Final Year Research Project Proposal

Association of ADIPOQ Polymorphisms with the Risk of

Developing Metabolic Syndrome Among Orang Asli Temiar

Mohd Haikal bin Mohd Hashim

(BHCL18049605)

A Proposal Submitted in Partial Fulfilment for the Degree of

Bachelor of Biomedical Science (Hons.)

Supervisor:

Dr. Mohd Nizam bin Zahary

Faculty of Health Sciences

Universiti Sultan Zainal Abidin

October 2021
 Table of Contents

Chapter 1: Introduction ................................................................................... 1

1.1 Study Background ....................................................................................... 1
1.2 Problem Statement ...................................................................................... 2
1.3 Significance of the Study ............................................................................. 2
1.4 Research Questions .................................................................................... 3
1.5 Hypothesis .................................................................................................. 3
1.6 General Objective ........................................................................................ 3
1.10 Specific Objective ...................................................................................... 3

Chapter 2: Literature Review .......................................................................... 4

2.1 Metabolic Syndrome .................................................................................... 4
2.1.1 NCEP ATP III Criteria in Diagnosing Metabolic Syndrome .................. 4
2.1.2 Metabolic Syndrome Pathogenesis ..................................................... 5
2.2 Orang Asli Temiar ........................................................................................ 5
2.3 Protein Adiponectin ...................................................................................... 6
2.3.1 Adiponectin Receptors ........................................................................ 6
2.3.2 Function of Adiponectin ....................................................................... 7
2.4 ADIPOQ Gene Polymorphisms ................................................................... 7
2.4.1 Association of ADIPOQ +45T>G (rs2241766) Polymorphisms with
Metabolic Syndrome ..................................................................................... 7
2.4.2 Association of ADIPOQ +276G>T (rs1501299) Polymorphisms with
Metabolic Syndrome ..................................................................................... 8
2.5 Polymerase Chain Reaction-Restriction Fragment Length Polymorphism
(PCR-RFLP) .................................................................................................. 8

Chapter 3: Methodology .................................................................................. 10

3.1 Study Design ............................................................................................... 10
3.2 Study Period ................................................................................................ 10
3.3 Study Location ............................................................................................. 10
3.4 Reference Population .................................................................................. 10

i
3.5 Source Population ....................................................................................... 10
3.6 Sampling Frame .......................................................................................... 10
3.7 Subjects Population and Site Selection ....................................................... 11
3.8 Inclusion Criteria .......................................................................................... 11
3.9 Exclusion Criteria ......................................................................................... 11
3.10 Estimation of Sample Size ......................................................................... 11
3.11 Study Procedures ...................................................................................... 12
3.11.1 Genotyping of ADIPOQ +45T>G (rs2241766) and +276G>T
(rs1501299) Polymorphisms ........................................................................ 12
3.12 Flowchart of the Study Activities ................................................................ 14

Chapter 4: Expected Results .......................................................................... 15

Chapter 5: Gantt Chart and Milestone ........................................................... 18

5.1 Gantt Chart .................................................................................................. 18
5.2 Milestone Table ........................................................................................... 19

Chapter 6: Reference ....................................................................................... 20

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 Chapter 1: Introduction

1.1 Study Background

Metabolic Syndrome (MetS) is a collection of risk factors for type 2 diabetes mellitus
(T2DM), coronary heart disease (CHD), and other atherosclerotic cardiovascular disease
(CVD) (Kassi et al., 2011). MetS has been recognized as a serious global health problem
with significant health implications (O'Neill et al., 2016). This major public-health issue is
escalating as a result of urbanization, increase in obesity and sedentary lifestyle routines
(Mohamud & Suraiami, 2010). The prevalence of MetS is different based on the
populations studied. The increased risk of both the MetS and its individual components is
influenced by variability in genetic makeup, diet, levels of physical activity, population age
and gender, levels of over- and undernutrition, and body habitus (Cameron et al., 2004).
MetS potentially increases the risk on developing T2DM up to five times, whereas in a
systematic review of 37 studies involving over 170,000 patients found that MetS doubles
the risk of cardiovascular events (Ramli et al., 2010). Despite of the previous history of
cardiovascular events, MetS patients are having two to four-fold of increased risk for
stroke, a three to four-fold increased chance for myocardial infarction (MI) and a two-fold
risk of dying to such event compared to those without MetS.

