Molecular Mechanisms Governing Sight Loss in Inherited Cone Disorders.

Inherited cone disorders (ICDs) are a heterogeneous sub-group of inherited retinal disorders (IRDs), the leading cause of sight loss in children and working-age adults. ICDs result from the dysfunction of the cone photoreceptors in the macula and manifest as the loss of colour vision and reduced visual acuity. Currently, 37 genes are associated with varying forms of ICD; however, almost half of all patients receive no molecular diagnosis. This review will discuss the known ICD genes, their molecular function, and the diseases they cause, with a focus on the most common forms of ICDs, including achromatopsia, progressive cone dystrophies (CODs), and cone-rod dystrophies (CORDs). It will discuss the gene-specific therapies that have emerged in recent years in order to treat patients with some of the more common ICDs.

Clinical course of two siblings with potassium voltage-gated channel modifier subfamily V member 2 (KCNV2)-associated retinopathy.

BACKGROUND: KCNV2-associated retinopathy causes a phenotype reported as "cone dystrophy with nyctalopia and supernormal rod responses (CDSRR; OMIM# 610356)," featuring pathognomonic findings on electroretinography (ERG). Here, we report the clinical courses of two siblings with CDSRR. CASE REPORTS: Patient 1: A 3-year-old boy with intermittent exophoria was referred to our hospital. The patient's decimal best-corrected visual acuity (BCVA) at age 6 was 0.7 and 0.7 in the right and left eyes, respectively. Photophobia and night blindness were also observed. Because the ERG showed a delayed and supernormal b-wave with a "squaring (trough-flattened)" a-wave in the DA-30 ERG, and CDSRR was diagnosed. The patient's vision gradually worsened, and faint bilateral bull's eye maculopathy was observed at the age of 27 years, although the fundi were initially unremarkable. Genetic examination revealed a homozygous missense variant, c.529T > C (p.Cys177Arg), in the KCNV2 gene. Patient 2: The second patient was Patient 1's younger sister, who was brought to our hospital at 3 years of age. The patient presented with exotropia, mild nystagmus, photophobia, night blindness, and color vision abnormalities. The patients' decimal BCVA at age 13 was 0.6 and 0.4 in the right and left eyes, respectively, and BCVA gradually decreased until the age of 24 years. The fundi were unremarkable. The siblings had similar ERG findings and the same homozygous missense variant in the KCNV2 gene. CONCLUSIONS: The siblings had clinical findings typical of CDSRR. High-intense flash ERG is recommended for identifying pathognomonic "squaring" a-waves in patients with CDSRR.

Natural history and biomarkers of KCNV2-associated retinopathy.

BACKGROUND: KCNV2-associated retinopathy is an autosomal recessive inherited retinal disease classically named cone dystrophy with supernormal rod response (CDSRR). This study aims to identify the best biomarker for evaluating the condition. METHODS: A retrospective review of eight patients from seven families with genetically confirmed KCNV2-associated retinopathy was performed. The best corrected visual acuity (BCVA), full-field electroretinogram (ffERG), pattern ERG (pERG), fundus imaging: retinal photograph and fundus autofluorescence (FAF), and optical coherence tomography (OCT) were analysed. RESULTS: There was a disproportionate increase in b-wave amplitude with a relatively small light intensity increase, especially between the two dimmest stimuli of DA 0.002 and 0.01 (-2.7 and -2.0 log cd.s/m2). The a-wave amplitude was normal. The a-wave peak time was delayed in all stimuli. The b-wave peak time was delayed compared to normal, but the gap tightened as intensity increased. The b:a wave ratio was above or at the upper limit for the reference values. FAF bull's eye maculopathy pattern was prominent and variable foveal disruption on OCT was apparent in all patients. Legal blindness was reached before the age of 25. CONCLUSIONS: We identified three potential electrophysiology biomarkers to assist in evaluating future therapies: the disproportionate b-wave amplitude jump, delayed a-wave and b-wave peak time, and the higher than normal b:a wave ratio. Any of these biomarkers found with photoreceptor ellipsoid zone foveal-perifoveal disruption should prompt consideration for KCNV2 retinopathy. The BCVA natural history data suggests the probable optimum therapeutic window in the first three decades of life.

