SHORT COMMUNICATION
NLRP1 L155H Polymorphism is a Risk Factor for Preeclampsia Development Alessandra Pontillo1, Edione C. Reis1, Pamela N. Bricher1, Priscila Vianna2, Solange Diniz3, Karla S. Fernandes3, Jose A. Chies2, Valeria Sandrim4 1
Laboratory of Immunogenetics, Department of Immunology, Institute of Biomedical Sciences, University of Sao Paulo/USP, Sao Paulo, SP, Brazil; Laboratory of Immunogenetics, Department of Genetics, Federal University of Rio Grande do Sul/UFRS, Porto Alegre, RS, Brazil; 3 Nucleo de Pos-Graduacß~ao e Pesquisa, Santa Casa de Belo Horizonte, Belo Horizonte, MG, Brazil; 4 Department of Pharmacology, Institute of Biosciences, University ‘Estadual Paulista’/UNESP, Botucatu, SP, Brazil 2
Keywords IL-1ß, inflammasome, NLRP1, preeclampsia Correspondence Alessandra Pontillo, Laboratory of Immunogenetics, Department of Immunology, Institute of Biomedical Sciences, University of Sao Paulo, Avenida Prof. Lineu Prestes 1730, 05508-000 Cidade Universitaria, Sao Paulo, SP, Brazil. E-mail: [email protected] Submission October 2, 2014; accepted November 28, 2014. Citation Pontillo A, Reis EC, Britcher PN, Vianna P, Diniz S, Fernandes KS, Chies JA, Sandrim V. NLRP1 L155H polymorphism is a risk factor for preeclampsia development. Am J Reprod Immunol 2015; 73: 577–581
Problem Augmented levels of IL-1ß have been pointed out as an important pathogenic factor for preeclampsia development. Inflammasome is the cytoplasmic complex responsible for pro-IL1ß cleavage and IL-1ß secretion. Aim of the study was to evaluate the association between polymorphisms in inflammasome’ genes and preeclampsia. Method of study Selected polymorphisms in inflammasome genes (NLRP1, NLRP3, CARD8, and IL1B) were analyzed in 286 Brazilian women with and 309 without preeclampsia. Results and Conlclusions The NLRP1 variant rs12150220 (L155H) was associated with the development of preeclampsia (OR = 1.58), suggesting a role of this inflammasome receptor in the pathogenesis of this multifactorial disorder.
doi:10.1111/aji.12353
Introduction Preeclampsia (PE) is a multifactorial disease affecting about 5–8% of pregnancies and is characterized by systemic inflammation,1 maternal hypertension, endothelial dysfunction,2 resulting in poor placentation3 with risk for mother and fetus.4,5 Augmented levels of IL-1ß during the first trimester of pregnancy have been pointed out as an important pathogenic factor for PE development.6 IL-1ß secretion depends on the activation of the cysteine-aspartic protease caspase-1, which in turn is cleaved in its active form as consequence of the assembling of a cytoplasmic receptor complex, known as inflammasome. Two nod-like receptors with a pyrin domain (NRLP), American Journal of Reproductive Immunology 73 (2015) 577–581 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
NLRP1 and NLRP3, are responsible for inflammasome formation in response to pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Upon recognition of PAMPs and/or DAMPs, NLRP1 or NLRP3 recruits the adaptor protein ASC and the caspase-1, leading to the processing of pro-IL-1ß and pro-IL-18. NLRP1 and NLRP3 are expressed in immune cells, but also in other cell types including placental cells.7–9 NLRP3 has been recognized as a sensor of uric acid, and Mulla et al. recently demonstrated that hyperuricemia associated with preeclampsia might be related with NLRP3 inflammasome activation in trophoblast and consequent augmented levels of IL-1ß in placenta,8 suggesting an important role of the inflammasome in PE pathogenesis.
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lows: no rise in blood pressure, the absence of hypertension or proteinuria. Healthy pregnant women were also matched for age and ethnicity with cases. Main clinical characteristics of women with and without preeclampsia are reported in Table I. Written informed consent was obtained according to the protocol of Ethical Committee of the hospitals.
Several genetic variants have been described as predisposing factors for PE development;10 however, to our knowledge, inflammasome genes have not yet been evaluated. All this considered frequency distribution of selected polymorphisms in inflammasome genes was analyzed in two Brazilian case/control cohorts of women with preeclampsia to evaluate the contribution of inflammasome genetics in the development of this pro-inflammatory condition.
