Genotype Impacts Axial Length Growth in Pseudophakic Eyes of Marfan Syndrome.

Purpose: The purpose of this study was to investigate the relationship between axial length (AL) growth and FBN1 genotype in patients with Marfan syndrome (MFS) after lens surgery and customize the selection of intraocular lens (IOL) power. Methods: Patients with MFS who had lens surgery and primary IOL implantation received panel-based next-generation sequencing (NGS). The rate of axial length growth (RALG) was calculated using pre- and postoperative AL measurements and corrected log10-transformed age. A multivariable regression model of RALG was developed after analyzing the effect of FBN1 genotypes and confounding factors. Results: A total of 139 probands of MFS with a median age at lens surgery of 6.25 years (interquartile range [IQR] = 4.67, 12.50 years) were followed up for a median duration of 2.08 years (IQR = 1.16, 3.00 years). The AL growth curve between the age of 3 and 15 years old was logarithmic. Dominant-negative (DN) variants affecting the disulfide-bridge forming cysteines and the conserved residues for calcium-binding had significantly higher RALG than DN variants affecting other structures (P = 0.001) but comparable to that of haplo-insufficiency variants (P = 1.000). Pre-operative AL (b = 0.563, P = 0.011) and genotype constant (b = 2.603, P = 0.011) were significantly associated with RALG in the final model. A Python-based calculator, Marfan IOL Calculator version 2.0, was programmed using the RALG to predict postoperative AL and customize IOL selection based on the ocular biometric parameters and FBN1 genotype. Conclusions: FBN1 genotype impacted the growth of AL in patients with MFS after IOL implantation. Knowing the FBN1 genotype could help cataract surgeons to customize IOL selection.

Morphometric Assessment of the Ciliary Body in Patients With Marfan Syndrome and Ectopia Lentis: A Quantitative Study Using Ultrasound Biomicroscopy.

PURPOSE: To explore the biometric characteristics of the ciliary body in patients with Marfan syndrome (MFS) and ectopia lentis (EL). DESIGN: Cross-sectional study. METHODS: Seventy-two consecutive patients with MFS and EL and 72 nondiseased control subjects were recruited. Ciliary body biometric parameters such as ciliary muscle cross-sectional area at 2000 µm from the scleral spur (CMA2000), ciliary muscle thickness at 1000 µm from the scleral spur (CMT1000), and maximum ciliary body thickness (CBTmax) were measured from multiple directions with ultrasound biomicroscopy (UBM). The relationship between ciliary body parameters and other ocular characteristics was also evaluated. RESULTS: Average CMA2000, CMT1000, and CBTmax were 0.692 ± 0.015 mm2, 0.405 ± 0.010 mm, and 0.855 ± 0.023 mm in eyes of patients with MFS, respectively, and were significantly smaller than these values in control subjects (all P < .001). The prevalence of ciliary body thinning was 22.2% in the MFS group vs 0 in the control group (P < .001); eyes with more severe EL had smaller CMA2000 (P = .050), thinner CMT1000 (P = .022), and shorter CBTmax (P = .015). Patients with microspherophakia (MSP) had even smaller CMA2000 (P = .033) and CMT1000 (P = .044) than those without MSP. The most common subluxation direction was in the superonasal quadrant (n = 25; 39.7%), which probably correlates with the thinnest CMT1000 in the inferotemporal quadrant (P = .005). CONCLUSIONS: Patients with MFS and EL had thinner ciliary muscles, shorter ciliary processes, and a higher prevalence of ciliary body thinning, especially those with MSP. Both the extent and direction of subluxation were associated with ciliary body biometry..

Predicting axial length in patients with Marfan syndrome and ectopia lentis after modified capsular tension ring and intraocular lens implantation.

