Polyalanine Repeat Expansion Mutation Research Articles

Congenital central hypoventilation syndrome in Chinese population: Analysis of three new cases and review of the literature.

Congenital central hypoventilation syndrome (CCHS) is a rare autosomal dominant disease that is mainly caused by PHOX2B mutations. The purpose of this study is to analyze and summarize the clinical and genetic characteristics of CCHS patients in the Chinese population from our study and previous literature. The potential pathogenic gene mutations of CCHS were identified and verified by next generation sequencing combined with Sanger sequencing, fluorescent probe PCR and capillary electrophoresis. The clinical characteristics and gene mutations of CCHS cases in Chinese population were summarized from our study and previous literature to explore the genotype-phenotype correlations. We identified 48 CCHS cases including three new cases from our report in China. Overall, 77.1% of the patients had PHOX2B polyalanine repeat expansion mutations (PARMs), and the remaining 22.9% had 10 distinct PHOX2B non-polyalanine repeat expansion mutations (NPARMs). Compared to those with PARMs, patients with NPARMs were more likely to have premature birth (54.5% vs. 2.8%, p < 0.001) and lower birth weight (33.3% vs. 3.2%, p = 0.030), with statistical significance. The patients with PARMs were more likely to have cardiovascular defects (64.9% vs. 27.3%, p = 0.063), cerebral hemorrhage (29.7% vs. 9.1%, p = 0.322) and seizures (37.8% vs. 9.1%, p = 0.151) than those with NPARMs, with no statistical significance. CCHS patients with PHOX2B NPARMs were more likely to have premature birth and low birth weight, while PHOX2B PARMs tended to be positively associated with the risk of cardiovascular defects, cerebral hemorrhage and seizures in Chinese population.

Heart rate variability in congenital central hypoventilation syndrome: relationships with hypertension and sinus pauses.

Autonomic nervous system (ANS) dysregulation has been described in congenital central hypoventilation syndrome (CCHS). The objectives were to describe heart rate variability (HRV) analyses in children suffering from CCHS both while awake and asleep and their relationships with both ambulatory blood pressure (BP) and ECG monitoring results. This retrospective study enrolled children with CCHS (n = 33, median age 8.4 years, 18 girls) who had BP and ECG monitored during the same 24 h. From the latter, HRV analyses were obtained during daytime and nighttime. The prevalences of hypertension and sinus pauses were 33% (95% confidence interval [CI]: 18-52) and 18% (95% CI: 7-35), respectively. The decrease in systolic BP at night negatively correlated with an increase in very low frequency (VLF) and LF powers at night, and the longest RR interval positively correlated with daytime VLF and LF powers. Among the three groups of children (polyalanine repeat expansion mutation [PARM], moderate [20/25 and 20/26], severe [20/27 and 20/33], and non-PARMs), the prevalence of elevated BP or hypertension was different: in PARM subjects: 6/18 moderate, 7/9 severe versus 0/6 in non-PARM (p = 0.002). Modifications of cardiac ANS are associated with systemic hypertension and the occurrence of sinus pauses in CCHS. Children with congenital central hypoventilation syndrome (CCHS) exhibit an increased prevalence of hypertension and sinus pauses that are linked to cardiac autonomic nervous system dysfunction. Sinus pauses are the main manifestation of sinus nodal dysfunction in children with CCHS. The increased prevalence of hypertension, especially at nighttime, is a new finding in CCHS. Sinus nodal dysfunction can be due to the sole impairment of the cardiac autonomic nervous system. Ambulatory blood pressure and ECG monitoring are mandatory in patients with CCHS.

Generation of two hiPSC lines (UMILi027-A and UMILi028-A) from early and late-onset Congenital Central hypoventilation Syndrome (CCHS) patients carrying a polyalanine expansion mutation in the PHOX2B gene

Congenital Central Hypoventilation Syndrome (CCHS) is a rare disorder of the autonomic nervous system (ANS), characterized by inadequate control of autonomic ventilation and global autonomic dysfunction. Heterozygous polyalanine repeat expansion mutations in exon 3 of the transcription factor Paired-like homeobox 2B (PHOX2B) gene occur in 90% of CCHS cases. In this study, we describe the generation and characterization of two human induced pluripotent stem cell (hiPSC) lines from female CCHS patients carrying a heterozygous + 5 alanine expansion mutation. The generated iPSC lines show a normal karyotype, express pluripotency markers and are able to differentiate into the three germ layers.