Single-nucleotide Polymorphisms Selection and Genotyping
Material and methods
Eightsingle-nucleotide polymorphisms (SNPs) in inflammasome genes (NLRP1, NLRP3, CARD8, and IL1B) were selected based on functional effect and/or previously reported association with human disorders.12–15 SNPs genotyping was performed using commercially available TaqMan assays (Applied Biosystems/AB, Carlsbad, CA, USA) using StepOne Real-Time platform (AB, Carlsbad, CA, USA). Allelic discrimination was performed using the StepOne software (AB).
Subjects Hundred and ninety-two healthy pregnant women with uncomplicated pregnancies (healthy controls, HC; 26.3 6.5 years) and 160 pregnant with primary preeclampsia (PE; 27.0 6.7 years) were recruited at Hospital Sofia Feldman in Belo Horizonte (Central-Southern Brazil). Hundred and twentysix PE (30.3 7.5 years) and 117 HC (28.1 7.4 years) were recruited at the Maternity Unit of at public hospital in Southern Brazil (Hospital Nossa Senhora Conceicß~ ao, PortoAlegre). Women were classified as European or African-derived according to phenotypic characteristics and ethnicity data of parents/grandparents reported by the participants in an appropriate questionnaire. Preeclampsia was defined for the presence of hypertension [blood pressure > 140 mm Hg (systolic) and/or 90 mm Hg (diastolic)] and proteinuria (>300 mg of protein every 24 hr).11 Healthy pregnant women inclusion criteria were as fol-
Data Analysis R software (www.r-project.org) was used to perform Fisher exact test and odds ratio (OR) calculation for alleles and haplotypes and for genotype association and inheritance modeling (package ‘SNP assoc’ version 1.5–2).16 A formal Bonferroni correction for the number of SNPs analyzed would require a significance threshold of P = 0.006 (P0/N, P0 = 0.05, N = 8 SNPs). The Haploview software 17 was used to investigate the association and linkage disequilibrium (LD) pattern and for deriving the haplotypes.
Table I Clinical Characteristics of Women with and Without Preeclampsia
Characteristics Maternal age Pre-BMI SBP (mmHg) DBP (mmHg) Delivery weeks Fetal weight (g) Ethnicity (% white European origin)
Controls (HC; n = 309)
Preeclampsia (PE; n = 286)
25.9 26.7 120.6 67.1 38.0 2851.9 16.7%
26.7 26.2 153.6 94.8 35.1 2212.7 20.6%
6.1 3.4 12.8 8.7 2.2 651.9
a
6.7 3.8a 17.3 14.1 2.8b 591.1b
Early delivery PE (n = 38) 25.6 26.3 160 100 31.3 1624.1
6.1 4.3 15.2 11.3 2.3 433.3
Preterm PE (n = 71) 28.3 24.8 154.7 93.8 35.9 2313.1
7.4 5.5 18.3 13.1 0.4 370.6
PE (n = 47) 27.2 25.8 149.3 90.3 38.3 2780.5
6.6 3.4 17.8 18.2 0.8 492.7
Pre-BMI, pre-pregnancy body mass index; SBP, blood pressure, systolic; DBP, blood pressure, diastolic. a P-value > 0.05 versus HC. b P-value < 0.05 versus HC.
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American Journal of Reproductive Immunology 73 (2015) 577–581 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
NLRP1 AND PREECLAMPSIA
Results and discussion Two hundred and eighty-six women with primary preeclampsia and 309 healthy pregnant women were genotyped for eight selected SNPs in four inflammasome genes (NLRP1, NLRP3, CARD8, and IL1B). SNPs allelic and genotypic frequencies were in Hardy–Weinberg equilibrium in both groups. While other SNPs appeared to be similarly distributed in PE and HC (results reported in Table S1), the NLRP1 (rs12150220) A/T polymorphism was significantly associated with PE (Table II). NLRP1 (rs12150220) minor T allele was significantly more frequent in PE than in HC women (0.45 versus 0.34; P = 3.2 exp5), suggesting a predisposing role of this allele in susceptibility to develop PE (OR = 1.58; 95% CI = 1.22–2.05). Rs12150220 genotypes distribution was also significantly different in the two groups, and according to the Akaike information criterion, predisposing T allele behaved according to a codominant model (P = 8.6 exp05). Even when we corrected the analysis for age, ethnicity, and geographic
Table II Allelic and Genotypic Frequencies of rs12150220 in Women with and Without Preeclampsia
PE (n = 286) HC (n = 309)
Alleles frequency (T/ A)
Pa
0.45/0.55
3.2 exp5
0.24/0.45/0.31 0.11/0.46/0.43
Table III Haplotypes Frequencies of SNPs in NLRP1, NLRP3, and CARD8 Genes in Women with and Without Preeclampsia
Haplotypes OR 1.58
95% CI 1.22–2.05
0.34/0.66
Genotypes frequency (TT/ AT/AA) PE (n = 286) HC (n = 309)
NLRP1
