Genetic Association of ERAP1 and ERAP2 With Eclampsia and Preeclampsia in Northeastern Brazilian Women

Genetic Association of ERAP1 and ERAP2 With Eclampsia and Preeclampsia in Northeastern Brazilian Women | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genetic Association of ERAP1 and ERAP2 With Eclampsia and Preeclampsia in Northeastern Brazilian Women LEONARDO CAPISTRANO FERREIRA, CARLOS EDUARDO MAIA GOMES, PRIYA DUGGAL, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-235628/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Mar, 2021 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract The clinical spectrum of hypertensive disorders of pregnancy (HDP) is determined by the interplay between environmental and genetic factors, most of which remains unknown. ERAP1, ERAP2 and LNPEP genes code for multifunctional aminopeptidases involved with antigen processing and degradation of small peptides such as angiotensin II (Ang II), vasopressin and oxytocin. We aimed to test for associations between genetic variants in aminopeptidases and HDP. A total of 1282 pregnant women (normotensive controls, n=693; preeclampsia, n=342; chronic hypertension with superimposed preeclampsia, n=61; eclampsia, n=74; and HELLP syndrome, n=112) were genotyped for variants in LNPEP (rs27300, rs38034, rs2303138), ERAP1 (rs27044, rs30187) and ERAP2 (rs2549796 rs2927609 rs11135484). We also evaluated the effect of ERAP1 rs30187 on plasma Ang II levels in an additional cohort of 65 pregnant women. The genotype C/C, in ERAP1 rs30187 variant (c.1583T>C, p.Lys528Arg), was associated with increased risk of eclampsia (OR=1.85, p=0.019) whereas ERAP2 haplotype rs2549796(C)-rs2927609(C)-rs11135484(G) was associated with preeclampsia (OR=1.96, corrected p-value=0.01). Ang II plasma levels did not differ across rs30187 genotypic groups (p=0.895). In conclusion, ERAP1 gene is associated with eclampsia whereas ERAP2 is associated with preeclampsia, although the mechanism by which genetic variants in ERAPs influence the risk of preeclampsia and eclampsia remain to be elucidated. Plant Molecular Biology and Genetics Medical Genetics Cardiac & Cardiovascular Systems Aminopeptidases preeclampsia ERAP genetic association Figures Figure 1 Figure 2 Figure 3 Introduction Hypertensive disorders of pregnancy (HDP) account for 14% of all maternal deaths 1 and contribute to increase the cardiovascular risk in both mothers 2 and offspring 3 . As other complex diseases, HDP have a broad clinical spectrum ranging from mild hypertension without proteinuria to severe proteinuria, and eventual seizures ( i.e. eclampsia), or with hemolysis elevated liver enzymes and low platelet liver disease and severe inflammation ( i.e. HELLP syndrome). Risk factors for preeclampsia, such as pre-gestational body mass index, nulliparity, change in partners, and advanced maternal age have been reported for different populations 4 . Regarding the genetics of preeclampsia, genes INHBP 5 , FLT1 6 and PLEKHG1 7 were identified from genome wide association studies, however, the genetic architecture underlying the disease mechanism remains largely unknown 8 . Endoplasmic reticulum aminopeptidases -1 (ERAP1), -2 (ERAP2) and leucyl/cystinyl aminopeptidase (LNPEP), also known as A-LAP, L-RAP and P-LAP, respectively, are multifunctional enzymes belonging to the M1 family of aminopeptidases 9 . These aminopeptidases act in concert to trim peptides to be presented by the major histocompatibility complex (MHC) class I molecules 10 and, in addition, they cleave a variety of bioactive peptides, including angiotensins, bradykinin, kallidin and oxytocin 11 . Not surprisingly, these enzymes are involved in several biological processes such as immune and inflammatory responses, blood pressure regulation and pregnancy maintenance 12,13 . There is also increasing evidence that LNPEP is involved with preterm delivery due to its oxytocinase activity 14 . Johnson and colleagues identified a quantitative trait locus (QTL) for preeclampsia on chromosome 5q, in a region harboring the aminopeptidases genes 15 and, subsequently, confirmed the genetic association between ERAP2 and preeclampsia 16 . The missense genetic variants in ERAP1 , rs27044 and rs30187, have consistently been reported as associated with ankylosing spondylitis, psoriasis, multiple sclerosis and Crohn’s disease 17 . Lastly, maternal LNPEP variants were reported as associated with increased risk of preterm birth 18 . Thus, the present study aimed to evaluate genetic variants in ERAP1 , ERAP2 and LNPEP for association with the full clinical spectrum of HDP. For the first time, eclampsia and HELLP phenotypes, which are the most severe and rare phenotypes, were tested for these genes. Methods Population and study design Our study population was recruited from Maternidade Escola Januário Cicco, a tertiary center for women’s health, located in Natal, Rio Grande do Norte state, Brazil. A total of 1693 women were recruited from 2002 to 2010, as part of a broader study aiming to investigate clinical, epidemiological and genetic aspects of hypertensive disorders of pregnancy. Clinical data as well as blood samples were collected at the time of enrollment. For the current study, we retrospectively selected 1282 women based on their pregnancy outcome: 693 normotensive women (control), 342 preeclampsia (PE), 61 superimposed preeclampsia (PEsuper), 74 eclampsia, and 112 HELLP syndrome cases. All Methods were performed in accordance with the Declaration of Helsink and followed the Brazilian ethical standards of scientific research. The research protocol was reviewed and approved by the Federal University of Rio Grande do Norte (CEP-UFRN 88) and Brazilian National Ethical Committee (CONEP 5059). All research participants or their legal guardian provided informed consent. Phenotype definition The diagnostic criteria followed the recommendations from the American College of Obstetrician and Gynecologists 19 . Preeclampsia was defined as the new onset hypertension (SBP ≥ 140 mmHg or DBP ≥ 90 mmHg) and proteinuria (≥ +1 on dipstick) after 20 weeks of gestation. Superimposed preeclampsia occurred when the woman had a previous diagnosis of chronic hypertension and developed proteinuria after 20 weeks of gestation. Eclampsia was defined by the presence of seizure, while HELLP syndrome diagnosis was based on Mississippi Class III system (AST > 40 IU/L and LDH > 600 IU/L and platelets < 150,000/μL) 20 . Controls were healthy pregnant women with no history of hypertension. Women with multiple pregnancies, diabetes or other chronic diseases were excluded from study. Genetic variants The variants in ERAP2 (rs2549796, rs2927609, rs11135484) and LNPEP (rs27300, rs38034, rs2303138) were all tag-variants, identified through a pairwise selection strategy with an r 2 threshold ≥ 0.8 in Haploview 4.2 21 using the HapMap CEU population genotype data (HapMap Rel 27 phase II+III). Variants rs30187 and rs27044, in ERAP1 , were chosen based on their effect on protein function 22,23 as well as their implication in other diseases 17 . Genotyping DNA extraction was carried out as previously described 24 . Samples were genotyped by SNaPshot® technique and the capillary electrophoresis performed on ABI PRISM® 3100 Avant Genetic Analyzer (Applied Biosystems). Technique standardization was carried out according to Lins and colleagues 25 . GeneMapper® software (Applied Biosystems, CA, USA) was used for the genotype calling. Population stratification assessment To avoid confounding by ethnicity we used a panel with 27 ancestry informative markers (AIMs) particularly designed for the Brazilian population 26 . A sub-sample of 756 women randomly selected was used to assess the genetic ancestry of our study population (n=1282) using principal component analysis in SNPRelate R package 27 . Samples from The 1000 Genomes Project 28 of European (IBS), African (ASW, MSL, YRI) and American (CLM) origins were used as reference populations. Functional validation Variant effects on mRNA and protein levels were assessed from GTEx (dbGaP Accession phs000424.v8.p2) 29 and single nucleotide polymorphisms annotator (SNiPA) 30 databases. Aiming to functionally validate the ERAP1 genetic finding, we recruited an additional cohort of 65 pregnant women, including 29 normotensive controls and 36 severe preeclampsia cases, that had their Ang II plasma concentration measured by ELISA commercial kit (MyBioSource, San Diego, CA, USA, Cat.Num. MBS453098). Briefly, blood samples were systematically collected between 7 and 9 am in EDTA tubes and immediately centrifuged. The obtained plasma was stored at -80 °C until assay. Statistical analysis Clinical and demographic data were analyzed through chi-squared and t-test for categorical and quantitative variables, respectively. With regard to the genetic data, allele frequencies were compared by Fisher exact test, whereas genotype and haplotype association tests were performed through logistic regression models including maternal age and parity (primigesta vs others) as covariates. Haplotype frequencies were estimated by Expectation-Maximization algorithm with a minor haplotype frequency threshold of 0.03. The p-values were corrected for family-wise error rate by permutation procedures (10,000x) implemented