Are genetic tests good for population screening?

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Genetic mapping studies - Asthma and allergy
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Nature of disease gene projects
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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

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How to think of gene effects in multifactorial
diseases?

• Pedigrees and penetrance
• The threshold model of susceptibility
• Quantitative gene effects
• Diversity of disease-associated variants

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How to find the asthma gene?
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Autosomal dominant, 100 penetrance
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67 penetrance
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33 penetrance
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Threshold model of susceptibility
Number of people
Quantitative measure
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Diversity of mutations
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A gene mappers lunchbasket for an excursion to
multifactorial diseases
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A brief population history
Rapid late population growth (10 x / 250 y)
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Why study a multifactorial

Why study a multifactorial
disease in a founder isolate?
disease in a founder isolate?
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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
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Genome scan

• A set of 312 microsatellite markers were chosen
  in order to find out genomic regions
  co-segregating with the disease status
• All markers genotyped in all individuals of the
  families recruited
• Linkage analysis was carried out

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Linkage results of the genome scan for asthma
with 304 autosomal and 8 X-chromosomal markers in
86 Finnish pedigrees.
Laitinen et al., Nature Genetics 2887, 2001
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A susceptibility gene for asthma in chromosome 7p

• Genome scan in Finnish families gave significant
  evidence for linkage to chromosome 7 (NPL3.9 for
  high IgE phenotype NPL3.0 for asthma)
• Result replicated in French-Canadian pedigrees
  from Saguenay-Lac-St-Jean (NPL2.7 for asthma)
• Second replication in North Karelian pedigrees
  (NPL1.9 for high IgE)

Laitinen et al., Nature Genetics 2887, 2001
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Linkage disequilibrium mapping
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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 Asthma-Related Traits.

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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
  gt2. 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.

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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).

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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.

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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.

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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

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Example an asthma gene in a population
Asthma Healthy Sum
Gene
Gene
Sum 100 000
Penetrance 20
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Association studies
Nr with disease Nr of healthy Nr of subjects
Gene A B AB
Gene C D CD
Sum AC BD ABCD
A(AC) B(BD)
Allele-specific risk
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Candidate gene regions in asthma

• Chromosome 5q31-q33
• Interleukin gene cluster no gene implicated so
  far
• Chromosome 11q13, FCER1B
• Initial results on effect largely unconfirmed
• Chromosome 16p12, IL4R
• Replicated in several studies, small effect
• Xq28, IL9R
• Replicated in several studies, small effect
• Chromosome 19p13, FCER2
• Unconfirmed
• At least 12 other more or less uncertain
  localizations

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