Dark continuous noise from mutant G90D-rhodopsin predominantly underlies congenital stationary night blindness.

Congenital stationary night blindness (CSNB) is an inherited retinal disease that causes a profound loss of rod sensitivity without severe retinal degeneration. One well-studied rhodopsin point mutant, G90D-Rho, is thought to cause CSNB because of its constitutive activity in darkness causing rod desensitization. However, the nature of this constitutive activity and its precise molecular source have not been resolved for almost 30 y. In this study, we made a knock-in (KI) mouse line with a very low expression of G90D-Rho (equal in amount to ~0.1% of normal rhodopsin, WT-Rho, in WT rods), with the remaining WT-Rho replaced by REY-Rho, a mutant with a very low efficiency of activating transducin due to a charge reversal of the highly conserved ERY motif to REY. We observed two kinds of constitutive noise: one being spontaneous isomerization (R*) of G90D-Rho at a molecular rate (R* s-1) 175-fold higher than WT-Rho and the other being G90D-Rho-generated dark continuous noise comprising low-amplitude unitary events occurring at a very high molecular rate equivalent in effect to ~40,000-fold of R* s-1 from WT-Rho. Neither noise type originated from G90D-Opsin because exogenous 11-cis-retinal had no effect. Extrapolating the above observations at low (0.1%) expression of G90D-Rho to normal disease exhibited by a KI mouse model with RhoG90D/WTand RhoG90D/G90D genotypes predicts the disease condition very well quantitatively. Overall, the continuous noise from G90D-Rho therefore predominates, constituting the major equivalent background light causing rod desensitization in CSNB.

Clinical and genetic studies for a cohort of patients with congenital stationary night blindness.

BACKGROUND: Congenital stationary night blindness (CSNB) is an inherited retinal disorder. Most of patients have myopia. This study aims to describe the clinical and genetic characteristics of fifty-nine patients with CSNB and investigate myopic progression under genetic cause. RESULTS: Sixty-five variants were detected in the 59 CSNB patients, including 32 novel and 33 reported variants. The most frequently involved genes were NYX, CACNA1F, and TRPM1. Myopia (96.61%, 57/59) was the most common clinical finding, followed by nystagmus (62.71%, 37/59), strabismus (52.54%, 31/59), and nyctalopia (49.15%, 29/59). An average SE of -7.73 ± 3.37 D progressed to -9.14 ± 2.09 D in NYX patients with myopia, from - 2.24 ± 1.53 D to -4.42 ± 1.43 D in those with CACNA1F, and from - 5.21 ± 2.89 D to -9.24 ± 3.16 D in those with TRPM1 during the 3-year follow-up; the TRPM1 group showed the most rapid progression. CONCLUSIONS: High myopia and strabismus are distinct clinical features of CSNB that are helpful for diagnosis. The novel variants identified in this study will further expand the knowledge of variants in CSNB and help explore the molecular mechanisms of CSNB.

Unusual OCT findings in a patient with CABP4-associated cone-rod synaptic disorder.

PURPOSE: Bi-allelic variants in CABP4 are associated with congenital cone-rod synaptic disorder, which has also been classified, electrophysiologically, as incomplete congenital stationary night blindness (iCSNB). We describe clinical findings in a patient who demonstrated an unusual macular optical coherence tomography (OCT) phenotype, not previously reported in this condition. METHODS: Our patient underwent multimodal retinal imaging, international standard full-field ERG testing and whole genome sequencing. RESULTS: The patient was a 60-year-old woman with non-progressive visual impairment since birth, nystagmus and preference for dim lighting. Clinical fundus examination was unremarkable. OCT imaging revealed a hypo-reflective zone under an elevated fovea in both eyes. ERGs showed an electronegative DA10 response, with severely abnormal light-adapted responses. Whole genome sequencing revealed homozygosity for a known pathogenic variant in CABP4. No variants were found in other genes that could explain the patient's phenotype. CONCLUSIONS: OCT findings of foveal elevation and an underlying hypo-reflective zone are novel in this condition. Whilst the clinical history was similar to achromatopsia and other cone dysfunction syndromes, ERG findings suggested disease associated with CACNA1F or CABP4. As CACNA1F is X-linked, CABP4 was more likely, and confirmed on genetic testing. The patient saw better in dim light, confirming that night blindness is not a feature of CABP4-associated disease. Our case highlights the value of ERGs in discriminating between causes of cone dysfunction, and extends the range of retinal imaging phenotypes reported in this disorder.

