RESUMO
PURPOSE: Retinoschisis is a distinctive condition characterized by intraretinal layer clefts, primarily associated with X-linked recessive inheritance due to RS1 gene mutations. This study aims to uncover the RS1 mutation spectrum in a cohort of 22 X-linked retinoschisis cases from South India and emphasizes the genotypic and phenotypic associations within patients harboring only RS1 mutations. METHODS: A total of 22 probands were suspected of having X-linked retinoschisis. All study subjects underwent ophthalmic investigations, including assessments of visual acuity, fundus examination, optical coherence tomography (OCT), and electroretinogram (ERG). RS1 gene screening was conducted using Sanger sequencing, and the pathogenicity of the variants was assessed through Sorting Intolerant from Tolerant (SIFT) and PolyPhen-2 in silico tools. RESULTS: The study found that the probands had an average visual acuity of 0.79 ± 0.39 log of minimum angle of resolution (logMAR), ranging from 0.17 to 1.77. During fundus examination, the probands exhibited a characteristic spoke wheel-like pattern in the macular region. Furthermore, OCT analysis revealed distinct alterations in the inner retinal microstructure, and ERG results consistently showed a reduction in b-wave amplitude. Eventually, Sanger sequencing results showed hemizygous mutations in the RS1 gene in only 12 probands, including a novel missense mutation in the RS1 gene's signal sequence. CONCLUSION: This study provides valuable insights into the spectrum of RS1 mutations in X-linked retinoschisis probands from South India. It reveals distinct genotypic-phenotypic associations and highlights the clinical manifestations associated with the disease pathogenesis.
Assuntos
Proteínas do Olho , Genótipo , Mutação , Fenótipo , Retinosquise , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , DNA/genética , Análise Mutacional de DNA , Eletrorretinografia , Proteínas do Olho/genética , Índia , Linhagem , Retina/diagnóstico por imagem , Retina/patologia , Retinosquise/diagnóstico , Retinosquise/genética , Retinosquise/patologia , Tomografia de Coerência Óptica , Acuidade Visual/fisiologiaRESUMO
Background: Familial exudative vitreoretinopathy (FEVR) is an inheritable retinal vascular disease, which often leads to severe vision loss and blindness in children. However, reported mutations can only account for 50-60% of patients with FEVR. The purpose of this study was to identify novel frizzled class receptor 4 (FZD4) and Norrin cystine knot growth factor NDP (NDP) mutations in a cohort of Indian patients with FEVR by whole-exome sequencing. Methods: We performed data filtering and bioinformatic analyses. Results: Two novel heterozygous mutations in FZD4 gene were identified, each in two different families: c.1499_1500del [p.500_500del] and c.G296C [p.C99S]. One novel mutation in NDP in another family was identified: c.A256G [p.K86E]. All FZD4 mutations affected conserved amino acid residues and were absent in 1000 control individuals. To assess the effect of these FZD4 mutations on the biological activity of the protein, we introduced each FZD4 mutation into FZD4 cDNA by the site-directed mutagenesis techniques. A Norrin/beta-catenin pathway-based luciferase reporter assay revealed that the c.1499_1500del failed to activate the luciferase reporter; in contrast, compared with the wild-type FZD4 protein, the, c.G296C [p.C99S] mutation exhibited increased luciferase reporter activity. Conclusion: Our study found two novel FZD4 mutations, with opposite effects regarding functional expression levels in Indian patients with FEVR and expands on the mutational spectrum of FZD4 in Indian FEVR patients.
