Transcript Kartagener`s Syndrome: a relentless triad

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
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–
–
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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
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•
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
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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??