Objective To detect characteristics and the pathogenesis of rhodopsin (RHO) gene mutation in an inbreeding family with autosomal recessive retinitis pigmentosa (ARRP). Methods Peripheral venous blood 5-8 ml was abstracted from 8 members in the inbreeding ARRP family and 10 control individuals. DNA gene group was picked. Extron 1-5 of RHO gene was amplified by polymerase chain reaction (PCR)，and the mutation of RHO gene was screened by direct DNA sequence measurement. Results The Gln-344-Arg mutation in the RHO gene was detected in 3 patients with ARRP and homozygotes of the mutation in 3 patients were found. Heterozygous of the mutation was detected in the parent of patients and 1 healthy family member. No mutation of RHO gene was found in 2 healthy family members and 10 control individuals. Conclusions The Gln-344-Arg mutation in the RHO gene may be the pathogenic factor of the ARRP family; the frequency of the mutation of RHO gene may increase in the in breeding ARRP family.(Chin J Ocul Fundus Dis，2004，20：145-148)
ObjectiveTo observe the changes of circumpapillary retinal nerve fiber layer (CP-RNFL) thickness and optic disk parameters in retinitis pigmentosa (RP) eyes. MethodsProspective clinical case-control study. A total of 25 patients (42 eyes) with RP were in the RP group, and 42 age matched healthy subjects (84 eyes) in the control group. All subjects underwent optical coherence tomography (OCT) examination, in which 37 eyes with 3D optic disk scanning and 5 eyes with circle optic disk scanning. The parameters included average thickness of entire CP-RNFL, thickness of nasal, superior, temporal and inferior quadrant of CP-RNFL, disc area, disc cup area, rim area, cup/disc (C/D) area ratio, C/D horizontal diameter ratio, C/D vertical diameter ratio, disc cup volume and disc rim volume. ResultsThe average thickness and the thickness of temporal and nasal quadrants of CP-RNFL in RP group were significantly thicker than the control group (t=2.27, 3.73, 6.44; P=0.027, 0.00, 0.00), while the thickness of inferior and superior areas were the same as control group(t=-1.49, -1.19; P=0.14, 0.24). The disc area, disc cup area, C/D area ratio, C/D horizontal diameter ratio, C/D vertical diameter ratio, disc cup volume in RP group were significantly bigger than control group (P < 0.05), while rim area and rim volume were not significant differences (t=1.75, 0.40; P=0.08, 0.59). ConclusionIn comparison with the healthy subjects, the average thickness and temporal and nasal areas of CP-RNFL in RP eyes were thicker, and the disc area, disc cup area, C/D area ratio, C/D horizontal diameter ratio, C/D vertical diameter ratio, disc cup volume in RP eyes were bigger.
Objective To investigate the feasibility of gene transfection into retinal pigment epithelial (RPE) cells and photoreceptors (PRs) in vivo electroporation. Methods A total of 147 Sprague-Dawley (SD) rats were divided into 5, 10, 15, 20, 25, 30 and 35 V group according to different voltage. The right eyes of rats underwent the injection of eukaryotic expressive plasmid of enhanced green fluorescent protein (EGFP) pEGFP-N1 into subretinal space as experimental eyes; the left eyes were injected with TE buffer as control eyes. Each group was divided into RPE and RP subgroups according to different transfection direction. There were same parameters of 99 ms pulse width, 0.5 s pulse interval and 5 consecutive pulses except different voltage in groups. With a negative charge in the electric field was transfected into RPE cell layer, reverse electrode set to be transfected into PR cell layer. Retina mounts were made on seven days after transfection and the fluorescence of EGFP was photographed by fluorescent microscope. The expression of EGFP mRNA and protein were detected by reverse transcription polymerase chain reaction technique (RT-PCR) and Western blot.Results On seven days after transfection, in RPE subgroups, there were no specific fluorescence expressions in RPE cell layer and retina mounts of control eyes, while there were fluorescence expressions in experimental eyes. Western blot showed that the grayscale ratio of EGFP protein and beta;actin protein bands rose with the increased voltage. RT-PCR showed that each group produced positive amplification bands, and the relative ratio of gray level of EGFP mRNA and GADPH mRNA amplified bands gradually increased with the increased voltage.Conclusion Electroporation is an effective method for gene delivery into RPE cells in vivo.
