Transcript Are genetic tests good for population screening?

Genetic mapping studies
- Asthma and allergy
Nature of disease gene projects
Clinical expertise
Genetic analysis
Diagnostic classification Disease modelling
Hopes and aims:
what does one want to find?
• Development of therapies
– New bioactive factors or immediate drug targets
– New pathways or disease mechanisms
– New associations for known pathways
• Development of diagnostics
– Specific assays for disease screening
– Specific diagnostic assays for clinical use
– Informative and useful new assays
How to think of gene effects in
multifactorial diseases?
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Pedigrees and penetrance
The threshold model of susceptibility
Quantitative gene effects
Diversity of disease-associated variants
How to find the asthma gene?
Map location
Polymorphism
Expression pattern
Tissue
Autosomal dominant, 100% penetrance
…67% penetrance…
…33% penetrance
Number of people
Threshold model of susceptibility
healthy disease
gene —
gene +
Quantitative measure
Promoter
Missense
variants
Splice
site
Diversity
of
mutations
• altered protein function
variants
variants
• altered
transcription
• altered
transcript
Nonsense variants
UTR variants
• truncated transcript
• transcript instability
Intron variants
• regulatory elements
A gene mapper’s lunchbasket for an
excursion to multifactorial diseases
Population simulation
and disease modelling
Linkage analysis
Multilocus
association analysis
Etc.
Etc.
Etc.
Genetic factors in atopy and asthma
• population differences in the prevalence of
asthma are wide: 1.2%-6.2%
• twin studies show widely varying results:
• concordance in monozygotic twins
19%-88%
• concordance in dizygotic twins 4%-63%
• relative risk estimates vary between 1.3 and 6
• estimates of genetic component vary up to 87%
Genetic factors in adolescent asthma
Laitinen et al., Am J Respir Crit Care Med 157:1073, 1998
• Finnish population-based twin-family study
• 2483 twin families, participation rate 82-93%
Offspring
Mother
Father
Asthmatic
Healthy
Asthmatic
Healthy
Asthmatic
19 (11%)
103 (2.7%)
15 (10%)
107 (2.7%)
Healthy
157
3759
129
3787
Rate ratio
3.9
1.0
3.8
1.0
A brief population history
Little later
immigration
Small permanent
settlement of
south and west
coasts >2000 y
Population
movement in
the 1500’s
Rapid late
population
growth
(10 x / 250 y)
Population of Kainuu
1560-1574: about 200 houses
1577: estimated 1444 inhabitants
1609: estimated 1649 inhabitants
1626: estimated 2788 inhabitants
1641: estimated 1794 inhabitants
1654: estimated 2912 inhabitants
1860 : 25636 inhabitants (1.5% pop.)
1991: 52519 inhabitants (1.0% pop.)
Why study a multifactorial
disease in a founder isolate?
population
bottleneck
population
expansion
fewer
disease
loci and
alleles
time
Kainuu
Asthma Study
15-20 generations
Department of Medical Genetics and
Department of Pulmonary Diseases,
University of Helsinki and HUCH
Department of Clinical Genetics, the Finnish
Family Federation (Väestöliitto)
Kainuu Central Hospital, Kajaani
Disease gene mapping project
Design of study
Obtaining permissions
Recruitment of families
Verification of diagnoses
Collection of samples
Genotyping
Analysis of data
Identification of gene
Functional analysis
Utilization
Kainuu Asthma Study
Radio and newspaper advertisements
Probands contact the research group
Interview for entry criteria:
• physician-diagnosed asthma (self-reported)
• nuclear family willing to participate
• parents/grandparents born in Kainuu
Proband
• informed consent
• questionnaire and interview
• blood sample
Review of medical records
• verification of asthma diagnosis
Family members
• informed consent
• questionnaire
• blood sample
Verification of genealogy
• population records
Genome scan
• A set of 312 microsatellite markers were chosen
in order to find out genomic regions cosegregating with the disease status
• All markers genotyped in all individuals of the
families recruited
• Linkage analysis was carried out
Linkage results of the genome scan for asthma with 304 autosomal and 8 Xchromosomal markers in 86 Finnish pedigrees.
Laitinen et al., Nature Genetics 28:87, 2001
A susceptibility gene for asthma in
chromosome 7p
• Genome scan in Finnish families gave significant
evidence for linkage to chromosome 7 (NPL=3.9 for
high IgE phenotype; NPL=3.0 for asthma)
• Result replicated in French-Canadian pedigrees from
Saguenay-Lac-St-Jean (NPL=2.7 for asthma)
• Second replication in North Karelian pedigrees
(NPL=1.9 for high IgE)
Laitinen et al., Nature Genetics 28:87, 2001
Linkage disequilibrium mapping
Haplotypes
Marker
A
B
C
D
E
F
1
1
1
1
1
1
2
1
1
1
1
1
3
2
1
1
1
1
4
1
1
1
1
2
1
1
1
1
1
3
1
1
1
1
2
4
3
3
1
1
2
2
3
3
1
2
3
4
most
likely
location
for the
gene
Fine mapping
• Exact location of the gene was mapped by
subsequent analysis of linked regions
• Laitinen et al. 2004: Science Vol 304, Issue
5668, pages 300-304. Characterization of a
Common Susceptibility Locus for AsthmaRelated Traits.
