PHOX2B mutations and ventilatory control

Respiratory Physiology & Neurobiology 164 (2008) 49–54

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PHOX2B mutations and ventilatory control Jorge Gallego a,b,∗ , Stéphane Dauger a,c a

INSERM, U676, Hôpital Robert Debré, 48 Bd Sérurier, 75019 Paris, France Université Denis Diderot, Paris, France c Service de Réanimation et Surveillance Continue Pédiatriques, Pôle de Pédiatrie Aiguë et Médecine Interne, Hôpital Robert Debré, 48 Bd Sérurier, 75019 Paris, France b

a r t i c l e

i n f o

Article history: Accepted 9 July 2008 Keywords: PHOX2B Sleep apneas Hypercapnia CCHS

a b s t r a c t The transcription factor PHOX2B is essential for the development of the autonomic nervous system. In humans, polyalanine expansion mutations in PHOX2B cause Congenital Central Hypoventilation Syndrome (CCHS), a rare life-threatening disorder characterized by hypoventilation during sleep and impaired chemosensitivity. CCHS is combined with comparatively less severe impairments of autonomic functions including thermoregulation, cardiac rhythm, and digestive motility. Respiratory phenotype analyses of mice carrying an invalidated Phox2b allele (Phox2b+/− mutant mice) or the Phox2b mutation (+7 alanine expansion) found in patients with CCHS (Phox2b27Ala/+ mice) have shed light on the role for PHOX2B in breathing control and on the pathophysiological mechanisms underlying CCHS. Newborn mice that lacked one Phox2b allele (Phox2b+/− ) had sleep apneas and depressed sensitivity to hypercapnia. However, these impairments resolved rapidly, whereas the CCHS phenotype is irreversible. Heterozygous Phox2b27Ala/+ pups exhibited a lack of responsiveness to hypercapnia and unstable breathing; they died within the first few postnatal hours. The generation of mouse models of CCHS provides tools for evaluating treatments aimed at alleviating both the respiratory symptoms and all other autonomic symptoms of CCHS. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Respiratory control impairments occurring early during development stages may compromise brain oxygenation, thereby leading to irreversible motor and cognitive disorders. The etiology and incidence of these impairments vary considerably, from apnea of prematurity, seen in 85% of infants born before 34 weeks of gestation (Schmidt et al., 2006), to Congenital Central Hypoventilation Syndrome (CCHS or Ondine’s syndrome), a rare disorder typically presenting in the newborn period (Weese-Mayer et al., 1999). The incidence was estimated at 1 per 200,000 live births based on the French cohort of patients (Trang et al., 2005) but a more accurate estimate will be obtained in the near future from on-going international epidemiological studies of CCHS populations. Mouse studies of respiratory phenotypes in mutant newborn mice have helped to identify candidate genes for developmental respiratory control disorders (Gaultier and Gallego, 2005). Regarding CCHS, several groups (Matera et al., 2002; Amiel et al., 2003b; Weese-Mayer et al., 2003) examined the RNX gene, as RNX knockout mice exhibited respiratory control disorders, but found no mutations. Weese-Mayer et al. (2002) analyzed brain-derived neu-

∗ Corresponding author at: INSERM U676, Hôpital Robert Debré, 48 Bd Sérurier, 75019 Paris, France. Tel.: +33 1 40 03 19 75. E-mail address: [email protected] (J. Gallego). 1569-9048/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2008.07.003

rotrophic factor (BDNF) as a potential candidate gene in CCHS after respiratory control disorders were observed in mice with Bdnf mutations (Erickson et al., 1996). Based on mouse studies showing cross-regulation of the Phox2b and Mash1 genes and impaired ventilatory responses to hypercapnia, the human ortholog of Mash1 (HASH-1) was considered an additional candidate gene for CCHS (de Pontual et al., 2003; Weese-Mayer et al., 2003). Although rare, CCHS provides a unique opportunity to identify genetic factors involved in respiratory control development. This syndrome is characterized by sleep-related hypoventilation and apneas (especially in severe cases) with severe abnormalities in chemosensitivity, as well as by various autonomic disorders of widely variable penetrance (Weese-Mayer et al., 1999; Marazita et al., 2001; Weese-Mayer et al., 2001; Vanderlaan et al., 2004). However, a general limitation of most clinical descriptions of CCHS is the lack of genetic testing. The considerable inter-individual variability in the number and severity of associated symptoms probably reflects differences in the nature of the genetic defect (polyalanine expansion, missense or frameshift mutations, see below). Affected patients have absent or markedly reduced ventilatory responses to sustained hypercapnia (Paton et al., 1989) and, to a lesser extent, to sustained hypoxia (Paton et al., 1989). These abnormalities are generally ascribed to impaired central integration of chemosensory inputs to the brainstem, rather than to failure of the chemoreceptors, which are at least partially active (Marcus et al., 1991; Gozal et al., 1993; Spengler et al., 2001). Respiratory control disorders are

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difficult to analyze in the most severe forms of CCHS (e.g., patients who cannot breathe spontaneously even when awake). For this reason, previous studies may not reflect the entire spectrum of respiratory disorders in CCHS. The identification of PHOX2B as the disease-causing gene in CCHS (Amiel et al., 2003a; Sasaki et al., 2003; Weese-Mayer et al., 2003; Matera et al., 2004) has stimulated considerable interest in the role for this gene not only in CCHS, but also in prevalent conditions such as sleep apnea. Recently, there have been several reports of adults exhibiting sleep-related hypoventilation and, in some instances, central apneas and severe hypoxemia, with PHOX2B mutations generally characterized by five additional alanine residues in PHOX2B (Matera et al., 2004; Trang et al., 2004; Trochet et al., 2005a; Weese-Mayer et al., 2005b; Antic et al., 2006; Barratt et al., 2007; Diedrich et al., 2007; Doherty et al., 2007; Trochet et al., 2008). These observations suggest that respiratory control disorders associated with PHOX2B mutations may be more prevalent than previously inferred from the very low incidence of CCHS in newborns. More generally, PHOX2B appears pivotal to the development of respiratory networks. To analyze the functional impact of PHOX2B mutations on breathing at the organism level, mouse models must be studied. The present review focuses on the respiratory phenotypes of mice lacking one Phox2b allele (Phox2b+/− mutants). The fact that Phox2b+/− pups develop normally is in striking contrast to the severe abnormalities seen in humans with CCHS. However, the purpose of this mouse model is to determine which components of respiratory control, if any, depend on Phox2b for normal development. This approach is critical to unraveling the pathogenesis of CCHS. The present review also describes the respiratory phenotype of mice carrying polyalanine expansions. We studied mice carrying a +7 alanine expansion (Phox2b27Ala/+ mice). This genotype was the most frequent in a cohort of 188 patients with CCHS occurring either as an isolated disorder or in combination with Hirschsprung disease (HSCR) and/or tumors of the sympathetic nervous system (Trochet et al., 2005a).

