Polyalanine expansion and frameshift mutations of the paired-like homeobox gene PHOX2B in congenital central hypoventilation syndrome.
ABSTRACT Congenital central hypoventilation syndrome (CCHS or Ondine's curse; OMIM 209880) is a life-threatening disorder involving an impaired ventilatory response to hypercarbia and hypoxemia. This core phenotype is associated with lower-penetrance anomalies of the autonomic nervous system (ANS) including Hirschsprung disease and tumors of neural-crest derivatives such as ganglioneuromas and neuroblastomas. In mice, the development of ANS reflex circuits is dependent on the paired-like homeobox gene Phox2b. Thus, we regarded its human ortholog, PHOX2B, as a candidate gene in CCHS. We found heterozygous de novo mutations in PHOX2B in 18 of 29 individuals with CCHS. Most mutations consisted of 5-9 alanine expansions within a 20-residue polyalanine tract probably resulting from non-homologous recombination. We show that PHOX2B is expressed in both the central and the peripheral ANS during human embryonic development. Our data support an essential role of PHOX2B in the normal patterning of the autonomous ventilation system and, more generally, of the ANS in humans.
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ABSTRACT: Congenital Central Hypoventilation Syndrome (CCHS) is characterized by hypoventilation during sleep and impaired ventilatory responses to hypercapnia and hypoxemia. Most cases are sporadic and caused by de novo PHOX2B gene mutations, which are usually polyalanine repeat expansions. Physiological and neuroanatomical studies of genetically engineered mice and analyses of cellular responses to mutated Phox2b have shed light on the pathophysiological mechanisms of CCHS. Findings in Phox2b(27Ala/+) knock-in mice consisted of unstable breathing with apneas, absence of the ventilatory response to hypercapnia, death within a few hours after birth, and absence of the retrotrapezoid nucleus (RTN). Conditional mouse mutants in which Phox2b(27Ala) was targeted to the RTN also lacked the ventilatory response to hypercapnia at birth but survived to adulthood and developed a partial hypercapnia response. The therapeutic effects of desogestrel are being evaluated in clinical trials, and recent analyses of cellular responses to polyAla Phox2b aggregates have suggested new pharmacological approaches designed to counteract the toxic effects of mutated Phox2b.Respiratory Physiology & Neurobiology 05/2013; · 2.05 Impact Factor
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ABSTRACT: Finding genes for complex diseases has been the goal of many genetic studies. Most of these studies have been successful by searching for genes and mutations in rare familial cases, by screening candidate genes and by performing genome wide association studies. However, only a small fraction of the total genetic risk for these complex genetic diseases can be explained by the identified mutations and associated genetic loci. In this review we focus on Hirschsprung disease (HSCR) as an example of a complex genetic disorder. We describe the genes identified in this congenital malformation and postulate that both common 'low penetrant' variants in combination with rare or private 'high penetrant' variants determine the risk on HSCR, and likely, on other complex diseases. We also discuss how new technological advances can be used to gain further insights in the genetic background of complex diseases. Finally, we outline some steps to develop functional assays in order to determine the involvement of these variants in disease development.Developmental Biology 05/2013; · 3.87 Impact Factor
Article: Rodent Models of Sleep APNEA.[show abstract] [hide abstract]
ABSTRACT: Rodent models of sleep apnea have long been used to provide novel insight into the generation and predisposition to apneas as well as to characterize the impact of sleep apnea on cardiovascular, metabolic, and psychological health in humans. Given the significant body of work utilizing rodent models in the field of sleep apnea, the aims of this review are three-fold: first, to review the use of rodents as natural models of sleep apnea; second, to provide an overview of the experimental interventions employed in rodents to simulate sleep apnea; third, to discuss the refinement of rodent models to further our understanding of breathing abnormalities that occur during sleep. Given mounting evidence that sleep apnea impairs cognitive function, reduces quality of life, and exacerbates the course of multiple chronic diseases, rodent models will remain a high priority as a tool to interrogate both the pathophysiology and sequelae of breathing related abnormalities during sleep and to improve approaches to diagnosis and therapy.Respiratory Physiology & Neurobiology 05/2013; · 2.05 Impact Factor
nature genetics • advance online publication1
A genetic origin of CCHS1has long been
suspected based on concordance in
monozygotic twins2, rare familial cases
(siblings, half-siblings, and mother-to-
child transmission)3and segregation
analysis suggesting an autosmal domi-
nant locus with low penetrance or a
multigenic model4. So far, only low-
penetrant predisposing mutations of the
RET-Glial cell line-derived neurotrophic
factor (GDNF), endothelin 3 (EDN3)
and brain-derived neurotrophic factor
(BDNF) pathways have been reported in
a few individuals with CCHS5–7. Consid-
ering the broad range of defects in the
ANS in CCHS on the one hand and the
key role of Phox2b in the ontogeny of the
ANS reflex circuits in mice8,9 on the
other hand, we regarded PHOX2B (also
called PMX2B and NBPHOX) as a candi-
date gene in the disease.