A common cause for nystagmus in different congenital stationary night blindness mouse models.

In Nyxnob mice, a model for congenital nystagmus associated with congenital stationary night blindness (CSNB), synchronous oscillating retinal ganglion cells (RGCs) lead to oscillatory eye movements, i.e. nystagmus. Given the specific expression of mGluR6 and Cav 1.4 in the photoreceptor to bipolar cell synapses, as well as their clinical association with CSNB, we hypothesize that Grm6nob3 and Cav 1.4-KO mutants show, like the Nyxnob mouse, oscillations in both their RGC activity and eye movements. Using multi-electrode array recordings of RGCs and measurements of the eye movements, we demonstrate that Grm6nob3 and Cav 1.4-KO mice also show oscillations of their RGCs as well as a nystagmus. Interestingly, the preferred frequencies of RGC activity as well as the eye movement oscillations of the Grm6nob3 , Cav 1.4-KO and Nyxnob mice differ among mutants, but the neuronal activity and eye movement behaviour within a strain remain aligned in the same frequency domain. Model simulations indicate that mutations affecting the photoreceptor-bipolar cell synapse can form a common cause of the nystagmus of CSNB by driving oscillations in RGCs via AII amacrine cells. KEY POINTS: In Nyxnob mice, a model for congenital nystagmus associated with congenital stationary night blindness (CSNB), their oscillatory eye movements (i.e. nystagmus) are caused by synchronous oscillating retinal ganglion cells. Here we show that the same mechanism applies for two other CSNB mouse models - Grm6nob3 and Cav 1.4-KO mice. We propose that the retinal ganglion cell oscillations originate in the AII amacrine cells. Model simulations show that by only changing the input to ON-bipolar cells, all phenotypical differences between the various genetic mouse models can be reproduced.

Comprehensive Genotyping and Phenotyping Analysis of GUCY2D-Associated Rod- and Cone-Dominated Dystrophies.

PURPOSE: To describe the genetic and clinical spectrum of GUCY2D-associated retinopathies and to accurately establish their prevalence in a large cohort of patients. DESIGN: Retrospective case series. METHODS: Institutional study of 47 patients from 27 unrelated families with retinal dystrophies carrying disease-causing GUCY2D variants from the Fundación Jiménez Díaz hospital dataset of 8000 patients. Patients underwent ophthalmological examination and molecular testing by Sanger or exome sequencing approaches. Statistical and principal component analyses were performed to determine genotype-phenotype correlations. RESULTS: Four clinically different associated phenotypes were identified: 66.7% of families with cone/cone-rod dystrophy, 22.2% with Leber congenital amaurosis, 7.4% with early-onset retinitis pigmentosa, and 3.7% with congenital night blindness. Twenty-three disease-causing GUCY2D variants were identified, including 6 novel variants. Biallelic variants accounted for 28% of patients, whereas most carried dominant alleles associated with cone/cone-rod dystrophy. The disease onset had statistically significant differences according to the functional variant effect. Patients carrying GUCY2D variants were projected into 3 subgroups by allelic combination, disease onset, and presence of nystagmus or night blindness. In contrast to patients with the most severe phenotype of Leber congenital amaurosis, 7 patients with biallelic GUCY2D had a later and milder rod form with night blindness in infancy as the first symptom. CONCLUSIONS: This study represents the largest GUCY2D cohort in which 4 distinctly different phenotypes were identified, including rare intermediate presentations of rod-dominated retinopathies. We established that GUCY2D is linked to about 1% of approximately 3000 molecularly characterized families of our cohort. All of these findings are critical for defining cohorts for inclusion in future clinical trials.