PURPOSE: To predict the growth of axial length (AL) in patients with Marfan syndrome (MFS) and ectopia lentis (EL). SETTING: Eye and ENT Hospital of Fudan University, Shanghai, China. DESIGN: Consecutive retrospective case series. METHODS: Eyes were evaluated that had modified capsular tension ring and intraocular lens (IOL) implantation. The rate of AL growth (RALG) was calculated using AL divided by log10-transformed age. A multivariate linear regression model of RALG was developed after validation. RESULTS: 128 patients with MFS and EL were enrolled with a median follow-up duration of about 3 years. RALG was independent of age between 3 years and 15 years old ( P = .799) and decreased to 0 thereafter ( P = .878). Preoperative AL was associated with RALG in patients under 15 years old ( P = .003). Beta values for the final model of RALG were as below: intercept (-9.794) and preoperative AL (0.664). The postoperative AL was predicted as: postAL = preAL + RALG × log 10 ([postAge + 0.6]/[preAge + 0.6]). The mean prediction error was -0.003 (95% CI, -0.386 to 0.3791) mm and the mean absolute percentage error was 1.93% (95% CI, 0.73% to 3.14%). A Python-based calculator was developed to use the predicted AL in selecting IOL power and setting undercorrection. CONCLUSIONS: The AL growth of patients with MFS followed a logarithmic pattern and ceased at about age 15. A prediction model of postoperative AL was established for individual MFS patients between 3 and 15 years old, which could potentially optimize the IOL power selection.

Supracapsular scleral sutured intraocular lens implantation in anterior segment dysgenesis patients with ectopia lentis.

OBJECTIVE: To describe a new strategy to manage ectopia lentis in ASD patients assessing the visual outcomes and safety of supracapsular scleral sutured intraocular lens implantation and analyzing the accuracy of different intraocular lens (IOL) power calculation formulae. METHODS: Eight patients with ASD (13 eyes) were underwent supracapsular scleral suture fixation of posterior chamber (PC) IOL without capsular extirpation. The preoperative and postoperative clinical features were compared. The prediction error values from four formulae (SRK/T, Holladay 1, Hoffer Q, Haigis), with or without Wang-Koch (WK) adjustment, were calculated for the cases. RESULTS: Zonulodialysis and premature cataracts could be the main reason for the decreased vision in patients with ASD. There was a significant improvement in best corrected visual acuity on 3-month follow-up after applying supracapsular scleral suture fixation of PC IOL. The prediction errors of the different formulae showed a slight tendency towards postoperative myopia. The Haigis formula with WK adjustment showed the best performance. CONCLUSIONS: Supracapsular scleral suture fixation of IOLs for retaining the capsule-zonule barrier is a good option for ASD patients. The Haigis formula is recommended for ASD patients treated with supracapsular scleral suture fixation of IOLs. The predicted IOL power should be reduced based on the effect of the new anatomic position of the IOL to achieve a satisfactory visual outcome.

Biallelic ADAMTSL4 variants in a Chinese cohort of congenital ectopia lentis: Implications for genotype-phenotype relationships.

ADAMTSL4 variants are one of the common causes of congenital ectopia lentis (EL), reported ocular comorbidities of which include iris anomalies, cataract, and glaucoma. However, a genotype-phenotype correlation has not been established. Potentially pathogenic ADAMTSL4 variants were screened from a Chinese cohort of congenital EL using panel-based next-generation sequencing followed by multiple bioinformatics analyses. The genotype-phenotype correlation was assessed via a systematic review of ADAMTSL4 variants within our data and those from the literature. A total of 12 variants of ADAMTSL4, including seven frameshift variants, one nonsense variant, two splicing variants, and two missense variants, were found in nine probands. Combing genetic and clinical information from 72 probands in the literature revealed 37 ADAMTSL4 variants known to cause EL, and the ethnic difference was prominent. The lens was inclined to dislocate inferior temporally (22, 27.16%), while the pupil was always located oppositely (9, 81.82%). Several anterior segments anomalies were identified, including ectopia pupillae (15, 18.52%), persistent pupillary membrane (9, 11.10%), poor pupil dilation (4, 30.8%), cataract (13, 24.10%), and glaucoma (8, 13.33%). Genotype-phenotype analysis revealed that truncation variants had higher risks of combined iris anomalies, including either ectopia pupillae or a persistent pupillary membrane (p = 0.007). The data from this study not only extend our knowledge of the ADAMTSL4 variant spectrum but also suggest that deleterious variants of ADAMTSL4 might be associated with severe ocular phenotypes.

