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Database Search: Mutation Interpretation

Database Search: Mutation Interpretation. Huong Le Senior Hospital Scientist Department of Molecular & Clinical Genetics Royal Prince Alfred Hospital Sydney , Australia. Outline. Introduction Applications Pre-analytical consideration Analytical process Post-analytical process.

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Database Search: Mutation Interpretation

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

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

  3. Pre-analytical Consideration • Database search: • Clinical utilities • Information search • Journals / Publication • 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

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

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

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

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

  8. Analytical Process: Gene search • Database search:

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

  10. Analytical Process: Mutation frequency search • Database search

  11. Free Software: Primer3 Perlprimer Analytical Process: Primer design • Database search • Primer design • Primer selection • - primer length • - melting temperature (Tm) • - G/C content • - complementary primer sequences • - 3’-end clamp

  12. Analytical Process: Testing technologies • Database search: • Testing technologies • ARMS-PCR • RFLP • Sequencing • MLPA • Others

  13. Analytical Process: Mutation analysis using SeqScape

  14. Analytical Process: Identifying mutation/variant

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

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

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

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

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

  20. Post-analytical Process:Data interpretation

  21. Post-analytical Process:Data interpretation

  22. Post-analytical Process: How to report a mutation?

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

  24. Post-analytical Process: An example of HGVS nomenclature

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

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

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

  28. Summary • 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.

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

  30. TASKS Using online databases to search for your gene of interest to search for reference sequence to search for relevant mutation databases

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