Adiponectin, encoded by the ADIPOQ gene, is a hormone that believed to be

strongly related to the development of MetS. This is because adiponectin exerts protective
effects against inflammation and able to enhance insulin sensitivity (Khoramipour et al.,
2021). In normal subjects, the hormone adiponectin has crucial physiological functions in
maintaining the metabolic balance; therefore, decreased serum adiponectin levels can be
found in MetS patients (Zlibut et al., 2019). Single nucleotide polymorphisms (SNPs) at
ADIPOQ loci are strongly related to hypoadiponectinemia, and shown to be associated
with risk of MetS involving variety of population groups (Lau & Muniandy, 2011). SNPs
ADIPOQ +45T>G (rs2241766) and +276G>T (rs1501299) are some of the most studied
ADIPOQ polymorphisms in developing MetS.

Orang Asli (OA) Temiar is an indigenous group that available in Peninsular

Malaysia. Since some decades ago, they have been involved in relocation program that
directly exposed them to the sedentary lifestyle of modern living. Due to the culture shock,

1
this relocated OA tends to develop lifestyle-associated diseases or health condition
including MetS. Therefore, this study aims to investigate the association of genetic
polymorphisms of ADIPOQ +45T>G and ADIPOQ +276G>T with susceptibility risk of
MetS among OA Temiar in Kuala Betis, Gua Musang, Kelantan.

1.2 Problem Statement

MetS is a health condition and a collection of risk factors, which include elevated
blood pressure, obesity and raised blood sugar level. It relates to most of the lifestyle-
associated diseases. Presence of SNPs of ADIPOQ in the genetic make-up of individuals
may increase the risk on developing this health condition. Early diagnosis of MetS by
detection of this polymorphisms would lead to improvement for the disease management
and prevention. Exposure of unhealthy lifestyle of urban living on the OA community has
increased the risk on getting MetS condition or its associated diseases. This would
eventually increase the expenditure and demand for healthcare services for this
population.

1.3 Significance of the Study

This study on the specified polymorphisms will provide new understandings about
the molecular mechanism of MetS and its pathophysiological implication on chronic
diseases such as CVD. Considering that MetS diagnosis is complicated, it is frequently
be unnoticed and eventually untreated until the condition become more severe. These
polymorphisms have potential to serve as the biomarker for early diagnosis of MetS
among OA Temiar community. Early diagnosis is possible through the detection of
potential biomarker in primary prevention strategy, such as early screening of MetS based
on individual’s genetic make-up. This will improve the management of MetS, enable early
intervention and prevention, preventing the progression of more severe diseases, and
improve quality-of-life.

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1.4 Research Questions

Is single nucleotide polymorphisms (SNPs) of ADIPOQ +45T>G (rs2241766) and

ADIPOQ +276G>T (rs1501299) in the ADIPOQ gene associated with the risk of MetS
development among OA Temiar.

1.5 Hypothesis

1. There is no association of genetic polymorphisms of ADIPOQ +45T>G and

ADIPOQ +276G>T with susceptibility risk of MetS among Orang Asli Temiar in
Kuala Betis, Gua Musang, Kelantan.
2. There is an association of genetic polymorphisms of ADIPOQ +45T>G and
ADIPOQ +276G>T with susceptibility risk of MetS among Orang Asli Temiar in
Kuala Betis, Gua Musang, Kelantan.

1.6 General Objective

To investigate the association of genetic polymorphism of ADIPOQ (ADIPOQ +45T>G
and ADIPOQ +276G>T) with MetS susceptibility among Orang Asli Temiar in Kuala Betis,
Gua Musang, Kelantan.

1.7 Specific Objective

1. To determine the genotype and allele frequency of ADIPOQ +45T>G and ADIPOQ
+276G>T among OA Temiar sub-tribe in RPS Kuala Betis, Gua Musang, Kelantan.
2. To investigate the risk association of variant genotypes of ADIPOQ +45T>G and
ADIPOQ +276G>T polymorphisms with susceptibility to MetS.

