Association between the p.Thr1406Asn polymorphism of the carbamoyl-phosphate synthetase 1 gene and necrotizing enterocolitis: A prospective multicenter study

The p.Thr1406Asn (rs1047891) polymorphism of the carbamoyl-phosphate synthetase 1 (CPS1) gene has been linked to functional consequences affecting the downstream availability of the nitric oxide precursor L-arginine. L-arginine concentrations are decreased in preterm infants with necrotizing enterocolitis (NEC). In this multicenter prospective study, we investigated the association of the p.Thr1406Asn polymorphism with NEC in 477 preterm infants (36 cases of NEC) from 4 European neonatal intensive care units (Maastricht, Las Palmas de Gran Canaria, Mantova, and Milan). Allele and genotype frequencies of the p.Thr1406Asn polymorphism did not significantly differ between the infants with and without NEC. In contrast, the minor A-allele was significantly less frequent in the group of 64 infants with the combined outcome NEC or death before 34 weeks of corrected gestational age than in the infants without the outcome (0.20 vs. 0.31, P = 0.03). In addition, a significant negative association of the A-allele with the combined outcome NEC or death was found using the dominant (adjusted odds ratio, aOR: 0.54, 95% CI 0.29–0.99) and the additive (aOR 0.58, 95% CI 0.36–0.93) genetic models. In conclusion, our study provides further evidence that a functional variant of the CPS1 gene may contribute to NEC susceptibility.

Scientific RepoRts | 6:36999 | DOI: 10.1038/srep36999 Arginine is a urea cycle intermediate. The first step in the urea cycle occurs inside the mitochondrion and is catalyzed by the enzyme, carbamoyl-phosphate synthetase I (CPS1, EC 6.3.4.16) 22 . CPS1 deficiency in humans is a rare autosomal recessive inborn error of the urea cycle leading to hyperammonemia. Deficiency can be primary, due to mutations in the CPS1 gene (OMIM 608307, HGNC:2323) or secondary, due to the lack of the essential cofactor N-acetyl-L-glutamate 22 . In addition, a number of functional single nucleotide polymorphisms (SNPs) have been identified in the CPS1 gene [22][23][24] . One of those SNPs (p.Thr1406Asn also published as T1405N; rs1047891 formerly designated as rs7422339) has been linked to functional consequences affecting the downstream availability of urea-cycle intermediates, including L-arginine [22][23][24] . The SNP p.Thr1406Asn is a C-to-A nucleotide transversion (c.4217 C > A) in exon 36, which results in the substitution of asparagine (Asn) for threonine (Thr) in the critical N-acetylglutamate-binding domain 22,25 . The C-encoded Thr form of the polymorphism is considered the evolutionarily conserved version and the less frequent, A-encoded, Asn variant appears to be a relatively new, gain-of-function mutation 15,25 . It has been suggested that the A-allele may confer an advantage in terms of NO production, especially under conditions of environmental stress 15 . Previous studies demonstrated the association of the CPS1 p.Thr1406Asn genotype with clinical situations where endogenous NO production is critically important, such as persistent pulmonary hypertension of the newborn 15 , pulmonary hypertension following surgical repair of congenital heart defects 26 , and hepatic veno-occlusive disease after bone marrow transplantation 23 .
Several years ago, we reported in a retrospective series of 17 preterm infants with NEC and 34 controls that patients with NEC showed an underrepresentation of the A-encoded variant of the p.Thr1406Asn polymorphism of CPS1 27 . Those results suggested that the A-allele conferred protection against NEC and warranted confirmation using a prospective design and larger sample size. Herein we report the results of such study, involving 477 preterm infants (36 cases of NEC) from four neonatal intensive care units located in three different European countries (Spain, Italy, and the Netherlands). Since death in the first weeks of life is a competing outcome for NEC, we also analyzed the association of the p.Thr1406Asn polymorphism with the combined outcome NEC or death before 34 weeks of corrected gestational age(GA).

Definition of clinical characteristics and outcomes. Data on clinical characteristics and outcomes
were obtained from the medical records. GA was determined by the last menstrual period and early ultrasounds (before 20 weeks of gestation). Small for GA was defined as BW for GA below the sex-specific 10 th percentile. Chorioamnionitis was defined as every clinical suspicion of infection of the chorion, amnion, amniotic fluid, placenta, or a combination as judged by the obstetrician. Prolonged rupture of membranes was defined as rupture of membranes > 24 hours before delivery. Prenatal exposure to a single course of antenatal steroids was defined as two doses of betamethasone administered 24 h apart and exposure to a partial course of antenatal steroids was defined as administration of a single dose of betamethasone < 24 h prior to delivery.
