Gene polymorphism of MTHFR rs1801133 and susceptibility to childhood leukemia in Chinese population

Background The purpose of this study is to investigate the genotype and allele distribution of MTHFR rs1801133 in the Chinese population, and to analyze the relationship between gene polymorphism of MTHFR rs1801133 and risk of childhood leukemia. Methods Blood samples and clinical data of childhood leukemia cases (n=1132) and age-matched healthy controls (n=1053) were collected. Genotypes and allele distribution of MTHFR rs1801133 were detected by PCR-RFLP. Logistic regression model was generated to analyze the relation between MTHFR rs1801133 and susceptibility to childhood leukemia and the chemotherapy response. Results Age, sex, BMI and family history of tumor were comparable between childhood leukemia cases and healthy controls. Genotypes and allele distribution of MTHFR rs1801133 were remarkably correlated to the risk of childhood leukemia. Genotype risk of MTHFR rs1801133 was parallel to the susceptibility to childhood leukemia. Specifically, compared with people carrying AA allele of MTHFR rs1801133, higher risk of childhood leukemia may occur in people carrying AG+GG allele of MTHFR rs1801133 with a younger age (<15 years) or complete remission from chemotherapy. Conclusions MTHFR rs1801133 gene polymorphism has a significant correlation with childhood leukemia. It is an important genetic susceptibility gene of childhood leukemia. The reliability of the results requires to be further validated by the high-quality research involving a large sample size in multi-center hospitals.


Summary
Background: The purpose of this study is to investigate the genotype and allele distribution of MTHFR rs1801133 in the Chinese population, and to analyze the relationship between gene polymorphism of MTHFR rs1801133 and risk of childhood leukemia.Methods: Blood samples and clinical data of childhood leukemia cases (n=1132) and age-matched healthy controls (n=1053) were collected.Genotypes and allele distribution of MTHFR rs1801133 were detected by PCR-RFLP.Logistic regression model was generated to analyze the relation between MTHFR rs1801133 and susceptibility to childhood leukemia and the chemotherapy response.Results: Age, sex, BMI and family history of tumor were comparable between childhood leukemia cases and healthy controls.Genotypes and allele distribution of MTHFR rs1801133 were remarkably correlated to the risk of childhood leukemia.Genotype risk of MTHFR rs1801133 was parallel to the susceptibility to childhood leukemia.Specifically, compared with people carrying AA allele of MTHFR rs1801133, higher risk of childhood leukemia may occur in people carrying AG+GG allele of MTHFR rs1801133 with a younger age (<15 years) or complete remission from chemotherapy.Conclusions: MTHFR rs1801133 gene polymorphism has a significant correlation with childhood leukemia.It is an important genetic susceptibility gene of childhood

Introduction
Leukemia is a malignant disease attacking the hematopoietic system.It is the mostly diagnosed tumor in children.Based on disease course and cell differentiation, leukemia is classified to acute and chronic type.Acute leukemia (AL) covers about 95% of childhood leukemia (1)(2)(3).Classified by malignant cell origins, AL is subgrouped to acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL).The incidence of ALL (3.4-4.1/100,000) is 5 times that of AML (4,5).At present, chemotherapy is preferred to childhood ALL, and its 5-year survival is up to 85% (3,6).Even for the standard risk group patients, the 5-year progression-free survival can reach 93%.Nevertheless, chemotherapyinduced adverse effects and drug toxins seriously affect life quality of childhood leukemia patients (7)(8)(9).
The protein encoded by MHTFR (NADPH) is a key enzyme of folate metabolism.NAPDH catalyzes the conversion of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate for homocysteine (hCY) remethylation to methionine (10,11).The mutation of MTHFR gene leads to increased plasma concentration of hCY and DNA hypomethylation, which further affect the production of folic acid and the synthesis of DNA (11).There are 14 mutations in the coding region of MTHFR gene, of which rs1801133 is the most common single nucleotide polymorphism (SNP) (12,13).Gene polymorphism of MTHFR in the development of childhood leukemia is rarely reported (14,15).In this case-control study, we aim to uncover the relation between MTHFR rs1801133 and susceptibility to childhood leukemia in Chinese population.

Study population
Leukemia children (n=1132) and age-matched healthy controls (n=1053) were randomly recruited in Anhui Provincial Children's Hospital.Eligible leukemia children did not have other malignancies or received pre-chemotherapy.Childhood leukemia was independently diagnosed by two pathologists based on clinical symptoms, physical, imaging and histological examinations.
Healthy controls were recruited from whom participated in physical examinations during the same period.They did not have blood connections to other participants.Family history of cancer was defined as the presence of cancer in any first-degree relatives (parents, siblings or offspring).This study was approved by the institutional review committee of our hospital.All participants and their parents were informed consent.