3
 Chapter 2: Literature Review

2.1 Metabolic Syndrome

Metabolic syndrome (MetS) can be generally defined as a disorder of energy

utilization and storage that reflects underlying insulin resistance condition (Kean Ghee &
Wee Kooi, 2016). It is also a commonly used term for a cluster of clinical and metabolic
factors that increase the risk in getting type 2 diabetes (T2DM), coronary artery disease
(CAD) and stroke (Zafar et al., 2018). MetS can be characterized based on the presence
of at least three out of five components, namely central obesity, hypertension, high fasting
blood sugar level, high serum triglycerides and low high-density lipoprotein cholesterol
(HDL-C) (Mendrick et al., 2018). However, there are a number of slightly different
diagnostic criteria regarding the measurement of those risk factors that has been
introduced from different organizations, including the modified World Health Organization
(WHO 1998), the International Diabetes Federation (IDF) criteria, the revised National
Cholesterol Education Program (NCEP ATP III) and the Joint Interim Statement (JIS 2009)
criteria (Kean Ghee & Wee Kooi, 2016). Thus, results that involved the study of prevalence
of MetS can be varied, depends on the guideline used. Apart from that, MetS prevalence
also varies across the populations due to factors like genotype, ethnicity and lifestyle
(Zafar et al., 2018).

2.1.1 NCEP ATP III Criteria in Diagnosing Metabolic Syndrome

In 2001, the National Cholesterol Education Program (NCEP) Adult Treatment

Panel III (ATP III) has introduced a list of specific criteria to determine MetS, and was
updated by the American Heart Association (AHA) and the National Heart, Lung, and
Blood Institute (NHLBI) in 2005 (Grundy et al., 2005). According to the modified NCEP
ATP III, the diagnosis of MetS required the fulfillment of at least any three from the five
following criteria: abdominal obesity which can be measured using waist circumference
(WC), suggesting the cut-off of 90 cm in Asian origin men and 80 cm in Asian origin
women; hypertriglyceridaemia (triglycerides ≥1.7 mmol/L); low HDL cholesterol (HDL-C ≤
1.03 mmol/L for men and ≤1.29 mmol/L for women); hypertension (systolic blood pressure

4
≥130 mmHg and/or diastolic blood pressure ≥85 mmHg) or currently use antihypertensive
drugs; impaired fasting blood glucose level (fasting plasma glucose ≥5.6 mmol/L) (Moy &
Bulgiba, 2010). The modified NCEP ATP III criteria are shown to be much reliable than the
IDF criteria as shown in a study of MetS diagnosis among Malays in Kuala Lumpur, as
7.6% of the Malays with MetS as defined by modified NCEP ATP III were failed to be
diagnosed with MetS when using IDF criteria (Moy & Bulgiba, 2010). Thus, modified NCEP
ATP III is believed to show reliable result even when applied in a study involving different
Malaysia ethnics.

2.1.2 Metabolic Syndrome Pathogenesis

The pathogenesis of Mets involves a complex interaction of pathways, genetic

variants, and environmental variables like sedentary lifestyle and unhealthy diet. The
exact etiology of MetS is still unclear, but studies have linked the MetS pathogenesis with
the pathways that lead to insulin resistance, oxidative stress, inflammation, obesity,
endothelial dysfunction, and cardiovascular diseases (Zlibut et al., 2019).

2.2 Orang Asli Temiar

Malaysia is a multiracial country consisting a major ethnic of Malay, Chinese,

Indians, and mixed ethnic groups including the indigenous people or Orang Asli (OA).
Temiar is a subtribe of Senois tribe with a population of 30,000 to 40,000. Most of them
can be found on the fringes of rainforests of Peninsular Malaysia, subsisting on traditional
activities like forest gathering. OA Temiar provides an excellent opportunity for gene-
environment interactions study. However, in recent decades, they have been subjected to
relocation initiatives which have exposed them to unhealthy lifestyle and diet of the
modern living. Despite that, their genetic materials are believed to be well preserved due
to infrequent inter-marriages with other ethnics (Zahary et al., 2020). In a study conducted
by Zahary et al. suggests a crucial role of ADIPOQ +45T > G (rs2241766) and +276G >
T (rs1501299) polymorphisms in predisposing OA Temiar to MetS. Not much study

5
involving the indigenous people with MetS had been conducted especially the study that
relate their genetic components with this health condition.

2.3 Protein Adiponectin

Adiponectin is a fat-derived hormone and adipokine that encoded by the ADIPOQ

gene, which exclusively synthesized by adipocytes. Adiponectin can be found in the
circulation as the form of low-molecular weight (LMW) of homotrimers and hexamers, and
full-length high-molecular weight (HMW) multimers. A smaller form of adiponectin consists
globular domain are available in plasma with insignificant amounts. Adiponectin plays an
important role in protection against insulin resistance and atherosclerosis (Arunkumar E.
Achari & Sushil K. Jain, 2017). Adiponectin deficiency is regarded to be a key factor in the
development of type 2 diabetes, obesity, and cardiovascular disease in humans.
Therefore, many studies have been done among various groups in different countries in
order to investigate the relationship between adiponectin with MetS, with most of them
demonstrate an inverse relationship between the serum adiponectin level and MetS
(Robberecht & Hermans, 2016).

