Alanine Expansions Associated with Congenital Central Hypoventilation Syndrome Impair PHOX2B Homeodomain-mediated Dimerization and Nuclear Import.

Heterozygous mutations of the human PHOX2B gene, a key regulator of autonomic nervous system development, lead to congenital central hypoventilation syndrome (CCHS), a neurodevelopmental disorder characterized by a failure in the autonomic control of breathing. Polyalanine expansions in the 20-residues region of the C terminus of PHOX2B are the major mutations responsible for CCHS. Elongation of the alanine stretch in PHOX2B leads to a protein with altered DNA binding, transcriptional activity, and nuclear localization and the possible formation of cytoplasmic aggregates; furthermore, the findings of various studies support the idea that CCHS is not due to a pure loss of function mechanism but also involves a dominant negative effect and/or toxic gain of function for PHOX2B mutations. Because PHOX2B forms homodimers and heterodimers with its paralogue PHOX2A in vitro, we tested the hypothesis that the dominant negative effects of the mutated proteins are due to non-functional interactions with the wild-type protein or PHOX2A using a co-immunoprecipitation assay and the mammalian two-hybrid system. Our findings show that PHOX2B forms homodimers and heterodimerizes weakly with mutated proteins, exclude the direct involvement of the polyalanine tract in dimer formation, and indicate that mutated proteins retain partial ability to form heterodimers with PHOX2A. Moreover, in this study, we investigated the effects of the longest polyalanine expansions on the homeodomain-mediated nuclear import, and our data clearly show that the expanded C terminus interferes with this process. These results provide novel insights into the effects of the alanine tract expansion on PHOX2B folding and activity.

Congenital central hypoventilation syndrome: mutation analysis of the receptor tyrosine kinase RET.

Congenital central hypoventilation syndrome (CCHS) usually occurs as an isolated phenotype. However, 16% of the index cases are also affected with Hirschsprung disease (HSCR). Complex segregation analysis suggests that CCHS is familial and has the same inheritance pattern with or without HSCR. We postulate that alteration of normal function of the receptor tyrosine kinase, RET, may contribute to CCHS based on RET's expression pattern and the identification of RET mutations in HSCR patients. To further explore the nature of the inheritance of CCHS, we have undertaken two main routes of investigation: cytogenetic analysis and mutation detection. Cytogenetic analysis of metaphase chromosomes showed normal karyotypes in 13 of the 14 evaluated index cases; one index case carried a familial pericentric inversion on chromosome 2. Mutation analysis showed no sequence changes unique to index cases, as compared to control individuals, and as studied by single strand conformational polymorphism (SSCP) analysis of the coding region of RET. We conclude that point mutations in the RET coding region cannot account for a substantial fraction of CCHS in this patient population, and that other candidate genes involved in neural crest cell differentiation and development must be considered.

Inhibition of erythrocyte Na,K-ATPase activity during anticipatory hypoventilation in micropigs.

Previous studies with micropigs showed that conditioned suppression of respiration preceding the onset of an avoidance task was associated with increased pCO2, decreased plasma pH, decreased hematocrit, and increased blood pressure with no change in heart rate. Voluntary hypoventilation by humans, which evoked similar effects, was found to elicit increases in plasma endogenous digitalis-like factors (EDLF) and decreases in erythrocyte Na,K-ATPase. The present study investigated plasma EDLF and Na,K-ATPase activity in micropigs preceding and during avoidance sessions. Compared with levels in a controlled environment, 1 h of quiet waiting for the onset of a 30-min avoidance task was associated with hypoventilation, acidification of the plasma, and a decrease in hematocrit with progressive increases in plasma EDLF, and decreased in erythrocyte Na,K-ATPase activity (1.67 +/- 0.35 v 2.73 +/- 0.24 mumol Pi/mL er/h). Systolic blood pressure increased (126.5 +/- 5.7 v 121.7 +/- 4.2 mm Hg) during preavoidance periods, with no changes in heart rate (89.5 +/- 3.9 v 89.4 +/- 4.0 beats/min). During the avoidance sessions, plasma EDLF, systolic blood pressure (126.7 +/- 4.5 mm Hg), and heart rate (107.3 +/- 4.8 beats/min) were elevated above the first 10 min of preavoidance, whereas Na,K-ATPase activity returned toward control values (2.46 +/- 0.83 mumol Pi/mL er/h). These findings are consistent with the view that elevation of blood pressure during behaviorally induced hypoventilation in micropigs is mediated in part by inhibition of Na,K-ATPase by increases in plasma EDLF due to expanded plasma volume.