Paired-like homeobox gene (PHOX2B) nonpolyalanine repeat expansion mutations (NPARMs): genotype–phenotype correlation in congenital central hypoventilation syndrome (CCHS)

PurposeCCHS is an extremely rare congenital disorder requiring artificial ventilation as life support. Typically caused by heterozygous polyalanine repeat expansion mutations (PARMs) in the PHOX2B gene, identification of a relationship between PARM length and phenotype severity has enabled anticipatory management. However, for patients with non-PARMs in PHOX2B (NPARMs, ~10% of CCHS patients), a genotype–phenotype correlation has not been established. This comprehensive report of PHOX2B NPARMs and associated phenotypes, aims at elucidating potential genotype–phenotype correlations that will guide anticipatory management. MethodsAn international collaboration (clinical, commercial, and research laboratories) was established to collect/share information on novel and previously published PHOX2B NPARM cases. Variants were categorized by type and gene location. Categorical data were analyzed with chi-square and Fisher’s exact test; further pairwise comparisons were made on significant results. ResultsThree hundred two individuals with PHOX2B NPARMs were identified, including 139 previously unreported cases. Findings demonstrate significant associations between key phenotypic manifestations of CCHS and variant type, location, and predicted effect on protein function. ConclusionThis study presents the largest cohort of PHOX2B NPARMs and associated phenotype data to date, enabling genotype–phenotype studies that will advance personalized, anticipatory management and help elucidate pathological mechanisms. Further characterization of PHOX2B NPARMs demands longitudinal clinical follow-up through international registries.

Improvement of hypoventilation in Phox2b mutant mice modelling the congenital central hypoventilation syndrome by etonogestrel, a potent progestin. Perspectives for a possible potentiation by serotoninergic drugs

Congenital central hypoventilation syndrome (CCHS) is a life-threatening disorder characterized by hypoventilation during sleep and absence of the ventilatory response to CO2/H+. The disease-causing mutations are generally polyalanine repeat expansion mutations of PHOX2B. Knock-in mice bearing the 7-alanine expanded allele of PHOX2B (the most frequent mutation in patients) exhibit massive loss of retrotrapezoid nucleus neurons, lack CO2/H + chemosensitivity, and die within hours after birth. No pharmacological treatment is available. Clinical observations revealed a recovery of CO2/H+ chemosensitivity in two adult female patients with CCHS, both under desogestrel, a potent progestin used for contraceptive purpose. We previously showed that in mice, etonogestrel (ETO), the active metabolite of desogestrel, increased baseline respiratory frequency (fR) by a medullary action involving the serotoninergic (5-HT) systems. Accordingly, we hypothesized that ETO may stimulate fR on Phox2b mutant mice modelling CCHS and that 5-HT drugs would improve this respiratory effect of ETO. Experiments were made on ex vivo medullary-spinal cord preparations from conditional RTN Phox2b mutant (Egr2cre/+;Phox2b27Alaki) and OF1 WT newborn mice (P0-P2). fR was assessed by recording the 4th spinal ventral root. Effect of ETO on fR under normopH and metabolic acidosis (MA) conditions was evaluated in presence or not of 5-HT reuptake inhibitor. Without ETO, the increase in fR induced by MA observed in WT mice was absent in Egr2cre/+;Phox2b27Alaki mutant mice. ETO restored a respiratory response to MA in Egr2cre/+;Phox2b27Alaki mutant mice. In OF1 WT mice, when ETO was associated with 5-HT reuptake inhibitor, the increase in fR induced by MA was enhanced. Surprisingly, this potentiation was not retrieved in Egr2cre/+;Phox2b27Alaki mutant mice at concentrations used in OF1 WT. ETO was able to alleviate hypoventilation in Phox2b mutant mice modelling CCHS by a medullary action. Modulation of 5-HT systems may improve the respiratory effect of ETO in WT mice, but further studies are necessary to determine whether this increase of ETO effect by 5-HT extends to mutant mice.