2.3.1 Adiponectin Receptors

There are two main receptors for adiponectin, namely AdipoR1 and AdipoR2.
AdipoR1 has high affinity for globular adiponectin and low affinity for full-length
adiponectin in skeletal muscle. Meanwhile, in the liver, AdipoR2 generates intermediate
affinity towards both globular and full-length HMW adiponectin. AdipoR1 can be found
mainly in skeletal muscle, synovial firoblasts, endothelium, and atrial cells, whereas
AdipoR2 is expressed mainly in the liver, which able to inhibit "peroxisome proliferator-
activated receptors type alpha" (PPAR-α) receptors, promotes insulin sensitivity (Kayvan
Khoramipour et al., 2021).

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2.3.2 Function of Adiponectin

Generally, adiponectin binds to its receptors to regulate energy metabolism,

inflammatory responses, insulin sensitivity, and fat-burning process. The actions exerted
by adiponectin can cause pleiotropic effects which leads to different influence or function
in different organs. In liver, adiponectin involved in the regulation of glucose-uptake and
lipids metabolism, by lowering gluconeogenesis, and promoting glycolysis and fatty acid
oxidation (Kayvan Khoramipour et al., 2021). Adiponectin positive effects on heart include
the increase translocation of cluster of differentiation 36 (CD36) and fatty acids absorption
together with raised insulin-stimulated glucose uptake and phosphorylation of Akt, in
cardiomyocytes (Kayvan Khoramipour et al., 2021).

2.4 ADIPOQ Gene Polymorphism

Human ADIPOQ gene is located on chromosome 3q27.3 that spans 15,790bp with
three exons and two introns, responsible to encode adiponectin hormone. Genetic
variants in ADIPOQ gene may cause substantial changes in adiponectin levels, which
provides convincing evidence that the genetic code alterations can significantly affect the
health (Sanchez et al., 2019). Single nucleotide polymorphisms (SNPs) at the ADIPOQ
loci are shown to be associated with the risk of T2DM and MetS in some prospective
epidemiological studies involving various population groups, but often with contrasting
results. The 276G>T (rs1501299) and 45T>G (rs2241766) SNPs of ADIPOQ gene are
the ones that are most studied (Laclaustra et al., 2007).

2.4.1 Association of ADIPOQ +45T>G (rs2241766) Polymorphisms with Metabolic

Syndrome

The relationship between ADIPOQ 45T>G polymorphism with MetS are variable
according to different studies. Some of the studies have reported the insignificance
association between 45T>G polymorphism and adiponectin concentrations, as it only
involves synonymous mutation (Gly15Gly) in exon 2. However, there are studies that
show the significance results between the 45T>G SNPs with MetS. For Example, Berthier
7
et al. and Vasseur et al. in Quebec and France respectively have reported a significant
association between the G minor allele with lower adiponectin level (Berthier et al., 2005;
Vasseur et al., 2005). Apart from that, a study involved a large cohort (SAFIR) of French
Caucasians by Fumeron et al. has found that the G minor allele was associated with
weight gain (Laclaustra et al., 2007).

2.4.2 Association of ADIPOQ +276G>T (rs1501299) Polymorphisms with Metabolic

Syndrome

Studies have reported, with some level of consistency, the minor T allele at the
276G>T polymorphism in intron 2 has been significantly associated with higher plasma
adiponectin level. However, there are still studies that correlate this polymorphism with
MetS components. Mohammadzadeh et al. have found that T allele of SNP 276G>T
associated with the risk of CAD in among subjects with T2DM (Mohammadzadeh et al.,
2016). A large case control study conducted by Filippi et al. also have shown a significant
association between SNP 276G>T in ADIPOQ gene with CAD (Filippi et al., 2005).

