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SINGLE NUCLEOTIDE POLYMORPHISMS (SNPs) OF ADIPONECTIN GENE AND ITS ASSOCIATION WITH SERUM ADIPONECTIN CONCENTRATION AND METABOLIC SYNDROME RISK FACTORS

AMONG MALAY ADULTS

by

NUR FIRDAUS ISA

Thesis submitted in fulfillment of the requirements for the degree of

Master of Science

August 2011

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ACKNOWLEDGEMENT

Alhamdulillah. Praise to ALLAH the Almighty.

First and foremost I offer my sincerest gratitude to my supervisor Dr Hamid Jan Jan Mohamed and my co-supervisor Associate Professor Zafarina Zainuddin for giving me an opportunity to be their postgraduate student, for the countless guidance and encouragement throughout my laboratory works, and for the patience and knowledge they have shared in completing this thesis. Special thanks to Professor Wan Abdul Manan Wan Muda, the head of the research project for his invaluable helps.

I would like to express gratitude to Ministry of Science, Technology and Innovation, Malaysia (MOSTI) for my sponsorship through the National Science Fellowship (NSF), and Research University Postgraduate Research Grant Scheme (USM-RU- PRGS) for partially funded the study. Also thank you to the Bachok population, staff and postgraduate students of Central Research Lab (CRL), School of Medical Sciences and Nutrition and Forensic Sciences Programmes, School of Health Sciences, Universiti Sains Malaysia for providing me with the help throughout data collection and facilities for biochemical and SNP analysis. Also, I have been grateful and blessed with a cheerful group of fellow postgraduate students.

Last but not least, very special thanks to my parents Haji Isa and Hajjah Siti Halimah and lovely siblings; Fattah, Faizal, Fathihah, Farhan, Firzanah, Fathurrahman, and Farwizah; of whom without fail, have provided me something much greater in all these years, the love.

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TABLE OF CONTENT

ACKNOWLEDGEMENT ... ii

TABLE OF CONTENT ... iii

LIST OF TABLES ... viii

LIST OF FIGURES ... xi

LIST OF ABBREVIATIONS ... xv

ABSTRAK ... xvii

ABSTRACT ... xix

CHAPTER ONE: INTRODUCTION ... 1

1.1 General Introduction ... 1

1.2 Literature Reviews ... 4

1.2.1 Metabolic Syndrome ... 4

1.2.2 Adipocytokines ... 7

1.2.3 Adiponectin ... 9

1.2.4 Adiponectin Gene ... 13

1.2.5 Association of Adiponectin, Adiponectin Gene and Metabolic Syndrome ... 16

1.3 Rationale of the Study ... 24

1.4 Objectives ... 24

1.4.1 General Objectives ... 24

1.4.2 Specific Objectives ... 25

CHAPTER TWO:MATERIALS AND METHODS ... 26

2.1 Research Methodology ... 26

2.1.1 Study Design ... 26

2.1.2 Sample Size ... 29

2.1.3 Inclusion and Exclusion Criteria ... 30

2.2 Structured Questionnaire ... 30

2.3 Metabolic Syndrome Definition Tool ... 31

2.4 Plasticware ... 31

2.5 Instruments ... 32

2.5.1 Pipettes ... 32

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2.5.2 Centrifuge ... 32

2.5.3 Vitalab Selectra E Chemistry Analyzer ... 33

2.5.4 Tecan GENios™ Microplate Reader ... 33

2.5.5 GeneAmp® PCR System 9700 Thermal Cycler ... 33

2.5.6 ABI PRISM 3130xl Genetic Analyzer ... 33

2.6 Field Examination ... 34

2.6.1 Blood Collection ... 34

2.6.2 Waist Circumference Measurement ... 34

2.6.3 Blood Pressure Measurement ... 35

2.7 Principles and Materials for Biochemical Tests ... 35

2.7.1 Principle of GOD-PAP Assay for Glucose ... 35

2.7.2 Glucose Reagent Test Kit ... 36

2.7.3 Principle of CHOL-PAP Assay for HDL Cholesterol ... 36

2.7.4 HDL Cholesterol Reagent Test Kit ... 38

2.7.5 Principle of GPO-PAP Assay for Triglycerides ... 38

2.7.6 Triglycerides Reagent Test Kit ... 39

2.7.7 Principle of Enzyme-linked Immunosorbent Assay (ELISA) for Human Adiponectin... 40

2.7.8 Human Adiponectin ELISA kit ... 41

2.8 Materials for SNP Analysis ... 41

2.8.1 QIAamp DNA Blood Mini Kit ... 41

2.8.2 Polymerase Chain Reaction (PCR) Master Mix ... 42

2.8.3 Exonuclease I (Exo I) ... 42

2.8.4 Shrimp Alkaline Phosphatase (SAP) ... 42

2.8.5 ABI PRISM® SNaPshotTM Multiplex kit ... 43

2.9 Methods for SNP Analysis... 43

2.9.1 DNA Extraction ... 44

2.9.2 Primer Design for Polymerase Chain Reaction (PCR)... 45

2.9.3 Polymerase Chain Reaction (PCR) ... 47

2.9.4 Agarose Gel Electrophoresis ... 47

2.9.5 Post Polymerase Chain Reaction (PCR) Treatment ... 49

2.9.6 Minisequencing Reaction ... 49

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2.9.7 Thermal Cycling ... 51

2.9.8 Post-Extension Treatment ... 51

2.9.9 Principle of Capillary Electrophoresis ... 56

2.9.10 Capillary Electrophoresis of Minisequencing Product ... 57

2.10 Statistical Analyses ... 57

CHAPTER THREE: RESULTS ... 59

3.1 Laboratory Assays ... 59

3.1.1 Optimizations for Glucose, HDL Cholesterol and Triglycerides ... 59

3.1.2 Optimizations for Adiponectin Concentration ... 59

3.2 Sociodemographic Characteristics ... 60

3.2.1 General Characteristics ... 60

3.2.2 Status of Metabolic Syndrome among the Subjects ... 60

3.2.3 Distribution of SNPs... 61

3.3 SNP Analyses... 70

3.3.1 DNA Extraction ... 70

3.3.2 Amplification and Post-PCR of the DNA Templates... 70

3.3.3 Capillary Electrophoresis of Size Standard and Controls ... 71

3.3.4 Singleplex Reaction of Minisequencing Product ... 80

3.3.5 Multiplex Reaction of Minisequencing Product... 80

3.4 Association of Metabolic Syndrome, Adiponectin Concentration and Single Nucleotide Polymorphisms ... 90

3.4.1 Association of Adiponectin Concentration with Metabolic Syndrome ... 90

3.4.2 Association of SNPs and Haplotypes with Adiponectin Concentration ... 90

3.4.3 Association of SNPs and Haplotypes with Metabolic Syndrome ... 90

3.4.4 Mean Difference of Adiponectin Concentration Based on SNPs and Metabolic Syndrome ... 91

3.4.5 Association of Adiponectin Concentration with Metabolic Syndrome Risk Factors... 91

3.4.6 Association of SNPs and Haplotypes with Metabolic Syndrome Risk Factors ... 100

3.4.7 Mean Difference of Adiponectin Concentration Based on SNPs and Metabolic Syndrome Risk Factors... 105

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CHAPTER FOUR: DISCUSSION ... 110

