Transcript a polyalanine repeat disorder

Congenital Central
Hypoventilation Syndrome;
a polyalanine repeat disorderthe UK cohort
Sarah Burton-Jones
Bristol Genetics Laboratory
Bristol Genetics Laboratory 2010
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
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
The PHOX2B Gene
Ala repeats
PHOX2B structure
5’
1
PHOX2B transcript
2
NH2
3
3’
COOH
Adapted : Amiel et al. (2003) Nature Genetics 33(4), 459-461
•
•
•
•
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
Bristol Genetics Laboratory 2010
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
Pathogenic PHOX2B Variants
Polyalanine expansion
(+4 to +13 Ala)
Frameshift / missense
mutation (NPARM)
20 alanine tract in exon 3
Exon 3 or
end of exon 2 (most)
Proportion of all pathogenic
variants
~90%
~10%
Present with Hirschprung’s
Disease
<20%
>87%
~1% (all ≥+11 Ala)
~50% over 1 year old
Up to 14%
Almost all de novo
Misfolding, cytoplasmic
aggregate formation, nuclear
exclusion
Nuclear sequestration
Feature
Location in PHOX2B
Develop neural crest
tumour
Parent carries mutation
Predicted effect on protein
Figures from published data
Bristol Genetics Laboratory 2010
Bristol PHOX2B Service
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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
PHOX2B Testing Strategy; BGL
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•
•
All diagnostic requests
Familial expansion testing
Exclusion testing
Polyalanine expansion
analysis (GC rich PCR)
+ve
Report, request
parental samples
-ve
Report
At request
of referring
clinician
•
Strong clinical suspicion of CCHS or LO-CHS
Bristol Genetics Laboratory 2010
PHOX2B sequence
analysis
Polyalanine Expansion Analysis
Normal control
+6 Ala expansion
Parent mosaic for +6 Ala
• All expansions are confirmed by sequencing of PHOX2B
exon 3 (fragment B)
Bristol Genetics Laboratory 2010
CCHS UK Diagnostic Results
CCHS diagnostic referrals to BGL 2005-2010
(UK and Ireland, n=230)
PolyAla and
sequence
analysis, no
mutation detected
PolyAla analysis
only, no mutation
detected
Polyalanine
expansion
mutation
detected
20
(9%)
46
(20%)
5 (2%)
159
(69%)
Bristol Genetics Laboratory 2010
Non-expansion
mutation
(NPARM)
detected
Collective Data; PHOX2B Mutations
Weese-Mayer
et al
Sasaki
et al
Trochet
et al
Parodi
et al
Weese-Mayer
et al
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
67
10
188*
63†
350
71
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%
Cohort size
Polyalanine
expansions
BGL
* Included 29 patients from earlier Amiel study, France, 2003
† Includes patients previously reported by Matera et al 2004
Bristol Genetics Laboratory 2010
BGL CCHS Data in Context
Comparison of PHOX2B global data with UK and Ireland data (BGL)
35%
WeeseMayer
review
2009
(n=640)
30%
25%
% all mutations
20%
detected
UK &
Ireland
(n=51)
15%
10%
5%
PHOX2B mutant allele
Bristol Genetics Laboratory 2010
RM
NP
A
+1
3
+1
2
+1
1
+1
0
+9
+8
+7
+6
+5
+4
/+
4
+4
Co
nt
-7
/- 7
0%
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:
Publication
Weese-Mayer et al (2003) Am J Med Genet 123A:267-268
Parodi et al (2008) Hum Mutat 29:206
Trochet et al (2008) Am J Respir Crit Care Med 177:906-911
Parental carrier rate
7.4% (4/54)
13.9% (6/43)
8.1% (10/124)
• 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
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, unaffected
1 Mother mosaic, unaffected
1 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)
Bristol Genetics Laboratory 2010
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
UK Germinal Mosaic Case?
N/N
CCHS,
N/+5 Ala
N/N
CCHS,
N/+5 Ala
• Urgent referral aged 9 days, ventilator dependent
• 5-alanine PHOX2B expansion in proband and twin brother
• Dizygous twins; fraternity and paternity confirmed by QFPCR zygosity analysis (AWMGLS Cardiff)
• Both parents N/N using DNA from peripheral blood
• Saliva samples requested
• Expansion test sensitivity determined to be 2%
Bristol Genetics Laboratory 2010
PHOX2B Mutations (NPARM); BGL
Mutation
type
BGL
examples
Phenotype
Published
data*
Frameshift
c.722_759del
c.722_759del
c.721_739del
c.861dupT
c.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%
p.Ala140Glu
Variable; late onset in father,
congenital with Hirschprung’s in son (LEIDEN)
19%
-
3%
Missense
(NB. Non-UK)
Nonsense
None to date
* from Weese-Mayer DE et al (2009) Pediatric Pulmonology 44:521-535
Bristol Genetics Laboratory 2010
Parental Studies; NPARMs
• Rarely reported
• Variable penetrance
• Single base deletions in exon 3 upstream of poly Ala tract
– c.618delC
– c.577delG
Matera et al (2004) J Med Genet 41:373-380
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:
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–
–
–
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
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
Bristol Genetics Laboratory 2010
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
Acknowledgements
Bristol Genetics Laboratory
All Wales Genetics Service
Maggie Williams
Claire Faulkner
Thais Simmonds
Teresa Lamb (Patrick)
Julie Evans
Rachel Butler
Julian Sampson
Rhianedd Ellwood-Thompson
Bristol Clinical Team
Leiden
Peter Fleming
Peter Lunt
Dietje Fransen van de Putte
Bristol Genetics Laboratory 2010