Amiel J, Laudier B, Attie-Bitach T, Trang H, de Pontual L, Gener B et al.. Polyalanine expansion and frameshift mutations of the paired-like homeobox gene PHOX2B in Congenital Central Hypoventilation Syndrome. Nat Genet 33: 459-461

Hospital Universitario Cruces, Bilbo, Basque Country, Spain
Nature Genetics (Impact Factor: 29.35). 05/2003; 33(4):459-61. DOI: 10.1038/ng1130
Source: PubMed


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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    • "Consistent with this hypothesis, RTN neurons are activated by hypercapnia or acidification ex vivo, including after isolation (Figure 2B) (Mulkey et al., 2004; Wang et al., 2013b). In 2003, mutations of homeodomain transcription factor Phox2b, were shown to cause CCHS (Amiel et al., 2003) and, in 2006, Phox2b was identified in all RTN CO 2 responsive neurons (Figures 1C–1F 1 ) (Stornetta et al., 2006). "
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    Neuron 09/2015; 87(5):946-61. DOI:10.1016/j.neuron.2015.08.001 · 15.05 Impact Factor
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    • "Interestingly, co-transfection of the deleted proteins with the wild-type protein reduced promoter activity in comparison with the full transactivation observed with PHOX2B WT alone only in the case of the PHOX2B Δaa 200–290 mutant (Fig. 7D, compare hatched bars with white bar). These data are in line with the fact that in-frame polyalanine contraction variants (with 7, 13, 14 or 15 repeats in the polyalanine tract) are also found in the control population (Amiel et al., 2003; Hung et al., 2007; Matera et al., 2004; Toyota et al., 2004). Finally, our data support the idea that the portion of the protein downstream of the homeodomain containing the polyalanine tract modulates the DNA binding and transactivation function of the PHOX2B protein, and it is reasonable to hypothesize that the deletion of a large C-terminal region of the protein encompassing the polyalanine stretch can affect the correct orientation, folding and/or protein–protein interaction properties of the remaining portion of the protein generating a mutant variant with dominant-negative effects on a subset of promoters. "
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    ABSTRACT: The PHOX2B transcription factor plays a crucial role in autonomic nervous system development. In humans, heterozygous mutations of the PHOX2B gene lead to Congenital Central Hypoventilation Syndrome (CCHS), a rare disorder characterized by a broad variety of symptoms of autonomic nervous system dysfunction including inadequate control of breathing. The vast majority of patients with CCHS are heterozygous for a polyalanine repeat expansion mutation involving a polyalanine tract of twenty residues in the C-terminus of PHOX2B. Although several lines of evidence support a dominant-negative mechanism for PHOX2B mutations in CCHS, the molecular effects of PHOX2B mutant proteins on the transcriptional activity of the wild-type protein have not yet been elucidated. As one of the targets of PHOX2B is the PHOX2B gene itself, we tested the transcriptional activity of wild-type and mutant proteins on the PHOX2B gene promoter, and found that the transactivation ability of proteins with polyalanine expansions decreased as a function of the length of the expansion, whereas DNA binding was severely affected only in the case of the mutant with the longest polyalanine tract (+13 alanine). Co-transfection experiments using equimolar amounts of PHOX2B wild-type and mutant proteins in order to simulate a heterozygous state in vitro and four different PHOX2B target gene regulatory regions (PHOX2B, PHOX2A, DBH, TLX2) clearly showed that the polyalanine expanded proteins alter the transcriptional activity of wild-type protein in a promoter-specific manner, without any clear correlation with the length of the expansion. Moreover, although reduced transactivation may be caused by retention of the wild-type protein in the cytoplasm or in nuclear aggregates, this mechanism can only be partially responsible for the pathogenesis of CCHS because of the reduction in cytoplasmic and nuclear accumulation when the +13 alanine mutant is co-expressed with wild-type protein, and the fact that the shortest polyalanine expansions do not form visible cytoplasmic aggregates. Deletion of the C-terminal of PHOX2B leads to a protein that correctly localizes in the nucleus but impairs PHOX2B wild-type transcriptional activity, thus suggesting that protein mislocalization is not the only mechanism leading to CCHS. The results of this study provide novel in vitro experimental evidence of a transcriptional dominant-negative effect of PHOX2B polyalanine mutant proteins on wild-type protein on two different PHOX2B target genes.
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    • "The homozygous disruption of the PHOX2B gene has been reported to result in the absence of enteric ganglia, a feature which is reminiscent of HSCR (Elworthy et al., 2005). Amiel et al. (2003) reported that PHOX2B is the primary disease locus in congenital central hypoventilation syndrome (CCHS). They found that mutations in PHOX2B not only exist in isolated cases of CCHS but also in individuals with a more complex neural crest involvement including CCHS and HSCR (Haddad syndrome). "
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