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Relationship between small-for-gestational age births and maternal thrombophilic mutations
Thrombosis Research (2008) 122, 175–178
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REGULAR ARTICLE
Relationship between small-for-gestational age births and maternal thrombophilic mutations N. Özbek a⁎ , F.B. Ataç b, ⁎, H. Verdi b , S. Çetintaş a , B. Gürakan, A. Haberal d Received 31 March 2007; received in revised form 10 October 2007; accepted 17 October 2007
Abstract Small gestational age (SGA) is one of the major causes of fetal mortality and morbidity. Altered maternal homeostasis as a result of point mutations in the coagulation cascade has been reported as an important risk factor for this adverse pregnancy outcome. This study aims to investigate the relationship between mother's thrombophilic mutations and SGA deliveries in our population. The study group was consisted of sixty-six women who gave birth to one or more SGA babies. 104 women who gave birth to appropriatefor-gestational age (AGA) babies were sampled for the control group. Restriction fragment size analysis were performed by visualizing digested PCR products for Factor V Leiden (G1691A), Factor V Cambridge (A1090G), Factor V A1299G, prothrombin G20210A, methylene tetrahydropholate reductase C677T, A1298C and T1317C mutations. The results of this study indicate that maternal C677T (p = 0.01) and A1298C (p b 0.01) mutations in MTHFR gene may be suggested as risk factors for SGA outcome in our population. Therefore, maternal screening of these two mutations in the first trimester of pregnancy could help in the assessment of patients. © 2007 Elsevier Ltd. All rights reserved.
Introduction Growth of the fetus from conception to birth results from complex interactions. Recently, several investigations concerning unfavorable genetic factors in human reproduction have progressed [1–3]. The development of the placental circulation is ensured ⁎ Corresponding author. Baskent University School of Medicine, Department of Medical Biology and Genetics, Baglica Kampusu, Eskişehir Yolu 20. km. Ankara 06530, Turkey. Tel.: +90 312 234 1010/ 1528. E-mail address: [email protected] (F.B. Ataçb).
by structural modifications of the spiral arteries and a hypercoagulable state, which is the result of an increase in procoagulant factors and the decrease in both anticoagulant factors and fibrinolysis. Disturbances in this hemostatic balance have been suggested to cause adverse pregnancy outcome such as pregnancy loss, preeclampsia, placental abruption, and fetal growth retardation [4]. Small-for-gestational-age (SGA) describes a neonate whose weight is less than estimated range based on gestational age as established by population studies. Although a number of causes have been linked to SGA deliveries, the exact pathophysiological
0049-3848/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2007.10.004
176 mechanism still remains to be elucidated. There is an increased morbidity and mortality among SGA infants born at term. Thrombophilic mutations have been thought to be the cause of increased thrombosis hazard and predisposition to gestational complications. A meta-analysis disclosed that both factor V Leiden (FVL) and prothrombin gene G20210A (PT G20210A) mutations confer an increased risk for SGA births [5]. Unfortunately, this finding is not confirmed by other reports. Hyperhomocysteinemia is an independent risk factor for arterial and venous thromboembolism [6]. Point mutations in methylenetetrahydropholate reductase (MTHFR) gene are among several factors that cause hyperhomocysteinemia. A recent study has shown that homocysteine (Hcy) levels have a graded effect on risk of fetal loss due to impaired placental circulation [7]. In another report, a possible link has been suggested between polymorphisms in folate metabolizing genes and risk for preterm and SGA birth [8]. This study aims to investigate the relationship between thrombophilic mutations of the mother and SGA deliveries in our population. We studied three polymorphic sites in both the factor V (FV) gene [FVL, FV Cambridge (A1090G), and FV A1299G] and the MTHFR gene (C677T, A1298C, and T1317C), and the PT G20210A mutation. Materials and methods The study population comprised 66 women who gave birth to one or more SGA babies (SGA group) and the controls were 104 women who gave birth to appropriate -for-gestational-age (AGA) babies. The Ethics Committee of Başkent University Faculty of Medicine approved the study protocol. Informed consent was obtained from all women. Demographical, and gestational history, nutrition and folate intake of mothers was noted. All the babies in this study, either SGA or AGA, were single births and delivered at term. SGA babies had gestational weight below the 10th percentile adjusted for the gestational age. Not only the premature babies in the SGA group but patients with preeclampsia, placental abruption or other obstetrical complications in the study and control groups were excluded.
