Search

Significance As a genetic malignancy, retinoblastoma (Rb) is caused by RB1 mutations; however, its developmental origin and drug agents for human Rb remain largely unexplored. Here we describe an innovative Rb organoid model derived from human embryonic stem cells with a biallelic mutagenesis of the RB1 gene. We identify tumorigenic growth in the Rb organoids, as well as properties consistent with human primary Rb. We confirm that the Rb cell of origin stemmed from ARR3+ maturing cone precursor cells and SYK inhibitors displaying a significant therapeutic response. Our elegant in-dish Rb organoid model can be used to efficiently and effectively dissect the origin of Rb and mechanisms of Rb tumorigenesis, as well as screen novel therapies., Retinoblastoma (Rb) is the most prevalent intraocular malignancy in children, with a worldwide survival rate

Summary A major challenge to the development of therapies for human retinal degenerative diseases is the lack of an ideal preclinical model because of the physiological differences between humans and most model animals. Despite the successful generation of a primate model through germline knockout of a disease-causing gene, the major issues restricting modeling in nonhuman primates (NHPs) are their relatively long lifespan, lengthy gestation, and dominant pattern of singleton births. Herein, we generated three cynomolgus macaques with macular in situ knockout by subretinal delivery of an adeno-associated virus (AAV)-mediated CRISPR-Cas9 system targeting CNGB3, the gene responsible for achromatopsia. The in vivo targeting efficiency of CRISPR-Cas9 was 12%–14%, as shown by both immunohistochemistry and single-cell transcriptomic analysis. Through clinical ophthalmic examinations, we observed a reduced response of electroretinogram in the central retina, which corresponds to a somatic disruption of CNGB3. In addition, we did not detect CRISPR-Cas9 residue in the heart, liver, spleen, kidney, brain, testis, or blood a year after administration. In conclusion, we successfully generated a NHP model of cone photoreceptor dysfunction in the central retina using an in situ CNGB3-knockout strategy., Graphical Abstract, Jin and colleagues delivered AAV-mediated CRISPR-cas9 targeting a cone-specific gene, CNGB3, to rapidly generate a non-human primate model with achromatopsia. This study provides a new approach and insights to establish a non-human primate model recapitulating human macular degeneration.

Circular RNAs (circRNAs) represent a class of noncoding RNAs with a wide expression pattern, and they constitute an important layer of the genome regulatory network. To date, the expression pattern and regulatory potency of circRNAs in the retina, a key part of the central nervous system, are not yet well understood. In this study, RNAs from five stages (E18.5, P1, P7, P14, and P30) of mouse retinal development were sequenced. A total of 9,029 circRNAs were identified. Most circRNAs were expressed in different stages with a specific signature, and their expression patterns were different from those of their host linear transcripts. Some circRNAs could act as sponges for several retinal microRNAs (miRNAs). Furthermore, circTulp4 could function as a competitive endogenous RNA (ceRNA) to regulate target genes. Remarkably, silencing circTulp4 in vivo led to mice having a thin outer nuclear layer (ONL) and defective retinal function. In addition, we found that circRNAs were dysregulated at a much earlier time point than that of disease onset in a retinal degeneration model (rd8 mice). In summary, we provide the first circRNA expression atlas during retinal development and highlight a key biological role for circRNAs in retinal development and degeneration.

Adenosine diphosphate (ADP)-ribosylation factor-like 2 (ARL2) protein participates in a broad range of cellular processes and acts as a mediator for mutant ARL2BP in cilium-associated retinitis pigmentosa and for mutant HRG4 in mitochondria-related photoreceptor degeneration. However, mutant ARL2 has not been linked to any human disease so far. Here, we identified a de novo variant in ARL2 (c.44G > T, p.R15L) in a Chinese pedigree with MRCS (microcornea, rod-cone dystrophy, cataract, and posterior staphyloma) syndrome through whole-exome sequencing and co-segregation analysis. Co-immunoprecipitation assay and immunoblotting confirmed that the mutant ARL2 protein showed a 62% lower binding affinity for HRG4 while a merely 18% lower binding affinity for ARL2BP. Immunofluorescence images of ARL2 and HRG4 co-localizing with cytochrome c in HeLa cells described their relationship with mitochondria. Further analyses of the mitochondrial respiratory chain and adenosine triphosphate production showed significant abnormalities under an ARL2-mutant condition. Finally, we generated transgenic mice to test the pathogenicity of this variant and observed retinal degeneration complicated with microcornea and cataract that were similar to those in our patients. In conclusion, we uncover ARL2 as a novel candidate gene for MRCS syndrome and suggest a mitochondria-related mechanism of the first ARL2 variant through site-directed mutagenesis studies.

