Caffeine Therapy for Apnea of Prematurity: Role of the Circadian CLOCK Gene Polymorphism

Abstract

Standard-dose caffeine citrate has been routinely prescribed for apnea of prematurity (AOP) management; however, some preterm infants respond well to the therapy while others do not. The AOP phenotype has been attributed solely to the immature control of the respiratory system consequent to preterm birth, but there are also important genetic influences. Based on our previous report, we tested the hypothesis that the human circadian locomotor output cycles kaput (CLOCK) gene polymorphisms play a role in the response to caffeine citrate therapy in preterm infants. We also studied the interactions of the circadian clock with aryl hydrocarbon receptor (AHR) signaling pathways in preterm babies who received caffeine citrate. This single-center study collected data from 112 preterm infants (<35 weeks gestational age) between July 2017 and July 2018, including apnea-free (n = 48) and apneic (n = 64) groups. Eighty-eight candidate single nucleotide polymorphisms (SNPs) were tested using the MassARRAY system. Association analysis was performed using the PLINK Whole Genome Data Analysis Toolset and SNPStats software. Linkage disequilibrium (LD) and haplotype analyses were performed using Hapview software. No significant intergroup differences in allele distributions or genotype frequencies of CYP1A2, CYP3A4, CYP3A5, and CYP3A7 were detected in our study on preterm babies. Two more SNPs in AHR were found to be associated with determining the response to caffeine citrate therapy in our pediatric patients. Of the 46 candidate SNPs in the CLOCK gene, 26 were found to be associated with determining the response to caffeine treatment in these babies. Interestingly, a significant association was retained for 18 SNPs in the CLOCK gene after false discovery rate correction. Moreover, strong LD formed in those variants in AHR, ADORA2A, and CLOCK genes was confirmed to be significantly associated with a better response to standard-dose caffeine therapy. In summary, CLOCK gene polymorphisms play a role in determining the response to caffeine therapy in premature neonates with AOP. However, whether the AHR and CLOCK signaling pathways crosstalk with each other during caffeine treatment remains largely unclear. Future clinical studies including more immature babies and basic research are needed to explore the mechanism by which circadian rhythms affect the response to caffeine therapy.

Keywords: AHR; apnea of prematurity; caffeine; circadian rhythm; clock; polymorphism; preterm infant.

FIGURE 1

Significances (plotted as−log10 of nominal p values, closed blue circles) resulting from the associations between the investigated SNPs and different response (apnea-free vs. apneic) to caffeine therapy in preterm infants (n = 112). The x-axis represents the relative genomic position/chromosome, and the y-axis represents the distribution of the nominal−log10(P) values in the two groups of infants. The 26 SNPs, which are associated with the response to caffeine treatment (p < 0.05; above stippled red line), map to the CLOCK gene located on chromosome 4.

FIGURE 2

Linkage disequilibrium (LD) structure (triangle plots) for all investigated single nucleotide polymorphisms (SNPs) (rs numbers below gene names). Red diamonds indicate strong historical LD, r ² -based haplotype boundaries, among SNPs.

FIGURE 3

A model of caffeine, AR signaling pathway, AHR signaling pathways, and human circadian clock. (A) Caffeine alters intracellular cAMP levels through binding to adenosine receptors (A₁, A_2A, A_2B and A₃) and inhibiting phosphodiesterases. In addition, caffeine mobilizes intracellular Ca²⁺ from the endoplasmic reticulum through activation of the RyR channels. Increased cytosolic cAMP/Ca²⁺ signaling culminates CREB activation acting in tandem with rhythmic transcriptional activation by CLOCK/BMAL1. CLOCK/BMAL1 binds to E-box elements in the PER promoter, where they act to stimulate PER transcription. (B) Inactive AHR complexes with HSP90, AIP, p23 and SRC in the cytosol. Upon agonist binding, AHR and some components of the chaperone complex translocate to the nucleus, where AHR forms a dimer with ARNT binding to the XRE to control gene expression (such as AHRR, CYP1A1, CYP1A2 and CYP1B1). (C) Activated AHR can physically interact with BMAL1, which exhibits high homology with ARNT, thereby reducing CLOCK-BMAL1 interactions and repressing PER transcription. In addition, activated AHR phosphorylates and activates JNK, which represses CRE-mediated transcriptional activity to suppress PER transcription (left). Rhythmic transcription of AHR is driven by CLOCK: BMAL1 at E-Box promoter elements. PER and CRY inhibit AHR transcript levels (right). Abbreviation: cAMP, cyclic adenosine monophosphate; RyR, ryanodine receptor; PDEs, phosphodiesterases; AHR, aryl hydrocarbon receptor; AHRR, AHR repressor; ARNT, AHR nuclear translocator; XRE, xenobiotic response element; HSP90, 90 kDa heat shock protein; AIP, AHR-interacting protein. CLOCK, circadian locomotor output cycles kaput; BMAL1, brain and muscle ARNT-Like 1; CREB, cAMP response element-binding; CBP, CREB binding protein; PER, periods; CRY, crypto-chromes.