Association Study between Macrophage Migration Inhibitory Factor -173 Polymorphism
and Acute Myeloid Leukemia in Taiwan.
Latha Ramireddya, Chien-Yu Lin b,c, Su-Ching Liud, Wan-Yu Lo e,f,g*, Rouh-Mei Hua, Yi-Chin
Pengd, Ching-Tien Peng d, h*
a
Department of Biomedical informatics, Asia University, Taichung, Taiwan
b
Department of Laboratory Medicine, China Medical University Hospital.
c
Graduate Institute of Clinical Medical Sciences, China Medical University, Taichung, Taiwan
d
Department of Pediatrics, Children’s Hospital, China Medical University, Taichung
e
Division of Surgery, Department of Medical Research, China Medical University Hospital,
Taichung, Taiwan
f
Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan
g
Department of life science, National Chung Hsing University, Taichung, Taiwan
h
Department of Biotechnology, Asia University, Taichung, Taiwan
Dr. Lo and Dr. Peng contributed equally to this work
*Corresponding author: Ching-Tien Peng, Department of pediatrics, China Medical
University and Hospital.No.2,Yuh-Der Road, Taichung, Taiwan. Tel:+886-4-22062121
ext.7645,Fax:22032798, E. mail: wylo@mail.cmu.edu.tw
ABSTRACT:
Acute Myeloid Leukemia (AML) is the most common acute leukemia diagnosed in adults.
Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that plays
significant role in pathogenesis and auto-immune diseases. The major function of MIF is to
promote the cell proliferation, migration and invasion. The aim of the present study is to identify
the association between MIF -173 (rs755662) Single Nucleotide Polymorphism (SNP) and Acute
Myeloid Leukemia in Taiwanese population.DNA samples extracted from 256 AML patients and
256 healthy controls were investigated using polymerase chain reaction (PCR) followed by
restriction fragment length polymorphism (RFLP) analysis. The association between MIF-173
SNP genotype and AML patients were assessed with SPSS software. The results show that the
GC genotype of MIF -173 SNP is significantly higher in AML patients than in the healthy
controls (OR: 1.58, 95% CI: 1.06, P =0.034). Carrier genotypes GC and CC may be a causative
factor for AML cancer (OR: 1.39, 95% CI: 0.95, P =0.085). White Blood Cell count (103/ µl)
were significantly associated with AML MIF-173 polymorphism patients (P=0.002). Our results
in this study provide the first evidence that the MIF -173 polymorphism is associated with
AML. MIF is a potential biomarker for development of AML cancer in male adult in Taiwanese
population. Further validations in other populations are warranted.
Key Words: Acute myeloid leukemia, Macrophage migration inhibitory factor, Single
nucleotide polymorphism, Susceptibility, Restriction Fragment length polymorphism.
2
INTRODUCTION:
Acute Myeloid Leukemia is associated with poor prognosis, particularly in older patients.
According to the American cancer society 2013, the estimated AML new cases to be 14590, the
median age at diagnosis is 66 years. Although much effort has been made concerning AML
treatment, overall survival has not improved substantially in the past decade and still remains
dismal [1, 2]. Older patients have the poor prognosis due to an increased chemo resistance and
concomitant medical co-morbidities resulting in poor forbearance to chemotherapy. Among
men, leukemia is the leading cause of cancer death in older adults. Novel therapeutic strategies
are needed for adults to improve the quality of life [3].