An early onset cone dystrophy due to CEP290 mutation: a case report.

PURPOSE: Biallelic mutations in the CEP290 gene cause early onset retinal dystrophy or syndromic disease such as Senior-Loken or Joubert syndrome. Here, we present an unusual non-syndromic case of a juvenile retinal dystrophy caused by biallelic CEP290 mutations imitating initially the phenotype of achromatopsia or slowly progressing cone dystrophy. METHODS: We present 13 years of follow-up of a female patient who presented first with symptoms and findings typical for achromatopsia. The patient underwent functional and morphologic examinations, including fundus autofluorescence imaging, spectral-domain optical coherence tomography, electroretinography, color vision and visual field testing. RESULTS: Diagnostic genetic testing via whole genome sequencing and virtual inherited retinal disease gene panel evaluation finally identified two compound heterozygous variants c.4452_4455del;p.(Lys1484Asnfs*4) and c.2414T > C;p.(Leu805Pro) in the CEP290 gene. CONCLUSIONS: CEP290 mutation causes a wide variety of clinical phenotypes. The presented case shows a phenotype resembling achromatopsia or early onset slowly progressing cone dystrophy.

A Novel GUCA1A Variant Associated with Cone Dystrophy Alters cGMP Signaling in Photoreceptors by Strongly Interacting with and Hyperactivating Retinal Guanylate Cyclase.

Guanylate cyclase-activating protein 1 (GCAP1), encoded by the GUCA1A gene, is a neuronal calcium sensor protein involved in shaping the photoresponse kinetics in cones and rods. GCAP1 accelerates or slows the cGMP synthesis operated by retinal guanylate cyclase (GC) based on the light-dependent levels of intracellular Ca2+, thereby ensuring a timely regulation of the phototransduction cascade. We found a novel variant of GUCA1A in a patient affected by autosomal dominant cone dystrophy (adCOD), leading to the Asn104His (N104H) amino acid substitution at the protein level. While biochemical analysis of the recombinant protein showed impaired Ca2+ sensitivity of the variant, structural properties investigated by circular dichroism and limited proteolysis excluded major structural rearrangements induced by the mutation. Analytical gel filtration profiles and dynamic light scattering were compatible with a dimeric protein both in the presence of Mg2+ alone and Mg2+ and Ca2+. Enzymatic assays showed that N104H-GCAP1 strongly interacts with the GC, with an affinity that doubles that of the WT. The doubled IC50 value of the novel variant (520 nM for N104H vs. 260 nM for the WT) is compatible with a constitutive activity of GC at physiological levels of Ca2+. The structural region at the interface with the GC may acquire enhanced flexibility under high Ca2+ conditions, as suggested by 2 µs molecular dynamics simulations. The altered interaction with GC would cause hyper-activity of the enzyme at both low and high Ca2+ levels, which would ultimately lead to toxic accumulation of cGMP and Ca2+ in the photoreceptor outer segment, thus triggering cell death.

Compound heterozygous KCNV2 variants contribute to cone dystrophy with supernormal rod responses in a Chinese family.

BACKGROUND: Cone dystrophy with supernormal rod response (CDSRR) is an autosomal recessive retinal disorder characterized by myopia, dyschromatopsia, nyctalopia, photophobia, and nystagmus. CDSRR is caused by mutations in KCNV2, the gene encoding for an electrically silent Kv subunit (Kvs) named Kv8.2. METHODS: A Chinese CDSRR family was recruited. Complete ophthalmology clinical examinations were performed to clarify the phenotype. Genetic examination was underwent using whole exome sequencing (WES). In addition, a candidate gene was validated by Sanger sequencing. Expression analysis in vitro including immunoblotting, quantitative real-time PCR (qRT-PCR), and co-immunoprecipitation experiments was performed to investigate the pathogenic mechanism of the identified gene variants. RESULTS: WES identified two KCNV2 heterozygous mutations from the proband. Sanger sequencing validated that the patient's parents had, respectively, carried those two mutations. Further in vitro functional experiments indicated that the mutated alleles had led the Kv8.2 proteins to fail in expressing and interacting with the Kv2.1 protein, respectively. CONCLUSIONS: This study expanded the KCNV2 mutation spectrum. It can also be deduced that CDSRR has a broad heterogeneity. It is further confirmed that the inability expression of Kv8.2 proteins and the failure of Kv8.2 proteins to interact with Kv2.1 may have accounted for the etiology of CDSRR based on previous studies and this study.

