Genetics of Asthma
The University of Toronto Program
A. S. SLUTSKY, N. ZAMEL, and the UNIVERSITY OF TORONTO GENETICS of ASTHMA RESEARCH GROUP
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, The Toronto Hospital, Women’s College Hospital, and University
of Toronto, Canada
In 1991, we initiated a study to identify susceptibility gene(s) predisposing individuals to the development of asthma. Our strategy was to focus on the collection of inbred populations because (1)
they are likely to exhibit greater homogeneity than outbred populations, potentially important in
multigenic diseases, (2) there may be fewer genes explaining the asthma diathesis in families with a
“founder effect,” (3) it is possible to follow the pattern of inheritance more closely, and (4) environmental factors are likely to be more uniform in geographically isolated poulations. We have identified, and in some cases collected data on inbred and/or isolated populations in Brazil, China, Easter
Island, Israel, and Tristan da Cunha. We have completed a genome-wide scan on samples from
Tristan da Cunha based on a coverage of approximately 20 cM between markers. In addition, we
have collected hundreds of outbred individuals from Canada; these data have been helpful in narrowing a linked region based on the transmission/disequilibrium test. Slutsky AS, Zamel N, and the University of Toronto Genetics of Asthma Research Group. Genetics of asthma: the University of
AM J RESPIR CRIT CARE MED 1997;156:S130–S132.
Toronto program.
In 1991, using a grant from the National Sanitarium Association, we initiated a program at the University of Toronto to
identify susceptibility genes predisposing individuals to the development of asthma. In 1994, we developed a collaboration
with Sequana Therapeutics, Inc., a gene searching company,
and in 1995, we developed a collaboration with Boehringer Ingelheim Ltd., a multinational pharmaceutical company.
Our initial strategy was to collect families for a genomewide scan looking for linkage. Although we started to collect
nuclear families with affected sibling pairs, a major focus of
our program over the past few years has been identifying and
collecting data from isolated populations that have a relatively
high prevalence of asthma. There are a number of theoretical
advantages to such an approach. First, since asthma is a multigenic disease, it is likely that there will be greater genetic heterogeneity in outbred populations than in inbred populations.
Second, there may be fewer genes in families in which there is
a “founder effect.” Third, it is possible to follow the pattern of
inheritance more closely in inbred populations (1). Finally,
environmental factors that contribute importantly to the development of asthma may be more uniform in geographically
isolated populations. To this end, we identified isolated populations in Tristan da Cunha, Easter Island, and Israel, as well
as extended families in Brazil and China. This report will
briefly summarize these data collections, focus on the data collected from Tristan da Cunha, and present an outline of our
genetic strategy. A more detailed description of the Tristan da
Cunha study has already been published (2).
CLINCIAL METHODS
Clinical histories were obtained using a questionnaire that
captured data on physician diagnosis of asthma; presence of
respiratory symptoms such as cough, sputum, and wheezing; the
presence of other chest disorders including recent respiratory
tract infections; allergic history; asthma attacks including age at
onset/offset, severity, and precipitatory factors; other illnesses;
smoking history; and all medications taken within the past 3 mo.
Wherever possible, we obtained details of the family pedigree.
Skin-prick tests to 15 common allergens were obtained,
along with saline and histamine controls. Wheal sizes were
corrected by subtracting the saline control wheal diameter,
and a corrected wheal size > 3 mm (recorded 10 min after application) was considered to be a positive response.
Spirometry was measured; if FEV1 was , 70% of predicted, airway responsiveness was assessed by delivering 400 mg
of salbutamol via metered-dose inhaler (MDI). An improvement in FEV1 > 15% at 15 min was considered a positive response. For patients in whom baseline FEV1 was > 70% of predicted, methacholine challenge response was determined using
the tidal breathing method (3). Doubling doses of methacholine from 0.03 to 16 (or greater) mg/ml were administered by a
Wright nebulizer at 4-min intervals to measure the provocative
concentration (PC20). In all studies, participants were asked to
withhold bronchodilators at least 8 h before testing; inhaled or
systemic steroids were maintained at the usual dosage.
Total serum immunoglobulin E (IgE), when measured, was
measured using a two-site chemiluminometric (sandwich) assay (Ciba Corning Automated Chemiluminescence System).
CLINICAL RESULTS
Correspondence and requests for reprints should be addressed to Arthur S.
Slutsky, M.D., Mount Sinai Hospital, 600 University Avenue, Room 656A, Toronto, ON, M5G 1X5 Canada.
