Severity of familial isolated retinitis pigmentosa across different inheritance patterns among an Asian Indian cohort.

PURPOSE: To predict the progression to legal blindness in patients with isolated inherited retinitis pigmentosa. METHODS: This retrospective study evaluated patients with isolated inherited retinitis pigmentosa for age at onset, duration of the disease, and best-corrected visual acuity in an Asian Indian cohort. The Mann­Whitney U test was used to analyze the variables. RESULTS: Of 134 patients evaluated, 72% were autosomal recessive, 17% were autosomal dominant, and 11% were X-linked recessive pedigrees. Median age at onset was 8 years for X-linked recessive, 11 years for autosomal recessive, and 21 years for autosomal dominant disease, which was statistically significant. The refractive error due to axial myopia was significantly high in X-linked recessive (−3.50 diopter sphere) compared to autosomal recessive (−1 diopter sphere) and autosomal dominant (0.00 diopter sphere) cases (P < .000). Legal blindness occurred in 50% of X-linked recessive and autosomal recessive cases but in only 32% of autosomal dominant cases. CONCLUSION: Identifying the gene defects involved in this cohort will help understand the phenotype variability.

High glucose induced differential expression of lysyl oxidase and its isoform in ARPE-19 cells.

PURPOSE: Lysyl oxidase (LOX) stabilizes the extracellular matrix (ECM) by cross-linking collagen and elastin molecules. In proliferative diabetic retinopathy (PDR), there is ECM remodeling with neovascularization and basement membrane changes. While protease activities are well reported, the role of LOX in the pathogenesis of diabetic retinopathy is less studied. This study was done to see the effect of high glucose on the activity and expression of LOX and its isoforms in ARPE-19 cells. MATERIALS AND METHODS: ARPE-19 cells were exposed to high glucose up to 48 h, and LOX activity was determined by N-acetyl-3,7-dihydroxyphenoxazine assay. The mRNA expression of LOX and its isoforms was done by real-time PCR and the protein expression by ELISA. Immunohistochemistry for LOX was done in epiretinal membrane from PDR. RESULTS: With an increase in glucose concentration LOX activity and protein was reduced significantly at 30 mM glucose at 48 h. mRNA expression of LOX, LOXL1, and LOXL2 varied with time and concentration of glucose. Vascular endothelial growth factor (VEGF) increased the LOX activity as well as the mRNA expression. Pigment epithelium-derived factor (PEDF) downregulated the mRNA expression of LOX, LOXL1, and LOXL2. The matrix metalloprotease (MMP) activity increased significantly with the increase in glucose concentration. The diabetic neovascular membrane showed increased immunostaining of LOX. CONCLUSIONS: This study suggests that although the LOX activity, which is composite of all the isoforms, was reduced under high glucose conditions, there was a differential mRNA expression with increased LOX and LOXL1 and decreased LOXL2 expression.

Ranibizumab for choroidal neovascular membrane in a rare case of Bietti's crystalline dystrophy: a case report.

We report a rare case of Bietti's crystalline dystrophy presenting with choroidal neovascular membrane (CNVM) which was treated with three injections of intravitreal ranibizumab. The CNVM underwent scarring after the injections with stabilization of visual acuity at a follow-up period of 12 months suggesting that intravitreal ranibizumab may have a role in the management of CNVM in these rare cases.

Molecular screening of the CYP4V2 gene in Bietti crystalline dystrophy that is associated with choroidal neovascularization.

