Risk of spontaneous preterm birth and fetal growth associates with fetal SLIT2

RESEARCH ARTICLE Risk of spontaneous preterm birth and fetal growth associates with fetal SLIT2 Heli Tiensuu 1 , Antti M. HaapalainenID 1 , Minna K. Karjalainen 1 , Anu Pasanen 1 , Johanna M. HuuskoID 1,2 , Riitta Marttila 1 , Marja OjaniemiID 1 , Louis J. MugliaID 2 , Mikko HallmanID 1☯ , Mika Ra ¨met 1,3☯ * 1PEDEGO Research Unit, Medical Research Center Oulu, University of Oulu, and Department of Children and Adolescents, Oulu University Hospital, Oulu, Finland, 2Division of Human Genetics, Center for Prevention of Preterm Birth, Perinatal Institute, Cincinnati Children’s Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, March of Dimes Prematurity Research Center Ohio Collaborative, Cincinnati, Ohio, United States of America, 3Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland ☯These authors contributed equally to this work. *[email protected] Abstract Spontaneous preterm birth (SPTB) is the leading cause of neonatal death and morbidity worldwide. Both maternal and fetal genetic factors likely contribute to SPTB. We performed a genome-wide association study (GWAS) on a population of Finnish origin that included 247 infants with SPTB (gestational age [GA] <36 weeks) and 419 term controls (GA 38–41 weeks). The strongest signal came within the gene encoding slit guidance ligand 2 (SLIT2; rs116461311, minor allele frequency 0.05, p= 1.6×10 −6 ). Pathway analysis revealed the top-ranking pathway was axon guidance, which includes SLIT2. In 172 very preterm-born infants (GA <32 weeks), rs116461311 was clearly overrepresented (odds ratio 4.06, p= 1.55×10 −7 ). SLIT2 variants were associated with SPTB in another European population that comprised 260 very preterm infants and 9,630 controls. To gain functional insight, we used immunohistochemistry to visualize SLIT2 and its receptor ROBO1 in placentas from spontaneous preterm and term births. Both SLIT2 and ROBO1 were located in villous and decidual trophoblasts of embryonic origin. Based on qRT-PCR, the mRNA levels of SLIT2 and ROBO1 were higher in the basal plate of SPTB placentas compared to those from term or elective preterm deliveries. In addition, in spontaneous term and preterm births, placental SLIT2 expression was correlated with variations in fetal growth. Knockdown of ROBO1 in trophoblast-derived HTR8/SVneo cells by siRNA indicated that it regulate expression of several pregnancy-specific beta-1-glycoprotein (PSG) genes and genes involved in inflammation. Our results show that the fetal SLIT2 variant and both SLIT2 and ROBO1 expression in placenta and trophoblast cells may be correlated with susceptibility to SPTB. SLIT2-ROBO1 signaling was linked with regulation of genes involved in inflammation, PSG genes, decidualization and fetal growth. We propose that this receptor-ligand couple is a component of the signaling network that promotes SPTB. PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008107 June 13, 2019 1 / 25 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Tiensuu H, Haapalainen AM, Karjalainen MK, Pasanen A, Huusko JM, Marttila R, et al. (2019) Risk of spontaneous preterm birth and fetal growth associates with fetal SLIT2. PLoS Genet 15 (6): e1008107. https://doi.org/10.1371/journal. pgen.1008107 Editor: Gregory S. Barsh, Stanford University School of Medicine, UNITED STATES Received: December 21, 2018 Accepted: March 18, 2019 Published: June 13, 2019 Copyright: ©2019 Tiensuu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The transcriptomics data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE119101. All other relevant data are available within the manuscript and its Supporting Information files. Funding: This work was supported by the Jane and Aatos Erkko Foundation (MH and MR), Sigrid Juse ´lius Foundation (MH) and Competitive State Research Financing of the Expert Responsibility Area of Oulu University Hospital (MR), March of Author summary Worldwide, more than 10% of babies are born prematurely without effective means of prevention. Premature birth is associated with mortality and lifelong comorbidities. Aggregation of spontaneous preterm birth in certain families suggests that there are underlying genetic factors that predispose to preterm birth. Both maternal and fetal genomes likely affect susceptibility. We set out to identify fetal genetic variants that predispose infants to premature birth in a population of Finnish origin. Our results from a genome-wide association study indicate that a variant of slit guidance ligand 2 (SLIT2) is associated with the risk of spontaneous preterm birth. Furthermore, SLIT2 and its receptor roundabout guidance receptor 1 (ROBO1) are expressed in placental cells, and their mRNA levels are higher in placentas from spontaneous preterm deliveries compared to term controls. Based on gene knockdown experiments in cultured placental tissue– derived cells, ROBO1 regulates expression of pregnancy-specific beta-1-glycoprotein (PSG) genes and genes involved in inflammation. Thus, our results indicate that the fetal SLIT2 variant and expression of both SLIT2 and ROBO1 in placental cells are correlated with susceptibility to spontaneous preterm birth. We propose that this receptor–ligand pair is a component of the signaling network that promotes spontaneous preterm birth. Introduction Preterm live births that take place before 37 completed weeks of gestation and even as early as 22–24 weeks are a global