Gel analysis and genotyping A total of 10 ml venous blood was drawn into tubes containing EDTA for genetic studies. Genomic DNA was prepared from leukocyte pellets by sodium dodecyl sulphate (SDS) lysis, ammonium acetate extraction and ethanol precipitation [9]. The primers and PCR conditions were performed as previously described [10–14]. In order to analyze FVL (G1691A) mutation, the 241 bp digested PCR products were separated by electrophoresis in 12% PAGE. The ethidium bromide stained gel showed a non-digested band of 241 bp
N. Özbek et al. for G allele. In the presence of A allele, the PCR product 241 bp was cut into two fragments of 209 bp and 32 bp. Factor V Cambridge (A1090G), AA homozygote produced two fragments at 162 bp and 66 bp, the AG heterozygote 228 bp, 162 bp, 66 bp and the GG homozygote one fragment of 228 bp which did not splice with BstNI restriction enzyme due to the substitution of A by G. A 877 bp PCR product was cut with RsaI for Factor V A1299G mutation. The uncut product 877 bp shows the presence of A allele. If the PCR product was cut into two fragments as 476 bp and 401 bp, it reveals the G allele. For the analysis of the PT G20210A mutation, a 345 bp PCR product digested with HindIII enzyme according to the manufacturer's instruction. The 20210G allele lacks the HindIII site. However, the 20210A variant has two bands corresponding to both 322 bp and 23 bp. A 198 bp PCR product was cut with HinfI for MTHFR C677T mutation. The uncut product 198 bp shows the presence of C allele. If the PCR product was cut into two fragments as 175 and 23 bp it reveals the T allele. For the genotyping of MTHFR A1298C mutation 163 bp PCR product was digested with MbOII enzyme. Wild type produced 5 fragments of 56, 31, 30, 28, 18 bp while the CC genotype reveals 4 fragments of 84, 31, 30, 18 bp. PCR product was digested with TaqI enzyme for MTHFR T1317C mutation. The 1317 T allele lacks the TaqI site. However, the 1317 C has two bands corresponding to both 202 bp and 25 bp. Results The mean age of mothers who gave birth to SGA and AGA babies were not different (26 ± 2.3 vs 27.4 ±1 years). There was no difference in ethnic background. They all had a diet containing sufficient amounts of vegetables and fruits during pregnancy. However, most women did not take supplemental folate and vitamins. Only 21 women in the study group and 34 women in the control group took supplemental folate and vitamins regularly. Means of the gestational periods did not differ between the study (38.7 ± 0.11 weeks) and control groups (39.4 ±0.13 weeks). On the other hand, there was a significant difference between the weight and height of SGA (2270 ±17 g, 47.8 ± 0.1 cm) and AGA (3286 ± 51 g, 50.4 ± 0.2 cm) babies at birth (p b 0.05 for both).
Since we could not get informed consent for some of the genetic analysis, the number of studied subjects was not equal for mutations studied. (Table 1). There was no difference between groups considering FVL, FV Cambridge, FV A1299G, PT G20210A, and MTHFR T1317C. However, significant differences between groups were found with regards to MTHFR C677T (p = 0.01) and MTHFR A1298C (p b 0.001). The
Relationship between small-for-gestational age births and maternal thrombophilic mutations Table 1
Genotype distribution FVL G1691A
SGA Total Control Total
177
GG 65 65 95 97
FVCAMB A1090G
GA –
AA –
2
–
AA 58 63 84 91
FVH1/H2 A1299G
AG 1
GG 4
5
2
AA 41 54 72 91
PRT G20210A
AG 13
GG –
19
–
number of mothers who had both MTHFR C677T and MTHFR A1298C mutations were higher in SGA group than in controls (8 vs 2).