2. PHOX2B, the main disease-causing gene for CCHS About 20% of patients with CCHS also have Hirschsprung disease, a developmental disorder of the enteric nervous system characterized by absence of ganglion cells in the distal colon (Weese-Mayer et al., 2001; Amiel et al., 2008). This association suggested shared pathophysiological mechanisms in CCHS and HSCR. Furthermore, most patients with CCHS exhibit a variety of autonomic disorders, and CCHS is associated with tumors developed from neural crest cells (neuroblastoma), in addition to chemosensitivity disorders. Taken together, these observations suggested abnormal neural crest development as the abnormality underlying CCHS. Therefore, human genome screening and mouse model studies were conducted to investigate genes involved in the development and migration of neural crest cells. Of special interest was the gene encoding the RET receptor tyrosine kinase signaling pathway, which involves the sequential expression of MASH1, PHOX2A/2B, RET, and TH (Pattyn et al., 1999). This pathway is responsible for the development of all transiently or permanently produced noradrenergic derivatives (Pattyn et al., 1999). The gene causing CCHS was found to be the transcription-factor gene PHOX2B (Amiel et al., 2003a; Sasaki et al., 2003; Weese-Mayer et al., 2003; Matera et al., 2004). Over 90% of patients carry a heterozygous PHOX2B mutation consisting, in most cases, in a polyalanine-repeat expansion (Trochet et al., 2005b; Berry-Kravis et al., 2006). A small proportion of patients with CCHS, however, carry other PHOX2B mutations instead, such as missense, nonsense, or frameshift mutations, which severely disrupted PHOX2B function (Trochet et al.,

2005a; Berry-Kravis et al., 2006). These important results pointed toward PHOX2B as a pivotal factor in the neurochemical control of breathing, and more generally, in autonomic nervous system disorders, Hirschsprung disease, and neuroblastoma. Several CCHS patients carry heterozygous mutations affecting seven genes generally involved in neural crest cell development (reviewed in Weese-Mayer et al., 2005a, see also www.genetests.org). However, the role in CCHS for mutations in genes other than PHOX2B remains unclear. The finding that PHOX2B was the main disease-causing gene for CCHS was consistent with previous knowledge on the role for this gene in neurodevelopmental processes, which was reviewed recently (Brunet and Goridis, 2008). The transcription factor Phox2b is required for the development of most neuronal types in the central and peripheral nervous systems (Tiveron et al., 1996; Pattyn et al., 1997; Brunet and Pattyn, 2002). In the peripheral nervous system, Phox2b is expressed in the neurons of all autonomic and sensory ganglia of the VIIth, IXth, and Xth cranial nerves. In the central nervous system, Phox2b is expressed in the hindbrain branchiomotor and visceromotor neurons, all noradrenergic neurons, and neurons in the nucleus tractus solitarius (Pattyn et al., 1999, 2000a,b; Dauger et al., 2003). Despite their diverse origins, most Phox2b-dependent neurons are part of the visceral nervous system and connect synaptically to form medullary visceral reflex arcs (Brunet and Pattyn, 2002). Furthermore, the specific role for Phox2b in central chemosensory pathways was clarified recently. In adult rats, Phox2b is expressed by a group of chemosensitive glutamatergic interneurons located in the retrotrapezoid nucleus (RTN) (Stornetta et al., 2006). A finding of major relevance to CCHS is Phox2b expression by a chain of neurons involved in the integration of peripheral and central chemoreception (Stornetta et al., 2006). The chain included the carotid bodies, chemoreceptor afferents, chemoresponsive projections of the NTS to the ventrolateral medulla, and central chemoreceptors located in the RTN (Stornetta et al., 2006). These important findings failed, however, to explain the contrast between the pervasive role for Phox2b in autonomous circuit development and the relatively targeted impact of PHOX2B mutations on respiratory control in CCHS. Genetic mouse models were needed to clarify the role for Phox2b in breathing control and, in particular, to investigate the emerging genotype–phenotype relationships in humans with CCHS. 3. Ventilatory phenotype in Phox2b+/− mice 3.1. Early assessment of respiratory control in mice To investigate the role for Phox2b in the development of respiratory control, the respiratory phenotype of neonatal mice with one invalidated Phox2b allele (Phox2b+/− ) was determined. Homozygous Phox2b knock-out mice (Phox2b−/− ) die in utero around embryonic day 14 (Pattyn et al., 2000b), whereas Phox2b+/− pups survive and are fertile. Body weight and mouth temperature were normal in 2-day-old Phox2b+/− mice (Dauger et al., 2003). At 5 days of age, however, the mutant pups had slightly lower body weights and lower temperatures than the wild-type pups, suggesting disorders of autonomic regulation such as described in patients with CCHS (Weese-Mayer et al., 1999; Marazita et al., 2001; WeeseMayer et al., 2001; Vanderlaan et al., 2004). Breathing control was assessed in vivo, in unrestrained, nonanaesthetized newborn mice. Breathing was quantified based on breath duration, inspiratory and expiratory durations, tidal volume, ventilation, and apneas. Tidal volume (VT ) and ventilation (VE ) were divided by body weight to adjust for inter- and intraindividual differences in growth, which are particularly marked

