Advertisement

BMC Medical Genetics

, 20:2 | Cite as

Factor-V Leiden G1691A and prothrombin G20210A polymorphisms in Sudanese women with preeclampsia, a case -control study

  • Nadir A. Ahmed
  • Ishag Adam
  • Salah Eldin G. Elzaki
  • Hiba A. Awooda
  • Hamdan Z. Hamdan
Open Access
Research article
  • 176 Downloads
Part of the following topical collections:
  1. Genetic epidemiology and genetic associations

Abstract

Background

Preeclampsia can lead to adverse maternal and perinatal outcomes. There are few studies on the association of preeclampsia with thrombophilia in Africa including Sudan.

Methods

A case –controls study was conducted at Saad Abualila Hospital in Khartoum, Sudan during the period of February through November 2017. The cases were women with preeclampsia and healthy pregnant women were the controls (180 women in each arm of the study). Genotyping for Factor-V Leiden 1691G/A and Prothrombin gene variation 20210G/A was done by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP).

Results

There was no significant difference in the age, parity, body mass index (BMI) and the other characteristics between the cases and the controls. Genotypes distribution of Factor V Leiden 1691G/A and prothrombin gene 20210G/A in controls was in accordance with the Hardy–Weinberg equilibrium (P > 0.05). The factor V Leiden-variation was present in 9.6% of the cases compared with 0.6% of the controls, P < 0.001 (OR = 18.60, 95% CI = 2.38–136.1). Only 4 patients with severe preeclampsia had homozygous variation A/A and it was not detected in the controls. Prothrombin G20210A variations not detected neither in the cases nor in the controls group.

Conclusions

High prevalence of Factor V Leiden 1691G/A variation in preeclamptic patients compared to controls suggest an involvement of this variation in predisposing to preeclampsia in this setting.

Keywords

Factor V Leiden1691G/A prothrombin20210G/A Polymorphism Factor II Factor V Preeclampsia Sudan 

Abbreviations

BMI

Body mass index

EDTA

Ethylene diamine tetra acetic acid

HELLP syndrome

Hemolysis, elevated liver enzymes, low platelet count

HWE

Hardy–Weinberg Equilibrium

PCR

Polymerase chain reaction

PCR–RFLP

Polymerase chain reaction–restriction fragment length polymorphism

TBE

Tris Base/Borate/EDTA

UV

Ultra Violet

Background

Preeclampsia is defined as occurrence of hypertension during pregnancy and the presence of proteinuria after the 20th week of gestation in previously normotensive woman [1]. Preeclampsia is a multisystem disorder and one of the most common encountered serious pregnancy-related disease that affects around 3–8% of all human pregnancies [2, 3].

Although the exact etiology of preeclampsia is not fully understood, the main pathophysiology is the inadequate development of the early placenta “poor placentation”, reduced capacity of the utero-placental circulation which can lead to endothelial dysfunction and the initiation of preeclampsia [4, 5].

Preeclampsia has gene variations; however the precise patho-mechanism and genetic basis of preeclampsia remains unknown [6]. The association of Factor-V (rs6025) and Factor II (Prothrombin) (rs1799963) variations with preeclampsia and their possible role in the pathogenesis of preeclampsia have been previously investigated [7]. The results, however, are still inconclusive and contradictory. While some studies reported association between preeclampsia and Factor-V Leiden [8, 9, 10], others fail to find an association [11, 12]. Likewise, prothrombin G20210A variation were associated with sever preeclampsia in some studies [13, 14], others found no association [15, 16].

Preeclampsia/eclampsia is the major health problem and associated with high maternal and perinatal mortality in Sudan [17, 18]. There are few published data on Factor-V Leiden, Prothrombin G20210A variations and preeclampsia in Africa and there is no published data on Factor-V Leiden, Prothrombin G20210A variations and preeclampsia in Sudan [19, 20]. Therefore, the current study was conducted to assess Factor-V Leiden and Prothrombin variation in Sudanese women with preeclampsia.