Assuntos
Proteínas do Olho/genética , Vitreorretinopatias Exsudativas Familiares/genética , Receptores Frizzled/genética , Mutação , Proteínas do Tecido Nervoso/genética , Criança , Feminino , Humanos , Índia , MasculinoRESUMO
PURPOSE: Primary open-angle glaucoma (POAG) is the leading cause of irreversible blindness worldwide and mutations in known genes can only explain 5-6% of POAG. This study was conducted to identify novel POAG-causing genes and explore the pathogenesis of this disease. METHODS: Exome sequencing was performed in a Han Chinese cohort comprising 398 sporadic cases with POAG and 2010 controls, followed by replication studies by Sanger sequencing. A heterozygous Ramp2 knockout mouse model was generated for in vivo functional study. RESULTS: Using exome sequencing analysis and replication studies, we identified pathogenic variants in receptor activity-modifying protein 2 (RAMP2) within three genetically diverse populations (Han Chinese, German, and Indian). Six heterozygous RAMP2 pathogenic variants (Glu39Asp, Glu54Lys, Phe103Ser, Asn113Lysfs*10, Glu143Lys, and Ser171Arg) were identified among 16 of 4763 POAG patients, whereas no variants were detected in any exon of RAMP2 in 10,953 control individuals. Mutant RAMP2s aggregated in transfected cells and resulted in damage to the AM-RAMP2/CRLR-cAMP signaling pathway. Ablation of one Ramp2 allele led to cAMP reduction and retinal ganglion cell death in mice. CONCLUSION: This study demonstrated that disruption of RAMP2/CRLR-cAMP axis could cause POAG and identified a potential therapeutic intervention for POAG.
Assuntos
Glaucoma de Ângulo Aberto/genética , Proteína 2 Modificadora da Atividade de Receptores/genética , Animais , Povo Asiático , Células COS , Proteína Semelhante a Receptor de Calcitonina/genética , Proteína Semelhante a Receptor de Calcitonina/metabolismo , China , Chlorocebus aethiops , Estudos de Coortes , AMP Cíclico/genética , Predisposição Genética para Doença/genética , Glaucoma de Ângulo Aberto/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Mutação/genética , Linhagem , Polimorfismo de Nucleotídeo Único , Proteína 2 Modificadora da Atividade de Receptores/metabolismo , Sequenciamento do Exoma/métodosRESUMO
BACKGROUND: Cataract is a major health burden in many countries and a significant problem in India. While observational studies show lower cataract risk with increasing dietary or plasma vitamin C, randomised controlled trials of supplements have been negative. Genetic variants in vitamin C transporter proteins (SLC23A1), especially rs33972313, may provide evidence on a causal association of vitamin C with cataract. METHODS: We used data from a randomly selected population-based study in people aged 60 years and above in north and south India. Of 7518 sampled, 5428 (72%) were interviewed for socioeconomic and lifestyle factors, attended hospital for lens imaging and blood collection and were subsequently genotyped for rs33972313 and rs6596473. Mixed or pure types of cataract were graded by the Lens Opacity Classification System III as nuclear (2404), cortical (494) or posterior subcapsular cataract (PSC) (1026); 1462 had no significant cataract and no history of cataract surgery and 775 had bilateral aphakia/pseudophakia. RESULTS: rs33972313 was associated with cortical (OR 2.16; 95% CI 1.34 to 3.49, p=0.002) and PSC (OR 1.68; 95% CI 1.06 to 2.65, p=0.03) but not with nuclear cataract. In analyses of pure cataracts, associations were found only between rs33972313 and pure cortical cataracts (OR 2.29; 95% CI 1.12 to 4.65, p=0.03) and with a standardised cortical opacity score. There was no association with rs6596473 and any cataract outcomes. CONCLUSIONS: Using an established genetic variant as a proxy for lifetime ascorbate concentrations, our results support a causal association of vitamin C with cataract.
