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Pulmonary Surfactant Metabolism Dysfunction types 1, 2 and 3

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. 1° Bronchus.

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Pulmonary Surfactant Metabolism Dysfunction types 1, 2 and 3

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  1. Pulmonary Surfactant Metabolism Dysfunction types 1, 2 and 3 Caroline Archer NE Thames Regional Molecular Genetics 03-Apr-2008

  2. Outline • Clinical overview • Physiological Role of the three genes • Phenotype of each gene • Service need • Methodology • Results • Case studies

  3. 1° Bronchus 2° Bronchus 3° Bronchus Alveoli Bronchiole Alveoli Terminal bronchiole Pulmonary Surfactant Metabolism Dysfunction (SMDP) http://static.howstuffworks.com/gif/adam/images/en/lungs-picture.jpg No surfactant • Decreased pulmonary compliance • Increased tendency for alveoli to collapse

  4. Alveola Alveolar space SMDP Types 1, 2 & 3 by genotype: Diagnosis defined SMDP1 Pulmonary Surfactant protein B (SFTPB) Pulmonary Surfactant protein C (SFTPC) ABCA3 SMDP2 SMDP3

  5. 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

  6. 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

  7. 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)

  8. Lung Biopsy • Chest CT • Bronchoalveolar lavage Other investigations • Chest X-ray

  9. 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

  10. 2006 Full screen: SFTPB - 10 exons SFTPC - 5 exons ABCA3 - 30 exons • SFTPB linkage – six markers Strategy Considerations • Turnaround Times • Low DNA requirement • Very low referral numbers expected • RNA not possible • Polymorphisms – known susceptibility to RDS • Mutations reported throughout coding region Diagnostic service • 2002 SFTPB ‘common’ mutation service: pick up 0%

  11. 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

  12. 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

  13. Still born 38/40 Still born 40/40 Hearing loss & cataracts died 4 weeks Died 4 weeks Alive & Well Miscarriage 10/40 Case Study 1 • Infant with severe respiratory distress • Dependency on ventilation • Consanguineous Asian • SFTPB c.484A>T homozygote (p.Lys162X)

  14. 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

  15. Asian c.127 C>T (p.Arg43Cys) Heterozygote Caucasian c.4747 C>T (p.Arg1583Trp) Heterozygote c. [127 C>T]+[4747 C>T] (p.[Arg1583Trp]+[p.Arg43Cys]) 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

  16. p.Arg1583Trp p.Arg43Cys Tryptophan Cysteine Arginine • Intrans & different physiochemical properties Case Study 3 - Evidence of pathogenicity • Missense change at p.Arg43 previously reported • Location: p.Arg43Cys at the first extracellular loop p.Arg1583Trp near the C-terminus • Highly conserved

  17. 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.

  18. Acknowledgements Quen Mok Paediatric Intensive Care Unit Gail Norbury, Lucy Jenkins, Vicky Aldridge, & All Staff 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 Larry Nogee Department of Paediatrics Johns Hopkins University School of Medicine Baltimore USA Sian Jenkins Evelina Children's Hospital St Thomas' Hospital Lambeth Palace Road London SE1 7EH

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