Macrophage Migration Inhibitory Factor (MIF) is a lymphatic factor of a pro-inflammatory
cytokine which plays an important role in inflammation, angiogenesis and acquisition of allergic
reactions [4]. MIF gene is localized on chromosome 22 (11q.23) and it has three exons and two
introns [5].MIF is highly expressed in tumor tissues and it involved in tumor proliferation,
differentiation, invasion[6].It has been found that over-expression of the MIF increases the
interleukins (IL) which leads to the susceptibility to the diseases such as lung and breast cancer
[7-10].The polymorphism in the MIF gene promoter region -173G/C and 7-CATT progresses the
development of the cancer [11,12]. Although a G to C polymorphism at the position-173 plays a
crucial role in inflammatory diseases, tumor genesis, and various cancers [11-15].In addition, the
MIF -173 G/C (rs755622) polymorphism influences the MIF promoter activity in T lymphoblast
cell lines [16]. Moreover a number of studies have been focused on the association between MIF
polymorphism and susceptibility to Acute lymphoblastic leukemia [16-18]. the relevance of this
mutation in AML patients is unclear. Since little data exists on the role of the MIF with AML
susceptibility, the aim of the present study is to investigate the potential association of the MIF3
173 SNP with AML and the risk of AML in Taiwanese population. The investigation reveals the
association of MIF-173G/C SNP with AML by Polymerase chain reaction (PCR) and Restriction
Fragment length polymorphism (RFLP) methods in the Taiwanese population.
PATIENTS AND METHODS:
Study population:
In this case-control study, the Frequency distribution of the MIF variant genotype was
evaluated by considering two hundred and fifty six (256)AML adult patients, and 256 healthy
controls adults recruited between 2000 to 2011 in China Medical University Hospital. The
median age of the recruited will be 53.44 and 55.8 years AML patients and controls respectively.
The age, sex, and ethnic of control group were matched with that of AML patients. The control
group considered was without history and clinical characteristic of AML and was examined in
the China Medical University Hospital. Patients with other hematological diseases or previous
cancer and who had undergone chemotherapy and radiation were excluded. Blood samples used
for the research purpose compiled with the regulations set of the Institutional Review Board
(DMR-100-IRB-032).All participants have completed the consent form before the examination.
The study was approved by the ethical committee of the China Medical University Hospital.
Genotyping:
Blood samples were collected from each subject; Genomic DNA was extracted from
peripheral blood lymphocytes by proteinase K digestion followed by Phenol-chloroform
extraction and ethanol precipitation from EDTA anti-coagulated blood samples. Polymerase
chain reaction (PCR) was carried out with the forward ( 5'-ACTAAGAAAG ACCCGAGGC-3')
and reverse (5'-GGGGCACGTTGGTGTTTAC-3) primers ( Genomics Biosci & tech,
4
Taiwan).PCR master mix with 50 µl of total volume had 50-100 ng/ µl of genomic DNA, 5 µl
10XPCR buffer, 4 µl dNTP (2.5 mM),1µl of each oligonucleotide primer (10 µM), and 0.5 µl of
Taq Polymerase (5 U/µl).The PCR conditioned followed were: 940C for 5 min, 940C for 30 Sec,
520 C for 40 Sec,720C for 40 Sec, 720C for 2 min .The total number of cycles were limited to 35
cycles. The PCR product displayed a distinct band of 366 bp after electrophoresis. Which were
digested with AluI restriction endonuclease (New England Biolabs Inc, Taiwan) kept at 370C
overnight. The digested product was loaded on 3.5% agarose gel and the electrophoresis was
conducted at 100V for 25 min, and then visualized under UV light staining with Ethidium
bromide. The DNA size was assessed by using 100bp DNA ladder (Biolabs Inc, Taiwan) as
control. The GG genotype contains 2 fragments 268bp and 98 bp. The CC genotype had a two
cutting sites, which contains 3 fragments at 206 bp, 98 bp and 62 bp sizes. The heterozygous GC
shows 4 bands at 268, 206, 98 and 62 bp sizes (Fig.1). FMS like Tyrosine Kinase 3(FLT3) gene
detection carried out as described previous studies [19].
Statistical analysis:
Genotype distributions of all cases and controls were tested for Hardy-Weinberg
equilibrium and were found to be in equilibrium. Chi-square test was applied to assess the
differences in the frequency distributions of genotypes of the MIF-173 G/C polymorphism
between cases and controls. Logistic regression analysis was performed to obtain the odds ratios
(ORs), 95% confidence intervals (95% CI s) determined associations between MIF genotypes
and MIF susceptibility. The χ2-test was also applied to compare the clinical characteristic of
AML patients SNP with MIF -173 G/C polymorphism. The P value (P<0.05) was considered as
the significant level at <0.05. Statistical analysis was performed using the SPSS software version
16.0 (SPSS Inc, Chicago IL).
