural history, and molecular analysis results of an early onset form of CRD. Methods: An otherwise healthy 25-year-old man from Sardinia, Italy, initially presented with subacute visual loss and central scotoma in both eyes. He underwent a complete ophthalmic examination, electrophysiologic testing, and genetic counseling. We first applied a candidate gene approach on ABCA4 to detect mutations; then, we performed exome sequencing (WES) on all family members to identify causative mutations. Results: The ophthalmic examination was unremarkable except the fundus examination, which revealed a well-circumscribed ring-shaped area of choroidal and RPE atrophy surrounding the fovea in the left eye and small white patches of atrophy around the fovea in the right eye. The ocular features and medical history were consistent with a diagnosis of CRD. Twenty years later, he showed a marked impairment in visual function, secondary to severe atrophic maculopathy associated with sparse pigmentary deposits. Molecular analysis identified two novel frameshift mutations in C2orf71: c.3039dupC: p.Ser1014Leufs*93 and c.1804_1805delAG:p. His603Argfs*77. Conclusions: The mutations in C2orf71 reported in this study comprise protein truncation mutations, which are likely to be involved in the pathogenesis of this severe form of early onset CRD.

Whole exome sequencing resolves complex phenotype and identifies CC2D2A mutations underlying non-syndromic rod-cone dystrophy.

Genetic investigations were performed in three brothers from a consanguineous union, the two oldest diagnosed with rod-cone dystrophy (RCD), the youngest with early-onset cone-rod dystrophy and the two youngest with nephrotic-range proteinuria. Targeted next-generation sequencing did not identify homozygous pathogenic variant in the oldest brother. Whole exome sequencing (WES) applied to the family identified compound heterozygous variants in CC2D2A (c.2774G>C p.(Arg925Pro); c.4730_4731delinsTGTATA p.(Ala1577Valfs*5)) in the three brothers with a homozygous deletion in CNGA3 (c.1235_1236del p.(Glu412Valfs*6)) in the youngest correcting his diagnosis to achromatopsia plus RCD. None of the three subjects had cerebral abnormalities or learning disabilities inconsistent with Meckel-Gruber and Joubert syndromes, usually associated with CC2D2A mutations. Interestingly, an African woman with RCD shared the CC2D2A missense variant (c.2774G>C p.(Arg925Pro); with c.3182+355_3825del p.(?)). The two youngest also carried compound heterozygous variants in CUBN (c.7906C>T rs137998687 p.(Arg2636*); c.10344C>G p.(Cys3448Trp)) that may explain their nephrotic-range proteinuria. Our study identifies for the first time CC2D2A mutations in isolated RCD and underlines the power of WES to decipher complex phenotypes.

Characterization of the cone-rod dystrophy retinal phenotype caused by novel homozygous DRAM2 mutations.

Cone-rod dystrophies (CRD) are a group of Inherited Retinal Dystrophies (IRD) characterized by the primary involvement of cone photoreceptors, resulting in the degeneration of the central retina, or macula. Although there are more than 55 CRD genes, a considerable percentage of cases remain unsolved. In this context, the present study aimed to describe and characterize the phenoptype and the genetic cause of 3 CRD families from a cohort of IRD cases. Clinical evaluation in each patient was supported by a complete ophthalmological examination, including visual acuity measurement, fundus retinography, fundus autofluorescence imaging, optical coherence tomography and full-field electroretinography. Molecular diagnoses were performed by whole exome sequencing analyzing a group of 279 IRD genes, and cosegregation of the identified pathogenic variants was confirmed by Sanger sequencing. Three novel homozygous mutations in the autophagy gene DRAM2 were identified as the molecular cause of disease in the three families: c.518-1G>A, c.628_629insAG and c.693+2T>A. Clinical data revealed that the 3 patients presented a shared CRD phenotype with adult-onset macular involvement and later peripheral degeneration, although the age of onset, evolution and severity were variable. In order to characterize the transcription effects of these variants, mRNA expression studies were performed. The results showed alterations in the DRAM2 transcription, including alternative splicing forms and lower levels of mRNA, which correlated with the phenotypic variability observed between patients. For instance, frameshift mutations were related to a less severe phenotype, with circumscribed mid-peripheral involvement, and lower levels of mRNA, suggesting an activation of the nonsense-mediated decay (NMD) pathway; while a more severe and widespread retinal degeneration was associated to the inframe alternative splicing variant reported, possibly due to a malfunctioning or toxicity of the resulting protein. Following these findings, DRAM2 expression was assessed in several human tissues by semi-quantitative RT-PCR and two isoforms were detected ubiquitously, yet with a singular tissue-specific pattern in retina and brain. Altogether, although the unique retinal phenotype described did not correlate with the ubiquitous expression, the retinal-specific expression and the essential role of autophagy in the photoreceptor survival could be key arguments to explain this particular DRAM2 phenotype.

