• Summary

    We aim to explore the relation of TCF7L2 gene polymorphism, inflammatory status,the disease control parameters, and the dietary habit of the diabetic patients type II. we also aim to  explore the relation of the genetic variants and the dietary paramenters in the patient's relatives and compare the results to those of the patients

  • Achievements

     

    1. Following Laboratory investigations were done:
    1. Random Blood Glucose:

    Assay was done using Beckman Coulter UniCel DxC 600 Synchron clinical system, (Instruments Inc., Scientific Instruments Division, Fullerton, CA 92634-3100, USA) applying enzymatic colorimetric method.

     

    1. Glycated Hemoglobin (HbA1c):

    HbA1c was measured using Roche cobas b 101 POC system (Roche Diagnostics International Ltd, CH-6343 Rotkreuz, Switzerland).

    1. Interleukin-6 Assay by ELISA

    Assay was performed using the commercially available Human IL-6 PicoKine ELISA Kit (Product code: EK0410) supplied by Boster Biological technology (3942 B Valley Ave, Pleasanton, CA 94566, United States).

    Assay Procedure:

    All reagents and samples were brought to room temperature (20–25°C) before use.

    • 100 µL ml per well of the 300 pg/ml, 150 pg/ml, 75 pg/ml, 37.5 pg/ml, 18.75 pg/ml, 9.375 pg/ml, and 4.6875 pg/ml human IL-6 standard solutions were pipetted into the precoated 96-well plate. 100 µL of the sample diluent buffer was added into the control well (Zero well).
    • 50 µL of sample diluent buffer was added to each well, then 50 μl of the serum samples was added to the corresponding wells.
    • The plate was sealed with the cover and incubated at 37°C for 90 min.
    • The cover was removed, the plate content was discarded, and the plate was blotted onto paper towels.
    • 100 µL of anti-human IL-6 antibody working solution (diluted 1:100 with antibody diluent buffer) was added into each well and incubated at 37°C for 60 min.
    • The plate was washed 3 times with 0.01 M of PBS, letting the washing buffer stay in the wells for 1 min. The washing buffer was discarded and the plate was blotted onto paper towels.
    • 100 µL of prepared substrate working solution (diluted 1:100 with substrate diluent buffer) was added into each well and incubated at 37°C for 30 min.
    • The plate was washed 5 times with 0.01 M of PBS, each time letting washing buffer stay in the wells for 1-2 min. The washing buffer was discarded and the plate was blotted onto paper towels.
    • 90 μl of color developing agent was added into each well and incubated at 37°C in dark for 20-25 min.
    • 100 µL of stop solution was added into each well. The color changed into yellow immediately.
    • The absorbance was read at 450 nm in a microplate reader within 30 min after adding the stop solution.

    Calculation of Results:

    A log-log curve was constructed on graph paper by plotting the absorbance obtained from each standard on the vertical (Y) axis against its concentration in pg/mL on the horizontal (X) axis. The Human IL-6 concentration of the samples was interpolated from the standard curve. The results from the interpolation were multiplied by 2 to account for the dilution factor of the samples and obtain the results before dilution.

    Assay Range: 4.69 pg/ml – 300 pg/ml

    Description: elisa curve.jpg

    Figure (1): Log-log curve of human IL-6 ELISA Kit

    1. Genotyping of TCF7L2 gene:

    Genomic DNA extraction and analysis for TCF7L2 gene polymorphisms was done by real-time PCR technique.

    This process was done through three steps which included:

    • Extraction of genomic DNA from peripheral blood leucocytes of EDTA anticoagulant blood.
    • Amplification of the extracted DNA.
    • Allelic discrimination.

     

     
    • Extraction of Genomic DNA from Peripheral Blood Leucocytes:

    Human genomic DNA was isolated from peripheral blood leukocytes using G-spin total DNA extraction kit from iNtRON Biotechnology, Inc. (Catalog no. 17045).

    Reagent preparation:

    • All reagents were brought to room temperature before use (20– 25°C).
    • 40 ml of absolute ethanol alcohol was added to washing buffer B before use.
    • Lyophilized proteinase K was dissolved in 1.1 ml of DNAse free water.
    • Lyophilized RNase A was dissolved in 0.3 ml of DNAse free water.

    Assay procedure:

    Frozen whole blood samples on EDTA tubes were allowed to thaw and brought to room temperature only before time of analysis. Repeated freezing and thawing was avoided.

