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

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 = 1.6x10-6). 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 = 1.55x10(-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. 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.