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during early development. Breathing variables were measured both under baseline conditions, i.e., while the animals were breathing air and during exposure to chemical stimuli such as hypercapnia, hypoxia, or hyperoxia, to test for chemosensitivity. Methods for in vivo assessment of newborn mice are discussed elsewhere (Gaultier et al., 2006). In newborn mice, birth weight (about 1–2 g) and tidal volume (3–4 ␮l) are too small for the measurement devices used in adult mice (e.g., pneumotachometers, thermistors, respiratory inductance spirometers, and magnetometers). Two methods provide valid measurements in newborn mice, namely, head-out plethysmography (e.g., Burton et al., 1997) and whole-body plethysmography (e.g., Matrot et al., 2005). Headout plethysmography gives a relatively direct measure of VT but requires that the animal be tightly restrained to prevent air from leaking around the neck. The effects of the neck collar on upper airway resistance in these tiny animals are difficult to control. Whole-body flow barometric plethysmography, which consists in measuring pressure changes in a chamber where the animal is placed, can be performed in unrestrained animals. Whole-body plethysmography, initially developed in large animals (Drorbaugh and Fenn, 1955), has been validated against pneumotachography in adult mice (Onodera et al., 1997) but not in newborn mice. Thus, in newborn mice, whole-body plethysmography provides semi-quantitative measurements of VT and ventilation while allowing valid measurements of breathing frequency and apnea. Despite its limitations, whole-body flow barometric plethysmography remains the only non-invasive method for studying unrestrained newborn mice and is therefore the method of choice for investigating respiratory control, whose marked variability across sleep–wake states is a prominent characteristic of newborn mammals. 3.2. Sleep-disordered breathing in Phox2b+/− newborn mice In patients with CCHS, hypoventilation episodes and apneas typically occur during sleep. In Phox2b+/− pups, the lack of a Phox2b allele was expected to disrupt the control of breathing at the brainstem level and its modulation by peripheral inputs, without necessarily altering the wakefulness drive to breathing produced by suprapontine structures, which do not express Phox2b. To investigate the impact of sleep–wake states on breathing control in Phox2b+/− mutant pups, new methods for classifying sleep–wake states in newborn mice were developed (Durand et al., 2005), as well as methods to assess arousal responses to chemical challenges based on behavioral criteria (Dauger et al., 2001). An important point is that sleep states cannot be determined by electroencephalography or electro-oculography in newborn rodents (Karlsson and Blumberg, 2002). In contrast, nuchal muscle tone, coordinated movements, and motor twitches in newborn rats proved reliable for identifying sleep–wake states between 2 and 8 days of age (Karlsson et al., 2004). Behavioral states (wakefulness, active sleep, quiet sleep) can be determined using these criteria in newborn mice as well (Durand et al., 2005). State-dependent analyses indicated that sleep apnea time was increased about 6-fold in Phox2b+/− mutant pups and that ventilation during active sleep was decreased by about 20%, compared to wild-type pups on P5 (Durand et al., 2005). A possible explanation to these abnormalities is a decrease in the tonic drive to breathe provided by chemosensitive sites that act predominantly during sleep (Nattie and Li, 2001, 2002a). In rats, the CO2 -sensitive neurons of the caudal medullar raphe are active only during sleep (Nattie and Li, 2001) and those of the rostral nucleus tractus solitarius (which express Phox2b) are more effective during sleep than during wakefulness (Nattie and Li, 2002a). However, these interpretations need confirmation.

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3.3. Diminished sensitivity to CO2 in Phox2b+/− newborn mice The ventilatory increase during hypercapnia (8% CO2 ) was about 40% smaller in 2-day-old Phox2b+/− than Phox2b+/+ pups, due to depression of the breath duration (TTOT ) response (Dauger et al., 2003). The number and the total duration of apneas were significantly higher in Phox2b+/− than in Phox2b+/+ pups during hypercapnia. CO2 -sensitive sites (locus ceruleus and area postrema (Ruggiero et al., 1999; Nattie, 2001), RTN (Stornetta et al., 2006)) and afferent pathways from the carotid bodies (which contribute to CO2 sensitivity) also depend on Phox2b for their development (Dauger et al., 2003). The prevalence of Phox2b in central chemosensory pathways may account for the disrupted ventilatory response to CO2 in Phox2b+/− pups. Interestingly, the postnatal impairment of the hypercapnic ventilatory response seen in Phox2b+/− pups was short-lived: no differences in these ventilatory responses to CO2 were detected between Phox2b+/+ and Phox2b+/− pups aged 10 days, whereas 6-day-old pups exhibited an intermediate phenotype between 2- and 10-day-old pups (Dauger et al., 2003). The mechanisms of this rapid functional recovery are unknown. Such mechanisms obviously do not operate in patients with CCHS, in whom the symptoms are severe and irreversible. Thus, heterozygous null mutation in mice yielded a less severe respiratory phenotype than the human CCHS phenotype. 3.4. Augmented hypoxic ventilatory decline in Phox2b+/− newborn mice In 2-day-old Phox2b+/− pups, a biphasic pattern of VE changes occurred in response to hypoxia (5% O2 , Dauger et al., 2003). This pattern is characteristic of the ventilatory response to hypoxia in newborn mammals. The immediate hyperpneic response to hypoxia (i.e., the ascending limb of the biphasic ventilatory response) was normal in mutant pups. Arousal responses to hypoxia were not significantly different between Phox2b+/+ and Phox2b+/− pups. In contrast, the hypoxic decline was markedly increased in the Phox2b+/− pups, mainly as a result of abnormal TTOT control. Apneas occurred chiefly during the post-hypoxic decline. Their numbers were similar in Phox2b+/+ and Phox2b+/− pups, but their total duration was considerably longer during the post-hypoxic decline in Phox2b+/− than in Phox2b+/+ pups. The hypoxic ventilatory decline has not been examined in CCHS patients. The hyperpneic response to hypoxia varied across patients (Paton et al., 1989). Those patients who were able to ventilate adequately during wakefulness had normal hyperpneic responses to hypoxia (Gozal et al., 1993), suggesting that defects in peripheral chemosensitivity may exist only in the most severe cases. However, ventilatory control disorders were studied in only a few patients with CCHS, usually before diagnostic genetic testing became available. 3.5. Sensitivity to hyperoxia in Phox2b+/− newborn mice Chemosensitivity to oxygen can be assessed by inducing “physiological chemodenervation” via inhalation of an oxygen bolus. The extent of ventilation inhibition in response to hyperoxia provides a functional estimate of the tonic chemoreceptor drive (Ungar and Bouverot, 1980; Mortola and Tenney, 1986; Kline et al., 1998). Hyperoxic and hypoxic tests are complementary and may show divergent results in newborn mammals (Milerad et al., 1995; Bamford and Carroll, 1999), raising the possibility that a decrease in tonic chemoreceptor drive might coexist with normal ventilatory responses to hypoxia. The ventilatory decrease caused by hyperoxia was larger in newborn Phox2b+/− mutant mice than in their wild-type littermates