Disease mechanisms of X-linked cone dystrophy caused by missense mutations in the red and green cone opsins.

Cone photoreceptors are responsible for the visual acuity and color vision of the human eye. Red/green cone opsin missense mutations N94K, W177R, P307L, R330Q, and G338E have been identified in subjects with congenital blue cone monochromacy or color-vision deficiency. Studies on disease mechanisms due to these cone opsin mutations have been previously carried out exclusively in vitro, and the reported impairments were not always consistent. Here we expressed these mutants via AAV specifically in vivo in M-opsin knockout mouse cones to investigate their subcellular localization, the pathogenic effects on cone structure, function, and cone viability. We show that these mutations alter the M-opsin structure, function, and localization. N94K and W177R mutants appeared to be misfolded since they localized exclusively in cone inner segments and endoplasmic reticulum. In contrast, P307L, R330Q, and G338E mutants were detected predominately in cone outer segments. Expression of R330Q and G338E, but not P307L opsins, also partially restored expression and correct localization of cone PDE6α' and cone transducin γ and resulted in partial rescue of M-cone-mediated light responses. Expression of W177R and P307L mutants significantly reduced cone viability, whereas N94K, R330Q, and G338E were only modestly toxic. We propose that although the underlying biochemical and cellular defects caused by these mutants are distinct, they all seem to exhibit a dominant phenotype, resembling autosomal dominant retinitis pigmentosa associated with the majority of rhodopsin missense mutations. The understanding of the molecular mechanisms associated with these cone opsin mutants is fundamental to developing targeted therapies for cone dystrophy/dysfunction.

The role of voltage-gated ion channels in visual function and disease in mammalian photoreceptors.

Light activation of the classical light-sensing retinal neurons, the photoreceptors, results in a graded change in membrane potential that ultimately leads to a reduction in neurotransmitter release to the post-synaptic retinal neurons. Photoreceptors show striking powers of adaptation, and for visual processing to function optimally, they must adjust their gain to remain responsive to different levels of ambient light intensity. The presence of a tightly controlled balance of inward and outward currents modulated by several different types of ion channels is what gives photoreceptors their remarkably dynamic operating range. Part of the resetting and modulation of this operating range is controlled by potassium and calcium voltage-gated channels, which are involved in setting the dark resting potential and synapse signal processing, respectively. Their essential contribution to visual processing is further confirmed in patients suffering from cone dystrophy with supernormal rod response (CDSRR) and congenital stationary night blindness type 2 (CSNB2), both conditions that lead to irreversible vision loss. This review will discuss these two types of voltage-gated ion channels present in photoreceptors, focussing on their structure and physiology, and their role in visual processing. It will also discuss the use and benefits of knockout mouse models to further study the function of these channels and what routes to potential treatments could be applied for CDSRR and CSNB2.

Clinical Characteristics of POC1B-Associated Retinopathy and Assignment of Pathogenicity to Novel Deep Intronic and Non-Canonical Splice Site Variants.