2.5 Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-

RFLP)

PCR-RFLP is a standard laboratory technique that are used in small-scale basic

scientific studies of complicated genetic conditions linked to SNPs (Hashim & Al-Shuhaib,
2019). Application of PCR-RFLP assay in SNPs studies crucially involves specific
restriction enzymes in order to discriminate SNPs genotype. The products generated from
the PCR-RFLP is then would be separated based on their size via electrophoresis before
the results can be observed. In the genotyping of ADIPOQ +45T>G and +276G>T
polymorphism carried-out by Zahary et al. in a study to determine risk of MetS with the
ADIPOQ polymorphisms among OA Temiar, DNA are firstly extracted from the collected
venous blood of the subjects. Amplicons with ADIPOQ genetic variants then amplified
using feasible forward and reverse primers designed. Then the amplicons containing the
interest polymorphic sites were digested using BspHI and BsmI restriction enzyme. After

8
the application of restriction enzyme, amplicons with ADIPOQ 45T>G that were cleaved
by BspHI would generate three genotypes: homozygous wild type (T/T) would be
produced as two fragments, heterozygous (T/G) would be shown as three fragments, and
homozygous variants (G/G) would be presented as one fragment only since the restriction
enzyme assigned would not cleave the G allele. The amplicons with ADIPOQ 276G>T
that were cleaved by BsmI would also generate three genotypes: homozygous wild type
(G/G) as three fragments, heterozygous (G/T) as four fragments, and homozygous
variants (T/T) as a single fragment since T allele would not being cleaved by BsmI (Zahary
et al., 2020). These fragments can only be observed after performing gel electrophoresis
and analysis.

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 Chapter 3: Methodology

3.1 Study Design

The study design for this project is cross-sectional study on relocated OA Temiar subtribe
in Rancangan Pengumpulan Semula (RPS) Kuala Betis, Gua Musang, Kelantan.

3.2 Study Period

The study period is estimated to be conducted within the whole two semester or eight
months of the final year: 15/10/2021 – 16/6/2022.

3.3 Study location

The participants blood sample are collected at RPS Kuala Betis, Gua Musang, Kelantan,
and will be processed at Universiti Sultan Zainal Abidin, Kampus Perubatan, Jalan Sultan
Mahmud, 20400 Kuala Terengganu, Terengganu.

3.4 Reference population

The reference population for this study is OA Temiar subtribe group in Malaysia.

3.5 Source population

The source population of this study is OA Temiar subtribe in RPS Kuala Betis, Gua
Musang, Kelantan.

3.6 Sampling frame

The sampling frame of this study is simple random sampling.

10
3.7 Subjects population and Site Selection

RPS Kuala Betis is located at around 30km from Bandar Gua Musang. It consists of 22
villages with a total population of over 2,000 people, and about 1260 of them are adults.
This site was pragmatically chosen due to the capability of ensuring appropriate
representation of Temiar sub-tribe population that had experience urbanization and
modernization. Application for ethical approval will be requested from UniSZA Human
Research Committee (UHREC) together with written permission from Jabatan Hal Ehwal
Orang Asli (JHEOA) and head of each village for the study to be conducted.

3.8 Inclusion criteria

The inclusion criteria for this study are the participants with:

1. Age > 18 years

3.9 Exclusion criteria

The exclusion criteria for this study are the participants with either one of the following
issues:

1. Psychiatric illness
2. Neurological deficit
3. Body dysmorphic
4. Individuals who refuse to participate in the study

3.10 Estimation of sample size

Sample size is calculated using single proportion formula:

𝑧 2
𝑛 = ( ) 𝑃(1 − 𝑃)
∆

n: sample size

z: value from table of probabilities of standard normal distribution of desired CI (95%)

11
∆: precision (generally at 0.05)

p: expected proportion of individuals in the sample with features of interest at the

determined CI. It can be obtained from literature, a pilot study or preliminary work.

The value of p was taken from Wan Nazaimoon and Suraiami, 2010.

1.96 2
n = (0.05) 0.22(1 − 0.22)

n = 264

However, for the purpose of FYP study, the sample size used will be reduced to 50. It
means that 50 individuals’ data will be randomly choose from the exact sample size.

3.11 Study Procedures

In order to reduce variability in data collecting methods, investigators and interviewers will
be trained in the standardization of research protocols prior to the start of the study.
Demographic data such as gender, age, occupation, and smoking status will be collected
using standardized interviewer-based questions. Recruitment of the participants will be
done with announcements and invitations through the villages’ leader via JHEOA. The
goal of the study will be informed to the participants. They will be asked to not eat or drink
for eight hours before screening. The purpose of screening is to sort-out eligible subjects.
The participants will be given information pamphlets about the study and written consent
will be obtained.

