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2. 2 Methods for detection of un known mutations BRCA

3. 3 BRCA1 Gene

4. 4 BRCA2 Gene

5. 5 SSCP single strand conformation polymorphism  simplicity  clearly by heteroduplex analysis (HA)

6. 6 SSCP  SSCP Gels  Prepare 0.5x MDE gel as follows:  MDE gel16.0mlddH2O44.2ml10X TBE3.84ml10% APS256µlTEMED25.6µlPour sequencing gel format with appropriate sharkstooth comb. Gel will polymerize in about 1 hour

7. 7 SSCP  Loading Buffer  95% formamide  10mM NaOH  0.025% Bromophenol Blue  0.025% Xylene Cyanol  Run gel in 0.6X TEB buffer.  Heat denature samples at 94°C for 5 minutes and place them on ice for 3-5 minutes. Load 2.0-4.0µl per sample. Include non-denatured controls

8. 8  Electrophoresis conditions  Fragment Size: 150-200 bp  6 Watts  10-12 hours  room temperature  Fragment Size: > 200 bp  8 Watts  10-12 hours  room temperature  Exposure  Dry gel and expose either at -80°C for 2 hours or at room temperature for 16-18 hours.

9. 9 Pedigree of a selected family with breast cancer

10. 10 SSCP Analysis BRCA1 Exon 15, 4650delCA

11. 11 Pedigree of a selected family with breast cancer

12. 12 SSCP Analysis BRCA1, Exon 20, Nt 5382

13. 13 SSCP Analysis Exon 11pi BRCA1 MS R1347G

14. 14 Protein truncation test PTT

15. 15 PTT For BRCA1/2 using the Protein Truncation Test (PTT) for exon 11 of BRCA1 & exon 10-11 of BRCA2 These exons cover approximately over 60% of each gene

16. 16 PTT  Coding sequence without introns  cDNA via RT-PCR from RNA  or large exons in genomic DNA

17. 17 cDNA  It is PCR amplified  The forward primer carries at its 5' end a T7 promoter  followed by a eukaryotic translation initiation sequence  which includes an ATG start codon  Next is a gene-specific sequence designed so that the sequence amplified reads in-frame from the ATG

18. 18 Protein truncation test (PTT)

19. 19 PTT  After amplification  the PCR product is added to a coupled in vitro transcription-translation system  For detection a labelled amino acid is included  which is usually methionine, leucine or cysteine  The label can either be a radionucleotide such as [35S]  which is visualised by autoradiography  Or biotin which is detected by a colorimetric Western blot employing a streptavidin-biotin- alkaline phosphatase complex

20. 20 PTT  The polypeptides produced are separated by size using an SDS-PAGE gel.  If the product is only full length  no truncating mutation is present  Truncating mutations result in shorter products  the size of which gives the approximate position of the mutation.

21. 21 Protein truncation test  used in diagnostic laboratories dealing with cancer genes because they often contain truncating mutations.

22. 22 Protein truncation test (PTT)

23. 23 A nonisotopic protein truncation test WT is wild-type DNA C1−C3 are mutant homozygous DNA samples from cell lines P1−P4 are the heterozygous DNA samples from patients diagnosed with FAP BL1/2: a cell-free translation performed lacking both tRNAs and DNA

24. 24 ) The protein truncation test (PTT)  First, RNA is reverse transcribed (RT) to generate a cDNA copy.  Second, the cDNA (or genomic DNA) is amplified using the polymerase chain reaction (PCR) in combination with a specifically tailed forward primer facilitating in vitro transcription by T7-RNA polymerase.  Products are analyzed on agarose gel to verify amplification  abnormally migrating products point to mutations  Deletions  Duplications  affecting splicing  Finally, in vitro transcription/translation is used to generate peptide fragments  analyzed on SDS-PAGE gel  to detect translation terminating mutations

25. 25 The protein truncation test (PTT)

26. 26 ADVANTAGES Detects truncating mutations  Allows the analysis of large stretches of coding sequence (up to 5 kb: 2kb:genomic DNA, 1.3-1.6kb cDNA is best)  Either: large single exons (DNA template) or multiple exons (RNA template).  Length of the truncated protein pinpoints the position of the mutation, thereby facilitating its confirmation by sequencing analysis  SENSITIVITY: the sensitivity of PTT is good

27. 27 DISADVANTAGES  Not applicable to all genes  E.g. APC, BRCA1, BRCA2 and Dystrophin all have approximately 90- 95% truncating mutations  but NF1 has only 50% truncating mutations respectively  Most powerful as a technique when RNA is used, however, most laboratories only have DNA stored.

28. 28 DISADVANTAGES  The most readily available source of RNA is blood.  However expression of the target gene in this tissue may be low, requiring technically more demanding nested amplification reactions to obtain sufficient signal.  Cannot detect mutations occurring outside the coding region, which affect control of expression and RNA stability

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30. 30 Deletions/insertions/duplications Out of frame In frame

31. 31 Deletions/insertions/duplications  Out of frame:  result in frameshifts giving rise to stop codons.  no protein product or truncated protein product  deletions/insertions in DMD patients : truncated dystrophins of decreased stability  RB1 gene - usually no protein product in retinoblastoma

32. 32 Deletions/insertions/duplications  In frame:  loss or gain of amino acid(s)  depending on the size and may give rise to altered protein product with changed properties  eg CF Delta F508 loss of single amino acid  In some genes loss or gain of a single amino acid: mild

33. 33 In frame:  In some regions of RB1 a single amino acid loss:  rise to mild retinoblastoma or incomplete penetrance  BMD patients:  Some times in-frame deletions/duplications  DMD deletions:  mostly disrupt the reading frame

34. 34 Deletions/insertions/duplications  In untranslated regions:  these might affect transcription/expression and/or stability of the message:  Fragile X  MD expansions.

35. 35 Mutation Databases

36. 36 Mutation Databases  Online Mendelian Inheritance in Man (OMIM)  problem of collecting mutations  if each out of approximately 50 000 genes can be subject to 100 mutations to cause disease  then there could be potentially five million mutations  it needed to get organised quickly to undertake

37. 37 Examples of central and locus- specific databases

38. 38 Current mutation detection methods

40. 40 Characteristics of the scanning methods

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