ASSOCIATION OF TCF7L2 GENE POLYMORPHISM WITH NON OBESETYPE 2 DIABETES MELLITUS IN JAVANESE POPULATION

gene polymorphism was analyzed by Chi-square and analysis of sample characteristics was carried out with Odds Ratio. The results showed that there were significant differences in the allele frequency of TCF7L2 gene polymorphism and in the characteristics of age, KGD and systole between case subjects and control subjects. From this study it was concluded that TCF7L2 gene polymorphism is associated with the type 2 DM through interactions between genetic factors (T allele) and life style.

Type 2 diabetes is characterized by insulin deficiency due to inadequate insulin secretion or decreased tissue response to insulin at one or more points.Three important factors that influence the pathogenesis of type 2 DM are individual factors or ethnic genetics, factors of pancreatic β cell damage and insulin resistance factors.The TCF7L2 gene is a gene that codes for TCF7L2 protein, a group of proteins that act as transcription factors in the Wnt signal pathway.TCF7L2 gene polymorphism occurs when cytosine is replaced with thymine in the intron 3 position in wildtype due to alternative splicing.The purpose study was to determine the frequency of TCF7L2 gene polymorphism in Javanese type 2 non-obese DM patients in Semarang.Detection of TCF7L2 gene polymorphism was carried out on 49 people with type 2 non-obese DM patients and 38 people not with type 2 DM using the PCR-RFLP method.The restriction enzyme used is Rsa1.The allele frequency of TCF7L2 gene polymorphism was analyzed by Chi-square and analysis of sample characteristics was carried out with Odds Ratio.The results showed that there were significant differences in the allele frequency of TCF7L2 gene polymorphism and in the characteristics of age, KGD and systole between case subjects and control subjects.From this study it was concluded that TCF7L2 gene polymorphism is associated with the type 2 DM through interactions between genetic factors (T allele) and life style.