Extended functional rescue following AAV gene therapy in a canine model of LRIT3-congenital stationary night blindness.

Congenital stationary night blindness (CSNB) is a group of inherited retinal diseases in which either rod-to-ON-bipolar cell (ON-BC) signaling, or rod function is affected leading to impaired vision under low light conditions. One type of CSNB is associated with defects in genes (NYX, GRM6, TRPM1, GPR179, and LRIT3) involved in the mGluR6 signaling cascade at the ON-BC dendritic tips. We have previously characterized a canine model of LRIT3-CSNB and demonstrated short-term safety and efficacy of an ON-BC targeting AAV-LRIT3 (AAVK9#4-shGRM6-cLRIT3-WPRE) gene therapy. Herein, we demonstrate long-term functional recovery and molecular restoration following subretinal injection of the ON-BC targeting AAV-LRIT3 vector in all eight treated eyes for up to 32 months. Following subretinal administration of the therapeutic vector, expression of the LRIT3 transgene, as well as restoration of mGluR6 signaling cascade member TRPM1, were confirmed in the outer plexiform layer (OPL) of the treated area. However, further investigation of the transgene LRIT3 transcript expression by RNA in situ hybridization (RNA-ISH) revealed off-target expression in non-BCs including the photoreceptors, inner nuclear, and ganglion cell layers, despite the use of a mutant AAVK9#4 capsid and an improved mGluR6 promoter designed to specifically transduce and promote expression in ON-BCs. While the long-term therapeutic potential of AAVK9#4-shGRM6-cLRIT3-WPRE is promising, we highlight the necessity for further optimization of AAV-LRIT3 therapy in the canine CSNB model prior to its clinical application.

A novel homozygous missense substitution p.Thr313Ile in the PDE6B gene underlies autosomal recessive retinitis pigmentosa in a consanguineous Pakistani family.

BACKGROUND: Retinitis pigmentosa (RP) is one of the most frequent hereditary retinal diseases that often starts with night blindness and eventually leads to legal blindness. Our study aimed to identify the underlying genetic cause of autosomal recessive retinitis pigmentosa (arRP) in a consanguineous Pakistani family. METHODS: Following a detailed ophthalmological examination of the patients by an ophthalmologist, whole-exome sequencing was performed on the proband's DNA to delineate the genetic cause of RP in the family. In-depth computational methods, in-silico analysis, and familial co-segregation study were performed for variant detection and validation. RESULTS: We studied an inbred Pakistani family with two siblings affected by retinitis pigmentosa. The proband, a 32 years old female, was clinically diagnosed with RP at the age of 6 years. A classical night blindness symptom was reported in the proband since her early childhood. OCT report showed a major reduction in the outer nuclear layer and the ellipsoid zone width, leading to the progression of the disease. Exome sequencing revealed a novel homozygous missense mutation (c.938C > T;p.Thr313Ile) in exon 12 of the PDE6B gene. The mutation p.Thr313Ile co-segregated with RP phenotype in the family. The altered residue (p.Thr313) was super conserved evolutionarily across different vertebrate species, and all available in silico tools classified the mutation as highly pathogenic. CONCLUSION: We present a novel homozygous pathogenic mutation in the PDE6B gene as the underlying cause of arRP in a consanguineous Pakistani family. Our findings highlight the importance of missense mutations in the PDE6B gene and expand the known mutational repertoire of PDE6B-related RP.

Shedding light on myopia by studying complete congenital stationary night blindness.

