NUCLEOTIDE SEQUENCES OF mtDNA ENCODING 16S rRNA AND tRNALeu FROM DIABETES MELLITUS AND CATARACT PATIENTS

• Main Author: Ubyaan, Richardo
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/35483
• Institution: Institut Teknologi Bandung

Mitochondrial disease can cause various disfunction both in morphology and genetics and various other biochemical functions. In 1988, the first disease caused by mutations in mitochondrial DNA (mtDNA) has been found. Since then, more than a dozen different diseases as a result of human mtDNA mutations have been reported. Some of the symptoms of abnormalities generate mitochondrial disorders, especially in the brain, muscle, liver, eyesight or hearing. mtDNA mutations are also associated with abnormal cells, such as diabetes mellitus (DM) and myopathy. Abnormal cell death leads to a multisystem disease. Another phenomenon is a symptom caused by a variety of the same mutation in different individuals or different mutations result in the same phenotype. Cataracts are often found as a secondary disease among DM patients. It is due to sorbitol accumulation in the lens of the eye. Based on the clinical phenotype data MITOMAP, there are several nucleotide mutation leading to diabetes; C1310T, A1438G, A3243G, T3264C, T3271C, G3316A, and A12026G. The molecular studies of mtDNA mutations as the causative agent of diabetes mellitus associated with cataracts have not been performed. Therefore, whether diabetic and cataract phenotypes are associated with abnormalities of mtDNA genotypes will be investigated in this study. This research is a small part of the human mtDNA genome studies related to diabetes and cataracts. The samples used in this research were urine of pure cataract and DM patients and the lens of the eye of a DM with cataract patient obtained from RSM Cicendo Bandung and RSCM Jakarta. The strategy of research started with amplification of 16S rRNA and tRNALeu genes of mtDNA by PCR, sequenced by Dideoxy Sanger using the Automatic DNA Sequencer method, and in silico analysis using the DNASTAR programs and macros of microsoft office excel. Amplification by PCR using two primers, namely Bfor and Brev with their respective sizes of 18 and 19 nucleotides. Primers for sequencing were Bfor, Brev, and 5F. Electrophoresis of amplified DNA fragments produced one band with approximately size 0f 2 kb. The numbers of nucleotides obtained for DM accompanied by cataracts, DM, and cataracts phenotypes are 1875, 955, and 1878 nucleotides, respectively. The nucleotide sequences were then analyzed using the DNASTAR program. The nucleotide numbers of three samples were 952, with the revised human mtDNA sequence (rCRS) as a reference. All phenotypes showed the differences in mtDNA sequences compared to the rCRS reference. DM and cataract phenotypes are different at the same point, namely a2706G and t3027C. For diabetic with cataract, the difference is only at a2706G. These results have been confirmed with calculation using microsoft office excel. All mutation results were also subjected to the MITOMAP database (updated May 16, 2010). Mutation of A2706G and t3027C have been reported as polymorphism in the gene encoding region of mtDNA. Based on these results, the cause of diabetes mellitus associated with cataracts might be on the other gene, which are probably the tRNAIle, tRNALys, tRNASer, ND1, and ND4.