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

Bristol Genetics Laboratory 2010

Congenital Central Hypoventilation Syndrome;

a polyalanine repeat disorder- the UK cohort

Sarah Burton-JonesBristol Genetics Laboratory

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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

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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’


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• 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

The PHOX2B Gene

5’ 3’1 2 3

NH2 COOH

PHOX2B structure

PHOX2B transcript

Adapted : Amiel et al. (2003) Nature Genetics 33(4), 459-461

Ala repeats

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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.

Pathogenic PHOX2B Variants

FeaturePolyalanine expansion

(+4 to +13 Ala)Frameshift / missense

mutation (NPARM)

Location in PHOX2B 20 alanine tract in exon 3Exon 3 or

end of exon 2 (most)

Proportion of all pathogenic variants

~90% ~10%

Present with Hirschprung’s Disease

<20% >87%

Develop neural crest tumour

~1% (all ≥+11 Ala) ~50% over 1 year old

Parent carries mutation Up to 14% Almost all de novo

Predicted effect on proteinMisfolding, cytoplasmic

aggregate formation, nuclear exclusion

Nuclear sequestration


Figures from published data


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.

PHOX2B Testing Strategy; BGL

• All diagnostic requests

• Familial expansion testing

• Exclusion testing

• Strong clinical suspicion of CCHS or LO-CHS

Polyalanine expansion analysis (GC rich PCR)

PHOX2B sequence analysis

At request of referring clinician

Report, request parental samples

Report

+ve -ve



Polyalanine Expansion Analysis

• All expansions are confirmed by sequencing of PHOX2B exon 3 (fragment B)

Normal control +6 Ala expansion Parent mosaic for +6 Ala


CCHS UK Diagnostic Results

PolyAla and sequence analysis, no mutation detected

PolyAla analysis only, no mutation detected

Polyalanine expansion mutation detected

Non-expansion mutation (NPARM) detected

46 (20%)

5 (2%)

159 (69%)

20 (9%)


Collective Data; PHOX2B Mutations

Weese-Mayer et al

Sasaki et al

Trochet et al

Parodi et al

Weese-Mayer et al

BGL

Source USA Japan US, UK, France,

Germany, Sweden

Italy, Spain, Germany,

Netherlands, Slovenia

USA UK and Ireland

Date published 2003 2003 2005 2007 2008 2005-2010

Cohort size67 10 188* 63† 350 71

Polyalanine expansions 98.5% 40% 85.6% 81% 88.3% 64.8%

NPARM 1.5% 10% 7% 11% 11.7% 7%

Detection 100% 50% 92.6% 92% 100% 71.8%

* Included 29 patients from earlier Amiel study, France, 2003† Includes patients previously reported by Matera et al 2004


BGL CCHS Data in Context

Comparison of PHOX2B global data with UK and Ireland data (BGL)

0%

5%

10%

15%

20%

25%

30%

35%

Cont -

7/-7 +4

+4/+

4 +5 +6 +7 +8 +9 +10

+11

+12

+13

NPARM

PHOX2B mutant allele

% all mutations detected

Weese-Mayerreview2009(n=640)

UK &Ireland(n=51)


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

Publication Parental carrier rate

Weese-Mayer et al (2003) Am J Med Genet 123A:267-268 7.4% (4/54)

Parodi et al (2008) Hum Mutat 29:206 13.9% (6/43)

Trochet et al (2008) Am J Respir Crit Care Med 177:906-911 8.1% (10/124)


Polyalanine Expansion Cases; BGL

Expansion size

Number of probands

Neither parent tested or

one tested and normal

Both parents tested or

one tested and positive

Parents positive for expansion

+5 10 6 4 1 Father carrier, affected

+6 15 4 11 2 Fathers mosaic, unaffected

+7 17 5 12

1 Father mosaic, unaffected1 Mother mosaic, unaffected1 Mother carrier, unaffected

(non-penetrant)

+10 1 0 1 -

+11 1 0 1 -

+12 1 0 1 -

+13 1 1 0 -

Total 46 16 30 6 (13% of 46, 20% of 30)


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


UK Germinal Mosaic Case?

• 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%

N/N N/N

CCHS,N/+5 Ala

CCHS, N/+5 Ala


PHOX2B Mutations (NPARM); BGL

* from Weese-Mayer DE et al (2009) Pediatric Pulmonology 44:521-535

Mutation type

BGL examples

Phenotype Published data*

Frameshift

c.722_759delc.722_759delc.721_739del

c.861dupTc.866dupG

Died 27 days. Ventilated from birth, Hirschprung’s. (OX)

Died 4 weeks. Colon aganglionosis. (NOTTS)

Apnoea. Colon aganglionosis. Treatment withdrawn. (OX)

Died 40 days. Ventilated from birth. Aganglionosis. (BRIS)

Hypotonia, apnoea, Hirschprung’s. Died (cardiac). (BRIS)

78%

Missensep.Ala140Glu

(NB. Non-UK)

Variable; late onset in father, congenital with Hirschprung’s in son (LEIDEN)

19%

Nonsense None to date - 3%


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


Clinical Data Questionnaire

Target groups:

1. Expansion mutation detected– Both parents tested (for

clinical info)– One or neither parent

tested (request samples)

2. Non-expansion PHOX2B mutation detected

3. No mutation detected by polyalanine tract or sequence analysis


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


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

Documents

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