in PLINK 31 . All analyses were performed by comparing each case phenotype ( i.e. PE, PEsuper, eclampsia and HELLP) against the normotensive control group. Results Demographics and clinical characteristics Table 1 summarizes the main clinical characteristics and demographics for our study population. Women with eclampsia and HELLP were the youngest and oldest, respectively, when compared to the control group, whereas the proportion of primiparas was higher in the preeclampsia and eclampsia groups. Women with HELLP syndrome delivered their babies earlier in pregnancy (mean gestational age=34.3 weeks), followed by eclampsia (mean gestational age=36.2 weeks) and preeclampsia (37.2 weeks) groups. Overall, the frequency of family members affected by chronic hypertension was higher in the case groups when compared to control group, suggesting shared genetic components between essential hypertension and hypertensive disorders of pregnancy (Table 1). Of note, the prevalence of chronic hypertension and eclampsia in family members was much higher in superimposed preeclampsia (PEsuper) and eclampsia groups, respectively. Table 1. Demographics and clinical characteristics. Characteristics Control PE PEsuper Eclampsia HELLP Sample size, n 693 342 61 74 112 Maternal age, y, mean (±SD) 24.4 (±6.2) 25.2 (±6.8) 31.1 (±6.9) a 20.5 (±6.1) a 27.0 (±6.7) a SBP, mmHg, mean (±SD) 117 (±12) 156 (±19) a 166 (±28) a 159 (±21) a 154 (±21) a DBP, mmHg, mean (±SD) 75 (±9) 105 (±13) a 108 (±14) a 108 (±16) a 101 (±13) a Proteinuria, n (%) Negative 693 (100) 0 (0.0) 0 (0.0) 2 (2.7) 7 (6.3) 1+ - 104 (30.4) 28 (45.9) 11 (14.9) 13 (11.6) ≥ 2+ - 238 (69.6) 16 (26.2) 43 (58.1) 85 (75.8) Missing - 0 (0.0) 17 (27.9) 18 (24.3) 7 (6.3) Primigestas, n (%) 300 (43.3) 180 (52.8) a 14 (23.3) a 54 (73.0) a 46 (41.4) Gestational age at delivery, w, mean (±SD) 38.8 (±3.1) 37.2 (±3.0) a 35.8 (±3.5) a 36.2 (±3.6) a 34.3 (±4.0) a Number of antenatal care visits, mean (±SD) 5.9 (±2.8) 6.2 (±2.3) 6.1 (±2.9) 4.5 (±2.5) a 5.3 (±2.3) a Family history of Chronic hypertension b 238 (35.5) 178 (56.9) a 46 (80.7) a 32 (46.4) 55 (49.6) a Preeclampsia c 22 (3.8) 41 (15.9) a 5 (10.9) a 12 (21.0) a 8 (8.3) a Eclampsia c 7 (1.2) 11 (4.4) a 3 (6.5) a 5 (8.8) a 2 (2.1) a P<0.05 for comparison with the control group. b If at least one first degree relative has the disease. c If the mother had had the disease. SBP/DBP: systolic blood pressure/diastolic blood pressure. PEsuper: chronic hypertension with superimposed preeclampsia. Genetic analysis All genetic variants achieved standard quality control thresholds ( i.e. genotyping error rates 0.01, and p-value > 0.05 for Hardy-Weinberg equilibrium test). In addition, there was no evidence of population stratification, since cases and controls were equally distributed across the reference ethnical groups (Supplementary Figure 1). Figure 1 shows the linkage disequilibrium (LD) pattern across the genomic region encompassing the studied variants. ERAP1 variants were moderately correlated (r 2 =0.65) while ERAP2 and LNPEP variants seemed to belong to the same haploblock. In order to analyze the combined effect of variants on disease risk, we performed haplotype-based tests (Table 2). As result, ERAP2 haplotype rs2549796 - rs2927609 - rs11135484 was associated with preeclampsia (corrected p=0.0109). There was no haplotype associated with the remaining case groups (corrected p>0.05). Table 2. Haplotype tests of association showing ERAP2 C-C-G haplotype associated with preeclampsia. Haplotypes Haplotype frequency Control PE PEsuper Eclampsia HELLP ERAP1 T-G 0.354 0.358 0.328 0.341 0.323 T-C 0.105 0.097 0.121 0.051 0.074 C-C 0.541 0.545 0.552 0.609 0.602 ERAP2 C-T-A 0.362 0.361 0.356 0.301 0.352 C-C-A 0.064 0.066 0.060 0.065 0.079 C-C-G 0.045 0.076 a 0.050 0.045 0.065 T-C-G 0.488 0.482 0.515 0.523 0.452 LNPEP T-C-A 0.123 0.130 0.105 0.132 0.135 C-T-G 0.398 0.376 0.375 0.345 0.397 T-C-G 0.479 0.494 0.520 0.521 0.468 ERAP1 : rs30187 - rs27044 ERAP2 : rs2549796 - rs2927609 - rs11135484 LNPEP : rs27300 - rs38034 – rs2303138 a PE vs Control (Uncorrected p=0.0013; P-value corrected for family-wise error rates by running 10,000 permutations: p=0.0109). There was no difference regarding allele frequencies between control and case groups (Supplementary Table 1), although the genotype distribution for ERAP1 variants in eclampsia group seemed to differ, when compared to controls (Table 3). The frequency of genotype C/C (rs30187) was notably higher in eclampsia group (40.9 %), what would be consistent with a recessive genetic model. Table 3. Genotype distribution for ERAP1 variants across phenotypic groups. SNP Genotypes Genotype distribution, n (%) Control PE PEsuper Eclampsia a HELLP rs30187 T/T 139 (20.5) 68 (20.5) 12 (19.7) 16 (22.5) 16 (14.6) C/T 343 (50.7) 167 (50.5) 32 (52.5) 26 (36.6) 56 (50.9) C/C 195 (28.8) 96 (29.0) 17 (27.8) 29 (40.9) 38 (34.6) rs27044 G/G 86 (12.7) 48 (14.5) 9 (15.5) 14 (19.2) 12 (10.9) G/C 307 (45.4) 141 (42.6) 20 (34.5) 23 (31.5) 48 (43.6) C/C 283 (41.9) 142 (42.9) 29 (50.0) 36 (49.3) 50 (45.5) a Chi-squared test of genotypic association for Control vs Eclampsia: p=0.055 for rs30187 and p=0.079 for rs27044. Of note, rs30187 C allele codes for Arg528 (instead of Lys528), resulting in an enzyme type characterized by lower peptidase activity against Ang II 32 . Therefore, we defined a recessive genetic model with C/C as the risk genotype for eclampsia. The model was implemented through logistic regression with genotype (T/T+T/C vs C/C) as the main explanatory variable and maternal age, and parity as covariates. As result, women homozygotes for rs30187 C/C risk genotype were more likely to develop eclampsia (OR=1.85, p=0.019) (Table 4). Table 4. Genetic effect of rs30187 on eclampsia risk under recessive genetic model. Genotypic group Control a Eclampsia a Eclampsia risk b n (%) n (%) OR (95% CI) p-value T/T + C/T 482 (71.2) 40 (58.0) 1.0 0.019 C/C 195 (28.8) 29 (42.0) 1.85 (1.11 – 3.11) a Chisquared test of association for genotype distribution between Control and Eclampsia (p=0.022) b Odds ratio (OR) and confidence interval (CI) estimated by logistic regression model adjusted for maternal age and parity. Functional validation According to GTEx and SNiPa data, rs30187 has a significant effect on both ERAP1 mRNA and protein levels in blood with C/C genotype associated with the lowest expression levels (Figure 2). Given the qualitative and quantitative effect of rs30187 on ERAP1 expression, we hypothesized that women homozygotes for C/C genotype have higher circulating levels of Ang II. In order to test that, plasma Ang II concentrations were determined in an additional cohort of women with severe preeclampsia (n=36) and normotensive pregnant controls (n=29). We rejected this hypothesis (p=0.895) since no difference was detected between genotypic groups (Figure 3). The intra-group analysis ( i.e. cases-only and controls-only) did not detect any difference in Ang II levels across genotypic groups as well (data not shown). Discussion Endoplasmic reticulum aminopeptidases (ERAPs), as well as leucyl/cystinyl aminopeptidase (LNPEP), play roles in antigen processing, inflammatory response, blood pressure regulation and angiogenesis, all processes potentially implicated in preeclampsia pathophysiology. The present study confirmed a genetic association between ERAP2 and preeclampsia, and, for the first time, reported an association between ERAP1 and eclampsia. Thus, our findings may help to disentangle the intricate association between the correlated phenotypes preeclampisa/eclampisa and the functionally and physically connected genes ERAP1 / ERAP2 . Johnson et al., tested ERAP1 , ERAP2 and LNPEP for associations with preeclampsia in Australian and Norwegian populations, and identified ERAP2 variants (rs2549782, rs2548538, rs2287988 and rs17408150) associated with preeclampsia 16 . In the same study, rs27044 and rs30187 ( ERAP1 ) were not associated with disease although borderline association with preeclampsia was found for markers rs3734016 and rs34750, both within ERAP1 gene. It is important to highlight that the Australian cohort contained both preeclampsia and eclampsia cases, but they were analyzed as a unique group 16 . Out study treated the two phenotypes as different entities. A recent study with 148 preeclamptic women and 133 controls from Iran investigated four variants in ERAP1 (including rs30187) and three variants in ERAP2 . None of the variants were associated with disease, but a haplotype encompassing the seven variants was associated with preeclampsia 33 . In another Iranian independent study, ERAP2 variants (rs2549782 and rs17408150) were also associated with preeclampsia 34 . Interestingly, the fetal minor allele for variant rs2549782 ( ERAP2 ) was associated with preeclampsia in African American population 35 . Besides the genetic association findings, Founds et al showed ERAP2 was differentially expressed in the first trimester placentas of women who later developed preeclampsia 36 . The ERAP1 rs30187 C allele codes an enzyme with Arg528 that causes a reduction on peptidase activity for angiotensin II degradation by approximately 60%, when compared to the enzyme with Lys528, coded by the T allele 22,32 . We failed to confirm the hypothesis that women