Congenital central hypoventilation syndrome in neonates: report of fourteen new cases and a review of the literature.

BackgroundCongenital central hypoventilation syndrome (CCHS) is a rare autosomal dominant disorder caused by pathogenic variants in paired-like homeobox 2B (PHOX2B) gene. Characteristics of neonatal-onset CCHS cases have not been well assessed. The aim of this study is to expand current knowledge of clinical and genetic features of neonates with CCHS and provide data on the genotype-phenotype correlation.MethodsWe made a retrospective analysis of 14 neonates carrying PHOX2B pathogenic variants from 2014 to 2019 and we reviewed previously published neonatal-onset cases. Clinical and genetic data were analyzed. Moreover, genotype-phenotype correlation analysis was performed.ResultsWe identified a total of 60 neonatal-onset CCHS cases (35 males and 25 females) including 14 novel cases from our local cohort. Nearly 20% (18.2%) of the patients were born prematurely. Nearly half (46.2%) of the patients had abnormal family history. Polyhydramnios was observed in 21.3% (10/47) of the patients. About 90% of the patients manifested symptoms of hypoventilation in the first week of life. Fourteen patients (23.3%) were classified as mild-CCHS and the rest were severe-CCHS. Gastrointestinal manifestations were observed in 71.7% of the patients. Approximately twofold more males than females were affected by Hirschprung disease (HSCR)/variant HSCR (75.8% vs. 35%, P=0.003). Neural crest tumor occurred in 9.1% (4/44) patients. Half patients had polyalanine repeat expansion mutations (PARMs) in PHOX2B (seven with 25 PARM, nine with 26 PARM, twelve with 27 PARM, one with 28 PARM and one with 31 PARM) and the other half patients had 23 distinct non-polyalanine repeat expansion mutations (NPARMs) with one novel pathogenic variant (c.684dup). The prevalence of HSCR and mild-CCHS among patients with NPARMs was significantly greater than that of the patients with PARMs.ConclusionsThis report provides a large cohort of neonatal-onset CCHS cases. The results indicate that severe hypoventilation and HSCR are frequently observed in this group. NPARMs accounted for half of the cohort with some genotypes tend to be associated with mild phenotype. Molecular testing in neonates with suspicion of CCHS and genetic counseling for CCHS families are highly recommended.

How the Management of Children With Congenital Central Hypoventilation Syndrome Has Changed Over Time: Two Decades of Experience From an Italian Center.

Background: Congenital central hypoventilation syndrome (CCHS) is a rare disorder whose clinical phenotype is closely related to genotype.Methods: A retrospective analysis has been conducted on 22 patients with CCHS, who were referred to the Pediatric Pulmonology and Respiratory Intermediate Care Unit of Bambino Gesù Children's Hospital (Italy) for a multidisciplinary follow-up program between 2000 and 2020.Results: Apnea and cyanosis were the most frequent symptoms at onset (91%). Overall, 59% of patients required tracheostomy and invasive mechanical ventilation (IMV) in the first months of life. Thirty-two percent of patients had Hirschsprung disease (HSCR) that was associated with longer polyalanine repetitions or non-polyalanine repeat expansion mutations (NPARMs). Polyalanine repeat expansion mutations (PARMs) were more frequent and two novel NPARMs (c.780dupT and C.225–256delCT) were described in 14% of patients. Focal epilepsy was first described in 14% of patients and neurocognitive and neuromotor impairment involved 27% and 23% of children, respectively. Symptoms due to autonomic nervous system dysfunction/dysregulation (ANSD)—including strabismus (27%), dysphagia (27%), abnormal heart rhythm (10%), breath-holding spells (9%), and recurrent seizures due to hypoglycemia (9%)—were associated with an increased number of polyalanine repetitions of exon 3 or NPARMs of PHOX2B gene. Overall, the number of patients with moderate to severe phenotype initially treated with non-invasive ventilation (NIV) increased over time, and the decannulation program was concluded with 3 patients who started with IMV.Conclusions: Our study confirms that more severe phenotypes of CCHS are related to the number of polyalanine repetitions or to NPARMs. Although invasive ventilation is often required by patients with severe genotype/phenotype, gradual acquisition of specific skills in the management of patients with CCHS and technological improvements in mechanical ventilation allowed us to improve our therapeutic approach in this population.