4.1 Adiponectin Concentration ... 110

4.2 Status of Metabolic Syndrome Among the Subjects ... 110

4.3 Analyses and Genotype Frequencies of SNPs ... 111

4.4 Association Studies ... 116

4.4.1 Association of Adiponectin Concentration with Metabolic Syndrome ... 116

4.4.2 Association of SNPs and Haplotypes with Adiponectin Concentration ... 116

4.4.3 Association of SNPs and Haplotypes with Metabolic Syndrome .... 117

4.4.4 Mean Difference of Adiponectin Concentration Based on SNPs and Metabolic Syndrome ... 118

4.4.5 Association of Adiponectin Concentration with Metabolic Syndrome Risk Factors... 119

4.4.5 (a) Association of Adiponectin Concentration with Central Obesity ... 119

4.4.5 (b) Association of Adiponectin Concentration with Hypertension ... 120

4.4.5 (c) Association of Adiponectin Concentration with Hyperglycemia ... 122

4.4.5 (d) Association of Adiponectin Concentration with Hypertriglyceridemia and Reduced HDL Cholesterol ... 123

4.4.6 Association of SNPs and Haplotypes with Metabolic Syndrome Risk Factors ... 124

4.4.6 (a) Association of SNPs and Haplotypes with Central Obesity ... 124

4.4.6 (b) Association of SNPs and Haplotypes with Hypertriglyceridemia and Hyperglycemia ... 125

4.4.6 (c) Association of SNPs and Haplotypes with Hypertension and Reduced HDL Cholesterol ... 127

4.4.7 Mean Difference of Adiponectin Concentration Based on SNPs and Metabolic Syndrome Risk Factors... 127

4.5 Limitation of the Study ... 129

4.6 Recommendation for Future Research ... 130

CHAPTER FIVE:SUMMARY AND CONCLUSION ... 131

REFERENCES ... 133

APPENDIX I ... 143

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APPENDIX II ... 146

APPENDIX III ... 151

APPENDIX IV ... 153

APPENDIX V ... 154

APPENDIX VI ... 159

APPENDIX VII ... 164

APPENDIX VIII ... 165

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LIST OF TABLES

Table 1 International Diabetes Federation definition of the metabolic

syndrome (Zimmet et al., 2005) ... 6

Table 2 Multimeric forms of adiponectin (Wiecek et al., 2007) ... 11

Table 3 Amino acid changes introduced by respective SNPs in adiponectin gene ... 20

Table 4 The genetic variants and adiponectin or MS risk factors with the respective ethnic group that has been studied (Gu, 2009) ... 23

Table 5 Primers used for the PCR reaction ... 46

Table 6 Reaction mixtures for PCR ... 48

Table 7 Reaction mixture for control reactions ... 52

Table 8 Primers used in minisequencing reaction ... 53

Table 9 Reaction mixtures for singleplex reactions ... 54

Table 10 Optimized reaction mixtures for multiplex reaction... 55

Table 11 Validation of biochemical assays against manufacturer’s recommended calibrators and controls ... 62

Table 12 General characteristics of the study subjects (n=298) ... 65

Table 13 Prevalence of risk factors and metabolic syndrome ... 66

Table 14 Genotype distribution of the selected SNPs of adiponectin gene in Malay adults (n=298)... 67

Table 15 Hardy-Weinberg Equilibrium (HWE) of the selected SNPs of adiponectin gene ... 68

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Table 16 The recommended and observed mobility shift for positive control reactions ... 77 Table 17 The recommended and observed mobility shift for the

minisequencing primers ... 81 Table 18 Association between SNPs and haplotypes with adiponectin

concentration ... 94 Table 19 Association between SNPs and haplotypes with status of MS ... 96 Table 20 Association between SNPs and haplotypes with the adjusted

adiponectin concentration ... 97 Table 21 Association between the MS risk factors with the adiponectin

concentration ... 99 Table 22 Association between SNP+276 with hypertriglyceridemia (n=298) ... 102 Table 23 Association between haplotype -11426/-11377 and status of

hyperglycemia (n=298)... 104 Table 24 Association of adjusted adiponectin concentration on status of

reduced HDL cholesterol and SNP-11426 ... 107 Table 25 Association of adjusted adiponectin concentration on status of

hpertriglyceridemia and haplotype -11426/+45 ... 109 Table 26 Adiponectin concentration of Malay population and other

populations... 113 Table 27 Distribution of the genotype frequencies for SNP+45 and SNP+276

of the Malay population and other reported populations ... 114

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Table 28 Distribution of the genotype frequencies for SNPs -11426, -11391 and -11377 of Malay population and other reported populations .. 115 Table 29 Association between SNPs and haplotypes with central obesity ... 154 Table 30 Association between SNPs and haplotypes with hypertension ... 155 Table 31 Association between SNPs and haplotypes with hyperglycemia ... 156 Table 32 Association between SNPs and haplotypes with hypertriglyceridemia ... 157 Table 33 Association between SNPs and haplotypes with reduced HDL

cholesterol ... 158 Table 34 Association of adiponectin concentration on status of central obesity

(CO) and SNP or haplotypes ... 159 Table 35 Association of adiponectin concentration on status of hypertension

(HT) and SNP or haplotypes ... 160 Table 36 Association of adiponectin concentration on status of hyperglycemia

(GL) and SNP or haplotypes ... 161 Table 37 Association of adiponectin concentration on status of

hypertriglyceridemia (TG) and SNP or haplotypes ... 162 Table 38 Association of adiponectin concentration on status of reduced HDL

cholesterol (rHDL) and SNP or haplotypes ... 163

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LIST OF FIGURES

Figure 1 Major adipokines secreted from white adipose tissue (Trayhurn et al., 2006) ... 8 Figure 2 Anti-atherogenic and of insulin-sensitizing actions of adiponectin

(Wiecek et al., 2007) ... 12 Figure 3 Ideogram of chromosome 3 where adiponectin spanned on 3q27

(National Center for Biotechnology Information, 2008) ... 14 Figure 4 Organization of the adiponectin gene on chromosome 3 (Vasseur et

al., 2006) ... 15

Figure 5 Flowchart showing an overview of the research methodology ... 27 Figure 6 Illustration map showing the study locations with no scale provided

(Majlis Daerah Bachok, 2008) ... 28 Figure 7 Standard curve showing the optical density measured at 450 nm

against the adiponectin concentration (ng/ml). ... 63 Figure 8 Histogram showing frequency distribution of adiponectin

concentration ... 64 Figure 9 Linkage Disequilibrium (LD) plot for the selected SNPs of

adiponectin gene ... 69 Figure 10 Agarose gel electrophoresis of the extracted genomic DNA.