Discussion Hemostatic imbalance is the key mechanism of all types of thrombosis. Thrombophilic risk factors are one of the leading causes of maternal mortality and morbidity. Physiological changes in pregnancy promote coagulation by affecting both procoagulant and fibrinolytic systems, thereby potentiating thrombophilias. Either acquired or mutations in the genes of coding the proteins in coagulation cascade may be associated with placental thrombosis which provides a common pathological link between poor pregnancy outcomes [15]. However, little is known about the role of thrombotic tendency in mothers who have SGA deliveries. FVL and PT G20210A mutations are among the first mutations studied in the etiology of SGA births. Recent studies have disclosed that thrombophilic disorders such as FVL, PT G20210A, or MTHFR C677Tor protein S deficiency may play a role in the etiology of fetal growth retardation, stillbirth, and preeclampsia [16–18] Furthermore, it has been reported that both FV Leiden and prothrombin G20210A mutations increased the risk of delivering a SGA baby two-fold [19]. However, a case-control study revealed no association between these mutations and SGA deliveries [20]. The results of our study indicate that, neither the mutations in FV gene nor the PT G20210A mutation are related with the SGA delivery. In Turkey, the prevalence of the FVL and the PT G20210A mutations in normal population are approximately 7.3% and 2.3%, respectively [21,22]. However, the percentage of these two mutations was lower than in the normal population in both SGA and control groups in our study. Also, there was no difference between the two groups in terms of percentage of mothers bearing this mutation. Increased risk for SGA may be due to thrombosis of placental circulation that is caused by hyperhomocysteinemia due to mutations in MTHFR gene. Recent studies showed a decrease in MTHFR enzyme levels in both C677T and A1298C mutations. However, hyper-
AA 63 63 93 94
MTHFR C677T
GA –
GG –
1
–
CC 36 64 78 104
MTHFR A1298C
CT 28
TT –
26
–
AA 25 60 65 93
MTHFR T1317C
AC 33
CC 2
26
2
TT 59 60 95 96
TC 1
CC –
1
–
homocysteinemia has been observed not only in MTHFR C677T mutation, but also in A1298C muation [23]. It is concluded that enzyme reduction may lead to hyperhomocysteinemia especially in folate deficient conditions such as pregnancy in MTHFR 1298C genotype carriers [24,25]. A recent article disclosed a close relationship between MTHFR A1298C mutation and coronary artery disease [26]. In Turkish patients, no relationship has been obtained between MTHFR T1317C mutation and deep vein thrombosis [27]. Recent reports include diverse results concerning the relationship between MTHFR gene mutations and SGA deliveries. Two studies revealed no relationship between MTHFR C677T mutation and pregnancy outcome [28]. Furthermore, Infante-Rivard et al. [29] measured plasma Hcy levels in mothers who gave birth to premature AGA and premature SGA babies. Interestingly, a negative correlation was found between maternal and newborn plasma homocysteine levels and babies' birth weight. However, the measured plasma Hcy values were below the dangerous level (N 15 μmol/L for mild homocysteinemia) in both AGA and SGA mothers (5.11 μmol/L vs 5.59 μmol/L). Results were even lower in both SGA and AGA babies. They concluded that plasma Hcy levels were not increased in patients with MTHFR C677T and A1298C mutations, however, the number of patients bearing these mutations were not given within the text body. Also, another