Mutation analysis of SUOX in isolated sulfite oxidase deficiency with ectopia lentis as the presenting feature: insights into genotype-phenotype correlation.

BACKGROUND: Isolated sulfite oxidase deficiency (ISOD) caused by sulfite oxidase gene (SUOX) mutations is a rare neurometabolic disease associated with ectopia lentis (EL). However, few genotype-phenotype correlations have been established yet. METHODS: Potentially pathogenic SUOX mutations were screened from a Chinese cohort of congenital EL using panel-based next-generation sequencing and analyzed with multiple bioinformatics tools. The genotype-phenotype correlations were evaluated via a systematic review of SUOX mutations within our data and from the literature. RESULTS: A novel paternal missense mutation, c.205G > C (p.A69P), and a recurrent maternal nonsense mutation, c.1200 C > G (p.Y400*), of SUOX were identified in a 4-year-old boy from 312 probands. The biochemical assays manifested elevated urine sulfite and S-sulfocysteine accompanied by decreased homocysteine in the blood. The patient had bilateral EL and normal fundus, yet minimal neurological involvement and normal brain structure. Molecular modeling simulation revealed the p.A69P mutant had an unstable structure but an unchanged affinity for sulfite, while the truncated p.Y400* mutant showed decreased binding capacity. Genotype-phenotype analysis demonstrated patients with biallelic missense mutations had milder symptoms (P = 0.023), later age of onset (P < 0.001), and a higher incidence of regression (P = 0.017) than other genotypes. No correlations were found regarding EL and other neurological symptoms. CONCLUSION: The data from this study not only enrich the known mutation spectrum of SUOX but also suggest that missense mutations are associated with mild and atypical symptoms.

Genotype-phenotype correlations of marfan syndrome and related fibrillinopathies: Phenomenon and molecular relevance.

Marfan syndrome (MFS, OMIM: 154700) is a heritable multisystemic disease characterized by a wide range of clinical manifestations. The underlying molecular defect is caused by variants in the FBN1. Meanwhile, FBN1 variants are also detected in a spectrum of connective tissue disorders collectively termed as 'type I fibrillinopathies'. A multitude of FBN1 variants is reported and most of them are unique in each pedigree. Although MFS is being considered a monogenic disorder, it is speculated that the allelic heterogeneity of FBN1 variants contributes to various manifestations, distinct prognoses, and differential responses to the therapies in affected patients. Significant progress in the genotype-phenotype correlations of MFS have emerged in the last 20 years, though, some of the associations were still in debate. This review aims to update the recent advances in the genotype-phenotype correlations of MFS and related fibrillinopathies. The molecular bases and pathological mechanisms are summarized for better support of the observed correlations. Other factors contributing to the phenotype heterogeneity and future research directions were also discussed. Dissecting the genotype-phenotype correlation of FBN1 variants and related disorders will provide valuable information in risk stratification, prognosis, and choice of therapy.

Comparison of Accuracy in Keratometries Measured by Different Camera Devices for Predicting the Intraocular Lens Power in Lens-Dislocated Patients.