Neurocognitive functioning in individuals with congenital central hypoventilation syndrome

BackgroundCongenital central hypoventilation syndrome (CCHS) is a rare disorder characterized by respiratory system abnormalities, including alveolar hypoventilation and autonomic nervous system dysregulation. CCHS is associated with compromised brain development and neurocognitive functioning. Studies that evaluate cognitive skills in CCHS are limited, and no study has considered cognitive abilities in conjunction with psychosocial and adaptive functioning. Moreover, the roles of pertinent medical variables such as genetic characteristics are also important to consider in the context of neurocognitive functioning.MethodsSeven participants with CCHS ranging in age from 1 to 20 years underwent neuropsychological evaluations in a clinic setting.ResultsNeurocognitive testing indicated borderline impaired neurocognitive skills, on average, as well as relative weaknesses in working memory. Important strengths, including good coping skills and relatively strong social skills, may serve as protective factors in this population.ConclusionCCHS was associated with poor neurocognitive outcomes, especially with some polyalanine repeat expansion mutations (PARMS) genotype. These findings have important implications for individuals with CCHS as well as medical providers for this population.

Life-threatening cardiac arrhythmias in congenital central hypoventilation syndrome.

Congenital central hypoventilation syndrome (CCHS) patients are at risk for life-threatening cardiac arrhythmias, and presentation is dependent on their PHOX2B gene mutation. We describe the presentation of life-threatening arrhythmias in our cohort of CCHS patients. We reviewed the records of 72 CCHS patients seen at CHLA from 2004 to 2018. Data collected included demographics, PHOX2B genotype, ventilatory support, clinical symptoms, ambulatory cardiac monitoring results, and presence of cardiac pacemaker. Sixteen of 72 patients had evidence of potential life-threatening cardiac arrhythmias. PHOX2B genotypes were 20/25 polyalanine repeat expansion mutation (PARM), 20/26 PARM, 20/27 PARM, 20/32 PARM, and c.245C > T non-polyalanine repeat mutation. 11/16 patients were ventilated during sleep only. Symptoms included syncope, dizziness, chest pain, tingling in the left arm, and palpitations. 15/16 patients had recorded ambulatory cardiac monitoring. 5/16 patients were symptomatic without significant sinus pauses. 12/16 patients had implantation of cardiac pacemakers. 9/12 had significant sinus pauses on ambulatory monitoring, and 7/12 patients were symptomatic.Conclusion: CCHS patients have potential life-threatening arrhythmias requiring cardiac pacemaker implantation. Many of these patients are symptomatic with significant sinus pauses on ambulatory monitoring. However, some symptomatic patients with no significant pauses on ambulatory monitoring may still require cardiac pacemaker implantation.What is Known:• CCHS patients are at risk for life-threatening sinus pauses and require cardiac pacemaker implantation.What is New:• CCHS patients regardless of PHOX2B genotype are at risk for significant sinus pauses. Many CCHS patients with significant sinus pause on ambulatory cardiac monitoring are symptomatic and most present with syncope. Some symptomatic patients do not have significant sinus pauses but may still require cardiac pacemaker implantation.

Progress of diagnosis and treatment of congenital central hypoventilation syndrome

Congenital central hypoventilation syndrome (CCHS) is a rare genetic disorder characterized by ventilatory insensitivity to hypercapnia and hypoxemia during sleep and/or wakefulness presenting in infancy, childhood or adulthood.Paired-like homeobox 2B (PHOX2B) is the disease-defining gene for CCHS, which includes 2 mutation types, polyalanine repeat expansion mutation(PARM) and non- PARM.The CCHS-phenotype is associated with PHOX2B genotype.Children with CCHS typically have normal breathing when awake, while have progressive shallow breathing, cyanosis, hypercapnia and hypoxemia when asleep, but they don′t response with respiratory effort.CCHS patients mainly present at birth, some may present in adult years.CCHS is also associated with generalized dysfunction of the autonomic nervous system, and Hirschsprung disease and tumors of neural crest origin are associated with the disease.Non-invasive ventilation is an effective treatment and it can increase the quality of life for CCHS patient. Key words: Congenital central hypoventilation syndrome; Infant; Paired-like homeobox 2B

