The Kallam Anji Reddy Molecular Genetics Laboratory, established with the generous support of Dr K Anji Reddy of Dr Reddy's Laboratories, Hyderabad, conducts research leading to the understanding of underlying molecular mechanisms in various inherited eye disorders. This laboratory has facilities for advanced research in molecular genetics and molecular biology and is equipped with thermal cyclers, gel documentation units, spectrophotometer, electrophoresis equipment, dHPLC - a Real Time PCR and two automated DNA sequencers.
The primary focus lies in identifying and characterizing the genes involved in ocular disorders, understanding their mutation spectrum, screening SNPs that could be potential risk factors for the disease condition and generating haplotypes to understand the origin, evolution and differentiation of the mutations. Based on this an attempt is made towards establishing a genotype-phenotype correlation that may be useful to monitor prognosis. Identification of gene mutations and variants can further lead to devising molecular diagnostics for rapid screening and early intervention. In addition, functional studies are also being undertaken by cloning and expressing the disease-causing genes, and studying the resultant proteins so as to determine the molecular phenotype based on the function of the proteins and their interactions.
Kallam Anji Reddy Molecular Genetics Laboratory And Champalimaud Translational Centre For Eye Research (C-TRACER)
Genomics Of Familial Exudative Vitreoretinopathy (FEVR)
Familial exudative vitreoretinopathy (FEVR, MIM # 133780) is a vasoproliferative disorder of the retina resulting from ischemia due to non-formation of peripheral retinal vasculature. The defects in candidate genes, which encode for a ligand (NDP) and receptor complex (FZD4, LRP5 and TSPAN12) involved in the Norrin β-catenin signaling pathway, have been associated with the disease pathogenesis. The present study aimed at understanding the role of these genes in FEVR patients from Southern India. The screening of NDP, FZD4 and TSPAN12 genes revealed 24 different mutations in 27 /115 FEVR families (24%). Additionally 3 novel and 6 reported single nucleotide polymorphisms (SNP) were detected. Among the observed mutations, 16 mutations were novel, including missense (n=8), frame shift (n=4) and 3’ UTR (n=4) changes. The missense mutations were mainly located in the domains, which are functionally crucial in formation of the ligand-receptor complex. The NDP, FZD4 and TSPAN12 gene mutations were identified in 10%, 8% and 7% of the patients, respectively, indicating their potential role in FEVR pathogenesis. The observed mutations segregated with the disease phenotype with variable expressivity.
Genetic And Phenotypic Comparison Of Primary Congenital Glaucoma In India And Brazil
Investigators: Subhabrata Chakrabarti, Anil K Mandal, Ivan M Tavares,1 Jose P C Vasconcellos,2 Monica Mello2
Primary congenital glaucoma (PCG) is largely attributed to mutations in the CYP1B1 gene resulting in irreversible blindness across children in the developing world. While the mutation frequencies widely vary across populations, there are subtle commonalities with respect to the prevalent mutation that occurs on a uniform genetic signature (haplotype). We looked for clinical indicators for common and local mutations in CYP1B1 across PCG patients in India and Brazil for undertaking a genotype-phenotype correlation. The initial results indicate that gender, consanguinity and presenting cup to disk ratios (CDR) among the Brazilian patients were strongly associated with the presence of a mutation. On the other hand, presenting IOP, CDR and family history conferred the maximum risk in PCG patients harboring a mutation in the Indian patients.
Central India Eye And Medical Diseases Study
Investigators: Subhabrata Chakrabarti, Jonas Jost,3 Vinay Nangia,4 Kollu N Rao, Meha Kabra, Inderjeet Kaur
Support: University of Heidelberg, Germany
The Central India Eye and Medical Diseases Study (CIEMS) is a clinical and population genomic study in a rural central Indian region (Nagpur) on 5000 individuals over 40 years of age. It aims to dissect the molecular mechanisms underlying certain quantitative traits associated with various ocular and systemic diseases. Initial epidemiological study have provided the prevalence and etiology of various conditions. Genomic profiling for risk factors for early AMD in this cohort have led to dissecting the role of common variants in the disease. It was also observed that none of the genomic variants conferred any additional risk to the clinical traits. Further, the molecular mechanism of these quantitative traits are being characterized by next generation sequencing, which will be followed up with dissecting the gene-environment interactions to devise means for predictive testing.
