PHOX2B mutations and ventilatory control

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Abstract

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.

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 knock-out mice exhibited respiratory control disorders, but found no mutations. Weese-Mayer et al. (2002) analyzed brain-derived neurotrophic 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 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).

Section snippets

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

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

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

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

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