Myopia is the most common eye disorder, caused by heterogeneous genetic and environmental factors. Rare progressive and stationary inherited retinal disorders are often associated with high myopia. Genes implicated in myopia encode proteins involved in a variety of biological processes including eye morphogenesis, extracellular matrix organization, visual perception, circadian rhythms, and retinal signaling. Differentially expressed genes (DEGs) identified in animal models mimicking myopia are helpful in suggesting candidate genes implicated in human myopia. Complete congenital stationary night blindness (cCSNB) in humans and animal models represents an ON-bipolar cell signal transmission defect and is also associated with high myopia. Thus, it represents also an interesting model to identify myopia-related genes, as well as disease mechanisms. While the origin of night blindness is molecularly well established, further research is needed to elucidate the mechanisms of myopia development in subjects with cCSNB. Using whole transcriptome analysis on three different mouse models of cCSNB (in Gpr179-/-, Lrit3-/- and Grm6-/-), we identified novel actors of the retinal signaling cascade, which are also novel candidate genes for myopia. Meta-analysis of our transcriptomic data with published transcriptomic databases and genome-wide association studies from myopia cases led us to propose new biological/cellular processes/mechanisms potentially at the origin of myopia in cCSNB subjects. The results provide a foundation to guide the development of pharmacological myopia therapies.

Genetic analysis and clinical features of three Chinese patients with Oguchi disease.

BACKGROUND: Oguchi disease is a rare autosomal recessive form of congenital stationary night blindness caused by disease-causing variants in the rhodopsin kinase gene (GRK1) or the arrestin gene (SAG). Our study aims to describe the clinical features and identify the genetic defects for three Chinese patients with Oguchi disease. METHODS: We conducted detailed ophthalmologic examinations for three patients from three unrelated non-consanguineous Chinese families. Targeted next-generation sequencing (targeted NGS) and copy number variations (CNVs) analysis were applied to screen pathogenic variants. Sanger sequencing validation, quantitative real-time PCR (qPCR), and segregation analysis were further performed for confirmation. Subsequently, a combined genetic and structural biology approach was used to infer the likely functional consequences of novel variants. RESULTS: All three patients presented with typical clinical features of Oguchi disease, including night blindness, characteristic fundus appearance (Mizuo-Nakamura phenomenon), attenuated rod responses, and negative ERG waveforms. Their visual acuity and visual field were normal. Genetic analysis revealed two pathogenic variants in SAG and four pathogenic variants in GRK1. Patient 1 was identified to harbor compound heterozygous SAG variants c.874C > T (p.R292*) and exon2 deletion. Compound heterozygous GRK1 variants c.55C > T (p.R19*) and c.1412delC (p.P471Lfs*52) were found in patient 2. In patient 3, compound heterozygous GRK1 variants c.946C > A (p.R316S) and c.1388 T > C (p. L463P) were detected. CONCLUSIONS: We reported the first two Chinese Oguchi patients with novel GRK1 pathogenic variants (P471Lfs*52, R316S, L463P) and one Oguchi case with SAG, indicating both GRK1 and SAG are important causative genes in Chinese Oguchi patients.

Multimodal imaging in Schubert-Bornschein congenital stationary night blindness.

BACKGROUND: Schubert-Bornschein (SB) is the most common type of people with congenital stationary night blindness (CSNB). The aim of the study is to describe the optical coherence tomography (OCT) and optical coherence tomography angiography (OCTA) findings in patients with SB CSNB. METHODS: Prospective, observational case series including three patients with genetically confirmed CSNB along with matched controls, who underwent complete ophthalmic examination and multimodal imaging. RESULTS: On SD-OCT, a significant focal outer plexiform layer (OPL) thickening and a corresponding focal outer nuclear layer (ONL) thinning were identified in the macular area (p < 0.001). OCTA analysis overall showed decreased density of macular deep capillary plexus (mDCP) and macular choriocapillaris (mCC) (p = 0.008 and p = 0.033, respectively). DCP vessel density in the area corresponding to OPL thickening was significantly increased compared to the remaining retina (p < 0.001). CONCLUSION: SB CSNB is characterized by retinal vascular impairment, as detected on OCTA.