Methods

A case –controls study (180 women in each arm) was conducted at Saad Abualila Maternity Tertiary Hospital in Khartoum, Sudan during the period of February through November 2017.

Pregnant women with preeclampsia (blood pressure ≥ 140/90 mmHg on 2 occasions, at least 6 h apart, and proteinuria of ≥300 mg/24 h) were the cases. Preeclampsia was classified in mild and severe form. Severe preeclampsia was considered in the occurrence of one or more of the followings; blood pressure ≥ 160/110 mmHg, proteinuria of ≥5 g/24 h and HELLP syndrome “hypertension, proteinuria and presence of hemolysis, elevated liver enzymes and low platelet count” [1].

The controls were healthy pregnant women without hypertension or proteinuria or diabetes or any underlying disease such as thyroid disorders.

After signing an informed consent, clinical and obstetrics history (age, parity, and gestational age) were gathered using a questionnaire. The Body Mass Index (BMI) was computed from the measured weight and height as weight in kg/ square height in meter. Then 3 mL of whole blood collected in Ethylene diamine tetra acetic acid (EDTA) was used to extract DNA using salting out method [21]. The detection of Factor-V Leiden G1691A and Prothrombin G20210A variations was based on examination of the size of the polymerase chain reaction (PCR) products following DNA amplification of the target sequence of the Factor-V gene and factor II gene, respectively. Oligonucleotides used as primers were: 5’-CATACTACAGTGACGTGGAC-3′ and 5’-TGTTCTCTTGAAGGAAATGC-3′ for Factor-V Leiden; 5’-TCTAGAAACAGTTGCCTGGC-3′ and 5’-ATAGCACTGGGAGCATTGAAGC-3′ for the G20210A variation of the Prothrombin T gene. A reaction mixture consist of DNA, forward and revered primer and nuclease free water were added into PCR tubes using ready mix (Maxime PCR PreMix,i-Taq for 20 μl). The mixture was loaded into thermocycler according to the specific temperature profile. The PCR was performed in 35 cycles consisting of denaturation at 94 °C for 30 s, annealing at 63 °C for 30 s, extension at 72 °C for 30 s, and final extension at 72 °C for 5 min. 1.5% agarose (iNtRON, Biotechnology) was prepared from 1x TBE and 5 μl PCR products were loaded, run on the gel for 30 mins and visualized on UV transllimantor. The resulting PCR product for Factor-V 206-bp digested with 10 μl of DNA restriction enzyme MnI1 (New England Biolabs®Inc.), and resulted in 3 fragments their length are 36, 47 and 123 bp for wild allele G/G, 2 fragments for mutant allele (47 and 159 bp) A/A and heterozygote allele resulted in four fragments (36,47,123 and 159 bp) A/G, Fig. 1. While, Prothrombin PCR product 345 bp digested with 20 U of Hind-III (New England Biolabs®Inc.), and resulted in undigested PCR product for wild type G/G. Two fragments for mutant allele A/A (322 and 23 bp) and 3 fragments for heterozygote allele A/G (322, 23 and 345 bp). Genotyping for factor V and prothrombin is further confirmed by DNA sequencing for the PCR product by using the same primers pairs and it is done in Macrogen Inc., South Korea.
Fig. 1

PCR-RFLP analysis for Factor-V Leiden G1691A polymorphism. Two fragments of 47 bp and 159 bp indicate mutant homozygous (A/A), three fragments of 36 bp, 47 bp and 123 bp for wild type homozygous (G/G) and four fragments of 36 bp, 47 bp, 123 bp and 159 bp indicate A/G genotype. Lane L show the 100 bp ladder; Lane 1 and 2 undigested PCR products. Lane 3 and 4 indicate wild type homozygous (G/G); 5, 7–9 homozygous mutant (A/A) and Lane 6 represents heterozygous (A/G) genotype

The sample size was calculated as for case -control study 1: 1ratio and the difference in the proportions of variation between the cases and controls. It was assumed that Factor-V Leiden variation would be in 7.0% of the pregnant women with preeclampsia and 1% in pregnant women with no complication. This rate was assumed from our previous report on Factor-V Leiden variation among pregnant Sudanese women with deep venous thrombosis [22]. This would give the study at least 80% power and the difference of 0.05 at α level [23].