Circular RNAs (circRNAs) belong to an endogenous class of RNA molecules with both ends covalently linked in a circle. Although their expression pattern in the mammalian brain has been well studied, the characteristics and functions of circRNAs in retinas remain unknown. To reveal the whole expression profiles of circRNAs in the neural retina, we investigated retinal RNAs of human, monkey, mouse, pig, zebrafish and tree shrew and detected thousands of circRNAs showing conservation and variation in the retinas across different vertebrate species. We further investigated one of the abundant circRNAs, circPDE4B, identified in human retina. Silencing of circPDE4B significantly inhibited the proliferation of human A549 cells. Functional assays demonstrated that circPDE4B could sponge miR-181C, thereby altering the cell phenotype. We have explored the retinal circRNA repertoires across human and different vertebrates, which provide new insights into the important role of circRNAs in the vertebrate retinas, as well as in related human diseases.

Abundant retinal microRNA-183 cluster (miR-183C) has been reported to be a key player in photoreceptor development and functionality in mice. However, whether there is a protagonist in this cluster remains unclear. Here, we used a mutant mouse model to study the role of miR-96, a member of miR-183C, in photoreceptor development and functionality.The mature miR-96 sequence was removed using the CRISPR/Cas9 genome-editing system. Electroretinogram (ERG) and optical coherence tomography (OCT) investigated the changes in structure and function in mouse retinas. Immunostaining determined the localization and morphology of the retinal cells. RNA sequencing was conducted to observe retinal transcription alterations.The miR-96 mutant mice exhibited cone developmental delay, as occurs in miR-183/96 double knockout mice. Immunostaining of cone-specific marker genes revealed cone nucleus mislocalization and exiguous Opn1mw/Opn1sw in the mutant (MT) mouse outer segments at postnatal day 10. Interestingly, this phenomenon could be relieved in the adult stages. Transcriptome analysis revealed activation of microtubule-, actin filament-, and cilia-related pathways, further supporting the findings. Based on ERG and OCT results at different ages, the MT mice displayed developmental delay not only in cones but also in rods. In addition, a group of miR-96 potential direct and indirect target genes was summarized for interpretation and further studies of miR-96-related retinal developmental defects.Depletion of miR-96 delayed but did not arrest photoreceptor development in mice. This miRNA is indispensable for mouse photoreceptor maturation, especially for cones.

Human visual acuity is anatomically determined by the retinal fovea. The ontogenetic development of the fovea can be seriously hindered by oculocutaneous albinism (OCA), which is characterized by a disorder of melanin synthesis. Although people of all ethnic backgrounds can be affected, no efficient treatments for OCA have been developed thus far, due partly to the lack of effective animal models. Rhesus macaques are genetically homologous to humans and, most importantly, exhibit structures of the macula and fovea that are similar to those of humans; thus, rhesus macaques present special advantages in the modeling and study of human macular and foveal diseases. In this study, we identified rhesus macaque models with clinical characteristics consistent with those of OCA patients according to observations of ocular behavior, fundus examination, and optical coherence tomography. Genomic sequencing revealed a biallelic p.L312I mutation in TYR and a homozygous p.S788L mutation in OCA2 , both of which were further confirmed to affect melanin biosynthesis via in vitro assays. These rhesus macaque models of OCA will be useful animal resources for studying foveal development and for preclinical trials of new therapies for OCA.

The etiology of the highly myopic condition has been unclear for decades. We investigated the genetic contributions to early-onset high myopia (EOHM), which is defined as having a refraction of less than or equal to −6 diopters before the age of 6, when children are less likely to be exposed to high educational pressures. Trios (two nonmyopic parents and one child) were examined to uncover pathogenic mutations using whole-exome sequencing. We identified parent-transmitted biallelic mutations or de novo mutations in as-yet-unknown or reported genes in 16 probands. Interestingly, an increased rate of de novo mutations was identified in the EOHM patients. Among the newly identified candidate genes, a BSG mutation was identified in one EOHM proband. Expanded screening of 1,040 patients found an additional four mutations in the same gene. Then, we generated Bsg mutant mice to further elucidate the functional impact of this gene and observed typical myopic phenotypes, including an elongated axial length. Using a trio-based exonic screening study in EOHM, we deciphered a prominent role for de novo mutations in EOHM patients without myopic parents. The discovery of a disease gene, BSG, provides insights into myopic development and its etiology, which expands our current understanding of high myopia and might be useful for future treatment and prevention.