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and was magnified by longer apnea durations (Ramanantsoa et al., 2006). Furthermore, compared to wild-type pups, mutant pups showed a more sustained ventilatory decrease, which outlasted the return to normoxia. These results suggest stronger tonic activity of oxygen-sensitive peripheral chemoreceptors in mutant pups. This augmented peripheral tonic input may be ascribable to low arterial PO2 levels, caused by low CO2 chemosensitivity (Dauger et al., 2003) and sleep-related apneas (Durand et al., 2005). 3.6. Effects of ambient temperature of breathing disorders in Phox2b+/− mutant mice The pervasive role for Phox2b in the development of autonomic functions raises the possibility that respiratory disorders in Phox2b+/− pups may be aggravated by abnormalities in autonomic functions, most notably thermoregulation (Ramanantsoa et al., 2007). Studies of 2-day-old Phox2b+/− mice exposed to three ambient temperatures (29 ◦ C, 32 ◦ C, and 35 ◦ C) indicated that ambient temperature influenced the ventilatory response to hypercapnia (8% CO2 ). The hypercapnic ventilatory response increased linearly with ambient temperature in both groups, while remaining smaller in mutant than in wild-type pups at all ambient temperatures. The differences between the absolute increases in ventilation in mutant and wild-type pups become more pronounced as temperature increased above 29 ◦ C. The ventilatory abnormalities in mutant pups were not associated with significant impairments of heart rate control. The hypothalamus, which is the primary locus for thermoregulation and exerts direct control over respiratory brainstem structures, does not express Phox2b (Dauger et al., 2003). However, hypothalamic abnormalities secondary to structural and neurological abnormalities in Phox2b-expressing brain regions cannot be ruled out. Many CCHS patients experience symptoms related to impaired thermoregulation (sporadic profuse sweating, decreased basal body temperature with cool extremities, and absence of fever with infections (Weese-Mayer et al., 1999; Marazita et al., 2001; Weese-Mayer et al., 2001; Vanderlaan et al., 2004)).

haploinsufficiency, is further supported by the extremely severe phenotype of Phox2b27Ala/+ mice, as detailed below. 4. The ventilatory phenotype of Phox2b27Ala/+ mice In contrast to Phox2b+/− mutant mice (carrying one invalidated Phox2b allele), Phox2b27Ala/+ mice carry a widespread CCHS-causing mutation, namely, a +7 alanine expansion of the 20-residue poly-Ala tract (the Phox2b27Ala allele) (Dubreuil et al., 2008). Heterozygous Phox2b27Ala/+ offspring of the chimeric founders (obtained using a knock-in approach) died within a few hours after birth. At birth, the weights and body temperatures (before and after plethysmographic recordings) of mutant and wild-type pups were closely similar. However, neonatal mortality was high and scores of adaptation to extra-uterine life (derived from the Apgar score in humans) were significantly lower in mutant than in wildtype pups. Plethysmographic recordings were done during the first 20 min after delivery. The breathing pattern showed considerable inter-individual variability, ranging from gasping or very unstable breathing interrupted by apneas to relatively shallow breathing at a slower rate compared to wild-type littermates (Fig. 1). The mean VE

3.7. The mild phenotype of Phox2b+/− mutant pups A possible explanation to the milder impairment of the hypercapnic ventilatory response in Phox2b+/− mutant pups, compared to CCHS patients, may be the presence of genetic abnormalities in CCHS patients, in addition to PHOX2B mutations. Abnormal ventilatory responses to hypercapnia have been reported in newborn mice lacking genes involved in the development of the autonomic nervous system, most notably via the endothelin-1 and the Mash1–Ret–Phox2a–Phox2b signaling pathways (Kuwaki et al., 1996; Burton et al., 1997; Dauger et al., 1999, 2003). However, the effects of multiple mutations have not been examined in mice. A more likely hypothesis is that the phenotype differences between Phox2b+/− mice and patients with CCHS are related to functional differences between the Phox2b-targeted mutation in mice (a null mutation) and the alanine expansion generally found in the PHOX2B gene of CCHS patients. Alanine expansion may result in a protein that binds to the correct targets in the genome but fails to exert its normal regulatory function and therefore competes with the product of the wild-type allele (Bachetti et al., 2005). In contrast, a single functional Phox2b allele may ensure correct protein function, leading to a less severe phenotype (Cross et al., 2004). Furthermore, alanine-expanded proteins may form toxic aggregates in vulnerable cells that express them (Brown and Brown, 2004; Trochet et al., 2005b; Bachetti et al., 2007). The possibility that the respiratory disorders typical of CCHS may be related to a toxic gain-of-function or to a dominant-negative effect, as opposed to

Fig. 1. Disrupted breathing in Phox2b27Ala/+ newborn mice. (A) Representative examples of plethysmographic recordings of wild-type (wt) and Phox2b27Ala/+ (ki) littermates breathing a normal or hypercapnic mixture (8% CO2 ) during the first 20 min after delivery. (B) Ventilatory (VE ) tracings of Phox2b27Ala/+ (black dots) pups and their wild-type (white dots) littermates breathing air or a hypercapnic mixture (8% CO2 ; shaded area). Each dot represents the mean value ± SEM over a 30-s period (n = 15 and n = 43 for mutant and wild-type pups, respectively, for all analyses). Baseline ventilation in air was depressed in the mutants, which did not increase VE during hypercapnia. (C) Total apnea duration summed over the 5-min baseline period in air for wild-type and Phox2b27Ala/+ pups, excluding pups that breathed only by gasping. (D) Average VE for wild-type and Phox2b27Ala/+ pups breathing air or a hypercapnic mixture. Normocapnic values were calculated as the mean value over the first 5 min and the last 5 min of the recording to take into account the overall increase in VE . Hypercapnic VE values were calculated as the mean values over the last 3 min of hypercapnic exposure. In contrast to wild-type pups, the mutants did not increase their ventilation in response to CO2 . From Dubreuil et al. (2008), with permission.

