Kartagener Syndrome: a relentless triad

1
Kartagener Syndrome a relentless triad

• Betsy Ott

2
1933 Dr. Kartagener

• Primary ciliary dyskinesia (PCD), formerly
  immotile cilia syndrome (ICS)
• PCD patients do not have situs inversus

• 4 cases
• Quantified the clinical triad
• Bronchiectasis
• Chronic sinusitis
• Situs inversus

3
Overview

• Whats 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 Inversus
Situs Solitus
5
Whats 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
DNAH5 (Omran et al. 2000)

• 1st to identify DNAH5 as candidate gene
• Homozygosity mapping strategy

• Whole genome scan with probes
• LOD score Zmax3.51

11
DNAH5 (Contd)

• 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 (Contd)2

• Total genome Linkage analysis
• 340 microsatellite markers
• Avg spacing of 11cM
• LOD scores calculated (Zmax3.51)
• Each individual evaluated to give physical map

13
Haplotypes and Recombination
Omran et al. 2000
14
Marker (D5S630) and DNAH5 Linked
Zmax2.96
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 chsome
• Used DNAI1 probe and localized to chsome 9

Pannarun et al. 1999
19
Step 2 (contd) 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
Whats 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.
  6515081519.
• 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. 39433954.
• Blair DF, Dutcher SK (1992) Flagella in
  prokaryotes and lower eukaryotes. Curr Opin Genet
  Dev 2756767
• 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 83489501
• 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. Humgt Genet.
  681030-1035.

31

• Thank You!!!
• Questions??