INTRODUCTION
Diabetes Mellitus (DM) is a non-communicable disease characterized by chronic hyperglycemia which is caused by an impaired insulin secretion (International Diabetes Ari Y., R. Susanti, Nugrahaningsih, Rahmatika S. R.. Association of TCF7L2... 24|BIOMA: Jurnal Ilmiah Biologi, 12 (2), October 2023 Federation, 2017).One type of DM is type 2 DM, this disease is characterized by insulin work deficiency due to inadequate insulin secretion or decreased tissue response to insulin at one point or more (International Diabetes Federation, 2017).
Type 2 Diabetes Mellitus (T2DM), one of the most common metabolic disorders, is caused by a combination of two primary factors: defective insulin secretion by pancreatic β-cells and the inability of insulin-sensitive tissues to respond appropriately to insulin (Garcia et al., 2020) Previously, health experts thought only obese people are at risk for type 2 diabetes yet a recent study denied it.According to the researcher from the University of Florida, thin people are also at risk of developing type 2 diabetes if they have sedentary lifestyles or sitting all day long (Mainous, 2016).
In 2017, the average prevalence of DM in Tawa Tengah was 19.22%, with the number of DM cases in Semarang City as many as 117037 (Dinas Kesehatan Provinsi Jawa Tengah, 2017).TCF7L2 is the most potent locus for type 2 diabetes (T2D) risk and the first locus to have been robustly reported by genomic linkage studies.TCF7L2 is a transcription factor that forms a basic part of the Wnt signaling pathway (Bosque-Plata et al., 2021).The TCF7L2 protein is a key transcriptional effector of the Wnt/β-catenin signaling pathway, which is an important developmental pathway that negatively regulates adipogenesis (Chen, 2018).TCF7L2 gene polymorphism occurs when cytosine is replaced with thymine in the intron 3 position in wild-type due to alternative splicing.
Polymorphism is a variance of DNA sequence that can cause change of protein function in the body.Polymorphism can be caused by environment exposure and mutagen.Polymorphism impact causes susceptible of a population against a disease.The polymorphism that occurs will continue to be lowered so that the frequency of polymorphisms in each ethnicity varies.Many controversies about TCF7L2 polymorphism in the case of type 2 DM, but Li et al. (2021) speculate that the TCF7L2 can promote GLP-1 expression and sensitize pancreatic β-cells to blood glucose, but Ari Y., R. Susanti, Nugrahaningsih, Rahmatika S. R.. Association of TCF7L2... 25|BIOMA: Jurnal Ilmiah Biologi, 12 (2), October 2023 TCF7L2 rs7903146 causes a decrease in protein levels.These underlying mechanisms are consistent with the function and pathological changes in the islets of T/T allele homozygotes.The presence of the risk allele (T) TCF7L2 causes the Wnt signal to be unable to regulate proglucagon gene expression, so the proglucagon gene cannot encode the GLP-1 protein.It results in a decrease in ATP concentration in pancreatic β cells, so that the K+ canal opens and depolarizes the pancreatic β cell membrane, and closes the Ca2+ canal.The low concentration of Ca2+ in pancreatic β cells results in insulin-filled granules ready to be released or Readily Releasable Granules (RRG) unable to fuse with the plasma membrane.As a consequent, insulin is not secreted outside pancreatic β cells.
Decreasing insulin secretion causes an increase in hepatic sugar production and a decrease in glucose use by tissues, resulting in chronic hyperglycemia.As β-cells are attacked, the body fails to produce insulin, resulting in insulin deficiency, chronic hyperglycemia, and long-term complications (Khin et al., 2023).Numerous studies worldwide have evaluated the link between TCF7L2 gene and a high risk of T2DM (Elhourch et al., 2021).In Indonesia, there is no data on the genotypic distribution of type 2 non-obese Javanese DM patients in Semarang based on TCF7L2 gene markers.TCF7L2 gene polymorphism can be used as a marker for early detection of non-obese type 2 DM disease, thus minimizing further risk of the disease.The purpose of this study was to determine the frequency of TCF7L2 gene polymorphism in non-obese Javanese patients of type 2 DM in Semarang City.Every patient who entered the health center was seen in his medical record, then patients who met the inclusion criteria were collected until 49 samples.Respondents as a case and control group after signing informed consent were taken blood by officers of the CITO Laboratory (laboratories that collaborated in the Prolanis program at Pegandan, Pandanaran, Ngaliyan, Gunungpati, Puskesmas, Unnes Polyclinic, and Nayaka Husada Semarang City).Inclusion criteria from the characteristics of the samples taken: type 2 DM patients, ages 30-65 years, BMI ≤ 25 kg/m2, fasting blood sugar levels ≤126 mg/dL, no hypertension (≤140 mmHg), and ethnic Javanese.3 ml of blood was taken from each respondent by the aseptic method using a sterile syringe, put in Vacutainer tube containing K3EDTA and absolute ethanol.Blood samples were stored in a freezer at -20ºC until DNA isolation is carried out.

DNA Isolation
The DNA extraction method is the GeneJET Genomic DNA Purification Kit.A spoonful of spatula blood sample was crushed until smooth using a mortar, then added 400 µL Lysis Solution and refined again.Then the mixture was transferred to a 1.5 mL microtube and added with 20 µL Proteinase K Solution.Then the solution is homogenized using vortex.The mixture was incubated at 56°C for 10 minutes and every 5 minutes the mixture was shaken to form a number 8.Precipitation of DNA was carried out by adding 200 µL of Ethanol 96% to the sample and being vortexed.Then the suspension was transferred into the column and collection tube, the suspension was centrifuged at a speed of 6000 x g for 1 minute.The liquid in the collection tube is removed and the collection tube is reinstalled.500 µL Wash Buffer I was added to the suspension, then centrifuged at 8000 x g for 1 minute.The liquid in the collection tube is removed and installed again.
After that, 500 µL of Wash buffer II was added to the suspension and centrifuged at a speed of 12000 x g for 3 minutes.The suspension was re-centrifuged at a speed of 12000 x g for 1 minute.Then the collection tube is removed and the column is moved to a 1.5 mL microtube.The column suspension was added to 100 µL Elution Buffer and incubated at room temperature for 2 minutes.The suspension was centrifuged at 8000 x g for 1 minute.Then the suspension was centrifuged again at a speed of 8000 x g for 1 minute.
The column was removed and the DNA is stored on a 1.5 mL microtube which has been labeled at -20°C.
The amplification process of TCF7L2 gene on the thermocycle PCR machine (eppendorf) are; first step, Hotstart with a temperature of 95oC for 10 minutes, second step denaturation at 95oC for 1 minute, annealing (attaching primer) at 58oC for 1 minute, the next step is extension at 72oC for 1 minute, the final step is the final extension at 72oC for 7 minutes.Gene TCF7L2 Amplification with specific primer will result DNA fragment size 113 bp.

