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Preeclampsia (PE) is a multisystem disorder complicating 5-7% pregnancies. It forms one of the deadly triad, along with hemorrhage and infection, which contributes greatly to maternal as well as fetal morbidity and mortality. The disease is characterized by the development of hypertension, to the extent of 140/90 mmHg or more with proteinuria 300 mg/24 hours after the 20th week of gestation in a previously normotensive and non-proteinuric pregnant mother. Preeclampsia causes multiorgan damage with generalized water retention, pulmonary edema, renal involvement, etc. This disease may deteriorate with severe signs such as eclampsia and HELLP syndrome.
Once the disease is evident clinically, it can be cured only by delivery. Preeclamptic women have higher predisposal to develop cardiovascular disease or hypertension in later life, thus contributing to another source of significant morbidity. The ability to predict women likely to develop preeclampsia before the onset of the disease is undoubtedly an important aim, as this would not only identify those women who require closer antenatal surveillance, also to counsel the women for lifestyle changes so as not to develop later onset cardiovascular, hypertension and related disorders. The genetic predisposition of the disease has been established by epidemiological and family based studies in different populations and has been elucidated that preeclampsia is multi-factorial with familial tendency and influenced by race, ethnicity and environment.
This study was conducted in the Department of Obstetrics & Gynecology, UCMS & GTB Hospital, Shahdara, Delhi-110095 and The Centre of Genomic Applications, Okhla, New Delhi.
Blood samples were collected in either ACD or EDTA vacutainers and were stored at 4°C. The samples were kept anonymous and a unique ID was assigned to each sample before processing. A database was prepared which incorporated the details of clinical history and laboratory parameters. Genomic DNA was isolated from approximately 625 whole blood samples using, Qiagen Flexigene Maxi kit.
In Phase I, 150 maternal and fetal samples were collected to study the effect of inter-individual variation with respect to polymorphisms in the genes of RAAS pathway to the contribution of preeclampsia/eclampsia. The case-control groups were well matched for age, BMI and socio-economic status. The subjects belonged to North India and can be grouped in the Indo European genetic cluster. Other than high blood pressure and proteinuria, headache, found in 55% of the cases of severe preeclampsia, was the commonest symptom. Serum creatine and liver
enzymes (AST and ALT) levels were found to be higher in the cases as compared to control mothers. There were 5 cases of partial HELLP (platelet count <1,00,000 and increased AST >72IU/I) in severe preeclampsia category. Fifty percent of severe preeclampsia mothers underwent caesarean sections because of non-reassuring fetal status. Number of low birth weight babies were more in preeclampsia than in controls (p=0.004).Also the incidence of NICU admission of fetus was more in Preeclampsia (16%) (p=0.031).Analysis of gene variations have found a polymorphism of VEGFA (rs25648, synonymous codon, upstream variant 2KB, 5’ UTR variant) to have a risk allele (C allele) which is prevalent in Preeclampsia mothers as compared to control mothers. There is a prevalence of the AGT (rs7079) polymorphism among women with severe preeclampsia as compared to mild preeclampsia (Chi-square= 5.771, p=0.016) with OR=0.238 (CI is 0.070 to 0.807). A polymorphism of RENIN (rs11240688) polymorphism among fetus of preeclampsia woman as compared to fetus from control woman (Chi-square= 9.673, p=0.002) with OR=3.042 (CI is 1.476 to 6.267) implying that the A allele is the disease causing allele for preeclampsia. Polymorphism of AGT gene (rs11122576) was found to be marginally significant among fetus of preeclampsia mothers with p=0.044, OR at 95% confidence interval is 2.292 (1.007-5.216). There is marked allelic difference in the prevalence of the rs8066276 (ACE;C/T, p=0.006), rs4354 (ACE;T/C, p=0.013), rs5443 (GNB3;T/C, p=0.03), rs928554 (ESR2;A/G, p=0.036), rs11571081 (REN;G/A, p=0.036) and rs7204560 (SCNNN1B;T/C, p=0.046) polymorphisms among the fetuses of preeclampsia as compared to fetus of mild preeclampsia women.
The result of the present study showed interesting cross talk between fetal and maternal genotypes especially when a plausible defect in maternal angiogenesis is present. The distribution of allele frequencies of VEGFA and Renin was statistically different in preeclampsia mothers and fetuses respectively, when compared to the normotensive mothers and their fetuses. The interplay of RAAS and angiogenic pathway along with other interacting partners have to be explored to provide interesting leads to this multi-systemic disease which has a burden on maternal and fetal mortality and morbidity. Further studies are needed to validate these findings with large maternal-fetal dyads. This gains significance with the occurrence of shared etiology of preeclampsia and cardiovascular and cerebrovascular events and its impact on public health well beyond the affected pregnancies.
In Phase II, a total of 100 pregnant women with preeclampsia/eclampsia and 210 normotensive women as controls were recruited for analysis of genetic polymorphisms of thrombophilia pathway.
There was significant difference (p<0.001) in the mean gestational age at birth in preeclampsia (36.097 ± 4.71 weeks) as compared to controls (38.69 ± 1.22 weeks). Significantly higher values of serum creatinine, AST, ALT and S. bilirubin were noted in preeclampsia. In this group also 50% of women with preeclampsia was delivered by caesarean section and 49.48% via vaginal route while all normotensive women delivered by vaginal route (p<0.001).Birth weights of newborns were significantly lower in the preeclampsia. All laboratory parameters were comparable in mild and severe preeclampsia cases except ALT (p<0.05) which was significantly high in the severe preeclampsia. Lower birth weight of newborns were also markedly more in severe preeclampsia group compared to mild preeclampsia group, p<0.01.
We have analyzed 97 SNPs from five genes of thrombophilia. SNP based analysis showed promising association of F5 and MTHFR with preeclampsia. In preeclampsia-control analysis, rs22998908; A>G of F5 gene showed significant association of G allele with preeclampsia (p=0.04). Analysis of subgroups of disease showed clear association of different genetic polymorphisms and allelic association with the severity of the disease. In eclampsia, a very strong association of four SNPs of F5 gene, rs10800456;A>G (p=0.018), rs1557572;A>C (p=0.28), rs4656687;T>C (p=0.033) and rs2298909;A>T (p=0.036) was observed.
This study suggests that molecular markers associated with thrombophilia may play a direct role in the severity of the disease as in the manifestation of severe preeclampsia and eclampsia. Further molecular studies are needed to elucidate the complex relationship between functional polymorphisms and preeclampsia. It has been shown that pooling mild preeclampsia, severe preeclampsia and eclampsia cases is likely to result in inconsistent association of thrombophilia SNPs. Hence for genetic analysis, it is very important to phenotype the cases and also the sub-phenotypes of the disease should be carried out. From our association analysis we found two genes (F5 and MTHFR) to be significantly important. Role of thrombophilia genes in preeclampsia has to be explored deeply as thrombophilia is an emerging concern and has known to be associated with severe pregnancy complications.
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