origin, the rs12150220 variation continued to be significantly associated with preeclampsia in a codominant way (P = 1.53 exp05) (Table II). Women with preeclampsia were then stratified for delivery time in three groups: early (0.05), which are similarly distributed between case and controls (P > 0.05) (Table III). SNPs in NLRP1 and NLRP3 genes did not appear to be in LD in the studied populations, but the com-
Pcodb (Padjust)
Pdomb (Padjust)
Precb (Padjust)
8.6 exp5 (1.5 exp4)
0.004 (5.3 exp4)
5.3 exp5 (6.3 exp4)
P, P-value; OR, odds ratio; 95% CI, 95% confidence interval. a Fisher test. b Likelihood ratio test. Padjust, P-value adjusted by age, ethnicity, and origin; Pcod, P-value of codominant model (three genotypes); Pdom, P-value of dominant model [(homozygotes of risk allele+heterozygotes) versus (homozygotes of non-risk allele)]; Prec, P-value of recessive model [(homozygotes of risk allele) versus (heterozygotes+homozygotes of non-risk allele)]. The T allele is the risk allele.
American Journal of Reproductive Immunology 73 (2015) 577–581 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Haplotypes frequency (PE, HC)
Pa
OR (95% CI)
NLRP1 rs11651270-rs12150220-rs2670660 (rs11651270-rs12150220: D0 = 0.11; r2 = 0.009; rs12150220-rs2670660: (D0 = 0.64; r2 = 0.30) T-A-A 0.237, 0.278 0.141 0.81 C-T-G 0.198, 0.186 0.641 1.08 C-A-A 0.140, 0.220 8.1 exp5 0.58 T-T-G 0.154, 0.131 0.333 1.20 T-A-G 0.088, 0.076 0.501 1.19 C-A-G 0.067, 0.069 1.0 0.98 NLRP3 rs35829419- rs10754558 (D0 = 0.89; r2 = 0.03) C-C 0.654, 0.678 0.395 0.89 C-G 0.320, 0.307 0.69 1.06 A-G 0.025, 0.013 0.016 2.98 CARD8 rs2043211-rs6509365 (D0 = 0.99; r2 = 0.84) A-A 0.666, 0.694 0.349 0.88 T-G 0.297, 0.268 0.298 1.16
(0.61–1.07) (0.79–1.48) (0.41–0.81) (0.84–1.72) (0.75–1.89) (0.59–1.62)
(0.69–1.17) (0.81–1.38) (1.19–8.45)
(0.67–1.15) (0.87–1.54)
P, P-value; OR, odds ratio; 95% CI, 95% confidence interval. a Fisher test. Linkage disequilibrium indexes (D0 : normalized disequilibrium index; r2: correlation coefficient). Statistically significant values are indicated in bold.
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bined effect of polymorphisms within the same genes was evaluated for those genes. NLRP1 SNPs combined to form 6 main haplotypes, and the rs11651270/Crs12150220/A-rs2670660/A combination was significantly less frequent in PE women as compared to controls (0.14 versus 0.22), suggesting of a protective effect against preeclampsia development (P = 8.1 exp5; OR = 0.58; 95% CI = 0.41–0.81), as expected because it contains the rs12150220 major allele A instead of the risk T allele (Table III). The frequencies of all other combined haplotypes in NLRP1 as well in NLRP3 genes were not statistically different in the two groups (Table III). Considering previously reported data about the combined effect of the two NLRP3 rs35829419 (Q705K) and CARD8 rs2043211 (C10X) variants,18 we also analyzed this combination in our case/control population. However, no statistical significant association was observed with preeclampsia (P > 0.05; data not shown). Even considering that the NLRP3-inflammasome was previously pointed out as a main factor in IL-1ß dysregulation in preeclampsia,8 due to its reported role in placenta physiology7 as well as in uric acid response8 and essential hypertension,19 and that rs35829419 (Q705K) and rs10754558 (30 UTR) have been associated with elevated IL-1ß level and inflammatory condition,20,21 those NLRP3 variants did not resulted associated with preeclampsia in our studied population. In contrast, a strong association with NLRP1 rs12150550 missense variation (L155H) was observed, suggesting a role of this inflammasome receptor in the pathogenesis of preeclampsia. NLRP1 is involved in caspase-1 and caspase-5 mediated IL-1ß secretion and pyroptose; however, the cognate stimulus leading to NLRP1-inflammasome activation is still not fully elucidated.22 NLRP1 rs12150550 has been reported to be associated with an increased secretion of IL-1ß.23 NLRP1 expression is seen in immune and other cell types, such as fibroblasts,24 as well as in brain and in the respiratory tract.25 Recently, NLRP1 has been described in endothelial cells,26 leading us to hypothesize that it is involved in the pathogenesis of preeclampsia mainly due to its role in endothelial cells. In conclusion, the NLRP1 variant rs12150220 was associated with preeclampsia development suggesting a role for this inflammasome receptor in the pathogenesis of this multifactorial disorder.