carrying two copies of the C allele have increased levels of Ang II in their blood, which in turn could cause blood pressure elevation and seizure. However, we cannot rule out a potential effect of rs30187 on local RAS ( e.g. brain and kidney). While Arg528 variant is associated with hypertensive disease 37 , the Lys528 variant is strongly associated with susceptibility to ankylosing spondylitis 38 and other autoimmune diseases 17 . Since both Arg528 and Lys528 alleles are associated with bad outcomes, it is likely that ERAP genes would be subject to balancing selection, a process where heterozygous individuals are more adaptive than either of the two types of homozygous 39 . In addition, these genes play key role in the maintenance of immunotolerance to self-peptides as well as protecting against infectious agents, such as HIV 40 . The small sample size for some of our case groups represents an important limitation for the present study, even though we should consider that eclampsia and HELLP are extremely rare phenotypes. On the other hand, the marker associated with eclampsia (rs30187) has been well characterized as affecting the protein function, what strengthens the biological plausibility for the genetic association reported here. Furthermore, we accounted for important confounders such as age, parity and ethnicity. The mechanism by which endoplasmic reticulum aminopeptidases (ERAPs) influence the risk of preeclampsia/eclampsia remains to be elucidated. In addition to Ang II degradation and peptide trimming for antigen presentation via MHC-1, ERAPs also play role in inflammatory response by shedding cytokine receptors ( e.g. IL-6R, IL-1R2 and TNFR) 41–43 . Lastly, ERAP1 plays crucial role in VEGF-stimulated proliferation and migration of endothelial cells, as well as angiogenesis, via the binding and modification of PDk1 44 . All the above-mentioned mechanisms are potentially involved in the causal pathway of preeclampsia/eclampsia. Conclusions In conclusion, we identified genetic variants in ERAP1 and ERAP2 associated with eclampsia and preeclampsia, respectively. Sequencing and functional studies are needed in order to elucidate the mechanisms underlying these genetic associations. Declarations Data availability The genetic data used in the present study is available from the corresponding author on reasonable request. Acknowledgement We thank the staff of Maternidade Escola Januário Cicco for their help in ascertaining patients’ clinical classification and sample collection. We do appreciate the collaboration of SNiPA team (Karsten Suhre and Arnold Matthias) that kindly generated rs30187 pQTL plot (Figure 2B) and Victor Lima (on behalf of Laboratório de Análises Clínicas DNA Center) to perform capillary electrophoresis needed to genotype the additional cohort (n=65) of pregnant women. Authors’ contributions LCF worked on the study design, carried out the genotyping and the genetic analysis, and the manuscript writing. CEMG contributed with the study design and genotyping. PD contributed with the genetic analysis and revising the manuscript. IPH and PRPN carried out sample processing and Ang II measurement. ASL contributed with the study participant recruitment and phenotype ascertainment. SMBJ contributed with the study design, recruitment of subjects and manuscript revision. Competing interests There is no conflict of interest from any of the authors. References Say, L. et al. Global causes of maternal death: a WHO systematic analysis. Lancet Glob. Health 2 , e323–e333 (2014). Benschop, L., Duvekot, J. J. & Lennep, J. E. R. van. Future risk of cardiovascular disease risk factors and events in women after a hypertensive disorder of pregnancy. Heart 105 , 1273–1278 (2019). Andraweera, P. H. & Lassi, Z. S. 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Identification of 33 polymorphisms in the adipocyte-derived leucine aminopeptidase (ALAP) gene and possible association with hypertension. Hum. Mutat. 19 , 251–257 (2002). Evans, D. M. et al. Interaction between ERAP1 and HLA-B27 in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27 in disease susceptibility. Nat. Genet. 43 , 761–767 (2011). Andrés, A. M. et al. Balancing Selection Maintains a Form of ERAP2 that Undergoes Nonsense-Mediated Decay and Affects Antigen Presentation. PLoS Genet. 6 , e1001157 (2010). Cagliani, R. et al. Genetic diversity at endoplasmic reticulum aminopeptidases is maintained by balancing selection and is associated with natural resistance to HIV-1 infection. Hum. Mol. Genet. 19 , 4705–4714 (2010). Cui, X. An Aminopeptidase, ARTS-1, Is Required for Interleukin-6 Receptor Shedding. J. Biol. Chem. 278 , 28677–28685 (2003). Cui, X., Rouhani, F. N., Hawari, F. & Levine, S. J. 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Supplementary Files SupplementaryInformationFerreiraLC.pdf Cite Share Download PDF Status: Published Journal Publication published 24 Mar, 2021 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 24 Feb, 2021 Reviews received at journal 22 Feb, 2021 Reviewers agreed at journal 18 Feb, 2021 Reviewers invited by journal 17 Feb, 2021 Editor assigned by journal 17 Feb, 2021 Editor invited by journal 16 Feb, 2021 Submission checks completed at journal 16 Feb, 2021 First submitted to journal 11 Feb, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-235628","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":12078675,"identity":"a926cc3a-97bc-4249-9893-60ab62cf5f55","order_by":0,"name":"LEONARDO CAPISTRANO FERREIRA","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYBAC9gYQacDAwMfAfIAZyOQHYja8WngOQLWwMbAlgLRINhCnBWwyjwGRWth7n334UHBPno39zMfPBQyHJcxnN7A9rsCnhee48cwZBsWGbTy5m6VnALXI3DnAbngGjxZ7iTRmZh6DBMY2Cd5tzDwMh+skJBLYQK7DbYv8M2bmPwYJ9m0SPM9AWiQIa5FgY2ZmMEhIBGphI1ILTxozY49BQnIbT5qx9AyDdKCWxHZDvFrYjzEz/PiTYNvPfvjh54IKa6CW5GMP8WlBAwYggpEEDaNgFIyCUTAKsAIAuPQ7SNkHMt0AAAAASUVORK5CYII=","orcid":"","institution":"Federal University of Rio Grande do Norte","correspondingAuthor":true,"prefix":"","firstName":"LEONARDO","middleName":"CAPISTRANO","lastName":"FERREIRA","suffix":""},{"id":12078676,"identity":"5aa9d444-0702-4046-ab95-b8c8e622216b","order_by":1,"name":"CARLOS EDUARDO MAIA GOMES","email":"","orcid":"","institution":"Federal University of Rio Grande do Norte","correspondingAuthor":false,"prefix":"","firstName":"CARLOS","middleName":"EDUARDO MAIA","lastName":"GOMES","suffix":""},{"id":12078677,"identity":"bec9af88-4804-4f90-8fd8-098bda0664e8","order_by":2,"name":"PRIYA DUGGAL","email":"","orcid":"","institution":"Johns Hopkins University","correspondingAuthor":false,"prefix":"","firstName":"PRIYA","middleName":"","lastName":"DUGGAL","suffix":""},{"id":12078678,"identity":"c9b8a504-e671-40d8-b9b8-9592e7b9f4b2","order_by":3,"name":"INGRID DE PAULA HOLANDA","email":"","orcid":"","institution":"Federal University of Rio Grande do Norte","correspondingAuthor":false,"prefix":"","firstName":"INGRID","middleName":"DE PAULA","lastName":"HOLANDA","suffix":""},{"id":12078679,"identity":"6b5e7b37-404b-4080-97fe-7cef68721457","order_by":4,"name":"AMANDA SAMARA DE LIMA","email":"","orcid":"","institution":"Federal University of Rio Grande do Norte","correspondingAuthor":false,"prefix":"","firstName":"AMANDA","middleName":"SAMARA","lastName":"DE LIMA","suffix":""},{"id":12078680,"identity":"7077c203-3e93-43dc-9cbe-51b439132a04","order_by":5,"name":"PAULO RICARDO PORFÍRIO DO NASCIMENTO","email":"","orcid":"","institution":"Federal University of Rio Grande do Norte","correspondingAuthor":false,"prefix":"","firstName":"PAULO","middleName":"RICARDO PORFÍRIO DO","lastName":"NASCIMENTO","suffix":""},{"id":12078681,"identity":"f9d033da-7735-4cd2-813a-6861e02f32b0","order_by":6,"name":"SELMA MARIA BEZERRA JERONIMO","email":"","orcid":"","institution":"Federal University of Rio Grande do Norte","correspondingAuthor":false,"prefix":"","firstName":"SELMA","middleName":"MARIA BEZERRA","lastName":"JERONIMO","suffix":""}],"badges":[],"createdAt":"2021-02-12 02:59:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-235628/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-235628/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-021-86240-z","type":"published","date":"2021-03-24T19:05:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":6373274,"identity":"54767205-2ae8-46aa-a27e-003952d29f36","added_by":"auto","created_at":"2021-02-25 22:45:13","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":50539,"visible":true,"origin":"","legend":"Linkage disequilibrium (LD) pattern among the studied markers. In the LD plot, the inside- square number represents the correlation coefficient value (r2). The genomic organization and LD pattern suggest ERAP2 and LNPEP markers as belonging to the same haploblock.","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-235628/v1/f4f1242eb4a009fe8364f534.jpg"},{"id":6373275,"identity":"1de7fffc-f76e-4a21-99f6-6aa5bce66dbf","added_by":"auto","created_at":"2021-02-25 22:45:13","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":44538,"visible":true,"origin":"","legend":"Effect of ERAP1 rs30187 variant on mRNA (Data source: GTEx) and protein levels (Data source: SNiPA) in blood. ERAP1 rs30187 CC genotype is associated with the lowest expression level. Each T allele additivelly increases ERAP1 mRNA in whole blood cells (a) and Erap1 protein concentration in plasma (b). NES: Normalized effect size; Norm. Expression: normalized expression; invnorm data: inverse-normal scaled data. \nFigure 2b was kindly provided by Karsten Suhre from SNiPA team.