It is a thesis presentation on " Genotyping of Thiopurine S-methyltransferase (TPMT) for its Variant TPMT*3B (G460A) in a Bangladeshi Population".

1. Genotyping of Thiopurine S-methyltransferase
(TPMT) for its Variant TPMT*3B (G460A) in a
Bangladeshi Population
Presented by
Md. Solayman
Session: 2014-2015
Department of Biochemistry & Molecular Biology
Faculty of Biological Sciences
Jahangirnagar University, Savar, Dhaka-1342

3. Thiopurine S-methyltransferase (TPMT)
 TPMT encodes TPMT enzyme that metabolizes thiopurine
drugs.
 TPMT is located in the 6p22.3.
6
Figure 1. Location of TPMT gene in the short (p) arm of
chromosome 6 at position 22.3
 Subcellular Expression: Human liver, Kidney and RBC.

4. TPMT is a Phase II Enzyme
Figure 2. Fraction of clinically used drugs metabolized by the
major phase II enzymes (left) and 3D structure of TPMT enzyme
(right)
 TPMTcatalyzes the S-methylation of thiopurine drugs

5. Thiopurine Drugs
Azathioprine (AZA) 6-Mercaptopurine 6-Thioguanine
Used in the treatment of acute lymphoblastic leukemia,
autoimmune disorders (e.g., Crohn's disease and Rheumatoid
Arthritis) and of organ transplant recipients
Figure 3. Structure of common thiopurine drugs

6. Polymorphism of TPMT
 Single Nucleotide Polymorphisms (SNPs) in TPMT gene
influence TPMT activity
 To date, 26 genetic variants of TPMT have been identified
 TPMT*3B (G460A) is one of the major SNPs found in TPMT
 TPMT*3B (G460A) SNP causes the amino acid change as
Ala154Thr
 TPMT*3B SNP causes intermediate activity in its heterozygous
variant [TPMT*3B (GA)]
 TPMT*3B SNP causes low activity in its homozygous variant
[TPMT*3B (AA)]

7. Although a number of studies have reported on the detection of
TPMT*3B (G460A) variants in several populations, presence or
absence of TPMT*3B (G460A) polymorphisms in any
Bangladeshi population is not yet known.
Hypothesis

8.  To examine the biochemical markers of liver and kidney
function status of the recruited subjects
 To include the healthy ones based on the status of liver and
kidney function markers
This study was aimed to anticipate possible drug dose-
response relationship among Bangladeshi subjects for drugs
to be metabolized by TPMT as well as to preview the
management of relevant diseases with the selection of
appropriate drug and its dose.
continued
Aims and Objectives

9.  To detect G460A mutation in TPMT leading to the
generation of TPMT*3B variants
 To estimate the genotype and allelic frequencies of
TPMT*3B variants
 To optimize suitable condition for PCR method to detect
TPMT*3B variants at the genomic level

11. Place of Study
The study was conducted in the Research Laboratory for
Biomedical Sciences, Department of Biochemistry and
Molecular Biology, Jahangirnagar University, Savar, Dhaka,
Bangladesh.
Study Design
It was a cross-sectional study.
Study Subjects
A total of symptomatically healthy unrelated 129 male and
female subjects were recruited irrespective of race, religion and
socioeconomic status.

12. Inclusion Criteria
 Undergraduate as well as Postgraduate Students of
Jahangirnagar University as study subjects
 Subjects representing different Divisions of Bangladesh.
 Symptomatically healthy subjects
 Subjects having levels of liver and kidney function markers
within the normal ranges
Exclusion Criteria
 Subjects unable to provide informed consent
 Subjects providing unreliable information
 Subjects having abnormal levels of liver and kidney function
markers

13. AST, ALT, ALP,
DBIL, TBIL, TP, Alb
Biochemical Parameters
Creatinine, Urea, TP,
Alb
 All of the biochemical parameters were measured by
Spectrophotometric method (QCA mini Biochemistry Auto
Analyzer, Spain)
 Glycemic status: Glucose
 Haemato-immunological status: ESR

14. Study of TPMT*3B (G460A) Polymorphism
i. DNA extraction from whole blood: Genomic DNA
Purification Kit (Promega, USA)
 Quantitative determination of DNA concentration: Nano
Drop Spectrophotometer
 Qualitative determination of DNA yield: Agarose gel (0.7%)
electrophoresis
ii. Polymerase chain reaction (PCR)
 Qualitative determination of amplified product: Agarose gel
(1.5%) electrophoresis
 Primer set:
Forward: 5ˈ- AGGCAGCTAGGGAAAAAGAAAG -3ˈand
Reverse: 5ˈ- CCTTATAGCCTTACACCCAG - 3ˈ
 PCR product size was 690 bp
continued

15. iii. RFLP of TPMT*3B (G460A) polymorphic marker in TPMT
gene was analyzed using MwoI restriction enzyme
Recognition site of MwoI restriction enzyme was
5′…GCNNNNN NNGC…3′
3′…CGNN NNNNNCG… 5′
 Enzyme digestion product was resolved in agarose gel (2.0%)
electrophoresis.