Mutations in POC1B are a rare cause of inherited retinal degeneration. In this study, we present a thorough phenotypic and genotypic characterization of three individuals harboring putatively pathogenic variants in the POC1B gene. All patients displayed a similar, slowly progressive retinopathy (cone dystrophy or cone-rod dystrophy) with normal funduscopy but disrupted outer retinal layers on optical coherence tomography and variable age of onset. Other symptoms were decreased visual acuity and photophobia. Whole genome sequencing revealed a novel homozygous frameshift variant in one patient. Another patient was shown to harbor a novel deep intronic variant in compound heterozygous state with a previously reported canonical splice site variant. The third patient showed a novel nonsense variant and a novel non-canonical splice site variant. We aimed to validate the effect of the deep intronic variant and the non-canonical splice site variant by means of in vitro splice assays. In addition, direct RNA analysis was performed in one patient. Splicing analysis revealed that the non-canonical splice site variant c.561-3T>C leads to exon skipping while the novel deep intronic variant c.1033-327T>A causes pseudoexon activation. Our data expand the genetic landscape of POC1B mutations and confirm the benefit of genome sequencing in combination with downstream functional validation using minigene assays for the analysis of putative splice variants. In addition, we provide clinical multimodal phenotyping of the affected individuals.

Cone Dystrophy Associated with a Novel Variant in the Terminal Codon of the RPGR-ORF15.

Mutations in RPGRORF15 are associated with rod-cone or cone/cone-rod dystrophy, the latter associated with mutations at the distal end. We describe the phenotype associated with a novel variant in the terminal codon of the RPGRORF15 c.3457T>A (Ter1153Lysext*38), which results in a C-terminal extension. Three male patients from two families were recruited, aged 31, 35, and 38 years. Genetic testing was performed by whole exome sequencing. Filtered variants were analysed according to the population frequency, ClinVar database, the variant's putative impact, and predicted pathogenicity; and were classified according to the ACMG guidelines. Examination included visual acuity (Snellen), colour vision (Ishihara), visual field, fundus autofluorescence (FAF), optical coherence tomography (OCT), and electrophysiology. All patients were myopic, and had central scotoma and reduced colour vision. Visual acuities on better eyes were counting fingers, 0.3 and 0.05. Electrophysiology showed severely reduced cone-specific responses and macular dysfunction, while the rod-specific response was normal. FAF showed hyperautofluorescent ring centred at the fovea encompassing an area of photoreceptor loss approximately two optic discs in diameter (3462-6342 µm). Follow up after 2-11 years showed enlargement of the diameter (avg. 100 µm/year). The novel c.3457T>A (Ter1153Lysext*38) mutation in the terminal RPGRORF15 codon is associated with cone dystrophy, which corresponds to the previously described phenotypes associated with mutations in the distal end of the RPGRORF15. Minimal progression during follow-up years suggests a relatively stable disease after the initial loss of the central cones.

Impaired Ca2+ Sensitivity of a Novel GCAP1 Variant Causes Cone Dystrophy and Leads to Abnormal Synaptic Transmission Between Photoreceptors and Bipolar Cells.

Guanylate cyclase-activating protein 1 (GCAP1) is involved in the shutdown of the phototransduction cascade by regulating the enzymatic activity of retinal guanylate cyclase via a Ca2+/cGMP negative feedback. While the phototransduction-associated role of GCAP1 in the photoreceptor outer segment is widely established, its implication in synaptic transmission to downstream neurons remains to be clarified. Here, we present clinical and biochemical data on a novel isolate GCAP1 variant leading to a double amino acid substitution (p.N104K and p.G105R) and associated with cone dystrophy (COD) with an unusual phenotype. Severe alterations of the electroretinogram were observed under both scotopic and photopic conditions, with a negative pattern and abnormally attenuated b-wave component. The biochemical and biophysical analysis of the heterologously expressed N104K-G105R variant corroborated by molecular dynamics simulations highlighted a severely compromised Ca2+-sensitivity, accompanied by minor structural and stability alterations. Such differences reflected on the dysregulation of both guanylate cyclase isoforms (RetGC1 and RetGC2), resulting in the constitutive activation of both enzymes at physiological levels of Ca2+. As observed with other GCAP1-associated COD, perturbation of the homeostasis of Ca2+ and cGMP may lead to the toxic accumulation of second messengers, ultimately triggering cell death. However, the abnormal electroretinogram recorded in this patient also suggested that the dysregulation of the GCAP1-cyclase complex further propagates to the synaptic terminal, thereby altering the ON-pathway related to the b-wave generation. In conclusion, the pathological phenotype may rise from a combination of second messengers' accumulation and dysfunctional synaptic communication with bipolar cells, whose molecular mechanisms remain to be clarified.