Restriction Fragment Length Polymorphism (RFLP)
Detection of polymorphism was done using the PCR-RFLP method.The amplified product was cut using the Rsa1 restriction enzyme (Biolabs Inc, New England).3 µL PCR products were taken using a micropipette and inserted into a 0.2 mL microtube, then added 0.5 µL RsaI enzyme, 1 µL NE Buffer and 5.5 µL ddH2O free nuclease.The mixture was incubated for 1 hour at 37oC.

Electrophoresis
The results of PCR-RFLP were electrophoresed using 3% agarose gel to determine whether DNA fragments were amplified and cut according to the target.Agarose gel electrophoresis performed at voltages of 100 volts for 30 minutes.The electrophoresis results were checked using UV illuminator [Alpha Innotech] then.

Data Analysis
Molecular data analysis of TCF7L2 gene polymorphism in Javanese ethnic type 2 non-obese DM patients included qualitative and quantitative analysis.Qualitatively by looking at the DNA band of PCR-RFLP results from TCF7L2 gene in gel electrophoresis.
While quantitatively is seeing whether there is a difference in frequency of TCF7L2 gene polymorphism in type 2 non-obese DM patients with controls using Chi-Square test and

RESULTS AND DISCUSSION
This study involved 87 subjects, consisting of 49 people with type 2 diabetes mellitus non obese as the case subject and 38 non-obese people who did not suffer from type 2 diabetes as control subjects.The characteristics of the research subjects can be seen in Table 1.Statistically, different factors between case and control subjects were age and blood sugar levels.While systolic blood pressure was considered different due to DM differences.High blood sugar levels cause thickening of blood vessels which causes narrowing of blood vessel diameter due to increased blood pressure and hypertension.
Amplification of the TCF7L2 gene using specific primers F: 5'-GAG AGC TAA GTA CTT AGG TA-3 'and R: 5'-CTG ACA TTG ACT AAG TTA CTT GC-3' was successfully carried out using DNA fragments in Figure 1.The results of the calculation of the frequency of TCF7L2 gene polymorphism in patients with type 2 DM and controls performed on all samples (49 sufferers and 38 controls) are seen in Table 2. Genotypes found in type 2 DM subjects were CC, CT and TT genotypes.While in non-type 2 DM subjects (controls) only found two genotypes, CC and CT genotypes, while TT genotypes were not found.Based on the results of the frequency analysis of genotypes and alleles in Table 3, the frequencies of C and T alleles from both samples showed significant differences.Thus, the T and C alleles play a role in the appearance of type 2 DM on Javanese ethnic, but it is likely not a major risk factor for Javanese type 2 DM.
Based on the results of allele frequency analysis in all samples showed that the frequencies of T and C alleles were significantly different with p = 0.044.This showed that the T and C alleles play a role in the appearance of type 2 DM in Javanese ethnic groups.Based on this study, the possibility of TCF7L2 gene polymorphism is a risk factor for the occurrence of non-obese type 2 DM in ethnic Javanese.The Asian population has shown positive results regarding the relationship of TCF7L2 gene polymorphism with DM type 2. Other studies with similar results where TCF7L2 gene polymorphisms are positively associated with the occurrence of type 2 DM in the French population (Cauchi et al., 2017), African-Americans (Sale et al., 2017), Finland (Scott et al., 2016), UK (Groves et al., 2016), Japan (Miyake et al., 2018), and China (Chang et al., 2017).Each population has a different frequency of polymorphism.
T allele in the TCF7L2 gene is referred to as risk alleles and is associated with impaired insulin secretion (Alibegovic et al., 2016).T alleles in the TCF7L2 gene are often associated with pancreatic β cell dysfunction and type 2 DM (Grants et al., 2016).(Meloni et al., 2016).
The low concentration of Ca2+ in pancreatic β cells results in insulin-filled granules ready to be released or Readily Releasable Granules (RRG) unable to fuse with the plasma membrane, consequently, insulin is not secreted outside pancreatic β cells.Decreasing insulin secretion causes an increase in hepatic sugar production and a decrease in glucose use by tissues, resulting in chronic hyperglycemia.Chronic hyperglycemia will lead to hyperstimulated metabolism, which will be constitutively coupled to hyperexcitability and chronically elevated Ca2+, factors that have all been suggested to play a role in diabetic loss of β-cell function (Shyr et al., 2019).
Based on the results of the study, the gender average of women was greater than men, both in the case subjects and in the control subjects.Based on the analysis of the odds ratio, the possibility of women having a risk of 0.869 times for the occurrence of type 2 DM compared to men.Type 2 diabetes mellitus, it is highlighted that it is more frequently diagnosed in men who have a lower age and body mass index than in women (Ciarambino et al., 2022), this occurs because men are influenced by body fat distribution, fat accumulation is concentrated around the abdomen so that it triggers central obesity which is more at risk of triggering metabolic disorders and the emergence of type 2 DM.