Optic nerve involvement in CACNA1F-related disease: observations from a multicentric case series.

BACKGROUND: Congenital Stationary Night Blindness (CSNB) constitutes a group of non-progressive retinal disorders characterized by disturbances in scotopic vision and/or by a delay in adaptation to darkness, as well as by low visual acuity, myopia, nystagmus, and strabismus. Color vision and fundus appearance tend to be normal. To date, several CACNA1F gene variants have been linked to a CSNB phenotype but only few reports have focused on the optic nerve in this disease. MATERIALS AND METHODS: Twelve patients underwent standard ophthalmological and genetic evaluation including spectral domain optical coherence tomography (SD-OCT), full-field electroretinography (ffERG), kinetic perimetry, fundus photography, magnetic resonance imaging (MRI), and next-generation sequencing (NGS). Bilateral thinning of the peripapillary nerve fiber layer (pRNFL) and the ganglion cell complex (GCC) supported involvement of the optic nerves. MRI, when available, was assessed for gross intracranial optic pathway abnormalities. RESULTS: All patients were shown to carry pathogenic variants in the CACNA1F gene, and all showed signs of optic nerve involvement. All patients showed a certain degree of myopic refractive error. Low average pRNFL thickness was evident in all patients. In three of them, pRNFL thickness was evaluated longitudinally and was proven to be stable over time. MRI imaging was unremarkable in all cases. CONCLUSION: Our data support the hypothesis that CACNA1F could be related to early-onset or congenital optic nerve involvement without any signs of a progressive optic neuropathy. Even though additional data from larger cohorts and longer follow-up periods are needed to further support and confirm our findings, there is a clear significance to our findings in the preparation for future CACNA1F gene therapy trials.

Congenital Stationary Night Blindness: Clinical and Genetic Features.

Congenital stationary night blindness (CSNB) is an inherited retinal disease (IRD) that causes night blindness in childhood with heterogeneous genetic, electrophysical, and clinical characteristics. The development of sequencing technologies and gene therapy have increased the ease and urgency of diagnosing IRDs. This study describes seven Taiwanese patients from six unrelated families examined at a tertiary referral center, diagnosed with CSNB, and confirmed by genetic testing. Complete ophthalmic exams included best corrected visual acuity, retinal imaging, and an electroretinogram. The effects of identified novel variants were predicted using clinical details, protein prediction tools, and conservation scores. One patient had an autosomal dominant CSNB with a RHO variant; five patients had complete CSNB with variants in GRM6, TRPM1, and NYX; and one patient had incomplete CSNB with variants in CACNA1F. The patients had Riggs and Schubert-Bornschein types of CSNB with autosomal dominant, autosomal recessive, and X-linked inheritance patterns. This is the first report of CSNB patients in Taiwan with confirmed genetic testing, providing novel perspectives on molecular etiology and genotype-phenotype correlation of CSNB. Particularly, variants in TRPM1, NYX, and CACNA1F in our patient cohort have not previously been described, although their clinical significance needs further study. Additional study is needed for the genotype-phenotype correlation of different mutations causing CSNB. In addition to genetic etiology, the future of gene therapy for CSNB patients is reviewed and discussed.

A Japanese boy with Bardet-Biedl syndrome caused by a novel homozygous variant in the ARL6 gene who was initially diagnosed with retinitis punctata albescens: A case report.

