The WHO defines preterm birth as birth before 37 weeks of gestation. Premature babies, especially those born very early, often have complicated medical problems. A preterm baby may have trouble breathing due to an immaturely developed respiratory control system. For example, some preterm babies may experience a cessation of breathing for 20 s or longer or a shorter pause accompanied by bradycardia (<100 beats per minute), cyanosis, or pallor, known as apnea of prematurity (AOP) (Eichenwald et al., 2016). Neonates with AOP experience frequent episodes of apnea, resulting in hypoxemia and bradycardia, all of which place the infant at risk for prolonged mechanical ventilation, retinopathy of prematurity (ROP), bronchopulmonary dysplasia (BPD), and long-term neurodevelopmental impairment (Puia-Dumitrescu et al., 2019). Frequent apnea may be one of the most troublesome problems in the neonatal intensive care unit (NICU).

Various mechanisms implicated in the pathogenesis of AOP have been identified, at least in the central nervous system and peripheral reflex pathways (Eichenwald et al., 2016). Ventilatory responses to hypoxia and inhibitory reflexes were exaggerated in preterm neonates. These unique vulnerabilities predispose the neonate to the development of apnea (Mathew, 2011; Martin and Wilson, 2012). Importantly, AOP may also have a critical genetic basis underlying this development-related disorder of respiratory control (Bloch-Salisbury et al., 2010). Methylxanthines have been the mainstay of pharmacological therapy for AOP for over 40 years, and caffeine is generally preferred over other methylxanthines because of its wider therapeutic index and lower incidence of serious complications (Morton and Smith, 2016). However, caffeine is not completely efficient, and in about half of treated infants their apnea frequency remains elevated; thus, aggressive interventions such as mechanical ventilation are needed (Laouafa et al., 2019; He et al., 2021).

The blockade of inhibitory adenosine A₁ receptors (A₁-AR; encoded by the ADORA1 gene), with the resultant respiratory neural output, as well as the blockade of excitatory adenosine A_2A receptors (A_2A-AR; encoded by ADORA2A gene) located on γ-aminobutyric acidergic neurons has been recognized as the primary mechanism of action of caffeine therapy (Eichenwald et al., 2016). Caffeine also competitively inhibits phosphodiesterases (PDEs) and binds to intracellular calcium channel ryanodine receptors, leading to intracellular Ca²⁺ release (Kumar and Lipshultz, 2019). Specific ADORA1 and ADORA2A polymorphisms have been associated with variability in response to caffeine therapy, as well as with a higher risk of AOP and BPD (Kumral et al., 2012). In our recent study, 88 single nucleotide polymorphisms (SNPs) in 19 genes encoding proteins involved in determining the disposition and pharmacological actions of caffeine were genotyped to evaluate the association between genetic mutations and the response to caffeine therapy in 112 preterm infants (He et al., 2021). The major findings of our previous study included the following: 1) No significant association between the plasma concentrations of caffeine and polymorphisms of caffeine-metabolism-related enzymes such as CYP1A2 and transcription factor aryl hydrocarbon receptor (AHR) was found; 2) polymorphisms of ADORA1, ADORA2A, ADORA3, and PDE4D, especially those of AHR and adenosine dehydrogenase (ADA) genes, play a critical role in determining interindividual variability to caffeine therapy. Interestingly, the influence of AHR polymorphisms on the response to caffeine therapy could not be explained by the mechanisms involving the AHR-CYP1A2 metabolic pathway (He et al., 2021). This AHR signaling pathway may play an important role in AOP development and the response to caffeine therapy.

In the respiratory control system, A₁-ARs are found at high densities in the brainstem and anterior hypothalamus, while A_2A-ARs are widely distributed in the medulla. Increased expression of ADORA1 and ADORA2A was observed in both the brainstem and hypothalamus of caffeine-treated neonatal rats. Increased adenosinergic maturation in the central cardiorespiratory areas could partly explain the pharmacological effects of caffeine observed in premature infants (Gaytan and Pasaro, 2012). Adenosine is an inhibitory neuromodulator involved in sleep-wake regulation. A_2A-ARs, but not A₁-ARs, mediate the arousal effect of caffeine (Huang et al., 2005). Intriguingly, caffeine has been shown to influence circadian timing in humans by an A₁-AR/cAMP-dependent mechanism (Burke et al., 2015). The basic mechanism of the clock is a cell-autonomous interlocked transcription-translation feedback loop sustained by transcriptional activators brain and muscle ARNT-Like 1 (BMAL1), also known as aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL1), Circadian Locomotor Output Cycles Kaput (CLOCK) or neuronal PAS domain protein (NPAS2), as well as period (PER) and cryptochrome (CRY) (Hardin and Panda, 2013). As the core molecular clock is present in nearly every cell in mammals (Mohawk et al., 2012), the circadian clock is likely involved in determining the tissue response to circulating factors, including drugs (Zhang et al., 2021). However, whether the human circadian clock affects the response to caffeine therapy in preterm infants is unknown.