problem. Up to 11.1% (15 million babies) of all births worldwide occur prematurely, and approximately 45–50% of them are idiopathic or spontaneous [1–3]. Complications caused by preterm birth are the most common cause of neonatal deaths and the largest direct cause of deaths of children <5 years of age [1,3]. The research focusing on spontaneous preterm birth (SPTB) has been complicated by etiological, pathophysiological, and genetic heterogeneities. Multiple events are associated with SPTB, either independently or in concert [4]. These include intrauterine inflammation, called chorioamnionitis, preterm premature rupture of fetal membranes (PPROM) and abnormal fetal growth relative to uterine size [5,6]. It is important to find new biomarkers for early detection of SPTB. Currently, our understanding of the early molecular pathways leading to SPTB is incomplete and there are no effective means to prevent SPTB. Knowledge of how maternal and fetal genomes contribute to the risk of SPTB could provide more personalized tools to prevent it [7]. Epidemiological studies have shown that both fetal and maternal genes affect fetal growth, birth weight, birth length, head circumference, and gestational age (GA) [8–11]. A recent study indicated that variants of the fetal and maternal genome independently affect normal variations in birth weight [12]. In addition, maternal and fetal genomes are also considered to affect the susceptibility to preterm birth and duration of pregnancy in general [13–15]. The intrauterine environment influences fetal growth, and adverse intrauterine events affect pregnancy length not only in elective preterm pregnancies but also in SPTB [11]. Genetic analysis of 244,000 Swedish births resulting in twins, full siblings, and half-siblings revealed that 13% and 21% of the variation in birth timing is explained by fetal and maternal genetic factors, respectively [9]. Overall, preterm birth is a phenotype with contributions from both, maternal and fetal genomes that may have separate contributions and together with environmental factors, interactively determine the outcome [7,13,16]. A recent study that included a population of >40,000 women and replication cohorts of >8000 women identified several common variants in EBF1,EEFSEC, and AGTR2 that SLIT2 and ROBO1 in spontaneous preterm birth PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008107 June 13, 2019 2 / 25 Dimes Prematurity Research Center Ohio Collaborative, March of Dimes, URL:http://www. marchofdimes.org/, the Bill and Melinda Gates Foundation, URL:http://www.gatesfoundation.org (OPP1113966, LJM) and NIH/The Eunice Kennedy Shriver National Institute of Child Health and Human Development, URL:https://www.nichd.nih. gov/ (HD091527, LJM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: No authors have competing interests. showed associations with preterm birth at a genome-wide significance level [13]. In addition, other genome-wide association studies (GWAS) and SPTB genetic studies focused on mother [17–19] or infant [19–21] genomic signals have discovered genetic loci associated with preterm birth and gestational length. Studies that focused on fetal genomes have not revealed replicable associations between fetal genetic factors and SPTB. Many pathways and cellular processes are reported to be associated with SPTB, including response to infection, regulation of inflammation, stress, and other immunologically mediated processes [3]. According to our current understanding, inflammatory pathways also have roles in the initiation of spontaneous term birth, as normal labor starts when there is a shift in signaling between anti-inflammatory and proinflammatory pathways in the myometrium. This shift appears to involve many chemokines such as interleukin 8 (IL8), cytokines such as IL1 and IL6, and contraction-associated proteins such as oxytocin receptor (OXTR), connexin 43 (CX43), and prostaglandin receptors [22]. Therefore, it is likely that changes in inflammationassociated pathways also contribute to preterm birth. Evidence from candidate gene studies supports the role of inflammation-related factors in SPTB. For example, polymorphisms of the genes encoding TLR4, TNF, IL1B, interferon gamma (IFNγ), IL6, and matrix metalloproteinases may be associated with increased risk of SPTB [5]. The aim of the present study was to use a GWAS to investigate fetal genetic variants that may predispose infants to SPTB in a homogeneous population of Finnish origin. A variant of slit guidance ligand 2 (SLIT2) had the most suggestive association with SPTB in the GWAS and in a genetic pathway analysis. Therefore, we characterized SPTB-associated expression of SLIT2 and its receptor ROBO1 in the placenta and subsequently conducted experiments with relevant placenta-associated cells. Results GWAS association signals for spontaneous preterm birth In order to find fetal genetic factors associated with predisposition to SPTB, we analyzed polymorphisms encompassing the entire genome for associations with SPTB. After quality control, 247 infants born spontaneously preterm and 419 infants born at term remained for inclusion in the GWAS. We performed the analysis for both GA (quantitative trait) and SPTB (dichotomous setting). However, due to sample collection bias resulting in skewed GA distribution, we acknowledge that the results of the quantitative trait analysis should be interpreted with caution. Fig 1 summarizes the study workflow. We detected