This study found that a specific genetic polymorphism—the presence of at least one G allele at the −449 position—within the DDAH2 gene was associated with low plasma ADMA concentrations in children with severe sepsis or septic shock but not in non-septic controls. This same polymorphism was also associated with an increased likelihood of presenting with vasoconstricted or “cold” shock, with nearly half exhibiting this clinical phenotype compared with exclusively “warm” or no shock seen in the patients with the −449CC homozygous genotype. To our knowledge, this pilot study is the first to associate genetic variability in the DDAH2 gene with different hemodynamic profiles observed clinically in pediatric sepsis. Our results suggest that genotypic differences in the regulation of NO metabolism may contribute to phenotypic variability in sepsis pathophysiology.
Marked hemodynamic variability occurs in pediatric septic shock. In one study using pulmonary artery catheter measurements in children with fluid-refractory septic shock, only 20% exhibited the classic high cardiac output-low SVR (“warm”) shock state, while 58% had low cardiac output-high SVR (“cold”) shock. Similar findings were reported in a more recent study, with the majority of children with community-acquired sepsis exhibiting “cold” shock 
. Reflecting these clinical observations, current guidelines recommend a stratified therapeutic approach in pediatric sepsis with vasopressors utilized for persistent “warm” shock and inotropes with or without vasodilators for “cold” shock 
. The factors underlying individual differences in shock states in sepsis, however, remain unknown.
Alterations in NO bioavailability affect both myocardial and vascular function and therefore directly impact the adequacy of microvascular blood flow, tissue perfusion, and organ function 
. ADMA decreases NO production through competitive inhibition of all NOS isoforms and intracellular arginine transport 
, and differences in circulating ADMA concentrations have been associated with risk for development of essential hypertension, atherosclerosis, and pulmonary vascular disease 
. In adults with sepsis, several studies have found elevated plasma ADMA levels 
, particularly in those with septic shock requiring vasoactive infusions 
, suggesting that alterations in ADMA may also have important hemodynamic effects in critical illness. However, in pediatric sepsis, where “cold” shock states are more common, we previously found plasma ADMA to be decreased but did not associate ADMA with shock type in that study 
The two DDAH enzymes are responsible for 80–90% of ADMA metabolism and provide an important regulatory mechanism for NO synthesis, and therefore, NO bioavailability 
. Prior studies have shown that changes in DDAH activity cause alterations in intracellular ADMA in concentrations sufficient to impact NO synthesis 
. The DDAH2 isoform is found predominantly in endothelial and immune cells, where it co-localizes with endothelial and inducible NOS, respectively. Pharmacologic inhibition of DDAH increases ADMA, which leads to endothelium-dependent vasoconstriction 
and alters the behavior of circulating lymphocytes 
, supporting the importance of the ADMA-DDAH pathway in regulating NO-mediated vascular and immune function. The identification of genetic polymorphisms that influence expression or activity of the DDAH2 enzyme is therefore intriguing as factors that could affect immune response, susceptibility to infection, and hemodynamic changes that may ultimately impact microvascular blood flow and organ perfusion in pediatric sepsis.
We focused on two polymorphisms within the promoter region of the DDAH2
gene with previously described functional significance. The −871 6g/7g insertion/deletion polymorphism has been shown to alter the expression and activity of the DDAH2 enzyme in vitro
and the −449G/C SNP has been associated with alterations in plasma ADMA 
and clinical outcomes 
. Despite the potential influence on immune function, neither polymorphism was associated with an increased susceptibility to severe sepsis or septic shock in our study, although we did find a higher percentage of patients with the rare −871 7g allele in the septic group. Jones et al. observed that this −871 7g allele led to an increase in basal DDAH2
expression in cultured human umbilical vein endothelial cells compared with the −871 6g variant 
. In contrast, we did not observe a difference in plasma ADMA, as a surrogate measure of DDAH2 activity, between −871 6g/7g genotypes, although the rare occurrence of the 7g allele may have limited our analysis. The −449G/C SNP was evaluated in a previous study of 47 adult patients with severe sepsis or septic shock and carriage of the −449G allele was associated with an increased serum ADMA concentration 
. These findings contrast with our study, in which we found the −449G allele associated with lower ADMA levels. These conflicting results may reflect that the DDAH2 isoform contributes little to circulating ADMA, as it is more likely that DDAH1 regulates systemic ADMA metabolism 
. Alternatively, as DDAH2 activity is influenced by multiple factors other than genetics, including availability of L-arginine 
, inflammatory cytokines 
, and negative feedback by NO itself 
, the association of DDAH2
genotype with plasma ADMA may be influenced by different factors in distinct populations.