Human visual acuity is anatomically determined by the retinal fovea. The ontogenetic development of the fovea can be seriously hindered by oculocutaneous albinism (OCA), which is characterized by a disorder of melanin synthesis. Although people of all ethnic backgrounds can be affected, no efficient treatments for OCA have been developed thus far, due partly to the lack of effective animal models. Rhesus macaques are genetically homologous to humans and, most importantly, exhibit structures of the macula and fovea that are similar to those of humans; thus, rhesus macaques present special advantages in the modeling and study of human macular and foveal diseases. In this study, we identified rhesus macaque models with clinical characteristics consistent with those of OCA patients according to observations of ocular behavior, fundus examination, and optical coherence tomography. Genomic sequencing revealed a biallelic p.L312I mutation in TYR and a homozygous p.S788L mutation in OCA2, both of which were further confirmed to affect melanin biosynthesis via in vitro assays. These rhesus macaque models of OCA will be useful animal resources for studying foveal development and for preclinical trials of new therapies for OCA.

Purpose MicroRNA-182 (miR-182) is abundantly expressed in mammalian retinas; however, the association between miR-182 and retinal function remains unclear. In this study, we explored whether miR-182 contributes to functional decline in retinas using a miR-182 depleted mouse. Methods Electroretinogram (ERG) amplitudes at different ages were measured in miR-182 knockout (KO) mice. The thickness and lamination of retinas were assessed using a color fundus camera and high-resolution optical coherence tomography. Expression levels of key photoreceptor-specific genes and the miR-183/96/182 cluster (miR-183C) were quantified using quantitative real-time PCR. RNA sequencing and light-induced damage were carried out to observe the changes in the retinal transcriptome and sensitivity to light damage in the miR-182 KO mice. Results The ERG recording reveals that the ERG response amplitude decreased both at early and later ages when compared with control littermates. The expression of some key photoreceptor-specific genes was down-regulated with deletion of miR-182 in retina. RNA sequencing indicated that some biological processes of visual system were affected, and the numbers of potential target genes of miR-182 were presented in the mouse retina using bioinformatics analysis. The miR-182 KO mice were characterized by progressively losing the outer segment after being treated with light-damage exposure. The thickness and lamination of retina as well as compensatory expression of miR-183C showed no apparent changes in retina of miR-182 KO mice under normal laboratory lighting condition. Conclusions Our findings provided new insights into the relationship between the miR-182 and retinal development and revealed that miR-182 may play a critical role in maintaining retinal function.

SummaryTargeting genes to specific neuronal or glial cell types is valuable both for understanding and for repairing brain circuits. Adeno-associated viral vectors (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is a challenge. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that ~11% of these AAVs specifically target expression to neuronal and glial cell types in the mouse retina, mouse brain, non-human primate retinain vivo, and in the human retinain vitro. We demonstrate applications for recording, stimulation, and molecular characterization, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast, and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.

Retinoblastoma (Rb) is the most prevalent intraocular malignant tumour in children with survival rate less than 30% globally. Its developmental origin and drug agents remain largely unexplored. Here, we developed the first organoid Rb model derived from human embryonic stem cells (hESCs) with a biallelic knockout (RB1-/-) or mutagenesis (RB1Mut/Mut). These organoid retinoblastomas exhibit extremely consistent properties of Rb tumourigenesis, transcriptome, and genome-wide methylation. We found Rb originated from ARR3+ maturing cone precursors during development and a new unfolded protein response cell type. A key PI3K-Akt pathway was aberrantly regulated, and its activator SYK was significantly upregulated. Furthermore, the SYK inhibitors induced a more significant therapeutic response from early-stage organoid Rb. In conclusion, we established a novel organoid Rb model derived from human ESCs in a dish and discovered its cell-of-origin and potential drug agents, thus shedding light on the development and therapeutics of other human cancers.