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measured during apnea-free periods in non-gasping mutant pups was lower than in wild-type pups. Interbreath interval variability expressed as the percent coefficient of variance was significantly greater in mutant than in wild-type pups. Apneic episodes were more frequent and lasted longer in the mutants compared to the wild-type pups, resulting in a 6.5-fold increase in total apnea duration. Importantly, the lack of response to hypercapnia indicated that central chemoreception was defective in mutant pups (Dubreuil et al., 2008). Further studies revealed a targeted loss of parafacial interneurons (pFRG) in Phox2b27Ala/+ mice. Previous work indicated that neurokinin-1 receptor (NK1R)-positive neurons in the RTN participated in CO2 sensitivity (Nattie and Li, 2002b). In newborn mutants, NK1R immunoreactivity was almost completely abolished. No abnormalities were found in other Phox2b-expressing structures known to play a role in respiratory control, including the carotid bodies, petrosal/nodose ganglionic complex, locus ceruleus, and NTS. Neither did the mutants exhibit abnormalities in two Phox2b-negative cell groups that are essential for normal breathing, namely, the preBötzinger complex (preBötC) (Smith et al., 1991; Feldman and Del Negro, 2006) and the medullary 5-HT neurons, which may be a crucial site of CO2 chemoreception (Richter et al., 2003). Thus, with the exception of the RTN/pFRG, the main neuronal groups involved in breathing control seemed normal (Dubreuil et al., 2008). In mice, the opiate-insensitive rhythm generator in RTN/pFRG may play a critical role in ensuring an adequate breathing pattern immediately after birth, when there is a short-lived surge of endogenous opiates that transiently depress the opiate-sensitive preBötC (Janczewski and Feldman, 2006). Naloxone administration may lift the opioid-induced preBötC inhibition, thereby prolonging survival in Phox2b27Ala/+ newborn mice. Preliminary studies indicated that naloxone administration in Phox2b27Ala/+ newborn mice reduced apnea duration, but the effects on survival await further studies. 5. Conclusion Over the last few years, important advances have shed light on the function of PHOX2B in respiratory control and on its role in CCHS. These achievements are the result of multidisciplinary efforts combining genetic studies in humans with neurobiological and physiological studies in mouse models. Phox2b is required for the development of all major homeostatic functions (Brunet and Goridis, 2008). In particular, studies in adult rats indicated that Phox2b was pivotal to the provision of central and peripheral chemosensory inputs to respiratory rhythm generators (Stornetta et al., 2006). In newborn mice, lack of a Phox2b allele caused transient chemosensitivity disorders (Dauger et al., 2003; Ramanantsoa et al., 2006, 2007). However, heterozygous Phox2b27Ala/+ exhibited a deficient response to hypercapnia, and most of them had highly unstable breathing interrupted by apneas, as well as loss of parafacial neurons (Dubreuil et al., 2008). Taken together, these results suggest a role for the RTN in respiratory rhythmogenesis and chemosensitivity during the early postnatal period. Also, they indicate that respiratory phenotype disorders in patients with CCHS may be related to a toxic gain-of-function or to a dominant-negative effect, rather than to haploinsufficiency. The generation of valid mouse models for CCHS has opened up new avenues of research toward therapeutic strategies aimed at alleviating the respiratory symptoms of CCHS. References Amiel, J., Laudier, B., Attie-Bitach, T., Trang, H., de Pontual, L., Gener, B., Trochet, D., Etchevers, H., Ray, P., Simonneau, M., Vekemans, M., Munnich, A., Gaultier,