5
RESULTS:
Characteristics of the Study subjects:
The frequency distribution of selected clinical characteristics of the cases and controls
was reported in table 1. The Median age of control and cases are 55.8 and 53.4 respectively. The
age range varied from 34-93 years in AML Patients and 40-84 in control. There was no
significance difference were observed in gender (P=0.418 >0.05). Significant different White
blood cell count were found between AML patients and control group (P=<0.001)
MIF 173G/C Polymorphism in the AML patients and controls:
The genotype and allele frequencies of the MIF polymorphism in controls and patients
and their association with AML were shown in Table 2. Statistically significant differences in the
distribution of the genotypes were observed between AML patients and healthy controls
(P=0.034).Hetero-genotype GC carrier shows the significantly higher OR and P values (OR: 1.58,
95%CI: 1.06, P= 0.034) than the homo-genotype (GG, CC). Furthermore, the combined variant
genotypes GC/CC were associated with a statistically significantly higher risk of AML compared
with the wild-type genotype GG (OR: 1.39, 95% CI: 0.95–2.03).
The relationship of MIF 173 (rs755662) SNP with AML Patients:
The Frequency distribution of MIF SNP polymorphism with gender, Fms like tyrosine
kinase 3 (FLT3) gene, white blood cell count, platelets count, and hemoglobin count was shown
in Table 3. The Heterozygous GC carrier genotype showed significantly higher in FLT3 negative,
WBC count, platelets count and hemoglobin count that of the homozygous CC carrier. MIF -173
G/C polymorphism was significantly higher in the FLT3 male patients (P=<0.001).MIF SNP
6
distribution has the significance with WBC count in males (P=0.002).Platelets and hemoglobin
counts were very low in AML patients (P= 0.882, Hb female P=0.552; and Male P=0.713).
These results indicate that the occurrence of MIF might be high in male patients .On conclusion
the MIF-173 G to C SNP plays an important role in the tumor progression in AML.
DISCUSSION:
In this study, we investigated the association of MIF -173 G/C polymorphism with the
risk of adult AML in Taiwanese population and found that MIF might be a potential risk factor.
As a pro-inflammatory cytokine MIF plays a key role in autoimmune diseases such as
inflammatory bowel disease [20, 21]. and inflammatory diseases [22].Previous studies have
shown that MIF gene contains 200 genetic polymorphisms, that are concentrated in four areas
namely: the intron regions +254 (T/C) and +656 (C/G), the promoter region -794 CATT repeat
sequences, and 5' flank area -173 G→C mutations [21, 24].Among them SNP in the promoter
region plays a significant role in altering the m-RNA and adjusting the protein expression. It is
also has been shown that MIF-173G/C is susceptible to many inflammatory, autoimmune
diseases and various cancers [23-26].Although the MIF-173-C allele carriers shows the high
serum levels and
increases the cervical cancer and lymph node metastasis[27].and also
associated with high synovial fluid levels in oligo articular juvenile idiopathic arthritis [28,29].
Recent findings suggest that MIF may promote the development of cancer. The absence
or inhibition of MIF may result in delay of the progression of chronic lymphocytic leukemia
(CLL) [30]. Furthermore, the MIF−173 G/C polymorphism is identified and showed to be
functional both in vitro and in vivo [1] Human T lymphoblast cell line has shown increased
7
promoter activity of MIF−173C-Luc compared to MIF−173G-Luc.The MIF−173C allele
subjects showed significantly higher serum MIF levels than the MIF−173GG genotype [31].
