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Presented by
Ms. Priya JoshiRegistration No. : 091701022
VI Semester III Year, B.Sc. BiotechnologyManipal Life Sciences Centre
Manipal University
Project Guide
Dr. Padmalatha Rai SProfessor
Division of BiotechnologyManipal Life Sciences Centre
Manipal University
Impact of TCF7L2 Gene Variant on Risk of Type 2 Diabetes
High blood sugar
Classic symptoms - Polyuria, polyphagia, polydypsia
300 million people will have diabetes world-wide by 2020 (1)
Treatable since insulin became available in 1921
Major long term complications include damage to blood vessels
Doubles the risk of cardiovascular disease
Diabetes
According to the WHO if the fasting glucose is > 126 mg/dL the person is Diabetic.
Type I
• Characterized by islet cell dysfunction
• Exact cause unknown, most likely autoimmune
• Failure to produce insulin (1)
Type II
• Defects in insulin secretion and action
• People suffering from T2D are generally obese and at a higher risk of heart diseases
• Peripheral insulin resistance (1)
Gestational
• Older than 25 when pregnant
• Family history of diabetes
• High blood pressure
• Increased amount of amniotic fluid (1)
Diabetes Classification
Type II Diabetes
Quantitative Traits• BMI• Gender• Age• Glucose
Environmental Factors
• Diet• Exercise• Style of living
Genetic Factors•SNPs•miRNAs•CNV
Factors Influencing Type II Diabetes
• The TCF7L2 gene product is a high mobility group (HMG) box containing transcription factor implicated in blood glucose homeostasis. (2)
• The risk conferring genes in TCF7L2 were associated with impaired beta-cell function but not with insulin resistance. (3)
• There are 9 exons in this gene.
• Both SNPs were in the intron.
• The previous reports suggest that TCF7L2 polymorphisms are associated with type 2 diabetes, hence studies were carried out to establish association amongst South Indian populations.
TCF7L2 GENE
Location : 10q25.3
Size: 217428bp
Exons: 9
Gene polymorphisms
Exon
Intron
TCF7L2 GENE
Functions:Participates in Wnt signaling pathway and modulates MYC by binding to its promoter.Acts as a repressor in the absence of CTNNB1 and as an activator in its presence.Expression of dominant negative mutants results in cell cycle arrest in G1.Necessary for the maintenance of the epithelial stem cell compartment of the small intestine.
Tissue specificity:Detected in epithelium from small intestineAlso detected in colon epithelium
TCF7L2 GENE
Molecular functions:DNA bindingβ-catenin binding Chromatin binding
Biological functions:Anti-apoptoticCell cycle arrestNegative regulation of Wnt regulator signaling pathwayResponse to glucose stimulusPositive secretion of insulin and pancreatic development
Post-translational modifications:
Phosphorylated at Thr-201 and/or Thr-212 by NLK (4)
Polysumoylated (5)
To evaluate the association of TCF7L2 gene variants with the risk of Type 2 Diabetes and related quantitative traits
Objective
Study Participants
Inclusion criteria
Clinically diagnosed Type 2 Diabetes mellitus
• Both Males & Females with in Age group of 25-70 years
• Cases with no history of diabetes for at least 1st degree relatives were taken as Controls
Exclusion criteria
Type 1 Diabetes
Gestational diabetes
Secondary diabetes
Hematological diseases
Ethical clearance was obtained from Kasturba Hospital ethical committee and written informed consent was taken from the participating subjects
Study Design
Methods
DNA Sequencing
1
4
3
2
Collection of blood sample from T2D and normal control subjects
Amplification of target sequence
Genotyping by PCR-RFLP
Amplification of target sequence and genotyping by Tetra ARMS PCR
2
Tetra ARMS PCR
ARMS – Amplification refractory mutation system.
Both inner primers encompass a deliberate mismatch at position -2 from the 3’ terminus.
Some use post PCR modifications such as RFLP.
In contrast Tetra ARMS requires no post PCR modifications. (6)
Statistical Analysis
Level of significance will be accepted only when P ≤ 0.05
Chi-square tests will be conducted to examine whether the genotype frequencies of the selected SNPs were in Hardy-Weinberg equilibrium (HWE).