Genetic Comparison Of Primary Congenital Glaucoma In India And Tunisia – Understanding The Possible Founder Effects
Investigators: Subhabrata Chakrabarti, Anil K Mandal, Guemira Fethi5, E A Mhd Ali6 , Douik Hayet6, G Abderraouf 7, Harzallah Latifa7, Jihene Bouassida7
Support: Department of Science and Technology, India (Indo-Tunisia collaborative program)
Primary congenital glaucoma (PCG), attributed to mutations in the CYP1B1 gene, affects different populations worldwide. These mutations exhibit a strong geographical clustering, based on a uniform haplotype background, as evidenced from the mutation spectrum among PCG patients in Morocco, Saudi Arabia and India. So far, there are no reports on the genetics of PCG in Tunisia. In the present study, we estimated the overall mutation spectrum of CYP1B1 in Tunisia. Further, we hypothesized that common and prevalent mutations, such as G61E and 4340delG among Tunisian patients may have a strong founder effect, as seen elsewhere. The current analysis addressed the similarities and dissimilarities in the genetic basis of PCG in Tunisia and India along with their origin and migration. We also established the founder effect for the G61E mutation with Saudi Arabians and Indians and 4340delG with other populations (Brazilians) and its diversification worldwide.
Vision Institute, Federal University of Sao Paolo, Brazil
Campinas State University, Sao Paolo, Bra
University of Heidelberg, Manheim, Germany
Suraj Eye Institute, Nagpur
Institut Salah Azaiz, Tunis, Tunisia
Service d'Ophthalmologie, Habib Thameur Hospital, Tunis, Tunisia
Service de Biologie Clinique, Institut Salah Azaiz, Tunis, Tunisia
Runs Of Homozygosity For Identification Of New Genes In Primary Congenital Glaucoma
Support: Indo-Australia Biotechnology Fund, Department of Biotechnology, India
While CYP1B1 happens to be the major gene in primary congenital glaucoma (PCG), it accounts for only 44% of all cases in India. The other gene (LTBP2) does not exhibit any major involvement in these cases. Thus, in order to discover the other causative genes in the remaining cases that were devoid of mutations in CYP1B1 and LTBP2, we used a microarray based approach using 1.8 million variants (Affymetrix) to unfold large regions of shared homozygosity in patients. Shared haplotypes across extended homozygous regions on different chromosomes that were unique to patients were searched using certain novel alogorithms. Using this approach, we identified two extended regions of homozygosity that harbored two potential candidate genes on chromosomes 19 and 20. These genes are now being characterized by resequencing for causal mutations in PCG.
Functional Genomics Of Primary Open Angle Glaucoma(POAG)
Investigators: Subhabrata Chakrabarti, Kollu N Rao, Meha Kabra, Farheen Amman, Anil K Mandal, Sirisha Senthil, Harsha BL Rao, Garudadri Chandra Sekhar, Muralidhara Ramappa, Virender S Sangwan, Rohit C Khanna, Inderjeet Kaur
Support: Department of Biotechnology, India
Based on global gene expression analysis of 45,000 genes and gene targets in our primary open angle glaucoma (POAG) cohort, we have selectively screened 40 candidate genes that are involved in two distinct pathways, namely, the complement activations and the extracellular matrix (ECM) regulations. A combined analysis of the genes and their quantifications in the aqueous humor revealed no apparent involvement of the complement activation genes. But specific ECM-associated genotypes/haplotypes exhibited a strong association with POAG that was also correlated with the protein levels in the aqueous humor. Further validation on immunohistochemistry indicated their localization in the relevant human tissues. Further these associations were analyzed against the background of responses to therapy and it was seen that patients with severe phenotypes exhibited the strongest associations of the ECM-related genes.