Purpose The microRNA cluster miR-183C, which includes miR-183 and two other genes, is critical for multiple sensory systems. In mouse retina, removal of this cluster results in photoreceptor defects in polarization, phototransduction, and outer segment elongation. However, the individual roles of the three components of this cluster are not clearly known. We studied the separate role of mouse miR-183 in in vivo. Methods miR-183 knockout mice were generated using the CRISPR/Cas9 genome-editing system. Electroretinography were carried out to investigate the changes of retinal structures and function. miR-183 was overexpressed by subretinal adeno-associated virus (AAV) injection in vivo. Rnf217, a target of miR-183 was overexpressed by cell transfection of the photoreceptor-derived cell line 661W in vitro. RNA sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to compare the gene expression changes in AAV-injected mice and transfected cells. Results The miR-183 knockout mice showed progressively attenuated electroretinogram responses. Over- or under-expression of Rnf217, a direct target of miR-183, misregulated expression of cilia-related BBSome genes. Rnf217 overexpression also led to compromised electroretinography responses in WT mice, indicating that it may contribute to functional abnormalities in miR-183 knockout mice. Conclusions miR-183 is essential for mouse retinal function mediated directly and indirectly through Rnf217 and cilia-related genes. Our findings provide valuable insights into the explanation and analysis of the regulatory role of the individual miR-183 in miR-183C.

Cell replacement therapy is a promising treatment for irreversible retinal cell death in diverse diseases, such as age-related macular degeneration (AMD), Stargardt's disease, retinitis pigmentosa (RP) and glaucoma. These diseases are all characterized by the degeneration of one or two retinal cell types that cannot regenerate spontaneously in humans. Aberrant retinal pigment epithelial (RPE) cells can be observed through optical coherence tomography (OCT) in AMD patients. In RP patients, the morphological and functional abnormalities of RPE and photoreceptor layers are caused by a genetic abnormality. Stargardt's disease or juvenile macular degeneration, which is characterized by the loss of the RPE and photoreceptors in the macular area, causes central vision loss at an early age. Loss of retinal ganglion cells (RGCs) can be observed in patients with glaucoma. Once the retinal cell degeneration is triggered, no treatments can reverse it. Transplantation-based approaches have been proposed as a universal therapy to target patients with various concomitant diseases. Both the replacement of dead cells and neuroprotection are strategies used to rescue visual function in animal models of retinal degeneration. Diverse retinal cell types derived from pluripotent stem cells, including RPE cells, photoreceptors, RGCs and even retinal organoids with a layered structure, provide unlimited cell sources for transplantation. In addition, mesenchymal stem cells (MSCs) are multifunctional and protect degenerating retinal cells. The aim of this review is to summarize current findings from preclinical and clinical studies. We begin with a brief introduction to retinal degenerative diseases and cell death in diverse diseases, followed by methods for retinal cell generation. Preclinical and clinical studies are discussed, and future concerns about efficacy, safety and immunorejection are also addressed.

MicroRNAs (miRNAs) are known to be essential for retinal maturation and functionality; however, the role of the most abundant miRNAs, the miR-183/96/182 cluster (miR-183 cluster), in photoreceptor cells remains unclear. Here we demonstrate that ablation of two components of the miR-183 cluster, miR-183 and miR-96, significantly affects photoreceptor maturation and maintenance in mice. Morphologically, early-onset dislocated cone nuclei, shortened outer segments and thinned outer nuclear layers are observed in the miR-183/96 double-knockout (DKO) mice. Abnormal photoreceptor responses, including abolished photopic electroretinography (ERG) responses and compromised scotopic ERG responses, reflect the functional changes in the degenerated retina. We further identify Slc6a6 as the cotarget of miR-183 and miR-96. The expression level of Slc6a6 is significantly higher in the DKO mice than in the wild-type mice. In contrast, Slc6a6 is down-regulated by adeno-associated virus-mediated overexpression of either miR-183 or miR-96 in wild-type mice. Remarkably, both silencing and overexpression of Slc6a6 in the retina are detrimental to the electrophysiological activity of the photoreceptors in response to dim light stimuli. We demonstrate that miR-183/96-mediated fine-tuning of Slc6a6 expression is indispensable for photoreceptor maturation and maintenance, thereby providing insight into the epigenetic regulation of photoreceptors in mice.