Fine mapping after linkage finding
•
Fig. 1. (A) Hierarchical gene mapping strategy. The
linkage region of 20 cM implicated by the genome
scan was refined by genotyping 76 microsatellite
markers in families from Kainuu. We used the HPM
algorithm for finding haplotypes associated with
high serum IgE. Haplotype patterns spanning 12
microsatellite markers within 3.5 cM were found
associated by a permutation test implemented in
HPM. At the next round of fine mapping, 10
additional microsatellites implicated a 301-kb
haplotype pattern (5 markers yielded the highest
associations). A further five microsatellites and 13
SNPs were genotyped next, implicating a 47-kb
haplotype pattern (10 markers) between NM51 and
SNP563704. All together, a 133-kb region was
sequenced around this segment from a homozygous
patient with asthma. Eighty polymorphisms were
identified by comparison to the public genomic
sequence. (D) Phylogenetic analysis of haplotypes
H1 to H7 within a 77-kb segment in Kainuu, North
Karelia, and Quebec. The same seven haplotypes
occur in all three populations at frequencies >2%.
H4 and H5 are the most common risk-associated
haplotypes in Kainuu, H7 in North Karelia, and H2
among French Canadians. H1, H3, and H6 are
nonrisk haplotypes in all three populations.
Gene structure in the 133-kb region
•
Fig. 2. Gene content around the conserved 133-kb
haplotype segment (gray box). (A) The 133-kb
segment spans from intron 2 to intron 5 of GPRA.
GPRA undergoes alternative splicing with multiple
variants; the three longest variants are shown (thin
lines joining exons marked E1 to E9b). Exon 2
donor site may join to alternative exon 3 acceptor
sites, separated by 33 bp in the same reading frame,
and there are two alternative 3' exons, 9a and 9b.
Further splice variants may skip exon 3 or 4 or both,
suggesting an involvement of the associated
polymorphisms in regulation of splicing and protein
isoform production. (B) In the opposite DNA strand,
there is a previously unknown gene, AAA1, with at
least 18 exons (numbered 1 to 18) with complex
alternative splicing. AAA1 spans a total of 500 kb of
genomic sequence. Eight exons of GPRA (E1 to E8)
are shown for orientation. (C) Northern blot
hybridization with a 1285-bp full-length GPRA-A
cDNA probe (left) and a mixed splice variant probe
for AAA1 (right). A 2.4-kb transcript is visible in all
nine lanes (upper arrow) and a 1.8-kb transcript
(lower arrow) in four tissues for GPRA. Several
alternative transcripts are seen for AAA1 (arrows).
GPRA expression patterns in tissues
Fig. 4. (A) Expression of GPRA isoform B
in bronchial biopsies from a healthy
control (left) and an asthma patient
(right). E, epithelium; BM, basement
membrane; LP, lamina propria; SM,
smooth muscle. (Top) The airway
epithelium in the control sample shows
only faint staining. Results are typical of
8 asthmatic and 10 control biopsies
studied. (B) Relative expression levels
of Gpra mRNA in lungs from sensitized
(n = 7) and control (n = 8) mice after
inhaled ovalbumin challenge. Gpra was
significantly up-regulated in sensitized
compared with control mice. (C)
Variable alternative splicing for AAA1
depending on genotype.
GPRA
• The properties of GPRA make it a strong candidate for
involvement in the pathogenesis of asthma and other IgE-mediated
diseases, as well as a possible drug target.
• GPRA might act as a receptor for an unidentified ligand
• The putative ligand, isoforms of GPRA, and their putative
downstream signaling molecules may define a new pathway
critically altered in asthma.
• GPRA encodes isoforms that are produced in distinct patterns by
bronchial epithelial cells and smooth muscle cells in asthmatic and
healthy individuals.
• GPRA is also expressed by gut epithelia and keratinocytes of the
skin, suggesting a potential role in a wider spectrum of allergic
diseases.
Acknowledgements
Key group members
• Asthma: Tarja Laitinen,
Siru Mäkelä, Anne Polvi,
Johanna Vendelin
• Computational methods:
Päivi Onkamo, Petteri
Sevon, Vesa Ollikainen
Collaborators
• Asthma mapping: Lauri A.
Laitinen, Mark Daly, Tom
Hudson, Eric Lander
• Computational methods: Heikki
Mannila, Hannu T.T. Toivonen
• Gene expression: Riitta
Lahesmaa
One day I’ll
mutate…