Identification Of Biomarkers For Risk Prediction And Disease Progression In Retinopathy Of Prematurity (ROP): A Potentially Blinding Neonatal Disorder
Retinopathy of prematurity (ROP) is a self-limiting, vascular vitreoretinopathy that affects premature infants with short gestational age and low birth-weight and remains a leading cause (5-8%) of vision impairment and blindness in children There is a great need for a comprehensive research work on neovascular eye disease which might result in identifying predictive markers for severe ROP and anti-angiogenic approaches that will arrest the development of ROP by specifically targeting the involved molecular mediators. The present study aims to dissect the underlying molecular mechanism in ROP through a combination of molecular genetics, transcriptomics and proteomics based approaches, which would then be assessed with clinical parameters to understand the genotype-phenotype correlation. Proteomic analysis of vitreous humor and plasma samples of ROP patients is being undertaken at the Centre for Cellular and Molecular Biology (CCMB), Hyderabad. The expected outcome of this research would be the identification of biomarkers for ROP that could be eventually helpful in developing a diagnostic test for risk prediction. Timely detection and molecular diagnosis of ROP would also help in better prognosis of the disease and avoid the complete visual loss among the preterm babies.
Identification Of Genomic Regions For The Detection And Prediction Of High Risk Tumors In Uveal Melanoma (UM)
Support: National Institute of Biomedical Genomics, Kalyani, India
Uveal melanoma (UM) is the most common intraocular malignant tumor, with an incidence of approximately six cases per million per year in the Caucasian population. It shows a high propensity (in 90% of cases) to metastasize to the liver. Its prognosis is poor, with a survival of approximately 50% at 10 to 15 years, despite successful treatment of the primary tumor. UM shares similarity with cutaneous melanoma (CM) as both are derived from melanocytes but show remarkable differences in tumorigenesis, mode of metastatic spread, genetic alterations, and therapeutic response. In this study, we wished to investigate the use of exome sequencing for the identification of regions of imbalance related to metastatic evolution in UM and for the search of genes involved in the development of this tumor. This is the first time that such a study has been carried out to find out somatic mutations in a Uveal Melanoma (UM) patient from India. Here, we have sequenced the whole exome of the UM patient by massively-parallel technology and identified somatic mutations in GNAQ and SF3B1 genes are to be involved in UM. In addition to that we have detected several other somatic mutations in few other genes which probably have a role in the development and progression of UM. Future studies on more such samples may show better identification of causal somatic mutations in the UM and better implications on the management of this cancer type in Indians.
Genetic Susceptibility To Contact Lens-Related Adverse Events
In order to understand the genetic susceptibility of the host to contact lens-related adverse events, specific immune regulatory genes comprising the interleukins, toll-like receptors and defensins are being analysed by customized genotyping microarray. The idea is to look for specific tag SNPs in the Indian cohort and determine the extent of genetic association. Further functional analysis would be carried out on subjects exhibiting the associated genotype/haplotype to determine the causal risk or protection conferred by these genes.
Molecular Mechanism Of Pathogenesis Of Glaucoma Caused By Mutation In Optineurin
Investigators: D Balasubramanian, Ghanshyam Swarup3, Subhabrata Chakrabarti
Support: Department of Biotechnology, India
Optineurin is a multifunctional protein involved in several functions such as vesicular trafficking from the Golgi to the plasma membrane, NF-kappaB regulation, signal transduction and gene expression. Mutations in optineurin are associated with glaucoma. However, functional alterations caused by mutations in optineurin are not known. Here, we have analyzed the role of optineurin in endocytic recycling and the effect of the severe disease-causing E50K mutant on this process. Our results show that the knockdown of optineurin impairs trafficking of transferrin receptor to the juxtanuclear region. Studies using cell lines transfected with the wild type and the E50K mutant cDNAs show that E50K impairs endocytic recycling of transferrin receptor as shown by enlarged recycling endosomes, slower dynamics of E50K vesicles and decreased transferrin uptake by the E50K-expressing cells. This impaired trafficking by the E50K mutant requires the function of its ubiquitin-binding domain. Compared to wild type optineurin, the E50K optineurin shows enhanced interaction and colocalization with transferrin receptor and Rab8. The velocity of Rab8 vesicles is reduced by co-expression of the E50K mutant. These results suggest that the E50K impairs trafficking at the recycling endosomes due to altered interactions with Rab8 and transferrin receptor. These results have implications for the pathogenesis of glaucoma caused by the E50K mutation because endocytic recycling is vital for maintaining homeostasis.
Molecular Biophysics Unit, Indian Institute of Science, Bangalore
Novarus Discoveries Pvt Ltd, Hyderabad 500 073
University of Miami Miller School of Medicine, Miami, FL, USA
Analysis Of The Molecular Properties Of Mutant Human Βγ-Crystallins Associated With Congenital Cataract.
