Angiotensinogen Gene ( M 235 T ) Variant and Pre-Eclampsia in Egyptian Pregnant Women

Association between the angiotensinogen gene (M235T) and pre-eclampsia has been confirmed in recent studies. Pre-eclampsia is a complication of pregnancy characterized by increased vascular resistance, higher blood pressure, proteinuria and oedema that appear in the second and third trimester of pregnancy. This study aimed at investigating the relationship between M235T gene polymorphism and pregnant women with different forms of pre-eclampsia. One hundred and fifteen pre-eclamptic women and 100 normal control group were recruited and evaluated for the frequency of M235T mutation using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). A positive association was found between maternal age over 35 years (OR= 6.67; CI: 2.09-23.59), previous family history of hypertension (OR= 3.01; CI: 1.18-7.66), previous pre-eclampsia (OR= 7.44; CI: 2.47-22.42), history of reproductive losses (OR = 53.98; CI: 3.23-90.88), fetal anomalies (OR= 8.4; CI: 1.06180.33), and pre-eclampsia. The frequency of heterozygous carriers of M235T mutation in preeclampsia (19.1%) was higher than that in control (14%) but the difference was statistically non-significant. Also, the frequency of M235T mutation was higher in mild pre-eclampsia women (63.6%) compared to women with severe pre-eclampsia (36.4%), however this was statistically non-significant. This study revealed that the frequency of M235T mutation was higher within women with mild pre-eclampsia.


INTRODUCTION
Pre-eclampsia is a disorder associated with pregnancy that consists of hypertension and proteinuria and manifests often after 20 th weeks of gestation. (1)It is a heterogenous disorder which complicates 5-7% of all pregnancy usually primigravida and in women with preexisting hypertension or vascular diseases . (2)though the etiology of pre-eclampsia remains unclear, it has been suggested that abnormal placentation and endothelial cell dysfunction are key features of pathogenesis of pre-Bull High Inst Public Health Vol.37 No. 3 [2007]   eclampsia. (3)It was observed that preeclampsia is associated with increased risks of placental abruptia, acute renal failure, cerebrovascular and cardiovascular complications, disseminated intravascular coagulation, and maternal death. (4)e fetus may be affected by growth retardation and about 20% cases die either in utero or as a result of prematurity. (5)e-eclampsia can be classified as mild or severe.Severe pre-eclampsia is characterized by hypertension greater than (160/110 mmHg) and the presence of significant proteinuria.It is sometimes associated with oliguria, cerebral or visual disturbances, pulmonary oedema, or cyanosis.In mild pre-eclampsia, hypertension and proteinuria are present, but not to these extreme levels, and the patients has no evidence of other organ dysfunctions. (6)e-eclampsia appears to have a genetic component and pedigree analysis suggests that it may be inherited as a single-gene disorder. (7)ndidate genes that determine blood pressure variation include those whose products have a direct role in vascular biology, such as components of the renin-angiotensin system. (8)he importance of the rennin-angiotensin system for maintenance of normal cardiovascular homeostasis and its participation in the pathophysiology of pre-eclampsia is well established.The plasma-renin activity is high and the level of angiotensinogen may be also increased, suggesting a possible role of angiotensiogen in pre-eclampsia. (9)e M235T angiotensinogen gene T235 allele represents the mutant allele and M235 allele represents the wild type. (10)e aim of this study was to investigate the relationship between M235T gene polymorphism and pregnant women with different forms of pre-eclampsia.

MATERIAL and METHODS
A total of 115 women with preeclampsia were recruited prospectively 2. General examination including: blood pressure and lower limb oedema.

Dip stick urine analysis testing for
protein.

Laboratory investigations
including: CBC, urea, creatinine, fasting blood sugar, serum uric acid and liver enzymes.

Ultrasonographic examination of
the pelvis .

