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1 Seminar 4 Part 1 Part 2 Part 3 What Is A Genetic Disease? A genetic disease is a disease caused in whole or in part by a "variation" (an unusual form) or "mutation" (alteration) of a gene. Genetic disorders can be passed on to family members who inherit the genetic abnormality. A small number of rare disorders are caused by a mistake in a single gene. But most disorders involving genetic factors - such as heart disease and most cancers - arise from a complex interplay of multiple genetic changes and environmental influences. 1 4 PART I Categories of Genetic Disorders? Single gene disorders (1) Autosomal dominant (AD) (2) Autosomal recessive (AR) (3) X-linked (XD or XR) (4) Y-linked Thalassemia, cystic fibrosis Chromosome disorders Down syndrome Multifactorial inheritance disorders Alzheimer's disease Somatic cell disorders most cancers Mitochondrial inherited disorders diabetes-deafness syndrome, maternally transmitted 2 All known disease are genetic disease? 5 Positional Cloning Genes & Diseases Positional Candidate Cloning Candidate Gene Approach Statistics: Genes & Diseases (Dated on 11/28/04) How many genes in Homo sapiens? 20000~25000 in estimation (Nature, 2004, Oct 21) records in LocusLink loci in RefSeq How many diseases (phenotypes) have been described? 3091 How many diseases are known to be the mutation of known genes? 1655 One Gene, One Disease? 3 6 1

2 How to find a disease gene? Genetic Mapping Phenotype Gene 1. Positional Candidate Cloning (map-based) Two-loci linkage analysis Whole genome scan 2. Candidate Gene Studies (no map-based) 3. Function Cloning (function-based) Gene Phenotype Mutagenesis (Knock-out) 7 Family-Based Backcross, Intercross Pedigree Point Estimation (MLE): recombination fraction: θ genetic distance: d (map f.) Linkage Tests (LRT/Lods) GeneHunter linkage MapManager Linkage (θ< 0.5, Lods > 3) Genetic map NPL: TDT, ASP Population-Based Case-control association Relative Risk (RR) Odds Ratio (OR) Correlation & Regression Y(phenotype) = f (x, genotype) 10 History of Positional Cloning The first gene identified using the positional cloning approach was the X-linked gene for chronic granulomatous disease in Genes for DMD (1986), retinoblastoma (1989) and CF (1989) followed soon after. CFTR was the first gene to be identified by positional candidate cloning without a cytogenetic abnormality to aid chromosomal localization. Over 100 genes have been identified by positional cloning so far. The information and resources generated by the Human Genome Project mean that a positional candidate approach will largely replace pure positional cloning. Positional cloning can find Mendelian traits, single-gene disorders with non-mendelian pattern, as well as complex traits. Case Study 1: Positional Candidate Cloning of Hermansky-Pudlak Syndrome Genes 8 11 Flowchart of positional candidate cloning Genetic Mapping Backcross ( progenies) or Pedigrees Physical Mapping Markers, Genome browser BAC Mapping BAC contig BAC Shotgun Sequencing & Sequence Annotation shortcut Genome Database Browsers Functional Studies Structures, expression, biochemistry, proteomics, cell biology Confirmative Studies BAC rescue, cell-line rescue Mutation Analysis RT-PCR/sequencing SSCP/HA, Blotting Candidate Selection Known genes or ESTs, predicted genes 9 Introduction to Hermansky-Pudlak Syndrome 1) Autosomal recessive inherited Mendelian trait. 2) A genetically heterogeneous disease both in human (HPS1, HPS2 HPS7) and mouse (16 loci). 3) Characterized by a triad: oculocutaneous albinism, bleeding tendency, and ceroid deposition. 4) Themes: Disrupted biogenesis and/or function of specialized lysosomes, such as melanosomes, dense granules, and conventional lysosomes. 12 2