Statistics

Data were entered in computer sing SPSS for Windows for data analyses. The clinical data were compared between women with preeclampsia and controls by t-test and chi-square test for continuous and categorized data, respectively. Hardy–Weinberg equilibrium (HWE) was conducted by comparing of the observed frequencies of the different genotype distribution with their expected frequency under HW in the controls using Pearson’s chi-square (χ2) statistical test [24]. Pearson’s chi-square (χ2) statistical test was used to compare the alleles difference between the cases and controls. A two-sided P value < 0.05 was considered statistically significant.

Results

There was no significant difference in the age, parity, BMI and the other characteristics between the cases (preeclampsia) and the controls (180 women in each arm), Table 1. There were 63 (35.0%) and 117 (65.0%) women with severe and mild preeclampsia, respectively. The cases of preeclampsia were early preeclampsia in 24 (13.3%) women only and the remaining women 156 (86.7%) had late preeclampsia. Genotypes distribution of Factor-V Leiden1691G/A in controls was in accordance with the HWE (P > 0.05).
Table 1

Comparing the mean (SD) of the sociodemographic characteristics between women with preeclampsia and the controls

Variables

Preeclampsia N = (180)

Controls N = (180)

P-value

Age, year

28.9 (5.2)

28.8 (4.9)

0.884

Parity

3.0 (1.8)

2.7 (1.7)

0.071

Body mass index, Kg/m2

24.5 (2.2)

24.5 (2.2)

0.995

Hemoglobin, g/dl

10.3 (1.4)

10.6 (1.6)

0.101

The Factor-V Leiden-variation was present in 9.6% of patients compared to 0.6% of the controls, P < 0.001 (OR = 18.60, 95% CI = 2.38–136.1). The homozygous variation A/A were detected Only in 4 patients (6.3%) with severe preeclampsia while, none of the controls, Table 2. Prothrombin G20210A variations were not detected, neither in cases nor in controls.
Table 2

Comparing the genotypes and alleles of Factor-V Leiden between women with preeclampsia and the controls

Genotypes

Preeclampsia (180)

Controls (180)

OR (95% CI)

P-value

N

%

N

%

GG

163

90.6

179

99.4

Reference

 

GA

13

7.2

1

0.6

14.2 (1.84–110.3)

0.011

AA

4

2.2

0

0

9.88 (0.52–185.1)

0.053

GA + AA

17

9.6

1

0.6

18.60 (2.38–136.1)

< 0.001

Allele A

21

5.8

1

0.3

22.24 (2.97–166.30)

< 0.001

Allele G

339

94.2

359

99.7

Reference

 

OR Odds ratio

Discussion

The main findings of the current study were the significant association between Factor-V Leiden-variation and preeclampsia. We have recently observed that Sudanese women with severe preeclampsia (compared with controls) had significantly higher levels of thrombin-activatable fibrinolysis inhibitor, and significantly lower plasminogen-activated inhibitor 2 level [25]. In line with our findings (association between Factor-V Leiden and preeclampsia) previous studies reported significant associations between the preeclampsia and Factor-V [8, 9, 10]. Interestingly in their a meta-analysis Dudding et al [26] showed that Factor-V Leiden increased the risk of preeclampsia by almost 50%. Likewise Lin and August [27] in their systemic review and meta-analyses which included 31 studies (7522 patients) reported that over all women with Factor-V Leiden were at 1.81 higher risk of preeclampsia. In contrary previous study found no association between preeclampsia and factor V Leiden [11, 12]. A systematically review showed no association of Factor-V Leiden with preeclampsia [28].