PHOX2B maps to chromosome 4p12
and encodes a highly conserved homeobox
transcription factor of 314 amino acids
with two short and stable polyalanine
Polyalanine expansion and frameshift
mutations of the paired-like
homeobox gene PHOX2B in congenital
central hypoventilation syndrome
Published online 17 March 2003, doi:10.1038/ng1130
Congenital central hypoventilation syndrome (CCHS or Ondine’s curse; OMIM 209880)
is a life-threatening disorder involving an impaired ventilatory response to hypercar-
bia and hypoxemia. This core phenotype is associated with lower-penetrance anom-
alies of the autonomic nervous system (ANS) including Hirschsprung disease and
tumors of neural-crest derivatives such as ganglioneuromas and neuroblastomas. In
mice, the development of ANS reflex circuits is dependent on the paired-like homeo-
box gene Phox2b. Thus, we regarded its human ortholog, PHOX2B, as a candidate
gene in CCHS. We found heterozygous de novo mutations in PHOX2B in 18 of 29
individuals with CCHS. Most mutations consisted of 5–9 alanine expansions within a
20-residue polyalanine tract probably resulting from non-homologous recombina-
tion. We show that PHOX2B is expressed in both the central and the peripheral ANS
during human embryonic development. Our data support an essential role of
PHOX2B in the normal patterning of the autonomous ventilation system and, more
generally, of the ANS in humans.
Fig. 1 Mutations of PHOX2B in CCHS. a, Genomic organization of PHOX2B and schematic representation of the PHOX2B protein. The homeobox domain and the
9- and 20-residue polyalanine tracts are indicated (blue and green boxes, respectively). b, DNA sequence electropherogram and PCR digestion showing the iden-
tified mutations in PHOX2B. Left, Heterozygous frameshift mutations. Right, Heterozygous alanine triplet expansions. Mutated alleles are seen as extra bands of
variable size between the 170- and 210-bp bands after enzymatic digestion with StuI of the 380-bp PCR product of PHOX2B exon 3 and migration on a 3%
agarose gel (see Supplementary Note 1 online). c, Mutant PHOX2B proteins. Top, 618–619insC and 722–759del37nt frameshift mutations. Bottom, various
polyalanine expansions observed. The duplicated codons are shown in green boxes.
© 2003 Nature Publishing Group http://www.nature.com/naturegenetics
2 nature genetics • advance online publication
repeats of 9 and 20 residues, respectively
(Fig. 1a). The length of the polyalanine
tracts is conserved in mice and humans.
We screened the coding sequence of
PHOX2B in a series of 29 unrelated indi-
viduals with CCHS (see Supplementary
Note 1 online). Direct DNA sequencing
showed heterozygosity with respect to
PHOX2B variations in 18 of 29 cases. In 16
of 18 cases, the nucleotide variation was a
triplet expansion of 15–27 nucleotides (nt
721–780; Fig. 1b and Supplementary Table
1 online) adding 5–9 alanines to the 20-
residue polyalanine tract (Fig. 1c). Most
mutant genotypes were different, suggest-
ing that they derived from independent
mutational events (Fig. 1c). In addition,
whenever parents were available, we found
that the polyalanine triplet expansion
occurred de novo, supporting its role in the
disease phenotype (see Supplementary
Table 1 online). In two other CCHS cases,
a de novo cytosine insertion in a stretch of
four cytosines (618–619insC) and a dele-
tion of 37 nucleotides (722–759del37nt)
resulted in a frame shift downstream of the
homeobox, predicting a mutant protein
with no known function or homology
(Fig. 1b,c). The 722–759del37nt mutation
could be regarded as an out-of-frame con-
traction of the polyalanine tract. We found
neither alanine triplet expansions nor
frameshift mutations in 250 control chro-
mosomes from various ethnic back-
grounds. Notably, 2 of 250 control alleles
had a small polyalanine contraction (5 and
6 triplets, data not shown), suggesting an
unequal crossing-over during meiosis
rather than a polymerase slippage during
replication. In the former case, polyalanine
expansions and contractions are equally
likely to occur; PHOX2Bcontractions were
found as a rare variant in controls.
