Systematic assessment of the contribution of structural variants to inherited retinal diseases.

Despite increasing success in determining genetic diagnosis for patients with inherited retinal diseases (IRDs), mutations in about 30% of the IRD cases remain unclear or unsettled after targeted gene panel or whole exome sequencing. In this study, we aimed to investigate the contributions of structural variants (SVs) to settling the molecular diagnosis of IRD with whole-genome sequencing (WGS). A cohort of 755 IRD patients whose pathogenic mutations remain undefined were subjected to WGS. Four SV calling algorithms including include MANTA, DELLY, LUMPY and CNVnator were used to detect SVs throughout the genome. All SVs identified by any one of these four algorithms were included for further analysis. AnnotSV was used to annotate these SVs. SVs that overlap with known IRD-associated genes were examined with sequencing coverage, junction reads and discordant read pairs. Polymerase Chain Reaction (PCR) followed by Sanger sequencing was used to further confirm the SVs and identify the breakpoints. Segregation of the candidate pathogenic alleles with the disease was performed when possible. A total of 16 candidate pathogenic SVs were identified in 16 families, including deletions and inversions, representing 2.1% of patients with previously unsolved IRDs. Autosomal dominant, autosomal recessive and X-linked inheritance of disease-causing SVs were observed in 12 different genes. Among these, SVs in CLN3, EYS and PRPF31 were found in multiple families. Our study suggests that the contribution of SVs detected by short-read WGS is about 0.25% of our IRD patient cohort and is significantly lower than that of single nucleotide changes and small insertions and deletions.

Cwc27, associated with retinal degeneration, functions as a splicing factor in vivo.

Previous in vitro studies indicate that CWC27 functions as a splicing factor in the Bact spliceosome complex, interacting with CWC22 to form a landing platform for eIF4A3, a core component of the exon junction complex. However, the function of CWC27 as a splicing factor has not been validated in any in vivo systems. CWC27 variants have been shown to cause autosomal recessive retinal degeneration, in both syndromic and non-syndromic forms. The Cwc27K338fs/K338fs mouse model was shown to have significant retinal dysfunction and degeneration by 6 months of age. In this report, we have taken advantage of the Cwc27K338fs/K338fs mouse model to show that Cwc27 is involved in splicing in vivo in the context of the retina. Bulk RNA and single cell RNA-sequencing of the mouse retina showed that there were gene expression and splicing pattern changes, including alternative splice site usage and intron retention. Positive staining for CHOP suggests that ER stress may be activated in response to the splicing pattern changes and is a likely contributor to the disease mechanism. Our results provide the first evidence that CWC27 functions as a splicing factor in an in vivo context. The splicing defects and gene expression changes observed in the Cwc27K338fs/K338fs mouse retina provide insight to the potential disease mechanisms, paving the way for targeted therapeutic development.

The neural representation of metacognition in preferential decision-making.

Humans regularly assess the quality of their judgements, which helps them adjust their behaviours. Metacognition is the ability to accurately evaluate one's own judgements, and it is assessed by comparing objective task performance with subjective confidence report in perceptual decisions. However, for preferential decisions, assessing metacognition in preference-based decisions is difficult because it depends on subjective goals rather than the objective criterion. Here, we develop a new index that integrates choice, reaction time, and confidence report to quantify trial-by-trial metacognitive sensitivity in preference judgements. We found that the dorsomedial prefrontal cortex (dmPFC) and the right anterior insular were more activated when participants made bad metacognitive evaluations. Our study suggests a crucial role of the dmPFC-insula network in representing online metacognitive sensitivity in preferential decisions.

Transcript isoforms of Reep6 have distinct functions in the retina.

