PHOX2B Mutations Research Articles

Phox2B mutations and the Delta–Notch pathway in neuroblastoma

We recently identified six neuroblastoma patients with constitutional or tumor-specific mutations in the homeobox gene Phox2B. Phox2B controls part of the differentiation program of the sympathetic nervous system (SNS). Mice with a homozygous inactivation of Phox2B fail in the proper differentiation of the chromaffin lineage of the SNS. Phox2B regulates HASH1 which can control expression of genes of the Delta–Notch pathway. We previously showed that a subset of neuroblastoma cell lines highly expresses Delta-like 1 (Dlk1), which is a marker for the chromaffin lineage of the SNS. Notch3 is expressed in another subset of neuroblastoma cell lines and marks tumors from an alternative differentiation lineage. Phox2B is also related to the TrkA differentiation pathway in neuroblastoma. Here we will review the role of Phox2B in differentiation programs of the SNS and in neuroblastoma pathogenesis.

In pursuit (and discovery) of a genetic basis for congenital central hypoventilation syndrome

Congenital central hypoventilation syndrome (CCHS) typically presents in the newborn period with a phenotype including alveolar hypoventilation, symptoms of autonomic nervous system dysregulation, and in a subset of cases Hirschsprung disease and later tumors of neural crest origin. Study of genes related to the autonomic dysregulation and the embryologic origin of the neural crest has led to identification of the genetic basis for CCHS, the mode of inheritance, and the presence of mosaicism in a subset of parents. Polyalanine expansion mutations in PHOX2B have been identified to be the disease-defining mutation in CCHS, with a small subset of patients having other mutations in PHOX2B. Further, the size of the polyalanine repeat mutation in PHOX2B is correlated with the severity of the phenotype in CCHS, and non-polyalanine repeat mutations appear to, in general, result in CCHS phenotypes at the severe end of the spectrum. These studies highlight the utility of PHOX2B genetic testing for confirmation of the CCHS diagnosis, for prenatal diagnosis, and for identification of previously undiagnosed adults with unexplained hypercarbia or control of breathing deficits. This diagnostic approach may be a consideration for other complex, seemingly undecipherable diseases that affect infants and children. The purpose of this article is to provide a comprehensive review of current research into the genetic basis for CCHS, an explanation for how these studies evolved, recent studies that begin to explain the mechanisms through which mutations in PHOX2B exert their effects, and clinical application of the genetic testing.

Distinct pathogenetic mechanisms for PHOX2B associated polyalanine expansions and frameshift mutations in congenital central hypoventilation syndrome

Congenital central hypoventilation syndrome (CCHS) is a rare neurocristopathy characterized by absence of adequate autonomic control of respiration with decreased sensitivity to hypoxia and hypercapnia. Frameshift mutations and polyalanine triplet expansions in the coding region of PHOX2B have been identified in the vast majority of CCHS patients and a correlation between length of the expanded region and severity of CCHS has been reported. In this work, we have undertaken in vitro analyses aimed at identifying the pathogenetic mechanisms which underlie the effects of PHOX2B mutations in CCHS. According to the known role of this gene, a transcription factor expressed during autonomic nervous system development, we have tested the transcriptional activity of WT and mutant PHOX2B expression constructs on the regulatory regions of two target genes, DbetaH and PHOX2A. We observed that the two sets of mutations play different roles in the transcriptional regulation of these genes, showing a correlation between the length of polyalanine expansions and the severity of reduced transcriptional activity. In particular, although reduced transactivation due to polyalanine expansions may be caused by retention of the mutated protein in the cytoplasm or in the nuclear aggregates, frameshift mutations did not impair the PHOX2B nuclear income, suggesting a different mechanism through which they would exert the observed effects on target promoters. Moreover, the frameshift due to deletion of a cytosine residue seems to cause sequestration of the corresponding mutant PHOX2B in the nucleolar compartment.