Electrophoresis was performed on 1.2 % agarose gel for 45 minutes at 80 V ... 72 Figure 11 Agarose gel electrophoresis of the amplified PCR products from two

randomly selected subjects A and B. Electrophoresis was performed on 1.2 % agarose gel for 45 minutes at 80 V ... 73

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Figure 12 Electropherogram showing the sequencing result of the amplified product using primer set F-34/R+404 (forward strand) ... 74 Figure 13 Sequence alignment using BLAST showing that the amplified

product of primer set F-34/R+404 is part of the human adiponectin precursor ... 75 Figure 14 Electropherogram of the GeneScan-120 LIZ size standard ... 76 Figure 15 Electropherogram of the positive control reaction products along

with GeneScan-120 LIZ size standard. ... 78 Figure 16 Electropherogram of the negative control reaction showing negative

amplification ... 79 Figure 17 (a) Electropherogram showing minisequencing product of primer

-11391. Blue peak indicates an addition of ddGTP to the minisequencing primer ... 82 Figure 17 (b) Electropherogram showing minisequencing product of primer +276.

Blue peak indicates an addition of ddGTP to the minisequencing primer... 83 Figure 17 (c) Electropherogram showing minisequencing product of primer

-11426. Green peak indicates an addition of ddATP to the minisequencing primer ... 84 Figure 17 (d) Electropherogram showing minisequencing product of primer +45.

Red peak indicates an addition of ddTTP to the minisequencing primer... 85 Figure 17 (e) Electropherogram showing minisequencing product of primer

R-11377 (a reverse primer). Blue peak indicates an addition of ddGTP to the minisequencing primer ... 86

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Figure 18 Electropherogram showing the multiplex minisequencing products of five minisequencing primers for a randomly selected sample ... 87 Figure 19 Electropherogram showing the multiplex minisequencing products of

five minisequencing primers for a randomly selected sample ... 88 Figure 20 Electropherogram showing the multiplex minisequencing products of

five minisequencing primers for a randomly selected sample ... 89 Figure 21 The median (IqR) of adiponectin concentration among those with

and without MS (Mann-Whitney U test) ... 92 Figure 22 The median (IqR) of adiponectin concentration by respective SNPs

and haplotypes (Mann-Whitney U test) ... 93 Figure 23 Proportion of subjects having SNP-11426 based on status of MS

(Chi-square test ) ... 95 Figure 24 The median (IqR) of adiponectin concentration based on MS risk

factors ... 98 Figure 25 Proportion of subjects having SNP+276 based on status of

hypertriglyceridemia ... 101 Figure 26 Proportion of subjects having haplotype -11426/-11377 based on

status of hyperglycemia ... 103 Figure 27 Adjusted adiponectin concentration based on status of reduced HDL

cholesterol and SNP-11426 ... 106 Figure 28 Adiponectin concentration based on status of hypertriglyceridemia

and haplotype -11426/+45 ... 108 Figure 29 Histogram showing normal frequency distribution of (a) waist

circumference, (b) systolic blood pressure, (c) diastolic blood

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pressure, (d) glucose concentration, (e) HDL cholesterol concentration and (f) triglycerides concentration. ... 153 Figure 30 Illustration on the association of SNPs and haplotypes with metabolic

syndrome and adiponectin concentration in Malay adults ... 164 Figure 31 Illustration on the association of SNPs and haplotypes with central

obesity and adiponectin concentration in Malay adults ... 165 Figure 32 Illustration on the association of SNPs and haplotypes with

hypertension and adiponectin concentration in Malay adults ... 166 Figure 33 Illustration on the association of SNPs and haplotypes with

hyperglycemia and adiponectin concentration in Malay adults ... 167 Figure 34 Illustration on the association of SNPs and haplotypes with

hypertriglyceridemia and adiponectin concentration in Malay adults ... 168 Figure 35 Illustration on the association of SNPs and haplotypes with reduced

HDL cholesterol and adiponectin concentration in Malay adults .. 169

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LIST OF ABBREVIATIONS

ATP III National Cholesterol Education Program’s Adult Treatment Program III

BAT Brown adipose tissue

BLAST Basic Local Alignment Search Tool

BMI Body mass index

CI Confidence interval

CV Coefficient of variability

ddNTPs Dideoxy nucleoside triphosphates

DNA Deoxyribonucleic acid

EDTA Ethylenediaminetetraacetic acid

EGIR European Group for the Study of Insulin Resistance

ELISA Enzyme-linked Immunosorbent Assay

Exo I Exonuclease I

GOD Glucose oxidase

H2O2 Hydrogen peroxide

HDL cholesterol High-density lipoprotein cholesterol

HMW High molecular weight

HPLC High-performance liquid chromatography

HWE Hardy-Weinberg equilibrium

IDF International Diabetes Federation

IqR Interquartile range

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LD Linkage disequilibrium

LDL Low density lipoprotein

LMW Low molecular weight

MAF Minor allele frequency

MMW Medium molecular weight

MS Metabolic syndrome

NCBI National Center for Biotechnology Information

OR Odds ratios

PCR Polymerase chain reaction

SAP Shrimp Alkaline Phosphatase

SAT Subcutaneous adipose tissue

QTL Quantitative trait locus

SD Standard deviation

SNPs Single nucleotide polymorphisms

T2D Type 2 diabetes

TBE Tris-Borate-EDTA

VAT Visceral adipose tissue

VSR Visceral subcutaneous ratio

WAT White adipose tissue

WHO World Health Organization

WHR Waist hip ratio

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POLIMORFISME NUKLEOTIDA TUNGGAL PADA GEN ADIPONEKTIN DAN PERKAITANNYA DENGAN KEPEKATAN SERUM ADIPONEKTIN