study reported that there was no relationship between idiopathic SGA pregnancies and some thrombophilic mutations (FVL, PT G20210a and MTHFR C677T) [30]. On the other hand, in a population based study on 5883 women, comparison of upper and lower quartile of plasma Hcy levels resulted in a 32% higher adjusted risk for preeclampsia, 38% higher adjusted risk for prematurity and 101% higher adjusted risk for very low birth weight baby deliveries [31]. Although it is not clear from that study whether the infants were AGA or SGA, a strong relationship between plasma Hcy levels and low birth weight has been underlined. A recent study by Engel et al. [8] revealed no relationship between MTHFR C677T and
178 A1298C mutations and SGA births. However, their study suggested a possibility of a direct or indirect role for another folate metabolizing gene polymorphism, cytosolic serine hydroxymethyltransferase C1420T, in SGA births. In our study, we found that the number of mothers who bear MTHFR C677T or MTHFR A1298C mutations were higher in SGA group than in controls. However, plasma Hcy levels are not measured in our mothers. From these results, we suggest C677T and A1298C mutations in mothers' MTHFR gene may result in SGA deliveries in our population. The reasons for such an association are currently unclear. Very few convincing arguments can be found in the literature for a real toxic effect of mildly increased homocysteine levels on the fetoplacental unit. The investigated functional mutations in the MTHFR gene, which encodes for an enzyme playing a crucial central role in the homocysteine re-methylation pathway, may lead to a maternal impaired metabolic use of folates, thus altering the methylation status and the transcription of genes involved in the maternal participation to placentation. The maternal genetic properties may also be informative only, though there are at least partial correlation with their fetus genetic profiles. In the fetuses, an impaired use of folates may impact on the efficiency of trophoblast properties. We believe that prospective multicenter studies in different ethnic groups that include MTHFR gene C677T and A1298C mutations, Hcy levels and other common mutations are necessary to reach a definite conclusion.
References [1] Kujovich JL. Thrombophilia and pregnancy complications. Am J Obstet Gynecol 2004;191:412–24. [2] Gibson CS, MacLennan AH, Janssen NG, Kist WJ, Hague WM, Haan EA, et al. Associations between fetal inherited thrombophilia and adverse pregnancy outcomes. Am J Obstet Gynecol 2006;194:947–53. [3] Regan L, Rai R. Epidemiology and the medical causes of miscarriage. Baillieres Best Pract Res Clin Obstet Gynaecol 2000;14:839–54. [4] McIntire DD, Bloom SL, Casey BM, Leveno KJ. Birth weight in relation to morbidity and mortality among newborn infants. N Engl J Med 1999;340:1234–8. [5] Howley HE, Walker M, Rodger MA. A systematic review of the association between factor V Leiden or prothrombin gene variant and intrauterine growth restriction. Am J Obstet Gynecol 2005;192:694–708. [6] Eichinger S, Stumpflen A, Hirschl M, Bialonczyk C, Herkner K, Stain M, et al. Hyperhomocysteinemia is a risk factor of recurrent venous thromboembolism. Thromb Haemost 1998;80:566–9. [7] Gris JC, Perneger TV, Quéré I, Mercier E, Fabbro-Peray P, Lavigne-Lissalde G, et al. Antiphospholipid/antiprotein antibodies, hemostasis-related autoantibodies, and plasma homocysteine as risk factors for a first early pregnancy loss: a matched case-control study. Blood 2003;102:3504–13.