NEC was defined as Bell stage II disease or greater. At the conclusion of the study, all cases of NEC were reviewed in a blinded fashion by a panel of 4 investigators of the study. Cases of spontaneous intestinal perforation (i.e., without pathologic evidence of NEC) were excluded from the investigation. Since it was considered that some infants who died in the first weeks may have developed NEC if they had survived, we also analyzed the composite outcome of NEC or death before 34 weeks of corrected GA.
Respiratory distress syndrome (RDS) was defined as requirement for oxygen supplementation or respiratory support due to tachypnea, grunting, nasal flaring, retractions, or cyanosis. Bronchopulmonary dysplasia (BPD) was defined as a supplemental oxygen requirement at 36 weeks of corrected GA to maintain oxygen saturation > 90% 29 . Arterial hypotension was defined as the need for volume expansion or inotropic support. Patent ductus arteriosus (PDA) was defined as a requirement for indomethacin or ibuprofen and/or surgical ligation. A diagnosis of sepsis required signs of generalized infection, a positive blood culture, and antibiotic therapy. Intraventricular hemorrhage (IVH) was classified by using the 4-level grading system 30 . Grade < 2 IVHs were not included in the analysis. Retinopathy of prematurity (ROP) was defined as stage II or higher.
Samples and genotyping. Buccal cell samples for DNA testing were obtained with a sterile OmniSwab Statistical Analysis. Sample size was calculated based on data from our previous study 27 . Given an expected population incidence of NEC of 5%, expected frequencies for CC homozygosity of 0.7 (NEC group) and 0.4 (control group), alpha level = 0.05, and power of 0.8, 440 infants were needed to detect an odds ratio (OR) significantly different from 1.
Categorical variables were expressed as counts or percentages and compared using the chi-square test. Continuous variables were expressed as mean (SD) if they followed a normal distribution and compared using unpaired, two-sided t-test. If not normally distributed, continuous variables were expressed as median values (interquartile range, IQR; 25 th -75 th percentile) and compared using the Mann-Whitney U test. The Kolmogorov-Smirnov test was used to test for normal distribution of continuous data.
Differences in allelic frequencies and genotype distributions between the investigated populations, as well as Hardy-Weinberg equilibrium (HWE) for genotype distribution were assessed using a chi-square test. The Hardy-Weinberg law states that q 2 + 2pq + p 2 = 1, where p and q are allele frequencies in a two-allele system. Logistic regression analysis was used to compute the ORs and their 95% confidence intervals (CI) for NEC and the combined outcome NEC or death before 34 weeks of corrected GA based on genotype after accounting for the covariates which were significantly different between the groups and are known risk factors for developing NEC. Different genetic models were used to analyze the effect of the risk allele, including the general allelic (multiplicative or codominant model), dominant, recessive and additive models. Assuming a genetic penetrance parameter γ (γ > 1), a multiplicative model indicates that the risk of disease is increased γ -fold with each additional copy of the risk allele; an additive model indicates that risk of disease is increased γ -fold for the genotype with one copy of the risk allele and 2γ -fold for the genotype with two copies of the risk allele; a common recessive model indicates that two copies of the risk allele are required for a γ -fold increase in disease risk, and a common dominant model indicates that either one or two copies of the risk allele are required for a γ -fold increase in disease risk 31 . The major allele was considered as a reference and the interactions were tested in the different models by multivariable logistic regression model. All the statistical analyses were performed using IBM SPSS Statistics for Windows, Version 22.0. (IBM Corporation, Armonk, NY, USA) and conducted at the P < 0.05 level of significance.

Results
Patient characteristics. From 615 eligible infants, 477 (36 with NEC Bell stage II or greater) were included in the study (Fig. 1). Stage II NEC was present in 23 infants and stage III in 13 infants. In 21 cases surgery was required. The median age at the onset of NEC was 20 days (range 4-87, . Nine of the cases of NEC occurred in Maastricht (5 stage II NEC, 4 stage III NEC), 16 in Las Palmas (9 stage II NEC, 7 stage III NEC), and 11 in Italy (9 stage II NEC, 2 stage III NEC). Single intestinal perforation was present in 5 infants (1 in Maastricht, 4 in Italy). Demographic and clinical characteristics of the infants with and without NEC are shown and compared in Table 1. Mean GA, mean BW and median Apgar score at 1 min of infants with NEC were significantly lower than in infants without NEC. In addition, infants with NEC showed a higher incidence of vaginal delivery, mechanical ventilation, BPD, hypotension, IVH, PVL, PDA, ROP and mortality. We adjusted for GA, BW, Apgar score at 1 min, mechanical ventilation, hypotension, and PDA in the subsequent logistic regression analysis.