DAN FAKTOR-FAKTOR RISIKO SINDROM METABOLIK DALAM KALANGAN ORANG MELAYU DEWASA

ABSTRAK

Sindrom metabolik ialah kelompok faktor risiko yang termasuk keobesan di bahagian pinggang dan perut, hipertrigliseridemia, kolestrol HDL rendah, hipertensi, and hiperglisemia. Bukti-bukti terkumpul menyokong hipotesis bahawa hipoadiponektinemia meningkatkan lagi risiko terhadap penyakit metabolik. Antara yang menarik, sesetengah daripada polimorfisme biasa dalam bahagian promoter, exon dan intron 2 pada gen adiponektin manusia mempunyai kaitan dengan faktor- faktor risiko sindrom metabolik. Kajian ini bertujuan untuk menyiasat perkaitan antara beberapa polimorfisme nukleotida tunggal (SNPs) pada gen adiponektin dengan kepekatan adiponektin dan faktor-faktor risiko sindrom metabolik dalam kalangan orang Melayu dewasa. Seramai 298 orang Melayu dewasa yang terlibat di dalam kajian ini. Lilitan pinggang dan tekanan darah telah diukur sebelum darah diambil daripada subjek yang telah berpuasa semalaman. Ujian-ujian biokimia untuk paras trigliserida, kolestrol HDL dan glukosa dalam darah dijalankan dengan menggunakan alatan-alatan komersil. Kepekatan adiponektin plasma diukur menggunakan alatan Human Adiponectin ELISA. Sebanyak lima lokasi polimorfisme nukleotida tunggal pada gen adiponektin (SNPs -11426, -11391 dan -11377 pada bahagian proximal promoter dan SNPs +276 dan +45 pada bahagian

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exon 2) telah disaring menggunakan kaedah minipenjujukan. Dapatan kajian ini menunjukkan bahawa kepekatan adiponektin pada subjek yang mempunyai sindrom metabolik lebih rendah berbanding dengan subjek yang tidak mempunyai sindrom metabolik (p < 0.05). Kepekatan adiponektin juga berkait rapat dengan hipertrigliseridemia (p < 0.001) dan paras rendah kolestrol HDL (p < 0.001). Tidak ada polimorfisme nukleotida tunggal atau haplotip yang menunjukkan perkaitan dengan kepekatan adiponektin. Hanya SNP-11426 yang mempunyai kaitan yang signifikan dengan sindrom metabolik (p < 0.05), manakala SNP+276 berkait rapat dengan hipertrigliseridemia (p < 0.05) dan haplotip -11426/-11377 mempunyai perkaitan dengan hiperglisemia (p < 0.05). Secara keseluruhannya, tidak ada interaksi yang signifikan dari segi statistic di antara status sindrom metabolik dan polimorfisme nukleotida tunggal atau haplotip dengan kepekatan adiponektin. Walau bagaimanapun, terdapat perkaitan yang signifikan antara kepekatan adiponektin dan status paras rendah kolestrol HDL dengan SNP-11426 (p < 0.05). Di samping itu, terdapat juga perkaitan yang signifikan antara kepekatan adiponektin dan haplotip -11426/+45 dengan hipertrigliseridemia (p < 0.05). Kesimpulannya, hipoadiponektin dan polimorfisme nukleotida tunggal dan haplotip pada gen adiponektin boleh menyumbang kepada perkembangan sindrom metabolik dan faktor-faktor risiko melalui mekanisma yang masih lagi tidak diketahui di dalam populasi Melayu.

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SINGLE NUCLEOTIDE POLYMORPHISMS (SNPs) OF ADIPONECTIN GENE AND ITS ASSOCIATION WITH SERUM ADIPONECTIN CONCENTRATION AND METABOLIC SYNDROME RISK FACTORS

AMONG MALAY ADULTS

ABSTRACT

Metabolic syndrome is a cluster of risk factors that include central obesity, hypertriglyceridaemia, reduced HDL cholesterol, hypertension, and hyperglycemia.

Accumulating evidences support the hypothesis that hypoadiponectinemia, a type of adipokine, confer increased risk for metabolic diseases. Of interest, some of the common polymorphisms in the promoter region, exon and intron 2 of the human adiponectin gene are associated with risk factors of metabolic syndrome. The present study aims to investigate the association of several single nucleotide polymorphisms in the adiponectin gene with serum adiponectin concentration and metabolic syndrome risk factors among Malay adults. A total of 298 Malay adults were recruited in this study. Measurements for waist circumference and blood pressure were taken before drawing an overnight fasting blood. Biochemical tests for triglycerides, HDL cholesterol and glucose were carried out by using commercially available kits. Plasma adiponectin concentration was measured using Human Adiponectin ELISA kit. A total of five sites of single nucleotide polymorphisms in adiponectin gene (SNPs -11426, -11391 and -11377 at proximal promoter and SNPs +276 and +45 at exon 2 regions) were screened using minisequencing method.

Findings from this study showed that the adiponectin concentration in the subjects

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with MS was significantly lower than those without MS (p < 0.05). The adiponectin concentration was also significantly associated with only hypertriglyceridemia (p <

0.001) and reduced HDL cholesterol (p < 0.001). None of the studied SNPs or haplotypes showed any significant association with the adiponectin concentration.

Moreover, only SNP-11426 was significantly associated with MS (p < 0.05), while SNP+276 was associated with hypertriglyceridemia (p < 0.05), and haplotype -11426/-11377 was associated with hyperglycemia (p < 0.05). Overall, there was no statistically significant interaction between the status of MS and SNPs or haplotypes with respect to the adiponectin concentration. However, there was a significant association between the adiponectin concentration and status of reduced HDL cholesterol with SNP-11426 (p < 0.05). Besides, a significant association was also observed in the adiponectin concentration and hypertriglyceridemia with haplotype -11426/+45 (p < 0.05). In conclusion, hypoadiponectinemia and SNPs and haplotypes of adiponectin gene may contribute to the development of metabolic syndrome and its risk factors, via unknown mechanisms in Malay population.

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CHAPTER ONE: INTRODUCTION 1.1 General Introduction

Metabolic syndrome (MS) is an emerging public health problem throughout the world due to increase in the prevalence of the individual metabolic abnormalities i.e central obesity, hypertriglyceridaemia (the elevated serum triglyceride), reduced high-density lipoprotein cholesterol (HDL cholesterol), hypertension, and hyperglycemia (the elevated blood glucose levels) (Alberti et al., 2005). The rise of MS cases was shown to be in parallel with the prevalence of obesity (Grundy, 2008).

A comprehensive overview on MS pandemic in the America, Europe and India indicates more than 20 % of the adult populations as having MS (Grundy, 2008). The prevalence of MS recorded was 22.2 % in Italy, 23.9 % in Portugal, 46 % in the Netherlands, 41.8 % in Greece, 37 % in Finland and Asians especially India was 41.1

%, Thailand was 12.8 %, China was 13.2 % and Japan was 14.9 % (Grundy, 2008).