    • 200 μl of whole blood was pipetted into a 1.5 ml microcentrifuge tube.
    • 20 μl of Proteinase K and 5 μl of RNase A were added into sample tube and gently mixed.
    • 200 μl of Lysis solution buffer was added into upper sample tube and mixed thoroughly.
    • Lysate was incubated at 56°C for 10 min.
    • The 1.5 ml tube was briefly centrifuged to remove drops from the inside of the lid.
    • 200 μl of absolute ethanol was added into the lysate, and mixed well by gently inverting 5 - 6 times or by pipetting. After mixing, the 1.5 ml tube was briefly centrifuged to remove drops from inside of the lid.
    • 700 μl of washing buffer A was added to the spin column without wetting the rim, and centrifuged for 1 min at 13,000 rpm. The flow-through was discarded while the collection tube was reused.
    • 700 μl of washing buffer B was added to the spin column without wetting the rim, and centrifuged for 1 min at 13,000 rpm. The flow-through was discarded and the column was placed into a 2.0 ml collection tube (reused), and then centrifuged for additionally 1 min to dry the membrane. The flow-through and collection tube were discarded altogether.
    • The spin column was placed into a new 1.5 ml tube, and 50 μl of elution buffer was added directly onto the membrane. The mixture was incubated for 1 min at room temperature and then centrifuged for 1 min at 13,000 rpm to elute.

     

    • Amplification of the Extracted DNA using Polymerase Chain Reaction (PCR):

    The extracted DNA was amplified using TaqMan Universal Master Mix II (Catalog number: 4440043) and ready-made TaqMan SNP genotyping assay for rs7903146 (Catalog number: 4351379) from Applied Biosystems.

    Reagents Supplied:

    1. The TaqMan Universal Master Mix , was supplied in a 2 concentration and contained:

    • AmpliTaq Gold DNA polymerase

    • dNTPs with dUTP

    • ROX dye as a Passive Reference

    • Optimized buffer components

    1. The 40TaqMan® SNP Genotyping Assay contains:

    • Sequence-specific forward and reverse primers to amplify the polymorphic sequence of interest:

    Context Sequence of the Tested Polymorphism [C Nucleotide to be detected by JOE Dye/T Nucleotide to be detected by FAM Dye]

    TAGAGAGCTAAGCACTTTTTAGATA[C/T]TATATAATTTAATTGCCGTATGAGG

    It is a ready to use kit.

    • Two TaqMan probes:

      • One probe labeled with JOE dye detects the C Allele sequence
      • One probe labeled with FAM dye detects the T Allele sequence

    • 1 TE buffer

    The Polymorphism is a missense mutation, C/T, transition substitution in intron 5 region of the TCF7L2 gene on chromosome (10q25.3.).

    Reagent Preparation:

    The ready-made SNP genotyping assay was diluted from 40X to a 20 working stock with 1 TE buffer. The mixture was vortexed, then centrifuged. The working stock was stored at –15 to –25 °C.

    Assay procedure:

    • The TaqMan Universal Master Mix II was thoroughly mixed by swirling the bottle.
    • The frozen working solution of TaqMan SNP genotyping assay was thawed by placing them on ice. The tubes were vortexed then centrifuged briefly.
    • Genomic DNA was thawed on ice, then centrifuged briefly.
    • The components from table (1) were combined in an appropriate tube and vortexed.

    Table (1): The PCR reaction mix (total volume of 25 µL).

    Component

    Volume (µL) per reaction

    TaqMan® Universal Master Mix II (2)

    12.5

    TaqMan® genotyping assay mix (20)

    1.25

    Genomic extracted DNA

    5

    DNAse free water

    6.25

    • The PCR reaction mix (total volume of 25 µL) was pipetted onto the reaction plates.
    • The plate was loaded into the real-time PCR system.
    • The program was set up for amplification under conditions in table (2).

    Table (2):       Thermal cycler programming for amplification and genotyping

    Initial heating

    Denaturation

    Annealing/Extension

    950C for 10 min.

    920C for 15 sec.

    600C for 45 sec.

    -

    40 amplification cycles.

    Description: C:\Users\HP\Desktop\srep30015-f2.jpg

    Figure (2): An example of an amplification curve with baseline set correctly, beginning after the maximum deadline. Different curves refer to different samples.