\n","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-235628/v1/6a599d646b44463a53e5b838.jpg"},{"id":6372918,"identity":"b76fac77-1ddc-49ba-b0e1-e39b14faac80","added_by":"auto","created_at":"2021-02-25 22:42:13","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":21949,"visible":true,"origin":"","legend":"Plasma Ang II levels across rs30187 genotypic groups. Analysis of plasma Ang II concentration in pregnant women (n=65). No difference was detected among genotypic groups (ANOVA, p=0.895).","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-235628/v1/d6a6337a8373528d28806310.jpg"},{"id":15670944,"identity":"8e6fba2b-6d5f-4ab7-88e8-3faedc52b054","added_by":"auto","created_at":"2021-11-18 14:03:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":484346,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-235628/v1/4cdb7d84-f2bf-43af-bbd3-1762ba260ecb.pdf"},{"id":6372921,"identity":"b8eb6f51-93da-4ca4-ac69-1da7b050151f","added_by":"auto","created_at":"2021-02-25 22:42:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":204421,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationFerreiraLC.pdf","url":"https://assets-eu.researchsquare.com/files/rs-235628/v1/6a0a6138fb03dc86181ba6b8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eGenetic Association of \u003cem\u003eERAP1 \u003c/em\u003eand \u003cem\u003eERAP2 \u003c/em\u003eWith Eclampsia and Preeclampsia in Northeastern Brazilian Women\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHypertensive disorders of pregnancy (HDP) account for 14% of all maternal deaths \u003csup\u003e1\u003c/sup\u003e and contribute to increase the cardiovascular risk in both mothers \u003csup\u003e2\u003c/sup\u003e and offspring \u003csup\u003e3\u003c/sup\u003e. As other complex diseases, HDP have a broad clinical spectrum ranging from mild hypertension without proteinuria to severe proteinuria, and eventual seizures (\u003cem\u003ei.e.\u003c/em\u003e eclampsia), or with hemolysis elevated liver enzymes and low platelet liver disease and severe inflammation (\u003cem\u003ei.e.\u003c/em\u003e HELLP syndrome). Risk factors for preeclampsia, such as pre-gestational body mass index, nulliparity, change in partners, and advanced maternal age have been reported for different populations \u003csup\u003e4\u003c/sup\u003e. Regarding the genetics of preeclampsia, genes \u003cem\u003eINHBP \u003c/em\u003e\u003csup\u003e5\u003c/sup\u003e, \u003cem\u003eFLT1\u003c/em\u003e\u003csup\u003e6\u003c/sup\u003e and \u003cem\u003ePLEKHG1 \u003c/em\u003e\u003csup\u003e7\u003c/sup\u003e were identified from genome wide association studies, however, the genetic architecture underlying the disease mechanism remains largely unknown \u003csup\u003e8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eEndoplasmic reticulum aminopeptidases -1 (ERAP1), -2 (ERAP2) and leucyl/cystinyl aminopeptidase\u0026nbsp;(LNPEP), also known as A-LAP, L-RAP and P-LAP, respectively, are multifunctional enzymes belonging to the M1 family of aminopeptidases \u003csup\u003e9\u003c/sup\u003e. These aminopeptidases act in concert to trim peptides to be presented by the major histocompatibility complex (MHC) class I molecules \u003csup\u003e10\u003c/sup\u003e and, in addition, they cleave a variety of bioactive peptides, including angiotensins, bradykinin, kallidin and oxytocin \u003csup\u003e11\u003c/sup\u003e. Not surprisingly, these enzymes are involved in several biological processes such as immune and inflammatory responses, blood pressure regulation and pregnancy maintenance \u003csup\u003e12,13\u003c/sup\u003e. There is also increasing evidence that LNPEP is involved with preterm delivery due to its oxytocinase activity \u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eJohnson and colleagues identified a quantitative trait locus (QTL) for preeclampsia on chromosome 5q, in a region harboring the aminopeptidases genes \u003csup\u003e15\u003c/sup\u003e and, subsequently, confirmed the genetic association between \u003cem\u003eERAP2\u003c/em\u003e and preeclampsia \u003csup\u003e16\u003c/sup\u003e. The missense genetic variants in \u003cem\u003eERAP1\u003c/em\u003e, rs27044 and rs30187, have consistently been reported as associated with ankylosing spondylitis, psoriasis, multiple sclerosis and Crohn\u0026rsquo;s disease \u003csup\u003e17\u003c/sup\u003e. Lastly, maternal \u003cem\u003eLNPEP\u003c/em\u003e variants were reported as associated with increased risk of preterm birth \u003csup\u003e18\u003c/sup\u003e. Thus, the present study aimed to evaluate genetic variants in \u003cem\u003eERAP1\u003c/em\u003e, \u003cem\u003eERAP2\u003c/em\u003e and \u003cem\u003eLNPEP\u003c/em\u003e for association with the full clinical spectrum of HDP. For the first time, eclampsia and HELLP phenotypes, which are the most severe and rare phenotypes, were tested for these genes.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePopulation and study design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur study population was recruited from Maternidade Escola Janu\u0026aacute;rio Cicco, a tertiary center for women\u0026rsquo;s health, located in Natal, Rio Grande do Norte state, Brazil. A total of 1693 women were recruited from 2002 to 2010, as part of a broader study aiming to investigate clinical, epidemiological and genetic aspects of hypertensive disorders of pregnancy. Clinical data as well as blood samples were collected at the time of enrollment. For the current study, we retrospectively selected 1282 women based on their pregnancy outcome: 693 normotensive women (control), 342 preeclampsia (PE), 61 superimposed preeclampsia (PEsuper), 74 eclampsia, and 112 HELLP syndrome cases. All Methods were performed in accordance with the Declaration of Helsink and followed the Brazilian ethical standards of scientific research. The research protocol was reviewed and approved by the Federal University of Rio Grande do Norte (CEP-UFRN 88) and Brazilian National Ethical Committee (CONEP 5059). All research participants or their legal guardian provided informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhenotype definition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe diagnostic criteria followed the recommendations from the American College of Obstetrician and Gynecologists \u003csup\u003e19\u003c/sup\u003e. Preeclampsia was defined as the new onset hypertension (SBP \u0026ge; 140 mmHg or DBP \u0026ge; 90 mmHg) and proteinuria (\u0026ge; +1 on dipstick) after 20 weeks of gestation. Superimposed preeclampsia occurred when the woman had a previous diagnosis of chronic hypertension and developed proteinuria after 20 weeks of gestation. Eclampsia was defined by the presence of seizure, while HELLP syndrome diagnosis was based on Mississippi Class III system (AST \u0026gt; 40 IU/L and LDH \u0026gt; 600 IU/L and platelets \u0026lt; 150,000/\u0026mu;L) \u003csup\u003e20\u003c/sup\u003e. Controls were healthy pregnant women with no history of hypertension. Women with multiple pregnancies, diabetes or other chronic diseases were excluded from study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenetic variants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe variants in \u003cem\u003eERAP2\u003c/em\u003e (rs2549796, rs2927609, rs11135484) and \u003cem\u003eLNPEP\u003c/em\u003e (rs27300, rs38034, rs2303138) were all tag-variants, identified through a pairwise selection strategy with an r\u003csup\u003e2\u003c/sup\u003e threshold \u0026ge; 0.8 in Haploview 4.2 \u003csup\u003e21\u003c/sup\u003e using the HapMap CEU population genotype data (HapMap Rel 27 phase II+III). Variants rs30187 and rs27044, in \u003cem\u003eERAP1\u003c/em\u003e, were chosen based on their effect on protein function \u003csup\u003e22,23\u003c/sup\u003e as well as their implication in other diseases \u003csup\u003e17\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenotyping\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA extraction was carried out as previously described\u003csup\u003e24\u003c/sup\u003e. Samples were genotyped by SNaPshot\u0026reg; technique and the capillary electrophoresis performed on ABI PRISM\u0026reg; 3100 Avant Genetic Analyzer (Applied Biosystems). Technique standardization was carried out according to Lins and colleagues \u003csup\u003e25\u003c/sup\u003e. GeneMapper\u0026reg; software (Applied Biosystems, CA, USA) was used for the genotype calling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePopulation stratification assessment \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo avoid confounding by ethnicity we used a panel with 27 ancestry informative markers (AIMs) particularly designed for the Brazilian population \u003csup\u003e26\u003c/sup\u003e. A sub-sample of 756 women randomly selected was used to assess the genetic ancestry of our study population (n=1282) using principal component analysis in SNPRelate R package \u003csup\u003e27\u003c/sup\u003e. Samples from The 1000 Genomes Project \u003csup\u003e28\u003c/sup\u003e of European (IBS), African (ASW, MSL, YRI) and American (CLM) origins