N. Özbek et al. [8] Engel SM, Olshan AF, Siega-Riz AM, Savitz DA, Chanock SJ. Polymorphisms in folate metabolizing genes and risk for spontaneous preterm and small-for-gestational age birth. Am J Obstet Gynecol 2006;195:e1–e11 [1231]. [9] Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucl Acid Res 1998;16:1215. [10] Jebeleanu G, Procopciuc L. G20210A prothrombin gene mutation identified in patients with venous leg ulcers. J Cell Mol Med 2001;5:397–401. [11] De Stefano V, Zappacosta B, Persichilli S, Rossi E, Casorelli I, Paciaroni K, et al. Prevalence of mild hyperhomocysteinaemia and association with thrombophilic genotypes [factor V Leiden and prothrombin G20210A] in Italian patients with venous thromboembolic disease. Br J Haematol 1999;106:564–8. [12] Alhenc-Gelas M, Nicaud V, Gandrille S, van Dreden P, Amiral J, Aubry ML, et al. The factor V gene A4070G mutation and the risk of venous thrombosis. Thromb Haemost 1999;81:193–7. [13] Williamson D, Brown K, Luddington R, Baglin C, Baglin T. Factor V Cambridge: a new mutation [Arg306→Thr] associated with resistance to activated protein C. Blood 1998;91:1140–4. [14] Meisel C, Cascorbi I, Gerloff T, Stangl V, Laule M, Muller JM, et al. Identification of six methylenetetrahydrofolate reductase [MTHFR] genotypes resulting from common polymorphisms: impact on plasma Hcy levels and development of coronary artery disease. Atherosclerosis 2001;154:651–8. [15] Doyle NM, Monga M. Thromboembolic disease in pregnancy. Obstet Gynecol Clin North Am 2004;31:319–44. [16] Kupferminc MJ, Many A, Bar-Am A, Lessing JB, AscherLandsberg J. Mid-trimester severe intrauterine growth restriction is associated with a high prevalence of thrombophilia. BJOG 2002;109:1373–6. [17] 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. [18] Kupferminc MJ, Peri H, Zwang E, Yaron Y, Wolman I, Eldor A. High prevalence of the prothrombin gene mutation in women with intrauterine growth retardation, abruptio placentae and second trimester loss. Acta Obstet Gynecol Scand 2000;79:963–7. [19] Martinelli P, Grandone E, Colaizzo D, Paladini D, Scianname N, Margaglione M, et al. Familial thrombophilia and the occurrence of fetal growth restriction. Haematologica 2001;86:428–42831. [20] Infante-Rivard C, Rivard GE, Yotov WV, Genin E, Guiguet M, Weinberg C, et al. Absence of association of thrombophilia polymorphisms with intrauterine growth restriction. N Engl J Med 2002;347:19–25. [21] Gurgey A, Mesci L. The prevalence of factor V Leiden [1691 G→A] mutation in Turkey. Turk J Pediatr 1997;39: 313–5. [22] Akar N, Misirlioglu M, Akar E, Avcu F, Yalcin A, Sozuoz A. Prothrombin gene 20210 G-A mutation in the Turkish population. Am J Hematol 1998;58:249. [23] Rey E, Kahn SR, David M, Shrier I. Thrombophilic disorders and fetal loss: a meta-analysis. Lancet 2003;361:901–8. [24] Infante-Rivard C, Rivard E, Gauthier R, Theoret Y. Unexpected relationship between plasma homocysteine and intrauterine growth retardation. Clin Chem 2003;49:1476–82. [25] McCowan LM, Craigie S, Taylor RS, Ward C, McLintock C, North RA. Inherited thrombophilias are not increased in “idiopathic” small-for-gestational-age pregnancies. Am J Obstet Gynecol 2003;188:981–5. [26] Vollset SE, Refsum H, Irgens LM, Emblem BM, Tverdal A, Gjessing HK, et al. Plasma total homocysteine, pregnancy complications, and adverse pregnancy outcomes: the Hordaland Homocysteine study. Am J Clin Nutr 2000;71:962–9628.
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REGULAR ARTICLE
Relationship between small-for-gestational age births and maternal thrombophilic mutations N. Özbek a⁎ , F.B. Ataç b, ⁎, H. Verdi b , S. Çetintaş a , B. Gürakan, A. Haberal d Received 31 March 2007; received in revised form 10 October 2007; accepted 17 October 2007
Abstract Small gestational age (SGA) is one of the major causes of fetal mortality and morbidity. Altered maternal homeostasis as a result of point mutations in the coagulation cascade has been reported as an important risk factor for this adverse pregnancy outcome. This study aims to investigate the relationship between mother's thrombophilic mutations and SGA deliveries in our population. The study group was consisted of sixty-six women who gave birth to one or more SGA babies. 104 women who gave birth to appropriatefor-gestational age (AGA) babies were sampled for the control group. Restriction fragment size analysis were performed by visualizing digested PCR products for Factor V Leiden (G1691A), Factor V Cambridge (A1090G), Factor V A1299G, prothrombin G20210A, methylene tetrahydropholate reductase C677T, A1298C and T1317C mutations. The results of this study indicate that maternal C677T (p = 0.01) and A1298C (p b 0.01) mutations in MTHFR gene may be suggested as risk factors for SGA outcome in our population. Therefore, maternal screening of these two mutations in the first trimester of pregnancy could help in the assessment of patients. © 2007 Elsevier Ltd. All rights reserved.