several suggestive associations (p<10 −5 ) in the GWAS (Fig 2,Table 1). The two most promising regions were within the genes encoding SLIT2 (rs116461311, p= 1.6 × 10 −6 ) and succinyl-CoA:glutarate-CoA transferase (SUGCT; rs57670997, p= 1.8 ×10 −6 ). We also detected suggestive associations for GA as a quantitative trait (S2 Table,S1 Fig). SNP rs116461311 within the SLIT2 gene showed the most significant signal for GA (p= 3.1 ×10 −7 , S1 Fig). In addition to SLIT2, four regions showed suggestive signals both in the primary setting and in the GWAS of GA; these signals were within SUGCT, an intergenic region in chromosome 6 (nearest loci LOC105377949 and LOC107986634), and within the genes encoding anaplastic lymphoma receptor tyrosine kinase (ALK) and DLC1 Rho GTPase activating protein (DLC1). We further analyzed very preterm and moderate-to-late preterm SPTB infants separately against term-born controls (S3 and S4 Tables). Three regions showed suggestive signals (p<10 −5 ) both in the primary setting and in the analysis of very preterm birth (S3 Table). These signals were within SLIT2, in an intergenic region on chromosome 2 (nearest genes THUMPD2 and SLC8A1), and within the region encompassing the EXOSC1,ZDHHC16, SLIT2 and ROBO1 in spontaneous preterm birth PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008107 June 13, 2019 3 / 25 Fig 1. Overview of the study workflow. https://doi.org/10.1371/journal.pgen.1008107.g001 Fig 2. Manhattan plot of GWAS of SPTB. Each dot represents −log 10 (p) value of a single SNP in association analysis. Dashed line denotes level of suggestive significance (−log 10 (p)>5). SLIT2,SUGCT, and EXOSC1-ZDHHC16-MMS19UBTD1 were among the best associating loci. https://doi.org/10.1371/journal.pgen.1008107.g002 SLIT2 and ROBO1 in spontaneous preterm birth PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008107 June 13, 2019 4 / 25 MMS19, and UBTD1 genes, which encode exosome component 1; zinc finger DHHC-type containing 16; MS19 homolog, cytosolic iron-sulfur assembly component; and ubiquitin domain containing 1, respectively. The minor allele of SLIT2, SNP rs116461311, was overrepresented (OR 4.06, p= 1.55 ×10 −7 ) in very preterm-born infants (GA <32 weeks) compared to term-born infants. In moderate-to-late preterm infants (GA 32–36 weeks), two regions showed suggestive signals (S4 Table) that were also evident in the primary analysis: an intergenic region on chromosome 3 (nearest loci LOC105377173 and ROBO1) and within ADAMTS14, which encodes ADAM metallopeptidase with thrombospondin type 1 motif 14. We also studied associations with SPTB within the contexts of PPROM and no PPROM. There were separate suggestive associations with SPTB-PPROM and with SPTB without PPROM (S5 and S6 Tables). For the SLIT2 region, the effects were similar for infants born after PPROM (rs116461311, OR = 3.5, p= 3.0 ×10 −5 ) and for those born after spontaneous onset of labor with intact fetal membranes (rs116461311, OR = 3.6, p= 1.3 ×10 −5 ). To investigate potential maternal transmission of the minor allele of SPTB-associated rs116461311 (in SLIT2), we checked the MAF of the variant in maternal samples. The frequency of C-rs116461311 was 0.059 in SPTB mothers (n= 230) and 0.035 in mothers with term delivery (n= 378). Transmission analysis was not feasible because of the low minor allele counts. Replication of SLIT2 association in a European population SNPs with suggestive association signals in the region of the top GWAS gene (SLIT2) were examined for association with SPTB in a European population (n= 9890) [20]. In a European Table 1. Single-nucleotide polymorphisms suggestively associated with spontaneous preterm birth. Chr Locus a SNP b Reference allele Allele frequency Odds ratio (95% confidence interval) p 4SLIT2 rs116461311 C 0.05 3.43 (2.01–5.86) 1.58E-6 7SUGCT rs57670997 Del 0.68 0.56 (0.44–0.71) 1.79E-6 9 Intergenic (LOC105376188,LOC105376190) rs35464758 Del 0.05 2.82 (1.67–4.80) 1.82E-6 5 Intergenic (LOC100128898,LOC100130177) rs4704916 A 0.19 1.91 (1.45–2.52) 2.22E-6 10 EXOSC1-ZDHHC16-MMS19-UBTD1 c rs10678727 Ins 0.58 1.77 (1.40–2.23) 2.39E-6 2 Intergenic (THUMPD2,SLC8A1) rs17480505 C 0.35 1.72 (1.36–2.17) 3.88E-6 21 DSCAM rs9974083 A 0.24 1.73 (1.34–2.24) 4.32E-6 2PPP1CB-SPDYA-TRMT61B-WDR43 c rs200334508 G 0.87 0.51 (0.37–0.71) 4.44E-6 11 DLG2 rs202170665 Ins 0.08 0.40 (0.25–0.65) 4.59E-6 10 Intergenic (RBM17,LOC1019280803) rs10905856 T 0.08 2.28 (1.53–3.41) 4.87E-6 10 ABI1 rs72385215 Del 0.33 0.56 (0.44–0.72) 4.89E-6 10 ADAMTS14 rs12765664 A 0.93 0.40 (0.26–0.61) 5.95E-6 8DLC1 rs7006225 A 0.77 0.56 (0.43–0.72) 6.99E-6 6 Intergenic (LOC105377949,LOC107986634) rs1418269 C 0.69 1.73 (1.34–2.22) 7.51E-6 18 DLGAP1 rs610269 G 0.54 0.61 (0.49–0.76) 7.97E-6 2NCKAP5 rs1966628 T 0.45 1.64 (1.31–2.05) 8.29E-6 20 HSPA12B rs58505239 T 0.20 1.75 (1.34–2.29) 8.66E-6 3 Intergenic (LOC105377173,ROBO1) rs115723230 T 0.03 3.33 (1.70–6.54) 8.70E-6 1EIF1P3 rs539974331 Del 0.04 3.57 (1.95–6.55) 8.92E-6 9SVEP1 rs199638820 Del 0.56 1.61 (1.29–2.02) 9.56E-6 2ALK rs7608573 T 0.20 0.52 (0.38–0.70) 9.99E-6 a Respective gene shown for SNPs within genes; two nearest genes shown in parentheses for intergenic SNPs. b SNP with the most significant signal shown for each locus. c Association signal spans the indicated genes. https://doi.org/10.1371/journal.pgen.1008107.t001 SLIT2 and ROBO1 in spontaneous preterm birth PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008107 June 13, 2019 5 / 25 replication