3.11.1 Genotyping of ADIPOQ +45T>G (rs2241766) and +276G>T (rs1501299)

Polymorphisms

This study uses the ADIPOQ genotyping procedure including the primers design
applied by Zahary et al. (2020) in their study of increased risk of metabolic syndrome with
genetic polymorphism of ADIPOQ among a Temiar population in Malaysia. The

12
genotyping procedure comprises three parts, namely DNA extraction, polymerase chain
reaction-restriction fragment length polymorphism (PCR-RFLP), and gel electrophoresis.
DNA extraction will be performed on collected venous blood of the participants
using QIAamp DNA Blood Mini kit to extract the genomic DNA.
The genotyping of the ADIPOQ polymorphisms by PCR-RFLP on the extracted
genomic sample will be performed using master mix, specific PCR primers, and specific
restriction enzymes (BspHI and BsmI).
Gel electrophoresis will be used for ADIPOQ polymorphisms analysis and
detection in a 2% agarose gel.

13
3.12 Flowchart of the Study Activities

Source population:
OA Temiar subtribe from RPS Kuala Betis,
Gua Musang, Kelantan.
Sampling frame:
Simple random
sampling
Study subjects identification.

Ethical clearance

Raw data (MetS criteria among OA Temiar)

obtained.

DNA extraction from venous blood.

Genotyping of SNPs ADIPOQ +45T>G

(rs2241766) and +276G>T (rs1501299)
Polymorphisms

Association of SNPs of ADIPOQ with MetS

susceptibility risk.

Statistical analysis (by SPSS software) and

analysis of results.

Figure 1 shows flow of data processing and interpretation.

14
 Chapter 4: Expected Results

These are the examples of gel electrophoresis image of PCR-RFLP analysis of ADIPOQ +45T>G
and ADIPOQ +276G>T. Both images retrieved from Zahary et al. (2020).

Figure 2 shows the products of gel electrophoresis of PCR-RFLP analysis of

ADIPOQ +45T>G polymorphism. Lane: M, 100 bp marker; Lane 1, heterozygous
genotype (490, 274 and 216 bp); Lane 2 and 3, homozygous variant genotype (490
bp); Lane 4, homozygous wild type genotype (274 and 216 bp).

Figure 3 shows the products of gel electrophoresis of PCR-RFLP analysis of

ADIPOQ +276G>T polymorphism. Lane: M, 100 bp marker; Lane 1,
heterozygous genotype (634, 265, 256 and 113 bp); Lane 2, homozygous
wild type genotype (265, 256 and 113 bp); Lane 3 and 4, homozygous
variant genotype (634 bp).
15
Table 1: Genotype and allele frequencies of ADIPOQ +45T>G (rs2241766) and
+276G>T (rs1501299) polymorphisms in study subject (n=50).

MetS Non-MetS P-value

n (%) n (%)
Genotype ADIPOQ +45T>G (rs2241766)
Homozygous wildtype
(TT)
Heterozygous (TG)
Homozygous variant
(GG)
Allele
T
G
Genotype ADIPOQ +276G>T (rs1501299)
Homozygous wildtype
(GG)
Heterozygous (GT)
Homozygous variant (TT)
Allele
G
T

16
Table 2: Risk association of ADIPOQ +45T>G (rs2241766) and +276G>T (rs1501299)
polymorphisms with MetS susceptibility

Genotype MetS Non-MetS OR P-value

n= n= (95% CI)
Homozygous
wildtype
(TT)
Heterozygous
(TA)
Homozygous
variant
(AA)

17
 Chapter 5: Gantt Chart and Milestone
5.1 Gantt chart
Period 2021 2022
Activities Oct. Nov. Dec. Jan. Feb. Mac. Apr. May. Jun.
Project discussion
Proposal writing
Proposal presentation
Purchasing chemicals,
reagents, accessories
DNA extraction
Genotyping of
ADIPOQ +45T>G
(rs2241766) and
+276G>T (rs1501299)
polymorphism via
PCR-RFLP
Gel electrophoresis
Report writing
Final result
presentation

18
5.2 Milestone Table
Activities Completion Date Cumulative Project
Completion Percentage
(%)
Project discussion November 2021 10
Proposal writing November 2021 30
Proposal presentation December 2021 35
Purchasing chemicals, December 2021 45
reagents, accessories
DNA extraction December 2021 50
Genotyping of ADIPOQ January 2022 65
+45T>G (rs2241766) and
+276G>T (rs1501299)
polymorphism via PCR-
RFLP
Gel electrophoresis January 2022 70
Report writing May 2022 95
Final result presentation June 2022 100

19
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