Just Accepted by Leukemia & Lymphoma
Significance of MTHFR gene variants in acute lymphoblastic leukemia in Indian population: An experimental, computational and meta-analysis Ravishankara Bellampalli, Nagaraja M. Phani, Kamalakshi G. Bhat, Krishna Prasad, Nalini Bhaskaranand, Kanive P. Guruprasad, Padmalatha S. Rai and Kapaettu Satyamoorthy doi: 10.3109/10428194.2014.953154 ABSTRACT Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
Acute lymphoblastic leukemia (ALL) arises due to several genetic alterations in progenitor cells and methotrexate is frequently used as part of the treatment regimen. Although there are evidences for the effect of MTHFR C677T and A1298C variations on the drug response in ALL, its risk association for ALL is still unresolved. In a case-control study of total 203 ALL patients and 246 controls and meta-analysis on Indian population, we show an insignificant association of MTHFR C677T and A1298C genotypes with childhood and adult ALL. A comprehensive insilico characterization of non-synonymous single nucleotide polymorphisms (nsSNPs) and SNPs of 3’UTR region revealed 9 nsSNPs as deleterious and 3SNPs in 3’UTR region could possibly alter the binding of miRNA. The study revealed that several overlooked SNPs may contribute to risk of ALL susceptibility and further studies of these SNPs with functional characterization in large sample size is required to understand the significant role of MTHFR gene in ALL development.
© 2014 Informa UK, Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
GLAL_A_953154_Coverpage.indd 1
8/12/2014 7:47:18 AM
Significance of MTHFR gene variants in acute lymphoblastic leukemia in Indian population: An experimental, computational and meta-analysis
PT ED
Ravishankara Bellampalli1, Nagaraja M. Phani1, Kamalakshi G. Bhat2, Krishna Prasad3, Nalini Bhaskaranand4, Kanive P. Guruprasad1, Padmalatha S. Rai1*and Kapaettu Satyamoorthy1
Division of Biotechnology, School of Life Sciences, Manipal University, Manipal, Karnataka,
India, 2Department of Pediatrics, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India, 3Department of Medicine, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India, 4Department of Pediatrics, Kasturba Medical College, Manipal University, Manipal, Karnataka, India
CE
*Corresponding author: Division of Biotechnology, School of Life Sciences, Manipal University, Planetarium Complex, Manipal – 576104, India . Tele: +91 820 2923505, +91 820 2922058. Fax Numbers: +91 820 2571919. Email:
[email protected]
AC
Short title: MTHFR and Acute Lymphoblastic Leukemia
Key words: MTHFR gene polymorphism, acute lymphoblastic leukemia; Metaanalysis, Indian population, insilico analysis ABSTRACT
ST
Acute lymphoblastic leukemia (ALL) arises due to several genetic alterations in progenitor cells and methotrexate is frequently used as part of the treatment regimen. Although there are evidences for the effect of MTHFR C677T and A1298C variations on the drug response in ALL, its risk association for ALL is still unresolved. In a case-control study of total 203 ALL patients and 246 controls and meta-analysis on Indian population, we show an insignificant association of MTHFR C677T and A1298C genotypes with childhood and adult ALL. A comprehensive insilico characterization of non-synonymous single nucleotide polymorphisms (nsSNPs) and SNPs of 3'UTR region revealed 9 nsSNPs as deleterious and 3SNPs in 3’UTR region could possibly alter the binding of miRNA. The study revealed that several overlooked SNPs may contribute to risk of ALL susceptibility and further studies of these SNPs with functional characterization in large sample size is required to understand the significant role of MTHFR gene in ALL development.
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
1
1
INTRODUCTION The folate levels and its role in acute lymphoblastic leukemia (ALL) malignancy have been well established [1]. The normal metabolism of folate may be disturbed due to various factors such as imbalance in folate level, folate
PT ED
malabsorption and variations in the folate metabolism genes like MTHFR [2-3]. Skibola et al. [4] hypothesized that the decreased risk of ALL by polymorphic
synthesis in the cell that reduces the uracil misincorporation into DNA. There are two well characterized polymorphisms, MTHFR C677T (rs1801133) and MTHFR A1298C (rs1801131) which have been reported to reduce the activity of
CE
the enzyme [5-6]. Along with these two polymorphisms, MTHFR gene is also regulated by a) alternative splicing [7-8], b) hsa-miR-149 due to a SNP
AC
(rs4846049) at the RNA level [9], c) increased AdoMet/AdoHcy ratio at the protein level [10] and d) phosphorylation of the MTHFR N-terminal domain that reduces the inhibition of MTHFR enzyme activity by AdoMet [11]. In addition to these two polymorphisms in MTHFR, there are many other variations such as
ST
2 splice donor variants, 2 splice acceptor variants, 9 stop gain variants, 6 frame shift variants, 1 initiator coding variant, 121 missense variants, 10 splice region variants, 73 synonymous variants, 46 coding sequence variants, 5 5'UTR region variants, 117 3'UTR variants, 297 intronic variants, 124 upstream gene variants,
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
MTHFR is due to increased pool of 5, 10-methylene tetrahydrofolate for DNA
and 129 downstream gene variants that were reported in the database [http://www.ensembl.org/Homo_sapiens/Info/Index]. Among this, the non-
2
synonymous SNPs (nsSNPs) and SNPs in regulatory region always play major role in the gene regulation and function [12-15]. Recently, several meta-analysis studies have been reported in an effort to draw conclusions on the association of MTHFR variants with ALL but the information
PT ED
is incomplete on the Asian population in general and Indian population in particular despite the distinct ethnicity[16-21]. Hence, we have evaluated the
lymphoblastic leukemia in Indian population and a systematic review with metaanalysis was conducted on previously published reports on Indian population. The study was also extended to analyze additional nsSNPs and SNPs in 3'UTR
CE
region by bioinformatic methods along with MTHFR C677T and A1298C polymorphisms.
AC
MATERIALS AND METHODS Case-Control Study Subjects
Unrelated 203 acute lymphoblastic leukemia (ALL) patients were recruited for
ST
this study. Among these 138 were males and 65 were females with a mean age of 20.2 years. Diagnosis of ALL was done by morphological and immunophenotypic criteria based on the WHO classification [38]. Out of 203 ALL patients analyzed in this study, 129 were childhood ALL patients (< 15
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
association of MTHFR C677T and A1298C polymorphisms with acute
years) with a mean age of 6.66 years (87 males and 42 females) and 74 were adult ALL patients (>15years) with a mean age of 40.58 years (51 males and 23 females). Unrelated 246 normal healthy individuals recruited from the similar
3
socioeconomic group which comprised of 119 males and 127 females with mean age of 25.5 years served as controls. Out of 246 normal control samples, 191 were young adult normal healthy individuals (T polymorphism and folate status affect one-carbon incorporation into human DNA deoxynucleosides. J Nutr 2005; 135:389-396.