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References 1 Redman CW, Sargent IL: Immunology of pre-eclampsia. Am J Reprod Immunol 2010; 63:534–543. 2 Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK: Preeclampsia: an endothelial cell disorder. Am J Obstet Gynecol 1989; 161:1200–1204. 3 Pijnenborg R, Bland JM, Robertson WB, Brosens I: Uteroplacental arterial changes related to interstitial trophoblast migration in early human pregnancy. Placenta 1983; 4:397–413. 4 Brown MA: The physiology of pre-eclampsia. Clin Exp Pharmacol Physiol 1995; 22:781–791. 5 National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy. Report of the national high blood pressure education program. Am J Obstet Gynecol 2000;183: S1–S22. 6 Siljee JE, Wortelboer EJ, Koster MP, Imholz S, Rodenburg W, Visser GH, de Vries A, Schielen PC, Pennings JL: Identification of interleukin-1 beta, but no other inflammatory proteins, as an early onset pre-eclampsia biomarker in first trimester serum by beadbased multiplexed immunoassays. Prenat Diagn 2013; 33:1183–1188. 7 Abrahams VM: The role of the Nod-like receptor family in trophoblast innate immune responses. J Reprod Immunol 2011; 88:112–117. 8 Mulla MJ, Myrtolli K, Potter J, Boeras C, Kavathas PB, Sfakianaki AK, Tadesse S, Norwitz ER, Guller S, Abrahams VM: Uric acid induces trophoblast IL-1b production via the inflammasome: implications for the pathogenesis of preeclampsia. Am J Reprod Immunol 2011; 65:542–548. 9 Pontillo A, Girardelli M, Agostinis C, Masat E, Bulla R, Crovella S: Bacterial LPS differently modulates inflammasome gene expression and IL-1b secretion in trophoblast cells, decidual stromal cells, and decidual endothelial cells. Reprod Sci 2013; 20:563–566. 10 Valenzuela FJ, Perez-Sep ulveda A, Torres MJ, Correa P, Repetto GM, Illanes SE: Pathogenesis of preeclampsia: the genetic component. J Pregnancy 2012; 2012:632732. 11 Sibai BM: Diagnosis and management of gestational hypertension and preeclampsia. Obstet Gynecol 2003; 102:181. 12 Pontillo A, Oshiro TM, Girardelli M, Kamada AJ, Crovella S, Duarte AJ: Polymorphisms in inflammasome genes and susceptibility to HIV-1 infection. J Acquir Immune Defic Syndr 2012; 59:121–125. 13 Pontillo A, Catamo E, Arosio B, Mari D, Crovella S: NALP1/NLRP1 genetic variants are associated with Alzheimer disease. Alzheimer Dis Assoc Disord 2012; 26:277–281. 14 Pontillo A, Girardelli M, Kamada AJ, Pancotto JA, Donadi EA, Crovella S, Sandrin-Garcia P: Polymorphisms in inflammasome genes are involved in the predisposition to systemic lupus erythematosus. Autoimmunity 2012; 45:271–278. 15 Pontillo A, Carvalho MS, Kamada AJ, Moura R, Schindler HC, Duarte AJ, Crovella S: Susceptibility to Mycobacterium tuberculosis infection in HIV-positive patients is associated with CARD8 genetic variant. J Acquir Immune Defic Syndr 2013; 63:147–151. 16 Gonzalez JR, Armengol L, Sole X, Guin o E, Mercader JM, Estivill X, Moreno V: SNPassoc: an R package to perform whole genome association studies. Bioinformatics 2007; 23:644–645. 17 Barrett JC: Haploview: visualization and analysis of SNP genotype data. Cold Spring Harb Protoc 2009; 2009:pdb.ip71. 18 Schoultz I, Verma D, Halfvarsson J, T€ orkvist L, Fredrikson M, Sj€ oqvist U, L€ ordal M, Tysk C, Lerm M, S€ oderkvist P, S€ oderholm JD: Combined polymorphisms in genes encoding the inflammasome
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components NALP3 and CARD8 confer susceptibility to Crohn’s disease in Swedish men. Am J Gastroenterol 2009; 104:1180–1188. Omi T, Kumada M, Kamesaki T, Okuda H, Munkhtulga L, Yanagisawa Y, Utsumi N, Gotoh T, Hata A, Soma M, Umemura S, Ogihara T, Takahashi N, Tabara Y, Shimada K, Mano H, Kajii E, Miki T, Iwamoto S: An intronic variable number of tandem repeat polymorphisms of the cold-induced autoinflammatory syndrome 1 (CIAS1) gene modifies gene expression and is associated with essential hypertension. Eur J Hum Genet 2006; 14:1295–1305. Verma D, Lerm M, Blomgran Julinder R, Eriksson P, S€ oderkvist P, S€ arndahl E: Gene polymorphisms in the NALP3 