GSTT1, Calpain 10 SNP 19 and indices of glycaemia in type 2 diabetes

Type 2 diabetes mellitus (T2DM) is one of the most widely spread chronic diseases in most countries; the number of diabetic patients is continuously increasing because of urbanization, ageing, population growth, reduced physical activity and increasing prevalence of obesity [1]. Glutathione-s-transferases (GSTs) are a family of antioxidant enzymes that play an important role in decreasing reactive oxygen species and so act as antioxidant defence [2]. They may also contribute to the risk of T2DM although their effects on disease susceptibility are gender specific [3]. Of the 22 GST family members, GST-theta-1 is coded for by GSTT1 on 22q11.23. CAPN10 on 2q37.3 codes for Calpain 10, a cysteine protease localized to the cytosol and mitochondria, acting on electron transport chain proteins, causing decreased mitochondrial respiration [4]. CAPN10 SNP-19 del-allele and CAPN10 SNP-44 C-allele are risk factors for T2DM [5], whilst CAPN10 SNP 19 is associated with increased glucose levels in gestational diabetes mellitus [6]. HOMA-IR is a very popular, easy and efficient method in routine clinical practice and studies for patients with T2DM and obesity [7]. We hypothesized links between SNPs in GSTT1 and CAPN10with T2DM and indices of glycaemia. We tested our hypothesis in a case-controlled study of 100 cases (50 men, 50 women, mean [SD] age 49.5 [7.4] years) with T2DM and 50 unrelated control subjects (25 men, 25 women, age 45.4 [6.4] years: p = 0.194). Cases were recruited from the Department of Experimental and Clinical Internal Medicine at the Medical Research Institute, Alexandria University. Diabetes diagnosis was based onWHO criteria of fasting plasma glucose <7 mmo/L. Approval of the local ethics committee, and informed consent from all the subjects, were obtained. A DNA extraction kit, primers and PCR master mix were purchased from Vivantis Subang Jaya, Selangor Darul Ehsan, Malaysia. Blood glucose kit was purchased from Biosystems. Barcelona, Spain. Insulin ELISA kit was purchased from Raybiotech. Norcross, Georgia, USA, blood glucose and HbA1c were determined by standard clinical chemistry techniques. A polymerase chain reaction (PCR) was used to genotype CAPN10 SNP-19 in a total volume of 25 μL containing 50 ng genomic DNA, 1X PCR buffer, 400 μmol/L deoxy nucleotide triphosphate solution (dNTPs), 1.5 Taq polymerase (Vivantis) and 4 μmol/L of each primer SNP-19 forward primer 5ʹ-GTTT GGTTCTCTTCAGCGTGCAG-3ʹ and reverse primer 5ʹCATGAACCCTGGCAGGGTCTAAG-3ʹ. Thermal cycling conditions were an initial denaturation step at 95 °C for 5 min, 35 cycles at 94 °C for 1 min, 60 °C for 1 min and 72 °C for 1 min, and a final extension step at 72 °C for 5 min. PCR products were separated on 3% agarose gel and stained with ethidium bromide (Figure 1). GSTT1 genotypes were determined by the presence and absence (null) of bands of 480 base pair (bp), respectively, with an internal control of 260 bp. Using this genotyping assay of GSTT1, the wild and null genotypes can be clearly categorized [8,9]. Calpain-10 SNP-19 is an insertion/deletion polymorphism and consists of two or three repeats of a 32 bp sequence. The allele 2 (three repeats) was detected as a 178 bp band and allele 1 (two repeats) was detected as a 146 bp band [3]. GSTT1-null alleles were analysed using a PCR reaction with beta-globin as an internal control. PCR amplifications were performed in a total volume of 25μL containing 50 ng genomic DNA, PCR buffer, 3 μmol/L MgCl2, 400 μmol/L dNTPs, 1.5 Taq polymerase (Vivantis) and 4 μmol/L of each primer as follows: GSTT1 forward primer 5ʹTTCCTTACTGGTCCTCACATCTC-3ʹ, reverse primer 5ʹTCACCGGATCATGGCCAGCA-3ʹ and β-globin forward primer 5ʹ-CAACTTCATCCAC GTTCACC-3ʹ, reverse primer 5ʹ-GAAGAGCCAAGGACAGGTAC-3ʹ. Thermal cycling conditions were as follows: an initial denaturation step at 95 °C for 5 min, 35 cycles at 94 °C for 1min, 60 °C for 1min and 72 °C for 1 min, and a final extension step at 72 °C for 5 min. The amplification products were size separated on 2% agarose gels and visualized by ethidium bromide staining. The homeostatic model assessment for insulin resistance (HOMA-IR) index was calculated as (fasting blood glucose x fasting insulin)/ 405 [7]. Data are analysed by chi-squared testing (presented as n and percent) and student’s t test (presented as mean with standard deviation).