    • Allelic Discrimination:

    Real-time PCR was performed on the Stratagene Mx3000P real-time thermal cycler (Corbett Research, Australia). The system software uses the fluorescence measurements from each well made during the plate read, and then plots signal values. The software determines which alleles are in each sample for later genotyping analysis. The system software represents the results of the genotyping on a scatter plot (two variables graph) of allele 1 versus allele 2. Each well is represented as an individual point on the plot.

    1. Results and statistics:

     

    A highly significant statistical difference between patients and controls as regards the chosen indicators for glycemic control was observed, as shown in table (3). Random blood glucose (RBG) showed a highly significant statistical difference between cases and controls (p<0.001) with the mean increased from controls (91.96 ± 16.48 mg/dl) to cases (212.48 ± 68.92 mg/dl), while glycated hemoglobin (HbA1c) showed a highly significant statistical difference between cases and controls (p<0.001) with the mean increased from controls (5.34 ± 0.48%) to cases (8.83 ± 1.77%) (Fig. 3).

    Table (3):         Comparison between cases and control groups as regards glycemic control indicators (HbA1c and random blood glucose)

     

    Cases

    Controls

    t test

    Mean ± SD

    Mean ± SD

    p value

    sig.

    HbA1c

    8.83  ± 1.77

    5.34  ± 0.48

    <0.001

    HS

    RBG

    212.48  ± 68.92

    91.96  ± 16.48

    <0.001

    HS

    HS: Highly Significant; HbA1c: Glycated Hemoglobin (%); RBG: random blood glucose (mg/dl). SD: standard deviation.

    A high statistically significant difference between cases and control groups (p= 0.003) was found as regards interleukin-6 serum level, with the mean value in controls (32.37 ± 24.13 pg/ml) and (54.28 ± 25.55 pg/ml) in cases, as shown in table (4)

     

    Table (4):       Comparison between cases and control groups as regards IL-6 serum levels.

     

    Cases

    Controls

    t test

    Mean ± SD

    Mean ± SD

    p value

    sig.

    Interleukin 6

    54.28 ± 25.55

    32.37 ± 24.13

    0.003

    HS

    HS: Highly Significant. IL-6 in pg/ml. SD: standard deviation

    Figure (3): Comparison between cases and control groups as regards HbA1c (%) and serum IL-6 (pg/ml).

     

    There was no statistically significant difference between the two groups in dietary habits (table 5).

    Table (5):         Comparison between cases and control groups as regards dietary habits.

     

    Cases

    Controls

    t test

    Mean ± SD

    Mean ± SD

    p value

    sig.

    Caloric intake

    2524.66 ± 900.94

    2272.36 ± 668.52

    0.266

    NS

    Carbohydrates

    317.66 ± 115.03

    290.80 ± 106.69

    0.396

    NS

    Fats

    91.46 ± 36.84

    83.00 ± 33.53

    0.401

    NS

    NS: Non-Significant. Calories in K-Cal. Carbohydrates and fats in gm. SD: standard deviation.

    As regards TCF7L2 genotyping, 18 out of all 50 subjects (36%) enrolled in the study carried the CC homozygous genotype, while 30 subjects (60%) carried the heterozygous CT genotype, with only 2 subjects (4%) carried the homozygous TT genotype. There was no statistically significant difference found regarding TCF7L2 gene genotypes between case and control groups (p =0.183) (table 6). Also there was no statistically significant difference found regarding T and C alleles’ expression between case and control groups (p =0.205) (table 7).

    Table (6): Comparison between case and control groups regarding TCF7L2 genotype.

     

    Controls

    Cases

    Fisher’s exact test

    Count

    %

    Count

    %

    p value

    sig.

    Genotype

    CC

    12

    48%

    6

    24%

    0.183

    NS

    CT

    12

    48%

    18

    72%

    TT

    1

    4%

    1

    4%

    NS: Non-Significant

    Table (7): Comparison between cases and control groups regarding T and C alleles’ expression.

     

    Controls

    Cases

    Chi square

    Count

    %

    Count

    %

    p value

    sig.

    T and C alleles’ expression

    C allele

    36

    72%

    30

    60%

    0.205

    NS

    T allele

    14

    28%

    20

    40%

    NS: Non-Significant

    There is significant positive statistical correlation between HbA1C as a cumulative glycemic control indicator, and BMI in the whole sample (p= 0.002) as well as in the cases group (p= 0.036). Moreover, random blood glucose was significantly positively correlated with BMI in both the whole sample and the cases group (p= 0.006 and 0.023 respectively), but not in the control group (p= 0.776).