Across the Southeast Asia, there are several countries such as Singapore and Thailand that are actively involved in evaluating the MS. Tan et al. (2004) reported that in Singapore, the Asian Indian had the highest prevalence of MS followed by the Malays and Chinese (Tan et al., 2004). In contrast, a different study in Singapore suggested that Malay women were more likely to develop hypertension in association with insulin resistance, as compared to Chinese and Asian Indian (Ang et al., 2005). Moreover, hyperuricemia and elevated levels of liver enzymes, which were claimed to have association with MS, have been reported among Thai adults (Lohsoonthorn et al., 2006; Perera et al., 2008). Although Malaysia is still lacking of

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the representative data on MS, but the neighboring countries somehow reflect the condition of the syndrome herein as we govern the same major ethnics groups i.e Malays, Asian Indians and Chinese.

The mechanisms underlying MS are still not fully understood; however, accumulating studies have proposed an association between adiponectin; a type of adipokines with MS risk factors. Several studies have demonstrated low adiponectin concentration (hypoadiponectinemia) as a common denominator for the MS risk factors. The negative correlation between visceral adiposity and adiponectin concentration suggests that hypoadiponectinemia is related to central obesity (Matsuzawa, 2010; Ryo et al., 2004). Clinical reports have pointed out earlier that there are positive associations between the circulating adiponectin concentration with the HDL cholesterol and inverse association with triglycerides (Kazumi et al., 2004;

Matsubara et al., 2002). In addition, lower concentrations of adiponectin were observed in individuals with hypertension (Adamczak et al., 2003; Iwashima et al., 2004) and type 2 diabetes (T2D) (Hotta et al., 2000; Lindsay et al., 2002). From these evidences, it can be seen that adiponectin is a potential biomarker for the MS and its risk factors. Moreover, researchers have suggested that adiponectin can be a promising therapeutic agent for MS and its risk factors. It was proposed that by increasing circulating adiponectin concentration or enhancing adiponectin signaling through its receptors could promisingly tackle the root that cause the MS (Zhu et al., 2008). Although the claims excited many researchers, a lot of future investigations are required to support the matter.

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Despite the regulation of adiponectin expression in targeting MS which remains ambiguous, there has been growing interest in adiponectin gene that is deemed as potential genetic contributor to MS. Several studies have recently reported the genetic polymorphisms and linkages between adiponectin gene and MS. For instance, a genotype screening that was performed on 811 Hispanic individuals has demonstrated an association between genetic variations or single nucleotide polymorphisms (SNPs) in the adiponectin gene particularly in the promoter region with obesity, especially the visceral obesity (Sutton et al., 2005). However, the genetic variants of adiponectin and hypertriglyceridaemia and reduced HDL cholesterol were less explored and scantly reported (Yang & Chuang, 2006). A study to determine the possible effects of variation in adiponectin gene has shown that the mutant allele T of SNP+276 was associated with the elevated diastolic blood pressure whereas the wild-type allele T of SNP+45 demonstrated its protective role as it was associated with high HDL cholesterol levels (Mousavinasab et al., 2006).

On the other hand, Zacharova et al. (2005) which aimed to investigate the selected SNPs of the adiponectin gene and T2D reported that mutant allele G of SNP+45 was a predictor for T2D (Zacharova et al., 2005).

Taking together, hypoadiponectinemia and genetic variants of adiponectin gene play an important role in the pathogenesis of MS. Studies on the influence of the different ethnic groups is crucial to promote in-depth understanding on the regulation of MS, adiponectin and adiponectin gene. In this context, this present study was designed to investigate the association of several SNPs in adiponectin gene with serum adiponectin concentration and MS risk factors among Malay adults.

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4 1.2 Literature Reviews

1.2.1 Metabolic Syndrome

The MS has become one of the major public-health challenges worldwide. Grundy (2008) mentioned in his review that MS has a long history as early as 1923 when scientists began to notice the cluster of insulin resistance, hyperglycaemia, hypertension, low HDL cholesterol, and raised triglycerides as common risk factors for cardiovascular disease (Grundy, 2008). Since then, several names appeared to describe the cluster. Waine (2005) has reviewed previous studies on MS and summarized the names that have been used over the decades, such as the insulin resistance syndrome, plurimetabolic syndrome, dysmetabolic syndrome, and the deadly quartet (Waine, 2005). The term MS was first used by the World Health Organization (WHO) in 1998 which proposed a unifying definition for the syndrome by taking abnormal glucose tolerance as a core factor. A year later, European Group for the Study of Insulin Resistance (EGIR) came out with a modified version of the WHO recommendation (Zimmet et al., 2005). The National Cholesterol Education Program’s Adult Treatment Program III (ATP III) in 2001 then clinically defined MS by proposing abdominal obesity, dyslipidemia, hypertension, insulin resistance and prothrombotic and inflammatory states as the key components (Gable et al., 2007).

This was followed by the International Diabetes Federation (IDF) recommendations on 2005 (Zimmet et al., 2005) which prioritized ethnicity-specific waist circumference cut-off points as a measure for central obesity apart from other known risk factors. The IDF definition was constructed in such a way that it would rapidly

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identify individuals at risk without eliminating the ethnic factor. Due to its simplicity and appropriateness to our population, the IDF definition was used throughout the present study. Table 1 listed the criteria used for diagnosing MS by IDF definition.

Note that throughout the thesis, hyperglycemia (excessive amount of glucose circulates in the blood plasma) and T2D (characterized by hyperglycemia and caused by insufficient insulin production to regulate the circulating glucose) (Sheng &

Yang, 2008) were used interchangeably.

Moreover, Lahiry et al, (2008) has described that the concept of MS helped to emphasize on the risk of vascular disease related to central obesity and previously overlooked biomarkers such as serum triglycerides (Lahiry et al., 2008). Intense studies devoted to understand the MS risk factors especially the central obesity, adipose tissue and adipokines have eventually lead the scientists to the discovery of the adiponectin; a type of adipose-specific serum protein.

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Table 1 International Diabetes Federation definition of the metabolic syndrome (Zimmet et al., 2005)

1Following the ethnic-specific measurement of South Asian; 2HDL cholesterol: high- density lipoprotein cholesterol.