Much of the complexity of the eukaryotic cell transcriptome is due to the alternative splicing of mRNA. However, knowledge on how transcriptome complexity is translated into functional complexity remains limited. For example, although different isoforms of a gene may show distinct temporal and spatial expression patterns, it is largely unknown whether these isoforms encode proteins with distinct functions matching their expression pattern. In this report, we investigated the function and relationship of the two isoforms of Reep6, namely Reep6.1 and Reep6.2, in rod photoreceptor cells. These two isoforms result from the alternative splicing of exon 5 and show mutually exclusive expression patterns. Reep6.2 is the canonical isoform that is expressed in non-retinal tissues, whereas Reep6.1 is the only expressed isoform in the adult retina. The Reep6.1 isoform-specific knockout mouse, Reep6E5/E5, is generated by deleting exon 5 and a homozygous deletion phenotypically displayed a rod degeneration phenotype comparable to a Reep6 full knockout mouse, indicating that the Reep6.1 isoform is essential for the rod photoreceptor cell survival. Consistent with the results obtained from a loss-of-function experiment, overexpression of Reep6.2 failed to rescue the rod degeneration phenotype of Reep6 knockout mice whereas overexpression of Reep6.1 does lead to rescue. These results demonstrate that, consistent with the expression pattern of the isoform, Reep6.1 has rod-specific functions that cannot be substituted by its canonical isoform. Our findings suggested that a strict regulation of splicing is required for the maintenance of photoreceptor cells.

Gene augmentation therapy to rescue degenerative photoreceptors in a Cwc27 mutant mouse model.

Previous reports have demonstrated that defects in the spliceosome-associated protein CWC27 can lead to the degeneration of retinal cells in Cwc27 mutant mouse models. However, it is unknown whether gene replacement therapy can rescue this phenotype. The purpose of this study was to evaluate whether AAV based gene therapy could rescue the retinal degeneration observed in Cwc27 mutant mice. By 6 months of age, Cwc27 mutant mice show a retinal degenerative phenotype, including morphological and functional abnormalities, primarily driven by the death of photoreceptors. We hypothesize that subretinal injection of AAV8 to drive exogenous CWC27 protein expression will improve the retinal phenotype. We evaluated these improvements after gene therapy with electroretinography (ERG) and histology, either hematoxylin and eosin (H&E) or immunostaining. In this study, we demonstrated that subretinal injection of AAV8-GRK-Cwc27-FLAG in mutant mice can improve the functionality and morphology of the retina. Immunostaining analyses revealed a notable decrease in photoreceptor degeneration, including cone cell degeneration, in the AAV-injected eyes compared to the PBS-injected eyes. Based on these results, gene replacement therapy could be a promising method for treating retinal degeneration caused by mutations in Cwc27.

Transdermal Delivery of a Hydrogen Sulphide Donor, ADT-OH Using Aqueous Gel Formulations for the Treatment of Impaired Vascular Function: an Ex Vivo Study.

PURPOSE: Hydrogen sulphide (H2S) is an important signalling molecule involved in the regulation of several physiological and pathophysiological processes. The objective of this study was to investigate the feasibility of transdermal delivery of ADT-OH, a H2S donor, by investigating the transdermal flux of aqueous gels loaded with penetration enhancers or liposomes. Furthermore, we explored the ability of permeated ADT-OH to promote angiogenesis and mitochondrial bioenergetics in HUVEC cells. METHODS: Aqueous hypromellose gels (5% w/v) were prepared with up to 10% v/v propylene glycol (PG) or deformable liposomes with 0.025% w/w ADT-OH. ADT-OH permeation from formulations across excised murine skin into PBS was quantified over 24 h using HPLC-UV detection. Media was collected and applied to HUVEC cells to evidence ADT-OH functionality following permeation. Tube formation assays were performed as indicative of angiogenesis and mitochondrial oxygen consumption was evaluated using a Seahorse XF24. RESULTS: Increasing the loading of PG caused an increase in ADT-OH permeation rate across skin and a decrease in dermal drug retention whereas liposomal gels produced a slow-release profile. Treatment of HUVEC's using conditioned media collected from the ADT-OH loaded permeation studies enhanced tube formation and the basal oxygen consumption rates after 30 min of treatment. CONCLUSIONS: These findings demonstrate that transdermal delivery of ADT-OH may provide a promising approach in the treatment of impaired vascular function. Gels prepared with 10% v/v PG have the potential for use in conditions requiring rapid H2S release whereas liposomal loaded gels for treatment requiring sustained H2S release.

Placental mitochondrial function as a driver of angiogenesis and placental dysfunction.