Precursor messenger RNA (Pre-mRNA) splicing is an essential biological process in eukaryotic cells. Genetic mutations in many spliceosome genes confer human eye diseases. Mutations in the pre-mRNA splicing factor, RP9 (also known as PAP1), predispose autosomal dominant retinitis pigmentosa (adRP) with an early onset and severe vision loss. However, underlying molecular mechanisms of the RP9 mutation causing photoreceptor degeneration remains fully unknown. Here, we utilize the CRISPR/Cas9 system to generate both the Rp9 gene knockout (KO) and point mutation knock in (KI) (Rp9, c.A386T, P.H129L) which is analogous to the reported one in the retinitis pigmentosa patients (RP9, c.A410T, P.H137L) in 661 W retinal photoreceptor cells in vitro. We found that proliferation and migration were significantly decreased in the mutated cells. Gene expression profiling by RNA-Seq demonstrated that RP associated genes, Fscn2 and Bbs2, were down-regulated in the mutated cells. Furthermore, pre-mRNA splicing of the Fscn2 gene was markedly affected. Our findings reveal a functional relationship between the ubiquitously expressing RP9 and the disease-specific gene, thereafter provide a new insight of disease mechanism in RP9-related retinitis pigmentosa.

Purpose Accumulating evidence has demonstrated that excessive immunoreaction plays a prominent role in the pathogenesis of dry AMD. Toll-like receptor 3 (TLR3) can be activated by double-stranded (ds)RNA in retinal pigment epithelia and trigger an innate immunity-mediated inflammatory response. However, its role in photoreceptor cells, the effectors of AMD geographic atrophy, remains unclear. Methods The expression of TLR3 was examined in mouse retina and in a murine photoreceptor cell line (661W). Retinal structure, function, and cell death in the polyinosine-polycytidylic acid (poly I:C)-treated retina were investigated by optical coherence tomography, electroretinography (ERG), and immunostaining. Cytokine and chemokine expression as well as cell death were measured in poly I:C-exposed 661W cells and explant retinas. By comparing the RNA sequencing (seq) data of 661W cells and murine retina, we comprehensively investigated the contribution of photoreceptor in poly I:C-induced retinal immune response. Results Toll-like receptor 3 was highly expressed in the inner segment of the photoreceptor and in 661W cells. We found poly I:C induced significant retinal structural damages and impairment of ERG responses. Focal ERG demonstrated that injected and parainjected zones were functionally damaged by poly I:C. In addition, poly I:C acted on cultured photoreceptor cells directly and evoked an inflammatory response that exhibited similarities with the immune response in mouse retina. Moreover, TLR3 activation initiated cell death in murine photoreceptor cells in vivo and in vitro. Additionally, poly I:C initiated immune response in explant retinas. Conclusions We deciphered the TLR3-mediated inflammatory response in photoreceptor cells. Our findings suggested TLR3-mediated inflammatory response in photoreceptor cells may play an important role in dry AMD, offering new insights of potential treatments targeting photoreceptor immunity.

Experimental, controlled, animal study. To evaluate the effects of calcitriol on oxidative stress, apoptosis, autophagy and locomotor recovery in rats after spinal cord injury (SCI). China. Ninety female rats were randomly divided into three groups. Laminectomy only was performed in the control group. The SCI group received laminectomy as well as spinal cord compression injury. In the calcitriol group, SCI rats received an intraperitoneal injection of calcitriol (2 μg kg−1day−1). Oxidative stress was assessed by the tissue superoxide dismutase (SOD) activity and the contents of glutathione (GSH) and malondialdehyde (MDA). The extent of apoptosis was assessed by immunohistochemistry for C-caspase3, TUNEL staining and western blotting for C-caspase3, Bax and Bcl2. Transmission electron microscopy was used to examine autophagosomes in the injured spinal cord of calcitriol-treated rats. Autophagy was detected by western blotting for LC3-II, Beclin1 and p62. Histological changes were assessed by haematoxylin and eosin staining and Nissl staining. Functional recovery was reflected by the Basso, Beattie and Bresnahan locomotion rating scale and the inclined plane test. With calcitriol treatment, oxidative stress was decreased, SOD activity and GSH content were increased and MDA content was decreased. Moreover, apoptosis was inhibited in the SCI plus calcitriol group. However, a higher level of autophagy was detected in the lesions of the calcitriol group compared with the SCI group. Histological damage and neuron loss after SCI were reduced in calcitriol-treated rats, and functional recovery was significantly promoted in the calcitriol group compared with controls. Calcitriol promotes locomotor recovery after SCI by reducing oxidative stress and inhibiting apoptosis, as well as promoting autophagy.