Assuntos
Catarata/genética , Transportadores de Sódio Acoplados à Vitamina C/genética , Idoso , Feminino , Genótipo , Humanos , Índia , Estilo de Vida , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Fatores SocioeconômicosRESUMO
PURPOSE: To compare the levels of cytokines and growth factor in aqueous humor of the patients with chronic primary angle closure glaucoma (PACG) and cataract. METHODS: Aqueous humor samples were collected from 19 chronic PACG patients and compared with 14 nonglaucomatous controls presenting for cataract surgery. The levels of 27 cytokines and growth factors were measured in the aqueous samples using multiplex bead immunoassay and compared across groups. RESULTS: Significantly higher levels of interleukin (IL)-8 (p < 0.001), eotaxin (p < 0.001), interferon gamma-induced protein (IP)-10 (p < 0.001) and macrophage inflammatory protein-1-beta (MIP-1ß; p < 0.001) were observed in aqueous of chronic PACG patients compared to controls. In comparison to controls, significantly lower levels of IL-9 (p = 0.001), IL-17 (p < 0.001), tumor necrosis factor-alpha (TNF-α; p < 0.001), granulocyte-macrophage colony-stimulating factor (GM-CSF; p < 0.001), and IL-5 (p = 0.001) were observed in chronic PACG eyes. All other assayed cytokines-IL-1ß, interleukin-1 receptor antagonist (IL-1rα), IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, fibroblast growth factor-basic (FGF-basic), granulocyte colony-stimulating factor (G-CSF), monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1-alpha (MIP-1α), and vascular endothelial growth factor (VEGF) -showed no significant difference between the groups. CONCLUSIONS: These results suggest that the aqueous cytokine levels of chronic PACG eyes differ significantly from nonglaucomatous eyes. This is the first study reporting significantly increased levels of eotaxin, MIP-1ß, and IP-10 and lower levels of TNF-α, IL-5, IL-9, IL-17, and GM-CSF in chronic PACG patients, suggesting a plausible role of these inflammatory cytokines in its pathogenesis.
Assuntos
Humor Aquoso/metabolismo , Citocinas/metabolismo , Glaucoma de Ângulo Fechado/metabolismo , Imunoensaio/métodos , Idoso , Catarata/metabolismo , Doença Crônica , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Primary congenital glaucoma (PCG) is a severe ocular disorder that presents early in life. Cytochrome P4501B1 (CYP1B1) and latent transforming growth factor-beta-binding protein 2 (LTBP2) are the most commonly mutated genes in PCG. AIM: To investigate the causative genetic mutations in eight Indian families with PCG. MATERIALS AND METHODS: Whole-exome sequencing was applied to analyze the genomic DNA samples from PCG probands. Sanger sequencing was utilized to confirm the identified mutations. RESULTS: We identified four homozygous missense mutations (c.1405C>T, p.R469W; c.1397G>T, p.G466V; c.1198C>T, p.P400S; and c.1103G>A, p.R368H) in CYP1B1 and one nonsense mutation (c.2421G>A, p.W807X) in LTBP2 in eight Indian families. Among the five mutations identified, G466V in CYP1B1 and W807X in LTBP2 represent novel mutations. CONCLUSIONS: Our study expands the mutational spectrum of PCG in the Indian population.
Assuntos
Citocromo P-450 CYP1B1/genética , Glaucoma/genética , Proteínas de Ligação a TGF-beta Latente/genética , Adulto , Povo Asiático/genética , Criança , Pré-Escolar , Consanguinidade , Citocromo P-450 CYP1B1/metabolismo , Análise Mutacional de DNA , Proteínas do Olho/genética , Feminino , Estudos de Associação Genética , Estudo de Associação Genômica Ampla , Haplótipos , Humanos , Índia , Lactente , Proteínas de Ligação a TGF-beta Latente/metabolismo , Masculino , Mutação de Sentido Incorreto/genética , Linhagem , Polimorfismo de Nucleotídeo Único , Adulto JovemRESUMO
PURPOSE: The aim of this study was to explore the potential association of genetic variants across clusterin (CLU) and tumor necrosis factor-alpha (TNF-α) genes in South Indian individuals with pseudoexfoliation syndrome (PEXS) and pseudoexfoliation glaucoma (PEXG). MATERIALS AND METHODS: A total of 523 individuals including 299 unrelated cases (150 PEXS and 149 PEXG) and 224 age- and ethnically-matched healthy controls were recruited for genetic analysis. Six single-nucleotide polymorphisms (SNPs) including, five CLU SNPs (rs11136000, rs2279590, rs9331888, rs9331931, rs3087554) and one promoter SNP (rs1800629) of TNF-α were genotyped in all study subjects. Genotyping of CLU SNPs were performed using the TaqMan allelic discrimination assay while TNF-α SNP was genotyped using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis. Association analysis was performed by determining the distributions of genotype and allele frequencies, Hardy-Weinberg equilibrium, and chi-square p values and odds ratios as implemented in the Golden Helix SNP & Variation Suite (SVS). RESULTS: Five CLU SNPs did not show any significant differences in allele frequencies between patients and control subjects (rs3087554, p = 0.919, OR = 1.01, 95% CI: 0.77-1.33; rs2279590, p = 0.432, OR = 1.12, 95% CI: 0.84-1.51; rs9331931, p = 0.310, OR = 1.24, 95% CI: 0.81-1.89; rs11136000, p = 0.072, OR = 1.31, 95% CI: 0.97-1.76; rs9331888, p = 0.911, OR = 1.01, 95% CI: 0.78-1.31). The investigation of TNF-α SNP established a significant association with PEXS and PEXG (p = 0.042, OR = 0.61, 95% CI: 0.38-0.99). However, this association did not remain significant after Bonferroni correction. CONCLUSIONS: Our data suggest that genetic variants in CLU and TNF-α genes do not play a major role in the development of PEXS and PEXG in the South Indian population.
