Novel mutations in CYP4V2 in Bietti corneoretinal crystalline dystrophy: Next-generation sequencing technology and genotype-phenotype correlations.

Purpose: To identify any novel mutations in CYP4V2 in 85 Chinese families with Bietti corneoretinal crystalline dystrophy (BCD) by using next-generation sequencing, and to summarize the mutation spectrum in this population, along with any genotype-phenotype correlations. Methods: A total of 90 patients with BCD from 85 unrelated Chinese families were recruited. All probands were analyzed by using gene chip-based next-generation sequencing, to capture and sequence all the exons of 57 known hereditary retinal degeneration-associated genes. The candidate variants were validated with PCR and Sanger sequencing. Results: Twenty-eight mutations were detected in all patients, including thirteen novel mutations (five missense, six deletions, one splicing and one frame-shift mutations) and 15 previously reported mutations. Mutations in 64 patients were inherited from their parents, while three patients had de novo mutations. c.802-8_810del17insGC was the most common mutation, accounting for 78% of the mutations. Although 16 patients were homozygous at this site, the clinical features of all 16 patients were highly heterogeneous. Conclusions: These results expand the spectrum of mutations in CYP4V2, and suggest that mutations in CYP4V2 may be common in the Chinese population. The phenotype of patients with the homozygous mutation (hom.c.802-8_810del17insGC) is highly heterogeneous.

Detecting genetic variations in hereditary retinal dystrophies with next-generation sequencing technology.

PURPOSE: To identify pathogenic mutations responsible for retinal dystrophies (RDs) in three unrelated Chinese families. METHODS: Three probands from unrelated families with RDs were recruited. Genomic DNA prepared from leukocytes was analyzed using gene chip-based next-generation sequencing (NGS) to capture and sequence all of the exons of 100 known RD-associated genes. Candidate variants were validated with PCR and Sanger sequencing in the respective families. Thorough ophthalmic examinations including best-corrected visual acuity, funduscopic examination, and full-field electroretinograms were performed in the affected individuals. RESULTS: We successfully identified causative mutations in patients from the Chinese families with RDS: the known mutation IMPDH1 c.942_944delGAA in a family with retinitis pigmentosa, the novel mutation ABCA4 c.1924T>A in a family with Stargardt disease, and the novel mutation NMNAT1 c.272A>G and known mutation NMNAT1 c.196C>T in a family with Leber congenital amaurosis. All variations segregated with the disease phenotypes in the respective families and were absent from ethnically matched control chromosomes. Prediction analysis demonstrated the two novel missense mutations might be damaging. CONCLUSIONS: The results strongly suggested these mutations were responsible for different RD phenotypes in the Chinese families. NGS technology provides an accurate and economic method for identifying causative genes for RDs.

Identification of novel CYP4V2 gene mutations in 92 Chinese families with Bietti's crystalline corneoretinal dystrophy.

PURPOSE: To characterize the spectrum of CYP4V2 gene mutations in 92 unrelated Chinese probands with Bietti's crystalline dystrophy (BCD) and to describe the molecular and clinical characteristics of four novel CYP4V2 mutations associated with BCD. METHODS: All study participants underwent a complete ophthalmological examination. Mutational screening of CYP4V2 coding regions and flanking intron sequences was examined via directional Sanger sequencing, with allele separation confirmed by screening other family members. Subsequent in silico analysis of the mutational consequence on protein function was undertaken, with the impact of the novel mutation on pre-mRNA splicing examined via RT-PCR. RESULTS: Fifteen disease-causing variants were identified in 92 probands with BCD, including four novel mutations and eleven previously reported mutations. The most prevalent mutation was c.802_810del17insGC, which was detected in 69 unrelated families, with an allele frequency of 52.7% (97/184). Homozygosity was revealed in 35 unrelated families, and compound heterozygosity was observed in 43 subjects. Four patients harbored four novel variants, with these mutations cosegregated within all affected individuals and were not found in unaffected family members and 100 unrelated controls. Transcriptional analysis of a novel splice mutation revealed altered RNA splicing. In silico analysis predicted that the missense variant, p.Tyr343Asp, disrupted the CYP4V2 surface electrostatic potential distribution and spatial conformation. Among the patients with four novel mutations, genotype did not always correlate with age at onset, disease course, or electroretinogram (ERG) changes, with phenotypic variations even noted within the same genotype. CONCLUSIONS: The c.802_810del17insCG mutation was the most common mutation in the 92 Chinese probands with BCD examined. Four novel mutations were identified, contributing to the spectrum of CYP4V2 mutations associated with BCD, with no clear link established between disease phenotype and genotype.

Application effect of diversified health-promoting models on rehabilitation exercises for cervical spondylotic myelopathy.