A duplication on chromosome 16q12 affecting the IRXB gene cluster is associated with autosomal dominant cone dystrophy with early tritanopic color vision defect.

Cone dystrophies are a rare subgroup of inherited retinal dystrophies and hallmarked by color vision defects, low or decreasing visual acuity and central vision loss, nystagmus and photophobia. Applying genome-wide linkage analysis and array comparative genome hybridization, we identified a locus for autosomal dominant cone dystrophy on chromosome 16q12 in four independent multigeneration families. The locus is defined by duplications of variable size with a smallest region of overlap of 608 kb affecting the IRXB gene cluster and encompasses the genes IRX5 and IRX6. IRX5 and IRX6 belong to the Iroquois (Iro) protein family of homeodomain-containing transcription factors involved in patterning and regionalization of embryonic tissue in vertebrates, including the eye and the retina. All patients presented with a unique progressive cone dystrophy phenotype hallmarked by early tritanopic color vision defects. We propose that the disease underlies a misregulation of the IRXB gene cluster on chromosome 16q12 and demonstrate that overexpression of Irx5a and Irx6a, the two orthologous genes in zebrafish, results in visual impairment in 5-day-old zebrafish larvae.

PDE6C: Novel Mutations, Atypical Phenotype, and Differences Among Children and Adults.

Purpose: Genetic variation in PDE6C is associated with achromatopsia and cone dystrophy, with only a few reports of cone-rod dystrophy in the literature. We describe two pediatric and two adult patients with PDE6C related cone and cone-rod dystrophy and the first longitudinal data of a pediatric patient with PDE6C-related cone dystrophy. Methods: This cohort of four patients underwent comprehensive ophthalmologic evaluation at the National Eye Institute's Ophthalmic Genetics clinic, including visual field testing, retinal imaging and electroretinogram (ERG). Next-generation sequencing-based genetic testing was performed and subsequent analysis of the variants was done through three-dimensional protein models generated by Phyre2 and Chimera. Results: All cases shared decreased best-corrected visual acuity and poor color discrimination. Three of the four patients had a cone-rod dystrophy, presenting with an ERG showing decreased amplitude on both photopic and scotopic waveforms and a mild to moderately constricted visual field. One of the children was diagnosed with cone dystrophy, having a preserved peripheral field. The children had none to minor structural retinal changes, whereas the adults had clear macular dystrophy. Conclusions: PDE6C-related cone-rod dystrophy consists of a severe phenotype characterized by early-onset nystagmus, decreased best-corrected visual acuity, poor color discrimination, progressive constriction of the visual field, and night blindness. Our work contributes with valuable information toward understanding the visual prognosis and allelic heterogeneity of PDE6C-related cone and cone-rod dystrophy.

Yap haploinsufficiency leads to Müller cell dysfunction and late-onset cone dystrophy.

Hippo signalling regulates eye growth during embryogenesis through its effectors YAP and TAZ. Taking advantage of a Yap heterozygous mouse line, we here sought to examine its function in adult neural retina, where YAP expression is restricted to Müller glia. We first discovered an unexpected temporal dynamic of gene compensation. At postnatal stages, Taz upregulation occurs, leading to a gain of function-like phenotype characterised by EGFR signalling potentiation and delayed cell-cycle exit of retinal progenitors. In contrast, Yap+/- adult retinas no longer exhibit TAZ-dependent dosage compensation. In this context, Yap haploinsufficiency in aged individuals results in Müller glia dysfunction, late-onset cone degeneration, and reduced cone-mediated visual response. Alteration of glial homeostasis and altered patterns of cone opsins were also observed in Müller cell-specific conditional Yap-knockout aged mice. Together, this study highlights a novel YAP function in Müller cells for the maintenance of retinal tissue homeostasis and the preservation of cone integrity. It also suggests that YAP haploinsufficiency should be considered and explored as a cause of cone dystrophies in human.

Constitutive Activation of Guanylate Cyclase by the G86R GCAP1 Variant Is Due to "Locking" Cation-π Interactions that Impair the Activator-to-Inhibitor Structural Transition.