inflammasome are associated with interleukin-1 production and severe inflammation: relation to common inflammatory diseases? Arthritis Rheum 2008; 58:888–894. Hitomi Y, Ebisawa M, Tomikawa M, Imai T, Komata T, Hirota T, Harada M, Sakashita M, Suzuki Y, Shimojo N, Kohno Y, Fujita K, Miyatake A, Doi S, Enomoto T, Taniguchi M, Higashi N, Nakamura Y, Tamari M: Associations of functional NLRP3 polymorphisms with susceptibility to food-induced anaphylaxis and aspirin-induced asthma. J Allergy Clin Immunol 2009; 124:779–785.e6. Masters SL: Specific inflammasomes in complex diseases. Clin Immunol 2013; 147:223–228. Levandowski CB, Mailloux CM, Ferrara TM, Gowan K, Ben S, Jin Y, McFann KK, Holland PJ, Fain PR, Dinarello CA, Spritz RA: NLRP1 haplotypes associated with vitiligo and autoimmunity increase interleukin-1b processing via the NLRP1 inflammasome. Proc Natl Acad Sci USA 2013; 110:2952–2956.
American Journal of Reproductive Immunology 73 (2015) 577–581 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
24 Fan C, Zhao X, Guo X, Cao X, Cai J: P2X4 promotes interleukin-1b production in osteoarthritis via NLRP1. Mol Med Rep 2014; 9:340–344. 25 Yin Y, Yan Y, Jiang X, Mai J, Chen NC, Wang H, Yang XF: Inflammasomes are differentially expressed in cardiovascular and other tissues. Int J Immunopathol Pharmacol 2009; 22:311–322. 26 Bleda S, de Haro J, Varela C, Esparza L, Ferruelo A, Acin F: NLRP1 inflammasome, and not NLRP3, is the key in the shift to proinflammatory state on endothelial cells in peripheral arterial disease. Int J Cardiol 2014; 172:e282–e284.
Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Linkage Disequilibrium analysis. Results for Linkage Disequilibrium (LD) analysis of studied SNPs. D’/LOD values and color scheme were reported according to Haploview software. Table S1. Allelic and genotypic frequencies in women with and without preeclampsia. Table S2. Association analysis for NLRP1 rs12150220 in women with and without preeclampsia stratified for delivery time and age.
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NLRP1 L155H Polymorphism is a Risk Factor for Preeclampsia Development Alessandra Pontillo1, Edione C. Reis1, Pamela N. Bricher1, Priscila Vianna2, Solange Diniz3, Karla S. Fernandes3, Jose A. Chies2, Valeria Sandrim4 1
Laboratory of Immunogenetics, Department of Immunology, Institute of Biomedical Sciences, University of Sao Paulo/USP, Sao Paulo, SP, Brazil; Laboratory of Immunogenetics, Department of Genetics, Federal University of Rio Grande do Sul/UFRS, Porto Alegre, RS, Brazil; 3 Nucleo de Pos-Graduacß~ao e Pesquisa, Santa Casa de Belo Horizonte, Belo Horizonte, MG, Brazil; 4 Department of Pharmacology, Institute of Biosciences, University ‘Estadual Paulista’/UNESP, Botucatu, SP, Brazil 2
Keywords IL-1ß, inflammasome, NLRP1, preeclampsia Correspondence Alessandra Pontillo, Laboratory of Immunogenetics, Department of Immunology, Institute of Biomedical Sciences, University of Sao Paulo, Avenida Prof. Lineu Prestes 1730, 05508-000 Cidade Universitaria, Sao Paulo, SP, Brazil. E-mail: [email protected] Submission October 2, 2014; accepted November 28, 2014. Citation Pontillo A, Reis EC, Britcher PN, Vianna P, Diniz S, Fernandes KS, Chies JA, Sandrim V. NLRP1 L155H polymorphism is a risk factor for preeclampsia development. Am J Reprod Immunol 2015; 73: 577–581
Problem Augmented levels of IL-1ß have been pointed out as an important pathogenic factor for preeclampsia development. Inflammasome is the cytoplasmic complex responsible for pro-IL1ß cleavage and IL-1ß secretion. Aim of the study was to evaluate the association between polymorphisms in inflammasome’ genes and preeclampsia. Method of study Selected polymorphisms in inflammasome genes (NLRP1, NLRP3, CARD8, and IL1B) were analyzed in 286 Brazilian women with and 309 without preeclampsia. Results and Conlclusions The NLRP1 variant rs12150220 (L155H) was associated with the development of preeclampsia (OR = 1.58), suggesting a role of this inflammasome receptor in the pathogenesis of this multifactorial disorder.
