Transcript Developing a diagnostic service for screening ABCA3, SFTPB

Pulmonary Surfactant Metabolism
Dysfunction types 1, 2 and 3
Caroline Archer
NE Thames Regional Molecular Genetics
03-Apr-2008
Outline
• Clinical overview
• Physiological Role of the three genes
• Phenotype of each gene
• Service need
• Methodology
• Results
• Case studies
Pulmonary
Surfactant
Metabolism
Dysfunction
1° Bronchus
2° Bronchus
3° Bronchus
(SMDP)
Alveoli
Bronchiole
Terminal bronchiole
Alveoli
http://static.howstuffworks.com/gif/adam/images/en/lungs-picture.jpg
No surfactant
• Decreased pulmonary compliance
• Increased tendency for alveoli to collapse
SMDP Types 1, 2 & 3
Diagnosis defined by genotype:
SMDP1 Pulmonary Surfactant
protein B (SFTPB)
SMDP2 Pulmonary Surfactant
protein C (SFTPC)
SMDP3 ABCA3
Alveola
Alveolar space
SMDP type 1
Autosomal Recessive mutations SFTPB
• 1 per million live births
• Term neonates with severe respiratory distress
presenting within hours of birth
• Refractory to ventilation & synthetic surfactant
replacement therapy
• Fatal in first three months of life - lung transplant
is the only successful intervention
• Partial deficiencies may be less severe
SMDP type 2
Autosomal Dominant mutations SFTPC
• Incidence unknown
• Familial and sporadic (55%)
• Highly variable clinical course & severity
• Later onset tachypnoea & cyanosis with
interstitial lung disease
• Rarely acute neonatal lung disease
SMDP type 3
Autosomal Recessive mutations in ABCA3
• Incidence unknown
• Phenotypic overlap with SMPD1 and SMDP2
• Severe hypoxic respiratory disease with death
in the first three months
• Milder paediatric lung disease with survival
past infancy
• Mutations in ABCA3 modifies phenotype of
SMDP2 (SFTPC)
Other investigations
• Chest X-ray
• Lung Biopsy
• Chest CT
• Bronchoalveolar lavage
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Clinical Utility
Supportive care
Consider lung transplantation
No rapid diagnostic test
Genotype critical for parental counselling
Clinical Overlap
• Transient symptoms in premature infants respiratory
distress syndrome (RDS)
• Short term treatment with synthetic surfactant
Proforma
• Clinical information required
• Clinical queries to an in-house Consultant Intensivist
Diagnostic service
• 2002 SFTPB ‘common’ mutation service: pick up 0%
• 2006 Full screen: SFTPB - 10 exons
SFTPC - 5 exons
ABCA3 - 30 exons
• SFTPB linkage – six markers
Strategy Considerations
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Turnaround Times
Low DNA requirement
Very low referral numbers expected
RNA not possible
Polymorphisms – known susceptibility to RDS
Mutations reported throughout coding region
Direct Sequencing
• 45 exons (all 3 genes)
• Robotic PCR set up using 8 span robot
• Each gene separate
• Touch-down PCR program for all three
genes
• Direct sequencing using 8 & 96 span robots
• Robotic Exosap-IT or Ampure beads
• Robotic sequencing set-up
• Tailed primers
• Robotic clean-up using clean seq beads
Results for Index cases to end 2007
• Overall mutation detection rate 15/33 patients
(3/14 patients referred for all 3 genes)
• SFTPB: 7/25
• SFTPC: 5/20
• ABCA3: 3/20
• 29 Carrier tests for these index cases
• 1 Prenatal diagnosis
• 1 Perinatal test
• 1 SFTPB Linkage
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Case Study 1
Infant with severe respiratory distress
Dependency on ventilation
Consanguineous Asian
SFTPB c.484A>T homozygote (p.Lys162X)
Alive &
Well
Died
4 weeks
Miscarriage
10/40
Still born Hearing loss & died
40/40
4 weeks
cataracts
Still born
38/40
Case Study 2
Child post lung transplant referred for SFTPC
• c.215G>A (p.Ser72Asn) heterozygote
• No mutation in ABCA3
• Highly conserved cross-species
• p.Ile73Thr common mutation
Case Study 3
• Infant with severe respiratory distress and evidence
of desquammating interstitial pneumonitis
• No mutation detected in
SFTPB and SFTPC
• ABCA3: Two unclassified
variants in trans
• Possible origin
deamination of C to U
• Collaborated on case
report for publication
Caucasian
c.4747 C>T
(p.Arg1583Trp)
Heterozygote
Asian
c.127 C>T
(p.Arg43Cys)
Heterozygote
c. [127 C>T]+[4747 C>T]
(p.[Arg1583Trp]+[p.Arg43Cys])
Case Study 3 - Evidence of pathogenicity
p.Arg43Cys
Cysteine
p.Arg1583Trp
Arginine
Tryptophan
• In trans & different physiochemical properties
• Missense change at p.Arg43 previously reported
• Location: p.Arg43Cys at the first extracellular loop
p.Arg1583Trp near the C-terminus
• Highly conserved
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Further Work
SFTPC linkage for de novo cases
Use proforma to develop a tiered strategy based
on genetic & clinical data.
Now charging for testing - Gene Dossier May 2008
Summary
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NE Thames RMG offers screening of SFTPB,
SFTPC, ABCA3 for SMDP1, 2 and 3.
This is a group of rare chronic respiratory
disorders typically seen in full term neonates and
younger children.
Acknowledgements
Gail Norbury, Lucy Jenkins,
Vicky Aldridge, & All Staff
Quen Mok
Paediatric Intensive Care Unit
NE Thames Regional Molecular Genetics laboratory
Great Ormond Street Hospital for Children
London WC1N 3JH
http://www.ich.ucl.ac.uk/gosh/clinicalservices/Molecular_Genetics
Sian Jenkins
Evelina Children's Hospital
St Thomas' Hospital
Lambeth Palace Road
London
SE1 7EH
Larry Nogee
Department of Paediatrics
Johns Hopkins University
School of Medicine
Baltimore
USA