The incidence of type 2 DM varies between the sexes in one population with another population.This difference is not significant unless it is affected by lack of physical Research conducted by Kurniawaty & Bella (2016) has a significant relationship between age and incidence of type 2 DM, where people > 45 years have 9 times the risk of the occurrence of type 2 DM compared to those aged < 45 years.The existence of the aging process causes a reduction in the ability of pancreatic β cells to produce insulin.In addition, there is a decrease in mitochondrial activity in muscle cells by 35% in older individuals.This is associated with an increase in muscle fat levels by 30% and triggers insulin resistance (Pardede et al., 2017).
Based on the results of the characteristic analysis between type 2 non-obese DM subjects and control subjects showed that KGD (blood sugar levels) was associated with the emergence of type 2 DM in Javanese ethnic.Various factors, including genetics, weight, age, sex, alcohol intake, coffee and caffeine intake, and smoking habits can affect KGD which triggers the occurrence of type 2 diabetes.An imbalance between highenergy food intake and energy expenditure for long-term activities allows the occurrence of insulin resistance and type 2 diabetes.Intake of high energy food can stimulate insulin resistance through increased blood sugar levels.
Some of the evidence underpinning these concerns is derived from cross-sectional surveys or ecological observations, but there are also prospective cohort studies and controlled intervention trials which have addressed this issue.This brief narrative review arises from a Symposium on Sugars and Health held at the European Nutrition Conference in October 2015 (Macdonald, 2016).Reducing carbohydrate intake can increase insulin sensitivity in healthy individuals and decrease fasting blood sugar levels in type 2 DM patients (Bolla et al., 2016).Too much reduction in the number of carbohydrates consumed is not allowed for people with type 2 diabetes.
The TCF7L2 gene is not the only gene that can cause DM type 2. Some gene variants that play a role in type 2 DM include the Peroxisome Proliferator-Activate Receptor gene PP (PPARG), Potassium inwardly-rectifying subfamily channel J member research was conducted at the Laboratory of Molecular Biology, Biology Department, FMIPA, Universitas Negeri Semarang, Semarang and the Laboratory of Biochemistry, Faculty of Medicine, Public Health and Nursing, Gadjah Mada University, Jogjakarta in January-October 2018.The equipment in this study were Sterile Spuit, Vacutainer tube containing K3EDTA, Ice-box, Ice gel/ice cool, tourniquet, digital scales, weigh paper, autoclave, erlenmeyer 250 ml, measuring cup, microtube, micropippete, tip, centrifuge, incubator, Thermocycler, spindown, microwave, -20oC deep freezer, horizontal electrophoresis and doc gel.The materials in the study were absolute ethanol, alcohol swab, DNA isolation kit [GENEJET Genomic Purification Kit]: Lysis Solution, Proteinase K, Wash Buffer I, Ari Y., R. Susanti, Nugrahaningsih, Rahmatika S. R.. Association of TCF7L2... 26|BIOMA: Jurnal Ilmiah Biologi, 12 (2), October 2023 Wash Buffer II, 96-100% Ethanol, Elution Buffer, 70% alcohol, tissue, blood sample, forward & reverse primer, PCR Mix and dH2O free nuclease (Thermoscientific), RsaI [Biolab] enzyme, NE Buffer, ddH2O free nuclease, 3% Agarose Gel, 1X TBE buffer, fluorosave, 50 bp DNA ladder, parafilm paper and loadingdye.The population in this study were non-obese type 2 DM patients, while the sample in this study was 49 patients non-obese type 2 DM and 49 not patients of non-obese type 2 DM who met the criteria.The determination of sample size in this study was based on inclusion and exclusion criteria.The determination of Puskesmas (health center) and clinics were based on the number of patients and the schedule of Prolanis, so that Puskesmas Pegandan, Pandanaran, Ngaliyan, Gunungpati, Unnes Polyclinic, and Nayaka Husada clinics are selected that meet the inclusion criteria: type 2 DM patients, nonobese, not hypertension, 30-65 years Javanese ethnic.Exclusion criteria is criteria from the research subject which should not be exist and if subject has exclusion criteria therefore subject must exclude from the research.Exclusion criteria in this research are: patients with type 2 DM who are not willing to be the subject of research and absent at the time of data collection.Blood Sampling Before this research is conducted beforehand, the proposal is sent to the Research Ethics Commission, Research and Development Unit of Semarang State University (UNNES) to obtain information on ethical appropriateness.Then asked for permission to conduct research to KESBANGLITMAS, Head of Semarang City Health Office and Head of Puskesmas to be used as a research place.The study is conducted after obtaining permission for Research and Ethical Clearance from the Biomedical Research Ethics Commission on Humans at the State University of Semarang and after Type 2 DM patients were asked for approval and willingness to take part in research and provide informed consent.In each patient who will be included in this study are explained the background and purpose of this study : a.Given the freedom to choose whether to take part or not in this research.b.Patients who are willing to take part in this study are asked to fill out informed consent.c.Prioritized of service by always heeding applicable ethics Ari Y., R. Susanti, Nugrahaningsih, Rahmatika S. R.. Association of TCF7L2... 27|BIOMA: Jurnal Ilmiah Biologi, 12 (2), October 2023 relationship between sample characteristics of type 2 DM patients nonobese with Chi-Square test and Odds Ratio quantitatively begins by calculating allele frequency and frequency of genotype.