INTRODUCTION: This is a cross-sectional cohort study focused on assessing the influence of ocular biometric parameters of different camera devices for accurately predicting the intraocular lens (IOL) power in the congenital ectopia lentis (EL) patients. METHODS: This study includes a total of 91 eyes of 60 patients with congenital EL from June 2018 to April 2021. All patients underwent lens subluxation surgery with Cionni modified capsular tension rings (MCTR) implantation. Ocular parameters measured by partial coherence interferometry (IOLMaster 700, Carl Zeiss Meditec AG, Jena, Germany) and rotating Scheimpflug camera (Pentacam HR system, Oculus Optikgeräte GmbH, Wetzlar, Germany) were acquired from the database. The authenticity of the different keratometries (K) were analyzed by comparing the prediction error in spherical equivalent under controlled formula SRK/T, Haigis, and after Wang-Koch (WK) adjustment. RESULTS: We observed significant greater K values were obtained in IOLMaster than Pentacam, resulting in more significant hyperopia error while calculating SRK/T. The IOL power calculated with the total corneal refractive power (TCRP) from Pentacam revealed the highest prediction accuracy, indicating that TCRP is the closest to the actual refractive power of the cornea. However, in an exceptional case for long eye patients, total keratometry from IOLMaster was better recommended when using formula Haigis with WK adjustment. CONCLUSIONS: For most instances, TCRP is the best-recommended source of K value while calculating IOL power for EL patients. However, the total keratometry from IOLMaster preferably fits for long eye patients, who require WK adjustment for Haigis formula.

Phacoemulsification Combined With Supra-Capsular and Scleral-Fixated Intraocular Lens Implantation in Microspherophakia: A Retrospective Comparative Study.

Background: Microspherophakia (MSP) is a rare ocular condition, the lens surgery of which is complicated by both insufficient zonules and undersized capsule. Methods: This study included MSP eyes managed with phacoemulsification combined with supra-capsular and scleral-fixated intraocular lens implantation (SCSF-IOL) and made the comparison with those treated by transscleral-fixated modified capsular tension ring and in-the-bag intraocular lens implantation (MCTR-IOL). Results: A total of 20 MSP patients underwent SCSF-IOL, and 17 patients received MCTR-IOL. The postoperative best corrected visual acuity was significantly improved in both groups (P < 0.001), but no difference was found between the groups (P = 0.326). The IOL tilt was also comparable (P = 0.216). Prophylactic Nd:YAG laser posterior capsulotomy was performed 1 week to 1 month after the SCSF-IOL procedure. In the SCSF-IOL group, two eyes (10.00%) needed repeated laser treatment and one eye (5.00%) had a decentered capsule opening. Posterior capsule opacification was the most common complication (6, 35.29%) in the MCTR group. No IOL dislocation, secondary glaucoma, or retinal detachment was observed during follow-up. Conclusions: SCSF-IOL is a viable option for managing MSP and is comparable with the MCTR-IOL. Nd:YAG laser posterior capsulotomy was necessary to prevent residual capsule complications after the SCSF-IOL procedure.

Combination of Panel-based Next-Generation Sequencing and Clinical Findings in Congenital Ectopia Lentis Diagnosed in Chinese Patients.

PURPOSE: To evaluate the diagnostic yield of congenital ectopia lentis (EL) in a Chinese cohort by combining panel-based next-generation sequencing with clinical findings. DESIGN: A cohort study. METHODS: In total, 175 patients with congenital EL and their available family members (n = 338) were enrolled. All patients with congenital EL underwent genetic testing. Genotype-phenotype analyses were conducted to assess the biometric and structural ocular manifestations of congenital EL. RESULTS: In total, 175 patients with congenital EL and 338 of their relatives were included in this study. In these patients, 92.57% (162 of 175) of disease-related variants were detected in FBN1 (83.43%), CPAMD8 (1.71%), COL4A5 (0.57%), ADAMTSL4 (3.43%), LTBP2 (1.71%), and CBS (2.29%). Based on genetic and clinical findings, the primary diagnostic rate was increased to 40.57% from 19.43% with the exception of the 91 diagnoses of potential Marfan syndrome, with a new diagnostic strategy for congenital EL, thus having been developed. Within this group of patients harboring FBN1 mutations, 16.44% (19 of 141) probands were diagnosed with EL syndrome and 2.13% (3 of 141) were diagnosed with Marfan syndrome. CONCLUSIONS: The results of this cohort study expand the genomic landscape associated with congenital EL in Chinese cohorts. FBN1 mutations represent the most common cause of congenital EL in this population, and we have developed a new diagnostic strategy for congenital EL subtypes via the use of a well-designed panel-based next-generation sequencing that can be used to efficiently and precisely diagnose patients with congenital EL in a cost-effective manner.

