MTHFR and F5 genetic variations have association with preeclampsia in Pakistani patients: a case control study

The proposed case-control study was conducted after approval from the Research Ethics Committee of Liaquat University of medical and health sciences (LUMHS), Jamshoro. Written informed consent was taken from all the participants. A total of 250 pregnant women (125 cases and 125 controls) were selected from labour room, wards and outpatient department of Gynaecology and Obstetrics units. It was attempted to recruit all preeclamptic patients admitted from March 2014 to Feb 2015. During this period 187 preeclamptic patients from 20 different districts of Sindh were admitted and after following exclusion criteria, 125 cases were recruited.

Inclusion criteria for the preeclamptic woman was defined according to the American college of obstetrics and gynaecologists as the development of gestational hypertension (blood pressure ≥ 140/90 mmHg on two events at least 6 h apart) and significant proteinuria (≥ 0.3 g protein in 24-h urine specimen or ≥ 1+ on dipstick test) after 20 weeks of gestation in previously normotensive women. Severe preeclampsia was defined on the basis of the presence of one of the following symptoms or signs in the presence of preeclampsia i.e. blood pressure ≥ than 160/110 mmHg (readings were taken on two events at least 6 h apart), proteinuria ≥5 g in 24 h urine specimen (or ≥ 3+ on two urine samples at least 4 h apart), oliguria (urine volume < 500 ml / 24 h), cerebral or visual disturbances, pulmonary edema or cyanosis, epigastric or right upper quadrant pain, platelet count less than 100,000/mm³, presence of haemolysis, elevated liver enzymes and low platelets (HELLP) syndrome and fetal growth restriction. Eclampsia was defined as the occurrence of convulsions in women with preeclampsia [17]. Early and late onset preeclampsia was defined as the development of preeclampsia before 34 weeks and at or after 34 weeks of gestation, respectively [18].

Inclusion criteria for controls were pregnant women greater than 20 weeks of gestation in the absence of diagnostic criteria for preeclampsia until discharge of pregnant women after delivery of the baby. Subjects with the history of chronic hypertension, renal diseases, multiple pregnancy, molar pregnancy, diabetes mellitus, chronic infectious diseases, thromboembolic events, and antiphospholipid syndrome were excluded from the study. Controls recruited were matched for age, ethnicity and parity.

Six SNVs F5:c.1601G > A(g.chr1:169549811), F5:c.6665A > G(g.chr1:169514323;rs6027), MTHFR:c.665C > T(g.chr1:11796321), MTHFR:c.1286A > C(g.chr1:11794419), VEGFA:c.-2055A > C (g.chr6:43768652) and VEGFA:c.*237C > T (g.chr6:43784799) were included in the current study. The selection of the SNVs was based on the role of the respective gene in the placenta and the association with preeclampsia in other world populations [3, 19, 20]. Secondly, there are very few published data on the genetic role of SNVs in preeclamptic Pakistani women [16] and the selected SNVs have not been studied in association with preeclampsia in Pakistani patients.

The sample size was calculated by taking the prevalence of combined thrombophilic mutations (F5:c.1601G > A and MTHFR:c.665C > T) as described by Mello G et al. [21]. Assuming the prevalence as 19.8% in cases and 5.3% in controls, the sample size was calculated at 95% confidence level with alpha = 0.05 and > 80% power, using Ausvet Epitools Epidemiological calculator (http://​epitools.​ausvet.​com.​au/​). The minimum sample size was calculated to be n = 95 in each group, however, to increase the confidence, 125 subjects were selected in each group.

Ten ml of venous blood was collected in 50 ml tube containing 400 μl of anticoagulant ethylene diamine tetra acetic acid (EDTA), 0.5 M from both preeclamptic and normal pregnant women. Genomic DNA was extracted by inorganic method, described as previously [22].

Genotyping of variants of selected genes was carried out by tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS PCR) [23]. Primers were designed using PRIMER1 web tool [24]. Primer sequences were confirmed by UCSC In-Silico PCR and Blat-UCSC genome browser websites [25]. Amplification conditions were optimized and desired fragments amplified using 2720 thermocycler (Applied Biosystems). For SNVs F5:c.1601G > A and VEGFA: c.*237C > T, PCR performed in 20 μl reaction containing 100 ng genomic DNA, 1.25 mM dNTP, 0.6 U Taq polymerase, 2.5 mM MgCl₂ buffer and10μM of each forward and reverse outer primers and forward and reverse inner primers. PCR reaction for remaining SNVs was carried out in 20 μl reaction containing 100 ng of genomic DNA, 1.25 μM of dNTP, 0.6 U Taq polymerase, 2 mM MgCl₂ buffer and 8 μM of each forward and reverse outer primers and forward and reverse inner primers. PCR conditions were 95 °C for 5 min, followed by 30 cycles at 94 °C for 30 s, annealing for 45 s, extension at 65 °C for 2 min and a final extension at 72 °C for 10 min. PCR products were separated on a 2% agarose gel (Fig. 1). The selected samples were Sanger sequenced for confirmation of the amplicon and to validate the ARMS assays. Forward and reverse outer primers were used to amplify desired segment for DNA sequencing. Sequences of primers for ARMS assay and product sizes are mentioned in (Table 1).

