Dr. Derakhshandeh, PhD Mutation Screening

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

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 genome mutations: changes in chromosome number changes in chromosome number

5 Techniques Karyotyping, conventional cytogenetics

6 Down Syndrome (Trisomy 21( Trisomy 2(

7 chromosome mutations: changes in chromosome structure

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

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

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

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

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

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

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 Modifications/Adaptions to the original ARMS methodology  Multiplex ARMS

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 Agarose gels showing the feasibility of the ARMS concept

19 ARMS-PCR N M N M N M N M N M N M

20 Methods for detection of unknown mutations

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

22 BRCA

23 BRCA1 Gene

24 BRCA2 Gene

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

26 Pedigree of a selected family with breast cancer

27 SSCP Analysis BRCA1 Exon 15, 4650delCA

28 Pedigree of a selected family with breast cancer

29 SSCP Analysis BRCA1, Exon 20, Nt 5382

30 SSCP Analysis Exon 11pi BRCA1 MS R1347G

31 A woman having amniocentesis