PURPOSE: Bardet-Biedl Syndrome (BBS) is an autosomal recessive systemic disorder characterized by retinitis pigmentosa, polydactyly, obesity, intellectual disability, renal impairments, and hypogonadism. The purpose of this study was to determine the ocular characteristics of a boy with BBS caused by a novel homozygous variant in the ARL6 (alternative named BBS3) gene who had been originally diagnosed with retinitis punctata albescens. METHODS: This was an observational case study. The patient underwent ophthalmological examinations, electroretinography, and genetic analyses using whole-exome sequencing. RESULTS: A 7-year-old boy was examined in our hospital with complaints of a progressive reduction of his visual acuity and night blindness in both eyes. There was no family history of eye diseases and no consanguineous marriage. Fundus examinations showed numerous white spots in the deep retina and retinal pigment epithelium. Fundus autofluorescence showed hypofluorescence consistent with these spots. Both the scotopic and photopic components of the full-field electroretinographies were non-detectable. Based on these clinical findings, this boy was suspected to have retinitis punctata albescens. Subsequent genetic testing using whole-exome sequencing revealed a novel homozygous variants in the ARL6/BBS3 gene (NM_001278293.3:c.528G>A, (p.Trp176Ter)). A systemic examination by the pediatric department revealed that this boy had a history of a surgical excision of polydactyly on his left foot when he was born, and that he was mildly obese. There were no prominent intellectual or gonadal dysfunctions, no craniofacial or dental abnormalities, no congenital heart disease, and no hearing impairment. He was then clinically and genetically diagnosed with BBS. CONCLUSION AND IMPORTANCE: In children with night blindness and progressive visual dysfunction, it is important for ophthalmologists to consult clinical geneticists and pediatricians to rule out the possibility of systemic diseases such as BBS.

Identification of a novel large multigene deletion and a frameshift indel in PDE6B as the underlying cause of early-onset recessive rod-cone degeneration.

A family, with two affected identical twins with early-onset recessive inherited retinal degeneration, was analyzed to determine the underlying genetic cause of pathology. Exome sequencing revealed a rare and previously reported causative variant (c.1923_1969delinsTCTGGG; p.Asn643Glyfs*29) in the PDE6B gene in the affected twins and their unaffected father. Further investigation, using genome sequencing, identified a novel ∼7.5-kb deletion (Chr 4:670,405-677,862del) encompassing the ATP5ME gene, part of the 5' UTR of MYL5, and a 378-bp (Chr 4:670,405-670,782) region from the 3' UTR of PDE6B in the affected twins and their unaffected mother. Both variants segregated with disease in the family. Analysis of the relative expression of PDE6B, in peripheral blood cells, also revealed a significantly lower level of PDE6B transcript in affected siblings compared to a normal control. PDE6B is associated with recessive rod-cone degeneration and autosomal dominant congenital stationary night blindness. Ophthalmic evaluation of these patients showed night blindness, fundus abnormalities, and peripheral vision loss, which are consistent with PDE6B-associated recessive retinal degeneration. These findings suggest that the loss of PDE6B transcript resulting from the compound heterozygous pathogenic variants is the underlying cause of recessive rod-cone degeneration in the study family.

Assessing the Pathogenicity of In-Frame CACNA1F Indel Variants Using Structural Modeling.

Small in-frame insertion-deletion (indel) variants are a common form of genomic variation whose impact on rare disease phenotypes has been understudied. The prediction of the pathogenicity of such variants remains challenging. X-linked incomplete congenital stationary night blindness type 2 (CSNB2) is a nonprogressive, inherited retinal disorder caused by variants in CACNA1F, encoding the Cav1.4α1 channel protein. Here, structural analysis was used through homology modeling to interpret 10 disease-correlated and 10 putatively benign CACNA1F in-frame indel variants. CSNB2-correlated changes were found to be more highly conserved compared with putative benign variants. Notably, all 10 disease-correlated variants but none of the benign changes were within modeled regions of the protein. Structural analysis revealed that disease-correlated variants are predicted to destabilize the structure and function of the Cav1.4α1 channel protein. Overall, the use of structural information to interpret the consequences of in-frame indel variants provides an important adjunct that can improve the diagnosis for individuals with CSNB2.

Association of Missense Variants in VSX2 With a Peculiar Form of Congenital Stationary Night Blindness Affecting All Bipolar Cells.