Carbamazepine Improves Apneic Episodes in Congenital Central Hypoventilation Syndrome (CCHS) With a Novel PHOX2B Exon 1 Missense Mutation

Pathogenic variants in Paired-Like Homeobox 2B (PHOX2B) gene cause congenital central hypoventilation syndrome (CCHS), a rare disorder of the nervous system characterized by absent or reduced ventilatory response to hypoxia and hypercapnia. The focus of management in CCHS is optimizing ventilation. Thus far, no medication has proved effective in improving ventilation. Most CCHS cases are caused by polyalanine repeat expansion mutations. Non-polyalanine repeat expansion mutations are the cause in 8% of cases and result in a more severe clinical presentation. PHOX2B has 3 exons. Exon 3 of PHOX2B is the most common location for CCHS-causing mutations. Thus far, only 9 CCHS-causing mutations have been reported in exon 1, 8 of which were nonsense mutations. We report a child with CCHS who was found to have a novel heterozygous missense variant in exon 1; c.95A > T. Improvement in his apneic episodes was observed following treatment with carbamazepine.

Congenital Central Hypoventilation Syndrome: Neurocognition Already Reduced in Preschool-Aged Children.

Congenital Central Hypoventilation Syndrome (CCHS) is a rare neurocristopathy characterized by severe hypoventilation and autonomic dysregulation, with typical presentation in the neonatal period, and deficient cognitive skills in school-aged patients. We hypothesized that younger (preschool) children with CCHS would also show neurocognitive delay and that CCHS-related physiologic factors would impact neurocognitive test results. We studied developmental (Bayley) test results collected during routine clinical care in 31 children (mean age 25.0 ± 8.5 months; range, 6-40 months) with PHOX2B mutation-confirmed CCHS by comparing them with the normative reference mean from the Bayley standardization sample; we also examined associations between Bayley scores and CCHS disease-related factors. Preschool patients with CCHS fell significantly below the normative mean of 100 on Bayley indices of mental (mean, 83.35 ± 24.75) and motor (mean, 73.33 ± 20.48) development (P < .001 for both). Significantly lower Bayley mental and motor scores were associated with severe breath-holding spells, prolonged sinus pauses, and need for 24 h/d artificial ventilation. Lower Bayley motor scores were also associated with seizures. Bayley scores differed among children with the three most common polyalanine repeat expansion mutation genotypes (mental, P = .001; motor, P = .006), being essentially normal in children with the 20/25 genotype but significantly lower in the other genotype groups (P < .05). These results confirm neurodevelopmental impairment of CCHS preschoolers, with severity related to physiologic compromise and PHOX2B genotype. These findings suggest that adverse effects begin early in the disease process, supporting the need for neurodevelopmental monitoring and intervention from early infancy.

Genotype-phenotype relationship in Japanese patients with congenital central hypoventilation syndrome.

Examine the genotype-phenotype relationship in Japanese congenital central hypoventilation syndrome (CCHS) patients and estimate the incidence of CCHS in Japan. Subjects were 92 Japanese patients with PHOX2B mutations; 19 cases carried 25 polyalanine repeat expansion mutations (PARMs); 67 cases carried 26 or more PARMs; and 6 had non-PARMs (NPARMs). We collected clinical data in all patients and estimated the development or intelligent quotients only in the patients carrying 25 PARM. The estimated incidence of CCHS was greater than one case per 148 000 births. Polyhydramnios was observed in three cases. Twelve infants exhibited depressed respiration at birth. In 19 cases carrying 25 PARM, the male-to-female ratio was ~3, no cases had Hirschsprung disease; 7 cases (37%) developed hypoventilation after the neonatal period, and 8 cases (42%) had mental retardation. In other 73 cases carrying 26 or more PARMs or NPARMs, male-to-female ratio was equal; patients frequently complicated with Hirschsprung disease and constipation, and all patients presented with hypoventilation in the neonatal period. Clinical symptoms were severe in most patients carrying long PARMs and NPARMs. In 25 PARM, additional genetic and/or epigenetic factors were required for CCHS development and male sex is likely a predisposing factor. The patients carrying 25 PARM frequently had mental retardation likely because they were not able to receive appropriate ventilation support following a definitive diagnosis owing to subtle and or irregular hypoventilation. Molecular diagnosis provides a definitive diagnosis and enables to receive appropriate ventilator support.