PURPOSE: Bietti crystalline dystrophy (BCD) is an autosomal recessive disease characterized by intraretinal deposits of multiple small crystals, with or without associated crystal deposits in the cornea. The disease is caused by mutation in the cytochrome p450, family 4, subfamily v, polypeptide 2 (CYP4V2) gene. Choroidal neovascularization (CNV) is a rare event in BCD. We report two cases of BCD associated with CNV. CYP4V2 and exon 5 of tissue inhibitor of metalloproteinase 3 (TIMP3) were screened in both cases. A patient with BCD, but without CNV, was also screened to identify pathogenic variations. METHODS: Three BCD families of Asian Indian origin were recruited after a comprehensive ophthalmic examination. Genomic DNA was isolated from blood leukocytes, and coding exons and flanking introns of CYP4V2 and exon 5 of TIMP3 were amplified via polymerase chain reaction (PCR) and were sequenced. Family segregation, control screening, and bioinformatics tools were used to assess the pathogenicity of the novel variations. RESULTS: Of the three BCD patients, two had parafoveal CNV. The patient with BCD, but without CNV had novel single base-pair duplication (c.1062_1063dupA). This mutation results in a structurally defective and unstable protein with impaired protein function. Four novel benign variations (three in exons and one in an intron) were observed in the cohort. Screening of exon 5 of TIMP3 did not reveal any variation in these families. CONCLUSIONS: A novel mutation was found in a patient with BCD but without CNV, while patients with BCD and CNV did not show any pathogenic variation. The modifier role of TIMP3 in the pathogenesis of CNV in BCD was partly ruled out, as no variation was observed in exon 5 of the gene. A larger BCD cohort with CNV needs to be studied and screened to understand the genetics of CNV in BCD.

CDKN1C (p57KIP2) mRNA expression in human retinoblastomas.

PURPOSE: Quantify cyclin dependent kinase inhibitor 1C (CDKN1C or p57KIP2) mRNA levels in human retinoblastoma tumors (RB) and associate with disease phenotype. METHODS: CDKN1C mRNA expression was quantified in 55 RB, 3 retinoblastoma cell lines, and 12 control retinas by real time PCR. Localization of CDKN1C protein was confirmed by immunohistochemistry. Tumor CDKN1C expression levels were correlated with phenotype. RESULTS: Fifty-three of 55 tumors showed significantly elevated levels of CDKN1C mRNA relative to age-matched or adult retina controls. No significant difference was seen relative to fetal retinal controls or retinoblastoma cell lines. Immunohistochemistry revealed heterogeneous staining of CDKN1C protein in tumor cells, which was mainly nuclear although some protein appeared to be cytoplasmic. No correlation was observed between the CDKN1C mRNA expression levels and tumor phenotype. CONCLUSION: High levels of CDKN1C expression are common in RB. It remains to be determined if elevated expression is a protective response to transformation, provides a selective advantage to tumor cells, or simply reflects the presence of dividing cells.

BBS7 and TTC8 (BBS8) mutations play a minor role in the mutational load of Bardet-Biedl syndrome in a multiethnic population.

Bardet Biedl syndrome is a genetically heterogeneous ciliopathy with fourteen genes currently identified. To date, mutations in BBS7 and TTC8 (BBS8) were reported in 4.2% and 2.8% of BBS families respectively. We sequenced the coding regions of BBS7 and TTC8 in 35 BBS families of diverse ancestral backgrounds. In addition, the role of putative modifier genes on phenotype severity; NXNL1 and MGC1203 c.430C>T, was assessed. Genotype-phenotype correlation was explored in patients with identified mutations. Four novel pathogenic BBS7 changes were identified in 2/35 families (5.7%). In one family with two affected individuals with BBS7 mutations, a more severe phenotype was observed in association with a third mutation in BBS4. The overall retinal phenotype appeared more severe than that seen in patients with BBS1 mutations. This study confirms the small role of BBS7 and TTC8 in the overall mutational load of BBS patients. The variability of the ocular phenotype observed, could not be explained by the putative modifier genes; NXNL1 and MGC1203 c.430C>T.

KIF14 and E2F3 mRNA expression in human retinoblastoma and its phenotype association.

PURPOSE: We quantified mRNA expression of candidate genes for proliferation (KIF14 and E2F3) in a large retinoblastoma tumor cohort and associated with disease phenotype. METHODS: KIF14 and E2F3 mRNA expression was quantified by real time PCR in 57 retinoblastoma (RB) tumors, 3 RB cell lines, and control samples that included 4 each fetal, age-matched, adult retinas. Immunohistochemistry was done to confirm KIF14 and E2F3 protein expression in tumor cells. The mRNA expression levels were correlated with disease phenotypes including the significance of chemotherapy on tumors. RESULTS: There was statistically significant overexpression of KIF14 and E2F3 mRNA in tumors compared with control retinas (p<0.0001). Further, E2F3 also showed a significant overexpression compared to RB cell lines (p=0.01). Immunohistochemistry confirmed KIF14 and E2F3 protein overexpression in tumor cells. KIF14 had significant mRNA overexpression with older age (p=0.01) in presenting patients and in unilateral RB patients (p=0.04). Chemotherapy-treated tumors showed a significant decrease in KIF14 and E2F3 expression compared to untreated tumors (p<0.01 and 0.001, respectively). CONCLUSIONS: This report confirms significant mRNA overexpression of KIF14 and E2F3 together in a large cohort of RB tumors. The decreased expression in chemotherapy treated cases needs further validation in a large chemotherapy-treated cohort.