Am J Respir Crit Care Med Vol 156. pp S130–S132, 1997
Canada
We had two goals in collecting patients from Canada. The first
was to obtain patients that could be used for linkage analysis.
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Slutsky and Zamel: University of Toronto Program
Therefore, we recruited families with at least one affected
child and parents who were available to be studied. We have
evaluated approximately 60 such families using all of the phenotypic tests decribed above. Approximately one third of
these families had affected sibling pairs. Over the past two
years, our focus has changed to the collection of families that
could be analyzed using TDT (transmission disequilibrium
tests) to help refine the linked region (4). We have studied approximately 200 families consisting of a proband who has had
most of the phenotypic testing described above and at least
one parent, in whom phenotyping is not usually done. In the
parent, blood is collected for DNA analysis.
China
In 1994, we collected data from a multigenerational family
that has a high prevalence of asthma (5). The family, initially
described in the Chinese literature, lives on Nantian Island, a
few hundred miles south of Shanghai. We collected data on
118 individuals from four generations (6). An asthma history
was reported by 28% of the family; 33% had a PC20 , 8 mg/ml
or improvement on postbronchodilator FEV1 > 15%; and
22% were atopic as defined by skin tests.
Figure 1. Distribution of methacholine PC20 in all participants (n 5
226); proportion with a positive challenge (PC 20 , 8 mg/ml) is
46.9%. (From Reference 2, by permission.)
Brazil
In 1995, we collected data on 205 individuals (141 family members and 64 spouses) in a four-generation Brazilian family. Approximately 35% of the family had hyperresponsiveness, 29%
had a history of asthma, and 41% had evidence of atopy (7).
Tristan da Cunha is a tiny volcanic island in the middle of the
South Atlantic Ocean that has been termed the most isolated
inhabited location on earth. It is 2,400 kilometers from the
nearest mainland, has no airport, and all of the island’s inhabitants can trace their origins to a few ancestors who settled the
island in the early 19th century. Two previous studies have
suggested that at least 30% of the inhabitants have asthma (9,
10). In addition to the inbred nature of this population, important environmental factors make this an excellent population
study. Virtually all of the current inhabitants were born on the
island. Because of the topography of the island, all inhabitants
live in a relatively small settlement of 90 homes clustered at
one end of a narrow plateau (, 2 square miles). These homes
are generally made of the same materials, and the islanders
share common occupations (farming, sheep shearing, fishing)
and have essentially the same diet (potatoes, fish, mutton,
beef, and poultry). Furthermore, there is virtually no air pollution on the island because of the lack of industry and the
strong prevailing winds. Thus, even through environmental
factors may play a role in the development of asthma in these
individuals, the differences in the environment alone are unlikely to explain the development of asthma in some subjects
and not in others, because the environmental background is so
constant.
Data collection on Tristan da Cunha was carried out by
Peter Sandell (Medical Officer on the island at the time), Noe
Zamel, and Pat McClean in September–October 1993. At the
time, there were 289 islanders residing on Tristan da Cunha.
In total, data were obtained on 282 subjects (including visiting
TABLE 1
TABLE 2
PREVALENCE OF ASTHMA AND OTHER CHARACTERISTICS
OF THE TRISTAN POPULATION WHO HAD
BRONCHIAL CHALLENGES (n 5 254)
PREVALENCE OF ASTHMA AND OTHER CHARACTERISTICS
OF THE TRISTAN POPULATION WHO HAD BRONCHIAL
CHALLENGES—NONSMOKERS ONLY (n 5 214)
Easter Island/Israel
We have identified approximately 60 families with at least one
asthmatic member in Easter Island and have recently analyzed questionnaire data from this family. We have also begun
a study in Israel of Coshin Jews. These individuals, who are
said to have an increased prevalence of asthma, are descended
from individuals who left Israel in approximately 1,000 B.C.E.
and moved to southern India. They lived in relative isolation
and were highly inbred until moving back to Israel over the
past 50 years. There are approximately 1,500 individuals in
four locations in Israel. Families that have been inbred for
long periods of time offer advantages in fine mapping of disease loci (8).
Tristan da Cunha
% of Population
Age†
Atopic, %
Baseline FEV1, % pred†
Smoking Hx , 1 pk yr, %
Asthma*
(AR1, Hx1)
AR1
(Hx2)
Hx1
(AR2)
AR2, Hx2
23
44 6 20
74
81 6 23
14
23
37 6 21
45‡
89 6 15†
9
13
45 6 21
55
92 6 22‡
21
42
41 6 19
32‡
96 6 15†
19
Reprinted from Reference 2, by permission.