Late-Onset Hypoventilation Without PHOX2B Mutation or Hypothalamic Abnormalities

Most children with idiopathic central hypoventilation have symptoms at birth or shortly thereafter and have mutations of the PHOX2B gene. Those whose symptoms appear later usually have obesity and hypothalamic abnormalities. We describe a case of a boy who presented at 5 years of age with severe idiopathic central hypoventilation, but no obesity or hypothalamic abnormalities, and who tested negative for mutation of the PHOX2B gene. This case illustrates the heterogeneity of childhood idiopathic central hypoventilation syndromes and indicates the multifactorial etiology of these syndromes.

The Phox2B homeobox gene is mutated in sporadic neuroblastomas

Neuroblastomas are embryonal tumours of the sympatho-adrenal lineage with a clinical course ranging from spontaneous regression to fatal progression. The Phox2B homeobox transcription factor functions in the differentiation of the sympatho-adrenal lineage. Targets of Phox2B are, for example, genes of the (nor)adrenalin synthesis route, like Dopamine Beta Hydroxylase (DBH). Congenital Central Hypoventilation Syndrome was recently found to result from Phox2B mutations and two such patients in addition developed neuroblastoma. A germline mutation in Phox2B was identified in a family with hereditary neuroblastoma. Here, we report the first analysis of Phox2B in a series of 237 sporadic neuroblastomas and 22 cell lines. Six frameshift mutations were found in exons 2 and 3; including one in cell line SK-N-SH. Two patients showed de novo constitutional mutations. One of them was diagnosed with Haddad syndrome. All analysed cases expressed the mutated and wild-type Phox2B alleles. Ectopic expression of TrkA, the Nerve Growth Factor receptor, strongly downregulated Phox2B and DBH expression in cell line SH-SY5Y. However, TrkA and Phox2B showed a positive correlation in a panel of 66 neuroblastoma tumours. Although Phox2B mutations are infrequent (2.3%), they implicate a role for the Phox2B pathway in oncogenesis.

Haploinsufficiency for Phox2b in mice causes dilated pupils and atrophy of the ciliary ganglion: mechanistic insights into human congenital central hypoventilation syndrome.

Dilp1 is a semi-dominant mouse mutation that causes dilated pupils when heterozygous and is lethal when homozygous. We report here that it is caused by a point mutation that introduces a stop codon close to the start of the coding sequence of the paired-like homeobox transcription factor Phox2b. Mice carrying a targeted allele of Phox2b also have dilated pupils and the two alleles do not complement. Phox2b is necessary for the development of the autonomic nervous system and when absent one of the consequences is that all parasympathetic ganglia fail to form. Constriction of the pupil is a parasympathetic response mediated by the ciliary ganglion and we find that in Phox2b heterozygous mutants it is highly atrophic. The development of other parasympathetic and sympathetic ganglia appears to be largely unaffected indicating that the ciliary ganglion is exquisitely sensitive to a reduction in dose of this transcription factor. PHOX2B has been implicated in human disease. Mutations, principally leading to polyalanine expansions within the protein, have been found in patients with congenital central hypoventilation syndrome (CCHS), the cardinal feature of which is an inability to breathe unassisted when asleep. Additionally, some CCHS patients have ocular abnormalities, including pupillary defects, although they principally have constricted rather than dilated pupils. The apparent phenotypic differences observed between mice carrying a loss-of-function mutation of Phox2b and CCHS patients indicate that PHOX2B mutations found in CCHS patients, all of which can produce proteins with intact DNA-binding domains, are gain-of-function mutations that alter rather than abolish protein function.

Phox2b controls the development of peripheral chemoreceptors and afferent visceral pathways.