We found that 2 of 29 individuals with
CCHS carried heterozygous variants in
genes involved in the same developmental
pathway, namely RET and GDNF (amino-
acid substitutions P1039L and R93W,
respectively; see Supplementary Table 1
online; ref. 5), in addition to polyalanine
expansions in PHOX2B. Notably, Phox2
genes control Ret expression in both sym-
pathetic and enteric neurons in mice8.
Unlike the polyalanine expansions, how-
ever, these variants are neither necessary
(most individuals with CCHS do not have
any RET or GDNF gene variant) nor suffi-
cient for the disease to occur (carrier par-
ents have no phenotypic expression).
These data suggest that PHOX2Bis the pri-
mary disease locus in CCHS. Moreover, we
found mutations in PHOX2B not only in
isolated cases of CCHS but also in individ-
uals with a more complex neural-crest
Hirschsprung disease (Haddad syndrome)
as well as early-onset neuroblastoma. We
did not find any correlation between the
size of the polyalanine tract and the com-
plexity of the disease in our analyses.
To confirm the involvement of muta-
tions in PHOX2B in the disease pheno-
type, we studied the expression pattern of
PHOX2B in early human development
(see Supplementary Note 1 online). In
accordance with the wide spectrum of
ANS dysfunction observed in individuals
with CCHS, the expression pattern of
PHOX2B involved both central auto-
nomic circuits and peripheral neural-
crest derivatives. From day 32 of
development, PHOX2B was expressed in
the seventh ganglion and the ninth/tenth
ganglionic complex (Fig. 2a–d). From day
33 of development, we observed strong
expression of PHOX2B in terminal rhom-
bomeres 4–8, in the presumptive enteric
ganglia and in the sympathetic chain gan-
glia (Fig. 2g–l). Finally, we detected
expression of PHOX2B in the presump-
tive carotid body at the carotid bifurca-
tion, ventral to the superior cervical
ganglion, which also expressed PHOX2B
(Fig. 2e,f). We did not detect any
PHOX2B expression in adrenal medulla
at the stages investigated.
involving homeodomain or non-home-
odomain transcription factors (HOXA13,
HOXD13, ARX, RUNX2, ZIC2, FOXL2)
have been described in several human
malformations10,11. In each of these cases,
both the normal and expanded alanine
tracts range in a similar size, suggesting a
common underlying mechanism. In the
case of HOXD13and ZIC2, the expansions
are not just loss-of-function mutations
but are responsible for a dominant nega-
tive effect12,13. In the case of PHOX2B, a
loss-of-function mutation with lower
penetrance for the enteric nervous system
anomalies is a possibility, as we found
frameshift mutations in two individuals.
Assuming that the protein is stable, how-
ever, the homeodomain is preserved in
Fig. 2 PHOX2B gene expression in developing human brainstem and enteric nervous system. Slides stained
with hematoxylin and eosin (a,c,e,g,i,k) and dark-field illumination of the hybridized adjacent sections
(b,d,f,h,j,l) at day 33 (a,b), day 54 (c–f) and day 47 of development (g–l). Parasagittal sections showing
expression of PHOX2B in rhombencephalon (rh; a,b), cervical spinal cord (sp; a,b), seventh (VII) ganglia and
ninth/tenth (IX+X) ganglionic complex (a–d), presumptive enteric ganglia of oesophagus (oe) and intestine
(in; i–l) and paravertebral sympathetic ganglia (sym), including the superior cervical ganglion (scg; g,h).
PHOX2B expression was also detected in the presumptive carotid body (arrow) at the carotid (c) bifurcation,
ventral to the superior cervical ganglion (e,f). Magnifications: a,b,g,h, ×15; c,d, ×25; i–l, ×30; e,f, ×40.
© 2003 Nature Publishing Group http://www.nature.com/naturegenetics
nature genetics • advance online publication3
both cases. As far as the ocular phenotype
is concerned, one can speculate that
PHOX2B mutant protein products exert a
dominant negative effect on PHOX2A,
considering the overlapping features
observed in CCHS and congenital fibrosis
of the extra ocular muscle type 2
(CFEOM2) resulting from homozygous
mutations of the gene PHOX2A (ref. 14).
