Genetic mapping studies

- Asthma and allergy

Nature of disease gene projects

Clinical expertiseDiagnostic classification

Genetic analysisDisease 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?

• Pedigrees and penetrance

• The threshold model of susceptibility

• Quantitative gene effects

• Diversity of disease-associated variants

How to find the asthma gene?

Expression pattern

Polymorphism Tissue

Map location

Autosomal dominant, 100% penetrance

…67% penetrance…

…33% penetrance

gene — gene +

healthy disease

Num

ber

of p

eopl

e

Quantitative measure

Threshold model of susceptibility

Diversity of mutationsPromoter variants• altered transcription

Splice site variants • altered transcript

Missense variants• altered protein function

Nonsense variants• truncated transcript

Intron variants• regulatory elements

UTR variants• transcript instability

A gene mapper’s lunchbasket for an excursion to multifactorial diseases

Linkage analysis

Population simulation and disease modelling

Multilocus association analysis

Etc.

Etc.Etc.

Genetic factors in atopy and asthmaGenetic 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%

• 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 asthmaGenetic factors in adolescent asthma

• Finnish population-based twin-family study

• 2483 twin families, participation rate 82-93%

• Finnish population-based twin-family study

• 2483 twin families, participation rate 82-93%

Laitinen et al., Am J Respir Crit Care Med 157:1073, 1998Laitinen et al., Am J Respir Crit Care Med 157:1073, 1998

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

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

Little later immigration

Rapid late population

growth (10 x / 250 y)

Small permanent settlement of

south and west coasts >2000 y

Population movement in

the 1500’s

A brief population history

Population of KainuuPopulation 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?Why study a multifactorial disease in a founder isolate?

timetime

population bottleneckpopulation bottleneck

population expansionpopulation expansion

fewer disease loci and alleles

fewer disease loci and alleles

15-20 generations15-20 generationsDepartment 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

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

Kainuu Asthma StudyKainuu Asthma Study

Disease gene mapping project

Recruitment of families Verification of diagnosesCollection of samples

GenotypingAnalysis of data

Design of studyObtaining permissions

Identification of geneFunctional analysis

Utilization

Kainuu Asthma StudyKainuu Asthma StudyRadio and newspaper advertisementsRadio and newspaper advertisements

Probands contact the research groupProbands contact the research groupInterview for entry criteria: • physician-diagnosed asthma (self-reported) • nuclear family willing to participate • parents/grandparents born in Kainuu

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

Proband • informed consent • questionnaire and interview • blood sample

Family members • informed consent • questionnaire • blood sample

Family members • informed consent • questionnaire • blood sample

Review of medical records • verification of asthma diagnosisReview of medical records • verification of asthma diagnosis

Verification of genealogy • population recordsVerification of genealogy • population records

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

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 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

most likely location for the gene

most likely location for the gene

HaplotypesHaplotypesMarker

A B C D E F

Marker

A B C D E F

1 1 1 1 1 1

1 1 1 1 1 1

2 1 1 1 1 1

2 1 1 1 1 1

3 2 1 1 1 1

3 2 1 1 1 1

4 1 1 1 1 2

4 1 1 1 1 2

1 1 1 1 1 3

1 1 1 1 1 3

1 1 1 1 2 4

1 1 1 1 2 4

3 3 1 1 2 2

3 3 1 1 2 2

3 3 1 2 3 4

3 3 1 2 3 4

Linkage disequilibrium mapping

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.

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.

AcknowledgementsKey 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…