3 Newly identified HPS protein complexes in vesicle trafficking: Biogenesis of Lysosomerelated Organelles Complexe (BLOC) The Road to Clone the HPS Genes Positional Candidate Cloning Approaches Mouse backcrosses (from mouse to human) HPS pedigrees HPS mouse mutants Positional Cloning of the sdy/hps7 gene Li W, Zhang Q, Oiso N, et al. Nature Genetics, 2003, 35 (1): Hps genes in mouse and 7 HPS genes in human have been cloned Mouse loci Human loci Year Major contributors pale-ear (ep) HPS Oh J (Dr. Spritz) Gardner JM (Dr. Brilliant) (Swank RT) mocha (mh) AP3D 1998 Kantheti P (Dr. Burmeister) pearl (pe) HPS2/AP3B Feng L (Dr. Swank) Seymour AB (Dr. Gorin) Dell Angelica (Dr. Bonifacino) pallid (pa) PLDN 1999 Huang L (Dr. Gitschier) gunmetal (gm) RABGGTA 2000 Detter JC (Dr. Kingsmore) Zhang Q (Dr. Swank) ashen (ash) RAB27A 2000 Wilson SM (Dr. Jenkins) (Swank RT) cocoa (coa) HPS Anikster Y (Dr. Gahl) Suzuki T (Dr. Spritz) Li W (Dr. Swank) muted (mu) MU 2002 Zhang Q, Li W (Dr. Swank) light-ear (le) HPS Suzuki T (Dr. Spritz) Li W (Dr. Swank) ruby-eye 2(ru2) HPS ruby-eye (ru) HPS Zhang Q, Zhao B, Li W (Dr. Swank) Oiso N (Dr. Spritz) buff (bf) VPS33A 2003 Suzuki T, Oiso N (Dr. Spritz) Gautam R (Dr. Swank) cappuccino (cno) CNO 2003 Ciciotte SL (Dr. Peters) sandy (sdy) HPS Li W, Zhang Q (Dr. Swank) Oiso N (Dr. Spritz) Reduced pigmentation (rp) BLOC1S Starcevic M (Dr. DellÁngelica) Subtle gray (sut) SLC7A Chintala S, Li W (Dr. Swank) 15 A Mouse Chr 13 B Celera Chr 13 D13MITs Positions mcts Genes C RPCI-23 BACs cm , Sdy,179,64, 245, , 308, 181, 91, M 43.0M 43.5M 44.0M D13Mit Jmj 257B17 248C17 152H18 395C kb 0.20 Dtnbp1 54F9 230P L13 151E D ,39 Genetic and Physical Mapping of the sdy Gene 18 3

4 Northern Blotting of Dtnbp1 cdna Probe Structure of human and mouse DTNBP1 Gene and Diagram of the Mutations Transgenic mice exhibits rescued platelet serotonin concentration Western Blotting of BLOC-1 Components in Multiple HPS Mutants RP23-54F9 microinjection C3H/B6 backcross sdy/sdy Tg-mice: 1.79 ±0.62 µg/10 9 platelet, n=6 sdy/sdy: 0.03 ±0.02 µg/10 9 platelet, n=5 (P=0.017) Dysbindin is expressed in the transgenic mice Mutation Detection of the DTNBP1 Gene in HPS Patients Yeast-two-hybrid Screening of Dysbindin Direct Interactions 22 unrelated non-puerto Rican HPS patients (no mutations in HPS1-HPS6) and 61 normal controls were screened. A homozygous nonsense mutation Q103X (CAG>TAG) was found in one HPS type-7 patient. His Trp Leu Adenine Trp Leu

5 Co-immunoprecipitation of Dysbindin HPS gene products are formed in complexes for the biogenesis of lysosome-related organelles AP1 Platelet Granule Co-transfection Endosome Melanosome IP: Dysbindin Lower/Higher Eukaryotic Transport AP3 Ap3b1, Ap3d E R G O L G I T G N Higher Eukaryotic Transport HOPS Vps33a BLOC1 Muted, Pldn, Cno, Hps7, Blos1, Blos2, Blos3, Snapap BLOC2 M V B Lysosome Lamellar Body MHCII C Hps3, Hps5, Hps6 BLOC3 Lytic Granule 25 Adapted from Li W, et al. BioEssays, 2004; 26: Hps1, Hps4 28 Dysbindin is a subunit of BLOC-1 Flowchart of the expression-based cloning method (Candidate Studies) 26 Clustering melenaocytes vs. controls cdna melanocyte cdna slides clusters mapping data candidate selection Comparison wild-type vs. mutant melanocytes cdna mouse Affychips expression difference mutation analysis functional studies29 Case Study 2: Candidate Cloning of the cht/rab38 gene by expression clustering Luftus SK, et al. PNAS, 2002; 99:

6 RAB38 is clustered as a pigmentation gene by its expression pattern Summary Comparisons of two cloning methods Labor Efficiency Cost Map-based Labor-intensive ~5 yrs (before 2000) ~2 yrs (after 2000) High Hundreds of genes have been cloned since late 1980 s Fairly low Animal facilities Microarray-based Labor-intensive in developing a home-made microarray, less work by using commercial one Low Only a few genes have been cloned since late 1990 s Fairly high Microarray facilities Limitation Sample size Genetic heterogeneity False positive False negative RAB38 is a candidate gene of cht by synteneic analysis 32 Key Points to the Success of Positional Candidate Cloning 1. Prerequisites: evidence of a gene is involved by twin studies, segregation analysis, or case-control studies. 2. For Mendelian traits: clear family history, non-recombinant meioses>10, LOD score> For non-mendelian traits: many small families with affected siblings, affected pedigree members. Robust study designs typically incorporate individuals. 4. Cytogenetic rearrangements will be big shortcuts to clone the gene. 5. Fine mapping is critical to narrow down the critical region within 1Mb. 6. More meiosis, consanguineous families, genetically isolated populations, heterozygote advantage and linkage disequilibrium (LD) pattern are key factors to narrow down the critical region. 7. Marker density can be improved by SNP genotyping. 8. Confounders: epigenetics, heterogeneity, phenocopy, gene-gene and gene-environment interactions. 35 Rab38 146G T (G19V) mutation in cht/cht results in the impaired melanosomes

