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Kartagener Syndrome: a relentless triad Betsy Ott 1933 Dr. Kartagener • 4 cases • Quantified the clinical triad: – Bronchiectasis – Chronic sinusitis – Situs inversus • Primary ciliary dyskinesia (PCD), formerly immotile cilia syndrome (ICS) – PCD patients do not have situs inversus Overview • What’s the problem? • Candidate Genes – DNAH5 and DNAI1 • DNAH5 – Whole Genome Scan – Homozygosity Mapping – LOD scores • DNAI1 – Cloning and Sequencing – Mapping – SSCP Mutation analysis • Inheritance • Diagnosis • Treatment Left is right? Situs Solitus Situs Inversus What’s the problem? • Dynein arms do not function – Immotile cilia/flagella • Consequences – 50% result in situs inversus – Symptoms similar to cystic fibrosis – Constant infection in lungs and sinuses • Dysfunctional cilia!! Dynein Arm defects • 18 ultrastructural defects identified • Most common – – – – Outer dynein arm Inner dynein arm Radial spokes Absence of nexin links Geremek et al. 2004 Find the problem! • Study genes selected based on ciliary function – Use model organisms – Identify candidate genes • Linkage studies – Evaluate candidate genes – Looking for markers linked with the disease in affected families Chlamydomonas reinharditii Model organism • Unicellular alga • 2 flagella similar in structure to human respiratory cilia • Used to identify candidate genes • Blair and Dutcher 1992 Identify Candidates Geremek et al. 2004 DNAH5 (Omran et al. 2000) • 1st to identify DNAH5 as candidate gene • Homozygosity mapping strategy • Whole genome scan with probes • LOD score Zmax=3.51 DNAH5 (Cont’d) • Encodes an exonemal heavy dynein chain of outer arm • DNAH5 homologous to Chalmydomonas gene Healthy • Studied Arabic Family – 10 members – 4 children affected – 1 with KS, 3 with PCD Mutant Omran et al. 2000 DNAH5 (Cont’d)2 • Total genome Linkage analysis – 340 microsatellite markers – Avg spacing of 11cM – LOD scores calculated (Zmax=3.51) • Each individual evaluated to give physical map Haplotypes and Recombination Omran et al. 2000 Marker (D5S630) and DNAH5 Linked Zmax=2.96 Omran et al. 2000 DNAI1 Dynein Axonemal Intermediate Chain 1 • Localized on 9p13-p21 • 20 exons and 699 aa • Codes for a motor protein DNAI1 (Pannarun et al. 1999) • Pennarun-1st to identify DNAI1 as candidate gene • Used Homolgous-gene approach in Chalmydomanas reinhardtii Step 1: Cloning and Sequencing of cDNA and genomic DNA 1. Primers designed from IC78 (Chlamydomonas) and IC2 (sea urchin) – Codes for intermediate dynein arm 2. RT-PCR – 975bp – More primers made from RT-PCR products 3. RACE experiments led to characterization of full length 2,526 bp DNAI1 coding sequence – Made more primers 4. Long-range PCRs determined genomic structure of DNAI1 – 20exons – 19 introns Step 2: Mapping of DNAI1 1. Screened 24 hybrid somatic cell lines (human/rodent) by PCR – – Each hybrid contained 1 human ch’some Used DNAI1 probe and localized to ch’some 9 Pannarun et al. 1999 Step 2 (cont’d): FISH 2. FISH and Rbanding – – Biotin labeled probe by nicktranslation and FTIC-avidin DNAI1 localized to p13-21 Pannarun et al. 1999 Step 3: SSCP Mutation Analysis 1. DNAI1 exons amplified PCR 2. Products run on mutation-detectionenhancement gel 3. Detected bandshifts were sequenced – 2 mutated sites found in patient II-1 • • Paternal (exon 1) mutation Maternal (exon 5) mutation Step 4: Mutation analysis Exon 5 • • Maternally (I-2) Inherited 4 bp insertion Creates Frameshift mutation SSCP VspI restriction site created Pannarun et al. 1999 Step 4: Mutation analysis Exon 5 • Digestion with VspI Pannarun et al. 1999 Step 4: Mutation analysis Exon 1 • Paternally (I-1) Inherited 1. I bp insertion SSCP 2. Produces a HpaI site Pannarun et al. 1999 Step 4: Mutation analysis Exon 1 • Treatment with HpaI – I-1 and II-1 have mutation 3. RT-PCR on total RNA – Alternate splicing Pannarun et al. 1999 Step 4: Mutation analysis Exon 1 • RT-PCR on total RNA – Alternate splicing – Intron 1 not spliced Pannarun et al. 1999 Inheritance • Autosomal Recessive • Incomplete Penetrance • Extensive heterogeneity Afzelius and Mossberg, 1995 New Kartagener Kids • 1/15,000-1/60,000 live PCD births • 1/30,000-1/120,000 live KS births What’s up Doc? Electron micrograph of dynein arms • Look for clinical triad: – Bronchiectasis – Chronic sinusitis – Situs inversus Treatment • Sputum culture to determine type of infection – – – – Prescribe effective antibiotics Chest Vest Inhaler Nebulizer STAY AWAY FROM THE BARS!!! References 1. 2. 3. 4. 5. 6. 7. Pennarun, G., E. Escudier, C. Chapelin, A. M. Bridoux, V. Cacheux, G. C., Roger, M. Goossens, S. Amselem, and B. Duriez. 1999. Loss-of-function mutations in a human gene related to Chlamydomonas reinhardtii dynein IC78 result in primary ciliary dyskinesia. Am. J. Hum. Genet. 65:1508–1519. Afzelius, B. A., and B. Mossberg. 1995. Immotile cilia syndrome (primary ciliary dyskinesia) including Kartagener Syndrome. In The Metabolic and Molecular Bases of Inherited Disease. C. R. Scriver, A. L. Beaudet, and W. S. Sly, editors. McGraw-Hill, Inc., New York. 3943–3954. Blair DF, Dutcher SK (1992) Flagella in prokaryotes and lower eukaryotes. Curr Opin Genet Dev 2:756–767 Geremek, M., and Witt, M. 2004. Primary ciliary dyskinesia: genes, candidate genes and chromosomal regions. J. Appl. Genet. 45(3): 347-361 Kartagener M (1933) Zur Pathologie der Bronchiektasien: Bronchiektasien bei Situs viscerum invertus. Beitr Klin Tuberk 83:489–501 Afzelius, B.A. & Mossberg, B. (1995) in The Metablolic and Molecular Bases of Inherited Disease, eds. Scriver, C.R., Beaudet, A. L., Sly, W.S. & Valle, D. (McGrawHill, Yew York), pp. 3943-3954. Guichard, C., Harricane, M., Lafitte, J., Godard, P., Zaegel, M, Tack, V., Lalau, G., and Bouvagnet, P. 2001. Axonemal Dynein Intermiediate-Chain Gene (DNAI1) Mutations Result in Situs Inversus and Primary Ciliary Dyskinesia (Kartagener Syndrome). Am. J. Hum> Genet. 68:1030-1035. Thank You!!! Questions??