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«Calpain 10 Gene Polymorphism in Type 2 Diabetes Mellitus Patients in Gaza Strip Prepared by Mazen M. El Zaharna Supervisors: Prof. Fadel A. Sharif ...»

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‫ة‬ – ‫ا‬ ‫ا‬

The Islamic University – Gaza

‫دة ا را ت ا‬

Deanery of Higher Education

‫م‬ ‫ا‬ ‫آ‬

Faculty of Science

‫ما‬ ‫ا‬

Master of Biological Sciences

Medical Technology

Calpain 10 Gene Polymorphism in Type 2 Diabetes Mellitus

Patients in Gaza Strip

Prepared by

Mazen M. El Zaharna

Supervisors:

Prof. Fadel A. Sharif Dr. Abdalla Abed Submitted in Partial Fulfillment of Requirements for the Degree of Master of Biological Sciences/Medical Technology Department of Medical Technology Faculty of Science ‫7002 م / 8241 هـ‬ Declaration I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person nor material which to a substantial extent has been accepted for the award of any other degree of the university of other institute, except where due acknowledgment has been made in the text.

Signature Name Date Mazen Medhat Moustafa El Zaharna March- 28 - 2007 Mazen Copy right.

All Rights Reserved: No part of this work can be copied, translated or stored in a retrieval system, without prior permission of the authors.

Dedication To my father and my mother who taught me how to give To my wife who supported me wholeheartedly To my children Medhat & Motasem To my brothers and sisters To all my friends who spare no effort to help To all of them I dedicate this work II Acknowledgements The work for this thesis was carried out at the Molecular biology Laboratory- Islamic University – Gaza.

Many individuals have contributed to the completion of this work, including the formation of this dissertation. I wish to thank all who made this work possible with their support, advice, and time.

I would like to express my gratitude to my supervisors Prof. Fadel A. Sharif and Dr. Abdalla Abed, for their support, patience, special insights and encouragement throughout my research. The technical and editorial advice of Prof. Fadel A.

Sharif was essential to the completion of this work and have taught me innumerable lessons and insights on the workings of academic research in general.

I am very much thankful to the staff at the Diabetic Clinic – Al Remal Clinic for their help in sample collection. Especially Dr. Soheal El Kichoui, Dr. Samy El Esawi, Dr. Mohammed Bolbol, Dr. Awny El Jaro and Mr. Yosef Abu Rahma. Their sincere contributions made this research attempt fruitful and possible.

I am deeply appreciate the assistance and support of Mr. Nasser Abu Shaaban, in the technical work.

I want to thank all the staff at the department of the biological sciences at the Islamic University- Gaza, for their support and help. I would like also to warmly thank Dr. Abboud El Kichaoui for his help and support during the course. I would like also to thank Mr. Mohamed Abou Oada for his assistance.

III I want to thank all my colleagues, especially Zaid, Iyad, and Hosam thank you so much for always being there for me offering a helping hand. Good luck with everything!

Last but not least, I want to thank my beloved father and mother, thank you for always believing in me and loving me unconditionally, without your support and love I would not be where I am today. My heartfelt thanks go to my loving wife;

your tender care and patience have not ceased even in my worst moments

–  –  –

Calpain-10 (CAPN10) is a member of a large family of intracellular proteases.

The polymorphisms at CAPN10 gene have recently been associated with complex forms of type 2 diabetes mellitus (T2DM). It was shown in MexicanAmericans and other populations that variants of polymorphisms, single nucleotide polymorphism (SNP)-44, SNP-43, deletion/insertion (del/ins)-19, and SNP-63, of this gene influence susceptibility to T2DM. However, substantial differences were shown between ethnic groups in at risk alleles and haplotypes as well as in their attributable risk.

Aim: To examine the role of CAPN10 SNPs -44, -43, -63, and del/ins-19 in genetic susceptibility to T2DM and to the levels of triglycerides and cholesterol in Gaza Strip.

Methods: Ninety six individuals were examined: 48 T2DM patients and 48 control individuals. The groups were genotyped for CAPN10 SNP-44, SNP-43, SNP-63, and del/ins-19. Mutagenically separated polymerase chain reaction (MS-PCR) was used to examine SNP-44, del/ins-19 was examined by electrophoresis of the PCR product on agarose gel by size, while the restriction fragment length polymorphism (RFLP) method was used for SNP-43 and SNP-63.