Dysregulation of locus coeruleus development in congenital central hypoventilation syndrome.

Human congenital central hypoventilation syndrome (CCHS), resulting from mutations in transcription factor PHOX2B, manifests with impaired responses to hypoxemia and hypercapnia especially during sleep. To identify brainstem structures developmentally affected in CCHS, we analyzed two postmortem neonatal-lethal cases with confirmed polyalanine repeat expansion (PARM) or Non-PARM (PHOX2B∆8) mutation of PHOX2B. Both human cases showed neuronal losses within the locus coeruleus (LC), which is important for central noradrenergic signaling. Using a conditionally active transgenic mouse model of the PHOX2B∆8 mutation, we found that early embryonic expression (<E10.5) caused failure of LC neuronal specification and perinatal respiratory lethality. In contrast, later onset (E11.5) of PHOX2B∆8 expression was not deleterious to LC development and perinatal respiratory lethality was rescued, despite failure of chemosensor retrotrapezoid nucleus formation. Our findings indicate that early-onset mutant PHOX2B expression inhibits LC neuronal development in CCHS. They further suggest that such mutations result in dysregulation of central noradrenergic signaling, and therefore, potential for early pharmacologic intervention in humans with CCHS.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-015-1441-0) contains supplementary material, which is available to authorized users.

Heterozygous 24-polyalanine repeats in the PHOX2B gene with different manifestations across three generations

Congenital central hypoventilation syndrome (CCHS) is an uncommon genetic disorder that is characterized by alveolar hypoventilation and autonomic dysregulation. More than 90% of the patients are heterozygous for polyalanine repeat expansion mutations in the paired-like homeobox 2b (PHOX2B) gene. The normal genotype has a 20-polyalanine sequence whereas expanded alleles are usually 25-33. Heterozygous 24-polyalanine repeat expansion mutation in the PHOX2B gene has rarely been reported. We report three consecutive generations harboring heterozygous 24-polyalanine repeats in the PHOX2B gene with manifestations ranging from apparently asymptomatic to alveolar hypoventilation and apnea requiring mechanical ventilation. The 3-year-old proband developed cor pulmonale and central hypoventilation following an upper respiratory tract infection. Our findings add to the accumulating evidence that the 24-polyalanine repeat in the PHOX2B is a disease-causing mutation. In addition, a high index of suspicion and careful monitoring after anesthesia, sedation, or respiratory illnesses should be exercised when evaluating asymptomatic family members with this genotype.

A Triple Threat: Down Syndrome, Congenital Central Hypoventilation Syndrome, and Hirschsprung Disease

Down syndrome (DS) is recognized by characteristic facial features, intellectual disability, and an increased risk for cardiac malformations and duodenal atresia. Recently, Hirschsprung disease (HSCR), or congenital aganglionic megacolon, has been seen more often among patients with DS. Given the systemic nature of DS-related features, it is natural to attribute neonatal complications to the chromosomal aberration. We describe a biracial male infant with DS who had significantly delayed defecation and required continuous ventilator support, but had no primary cardiac or lung disease. Subsequent evaluations confirmed total colonic aganglionosis. Because we were unable to safely extubate the infant, a diagnosis of congenital central hypoventilation syndrome (CCHS) was considered and confirmed by molecular analysis of the PHOX2B gene, revealing a heterozygous polyalanine repeat-expansion mutation containing 27 repeats (normal gene contains 20 repeats). HSCR coexisting with CCHS is known as Haddad syndrome. This is the first reported case with co-occurrence of DS, CCHS, and HSCR.

