Subjects
This study enrolled 106 maternal blood samples of premature infants admitted to the Department of Neonatology, Jilin Province Maternal and Child Health Hospital from April 2019 to April 2021 as the case group.168 maternal blood samples of normal births admitted to obstetrics during the same period were selected as the control group.Maternal inclusion criteria: a.No obstetric complications such as eclampsia, diabetes, placenta previa; b.Take multivitamins instead of conventional folic acid during pregnancy. The criteria for selection of case parturients who gave birth to low birth weight infants: a. Gestational age >28 weeks, body weight < 2 500 g; b. Normal delivery, exclude premature babies caused by accidents; c.Eliminate twins or multiple births and congenital malformations. Selection criteria of control parturients who gave birth to infants with normal weight: a.Choose the gestational week born in the same hospital >28 weeks, body weight >2 500 g, difference in birth time <7 d, singleton normal live birth infants; b.Excluding pregnant women and pregnant women with complications and complications during pregnancy newborn. Comparing the general data of the two groups, the difference was not statistically significant (P ༞0.05).
Serum folate levels
The fasting venous blood of the subjects was extracted, the serum was separated in time and placed in the refrigerator at - 20 ℃ for examination. The serum folate level was measured by chemiluminescence method with Abbott i2000R(Abbott ,USA) .
DNA Extraction and Genotyping
We extracted DNA from 2 mL whole blood, which was collected in ethylenediaminetetraacetic acid (EDTA) and stored at -20 ℃based on provided instructions(Nucleic Acid Extraction and Purification Kit, Kuangyuan Bio.,Suzhou,China). Polymorphisms MTHFR C677T, A1298C and MTRR A66G were typed in an allele-specific polymerase chain reaction (ASPCR) assay combined with TaqMan probe technique (Applied Biosystems, Foster City, CA,USA) with the ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA,USA). A sample of the PCR system contained 2 µl of genomic DNA, 22 µl of amplification reagent that included labelled primer pairs (MTHFR gene detection kit; Kuangyuan Bio.,Suzhou,China), and 1 µl of reaction solution A (MTHFR gene detection kit; Kuangyuan Bio.,Suzhou,China). The positive controls (TT genotype) and negative controls (MTHFR gene detection kit; Kuangyuan Bio.,Suzhou,China) were amplified at the same time.The amplification steps were 50℃ for 2 min, 95℃for 3 min, and 45 cycles of the following steps: 95℃ for 10 s, 56℃for 30 s, and 60℃ for 30 s.Fluorescence signal was collected in the last 35 amplification cycles.Finally, the type of MTHFR C677T, A1298C and MTRR A66G genotype was determined according to the number of cycles (CT value) of the fluorescence signal forming the amplification curve at a specific threshold.The main parameters for ASPCR of the three single nucleotide polymorphisms (SNPs) were shown in Table 1.
Table 1
Primer sequences for MTHFR C677T, A1298C and MTRR A66G polymorphisms.
Gene
|
Primer sequence 1
|
Product Size
|
MTHFR C677T
|
F: 5’-TGAAGGAGAAGGTGTCTGCGGGA-3’
R: 5’-AGGACGGTGCGGTGAGAGTG-3’
|
198bp
|
MTHFRA1298C
|
F: 5’-CTTTGGGGAGCTGAAGGACTACTAC-3’
R: 5’-CACTTTGTGACCATTCCGGTTTG-3’
|
163bp
|
MTRR A66G
|
F: 5’-GCAAAGGCCATCGCAGAAGACAT-3’
R: 5’-GTGAAGATCTGCAGAAAATCCATGTA-3’
|
151bp
|
1 F: forward primer; R: reverse primer. |
Statistical Analysis
The relationship between MTHFR C677T, A1298C and MTRR A66G polymorphisms and LBW used SPSS v22.0 (IBM, Inc., Armonk, NY, USA) for statistical testing in our study.Quantitative data were expressed as mean±standard deviation (‾x±s) and qualitative data as n (%).Comparisons of difference between the case and control with respect to general characteristics were performed by two-sample t test and Chi-square (χ2)test, respectively.Analysis of variance was used for multi group comparison. Unconditional logistic regression analysis was used to evaluate the associations of multi-factors and the three polymorphisms by Odds ratios (OR) and 95% confidence interval (CI). The trend test was used to verify the above results.Hardy-Weinberg equilibrium (HWE) was also assessed for the distributions of genotypes withχ2 test between cases and controls. A two sided p value below 0.05 was considered statistical significance.