AC
[43] Damnjanovic T, Milicevic R, Novkovic T, et al. Association between the methylenetetrahydrofolate
reductase
polymorphisms
and
risk
of
acute
lymphoblastic leukemia in Serbian children. J Pediatr Hematol Oncol 2010;
ST
32:e148-150.
[44] Franco RF, Simoes BP, Tone LG, Gabellini SM, Zago MA, Falcao RP. The methylenetetrahydrofolate reductase C677T gene polymorphism decreases the risk of childhood acute lymphocytic leukaemia. Br J Haematol 2001; 115:616-
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
[41] Sohn KJ, Jang H, Campan M, et al. The methylenetetrahydrofolate
618. 2.
[45] Lightfoot TJ, Johnston WT, Painter D, et al. Genetic variation in the folate metabolic pathway and risk of pediatric leukemia. Blood. 2010;115:3923-3929.
21
[46] Koppen IJ, Hermans FJ, Kaspers GJ. Folate related gene polymorphisms and susceptibility to develop childhood acute lymphoblastic leukaemia. Br J Haematol 2010; 148:3-14. [47] Gast A, Bermejo JL, Flohr T, et al. Folate metabolic gene polymorphisms
2007;21:320-325.
PT ED
and pediatric acute lymphoblastic leukemia: a case-control study. Leukemia.
association analysis of microRNA target sites in one-carbon metabolism genes. PLoS One 2011;6(7):e21851.
[49] Rubie C, Kruse B, Frick VO, et al. Chemokine receptor CCR6 expression is
CE
regulated by miR-518a-5p in colorectal cancer cells. J Transl Med 2014; 12:48. [50] Rai PS, Pai GC, Alvares JF, Bellampalli R, Gopinath PM, Satyamoorthy K. Intraindividual somatic variations in MTHFR gene polymorphisms in relation to
AC
colon cancer. Pharmacogenomics 2014; 15:349-359.
[51] El Awady MK, Karim AM, Hanna LS, et al. Methylenetetrahydrofolate reductase gene polymorphisms and the risk of colorectal carcinoma in a sample
ST
of Egyptian individuals. Cancer Biomark 2009;5:233-240.
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
[48] Stone N, Pangilinan F, Molloy AM, et al. Bioinformatic and genetic
22
PT ED CE
ST
AC
Figure 1.The flowchart representing the process of inclusion and exclusion of published articles for metaanalysis study.
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
FIGURE LEGENDS
23
PT ED CE
AC
ST JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
Figure 2. Association of MTHFR C677T and MTHFR A1298C polymorphism with risk of childhood and adult ALL development in Indian population. In forest plot pooled OR is shown under a random-effects model for the 677T and 1298C alleles. (A) Association of MTHFR 677T allele with childhood and adult ALL (B) Association of MTHFR 1298C allele with childhood and adult ALL. In the plot studies are represented by ascending order of reported year. The size of the square in the plot represents the weight of the corresponding study. Weights are given for the overall combined OR. The diamond in the plot represents overall effect estimate OR and lateral tips of diamond represents the associated 95%CI. (A: adult ALL, C: childhood ALL, C+A: childhood + adult ALL).
24
PT ED CE
AC ST JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
Figure 3. Bioinformatic analysis of nsSNPs in MTHFR gene.(A) Percentage of deleterious and tolerated nsSNPs of MTHFR gene predicted by 8 different bioinformatic tools.(B) Schematic representation of MTHFR gene structure displaying the position of 9 deleterious nsSNPs.
25
TABLE LEGENDS Table I. Distribution of genotypes and allele frequencies of MTHFR C677T (rs1801133) and MTHFR A1298C (rs1801131) polymorphisms in childhood acute lymphoblastic leukemia patients and normal healthy individuals
MTHFR A1298C/ rs1801131
ALL Cases n=129 (%)
Healthy Individuals n=191 (%)
OR (95% CI)
p Value
677CC 677CT 677TT 677CT+TT C allele T allele 1298AA 1298AC 1298CC 1298AC+CC A allele C allele
107 (82.95) 21(16.28) 1 (00.77) 22 (17.05) 0.91 0.09 46 (35.66) 60 (46.51) 23 (17.83) 83 (64.34) 0.59 0.41
156 (81.68 ) 31(16.23) 4 (02.09) 35 (18.32) 0.9 0.1 64 (33.51) 92 (48.17) 35 (18.32) 127 (66.49) 0.58 0.42
1.00 (Reference) 0.98 (0.53-1.81) 0.36 (0.04-3.30) 0.91 (0.50-1.64) 1.00 (Reference) 0.86 (0.50-1.47) 1.00 (Reference) 0.90 (0.55-1.49) 0.91 (0.47-1.74) 0.90 (0.56-1.45) 1.00 (Reference) 0.94 (0.68-1.30)
-0.96 0.35 0.77 -0.58 -0.70 0.78 0.69 -0.73
OR and 95% CI were calculated with MTHFR 677CC and MTHFR 1298AA genotype as reference group. MTHFR: 5, 10-Methylenetetrahydrofolate reductase, ALL: acute lymphoblastic leukemia, OR: odds ratio; CI: confidence interval, p value less than 0.05 is significant
CE
Table II. Combined genotype frequencies of MTHFR C677T (rs1801133) and MTHFR A1298C (rs1801131) polymorphisms in childhood acute lymphoblastic leukemia patients and normal healthy individuals ALL Cases n =129(%)
Healthy Controls n=191 (%)
OR (95% CI)
p Value
677CC/1298AA 677CT/1298AA 677TT/1298AA 677CC/1298AC 677CT/1298AC 677TT/1298AC 677CC/1298CC 677CT/1298CC 677TT/1298CC
36 (27.91) 9 (6.98) 1 (0.77) 51 (39.53) 9 (6.98) 0 (0) 20 (15.50) 3 (2.33) 0 (0)