Investigators: D. Balasubramanian, Venkata Pullarao Vendra, Garima Agarwal1, N. Srinivasan1, Sushil Chandani2, VenuTalla3
Support: Department of Biotechnology, India
Congenital cataract, associated with 28 mutations in γ-crystallins, occurs in two major phenotypes. About half of them cause nuclear cataract, while the other half cause milder peripheral progressive cataract. In order to understand this phenotypic dichotomy at the molecular level, we have studied the structural and aggregation features of representative mutations of human γ-crystallins, which are folded using a double domain structure containing a series of four highly stable “Greek key” motif, which offers structural compactness and high intrinsic stability against stress. How this Greek key-derived dense packing of these proteins in the eye lens translates into transparency is an issue of biological functional interest.
To this end, wild type and several representative mutant proteins were cloned, expressed and purified and their structural details and stability were compared in solution using spectroscopy and molecular modeling in silico.
Results revealed that those mutants wherein the Greek key is distorted display lowered solubility and structural stability, expose several buried residues to the surface, aggregate in vitro and in cellulo, and they are associated with nuclear cataract. In contrast, mutants where the motif is left undisturbed behave quite similar to the wild type molecule, and are associated with peripheral cataract.
We conclude that when a mutation distorts even one of the four Greek key motifs, the protein readily self-aggregates and precipitates, consistent with the phenotype of nuclear cataract, while mutations not affecting the motif display ‘native state aggregation’, leading to peripheral cataract, thus offering a protein structural rationale for the cataract phenotypic dichotomy.
Support: Champalimaud Foundation, Portugal, Department of Biotechnology, Govt of India
Retinal dystrophies are degenerative disorders of the retinal photoreceptors, resulting in irreversible blindness. They are caused by single gene defects and are extremely heterogeneous in genetic causation. Using a candidate gene approach with LCA, we tested the NMNAT1 gene (nicotinamide mononucleotide adenylyl transferase 1) in collaboration with Eric Pierce (Harvard Medical School, Boston) by screening 100 probands for pathogenic mutations. We found pathogenic changes in NMNAT1 in 4 out of 100 patients screened [Nat Genet 2012 Sep; 44:1040-5]. Further studies will help determine other associated LCA genes in Indian patient populations.
National Institute of Biomedical Genomics, Kalyani, West Bengal,
University of New South Wales, Australia,
Centre for Cellular and Molecular Biology, Hyderabad
Identification Of Genes Causing Autosomal Recessive Retinitis Pigmentosa
Support: Champalimaud Foundation, Portugal, Department of Biotechnology, Govt of India
Retinitis pigmentosa (RP) is one of the major forms of incurable blindness across the world. It is a genetic disorder that is inherited in different Mendelian and mitochondrial forms and is extremely genetically heterogeneous. We employed homozygosity screening on SNP arrays to localize and identify genes responsible for ARRP. Screening of several known candidates mapping to homozygous regions of the genome in 26 families revealed disease-associated mutations in 4 families. Novel mutations were identified in various genes including MFRP (membrane frizzled related protein), NR2E3 (nuclear receptor subfamily 2, group E, member 3), TULP1 (tubby like protein 1) in 3 separate families [Mol Vis 2012; 18:1165-1174]. In addition, we also detected a pathogenic missense change in the RPGRIP1 gene in one family. Further studies are in progress to identify genes for both dominant and recessive RP in Indian families.
Molecular Genetics Of Congenital Cataract
Investigators: Chitra Kannabiran, Surya Prakash G Ponnam
Support: Council of Scientific & Industrial Research, India Hyderabad Eye Research Foundation
Congenital hereditary cataracts arise in infancy or childhood and are genetically heterogeneous. We screened 40 families comprising 184 individuals for mutations in 10 different genes that have a known or potential pathogenic role in hereditary cataracts. Mutations were identified in the connexin genes (Cx46 and Cx50) including a homozygous missense mutation (Val196Met) in connexin 50 in one family having autosomal recessive cataract, and heterozygous missense changes in Cx46 and Cx50 in 3 families with dominant cataracts [Mol Vis 2013; 19:1141-1148]. Together with mutations in Cx50 and LIM2 genes detected earlier in our study, 6/40 families are found to have mutations in the selected genes in this study.