Abstract
Background/Aim: A single study has shown positive association and genotype-phenotype correlation between metalloproteinase-9 (MMP-9) promoter genotypes and adult acute lymphocytic leukemia (ALL). However, there is no report about childhood ALL. Thus, this study aimed at examining the role of MMP-9 rs3918242 genotypes in childhood ALL risk. Patients and Methods: A total of 266 childhood ALL cases and 266 healthy controls in Taiwan were examined for their MMP-9 rs3918242 genotypes via polymerase chain reaction-restriction fragment length polymorphism methodology. Results: The MMP-9 rs3918242 CT or TT genotype carriers only had a slightly increased risk compared with CC carriers (p=0.6386 and 0.6005, respectively). The allelic frequency analysis also supported the idea that the variant T allele at MMP-9 rs3918242 is not differentially distributed between the case and control groups (p=0.4834). Conclusion: MMP-9 rs3918242 genotypes may indirectly influence the risk of childhood ALL. Further validations in other populations and analysis of the detail mechanisms are needed.
Childhood acute lymphoblastic leukemia (also called acute lymphocytic leukemia, ALL) is the most prevalent type of malignancy among children all over the world. The molecular background and etiology of childhood ALL are not fully revealed. In the clinic, diagnostic tests examining the blood and/or bone marrow are frequently used to detect and diagnose childhood ALL. The disease progresses rapidly if the patients are not properly treated. Thus, the early detection and prediction of childhood ALL may play a critical role in the treatment and survival of childhood ALL patients. In recent years, several genome-wide association studies in addition to candidate-gene association ones have shown that genetic polymorphisms in specific susceptibility genes are associated with personal susceptibility to childhood ALL (1-5).
Up to now, at least 28 subtypes of matrix metalloproteinases (MMPs) have been detected and identified (6-10), while these MMPs are believed to be responsible for regulating the composition of the extracellular matrix (ECM) that are closely related to tumor metastatic behaviors. Among the 28 subtypes of MMPs, the down-regulation of MMP-2 and -9 expression is most frequently reported to enhance the metastatic capacity of cancer cells (11-13). MMP-9 is one of the key enzymes in the extracellular matrix, that proteolyzes type IV collagens, type V collagens, and other extracellular matrix proteins (14). Since the expression levels of MMP-9 are largely controlled by the promoter region of MMP-9, we focused on identifying single-nucleotide polymorphisms (SNPs) constituting substitutions of single bases in this region. Among the MMP-9 promoter SNPs, C-1562T (rs3918242) is the most frequently studied one, where the substitution of C by T, causes a dramatic decrease in the binding capacity between nuclear proteins, such as CCAAT/enhancer binding protein and homeobox D clusters, with the sequences of the MMP-9 promoter (15). In the literature, MMP-9 rs3918242 genotypes can serve as predictive biomarkers for personal susceptibility for some types of cancer, including lung cancer (16), breast cancer (17), prostate cancer (18), gastric (19, 20), and colorectal cancer (21-23). However, there is no investigation about the contribution of MMP-9 genotypes to childhood ALL risk in Taiwan or other countries. There is only one investigation examining the contribution of MMP-9 rs3918242 genotypes to adult ALL in 376 ALL patients and 352 controls in South China (24). In that study, the authors found that the CT+TT genotypes of MMP-9 rs3918242 are at higher percentages among the cases compared to controls. In addition, they provided phenotypic evidence that ALL patients of MMP-9 rs3918242 with CT+TT genotypes had higher MMP-9 levels in their serum compared to patients with the CC wild-type genotype (24). According to the above information, the genotype of MMP-9 rs3918242 may play a critical role in the etiology of childhood ALL. Thus, in the current case–control study, we aimed to investigate the distribution of genotypes of MMP-9 rs3918242 polymorphism, and evaluate its association with childhood ALL risk in a representative Taiwanese population.
Distribution of age and gender of the 266 childhood ALL patients and the 266 matched controls.
Patients and Methods
Collection of childhood ALL patients and healthy controls. The research design and experimental procedures were approved by the Institutional Review Board of China Medical University Hospital (DMR106-IRB-142). First, patients admitted to the Department with childhood leukemia were well ascertained and recruited into the study by expert pediatric clinicians with pathologic confirmation. Written informed consents from all the participants in the case group were obtained with the help of one or both parents of all children. Totally, 266 patients were initially recruited. All investigated subjects voluntarily participated in this study, completed a questionnaire form with the help of their parents or guardians, and provided their peripheral blood samples of less than 5 ml. An equal number of age- and gender-matched healthy subjects were recruited as the control group. All the detailed procedures can be found in our previous publications (2, 3, 25, 26). The concise summary and comparison of the age and gender for the case and control groups are shown in Table I.