Correlation between FBN1 mutations and ocular features with ectopia lentis in the setting of Marfan syndrome and related fibrillinopathies.

Mutations of fibrillin-1 (FBN1) have been associated with Marfan syndrome and pleiotropic connective tissue disorders, collectively termed as "type I fibrillinopathy". However, few genotype-phenotype correlations are known in the ocular system. Patients with congenital ectopia lentis (EL) received panel-based next-generation sequencing, complemented with multiplex ligation-dependent probe amplification. In a total of 125 probands, the ocular phenotypes were compared for different types of FBN1 mutations. Premature termination codons were associated with less severe EL and a thinner central corneal thickness (CCT) than the inframe mutations. The eyes of patients with mutations in the C-terminal region had a higher incidence of posterior staphyloma than those in the middle and N-terminal regions. Mutations in the TGF-ß-regulating sequence had larger horizontal corneal diameters (white-to-white [WTW]), higher incidence of posterior staphyloma, but less severe EL than those with mutations in other regions. Mutations in the neonatal region were associated with thinner CCT. Longer axial length (AL) was associated with mutations in the C-terminal region or TGF-ß regulating sequence after adjusting for age, EL severity, and corneal curvature radius. FBN1 genotype-phenotype correlations were established for some ocular features, including EL severity, AL, WTW, CCT, and so forth, providing novel perspectives and directions for further mechanistic studies.

Evaluation of axial length/total corneal refractive power ratio as a potential marker for ocular diagnosis of Marfan's syndrome in children.

AIM: To investigate whether the axial length (AL)/total corneal refractive power (TCRP) ratio is a sensitive and simple factor that can be used for the early diagnosis of Marfan's syndrome (MFS) in children. METHODS: The relationship between the AL/TCRP ratio and the diagnosis of MFS for 192 eyes in 97 children were evaluate. The biological characteristics, including age, sex, AL, and TCRP, were collected from medical records. Receiver operating characteristic (ROC) curve analysis was performed to investigate whether the AL/TCRP ratio effectively distinguishes MFS from other subjects. The Youden index was used to re-divide the whole population into two groups according to an AL/TCRP ratio of 0.59. RESULTS: Of 96 subjects (mean age 7.46±3.28y) evaluated, 56 (110 eyes) had a definite diagnosis of MFS in childhood based on the revised Ghent criteria, 41 (82 eyes) with diagnosis of congenital ectopia lentis (EL) were included as a control group. AL was negatively correlated with TCRP, with a linear regression coefficient of -0.36 (R 2=0.08). A significant correlation was found between age and the AL/TCRP ratio (P=0.023). ROC curve analysis showed that the AL/TCRP ratio distinguished MFS from the other patients at a threshold of 0.59. MFS patients were present in 24/58 (41.38%) patients with an AL/TCRP ratio of ≤0.59 and in 34/39 (87.18%) patients with an AL/TCRP ratio of >0.59. CONCLUSION: An AL/TCRP ratio of >0.59 is significantly associated with the risk of MFS. The AL/TCRP ratio should be measured as a promising marker for the prognosis of children MFS. Changes in the AL/TCRP ratio should be monitored over time.

Analysis of Axial Length in Young Patients with Marfan Syndrome and Bilateral Ectopia Lentis by Z-Scores.