Indeed, it has been shown that the third
and fourth motor nuclei express both
Phox2A and Phox2b in mice and are
Phox2a-dependent8. The mechanism for a
putative involvement of PHOX2B in iso-
lated neural-crest tumors is undefined at
present, but it is worth mentioning that
Phox2b has been shown to regulate neu-
ronal cell cycle15.
Little is known regarding the bases of
ventilatory control anomalies in CCHS.
It has been speculated that the disease
involves a defect in the integration by the
nucleus of the solitary tract and
interneurons of the inputs from the cen-
tral CO2/pH-sensitive chemoreceptors
(medulla oblongata) and the peripheral
O2, CO2and pH-sensitive chemorecep-
tors in the carotid bodies1. Notably, sev-
eral of these structures express Phox2b in
mice8,9and humans (this study) and fail
to form or degenerate in Phox2b–/–
mouse mutants (refs. 8,9 and J.-F. Brunet,
pers. comm.). So far, no phenotype has
been reported in Phox2b+/–mice. Our
mutation and expression studies strongly
support the view that PHOX2B is a mas-
ter gene for the formation and/or func-
tion of the neuronal network for
autonomous control of ventilation and
further suggests that PHOX2B mutations
trigger a wide spectrum of ANS disorders
ranging from dysgenetic malformations
to tumor predisposition.
Note: Supplementary information is avail-
able on the Nature Genetics website.
We thank the individuals with CCHS, their
families and the Association Française du
Syndrome d’Ondine who participated in this
study, J.-F. Brunet and C. Goridis for helpful
comments and discussions and G. Mattéi for
technical help. This study was supported by grants
from the European Community, Association
Française contre les Myopathies-INSERM
(Maladies Rares) and Hoechst-Marion-Roussel.
B.L. is a recipient of a Sanofi-Synthelabo grant.
Competing interests statement
The authors declare that they have no competing
Jeanne Amiel1, Béatrice Laudier1, Tania
Attié-Bitach1, Ha Trang2, Loïc de
Pontual1, Blanca Gener3, Delphine Tro-
chet1, Heather Etchevers1, Pierre Ray1,
Michel Simonneau2, Michel Vekemans1,
Arnold Munnich1, Claude Gaultier2
& Stanislas Lyonnet1
1Unité de Recherches sur les Handicaps
Génétiques de l’Enfant INSERM U-393, et
Département de Génétique, Hôpital Necker-
Enfants Malades, 149, rue de Sèvres, 75743
Paris Cedex 15, France. 2Service de Physiologie
CIC INSERM 9202, et Equipe INSERM E9935,
Hôpital Robert Debré, Paris, France. 3Clinica
Materno-Infantil, Hospital de Cruces, 48903
Barakaldo, Spain. Correspondence should be
addressed to J.A. (e-mail: firstname.lastname@example.org).
Received 31 October 2002; accepted 20 February 2003.
Gozal, D. Pediatr. Pulmonol. 26, 273–282 (1998).
Khalifa, M.M., Flavin, M.A. & Wherrett, B.A. J.
Pediatr. 113, 853–855 (1988).
Sritippayawan, S. et al. Am. J. Respir. Crit. Care
Med. 166, 367–369 (2002).
Weese-Mayer, D.E., Silvestri, J.M., Marazita, M.L. &
Hoo, J.J. Am. J. Med. Genet. 47, 360–367 (1993).
Amiel, J. et al. Am. J. Hum. Genet. 62, 715–717
Bolk, S. et al. Nat. Genet. 13, 395–396 (1996).
Weese-Mayer, D.E., Bolk, S., Silvestri, J.M. &
Chakravarti, A. Am. J. Med. Genet. 107, 306–310
Brunet, J.F. & Pattyn, A. Curr. Opin. Genet. Dev. 12,
Pattyn, A., Morin, X., Cremer, H., Goridis, C. &
Brunet, J.F. Nature 399, 366–370 (1999).
10. Goodman, F.R. & Scambler, P.J. Clin. Genet. 59, 1–11
11. Stromme, P. et al. Nat. Genet. 30, 441–445 (2002).
12. Bruneau, S., Johnson, K.R., Yamamoto, M.,
Kuroiwa, A. & Duboule, D. Dev. Biol. 237, 345–353
13. Brown, S.A., Abigani, M. & Brown, L.Y. Am. J. Hum.
Genet. 71 Supp. 166 (2002).
14. Nakano, M. et al. Nat. Genet. 29, 315–320 (2001).
15. Dubreuil, V., Hirsch, M.R., Pattyn, A., Brunet, J.F. &
Goridis, C. Development 127, 5191–5201 (2000).
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