The placenta is a highly vascularized and complex foetal organ that performs various tasks, crucial to a healthy pregnancy. Its dysfunction leads to complications such as stillbirth, preeclampsia, and intrauterine growth restriction. The specific cause of placental dysfunction remains unknown. Recently, the role of mitochondrial function and mitochondrial adaptations in the context of angiogenesis and placental dysfunction is getting more attention. The required energy for placental remodelling, nutrient transport, hormone synthesis, and the reactive oxygen species leads to oxidative stress, stemming from mitochondria. Mitochondria adapt to environmental changes and have been shown to adjust their oxygen and nutrient use to best support placental angiogenesis and foetal development. Angiogenesis is the process by which blood vessels form and is essential for the delivery of nutrients to the body. This process is regulated by different factors, pro-angiogenic factors and anti-angiogenic factors, such as sFlt-1. Increased circulating sFlt-1 levels have been linked to different preeclamptic phenotypes. One of many effects of increased sFlt-1 levels, is the dysregulation of mitochondrial function. This review covers mitochondrial adaptations during placentation, the importance of the anti-angiogenic factor sFlt-1in placental dysfunction and its role in the dysregulation of mitochondrial function.

Noncoding mutation in RPGRIP1 contributes to inherited retinal degenerations.

Purpose: Despite the extensive use of next-generation sequencing (NGS) technology to identify disease-causing genomic variations, a major gap in our understanding of Mendelian diseases is the unidentified molecular lesion in a significant portion of patients. For inherited retinal degenerations (IRDs), although currently close to 300 disease-associated genes have been identified, the mutations in approximately one-third of patients remain unknown. With mounting evidence that noncoding mutations might contribute significantly to disease burden, we aimed to systematically investigate the contributions of noncoding regions in the genome to IRDs. Methods: In this study, we focused on RPGRIP1, which has been linked to various IRD phenotypes, including Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), and macular dystrophy (MD). As several noncoding mutant alleles have been reported in RPGRIP1, and we observed that the mutation carrier frequency of RPGRIP1 is higher in patient cohorts with unsolved IRDs, we hypothesized that mutations in the noncoding regions of RPGRIP1 might be a significant contributor to pathogenicity. To test this hypothesis, we performed whole-genome sequencing (WGS) for 25 patients with unassigned IRD who carry a single mutation in RPGRIP1. Results: Three noncoding variants in RPGRIP1, including a 2,890 bp deletion and two deep-intronic variants (c.2710+233G>A and c.1468-263G>C), were identified as putative second hits of RPGRIP1 in three patients with LCA. The mutant alleles were validated with direct sequencing or in vitro assays. Conclusions: The results highlight the significance of the contribution of noncoding pathogenic variants to unsolved IRD cases.

Angiogenic Properties of Placenta-Derived Extracellular Vesicles in Normal Pregnancy and in Preeclampsia.

Angiogenesis is one of the main processes that coordinate the biological events leading to a successful pregnancy, and its imbalance characterizes several pregnancy-related diseases, including preeclampsia. Intracellular interactions via extracellular vesicles (EVs) contribute to pregnancy's physiology and pathophysiology, and to the fetal-maternal interaction. The present review outlines the implications of EV-mediated crosstalk in the angiogenic process in healthy pregnancy and its dysregulation in preeclampsia. In particular, the effect of EVs derived from gestational tissues in pro and anti-angiogenic processes in the physiological and pathological setting is described. Moreover, the application of EVs from placental stem cells in the clinical setting is reported.

Integrative genomic analysis reveals novel regulatory mechanisms of eyeless during Drosophila eye development.

Eyeless (ey) is one of the most critical transcription factors for initiating the entire eye development in Drosophila. However, the molecular mechanisms through which Ey regulates target genes and pathways have not been characterized at the genomic level. Using ChIP-Seq, we generated an endogenous Ey-binding profile in Drosophila developing eyes. We found that Ey binding occurred more frequently at promoter compared to non-promoter regions. Ey promoter binding was correlated with the active transcription of genes involved in development and transcription regulation. An integrative analysis revealed that Ey directly regulated a broad and highly connected genetic network, including many essential patterning pathways, and known and novel eye genes. Interestingly, we observed that Ey could target multiple components of the same pathway, which might enhance its control of these pathways during eye development. In addition to protein-coding genes, we discovered Ey also targeted non-coding RNAs, which represents a new regulatory mechanism employed by Ey. These findings suggest that Ey could use multiple molecular mechanisms to regulate target gene expression and pathway function, which might enable Ey to exhibit a greater flexibility in controlling different processes during eye development.

A Dipeptide-Based Hierarchical Nanoarchitecture with Enhanced Catalytic Activity.