INTRODUCTION: Marfan syndrome (MFS) is characterized by ectopia lentis (EL) and elongated axial length (AL). The characteristics of AL in young patients with MFS and bilateral EL before the lens surgery are not fully understood. METHODS: This study reviewed MFS patients under 20 years old with bilateral EL from January 2015 to October 2020. The Z-scores were introduced in terms of the number of standard deviations from the mean of age-matched normative data. Using Z-scores, the distribution of AL and influence factors were evaluated. The correlations between AL and other biometrics were analyzed. RESULTS: We reviewed 183 patients and enrolled both eyes. The mean age was 8.44 ± 4.69 years. About 36% of the patients were children under 6 years old. The median AL increased from 23.16 mm under 5 years old to 26.20 mm in the 16-20 age group, and when plotted, the trend presented a logarithmic curvature (R2 = 0.145, p < 0.001). The median Z-AL score was 1.24. One-third of eyes had Z-score <0. About 20% of the patients had AL difference over 1 mm between the right and left eyes, and the right one had longer Z-AL scores (p = 0.013). The eye complicated with megalocornea (10, 7.04%) had larger Z-AL scores (4.72 ± 3.51 vs. 1.10 ± 2.25, p = 0.002). A positive correlation was found between Z-AL and Z-corneal curvature radius (r = 0.265, p < 0.001). CONCLUSION: Young patients with bilateral EL but small AL should not be excluded from MFS without systematic examination. The age-adjusted Z-score will facilitate further study of the individual variations in AL across different ages.

Lens Biometry in Congenital Lens Deformities: A Swept-Source Anterior Segment OCT Analysis.

Aims: To investigate the lens biometric parameters in congenital lens deformities, using a novel technique of swept-source anterior segment optical coherence tomography (SS-ASOCT). Methods: This prospective study included patients with microspherophakia (MSP), coloboma lentis (CL), and posterior lenticonus (PL). For this cohort, 360-degree high-resolution lens images were obtained using the latest SS-ASOCT (CASIA2, Tomey Corp, Nagoya, Japan). The lens biometric parameters were calculated by the CASIA2 built-in software for anterior lens radius (ALR), posterior lens radius (PLR), anteroposterior distance (APD), anterior chamber depth (ACD), equatorial diameter (Eq Dia), rear projection length (RPL), and maximum diameter of the lesion (MDL). Results: This study included two eyes each with MSP and CL and one eye with PL. The lens of MSP was spherical and posteriorly dislocated, with decreased ALR and PLR, Eq Dia, but increased APD. In patients with CL, the coloboma was isolated, bilateral, inferior, and located toward the maldeveloped ciliary body. High astigmatism was mainly lenticular, and this was calculated by the ALR and PLR. Regarding the site of coloboma, a significant decrease in ALR was observed, while the PLR and APD were not affected. The PL eyes had a cone-shaped protrusion of the posterior lens surface with a subtle cataractous region around the apex. An extremely high posterior surface curvature was observed with a mean PLR of 1.67 mm. The RPL and MDL were about 1.80 and 0.4 mm, respectively, which were homogenous at different sections. Conclusions: The CASIA2 is a valuable option for in vivo crystalline lens measurement for congenital lens deformities, enabling the accurate diagnosis and providing illuminating insights into the pathogenesis of MSP, CL, and PL.

Long-range surface plasmon resonance sensor based on dielectric/silver coated hollow fiber with enhanced figure of merit.

A long-range surface plasmon resonance (LRSPR) sensor based on dielectric/silver-coated hollow fiber (HF) is proposed. It can detect the refractive index (RI) of sensed liquid filled in the hollow core of the sensor. A HF LRSPR sensor with 90-nm-thick silver layer and 260-nm-thick OC300 layer is fabricated. Experiments are taken to evaluate the performance of the sensor by measuring the transmission spectra. Theoretical analysis based on a ray model is also taken, and the results agree well with the experimental results. The proposed sensor has similar sensitivity but much smaller SPR dip width than the silver-coated HF SPR sensor. Thus figure of merit of the sensor is enhanced approximately five times. The stability of the sensor is also improved because the dielectric layer acts as a protection layer for the damageable silver layer.