Guanylate Cyclase activating protein 1 (GCAP1) mediates the Ca2+-dependent regulation of the retinal Guanylate Cyclase (GC) in photoreceptors, acting as a target inhibitor at high [Ca2+] and as an activator at low [Ca2+]. Recently, a novel missense mutation (G86R) was found in GUCA1A, the gene encoding for GCAP1, in patients diagnosed with cone-rod dystrophy. The G86R substitution was found to affect the flexibility of the hinge region connecting the N- and C-domains of GCAP1, resulting in decreased Ca2+-sensitivity and abnormally enhanced affinity for GC. Based on a structural model of GCAP1, here, we tested the hypothesis of a cation-π interaction between the positively charged R86 and the aromatic W94 as the main mechanism underlying the impaired activator-to-inhibitor conformational change. W94 was mutated to F or L, thus, resulting in the double mutants G86R+W94L/F. The double mutants showed minor structural and stability changes with respect to the single G86R mutant, as well as lower affinity for both Mg2+ and Ca2+, moreover, substitutions of W94 abolished "phase II" in Ca2+-titrations followed by intrinsic fluorescence. Interestingly, the presence of an aromatic residue in position 94 significantly increased the aggregation propensity of Ca2+-loaded GCAP1 variants. Finally, atomistic simulations of all GCAP1 variants in the presence of Ca2+ supported the presence of two cation-π interactions involving R86, which was found to act as a bridge between W94 and W21, thus, locking the hinge region in an activator-like conformation and resulting in the constitutive activation of the target under physiological conditions.

Characterization of GUCA1A-associated dominant cone/cone-rod dystrophy: low prevalence among Japanese patients with inherited retinal dystrophies.

GUCA1A gene variants are associated with autosomal dominant (AD) cone dystrophy (COD) and cone-rod dystrophy (CORD). GUCA1A-associated AD-COD/CORD has never been reported in the Japanese population. The purpose of this study was to investigate clinical and genetic features of GUCA1A-associated AD-COD/CORD from a large Japanese cohort. We identified 8 variants [c.C50_80del (p.E17VfsX22), c.T124A (p.F42I), c.C204G (p.D68E), c.C238A (p.L80I), c.T295A (p.Y99N), c.A296C (p.Y99S), c.C451T (p.L151F), and c.A551G (p.Q184R)] in 14 families from our whole exome sequencing database composed of 1385 patients with inherited retinal diseases (IRDs) from 1192 families. Three variants (p.Y99N, p.Y99S, and p.L151F), which are located on/around EF-hand domains 3 and 4, were confirmed as "pathogenic", whereas the other five variants, which did not co-segregate with IRDs, were considered "non-pathogenic". Ophthalmic findings of 9 patients from 3 families with the pathogenic variants showed central visual impairment from early to middle-age onset and progressive macular atrophy. Electroretinography revealed severely decreased or non-recordable cone responses, whereas rod responses were highly variable, ranging from nearly normal to non-recordable. Our results indicate that the three pathogenic variants, two of which were novel, underlie AD-COD/CORD with progressive retinal atrophy, and the prevalence (0.25%, 3/1192 families) of GUCA1A-associated IRDs may be low among Japanese patients.

Phenotypical Characteristics of POC1B-Associated Retinopathy in Japanese Cohort: Cone Dystrophy With Normal Funduscopic Appearance.