BACKGROUND: With improving living standards, the incidence of cervical spondylotic myelopathy (CSM) has become increasingly high. OBJECTIVE: The study aims to explore the effect of diversified health-promoting models on rehabilitation exercises in patients with CSM after an operation. METHOD: This was a randomized controlled trial, wherein 107 patients with CSM treated by neurosurgery were selected as the subjects. Of those, 52 patients in the control group adopted the conventional health-promoting model, while the remaining 55 patients in the intervention group adopted diversified health-promoting models. The effect of rehabilitation exercises in the two groups was evaluated according to the self-efficacy rehabilitation outcome scale, grip strength measurement of the affected limb, and Barthel index. RESULTS: At Day 3 post-operation and before discharge, the self-efficacy management of rehabilitation exercises in the intervention group was better than that of the control group (P< 0.05). The grip strength measurement of the affected limb, Japanese Orthopedic Association score of the cervical vertebra, and Barthel index of the two groups at Day 3 post-operation were lower than before the intervention and were not statistically significant (P> 0.05). However, these three items before discharge were improved when compared with those of before intervention and were statistically significant (P< 0.05). CONCLUSION: Postoperative rehabilitation exercises guided by the diversified health-promoting models for patients with CSM can improve the patients' self-efficacy management ability in rehabilitation exercises, help improve grip strength, and promote the recovery of cervical vertebra function, thereby improving the patients' quality of life.

The characterization of functional disturbances in Chinese patients with Bietti's crystalline dystrophy at different fundus stages.

PURPOSE: The aim of this study was to characterize the functional and clinical disturbances and screen the optimal functional tests in assessing Bietti's crystalline dystrophy (BCD) patients by a cross-sectional method. METHODS: The clinical characteristics of BCD were studied in 15 Chinese patients using fundoscopy, fundus fluorescein angiography (FFA), and autofluorescence (AF). The functional features were evaluated by full-field electroretinography (fERG), 85º and 30º perimetry, multifocal ERG (mERG), and chromatic pupillometry. RESULTS: The 15 patients were separated into three clinical stages according to their fundus features. fERG- and mERG- showed reduced reponses in the early stages. Substages could be further defined according to the fERG results in the intermediate stages. Reduced pupillary light reflex (PLR) activities with blue-and white-light stumili existed in all patients. The most reduced PLR activities were elicited in the advanced stage of patients who had other nonresponsive functional tests. CONCLUSIONS: This study identified the most sensitive functional methods for assessing BCD patients, and the significance of PLR in the advanced stages. In addition, the defined-substages can help us understand the disease more clearly.

Transient pupillary light reflex in relation to fundus autofluorescence and dark-adapted perimetry in typical retinitis pigmentosa.

PURPOSE: To determine whether the pupillary light reflex (PLR) can serve as an indicator of photoreceptor function in patients with advanced typical retinitis pigmentosa (RP). METHODS: Dark-adapted transient PLRs elicited by blue or white light over a luminance range of 4 log units were recorded from 27 eyes of 19 patients with advanced RP. Retinas were characterized according to fundus autofluorescence (AF) and dark-adapted perimetry. We qualitatively analyzed whether PLR thresholds were correlated with AF patterns or scotopic sensitivity. Quantitative analysis included correlations between relative pupillary constrictions (RPCs) elicited by blue light (≤-1 log cd/m(2)) and the area of abnormal ring or central AF, and between RPCs elicited by white light and perimetric mean sensitivity. RESULTS: The PLRs of all patients showed varying degrees of threshold elevation and relative afferent pupil defects. We classified three types of abnormal fundus AF: abnormal ring AF, abnormal central AF, and fragmentary AF. PLR thresholds were largely consistent with the patterns of AF and scotopic sensitivity. Rod-mediated RPCs were not correlated with the area of the abnormal ring AF (p > 0.05), but were correlated with the area of abnormal central AF (p < 0.05). RPCs elicited with a white stimulus (-0.3 or 0.7 log cd/m(2)) were significantly correlated with the mean sensitivity of the dark-adapted perimetry. CONCLUSIONS: PLR testing is a powerful technique for assessing photoreceptor dysfunction. The high correlation with AF and dark-adapted perimetry suggests that the key to quantifying photoreceptor function using the transient PLR is to optimize the luminance of the stimulus.

A phase I clinical trial of human embryonic stem cell-derived retinal pigment epithelial cells for early-stage Stargardt macular degeneration: 5-years' follow-up.

