NUCLEOTIDE SEQUENCES OF mtDNA ENCODING GENES tRNA-Phe AND 12S rRNA WHICH HAS PURE CATARACT AND DIABETES MELLITUS TIPE II ASSOCIATED TO CATARACT PHENOTYPES

• Main Author: Ilhamuddin
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/35451
• Institution: Institut Teknologi Bandung

Cataract is the main cause of blindness, which can be formed as a primary disease (pure cataract) and as a secondary disease as a complication of sistemic diseases, such as Diabetes Mellitus (DM). Both of cataract and DM are still the major health problem in Indonesia. The increasing of morbidity and mortality due to DM, makes this problem worse. This affects human life quality. Ethiology and pathogenesis of those diseases are still investigated, including mitochondrial dysfunction as an energy producer (in a form of ATP). Decreasing of its function at the molecular level can be caused by mutations in mitochondrial DNA (mtDNA). Previous study shows that there are several various sequences of mtDNA of the pure cataract phenotype and the DM type II associated with cataract, compared with revised Cambridge Reference Sequence (rCRS). Generally, these differences are conserved for both phenotypes, either in the form of homoplasmy or heteroplasmy. Up to date, molecular correlation between mtDNA mutation and both disease are still unclear. There are 87 samples collection, consisting of cataract lens and urine speciment from both phenotypes. Only 16 of them have been investigated. Therefore, this research is aim to analyze the variation between mtDNA sequences of both disease, using rRCS and the normal phenotype as standards. This research is focused on tRNA-Phe and 12S rRNA encoding genes of mtDNA. There are four samples; two eye lenses of cataract patients and two urine of DM associated with cataract patients. All samples were lysed for their template DNA. Thus, the template DNA was used for the amplification using the Polymerase Chain Reaction (PCR) method. Primers used in this research were at position 458-479 for forward (A for) and at 2491-2473 for reverse (A rev). The PCR product was then separated by gel agarose 0,7% (b/v) electrophoresis and was visualized under ultraviolet light. It is shown that there was a single band, corresponding to design primers. Subseqeuntly, sequencing with the dideoxy Sanger method was applied. In silico analysis of the sequencing result were than carried out by the SeqMan program of DNA Star. The nucleotide variances were then analyzed according to rRCS and mitomap database. The PCR results showed a single band of approxiamately 2 kilobase. The nucleotides have sequenced by using A for primer. Numbers of sequenced nucleotides were 950 bases and 508 of them were analyzed, corresponding to positions 554-1062. These sequences were related to genes of tRNA-Phe and partial gene of 12S rRNA. Homology analysis of all sequences showed that both genes are highly conserved, except for position709 and 750. The differences were g709A and a750G. Both variances are already reported as plymorphisms on the 12S rRNA gene of mtDNA. Hence, specific genes associated with DM and cataract are probably at the other genes of mtDNA. Whole genome sequences and analysis of mtDNA supposed to be carried out to study the specific genes related to both DM and cataract. Further, histochemical analysis of both diseases are also important to relate mtDNA disorder and the disease. This information is important for rational therapy of both diseases in order to develop a gene therapy method