nature publishing group Articles Clinical Investigation Genetic variation in CYB5R3 is associated with methemoglobin levels in preterm infants receiving nitric oxide therapy Tyson D. Fuller1, Cassandra N. Spracklen2, Kelli K. Ryckman2, Lindsey A. Knake1, Tamara D. Busch1, Allison M. Momany1, Jeffrey C. Murray1 and John M. Dagle1 BACKGROUND: In recent years, increasing numbers of pre- to functional hemoglobin, which puts them at increased risk term infants have been exposed to inhaled nitric oxide (iNO). for developing MetHb toxicity (9). At the same time, however, This population has decreased methemoglobin (MetHb) preterm infants are being treated with inhaled NO (iNO) as reductase activity in their erythrocytes, which may increase the therapy to prevent chronic lung disease (1,8–10). In order to risk of MetHb toxicity. We sought to determine if genetic fac- prevent MetHb toxicity, MetHb levels are frequently measured tors are associated with the observed variance in MetHb levels. while the preterm infant is receiving iNO therapy (8,9). METHODS: A population of 127 preterm infants was geno- CYB5A (cytochrome B5 type A) and CYB5R3 (cytochrome typed for five single-nucleotide polymorphisms (SNPs) in the B5 reductase 3) are genes that encode proteins with MetHb CYB5A and CYB5R3 genes. iNO dose and levels of MetHb were reductase activity, each of which have two major splice vari- obtained by chart abstraction. ANOVA was performed to iden- ants. A soluble form of these proteins in the erythrocytes tify genetic associations with MetHb levels. is responsible for reducing MetHb back into functional RESULTS: An association was found between the heterozy- hemoglobin. Several studies have demonstrated that methe- gous genotype (GA) of rs916321 in the CYB5R3 gene and the moglobinemia is associated with genetic variations in both mean of the first recorded MetHb levels in Caucasian infants (P CYB5A and CYB5R3, resulting in decreased enzyme activity = 0.01). This result remained significant after adjustment for the (7,11–15). iNO dose (P = 0.009), gender (P = 0.03), multiple gestation (P = Although genetic influences may play a role in a preterm 0.03), birth weight (P = 0.02), and gestational age (P = 0.02). No infant’s susceptibility to elevated MetHb levels following iNO significant associations were found with the other SNPs. exposure, this association has not yet been studied. Thus, the CONCLUSION: We demonstrate a novel genetic association objective of this study was to evaluate the association between with neonatal MetHb levels. Identification of genetic risk fac- two single-nucleotide polymorphisms (SNPs) in CYB5A and tors may be useful in determining which preterm infants are three SNPs in CYB5R3, two genes known to influence MetHb most at risk of developing MetHb toxicity with the use of iNO. levels, and MetHb levels in preterm infants receiving iNO. This information will give us a better understanding of the genetic influence on MetHb levels and may help to determine more itric oxide (NO) is naturally produced in the vascular endo- appropriate iNO administration and monitoring of MetHb Nthelium by the enzyme NO synthase (1). NO enters the pul- levels in this vulnerable population. monary vascular smooth muscle cells either by simple diffusion or through extracellular formation of SNO-l-cysteine which RESULTS enters through the L-type amino acid transporter (2). There, it Characteristics of our primary study population can be seen in stimulates soluble guanylate cyclase, which produces the intra- Table 1. Of the 109 Caucasian preterm infants receiving iNO cellular second messenger cyclic guanosine monophosphate, a therapy, the majority of the infants were male, non-Hispanic, mediator in the vasodilatory pathway (1,3). When NO enters of singleton gestation, born weighing <1,000 g, and born at the blood stream, it quickly reacts with hemoglobin to form ≤28 wk gestation. There was no statistical difference in MetHb methemoglobin (MetHb), and as a result, the hemoglobin is no levels with respect to gender, multiple gestation, birth weight, longer able to bind oxygen (1,4,5). The concentration of MetHb gestational age, or ethnicity. in the blood under normal conditions is typically less than 2% The average starting dose of iNO in this cohort was 12.60 of all hemoglobin (6). When levels of MetHb in the blood reach ppm. The first MetHb level was selected for analysis as this was elevated concentrations, MetHb toxicity can occur (6–9). assumed to be the closest to the baseline MetHb levels for the Preterm infants have lower than normal levels of MetHb infants. The time interval from