Lysyl oxidase activity in the ocular tissues and the role of LOX in proliferative diabetic retinopathy and rhegmatogenous retinal detachment.

PURPOSE: Lysyl oxidase (LOX) cross-links the side chain of collagen and elastin and thereby contributes to extracellular matrix (ECM) integrity. ECM remodeling is seen in various ocular diseases. Until now, there have been no reports on the LOX enzyme's activity in ocular tissues. The purpose of this study was to estimate LOX activity and expression in human donor ocular tissues and to measure the specific activity of LOX in the vitreous of proliferative diabetic retinopathy (PDR) and rhegmatogenous retinal detachment (RRD). METHOD: Human donor eyeballs obtained from an eye bank were used to study tissue distribution of LOX. Human vitreous specimens were obtained during vitreoretinal surgery from PDR (n = 16) and RRD (n = 10). LOX activity was estimated by N-acetyl-3,7-dihydroxyphenoxazine assay, immunohistochemistry, and real-time polymerase chain reaction (RT-PCR). Matrix metalloprotease (MMP)-2 and -9 were quantified in the vitreous by sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: The specific activity of LOX in ocular tissues was on the order of vitreous, iris ciliary body, lens, choroid RPE, and retina, which were comparable by mRNA expression and immunolocalization. The vitreous level of LOX activity decreased significantly in PDR and RRD, with an increase in total MMP-2 and -9 levels compared with normal donor vitreous. CONCLUSIONS: LOX activity showed a statistically significant decrease in the vitreous of PDR and RRD relative to control specimens. This effect can contribute to the inadequate collagen cross-linking that causes the ECM changes that occur in these diseases.

Retinoblastoma: from disease to discovery.

Retinoblastoma has contributed much to the understanding of cancer. It provided the classic 'two-hit model' for oncogenesis and helped to identify the first tumor suppressor gene RB1. Thirty years since then, the search for additional events underlying disease progression continues. Phenotypic variations in retinoblastoma offer numerous clues to disease pathogenesis. Understanding their molecular biological basis will provide insight into mechanisms underlying tumor progression. These not fully understood genetic and stochastic events play a major role in uncontrolled retinal precursor cell proliferation. Comparative genomic hybridization and gene expression studies have facilitated probing of genes controlling basic events in cellular development, i.e. proliferation, differentiation and apoptosis. Research to determine the cell of origin that underlies the evolution of retinoblastoma can lead to understanding of the stochastic events underlying the genesis of this cancer, which currently remains unclear. In this review, we discuss the recent developments in retinoblastoma and describe how they are beginning to shape a new and revised picture of retinoblastoma pathogenesis and progression.

The relationship between tumor cell differentiation and age at diagnosis in retinoblastoma.

PURPOSE: To determine the relationship between tumor cell differentiation and age at diagnosis in retinoblastoma. METHODS: Medical records of 170 patients with retinoblastoma treated by enucleation were reviewed retrospectively. Age at diagnosis and histopathological features were analyzed. RESULTS: Well-differentiated tumors presented earlier than poorly differentiated tumors. The frequency of presentation was highest in the first year of age for well-differentiated tumors and in the third year of age for poorly differentiated tumors (P < .0001). Bilateral well-differentiated tumors presented earlier than bilateral poorly differentiated tumors. Similarly, unilateral well-differentiated tumors presented earlier than unilateral poorly differentiated tumors. CONCLUSIONS: Differentiated tumors present earlier than poorly differentiated tumors, irrespective of laterality in retinoblastoma.

High expression of KIF14 in retinoblastoma: association with older age at diagnosis.