Expression profiling of microRNAs in hippocampus of rats following traumatic brain injury.

The changes of microRNA expression in rat hippocampus after traumatic brain injury (TBI) were explored. Adult SD rats received a single controlled cortical impact injury, and the ipsilateral hippocampus was harvested for the subsequent microarray assay at three time points after TBI: 1st day, 3rd day and 5th day, respectively. We characterized the microRNA expression profile in rat hippocampus using the microRNA microarray analysis, and further verified microarray results of miR-142-3p and miR-221 using quantitative real-time PCR. Totally 205 microRNAs were identified and up-/down-regulated more than 1.5 times. There were significant changes in 17 microRNAs at all three time points post-TBI. The quantitative real-time PCR results of miR-142-3p and miR-221 indicated good consistency with the results of the microarray method. MicroRNAs altered at different time points post-TBI. MiR-142-3p and miR-221 may be used as potentially biological markers for TBI assessment in forensic practice.

Hollow fiber surface plasmon resonance sensor for the detection of liquid with high refractive index.

A new kind of surface plasmon resonance (SPR) sensor based on silver-coated hollow fiber (HF) structure for the detection of liquids with high refractive index (RI) is presented. Liquid sensed medium with high RI is filled in the hollow core of the HF and its RI can be detected by measuring the transmission spectra of the HF SPR sensor. The designed sensors with different silver thicknesses are fabricated and the transmission spectra for filled liquids with different RI are measured to investigate the performances of the sensors. Theoretical analysis is also carried out to evaluate the performance. The simulation results agree well with the experimental results. Factors that might affect sensitivity and detection accuracy of the sensor are discussed. The highest sensitivity achieved is 6,607 nm/RIU, which is comparable to the sensitivities of the other reported fiber SPR sensors.

[The cell-type-specific HSV-tk gene expression of suicide gene therapy system regulated by lens-specific promoter LEP503].

OBJECTIVE: To explore the specific expression of HSV-tk gene and killing effects on ocular leading cells of the enhanced specific HSV-tk/GCV gene therapy system regulated by lens-specific promoter LEP503. METHODS: Experimental research. The enhanced specific HSV-tk/GCV gene system of two vectors were constructed (Lenti-LEP503-HSVtk-Cre and Lenti-HPGK-Loxp-EGFP-pA-Loxp-HSVtk). The lentiviral vectors were produced by transient transduction of transfering vectors, packaging vectors and enveloping vector into 293T cells. Virus was collected with ultracentrifugation and resuspended with 1 ml phosphate buffered saline and stored at -80°C. The HLEC and RPEC, NIH3T3, 293T cells were transduced with the enhanced specific HSV-tk gene system. The specific expressions of EGFP and HSV-tk were detected by fluorescence microscopy, flow cytometry and RT-PCR. The killing effects of HLEC and RPEC at the concentration of 20 mg/L GCV were assayed and compared by flow cytometry and CCK-8 kit. Difference of RPE cell viability among groups was evaluated by analysis of variance (ANOVA). RESULTS: Expression efficiency of EGFP in RPEC group was 62.3%, 68.3% in NIH3T3 group, 75.8% in 293T group, whereas 17.5% in HLEC group. There was higher expression of HSV-tk at mRNA level in HLEC group than that in RPEC group. The relative intensity of HSV-tk mRNA in HLEC group transduced with the enhanced specific HSV-tk gene system was 4.01, whereas 0.29 in RPEC group. At the concentration of 20 mg/L GCV after 72 hours, the percentage of apoptosis detected by the flow cytometry in HLEC group transduced by the enhanced specific HSV-tk gene system was 76.51%, and 2.44% in RPEC group. There was no significant difference in the RPE cell viability among the enhanced specific HSV-tk gene combination-RPE group, normal-RPE group and negative-RPE control group at the concentration of 20 mg/L GCV after 72 hours (MD(1) = -0.047, P = 0.671; MD(2) = 0.027, P = 0.912). CONCLUSIONS: The enhanced specific HSV-tk gene system express HSV-tk selectively in HLEC. At the concentration of 20 mg/L GCV, it is effective against the proliferation of HLEC in vitro, but has less kill effect on RPEC.