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C., Lyonnet, S., 2003a. Polyalanine expansion and frameshift mutations of the paired-like homeobox gene PHOX2B in congenital central hypoventilation syndrome. Nat. Genet. 33, 459–461. Amiel, J., Pelet, A., Trang, H., de Pontual, L., Simonneau, M., Munnich, A., Gaultier, C., Lyonnet, S., 2003b. Exclusion of RNX as a major gene in congenital central hypoventilation syndrome (CCHS, Ondine’s curse). Am. J. Med. Genet. A 117, 18–20. Amiel, J., Sproat-Emison, E., Garcia-Barcelo, M., Lantieri, F., Burzynski, G., Borrego, S., Pelet, A., Arnold, S., Miao, X., Griseri, P., Brooks, A.S., Antinolo, G., de Pontual, L., Clement-Ziza, M., Munnich, A., Kashuk, C., West, K., Wong, K.K., Lyonnet, S., Chakravarti, A., Tam, P.K., Ceccherini, I., Hofstra, R.M., Fernandez, R., 2008. Hirschsprung disease, associated syndromes and genetics: a review. J. Med. Genet. 45, 1–14. Antic, N.A., Malow, B.A., Lange, N., McEvoy, R.D., Olson, A.L., Turkington, P., Windisch, W., Samuels, M., Stevens, C.A., Berry-Kravis, E.M., Weese-Mayer, D.E., 2006. PHOX2B mutation-confirmed congenital central hypoventilation syndrome: presentation in adulthood. Am. J. Respir. Crit. Care Med. 174, 923–927. Bachetti, T., Matera, I., Borghini, S., Duca, M.D., Ravazzolo, R., Ceccherini, I., 2005. Distinct pathogenetic mechanisms for PHOX2B associated polyalanine expansions and frameshift mutations in congenital central hypoventilation syndrome. Hum. Mol. Genet. 14, 1815–1824. Bachetti, T., Bocca, P., Borghini, S., Matera, I., Prigione, I., Ravazzolo, R., Ceccherini, I., 2007. Geldanamycin promotes nuclear localisation and clearance of PHOX2B misfolded proteins containing polyalanine expansions. Int. J. Biochem. Cell Biol. 39, 327–339. Bamford, O.S., Carroll, J.L., 1999. Dynamic ventilatory responses in rats: normal development and effects of prenatal nicotine exposure. Respir. Physiol. 117, 29–40. Barratt, S., Kendrick, A.H., Buchanan, F., Whittle, A.T., 2007. Central hypoventilation with PHOX2B expansion mutation presenting in adulthood. Thorax 62, 919–920. Berry-Kravis, E.M., Zhou, L., Rand, C.M., Weese-Mayer, D.E., 2006. Congenital central hypoventilation syndrome: PHOX2B mutations and phenotype. Am. J. Respir. Crit. Care Med. 174, 1139–1144. Brown, L.Y., Brown, S.A., 2004. Alanine tracts: the expanding story of human illness and trinucleotide repeats. Trends Genet. 20, 51–58. Brunet, J.F., Goridis, C., 2008. Phox2b and the homeostatic brain. In: Gaultier, C. (Ed.), Genetic Basis for Respiratory Control Disorders. Springer, New York, pp. 25–38. Brunet, J.F., Pattyn, A., 2002. Phox2 genes—from patterning to connectivity. Curr. Opin. Genet. Dev. 12, 435–440. Burton, M.D., Kawashima, A., Brayer, J.A., Kazemi, H., Shannon, D.C., Schuchardt, A., Costantini, F., Pachnis, V., Kinane, T.B., 1997. RET proto-oncogene is important for the development of respiratory CO2 sensitivity. J. Auton. Nerv. Syst. 63, 137–143. Cross, S.H., Morgan, J.E., Pattyn, A., West, K., McKie, L., Hart, A., Thaung, C., Brunet, J.F., Jackson, I.J., 2004. Haploinsufficiency for Phox2b in mice causes dilated pupils and atrophy of the ciliary ganglion: mechanistic insights into human congenital central hypoventilation syndrome. Hum. Mol. Genet. 13, 1433–1439. Dauger, S., Renolleau, S., Vardon, G., Nepote, V., Mas, C., Simonneau, M., Gaultier, C., Gallego, J., 1999. Ventilatory responses to hypercapnia and hypoxia in Mash-1 heterozygous newborn and adult mice. Pediatr. Res. 46, 535–542. Dauger, S., Aizenfisz, S., Renolleau, S., Durand, E., Vardon, G., Gaultier, C., Gallego, J., 2001. Arousal response to hypoxia in newborn mice. Respir. Physiol. 128, 235–240. Dauger, S., Pattyn, A., Lofaso, F., Gaultier, C., Goridis, C., Gallego, J., Brunet, J.F., 2003. Phox2b controls the development of peripheral chemoreceptors and afferent visceral pathways. Development 130, 6635–6642. de Pontual, L., Nepote, V., Attie-Bitach, T., Al Halabiah, H., Trang, H., Elghouzzi, V., Levacher, B., Benihoud, K., Auge, J., Faure, C., Laudier, B., Vekemans, M., Munnich, A., Perricaudet, M., Guillemot, F., Gaultier, C., Lyonnet, S., Simonneau, M., Amiel, J., 2003. Noradrenergic neuronal development is impaired by mutation of the proneural HASH-1 gene in congenital central hypoventilation syndrome (Ondine’s curse). Hum. Mol. Genet. 12, 3173–3180. Diedrich, A., Malow, B.A., Antic, N.A., Sato, K., McEvoy, R.D., Mathias, C.J., Robertson, D., Berry-Kravis, E.M., Weese-Mayer, D.E., 2007. Vagal and sympathetic heart rate and blood pressure control in adult onset PHOX2B mutation-confirmed congenital central hypoventilation syndrome. Clin. Auton. Res. 17, 177–185. Doherty, L.S., Kiely, J.L., Deegan, P.C., Nolan, G., McCabe, S., Green, A.J., Ennis, S., McNicholas, W.T., 2007. Late-onset central hypoventilation syndrome: a family genetic study. Eur. Respir. J. 29, 312–316. Drorbaugh, J.E., Fenn, W.O., 1955. A barometric method for measuring ventilation in newborn infants. Pediatrics 16, 81–87. Dubreuil, V., Ramanantsoa, N., Trochet, D., Vaubourg, V., Amiel, J., Gallego, J., Brunet, J.F., Goridis, C., 2008. A human mutation in Phox2b causes lack of CO2 chemosensitivity, fatal central apnea, and specific loss of parafacial neurons. Proc. Natl. Acad. Sci. U.S.A. 105, 1067–1072. Durand, E., Dauger, S., Pattyn, A., Gaultier, C., Goridis, C., Gallego, J., 2005. Sleepdisordered breathing in newborn mice heterozygous for the transcription factor Phox2b. Am. J. Respir. Crit. Care Med. 172, 238–243. Erickson, J.T., Conover, J.C., Borday, V., Champagnat, J., Barbacid, M., Yancopoulos, G., Katz, D.M., 1996. Mice lacking brain-derived neurotrophic factor exhibit visceral sensory neuron losses distinct from mice lacking NT4 and display a severe developmental deficit in control of breathing. J. Neurosci. 16, 5361–5371. Feldman, J.L., Del Negro, C.A., 2006. Looking for inspiration: new perspectives on respiratory rhythm. Nat. Rev. Neurosci. 7, 232–242. Gaultier, C., Gallego, J., 2005. Development of respiratory control: evolving concepts and perspectives. Respir. Physiol. Neurobiol. 149, 3–15.