We report that the afferent relays of visceral (cardiovascular, digestive and respiratory) reflexes, differentiate under the control of the paired-like homeobox gene Phox2b: the neural crest-derived carotid body, a chemosensor organ, degenerates in homozygous mutants, as do the three epibranchial placode-derived visceral sensory ganglia (geniculate, petrosal and nodose), while their central target, the nucleus of the solitary tract, which integrates all visceral information, never forms. These data establish Phox2b as an unusual 'circuit-specific' transcription factor devoted to the formation of autonomic reflex pathways. We also show that Phox2b heterozygous mutants have an altered response to hypoxia and hypercapnia at birth and a decreased tyrosine hydroxylase expression in the petrosal chemosensory neurons, thus providing mechanistic insight into congenital central hypoventilation syndrome, which is associated with heterozygous mutations in PHOX2B.

Idiopathic congenital central hypoventilation syndrome: analysis of genes pertinent to early autonomic nervous system embryologic development and identification of mutations in PHOX2b.

Idiopathic congenital central hypoventilation syndrome (CCHS) has been linked to autonomic nervous system dysregulation and/or dysfunction (ANSD) since it was first described in 1970. A genetic basis of CCHS has been proposed because of the reports of four families with two affected children, because of mother-child transmission, and because of a recent report of a polyalanine expansion mutation in PHOX2b in a subset of CCHS subjects. We, therefore, studied genes pertinent to early embryologic development of the ANS including mammalian achaete-scute homolog-1 (MASH1), bone morphogenic protein-2 (BMP2), engrailed-1 (EN1), TLX3, endothelin converting enzyme-1 (ECE1), endothelin-1 (EDN1), PHOX2a, and PHOX2b in 67 probands with CCHS, and gender- and ethnicity-matched controls. No disease-defining mutations were identified in MASH1, BMP2, EN1, TLX3, ECE1, EDN1, or PHOX2a. The 65/67 CCHS probands (97%) were found to be heterozygous for the exon 3 polyalanine expansion mutation identified previously in PHOX2b. Further, there was an association between repeat mutation length and severity of the CCHS/ANSD phenotype. Of the two probands who did not carry the expansion mutation, one had a nonsense mutation in exon 3 which truncated the protein and the other had no mutation in PHOX2b but had a previously reported EDN3 frameshift point mutation. The polyalanine expansion mutation was not found in any of 67 matched controls. Of 54 available families (including 97 unaffected parents), whose child carried the PHOX2b mutation, 4 parents demonstrated mosaicism for an expansion mutation identical to that seen in the CCHS cases, suggesting that not all mutations in affected probands with unaffected parents are de novo. We also studied four women with CCHS who were heterozygous for the PHOX2b mutation, each with one child. Three of the four children were also affected and had the same mutation, demonstrating autosomal dominant inheritance of the mutation. Assay of the PHOX2b polyalanine repeat mutation represents a highly sensitive and specific technique for confirming the diagnosis of CCHS. Identification of the CCHS mutation will lead to clarification of the phenotype, allow for prenatal diagnosis for parents of CCHS probands and adults with CCHS in future pregnancies, and potentially direct intervention strategies for the treatment of CCHS.

Polyalanine expansion and frameshift mutations of the paired-like homeobox gene PHOX2B in congenital central hypoventilation syndrome.

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.

PHOX2A and PHOX2B genes are highly co-expressed in human neuroblastoma

The detection of PHOX2B mutations in a small proportion of patients affected with either familial or sporadic neuroblastoma (NB), has arisen interest on the possible pathogenic role of this gene in the disease determination. In this light, we have carried out a quantitative expression analysis of PHOX2B and its paralogue PHOX2A on a panel of NB cell lines and NB tumour samples to identify a possible differential expression between NB cells and their normal counterpart (adrenal medulla cells). Our results revealed that both PHOX2A and PHOX2B are over-expressed in tumour samples and NB cell lines. Particularly, the expression levels of the two genes in NB cell lines show a highly significant correlation, suggesting their possible synergistic role or a coordinated expression regulation. Furthermore, PHOX2 gene over-expression in NB tumours and cell lines suggests these genes may be widely involved in NB development through either a direct mechanism of up-regulation or a failure in maintaining proper transcript levels after embryonic development.