Of Mice and Babies: PHOX2B and Obstructive Apneas in Congenital Central Hypoventilation Syndrome.

From birth to death, breathing and upper airway function are precisely regulated by a neural network located in the brainstem and pons that generates respiratory rhythm and pattern in a sleep–wake state-dependent manner (1). Central respiratory chemoreceptors sense very small changes in brain Pco2 and activate the respiratory centers maintaining the stability of CO2 (2). The retrotrapezoid nucleus (RTN) resides at the ventral medullary surface and includes a well-defined subset of CO2-sensitive neurons characterized by the expression of Phox2b (paired-like homeobox gene 2b), Vglut2 (vesicular glutamate transporter 2), Nmb (neuromedin B), TASK-2 (two-pore-domain potassium channel 2), and GPR4 (G-protein–coupled receptor 4) (3). Failure to detect Pco2 fluctuations by RTN neurons and other central chemoreceptors leads to severe diseases such as congenital central hypoventilation syndrome (CCHS), which result in hypercapnic respiratory failure. The CO2 chemoreflex plays an important role in defense mechanisms against obstructive sleep apnea by activating hypoglossal motoneurons and the main pharyngeal dilator, the genioglossus muscle (4).

CCHS is a rare, autosomal dominant, and life-threatening disorder first described in 1970 by Mellins and colleagues, who suggested that “injury to medullary chemoreceptors is responsible for alveolar hypoventilation in this syndrome” (5). CCHS is characterized by an impaired ventilatory response to CO2, mainly during sleep, hypoxemia, facial dysmorphology, cardiac asystoles, Hirschsprung disease, and neuroblastoma (6). CCHS results from the mutation of Phox2b on chromosome 4p12, which is essential for the development of the respiratory control centers (7). The incidence of CCHS is estimated at 1/148,000 live births (8). There is no approved therapy for CCHS and its devastating complications. Like humans with CCHS, transgenic mice with Phox2b mutation develop hypoventilation during sleep, central sleep apneas, and decreased hypercapnic ventilatory response (7, 9). However, the effect of Phox2b deficiency on upper airway obstruction during sleep is unknown. Experiments in transgenic mice allow us to address two important questions: 1) Can a mouse model offer an insight into the mechanisms of different types of apneas in neonates? 2) Does Phox2b deficiency in CO2-sensing neurons predispose to obstructive apneas?

Investigators previously developed and validated plethysmographic methods for monitoring airflow and respiratory effort, which allowed the detection of upper airway obstruction during sleep in adult mice (10, 11). However, technology allowing us to differentiate between obstructive and central events in newborn animals did not previously exist (12).

In this issue of the Journal, Madani and colleagues (pp. 1200–1210) report that the Phox2b mutation predisposes neonatal mice to CCHS, manifested by central as well as obstructive and mixed apneas caused by hypoglossal dysgenesis (13). Madani and colleagues generated two mouse strains that carry the most common of the CCHS-causing mutations, a +7 alanine expansion of the 20-residue polyAla tract (the Phox2b allele), by a knock-in approach. The first strain carried the Phox2b transgene globally, whereas in the second strain, Phox2b mice, the mutation was expressed exclusively in the RTN neurons. The investigators developed the state-of-the-art technology to detect obstructive events. They performed three-dimensional (3D) scans of newborn mice and created custom-made pneumotachograph/face masks using 3D printing (Figure 1). This interesting approach was combined with a laser profilometer pointing radially at the lateral abdominal wall to detect abdominal effort, which allowed the differentiation of central, obstructive, and mixed events. In vivo data were complemented by morphometry of the hypoglossal nucleus and electrophysiology in brain slices. The authors provided convincing evidence that newborn mice with the global Phox2b mutation have markedly increased obstructive apneas in addition to central and mixed events. In contrast, Phox2b deficiency localized to the RTN did not induce obstructive events. Morphometry showed that global Phox2b deficiency led to hypoplasia of the hypoglossal nucleus, and an electrophysiology study demonstrated that the mutation was associated with a decreased burst frequency of the hypoglossal motoneurons and discoordination of hypoglossal and phrenic nerve activity.

Figure 1.

Summary of the main findings of Madani and colleagues. The investigators generated a mouse model of congenital central hypoventilation syndrome and validated the custom-made pneumotachograph/facemasks combined with a laser profilometer to detect abdominal effort. Putative monosynaptic or polysynaptic projections from Phox2b neurons in the NTS or RTN to the 12N may be necessary for upper airway patency during sleep. 7N = facial nucleus; 12N = hypoglossal nucleus; AP = area postrema; NTS = nucleus tractus solitarii; PHOX2B = paired-like homeobox gene; RTN = retrotrapezoid nucleus.

The hypoglossal nerve (12N) maintains upper airway muscle tone and pharyngeal patency by modulating activity of the tongue protruder genioglossus and retractors styloglossus and hyoglossus (14). The major finding of the paper is that global, but not RTN-specific, Phox2b mutation leads to hypoplasia and dysfunction of hypoglossal motoneurons as a likely cause of obstructive apneas in neonate mice.

The study has several limitations. First, mechanisms of the Phox2b defect in the hypoglossal motoneurons and upper airway function remain unclear. The investigators attributed the deficit to primary dysfunction of hypoglossal motoneurons, but the cause of this dysfunction is not clear, besides that it occurs outside of the RTN. In fact, hypoglossal motoneurons do not express PHOX2B, but this protein is found in the other brain regions, such as the nucleus tractus solitarii, which contributes to the hypercapnic ventilatory response (15). Whether or not PHOX2b-expressing neurons in the nucleus tractus solitarii are involved in obstructive apnea in CCHS is a matter deserving further investigation (Figure 1). Second, global Phox2b deficiency appeared to have a much more impressive impact on the apnea duration compared with the RTN-specific defect, which suggests severe impairment of control of breathing. Obstructive and mixed apnea in these animals are much longer than central events. Given technical challenges to differentiate between obstructive and mixed events, it opens a possibility that the airway may simply close at the end of a very long central event, as it is described in human obstructive sleep apnea (16). Third, although in vivo data suggest the lack of RTN Phox2b involvement in hypoglossal motoneuron dysfunction, future mechanistic studies using morphometry and electrophysiology in mice with Phox2b knockout specific to RTN are necessary to test this hypothesis.

In conclusion, despite its limitations, the study by Madani and colleagues represents a significant advancement in the field because it raises the possibility that Phox2b mutation may lead to obstructive apneas along with centrals. This seminal observation together with the elegant animal model developed in this study may allow further investigation of the individual role of brain regions in sleep-disordered breathing beyond CCHS.