DNA extraction and storage. Genomic DNA was extracted within 12 h after getting the blood from the peripheral blood leukocytes using QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan, ROC) as we have previously described (27, 28). Briefly, peripheral leukocyte genomic DNA from each subject was extracted, its concentration was measured with a nano-spectrophotometer (Thermo Scientific, Wilmington, USA), aliquoted, and stored at −20°C prior to use, or at −80°C for longer period.
MMP-9 rs3918242 genotyping methodology. The primer pairs for the MMP-9 rs3918242 PCR-RFLP genotyping methodology were designed as follows: forward: 5’-TGGTCAACGTAGTGAAACCC-3’; reverse: 5’-TCCAGCCCCAATTATCACAC-3’. The PCR conditions for childhood leukemia MMP-9 rs3918242 genotyping were: originally one cycle at 94°C for 5 min; 35 cycles at 94°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec, and a final extension at 72°C up to 10 min. The restriction endonuclease, SphI (New England Biolabs, Taipei, Taiwan) recognizes and distinguishes the SNP patterns of MMP-9 rs3918242. According to the fact that the C-allele and T-allele DNA sequences are non-digestible and digestible with SphI, respectively, the DNA sequences CC, CT and TT genotypes will produce fragments of 386 bps, 386+320+66 bps, and 320+66 bps, respectively. Then, after complete enzymatic digestion, each sample was immediately loaded into a 2.8% agarose gel and electrophoresed for 30-min under 100 volts.
Statistical analysis. The Student's t-test was used for the statistical analysis of age differences between the childhood ALL case and control groups. The Pearson's Chi-square was used to compare the distributions of the MMP-9 rs3918242 genotypes in subgroups. The associations between MMP-9 rs3918242 genotypes and childhood ALL risk were estimated with the indexes of adjusted odds ratios (ORs) after adjusting the age and gender of the subjects, together with the corresponding 95% confidence intervals (CIs). p<0.05 was regarded as statistically significant.
Results
The age and gender for the 532 participants in the current study, including 266 childhood ALL patients and 266 healthy controls, are presented and compared in Table I. Since we adopted the strategy of matching the age and gender while recruiting the controls, there was no significant difference regarding these two indexes (both p>0.05) (Table I).
The frequency of distributions of the MMP-9 rs3918242 genotypes among the 266 age- and gender-matched healthy controls and the 266 patients with childhood ALL are shown in Table II. First, the distribution of MMP-9 rs3918242 genotypes among the controls fitted well with the Hardy-Weinberg Equilibrium (p for HWE=0.1012). Second, the genotypes of MMP-9 rs3918242 were not differently distributed between the case and control groups (p for trend=0.7983) (Table II). In detail, the MMP-9 rs3918242 variant CT and TT genotypes were associated with childhood ALL risk compared with the wild-type CC genotype (adjusted OR=1.08 and 1.14, 95%CI=0.77-1.43 and 0.67-3.09, p=0.6386 and 0.5490, respectively). In addition, in the dominant analysis model, there was also no significant association between MMP-9 rs3918242 T-allele carriers (CT+TT) and childhood ALL risk, compared with those carrying the CC genotype (adjusted OR=1.10, 95%CI=0.82-1.53, p=0.5490).
In order to confirm the results and findings in Table II, we further analyzed the allelic frequency distributions of MMP-9 rs3918242 between the control and case groups. The results are presented in Table III. Supporting the findings that the variant genotypes of MMP-9 rs3918242 are not associated with childhood ALL risk in neither the co-dominant or dominant models, the frequency of variant allele T was 15.0% in the case group, not significantly higher than that of 13.5% in the control group (adjusted OR=1.12, 95%CI=0.82-1.49, p=0.4834) (Table III).
Discussion
MMP-9, also known as gelatinase B or type IV collagenase, belongs to the MMP family which is also involved in the regulation of ECM components and their metabolism (29). In cancer, MMP-9 plays a central role in carcinogenesis, from angiogenesis, to stromal remodeling, and ultimately in metastatic behaviors of cancer cells (30, 31). The significantly increased expression of MMP-9 has been reported in many types of cancer, such as breast (32), esophageal (33), gastric (34), colorectal cancer (35, 36) and in cancer cells with high metastatic capacity (37). As for leukemia, over-expression of MMP-9 has been found in the blood cells of adult leukemia patients in South China, especially those with poor prognosis (24).