Introduction Growth of the fetus from conception to birth results from complex interactions. Recently, several investigations concerning unfavorable genetic factors in human reproduction have progressed [1–3]. The development of the placental circulation is ensured ⁎ Corresponding author. Baskent University School of Medicine, Department of Medical Biology and Genetics, Baglica Kampusu, Eskişehir Yolu 20. km. Ankara 06530, Turkey. Tel.: +90 312 234 1010/ 1528. E-mail address: [email protected] (F.B. Ataçb).
by structural modifications of the spiral arteries and a hypercoagulable state, which is the result of an increase in procoagulant factors and the decrease in both anticoagulant factors and fibrinolysis. Disturbances in this hemostatic balance have been suggested to cause adverse pregnancy outcome such as pregnancy loss, preeclampsia, placental abruption, and fetal growth retardation [4]. Small-for-gestational-age (SGA) describes a neonate whose weight is less than estimated range based on gestational age as established by population studies. Although a number of causes have been linked to SGA deliveries, the exact pathophysiological
0049-3848/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2007.10.004
176 mechanism still remains to be elucidated. There is an increased morbidity and mortality among SGA infants born at term. Thrombophilic mutations have been thought to be the cause of increased thrombosis hazard and predisposition to gestational complications. A meta-analysis disclosed that both factor V Leiden (FVL) and prothrombin gene G20210A (PT G20210A) mutations confer an increased risk for SGA births [5]. Unfortunately, this finding is not confirmed by other reports. Hyperhomocysteinemia is an independent risk factor for arterial and venous thromboembolism [6]. Point mutations in methylenetetrahydropholate reductase (MTHFR) gene are among several factors that cause hyperhomocysteinemia. A recent study has shown that homocysteine (Hcy) levels have a graded effect on risk of fetal loss due to impaired placental circulation [7]. In another report, a possible link has been suggested between polymorphisms in folate metabolizing genes and risk for preterm and SGA birth [8]. This study aims to investigate the relationship between thrombophilic mutations of the mother and SGA deliveries in our population. We studied three polymorphic sites in both the factor V (FV) gene [FVL, FV Cambridge (A1090G), and FV A1299G] and the MTHFR gene (C677T, A1298C, and T1317C), and the PT G20210A mutation. Materials and methods The study population comprised 66 women who gave birth to one or more SGA babies (SGA group) and the controls were 104 women who gave birth to appropriate -for-gestational-age (AGA) babies. The Ethics Committee of Başkent University Faculty of Medicine approved the study protocol. Informed consent was obtained from all women. Demographical, and gestational history, nutrition and folate intake of mothers was noted. All the babies in this study, either SGA or AGA, were single births and delivered at term. SGA babies had gestational weight below the 10th percentile adjusted for the gestational age. Not only the premature babies in the SGA group but patients with preeclampsia, placental abruption or other obstetrical complications in the study and control groups were excluded.