population of very preterm and term-born controls, rs12503652 and rs79034379, which correlate with the best GWAS SNP of the SLIT2 region (rs116461311), were associated with SPTB (Table 2). The two SNPs were in high LD with one another (r 2 = 0.85, D0= 0.95) in the Finnish individuals in 1000 Genomes phase3 data. Functional annotation of the most promising genetic variants To investigate if the SNPs within regions with the most promising signals in the GWAS (p<10 −5 ) have functional consequences, we screened the GTEx data to examine whether any of the suggestively associated SNPs colocalize with cis eQTLs; that is, whether they correlate with mRNA levels in the analyzed tissues. In the GTEx data, 26 of the SNPs with p<10 −5 for association with SPTB overlapped with significant eQTLs (Table 3). These SNPs were located within five different regions; the majority were in the region encompassing EXOSC1, ZDHHC16,MMS19, and UBTD1 (Tables 1,S3 and S5). These SNPs correlated with mRNA levels of the following genes in different tissues: ZDHHC16,MMS19,FRAT1,ANKRD2, UBTD1, and RRP12. Associated SNPs within the top GWAS region, SLIT2, were not associated with mRNA levels. According to the GWAS catalog, none of the suggestively associated SNPs had been significantly associated with any phenotype. We further functionally annotated the SNPs (p<10 −4 ) within the SLIT2 region with HaploReg, v 4.1. Some of the most promising SNPs within SLIT2 (including the best associating variant rs116461311, as well as rs60126904, rs115707845, and rs16869667) were located within regions that contain histone marks and DNase-hypersensitive sites in several tissues. Table 2. Two SNPs that correlated with the top GWAS SNP rs116461311 in SLIT2 showed association with SPTB in European replication data. R 2 in 1000 Genomes FIN Discovery GWAS European replicate SNP rs116461311 Reference allele Odds ratio POdds ratio p rs116461311 1 C 3.43 1.58E-6 NA NA rs12503652 0.58 G 2.11 3.83E-4 1.47 0.023 rs79034379 0.61 G 2.02 3.49E-4 1.46 0.032 https://doi.org/10.1371/journal.pgen.1008107.t002 Table 3. Suggestively associated SNPs that overlap with cis eQTLs. SNP information cis-eQTL information GWAS region SNP a pin GWAS eGene eQTL p b Tissue EXOSC1-ZDHHC16-MMS19-UBTD1 rs7897727 c 2.03E-06 ZDHHC16 MMS19 FRAT1 2.0E-18 1.7E-07 2.0E-05 2.6E-06 5.0E-06 Testis Adipose–Subcutaneous Breast–Mammary tissue Lung Cells–Transformed fibroblasts DSCAM rs9974083 4.32E-06 PCP4 2.3E-07 Artery—Tibial Intergenic, chr6 rs1418269 7.51E-06 RP1-142L7.53.4E-05 Esophagus—Muscularis NCKAP5 rs1966628 d 8.29E-06 NCKAP5 1.3E-05 Testis HSPA12B rs58505239 8.66E-06 CDC25B ADAM33 1.8E-07 1.3E-06 2.9E-06 Adipose–Subcutaneous Artery—Tibial Esophagus—Muscularis a Top eQTL SNP shown for each region. b eQTL data from GTEx Portal V7. c Altogether, 15 suggestively associated SNPs within EXOSC1-ZDHHC16-MMS19-UBTD1 region colocalized with eQTLs; these SNPs are eQTLs for ZDHHC16, MMS19,FRAT1,ANKRD2,UBTD1, and RRP12 d Altogether, eight suggestively associated SNPs within NCKAP5 region are eQTLs for NCKAP5. https://doi.org/10.1371/journal.pgen.1008107.t003 SLIT2 and ROBO1 in spontaneous preterm birth PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008107 June 13, 2019 6 / 25 Furthermore, SLIT2 SNPs (rs60126904 and rs115707845) mapped to predicted enhancers in several cell types and tissues, including neuronal cells, different cells in the brain, lung, spleen, adipose cells, colon and duodenum smooth muscle cells, fetal lung and kidney, and fetal membranes (amnion). Thus, there is some evidence of the putative regulatory effects of SLIT2 SNPs in several tissues, including fetal tissues such as placenta. Biological pathways genetically associated with spontaneous preterm birth To identify biological pathways associated with SPTB, we performed pathway analysis of the GWAS data to search for gene set enrichment. SPTB was associated with 16 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways [corrected (S7 Table) and FDR adjusted p<0.05, Table 4]. The most significant pathways were axon guidance (p= 8.6 ×10 −10 ), focal adhesion (p= 6.6 ×10 −7 ), and vascular smooth muscle contraction (p= 1.4 ×10 −6 ). Axon guidance, the most significant pathway, included SLIT2 and the gene encoding its receptor, ROBO1. A Gene Ontology (GO) search revealed 35 GO terms associated with SPTB, with a false-discovery rate (FDR) of <0.05 (S8 Table). GO sets that included SLIT2 and ROBO1 are listed in S9 Table. The three most significant GO sets that included SLIT2 were retinal ganglion cell axon guidance, telencephalon development, and negative chemotaxis. The three most significant GO sets that included ROBO1 were telencephalon development, neuron recognition, and negative chemotaxis. These results led us to a more-detailed investigation of the roles of SLIT2 and ROBO1 in SPTB. SLIT2 and its receptor ROBO1 are localized in villous and decidual trophoblasts We detected suggestive association signals for SNPs in a region that encompasses SLIT2 and in a region downstream of ROBO1. Protein Slit2 binds to Robo proteins specifically and with high affinity [23,24]. Therefore, we analyzed the localization of SLIT2 and its receptor ROBO1 in human placenta by immunohistochemical staining of placentas from SPTB and spontaneous term birth (STB) with anti-human SLIT2 and ROBO1 antibodies (Fig 3). Both SLIT2 and ROBO1 localized to cytotrophoblasts, syncytiotrophoblasts, and decidual trophoblasts. In addition, we observed strong