doi:10.1111/aji.12353
Introduction Preeclampsia (PE) is a multifactorial disease affecting about 5–8% of pregnancies and is characterized by systemic inflammation,1 maternal hypertension, endothelial dysfunction,2 resulting in poor placentation3 with risk for mother and fetus.4,5 Augmented levels of IL-1ß during the first trimester of pregnancy have been pointed out as an important pathogenic factor for PE development.6 IL-1ß secretion depends on the activation of the cysteine-aspartic protease caspase-1, which in turn is cleaved in its active form as consequence of the assembling of a cytoplasmic receptor complex, known as inflammasome. Two nod-like receptors with a pyrin domain (NRLP), American Journal of Reproductive Immunology 73 (2015) 577–581 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
NLRP1 and NLRP3, are responsible for inflammasome formation in response to pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Upon recognition of PAMPs and/or DAMPs, NLRP1 or NLRP3 recruits the adaptor protein ASC and the caspase-1, leading to the processing of pro-IL-1ß and pro-IL-18. NLRP1 and NLRP3 are expressed in immune cells, but also in other cell types including placental cells.7–9 NLRP3 has been recognized as a sensor of uric acid, and Mulla et al. recently demonstrated that hyperuricemia associated with preeclampsia might be related with NLRP3 inflammasome activation in trophoblast and consequent augmented levels of IL-1ß in placenta,8 suggesting an important role of the inflammasome in PE pathogenesis.
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PONTILLO ET AL.
lows: no rise in blood pressure, the absence of hypertension or proteinuria. Healthy pregnant women were also matched for age and ethnicity with cases. Main clinical characteristics of women with and without preeclampsia are reported in Table I. Written informed consent was obtained according to the protocol of Ethical Committee of the hospitals.
Several genetic variants have been described as predisposing factors for PE development;10 however, to our knowledge, inflammasome genes have not yet been evaluated. All this considered frequency distribution of selected polymorphisms in inflammasome genes was analyzed in two Brazilian case/control cohorts of women with preeclampsia to evaluate the contribution of inflammasome genetics in the development of this pro-inflammatory condition.
Single-nucleotide Polymorphisms Selection and Genotyping
Material and methods
Eightsingle-nucleotide polymorphisms (SNPs) in inflammasome genes (NLRP1, NLRP3, CARD8, and IL1B) were selected based on functional effect and/or previously reported association with human disorders.12–15 SNPs genotyping was performed using commercially available TaqMan assays (Applied Biosystems/AB, Carlsbad, CA, USA) using StepOne Real-Time platform (AB, Carlsbad, CA, USA). Allelic discrimination was performed using the StepOne software (AB).
Subjects Hundred and ninety-two healthy pregnant women with uncomplicated pregnancies (healthy controls, HC; 26.3 6.5 years) and 160 pregnant with primary preeclampsia (PE; 27.0 6.7 years) were recruited at Hospital Sofia Feldman in Belo Horizonte (Central-Southern Brazil). Hundred and twentysix PE (30.3 7.5 years) and 117 HC (28.1 7.4 years) were recruited at the Maternity Unit of at public hospital in Southern Brazil (Hospital Nossa Senhora Conceicß~ ao, PortoAlegre). Women were classified as European or African-derived according to phenotypic characteristics and ethnicity data of parents/grandparents reported by the participants in an appropriate questionnaire. Preeclampsia was defined for the presence of hypertension [blood pressure > 140 mm Hg (systolic) and/or 90 mm Hg (diastolic)] and proteinuria (>300 mg of protein every 24 hr).11 Healthy pregnant women inclusion criteria were as fol-
Data Analysis R software (www.r-project.org) was used to perform Fisher exact test and odds ratio (OR) calculation for alleles and haplotypes and for genotype association and inheritance modeling (package ‘SNP assoc’ version 1.5–2).16 A formal Bonferroni correction for the number of SNPs analyzed would require a significance threshold of P = 0.006 (P0/N, P0 = 0.05, N = 8 SNPs). The Haploview software 17 was used to investigate the association and linkage disequilibrium (LD) pattern and for deriving the haplotypes.
