Type 2 Diabetes in a Subpopulation of Saudi Arabian Women - Essay Example

Study of Genetic Basis of Resistance to Type 2 Diabetes in a Subpopulation of Saudi Arabian Women Type 2 diabetes, associated with the development ofmetabolic resistance to insulin, is one of the most common diseases worldwide (Wild et al, 2004). The most important environmental risk factor is obesity; however, this is not the only determinant of population or individual risk (Al-Harithy & Al-Ghamdi, 2005). Genetic predisposition for the disorder is also a contributing factor, and decreased susceptibility to the development of Type 2 diabetes may be correlated with certain genotypic factors. At the genetic level, the link between diabetes and obesity appears to involve genes associated with the production bioactive peptides called adipocytokines (Pittas & Greenberg, 2004). Among these are the genes encoding leptin, tumor necrosis factor-alpha and adiptonectin. These genes may be involved in the development of insulin resistance. Therefore, to address the potential genetic association between resistance to type 2 diabetes, it is important to assess whether individuals whose phenotype shows resistance to the disorder are genetically screened to see if differences exist between the structure and/or activity of adipocytokine genes in groups women with similar risk factors (viz. obesity) and markedly different incidence rates for diabetes. A subpopulation of Saudi Arabian women has been identified that is resistant to the development of type 2 diabetes, despite incidence rates of obesity at the same level as the entire population of women in Saudi Arabia. The purpose of this research was to identify whether or not there was a genetic basis to this phenotypic observation. The present study involves a genetic assessment of the gene called resistin, previously identified in mice on chromosome 8 (Steppan et al, 2001). To study this gene in humans it was necessary to clone the gene. This process is initiated by a technique called fluorescent in situ hybridization (FISH). The mouse gene is used as a probe to identify the chromosomal location of its human gene counterpart. Since gene sequences are frequently homologous among different species, the related genes or orthologues can be used to identify similar genes in different species (Gregory & Hebert, 1999). The mouse gene is attached to a fluorescent probe and mixed with human chromosomal DNA that has been denatured (connverted to single stranded form). The fluorescent band identifies the chromosomal location of the gene in humans. The chromosome segment can then be cut with restriction enzymes and linked to an expression vector to generate a recombinant DNA. This DNA is then used to transform bacteria to produce many copies of the inserted gene (see Figure 1). The DNA can then be purified from the bacterial cells and then sequenced to determine the base sequence of the human resistin gene. The gene sequence can be compared between women who have diabetes and those who do not in order to determine whether there are any genotypic differences between the two groups (Mukai et al, 2007). These genotypic differences will result in the presence of different allelic forms of the gene in the two populations (Gregory & Hebert, 1999). The allelic differences are the result of differences in the DNA base sequences of the genes in the two groups. The putative protein sequence of the gene can be determined also from the DNA sequence by using bioinformatics to scan the gene, identify the open reading frames and to identify the sequence of amino acids specified by the genetic code (Gregory & Hebert, 1999). Population studies are then done to compare many women from each group. Once the gene has been isolated and sequenced, primers can be made so that polymerase chain reaction (PCR) can be used to facilitate the rapid characterization of the gene structure in many different women (Hastings, Lupski & Rosenberg, 2009). In this way, one can determine whether a specific allelic variant of the resistin gene is correlated with a specific phenotype, that of resistance to the development of type 2 diabetes in the subpopulation of Saudi Arabian women. In addition, this type of study can reveal the incidence of the different allelic forms of the gene in these two populations and determine which allelic forms are associated with genotypes that produce phenotypic resistance to diabetes. The use of expression vectors allows for the transcription and translation of cloned genes to produce high levels of gene product. This method permits the isolation and characterization of the resistin protein (see Figure 2). The protein analysis involves a