ROBO1 and faint SLIT2 staining in capillary endothelial cells. We also detected both proteins in the basal and chorionic plates of the placenta. We did not see apparent differences in staining intensities or cellular localization between placentas from SPTB and STB. This indicates that SLIT2 and ROBO1 are expressed in the placenta at the interface between mother and fetus during pregnancy. Table 4. Pathways associated with SPTB. Pathway Pathway name pCorrected p a Top genes in pathway b hsa04360 Axon guidance 8.6E-10 1.6E-07 SLIT2,PAK6,NRP1,SEMA3E,ABLIM2 hsa04510 Focal adhesion 6.6E-07 6.2E-05 PPP1CB,AKT3,PAK6,EGFR,PARVB hsa04270 Vascular smooth muscle contraction 1.4E-06 8.9E-05 PPP1CB,PLA2G4A,KCNMA1,ADRA1B,PRKG1 hsa04720 Long-term potentiation 2.3E-05 1.1E-03 PPP1CB,GRIN2B,GRM5,CAMK2G,PLCB1 hsa04730 Long-term depression 7.0E-05 2.6E-03 PLA2G4A,GRM5,PRKG1,LYN,PLCB1 hsa04012 ErbB signaling pathway 4.1E-04 0.01 AKT3,PAK6,EGFR,PLCG1,NRG2 hsa04514 Cell adhesion molecules (CAMs) 4.4E-04 0.01 HLA-DOA,NLGN1,CDH4,ITGAM,NCAM2 hsa05412 Arrhythmogenic right ventricular cardiomyopathy (ARVC) 4.7E-04 0.01 CACNA2D3,CACNB2,CACNA1D,ACTN1 a KEGG pathways with corrected and FDR adjusted p<0.05 shown. b Top five genes or all genes with SNPs with p<1E-02 in GWAS shown. https://doi.org/10.1371/journal.pgen.1008107.t004 SLIT2 and ROBO1 in spontaneous preterm birth PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008107 June 13, 2019 7 / 25 SLIT2 and ROBO1 mRNA level elevation in SPTB placentas Immunohistochemistry demonstrated SLIT2 and ROBO1 in different types of trophoblasts in human placenta (Fig 3). To obtain more quantitative data about placental expression of these proteins, we analyzed SLIT2 and ROBO1 mRNA levels by qRT-PCR in samples collected from the basal and chorionic plates of placentas from SPTB (n= 23), STB (n= 23), and elective preterm birth (EPTB) (n= 34). We first compared SLIT2 and ROBO1 expression levels between SPTB (n= 23) and STB (n= 23) placentas (Fig 4). Both SLIT2 and ROBO1 mRNA levels were higher in the basal plate of SPTB placentas (SLIT2 fold change [FC] = 1.679, SD = 0.667; ROBO1 FC = 1.387, SD = 0.670) compared to those of STB (SLIT2 p = 0.004, ROBO1 p = 0.013; Fig 4A). There were no differences in mRNA levels of SLIT2 (p= 0.173) and ROBO1 (p= 0.297) between the chorionic plates of SPTB and STB placentas (Fig 4B). To explore the effects of mode of delivery and GA on SLIT2 and ROBO1 mRNA levels, we compared SPTB with EPTB placentas. SLIT2 and ROBO1 mRNA levels were significantly higher for SPTB (SLIT2 FC = 1.595, SD = 0.580; ROBO1 FC = 1.282, SD = 0.577) compared to EPTB (SLIT2 p = 0.005, ROBO1 p = 0.031) in basal plate samples (Fig 4A). There were no significant differences in these levels in EPTB and STB placentas (SLIT2 p = 0.216, ROBO1 p = 0.328; Fig 4A). These results suggest that higher SLIT2 and ROBO1 expression levels are associated with SPTB. Fig 3. Placental localization of SLIT2 and ROBO1 in spontaneous preterm and term births. Samples from 18 placentas were immunostained with anti-human SLIT1 (A) and ROBO1 (B) antibodies. Twelve of the placentas were from SPTB deliveries, and six were from spontaneous term deliveries. Two samples from each placenta were stained; one sample from the basal plate (maternal side) and the other from the chorionic plate (fetal side). Immunostaining is indicated by filled large arrows in cytotrophoblasts, unfilled large arrows in syncytiotrophoblasts, filled small arrows in decidual trophoblasts, and filled arrowheads in capillary endothelial cells. Original magnification is 20×in all figures. Control represents isotype controls for immunostaining. Scale bar, 100 μm. https://doi.org/10.1371/journal.pgen.1008107.g003 SLIT2 and ROBO1 in spontaneous preterm birth PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008107 June 13, 2019 8 / 25 Association of SLIT2 expression with fetal growth SLIT/ROBO are involved in many processes that involve cell migration, including axon guidance; thus, they could affect trophoblast cell invasion and decidualization. To this end, we Fig 4. mRNA levels of SLIT2 and ROBO1 in spontaneous preterm, spontaneous term, and elective term placentas. Relative mRNA levels from the basal (A) or chorionic (B) plates were normalized to mRNA levels of the housekeeping gene CYC1. Differences were analyzed with nonparametric Mann–Whitney U-test. Asterisk indicates statistically significant changes. Box and whiskers display quartiles. Band inside the box is the median, and ends of whiskers show the minimum and maximum values; square inside the box represents the mean value. https://doi.org/10.1371/journal.pgen.1008107.g004 SLIT2 and ROBO1 in spontaneous preterm birth PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008107 June 13, 2019 9 / 25 DNA sample preparation, genotyping, and SNP inclusion criteria Umbilical cord blood, umbilical cord tissue, or saliva was obtained from the study subjects. Commercial kits were used to extract genomic DNA from blood (UltraClean Blood DNA Isolation Kit; MO BIO Laboratories, Inc., Carlsbad, CA, USA or Puregene Blood Core Kit; Qiagen, Hilden, Germany) and cord tissue (Gentra Puregene Tissue Kit, Qiagen). OraGene DNA collection kits (DNA Genotek, Ontario, Canada) were used for collecting saliva, and DNA was extracted with the prepIT-L2P kit (DNA Genotek). Genome-wide SNP genotyping was performed with the Infinium HumanCoreExome BeadChip (Illumina, San Diego, CA, USA) by the Technology