No Risk Factors Measurement Sex Readings 1. Central obesity Waist circumference1

Men ≥ 90 cm

Women ≥ 80 cm

Plus any 2 of the following

2. Hypertension

Systolic blood pressure / Diastolic blood pressure (or received treatment of

previously diagnosed hypertension)

Both ≥ 130/85 mmHg

3. Hyperglycemia

Fasting plasma glucose (or previously diagnosed

type 2 diabetes)

Both ≥ 5.6 mmol/L (100 mg/dL)

4. Reduced HDL

cholesterol2

HDL cholesterol (or specific treatment for

this lipid abnormality)

Men <1.0 mmol/L (40 mg/dL) Women <1.3 mmol/L

(50 mg/dL)

5. Hypertriglyceridemia

Triglycerides (or specific treatment for

this lipid abnormality)

Both ≥ 1.7 mmol/L (150 mg/dL)

(27)

7 1.2.2 Adipocytokines

Adipose tissue has been considered as an active endocrine due to the production of vital hormones called adipocytokines (also known as adipokines) which significantly involved in the regulation of body’s homeostasis (Koerner et al., 2005). There are two types of adipose tissue i.e the brown adipose tissue (BAT) and white adipose tissue (WAT) (Gu, 2009). BAT mainly involves in generating the heat by triggering the combustion of lipids and glucose in the tissue (Cannon & Nedergaard, 2004). On the other hand, WAT is of interest due to its role as the primary site in producing the most important adipocytokines such as adiponectin, leptin, zinc-α2-glycoprotein (ZAG), various interleukins, transforming growth factor-β (TGF-β), monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), tumor necrosis factor-α (TNF-α), and plasminogen activator inhibitor-1 (PAI-1) (Figure 1) (Trayhurn et al., 2006). Leptin, for instance, has been investigated for its role in energy homeostasis, glucose and lipid metabolism, and immune and neuroendocrine function (Ahima, 2006), while the interleukins have been emphasized as markers for inflammation (Trayhurn et al., 2006).

The WAT is mainly located in the subcutaneous region and viscera, hence closely associated with the pathogenesis of obesity-related disorders (Ahima, 2006).

The adiponectin that is abundantly secreted by WAT has received much attention due to its protective role against the obesity-related diseases (Gu, 2009; Koerner et al., 2005).

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8

Figure 1 Major adipokines secreted from white adipose tissue (Trayhurn et al., 2006)

Note:

IL-1β Interleukins 1β IL-6 Interleukins 6 IL-8 Interleukins 8 IL-10 Interleukins 10

MCP-1 Monocyte chemoattractant protein-1 MIF Macrophage migration inhibitory factor NGF Nerve growth factor

PAI-1 Plasminogen activator inhibitor-1 TGF-β Transforming growth factor-β TNF-α Tumor necrosis factor-α

VEGF Vascular endothelial growth factor ZAG Zinc-α2-glycoprotein

(29)

9 1.2.3 Adiponectin

Various adipocyte-derived secretory proteins have been increasingly linked to MS.

Adiponectin, another type of adipokines recently attracts much attention among scientists. It is a hormone extensively secreted by adipocytes, expressed inversely to total fat (Comuzzie et al., 2001) and acted as anti-diabetic, anti-inflammatory and anti-atherogenic agents (Broedl et al., 2006). Adiponectin belongs to the complement 1q family and comprises four domains; an amino-terminal collagen-like sequence, a variable region, a collagenous domain and a carboxy-terminal globular domain (Garaulet et al., 2007; Kadowaki & Yamauchi, 2005). This protein is constructed by 244 amino acids with a molecular weight of 26,414 Da and typically exists in three forms; low molecular weight (LMW) hexamers, medium molecular weight (MMW) and high molecular weight (HMW) multimeric structures (Gu, 2009; Kadowaki &

Yamauchi, 2005). Adiponectin can be found extensively in serum ranging from 5 to 30 μgml-1 (Garaulet et al., 2007). Table 2 represents the variety of multimeric forms of adiponectin.

This hormone was reported to function in two ways; 1) anti-atherosclerotic actions and 2) insulin-sensitizing actions, through a number of mechanisms. As shown in Figure 2, Wiecek et al. (2007) in a review paper on adiponectin has summarized the anti-atherogenic actions of adiponectin that suppresses the production of tumour necrosis factor-α (TNF- α), modulates biological actions of growth factors by binding with platelet-derived growth factor BB (PDGF-BB), basic fibroblast growth factor (FGF), and heparin-binding epidermal growth factor-like

(30)

10

growth factor (HB EGF), reduces accumulation of lipids in human monocyte-derived macrophages, inhibits transformation of macrophages into foam cells by down-regulating scavenger receptors, suppresses superoxide generation, decreases the expression of adhesion molecules (VCAM-1; ICAM-1, E-selectin), increases expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in infiltrating macrophages, and last but not least increases activity of endothelial nitric oxide (NO) synthase (Wiecek et al., 2007). Figure 2 summarizes the insulin-sensitizing action of adiponectin which stimulates glucose utilization and fatty acid oxidation in skeletal muscles and in the liver, enhances insulin signalling in skeletal muscle, facilitates glucose uptake and suppresses gluconeogenesis in the liver (Wiecek et al., 2007).

This mechanism of actions of adiponectin ultimately results in increasing insulin sensitivity and suppression of atherosclerosis (Kadowaki & Yamauchi, 2005). Since this type of protein characterizes a number of metabolic derangements, it is crucial to have insights into its molecular level.

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11

Table 2 Multimeric forms of adiponectin (Wiecek et al., 2007)

Structures Multimeric Forms Molecular Weight Globular C-terminal

domain fragments

None Monomer

Trimer Low Molecular Weight

(LMW)

Hexamer Medium Molecular

Weight (MMW)

12-mers

High Molecular Weight (HMW)

18-mers

(32)

12

Figure 2 Anti-atherogenic and of insulin-sensitizing actions of adiponectin (Wiecek et al., 2007)

Note:

FGF Fibroblast growth factor

HB EGF Heparin-binding epidermal growth factor ICAM-1 Intercellular adhesion molecule-1

NO Nitric oxide

PDGF-BB Platelet-derived growth factor BB TIMP-1 Tissue inhibitor of metalloproteinase-1 TNF- α Tumour necrosis factor-α

VCAM-1 Vascular cell adhesion molecule-1

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13 1.2.4 Adiponectin Gene

Despite of extensive studies correlating environmental factors such as high fat diet and physical inactivity with the pathogenesis of MS, there is also strong evidence showing that the syndrome is possibly inheritable (Lyssenko et al., 2008). Groop (2000) listed possible genes which are associated with fat and glucose metabolism such as genes for leptin/leptin receptor, β2- and β3- adrenergic receptors, lipases, TNF-α, PPAR-γ, glycoprotein PC-1, IRS-1 and lycogen synthase (Groop, 2000).

Apart from these genes, there is an increase of interest among scientists to focus more onto adiponectin gene recently.

The 16-kb structural gene, which has been mapped to chromosome 3 (3q27) in the human APM1/ACDC/ADIPOQ gene, encodes the protein product adiponectin (Comuzzie et al., 2001). This particular region of chromosome 3 (Accession ID D45371) has also been found to contain a quantitative trait locus (QTL) with a strong

influence on phenotypes of the MS (Kissebah et al., 2000). The adiponectin gene contains three exons and two introns (Gable et al., 2006; Gable et al., 2007;

Kadowaki & Yamauchi, 2005). Exon 1 and 2 are 76 and 222 bp, respectively, with 10.3 kb of intron 1 lies in between them while exon 3 is approximately 4.28 kb. The translation starts at exon 2 and ends at exon 3, leaving exon 1 and part of exon 3 untranslated (Takahashi et al., 2000). Figure 3 and 4 show the ideogram and genomic organization of the adiponectin gene, respectively.