Purpose: Cone/cone-rod dystrophy is a large group of retinal disorders with both phonotypic and genetic heterogeneity. The purpose of this study was to characterize the phenotype of eight patients from seven families harboring POC1B mutations in a cohort of the Japan Eye Genetics Consortium (JEGC). Methods: Whole-exome sequencing with targeted analyses identified homozygous or compound heterozygous mutations of the POC1B gene in 7 of 548 families in the JEGC database. Ophthalmologic examinations including the best-corrected visual acuity, perimetry, fundus photography, fundus autofluorescence imaging, optical coherence tomography, and full-field and multifocal electroretinography (ERGs) were performed. Results: There were four men and four women whose median age at the onset of symptoms was 15.6 years (range, 6-23 years) and that at the time of examination was 40.3 years (range, 22-67 years). The best-corrected visual acuity ranged from -0.08 to 1.52 logMAR units. The funduscopic appearance was normal in all the cases except in one case with faint mottling in the fovea. Optical coherence tomography revealed an absence of the interdigitation zone and blurred ellipsoid zone in the posterior pole, but the foveal structures were preserved in three cases. The full-field photopic ERGs were reduced or extinguished with normal scotopic responses. The central responses of the multifocal ERGs were preserved in two cases. The diagnosis was either generalized cone dystrophy in five cases or cone dystrophy with foveal sparing in three cases. Conclusions: Generalized or peripheral cone dystrophy with normal funduscopic appearance is the representative phenotype of POC1B-associated retinopathy in our cohort.

The identification of a RNA splice variant in TULP1 in two siblings with early-onset photoreceptor dystrophy.

BACKGROUND: Early-onset photoreceptor dystrophies are a major cause of irreversible visual impairment in children and young adults. This clinically heterogeneous group of disorders can be caused by mutations in many genes. Nevertheless, to date, 30%-40% of cases remain genetically unexplained. In view of expanding therapeutic options, it is essential to obtain a molecular diagnosis in these patients as well. In this study, we aimed to identify the genetic cause in two siblings with genetically unexplained retinal disease. METHODS: Whole exome sequencing was performed to identify the causative variants in two siblings in whom a single pathogenic variant in TULP1 was found previously. Patients were clinically evaluated, including assessment of the medical history, slit-lamp biomicroscopy, and ophthalmoscopy. In addition, a functional analysis of the putative splice variant in TULP1 was performed using a midigene assay. RESULTS: Clinical assessment showed a typical early-onset photoreceptor dystrophy in both the patients. Whole exome sequencing identified two pathogenic variants in TULP1, a c.1445G>A (p.(Arg482Gln)) missense mutation and an intronic c.718+23G>A variant. Segregation analysis confirmed that both siblings were compound heterozygous for the TULP1 c.718+23G>A and c.1445G>A variants, while the unaffected parents were heterozygous. The midigene assay for the c.718+23G>A variant confirmed an elongation of exon 7 leading to a frameshift. CONCLUSION: Here, we report the first near-exon RNA splice variant that is not present in a consensus splice site sequence in TULP1, which was found in a compound heterozygous manner with a previously described pathogenic TULP1 variant in two patients with an early-onset photoreceptor dystrophy. We provide proof of pathogenicity for this splice variant by performing an in vitro midigene splice assay, and highlight the importance of analysis of noncoding regions beyond the noncanonical splice sites in patients with inherited retinal diseases.

A nonhuman primate model of inherited retinal disease.

Inherited retinal degenerations are a common cause of untreatable blindness worldwide, with retinitis pigmentosa and cone dystrophy affecting approximately 1 in 3500 and 1 in 10,000 individuals, respectively. A major limitation to the development of effective therapies is the lack of availability of animal models that fully replicate the human condition. Particularly for cone disorders, rodent, canine, and feline models with no true macula have substantive limitations. By contrast, the cone-rich macula of a nonhuman primate (NHP) closely mirrors that of the human retina. Consequently, well-defined NHP models of heritable retinal diseases, particularly cone disorders that are predictive of human conditions, are necessary to more efficiently advance new therapies for patients. We have identified 4 related NHPs at the California National Primate Research Center with visual impairment and findings from clinical ophthalmic examination, advanced retinal imaging, and electrophysiology consistent with achromatopsia. Genetic sequencing confirmed a homozygous R565Q missense mutation in the catalytic domain of PDE6C, a cone-specific phototransduction enzyme associated with achromatopsia in humans. Biochemical studies demonstrate that the mutant mRNA is translated into a stable protein that displays normal cellular localization but is unable to hydrolyze cyclic GMP (cGMP). This NHP model of a cone disorder will not only serve as a therapeutic testing ground for achromatopsia gene replacement, but also for optimization of gene editing in the macula and of cone cell replacement in general.