Transcriptional dysregulation and impairment of PHOX2B auto-regulatory mechanism induced by polyalanine expansion mutations associated with congenital central hypoventilation syndrome

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.

Congenital Central Hypoventilation Syndrome with PHOX2B Gene Mutation: Are We Missing the Diagnosis?

Congenital Central Hypoventilation Syndrome is a rare disorder of autonomic and central nervous system dysfunction with impaired control of breathing. The authors report a 37- d-old girl infant with recurrent apnea requiring repeated mechanical ventilation with no evidence of neuromuscular, cardiac or lung disease. A mutation analysis of PHOX2B gene revealed 25 polyalanine repeat expansion mutation on chromosome 4p12. This article aims at raising awareness among pediatricians about molecular basis and availability of confirmatory genetic testing for diagnosis and to help with prognosis in this disorder.

Germline mosaicism of PHOX2B mutation accounts for familial recurrence of congenital central hypoventilation syndrome (CCHS)

Congenital central hypoventilation syndrome (CCHS), a rare disorder characterized by alveolar hypoventilation and autonomic dysregulation, is caused by mutations in the PHOX2B gene. Most mutations occur de novo, but recent evidence suggests that up to 25% are inherited from asymptomatic parents with somatic mosaicism for these mutations. However, to date, germline mosaicism has not been reported. This report describes a family with recurrence of PHOX2B mutation-confirmed CCHS due to germline mosaicism. The first occurrence was a baby girl, noted on day 2 of life to have multiple episodes of apnea, bradycardia, and cyanosis while breathing room air. PHOX2B gene testing confirmed the diagnosis of CCHS with a heterozygous polyalanine repeat expansion mutation (PARM); genotype 20/27 (normal 20/20). Both parents tested negative for this mutation using fragment analysis (limit of detection<1%). Upon subsequent pregnancy [paternity confirmed using short tandem repeat (STR) analysis], amniocentesis testing identified the PHOX2B 20/27 genotype, confirmed with repeat testing. Elective abortion was performed at 21.5 weeks gestation. Testing of abortus tissue confirmed amniocentesis testing. The PHOX2B 20/27 expansion was not observed in a paternal sperm sample. This case represents the first reported family with recurrence of PHOX2B mutation-confirmed CCHS without detection of a parental carrier state or mosaicism, confirming the previously hypothesized possibility of germline mosaicism for PHOX2B mutations. This is an important finding for genetic counseling of CCHS families, suggesting that even if somatic mosaicism is not detected in parental samples, there is still reason for careful genetic counseling and consideration of prenatal testing during subsequent pregnancies.

Le syndrome d’hypoventilation alvéolaire central congénital (CCHS) : un cas de révélation tardive

The congenital central hypoventilation syndrome (CCHS) or Ondine's curse is a rare autosomal dominant disease, characterized by disorders of the autonomic nervous system, with abnormal ventilatory responses to hypercapnia and hypoxia. PHOX2B has been identified as the major gene causing CCHS. It results from polyalanine repeat expansion mutations. It typically presents in the newborn period but some cases have been described in adults (late onset CCHS) reflecting the variable penetrance of PHOX2B mutations. A 48 year-old woman presented, after ovarian cyst surgery, with severe hypoventilation requiring intubation. Arterial blood gases revealed a PaO2 of 6.6kPa (50mmHg), a PaCO2 of 10kPa (80mmHg) and a pH of 7.22. The past medical history revealed nocturnal symptoms for a few years. These included apnoeas, fitful sleep and awakening with headaches. Physical examination, pulmonary function tests, lung tomography and magnetic resonance imaging of the brainstem were all normal. Polysomnography revealed numerous central and obstructive apnoeas and hypopnoeas, with severe hypoxaemia and hypercapnia. Hypoxic and hypercapnic stimulation tests showed no adaptation of the ventilatory responses. Genetic analysis showed a heterozygous five alanine expansion mutation of the 20-residue polyalanine tract in exon 3 of the PHOX2B gene. The diagnosis of late onset CCHS should be considered in patients with unexplained hypoventilation, and physiological evaluation should be undertaken to document the abnormal ventilatory responses. The presence of a PHOX2B mutation confirms the diagnosis.