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J. Gallego, S. Dauger / Respiratory Physiology & Neurobiology 164 (2008) 49–54

Gaultier, C., Matrot, B., Gallego, J., 2006. Transgenic models to study disorders of respiratory control in newborn mice. ILAR J. 47, 15–21. Gozal, D., Marcus, C.L., Shoseyov, D., Keens, T.G., 1993. Peripheral chemoreceptor function in children with the congenital central hypoventilation syndrome. J. Appl. Physiol. 74, 379–387. Janczewski, W.A., Feldman, J.L., 2006. Novel data supporting the two respiratory rhythm oscillator hypothesis. Focus on “respiration-related rhythmic activity in the rostral medulla of newborn rats”. J. Neurophysiol. 96, 1–2. Karlsson, K.A., Blumberg, M.S., 2002. The union of the state: myoclonic twitching is coupled with nuchal muscle atonia in infant rats. Behav. Neurosci. 116, 912–917. Karlsson, K.A., Kreider, J.C., Blumberg, M.S., 2004. Hypothalamic contribution to sleep–wake cycle development. Neuroscience 123, 575–582. Kline, D.D., Yang, T., Huang, P.L., Prabhakar, N.R., 1998. Altered respiratory responses to hypoxia in mutant mice deficient in neuronal nitric oxide synthase. J. Physiol. 511 (Pt 1), 273–287. Kuwaki, T., Cao, W.H., Kurihara, Y., Kurihara, H., Ling, G.Y., Onodera, M., Ju, K.H., Yazaki, Y., Kumada, M., 1996. Impaired ventilatory responses to hypoxia and hypercapnia in mutant mice deficient in endothelin-1. Am. J. Physiol. 270, R1279–1286. Marazita, M.L., Maher, B.S., Cooper, M.E., Silvestri, J.M., Huffman, A.D., Smok-Pearsall, S.M., Kowal, M.H., Weese-Mayer, D.E., 2001. Genetic segregation analysis of autonomic nervous system dysfunction in families of probands with idiopathic congenital central hypoventilation syndrome. Am. J. Med. Genet. 100, 229–236. Marcus, C.L., Livingston, F.R., Wood, S.E., Keens, T.G., 1991. Hypercapnic and hypoxic ventilatory responses in parents and siblings of children with congenital central hypoventilation syndrome. Am. Rev. Respir. Dis. 144, 136–140. Matera, I., Bachetti, T., Cinti, R., Lerone, M., Gagliardi, L., Morandi, F., Motta, M., Mosca, F., Ottonello, G., Piumelli, R., Schober, J.G., Ravazzolo, R., Ceccherini, I., 2002. Mutational analysis of the RNX gene in congenital central hypoventilation syndrome. Am. J. Med. Genet. 113, 178–182. Matera, I., Bachetti, T., Puppo, F., Di Duca, M., Morandi, F., Casiraghi, G., Cilio, M., Hennekam, R., Hofstra, R., Schöber, J., Ravazzolo, R., Ottonello, G., Ceccherini, I., 2004. PHOX2B mutations and polyalanine expansions corellate with the severity of the respiratory phenotype and associated symptoms in both congenital and late onset Central Hypoventilation syndrome. J. Med. Genet. 41, 373–380. Matrot, B., Durand, E., Dauger, S., Vardon, G., Gaultier, C., Gallego, J., 2005. Automatic classification of activity and apneas using whole body plethysmography in newborn mice. J. Appl. Physiol. 98, 365–370. Milerad, J., Larsson, H., Lin, J., Sundell, H.W., 1995. Nicotine attenuates the ventilatory response to hypoxia in the developing lamb. Pediatr. Res. 37, 652–660. Mortola, J.P., Tenney, S.M., 1986. Effects of hyperoxia on ventilatory and metabolic rates of newborn mice. Respir. Physiol. 63, 267–274. Nattie, E.E., 2001. Central chemosensitivity, sleep, and wakefulness. Respir. Physiol. 129, 257–268. Nattie, E.E., Li, A., 2001. CO2 dialysis in the medullary raphe of the rat increases ventilation in sleep. J. Appl. Physiol. 90, 1247–1257. Nattie, E.E., Li, A., 2002a. CO2 dialysis in nucleus tractus solitarius region of rat increases ventilation in sleep and wakefulness. J. Appl. Physiol. 92, 2119–2130. Nattie, E.E., Li, A., 2002b. Substance P-saporin lesion of neurons with NK1 receptors in one chemoreceptor site in rats decreases ventilation and chemosensitivity. J. Physiol. 544, 603–616. Onodera, M., Kuwaki, T., Kumada, M., Masuda, Y., 1997. Determination of ventilatory volume in mice by whole body plethysmography. Jpn. J. Physiol. 47, 317–326. Paton, J.Y., Swaminathan, S., Sargent, C.W., Keens, T.G., 1989. Hypoxic and hypercapnic ventilatory responses in awake children with congenital central hypoventilation syndrome. Am. Rev. Respir. Dis. 140, 368–372. Pattyn, A., Morin, X., Cremer, H., Goridis, C., Brunet, J.F., 1997. Expression and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis. Development 124, 4065–4075. Pattyn, A., Morin, X., Cremer, H., Goridis, C., Brunet, J.F., 1999. The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives. Nature 399, 366–370. Pattyn, A., Goridis, C., Brunet, J.F., 2000a. Specification of the central noradrenergic phenotype by the homeobox gene Phox2b. Mol. Cell Neurosci. 15, 235–243. Pattyn, A., Hirsch, M., Goridis, C., Brunet, J.F., 2000b. Control of hindbrain motor neuron differentiation by the homeobox gene Phox2b. Development 127, 1349–1358. Ramanantsoa, N., Vaubourg, V., Dauger, S., Matrot, B., Vardon, G., Chettouh, Z., Gaultier, C., Goridis, C., Gallego, J., 2006. Ventilatory response to hyperoxia in newborn mice heterozygous for the transcription factor Phox2b. Am. J. Physiol. Regul. Integr. Comp. Physiol. 290, R1691–1696. Ramanantsoa, N., Vaubourg, V., Matrot, B., Vardon, G., Dauger, S., Gallego, J., 2007. Effects of temperature on ventilatory response to hypercapnia in newborn mice