Centre, Institute for Molecular Medicine Finland (FIMM), University of Helsinki. Processing of genome-wide SNP data Genome-wide SNP data were processed with PLINK, v. 1.9 [74]. SNPs with minor allele frequency (MAF) <0.01, genotyping rate <0.9, or deviation from Hardy–Weinberg equilibrium (p<0.0001) were excluded. Individuals with >0.1 missing genotypes were excluded. Identical by descent (IBD) clustering and multidimensional scaling (MDS) analyses were performed with a linkage disequilibrium–pruned SNP set; population outliers and close relatives (pihat >0.2) were excluded. Prephasing of genotypes was performed with SHAPEIT2 [75], followed by statistical imputation with IMPUTE2 [76] using the 1000 Genomes Phase 3 variant set (October 2014) as the reference panel. Before association analysis, SNPs with impute info score <0.8 or MAF <0.05 in cases or controls were excluded. Altogether 6,778,521 SNPs or short insertions/deletions remained for analysis after these quality control steps. Statistical analyses in genetic studies Associations between SPTB or GA and SNPs were assessed with the frequentist test under the additive model with SNPtest, v. 2.5.2 [77]. After the primary analysis, the following subgroups of SPTB infants were assessed: (1) very preterm infants (GA 23–31 wk + 6 d), (2) moderate-tolate SPTB infants (GA 32 wk + 0 d to 36 wk + 0 d), (3) PPROM before onset of labor, and (4) no PPROM before onset of labor. To account for population substructure in the GWAS, the first two MDS dimensions were included as covariates. In the GWAS, the effect of population stratification was minimal (λ= 1.03). Gene set analysis (GSA)-SNP was used to search for gene set enrichment in pathway analysis [78]. We included only genotyped SNPs located within genes in this analysis to avoid the complicating effects of SNPs in linkage disequilibrium. R, v. 3.2.2 (https://www.r-project.org) was used to create Manhattan plots. LocusZoom [79] was used to create regional association plots. Functional annotation of SNPs We annotated SNPs with three approaches: (1) We used Genotype-Tissue Expression (GTEx) data to analyze whether the SNPs overlap with cis expression quantitative trait loci (eQTLs) [80]; 2) we screened whether the SNPs had been associated with any phenotypes in previous GWA studies using the GWAS catalog[81]; and (3) we assessed whether the SNPs were located within putative regulatory regions using HaploReg, v. 4.1 [82]. Placental samples for immunohistochemistry and qRT-PCR Samples from human placenta were collected at Oulu University Hospital during 2010–2016 as described [16]. The placental samples used in immunohistochemical staining and in qRT-PCR analysis of SLIT2 and ROBO1 expression were subject to similar inclusion criteria as SLIT2 and ROBO1 in spontaneous preterm birth PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008107 June 13, 2019 16 / 25 the samples used in GWAS. The inclusion criteria of gestational age for preterm placental samples was from 25 weeks to 36 weeks+ 6 days and 39 weeks to 41 weeks + 6 days for term samples. The same conditions (multiple gestation, intrauterine growth restriction, placental abruption, polyhydramnios, fetuses with anomalies or requirements for special care of the newborn) as in GWAS were used as exclusion criteria for term controls. Spontaneous preterm samples had almost the same exclusion criteria except the population included few cases with chorioamnionitis or oligohydramnion. The control group of elective preterm samples included cases with various pregnancy complications like IUGR or pre-eclampsia resulting in elective preterm delivery without labor. Specifically, in total, 18 placental samples were analyzed by immunohistochemistry. Twelve samples were from SPTB deliveries (GA from 25 wk + 2 d to 35 wk + 2 d), and six were from spontaneous term deliveries (GA from 39 wk + 4 d to 41 wk +1 d). Samples from both basal and chorionic plates were included in the study. RT-qPCR was performed with 23 placental samples from SPTB (GA from 25 wk + 2 d to 36 wk + 0 d), 34 from elective preterm birth (EPTB) (GA from 25 wk + 1 d to 36 wk + 6 d), and 23 from spontaneous term birth (STB) (GA from 39 wk + 1 d to 41 wk + 6 d). Sample preparation and immunohistochemical staining Localization of encoded proteins was visualized in placental tissues by immunohistochemical staining. Samples were embedded in paraffin and cut into 4-μm slices, deparaffinized, and rehydrated. Antigen retrieval was done in Tris-EDTA buffer. Endogenous peroxidase activity was blocked in blocking solution (Agilent, Santa Clara, CA, USA). Samples from the chorionic plate were incubated with mouse anti-human SLIT2 antibody (1:4000 dilution, PA5-3113; ThermoFisher Scientific, Waltham, Massachusetts, USA) or mouse anti-human ROBO1 antibody (1:2000, PA5-34931; ThermoFisher Scientific). Samples from the basal plate of the placenta were incubated in a 1:5000 dilution of mouse anti-human SLIT2 antibody and 1:1000 dilution of mouse anti-human ROBO1 antibody. Bound antibodies were detected with the Envision kit (Agilent). Quantitative PCR Tissue samples were homogenized, RNA was isolated with the RNeasy Mini Kit (Qiagen), and cDNA was synthetized as described previously [16]. After the RT-PCR, cDNA samples were diluted 1:2 using Rnase-free H 2 0. SLIT2 and ROBO1 were relatively quantified by intron spanning assays with Light-Cycler96 (Roche Diagnostics, Risch-Rotkreuz, Switzerland) and cytochrome c-1 (CYC1) as a