In the current study Factor-V Leiden-variation was present in 9.6% of women with preeclampsia compared with 0.6% of the controls. A similar rate (9.1%) of Factor-V Leiden was reported in four of 44 preeclamptic women in Tunisia [19]. This rate (9.6%) of Factor V Leiden in our study was lower than rate of the Factor-V Leiden variation reported among patients with preeclampsia in different setting e.g. 15 and 20% in Germany [29, 30], 26% in Israel [31], 15% in Sweden [32] and 18.8% in Hungaria [10]. However, a lower rate in Lindqvist’s prospective study, only 1.9% of pregnant women with Factor-V Leiden variation developed preeclampsia, compared to 1.5% without the variation [32]. Likewise, a lower rate of Factor-V Leiden was reported in preeclamptic women in Italy 7.2–5.2% [8, 33]. In south Africa Factor-V Leiden was not detected in either the preeclamptic women or the control groups [20]. It is worth to be mentioned that our results should be compared with cautious with the results of these studies. Firstly, the difference in ethnicity in the different settings should be remembered. Secondly, types of preeclampsia were enrolled in the different studies e.g. severe and mild, early and late preeclampsia.

We did not detect Prothrombin gene variations neither in the cases nor in the controls. This in line with previous studies reported results [8, 9, 12, 31, 33, 34]. Furthermore, a systematic reviews of only prospective cohort studies [28] showed no associations. Interestingly in South African prothrombin polymorphisms, the variant gene allele was not detected in the investigated cases (preeclampsia) or control groups [20].

Conclusions

In this study high prevalence of Factor V Leiden gene variation G1691A compared to healthy controls is observed, which provide an evidence of involvement of Factor V Leiden in the precipitations of preeclampsia in Sudanese women. Prothrombin gene variation G20210A is not associated with preeclampsia in this setting.

Notes

Acknowledgements

The authors are very grateful to all the patients for their cooperation. Authors wish to thank Dr. Motasim Badri for re-checking the statistics.

Funding

Not received.

Availability of data and materials

All data generated or produced from this study are included in this manuscript. Raw data of the study will be available upon request after publication.

Authors’ contributions

NAA, HAA and IEA carried out the study and participated in the statistical analysis and procedures. NAA and SEGE carried out the practical part of the study. HZH and IA coordinated and participated in the design of the study, statistical analysis and the drafting of the manuscript. All the authors read and approved the final version.