Objective To observe the expression of cyclin dependent kinase 5 (Cdk5) and p25 in the pathogenesis of retinitis pigmentosa (RP) in Royal College of Surgeon (RCS) rats and its relationships with apoptosis. To explore the mechanism of Cdk5 and p25 induced photoreceptor apoptosis in the pathogenesis of RP. Methods Retinas of RCS and RCS-rdy+ rats were obtained at the ages of postnatal day 17, 25, 35, 60. The retinal structure and thickness of outer nuclear layer were measured by optical microscopy. The expression of Cdk5, p25, cleave-caspase 3 in the retina was evaluated by immunohistochemistry. The protein expression of cleave-caspase 3 in the retina was determined by Western blot. The apoptosis of retinal cells was examined by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL). The mean absorbance value of apoptotic cells was analyzed by SPSS 17.0 software. Results The retinal thickness of the RCS rats was significantly reduced in comparison to the RCS-rdy+ rats as the postnatal days progressed, particularly in the layer of rods and cones and the outer nuclear layer. The expression level of Cdk5, p25, cleave-caspase 3 of RCS rats increased from postnatal 17 days to postnatal 35 days, while decreased on postnatal 60 days; but there was no obvious change of above indexes in RCS-rdy+ rats. The protein expression of cleave-caspase 3 in the RCS rats was significantly increased with progression of postnatal days to postnatal 35; but there was no obvious similar change in RCS-rdy+ rats. The results of TUNEL showed that the apoptotic cells significantly increased in the outer nuclear layer of RCS rats from postnatal 17 days to postnatal 35 days, while decreased on postnatal 60 days; but there was no obvious change of above index in RCS-rdy+ rats. This study showed that there were significant correlations between the following variables: Cdk5 expression and p25 expression, Cdk5 expression and cleave-caspase 3 expression, Cdk5 expression and apoptotic cells, p25 expression and cleave-caspase 3 expression, p25 expression and apoptotic cells, cleave-caspase 3 expression and apoptotic cells. The partial correlation coefficients were 0.949, 0.808, 0.959, 0.887, 0.979, 0.852, respectively and the P value was 0.000. Conclusions The apoptotic cells significantly increases and the expression level of Cdk5, p25, cleave-caspase 3 of RCS rats increases from postnatal 17 days to postnatal 35 days. The tendency of apoptotic cells to increase is consistent with the change of Cdk5, p25, cleave-caspase 3 expression. The apoptosis of photoreceptor cells is related to increasing expression of Cdk5 and p25 in RCS rats. Cdk5 may be involved in the development of RP in RCS rats.
ObjectiveTo analyze the subfoveal choroidal thickness in retinitis pigmentosa (RP) patients and to evaluate the correlation between the subfoveal choroidal thickness (SCT) and visual function. MethodsTotally 42 RP patients (84 eyes) and 49 age and diopter-matched normal controls (98 eyes) were enrolled in this study. All the patients were taken the enhanced depth imaging technique of optical coherence tomography (EDI-OCT) examination for the measurement of the SCT. The covariate analysis was used to analyze the interaction effect between age and group. Then the SCT was amended. The RP patients were examined by 30°visual field test (T32 or LVC program) and electroretinogram (ERG) test. 32 eyes examined by T32 program, 52 eyes examined by LVC program. The waveform of ERG, mean sensitivity (MS) and mean defect (MD) were recorded. The relationship of SCT, MS and MD were analyzed by Pearson correlation analysis. ResultsThe SCT of RP patients and controls were (223.12±69.59), (288.29±52.36) μm. The covariate analysis of covariance with different age group interaction was not statistically significant (F=1.619, P=0.205), as amended SCT of RP patients and controls were (217.34±6.60), (293.20±6.00) μm, respectively. The SCT was decreased in RP patients (t=7.042, P < 0.001). Among 84 eyes, bright cone response weaken in 35 eyes, scotopic rod response weaken in 31 eyes. The difference of SCT in different ERG waveform was not significant (t=-0.976, -1.584; P=0.332, 0.117). The MS and MD of 32 eyes using T32 program were (9.05±6.42), (18.84±6.30) dB, the SCT was (209.83±71.48) μm; the MS and SCT of 52 eyes using LVC program were (7.14±5.03) dB and (228.32±66.32) μm. The SCT was related to MS (r=0.494, P=0.003) and MD (r=-0.448, P=0.009) in eyes using T32 program. There was no correlation between SCT and MD in eyes using LVC program (r=-0.232, P=0.095). ConclusionsThe SCT of RP patients is thinner than that of normal controls. The SCT of RP patients is related to MS and MD of T32 program, but not correlated to ERG waveform and MS of LVC program.