Molecular analysis:
a-Genomic DNA was extracted from peripheral EDTA-treated blood cells by DNA salting out as described by Sambrook et al., (1999) (11) with some modifications.

b-Detection of M235T angiotensinogen gene polymorphism:
The

1-DNA amplification
In order to amplify the 165 bp fragment that contains the M235T polymorphism, the first set of second exon primers described by Russ et al., (12) and Jeunemaitre et al., (13) were used.The forward primer was:

5CAGGGTGCTGTCCACACTGGACC
CC-3, and the reverse primer was: Genomic DNA (500 ng) was amplified in a reaction containing 0.

DISCUSSION
Pre-eclampsia is a multifactorial complication of pregnancy and it remains one of the most important causes of maternal and fetal mortality, and morbidity in developed countries. (14)metimes, it progresses to eclampsia in which potentially life-threatening seizures result.Thus, there is critical need for strategies to predict, prevent, and improve management of this disorder that safely prolongs gestation and would be a major advance in prenatal care. (15)ternal age greater than 35 years is one of the maternal specific risk factors for pre-eclampsia. (16)In the present study, maternal age over 35 years was found to have a significant association with pre-eclampsia.This coincides with the values obtained by Tan and Tan, (1994) (17) who reported that 17% of pregnant women over the age of 35 years developed preeclampsia.On the contrary, Choi et al., (2004) (2) found that most of the studied Bull High Inst Public Health Vol.37 No. 3 [2007]   women were below 35 years.These differences might be due to different ethnic background .
Women with familial history of hypertension and pre-eclampsia are at risk to develop different forms of preeclampsia during pregnancy. (10)Familial history of hypertension was observed in the current study in 17 (14.8%)women with different forms of hypertension.This is in agreement with other studies. (1,4)att and Miodovnick, (1999) (18) reported that the risk of pre-eclampsia is 4 times higher the relative risk-being the daughter or a sister of a women who has had pre-eclampsia.In the present study, family history of pre-eclampsia had a significant higher risk for pre-eclampsia (OR = 3.01, CI: 2.47-22.42).
This may be attributed to the fact that pre-eclampsia is associated with high perinatal morbidity and mortality rates as a result of iatrogenic prematurity. (20)though the etiology is unclear, a strong genetic component have been suggested. (2)re-eclampsia is associated with a common molecular variant of angiotensinogen (ATG) gene (Met 235 → Thr 235). (21)Several studies, (7,10,22,23) found that a molecular variant of angiotensinogen (M235T) in Japanese, Romenians, Caucasians, and Australians was significantly associated with pre-eclampsia and in part influenced the development of preeclampsia.
Considering the total number of patients in this study, a high frequency (33.9%) of M235T mutation in angiotensinogen gene in pregnant women with different forms of hypertension as compared to the frequency of this mutation in women with normal pregnancies (22%).
The present study revealed that 22 (19.1%)women with pre-eclampsia were heterozygous affected (MT) and 17 (14.8%)were homozygous affected (TT) for this mutation.Lucia et al., (2002) (10) reported in their study that the frequency of heterozygous women for the mutation (64.28%) was higher than that of the homozygous (14.28%) ones.

CONCLUSION
The frequency of M235T mutation was higher in women with mild preeclampsia and there was no significant association between the mutation and pre-eclamptic patients.
This is a preliminary study and more hypertensive and normotensive pregnant women need to be comparatively studied in order to improve statistical significance and to confirm that the presence of M235T angiotensinogen gene polymorphism can be considered as a risk factor in pre-eclamptic women.
mutation is a single base pair substitution of thymine (T) with cytosine (c) at nucleotide 704 (T704 → c) in exon 2 of the angiotensinogen gene (chromosome 1q42-43), leading the substitution of methionine with threonine at amino acid

M235T polymorphism in exon 2 of
the angiotensinogen was examined by using polymerase chain reaction, restriction fragment length polymorphism method (PCR-RFLP).The M235T allele creates a restriction site for Tth 1111 enzyme that removes 165 base pairs of the PCR products.Fragments were finally size fractionated on agarose gel to allow allele assignment.