However the association with the prednisone and poor MIF −173G/C polymorphism in
childhood ALL was not explained correctly [17]. Moreover after allo-stem cell transplantation
the presence of the MIF−173C allele in the patient reduces relapse, thereby leading to improved
survival [16].Furthermore, researches suggested that the MIF−173C allele was a potential risk
factor in tumor genesis and plays different roles in other pathological environments [18].This
data support that the MIF−173C allele was a potential risk factor for developing AML. In
developed countries like US, AML occurs 4% in children aged 14 years and younger, and is very
common in middle age and older people. AML survival rate decreases markedly with age at
diagnosis [32, 33]. Many drugs are available to diagnose the AML and Liang et al introduced a
new protocol to improve the treatment results for AML in Taiwan [34]. But new therapies are
needed to improve the prognosis [35].
Very less literature is available on the study of MIF polymorphism and their relationship
with AML susceptibility. For the first time, we investigated whether there is an association
between theMIF−173G/C (rs755662) polymorphism and risk of adult AML patients in
Taiwanese population It is found that the GC carriers are closely associated with the AML
susceptibility. However, the CC genotype was rare in this study population and no evidence of a
significant association between the homozygous CC genotype and the risk of AML was observed.
Heterogeneity of AML is related to the several clinical features that reflect the treatment
outcome of the AML. We investigated the prognostic factors includes Age, gender, WBC count,
Hb count, platelets count and FLT3 gene. Mutations in the Fms like tyrosine kinase 3 genes
associated with poor prognosis in AML in adults [36]. This study we also evaluated the FLT3
8
gene with MIF-173 plyorphism. The results show FLT3 gene and MIF-173 polymorphism has
significantly associated in older male patients.
In the present study, Male Patients showed significantly higher MIF polymorphism than
the female patients. AML MIF -173 G/C polymorphism was strongly associated with the FLT3
negative patients than the positive and was associated with Males (P=<0.001). The WBC count
was high (more than 10.39) in many patients than the normal (3.39-10.39); WBC count in male
patients shows significant association with MIF-173SNP polymorphism (P=0.002).Moreover,
platelets and hemoglobin content were very low (platelets <130, Hb <12.0 in females, Hb < 14.0
in Male) in many patients than Normal condition. (Platelets: 130-400, Hb Female: 12-16, Hb
Male: 14-18). GC genotype exhibited in high levels in all AML patients. GG/GC confirmed a
significantly higher risk of AML compared with wild genotype GG. The MIF-173 polymorphism
was the potential risk factor in Acute Myeloid Leukemia and especially for adult patients.
Some limitations in this study should be noted. This study was hospital based and limited
to China medical university, Taichung providence, Taiwan. Our study did not focus on the MIF
clustered polymorphic sites and the associated genes. Our study should be replicated in other
populations and larger cohorts to further validate our findings. Plasma levels of MIF were not
measured in the study subjects. To our knowledge this is the first report that tells the association
between a MIF -173 G to C polymorphism with AML patients in the Taiwanese population. Our
study suggests that the MIF is a susceptibility marker for adult's males among Taiwanese
patients. Our findings suggest that MIF might be a clinical marker for the prevention and further
treatment for AML cancer.
9
Competing interests:
The authors declare no conflict of interest to report.
Abbreviations:
AML: Acute Myeloid Leukemia; ALL: Acute Lymphoblastic Leukemia; SNP: Single Nucleotide
Polymorphism; MIF: Macrophage migration inhibitory factor; PCR: Polymerase chain reaction;
RFLP: Restriction fragment length polymorphism
Author's Contribution:
CYL, SCL: Collected the patient samples and performed the genomic DNA extraction, LR:
Performed the PCR and RFLP for SNP study and performed the statistical analysis and table
drawing. PCT, YCP, and RMH: revised the manuscript. LR, WYL: Helped to draw the tables,
figures and drafted the manuscript. All authors read and approved the final manuscript.
Acknowledgments:
The authors acknowledge the Research laboratory of Pediatrics, Children's Hospital,
China Medical University and National science council of Taiwan for the financial Support
sanctioned under grant no. 101-2221-E-468-022 and China Medical University Hospital (DMR99-058).