reference gene. CYC1 was chosen as a reference gene because it is one of the most stably expressed genes in the placenta [16,83–85]. Primers and probes were: forward 50-CTTCCAGAGACCATCAC AGAAA-30and reverse 50-CGTCTAAGCTTTTTATATGGTGAGAA-30for SLIT2 (with UPL probe #79), forward 50-CGCAGAGAAACCTACACAGATG-30and reverse 50-GGATTGGG CAGTAGGTGACT-30for ROBO1 (with UPL probe #31), and forward 50-ATAAAGCGGCA CAAGTGGTCA-30and reverse 50-GATGGCTCTTGGGCTTGAGG-30for CYC1 (with UPL probe #47). Probes were from the Universal Probe library (UPL) Set (Roche Diagnostics). Each qPCR measurement was done in triplicate. Levels of SLIT2 and ROBO1 were normalized against the CYC1 level, and relative quantifications were then assessed with the ΔΔ cycle threshold method. A few randomly chosen qPCR products were also verified by agarose gel electrophoresis and Sanger sequencing. Primers and probes genes for transforming growth factor alpha (TGFA), C-X-C motif chemokine ligand 6 (CXCL6), and SLIT-ROBO Rho GTPase activating protein 3 (SRGAP3)were: SLIT2 and ROBO1 in spontaneous preterm birth PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008107 June 13, 2019 17 / 25 forward 50-CCCTGGCTGTCCTTATCATC-30and reverse 50-GGCACCACTCACAGTGTT TTC-30for TGFA (with UPL probe #74), forward 50-CCAGAAAATTTTGGACAGTGG-30 and reverse 50-GGGATCTCCAGAAAACTGCTC-30for CXCL6 (with UPL probe #61), and forward 50-GAAGGGCACTCGATGAGGT-30and reverse 50-GCTCATGGTCTTCTCGAT GTC-30for SRGAP3 (with UPL probe #66). Total mRNA expression of different PSGs was measured with PCR primers: forward 50-CC TCTCAGCCCCTCCCTG-30and reverse 50-GGCAAATTGTGGACAAGTAGAAGA-30(with UPL probe #15), which are complementary to sequences conserved in all but two PSG transcript variants that both lack the N domain [26]. IL6 and TNFA were analyzed with primers forward 50-GCCCAGCTATGAACTCCTTCT-30and reverse 50-GCGGCTACATCTTTGGA ATC-30for IL-6 (with UPL probe #43) and forward 50-CAGCCTCTTCTCCTTCCGAT-30and reverse 50-GCCAGAGGGCTGATTAGAGA-30for TNFA (with UPL probe #40). Gene knockdown of SLIT2 and ROBO1 by transfection with small interfering RNAs (siRNAs) Human placental trophoblast cells HTR-8/SVneo (CRL-3271™; ATCC, Manassas, Virginia, USA). were grown in RPMI-1640 culture media (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and 1×penicillin/ streptomycin (Sigma-Aldrich). Cells were cultured under standard culturing conditions (37˚C, 5% CO 2 , humidified atmosphere), and subculturing was performed with 0.05% trypsin/ 0.02% EDTA. siRNAs targeting SLIT2 (s GUCAUAUCAAGAACUGUGAdTdT, a UCACAGUUCUUG AUAUGACdTdT) and ROBO1 (s CAUACCUAUGGCUACAUUUdTdT, a AAAUGUAGCC AUAGGUAUGdTdT) (Sigma-Aldrich) were reverse transfected and then forward transfected in HTR-8/SVneo cells with Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) [86]. MISSION siRNA Universal Negative Control #1 (Sigma-Aldrich) was used as a negative control for siRNA transfection and was transfected in the same manner as siRNAs targeting SLIT2 and ROBO1. In the reverse transfection, the cells (70,000 cells/well) were incubated with siRNAs at a final concentration of 30 nM. The cells were transfected again after 24 h of incubation. The second transfection was done as a forward transfection in the presence of 40 nM siRNAs. Cells were incubated with siRNAs for 24 h after the second transfection, and then fresh medium was added and cells were incubated for an additional 24 h. Cells were harvested with 1×Trypsin-EDTA (Sigma-Aldrich). Transcriptomic analysis of SLIT2and ROBO1-silenced HTR8/SVneo cells Cells were disrupted with a 25 G needle and 1 ml syringe, and RNA was isolated in accordance with the manufacturer’s instructions (RNeasy Micro Kit, Qiagen). The quality of isolated RNA was checked with an Agilent 2100 Bioanalyzer system at the Biocenter Oulu Sequencing Center, Finland. Samples containing total RNA were sent for transcriptomic analysis to the Finnish Functional Genomics Centre (FFGC; Turku, Finland), where transcriptional profiles of SLIT2 (n= 3) and ROBO1 (n= 3) -silenced cells and negative-control cells (n = 3) were detected with the Illumina HiSeq high-throughput sequencing system. Whole cell RNA sequencing data was analyzed by the Bioinformatics Unit Core Service at the Turku Centre for Biotechnology, Finland. The transcriptomics data have been deposited in NCBI’s Gene Expression Omnibus [87] and are accessible through GEO Series accession number GSE119101 (https://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc=GSE119101) SLIT2 and ROBO1 in spontaneous preterm birth PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008107 June 13, 2019 18 / 25 qRT-PCR verification of transcriptomic data from SLIT2and ROBO1silenced HTR8/SVneo cells Knockdown of SLIT2 and ROBO1 with siRNAs, total RNA isolation, cDNA synthesis, and qPCR were done as described above, except 30 nM siRNAs were used in the forward transfection instead of 40 nM. Statistical analysis of RT-qPCR results Differences in mRNA expression levels among the phenotypes were assessed by nonparametric Mann–Whitney U-test with SPSS Statistics 20.0 (IBM Corporation). Supporting information S1 Fig. Manhattan plot showing the results of genome-wide association study of gestational age. Each dot represents the–log 10 (p) value of a single SNP in association analysis. Blue line denotes the level of suggestive significance (–log 10 (p)>5). Loci are indicated for regions with suggestive significance. (TIF) S2 Fig. Regional association plots for SLIT2 region