46 (24.08) 14 (7.33) 4 (2.10) 80 (41.88) 12 (6.28) 0 (0) 30 (15.71) 5 (2.62) 0 (0)
1.00 (Reference) 0.82 (0.32-2.11) 0.31 (0.03-2.98) 0.81 (0.46-1.42) 0.95 (0.36-2.52) NA 0.85 (0.41-1.74) 0.76 (0.17-3.42) NA
-0.68 0.29 0.47 0.93 NA 0.65 0.72 NA
AC
Variant MTHFR, C677T/A1298C
ST
OR and 95% CI were calculated with MTHFR 677CC/MTHFR 1298AA genotype as reference group. MTHFR: 5, 10-Methylenetetrahydrofolate reductase, ALL: acute lymphoblastic leukemia; OR: odds ratio; CI: confidence interval, p value less than 0.05 is significant
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
MTHFR C677T/ rs1801133
Genotypes/ Alleles
PT ED
SNP/rsID
26
Table III. Distribution of genotypes and allele frequencies of MTHFR C677T (rs1801133) and MTHFR A1298C (rs1801131) polymorphisms in adult acute lymphoblastic leukemia patients and normal healthy individuals
MTHFR C677T/ rs1801133
ALL Cases n=74 (%)
Healthy Individuals n=55 (%)
OR (95% CI)
p Value
677CC 677CT 677TT 677CT+TT C allele T allele 1298AA 1298AC 1298CC 1298AC+CC A allele C allele
59 (79. 73) 13 (17.57) 2 (02.70) 15 (20.27) 0.88 0.12 25 (33.78) 30 (40.54) 19 (25.68) 49 (66.22) 0.54 0.46
46 (83.64) 8 (14.54) 1 (1.82) 9 (16.36) 0.91 0.09 14 (25.45) 34 (61.82) 7 (12.73) 41(74.55) 0.56 0.44
1.00 (Reference) 1.26 (0.48-3.31) 1.55 (0.13-17.73) 1.29 (0.52-3.23) 1.00 (Reference) 1.29 (0.57-2.95) 1.00 (Reference) 0.49 (0.21-1.12) 1.52 (0.51-4.50) 0.66 (0.30-1.45) 1.00 (Reference) 1.09 (0.66-1.80)
-0.62 0.71 0.57 -0.53 -0.08 0.48 0.30 -0.71
OR and 95% CI were calculated with MTHFR 677CC and MTHFR 1298AA genotype as reference group. MTHFR: 5, 10-Methylenetetrahydrofolate reductase, ALL: acute lymphoblastic leukemia, OR: odds ratio; CI: confidence interval, p value less than 0.05 is significant
CE
Table IV. Combined genotype frequencies of MTHFR C677T (rs1801133) and MTHFR A1298C (rs1801131) polymorphisms in adult acute lymphoblastic leukemia patients and normal healthy individuals ALL Cases n =74 (%)
Healthy Controls n=55 (%)
OR (95% CI)
p Value
677CC/1298AA 677CT/1298AA 677TT/1298AA 677CC/1298AC 677CT/1298AC 677TT/1298AC 677CC/1298CC 677CT/1298CC 677TT/1298CC
18 (24.32) 5 (6.75) 2 (2.70) 23 (31.10) 7 (9.46) 0 (0) 18 (24.32) 1 (1.35) 0 (0)
9 (16.37) 4 (7.27) 1 (1.81) 30 (54.55) 4 (7.27) 0 (0) 7 (12.73) 0 (0) 0 (0)
1.00 (Reference) 0.62 (0.13-2.91) 1.00 (0.08-12.55) 0.38 (0.14-1.00) 0.87 (0.20-3.79) NA 1.28 (0.39-4.20) NA NA
-0.54 1.00 0.048 0.85 NA 0.67 NA NA
AC
Variant MTHFR, C677T/A1298C
ST
OR and 95% CI were calculated with MTHFR 677CC/MTHFR 1298AA genotypes as reference group. MTHFR: 5, 10-Methylenetetrahydrofolate reductase, ALL: acute lymphoblastic leukemia; OR: odds ratio; CI: confidence interval, p value less than 0.05 is significant
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
MTHFR A1298C/ rs1801131
Genotypes/Allel es
PT ED
SNP/rsID
27
Table V: ORs with 95% CI and heterogeneity results for the genetic contrasts of MTHFR rs1801133 and rs1801131 polymorphisms for ALL risk in Indian population All: all the studies meet the inclusion criteria. Q test: Test to estimate heterogeneity between studies,
Adult
I2 (%)
p, Q test
Random-Effect OR
Fixed-Effect OR
9 9 8 8 9 6 6 5 5 6 2 2 2 2 2
23 42 2 0 51 30 39 0 0 31 51 64 0 0 67
0.24 0.09 0.41 0.43 0.04 0.21 0.15 0.46 0.62 0.20 0.15 0.10 0.72 0.72 0.08
1.12 (0.93-1.36) 1.13 (0.86-1.48) 1.25 (0.79-2.01) 1.27 (0.81-2.00) 1.10 (0.81-1.49) 1.15 (0.92-1.43) 1.25 (0.94-1.68) 0.98 (0.55-1.72) 0.98 (0.57-1.70) 1.30 (0.98-1.73) 0.85 (0.37-1.92) 0.74 (0.25-2.22) 1.08 (0.25-4.77) 0. 96 (0.22-4.28) 0.68 (0.20-2.33)
1.14 (0.97-1.34) 1.17 (0.96-1.43) 1.26 (0.79-2.00) 1.27 (0.81-2.00) 1.17 (0.95-1.44) 1.16 (0.96-1.39) 1.28 (1.02-1.60) 0.98 (0.55-1.72) 0.98 (0.57-1.70) 1.33 (1.05-1.67) 0.84 (0.47-1.49) 0.75 (0.39-1.46) 1.08 (0.25-4.77) 0.96 (0.22-4.28) 0.71 (0.35-1.43)
Random-Effect OR
Fixed-Effect OR
0.97 (0.79-1.19) 0.84 (0.57-1.26) 0.98 (0.71-1.37) 0.97 (0.74-1.28) 0.80 (0.51-1.23) 1.03 (0.87-1.22) 1.05 (0.77-1.42) 1.01 (0.72-1.40) 1.05 (0.78-1.42) 1.04 (0.75-1.43) 0.66 (0.24-1.85) 0.33 (0.08-1.34) 0.76 (0.18-3.18) 0.59 (0.26-1.32) 0.24 (0.06-1.05)
0.99 (0.86-1.14) 0.93 (0.76-1.14) 0.98 (0.72-1.33) 0.97 (0.74-1.28) 0.91 (0.74-1.13) 1.03 (0.89-1.20) 1.04 (0.84-1.30) 1.01 (0.72-1.40) 1.05 (0.78-1.42) 1.04 (0.83-1.30) 0.75 (0.50-1.12) 0.37 (0.20-0.67) 0.83 (0.36-1.89) 0.58 (0.28-1.23) 0.28 (0.15-0.55)
MTHFR A1298C Studies I2 (%) p, Q test (rs1801131) Contrast Allele 8 52 0.04 All Dominant 8 73 0.00 Additive 8 11 0.34 Recessive 8 0 0.55 CT v/s CC 8 75 0.00 Allele 6 21 0.28 Childhood Dominant 6 51 0.07 Additive 6 0 0.44 Recessive 6 0 0.74 CT v/s CC 6 51 0.07 Allele 2 84 0.01 Adult Dominant 2 81 0.02 Additive 2 65 0.09 Recessive 2 13 0.28 CT v/s CC 2 78 0.03 p30% indicates the heterogeneity between the studies
ST
AC
Populations
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