OBJECTIVES: To evaluate the long-term biosafety and efficacy of transplantation of human embryonic stem cells-derived retinal pigment epithelial (hESC-RPE) cells in early-stage of Stargardt macular degeneration (STGD1). MATERIALS AND METHODS: Seven patients participated in this prospective clinical study, where they underwent a single subretinal transplantation of 1 × 105 hESC-RPE cells in one eye, whereas the fellow eye served as control. These patients were reassessed for a 60-month follow-up through systemic and ophthalmic examinations. RESULTS: None of the patients experienced adverse reactions systemically or locally, except for two who had transiently high intraocular pressure post-operation. Functional assessments demonstrated that all of the seven operated eyes had transiently increased or stable visual function 1-4 months after transplantation. At the last follow-up visit, two of the seven eyes showed visual function loss than the baseline; however, one of them showed a stable visual acuity when compared with the change of fellow eye. Obvious small high reflective foci in the RPE layer were displayed after the transplantation, and maintained until the last visit. Interestingly, three categories of patients who were classified based on autofluorescence, exhibited distinctive patterns of morphological and functional change. CONCLUSIONS: Subretinal transplantation of hESC-RPE in early-stage STGD1 is safe and tolerated in the long term. Further investigation is needed for choosing proper subjects according to the multi-model image and function assessments.

Identification of 13 novel USH2A mutations in Chinese retinitis pigmentosa and Usher syndrome patients by targeted next-generation sequencing.

BACKGROUND: The USH2A gene encodes usherin, a basement membrane protein that is involved in the development and homeostasis of the inner ear and retina. Mutations in USH2A are linked to Usher syndrome type II (USH II) and non-syndromic retinitis pigmentosa (RP). Molecular diagnosis can provide insight into the pathogenesis of these diseases, facilitate clinical diagnosis, and identify individuals who can most benefit from gene or cell replacement therapy. Here, we report 21 pathogenic mutations in the USH2A gene identified in 11 Chinese families by using the targeted next-generation sequencing (NGS) technology. METHODS: In all, 11 unrelated Chinese families were enrolled, and NGS was performed to identify mutations in the USH2A gene. Variant analysis, Sanger validation, and segregation tests were utilized to validate the disease-causing mutations in these families. RESULTS: We identified 21 pathogenic mutations, of which 13, including 5 associated with non-syndromic RP and 8 with USH II, have not been previously reported. The novel variants segregated with disease phenotype in the affected families and were absent from the control subjects. In general, visual impairment and retinopathy were consistent between the USH II and non-syndromic RP patients with USH2A mutations. CONCLUSIONS: These findings provide a basis for investigating genotype-phenotype relationships in Chinese USH II and RP patients and for clarifying the pathophysiology and molecular mechanisms of the diseases associated with USH2A mutations.

Long-term safety of human retinal progenitor cell transplantation in retinitis pigmentosa patients.

BACKGROUND: Retinitis pigmentosa is a common genetic disease that causes retinal degeneration and blindness for which there is currently no curable treatment available. Vision preservation was observed in retinitis pigmentosa animal models after retinal stem cell transplantation. However, long-term safety studies and visual assessment have not been thoroughly tested in retinitis pigmentosa patients. METHODS: In our pre-clinical study, purified human fetal-derived retinal progenitor cells (RPCs) were transplanted into the diseased retina of Royal College of Surgeons (RCS) rats, a model of retinal degeneration. Based on these results, we conducted a phase I clinical trial to establish the safety and tolerability of transplantation of RPCs in eight patients with advanced retinitis pigmentosa. Patients were studied for 24 months. RESULTS: After RPC transplantation in RCS rats, we observed moderate recovery of vision and maintenance of the outer nuclear layer thickness. Most importantly, we did not find tumor formation or immune rejection. In the retinis pigmentosa patients given RPC injections, we also did not observe immunological rejection or tumorigenesis when immunosuppressive agents were not administered. We observed a significant improvement in visual acuity (P < 0.05) in five patients and an increase in retinal sensitivity of pupillary responses in three of the eight patients between 2 and 6 months after the transplant, but this improvement did not appear by 12 months. CONCLUSION: Our study for the first time confirmed the long-term safety and feasibility of vision repair by stem cell therapy in patients blinded by retinitis pigmentosa. TRIAL REGISTRATION: WHO Trial Registration, ChiCTR-TNRC-08000193 . Retrospectively registered on 5 December 2008.

Novel compound heterozygous mutation in the CNGA1 gene underlie autosomal recessive retinitis pigmentosa in a Chinese family.

Retinitis pigmentosa (RP) describes a group of inherited retinopathies that are characterized by the progressive degeneration of photoreceptor neurons, which causes night blindness, a reduction in the peripheral visual field and decreased visual acuity. More than 50 RP-related genes have been identified. In the present study, we analysed a Chinese family with autosomal recessive RP. We identified a compound heterozygous mutation, c.265delC and c.1537G>A, in CNGA1 using targeted next-generation sequencing (NGS) of RP-causing genes. The mutations were validated in the family members by Sanger sequencing. The mutations co-segregated with the RP phenotype and were absent from ethnically-matched control chromosomes. The mutant (mut) CNGA1 p.(G513R) protein caused by the mis-sense novel mutation c.1537G>A was expressed in vitro. The mut CNGA1 p.(G513R) protein was largely retained inside the cell rather than being targeted to the plasma membrane, suggesting the absence of cGMP-gated cation channels in the plasma membrane would be deleterious to rod photoreceptors, leading lead to RP.