The demographic and clinical characteristics of the infants with and without the combined outcome NEC or death before 34 weeks of corrected GA are shown and compared in Table 2. Mean GA, mean BW and median  Apgar score at 1 and 5 min of infants with NEC or death were significantly lower than in infants without the combined outcome. In addition, infants with the combined outcome NEC or death showed a higher incidence of vaginal delivery, mechanical ventilation, hypotension, IVH and PDA. We adjusted for GA, BW, Apgar score after at 1 and 5 min, mechanical ventilation, hypotension and PDA in the subsequent logistic regression analysis.

Analysis of genotypes.
Allele and genotype frequencies of the p.Thr1406Asn polymorphism in the total preterm population did not significantly differ from the allele and genotype frequencies observed in the population of 96 healthy term infants (P = 0.925, Table 3) 28 . The distribution of the genotypes p.Thr1406Asn did not fulfill Hardy-Weinberg criteria in the preterm population. The minor allele frequency (MAF) in the NEC group (0.208) was not significantly different from the MAF among the infants without NEC (0.303, P = 0.09; Table 4). The MAF in the NEC group with the combined outcome NEC or death before 34 weeks (0.200) was significantly different from the MAF among the infants without the outcome (0.311, P = 0.03; Table 5). We further analyzed the effect of the CPS1 p.Thr1406Asn polymorphism on the occurrence of NEC and NEC or death under different genetic models. As shown in Table 4, logistic regression analysis could not detect any significant association between the p.Thr1406Asn polymorphism and NEC in any of the genetic models. In contrast, the dominant and the additive model showed a negative significant association of the A-allele of the CPS1 p.Thr1406Asn polymorphism with the combined outcome NEC or death before 34 weeks of corrected GA (Table 5). Finally, we analyzed the correlation of the CPS1 p.Thr1406Asn polymorphism genotype with other neonatal outcomes. As shown in Table 6, we could not find any significant association.

Discussion
This is one of the largest prospective studies investigating the association of a SNP with NEC. Our study could not detect a significant association between the p.Thr1406Asn genotype of the CPS1 gene and the risk of developing NEC in very preterm infants (GA ≤ 30 weeks and BW ≤ 1500 g). However, when the combined outcome NEC or death before 34 weeks of corrected GA was analyzed, it was observed that the minor A-allele of the p.Thr1406Asn polymorphism was significantly less frequent in the group of infants who developed NEC or died than in those who survived without NEC. In addition, a significant negative association of the A-allele with the combined outcome NEC or death was found using the dominant and the additive genetic models.
In our previous retrospective case-control study, we examined the relationship between the CPS1 p.Thr1406Asn polymorphism and the presence of NEC in preterm infants and we found that patients with NEC showed an overrepresentation of the C-encoded variant of the CPS1 27 . Consequently, the A-encoded variant of the p.Thr1406Asn polymorphism was underrepresented in the infants with NEC. However, although the cases and the controls were well matched for GA and BW, when adjusted for these two known risk factors for NEC, the association between NEC and the p.Thr1406Asn polymorphism did not remain significant 27 . In the present   prospective cohort study, we observed that the A-allele was less frequent in the infants with NEC in a proportion close to reach statistical significance (P = 0.09). Moreover, only one infant with NEC was homozygous for the A-allele. Nevertheless, none of the genetic models could demonstrate a statistically significant association between the p.Thr1406Asn genotype and NEC. An important issue in designing studies involving high-risk patients is the selection of an appropriate primary outcome when death is a competing outcome 32 . In this situation, some patients will die before the outcome of interest can occur. For this reason, a composite outcome including death is often used when complications of prematurity such as BPD or NEC are studied 32 . Since the onset of 'classical' NEC takes place around the end of the   33,34 , we performed an additional analysis in which the infants with NEC were combined with the infants who died before 34 weeks of corrected GA. Interestingly, we observed that the minor A-allele was significantly less frequent among the infants with the combined outcome NEC or death. Moreover, the dominant and additive model demonstrated that the A variant of the p.Thr1406Asn polymorphism significantly decreased the risk of developing the combined outcome of NEC or death before 34 weeks of corrected GA. Thus, in other words, the minor A variant of the polymorphism might protect against this combined outcome.