10
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16
Figure 1. Restriction fragment length polymorphism (RFLP) analysis of MIF-173
genotypes. M: 1-KB molecular weight marker. S1-S2, S6-S7: MIF-173 GC heterozygote
(268 bp, 206 bp, 98 bp, and 62 bp), S4: MIF-173 CC homozygote (206 bp, 98 bp, and 62 bp).
S3, S5: MIF-173 GG homozygote (268 bp, and 98bp) PD: primer dimer.
Table 1. Clinical characteristics of the Acute Myeloid Leukemia (AML, n=256) and healthy
subjects (Control, n=256).
Table 2. Genotypic and allelic frequencies of MIF genetic polymorphism in patients with AML
and controls. The values within brackets are percentage and the ‘Ref’ stands for each
compared template.
Table 3. Stratified analysis of clinical characteristics of the 256 AML patients according to MIF
polymorphism (rs 755662). The values within brackets are percentage.
17
18
Table 1.
Characteristics
AML group
(n=256)
Control group
(n=256)
sex
P value
0.418
Male
147
156
female
109
100
Mean age (years)
53.44
55.8
[range]
[34-93
[40-84]
WBC (103/ul)
0.798
<0.001*
<3.99
44
17
3.99 -10.39
79
234
≥10.39
133
5
19
Table 2.
AML
Control
(n=256)
(n= 256)
dbSNP ID
OR (95% CI)
rs 755622
P value
0.034*
GG
168(65.6)
186(72.7)
0.63(0.26-1.55)
GC
80(31.2)
56(21.9)
1.58(1.06-2.36)
CC
8(3.1)
14(5.5)
Ref
Genotype (carrier)
0.191
GG+GC
248(96.8)
242(94.5)
0.56(0.23-1.35)
CC
8(3.1)
14(5.5)
Ref
GC+CC
88(34.3)
70(27.3)
1.39(0.95-2.03)
GG
168(65.6)
186(72.6)
Ref
Allele frequency
0.085
0.325
G
416(81.2)
428(83.6)
1.18(0.85-1.62)
C
96(18.8)
84(16.4)
Ref
* p<0.05. CI, confidence interval; OR, odds ratio
20
Table 3.
Clinical parameters
MIF (rs755662)
GG(n=168)
P value
GC(n=80)
CC(n=8)
FLT3 Male( >50 yr)
<0.001*
Positive
14(53.2)
12(46.1)
0(0.0)
Negative
45(84.9)
4(7.5)
4(7.5)
FLT3 Female(>50yr)
Positive
Negative
5(83.6)
27(58.7)
1(16.4)
18(39.1)
0(0.0)
1(2.2)
0.500
Gender
Male (n=147)
Female (n=109)
0.268
102(69.3)
66(60.5)
40(27.2)
40(36.6)
5(3.5)
3(2.9)
Platelets (103/ul)
< 130
130 - 400
≥400
102(68.0)
42(62.7)
24(61.5)
43(28.7)
23(34.3)
14(35.9)
5 (3.3)
2(3.0)
1(2.6)
Hb Male(gm/dL)
< 14.0
14.0 - 18.0
≥18.0
77(68.8)
17(63.0)
4(50.0)
31(27.7)
9(33.3)
4(50.0)
4(3.5)
1(3.7)
0(0.0)
Hb Female (gm/dL)
< 12.0
12.0- 16.0
≥16.0
57(67.9)
12(54.5)
1(33.3)
25(29.8)
2(66.7)
9(40.9)
2(2.4)
1(4.5)
0(0.0)
WBC Male (103/ µl)
< 3.99
3.99 - 10.39
≥10.39
50(70.4)
28(58.3)
20(71.4)
21(29.6)
19(39.6)
4(14.2)
0(0.0)
1(2.1)
4(14.2)
0.882
0.713
0.552
0.002*
0.348
WBC Female(103/µl)
< 3.99
3.99 - 10.39
≥10.39
13(59.0)
20(64.5)
37(66.0)
7(31.2)
11(34.5)
18(32.2)
21
2(9.1)
0(0.0)
1(1.8