Molecular consequences of PHOX2B missense, frameshift and alanine expansion mutations leading to autonomic dysfunction

Abstract

Heterozygous mutations of the PHOX2B gene account for a broad variety of disorders of the autonomic nervous system, either isolated or combined, including congenital central hypoventilation syndrome (CCHS), tumours of the sympathetic nervous system and Hirschsprung disease. In CCHS, the prevalent mutation is an expansion of a 20-alanine stretch ranging from +5 to +13 alanines, whereas frameshift and missense mutations are found occasionally. To determine the molecular basis of impaired PHOX2B function, we assayed the transactivation and DNA binding properties of wild-type and mutant PHOX2B proteins. Furthermore, we investigated aggregate formation by proteins with polyalanine tract expansions ranging from +5 to +13 alanines using immunofluorescence of transfected cells and gel filtration of in vitro translated proteins. We found that transactivation of the dopamine beta-hydroxylase promoter by PHOX2B proteins with frameshift and missense mutations was abolished or severely curtailed, as was in vitro DNA binding although the proteins localized to the nucleus. The transactivation potential of proteins with polyalanine tract expansions declined with increasing length of the polyalanine stretch, and DNA binding was affected for an expansion of +9 alanines and above. Cytoplasmic aggregation in transfected cells was only observed for the longest expansions, whereas even the short expansion mutants were prone to form multimers in vitro. Such a tendency to protein misfolding could explain loss of transactivation for alanine expansion mutations. However, additional mechanisms such as toxic gain-of-function may play a role in the pathogenic process.

The mutations and potential targets of the forkhead transcription factor FOXL2

“…These observations are consistent with the results obtained with FOXL2 and point to a common pathogenic mechanism. A similar behavior has been observed for PHOX2B in cellular models (Bachetti et al, 2005;Trochet et al, 2005 A c c e p t e d M a n u s c r i p t 8 expansion in Arx transcription factor forms intranuclear inclusions and results in increased cell death suggesting a toxic effect (Nasrallah et al, 2004). Thus, it appears that this expansion lead to cellular toxicity as it was suggested for PABPN1.…”

A human mutation in Phox2b causes lack of CO 2 chemosensitivity, fatal central apnea, and specific loss of parafacial neurons

“…Thus, understanding the molecular and cellular underpinnings of CCHS offers the promise of illuminating the mechanisms that are essential for CO 2 sensitivity and, more generally, proper breathing at birth. CCHS is known to be caused by heterozygous mutations of the PHOX2B transcription factor, mainly expansions of a polyalanine (polyAla) tract (13)(14)(15). The respiratory symptoms of CCHS patients point to a defect in the metabolic regulation of breathing and have been attributed to a failure of central chemosensory integration (11,12), but the neural structures affected by the disease have remained obscure.…”

Polyalanine expansion of PHOX2B in congenital central hypoventilation syndrome: rs17884724:A>C is associated with 7-alanine expansion

“…About 90% of patients have de novo polyalanine expansion mutations in the polyalanine tract of 20 residues. [1][2][3][4][5][6] Approximately 5% of patients inherit polyalanine expansion mutations mostly from asymptomatic parents with somatic mosaicism and rarely from affected parents. Polyalanine expansion disorders constitute one family of homopolymer expansion disorders, including at least nine disorders.…”