Assuntos
Clusterina/genética , Síndrome de Exfoliação/genética , Glaucoma/genética , Polimorfismo de Nucleotídeo Único , Fator de Necrose Tumoral alfa/genética , Idoso , Idoso de 80 Anos ou mais , Síndrome de Exfoliação/etnologia , Feminino , Frequência do Gene , Técnicas de Genotipagem , Glaucoma/etnologia , Humanos , Índia/epidemiologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Fragmento de Restrição , População Branca/etnologiaRESUMO
We have previously reported low concentrations of plasma ascorbate and low dietary vitamin C intake in the older Indian population and a strong inverse association of these with cataract. Little is known about ascorbate levels in aqueous humor and lens in populations habitually depleted of ascorbate and no studies in any setting have investigated whether genetic polymorphisms influence ascorbate levels in ocular tissues. Our objectives were to investigate relationships between ascorbate concentrations in plasma, aqueous humor and lens and whether these relationships are influenced by Single Nucleotide Polymorphisms (SNPs) in sodium-dependent vitamin C transporter genes (SLC23A1 and SLC23A2). We enrolled sixty patients (equal numbers of men and women, mean age 63 years) undergoing small incision cataract surgery in southern India. We measured ascorbate concentrations in plasma, aqueous humor and lens nucleus using high performance liquid chromatography. SLC23A1 SNPs (rs4257763, rs6596473) and SLC23A2 SNPs (rs1279683 and rs12479919) were genotyped using a TaqMan assay. Patients were interviewed for lifestyle factors which might influence ascorbate. Plasma vitamin C was normalized by a log10 transformation. Statistical analysis used linear regression with the slope of the within-subject associations estimated using beta (ß) coefficients. The ascorbate concentrations (µmol/L) were: plasma ascorbate, median and inter-quartile range (IQR), 15.2 (7.8, 34.5), mean (SD) of aqueous humor ascorbate, 1074 (545) and lens nucleus ascorbate, 0.42 (0.16) (µmol/g lens nucleus wet weight). Minimum allele frequencies were: rs1279683 (0.28), rs12479919 (0.30), rs659647 (0.48). Decreasing concentrations of ocular ascorbate from the common to the rare genotype were observed for rs6596473 and rs12479919. The per allele difference in aqueous humor ascorbate for rs6596473 was -217 µmol/L, p < 0.04 and a per allele difference in lens nucleus ascorbate of -0.085 µmol/g, p < 0.02 for rs12479919. The ß coefficients for the regression of log10 plasma ascorbate on aqueous humor ascorbate were higher for the GG genotype of rs6596473: GG, ß = 1460 compared to carriage of the C allele, CG, ß = 1059, CC, ß = 1132, p interaction = 0.1. In conclusion we found that compared to studies in well-nourished populations, ascorbate concentrations in the plasma, aqueous humor and lens nucleus were low. We present novel findings that polymorphisms in SLC23A1/2 genes influenced ascorbate concentration in aqueous humor and lens nucleus.