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14

Figure 3 Ideogram of chromosome 3 where adiponectin spanned on 3q27 (National Center for Biotechnology Information, 2008)

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15

Figure 4 Organization of the adiponectin gene on chromosome 3 (Vasseur et al., 2006)

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16

1.2.5 Association of Adiponectin, Adiponectin Gene and Metabolic Syndrome The discovery of adiponectin back in 1995 (Scherer et al., 1995) did not receive major attention in the scientific community for the next few years until its markedly protective role in the pathogenesis of obesity-related disorders was acknowledged in the new millennium. Yang and Chuang (2006) in their review article on human genetics of adiponectin in the MS claimed that this adipose-derived serum protein is an important biomarker for MS.

The human genetic studies not only allow scientists to have an insight into various SNPs and genetic make-up of adiponectin, but also provide information and strong evidence to support the fact that this gene is one of the contributing factors to MS (Yang et al., 2007). Studies demonstrated that serum adiponectin concentration is significantly low in subjects with obesity, insulin resistance, MS, T2D, and coronary heart disease (Broedl et al., 2006; Yang & Chuang, 2006; Yang et al., 2007). Scientists also found a number of variants of SNPs and missense mutations in European, North American and Japanese populations that are closely linked to the MS risk factors (Broedl et al., 2006; Gable et al., 2006). However, the association of adiponectin genetic variations with dyslipidemia and blood pressure was poorly explored (Yang & Chuang, 2006).

Three regions that are closely linked to MS have been identified on the highly polymorphic adiponectin gene; 1) the 5’ sequences; 2) the intron 2-exon 2 region, and 3) exon 3 (Vasseur et al., 2006). This is supported by Gable et al. (2007) which claimed that the promoter variants -11391G>A and -11377C>G, +45T>G variant of

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17

exon 2 and +276G>T variant of intron 2 are the most widely studied; all mentioned variants were selected in this present study. Yang and Chuang (2006) also agreed with the fact that promoter region, exon and intron 2, and the rare non-synonymous mutations in exon 3 harbor a number of common polymorphisms across different ethnic population (Yang & Chuang, 2006). When these polymorphisms were translated, all the mentioned SNPs excluding the -11391G>A introduced changes to the amino acid (Table 3).

Various studies demonstrated that SNP+276 is associated with increased risk of T2D, higher insulin resistance index, lower adiponectin concentration, cardiovascular disease risk, higher BMI, higher glucose level and obesity (Filippi et al., 2004; Hara et al., 2002; Jang et al., 2006; Ukkola et al., 2003; Xita et al., 2005;

Yang et al., 2007). Kadowaki and Yamauchi (2005) addressed that SNP+276 as one of the factors in reducing adiponectin concentration and in turn promote the development of insulin resistance, MS and atherosclerosis (Kadowaki & Yamauchi, 2005).

Haplotype of a variant with other variants perhaps doubled the effect on MS susceptibility. The haplotype +276/+45 was the most common haplotype studied that linked with higher body weight, waist circumference, blood pressure, HDL cholesterol/total cholesterol ratio, lower serum adiponectin and higher risk of T2D (Gable et al., 2006). SNP+45, located in exon 2 is a silent mutation which could possibly alter a putative enhancer or silencer of splicing (Vasseur et al., 2003). On the other hand, SNP+276 is located in intronic region (intron 2) which is well

(38)

18

documented as non-coding region. However, studies have proposed that unknown mechanism(s) of these intronic SNPs perhaps involved in the expression level of the adiponectin gene that ultimately cause the development of MS (Hara et al., 2002;

Jang et al., 2006).

Moreover, a study on French Caucasian population disclosed association between SNP-11391 and SNP-11377 with serum adiponectin concentration and diabetic status (Vasseur et al., 2003). The SNP-11391 was also associated with hypoadiponectinemia in Amish (people devoted to Christian living in Canada or United States) and Swedish population while SNP-11377 was associated with T2D in Japanese population. SNP-11426 has been shown to be associated with variation in insulin sensitivity in French and Swedish population (Gable et al., 2006). Gu (2009) has summarized SNPs of adiponectin gene that have been associated with MS risk factors in various ethnic populations as shown in Table 4 (Gu, 2009).

Although the genetic variation in 5’ sequences has not yet been defined to cause the development of MS, but these variants were shown to be associated with adverse metabolic features. Furthermore, SNPs in adiponectin gene have been demonstrated to contribute to hypoadiponectinemia, decreased insulin sensitivity and T2D in several populations. Therefore, these SNPs are thought to either modulate the expression of the adiponectin gene or in linkage disequilibrium with functional variants to modulate the expression of the gene (Vimaleswaran et al., 2008). There are several other variants contributing to the MS, with some probably still unknown.

Thus, in depth investigations are crucial to precisely determine which variant(s) leads

(39)

19

a significant role to the risk of MS. Taken all the consideration from previous studies as a whole, therefore the presence of SNPs +45, +276, -11426, -11391 and -11377 were shown to be worthy predictors to indicate the characteristics of MS in Malay population.

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20

Table 3 Amino acid changes introduced by respective SNPs in adiponectin gene

SNP Nucleotide Amino Acid Sequence

+45T>G

T

91 TTG CTG GGA GCT GTT CTA CTG CTA TTA GCT CTG CCC GGT CAT GAC 135 31 Leu Leu Gly Ala Val Leu Leu Leu Leu Ala Leu Pro Gly His Asp 45 136 CAG GAA ACC ACG ACT CAA GGG CCC GGA GTC CTG CTT CCC CTG CCC 180 46 Gln Glu Thr Thr Thr Gln Gly Pro Gly Val Leu Leu Pro Leu Pro 60

G

91 TTG CTG GGA GCT GTT CTA CTG CTA TTA GCT CTG CCC GGG CAT GAC 135 31 Leu Leu Gly Ala Val Leu Leu Leu Leu Ala Leu Pro Gly His Asp 45 136 CAG GAA ACC ACG ACT CAA GGG CCC GGA GTC CTG CTT CCC CTG CCC 180 46 Gln Glu Thr Thr Thr Gln Gly Pro Gly Val Leu Leu Pro Leu Pro 60 Note: The single nucleotide change from T to G at SNP+45 does not alter the amino acid in the protein chain.