Caffeine citrate was routinely prescribed for AOP management in the NICU. Some preterm infants respond well to caffeine, while others do not. We hypothesized that the human circadian clock is involved in the response to caffeine citrate therapy in preterm infants. The overarching goal of this study was to determine the association between circadian CLOCK gene polymorphisms in preterm babies and the clinical outcomes after caffeine treatment. In addition, this study touches on the crosstalk between the circadian clock and AHR signaling pathways based on our findings in preterm babies who received caffeine citrate treatment.

A comprehensive description of the AOP, BPD, ROP, intraventricular hemorrhage (IVH), patent ductus arteriosus (PDA), and necrotizing enterocolitis (NEC), as well as the methods for determining the plasma caffeine concentration and collecting clinical data for a single-center, retrospective study in Chinese preterm infants has been published elsewhere (He et al., 2021). The caffeine citrate treatment protocol, clinical outcomes, and grouping criteria have also been clearly described (He et al., 2021). Briefly, the apneic group included patients with apnea episodes that occurred even once despite the combined use of necessary nonpharmacological therapies, and the apnea-free group included those without apnea following the administration of caffeine and necessary respiratory support options. The study protocol was approved by the hospital ethics committee of the Children’s Hospital of Nanjing Medical University (protocol number 201902082-1).

The clear benefits and safety of caffeine citrate based on a standard-dose regimen have led to its wide and early use in very low birth weight infants. However, caffeine is not completely efficient, and in approximately 50% of treated infants, the apnea frequency remains elevated (Laouafa et al., 2019; He et al., 2021). In fact, studies are warranted to better understand why some preterm neonates persist with apnea after caffeine therapy, even after increasing the caffeine dose. The AOP phenotype has been attributed solely to immature respiratory system control consequent to preterm birth, but there may also be important genetic influences (Erickson et al., 2021). Therefore, the influence of genetics on the efficacy of caffeine in preterm infants should be better explored (Long et al., 2021).

In the present study, genetic polymorphisms of CYP3A genes, including 3A4, 3A5, 3A7, and 3A43, were also assessed (Table 1). Especially for CYP3A7, this isoform of CYP3A has been demonstrated to metabolize endogenous compounds that are known to be important in the growth and development of the fetus and neonate. In addition, CYP3A7 is an important component in the development and protection of the fetal liver and plays a role in certain disease status (Li and Lampe, 2019). However, because of MAF values < 0.001 or genotyping failure (Table 1), most of the selected SNPs were excluded from further association analysis.

A major finding of this study was that AHR genetic variations (rs1476080 and rs2066853), but not AHRR or ARNT genes, were found to be associated with the response to caffeine therapy (Tables 2, 3). Our previous study showed that two particular polymorphisms of the AHR gene (rs6968865 and rs4410790) were significantly associated with the response to caffeine treatment between the two groups of preterm infants (He et al., 2021). Furthermore, these variants in the AHR gene, located on chromosome 7, formed strong LD, with increased ORs for haplotypes CT (p = 0.00834), TA (p = 0.0122), and GACGG (p = 0.00243), which in part determined a better response to caffeine treatment (Figure 2; Table 4). Based on the plasma concentration data of caffeine, we excluded that the AHR-CYP1A2 metabolic pathway was responsible for the variable response to caffeine therapy (He et al., 2021). However, AHR is necessary to protect fetal human pulmonary microvascular endothelial cells against hyperoxic injury (Zhang et al., 2015) and plays a critical role in the maintenance of lung health (Guerrina et al., 2018). Therefore, the findings of our study suggest the potential role of AHR signaling in preterm infants who experience AOP episodes. The AHR signaling pathway may act alone or in combination with adenosine receptors and circadian CLOCK.

Another important finding in the present study was the significant association between circadian CLOCK polymorphisms, but not BMAL1 variants (also known as ARNTL1; Table 1), and the response to caffeine citrate therapy in these preterm babies (Table 2). Notably, after adjustment for clinically important variables, such as birth weight, birth height, respiratory support therapy, repeat intubation, and caffeine therapy duration, the significance was retained (Table 3). Moreover, 40 candidate CLOCK SNPs formed one small block and one big block with strong LD (Figure 2). Especially for the small block, the haplotype TTAAAG was found to be associated with a better response to caffeine therapy (OR = 3.1, p = 0.0436; Table 4).