Assuntos
Humor Aquoso/química , Ácido Ascórbico/metabolismo , Catarata/genética , Núcleo do Cristalino/química , Plasma/química , Polimorfismo Genético , Transportadores de Sódio Acoplados à Vitamina C/genética , Adulto , Idoso , Alelos , Catarata/metabolismo , Cromatografia Líquida de Alta Pressão , DNA/genética , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Transportadores de Sódio Acoplados à Vitamina C/metabolismoRESUMO
BACKGROUND: Leber congenital amaurosis (LCA) is the most severe form of inherited retinal visual impairment in children. So far, mutations in more than 20 genes have been known to cause LCA and among them, RPE65 is a suitable candidate for gene therapy. The mutational screenings of RPE65 and other LCA genes are requisite in support of emerging gene specific therapy for LCA. Therefore, we have carried out a comprehensive LCA genes screening using a combined approach of direct sequencing and DNA microarray based Asper chip analysis. METHODOLOGY/PRINCIPAL FINDINGS: Thirty clinically diagnosed index LCA cases from Southern India were screened for coding and flanking intronic regions of RPE65 through direct sequencing. Among thirty, 25 cases excluded from RPE65 mutations were subjected to Asper chip analysis, testing 784 known pathogenic variations in 15 major LCA genes. In RPE65 screening, four different pathogenic variations including two novel (c.361insT & c.939T>A) and two known (c.394G>A & c.361delT) mutations were identified in five index cases. In the chip analysis, seven known pathogenic mutations were identified in six index cases, involving genes GUCY2D, RPGRIP1, AIPL1, CRX and IQCB1. Overall, 11 out of 30 LCA cases (36.6%) revealed pathogenic variations with the involvement of RPE65 (16.6%), GUCY2D (10%), RPGRIP1 (3.3%), AIPL1 (3.3%) and CRX & IQCB1 (3.3%). CONCLUSIONS/SIGNIFICANCE: Our study suggests that such combined screening approach is productive and cost-effective for mutation detection and can be applied in Indian LCA cohort for molecular diagnosis and genetic counselling.
Assuntos
Amaurose Congênita de Leber/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ligação a Calmodulina/genética , Proteínas de Transporte/genética , Biologia Computacional , Proteínas do Citoesqueleto , Proteínas do Olho/genética , Feminino , Guanilato Ciclase/genética , Humanos , Índia , Masculino , Mutação , Proteínas/genética , Receptores de Superfície Celular/genética , cis-trans-Isomerases/genéticaRESUMO
Retinal dystrophies are predominantly caused by mutations affecting the visual phototransduction system and cilia, with few genes identified that function to maintain photoreceptor survival. We reasoned that growth factors involved with early embryonic retinal development would represent excellent candidates for such diseases. Here we show that mutations in the transforming growth factor-ß (TGF-ß) ligand Growth Differentiation Factor 6, which specifies the dorso-ventral retinal axis, contribute to Leber congenital amaurosis. Furthermore, deficiency of gdf6 results in photoreceptor degeneration, so demonstrating a connection between Gdf6 signaling and photoreceptor survival. In addition, in both murine and zebrafish mutant models, we observe retinal apoptosis, a characteristic feature of human retinal dystrophies. Treatment of gdf6-deficient zebrafish embryos with a novel aminopropyl carbazole, P7C3, rescued the retinal apoptosis without evidence of toxicity. These findings implicate for the first time perturbed TGF-ß signaling in the genesis of retinal dystrophies, support the study of related morphogenetic genes for comparable roles in retinal disease and may offer additional therapeutic opportunities for genetically heterogeneous disorders presently only treatable with gene therapy.