Pathogenicity discrimination and genetic test reference for CRX variants based on genotype-phenotype analysis.

The cone-rod homeobox (CRX) gene is specifically expressed in developing and mature photoreceptors and is relatively conserved, with limited polymorphisms in coding regions. Rare variants in CRX are usually considered causative for different forms of retinal degeneration, but this might be problematic based on recent data. This study aimed to classify CRX variants based on a genotype-phenotype analysis of our data and the literature. Twenty-four CRX variants, including 14 novel variants, were detected in 37 Chinese families based on exome sequencing data obtained from 4971 Chinese probands with different forms of eye diseases. After detailed phenotypic analysis and cosegregation analysis in families with CRX variants, the 24 variants could be classified into three groups: benign (six), likely benign (six), and pathogenic (12). Somatic mosaicism was identified in a family with unaffected parents (the father had a mutant allele that was detected in approximately 17% of his leukocyte DNA) and two affected sons. Furthermore, a thorough reassessment was systematically performed for all 113 heterozygous variants as well as for their associated phenotypes from our cohort and patients previously reported. Two critical findings on the pathogenicity of CRX variants were obtained based on the genotype-phenotype correlation, family segregation and ensemble predicting methods: 1) approximately half of heterozygous missense variants are likely benign, and 2) heterozygous truncating variants affecting the homeodomain are likely benign. Truncating mutations after the homeodomain are likely associated with a more severe phenotype. Although most heterozygous pathogenic variants in CRX are associated with autosomal dominant retinal degeneration, a homozygous c.268C> T (p.Arg90Trp) substitution and homozygous complete deletion of CRX have been reported to cause Leber congenital amaurosis. In conclusion, many rare missense variants and some truncating variants in CRX are likely benign, although previously, they might have been predicted to be damaging by some online tools. Evaluation of the pathogenicity of a CRX variant should consider both its nature and location. The information obtained in this study is critical in the era of routine clinical genetic test, not only for CRX but also for many other genes with many more variants. Functional studies and additional genotype-phenotype analyses are expected to confirm these associations.

Identification of novel PROM1 mutations responsible for autosomal recessive maculopathy with rod-cone dystrophy.

PURPOSE: To characterize two patients with macular and rod-cone dystrophy and identify the genetic basis for disease. METHOD: Ophthalmic examinations were performed for the family and the peripheral blood samples were collected for whole exome sequencing. The mutated sequences of PROM1 gene were cloned and expressed in cultured cell lines after transient transfection followed by analysis with confocal microscopy and bridge-PCR. RESULT: We reported that two patients, brothers in a family, were diagnosed with macular and rod-cone dystrophy. Phenotypically, both patients experience progressive visual impairment and nyctalopia. The fundus examination showed macular and choroid dystrophy with pigment deposits in the macular region. Functionally, photoreceptor response to electrophysiological stimulation was significantly compromised with more severe decline in rods. Genetic analysis by whole exome sequencing revealed two novel compound heterogeneous point mutations in PROM1 gene that co-segregate with patients in an autosomal recessive manner. Specifically, the c.C1902G(p.Y634X) nonsense mutation results in a truncated, labile, and mislocalized protein, while the c.C1682+3A>G intronic mutation disrupts messenger RNA splicing. CONCLUSION: Our findings have identified two novel deleterious mutations in PROM1 gene that are associated with hereditary macular and rod-cone dystrophy in human.

Phenotype Analysis of Retinal Dystrophies in Light of the Underlying Genetic Defects: Application to Cone and Cone-Rod Dystrophies.

Phenotypes observed in a large cohort of patients with cone and cone-rod dystrophies (COD/CORDs) are described based on multimodal retinal imaging features in order to help in analyzing massive next-generation sequencing data. Structural abnormalities of 58 subjects with molecular diagnosis of COD/CORDs were analyzed through specific retinal imaging including spectral-domain optical coherence tomography (SD-OCT) and fundus autofluorescence (BAF/IRAF). Findings were analyzed with the underlying genetic defects. A ring of increased autofluorescence was mainly observed in patients with CRX and GUCY2D mutations (33% and 22% of cases respectively). "Speckled" autofluorescence was observed with mutations in three different genes (ABCA4 64%; C2Orf71 and PRPH2, 18% each). Peripapillary sparing was only found in association with mutations in ABCA4, although only present in 40% of such genotypes. Regarding SD-OCT, specific outer retinal abnormalities were more commonly observed in particular genotypes: focal retrofoveal interruption and GUCY2D mutations (50%), foveal sparing and CRX mutations (50%), and outer retinal atrophy associated with hyperreflective dots and ABCA4 mutations (69%). This study outlines the phenotypic heterogeneity of COD/CORDs hampering statistical correlations. A larger study correlating retinal imaging with genetic results is necessary to identify specific clinical features that may help in selecting pathogenic variants generated by high-throughput sequencing.