© 2014 Elsevier Ltd. Various artificial membranes have been used as scaffolds for retinal pigment epithelium cells (RPE) for monolayer reconstruction, however, long-term cell viability and functionality are still largely unknown. This study aimed to construct an ultrathin porous nanofibrous film to mimic Bruch's membrane, and in particular to investigate human RPE cell responses to the resultant substrates. An ultrathin porous nanofibrous membrane was fabricated by using regenerated wild Antheraea pernyi silk fibroin (RWSF), polycaprolactone (PCL) and gelatin (Gt) and displayed a thickness of 3-5 μm, with a high porosity and an average fiber diameter of 166 ± 85 nm. Human RPE cells seeded on the RWSF/PCL/Gt membranes showed a higher cell growth rate (p < 0.05), and a typical expression pattern of RPE signature genes, with reduced expression of inflammatory mediators. With long-term cultivation on the substrates, RPE cells exhibited characteristic polygonal morphology and development of apical microvilli. Immunocytochemisty demonstrated RPE-specific expression profiles in cells after 12-weeks of co-culture on RWSF/PCL/Gt membranes. Interestingly, the cells on the RWSF/PCL/Gt membranes functionally secreted polarized PEDF and phagocytosed labeled porcine POS. Furthermore, RWSF/PCL/Gt membranes transplanted subsclerally exhibited excellent biocompatibility without any evidence of inflammation or rejection. In conclusion, we established a novel RWSF-based substrate for growth of RPE cells with excellent cytocompatibility in vitro and biocompatibility in vivo for potential use as a prosthetic Bruch's membrane for RPE transplantation.

Inherited retinal dystrophy (IRD) is a leading cause of blindness worldwide. Because of extreme genetic heterogeneity, the etiology and genotypic spectrum of IRD have not been clearly defined, and there is limited information on genotype–phenotype correlations. The purpose of this study was to elucidate the mutational spectrum and genotype–phenotype correlations of IRD. We developed a targeted panel of 164 known retinal disease genes, 88 candidate genes, and 32 retina-abundant microRNAs, used for exome sequencing. A total of 179 Chinese families with IRD were recruited. In 99 unrelated patients, a total of 124 mutations in known retinal disease genes were identified, including 79 novel mutations (detection rate, 55.3%). Moreover, novel genotype–phenotype correlations were discovered, and phenotypic trends noted. Three cases are reported, including the identification of AHI1 as a novel candidate gene for nonsyndromic retinitis pigmentosa. This study revealed novel genotype–phenotype correlations, including a novel candidate gene, and identified 124 genetic defects within a cohort with IRD . The identification of novel genotype–phenotype correlations and the spectrum of mutations greatly enhance the current knowledge of IRD phenotypic and genotypic heterogeneity, which will assist both clinical diagnoses and personalized treatments of IRD patients. Genet Med 17 4, 271–278.

Bardet-Biedl syndrome (BBS) is an autosomal recessive disorder with significant genetic heterogeneity. BBS is linked to mutations in 17 genes, which contain more than 200 coding exons. Currently, BBS is diagnosed by direct DNA sequencing for mutations in these genes, which because of the large genomic screening region is both time-consuming and expensive. In order to develop a practical method for the clinic diagnosis of BBS, we have developed a high-throughput targeted exome sequencing (TES) for genetic diagnosis. Five typical BBS patients were recruited and screened for mutations in a total of 144 known genes responsible for inherited retinal diseases, a hallmark symptom of BBS. The genomic DNA of these patients and their families were subjected to high-throughput DNA re-sequencing. Deep bioinformatics analysis was carried out to filter the massive sequencing data, which were further confirmed through co-segregation analysis. TES successfully revealed mutations in BBS genes in each patient and family member. Six pathological mutations, including five novel mutations, were revealed in the genes BBS2, MKKS, ARL6, MKS1. This study represents the first report of targeted exome sequencing in BBS patients and demonstrates that high-throughput TES is an accurate and rapid method for the genetic diagnosis of BBS.