In accordance with our results, it has been reported a protective role of the A-allele toward persistent pulmonary hypertension of the newborn 15 , pulmonary hypertension following surgical repair of congenital heart defects 26 , and hepatic veno-occlusive disease after bone marrow transplantation 23 . Moreover, the haplotype formed by the SNPs of CPS1 rs715 and rs1047891 (p.Thr1406Asn) yield a protective association with decreased risk of coronary artery disease in women 35 . As mentioned in the introduction, those findings led to the speculation that individuals with the A-allele may have an advantage in terms of availability of the NOS substrate L-arginine, especially under conditions of environmental stress 15,23,26 . Accordingly, the serum levels of arginine were higher in term newborns carrying the AA genotype 15 . However, activity reports on the in vitro activity of the p.Thr1406Asn variants are contradictory 22 . Thus, natural (A-encoded) Asn1406 CPS has been reported to have higher enzymatic activity than the (C-encoded) Thr1406 variant 15,23,26 but recombinant Asn1406 CPS1 showed inferior catalytic properties 36 . In order to evaluate whether p.Thr1406Asn genotypes correlated with urea cycle intermediates levels in preterm infants, we measured the concentrations of arginine and citrulline in the first 128 infants included in the present cohort and we did not observe any significant differences 28 . This lack of effect of CPS1 genotype on L-arginine concentrations has been also reported in adults 25 . Nevertheless, one limitation of our study was that L-arginine levels were determined between 6 and 12 hours after birth, whereas NEC has its onset later in life 33,34 . It can be speculated that the alterations in arginine levels related to the p.Thr1406Asn polymorphism may be only relevant under the stress conditions generated around the time of NEC onset. At that moment, being carrier of a genetic variant that potentially increases NO production (i.e., the A-allele) might be of critical relevance because NO is a key regulator intestinal blood flow, protector of the mucosa, and modulator of the inflammatory response [10][11][12] . In addition, the infants homozygous for the C-allele might be more susceptible to NEC and arginine supplementation 16,20,21 might be particularly indicated in this group.
One limitation of our study is that we did not collect information on the feeding practices. Human milk protects against NEC 1-3 and, although the preterm formulas currently used have concentrations of arginine similar to the human milk 16 , arginine intake might be different depending on the infants diet. Plasma arginine levels are likely to represent a balance between arginine intake, arginine synthesis, and the demands of protein synthesis and the multiple metabolic pathways for arginine utilization 14,37 . Enteric arginine synthesis appears to be necessary to cover neonatal requirements, because mammalian milk is a relatively poor source of arginine, whereas its precursors proline and glutamine are abundant 38 . In fact, proline is the major contributor to arginine synthesis in human preterm infants 39 . CPS, ornithine aminotransferase, and argininosuccinate synthetase are key enzymes in the control of de novo intestinal synthesis of arginine, which are already expressed in the mid gestation human intestine 38 . However, hypoargininemia often develops in preterm infants, in particular if they are maintained on total parenteral nutrition, and it has been suggested that the intestine only produces arginine if substrate is supplied through enteral nutrition 14,[37][38][39] . In situations of reduced availability of the substrate L-arginine or the cofactor BH 4 , NOS enzymatic activity becomes uncoupled, resulting in the production of superoxide instead of NO 40 . Therefore, besides resulting in a paucity of NO, the uncoupled enzyme will generate free radicals resulting in further intestinal damage. In addition, it should be taken into account that, besides NO, arginine is a substrate for synthesis of many biologically important molecules including agmatine, polyamines, and creatine 14,38,41 . These metabolites have roles in energy metabolism, gene expression, apoptosis, and cell proliferation and differentiation, which are crucial in intestinal homeostasis 14,38,41 . Our present results suggest that functional genetic variations in the CPS enzyme might be, at least partially, the link between hypoarginimemia and NEC in preterm infants 14,[16][17][18][19] . Alternatively, recent experimental evidences highlighted the role of argininosuccinate lyase, another enzyme involved in the intestinal synthesis of arginine, in the pathogenesis of NEC 41 .
Concluding remarks. NEC affects only a minority of preterm infants, which suggest an individual susceptibility toward the disease. Genetic polymorphisms might be an important factor in this individual susceptibility 2,4,5,9,[42][43][44][45][46][47][48][49][50][51] . Our study provides further evidence that a functional variant of the CPS1 gene may contribute to NEC susceptibility. Nevertheless, NEC is a complex multifactorial disease and an isolated genetic derangement may not be sufficient to account for the entire spectrum of its pathophysiology.
Given the importance of prematurity, intestinal function, immune defense, inflammatory signaling, and microcirculatory regulation mechanisms, potential variations in many genes could protect or leave a host infant susceptible to NEC 5,9,[42][43][44][45][46][47][48][49][50][51] . Future studies investigating the association of multiple SNPs and NEC may allow for the development of a laboratory genetic test that could predict, when environmental factors are properly assessed, the risk/probability of preterm infants developing NEC and lead to more targeted therapies.