Caffeine has been reported to affect the phase of the human circadian clock and primarily affect human cellular circadian clocks via an A₁-R/cAMP-dependent mechanism (Burke et al., 2015). Recent studies have revealed that caffeine and adenosine alter clock gene expression and circadian rhythms in vitro and in vivo via the Ca²⁺–ERK–AP-1 pathway (Jagannath et al., 2021). The present study is the first to show that genetic polymorphisms of CLOCK, encoding one of the two core components of the circadian rhythm, were significantly associated with the response to caffeine therapy. These findings suggest that the circadian rhythm may play critical roles in the response to caffeine citrate therapy in babies experiencing AOP episodes. Interestingly, caffeine has been reported to increase the light responsiveness of the mouse circadian pacemaker (van Diepen et al., 2014) and also affects the human circadian clock in vivo and in vitro (Burke et al., 2015). Therefore, synchronizing caffeine with the circadian rhythm may be useful for optimizing its treatment efficacy. In the meantime, investigations into essential mechanisms may provide therapies to reset or amplify circadian signals. However, very few studies have been performed to evaluate the proper timing of methylxanthine dosing (chronotherapy) as a means to maximize its efficacy and possibly reduce its adverse effects. Further research in this field is warranted to provide new insights and clinical advantages.

One of the major strengths of the present study was our ability to assess how genetics affect the response to caffeine based on 147 candidate SNPs in more than 40 human genes (Table 1), encoding various proteins related to the disposition of caffeine and/or pharmacological mechanisms of caffeine’s actions, as well as circadian control functions. Along with our previous report (He et al., 2021), we observed significant associations between ADORA1, ADORA2A, ADORA3, PDE4D, ADA, AHR, and CLOCK polymorphisms and the response to caffeine treatment (Tables 2, 3). Notably, the haplotypes of AHR, CLOCK, and ADORA2A were found to be associated with a better response to caffeine therapy (Table 4). A_2A-AR regulates phrenic nerve activity, and blockage with caffeine may be one mechanism responsible for the efficacy of xanthine against AOPs. Additional A₁-AR in the brainstem may regulate hypoxic ventilator drive. Such central stimulation results in increased respiratory drive, increased sensitivity to hypercarbia, decreased hypoxic suppression of respiration, and increased diaphragmatic contractility (Morton and Smith, 2016). Caffeine competitively inhibits PDEs, a group of enzymes that degrade cAMP (Burke et al., 2015). In addition, intracellular adenosines undergo metabolism to inosine by ADA, a deamination that occurs preferentially under pathological conditions featuring raised adenosine levels (Borea et al., 2018). Collectively, caffeine may play a critical role in AOP treatment by directly and/or indirectly agonizing the adenosine-AR-cAMP pathway, rather than the AHR-CYP1A2 pathway involved in the disposition of caffeine, an XRE-dependent control of the target gene expression pathway by AHR. One outstanding question is how to explain ADORA, AHR, and CLOCK gene polymorphisms that were observed to be associated simultaneously with the response to caffeine therapy. However, whether the AR, AHR, and CLOCK signaling pathways crosstalk with each other during caffeine treatment remains unclear. The potential model relationship between them is discussed below and illustrated in Figure 3.

Adenosine is a ubiquitous endogenous autacoid and functions as a signaling molecule through the activation of four distinct ARs (i.e., A₁, A_2A, A_2B, and A₃) (Borea et al., 2018). Adenosine encodes a sleep history and modulates circadian entrainment by light. However, caffeine has the opposite effect on sleep as adenosine, and it promotes wakefulness via A_2A-AR, rather than A₁-AR (Huang et al., 2005). Additionally, caffeine inhibits PDEs or promotes intracellular Ca²⁺ release (Kumar and Lipshultz, 2019). Pharmacological and genetic approaches demonstrate that adenosine acts on the circadian clock via A₁-AR/A_2A-AR signaling through the activation of the Ca²⁺–ERK–AP-1 and CREB/CRCT1–CRE pathways to regulate the clock genes PER 1 and PER 2 (Figure 3A). Interestingly, adenosine integrates light and sleep signaling for regulating the circadian rhythms in mice (Jagannath et al., 2021).

