Pulmonary Surfactant Metabolism Dysfunction types 1, 2 and 3

Caroline ArcherNE Thames Regional Molecular Genetics

03-Apr-2008

• Clinical overview

• Physiological Role of the three genes

• Phenotype of each gene

• Service need

• Methodology

• Results

• Case studies

Outline

Pulmonary Surfactant Metabolism Dysfunction

No surfactant• Decreased pulmonary compliance• Increased tendency for alveoli to collapse

(SMDP)

1° Bronchus

Alveoli

Terminal bronchiole

Bronchiole

3° Bronchus

Alveoli

2° Bronchus

http://static.howstuffworks.com/gif/adam/images/en/lungs-picture.jpg

SMDP Types 1, 2 & 3Diagnosis defined

Pulmonary Surfactantprotein B (SFTPB)

Pulmonary Surfactantprotein C (SFTPC)

ABCA3

by genotype:

SMDP1

SMDP3

SMDP2

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

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 2

SMDP type 3Autosomal 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• Lung Biopsy

• Bronchoalveolar lavage

• Chest X-ray

• Chest CT

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%

• Turnaround Times • Low DNA requirement• Very low referral numbers expected• RNA not possible• Polymorphisms – known susceptibility to RDS• Mutations reported throughout coding region

Strategy Considerations

SFTPB - 10 exons SFTPC - 5 exonsABCA3 - 30 exons

• SFTPB linkage – six markers

• 2006 Full screen:

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

Case Study 1• Infant with severe respiratory distress • Dependency on ventilation• Consanguineous Asian• SFTPB c.484A>T homozygote (p.Lys162X)

Died4 weeks

Still born 38/40

Miscarriage 10/40

Alive & Well

Still born 40/40

Hearing loss & cataracts

died 4 weeks

Case Study 2Child 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 c. [127 C>T]+[4747 C>T]

(p.[Arg1583Trp]+[p.Arg43Cys])

Caucasianc.4747 C>T

(p.Arg1583Trp)Heterozygote

Asianc.127 C>T

(p.Arg43Cys)Heterozygote

Case Study 3 - Evidence of pathogenicity

p.Arg1583Trp near the C-terminus

ArginineCysteine

p.Arg43Cys

Tryptophan

p.Arg1583Trp

• In trans & different physiochemical properties• Missense change at p.Arg43 previously reported • Location: p.Arg43Cys at the first extracellular loop

• Highly conserved

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• 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.

AcknowledgementsQuen MokPaediatric Intensive Care Unit

Gail Norbury, Lucy Jenkins, Vicky Aldridge, & All Staff

Larry NogeeDepartment of PaediatricsJohns Hopkins University School of MedicineBaltimoreUSA

NE Thames Regional Molecular Genetics laboratory

Great Ormond Street Hospital for Children London WC1N 3JHhttp://www.ich.ucl.ac.uk/gosh/clinicalservices/Molecular_Genetics

Sian Jenkins Evelina Children's HospitalSt Thomas' HospitalLambeth Palace RoadLondon SE1 7EH