Gel analysis and genotyping A total of 10 ml venous blood was drawn into tubes containing EDTA for genetic studies. Genomic DNA was prepared from leukocyte pellets by sodium dodecyl sulphate (SDS) lysis, ammonium acetate extraction and ethanol precipitation [9]. The primers and PCR conditions were performed as previously described [10–14]. In order to analyze FVL (G1691A) mutation, the 241 bp digested PCR products were separated by electrophoresis in 12% PAGE. The ethidium bromide stained gel showed a non-digested band of 241 bp
N. Özbek et al. for G allele. In the presence of A allele, the PCR product 241 bp was cut into two fragments of 209 bp and 32 bp. Factor V Cambridge (A1090G), AA homozygote produced two fragments at 162 bp and 66 bp, the AG heterozygote 228 bp, 162 bp, 66 bp and the GG homozygote one fragment of 228 bp which did not splice with BstNI restriction enzyme due to the substitution of A by G. A 877 bp PCR product was cut with RsaI for Factor V A1299G mutation. The uncut product 877 bp shows the presence of A allele. If the PCR product was cut into two fragments as 476 bp and 401 bp, it reveals the G allele. For the analysis of the PT G20210A mutation, a 345 bp PCR product digested with HindIII enzyme according to the manufacturer's instruction. The 20210G allele lacks the HindIII site. However, the 20210A variant has two bands corresponding to both 322 bp and 23 bp. A 198 bp PCR product was cut with HinfI for MTHFR C677T mutation. The uncut product 198 bp shows the presence of C allele. If the PCR product was cut into two fragments as 175 and 23 bp it reveals the T allele. For the genotyping of MTHFR A1298C mutation 163 bp PCR product was digested with MbOII enzyme. Wild type produced 5 fragments of 56, 31, 30, 28, 18 bp while the CC genotype reveals 4 fragments of 84, 31, 30, 18 bp. PCR product was digested with TaqI enzyme for MTHFR T1317C mutation. The 1317 T allele lacks the TaqI site. However, the 1317 C has two bands corresponding to both 202 bp and 25 bp. Results The mean age of mothers who gave birth to SGA and AGA babies were not different (26 ± 2.3 vs 27.4 ±1 years). There was no difference in ethnic background. They all had a diet containing sufficient amounts of vegetables and fruits during pregnancy. However, most women did not take supplemental folate and vitamins. Only 21 women in the study group and 34 women in the control group took supplemental folate and vitamins regularly. Means of the gestational periods did not differ between the study (38.7 ± 0.11 weeks) and control groups (39.4 ±0.13 weeks). On the other hand, there was a significant difference between the weight and height of SGA (2270 ±17 g, 47.8 ± 0.1 cm) and AGA (3286 ± 51 g, 50.4 ± 0.2 cm) babies at birth (p b 0.05 for both).
Since we could not get informed consent for some of the genetic analysis, the number of studied subjects was not equal for mutations studied. (Table 1). There was no difference between groups considering FVL, FV Cambridge, FV A1299G, PT G20210A, and MTHFR T1317C. However, significant differences between groups were found with regards to MTHFR C677T (p = 0.01) and MTHFR A1298C (p b 0.001). The
Relationship between small-for-gestational age births and maternal thrombophilic mutations Table 1
Genotype distribution FVL G1691A
SGA Total Control Total
177
GG 65 65 95 97
FVCAMB A1090G
GA –
AA –
2
–
AA 58 63 84 91
FVH1/H2 A1299G
AG 1
GG 4
5
2
AA 41 54 72 91
PRT G20210A
AG 13
GG –
19
–
number of mothers who had both MTHFR C677T and MTHFR A1298C mutations were higher in SGA group than in controls (8 vs 2).
Discussion Hemostatic imbalance is the key mechanism of all types of thrombosis. Thrombophilic risk factors are one of the leading causes of maternal mortality and morbidity. Physiological changes in pregnancy promote coagulation by affecting both procoagulant and fibrinolytic systems, thereby potentiating thrombophilias. Either acquired or mutations in the genes of coding the proteins in coagulation cascade may be associated with placental thrombosis which provides a common pathological link between poor pregnancy outcomes [15]. However, little is known about the role of thrombotic tendency in mothers who have SGA deliveries. FVL and PT G20210A mutations are among the first mutations studied in the etiology of SGA births. Recent studies have disclosed that thrombophilic disorders such as FVL, PT G20210A, or MTHFR C677Tor protein S deficiency may play a role in the etiology of fetal growth retardation, stillbirth, and preeclampsia [16–18] Furthermore, it has been reported that both FV Leiden and prothrombin G20210A mutations increased the risk of delivering a SGA baby two-fold [19]. However, a case-control study revealed no