Achieving synthetic architectures with simple structures and robust biomimetic catalytic activities remains a great challenge. Herein, we explore a facile supramolecular assembly approach to construct a dipeptide-based hierarchical nanoarchitecture with enhanced enzyme-like catalytic activity. In this nanoarchitecture, nanospheres are put in a chain-like arrangement through coordination-driven directional self-assembly. The reversible transformation of anisotropic nanochains to isotropic nanospheres switches biomimetic activity. Notably, the assembled nanoarchitecture exhibits a high enzyme-like activity and remarkable long-term stability to promote hydroquinone oxidation, superior to the natural counterpart. This work will pave the way to develop reversible and reusable supramolecular biocatalysts with ordered hierarchical structures for accelerating chemical transformations.

REEP6 deficiency leads to retinal degeneration through disruption of ER homeostasis and protein trafficking.

Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy. We recently identified mutations in REEP6, which encodes the receptor expression enhancing protein 6, in several families with autosomal recessive RP. REEP6 is related to the REEP and Yop1p family of ER shaping proteins and potential receptor accessory proteins, but the role of REEP6 in the retina is unknown. Here we characterize the disease mechanisms associated with loss of REEP6 function using a Reep6 knockout mouse generated by CRISPR/Cas9 gene editing. In control mice REEP6 was localized to the inner segment and outer plexiform layer of rod photoreceptors. The Reep6-/- mice exhibited progressive photoreceptor degeneration from P20 onwards. Ultrastructural analyses at P20 by transmission electron microscopy and 3View serial block face scanning EM revealed an expansion of the distal ER in the Reep6-/- rods and an increase in their number of mitochondria. Electroretinograms revealed photoreceptor dysfunction preceded degeneration, suggesting potential defects in phototransduction. There was no effect on the traffic of rhodopsin, Rom1 or peripherin/rds; however, the retinal guanylate cyclases GC1 and GC2 were severely affected in the Reep6 knockout animals, with almost undetectable expression. These changes correlated with an increase in C/EBP homologous protein (CHOP) expression and the activation of caspase 12, suggesting that ER stress contributes to cell death. Collectively, these data suggest that REEP6 plays an essential role in maintaining cGMP homeostasis though facilitating the stability and/or trafficking of guanylate cyclases and maintaining ER and mitochondrial homeostasis.

Fabrication of a Flexible and Stretchable Nanostructured Gold Electrode Using a Facile Ultraviolet-Irradiation Approach for the Detection of Nitric Oxide Released from Cells.

We developed a simple and environmentally friendly ultraviolet (UV)-irradiation-assisted technique to fabricate a stretchable, nanostructured gold film as a flexible electrode for the detection of NO release. The flexible gold film endows the electrode with desirable electrochemical stability against mechanical deformation, including bending to different curvatures and bearing repeated bending circumstances (200 times). The flexible nanostructured gold electrodes can catalyze NO oxidation at 0.85 V (as opposed to Ag/AgCl) and detect NO within a wide linearity in the range of 10 nM to 1.295 µM. Its excellent NO-sensing ability and its stretchability together with its biocompatibility allows the electrode to electrochemically monitor NO release from mechanically sensitive HUVECs in both their unstretched and stretched states. This result paves the way for an effective and easily accessible platform for designing stretchable and flexible electrodes and opens more opportunities for sensing chemical-signal molecules released from cells or other biological samples during mechanical stimulation.

NMNAT1 E257K variant, associated with Leber Congenital Amaurosis (LCA9), causes a mild retinal degeneration phenotype.

NMNAT1 (nicotinamide mononucleotide adenylyltransferase 1) encodes a rate-limiting enzyme that catalyzes the biosynthesis of NAD+ and plays a role in neuroprotection. Mutations in NMNAT1 have been identified to cause a recessive, non-syndromic early form of blindness genetically defined as Leber Congenital Amaurosis 9 (LCA9). One of the most common alleles reported so far in NMNAT1 is the c.769G > A (E257K) missense mutation, which occurs in 70% of all LCA9 cases. However, given its relatively high population frequency and the observation of individuals with homozygous E257K variant without phenotype, the pathogenicity of this allele has been questioned. To address this issue, we have studied the pathogenic effects of this allele by generating a knock-in mouse model. Interestingly, no obvious morphological or functional defects are observed in Nmnat1 E257K homozygous mice up to one year old, even after light-damage. Together with the previous clinical reports, we propose that the E257K allele is a weak hypomorphic allele that has significantly reduced penetrance in the homozygous state. In contrast, compound heterozygous Nmnat1E257K/- mice exhibit photoreceptor defects which are exacerbated upon exposure to light. Furthermore, retina tissue- specific Nmnat1 conditional knockout mice exhibit photoreceptor degeneration before the retina has terminally differentiated. These findings suggest that NMNAT1 plays an important role in photoreceptors and is likely involved in both retinal development and maintenance of photoreceptor integrity.