Childhood
Studies
PT ED
All
MTHFR C677T ( rs1801133) Contrast Allele Dominant Additive Recessive CT v/s CC Allele Dominant Additive Recessive CT v/s CC Allele Dominant Additive Recessive CT v/s CC
CE
Populations
28
Table VI: The distribution genotype and allelic frequency of MTHFR rs1801133 and rs1801131 polymorphism in acute lymphoblastic leukemia patients and their normal healthy individuals in selected reports for metaanalysis Distribution of MTHFR C677T Genotype Case
First Author and Year
Case
CC
Reddy et al.,2006
Control
Case
CT
51
79
Case
05
0.67
TT
77
58
07
Control
Case
0.76
0.33
C
Control T 0.24
73
85
13
14
00
00
0.92
0.93
0.08
0.07
Sood et al.,2010 Nikbhakt et al.,2012 Hussain et al.,2012 Sazawal (C) et al.,2013 Sazawal (A)et al.,2013 Present Study (C)
54
173
38
71
03
11
0.77
0.82
0.23
0.18
54
40
62
49
09
11
0.68
0.64
0.32
0.36
59
180
13
61
09
10
0.81
0.84
0.19
0.16
94
63
49
30
08
04
0.78
0.8
0.22
0.12
25
33
06
22
02
03
0.85
0.76
0.15
0.24
107
156
21
31
01
04
0.90
0.90
0.10
0.10
Present Study (A)
59
46
13
08
02
01
0.88
0.91
0.12
0.09
576
855
292
344
41
49
Total
Distribution of MTHFR A1298C Genotype Control AA
Case
Control AC
Frequency of MTHFR A1298C Alleles
Case
Control
Case
9
0.61
0.7
0.39
0.3
CC
Control
Case
A
Control C
Nikbhakt et al.,2012
52
40
65
49
08
11
0.68
0.64
0.32
0.36
Sazawal (C) et al.,2013
57
36
72
51
22
10
0.62
0.63
0.38
0.37
Sazawal (A) et al.,2013
22
14
CE
First Author and Year
Case
PT ED
Adiga et al.,2010
6
35
05
9
0.76
0.54
0.24
0.46
Present Study (C)
46
64
60
92
23
35
0.59
0.58
0.41
0.42
Present Study(A)
25
14
30
34
19
7
0.54
0.56
0.46
0.44
413
530
122
147
Reddy et al.,2006
41
65
83
68
11
20
28
51
55
15
16
0.53
0.56
0.47
0.44
Sood et al.,2010
30
59
46
146
19
50
0.56
0.52
0.44
0.48
AC
Adiga et al.,2010
ST
Total 293 320 A: Adult ALL and C: Childhood ALL
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
Control
Frequency of MTHFR C677T Alleles Contr ol
29
Table VII: List of polymorphisms which alter the binding of miRNA in 3'UTR region of MTHFR gene. The computational modification of MTHFR gene 3'UTR region SNPs to miRNAs binding. The insilico modeling of the interaction between different predicted miRNA and MTHFR 3’UTR region carrying different alleles by using RNAhybrid tool. Calculated free energy change and polymorphism site is also mentioned in the table. miRNA SNP Allele Energy Binding Map
rs1537514
[MAF: 0.1]
A
-27.5 kcal/mol
G
-19.1 kcal/mol
A
-22.9 kcal/mol
C
-22.7 kcal/mol
ST
AC
hsa-miR-518a-5p [LOSS]
-25.1 kcal/mol
hsa-miR-302a-5p [GAIN]
rs1537515 [MAF:0.1]
target 5' U CUCCCA CUU CCU C 3' GGGA GUGAA AC UGGAGCC CCCU CACUU UG GCCUCGG miRNA 3' C U UCU 5' target 5' U CUCCCA CUU UU C 3' GGGA GUGAA ACAC GGAGCC CCCU CACUU UGUG CCUCGG miRNA 3' C UCU 5' target 5' U UCCCA ACUUACC A 3' GGGAC GUGA CUUGG CCCUG UACU GAACU miRNA 3' GAU UC GU 5' target 5' U UCCCA ACUUA A 3' GGGAC GUGA CACUUGG CCCUG UACU GUGAACU miRNA 3' GAU UC 5'
CE
hsa-miR-6513-3p [GAIN]
G
PT ED
rs4846049 [MAF: 0.19]
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
hsa-miR-149 [GAIN]
G
-20.4 kcal/mol
G
-20.2 kcal/mol
T
-22.1 kcal/mol
30
target 5' U CUUG UUGGG G 3' GGGC CU CUUUGCAG CCCG GG GAAACGUC miRNA 3' CUUU AA 5' target 5' U UGGAGG GG U 3' GGGCUU CUCU GC CCCGAA GGGA CG miRNA 3' CUUU AA UC 5' target 5' A UA G G A 3' GGU AGGUG GUCCA G UCG UUCAU UAGGU C miRNA 3' G G AAAUUCA 5' target 5' A G UG G AAGA G 3' GG GAGU A UCAC GUUUAAG UC UUCA U GGUG CAAAUUC miRNA 3' G UG A A 5'
Significance of MTHFR gene variants in acute lymphoblastic leukemia in Indian population: An experimental, computational and meta-analysis Ravishankara Bellampalli, Nagaraja M. Phani, Kamalakshi G. Bhat, Krishna Prasad, Nalini Bhaskaranand, Kanive P. Guruprasad, Padmalatha S. Rai and Kapaettu Satyamoorthy doi: 10.3109/10428194.2014.953154 ABSTRACT Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
Acute lymphoblastic leukemia (ALL) arises due to several genetic alterations in progenitor cells and methotrexate is frequently used as part of the treatment regimen. Although there are evidences for the effect of MTHFR C677T and A1298C variations on the drug response in ALL, its risk association for ALL is still unresolved. In a case-control study of total 203 ALL patients and 246 controls and meta-analysis on Indian population, we show an insignificant association of MTHFR C677T and A1298C genotypes with childhood and adult ALL. A comprehensive insilico characterization of non-synonymous single nucleotide polymorphisms (nsSNPs) and SNPs of 3’UTR region revealed 9 nsSNPs as deleterious and 3SNPs in 3’UTR region could possibly alter the binding of miRNA. The study revealed that several overlooked SNPs may contribute to risk of ALL susceptibility and further studies of these SNPs with functional characterization in large sample size is required to understand the significant role of MTHFR gene in ALL development.