Importance: Congenital stationary night blindness (CSNB) is an inherited stationary retinal disorder that is clinically and genetically heterogeneous. To date, the genetic association between some cases with CSNB and an unusual complex clinical picture is unclear. Objective: To describe an unreported CSNB phenotype and the associated gene defect in 3 patients from 2 unrelated families. Design, Setting, and Participants: This retrospective case series was conducted in 2021 and 2022 at a national referral center for rare ocular diseases. Data for 3 patients from a cohort of 140 genetically unsolved CSNB cases were analyzed clinically and genetically. Exposures: Complete ocular examination including full-field electroretinography and multimodal fundus imaging (spectral-domain optical coherence tomography, color, infrared reflectance, and short-wavelength autofluorescence photographs) were performed. The gene defect was identified by exome sequencing and confirmed by Sanger sequencing and co-segregation analysis in 1 family. Screening was performed for genetically unsolved CSNB cases for VSX2 variants by direct Sanger sequencing. Main Outcomes and Measures: Ocular and molecular biology findings. Results: The series included 3 patients whose clinical investigations occurred at ages in the early 30s, younger than 12 years, and in the mid 40s. They had nystagmus, low stable visual acuity, and myopia from birth and experienced night blindness. Two older patients had bilateral lens luxation and underwent lens extraction. Full-field electroretinography revealed an electronegative Schubert-Bornschein appearance, combining characteristics of incomplete and complete CSNB, affecting the function of rod and cone ON- and OFF-bipolar cells. Exome sequencing and co-segregation analysis in a consanguineous family with 2 affected members identified a homozygous variant in VSX2. Subsequently, screening of the CSNB cohort identified another unrelated patient harboring a distinct VSX2 variant. Conclusions and Relevance: This case series revealed a peculiar pan-bipolar cell retinopathy with lens luxation associated with variants in VSX2. Clinicians should be aware of this association and VSX2 added to CSNB diagnostic gene panels.

Exome Sequencing Identified Molecular Determinants of Retinal Dystrophies in Nine Consanguineous Pakistani Families.

Inherited retinal dystrophies (IRDs) are a heterogeneous group of degenerative disorders of the retina. Retinitis Pigmentosa (RP) is a common type of IRD that causes night blindness and loss of peripheral vision and may progress to blindness. Mutations in more than 300 genes have been associated with syndromic and non-syndromic IRDs. Recessive forms are more frequent in populations where endogamy is a social preference, such as Pakistan. The aim of this study was to identify molecular determinants of IRDs with the common presentation of night blindness in consanguineous Pakistani families. This study included nine consanguineous IRD-affected families that presented autosomal recessive inheritance of the night blindness phenotype. DNA was extracted from blood samples. Targeted exome sequencing of 344 known genes for retinal dystrophies was performed. Screening of nine affected families revealed two novel (c.5571_5576delinsCTAGATand c.471dup in EYS and SPATA7 genes, respectively) and six reported pathogenic mutations (c.304C>A, c.187C>T, c.1560C>A, c.547C>T, c.109del and c.9911_11550del in PDE6A, USH2A, USH2A, NMNAT1, PAX6 and ALMS1 genes, respectively) segregating with disease phenotype in each respective family. Molecular determinants of hereditary retinal dystrophies were identified in all screened families. Identification of novel variants aid future diagnosis of retinal dystrophies and help to provide genetic counseling to affected families.

A clinical and electrophysiological case study of a child with a novel frame shift mutation in the CACNA1F and missense variation of RIMS1 genes.