CEP78 is mutated in a distinct type of Usher syndrome.

BACKGROUND: Usher syndrome is a genetically heterogeneous disorder featured by combined visual impairment and hearing loss. Despite a dozen of genes involved in Usher syndrome having been identified, the genetic basis remains unknown in 20-30% of patients. In this study, we aimed to identify the novel disease-causing gene of a distinct subtype of Usher syndrome. METHODS: Ophthalmic examinations and hearing tests were performed on patients with Usher syndrome in two consanguineous families. Target capture sequencing was initially performed to screen causative mutations in known retinal disease-causing loci. Whole exome sequencing (WES) and whole genome sequencing (WGS) were applied for identifying novel disease-causing genes. RT-PCR and Sanger sequencing were performed to evaluate the splicing-altering effect of identified CEP78 variants. RESULTS: Patients from the two independent families show a mild Usher syndrome phenotype featured by juvenile or adult-onset cone-rod dystrophy and sensorineural hearing loss. WES and WGS identified two homozygous rare variants that affect mRNA splicing of a ciliary gene CEP78. RT-PCR confirmed that the two variants indeed lead to abnormal splicing, resulting in premature stop of protein translation due to frameshift. CONCLUSIONS: Our results provide evidence that CEP78 is a novel disease-causing gene for Usher syndrome, demonstrating an additional link between ciliopathy and Usher protein network in photoreceptor cells and inner ear hair cells.

Leveraging splice-affecting variant predictors and a minigene validation system to identify Mendelian disease-causing variants among exon-captured variants of uncertain significance.

The genetic heterogeneity of Mendelian disorders results in a significant proportion of patients that are unable to be assigned a confident molecular diagnosis after conventional exon sequencing and variant interpretation. Here, we evaluated how many patients with an inherited retinal disease (IRD) have variants of uncertain significance (VUS) that are disrupting splicing in a known IRD gene by means other than affecting the canonical dinucleotide splice site. Three in silico splice-affecting variant predictors were leveraged to annotate and prioritize variants for splicing functional validation. An in vitro minigene system was used to assay each variant's effect on splicing. Starting with 745 IRD patients lacking a confident molecular diagnosis, we validated 23 VUS as splicing variants that likely explain disease in 26 patients. Using our results, we optimized in silico score cutoffs to guide future variant interpretation. Variants that alter base pairs other than the canonical GT-AG dinucleotide are often not considered for their potential effect on RNA splicing but in silico tools and a minigene system can be utilized for the prioritization and validation of such splice-disrupting variants. These variants can be overlooked causes of human disease but can be identified using conventional exon sequencing with proper interpretation guidelines.

Spata7 is a retinal ciliopathy gene critical for correct RPGRIP1 localization and protein trafficking in the retina.

Leber congenital amaurosis (LCA) and juvenile retinitis pigmentosa (RP) are severe hereditary diseases that causes visual impairment in infants and children. SPATA7 has recently been identified as the LCA3 and juvenile RP gene in humans, whose function in the retina remains elusive. Here, we show that SPATA7 localizes at the primary cilium of cells and at the connecting cilium (CC) of photoreceptor cells, indicating that SPATA7 is a ciliary protein. In addition, SPATA7 directly interacts with the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1), a key connecting cilium protein that has also been linked to LCA. In the retina of Spata7 null mutant mice, a substantial reduction of RPGRIP1 levels at the CC of photoreceptor cells is observed, suggesting that SPATA7 is required for the stable assembly and localization of the ciliary RPGRIP1 protein complex. Furthermore, our results pinpoint a role of this complex in protein trafficking across the CC to the outer segments, as we identified that rhodopsin accumulates in the inner segments and around the nucleus of photoreceptors. This accumulation then likely triggers the apoptosis of rod photoreceptors that was observed. Loss of Spata7 function in mice indeed results in a juvenile RP-like phenotype, characterized by progressive degeneration of photoreceptor cells and a strongly decreased light response. Together, these results indicate that SPATA7 functions as a key member of a retinal ciliopathy-associated protein complex, and that apoptosis of rod photoreceptor cells triggered by protein mislocalization is likely the mechanism of disease progression in LCA3/ juvenile RP patients.