© 2014 Informa UK, Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
GLAL_A_953154_Coverpage.indd 1
8/12/2014 7:47:18 AM
Significance of MTHFR gene variants in acute lymphoblastic leukemia in Indian population: An experimental, computational and meta-analysis
PT ED
Ravishankara Bellampalli1, Nagaraja M. Phani1, Kamalakshi G. Bhat2, Krishna Prasad3, Nalini Bhaskaranand4, Kanive P. Guruprasad1, Padmalatha S. Rai1*and Kapaettu Satyamoorthy1
Division of Biotechnology, School of Life Sciences, Manipal University, Manipal, Karnataka,
India, 2Department of Pediatrics, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India, 3Department of Medicine, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India, 4Department of Pediatrics, Kasturba Medical College, Manipal University, Manipal, Karnataka, India
CE
*Corresponding author: Division of Biotechnology, School of Life Sciences, Manipal University, Planetarium Complex, Manipal – 576104, India . Tele: +91 820 2923505, +91 820 2922058. Fax Numbers: +91 820 2571919. Email:
[email protected]
AC
Short title: MTHFR and Acute Lymphoblastic Leukemia
Key words: MTHFR gene polymorphism, acute lymphoblastic leukemia; Metaanalysis, Indian population, insilico analysis ABSTRACT
ST
Acute lymphoblastic leukemia (ALL) arises due to several genetic alterations in progenitor cells and methotrexate is frequently used as part of the treatment regimen. Although there are evidences for the effect of MTHFR C677T and A1298C variations on the drug response in ALL, its risk association for ALL is still unresolved. In a case-control study of total 203 ALL patients and 246 controls and meta-analysis on Indian population, we show an insignificant association of MTHFR C677T and A1298C genotypes with childhood and adult ALL. A comprehensive insilico characterization of non-synonymous single nucleotide polymorphisms (nsSNPs) and SNPs of 3'UTR region revealed 9 nsSNPs as deleterious and 3SNPs in 3’UTR region could possibly alter the binding of miRNA. The study revealed that several overlooked SNPs may contribute to risk of ALL susceptibility and further studies of these SNPs with functional characterization in large sample size is required to understand the significant role of MTHFR gene in ALL development.
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
1
1
INTRODUCTION The folate levels and its role in acute lymphoblastic leukemia (ALL) malignancy have been well established [1]. The normal metabolism of folate may be disturbed due to various factors such as imbalance in folate level, folate
PT ED
malabsorption and variations in the folate metabolism genes like MTHFR [2-3]. Skibola et al. [4] hypothesized that the decreased risk of ALL by polymorphic
synthesis in the cell that reduces the uracil misincorporation into DNA. There are two well characterized polymorphisms, MTHFR C677T (rs1801133) and MTHFR A1298C (rs1801131) which have been reported to reduce the activity of
CE
the enzyme [5-6]. Along with these two polymorphisms, MTHFR gene is also regulated by a) alternative splicing [7-8], b) hsa-miR-149 due to a SNP
AC
(rs4846049) at the RNA level [9], c) increased AdoMet/AdoHcy ratio at the protein level [10] and d) phosphorylation of the MTHFR N-terminal domain that reduces the inhibition of MTHFR enzyme activity by AdoMet [11]. In addition to these two polymorphisms in MTHFR, there are many other variations such as
ST
2 splice donor variants, 2 splice acceptor variants, 9 stop gain variants, 6 frame shift variants, 1 initiator coding variant, 121 missense variants, 10 splice region variants, 73 synonymous variants, 46 coding sequence variants, 5 5'UTR region variants, 117 3'UTR variants, 297 intronic variants, 124 upstream gene variants,
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
MTHFR is due to increased pool of 5, 10-methylene tetrahydrofolate for DNA
and 129 downstream gene variants that were reported in the database [http://www.ensembl.org/Homo_sapiens/Info/Index]. Among this, the non-
2
synonymous SNPs (nsSNPs) and SNPs in regulatory region always play major role in the gene regulation and function [12-15]. Recently, several meta-analysis studies have been reported in an effort to draw conclusions on the association of MTHFR variants with ALL but the information
PT ED
is incomplete on the Asian population in general and Indian population in particular despite the distinct ethnicity[16-21]. Hence, we have evaluated the
lymphoblastic leukemia in Indian population and a systematic review with metaanalysis was conducted on previously published reports on Indian population. The study was also extended to analyze additional nsSNPs and SNPs in 3'UTR
CE
region by bioinformatic methods along with MTHFR C677T and A1298C polymorphisms.
AC
MATERIALS AND METHODS Case-Control Study Subjects
Unrelated 203 acute lymphoblastic leukemia (ALL) patients were recruited for
ST
this study. Among these 138 were males and 65 were females with a mean age of 20.2 years. Diagnosis of ALL was done by morphological and immunophenotypic criteria based on the WHO classification [38]. Out of 203 ALL patients analyzed in this study, 129 were childhood ALL patients (< 15
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
association of MTHFR C677T and A1298C polymorphisms with acute
years) with a mean age of 6.66 years (87 males and 42 females) and 74 were adult ALL patients (>15years) with a mean age of 40.58 years (51 males and 23 females). Unrelated 246 normal healthy individuals recruited from the similar
3
socioeconomic group which comprised of 119 males and 127 females with mean age of 25.5 years served as controls. Out of 246 normal control samples, 191 were young adult normal healthy individuals (T polymorphism and folate status affect one-carbon incorporation into human DNA deoxynucleosides. J Nutr 2005; 135:389-396.