    Table (8): Correlation between glycemic control indicators and BMI

     

    All subjects (n=50)

    Cases     (n=25)

    Controls (n=25)

    BMI

    BMI

    BMI

    HbA1c

    r

    0.433

    0.422

    0.139

    p value

    0.002

    0.036

    0.509

    Sig.

    HS

    S

    NS

    RBG

    r

    0.384

    0.454

    0.060

    p value

    0.006

    0.023

    0.776

    Sig.

    HS

    S

    NS

    HS: Highly Significant; S: Significant; NS: Non-Significant; HbA1c: Glycated Hemoglobin; RBG: random blood glucose; BMI: Body mass index.

     

    For clear comparisons between the genotypes and other variables, the whole sample was divided into two major groups; negative for T allele (CC) and positive for T allele (CT+TT). A strong statistically significant difference was found between the genotypes regarding BMI (p=0.014), as well as HbA1c (p=0.031). Nevertheless, no statistically significant difference was found between the two genotypes groups as regards the RBS (p=0.069) as well as IL 6 serum levels (p=0.562). Table (9) demonstrates all mentioned comparisons.

     

     

    Table (9):         Comparison between the two genotypes groups regarding BMI, HbA1c, RBS, and IL 6 serum level respectively.

     

    CC (18)

    CT+TT (32)

    t test

    Mean ± SD

    Mean ± SD

    p value

    sig.

    BMI (kg/m2)

    27.928 ± 5.529

    32.978 ± 7.296

    0.014

    S

    HbA1c (%)

    6.28 ± 1.61

    7.53 ± 2.35

    0.031

    S

    RBS (mg/dl)

    125.39 ± 58.38

    167.31 ± 84.99

    0.069

    NS

    Interleukin 6 (pg/ml)

    46.31 ± 26.79

    41.64 ± 27.36

    0.562

    NS

    S: significant. NS: not significant. SD: standard deviation.

    The results in table (10) demonstrate the correlations between glycaemic control indicators (HbA1c and RBG) and IL-6 in the whole sample, cases group and control group. This correlation was statistically significant in the whole sample, but non-significant in cases group or in control group (fig. 4).

    Table (10):        Correlation between glycaemic control and interleukin 6 serum level in the whole sample, cases group and control group respectively

     

    All subjects
    (n=50)

    Cases
    (n=25)

    Controls (n=25)

    IL-6

    IL-6

    IL-6

    HbA1c

    R

    0.437

    0.275

    0.015

    p value

    0.002

    0.183

    0.941

    Sig.

    HS

    NS

    NS

    RBG

    R

    0.387

    0.221

    0.231

    p value

    0.005

    0.287

    0.266

    Sig.

    HS

    NS

    NS

     HS: Highly Significant; NS: Non Significant; HbA1c: Glycated Hemoglobin; RBG: random blood glucose

     

    Figure (4): Highly significant upward trend in the correlation between serum IL-6 levels (in pg/ml) and HbA1c (in %) in the whole sample (p= 0.002).

    There was a significant positive correlation between BMI and levels of serum IL-6 in the case group. However, the same correlation was not statistically significant in control group (table 11).

    Table (11): Correlation between BMI and IL6 serum level in cases and control groups

     

    Cases

    Controls

    BMI

    BMI

    Interleukin 6

    R

    0.416

    0.005

    p value

    0.038

    0.981

    Sig.

    S

    NS

    S: Significant; NS: Non significant; BMI: body mass index

    There was a significant positive correlation between levels of serum IL-6 and dietary fat intake in the case group as shown in (fig. 5). However, the same correlation was not statistically significant with caloric or carbohydrate intakes.

     

    Figure (5): Significant upward trend in the correlation between serum IL-6 levels (in pg/ml) and dietary fat intake (in grams) in the case group (p=0.042).

     

    1. The TCF7L2 genotyping results were submitted for publish. The paper was accepted to be published at the Acta Scientific Pharmaceutical Sciences  (ASPS) Journal (ISSN: 2581-5423) on the issue release in February 2020

     

  • List of Publications from the Project

    Mahmoud Ibrahim Mohamed Hassanein Elghazawy., et al. “Assessment of TCF7L2 Polymorphism in Type II Diabetes in Egyptian Population". Acta Scientific Pharmaceutical Sciences 4.2 (2020): 02-08.

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