ObjectiveTo observe the disease-causing genes and the inheritance in sporadic retinitis pigmentosa (sRP) in Ningxia region. Methods49 sRP patients and 128 family members were recruited for this study. All the patients and family members received complete ophthalmic examinations including best corrected visual acuity, slit-lamp microscope, indirect ophthalmoscopy, fundus color photography, visual field, optic coherence tomography, full view electroretinogram. DNA was extracted from patients and family members. Using exon combined target region capture sequencing chip to screen the 230 candidate disease-causing gene mutations, polymerase chain reaction and direct sequencing were used to confirm the disease-causing mutations. Results24/49 patients (49.0%) had identified disease-causing genes, totally 16 genes were involved. There were 41 mutation sites were found, including 32 new mutations (78.0%). The disease-causing genes include USH2A, C2orf71, GNGA1, RPGR1, IFT140, TULP1, CLRN1, RPE65, ABCA4, GUCA1, EYS, CYP4V2, GPR98 and ATXN7. Based on pedigree analysis, 20 patients were autosomal recessive retinitis pigmentosa, 3 patients were autosomal dominant retinitis pigmentosa and 1 patient was X linked retinitis pigmentosa. 3/7 patients with USH2A mutations were identified as Usher syndrome. ConclusionsUSHZA is the main disease-causing of sRP patients in Ningxia region. 83.3% of sRP in this cohort are autosomal recessive retinitis pigmentosa.
Objective To analyze the pathogenic gene and clinical phenotypes of a family affected with rare sector retinitis pigmentosa (sector RP). Methods A retrospective clinical study. A patient with sector RP diagnosed in Renmin Hospital of Wuhan University and his parents were included in the study. Detailed medical history was collected; best corrected visual acuity (BCVA), fundus color photography, autofluorescence (AF), visual field, optical coherence tomography (OCT), electroretinogram, fluorescein fundus angiography (FFA), indocyanine green angiography (ICGA) examination were performed. The peripheral venous blood of the patient and his parents were collected, and DNA was extracted. A whole exon sequencing was used for the proband. The mutations were verified by targeted Sanger sequencing and quantitative polymerase chain reaction. Bioinformatics analysis and cosegregation analysis were performed. ResultsThe proband, a 17-year-old male, had presented with gradually decreased vision in the past 2 years with BCVA of 0.4 in both eyes. Retinal vessels attenuation and macular dystrophy without obvious pigmentation on the fundus were observed. AF showed, in bilateral eyes, a symmetrical hypo-autofluorescent region only in the inferonasal quadrant and “petal-like” hyper-AF macula. The visual field examination showed defects in the superotemporal quadrant corresponding to the affected retina. OCT showed loss of the photoreceptor layer except for the foveal region. Electroretinogram examination presented reduced scotopic wave peaks and extinct photopic response. FFA and ICGA showed the atrophy retinal pigment epithelium around the optic disk and in the inferior retina. The clinical phenotypes of the parents were normal. The whole exon sequencing identified one mutation in SPATA7 gene, c.1112T>C (p.Ile371Thr) in exon10 and a copy number variation in trans. The missense mutation resulted in the change of isoleucine to threonine at amino acid 371 in the encoded SPATA7 protein, and the mother carried this heterozygous mutation c.1112T>C. According to the guidelines of the American College of Medical Genetics and Genomics (ACMG) criteria and guidelines for classification of genetic variants, the missense mutation was classified as the uncertain significance. The CNV, originating from his father, contributed to the loss of exon10 and was confirmed as the likely pathogenic variant. ConclusionsThe macula can be involved in sector RP, leading to the macular dystrophy. The missense variant in SPATA7 gene, c.1112T>C (p.Ile371Thr), might be a pathogenic mutation site in this pedigree.
