091701022 Priya

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.