association between these mutations and SGA deliveries [20]. The results of our study indicate that, neither the mutations in FV gene nor the PT G20210A mutation are related with the SGA delivery. In Turkey, the prevalence of the FVL and the PT G20210A mutations in normal population are approximately 7.3% and 2.3%, respectively [21,22]. However, the percentage of these two mutations was lower than in the normal population in both SGA and control groups in our study. Also, there was no difference between the two groups in terms of percentage of mothers bearing this mutation. Increased risk for SGA may be due to thrombosis of placental circulation that is caused by hyperhomocysteinemia due to mutations in MTHFR gene. Recent studies showed a decrease in MTHFR enzyme levels in both C677T and A1298C mutations. However, hyper-
AA 63 63 93 94
MTHFR C677T
GA –
GG –
1
–
CC 36 64 78 104
MTHFR A1298C
CT 28
TT –
26
–
AA 25 60 65 93
MTHFR T1317C
AC 33
CC 2
26
2
TT 59 60 95 96
TC 1
CC –
1
–
homocysteinemia has been observed not only in MTHFR C677T mutation, but also in A1298C muation [23]. It is concluded that enzyme reduction may lead to hyperhomocysteinemia especially in folate deficient conditions such as pregnancy in MTHFR 1298C genotype carriers [24,25]. A recent article disclosed a close relationship between MTHFR A1298C mutation and coronary artery disease [26]. In Turkish patients, no relationship has been obtained between MTHFR T1317C mutation and deep vein thrombosis [27]. Recent reports include diverse results concerning the relationship between MTHFR gene mutations and SGA deliveries. Two studies revealed no relationship between MTHFR C677T mutation and pregnancy outcome [28]. Furthermore, Infante-Rivard et al. [29] measured plasma Hcy levels in mothers who gave birth to premature AGA and premature SGA babies. Interestingly, a negative correlation was found between maternal and newborn plasma homocysteine levels and babies' birth weight. However, the measured plasma Hcy values were below the dangerous level (N 15 μmol/L for mild homocysteinemia) in both AGA and SGA mothers (5.11 μmol/L vs 5.59 μmol/L). Results were even lower in both SGA and AGA babies. They concluded that plasma Hcy levels were not increased in patients with MTHFR C677T and A1298C mutations, however, the number of patients bearing these mutations were not given within the text body. Also, another study reported that there was no relationship between idiopathic SGA pregnancies and some thrombophilic mutations (FVL, PT G20210a and MTHFR C677T) [30]. On the other hand, in a population based study on 5883 women, comparison of upper and lower quartile of plasma Hcy levels resulted in a 32% higher adjusted risk for preeclampsia, 38% higher adjusted risk for prematurity and 101% higher adjusted risk for very low birth weight baby deliveries [31]. Although it is not clear from that study whether the infants were AGA or SGA, a strong relationship between plasma Hcy levels and low birth weight has been underlined. A recent study by Engel et al. [8] revealed no relationship between MTHFR C677T and
178 A1298C mutations and SGA births. However, their study suggested a possibility of a direct or indirect role for another folate metabolizing gene polymorphism, cytosolic serine hydroxymethyltransferase C1420T, in SGA births. In our study, we found that the number of mothers who bear MTHFR C677T or MTHFR A1298C mutations were higher in SGA group than in controls. However, plasma Hcy levels are not measured in our mothers. From these results, we suggest C677T and A1298C mutations in mothers' MTHFR gene may result in SGA deliveries in our population. The reasons for such an association are currently unclear. Very few convincing arguments can be found in the literature for a real toxic effect of mildly increased homocysteine levels on the fetoplacental unit. The investigated functional mutations in the MTHFR gene, which encodes for an enzyme playing a crucial central role in the homocysteine re-methylation pathway, may lead to a maternal impaired metabolic use of folates, thus altering the methylation status and the transcription of genes involved in the maternal participation to placentation. The maternal genetic properties may also be informative only, though there are at least partial correlation with their fetus genetic profiles. In the fetuses, an impaired use of folates may impact on the efficiency of trophoblast properties. We believe that prospective multicenter studies in different ethnic groups that include MTHFR gene C677T and A1298C mutations, Hcy levels and other common mutations are necessary to reach a definite conclusion.
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