determination of its molecular weight by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In this procedure, the protein is chemically denatured and treated with sodium dodecyl sulfate before subjecting the protein to polyacrylamide gel electrophoresis. SDS-PAGE is used to determine the molecular weight of the protein since the rate of migration through the gel is inversely proportional to the molecular weight of the protein (Laemmli, 1970). A comparison with standard proteins of known molecular weight can be used to make an exact determination of the molecular weight of the resistin protein. The purified protein can then be injected into rabbits in order to elicit the formation of antibodies that can be used to precipitate the protein from cells and identify its properties and activities (Orengo & Thornton, 2005). The adipose cells are purified from skin samples taken from women participating in a research study. The selection is based on the unique lipid composition of the lipid comprising the membranes of this tissue that allow them to be separated easily from other cell types. The cells are then lysed using detergents and then the lysate is treated with antibody to the resistin protein. The antibody precipitate is then subject to electrophoresis to determine the molecular weight of the protein in samples from the two population groups (Harrison & Gerstein, 2002). Also, the amount of the protein produced is determined by western blot analysis, in which the SDS Page products are transferred to nitrocellulose filters and hybridized to a fluorescent anti-antibody that recognizes the precipitated resistin protein. The intensity of the fluorescence of the bound probe can be used to determine the cellular levels of the protein in the two groups of women (Wang & Caetano-Anollés, 2009), In this study the analysis of the two population groups revealed some interesting genotypic differences between the two groups that may account for differences in the incidence of diabetes in these two groups of women. It was found that in the subgroup of women in whom the disease is less common, the most common allele of the resistin gene contains a mutation in the promoter region that reduces the amount of transcription from this gene. This finding is consistent with the observation that the levels of the resistin protein were lower in this population subgroup. Since resistin protein appears to play a role in blocking glucose uptake into adipose tissue, this finding suggests that decreased levels of resistin may be responsible for the decreased occurrence of insulin-resistant diabetes in this group of Saudi Arabian women. References Al-Harithy R & Al-Ghamdi S. 2005, ‘Serum resistin, adiposity and insulin resistance in Saudi women with type 2 diabetes mellitus.’ Annals of Saudi Medicine, vol. 25, no. 4, pp. 283-287 Gregory TR & Hebert PD. 1999, ‘The modulation of DNA content:proximate causes and ultimate consequences. "The modulation of DNA content: proximate causes and ultimate consequences.’ Genome Res, vol. 9, no. 4, pp. 317–24. Harrison P & Gerstein M. 2002, "Studying genomes through the aeons: protein families, pseudogenes and proteome evolution". J Mol Biol, vol. 318, no. 5, pp. 1155–74. Hastings P J, Lupski JR & Rosenberg SM. 2009, ‘Mechanisms of change in gene copy number . Nature Reviews. Genetics, vol. 10, no. 8, pp. 551–564. Laemmli, U. K. 1970, ‘Cleavage of structural proteins during the assembly of the head of bacteriophage T4.’ Nature, vol. 227, pp. 680-68. Mukai H, et al. 2007, ’ Highly Efficient Isothermal DNA Amplification System Using Three Elements of 5-DNA-RNA-3 Chimeric Primers, RNaseH and Strand-displacing DNA Polymerase.’ J Biochem, vol. 142, pp. 273-281. Orengo CA & Thornton JM. 2005, ‘Protein families and their evolution-a structural perspective.’ Annu. Rev. Biochem, vol. 74, pp. 867–900. Pittas A, Joseph N & Greenberg A. 2004, ‘Adipocytokines and insulin resistance.’ J Clin Endocrinol Metab. Vol.89, no.2, pp.447-52.   Steppan C, Bailey S, Bhat S, Brown E, Banerjee R, Wright C, Patel H, Ahima R & Lazar M. 2001, ‘The hormone resistin links obesity to diabetes.’ Nature, vol. 409, pp. 307-312. Wang M & Caetano-Anollés G. 2009, ‘The evolutionary mechanics of domain organization in proteomes and the rise of modularity in the protein world.’ Structure, vol. 17, no.1, pp. 66–78. Wild S, Roglic G, Green A, Sicree R & King H. 2004, ‘Global prevalence of diabetes: estimates for the year 2000 and projections for 2030.’ Diabetes Care. Vol. 27, no. 5, pp.1047-53. Figure 1. Methods used to identify gene associated with resistance to type2 diabetes. Figure 2. Methods used in the isolation and characterization of the resistin protein. Read More