Results: There was no association between T2DM and allele frequencies of SNP-43, del/ins-19 and SNP-63. Distributions of genotypes, haplotypes and haplotypes combinations in the studied variants were not significantly different between the groups. However, there was evidence that the C-allele at SNP-44, plays a possible role in the susceptibility to T2DM (p=0.01). T2DM patients with G/A genotype were found to have higher cholesterol levels in comparison to those homozygous for allele 1 (G/G) in SNP-43. We found also that T2DM patients who are homozygous for del/ins-19 allele 2 have higher cholesterol levels in comparison to other genotypes. In addition, T2DM patients having the 121/221 haplotypes combination have higher serum cholesterol level than those with other haplotypes combinations. And that control subjects with haplotypes V combination 111/121 have higher serum cholesterol level than those with other haplotypes combinations.





Conclusion: The polymorphism SNP-44 at CAPN10 has an association with T2DM. CAPN10 also has an association with the levels of cholesterol in both T2DM patients and control individuals.

Keywords: Calpain 10, T2DM, Triglycerides, Cholesterol, Gaza strip.

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‫ا‬

–  –  –

Page 10 FIigure 2.1.

Mechanisms of insulin resistance………………………...

14 Figure 2.2.

Schematic representation of the etiology of T2DM. ……….

26 Figure 2.3. Location of CAPN10 on the human genome……………… Figure 2.4 Exon-intron organization of CAPN10 and the locations of the 27 SNPs………………………...………………………

27 Figure 2.5 Alternative splicing of CAPN10………………………........

Figure 2.6.

Haplotype combination associated with the highest risk of 29 T2DM in Mexican Americans………………………...………………..

Figure 4.1.

A Photograph representing the quality of 10 DNA samples 55 extracted from whole blood samples…………………………………..

Figure 4.2.

A photograph of ethidium bromide stained 3% agarose gel 56 showing the MS-PCR product for SNP-44……………………………..

Figure 4.3.

A photograph of ethidium bromide stained 2% agarose gel 56 showing the PCR product of SNP-43………………………………….

Figure 4.4.

A photograph of ethidium bromide stained 3% agarose gel 57 showing the PCR product of del/ins-19……………………………….

Figure 4.5.

A photograph of ethidium bromide stained 2% agarose gel 57 showing the PCR product of SNP-63………………………………… Figure 4.6. A photograph of PCR-RFLP products of SNP-43 run on 4% 58 ethidium bromide stained 4% agarose gel……………………………..

Figure 4.7.

A photograph of PCR-RFLP products of SNP-63 run on 4% 59 ethidium bromide s tained agarose gel………………………………..

–  –  –

XV Abbreviations ATP Adenosine triphosphate BMI Body mass index CAPN10 Calpain 10 CVD Cardiovascular disease Del/ins Deletion/insertion DM Diabetes mellitus DZ Dizygotic FABP2 Fatty-acid binding protein 2 FFA Free fatty acids GDM Gestational diabetes mellitus HGP Hepatic glucose production HLA Human leukocyte antigen HNF4A Hepatocyte nuclear factor 4, alpha HW Hardy-Weinberg IDDM Insulin-dependent diabetes mellitus IFG Impaired fasting glucose IGF-1 Insulin-growth factor 1 IGT Impaired glucose tolerance K-ATP Sensitive potassium channel adenosine triphosphate KCNJ11 Potassium inwardly-rectifying channel, subfamily J, member 11 kD Kilo dalton LD Linkage disequilibrium MODY Maturity-onset diabetes in the young MZ Monozygotic NGT Normal glucose tolerance NIDDM1 Non-insulin-dependent diabetes mellitus-1 OGTT Oral glucose tolerance test OR Odds ratio PCR Polymerase chain reaction PPAR Peroxisome proliferator-activated receptor PPARG Peroxisome proliferative activated receptor, gamma RFLP Restriction fragment length polymorphism rpm Round per minute RT Room temperature RT–PCR Reverse transcription polymerase chain reaction SNP Single nucleotide polymorphism T1DM Type 1 diabetes mellitus T2DM Type 2 diabetes mellitus UCP2 Uncoupling protein 2 WBC White blood cells WHO The world health organization UV Ultraviolet

XVIChapter 1 Introduction

Diabetes Mellitus (DM) is a progressive and chronic endocrine disorder which results primarily in a hyperglycemic (excess glucose in the blood) condition (1). DM affects the body's ability to metabolize fat, carbohydrates and proteins and often leads to a serious micro- and macrovascular complications, including cardiovascular diseases (2). The primary hormone, insulin, which maintains homeostasis of body glucose levels, is either insufficient or ineffective in individuals with DM (3).