PURPOSE: KIF14 a mitotic kinesin gene plays an important role in cytokinesis. Deregulation of KIF14 may be a pathway of tumor progression and results in decreased patient survival as seen in breast tumors. Recently, KIF14, a possible gene that drives gain of chromosome arm 1q (the most commonly gained chromosomal region in retinoblastoma), has been shown to be a strong oncogene candidate overexpressed by more than two orders of magnitude in retinoblastoma. This study was conducted to quantify the expression of KIF14 in human retinoblastoma tumors and correlate it with disease phenotype. METHODS: KIF14 expression was examined by using real-time RT-PCR in 30 retinoblastoma tumors with age at diagnosis between 3 and 68 months. Two 18-month-old, three adult (55-62 years), and three fetal (one 18 weeks' and another pooled retina of 18 and 20 weeks' gestation) retinas were used as the control. KIF14 expression was normalized to the housekeeping control gene TBP and compared with that in an 18-month-old control retina. The protein expression was confirmed in tumor cells by immunohistochemistry and phenotypic correlation was performed. RESULTS: KIF14 was expressed between 3- and 207-fold greater than 18-month-old retina in 30 retinoblastoma tumors (P < 0.0001). Immunohistochemistry revealed KIF14 localization to both nucleus and cytoplasm of tumor cells. KIF14 mRNA overexpression correlated significantly with older age at diagnosis (P = 0.006). There was no association with differentiation, invasion, or duration of the disease with KIF14 overexpression. CONCLUSIONS: Overexpression of KIF14 was confirmed in primary human retinoblastoma and showed that patients with an older age at diagnosis express significantly higher levels of KIF14.

Retinoblastoma in India : microsatellite analysis and its application in genetic counseling.

OBJECTIVES: This study was conducted with two objectives. The first was to estimate the frequency of loss of heterozygosity (LOH) of the RB1 gene as a mechanism in disease causation in tumors of patients from India. The second objective was to employ RB1 molecular deletion and microsatellite-based linkage analysis as laboratory tools, while counseling families with a history of retinoblastoma (RB). METHODS: DNA was extracted from peripheral blood and tumors of 54 RB patients and their relatives. Eight fluorescent microsatellite markers, both intragenic and flanking the RB1 gene, were used. After PCR amplification, samples were run on an ABI PRISM 310 genetic analyzer for LOH, deletion detection, and haplotype generation. RESULTS: LOH was found in conjunction with tumor formation in 72.9% of RB patients (39/54 patients; p=0.001; 95% CI 0.6028, 0.8417); however, we could not associate various other clinical parameters of RB patients with the presence or absence of RB1 LOH. Seven germline deletions (13% of RB patients) were identified, and the maternal allele was more frequently lost (p=0.01). A disease co-segregating haplotype was detected in two hereditary autosomal dominant cases. CONCLUSION: LOH of the RB1 gene could play an important role in tumor formation. Large deletions involving RB1 were observed, and a disease co-segregating haplotype was used for indirect genetic testing. This is the first report from India where molecular testing has been applied for RB families in conjunction with genetic counseling. In tertiary ophthalmic practice in India, there is an emerging trend towards the application of genetical knowledge in clinical practice.

Retinoblastoma: a diagnostic model for India.

PURPOSE: Molecular genetic diagnostics for retinoblastoma are prerequisite for accurate risk prediction and effective management. Developing a retinoblastoma diagnostic model to establish a flow for laboratory tests is thus a necessity for tertiary ophthalmic institutions. An efficient diagnostic model could reduce the overall health care costs, redirect the resources to the high risk group and also avoid unnecessary worry for families. To the best of our knowledge there has hitherto been no comprehensive diagnostic model for retinoblastoma implemented in any institution in India. METHODS AND DISCUSSION: The diagnostic model demonstrates the logical and practical flow of various genetics tests like karyotyping, loss of heterozygosity analysis, molecular deletion, linkage analysis (familial cases), mutation screening of -CGA exons first and then non-CGA exons, methylation screening of RB1 and essential promoter regions screening in a laboratory. CONCLUSIONS: The diagnostic model proposed offers acomprehensive methodology to identify the causative two-hits for retinoblastomas that could be used while genetic counseling families. This model is applicable in tertiary hospitals in India and neighboring countries, which have the highest incidence of retinoblastoma and fertility rates in the world. We suggest that this diagnostic model could also be applied with modification for other cancers.