Effects of factor v Leiden polymorphism on the pathogenesis and outcomes of preeclampsia

Preeclampsia (PE) is a disease of major public health concern with high incidence of maternal and perinatal mortality [1‐3]. Its etiology is multifactorial with no reliable test for predicting preeclampsia among a cohort of pregnant women. This has been complicated by the recent subclassification of preeclampsia as mild, moderate and severe or early and late onset [4]. According to the World Health Organization (WHO), maternal mortality ratio in Ghana is 350 per 100,000 live births, which is unacceptably high compared to the global ratio of 216 per 100,000 live births. Also, the maternal mortality in Korle-Bu Teaching Hospital (KBTH), which is the current study site, has been increasing since the mid-1990s from 734.4 [5] to 915.3 per 100,000 live births in 2012 [6]. This is clearly an aggravation, so it is imperative that investigations into the etiology and pathogenesis of preeclampsia is carried out in our indigenous women to help minimize its occurrence and the associated complications. We examined if mutations in Factor V Leiden (FVL) [7], contributes to the observed upward trend in Ghana. Factor V leiden, have currently been well associated with PE development. The factor V Leiden variant resists cleavage by activated protein C due to the substitution of glutamine residue with arginine at position 506 (Gln⁵⁰⁶), which is the cleavage site for activated protein C. The resistance is seen in inherited familial thrombophilia and venous thrombosis [8] and has recently been implicated in rampant fetal loss [9]. There is therefore anticipation that evolutionary signatures such as geographical origin, mutations, cis – acting elements, trans – acting factors and natural selection might influence genetic prediction of PE and its deadly complications. The current study sought to identify factor V leiden mutation in PE in the Ghanaian population and examine if these are associated with PE, to provide insight into a probable factor in the rise in PE, seen in the general population in pregnant women attending anti-natal care at the Korle-Bu Teaching Hospital in particular.

At recruitment, each subject completed a questionnaire (Additional file 1) after which demographic and anthropometric data were obtained. Spot urine samples were collected to determine protein-creatinine ratio using a Clinitek analytic strips. Also, 24-h urine samples were collected from PE patients, starting from the time of diagnosis, to determine the level of proteinuria. Venous blood (5 ml) were taken from the antecubital vein into vacutainers. All pregnant women were observed till delivery, for outcomes of PE. For the purposes of this study, PE was defined based on American College of Obstetricians and Gynecologists criteria. Accordingly, PE was defined as diastolic blood pressure (DBP) of ≥90 mmHg and or systolic blood pressure (SBP) of ≥140 mmHg with proteinuria ≥300 mg/dl occurring after 20 weeks of gestation [3]. Early or late onset preeclampsia occurs before and at or after 34 weeks of gestation respectively [4]. The preeclamptic cases were managed strictly according to the standard management protocols for preeclampsia at the KBTH including the hospital admission, monitoring of maternal and fetal parameters, the use of antihypertensives and or magnesium sulphate and timely delivery of the baby.

The study was completed by eighty-one (81) subjects out of the total of 96 who were enrolled. Those who did not complete or had missing data were either ill or decided not to have their blood sample taken (Additional file 2: Table S1). The average blood pressure and weeks of maternal gestation in PE ranged from early onset (142/90 mmHg; 34 weeks) to late onset (159/96 mmHg, ≥34 weeks) respectively. The PCR product size for exon 8 and exon 10 were 150 bp and 276 bp respectively. A digest in exon 8 yielded 100 bp and 50 bp. Normal alleles in Exon 10 yielded 176 bp, 69 bp and 31 bp. Overall allelic frequencies found in FVL exon 10 were 0.67 and 0.33 for G and A alleles respectively (Table 1). There were increased FVL mutations in PE and hypertensive patients. However, the frequencies in PE and hypertensive subject were not in agreement with Hardy-Weinberg Equilibrium due to loss of heterozygosity. FVL homozygotes had lower age (p < 0.03), high systolic blood pressure, diastolic blood pressure (DBP), liver enzymes, white blood cells (wbc) and hemoglobin levels (Tables 2 and 3). The ratio of aspartate transferase (AST) to alanine transferase (ALT) had a 3-fold increase in PE upon stratification. Also, uric acid, white blood cell count (wbc), urine protein, systolic blood pressure, diastolic blood pressure was elevated under the same stratification in PE cases while platelet count decreased (Table 2 and 3). Post hoc analysis confirmed where differences occurred between groups (Table 4, Additional file 2: Table S2).

In this study, a report on the effects of FVL on the outcomes of PE were presented. This study was the first to report baseline FVL genotypic and allelic frequencies as well as relating clinical variables with it. The study also reports for the first time that in the pregnant women studied, FVL mutation allele frequency being 0.33, is consistent with reports elsewhere [9]. The loss of heterozygosity in the PE and hypertensive subjects could be due to founder effects or genetic drifts [10]. These could be critically examined in a follow up study.

The strikingly three – fold increment of AST / ALT ratio, increased white blood cell (wbc) and increased uric acid levels observed in PE cases when stratified by both exons indicates that genetic disposition to FVL could be associated with susceptibility to PE and might confer poor PE outcomes. The underlying pathophysiology appears to be acute inflammation as all blood cell indices were elevated including uric acid. These were also consistent with previous findings in other populations [11‐14].

Although, wbc counts [11‐14], liver enzymes [11‐14] and uric acid [15‐19] had been suggested to increase in PE, it was notable to mention that no study associated FVL to such parameters in Ghana. More so, there had been scanty information on the impact of FVL in clinical events where inflammation was present in the country. FVL referred to a specific gene mutation which led to hypercoagulability with serious clinical consequences.

Much work has been done elsewhere on FVL [20‐26] in PE; however, while others never found FVL mutation in their cohort [23], others strongly cited indications of FVL [22, 24] being a unique predictor of PE. Even though sample size in the current study was small, findings of heterozygosity and normal homozygous in FVL exon 10 seemed to be consistent with Dizon – Townson et al. [9] We could not however extrapolate the findings to the general population unless this was further interrogated with a larger sample size. We tried to minimize selection bias using two control(s) e.g. pregnant normotensive and non-pregnant normotensive per case to improve the statistical power of the study since those from the hospital setting had outcomes related to the exposure being studied.

The inflammatory response attributed to FVL might be due to platelets activating the inflammation, with uric acid channeling macrophage and dendritic cell activation, thereby increasing proinflammatory cytokines to sustain inflammation and adverse outcomes [25]. The absence of FVL mutation in exon 8 was consistent with O’Shaughnessy et al. [26] Nevertheless, the current study was able to identify mutant homozygous in FVL exon 10.

FVL mutation could lead to an inflammatory state in PE. We have for the first time reported the allelic frequency in a section of the Ghanaian population.