Objective To observe the effect of Minocycline on RP process of retinal pigmentary degeneration rd mice［C3H/HeN (Pde6brd-/rd-)］. Methods 40 rd mice were divided into ten groups randomly: 5 experimental groups and 5 control groups, 4 rd mice in each group. The experimental group received intraperitoneal injection of minocycline 22.5mg/kg while the control group received saline 10ml/kg every day from the postnatal day 1 (P1) . Mice were sacrificed at P1, P7, P14, P21 and P28 respectively. Eyeballs were enucleated to carry out histology observation and apoptosis cell detection. Meanwhile, to statistically analyze the number of retinal photoreceptor cells,the thickness of outer nuclear layer (ONL)and the number of apoptosis cells. Results （1）Photoreceptor cell began to apoptosis on P7, peaked on P14, and totally disappeared on P28. （2）No statistically significant differences were found of the number of photoreceptor cells and the thickness of ONL on P7 between the experimental group and the control group. (3) The number of photoreceptor cells and the thickness of ONL in the experimental were more than that in the control group at P14, P21, P28 respectively, the differences are statistically significant（Plt;0.05）. (4) The apoptotic cells on ONL were less in the experimental group than that in the control group on P7 and P14 respectively, the difference are statistically significant（Plt;0.05）. Conclusions Minocycline appears to protect photoreceptor cell from apoptosis in the early stage of the retinal degeneration mice, but it may not completely prevent RP from occurrence.
ObjectiveTo observe the gene mutations and clinical phenotypes in patients with Usher syndrome type 2 (USH2) and retinitis pigmentosa (RP).Methods From August 2018 to January 2019, 4 patients and 11 normal family members from 3 families of USH2 and RP who visited Henan Eye Hospital were enrolled in the study. Detailed medical history was obtained and visual acuity, fundus color photography, OCT, visual field, full field ERG examination were performed. Among the three families, pedigree 1 was diagnosed with USH2, pedigree 2 and pedigree 3 were diagnosed with RP. The peripheral venous blood of patients and their family members were collected, and the whole genomic DNA was extracted. Targeted capture next generation sequencing analysis was performed on these members, and Sanger sequencing and family co-segregation were verified.ResultsIn the family F1, the proband had symptoms of RP and sensorineural deafness. Sequencing revealed two heterozygous frameshift variants: c.13877-13880 del AGAC (p. Q4626P) in exon 64 and c.798 del T (p. F266L) in exon 5 of USH2A. Both patients of family 2 and 3 showed RP signs without deafness. Two heterozygous variants c.15178T＞C (p. S5060 P) in exon 70 and c.6986C＞A (p. P2329H) in exon 37, and a pathogenic heterozygous variant c.5836C＞T (p. R1946X) in exon 29 of USH2A were identified in family F2. A heterozygous missense variant c.14951C＞T (p. P4984L) in exon 68 and a variant c.11156G＞A (p. R3719H) in exon 57 of USH2A were found in family F3. The results of conservation analysis showed that the corresponding amino acid sites of USH2A p.Q4626P, p.F266L, p.S5060P, p.P2329H and p.P4984L were highly conserved in many species. Among these 7 pathogenic variants detected, M1-M4 and M6 were novel.ConclusionsMutation USH2A gene are the main cause of USH2 and non-syndromic RP. Different variants affect protein translation and synthesis, consequently causing different clinical phenotypes.
Objective To detect and analyse the mutations in rhodopsin gene of members in a family affected by autosomal dominant retinitis pigmentosa (ADRP). Methods Using the polymerase chain reaction (PCR), we amplified exon 1-5 of rhodopsin gene in patients with ADRP,and analyzed it with direct sequence measuement. Results The Gly-182-Asp mutation in the rhodopsin gene was detected in most of affected members of this ADRP family, but no mutation was detected in two affected members and the control ones. Conclusion We cannot regard the Gly-182-Asp mutation in the rhodopsin gene as the pathagenic factor of the ADRP family. It is likely there is a new gene next to the rhodopsin gene. (Chin J Ocul Fundus Dis, 2002, 18: 256-258)