Table I Clinical Characteristics of Women with and Without Preeclampsia
Characteristics Maternal age Pre-BMI SBP (mmHg) DBP (mmHg) Delivery weeks Fetal weight (g) Ethnicity (% white European origin)
Controls (HC; n = 309)
Preeclampsia (PE; n = 286)
25.9 26.7 120.6 67.1 38.0 2851.9 16.7%
26.7 26.2 153.6 94.8 35.1 2212.7 20.6%
6.1 3.4 12.8 8.7 2.2 651.9
a
6.7 3.8a 17.3 14.1 2.8b 591.1b
Early delivery PE (n = 38) 25.6 26.3 160 100 31.3 1624.1
6.1 4.3 15.2 11.3 2.3 433.3
Preterm PE (n = 71) 28.3 24.8 154.7 93.8 35.9 2313.1
7.4 5.5 18.3 13.1 0.4 370.6
PE (n = 47) 27.2 25.8 149.3 90.3 38.3 2780.5
6.6 3.4 17.8 18.2 0.8 492.7
Pre-BMI, pre-pregnancy body mass index; SBP, blood pressure, systolic; DBP, blood pressure, diastolic. a P-value > 0.05 versus HC. b P-value < 0.05 versus HC.
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American Journal of Reproductive Immunology 73 (2015) 577–581 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
NLRP1 AND PREECLAMPSIA
Results and discussion Two hundred and eighty-six women with primary preeclampsia and 309 healthy pregnant women were genotyped for eight selected SNPs in four inflammasome genes (NLRP1, NLRP3, CARD8, and IL1B). SNPs allelic and genotypic frequencies were in Hardy–Weinberg equilibrium in both groups. While other SNPs appeared to be similarly distributed in PE and HC (results reported in Table S1), the NLRP1 (rs12150220) A/T polymorphism was significantly associated with PE (Table II). NLRP1 (rs12150220) minor T allele was significantly more frequent in PE than in HC women (0.45 versus 0.34; P = 3.2 exp5), suggesting a predisposing role of this allele in susceptibility to develop PE (OR = 1.58; 95% CI = 1.22–2.05). Rs12150220 genotypes distribution was also significantly different in the two groups, and according to the Akaike information criterion, predisposing T allele behaved according to a codominant model (P = 8.6 exp05). Even when we corrected the analysis for age, ethnicity, and geographic
Table II Allelic and Genotypic Frequencies of rs12150220 in Women with and Without Preeclampsia
PE (n = 286) HC (n = 309)
Alleles frequency (T/ A)
Pa
0.45/0.55
3.2 exp5
0.24/0.45/0.31 0.11/0.46/0.43
Table III Haplotypes Frequencies of SNPs in NLRP1, NLRP3, and CARD8 Genes in Women with and Without Preeclampsia
Haplotypes OR 1.58
95% CI 1.22–2.05
0.34/0.66
Genotypes frequency (TT/ AT/AA) PE (n = 286) HC (n = 309)
NLRP1
origin, the rs12150220 variation continued to be significantly associated with preeclampsia in a codominant way (P = 1.53 exp05) (Table II). Women with preeclampsia were then stratified for delivery time in three groups: early (0.05), which are similarly distributed between case and controls (P > 0.05) (Table III). SNPs in NLRP1 and NLRP3 genes did not appear to be in LD in the studied populations, but the com-
Pcodb (Padjust)
Pdomb (Padjust)
Precb (Padjust)
8.6 exp5 (1.5 exp4)
0.004 (5.3 exp4)
5.3 exp5 (6.3 exp4)
P, P-value; OR, odds ratio; 95% CI, 95% confidence interval. a Fisher test. b Likelihood ratio test. Padjust, P-value adjusted by age, ethnicity, and origin; Pcod, P-value of codominant model (three genotypes); Pdom, P-value of dominant model [(homozygotes of risk allele+heterozygotes) versus (homozygotes of non-risk allele)]; Prec, P-value of recessive model [(homozygotes of risk allele) versus (heterozygotes+homozygotes of non-risk allele)]. The T allele is the risk allele.