were used as reference populations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVariant effects on mRNA and protein levels were assessed from GTEx (dbGaP Accession phs000424.v8.p2) \u003csup\u003e29\u003c/sup\u003e and single nucleotide polymorphisms annotator (SNiPA) \u003csup\u003e30\u003c/sup\u003e databases. Aiming to functionally validate the \u003cem\u003eERAP1\u003c/em\u003e genetic finding, we recruited an additional cohort of 65 pregnant women, including 29 normotensive controls and 36 severe preeclampsia cases, that had their Ang II plasma concentration measured by ELISA commercial kit (MyBioSource, San Diego, CA, USA, Cat.Num. MBS453098). Briefly, blood samples were systematically collected between 7 and 9 am in EDTA tubes and immediately centrifuged. The obtained plasma was stored at -80 \u0026deg;C until assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical and demographic data were analyzed through chi-squared and t-test for categorical and quantitative variables, respectively. With regard to the genetic data, allele frequencies were compared by Fisher exact test, whereas genotype and haplotype association tests were performed through logistic regression models including maternal age and parity (primigesta \u003cem\u003evs\u003c/em\u003e others) as covariates. Haplotype frequencies were estimated by Expectation-Maximization algorithm with a minor haplotype frequency threshold of 0.03. The p-values were corrected for family-wise error rate by permutation procedures (10,000x) implemented in PLINK \u003csup\u003e31\u003c/sup\u003e. All analyses were performed by comparing each case phenotype (\u003cem\u003ei.e.\u003c/em\u003e PE, PEsuper, eclampsia and HELLP) against the normotensive control group.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDemographics and clinical characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 1 summarizes the main clinical characteristics and demographics for our study population. Women with eclampsia and HELLP were the youngest and oldest, respectively, when compared to the control group, whereas the proportion of primiparas was higher in the preeclampsia and eclampsia groups. Women with HELLP syndrome delivered their babies earlier in pregnancy (mean gestational age=34.3 weeks), followed by eclampsia (mean gestational age=36.2 weeks) and preeclampsia (37.2 weeks) groups. Overall, the frequency of family members affected by chronic hypertension was higher in the case groups when compared to control group, suggesting shared genetic components between essential hypertension and hypertensive disorders of pregnancy (Table 1). Of note, the prevalence of chronic hypertension and eclampsia in family members was much higher in superimposed preeclampsia (PEsuper) and eclampsia groups, respectively.\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"6\" width=\"747\"\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Demographics and clinical characteristics.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"228\"\u003e\n\u003cp\u003e\u003cstrong\u003eCharacteristics\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e\u003cstrong\u003ePE\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e\u003cstrong\u003ePEsuper\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e\u003cstrong\u003eEclampsia\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e\u003cstrong\u003eHELLP\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"228\"\u003e\n\u003cp\u003eSample size, n\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e693\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e342\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e61\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e74\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e112\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"228\"\u003e\n\u003cp\u003eMaternal age, y, mean (\u0026plusmn;SD)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e24.4 (\u0026plusmn;6.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e25.2 (\u0026plusmn;6.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e31.1 (\u0026plusmn;6.9) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e20.5 (\u0026plusmn;6.1) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e27.0 (\u0026plusmn;6.7) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"228\"\u003e\n\u003cp\u003eSBP, mmHg, mean (\u0026plusmn;SD)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e117 (\u0026plusmn;12)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e156 (\u0026plusmn;19) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e166 (\u0026plusmn;28) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e159 (\u0026plusmn;21)\u003csup\u003e a\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e154 (\u0026plusmn;21)\u003csup\u003e a\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"228\"\u003e\n\u003cp\u003eDBP, mmHg, mean (\u0026plusmn;SD)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e75 (\u0026plusmn;9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e105 (\u0026plusmn;13)\u003csup\u003e a\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e108 (\u0026plusmn;14)\u003csup\u003e a\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e108 (\u0026plusmn;16)\u003csup\u003e a\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e101 (\u0026plusmn;13)\u003csup\u003e a\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"228\"\u003e\n\u003cp\u003eProteinuria, n (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"228\"\u003e\n\u003cp\u003eNegative\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e693 (100)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e0 (0.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e0 (0.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e2 (2.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e7 (6.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"228\"\u003e\n\u003cp\u003e1+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e104 (30.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e28 (45.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e11 (14.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e13 (11.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"228\"\u003e\n\u003cp\u003e\u0026ge; 2+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e238 (69.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e16 (26.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e43 (58.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e85 (75.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"228\"\u003e\n\u003cp\u003eMissing\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e0 (0.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e17 (27.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e18 (24.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e7 (6.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"228\"\u003e\n\u003cp\u003ePrimigestas, n (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e300 (43.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e180 (52.8)\u003csup\u003e a\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e14 (23.3) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e54 (73.0)\u003csup\u003e a\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e46 (41.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"228\"\u003e\n\u003cp\u003eGestational age at delivery, w, mean (\u0026plusmn;SD)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e38.8 (\u0026plusmn;3.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e37.2 (\u0026plusmn;3.0)\u003csup\u003e a\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e35.8 (\u0026plusmn;3.5)\u003csup\u003e a\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e36.2 (\u0026plusmn;3.6)\u003csup\u003e a\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e34.3 (\u0026plusmn;4.0)\u003csup\u003e a\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"228\"\u003e\n\u003cp\u003eNumber of antenatal care visits,\u003c/p\u003e\n\u003cp\u003emean (\u0026plusmn;SD)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e5.9 (\u0026plusmn;2.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e6.2 (\u0026plusmn;2.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e6.1 (\u0026plusmn;2.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e4.5 (\u0026plusmn;2.5)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e5.3 (\u0026plusmn;2.3)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"228\"\u003e\n\u003cp\u003eFamily history of\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"228\"\u003e\n\u003cp\u003eChronic hypertension \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e238 (35.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e178 (56.9)\u003csup\u003e a\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e46 (80.7) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e32 (46.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e55 (49.6)\u003csup\u003e a\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"228\"\u003e\n\u003cp\u003ePreeclampsia \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e22 (3.