heterozygous for transcription factor Phox2b. Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R2027–2035. Richter, D.W., Manzke, T., Wilken, B., Ponimaskin, E., 2003. Serotonin receptors: guardians of stable breathing. Trends Mol. Med. 9, 542–548. Ruggiero, D.A., Gootman, P.M., Ingenito, S., Wong, C., Gootman, N., Sica, A.L., 1999. The area postrema of newborn swine is activated by hypercapnia: relevance to sudden infant death syndrome? J. Auton. Nerv. Syst. 76, 167–175. Sasaki, A., Kanai, M., Kijima, K., Akaba, K., Hashimoto, M., Hasegawa, H., Otaki, S., Koizumi, T., Kusuda, S., Ogawa, Y., Tuchiya, K., Yamamoto, W., Nakamura, T., Hayasaka, K., 2003. Molecular analysis of congenital central hypoventilation syndrome. Hum. Genet. 114, 22–26. Schmidt, B., Roberts, R.S., Davis, P., Doyle, L.W., Barrington, K.J., Ohlsson, A., Solimano, A., Tin, W., 2006. Caffeine therapy for apnea of prematurity. N. Engl. J. Med. 354, 2112–2121. Smith, J.C., Ellenberger, H.H., Ballanyi, K., Richter, D.W., Feldman, J.L., 1991. PreBotzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science 254, 726–729. Spengler, C.M., Gozal, D., Shea, S.A., 2001. Chemoreceptive mechanisms elucidated by studies of congenital central hypoventilation syndrome. Respir. Physiol. 129, 247–255. Stornetta, R.L., Moreira, T.S., Takakura, A.C., Kang, B.J., Chang, D.A., West, G.H., Brunet, J.F., Mulkey, D.K., Bayliss, D.A., Guyenet, P.G., 2006. Expression of Phox2b by brainstem neurons involved in chemosensory integration in the adult rat. J. Neurosci. 26, 10305–10314. Tiveron, M.C., Hirsch, M.R., Brunet, J.F., 1996. The expression pattern of the transcription factor Phox2 delineates synaptic pathways of the autonomic nervous system. J. Neurosci. 16, 7649–7660. Trang, H., Laudier, B., Trochet, D., Munnich, A., Lyonnet, S., Gaultier, C., Amiel, J., 2004. PHOX2B gene mutation in a patient with late-onset central hypoventilation. Pediatr. Pulmonol. 38, 349–351. Trang, H., Dehan, M., Beaufils, F., Zaccaria, I., Amiel, J., Gaultier, C., 2005. The French Congenital Central Hypoventilation Syndrome Registry: general data, phenotype, and genotype. Chest 127, 72–79. Trochet, D., Hong, S.J., Lim, J.K., Brunet, J.F., Munnich, A., Kim, K.S., Lyonnet, S., Goridis, C., Amiel, J., 2005a. Molecular consequences of PHOX2B missense, frameshift and alanine expansion mutations leading to autonomic dysfunction. Hum. Mol. Genet. 14, 3697–3708. Trochet, D., O’Brien, L.M., Gozal, D., Trang, H., Nordenskjold, A., Laudier, B., Svensson, P.J., Uhrig, S., Cole, T., Niemann, S., Munnich, A., Gaultier, C., Lyonnet, S., Amiel, J., 2005b. PHOX2B genotype allows for prediction of tumor risk in congenital central hypoventilation syndrome. Am. J. Hum. Genet. 76, 421–426. Trochet, D., de Pontual, L., Straus, C., Gozal, D., Trang, H., Landrieu, P., Munnich, A., Lyonnet, S., Gaultier, C., Amiel, J., 2008. PHOX2B germline and somatic mutations in late-onset central hypoventilation syndrome. Am. J. Respir. Crit. Care Med. 177, 906–911. Ungar, A., Bouverot, P., 1980. The ventilatory responses of conscious dogs to isocapnic oxygen tests. a method of exploring the central component of respiratory drive and its dependence on O2 and CO2 . Respir. Physiol. 39, 183–197. Vanderlaan, M., Holbrook, C.R., Wang, M., Tuell, A., Gozal, D., 2004. Epidemiologic survey of 196 patients with congenital central hypoventilation syndrome. Pediatr. Pulmonol. 37, 217–229. Weese-Mayer, D., Shannon, D.C., Keens, T.G., Silvestri, J.M., 1999. Idiopathic congenital central hypoventilation syndrome: diagnosis and management, American Thoracic Society. Am. J. Respir. Crit. Care Med. 160, 368–373. Weese-Mayer, D.E., Silvestri, J.M., Huffman, A.D., Smok-Pearsall, S.M., Kowal, M.H., Maher, B.S., Cooper, M.E., Marazita, M.L., 2001. Case/control family study of autonomic nervous system dysfunction in idiopathic congenital central hypoventilation syndrome. Am. J. Med. Genet. 100, 237–245. Weese-Mayer, D.E., Bolk, S., Silvestri, J.M., Chakravarti, A., 2002. Idiopathic congenital central hypoventilation syndrome: evaluation of brain-derived neurotrophic factor genomic DNA sequence variation. Am. J. Med. Genet. 107, 306–310. Weese-Mayer, D.E., Berry-Kravis, E.M., Zhou, L., Maher, B.S., Silvestri, J.M., Curran, M.E., Marazita, M.L., 2003. Idiopathic congenital central hypoventilation syndrome: analysis of genes pertinent to early autonomic nervous system embryologic development and identification of mutations in PHOX2b. Am. J. Med. Genet. 123A, 267–278. Weese-Mayer, D.E., Berry-Kravis, E.M., Marazita, M.L., 2005a. In pursuit (and discovery) of a genetic basis for congenital central hypoventilation syndrome. Respir. Physiol. Neurobiol. 142, 73–82. Weese-Mayer, D.E., Berry-Kravis, E.M., Zhou, L., 2005b. Adult identified with congenital central hypoventilation syndrome–mutation in PHOX2b gene and late-onset CHS. Am. J. Respir. Crit. Care Med. 171, 88.