16.  Statistical analysis was performed using Statistical Package
for Social Sciences (SPSS) software.
 Data were expressed as mean±SD and number (percentage)
as appropriate.
 Quantile- quantile (Q-Q) plot was performed to determine
normal distribution of studied variables.
 Chi-square test was performed to calculate the statistical
association.
 ‘p’ value <0.05 was considered statistically significant.
Statistical analyses

18. Study Subjects
n=20
n=18
n=26
n=19
n=12
n=4n=19
n=11
Total: (n=129)
Male: [n=81, (63%)]
Female: [n=48, (37%)]
Figure 4. Unrelated recruited subjects representative of
different divisions of Bangladesh

19. Assessment of Biochemical Parameters
Parameters Serum Levels (mean±SD) Normal Range
AST
27.12±6.54 U/L in male
31.97±7.78 U/L in female
≤34.00U/L in male
≤40.00 U/L in female
ALT 24.74±14.22 U/L 20.00-60.00 U/L
ALP 172.92±47.37 U/L 98.00-279.00 U/L
DBIL 0.32±0.50 mg/dL ≤0.40 mg/dL
TBIL 0.68±0.55 mg/dL ≤1.20 mg/dL
Alb 4.57±0.40 g/dL 3.50-5.00 g/dL
TP 7.37±1.60 g/dL 6.60-8.700 g/dL
Creatinine
0.77±1.45 mg/dL in male
0.69±0.17 mg/dL in female
0.60-1.20 mg/dL in male
0.50-1.10 mg/dL in female
Urea 24.25±6.34 mg/dL 10.00-50.00 mg/dL
Glucose 87.75±21.17 mg/dL 75.00-115.00 mg/dL
ESR
9.37±5.12 mm/hr in male
11.26±7.02 mm/hr in female
<15 mm/hr in male
<20 mm/hr in female
Table 1. Serum Levels (mean±SD) of the Biochemical and Haemato-
immunological Markers in the Study Subjects

20. Genomic Analyses of the Study Subjects
Figure 5. Representative agarose gel (0.7%) electrophoresis
image showing extracted gDNA from whole blood
L1
L2
L3
L4
L5
L6
L8
L7
L10
L17
L15
L13
L11
L9
L16
L12
L14
continued

21. Figure 6. Agarose (1.5%) gel image analysis of PCR
products having TPMT*3B (G460A) variant. L2 contains
negative control; L1, L3-L16 contains PCR products; and L17 contains
Molecular Weight Marker (MWM). L represents for Lane.
L1
L2
L3
L4
L5
L6
L8
L7
L10
L17
L15
L13
L11
L9
L16
L12
L14
200 bp
100 bp
1500 bp
600 bp690 bp
continued

22. Figure 7. Representative agarose gel (2%) electrophoresis image of MwoI
digested PCR products showing detection of TPMT*3B (G460A) variants.
Lane L1, L2, L3, L5, L6, L8, L9, L10, L11, L12, L13, L15 and L16 showed samples
detected as homozygous wild genotype (GG) (digested product sizes are 459 bp and
231bp). Lane L4 showed samples detected heterozygous (Ht) variant (GA) (digested
product sizes are 690 bp, 459 bp and 231bp).
Sample in Lane L7 showed homozygous (Hz) variant (AA) (690 bp).
Lane L14 showed a negative control (without any PCR product) and Lane L17 showed
molecular weight markers (MWM) of 100 bp DNA ladder.
200 bp
100 bp
1500 bp
600 bp690 bp
231 bp
459 bp
L1
L2
L3
L4
L5
L6
L8
L7
L10
L17
L15
L13
L11
L9
L16
L12
L14

23. Analysis of the Genotype and Allele Frequencies of
TPMT*3B
Table 2. Distribution of TPMT*3B (G460A) Genotypes in the Study
Subjects
Genotype Frequency (%) (n=129)
Wild type (GG) 96.90 (n=125)
Heterozygous mutant (GA) 0.80 (n=1)
Homozygous mutant (AA) 2.30 (n=3)
Allele Frequency (%) (n=129)
G 97.29 (n=251)
A 2.71 (n=7)
Table 3. Distribution of TPMT*3B (G460A) Alleles

24. Results were expressed as frequency (number). Chi-square test was
performed to calculate the statistical association. p value <0.05 was
considered as statistically significant level.
Gender-wise Association of TPMT*3B (G460A)
Genotypes in the Study Subjects
Table 4. Gender-wise Association of TPMT*3B (G460A) Genotypic
Variants in the Study Subjects
Genotype
Frequency of Genotypes (%)
(n=129) χ2 /p
Male (n=81) Female (n=48)
Wild type (GG) 97.53% (n=79) 95.83% (n=46)
0.015/
0.902
Heterozygous mutant (GA) 0.00 (n=0) 2.08% (n=1)
Homozygous mutant (AA) 2.47% (n=2) 2.08% (n=1)

25. Discussion
 Nothing is known about the presence of 26 variants of TPMT
gene polymorphisms in Bangladeshi population, which have
been reported in a number of populations in earlier studies.
 In this study, genotypic and allelic variants of TPMT*3B
(G460A) in a Bangladeshi population of healthy adults were
studied, who may require treatment with thiopurine drugs at
any time of their lifetime ahead.
 The frequency of the occurrences of TPMT*3B (G460A)
variant genotypes is remarkably higher in the studied
population of Bangladesh (3.10%) compared to that of Indian
(0.61%), American Caucasian (1.3%), Brazilian (0.3%),
French Caucasian (0.5%) populations.

26. Conclusions
1. Overall, 96.90% was wild type and 3.10% was mutants in
the study subjects.
2. Among 3.10% of mutants, 2.30% was homozygous and
0.80% was heterozygous variants.
3. This finding may help to select the dose of thiopurine drugs
 Wild genotype: normal dose
 Heterozygous mutant: low dose
 Homozygous mutant: need to take alternative therapy.
4. Screening of TPMT*3B (G460A) requiring treatment with
thiopurine drugs should be mandatory of the Bangladeshi
patients.