Supplementary Material for: A Potential Role for the NOD1 Variant (rs6958571) in Gram-Positive Blood Stream Infection in ELBW Infants

<p><b><i>Background:</i></b> The genetic basis of sepsis susceptibility in preterm infants remains understudied. Herein, we investigated the nucleotide binding-oligomerization domain (NOD)-like receptor (NLR) family of immune receptors as putative loci for preterm sepsis susceptibility. <b><i>Objective:</i></b> To determine whether single nucleotide polymorphisms (SNPs) in NLR genes are associated with blood stream infections (BSI) in premature infants. <b><i>Methods:</i></b> An international cohort of infants with gestational age (GA) <35 weeks were genotyped for SNPs in the <i>ATG16L1</i>, <i>CARD8</i>, <i>NLRP3</i>, <i>NOD2</i>, and <i>NOD1 </i>genes. χ² and logistic regression analyses were used to examine relationships between NLR variants and BSI. <b><i>Results:</i></b> Among 764 infants, 138 developed BSI, 113 had gram-positive bacterial (GPB) BSI, and 28 had gram-negative bacterial (GNB) BSI. Infants with BSI had a lower birth weight and GA (<i>p</i> < 0.001), but did not differ in gender, race, or chorioamnionitis. <i>NLR</i> variants were not associated with GPB or GNB BSI in the entire cohort. The CC genotype of the NOD1 SNP <i>(</i>rs6958571) was associated with increased GPB BSI in extremely low birth weight (ELBW, birth weight <1,000 g) infants (OR = 3.3, 95% CI: 1.4-7.5, <i>p</i> = 0.003, <i>n</i> = 362) and in Caucasian infants (OR = 2.5, 95% CI: 1.2-5.4, <i>p</i> = 0.016, <i>n</i> = 535). Regression models adjusting for clinical variables identified ELBW status and the <i>NOD1</i> CC genotype as risk factors for GPB BSI in Caucasian infants. <b><i>Conclusions:</i></b> In this study investigating relationships between <i>NLR</i> variants and sepsis in infants with GA <35 weeks, the <i>NOD1 (</i>rs6958571) SNP was associated with GPB BSI in Caucasian infants and ELBW infants. Replication of our results in an independent cohort would support a role for NLR variants in determining sepsis risk in ELBW infants.</p

Supplementary Material for: Genes Encoding Vascular Endothelial Growth Factor A (VEGF-A) and VEGF Receptor 2 (VEGFR-2) and Risk for Bronchopulmonary Dysplasia

<b><i>Background:</i></b> Bronchopulmonary dysplasia (BPD) is one of the main consequences of prematurity, with notably high heritability. Vascular endothelial growth factor A (VEGF-A) and its main receptor, vascular endothelial growth factor receptor 2 (VEGFR-2), have been implicated in the pathogenesis of BPD. <b><i>Objective:</i></b> To study whether common polymorphisms of the genes encoding VEGF-A and VEGFR-2 are associated with BPD. <b><i>Methods:</i></b> In this association study, six tagging single nucleotide polymorphism (tSNPs) for <i>VEGFA</i> and 25 tSNPs for <i>VEGFR2</i> were genotyped in a prospectively collected, genetically homogeneous discovery population of 160 infants (44 infants with grade 2-3 BPD) born before 30 completed gestational weeks. The replication population of 328 infants included 120 cases of BPD. <b><i>Results:</i></b><i>VEGFR2</i> SNP rs4576072 was associated with BPD grade 2-3 with a minor allele frequency in 23.9% of the cases compared to 9.1% in controls (p = 0.0005, odds ratio 3.15, 95% CI: 1.62-6.12) in the discovery population. This association was not observed in the more heterogeneous replication population. <b><i>Conclusions:</i></b> In line with the results of recent large-scale genetic studies, our findings indicate that common polymorphisms of the genes encoding VEGF-A and VEGFR-2 are not consistently associated with BPD. This finding does not rule out the involvement of <i>VEGFA</i> and <i>VEGFR2</i> in BPD pathogenesis since, in addition to common variations within the gene region, other mechanisms also play important roles in the regulation of gene function

Supplementary Material for: Hierarchical Maturation of Innate Immune Defences in Very Preterm Neonates

<b><i>Background:</i></b> Preterm neonates are highly vulnerable to infection. <b><i>Objectives:</i></b> To investigate the developmental contribution of prematurity, chorioamnionitis and antenatal corticosteroids (ANS) on the maturation of neonatal microbial pathogen recognition responses. <b><i>Methods:</i></b> Using standardized protocols, we assayed multiple inflammatory cytokine responses (IL-1β, IL-6, TNF-α and IL-12/23p40) to three prototypic Toll-like receptor (TLR) agonists, i.e. TLR4 (lipopolysaccharide), TLR5 (flagellin) and TLR7/8 (R848), and to the non-TLR retinoic acid-inducible gene I (RIG-I)-like receptor agonist, in cord blood mononuclear cells from neonates born before 33 weeks of gestation and at term. <b><i>Results:</i></b> TLR responses develop asynchronously in preterm neonates, whereby responses to TLR7/8 were more mature and were followed by the development of TLR4 responses, which were also heterogeneous. Responses to TLR5 were weakest and most immature. Maturity in TLR responses was not influenced by sex. Overall, we detected no significant contribution of ANS and chorioamnionitis to the developmental attenuation of either TLR or RIG-I responses. <b><i>Conclusions:</i></b> The maturation of anti-microbial responses in neonates born early in gestation follows an asynchronous developmental hierarchy independently of an exposure to chorioamnionitis and ANS. Our data provide an immunological basis for the predominance of specific microbial infections in this age group