DEFECTS IN DNA REPAIR GENES AND THEIR ASSOCIATION WITH SUSCEPTIBILITY OF AGE-RELATED CATARACT

Cataract is an age-related disorder and one of the leading causes of visual impairments worldwide. Progression of aging augmented due to oxidative stress which causes DNA damage. Hence, malfunctioning of DNA repair enzymes are hypothesized to contribute in the pathogenesis of age-related cataract (A

Project Title

DEFECTS IN DNA REPAIR GENES AND THEIR ASSOCIATION WITH SUSCEPTIBILITY OF AGE-RELATED CATARACT

Project Area of Specialization

Biomedical Engineering

Project Summary

Cataract is an age-related disorder and one of the leading causes of visual impairments worldwide. Progression of aging augmented due to oxidative stress which causes DNA damage. Hence, malfunctioning of DNA repair enzymes are hypothesized to contribute in the pathogenesis of age-related cataract (ARC). Many genes are involved in repair pathway, such as the X-ray cross-complementing group 4 (XRCC4) gene. XRCC4 is an important member of the non-homologous end-joining (NHEJ) repair system, and play a major role in the precision end joining of blunt DNA double strand breaks. XRCC4 gene has been associated with several types of cancer. Genetic variations in DNA repair XRCC4 gene could play a critical role in the diverse processes of ARC progression. Aim of this study is to investigate the role of XRCC4 intron 3 (rs28360071) and intron 7 (rs28360317) variants to the susceptibility of cataractogenesis in local population. It is a case-control study design comprised of approximately n=100 ARC cases and n=100 controls. DNA extraction will be performed using blood samples. Genotyping of respective XRCC4 insertion/deletion variants will be carried out using conventional polymerase chain reaction (PCR) and gel electrophoresis. Observed data will be analyzed by statistical and bioinformatics software. To the best of our knowledge, it is the first study to investigate the association of XRCC4 gene variants in age-related cataract. These genetic variants may increase the likelihood of genomic instability and lead to carcinogenesis with increasing age in ARC patients.

Project Objectives

• To investigate XRCC4 intron 3 (rs28360071) mutation in cataract patients and control groups.
• To examine XRCC4 intron 7 (rs28360317) mutation in cataract patients and control groups.
• To compare clinical variables between the two groups
• To perform association analysis between mutations and disease
• To determine linkage between XRCC4 intron 3 and intron 7 mutations in disease group.
• To evaluate relationship of XRCC4 genotypes and clinical variables.

Project Implementation Method

• DNA extraction: to extract whole genome from WBCs using blood samples of cases and controls.
• DNA quantitative analysis: to estimate concentration and purity of DNA samples
• DNA qualitative analysis: to evaluate integrity of isolated DNA through electrophoresis.
• Conventional polymerase chain reaction (PCR): for amplification and genotyping of XRCC4 intron 3 (rs28360071) mutation. This type of PCR is used for the detection of large fragent of DNA upto 30bp.
• Allele-specific polymerase chain reaction (PCR): for amplification and genotyping of XRCC4 intron 7 (rs28360317) mutation. This type of PCR is used for the detection of specific variations even in 3bp region.
• Gel electrophoresis: for resolving amplified products of both mutations on the basis of size and identification of specific genotypes.
• Data Analysis: observed data will be analyzed by statistical and bioinformatics software which include SPSS, MedCal, SNPstats, and Haploview respectively.

Benefits of the Project

XRCC4 gene play a key role in end-joining of non-homologous DNA fragments and its repair pathway. Alterations in DNA repair genes have been shown to reduce DNA repair efficacy thereby inflicting higher susceptibility to impairment in repairing process in patients with high level of oxidative stress. As the oxidative stress is majorly responsible for the dissociation of eye lens proteins and thus induces the formation of cataract. Therefore, the significance of present study is that it will investigate a potential genetic biomarker which could be used for early prognosis of cataract with the process of aging. This could contribute to the development of therapeutic strategies for delaying and preventing the process of cataract formation by keeping the lens proteins intact and healthy with adequate amount of antioxidants to fight against the oxidative stress in eye lens. It is a baseline study which will provide essential data in the form of biomarker which could be targeted in pharmacogenomics studies. Furthermore, the significant genetic biomarker could play an integral part in the implementation approach of gene therapy for targeted diseases in future.

Technical Details of Final Deliverable

This project involve the genetic investigation of two insertion/deletion mutations localized in intron region of 3-30 base pairs in one of the essential DNA repair enzyme XRCC4 gene of human genome. These mutation are situated in intergenic sequence of DNA therefore, it can affect the precise splicing of mRNA and thus could lead to the translation of nonfunctional variant of gene. The linkage disequilibrium will be utilize to analyze the linkage and pattern of co-inheritance during recombination event of cell division between the two targeted mutations with the pathogenicity of disease which are confined on the same chromosome. If these genetic mutations are frequently evident among the population of age related cataract patients in Pakistan then it can be concluded that they inferred an association with higher predisposition of cataract risk.

Final Deliverable of the Project

HW/SW integrated system

Core Industry

Health

Other Industries

Core Technology

Others

Other Technologies

Sustainable Development Goals

Good Health and Well-Being for People, Life on Land, Partnerships to achieve the Goal

Required Resources

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