Multiple logistic regression analysis will be carried out to assess the strength of the association between polymorphic genes in terms of odd ratio (ORs) with 95% confidence intervals (CIs)
346 bp
211 bp 135 bp
Results: Genotyping of TCF7L2 (rs12243326) Polymorphisms by PCR-RFLP and DNA Sequencing
TCF7L2 T>C
1 2 3
TT TC
Electropherogram of TCF7L2 TC
Electropherogram of TCF7L2 TT
1. TC (HZ) 2. TT (WT)3. 100bp marker
Results: Genotyping of TCF7L2 (rs7903146) Polymorphisms by Tetra ARMS PCR and DNA
Sequencing
Electropherogram of TCF7L2 CT
Electropherogram of TCF7L2 TT
TCF7L2 C>T
1. CC (WT)2. TT (WT)3. CT (HZ)4. 100bp marker
1 2 3 4
CLINICAL CHARACTERS
T2D SUBJECTS (n = 130)
CONTROL SUBJECTS (n = 120)
*P
n(men/women) 50 (32/18) 50(27/23)
Age (years) 54.2±9.352 45.9±14.2 *<0.001
Time of Disease onset (years) 8.5±7.0
BMI (Kg/m2) 24.56±2.45 20.58±2.13 *<0.001
HbA1c (%) 9.98±2.75 5.09±0.65 *<0.0001
FPG (mg/dl) 227.3±80.14 87.40±11.27 *<0.0001
RPG (mg/dl) 235.3±129.4 112±22.12 *<0.0001
PPG (mg/dl) 254.7±88.44 121.2±22.9 *<0.0001
Total cholesterol (mg/dl) 178.5±40.41 168.7±25.5 *0.0511
Triglycerides (mg/dl) 145.3±58.7 103.4±25.8 *<0.0001
HDL Cholesterol (mg/dl) 31.51±11.36 47.8±11.21 *<0.0001
LDL Cholesterol (mg/dl) 117.94±37.90 108.8±64.1 0.252
*Pearson’s chi square test
Clinical Characteristics of the T2D Subjects and Control
Level of significance < 0.05
Association of Candidate SNP loci with Type 2 Diabetes
T2D Control T2D Control0
1020304050607080
WT HZMT
SNP (Gene)
Chromosome Chromosome position
*Risk/Non risk allele
Case/Control
RAF in case
RAF in controls
OR (95% CI)
P
rs12243326(TCF7L2)
10 114788815 T/C 70/60 0.24 0.218 0.7642(0.3714-1.573)
0.464
rs7903146(TCF7L2)
10 114758349 C/T 60/60 0.89 0.30 0.213(0.09-0.470)
<0.0005
*Risk alleles as identified in earlier European studies, (7) *RAF- risk allele frequency
% G
enot
ype
freq
uenc
y rs 12243326 rs 7903146TT
TC
CC
TT
CC
TT
CC
CC
TT
CTCT
TC
Subjects
• We studies 60 case and 60 control samples for rs7903146 and 70 cases and 60 controls for rs12243326.
• Case and control samples for both SNPs were found to be in HWE.
• The results obtained were not in accordance to previously published work. This can be attributed to small sample size.
• rs7903146 showed a significant reduced risk towards the development of the disease in South Indian population, whereas, rs12243326 showed non-significant reduced risk.
SUMMARY
REFERENCES 1. Jonathan, E. S, Paul, Z., Daniel, et al. Type 2 Diabetes Worldwide According to the New
Classification and Criteria. Diabetes Care (2010); 23 : B5–B10.2. Yi, F., Brubaker, P. L., Jin, T. TCF-4 mediates cell type-specific regulation of proglucagon
gene expression by beta-catenin and glycogen synthase kinase-3-beta. J. Biol. Chem (2005); 280: 1457-1464.
3. Shu, L., Matveyenko, A. V., Kerr-Conte, J., et al. Decreased TCF7L2 protein levels in type 2 diabetes mellitus correlate with downregulation of GIP- and GLP-1 receptors and impaired beta-cell function. Hum. Molec. Genet (2009); 18: 2388-2399.
4. Ishitani, T., J. Ninomiya-Tsuji, et al. Regulation of lymphoid enhancer factor 1/T-cell factor by mitogen-activated protein kinase-related Nemo-like kinase-dependent phosphorylation in Wnt/beta-catenin signaling. Mol Cell Biol (2003); 23(4): 1379-89.
5. Yamamoto, H., M. Ihara, et al. Sumoylation is involved in beta-catenin-dependent activation of Tcf-4. EMBO (2003); J 22(9): 2047-59.
6. Shu, Y., Sahar, D., Xiayi, K., et al D. An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Research (2001); 29 : 1-8.
7. Saxena R, Voight BF, Lyssenko V., et al. Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels. Science (2007); 316:1331–1336.