PCR-RFLP (polymerase chain reaction -restriction fragment length polymorphism)
Genomic DNA was isolated and purified from peripheral blood lymphocytes by proteinase K digestion and phenol-chloroform method.MTHFR rs1801133 and its alleles were detected using PCR-RFLP (Applied Biosystems, Foster City, CA, USA).SNP primers were amplified at 95 °C for 10 min, followed by 45 cycles at 95 °C for 15 s and 60 °C for 1 min.PCR products were cleaved by BccI, loaded on 1% DNA agarose gel containing C 21 H 20 BrN 3 , and analyzed.

Statistical analysis
Statistical Product and Service Solutions (SPSS) 22.0 (IBM, Armonk, NY, USA) was utilized for statistical analysis.Enumeration data were expressed as frequency (%).The HWE of control genotype distribution, and comparison of enumeration data were evaluated using the c 2 test.Risk factors of childhood leukemia were assessed by Logistic regression test, and results were expressed as OR and 95% CI.P<0.05 considered as statistically significant.

Characteristics of childhood leukemia cases and healthy controls
A total of 1132 eligible leukemia children and 1053 age-matched healthy controls were recruited.By analyzing their clinical data, it is shown that age, sex, BMI and family history of tumor were comparable between childhood leukemia cases and healthy controls (P>0.05,Table I).
leukemia.The reliability of the results requires to be further validated by the high-quality research involving a large sample size in multi-center hospitals.

Association between childhood leukemia risk and MTHFR rs1801133
Genotypes and allele distribution of MTHFR rs1801133 were detected to be remarkably correlated to the risk of childhood leukemia (Table II).In particular, a higher susceptibility to childhood leukemia in people carrying GG (P=0.005,OR=1.56 (1.15-2.13))or AG+GG allele (P=0.032,OR=1.17 (1.01-1.35))was identified in comparison to whom carrying AA allele.G allele of MTHFR rs1801133 (P=0.006,OR=1.12 (1.06-2.30))predicted a higher risk of suffering from childhood leukemia than A allele.

Association between childhood leukemia pathology and MTHFR rs1801133
Logistic regression model was generated to analyze the relation between MTHFR rs1801133 and susceptibility to childhood leukemia and the pathology (Table III sis suggested that MTHFR rs1801133 enhanced the risk of childhood leukemia, especially AG, GG or AG+GG allele.To our knowledge, this study for the first time evaluated the role of MTHFR rs1801133 in the pathogenesis of childhood leukemia. Classified by genotypes of MTHFR rs1801133, we detected a higher susceptibility to childhood leukemia in people carrying GG or AG+GG Moreover, people carrying G allele of MTHFR rs1801133 had a higher risk of suffering from childhood leukemia than whom carrying A allele.Potential influences of MTHFR rs1801133 polymorphism on clinical data of childhood leukemia cases and their chemotherapy response were further assessed.It is shown that higher risk of childhood leukemia may occur in people carrying AG+GG allele of MTHFR rs1801133 with a younger age (<15 years) or complete remission compared with people carrying AA allele of MTHFR rs1801133.
Three limitations in the present study main limit the reliability of our conclusion.First of all, a relatively small sample size wound affect the results.Secondly, potential influences of the internal environment of the embryo, nutritional intake of the mother during pregnancy or other important factors are not taken into considerations.Thirdly, a possible effect of genotypes carried by parents on the risk of their offspring remains unclear.Collectively, our finding requires to be further validated by the high-quality research involving a large sample size in multi-center hospitals.

Conclusion
MTHFR rs1801133 gene polymorphism has a significant correlation with childhood leukemia.It is an important genetic susceptibility gene of childhood leukemia.

Table I
Student's t-test for age and BMI distributions between cases and controls; two sided c 2 test for other selected variables between cases and controls.Distribution of selected variables between the childhood leukemia cases and the control subjects.TableIIThe basic information of the genotyped polymorphisms in MTHFR rs1801133 Polymorphism associated with the risk of Childhood leukemia.Table III Association between MTHFR rs1801133 Polymorphism and Childhood leukemia in stratified analysis.Table IV Association between MTHFR rs1801133 Polymorphism and clinicopathologic characteristics of Childhood leukemia.*Two-sidedc 2 test for number of risk alleles in cases and controls; 95% CI: 95% confidence.Adjusted for age, pack-years of smoking, drinking status, and family history of cancer in logistic regression model. *