starting iNO to checking the reductase, the enzyme responsible for converting MetHb back first MetHb value varied between 1 and 24 h. Using a Pearson’s 1Department of Pediatrics, University of Iowa, Iowa City, Iowa; 2Department of Epidemiology, University of Iowa, Iowa City, Iowa. Correspondence: John M. Dagle ([email protected]) Received 16 May 2014; accepted 10 September 2014; advance online publication 14 January 2015. doi:10.1038/pr.2014.206 472 Pediatric REsEarch Volume 77 | Number 3 | March 2015 Copyright © 2015 International Pediatric Research Foundation, Inc. Genetic variation in MetHb levels Articles Table 1. Characteristics of Caucasian study participants correlation analysis, we found an association between the first Covariate N (%) Methemoglobin value Pa iNO dose and the first metHb value measured (P = 0.04). Gender The maximum iNO dose and corresponding MetHb levels Male 68 (62.4) 0.837 (0.361) 0.98 were also found to be correlated (P = 0.0006). Finally, we found no evidence of a consistent increase in MetHb levels with dura- Female 41 (37.6) 0.839 (0.356) tion of iNO exposure. Multiple gestation Table 2 compares the mean of the first recorded MetHb No 86 (78.9) 0.843 (0.348) 0.74 level with each genotype for the five analyzed SNPs. Infants Yes 23 (21.1) 0.817 (0.399) heterozygous (GA) for SNP rs916321 in the CYB5R3 gene Birth weight (g) have significantly lower MetHb levels than either homozygote (P = 0.01) (Figure 1). This association remained significant <1,000 67 (61.5) 0.820 (0.353) 0.84 after adjustment for the first iNO level (P = 0.009), gender 1,000–1,499 10 (9.2) 0.872 (0.366) (P = 0.03), multiple gestation (P = 0.03), birth weight 1,500–2,499 15 (13.8) 0.907 (0.485) (P = 0.02), and gestational age (P = 0.02). The association ≥2,500 17 (15.6) 0.846 (0.266) also remained significant when rs916321 variants were com- Gestational age (wk) pared with the average metHb levels in the first week of iNO ≤28 73 (68.9) 0.809 (0.347) 0.37 exposure (adjusting for iNO dose). Although the difference found in the MetHb levels between rs916321 variants was 29–32 9 (8.5) 0.911 (0.404) small, it was statistically significant and may show a larger 33–36 24 (22.6) 0.912 (0.394) effect in interactions with other genetic or environmental Ethnicity risk factors. No significant associations were found between Non-Hispanic 103 (94.5) 0.840 (0.355) 0.96 the first measured MetHb level and the other four genotyped Hispanic or Latino 6 (5.5) 0.834 (0.435) SNPs. All values presented as mean (SD). Additional analyses show that the highest recorded MetHb aP value for t-test comparing mean methemoglobin value. level measured was also associated with rs916321 genotype Table 2. Methemoglobin means and SDs for evaluated SNPs among Caucasians only Adjusted P valuea MetHb level, SNP N mean (SD) P valuea First NO level Gender Multiple gestation Birth weight Gestational age rs8096066 AA 58 0.853 (0.31) 0.69 0.04 0.80 0.67 0.83 0.84 AG 46 0.893 (0.42) GG 3 0.733 (0.31) rs7238784 GG 48 0.836 (0.33) 0.68 0.04 0.83 0.57 0.80 0.81 GT 50 0.897 (0.41) TT 11 0.845 (0.26) rs6002829 CC 34 0.878 (0.32) 0.95 0.05 0.98 0.72 0.97 0.96 CT 58 0.855 (0.40) TT 16 0.873 (0.28) rs916321 GG 46 0.934 (0.39) 0.01 0.009 0.03 0.03 0.02 0.02 GA 47 0.752 (0.31) AA 15 1.006 (0.30) rs2285138 TT 34 0.845 (0.32) 0.68 0.08 0.84 0.60 0.82 0.83 TC 55 0.855 (0.38) CC 20 0.927 (0.34) MetHb, methemoglobin; NO, nitric oxide; SNP, single-nucleotide polymorphism. aP values from ANOVA testing difference between mean methemoglobin values among the three different genotypes for each SNP. Copyright © 2015 International Pediatric Research Foundation, Inc. Volume 77 | Number 3 | March 2015 Pediatric RESEarch 473 Articles Fuller et al. 2.0 instability. In most cases, type I methemoglobinemia can be corrected with methylene blue or ascorbic acid and riboflavin, and patients with type I methemoglobinemia are expected to 1.5 have a normal life expectancy (7,13). Type II is a more severe form of methemoglobinemia that affects all cells, resulting in developmental defects, neurological disabilities, and early 1.0 mortality (13,14). Studies have shown different genetic poly- morphisms linked to type I and type II methemoglobinemia (12–14). Recent studies have also found genetic polymor- Methemoglobin percent 0.5 phisms associated with atypical type II disease phenotypes (14,16). Thus, investigation of common polymorphisms pres- ent in these genes was expected to provide information regard- 0.0 ing MetHb levels. GG GA AA We have shown one SNP (rs916321) to have an associa- rs916321 Genotype tion with MetHb levels of preterm infants receiving iNO.