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e41 (15.9)\u003csup\u003e a\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e5 (10.9) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e12 (21.0)\u003csup\u003e a\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e8 (8.3)\u003csup\u003e a\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"228\"\u003e\n\u003cp\u003eEclampsia \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e7 (1.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e11 (4.4)\u003csup\u003e a\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e3 (6.5) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e5 (8.8)\u003csup\u003e a\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e2 (2.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"6\" width=\"747\"\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e P\u0026lt;0.05 for comparison with the control group.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003e If at least one first degree relative has the disease.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ec\u003c/sup\u003e If the mother had had the disease.\u003c/p\u003e\n\u003cp\u003eSBP/DBP: systolic blood pressure/diastolic blood pressure.\u003c/p\u003e\n\u003cp\u003ePEsuper: chronic hypertension with superimposed preeclampsia.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr /\u003eGenetic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll genetic variants achieved standard quality control thresholds (\u003cem\u003ei.e.\u003c/em\u003e genotyping error rates \u0026lt; 0.05, minor allele frequency \u0026gt; 0.01, and p-value \u0026gt; 0.05 for Hardy-Weinberg equilibrium test). In addition, there was no evidence of population stratification, since cases and controls were equally distributed across the reference ethnical groups (Supplementary Figure 1).\u003c/p\u003e\n\u003cp\u003eFigure 1 shows the linkage disequilibrium (LD) pattern across the genomic region encompassing the studied variants. \u003cem\u003eERAP1\u003c/em\u003e variants were moderately correlated (r\u003csup\u003e2\u003c/sup\u003e=0.65) while \u003cem\u003eERAP2\u003c/em\u003e and \u003cem\u003eLNPEP\u003c/em\u003e variants seemed to belong to the same haploblock.\u003c/p\u003e\n\u003cp\u003eIn order to analyze the combined effect of variants on disease risk, we performed haplotype-based tests (Table 2). As result, \u003cem\u003eERAP2\u003c/em\u003e haplotype rs2549796 - rs2927609 - rs11135484 was associated with preeclampsia (corrected p=0.0109). There was no haplotype associated with the remaining case groups (corrected p\u0026gt;0.05).\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"6\" width=\"539\"\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Haplotype tests of association showing \u003cem\u003eERAP2 C-C-G\u003c/em\u003e haplotype associated with preeclampsia.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"103\"\u003e\n\u003cp\u003e\u003cstrong\u003eHaplotypes\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"5\" width=\"436\"\u003e\n\u003cp\u003e\u003cstrong\u003eHaplotype frequency\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"77\"\u003e\n\u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u003cstrong\u003ePE\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e\u003cstrong\u003ePEsuper\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e\u003cstrong\u003eEclampsia\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u003cstrong\u003eHELLP\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e\u003cem\u003eERAP1\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"77\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003eT-G\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"77\"\u003e\n\u003cp\u003e0.354\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.358\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e0.328\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.341\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.323\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003eT-C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"77\"\u003e\n\u003cp\u003e0.105\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.097\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e0.121\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.051\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.074\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003eC-C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"77\"\u003e\n\u003cp\u003e0.541\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.545\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e0.552\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.609\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.602\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e\u003cem\u003eERAP2\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"77\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003eC-T-A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"77\"\u003e\n\u003cp\u003e0.362\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.361\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e0.356\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.301\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.352\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003eC-C-A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"77\"\u003e\n\u003cp\u003e0.064\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.066\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e0.060\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.065\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.079\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003eC-C-G\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"77\"\u003e\n\u003cp\u003e0.045\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.076 \u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e0.050\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.045\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.065\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003eT-C-G\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"77\"\u003e\n\u003cp\u003e0.488\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.482\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e0.515\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.523\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.452\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003e\u003cem\u003eLNPEP\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"77\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003eT-C-A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"77\"\u003e\n\u003cp\u003e0.123\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.130\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e0.105\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.132\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.135\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003eC-T-G\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"77\"\u003e\n\u003cp\u003e0.398\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.376\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e0.375\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.345\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.397\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"103\"\u003e\n\u003cp\u003eT-C-G\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"77\"\u003e\n\u003cp\u003e0.479\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.494\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e0.520\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.521\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.468\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"6\" width=\"539\"\u003e\n\u003cp\u003e\u003cem\u003eERAP1\u003c/em\u003e: rs30187 - rs27044\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eERAP2\u003c/em\u003e: rs2549796 - rs2927609 - rs11135484\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLNPEP\u003c/em\u003e: rs27300 - rs38034 \u0026ndash; rs2303138\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e PE vs Control (Uncorrected p=0.0013; P-value corrected for family-wise error rates by running 10,000 permutations: p=0.0109).\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr /\u003eThere was no difference regarding allele frequencies between control and case groups (Supplementary Table 1), although the genotype distribution for \u003cem\u003eERAP1\u003c/em\u003e variants in eclampsia group seemed to differ, when compared to controls (Table 3). The frequency of genotype C/C (rs30187) was notably higher in eclampsia group (40.9 %), what would be consistent with a recessive genetic model.\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"7\" width=\"725\"\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e Genotype distribution for \u003cem\u003eERAP1\u003c/em\u003e variants across phenotypic groups.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"73\"\u003e\n\u003cp\u003e\u003cstrong\u003eSNP\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"98\"\u003e\n\u003cp\u003e\u003cstrong\u003eGenotypes\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"440\"\u003e\n\u003cp\u003e\u003cstrong\u003eGenotype distribution, n (%)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u003cstrong\u003ePE\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"110\"\u003e\n\u003cp\u003e\u003cstrong\u003ePEsuper\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e\u003cstrong\u003eEclampsia \u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u003cstrong\u003eHELLP\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" width=\"73\"\u003e\n\u003cp\u003ers30187\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003eT/T\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e139 (20.