Assuntos
Sobrevivência Celular , Fator 6 de Diferenciação de Crescimento/genética , Amaurose Congênita de Leber/genética , Retinose Pigmentar/genética , Sequência de Aminoácidos , Animais , Apoptose , Análise Mutacional de DNA , Estudos de Associação Genética , Fator 6 de Diferenciação de Crescimento/fisiologia , Humanos , Amaurose Congênita de Leber/patologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Linhagem , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/fisiologia , Retina/patologia , Retinose Pigmentar/patologia , Peixe-ZebraRESUMO
PURPOSE: The purposes of this study were to describe the clinical characteristics of corneal patients with mutations in the SLC4A11 gene and to determine if these characteristics could be correlated with specific genetic mutations. METHODS: A retrospective case series review was conducted. Baseline demographic data, including gender, age at diagnosis of congenital hereditary endothelial dystrophy, family history, and pedigree information, were obtained. Information from clinical examination, including intraocular pressure, ultrasonic pachymetry, best spectacle-corrected visual acuity, axial length, and slit-lamp biomicroscopic evaluation, including corneal diameter and fundus examination, were also documented from the notes. History of corneal surgery was also recorded. Hearing loss was assessed by audiometry. Genetic analysis was performed by polymerase chain reaction amplification and sequencing. RESULTS: Seven patients were identified. Four of the seven had associated hearing loss; all of the patients had undergone or were awaiting penetrating keratoplasty to one or both eyes. No correlation could be reached between the ocular phenotype and the gene mutation in this small sample. Individuals with the same mutation had different degrees of hearing loss within their respective families. CONCLUSIONS: Corneal endothelial cells are more vulnerable to defects in the functional activity of SLC4A11 than cells of the striae vascularis of the inner ear. Both congenital hereditary endothelial dystrophy 2 and Harboyan syndrome have similar ocular phenotypes, ie, diffuse bilateral corneal edema present at birth or within the neonatal period; hence, audiometry must be performed to differentiate the two conditions.
Assuntos
Proteínas de Transporte de Ânions/genética , Antiporters/genética , Distrofias Hereditárias da Córnea/diagnóstico , Distrofias Hereditárias da Córnea/genética , Endotélio Corneano/anormalidades , Perda Auditiva Neurossensorial/genética , Mutação , Adolescente , Audiometria , Criança , Pré-Escolar , Distrofias Hereditárias da Córnea/cirurgia , Endotélio Corneano/patologia , Feminino , Genótipo , Perda Auditiva Neurossensorial/diagnóstico , Humanos , Ceratoplastia Penetrante , Masculino , Linhagem , Fenótipo , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
The blepharophimosis syndrome (BPES) is an autosomal dominant developmental disorder in which craniofacial/eyelid malformations are associated (type I) or not (type II) with premature ovarian failure (POF). Mutations in the FOXL2 gene, encoding a forkhead transcription factor, are responsible for both types of BPES. Heterozygous polyalanine expansions of +10 residues (FOXL2-Ala24) account for 30% of FOXL2 mutations and are fully penetrant for the eyelid phenotype. Here we describe the first homozygous FOXL2 mutation leading to a polyalanine expansion of +5 residues (FOXL2-Ala19). This novel mutation segregates in an Indian family where heterozygous mutation carriers are unaffected whereas homozygous individuals have the typical BPES phenotype, with proven POF in one female. Expression of the FOXL2-Ala19 protein in COS-7 cells revealed a significantly higher cytoplasmic retention compared to the wild-type protein. This is the first study providing genetic evidence for a recessive inheritance of BPES associated with ovarian dysfunction.
Assuntos
Blefarofimose/genética , Expansão das Repetições de DNA/genética , Fatores de Transcrição Forkhead/genética , Genes Recessivos , Peptídeos/genética , Insuficiência Ovariana Primária/genética , Adulto , Animais , Blefarofimose/fisiopatologia , Células COS , Chlorocebus aethiops , Feminino , Proteína Forkhead Box L2 , Humanos , Índia , Masculino , Mutação , Linhagem , Insuficiência Ovariana Primária/fisiopatologia , SíndromeRESUMO
PURPOSE: To identify novel mutations in FZD4 gene that cause familial exudative vitreoretinopathy (FEVR) in Indian patients. METHODS: The study was conducted on 75 subjects from 53 Indian families. These families were clinically diagnosed to have FEVR by fundus examination and fluorescein angiography. The candidate gene FZD4 was amplified from genomic DNA and PCR products were screened for mutations by single strand conformational polymorphism (PCR-SSCP), TA-cloning followed by bi-directional sequencing. RESULTS: For the FZD4 exonic region, three mutations were identified, including two novel sequence variations (C204R, F82fsX135) and one reported (P33S) mutation. These sequence changes were not observed in 100 normal controls and clinically unaffected family members analyzed. CONCLUSIONS: Mutations in FZD4 were observed in 5.6% of the clinically diagnosed FEVR, in the studied Indian population. The identified genetic variations of FZD4 could play a vital role in pathogenesis and provide greater insight in to the genotype/phenotypic functions of FZD4 gene.
