Methods for detection of un known mutations BRCA

2. Methods for detection of un known mutations BRCA

3. BRCA1 Gene

4. BRCA2 Gene

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

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. 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. 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. Pedigree of a selected family with breast cancer

10. SSCP AnalysisBRCA1 Exon 15, 4650delCA

11. Pedigree of a selected family with breast cancer

12. SSCP AnalysisBRCA1, Exon 20,Nt 5382

13. SSCP AnalysisExon 11pi BRCA1 MS R1347G

14. Protein truncation testPTT

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

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. Protein truncation test (PTT)

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

22. Protein truncation test (PTT)

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. 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. The protein truncation test (PTT)

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. 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. 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

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

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

35. Mutation Databases

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. Examples of central and locus-specific databases

38. Current mutation detection methods

40. Characteristics of the scanning methods