AHR is a ligand-activated transcription factor that is a member of the periodic circadian protein (PER)–AHR nuclear translocator (ARNT)–single-minded protein (SIM) superfamily of transcription factors, in which the PER–ARNT–SIM (PAS) domain senses both the endogenous and exogenous factors (Figure 3B). AHR senses oxygen levels, redox potential, and changes in the circadian rhythm and control adaptation in the cellular environment (Rothhammer and Quintana, 2019). AHR is involved in controlling the cardiovascular and respiratory functions (Sauzeau et al., 2011). There is high constitutive AHR expression in lung epithelial/endothelial cells and fibroblasts (Rico de Souza et al., 2021). AHR promotes lipid droplet biogenesis and metabolic shift in respiratory club cells, which are metabolically active cells that are involved in xenobiotic metabolism and host defense as well as the maintenance of airway integrity (Wang et al., 2021). AHR deficiency enhanced airway inflammation and remodeling in a murine chronic asthma model (Chang et al., 2020), and also caused the development of chronic obstructive pulmonary disease (Guerrina et al., 2021), indicating its importance as a central player in maintaining normal lung function and determining disease severity. In addition, AHR activity in both endothelial and hematopoietic cells is necessary for vascular development and closure of the ductus venosus (Guerrina et al., 2018). Therefore, the AHR signaling pathway may play a critical role in AOP and BPD development, as well as in the response to caffeine treatment in preterm infants.

Intrinsic clocks determine nearly all circadian cycles, such as respiratory and exercise capacity (Allada and Bass, 2021). More importantly, AHR and the circadian signaling pathways are highly integrated and reciprocally regulated (Figure 3C). AHR exhibits a rhythmic expression and time-dependent sensitivity to activation by AHR agonists. Conversely, AHR influences the amplitude and phase of rhythms in circadian clock genes, hormones, and behavior (Tischkau, 2020). AHR directly interacts with the core circadian clock. In the canonical signaling pathway, activated AHR forms a heterodimer with ARNT, which exhibits sequence homology with BMAL1. However, activated AHR can also heterodimerize with BMAL1, thereby disrupting the normal binding of CLOCK/BMAL1 to E-box elements to drive the transcription of the target genes (Jaeger et al., 2017). As one of the potential mechanisms, AHR-CLOCK might set a pathological precondition for the efficacy of caffeine treatment to manifest. Thus, investigation into the mechanism by which the AHR-CLOCK interaction affects the response to caffeine treatment may provide insights into the pathological mechanisms of AOP and provide an innovative strategy to reset the treatment protocol in preterm neonates.

However, our study has several limitations. In this study, only the CLOCK and BMAL1 polymorphisms were assessed for their potential roles in the circadian rhythm. In mammals, at the cellular level, the transcription factors CLOCK and neuronal NPAS2 form heterodimers with ARNTL1 (i.e., BMAL1) to drive the expression of the genes encoding period circadian protein homologues PER1, PER2, and PER3 and crypto-chromes (CRY) 1 and 2 via direct binding to the E-box enhancer element during the day. In the late afternoon or evening, PER and CRY proteins heterodimerize, translocate into the nucleus, and interact with CLOCK and BMAL1, thus suppressing their transcriptional activity. In the meantime, the protein levels of PER1, PER2, CRY1, and CRY2 also decline by poly-ubiquitination and subsequent degradation via specific E3 ligase complexes (Ruan et al., 2021). However, we did not determine the roles of the genes encoding PER and CRY proteins in caffeine therapy. Another limitation is that this study was performed in a single center with a small sample size (n = 112), which may increase the risk that some significant changes will not be captured. The missed significant association between CYP1A2*1B (rs2470890) variant and response to caffeine therapy (p = 0.058; codominant model) in preterm babies may be due to the small study cohort. In addition, as described in our previous report (He et al., 2021), the grouping criteria may limit the reliability and accuracy of the present study. Lastly, in terms of statistical approach, within an FDR threshold of <0.05, 5% of the variants (i.e., 4-5 SNPs) were estimated to be false positives, which was generally acceptable.

Our study found that candidate CYPs 1A2, 3A4, 3A5, 3A7, 3A43, AHRR, ARNT, and BMAL1 polymorphisms had no effect on the response to caffeine therapy in the apnea-free and apneic groups. However, genetic candidate variants in AHR and CLOCK genes were found to be associated with variable responses to caffeine treatment in these preterm babies. Moreover, the variants in AHR, ADORA2A, and CLOCK genes formed strong LD with increased ORs, which were associated with a significantly better response to standard-dose caffeine therapy. Future and larger studies, as well as basic research, are required to understand how these SNPs affect the response to caffeine therapy. Our findings also indicate that circadian rhythm may play an essential role in the response to caffeine therapy in babies experiencing AOP episodes.

• Caffeine citrate therapy is a preferable choice for preterm infants with AOP in the NICU.

• No significant intergroup differences in allele distributions or genotype frequencies of CYP1A2, CYP3A4, CYP3A5, and CYP3A7 were found in our study on preterm babies.