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e68 (20.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"110\"\u003e\n\u003cp\u003e12 (19.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e16 (22.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e16 (14.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003eC/T\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e343 (50.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e167 (50.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"110\"\u003e\n\u003cp\u003e32 (52.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e26 (36.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e56 (50.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003eC/C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e195 (28.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e96 (29.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"110\"\u003e\n\u003cp\u003e17 (27.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e29 (40.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e38 (34.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"73\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"110\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" width=\"73\"\u003e\n\u003cp\u003ers27044\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003eG/G\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e86 (12.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e48 (14.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"110\"\u003e\n\u003cp\u003e9 (15.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e14 (19.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e12 (10.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003eG/C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e307 (45.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e141 (42.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"110\"\u003e\n\u003cp\u003e20 (34.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e23 (31.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e48 (43.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003eC/C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e283 (41.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e142 (42.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"110\"\u003e\n\u003cp\u003e29 (50.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e36 (49.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e50\u0026nbsp; (45.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"7\" width=\"725\"\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Chi-squared test of genotypic association for Control vs Eclampsia: p=0.055 for rs30187 and p=0.079 for rs27044.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr /\u003eOf note, rs30187 C allele codes for Arg528 (instead of Lys528), resulting in an enzyme type characterized by lower peptidase activity against Ang II \u003csup\u003e32\u003c/sup\u003e. Therefore, we defined a recessive genetic model with C/C as the risk genotype for eclampsia. The model was implemented through logistic regression with genotype (T/T+T/C vs C/C) as the main explanatory variable and maternal age, and parity as covariates. As result, women homozygotes for rs30187 C/C risk genotype were more likely to develop eclampsia (OR=1.85, p=0.019) (Table 4).\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\" width=\"671\"\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u003c/strong\u003e Genetic effect of rs30187 on eclampsia risk under recessive genetic model.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"170\"\u003e\n\u003cp\u003e\u003cstrong\u003eGenotypic group\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u003cstrong\u003eControl \u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"112\"\u003e\n\u003cp\u003e\u003cstrong\u003eEclampsia \u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"257\"\u003e\n\u003cp\u003e\u003cstrong\u003eEclampsia risk \u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u003cstrong\u003en (%)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"112\"\u003e\n\u003cp\u003e\u003cstrong\u003en (%)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u003cstrong\u003eOR (95% CI)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"106\"\u003e\n\u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"170\"\u003e\n\u003cp\u003eT/T + C/T\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e482 (71.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"112\"\u003e\n\u003cp\u003e40 (58.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"106\"\u003e\n\u003cp\u003e0.019\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"170\"\u003e\n\u003cp\u003eC/C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e195 (28.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"112\"\u003e\n\u003cp\u003e29 (42.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e1.85 (1.11 \u0026ndash; 3.11)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\" width=\"671\"\u003e\n\u003cp\u003e\u003csup\u003ea \u003c/sup\u003eChisquared test of association for genotype distribution between Control and Eclampsia (p=0.022)\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003e Odds ratio (OR) and confidence interval (CI) estimated by logistic regression model adjusted for maternal age and parity.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr /\u003eFunctional validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to GTEx and SNiPa data, rs30187 has a significant effect on both ERAP1 mRNA and protein levels in blood with C/C genotype associated with the lowest expression levels (Figure 2).\u003c/p\u003e\n\u003cp\u003eGiven the qualitative and quantitative effect of rs30187 on ERAP1 expression, we hypothesized that women homozygotes for C/C genotype have higher circulating levels of Ang II. In order to test that, plasma Ang II concentrations were determined in an additional cohort of women with severe preeclampsia (n=36) and normotensive pregnant controls (n=29). We rejected this hypothesis (p=0.895) since no difference was detected between genotypic groups (Figure 3). The intra-group analysis (\u003cem\u003ei.e.\u003c/em\u003e cases-only and controls-only) did not detect any difference in Ang II levels across genotypic groups as well (data not shown).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEndoplasmic reticulum aminopeptidases (ERAPs), as well as leucyl/cystinyl aminopeptidase (LNPEP), play roles in antigen processing, inflammatory response, blood pressure regulation and angiogenesis, all processes potentially implicated in preeclampsia pathophysiology. The present study confirmed a genetic association between \u003cem\u003eERAP2\u003c/em\u003e and preeclampsia, and, for the first time, reported an association between \u003cem\u003eERAP1\u003c/em\u003e and eclampsia. Thus, our findings may help to disentangle the intricate association between the correlated phenotypes preeclampisa/eclampisa and the functionally and physically connected genes \u003cem\u003eERAP1\u003c/em\u003e/\u003cem\u003eERAP2\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eJohnson \u003cem\u003eet al.,\u003c/em\u003e tested \u003cem\u003eERAP1\u003c/em\u003e, \u003cem\u003eERAP2\u003c/em\u003e and \u003cem\u003eLNPEP\u003c/em\u003e for associations with preeclampsia in Australian and Norwegian populations, and identified \u003cem\u003eERAP2\u003c/em\u003e variants (rs2549782, rs2548538, rs2287988 and rs17408150) associated with preeclampsia \u003csup\u003e16\u003c/sup\u003e. In the same study, rs27044 and rs30187 (\u003cem\u003eERAP1\u003c/em\u003e) were not associated with disease although borderline association with preeclampsia was found for markers rs3734016 and rs34750, both within \u003cem\u003eERAP1\u003c/em\u003e gene. It is important to highlight that the Australian cohort contained both preeclampsia and eclampsia cases, but they were analyzed as a unique group \u003csup\u003e16\u003c/sup\u003e. Out study treated the two phenotypes as different entities. A recent study with 148 preeclamptic women and 133 controls from Iran investigated four variants in \u003cem\u003eERAP1 \u003c/em\u003e(including rs30187) and three variants in \u003cem\u003eERAP2\u003c/em\u003e. None of the variants were associated with disease, but a haplotype encompassing the seven variants was associated with preeclampsia \u003csup\u003e33\u003c/sup\u003e. In another Iranian independent study, \u003cem\u003eERAP2\u003c/em\u003e variants (rs2549782 and rs17408150) were also associated with preeclampsia \u003csup\u003e34\u003c/sup\u003e. Interestingly, the fetal minor allele for variant rs2549782 (\u003cem\u003eERAP2\u003c/em\u003e) was associated with preeclampsia in African American population \u003csup\u003e35\u003c/sup\u003e. Besides the genetic association findings, Founds \u003cem\u003eet al\u003c/em\u003e showed \u003cem\u003eERAP2\u003c/em\u003e was differentially expressed in the first trimester placentas of women who later developed preeclampsia \u003csup\u003e36\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eERAP1\u003c/em\u003e rs30187 C allele codes an enzyme with Arg528 that causes a reduction on peptidase activity for angiotensin II degradation by approximately 60%, when compared to the enzyme with Lys528, coded by the T allele \u003csup\u003e22,32\u003c/sup\u003e. We failed to confirm the hypothesis that women carrying two copies of the C allele have increased levels of Ang II in their blood, which in turn could cause blood pressure elevation and seizure. However, we cannot rule out a potential effect of rs30187 on local RAS (\u003cem\u003ee.g.