AC
[43] Damnjanovic T, Milicevic R, Novkovic T, et al. Association between the methylenetetrahydrofolate
reductase
polymorphisms
and
risk
of
acute
lymphoblastic leukemia in Serbian children. J Pediatr Hematol Oncol 2010;
ST
32:e148-150.
[44] Franco RF, Simoes BP, Tone LG, Gabellini SM, Zago MA, Falcao RP. The methylenetetrahydrofolate reductase C677T gene polymorphism decreases the risk of childhood acute lymphocytic leukaemia. Br J Haematol 2001; 115:616-
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
[41] Sohn KJ, Jang H, Campan M, et al. The methylenetetrahydrofolate
618. 2.
[45] Lightfoot TJ, Johnston WT, Painter D, et al. Genetic variation in the folate metabolic pathway and risk of pediatric leukemia. Blood. 2010;115:3923-3929.
21
[46] Koppen IJ, Hermans FJ, Kaspers GJ. Folate related gene polymorphisms and susceptibility to develop childhood acute lymphoblastic leukaemia. Br J Haematol 2010; 148:3-14. [47] Gast A, Bermejo JL, Flohr T, et al. Folate metabolic gene polymorphisms
2007;21:320-325.
PT ED
and pediatric acute lymphoblastic leukemia: a case-control study. Leukemia.
association analysis of microRNA target sites in one-carbon metabolism genes. PLoS One 2011;6(7):e21851.
[49] Rubie C, Kruse B, Frick VO, et al. Chemokine receptor CCR6 expression is
CE
regulated by miR-518a-5p in colorectal cancer cells. J Transl Med 2014; 12:48. [50] Rai PS, Pai GC, Alvares JF, Bellampalli R, Gopinath PM, Satyamoorthy K. Intraindividual somatic variations in MTHFR gene polymorphisms in relation to
AC
colon cancer. Pharmacogenomics 2014; 15:349-359.
[51] El Awady MK, Karim AM, Hanna LS, et al. Methylenetetrahydrofolate reductase gene polymorphisms and the risk of colorectal carcinoma in a sample
ST
of Egyptian individuals. Cancer Biomark 2009;5:233-240.
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
[48] Stone N, Pangilinan F, Molloy AM, et al. Bioinformatic and genetic
22
PT ED CE
ST
AC
Figure 1.The flowchart representing the process of inclusion and exclusion of published articles for metaanalysis study.
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
FIGURE LEGENDS
23
PT ED CE
AC
ST JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
Figure 2. Association of MTHFR C677T and MTHFR A1298C polymorphism with risk of childhood and adult ALL development in Indian population. In forest plot pooled OR is shown under a random-effects model for the 677T and 1298C alleles. (A) Association of MTHFR 677T allele with childhood and adult ALL (B) Association of MTHFR 1298C allele with childhood and adult ALL. In the plot studies are represented by ascending order of reported year. The size of the square in the plot represents the weight of the corresponding study. Weights are given for the overall combined OR. The diamond in the plot represents overall effect estimate OR and lateral tips of diamond represents the associated 95%CI. (A: adult ALL, C: childhood ALL, C+A: childhood + adult ALL).
24
PT ED CE
AC ST JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
Figure 3. Bioinformatic analysis of nsSNPs in MTHFR gene.(A) Percentage of deleterious and tolerated nsSNPs of MTHFR gene predicted by 8 different bioinformatic tools.(B) Schematic representation of MTHFR gene structure displaying the position of 9 deleterious nsSNPs.
25
TABLE LEGENDS Table I. Distribution of genotypes and allele frequencies of MTHFR C677T (rs1801133) and MTHFR A1298C (rs1801131) polymorphisms in childhood acute lymphoblastic leukemia patients and normal healthy individuals
MTHFR A1298C/ rs1801131
ALL Cases n=129 (%)
Healthy Individuals n=191 (%)
OR (95% CI)
p Value
677CC 677CT 677TT 677CT+TT C allele T allele 1298AA 1298AC 1298CC 1298AC+CC A allele C allele
107 (82.95) 21(16.28) 1 (00.77) 22 (17.05) 0.91 0.09 46 (35.66) 60 (46.51) 23 (17.83) 83 (64.34) 0.59 0.41
156 (81.68 ) 31(16.23) 4 (02.09) 35 (18.32) 0.9 0.1 64 (33.51) 92 (48.17) 35 (18.32) 127 (66.49) 0.58 0.42
1.00 (Reference) 0.98 (0.53-1.81) 0.36 (0.04-3.30) 0.91 (0.50-1.64) 1.00 (Reference) 0.86 (0.50-1.47) 1.00 (Reference) 0.90 (0.55-1.49) 0.91 (0.47-1.74) 0.90 (0.56-1.45) 1.00 (Reference) 0.94 (0.68-1.30)
-0.96 0.35 0.77 -0.58 -0.70 0.78 0.69 -0.73
OR and 95% CI were calculated with MTHFR 677CC and MTHFR 1298AA genotype as reference group. MTHFR: 5, 10-Methylenetetrahydrofolate reductase, ALL: acute lymphoblastic leukemia, OR: odds ratio; CI: confidence interval, p value less than 0.05 is significant