American Journal of Reproductive Immunology 73 (2015) 577–581 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Haplotypes frequency (PE, HC)
Pa
OR (95% CI)
NLRP1 rs11651270-rs12150220-rs2670660 (rs11651270-rs12150220: D0 = 0.11; r2 = 0.009; rs12150220-rs2670660: (D0 = 0.64; r2 = 0.30) T-A-A 0.237, 0.278 0.141 0.81 C-T-G 0.198, 0.186 0.641 1.08 C-A-A 0.140, 0.220 8.1 exp5 0.58 T-T-G 0.154, 0.131 0.333 1.20 T-A-G 0.088, 0.076 0.501 1.19 C-A-G 0.067, 0.069 1.0 0.98 NLRP3 rs35829419- rs10754558 (D0 = 0.89; r2 = 0.03) C-C 0.654, 0.678 0.395 0.89 C-G 0.320, 0.307 0.69 1.06 A-G 0.025, 0.013 0.016 2.98 CARD8 rs2043211-rs6509365 (D0 = 0.99; r2 = 0.84) A-A 0.666, 0.694 0.349 0.88 T-G 0.297, 0.268 0.298 1.16
(0.61–1.07) (0.79–1.48) (0.41–0.81) (0.84–1.72) (0.75–1.89) (0.59–1.62)
(0.69–1.17) (0.81–1.38) (1.19–8.45)
(0.67–1.15) (0.87–1.54)
P, P-value; OR, odds ratio; 95% CI, 95% confidence interval. a Fisher test. Linkage disequilibrium indexes (D0 : normalized disequilibrium index; r2: correlation coefficient). Statistically significant values are indicated in bold.
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bined effect of polymorphisms within the same genes was evaluated for those genes. NLRP1 SNPs combined to form 6 main haplotypes, and the rs11651270/Crs12150220/A-rs2670660/A combination was significantly less frequent in PE women as compared to controls (0.14 versus 0.22), suggesting of a protective effect against preeclampsia development (P = 8.1 exp5; OR = 0.58; 95% CI = 0.41–0.81), as expected because it contains the rs12150220 major allele A instead of the risk T allele (Table III). The frequencies of all other combined haplotypes in NLRP1 as well in NLRP3 genes were not statistically different in the two groups (Table III). Considering previously reported data about the combined effect of the two NLRP3 rs35829419 (Q705K) and CARD8 rs2043211 (C10X) variants,18 we also analyzed this combination in our case/control population. However, no statistical significant association was observed with preeclampsia (P > 0.05; data not shown). Even considering that the NLRP3-inflammasome was previously pointed out as a main factor in IL-1ß dysregulation in preeclampsia,8 due to its reported role in placenta physiology7 as well as in uric acid response8 and essential hypertension,19 and that rs35829419 (Q705K) and rs10754558 (30 UTR) have been associated with elevated IL-1ß level and inflammatory condition,20,21 those NLRP3 variants did not resulted associated with preeclampsia in our studied population. In contrast, a strong association with NLRP1 rs12150550 missense variation (L155H) was observed, suggesting a role of this inflammasome receptor in the pathogenesis of preeclampsia. NLRP1 is involved in caspase-1 and caspase-5 mediated IL-1ß secretion and pyroptose; however, the cognate stimulus leading to NLRP1-inflammasome activation is still not fully elucidated.22 NLRP1 rs12150550 has been reported to be associated with an increased secretion of IL-1ß.23 NLRP1 expression is seen in immune and other cell types, such as fibroblasts,24 as well as in brain and in the respiratory tract.25 Recently, NLRP1 has been described in endothelial cells,26 leading us to hypothesize that it is involved in the pathogenesis of preeclampsia mainly due to its role in endothelial cells. In conclusion, the NLRP1 variant rs12150220 was associated with preeclampsia development suggesting a role for this inflammasome receptor in the pathogenesis of this multifactorial disorder.
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American Journal of Reproductive Immunology 73 (2015) 577–581 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Linkage Disequilibrium analysis. Results for Linkage Disequilibrium (LD) analysis of studied SNPs. D’/LOD values and color scheme were reported according to Haploview software. Table S1. Allelic and genotypic frequencies in women with and without preeclampsia. Table S2. Association analysis for NLRP1 rs12150220 in women with and without preeclampsia stratified for delivery time and age.
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