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DNA analysis Molecular genetic testing for cystic fibrosis

DNA analysis Molecular genetic testing for cystic fibrosis. Carolyn Tysoe Principal Clinical Scientist Royal Devon & Exeter NHS Foundation Trust. Outline. DNA basics – structure, function, types of mutation Mutation detection Introduction to cystic fibrosis and CFTR gene CFTR mutations

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DNA analysis Molecular genetic testing for cystic fibrosis

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  1. DNA analysisMolecular genetic testing for cystic fibrosis Carolyn TysoePrincipal Clinical ScientistRoyal Devon & Exeter NHS Foundation Trust

  2. Outline • DNA basics – structure, function, types of mutation • Mutation detection • Introduction to cystic fibrosis and CFTR gene • CFTR mutations • Testing strategy • Case study

  3. T T T T G G G G G G C C C A A A A C U C U U C A C G A A G A C G C U C AAA Glu Glu Lys Cys Phe Cys T T A G G T G A T A A G C C G G C G Glu Lys Cys Phe Cys Glu DNA transcription and translation 3’ 5’ DNA G 5’ 3’ mRNA nucleus cytosol

  4. Nonsense Glu Lys Lys Cys Cys Phe Phe Premature protein termination Met ATG AAG TGC TTC TGA Pathogenic? Glu Glu Lys Lys Arg Phe Phe Cys Cys Missense Met ATG AAG CGC TTC TGC GAG Effects of single base substitutions Wild type Glu Glu Lys Lys Cys Cys Phe Phe Cys Cys Met ATG AAG TGC TTC TGC GAG Stop

  5. Splice site mutations AG GT AG GT AG GT DNA DNA Normal spliced mRNA DNA Exon skipping DNA Intron inclusion DNA Use of a cryptic splice site

  6. Wild type Glu Glu Lys Lys Cys Cys Phe Phe Cys Cys Met 2 2 2 3 3 3 3 ATG AAG TGC TTC TGC GAG 1 1 1 1 …to one or a few exons Normal Single exon deletion Deletions and insertions of one or a few base pairs… Frameshift Arg Glu Lys Lys Cys Cys Phe Ser Cys Met Ala ATG AAG TGT TCT GCG AGG

  7. Search for unknown mutations eg sequencing Look for known mutations eg OLA Mutation detection methods Look for single or multi-exon deletions eg MLPA

  8. Polymerase Chain Reaction (PCR) • Primers can be fluorescently labelled – fragments separated by size and colour • PCR primers have a common tail – use one primer to sequence all fragments • Designed to work under the same conditions using MegaMix (mostly!) • PCR setup on 96-well plate by Biomek robot • Reagent lots recorded using 2D-barcoded tubes

  9. Method depends on mutation spectrum of gene CFTR gene and cystic fibrosis

  10. Cystic fibrosis • What is the mode of inheritance? • What is the incidence and carrier frequency? • Who does it affect? • What is the disorder characterised by?

  11. Cystic Fibrosis • Autosomal recessive • Incidence 1: 2500 • Affects children and young adults • Carrier frequency 1: 25 • Production of viscous mucus  obstructs ducts and glands  affects many organs  multisystem disease

  12. Cystic Fibrosis • What are the major clinical features? • Any additional features?

  13. Major Clinical features Lungs: Obstructive pulmonary disease Bacterial infection (Pseudomonas) Pancreas: Impaired exocrine pancreatic function • Insufficient secretion of lipolytic and proteolytic enzymes • Malabsorption, steatorrhoea, failure to thrive

  14. Other clinical features • Meconium ileus • Rectal prolapse • Obstructive jaudice • Nasal polyps • Sinusitis • Clubbing of fingers • Congenital bilateral absence of Vas Deferens (CBAVD) in males • Reduced fertility in females

  15. Identified in 1989 Long arm chromosome 7 (7q31.2) 230kb of DNA 27 exons 6.1kb mRNA 1480 amino acids Name: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) The CFTR gene

  16. mRNA CFTR gene to protein

  17. CFTR mutations • What is the mutation spectrum? • What is the most common mutation?

  18. CFTR mutations 42 10 16 2 13 0.5 15 1546 mutations listed on mutation database www.genet.sickkids.on.ca/cftr

  19. CFTR mutation spectrum

  20. Most common CFTR mutation p.Phe508del (F508) • 3bp deletion (CTT) • Deletes phenylalanine at codon 508 • 75% in UK population • 66% in world population

  21. Mutation classes Increasing severity in phenotype

  22. Variable splicing of exon 9 5T or 7T or 9T GTGTG(T)AACAG Exon 8 DNA Exon 9 Exon10 Intron 8 Intron 9

  23. Variable splicing of exon 9 Exon 8 Exon 9 Exon10 Exon 8 Exon10 FunctionalCFTR Non-functional CFTR 9T 100% 0% 7T 90% 10% 5T 40% 60%

  24. F508/R117H (5T) PS CF F508/R117H genotypes F508/R117H (7T) CBAVD or Pancreatic sufficient CF

  25. Estivill et al Nature Genetics 1996 Spectrum of CFTR disease

  26. CF testing at Exeter • What referral reasons do we see? • What molecular tests do we offer?

  27. Referral reasons • Establish or confirm the diagnosis of CF in symptomatic individuals • Failure to thrive • Chronic cough • Persistent chest infections • For carrier detection in at-risk relatives and their reproductive partners • In prenatal testing of at-risk pregnancies and in which foetal echogenic bowel has been identified • Infertility investigations (CBAVD) • Sperm and egg donor screening