Ethics approval and consent to participate

The study received ethical clearance from Al-Neelain University Ethics review board. All participants provide signed informed consent before enrolment.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.
    American College of Obstetricians and Gynecologists. ACOG practice bulletin. Diagnosis and management of preeclampsia and eclampsia. Number 33, January 2002. American College of Obstetricians and Gynecologists. Int J Gynaecol Obstet. 2002;77:67–75.CrossRefGoogle Scholar
  2. 2.
    Lo JO, Mission JF, Caughey AB. Hypertensive disease of pregnancy and maternal mortality. Curr Opin Obstet Gynecol. 2013;25:124–32.CrossRefGoogle Scholar
  3. 3.
    Abalos E, Cuesta C, Grosso AL, Chou D, Say L. Global and regional estimates of preeclampsia and eclampsia: a systematic review. Eur J Obstet Gynecol Reprod Biol. 2013;170:1–7.CrossRefGoogle Scholar
  4. 4.
    Redman CW, Sargent IL. Latest advances in understanding preeclampsia. Science. 2005;308:1592–4.CrossRefGoogle Scholar
  5. 5.
    Redman CW. Current topic: pre-eclampsia and the placenta. Placenta. 1991;12:301–8.CrossRefGoogle Scholar
  6. 6.
    Lisowska M, Pietrucha T, Sakowicz A. Preeclampsia and related cardiovascular risk: common genetic background. Curr Hypertens Rep. 2018;20:71.CrossRefGoogle Scholar
  7. 7.
    Agorastos T, Karavida A, Lambropoulos A, Constantinidis T, Tzitzimikas S, Chrisafi S, et al. Factor V Leiden and prothrombin G20210A mutations in pregnancies with adverse outcome. J Matern Neonatal Med. 2002;12:267–73.CrossRefGoogle Scholar
  8. 8.
    Benedetto C, Marozio L, Salton L, Maulà V, Chieppa G, Massobrio M. Factor V Leiden and factor II G20210A in preeclampsia and HELLP syndrome. Acta Obstet Gynecol Scand. 2002;81:1095–100.CrossRefGoogle Scholar
  9. 9.
    Muetze S, Leeners B, Ortlepp JR, Kuse S, Tag CG, Weiskirchen R, et al. Maternal factor V Leiden mutation is associated with HELLP syndrome in Caucasian women. Acta Obstet Gynecol Scand. 2008;87:635–42.CrossRefGoogle Scholar
  10. 10.
    Nagy B, Tóth T, Rigó J, Karádi I, Romics L, Papp Z. Detection of factor V Leiden mutation in severe pre-eclamptic Hungarian women. Clin Genet. 1998;53:478–81.CrossRefGoogle Scholar
  11. 11.
    Berks D, Duvekot JJ, Basalan H, De Maat MPM, Steegers EAP, Visser W. Associations between phenotypes of preeclampsia and thrombophilia. Eur J Obstet Gynecol Reprod Biol. 2015;194:199–205.CrossRefGoogle Scholar
  12. 12.
    Kahn SR, Platt R, McNamara H, Rozen R, Chen MF, Genest J, et al. Inherited thrombophilia and preeclampsia within a multicenter cohort: the Montreal Preeclampsia Study. Am J Obstet Gynecol. 2009;200:151 e1–9; discussion e1–5.CrossRefGoogle Scholar
  13. 13.
    Mello G, Parretti E, Marozio L, Pizzi C, Lojacono A, Frusca T, et al. Thrombophilia is significantly associated with severe preeclampsia: results of a large-scale, case-controlled study. Hypertens (Dallas, Tex 1979). 2005;46:1270–4.  https://doi.org/10.1161/01.HYP.0000188979.74172.4d.CrossRefGoogle Scholar
  14. 14.
    Wang X, Bai T, Liu S, Pan H, Wang B. Association between thrombophilia gene polymorphisms and preeclampsia: a meta-analysis. PLoS One. 2014;9:e100789.  https://doi.org/10.1371/journal.pone.0100789.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Silver RM, Zhao Y, Spong CY, Sibai B, Wendel G, Wenstrom K, et al. Prothrombin gene G20210A mutation and obstetric complications. Obstet Gynecol. 2010;115:14–20.  https://doi.org/10.1097/AOG.0b013e3181c88918.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Malek-Khosravi S, Rahimi Z, Rahimi Z, Jalilvand F, Parsian A. Thrombophilic mutations and susceptibility to preeclampsia in Western Iran. J Thromb Thrombolysis. 2012;33:109–15.  https://doi.org/10.1007/s11239-011-0653-y.CrossRefPubMedGoogle Scholar
  17. 17.
    Ali AA, Okud A, Khojali A, Adam I. High incidence of obstetric complications in Kassala Hospital, Eastern Sudan. J Obstet Gynaecol. 2012;32:148–9.CrossRefGoogle Scholar
  18. 18.