in GWAS of SPTB (A) and gestational age (B). Each dot represents −log 10 (p) value of a single SNP in association analysis. Blue line denotes level of suggestive significance (−log 10 (p)>5). (TIF) S3 Fig. SLIT2 mRNA expression correlation with Z-score. SLIT2 mRNA expression in the basal plate of the placenta compared to birthweight-for-gestational age Z-scores of spontaneously delivered infants (SPTB and STB samples together). SLIT2 mRNA levels correlated with Z-scores (p= 0.023). (TIF) S4 Fig. Gene expression changes after SLIT2 and ROBO1 silencing verified by qRT-PCR. (A) Four genes/gene families affected by ROBO1 silencing according to RNA sequencing were verified by qRT-PCR. (B) Expression changes of inflammation-associated cytokines IL-6 and TNF-A after SLIT2 and ROBO1 silencing were also measured with qRT-PCR. SLIT2 and ROBO1 were post-transcriptionally silenced in HTR8/SVneo cell line by siRNA. mRNA levels of selected genes compared between SLIT2or ROBO1-silenced cells and mRNA levels of untreated control cells. All mRNA levels normalized against mRNA levels of housekeeping gene CYC1. Columns represent median and SD values of the sample groups. (TIF) S1 Table. Clinical characteristics of discovery GWAS study population. (DOCX) S2 Table. Suggestive association signals in GWAS of gestational age. (DOCX) S3 Table. Suggestive association signals in GWAS of very preterm birth. (DOCX) S4 Table. Suggestive association signals in GWAS of moderate-to-late preterm birth. (DOCX) S5 Table. Suggestive association signals in GWAS of infants born after SPTB with PPROM. (DOCX) SLIT2 and ROBO1 in spontaneous preterm birth PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008107 June 13, 2019 19 / 25 S6 Table. Suggestive association signals in GWAS of infants born after onset of preterm labor with intact fetal membranes. (DOCX) S7 Table. Pathways associated with spontaneous preterm birth. (DOCX) S8 Table. Gene ontology terms associated with SPTB. (DOCX) S9 Table. Significant gene ontology terms that include SLIT2 and ROBO1 genes. (DOCX) S10 Table. Upregulated genes after SLIT2 silencing in HTR8/SVneo cells. SLIT2 silenced in HTR8/SVneo commercial cell line by siRNA. Transcriptome of these cells compared with transcriptome of cells treated with negative siRNA. Differentially expressed genes ranked based on FDR-adjusted pvalue and fold change (FC). Threshold of fold change was >2.0, and threshold of FDR-adjusted pvalue was <0.05. (DOCX) S11 Table. Downregulated genes after SLIT2 silencing in HTR8/SVneo cells. SLIT2 silenced in HTR8/SVneo commercial cell line by siRNA. Transcriptome of these cells compared with transcriptome of cells treated with negative siRNA. Differentially expressed genes ranked based on FDR-adjusted pvalue and fold change. Threshold of fold change was >2.0, and threshold of FDR-adjusted pvalue was <0.05. (DOCX) S12 Table. Upregulated genes after ROBO1 silencing in HTR8/SVneo cells. ROBO1 silenced in HTR8/SVneo cell line by siRNA. Transcriptome of these cells compared with transcriptome of cells treated with negative siRNA. Differentially expressed genes ranked based on FDRadjusted pvalue and fold change. Threshold of fold change was >2.0, and threshold of FDRadjusted pvalue was <0.05. (DOCX) S13 Table. Downregulated genes after ROBO1 silencing in HTR8/SVneo cells. ROBO1 silenced in HTR8/SVneo cell line by siRNA. Transcriptome of these cells compared with transcriptome of cells treated with negative siRNA. Differentially expressed genes ranked based on FDR-adjusted pvalue and fold change. Threshold of fold change was >2.0, and threshold of FDR-adjusted pvalue was <0.01. (DOCX) S14 Table. Biological pathways affected by SLIT2 silencing in HTR8/SVneo cells. (DOCX) S15 Table. Biological pathways affected by ROBO1 silencing in HTR8/SVneo cells. (DOCX) Acknowledgments We acknowledge the technical assistance of Maarit Haarala and sample collection by Dr. Mari Mahlman, Riitta Vikeva¨inen, Miia Lehto, and Sonja Eeli. We thank the Medical Bioinformatics Centre of Turku Centre for Biotechnology for the miRNA-seq data analysis. The Centre is SLIT2 and ROBO1 in spontaneous preterm birth PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008107 June 13, 2019 20 / 25 supported by University of Turku, Åbo Akademi University, and Biocenter Finland. CSC–IT Center for Science, Finland, is acknowledged for computational resources. Author Contributions Conceptualization: Mikko Hallman, Mika Ra¨met. Data curation: Heli Tiensuu, Minna K. Karjalainen, Riitta Marttila, Marja Ojaniemi, Mikko Hallman, Mika Ra¨met. Formal analysis: Heli Tiensuu, Antti M. Haapalainen, Minna K. Karjalainen, Anu Pasanen. Funding acquisition: Mikko Hallman, Mika Ra¨met. Investigation: Heli Tiensuu, Antti M. Haapalainen, Minna K. Karjalainen, Mikko Hallman, Mika Ra¨met. Methodology: Heli Tiensuu, Antti M. Haapalainen, Minna K. Karjalainen, Johanna M. Huusko, Riitta Marttila, Marja Ojaniemi, Louis J. Muglia, Mikko Hallman, Mika Ra¨met. Project administration: Mikko Hallman, Mika Ra¨met. Resources: Mikko Hallman, Mika Ra¨met. Software: Minna K. Karjalainen, Anu Pasanen. Supervision: Antti M. Haapalainen, Minna K. Karjalainen, Mikko Hallman, Mika Ra¨met. Validation: Heli Tiensuu, Antti M. Haapalainen. Visualization: Heli Tiensuu, Minna K. Karjalainen. Writing – original draft: Heli Tiensuu, Minna K. Karjalainen. Writing – review & editing: Antti M. Haapalainen, Anu Pasanen, Johanna M. 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