Kartagener Syndrome: a relentless triad

1. Kartagener Syndrome: a relentless triad Betsy Ott

2. 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 1933 Dr. Kartagener

3. 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

4. Left is right? Situs Solitus Situs Inversus

5. 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!!

6. 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

7. 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

8. Chlamydomonas reinharditiiModel organism • Unicellular alga • 2 flagella similar in structure to human respiratory cilia • Used to identify candidate genes • Blair and Dutcher 1992

9. Identify Candidates Geremek et al. 2004

10. 1st to identify DNAH5 as candidate gene Homozygosity mapping strategy Whole genome scan with probes LOD score Zmax=3.51 DNAH5 (Omran et al. 2000)

11. DNAH5 (Cont’d) • Encodes an exonemal heavy dynein chain of outer arm • DNAH5 homologous to Chalmydomonas gene • Studied Arabic Family • 10 members • 4 children affected • 1 with KS, 3 with PCD Healthy Mutant Omran et al. 2000

12. 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

13. Haplotypes and Recombination Omran et al. 2000

14. Marker (D5S630) and DNAH5 Linked Zmax=2.96 Omran et al. 2000

15. DNAI1 Dynein Axonemal Intermediate Chain 1 • Localized on 9p13-p21 • 20 exons and 699 aa • Codes for a motor protein

16. DNAI1 (Pannarun et al. 1999) • Pennarun-1st to identify DNAI1 as candidate gene • Used Homolgous-gene approach in Chalmydomanas reinhardtii

17. Step 1: Cloning and Sequencing of cDNA and genomic DNA • Primers designed from IC78 (Chlamydomonas) and IC2 (sea urchin) • Codes for intermediate dynein arm • RT-PCR • 975bp • More primers made from RT-PCR products • RACE experiments led to characterization of full length 2,526 bp DNAI1 coding sequence • Made more primers • Long-range PCRs determined genomic structure of DNAI1 • 20exons • 19 introns

18. Step 2: Mapping of DNAI1 • 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

19. Step 2 (cont’d): FISH • FISH and R-banding • Biotin labeled probe by nick-translation and FTIC-avidin • DNAI1 localized to p13-21 Pannarun et al. 1999

20. Step 3: SSCP Mutation Analysis • DNAI1 exons amplified PCR • Products run on mutation-detection-enhancement gel • Detected bandshifts were sequenced • 2 mutated sites found in patient II-1 • Paternal (exon 1) mutation • Maternal (exon 5) mutation

21. Step 4: Mutation analysisExon 5 • Maternally (I-2) Inherited • 4 bp insertion Creates Frameshift mutation SSCP VspI restriction site created Pannarun et al. 1999

22. Step 4: Mutation analysisExon 5 • Digestion with VspI Pannarun et al. 1999

23. Step 4: Mutation analysisExon 1 • Paternally (I-1) Inherited • I bp insertion • Produces a HpaI site SSCP Pannarun et al. 1999

24. Step 4: Mutation analysisExon 1 • Treatment with HpaI • I-1 and II-1 have mutation • RT-PCR on total RNA • Alternate splicing Pannarun et al. 1999

25. Step 4: Mutation analysisExon 1 • RT-PCR on total RNA • Alternate splicing • Intron 1 not spliced Pannarun et al. 1999

26. Inheritance • Autosomal Recessive • Incomplete Penetrance • Extensive heterogeneity Afzelius and Mossberg, 1995

27. New Kartagener Kids • 1/15,000-1/60,000 live PCD births • 1/30,000-1/120,000 live KS births

28. What’s up Doc? Electron micrograph of dynein arms • Look for clinical triad: • Bronchiectasis • Chronic sinusitis • Situs inversus

29. Treatment • Sputum culture to determine type of infection • Prescribe effective antibiotics • Chest Vest • Inhaler • Nebulizer STAY AWAY FROM THE BARS!!!

30. References • 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. (McGraw-Hill, 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.

31. Thank You!!! Questions??