CE
Table II. Combined genotype frequencies of MTHFR C677T (rs1801133) and MTHFR A1298C (rs1801131) polymorphisms in childhood acute lymphoblastic leukemia patients and normal healthy individuals ALL Cases n =129(%)
Healthy Controls n=191 (%)
OR (95% CI)
p Value
677CC/1298AA 677CT/1298AA 677TT/1298AA 677CC/1298AC 677CT/1298AC 677TT/1298AC 677CC/1298CC 677CT/1298CC 677TT/1298CC
36 (27.91) 9 (6.98) 1 (0.77) 51 (39.53) 9 (6.98) 0 (0) 20 (15.50) 3 (2.33) 0 (0)
46 (24.08) 14 (7.33) 4 (2.10) 80 (41.88) 12 (6.28) 0 (0) 30 (15.71) 5 (2.62) 0 (0)
1.00 (Reference) 0.82 (0.32-2.11) 0.31 (0.03-2.98) 0.81 (0.46-1.42) 0.95 (0.36-2.52) NA 0.85 (0.41-1.74) 0.76 (0.17-3.42) NA
-0.68 0.29 0.47 0.93 NA 0.65 0.72 NA
AC
Variant MTHFR, C677T/A1298C
ST
OR and 95% CI were calculated with MTHFR 677CC/MTHFR 1298AA genotype as reference group. MTHFR: 5, 10-Methylenetetrahydrofolate reductase, ALL: acute lymphoblastic leukemia; OR: odds ratio; CI: confidence interval, p value less than 0.05 is significant
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
MTHFR C677T/ rs1801133
Genotypes/ Alleles
PT ED
SNP/rsID
26
Table III. Distribution of genotypes and allele frequencies of MTHFR C677T (rs1801133) and MTHFR A1298C (rs1801131) polymorphisms in adult acute lymphoblastic leukemia patients and normal healthy individuals
MTHFR C677T/ rs1801133
ALL Cases n=74 (%)
Healthy Individuals n=55 (%)
OR (95% CI)
p Value
677CC 677CT 677TT 677CT+TT C allele T allele 1298AA 1298AC 1298CC 1298AC+CC A allele C allele
59 (79. 73) 13 (17.57) 2 (02.70) 15 (20.27) 0.88 0.12 25 (33.78) 30 (40.54) 19 (25.68) 49 (66.22) 0.54 0.46
46 (83.64) 8 (14.54) 1 (1.82) 9 (16.36) 0.91 0.09 14 (25.45) 34 (61.82) 7 (12.73) 41(74.55) 0.56 0.44
1.00 (Reference) 1.26 (0.48-3.31) 1.55 (0.13-17.73) 1.29 (0.52-3.23) 1.00 (Reference) 1.29 (0.57-2.95) 1.00 (Reference) 0.49 (0.21-1.12) 1.52 (0.51-4.50) 0.66 (0.30-1.45) 1.00 (Reference) 1.09 (0.66-1.80)
-0.62 0.71 0.57 -0.53 -0.08 0.48 0.30 -0.71
OR and 95% CI were calculated with MTHFR 677CC and MTHFR 1298AA genotype as reference group. MTHFR: 5, 10-Methylenetetrahydrofolate reductase, ALL: acute lymphoblastic leukemia, OR: odds ratio; CI: confidence interval, p value less than 0.05 is significant
CE
Table IV. Combined genotype frequencies of MTHFR C677T (rs1801133) and MTHFR A1298C (rs1801131) polymorphisms in adult acute lymphoblastic leukemia patients and normal healthy individuals ALL Cases n =74 (%)
Healthy Controls n=55 (%)
OR (95% CI)
p Value
677CC/1298AA 677CT/1298AA 677TT/1298AA 677CC/1298AC 677CT/1298AC 677TT/1298AC 677CC/1298CC 677CT/1298CC 677TT/1298CC
18 (24.32) 5 (6.75) 2 (2.70) 23 (31.10) 7 (9.46) 0 (0) 18 (24.32) 1 (1.35) 0 (0)
9 (16.37) 4 (7.27) 1 (1.81) 30 (54.55) 4 (7.27) 0 (0) 7 (12.73) 0 (0) 0 (0)
1.00 (Reference) 0.62 (0.13-2.91) 1.00 (0.08-12.55) 0.38 (0.14-1.00) 0.87 (0.20-3.79) NA 1.28 (0.39-4.20) NA NA
-0.54 1.00 0.048 0.85 NA 0.67 NA NA
AC
Variant MTHFR, C677T/A1298C
ST
OR and 95% CI were calculated with MTHFR 677CC/MTHFR 1298AA genotypes as reference group. MTHFR: 5, 10-Methylenetetrahydrofolate reductase, ALL: acute lymphoblastic leukemia; OR: odds ratio; CI: confidence interval, p value less than 0.05 is significant
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
MTHFR A1298C/ rs1801131
Genotypes/Allel es
PT ED
SNP/rsID
27
Table V: ORs with 95% CI and heterogeneity results for the genetic contrasts of MTHFR rs1801133 and rs1801131 polymorphisms for ALL risk in Indian population All: all the studies meet the inclusion criteria. Q test: Test to estimate heterogeneity between studies,
Adult
I2 (%)
p, Q test
Random-Effect OR
Fixed-Effect OR
9 9 8 8 9 6 6 5 5 6 2 2 2 2 2
23 42 2 0 51 30 39 0 0 31 51 64 0 0 67
0.24 0.09 0.41 0.43 0.04 0.21 0.15 0.46 0.62 0.20 0.15 0.10 0.72 0.72 0.08
1.12 (0.93-1.36) 1.13 (0.86-1.48) 1.25 (0.79-2.01) 1.27 (0.81-2.00) 1.10 (0.81-1.49) 1.15 (0.92-1.43) 1.25 (0.94-1.68) 0.98 (0.55-1.72) 0.98 (0.57-1.70) 1.30 (0.98-1.73) 0.85 (0.37-1.92) 0.74 (0.25-2.22) 1.08 (0.25-4.77) 0. 96 (0.22-4.28) 0.68 (0.20-2.33)
1.14 (0.97-1.34) 1.17 (0.96-1.43) 1.26 (0.79-2.00) 1.27 (0.81-2.00) 1.17 (0.95-1.44) 1.16 (0.96-1.39) 1.28 (1.02-1.60) 0.98 (0.55-1.72) 0.98 (0.57-1.70) 1.33 (1.05-1.67) 0.84 (0.47-1.49) 0.75 (0.39-1.46) 1.08 (0.25-4.77) 0.96 (0.22-4.28) 0.71 (0.35-1.43)
Random-Effect OR
Fixed-Effect OR