A complete screen for mutations of the rhodopsin gene in a panel of Chinese patients with autosomal dominant retinitis pigmentosa.

OBJECTIVE: To evaluate the prevalence of rhodopsin (RHO) mutations and the genotype-phenotype relationships in Chinese patients with autosomal dominant retinitis pigmentosa (ADRP) by conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing. METHODS: We have screened the five coding exons and splice sites of RHO gene in 27 probands who had no relativity from Chinese ADRP families and 100 normal controls to identify disease-associated mutations, using CSGE and direct DNA sequencing. Family members of some probands with disease-associated mutations were also genotyped to determine whether the RHO mutations segregated with retinitis pigmentosa (RP) in their families. RESULTS: Two RHO mutations, Pro347Leu and Pro327 (1-bp del), were identified separately in two families, thus the frequency of RHO mutations among this set of Chinese ADRP families is about 7.4% (2/27). Pro347Leu mutation was found in one ADRP proband as well as three her children who also had RP. She had relatively early onset at about 17 years. The only one child without this mutation had no symptom or sign of RP at age of 34. Pro327 (1-bp del) was identified in a late-onset ADRP patient, who appeared night blindness around 30 years old and in her fifties electroretinogram (ERG) has been flat in both scotopic and photopic phases. Family analysis showed that this mutation also existed in her younger daughter and her elder sister, both of them also had RP. Three other family members were genotypically and phenotypically normal. Neither of the two mutations was detected in 100 normal controls. CONCLUSIONS: The frequency of RHO mutations in Chinese patients was lower than that in Europe and North America. The phenotype of the patients with Pro347Leu corresponded to type 1 ADRP, with severe rod degeneration and some cone preservation later, while the phenotype of the patients carrying Pro327 (1-bp del) corresponded to type 2 ADRP, with a concomitant loss of rod and cone visual function. CSGE was found to be a sensitive, simple, and practical method for the screening of a large number of samples under highly reproducible conditions, and could be utilized in routine molecular diagnostic laboratories.

[Study on exons 3 and 4 of alpha-synuclein gene in Chinese familial Parkinson disease patients].

OBJECTIVE: To study the relationship of Chinese familial Parkinson disease with alpha-synuclein gene. METHODS: Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and polymerase chain reaction-heteroduplex analysis(PCR-HA) were employed to detect the abnormal mobilization in the familial Parkinson disease and sporadic Parkinson disease patients, then it was verified by gene sequencing. RESULTS: No mutation was found in alpha-synuclein gene exons 3 and 4 by PCR-SSCP together with PCR-HA. An inserted c and an inserted t were found in intron 4, position 23 and position 67 respectively. CONCLUSION: (1) Exons 3 and 4 of alpha-synuclein gene are not the mutational hot spots of Chinese familial Parkinson disease. (2) Two polymorphisms were found in intron 4 of alpha-synuclein gene. They are 23 ins c and 67 ins t.

Spectral-domain optical coherence tomography in patients with congenital nystagmus.

AIM: To study macular features in patients with congenital nystagmus and to assess the utility of spectral-domain optical coherence tomography (SD-OCT) in nystagmus. METHODS: The macular areas of 51 outpatients with congenital nystagmus were examined using SD-OCT. Morphological changes in the retinal layers of the macular area were analysed. RESULTS: Macular images were successfully obtained with SD-OCT from 50 (98%) patients. Patients with ocular albinism mainly have macular hypoplasia, abnormal foveal depression, and increased foveal thickness with persistence of an inner nuclear layer, an inner plexiform layer, a ganglion cell layer and a nerve fiber layer. Macular morphology similar to albinism was observed in three patients with idiopathic macular hypoplasia. The OCT findings of cone dystrophy included unclear, disrupted or invisible photoreceptor outer segment/inner segment in the fovea; fusion, thickening and uneven reflection of the outer segment/inner segment with external limiting membrane. Some patients with congenital idiopathic nystagmus showed normal macular morphology and structure, and others showed indistinct macular external limiting membrane reflection. CONCLUSION: SD-OCT is an effective and reliable method to detect the macular morphology of congenital nystagmus patients. This technique has diagnostic value in particular for patients with macular hypoplasia and cone cell dystrophy with no distinct abnormality on fundoscopy.