Assuntos
Povo Asiático/genética , Exsudatos e Transudatos/metabolismo , Oftalmopatias/genética , Receptores Frizzled/genética , Mutação , Receptores Acoplados a Proteínas G/genética , Doenças Retinianas/genética , Corpo Vítreo , Adolescente , Adulto , Sequência de Aminoácidos , Arginina , Cisteína , Éxons , Oftalmopatias/diagnóstico , Oftalmopatias/metabolismo , Angiofluoresceinografia , Fundo de Olho , Humanos , Índia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Fenilalanina , Prolina , Doenças Retinianas/diagnóstico , Doenças Retinianas/metabolismo , SerinaRESUMO
PURPOSE: Vascular endothelial growth factor (VEGF) is a major mediator of angiogenesis, and nitric oxide (NO) is an upstream and downstream regulator of VEGF mediated angiogenesis. VEGF and NO have been suggested to play an important role in the pathogenesis of microvascular complications in diabetic retinopathy (DR). The objective of this study was to examine the genetic variations of the VEGF and eNOS gene and assess their possible relationship to DR in type 2 diabetic patients in the Indian population. METHODS: In this study, 210 unrelated patients were enrolled and categorized into two study groups: a DR group, consisting of patients with proliferative diabetic retinopathy, and a diabetic without retinopathy (DWR) group comprised of patients with type 2 diabetes of more than 15 years duration who showed no signs of DR or had fewer than five dots or blot hemorrhages. Association of the genetic polymorphisms in the promoter and 5' UTR region of VEGF and the intron4 region of eNOS were studied. Total genomic DNA was isolated from peripheral blood leukocytes. PCR-RFLP analysis was performed for all samples to evaluate the genotypes. The distributions of the genotypes were compared using the chi2 test. Haplotype estimation and multiple logistic regression analysis were carried out to analyze the significance of polymorphisms. RESULTS: We investigated four reported polymorphisms in the VEGF (5' UTR, promoter) and one reported polymorphism (intron 4) in the eNOS gene in Type 2 diabetes patients with (n=120) and without (n=90) retinopathy. The genotype distribution of the C(-7)T, T(-1498)C, and C(-634)G polymorphisms of VEGF differed significantly between patients with DR and DWR (p=0.001, p=0.0001, and p=0.021, respectively). Allele C in the -1498 region (p=0.0001) and T in -7 region (p=0.002) were also found to be significantly increased in patients with retinopathy. Calculated odds ratios (OR) for three heterozygous genotypes of C(-7)T, T(-1498)C, and C(-634)G regions were 4.17 (95% CI: 1.90-9.18, p=0.0001), 4.37 (95% CI: 2.44-7.84, p=0.0001), and 2.33 (95% CI: 1.24-4.36, p=0.008), respectively, and was found to be significantly higher in the DR group when compared with the DWR group. Multiple logistic regression analysis revealed that the nongenetic parameters, age (p=0.024) and duration of diabetes (p=0.009), and the genetic parameters, like VEGF C(-7)T (p=0.002) and T(-1498)C (p=0.001) polymorphisms, were significantly associated with DR. The frequencies of haplotype consisting of the majority of alleles in VEGF were found to be significantly associated with DR. The genotype distribution of eNOS did not differ significantly between the two study groups, and therefore the eNOS intron 4 polymorphism was considered to be less significant. CONCLUSIONS: This is the first study to report VEGF and eNOS gene polymorphisms in patients with DR in the Indian population. The data suggest that the polymorphisms in the 5' UTR and promoter region of VEGF could be regarded as a major genetic risk factor for DR.