The genotype-phenotype correlation evidence for the SNPs on MMP-9 promoter sequence may differ among different tissues investigated. The first study we are going to discuss is the influence of MMP-9 rs3918242 on the expression levels of MMP-9 in non-small cell lung carcinoma (38). There was no positive MMP-9 rs3918242 genotype-phenotype correlation among 243 non-small cell lung carcinoma patients. The second study was conducted by Xing and his colleagues in colorectal cancer patients (21). They found that the T allele at MMP-9 rs3918242 was associated with an elevated colorectal cancer risk only among the elder people (>60 years old), not all colorectal cancer patients. Interestingly, the variant T allele at MMP-9 rs3918242 was associated with increased risk of lymph node (21) and distant metastasis (23). The third study has examined the genotype-phenotype correlation in colorectal cancer patients (22). They found that colorectal cancer patients had higher levels of MMP-9 in their peripheral blood, independently of their MMP-9 rs3918242 genotype. More important, the patients with the CT genotype at MMP-9 rs3918242 had significantly higher levels of MMP-9 than those with the CC genotype (22). The fourth study has examined the contribution of MMP-9 rs3918242 genotypes to adult ALL in 376 ALL patients and 352 controls in South China (24). In that study, the authors found that the CT+TT genotypes of MMP-9 rs3918242 were at higher percentages among the cases than among the controls. In addition, they provided evidence showing that ALL patients carrying the MMP-9 rs3918242 CT+TT genotypes had higher MMP-9 levels in their serum compared to patients with the CC wild-type genotype (24). The fifth study was performed by Medley and his colleagues (39), who have shown that in patients with coronary artery disease the expression levels of MMP-9 protein were significantly higher among the -1562T carriers compared to -1562C carriers. Although the fourth study has shown us a positive genotype-phenotype correlation for MMP-9 rs3918242 among adult leukemia patients, we are still interested in revealing the genotype-phenotype correlation for MMP-9 rs3918242 among childhood leukemia patients. Overall, the importance of MMP-9 rs3918242 genotype for childhood ALL risk was examined by the current study for the first time. There is still the question whether the children carrying CT and TT genotypes at MMP-9 rs3918242 have higher MMP-9 levels in their blood and tumor sites. The increased levels of MMP-9 in the extracellular microenvironment may result in the local breakage of the cellular membranes and the components of extracellular matrix, which lead to poor prognosis.
Distributions of matrix metalloproteinase-9 rs3918242 genotypic frequencies among the patients with childhood ALL and healthy controls.
Allelic frequencies for matrix metalloproteinase-9 rs3918242 polymorphisms among the patients with childhood ALL and healthy controls.
In conclusion, the solid evidence provided in this study within a moderate and representative population shows that MMP-9 rs3918242 genotypes do not directly affect the childhood ALL risk in Taiwanese population. To reach a more comprehensive conclusion, enlarged, representative and multi-ethic comparative studies involving larger sample sets, in addition to environmental interactions, are required to further investigate the role of MMP-9 rs3918242 genotypes and the mechanisms of childhood ALL.
Acknowledgements
The Authors would like to thank the personnel of the Tissue-Bank of China Medical University Hospital for their excellent technical assistance including all, doctors, nurses and colleagues. The excellent technical expertise and efforts from Yu-Chen Hsiau, Kai-Cheng Chan, Yu-Ting Chin and Tai-Lin Huang are also appreciated. This study was supported mainly by the China Medical University and Hospital (DMR-108-BC-1) to Dr. Kuo and Dr. Shen.
Footnotes
↵* These Authors contributed equally to this study.
Authors' Contributions
Research Design: Kuo CC, Tsai CW and Chang WS; Patient and Questionnaire Summarize: Shen TC, Li CH and Tsai FJ; Experiment Performance: Wang YC and Tsai CW; Statistical Analysis: Shen TC, Tzeng HE and Chang WS; Manuscript Writing: Tsai CW and Bau DT; Reviewing and Revising: Bau DT, Chang WS and Tsai CW.
Conflicts of Interest
All Authors declare no conflicts of interest in regard to the current study.
- Received August 19, 2020.
- Revision received August 31, 2020.
- Accepted September 1, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
References
In this issue
Jump to section
Related Articles
Cited By...
- The Genetic Role of Matrix Metalloproteinase-12 in Determining Childhood Acute Lymphocytic Leukemia Risk
- Significant Association of Matrix Metalloproteinase-9 Polymorphisms With Triple Negative Breast Cancer Risk
- Impacts of Matrix Metalloproteinase-9 Promoter Genotypes on Asthma Risk
- Association of Matrix Metalloproteinase-9 Genotypes With Nasopharyngeal Carcinoma Risk
- Association of Matrix Metalloproteinase-9 Genotypes With Lung Cancer Risk in Taiwan
- Impacts of Matrix Metalloproteinase 9 Genotypes on Renal Cell Carcinoma
- Significant Association of CCND1 Genotypes With Susceptibility to Childhood Acute Lymphoblastic Leukemia