  28. Molecular testing at Exeter CF1 – detection of p.Phe508del (F508) by sequencing exon 10 CF33 – detection of panel of 33 different mutations using the Oligonucleotide ligation assay (OLA) • CF33 OLA • Multiplex PCR • Ligation • Electrophoresis • Genemapper analysis OLA product has unique combination of electrophoretic mobility and fluorescence and permits identification of CFTR genotype

  29. G XXXX C Normal/Normal C G XXXX Normal G XXXX C Normal/Mutant T Normal A Mutant XXXXXX A XXXXXX T Mutant/Mutant T A XXXXXX Multiplex of 15 PCR reactions 1 9 10 20 2 3 11 12 13 14a 19 21 18 22 23 24 4 7 8 16 17a 17b 14b 15 5 6a 6b Mutant

  30. 01. Patient1 Blue Q493 1717-G 883 1398 01. Patient1 Green 1500 1000 500 1500 1500 3849+4 A 3905 W1282 R334 R347 3849+10kb C 1780 1100 1078 1628 1412 2195 1665 R1162 1226 2244 1000 1000 01. Patient1 Yellow 500 500 2789+5 G A455 R117 Y122 711+1 G 621+1 G 1067 1092 1339 1071 1558 1259 2183 AA 1898+1 G 700 1071 R553 I507 S549 G551 V520 F508 G542 G85 N1303 3659 G542 965 1535 1211 1024 820 1257 1257 1408 1303 1484 1348 Normal result

  31. Q493 1717-G F508 883 1398 820 1500 1500 3849+4 A 3905 W1282 R334 R347 3849+10kb C 1780 1100 1078 1628 1412 2195 1665 R1162 1226 2244 1000 1000 500 500 2789+5 G A455 R117 Y122 711+1 G 621+1 G 1067 1092 1339 1071 1558 1259 2183 AA 1898+1 G 700 1071 R553 S549 G551 3659 G542 G542 N1303 G85 965 1211 1257 1257 1303 1484 1408 1348 Heterozygous p.Phe508del 01. Patient1 Blue I507 V520 1535 1024 F508 01. Patient1 Green 803 1500 1000 500 01. Patient1 Yellow

  32. Q493 1717-G 883 1398 1500 1500 3849+4 A 3905 W1282 R334 R347 3849+10kb C 1780 1100 1078 1628 1412 2195 1665 R1162 1226 2244 1000 1000 500 500 2789+5 G A455 R117 Y122 711+1 G 621+1 G 1067 1092 1339 1071 1558 1259 2183 AA 1898+1 G 700 1071 R553 S549 G551 G542 G542 3659 N1303 G85 965 1211 1303 1484 1257 1408 1257 1348 Homozygous p.Phe508del 01. Patient1 Blue I507 V520 1535 1024 F508 01. Patient1 Green 1623 1500 1000 500 01. Patient1 Yellow

  33. Genetics White Paper 2003 “Our inheritance, our future”Realising the potential of genetics in the NHS “The NHS should lead the world in taking maximum advantage of the application of the new genetic knowledge for the benefit of all patients”

  34. Investment in genetics2003 • £50 million funding including: • £5.5M for gene therapy (including £2.5M for CF) • £3.5M to train up to 90 scientists • £18M capital to upgrade NHS genetics laboratories

  35. As a result of this investment • By 2006, genetic test results should be available: • Within 3 days for urgent samples (eg. Prenatal) • Within 2 weeks where the potential mutation is known • Within 8 weeks for unknown mutations in a large gene • All laboratories to secure accreditation with CPA or equivalent within 18 months

  36. Increased efficiency • rationalisation of tests • introduction of robotics • new IT system • Increased capacity • White Paper reporting times • CPA accreditation • Integration of genetics in pathology Testing strategy – extended CFTR analysis for SCOBEC network £6 million to achieve: Salisbury Cambridge Oxford Cardiff Bristol Exeter

  37. Modernisation of Exeter Lab DNA extraction PCR Sequencing Sequence analysis streamlined

  38. Salisbury C S O Cambridge C B E Oxford Cardiff Bristol Exeter Reporting time data • 3 days for urgent samples • 10 days for known mutation • 40 days for unknown mutations

  39. Testing strategy – extended CFTR analysis for SCOBEC network

  40. Extended CFTR testing • Sequencing of entire gene (27 exons) 2. Dosage analysis

  41. CTTCAAG CTTCAAG CTTCAAG CTTCAAG CTTAAG Partial or whole gene deletions are not detected by sequencing • When you sequence an exon – how do you know how many copies there are? • Need a quantitative (dosage) test

  42. 1 9 10 20 2 3 11 12 13 14a 19 21 18 22 23 24 4 7 8 16 17a 17b 14b 15 5 6a 6b CFTR deletions and duplications 44 reported out of 1546 CFTR mutations (2.9%) (CF mutation database) Deletion Duplication

  43. PCR primer sequence X FAM P Stuffer sequence MLPA probes PCR primer sequence Y 24 bp sequence specific probes

  44. PCR primer sequence Y PCR primer sequence X Stuffer sequence Annealing of probes

  45. PCR primer sequence X Stuffer sequence Ligation of probes PCR primer sequence Y A ligase enzyme ligates the 2 probes together – Only annealed probes will be ligated

  46. Samples are heated to denature the probe from the DNA

  47. Probe amplification The probe is amplified using the common primer pair

  48. All the probes can be amplified using the same primer pair and PCR conditions

  49. MLPA Results - Electrophoresis Normal Control CFTR Duplication Exons 6b-10

  50. MLPA Results – Spreadsheet analysis Normal Control Duplication Exons 6b-10 (Red) Deletion (Blue)

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