database search: mutation interpretation huong le senior hospital scientist department of molecular...

Database Search:Database Search:Mutation InterpretationMutation Interpretation

Huong LeSenior Hospital Scientist

Department of Molecular & Clinical GeneticsRoyal Prince Alfred Hospital

Sydney , Australia

OutlineOutline

IntroductionIntroduction ApplicationsApplications

1.1. Pre-analytical considerationPre-analytical consideration

2.2. Analytical processAnalytical process

3.3. Post-analytical processPost-analytical process

Pre-analytical ConsiderationPre-analytical Consideration

Database search: Database search:

– Clinical utilitiesClinical utilities Information searchInformation search

– Journals / PublicationJournals / Publication– http://www.ncbi.nlm.nih.gov/pubmed/– http://www.ncbi.nih.gov/sites/entrez?db= OMIMhttp://www.ncbi.nih.gov/sites/entrez?db= OMIM

– Clinical validitiesClinical validities Type of testType of test Benefit of test result Benefit of test result

Pre-analytical ConsiderationPre-analytical Consideration

Database search: Database search: – http://www.ncbi.nih.gov/sites/entrez?db= OMIMhttp://www.ncbi.nih.gov/sites/entrez?db= OMIM

Analytical Process: Analytical Process: Gene searchGene search

Database search:Database search:– Gene searchGene search

GenBank GenBank – ((U01317 for the beta-like globin genes for the beta-like globin genes

– Z84721 for the alpha-like globin genes) for the alpha-like globin genes)

– Reference sequence searchReference sequence search http://www.ensembl.org/index.htmlhttp://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/

Analytical Process: Analytical Process: Gene searchGene search

Database searchDatabase search– National Centre for Biotechnology InformationNational Centre for Biotechnology Information

http://www.ncbi.nlm.nih.gov/http://www.ncbi.nlm.nih.gov/

Analytical Process: Analytical Process: Gene searchGene search

Database search:Database search: The GDB Human Genome DatabaseThe GDB Human Genome Database

http://www.gdb.org/http://www.gdb.org/

Analytical Process: Analytical Process: Gene searchGene search

Database search:Database search:

Analytical Process: Analytical Process: Mutation frequency searchMutation frequency search

Database search:Database search:– Mutation frequency / relate to ethnic Mutation frequency / relate to ethnic

backgroundbackground

1.1. http://www.ncbi.nlm.nih.gov/pubmed/http://www.ncbi.nlm.nih.gov/pubmed/

2.2. http://www.ncbi.nih.gov/sites/entrez?db= http://www.ncbi.nih.gov/sites/entrez?db= OMIMOMIM

3.3. http://globin.cse.psu.edu/globin/ http://globin.cse.psu.edu/globin/ Globin Gene ServerGlobin Gene Server

Analytical Process: Analytical Process: Mutation frequency searchMutation frequency search

Database searchDatabase search

Analytical Process: Primer designAnalytical Process: Primer design

Database search Database search –Primer designPrimer design

Primer selection1. - primer length 2. - melting temperature (Tm) 3. - G/C content4. - complementary primer sequences 5. - 3’-end clamp

Free Software:– Primer3– Perlprimer

Analytical Process: Analytical Process: Testing technologiesTesting technologies

Database search:Database search:

– Testing technologiesTesting technologiesARMS-PCRARMS-PCRRFLPRFLPSequencingSequencingMLPA MLPA OthersOthers

Analytical ProcessAnalytical Process: : Mutation analysis using SeqScapeMutation analysis using SeqScape

Analytical ProcessAnalytical Process:: Identifying mutation/variant Identifying mutation/variant

Post-analytical Process:Post-analytical Process:Data interpretationData interpretation

Database search:Database search:

– Interpreter data into meaningful resultsInterpreter data into meaningful results

Mutation / variant classificationMutation / variant classification

– Known mutationsKnown mutations

– Unknown variantsUnknown variants

Post-analytical Process:Post-analytical Process:Data interpretationData interpretation

Data analysis bData analysis base on:ase on: 1.1. HGVS nomenclatureHGVS nomenclature

http://www.hgvs.org/mutnomen/http://www.hgvs.org/mutnomen/

2.2. Public DatabasesPublic Databaseshttp://www.hgmd.cf.ac.uk/ac/index.phphttp://www.hgmd.cf.ac.uk/ac/index.php Cardiff- Human Gene Mutation DatabaseCardiff- Human Gene Mutation Databasehttp://globin.bx.psu.edu/hbvar/menu.htmlhttp://globin.bx.psu.edu/hbvar/menu.html Globin Gene ServerGlobin Gene Server