Figure 2 .
Figure 2.Visualization and illustration of polymorphism geneTCF7L2 after Rsal enzyme restriction.DNA Ladder Marker50 bp (M).UC= uncut(PCR product before restriction with enzyme).Red ribbon: DNA marker, green ribbon: 91 bp DNA fragment and blue ribbon: 113 bp DNA fragmentsThe results of visualization of restriction products in Figure2showed that the Wild type (CC) had two DNA fragments measuring 91 bp and 22 bp (but 22 bp is not visible), The presence of the risk allele (T) TCF7L2 causes the Wnt signal to be unable to regulate proglucagon gene expression.As a result, the glucagon gene cannot code GLP-1 protein and results in decreased cAMP levels in pancreatic β cells.The reduced cAMP concentration in pancreatic β cells cannot activate Protein Kinase A (PKA) and The cAMP regulated guanine nucleotide exchange factor (cAMP-GEF) (Epac) which causes the K+ canal to remain open and the Ca2+ canal remains closed.This results in high concentrations of K+ in pancreatic β cells and vice versa the concentration of Ca2+ in pancreatic β cells is low activity and central obesity.Changes in lifestyle are associated with an increase in the frequency of type 2 DM.The appearance of type 2 DM is often associated with age factors.The results of the analysis showed that the average Javanese type 2 DM subjects aged > 45, while the average control subject was <45 years old.Based on the statistical analysis in this study, people aged > 45 years had a risk of 4,149 times for the occurrence of type 2 diabetes aged <45 years.Research conducted byMagliano et al., (2020) showed an accumulating data suggest that type 2 diabetes mellitus (T2DM) in younger people (aged <40 years), referred to as young-onset T2DM, has a more rapid deterioration of βcell function than is seen in later-onset T2DM.

Table 1 .
Characteristics of subjects with type 2 DM and non-type 2 DM

Table 2 .
Frequency of Genotype and Allele Gene TCF7L2