0.97 (0.79-1.19) 0.84 (0.57-1.26) 0.98 (0.71-1.37) 0.97 (0.74-1.28) 0.80 (0.51-1.23) 1.03 (0.87-1.22) 1.05 (0.77-1.42) 1.01 (0.72-1.40) 1.05 (0.78-1.42) 1.04 (0.75-1.43) 0.66 (0.24-1.85) 0.33 (0.08-1.34) 0.76 (0.18-3.18) 0.59 (0.26-1.32) 0.24 (0.06-1.05)
0.99 (0.86-1.14) 0.93 (0.76-1.14) 0.98 (0.72-1.33) 0.97 (0.74-1.28) 0.91 (0.74-1.13) 1.03 (0.89-1.20) 1.04 (0.84-1.30) 1.01 (0.72-1.40) 1.05 (0.78-1.42) 1.04 (0.83-1.30) 0.75 (0.50-1.12) 0.37 (0.20-0.67) 0.83 (0.36-1.89) 0.58 (0.28-1.23) 0.28 (0.15-0.55)
MTHFR A1298C Studies I2 (%) p, Q test (rs1801131) Contrast Allele 8 52 0.04 All Dominant 8 73 0.00 Additive 8 11 0.34 Recessive 8 0 0.55 CT v/s CC 8 75 0.00 Allele 6 21 0.28 Childhood Dominant 6 51 0.07 Additive 6 0 0.44 Recessive 6 0 0.74 CT v/s CC 6 51 0.07 Allele 2 84 0.01 Adult Dominant 2 81 0.02 Additive 2 65 0.09 Recessive 2 13 0.28 CT v/s CC 2 78 0.03 p30% indicates the heterogeneity between the studies
ST
AC
Populations
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
Childhood
Studies
PT ED
All
MTHFR C677T ( rs1801133) Contrast Allele Dominant Additive Recessive CT v/s CC Allele Dominant Additive Recessive CT v/s CC Allele Dominant Additive Recessive CT v/s CC
CE
Populations
28
Table VI: The distribution genotype and allelic frequency of MTHFR rs1801133 and rs1801131 polymorphism in acute lymphoblastic leukemia patients and their normal healthy individuals in selected reports for metaanalysis Distribution of MTHFR C677T Genotype Case
First Author and Year
Case
CC
Reddy et al.,2006
Control
Case
CT
51
79
Case
05
0.67
TT
77
58
07
Control
Case
0.76
0.33
C
Control T 0.24
73
85
13
14
00
00
0.92
0.93
0.08
0.07
Sood et al.,2010 Nikbhakt et al.,2012 Hussain et al.,2012 Sazawal (C) et al.,2013 Sazawal (A)et al.,2013 Present Study (C)
54
173
38
71
03
11
0.77
0.82
0.23
0.18
54
40
62
49
09
11
0.68
0.64
0.32
0.36
59
180
13
61
09
10
0.81
0.84
0.19
0.16
94
63
49
30
08
04
0.78
0.8
0.22
0.12
25
33
06
22
02
03
0.85
0.76
0.15
0.24
107
156
21
31
01
04
0.90
0.90
0.10
0.10
Present Study (A)
59
46
13
08
02
01
0.88
0.91
0.12
0.09
576
855
292
344
41
49
Total
Distribution of MTHFR A1298C Genotype Control AA
Case
Control AC
Frequency of MTHFR A1298C Alleles
Case
Control
Case
9
0.61
0.7
0.39
0.3
CC
Control
Case
A
Control C
Nikbhakt et al.,2012
52
40
65
49
08
11
0.68
0.64
0.32
0.36
Sazawal (C) et al.,2013
57
36
72
51
22
10
0.62
0.63
0.38
0.37
Sazawal (A) et al.,2013
22
14
CE
First Author and Year
Case
PT ED
Adiga et al.,2010
6
35
05
9
0.76
0.54
0.24
0.46
Present Study (C)
46
64
60
92
23
35
0.59
0.58
0.41
0.42
Present Study(A)
25
14
30
34
19
7
0.54
0.56
0.46
0.44
413
530
122
147
Reddy et al.,2006
41
65
83
68
11
20
28
51
55
15
16
0.53
0.56
0.47
0.44
Sood et al.,2010
30
59
46
146
19
50
0.56
0.52
0.44
0.48
AC
Adiga et al.,2010
ST
Total 293 320 A: Adult ALL and C: Childhood ALL
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
Control
Frequency of MTHFR C677T Alleles Contr ol
29
Table VII: List of polymorphisms which alter the binding of miRNA in 3'UTR region of MTHFR gene. The computational modification of MTHFR gene 3'UTR region SNPs to miRNAs binding. The insilico modeling of the interaction between different predicted miRNA and MTHFR 3’UTR region carrying different alleles by using RNAhybrid tool. Calculated free energy change and polymorphism site is also mentioned in the table. miRNA SNP Allele Energy Binding Map
rs1537514
[MAF: 0.1]
A
-27.5 kcal/mol
G
-19.1 kcal/mol
A
-22.9 kcal/mol
C
-22.7 kcal/mol
ST
AC
hsa-miR-518a-5p [LOSS]
-25.1 kcal/mol
hsa-miR-302a-5p [GAIN]
rs1537515 [MAF:0.1]
target 5' U CUCCCA CUU CCU C 3' GGGA GUGAA AC UGGAGCC CCCU CACUU UG GCCUCGG miRNA 3' C U UCU 5' target 5' U CUCCCA CUU UU C 3' GGGA GUGAA ACAC GGAGCC CCCU CACUU UGUG CCUCGG miRNA 3' C UCU 5' target 5' U UCCCA ACUUACC A 3' GGGAC GUGA CUUGG CCCUG UACU GAACU miRNA 3' GAU UC GU 5' target 5' U UCCCA ACUUA A 3' GGGAC GUGA CACUUGG CCCUG UACU GUGAACU miRNA 3' GAU UC 5'
CE
hsa-miR-6513-3p [GAIN]
G
PT ED
rs4846049 [MAF: 0.19]
JU
Leuk Lymphoma Downloaded from informahealthcare.com by Michigan University on 10/11/14 For personal use only.
hsa-miR-149 [GAIN]
G
-20.4 kcal/mol
G
-20.2 kcal/mol
T
-22.1 kcal/mol
30
target 5' U CUUG UUGGG G 3' GGGC CU CUUUGCAG CCCG GG GAAACGUC miRNA 3' CUUU AA 5' target 5' U UGGAGG GG U 3' GGGCUU CUCU GC CCCGAA GGGA CG miRNA 3' CUUU AA UC 5' target 5' A UA G G A 3' GGU AGGUG GUCCA G UCG UUCAU UAGGU C miRNA 3' G G AAAUUCA 5' target 5' A G UG G AAGA G 3' GG GAGU A UCAC GUUUAAG UC UUCA U GGUG CAAAUUC miRNA 3' G UG A A 5'
