Congenital Central Hypoventilation Syndrome; a polyalanine repeat disorder- the UK cohort

1. Congenital Central Hypoventilation Syndrome; a polyalanine repeat disorder- the UK cohort Sarah Burton-Jones Bristol Genetics Laboratory Bristol Genetics Laboratory 2010

2. Congenital Central Hypoventilation Syndrome(Formerly called ‘Ondine’s Curse’) • Autonomic nervous system disorder • Incidence ? 1 in 20000-50000 live births • Diagnosis in infancy / early childhood (NB. can be later onset) • Characteristic respiratory phenotype Failure of autonomic control of ventilation • Same respiratory rate awake and asleep  hypoventilation • No automatic response to hypoxia/hypercarbia • ‘Asphyxia’ when awake, without exertion Bristol Genetics Laboratory 2010

3. Congenital Central Hypoventilation Syndrome More severe phenotype: • Hirschprung’s Disease • colon aganglionosis • Neural crest tumours • e.g. neuroblastoma, ganglioneuroma • Can also present with: cardiac irregularities, dysphagia, eye abnormalities, temperature regulation problems, altered perception of anxiety and pain, and other symptoms PHOX2B now known as ‘the disease-defining gene for CCHS’ Bristol Genetics Laboratory 2010

4. 5’ 3’ 1 2 3 NH2 COOH The PHOX2B Gene Ala repeats PHOX2B structure • Paired-Like Homeobox • 4p12, 3 exons, 314 amino acids • Highly conserved homeodomain transcription factor • 2 polyalanine repeat tracts (9 and 20 Ala) • Imperfect repeats; can expand by unequal allelic recombination PHOX2B transcript Adapted : Amiel et al. (2003) Nature Genetics 33(4), 459-461 Bristol Genetics Laboratory 2010

5. Distribution of PHOX2B Mutations Figure from Weese-Mayer DE et al (2009) Pediatric Pulmonology 44:521-535 A de novo interstitial 4p12 deletion encompassing the PHOX2B gene has also been reported Benailly HK et al Clin Genet 2003; 64: 204-209. Bristol Genetics Laboratory 2010

6. Pathogenic PHOX2B Variants Figures from published data Bristol Genetics Laboratory 2010

7. Bristol PHOX2B Service • Developed as a trainee project at Bristol • Gene dossier submitted to the UKGTN Steering Group • Approved as a NHS diagnostic service test Feb 2005 Benefits of molecular genetic analysis: • Fast confirmation of diagnosis, aids clinical decision making • Parents can be tested for carrier status • Prenatal diagnosis offered Diagnostic referrals from: Neonatology, Respiratory Medicine, General Paediatrics, Neurology, Clinical Genetics. Bristol Genetics Laboratory 2010

8. PHOX2B Testing Strategy; BGL Polyalanine expansion analysis (GC rich PCR) • All diagnostic requests • Familial expansion testing • Exclusion testing • Strong clinical suspicion of CCHS or LO-CHS +ve -ve Report Report, request parental samples At request of referring clinician PHOX2B sequence analysis Bristol Genetics Laboratory 2010

9. Polyalanine Expansion Analysis • All expansions are confirmed by sequencing of PHOX2B exon 3 (fragment B) Bristol Genetics Laboratory 2010

10. 20 (9%) 46 (20%) 5 (2%) 159 (69%) CCHS UK Diagnostic Results Polyalanine expansion mutation detected Non-expansion mutation (NPARM) detected PolyAla and sequence analysis, no mutation detected PolyAla analysis only, no mutation detected Bristol Genetics Laboratory 2010

11. Collective Data; PHOX2B Mutations * Included 29 patients from earlier Amiel study, France, 2003 † Includes patients previously reported by Matera et al 2004 Bristol Genetics Laboratory 2010

12. BGL CCHS Data in Context Bristol Genetics Laboratory 2010

13. Parental Studies; Expansion Mutations • Autosomal dominant, incomplete penetrance • However, majority de novo occurrence • Possible paternal origin bias Arai et al J Hum Genet (2007) 52:921-925 and (2010) 55:4-7 • Studies indicate 7-14% cases are inherited: • Carriers who report no symptoms may show respiratory anomalies in sleep studies Parodi et al (2010) Clin Genet (epub ahead of print) • Somatic mosaicism common • No reports of germinal mosaicism in the literature to date Bristol Genetics Laboratory 2010

14. Polyalanine Expansion Cases; BGL Bristol Genetics Laboratory 2010

15. Parents of Expansion Cases; BGL • In 13% all probands, or 20% where we have complete data, the expansion was also detected in a parent. (Published figures up to 14% with complete data) • No somatic mosaicism seen in +5 Ala expansion cases, consistent with published data* • Expansions of ≥ 6 alanines postulated to be fully penetrant*, but note unaffected +7 carrier mother • New evidence for germinal mosaicism from a UK case... * Parodi S et al (2008) Hum Mut 29(1) 206 Bristol Genetics Laboratory 2010

16. UK Germinal Mosaic Case? N/N N/N • Urgent referral aged 9 days, ventilator dependent • 5-alanine PHOX2B expansion in proband and twin brother • Dizygous twins; fraternity and paternity confirmed by QF-PCR zygosity analysis (AWMGLS Cardiff) • Both parents N/N using DNA from peripheral blood • Saliva samples requested • Expansion test sensitivity determined to be 2% CCHS, N/+5 Ala CCHS, N/+5 Ala Bristol Genetics Laboratory 2010

17. PHOX2B Mutations (NPARM); BGL * from Weese-Mayer DE et al (2009) Pediatric Pulmonology 44:521-535 Bristol Genetics Laboratory 2010

18. Parental Studies; NPARMs • Rarely reported • Variable penetrance • Single base deletions in exon 3 upstream of poly Ala tract • c.618delC Matera et al (2004) J Med Genet 41:373-380 • c.577delG Berry-Kravis et al (2006) Am J Respir Crit Care Med 174:1139-1144 • Exon 2 missense substitution • c.422G>A (p.Arg141Gln), 2 families Berry-Kravis et al (2006) as above • Recent Bristol case referred from Netherlands: • Father presented with late-onset Central Hypoventilation Syndrome • Newborn son affected from birth, also has Hirschprung’s • Exon 2 missense mutation p.Ala140Glu detected in both • Previously reported only in isolated LO-CHS cases Bristol Genetics Laboratory 2010

19. Clinical Data Questionnaire Target groups: • Expansion mutation detected • Both parents tested (for clinical info) • One or neither parent tested (request samples) • Non-expansion PHOX2B mutation detected • No mutation detected by polyalanine tract or sequence analysis Bristol Genetics Laboratory 2010

20. Conclusions • UK PHOX2B mutation data closely matches distribution reported elsewhere • Sequencing of PHOX2B relevant even if late onset CHS • Majority of PHOX2B referrals (up to 69%) now seemingly for exclusion of CCHS • Clinical detail often lacking; questionnaire to refine genotype-phenotype data • Higher than expected parental carrier rate (20% vs 7-14%) • Carrier parents may be unaware of symptoms • Evidence for germinal mosaicism in a UK family • Prenatal diagnosis available Bristol Genetics Laboratory 2010

21. Acknowledgements Bristol Genetics Laboratory Maggie Williams Claire Faulkner Thais Simmonds Teresa Lamb (Patrick) Julie Evans Bristol Clinical Team Peter Fleming Peter Lunt All Wales Genetics Service Rachel Butler Julian Sampson Rhianedd Ellwood-Thompson Leiden Dietje Fransen van de Putte Bristol Genetics Laboratory 2010