7 PART II G6PD ( ) G6PD G6PD

8 APOE4/APOE 491A / BRCA1/BRCA2 AIDS CCR5 PPARγ/MitoDNA HLA-DQα HLA-G MSH1/MSH2/MSH3 HDL/ ABCA1 FAT/CD36 MitoDNA (LVH) PPAR α

9 1) ASO/RDB/Southern 2) ARMS 3) Restriction enzyme 4) AFLP 5) Gap-PCR 6) Primer extension 7) OLA/LCR 8) Sequencing 9) Pyrosequencing 10) DGGE/SSCP/dHPLC 11) Quantitative PCR 12) DNA chips PART III PCR (FQ-PCR) DNA SNP Pyro dhplc Gene-Oriented File GOF HPSD Devices (e.g. reagents, instruments, softwares)--safety and efficacy (FDA) Standards and analytes (CDC) Proficiency test (PT), quality control (QC), quality assurance (QA), patient test management (Clinical Laboratory Improvement Amendments, CLIA, 1988) Laboratory practice (Accreditation, CLIA) Personnel standards (Board-certified, degree, experience, CLIA) Test fee reimbursement (HCFA, HMO) Define A Gene at Genome Bioinformatics Level Gene Mapping Gene Structure Regulatory Elements Comparative Genomics Variations (polymorphisms)

10 Define A Gene at Structural Bioinformatics Level Primary structure: amino acid sequence Secondary structure: local conformation of backbone, α-helix, β-sheet, loop Tertiary structure: 3D conformation of backbone and side chains Quaternary structure: spatial arrangement of protein subunits 55 Materials & Methods Programming Languages HyperText Markup Language (HTML), JavaScript (JS) Web page Browsers & Plug-ins Internet Explorer (IE), Netscape Communicator Plug-ins: MDL Chime, Adobe Acrobat Reader, Mac PictureViewer or Windows QuickTime Player Other Softwares Adobe Photoshop, Microsoft Word, Microsoft PowerPoint, Adobe Acrobat Distiller, Bioinformatics servers Database Server 58 Database: GOF Structure & Data Resourses UCSC/NCBI/Ensembl_Genome Genomic Mapping Structure Regulatory element Transcript RefSeq Expression pattern Protein Sequence Ortholog Domain Motif /Site 3D model 2D-PAGE Function Ontology Location Interaction Pathway Mutation Allele&SNP Distribution Effect Phenotype Reference CONREAL, MEME NCBI_RefSeq GNF_microarray, NCBI_GEO, GeneCards, NCBI_Protein, SWISS-Prot HomoloGene, Ensembl, UCSC_GeneSorter, ClustalW, GeneDoc SMART, SOSHI, COG, InterPro, Pfam ScanProsite, PSORT, PRINTS, BLOCKS Swiss-Model, ModBase, UCSC_GeneSorter, Sparks, ProteinExplorer SWISS-2DPAGE, ProtParam GO, GeneCard PubMed, ScanProsite PubMed, Yeast GRID, CuraGen PubMed, KEGG PubMed, OMIM, HGMD, NCBI_SNP PubMed, OMIM, MGI 56 DEMO of the HPSD 1. Homepage 2. GOF: Dtnbp1 HPSD/HPSD.htm 59 Objectives & Goals: Data Integration in HPSD Data Collection: searching databases Data Prediction: using Bioinformatics servers Data Annotation: reviewing papers

11 Features of the HPSD Gene-Oriented File: unique Integrated Data Interface: user-friendly & Comprehensive Curated Data: peer-reviewed Future Directions Update & Maintenance Expand the entries Queries & Submissions Thanks

Microsoft PowerPoint - NCBA_Cattlemens_College_Darrh_B

Microsoft PowerPoint - NCBA_Cattlemens_College_Darrh_B Introduction to Genetics Darrh Bullock University of Kentucky The Model Trait = Genetics + Environment Genetics Additive Predictable effects that get passed from generation to generation Non-Additive Primarily