3.3. Published source Published source http://www.ncbi.nlm.nih.gov/pubmed/http://www.ncbi.nlm.nih.gov/pubmed/

Post-analytical Process:Post-analytical Process:Data interpretationData interpretation

Checking database for report of a known Checking database for report of a known mutationmutation

Checking any publication that report the Checking any publication that report the mutation and use it as referencemutation and use it as reference

Post-analytical Process:Post-analytical Process:Data interpretationData interpretation

Thalassaemia casehttp://www.hgvs.org/mutnomen/http://www.hgvs.org/mutnomen/

Post-analytical Process:Post-analytical Process:Data interpretationData interpretation

http://globin.bx.psu.edu/hbvar/menu.html

Post-analytical Process:Post-analytical Process:Data interpretationData interpretation

Post-analytical Process:Post-analytical Process:Data interpretationData interpretation

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

NameName MutationMutation HGVSHGVS

Hb Constant Hb Constant Spring (Hb CS)Spring (Hb CS)

Codon 142 Codon 142 (TAA->CAA) of (TAA->CAA) of the the αα2 gene 2 gene

HBA2:c.427T>C HBA2:c.427T>C

Codon 17 Codon 17

(A->T) (A->T)

ββ 17(A14) 17(A14) Lys>Stop Lys>Stop

HBB:c.52A>T HBB:c.52A>T

http://www.hgvs.org/mutnomen/recs-DNA.html

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

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

GenomicGenomic g.2490327G>Ag.2490327G>A

cDNAcDNA c.1706-10G>Ac.1706-10G>A

GenomicGenomic g.2479070A>Tg.2479070A>T

ProteinProtein p.E229Vp.E229V

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

Protocol for classification of variants as Protocol for classification of variants as

deleterious or neutraldeleterious or neutral Nearly 95% mutations have been described and 5% remains as Nearly 95% mutations have been described and 5% remains as

novel mutationnovel mutation

1.1. Frequency of a variant in normal population Frequency of a variant in normal population

2.2. Co-segregation of a sequence variant with the disease in Co-segregation of a sequence variant with the disease in familyfamily

3.3. Assess degree of conservation among different species (with Assess degree of conservation among different species (with or without Grantham calculation)or without Grantham calculation)

4.4. Assess type of amino acid substitutionAssess type of amino acid substitution

5.5. Protein modelingProtein modeling

6.6. Splicing studies if relevantSplicing studies if relevant

Automated splice site analysis http://splice.cmh.edu Automated splice site analysis http://splice.cmh.edu

7.7. Functional analysis Functional analysis

Follow the protocol for classification of variants as Follow the protocol for classification of variants as deleterious or neutraldeleterious or neutral

Result interpretation should considerResult interpretation should consider– Family studies Family studies – Other genes or other mutations on the regions Other genes or other mutations on the regions

that have not been tested for.that have not been tested for.

Post-analytical Process:Post-analytical Process: DNA study report of an unknown DNA study report of an unknown

mutationmutation

SummarySummary

Advance technologies result from the Advance technologies result from the Human Genome Project has a huge impact Human Genome Project has a huge impact on increasing in free access to public online on increasing in free access to public online databases databases – Allow for text-based search on human gene and Allow for text-based search on human gene and

diseases as well as nucleotide and protein diseases as well as nucleotide and protein sequences, protein structures & complete sequences, protein structures & complete genomesgenomes. .

List of useful database sourcesList of useful database sources

Phenotype source Phenotype source – NCBI (http://www.ncbi.nlm.nih.gov/)NCBI (http://www.ncbi.nlm.nih.gov/)– OMIM OMIM

Gene searchGene search– NCBI (RefSeq)NCBI (RefSeq)– Ensembl (http://www.ensembl.org/)Ensembl (http://www.ensembl.org/)– UCSC (http://genome.ucsc.edu/)UCSC (http://genome.ucsc.edu/)

Mutation databaseMutation database– http://globin.bx.psu.edu/hbvar/menu.htmlhttp://globin.bx.psu.edu/hbvar/menu.html– http://www.hgmd.cf.ac.uk/ac/http://www.hgmd.cf.ac.uk/ac/

TASKSTASKS

Using online databases Using online databases

1.1. to search for your gene of to search for your gene of interestinterest

2.2. to search for reference sequenceto search for reference sequence

3.3. to search for relevant mutation to search for relevant mutation databasesdatabases