+276G>T

G

586 ATG AAG GAT GTG AAG GTC AGC CTC TTC AAG AAG GAC AAG GCT ATG 630 196 Met Lys Asp Val Lys Val Ser Leu Phe Lys Lys Asp Lys Ala Met 210 631 CTC TTC ACC TAT GAT CAG TAC CAG GAA AAT AAT GTG GAC CAG GCC 675 211 Leu Phe Thr Tyr Asp Gln Tyr Gln Glu Asn Asn Val Asp Gln Ala 225

T

586 ATG AAG GAT GTG AAG TTC AGC CTC TTC AAG AAG GAC AAG GCT ATG 630 196 Met Lys Asp Val Lys Phe Ser Leu Phe Lys Lys Asp Lys Ala Met 210 631 CTC TTC ACC TAT GAT CAG TAC CAG GAA AAT AAT GTG GAC CAG GCC 675 211 Leu Phe Thr Tyr Asp Gln Tyr Gln Glu Asn Asn Val Asp Gln Ala 225 Note: The single nucleotide change from G to T at SNP+276 alters the amino acid from valine to phenylalanine in the protein chain.

(41)

21 +11426A>G

A

2746 TAG TAA AGA CAG GGT TTC ACC ATA TTG GCC AGG CTG GTC TCG AAC 2790 916 End End Arg Gln Gly Phe Thr Ile Leu Ala Arg Leu Val Ser Asn 930 2791 TCC TGA CCT TGT GAT CTG CCC GCC TCC ATT TTT GTT GTT ATT TTT 2835 931 Ser End Pro Cys Asp Leu Pro Ala Ser Ile Phe Val Val Ile Phe 945

G

2746 TAG TAA AGA CAG GGT TTC ACC ATA TTG GCC AGG CTG GTC TCG AGC 2790 916 End End Arg Gln Gly Phe Thr Ile Leu Ala Arg Leu Val Ser Ser 930 2791 TCC TGA CCT TGT GAT CTG CCC GCC TCC ATT TTT GTT GTT ATT TTT 2835 931 Ser End Pro Cys Asp Leu Pro Ala Ser Ile Phe Val Val Ile Phe 945 Note: The single nucleotide change from A to G at SNP+11426 alters the amino acid from asparagine to serine in the protein chain.

-11391G>A

G

3151 GTT TCC CTC CCG ATA TCA AAA AGA CTG TGG CCT GCC CAG CTC TCG 3195 1051 Val Ser Leu Pro Ile Ser Lys Arg Leu Trp Pro Ala Gln Leu Ser 1065 3196 TAT CCC CAA GCC ACA CCA TCT GGC TAA ATG GAC ATC ATG TTT TCT 3240 1066 Tyr Pro Gln Ala Thr Pro Ser Gly End Met Asp Ile Met Phe Ser 1080

A

3151 GTT TCC CTC CCG ATA TCA AAA AGA CTG TGG CCT GCC CAG CTC TCA 3195 1051 Val Ser Leu Pro Ile Ser Lys Arg Leu Trp Pro Ala Gln Leu Ser 1065 3196 TAT CCC CAA GCC ACA CCA TCT GGC TAA ATG GAC ATC ATG TTT TCT 3240 1066 Tyr Pro Gln Ala Thr Pro Ser Gly End Met Asp Ile Met Phe Ser 1080 Note: The single nucleotide change from G to A at SNP-11391 does not alter the amino acid in the protein chain.

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22 -11377C>G

C

1396 ACG TCC TGT CTT GGA AGG ACT ACT ACT CAA TGG CCC CTG CAC TAC 1440 466 Thr Ser Cys Leu Gly Arg Thr Thr Thr Gln Trp Pro Leu His Tyr 480 1441 TCT ACT TCC TCT TAC CTA TGT CCC TTC TCA TGC CTT TCC CTC CAA 1485 481 Ser Thr Ser Ser Tyr Leu Cys Pro Phe Ser Cys Leu Ser Leu Gln 495 G

1396 ACG TCC TGT CTT GGA AGG ACT ACT ACT CAA TGG CCC CTG GAC TAC 1440 466 Thr Ser Cys Leu Gly Arg Thr Thr Thr Gln Trp Pro Leu Asp Tyr 480 1441 TCT ACT TCC TCT TAC CTA TGT CCC TTC TCA TGC CTT TCC CTC CAA 1485 481 Ser Thr Ser Ser Tyr Leu Cys Pro Phe Ser Cys Leu Ser Leu Gln 495 Note: The single nucleotide change from C to G at SNP-11377 alters the amino acid from histidine to aspartic acid in the protein chain.

\

(43)

23

Table 4 The genetic variants and adiponectin or MS risk factors with the respective ethnic group that has been studied (Gu, 2009)

SNP Adiponectin or MS Risk Factors Ethnic Group

+45

Adiponectin concentration Japanese, Chinese

Type 2 diabetes Korean, Italian, Quebec family study

Obesity Swedish, Finnish

+276

Adiponectin concentration European Caucasians

Type 2 diabetes Japanese, Italian, German

Obesity Chinese, Korean

-11377

Adiponectin concentration French Caucasians

Type 2 diabetes Swedish, Danish

Obesity German, Italian

-11391

Adiponectin concentration French Caucasians

Type 2 diabetes UK Caucasian women

Obesity German, Italian

-11426 Adiponectin concentration French Caucasians, Swedish,

Type 2 diabetes European Caucasians

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24 1.3 Rationale of the Study

In regards to the gradual increase in prevalence of MS risk factors, Song et al. (2006) have pointed out the needs to identify susceptibility genes of MS and its mechanism of actions, which may enable the investigators to design preventive strategies and targeted treatments (Song et al., 2006). To date, there is still lack of nationally representative figures on occurrence and association of adiponectin and MS among Malay adults in Malaysia, let alone their association with the polymorphisms.

Human genetic epidemiological studies on adiponectin and parameters of MS would be helpful to understand the molecular mechanisms involved in regulating metabolism susceptibility hence protect against the development of metabolic diseases. Moreover, metabolic diseases appear to be varied across different population and ethnicity. As the ethnic differences strongly suggest a genetic component in the pathogenesis of metabolic syndrome (Song et al., 2006), therefore, this study perhaps will provide an insight on the underlying causes to prevent or reduce the long-term risks for MS among Malay population.

1.4 Objectives

1.4.1 General Objectives

To investigate the association of the selected SNPs in adiponectin gene with serum adiponectin concentration and MS risk factors among Malay adults.

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25 1.4.2 Specific Objectives

1. To determine the prevalence of SNPs +45, +276, -11426, -11391 and -11377 of the adiponectin gene among Malay adults.

2. To investigate the association between serum adiponectin concentration with the MS and MS risk factors.

3. To investigate the association between the selected SNPs and haplotypes with serum adiponectin concentration.

4. To investigate the association between the selected SNPs and haplotypes with the MS and MS risk factors.

Ethylenediaminetetraacetic acid phenylalanine om asparagine to serine om histidine to aspartic acid

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