Ultrasonic-Aided Fabrication of Nanostructured Au-Ring Microelectrodes for Monitoring Transmitters Released from Single Cells.

We report a novel ultrasonic-aided fast and straightforward approach to fabricate Au microelectrodes by electroless deposition of nanostructured gold films on the rigid outer surface of pulled glass capillaries. Microelectrodes with tip diameters ranging from several hundred nanometers to several micrometers were fabricated within 20 min via three sequential ultrasonication steps. The ultrasonication technique has been validated to be a very effective route in engineering the morphology of Au film surfaces and improves the fabrication efficiency of Au microelectrodes. The nanostructured surfaces of the Au microelectrodes demonstrate excellent sensing activity and antifouling for dopamine oxidation. The microelectrodes were applied for measurement of catecholamines released from exocytosis events from single chromaffin cells and exhibited faster dynamic peak parameters, compared with carbon fiber microelectrodes. This report provides a generally accessible and complementary platform for analyzing catecholamines release events, which should be useful for new electrode designs and neurochemical sensing.

Ultrasensitive and facile electrochemical detection of hydrogen sulfide in rat brain microdialysate based on competitive binding reaction.

In this study, a sensitive and facile method with wide linear range and low detection limit for detecting hydrogen sulfide in rat brain microdialysate was developed. The design of the sensor is based on the competitive binding reaction principle, in which cysteine was self-assembly immobilized on the surface of gold electrode, and then the Cu2+ as the electrochemical probe was anchored to the cysteine film through coordination bonding with carboxyl (-COOH) and amino group (-NH2) to form the Cu2+/Cys/Au electrode. The Cu2+/Cys/Au electrode can serve as an electrochemical H2S sensor through a ligand exchange reaction, which may come from the greater affinity of H2S than cysteine to the gold surface due to a steric hindrance reason. The hydrogen sulfide cuts off the S-Au bonds between cysteine and Au electrode and leads to the Cu2+ drop off from electrode, resulting in a decrease in the redox signal of Cu2+, thereby creating a current that is indirectly proportional to the logarithm of the concentration of H2S dissolved at the sensor surface. The current response, i.e., signal output, is in wide linearity to logarithm of the concentration of H2S in the range of 0.01-100.0 µM with ΔI/µA = 0.0857 lgCH2S(nM) +0.124 and very low detection limit 5 nM (S/N = 3). The assay demonstrated here is highly selective with respect to alleviating the interference of other thiol-containing species such as glutathione (GSH), homocysteine (Hcy), and cysteine commonly existing in the brain. The basal level of H2S in the microdialysate from the hippocampus of rats is determined to be around 8.6 ± 3.2 µM. The method demonstrated here is facile but reliable and durable and is envisaged to be applicable to understanding the chemical essence involved in physiological and pathological events associated with H2S. Graphical abstract By rationally tailoring the gold electrode surface through the competitive binding interaction of gold electrode between cysteine and H2S, we have successfully designed a simple, highly sensitive, and selective method for electrochemical sensing of H2S in brain microdialysate.

Mutations in human IFT140 cause non-syndromic retinal degeneration.

Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP) are two genetically heterogeneous retinal degenerative disorders. Despite the identification of a number of genes involved in LCA and RP, the genetic etiology remains unknown in many patients. In this study, we aimed to identify novel disease-causing genes of LCA and RP. Retinal capture sequencing was initially performed to screen mutations in known disease-causing genes in different cohorts of LCA and RP patients. For patients with negative results, we performed whole exome sequencing and applied a series of variant filtering strategies. Sanger sequencing was done to validate candidate causative IFT140 variants. Exome sequencing data analysis led to the identification of IFT140 variants in multiple unrelated non-syndromic LCA and RP cases. All the variants are extremely rare and predicted to be damaging. All the variants passed Sanger validation and segregation tests provided that the family members' DNA was available. The results expand the phenotype spectrum of IFT140 mutations to non-syndromic retinal degeneration, thus extending our understanding of intraflagellar transport and primary cilia biology in the retina. This work also improves the molecular diagnosis of retinal degenerative disease.