While we did not find a relationship between plasma ADMA and type of shock, patients with at least one −449G allele were more likely to exhibit “cold” shock compared with CC homozygotes. Given that the anticipated outcome of ADMA-induced inhibition of NO synthesis should be to oppose vascular relaxation, it is reasonable to expect that a polymorphism associated with decreased plasma ADMA should predispose to increased NO availability, vasodilatation, and “warm” shock. Since patients with the −449G allele exhibited lower plasma ADMA levels irrespective of shock type (), the association between this polymorphism and low ADMA may be more of an epiphenomenon and DDAH2
genotype may influence hemodynamics through a mechanism other than circulating ADMA. The disassociation between DDAH2
genotype, plasma ADMA, and hemodynamic phenotype is supported by previous studies. Wang et al. reported no difference in serum ADMA with DDAH2
silencing, but still observed reduced NO-regulated vascular changes 
. Maas et al. found an increased risk for hypertension in patients with the −449 GG genotype despite no association with plasma ADMA 
. The −449G allele was also shown to be less common than the CC homozygous genotype in adult patients needing vasopressor support following cardiac surgery 
, and although ADMA levels were not measured, the increased prevalence of the −449G allele in patients with a higher SVR in that study is consistent with our finding that patients with at least one −449G allele were more likely to exhibit “cold”, or high-SVR, shock.
Limitations of our study include reliance on clinical assessment to differentiate “warm” versus “cold” shock rather than direct hemodynamic measurements of cardiac output and SVR and lack of standardization of vasoactive therapy. Since patients may evolve from a warm to a cold shock state (or vice versa), either through natural progression of the septic response or as a consequence of pharmacological intervention, we chose to focus on the association of DDAH2 polymorphisms and shock state at initial presentation to minimize the influence of vasoactive therapy. However, differences in timing of presentation after onset of infection may itself have influenced hemodynamic state at PICU admission. The association of DDAH2 polymorphisms with hemodynamic changes after precise initiation of a septic insult and the natural evolution of the septic response in the absence of pharmacologic intervention would be best studied in animal models. Furthermore, we did not differentiate between use of inotrope, vasopressor, and vasodilator agents, which may have contributed to the lack of association between DDAH2 genotype and vasoactive infusion requirements.
We also recognize that the most important limitation of our study is the small sample size, which increases the risk for spurious conclusions about the association of genetic polymorphisms with clinical variables. To address this concern, three important questions need to be answered 
: Are the studied populations homogeneous? Does the polymorphism of the gene under study cause a relevant alteration in the level or function of the gene product? Does the product of the studied gene play an important role in the pathogenesis of the disease? The study cohort was derived from an investigation of arginine and ADMA in a heterogeneous population of septic children with controls matched only for age 
. Given the association of Black race with both the −449CC genotype and “warm” shock, we are not able to differentiate whether the −449G/C SNP itself influences type of shock or is simply a marker for an alternative genetic or environmental difference between racial groups. Further studies in larger, more homogeneous populations are necessary. Second, although the −871 6g/7g polymorphism has been shown to directly affect enzyme expression in vitro
and the −449G/C SNP has been associated with variable ADMA concentrations in clinical studies, it is not known to what extent genetic differences directly alter expression or activity of the DDAH2 enzyme in vivo
compared with other regulatory mechanisms, such as oxidative stress, arginine availability, and NO-induced inhibition. Finally, while increasing evidence supports the importance of DDAH in the regulation of NO bioactivity through ADMA metabolism in cardiovascular disease 
, the direct effects of DDAH2-induced changes in intracellular ADMA concentration on NO bioactivity and outcomes in sepsis has not been adequately studied. Therefore, we present this pilot study as a first step, exploratory analysis of a biologically plausible mechanism linking genetic differences to variability in hemodynamic shock in pediatric sepsis, a clinically observed phenomenon which remains poorly understood.
In conclusion, we studied whether two known functional polymorphisms in the DDAH2 gene are associated with plasma ADMA concentration, distinct hemodynamic states, and cardiovascular dysfunction in pediatric septic shock. We found that the −449G SNP was associated with both decreased plasma ADMA and an increased likelihood of presenting with “cold” shock in pediatric sepsis. Although racial differences emerged as an important confounder that mitigated the association of genotype with shock type, these results support and justify the need to study DDAH2 polymorphisms in larger, more homogeneous cohorts to examine whether genotypic differences in NO metabolism contribute to phenotypic variability in sepsis pathophysiology. An improved understanding of individual differences in NO metabolism could help to better target therapeutic interventions to critically ill children with hemodynamic compromise.