    Ali AAA, Rayis DA, Abdallah TM, Abdullahi H, Adam I. Hypertensive disorders in pregnancy in Kassala hospital, Eastern Sudan. 2011;04:656–9.Google Scholar
  19. 19.
    Klai S, Fekih-Mrissa N, Rachdi R, Gritli N. The status of Thrombophilic defects and non-O blood group as risk factors for gestational vascular complications among Tunisian women. Acta Haematol. 2011;125:115–20.CrossRefGoogle Scholar
  20. 20.
    Hira B, Pegoraro RJ, Rom L, Moodley J. Absence of factor V Leiden, thrombomodulin and prothrombin gene variants in black south African women with pre-eclampsia and eclampsia. BJOG. 2003;110:327–8.CrossRefGoogle Scholar
  21. 21.
    Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16:1215.CrossRefGoogle Scholar
  22. 22.
    Awad-Elkareem A, Elzaki SG, Khalid H, Abdallah MS, Adam I. A low rate of factor V Leiden mutation among Sudanese women with deep venous thrombosis during pregnancy and puerperium. J Obstet Gynaecol (Lahore). 2017;37:963–4.CrossRefGoogle Scholar
  23. 23.
    Das S, Mitra K, Mandal M. Sample size calculation: Basic principles. Indian J Anaesth. 2016;60:652.CrossRefGoogle Scholar
  24. 24.
    Michael C. A simple calculator to determine whether observed genotype frequencies are consistent with Hardy-Weinberg equilibrium. 2008;2008. https://view.officeapps.live.com/op/view.aspx?src=http%3A%2F%2Fwww.dr-petrek.eu%2Fdocuments%2FHWE.xls.
  25. 25.
    Elzein HO, Muddathir ARM, Rida M, Rayis DA, Elhassan EM, Adam I. Fibrinolysis parameters in Sudanese women with severe preeclampsia. Hypertens Pregnancy. 2016;35:559–64.CrossRefGoogle Scholar
  26. 26.
    DUDDING T, HERON J, THAKKINSTIAN A, NURK E, GOLDING J, PEMBREY M, et al. Factor V Leiden is associated with pre-eclampsia but not with fetal growth restriction: a genetic association study and meta-analysis. J Thromb Haemost. 2008;6:1868–75.CrossRefGoogle Scholar
  27. 27.
    Lin J, August P. Genetic thrombophilias and preeclampsia: a meta-analysis. Obstet Gynecol. 2005;105:182–92.CrossRefGoogle Scholar
  28. 28.
    Rodger MA, Betancourt MT, Clark P, Lindqvist PG, Dizon-Townson D, Said J, et al. The association of factor V Leiden and prothrombin gene mutation and placenta-mediated pregnancy complications: a systematic review and meta-analysis of prospective cohort studies. PLoS Med. 2010;7:e1000292.CrossRefGoogle Scholar
  29. 29.
    Von Tempelhoff GF, Heilmann L, Spanuth E, Kunzmann E, Hommel G. Incidence of the factor V Leiden-mutation, coagulation inhibitor deficiency, and elevated antiphospholipid-antibodies in patients with preeclampsia or HELLP-syndrome. Thromb Res. 2000;100:363–5.CrossRefGoogle Scholar
  30. 30.
    Krauss T, Augustin HG, Osmers R, Meden H, Unterhalt M, Kuhn W. Activated protein C resistance and factor V Leiden in patients with hemolysis, elevated liver enzymes, low platelets syndrome. Obstet Gynecol. 1998;92:457–60.PubMedGoogle Scholar
  31. 31.
    Kupferminc MJ, Eldor A, Steinman N, Many A, Bar-Am A, Jaffa A, et al. Increased frequency of genetic thrombophilia in women with complications of pregnancy. N Engl J Med. 1999;340:9–13.CrossRefGoogle Scholar
  32. 32.
    Lindqvist PG, Svensson PJ, Marsaál K, Grennert L, Luterkort M, Dahlbäck B. Activated protein C resistance (FV:Q506) and pregnancy. Thromb Haemost. 1999;81:532–7.PubMedGoogle Scholar
  33. 33.
    D’Elia AV, Driul L, Giacomello R, Colaone R, Fabbro D, Di Leonardo C, et al. Frequency of factor V, prothrombin and methylenetetrahydrofolate reductase gene variants in preeclampsia. Gynecol Obstet Investig. 2002;53:84–7.CrossRefGoogle Scholar
  34. 34.
    Alfirevic Z, Mousa HA, Martlew V, Briscoe L, Perez-Casal M, Toh CH. Postnatal screening for thrombophilia in women with severe pregnancy complications. Obstet Gynecol. 2001;97(5 Pt 1):753–9.PubMedGoogle Scholar

Copyright information

© The Author(s). 2019

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular Biology, Faculty of MedicineAl-Neelain UniversityKhartoumSudan
  2. 2.Faculty of MedicineUniversity of KhartoumKhartoumSudan
  3. 3.Department of EpidemiologyTropical Medicine Research InstituteKhartoumSudan

Personalised recommendations