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

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.

Fig 1. Overview of the study workflow.

Fig 2. Manhattan plot of GWAS of SPTB.

Each dot represents −log₁₀(p) value of a single SNP in association analysis. Dashed line denotes level of suggestive significance (−log₁₀(p) > 5). SLIT2, SUGCT, and EXOSC1-ZDHHC16-MMS19-UBTD1 were among the best associating loci.

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.

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.

Fig 5. mRNA expression level changes after gene knockdown.

SLIT2 (A) and ROBO1 (B) were post-transcriptionally knocked down with siRNA in human placental trophoblast continuous cell line HTR8/SVneo. mRNA levels of cells in which SLIT2 or ROBO1 were knocked down was compared with mRNA levels from siRNA negative treated control cells. Expression levels of the genes were first determined by qRT-PCR (levels normalized against housekeeping gene CYC1 mRNA levels). According to qRT-PCR, silencing percentage of SLIT2 was 60% and silencing percentage of ROBO1 was 85%. Gene expression levels also determined with high-throughput RNA sequencing. Reads per kilobase of exon per million reads mapped (RKPM) is a normalized gene count value determined in transcriptomic analysis. According to transcriptomics data, silencing percentages were 75% for SLIT2 and 74% for ROBO1. Columns represent median and SD values.