Student’s t-test, two-sided Fisher exact test/Chi-square test were applied to the continuous and categorical variables, respectively. A p-value of ≤0.05 was considered as statistically significant. Genotype, allele and haplotype frequencies and Hardy-Weinberg equilibrium (HWE) were calculated. The SNVs association between cases and controls in codominant, dominant, recessive, overdominant and log-additive genetic models, and haplotype association test were performed by logistic regression by using SNPStat software [26]. Odds ratio (OR) and 95% confidence interval were determined to find the association between allelic frequencies between the two groups. To overcome the adjustment of multiple comparisons; false discovery rate (FDR), which is the expected proportion of type 1 errors among all positive tests, controlled by applying the step-up approach of Benjamini and Hochberg was used [27]. For the calculation, the FDR online calculator was used (https://​tools.​carbocation.​com/​FDR). To assess the genetic association of onset and severity of preeclampsia with SNVs that were found significant; odds ratios were estimated by multinomial logistic regression model by using SPSS version 20.

The demographic and clinical characteristics of the participants are shown in Table 2.

Among the demographic variables; age at marriage, height, weight and body mass index (BMI) were not different between preeclamptic and control groups (P > 0.05). The gestational age at presentation and family history were found significantly different between both groups. The preeclamptic and control groups were matched for ethnicity; the ethnic distribution of the preeclamptic patients is presented in Table 2. Fifty (40%) patients presented with early onset and 75 (60%) with late onset preeclampsia whereas, 55 (44%) preeclamptic women developed mild preeclampsia and 70 (56%) presented with severe preeclampsia.

The genotype distribution of the six SNVs in both control and preeclamptic groups were concordant with HWE. The genotype frequencies and association test are presented in Table 3.

The F5:c.6665A > G variant showed 0.42 fold decreased risk for preeclampsia (A/G vs AA/GG: 95% CI = 0.21–0.84; P* = 0.038) in the overdominant model. It was also found to be associated with preeclampsia in the codominant and dominant models; however, the association lost after FDR correction.

Furthermore, significantly increased risk for preeclampsia was found with MTHFR: c.665C > T, in dominant (CT/TT versus CC: OR = 2.91, 95% CI = 1.29–6.57; P* = 0.0497), overdominant (CT versus CC/TT: OR = 2.79, 95% CI = 1.18–6.59; P* = 0.046) and log-additive (OR = 2.48, 95% CI = 1.20–5.13; P* = 0.0396) models.

The significantly decreased risk was associated with CC genotype of MTHFR: c.1286A > C variant in codominant (CC versus AA: OR = 0.36, 95% CI = 0.18–0.72; P* = 0.0392) and log-additive (OR = 0.60, 95% CI = 0.42–0.84; P* = 0.0336) models whereas association in dominant and recessive models did not remain significant after applying FDR. We did not find a significant association of VEGFA: c.-2055A > C, VEGFA: c.*237C > T and F5:c.1601G > A with preeclampsia in Pakistani women.

Allele frequencies for MTHFR: c.665C > T and c.1286A > C were significantly different among cases and control groups (P* = 0.0425; P* = 0.0308, respectively). The frequencies of preeclamptics according to the onset and severity of preeclampsia were further compared with the genotype distribution of F5:c.6665A > G and MTHFR variants (Table 4).

It was observed that the frequency of heterozygous MTHFR:c.1286A > C genotype was different between preeclamptics presented at less than 34 weeks of gestation as compared to patients presented at or after 34 weeks. Preeclamptics with MTHFR: c.1286A > C, AC genotype were found to be 0.19 times less susceptible to present with early onset preeclampsia (95% CI = 0.08–0.49; P* = 0.012) as compared to late onset. However; MTHFR: c.1286A > C, association with severity of preeclampsia did not remain significant at Benjamini-Hochberg adjusted P-value. The haplotype frequencies of variants in preeclamptic and controls; and linkage disequilibrium are shown in Table 5.

The G-G haplotype of F5:c.1601G > A and c.6665A > GG, and C-C and T-C haplotypes of MTHFR: c.665C > T and c.1286A > C variants showed significant association with preeclampsia.