PURPOSE: The purpose of this paper is to present a case study illustrating the importance of electrophysiological investigation in the diagnosis and serial monitoring of isolated congenital nystagmus. RESULTS: Serial electophysiological monitoring was undertaken in the male proband over a 9-year period commencing with initial assessment at 12 weeks of age: Skin electroretinograms (sERGs) were initially absent but subsequently revealed low-amplitude responses, electronegative morphologies and notched flicker responses suggestive of incomplete congenital stationary night blindness (CSNB2), but with an absent dark-adapted rod-specific response, while flash visual evoked potentials (fVEPs) demonstrated persistent crossed asymmetry, typical of albinoid misrouting of the optic nerves. Molecular investigation confirmed a novel hemizygous frame shift mutation in the CACNA1F gene, considered to be pathogenic and causative of X-linked CSNB2; additionally, a novel heterozygous missense variation in one copy of the RIMS1 gene was identified, pathogenic mutations of which underpin late-onset autosomal dominant cone-rod dystrophy (type 7). Segregation studies confirmed maternal inheritance of both mutations in the clinically asymptomatic mother in whom depressed rod-specific responses were confirmed on sERG. The child's visual acuity has remained stable as have the sERGs which have been verified by recordings using scleral electrodes. CONCLUSIONS: The importance of recording ERGs as part of evaluating infants who present with nystagmus, even with a normal fundus appearance, is supported. Further, sERGs were able to distinguish an apparent variant of CSNB2 and could give consistent results over many years. FVEP results add to the evidence that albinoid misrouting of the optic nerves may occur in cases of CSNB2. ERGs and fVEPs can provide valuable information in discriminating the relative diagnostic importance of multiple genetic abnormalities.

Unilateral cataract and congenital stationary night blindness in a child with novel variants in TRPM1.

Unilateral cataract can cause pediatric vision impairment. Although the majority of unilateral cataracts are idiopathic in nature, genetic causes have been reported. We present the case of a 4-week-old child of nonconsanguineous parents who was affected with unilateral cataract. Whole-genome sequencing using DNA extracted from blood and the lens epithelial cells following cataract surgery revealed two presumed pathogenic variants in the TRPM1 gene, the founding member of the melanoma-related transient receptor potential (TRPM) subfamily. TRPM1 is responsible for regulating cation influx to hyperpolarized retinal ON bipolar cells, and mutations in this gene are a major cause of autosomal recessive congenital stationary night blindness (CSNB). Electroretinography revealed findings consistent with CSNB, a phenotype that was not initially suspected, and which would likely have been missed without genome sequencing. It remains unclear whether the TRPM1 variants are associated with the cataract phenotype.

Two novel CACNA1F gene mutations cause two different phenotypes: Aland Eye Disease and incomplete Congenital Stationary Night Blindness.

Congenital Stationary Night Blindness type 2 (CSNB2) and Aland island Eye Disease (AIED) associated with CACNA1F mutation demonstrate a significant phenotype overlapping. We report two cases with different clinical presentation carrying two novel mutations in CACNA1F gene. Subjects underwent a complete neurophtahlmological examination associated with structural and electrofunctional insight. Next Generation Sequencing (NGS) analysis of 31 genes previously associated with retinal dystrophy (RD) was performed. Messenger RNAs derived from probands 'peripheral blood samples were analyzed by RT-PCR and cDNA sequencing. The neuro-ophthalmological examinations revealed different clinical, structural and morphological presentations, more severe in patient 1 compared with patient 2. Molecular analysis revealed, that both patients had the hemizygous form of two novel mutations in CACNA1F gene. Patient 1 presented a duplication (c.425dupC) in exon 4, resulting in shifting of the reading frame with the insertion of a premature Stop codon. In Patient 2 variant c.5156G > C localized in the donor's splicing site of exon 43 was identified. Complementary DNA sequencing demonstrated skipping of exon 43 with a deletion of 55 amino acids that causes a frame shift with insertion of a Stop codon. These findings suggest that the effect and the localization of the mutations in the CACNA1F gene can explain different clinical phenotypes. Clinical spectrum is more severe and resembles the AIED phenotype when the mutation affects the first part of the protein, while it is more similar to CSNB2 if the mutation is localized at the end of the protein. Genetic testing results to be an essential tool to provide more accurate diagnosis and prognosis in patients with inherited retinal degenerative disorders and could help, in the future, to develop more specific therapeutic strategies.