1. Database Search: Mutation Interpretation Huong Le Senior Hospital Scientist Department of Molecular & Clinical Genetics Royal Prince Alfred Hospital Sydney, Australia

2. Outline Introduction Introduction Applications Applications 1.Pre-analytical consideration 2.Analytical process 3.Post-analytical process

4. Pre-analytical Consideration Database search: Database search: –Clinical utilities  Information search –Journals / Publication –http://www.ncbi.nlm.nih.gov/pubmed/ http://www.ncbi.nlm.nih.gov/pubmed/ –http://www.ncbi.nih.gov/sites/entrez?db= OMIM –Clinical validities  Type of test  Benefit of test result

5. Pre-analytical Consideration Database search: Database search: –http://www.ncbi.nih.gov/sites/entrez?db= OMIM

6. Analytical Process: Gene search Database search: Database search: –Gene search  GenBank –(U01317 for the beta-like globin genes U01317 –Z84721 for the alpha-like globin genes) Z84721 –Reference sequence search  http://www.ensembl.org/index.html http://www.ensembl.org/index.html  http://genome.ucsc.edu/ http://genome.ucsc.edu/  http://www.ncbi.nlm.nih.gov/ http://www.ncbi.nlm.nih.gov/

7. Analytical Process: Gene search Database search Database search –National Centre for Biotechnology Information http://www.ncbi.nlm.nih.gov/ http://www.ncbi.nlm.nih.gov/http://www.ncbi.nlm.nih.gov/

8. Analytical Process: Gene search Database search: Database search: The GDB Human Genome Database The GDB Human Genome Database  http://www.gdb.org/

9. Analytical Process: Gene search Database search: Database search:

10. Analytical Process: Mutation frequency search Database search: Database search: –Mutation frequency / relate to ethnic background 1.http://www.ncbi.nlm.nih.gov/pubmed/ http://www.ncbi.nlm.nih.gov/pubmed/ 2.http://www.ncbi.nih.gov/sites/entrez?db= OMIM 3.http://globin.cse.psu.edu/globin/ http://globin.cse.psu.edu/globin/  Globin Gene Server

11. Analytical Process: Mutation frequency search Database search Database search

12. Analytical Process: Primer design Database search Database search –Primer design Primer selection 1. 1.- primer length 2. 2.- melting temperature (Tm) 3. 3.- G/C content 4. 4.- complementary primer sequences 5. 5.- 3’-end clamp Free Software: – –Primer3 – –Perlprimer

13. Analytical Process: Testing technologies Database search: Database search: –Testing technologies  ARMS-PCR  RFLP  Sequencing  MLPA  Others

14. Analytical Process : Mutation analysis using SeqScape

15. Analytical Process : Identifying mutation/variant

16. Post-analytical Process: Data interpretation Database search: Database search: –Interpreter data into meaningful results  Mutation / variant classification –Known mutations –Unknown variants

17. Post-analytical Process: Data interpretation Data analysis base on: Data analysis base on: 1.HGVS nomenclature http://www.hgvs.org/mutnomen/ 2.Public Databases http://www.hgmd.cf.ac.uk/ac/index.php Cardiff- Human Gene Mutation Database Cardiff- Human Gene Mutation Database http://globin.bx.psu.edu/hbvar/menu.html Globin Gene Server Globin Gene Server 3.Published source http://www.ncbi.nlm.nih.gov/pubmed/

18. Post-analytical Process: Data interpretation Checking database for report of a known mutation Checking database for report of a known mutation Checking any publication that report the mutation and use it as reference Checking any publication that report the mutation and use it as reference

19. Post-analytical Process: Data interpretation Thalassaemia case http://www.hgvs.org/mutnomen/

20. Post-analytical Process: Data interpretation http://globin.bx.psu.edu/hbvar/menu.html

21. Post-analytical Process: Data interpretation

23. Post-analytical Process: How to report a mutation? NameMutationHGVS Hb Constant Spring (Hb CS) Codon 142 (TAA->CAA) of the α2 gene HBA2:c.427T>C Codon 17 (A->T) β 17(A14) Lys>Stop HBB:c.52A>T

24. http://www.hgvs.org/mutnomen/recs-DNA.html Post-analytical Process: How to report a mutation?

25. Post-analytical Process: An example of HGVS nomenclature Genomicg.2490327G>A cDNAc.1706-10G>AGenomicg.2479070A>TProteinp.E229V

26. Post-analytical Process: DNA study report of a known mutation

27. Protocol for classification of variants as deleterious or neutral Nearly 95% mutations have been described and 5% remains as novel mutation 1.Frequency of a variant in normal population 2.Co-segregation of a sequence variant with the disease in family 3.Assess degree of conservation among different species (with or without Grantham calculation) 4.Assess type of amino acid substitution 5.Protein modeling 6.Splicing studies if relevant Automated splice site analysis http://splice.cmh.edu http://splice.cmh.edu 7.Functional analysis

28. Follow the protocol for classification of variants as deleterious or neutral Follow the protocol for classification of variants as deleterious or neutral Result interpretation should consider Result interpretation should consider –Family studies –Other genes or other mutations on the regions that have not been tested for. Post-analytical Process: DNA study report of an unknown mutation

29. Summary Advance technologies result from the Human Genome Project has a huge impact on increasing in free access to public online databases Advance technologies result from the Human Genome Project has a huge impact on increasing in free access to public online databases –Allow for text-based search on human gene and diseases as well as nucleotide and protein sequences, protein structures & complete genomes.

30. List of useful database sources Phenotype source Phenotype source –NCBI (http://www.ncbi.nlm.nih.gov/) http://www.ncbi.nlm.nih.gov/ –OMIM Gene search Gene search –NCBI (RefSeq) –Ensembl (http://www.ensembl.org/) http://www.ensembl.org –UCSC (http://genome.ucsc.edu/) http://genome.ucsc.edu Mutation database Mutation database –http://globin.bx.psu.edu/hbvar/menu.html http://globin.bx.psu.edu/hbvar/menu.html –http://www.hgmd.cf.ac.uk/ac/ http://www.hgmd.cf.ac.uk/ac/

31. TASKS Using online databases 1. to search for your gene of interest 2. to search for reference sequence 3. to search for relevant mutation databases