ATRA enhances the bystander effect of suicide gene therapy driven by the specific promoter LEP 503 in human lens epithelial cells.

PURPOSE: To establish a novel, targeted lentivirus-mediated LEP503-HSV-tk/GCV suicide gene therapy system combined with all trans-retinoic acid (ATRA) for the inhibition of human lens epithelial cell (HLEC) proliferation and treatment of posterior capsular opacification (PCO) after cataract surgery; to estimate the enhancement of the bystander effect by ATRA; and to explore the role of Connexin43 (Cx43) mediated gap junctional intercellular communication (GJIC) in the bystander effect of the HSV-K/GCV system. METHODS: A Lenti-LEP503-HSV-tk-EGFP vector was generated by cloning the lens-specific promoter LEP503 (lens specific promoter 503) from genomic DNA of HLECs by PCR. The vector was then inserted into the promoter-less vector from lentivirus-based (CMV)-HSV-tk-EGFP. The expressional specificity of the LEP503 promoter was assessed by investigating the expression of EGFP (enhanced green fluorescent protein) and HSV-tk (herpes simplex virus thymidine kinase) mRNA, both driven by Lenti-LEP503-HSV-tk-EGFP vector, by fluorescence microscopy, RT-PCR, flow cytometry, and western blot assays in HLECs, human adult retinal pigment epithelium cells (RPECs), human adult skin fibroblast cells (ASFCs), and Hela cells. Morphological changes were observed by fluorescence microscopy and cell viability was determined using the Cell Counting kit-8 Cell Proliferation (CCK-8) and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays after Lenti-LEP503-HSV-tk/GCV system combined with ATRA treatment on HLECs. Flow cytometry, DNA fragmentation, and western blot assays were employed to analyze the mechanisms of bystander effects. RESULTS: The promoter LEP503-mediated HSV-tk was specifically expressed in HLECs, and ATRA dose-dependently strengthened the bystander effect following LEP503-mediated HSV-tk/GCV gene therapy against lens cells by upregulating the expression of the gap junction protein Cx43. CONCLUSIONS: The Lenti-LEP503-HSV-tk/GCV suicide gene therapy system, combined with ATRA as an adjuvant, may be a feasible supplementary method for PCO treatment that targets residual lens cells.

αA crystallin may protect against geographic atrophy-meta-analysis of cataract vs. cataract surgery for geographic atrophy and experimental studies.

BACKGROUND: Cataract and geographic atrophy (GA, also called advanced "dry" age-related macular degeneration) are the two major causes of visual impairment in the developed world. The association between cataract surgery and the development of GA was controversial in previous studies. METHODS/PRINCIPAL FINDINGS: We performed a meta-analysis by pooling the current evidence in literature and found that cataract is associated with an increased risk of geographic atrophy with a summary odds ratio (OR) of 3.75 (95% CI: 95% CI: 1.84-7.62). However, cataract surgery is not associated with the risk of geographic atrophy (polled OR=3.23, 95% CI: 0.63-16.47). Further experiments were performed to analyze how the αA-crystallin, the major component of the lens, influences the development of GA in a mouse model. We found that theαA-crystallin mRNA and protein expression increased after oxidative stress induced by NaIO(3) in immunohistochemistry of retinal section and western blot of posterior eyecups. Both functional and histopathological evidence confirmed that GA is more severe in αA-crystallin knockout mice compared to wild-type mice. CONCLUSIONS: Therefore, αA-crystallin may protect against geographic atrophy. This study provides a better understanding of the relationship between cataract, cataract surgery, and GA.