Calpain-10 Gene Polymorphism in Type 2 Diabetes Mellitus Patients in the Gaza Strip

Objective: To examine the role of calpain-10 SNP-44, -43, -63 and del/ins-19 in genetic susceptibility to type 2 diabetes mellitus (T2DM) and associations with triglycerides and total cholesterol in a group of subjects residing in the Gaza Strip. Subjects and Methods: Ninety-six individuals were examined: 48 T2DM patients and 48 controls. The groups were genotyped for calpain-10 SNP-44, -43, -63, and del/ins-19. Mutagenically separated polymerase chain reaction was used to examine SNP-44; del/ins-19 was examined by electrophoresis of the PCR product on agarose gel, while the restriction fragment length polymorphism method was used for SNP-43 and -63. Results: There was evidence that the C allele at SNP-44 played a possible role in susceptibility to T2DM (p = 0.01). T2DM patients with G/A genotype were found to have higher levels of total cholesterol in comparison to those homozygous for allele 1 (G/G) in SNP-43. Total cholesterol levels increased in T2DM patients who are homozygous for del/ins-19 allele 2, in T2DM patients with the 121/221 haplotype combination, and in control subjects with the haplotype combination 111/121. Conclusion: SNP-44 Received: February 15, 2009 Revised: August 13, 2009 Mr. Mazen M. Zaharna Department of Medical Technology Islamic University of Gaza PO Box 108, Gaza City (Palestine) Tel. +970 59 960 4870, Fax +970 8 286 0800, E-Mail mzaharna @ iugaza.edu.ps © 2010 S. Karger AG, Basel 1011–7571/10/0196–0457$26.00/0 Accessible online at: www.karger.com/mpp Zaharna /Abed /Sharif Med Princ Pract 2010;19:457–462 458 the typical patient with new-onset T2DM has had diabetes for at least 4–7 years before it is diagnosed [3] . Among patients with T2DM, 25% are believed to have retinopathy, 9% neuropathy and 8% nephropathy at the time of diagnosis [3] . In Palestine, DM seems to be a serious health problem among the population – especially the refugees – because the prevalence rate of DM in Palestine was about 9% in 2002, while the rate in other populations in the same year was about 5.2% (in the age group 20–79 years) [4] . T2DM is a heterogeneous disorder that may result from defects in one or more diverse molecular pathways [5] . It is a classic example of a multifactorial disorder, its etiology combining both genetic and environmental factors [6] . Identification of the genetic components of T2DM is the most important area of diabetes research because elucidation of the diabetes genes (alleles) will influence all efforts toward a mechanistic understanding of the disease, its complications, treatment, cure and prevention [5] . Recently the calpain-10 gene has been identified as a diabetes susceptibility gene. Variation in calpain-10 has been associated with a threefold increased risk of T2DM in Mexican-Americans and an increased risk of diabetes in Northern European populations [7] . Variation in the calpain-10 gene has also been associated with increased levels of total cholesterol and triglycerides [8, 9] . The calpains are a family of calcium-dependent nonlysosomal cysteine proteases [10] . The presence of calpains in mammalian cells was first reported over 30 years ago. Since then, at least 14 members of the calpain family have been identified and their chemistry and biology extensively studied. Although their physiological function is still not fully understood, they are implicated in a variety of calcium-regulated cellular processes, such as signal transduction, cell proliferation, cell cycle progression, differentiation, apoptosis, membrane fusion and platelet activation [11] . Calpain-10 is located on chromosome 2q37 and consists of 15 exons spanning 31 kb. Analysis of human cDNA clones revealed a complex pattern of alternative splicing, generating proteins of 672, 544, 517, 513, 444, 274, 139 and 138 amino acids. There is a complex relationship between susceptibility to T2DM and polymorphisms in calpain-10. Susceptibility is attributable not to a single polymorphism or allele, but rather to multiple polymorphisms (e.g. SNP-44, -43, -63 and del/ins-19), whose collective effects are not easily predicted without having the full genotype/haplotype information at all contributing sites [7] . The haplotypes in CAPN10 were defined from 4 polymorphisms spread across the gene: SNP-43 (G/A within intron 3: allele 1 = G and allele 2 = A), SNP-19 (two repeats of 32-bp sequence or 3 repeats of 32-bp sequence within intron 6; allele 1 = 2 repeats and allele 2 = 3 repeats), SNP-63 (C/T within intron 13: allele 1 = C and allele 2 = T), and SNP-44 (T/C within intron 3: allele 1 = T and allele 2 = C) [12, 13] . Genetic variation in calpain-10 seems to affect susceptibility to T2DM in both MexicanAmericans and Europeans. The 112/121 haplotype combination is associated with a similarly increased risk (threefold) of T2DM in both groups [7] . The knowledge of T2DM genetic background becomes very important due to its scientific, prognostic, prophylactic and also therapeutic significance [14] . Therefore, the aim of this study was to examine the role of calpain-10 SNP-44, -43, -63 and del/ins-19 in genetic susceptibility to T2DM and to the levels of triglycerides and total cholesterol in subjects in the Gaza Strip. Subjects and Methods Ninety-six individuals were included in this study: 48 T2DM patients (males/females: 10/38; age: 56 8 9 years; duration of disease: 14 8 10 years) and another 48 non-diabetic controls (males/ females: 12/36; age: 41 8 9 years). The control group contained only individuals with a normal fasting glucose level and negative family history among first-degree relatives. The study was performed according to the ethical guidelines in the Declaration of Helsinki after approval from the institution’s ethics committee. Written informed consent was obtained from all participants. Blood samples were collected from patients and controls in the morning after 12–14 h fasting; 3 ml of venous blood were withdrawn from the antecubital vein into EDTA Vacutainer tubes. Genotyping DNA was extracted from peripheral blood lymphocytes by using the Wizard Genomic DNA Purification kit (Promega Corporation, Madison, Wisc., USA). We first genotyped the 4 polymorphisms indicating the highest risk for T2DM in the MexicanAmericans, SNP-44, -43, -63 and del/ins-19. Allele distribution of calpain-10 SNP-44, -43, -63 and del/ins-19 was examined in the T2DM patients and control subjects. We then examined the distribution of haplotype and haplotype combinations comprising these alleles. SNP-44 The mutagenically separated PCR (MS-PCR) method was used to genotype for SNP. A common reverse primer and 2 allelespecific forward primers of different lengths were used: common reverse primer, 5 -CTCATCCTCACCAAGTCAAGGC-3 ; allele 1 (T) primer, 5 -CAGGGCGCTCACGCTTGCTAT-3 ; allele 2 (C) primer, 5 -GTGGGCAGAGGACTGGTGGGCGCTCA CG CTTGCTTC-3 . PCR products were separated on 3.0% NuSieve agarose gel and were visualized by ethidium bromide staining. Allele 1 (T) should give a band of 71 bp and allele 2 (C) 86 bp [13] .