1. Dr. Derakhshandeh, PhD Mutation Screening

2. 2 TYPE OF MUTATIONS WHICH TECHNIQUES DETECT WHAT TYPE OF MUTATIONS In classical genetics, three types of mutations are distinguished:

3. 3 Different types of mutations  genome mutations: changes in chromosome number  chromosome mutations: changes in chromosome structure  gene or point mutations: mutations where changes are at molecular level

4. 4 genome mutations: changes in chromosome number changes in chromosome number

5. 5 Techniques Karyotyping, conventional cytogenetics

6. 6 Down Syndrome (Trisomy 21( Trisomy 2(

7. 7 chromosome mutations: changes in chromosome structure

8. 8 CHANGES IN CHROMOSOME STRUCTURE  Translocations  Large Deletions/Insertions  Inversions  Duplications/Amplifications

9. 9 Techniques  Conventional cytogenetics  molecular cytogenetics FISH  Molecular:  PFGE, Southern blotting, Northern Blotting Fluorescence Dosage analysis  large deletions  Insertions  duplications

10. 10 Interphase FISH Examples 13 (green), and 21 (red) 18 (aqua), X (green), and Y (red).

11. 11 gene or point mutations: mutations where changes are at molecular level

12. 12  Methods for detection of known mutations  Methods for detection of unknown mutations

13. 13 Methods for detection of known mutations  PCR and size separation eg. DMD  PCR and restriction enzyme digestion eg. SMN exon 7 & 8  Allele specific amplification (ASA)  Allele refactory mutation system (ARMS) eg. CF  Allele specific oligonucleotide hybridisation (ASO)  Dot Blot eg. CF  DNA chips eg. Brca1  Genomic DNA sequencing

14. 14 ARMS Amplification Refractory Mutation System Allele Specific PCR (ASPCR) PCR Amplification of Specific Alleles (PASA)

15. 15 Uses  Population screening  rapid (1 working day)  inexpensive  non-isotopic  Used for testing for  B-thalassaemia  Cystic Fibrosis  Sickle-cell anaemia  Phenylketonuria  Apolipoprotein E, etc

16. 16 Modifications/Adaptions to the original ARMS methodology  Multiplex ARMS

17. 17 To set up multiplex ARMS  Determine commonest mutations in the population  develop the muliplex ARMS these mutations  validate the results of the multiplex test on samples with another methology

18. 18 Agarose gels showing the feasibility of the ARMS concept

19. 19 ARMS-PCR 1 2 3 4 5 6 7 8 9 10 11 12 N M N M N M N M N M N M

20. 20 Methods for detection of unknown mutations

21. 21 Methods for unknown mutations (diagnostic methods)  These methods are relatively simple, but still require:  experience and skill to perform.

22. 22 BRCA

23. 23 BRCA1 Gene

24. 24 BRCA2 Gene

25. 25 SSCP single strand conformation polymorphism  simplicity  clearly by heteroduplex analysis (HA)

26. 26 Pedigree of a selected family with breast cancer

27. 27 SSCP Analysis BRCA1 Exon 15, 4650delCA

28. 28 Pedigree of a selected family with breast cancer

29. 29 SSCP Analysis BRCA1, Exon 20, Nt 5382

30. 30 SSCP Analysis Exon 11pi BRCA1 MS R1347G

31. 31 A woman having amniocentesis