\u003c/em\u003e brain and kidney). While Arg528 variant is associated with hypertensive disease \u003csup\u003e37\u003c/sup\u003e, the Lys528 variant is strongly associated with susceptibility to ankylosing spondylitis \u003csup\u003e38\u003c/sup\u003e and other autoimmune diseases \u003csup\u003e17\u003c/sup\u003e. Since both Arg528 and Lys528 alleles are associated with bad outcomes, it is likely that \u003cem\u003eERAP\u003c/em\u003e genes would be subject to balancing selection, a process where heterozygous individuals are more adaptive than either of the two types of homozygous \u003csup\u003e39\u003c/sup\u003e. In addition, these genes play key role in the maintenance of immunotolerance to self-peptides as well as protecting against infectious agents, such as HIV \u003csup\u003e40\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe small sample size for some of our case groups represents an important limitation for the present study, even though we should consider that eclampsia and HELLP are extremely rare phenotypes. On the other hand, the marker associated with eclampsia (rs30187) has been well characterized as affecting the protein function, what strengthens the biological plausibility for the genetic association reported here. Furthermore, we accounted for important confounders such as age, parity and ethnicity.\u003c/p\u003e\n\u003cp\u003eThe mechanism by which endoplasmic reticulum aminopeptidases (ERAPs) influence the risk of preeclampsia/eclampsia remains to be elucidated. In addition to Ang II degradation and peptide trimming for antigen presentation via MHC-1, ERAPs also play role in inflammatory response by shedding cytokine receptors (\u003cem\u003ee.g.\u003c/em\u003e IL-6R, IL-1R2 and TNFR) \u003csup\u003e41\u0026ndash;43\u003c/sup\u003e. Lastly, \u003cem\u003eERAP1\u003c/em\u003e plays crucial role in VEGF-stimulated proliferation and migration of endothelial cells, as well as angiogenesis, via the binding and modification of PDk1 \u003csup\u003e44\u003c/sup\u003e. All the above-mentioned mechanisms are potentially involved in the causal pathway of preeclampsia/eclampsia.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, we identified genetic variants in \u003cem\u003eERAP1\u003c/em\u003e and \u003cem\u003eERAP2\u003c/em\u003e associated with eclampsia and preeclampsia, respectively. Sequencing and functional studies are needed in order to elucidate the mechanisms underlying these genetic associations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genetic data used in the present study is available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the staff of Maternidade Escola Janu\u0026aacute;rio Cicco for their help in ascertaining patients\u0026rsquo; clinical classification and sample collection. We do appreciate the collaboration of SNiPA team (Karsten Suhre and Arnold Matthias) that kindly generated rs30187 pQTL plot (Figure 2B) and Victor Lima (on behalf of Laborat\u0026oacute;rio de An\u0026aacute;lises Cl\u0026iacute;nicas DNA Center) to perform capillary electrophoresis needed to genotype the additional cohort (n=65) of pregnant women.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLCF worked on the study design, carried out the genotyping and the genetic analysis, and the manuscript writing. CEMG contributed with the study design and genotyping. PD contributed with the genetic analysis and revising the manuscript. IPH and PRPN carried out sample processing and Ang II measurement. ASL contributed with the study participant recruitment and phenotype ascertainment. SMBJ contributed with the study design, recruitment of subjects and manuscript revision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no conflict of interest from any of the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSay, L. \u003cem\u003eet al.\u003c/em\u003e Global causes of maternal death: a WHO systematic analysis. \u003cem\u003eLancet Glob. Health\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, e323\u0026ndash;e333 (2014).\u003c/li\u003e\n\u003cli\u003eBenschop, L., Duvekot, J. J. \u0026amp; Lennep, J. E. R. van. Future risk of cardiovascular disease risk factors and events in women after a hypertensive disorder of pregnancy. \u003cem\u003eHeart\u003c/em\u003e \u003cstrong\u003e105\u003c/strong\u003e, 1273\u0026ndash;1278 (2019).\u003c/li\u003e\n\u003cli\u003eAndraweera, P. H. \u0026amp; Lassi, Z. S. 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Puromycin-insensitive leucyl-specific aminopeptidase (PILSAP) binds and catalyzes PDK1, allowing VEGF-stimulated activation of S6K for endothelial cell proliferation and angiogenesis. \u003cem\u003eBlood\u003c/em\u003e \u003cstrong\u003e104\u003c/strong\u003e, 2345\u0026ndash;2352 (2004).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Aminopeptidases, preeclampsia, ERAP, genetic association","lastPublishedDoi":"10.21203/rs.3.rs-235628/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-235628/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe clinical spectrum of hypertensive disorders of pregnancy (HDP) is determined by the interplay between environmental and genetic factors, most of which remains unknown. \u003cem\u003eERAP1, ERAP2\u003c/em\u003e and \u003cem\u003eLNPEP\u003c/em\u003e genes code for multifunctional aminopeptidases involved with antigen processing and degradation of small peptides such as angiotensin II (Ang II), vasopressin and oxytocin. We aimed to test for associations between genetic variants in aminopeptidases and HDP. A total of 1282 pregnant women (normotensive controls, n=693; preeclampsia, n=342; chronic hypertension with superimposed preeclampsia, n=61; eclampsia, n=74; and HELLP syndrome, n=112) were genotyped for variants in \u003cem\u003eLNPEP \u003c/em\u003e(rs27300, rs38034, rs2303138), \u003cem\u003eERAP1 \u003c/em\u003e(rs27044, rs30187) and \u003cem\u003eERAP2\u003c/em\u003e (rs2549796 rs2927609 rs11135484). We also evaluated the effect of \u003cem\u003eERAP1\u003c/em\u003e rs30187 on plasma Ang II levels in an additional cohort of 65 pregnant women. The genotype C/C, in \u003cem\u003eERAP1\u003c/em\u003e rs30187 variant (c.1583T\u0026gt;C, p.Lys528Arg), was associated with increased risk of eclampsia (OR=1.85, p=0.019) whereas \u003cem\u003eERAP2 \u003c/em\u003ehaplotype rs2549796(C)-rs2927609(C)-rs11135484(G) was associated with preeclampsia (OR=1.96, corrected p-value=0.01). Ang II plasma levels did not differ across rs30187 genotypic groups (p=0.895).\u003cstrong\u003e \u003c/strong\u003eIn conclusion,\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eERAP1\u003c/em\u003e gene is associated with eclampsia whereas \u003cem\u003eERAP2\u003c/em\u003e is associated with preeclampsia, although the mechanism by which genetic variants in ERAPs influence the risk of preeclampsia and eclampsia remain to be elucidated.\u003c/p\u003e","manuscriptTitle":"Genetic Association of ERAP1 and ERAP2 With Eclampsia and Preeclampsia in Northeastern Brazilian Women","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-25 22:42:11","doi":"10.21203/rs.3.rs-235628/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-02-24T14:10:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-02-22T16:05:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"cf0c5159-1cfe-41b4-aa1f-0e867f27c961","date":"2021-02-18T06:09:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-02-17T21:33:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-02-17T21:24:16+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-02-16T16:46:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-02-16T15:51:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2021-02-12T02:55:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"10aa1fee-52fc-47a9-ad46-60d776453288","owner":[],"postedDate":"February 25th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":2617071,"name":"Plant Molecular Biology and Genetics"},{"id":2617072,"name":"Medical Genetics"},{"id":2617073,"name":"Cardiac \u0026 Cardiovascular Systems"}],"tags":[],"updatedAt":"2021-08-18T19:40:13+00:00","versionOfRecord":{"articleIdentity":"rs-235628","link":"https://doi.org/10.1038/s41598-021-86240-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2021-03-24 19:05:31","publishedOnDateReadable":"March 24th, 2021"},"versionCreatedAt":"2021-02-25 22:42:11","video":"","vorDoi":"10.1038/s41598-021-86240-z","vorDoiUrl":"https://doi.org/10.1038/s41598-021-86240-z","workflowStages":[]},"version":"v1","identity":"rs-235628","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-235628","identity":"rs-235628","version":["v1"]},"buildId":"J0_U0BvcaRcwD8yVFaRlm","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}