1.1 Classification of diabetes mellitus

The most common types of diabetes mellitus are:

1. Type 1 diabetes dellitus (T1DM) results from autoimmunological destruction of the insulin-producing cells of the pancreas and accounts for 5–10% of all cases of diabetes, with the major susceptibility gene mapping to the human leukocyte antigen (HLA) region of chromosome 6 (4).

2. Type 2 diabetes mellitus (T2DM) is the most common form of diabetes, accounting for approximately 90% of cases and affecting 10–20% of those over 45 years of age in many developed countries (4). T2DM indicates an individual who has a physiological resistance to the effects of insulin within the peripheral tissues. Basically, the insulin, which the body is still capable of producing, is not physiologically effective (5).

1.2 Etiology of type 2 diabetes mellitus T2DM is a classic example of a complex disorder. It is strongly familial, but clearly arises as a consequence of the actions and interactions of many genetic and non-genetic factors with some forms of the disease resulting from mutations in a single gene, others are multifactorial in origin (4,6).

1.2.1 Risk factors of T2DM The risk factors for T2DM include environmental influences such as obesity (especially abdominal obesity), aging, ethnicity, family history of diabetes, history of gestational diabetes, sedentary lifestyle, low birth weight, and polycystic ovary syndrome (7). Eighty percent or more of the people with T2DM are obese with the remaining twenty percent considered above ideal weight indicating obesity as a predominant link to the development of T2DM (8).

1.2.2 Type 2 diabetes mellitus and genetics

Although insulin resistance and progressive pancreatic β-cell dysfunction have been established as the two fundamental features in the pathogenesis of T2DM, the specific molecular defects affecting insulin sensitivity and/or β-cell function remain largely undefined (2).

Substantial scientific evidence exists for the role of genetic factors in the pathogenesis of T2DM. For example, T2DM clusters in families, its concordance rate in monozygotic twins is higher than in dizygotic ones, and there are ethnic groups with a very high prevalence of this disease (9).

Although genomewide scans have identified several potential chromosomal susceptibility regions in several human populations, finding a causative gene for T2DM has remained elusive. Hanis et al. (1996) reported linkage to a region on chromosome 2q37.3 among Mexican Americans and identified a major susceptibility locus, located in the interval that spans markers D2S125–D2S140 (10). Following a combined strategy of positional cloning and a newly developed statistical method of partitioning linkage, these investigators identified a novel gene, Calpain 10 (CAPN10, as a putative T2DM susceptibility gene in this region (Horikawa et al. 2000) (4). Polymorphisms in this gene (SNP-44, SNPdel/ins-19, and SNP-63), all located in intronic sequences, were found to be involved in increased risk of the disease. Although the common G allele at SNPwas initially found to be significantly associated with the phenotype in families that showed linkage to NIDDM1 region on chromosome 2 (11).

Variation in CAPN10 has been associated with a threefold increased risk of T2DM in Mexican-Americans and an increased risk of diabetes in Northern European populations (12).

The understanding of the genetic basis of T2DM would assist in the development of screening tests to identify subjects at risk of developing T2DM at an early stage so that prevention strategies, lifestyle advice, and medical treatment can be commenced at the earliest possible stage. In addition, an understanding of the genetic basis of T2DM will pave the way for discovering new approaches for the prevention and more effective treatment of this condition (13).

1.3 Aim of the study The overall aim of the present study is to assess calpain-10 gene polymorphisms in type 2 diabetes mellitus patients in Gaza strip.

The specific Objectives are:

1. To study the prevalence of common variants in CAPN10 gene, SNPs – 44, -43, -63 and del/ins-19 in T2DM as compared to control subjects

2. To evaluate the relation of these variants in the development of T2DM in Gaza Strip population

3. To investigate the influence of these variants on the lipid profile in T2DM patients and control group

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