PRENATAL DIAGNOSIS OF HAEMOPHILA

1. PRENATAL DIAGNOSIS OF HAEMOPHILA Huong Le Senior Hospital Scientist Department of Molecular & Clinical Genetics Royal Prince Alfred Hospital Sydney , Australia

2. OUTLINE • Introduction • Haemophilia A (Factor VIII) • Haemophilia B (Factor IX) • The related genes • Genetic testing

3. INTRODUCTION • Haemophilia A (Factor VIII) • Recessive X linked hereditary bleeding disorders • Severe form (Factor VIII level <1%) • Moderate form (Factor VIII level 1-5%) • Mild form (Factor VIII level >5%) • Caused by deficiency or dysfunction of factor VIII, affect 1 in 5000 males and rare in females

4. INTRODUCTION • Haemophilia B - Factor IX (Christmas disease) • Recessive X linked hereditary bleeding disorders results from deficiency of pro-coagulant factor IX • Characterised by prolong oozing after injuries or surgery, delay bleeding • Less common than in haemophilia A with a frequency of 1 in 25000 males world wide

5. INTRODUCTION • Biochemistry

6. INTRODUCTION • Inheritance mode

7. FACTOR VIII GENE • Length: 186KB, 26 exons, 2332 amino acid residues • Intron 22 inversion account for 45-50% of severe haemophilia A • Intron 1 inversion account for 3-5% of severe haemophilia A • 943 point mutations

8. FACTOR VIII GENE

9. Factor VIII gene: inversion 22

10. Factor VIII gene: mutations

11. Factor VIII gene: mutations • Mutation frequency

12. FACTOR IX GENE • Length: 34KB, 8 exons, 415 amino acid residues • Exon 8 is the largest and half of mutations are found in this exon • Small change and short insertion or deletion account for most mutations • 962 point mutations

13. Factor IX gene: mutations • Mutation frequency

14. GENETIC TESTING- Indication • Identify mutation in known patients • To establish carrier status in women of reproductive age (family history) • Prenatal diagnosis

15. Direct testing Screening dHPLC SSCP Sequencing Indirect testing Linkage RFLP Can (intron13&intron22) GENETIC TESTING

16. GENETIC TESTING:Haemophilia A

17. GENETIC TESTING:Haemophilia A Steve Keeney's Primers for PCR amplification http://europium.csc.mrc.ac.uk

18. GENETIC TESTING:Factor VIII - intron 22 inversion

19. GENETIC TESTING:Haemophilia B

20. SEQUENCINGANALYSIS • Provide complete information about the location and nature of all DNA variants • Best approach for mutation analysis including coding regions, splice junctions, the 5’ and 3’ UTR regions. • Sequencing should be performed in both directions • Accompany with sequencing software analysis for automated rapid mutation identification and result reporting

21. MUTATION VALIDATION • What to do when an alteration in DNA sequence is found • Haemophilia mutation database • http://europium.csc.mrc.ac.uk/WebPages/Main/main.htm • Confirm • known mutation (previously report) • unknown variant (pathogenic or SNP)

22. LINKAGE ANALYSIS • Mutation has not been identified • The use of combine intragenic markers can give an informative result up to ~95% depending on the ethnic group

23. LINKAGE ANALYSIS

24. LINKAGE ANALYSIS

25. LINKAGE ANALYSIS • Factor IX • TaqI PCR • DdeI • MseI • HhaI • XmnI PCR • Cryptic Dinucleotide Repeat PCR

26. PRENAL DIAGNOSIS • CVS at 11-13 weeks of pregnancy • Determine fetal gender • Direct karyotyping • SRY/Amelogenin PCR • Female fetus requires no further testing • Male fetus • Test for known mutation • Informative markers

27. DIFFICULT CASES OF HAEMOPHILIA • These cases usually are referred to other laboratories for more extensive studies • Sequencing the whole gene including all exons, exon–intron boundaries, 5’UTR and 3’UTR or even sequencing of mRNA • Southern blotting

28. RESULTS AND REPORTS • Results + Reports received from Hospital Scientist • Checked by Senior Scientist/Medical Registrar • Checked by Head of Department