* PC20 , 4 mg/ml or bronchodilator response > 15%.
†
Mean 6 SD.
‡
p , 0.01 for difference from asthmatic group.
% of Population
Age†
Atopic, %
Baseline FEV1, % pred†
Asthma*
(AR1, Hx1)
AR1
(Hx2)
Hx1
(AR2)
AR2, Hx2
23
43 6 20
78
84 6 19
25
35 6 21
48‡
91 6 13§
12
39 6 19
54
100 6 13‡
40
38 6 18
30‡
98 6 13‡
Reprinted from Reference 2, by permission.
* PC20 , 4 mg/ml or bronchodilator response > 15%.
†
Mean 6 SD.
‡
p , 0.01 for difference from asthmatic group.
§
p , 0.05 for difference from asthmatic group.
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AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE
Figure 2. Methacholine dose-response curves in nonsmoking subjects (total n 5 199) with AR1, Hx1, (n 5 38); AR1, Hx2 (n 5
36); AR2, Hx1 (n 5 25); AR2, Hx2 (n 5 100); compared with nonsmoking controls (n 5 49). (From Reference 2, by permission.)
expatriates and 9 islanders who were temporarily off the island). The age of the subjects ranged from 3–94 yr, and the
participation level was 97% of the eligible population. Of these,
226 completed a methacholine challenge and 28, a bronchodilator response. Data were grouped based on the history of
asthma and airway hyperresponsiveness. Definitive evidence
of asthma included those with a positive history of asthma
(Hx1) and airway hyperresponsiveness (AR1). Partial evidence of asthma was defined as either AR1 with no history
(Hx–), or AR– with Nx1. No evidence of asthma was defined
as AR– and Hx–. Of the 282 islanders tested, 56.7% had at
least some evidence of asthma (Table 1). Twenty-three percent had a definitive diagnosis of asthma using the AR criteria
of PC20 , 4 mg/ml. Atopy was common, with 47% of the population testing positive to one or more extracts. The most
common allergens were house dust mites, Dermatophagoides
pteronyssinus and Dermatophagoides farinae. Atopy was more
prevalent in asthmatics (74%) than nonasthmatics (32%).
Forty-seven percent had a positive methacholine challenge
(Figure 1).
To examine the influence of smoking history, we reanalyzed the data to exclude 40 islanders who had a current or
past smoking history of > 1 pack-years. The resulting prevalence of asthma, atopy, and baseline pulmonary function was
essentially the same when these patients were excluded (Table
2). The average methacholine dose-response curve for each of
the groups (nonsmokers only; n 5 199) is given in Figure 2,
which also includes control subjects who were recruited in
Toronto for a different study. These control subjects were
healthy nonsmokers with no history of respiratory disease.
Their mean age was 31.2 6 8.3 yr and 29% were atopic.
GENETIC STUDIES
Our global strategy has been to complete a genome-wide scan
on samples from Tristan da Cunha looking for linkage. Therefore, we set out to replicate these findings with our data from
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1997
the other populations, specifically from the Canadian families.
We then saturated the linked regions with more markers and
began the physical mapping. The TDT families collected from
Toronto were used to narrow the region. When it was sufficiently small, the region was sequenced, gene identification
carried out, and the genes examined to try to identify any mutations.
The genome-wide scan is now complete using . 250 markers. This gave a coverage of approximately 20 cM between
markers. The microsatellite markers were mostly di-nucleotides, and 30–40% were tri- and tetra-nucleotides. An affected
sibling pair analysis was used, and we found 13 possible linkages (p , 0.05) to the phenotypes of asthma or bronchial hyperresponsiveness. Two of these regions had p values , 0.0001.
There were 10 possible linkages (p , 0.05) for atopy. None of
the atopy phenotypes was significant at the p , 0.001 level,
but it should be noted that at the time we did not have IgE levels on the subjects from Tristan. One of the two linkages to the
asthmatic phenotype was replicated in the family material collected from the Toronto families.
We initially narrowed the region of this one linkage to approximately 1,000 Kb and have determined that this region
contains approximately 16 genes, 13 of which are novel. Using
the TDT families, we have further narrowed this region to approximately 300–500 Kb containing approximately three
genes. Further studies will be required to determine if one of
these three genes indeed represents a susceptibility gene for
asthma.
Acknowledgment : We would like to acknowledge the following individuals
who made these studies possible: in Toronto, Dr. Meyer Balter, Dr. Ken
Chapman, Mr. Bruce Dzyngel, Dr. Kezheng Gan, Ms. Pat McClean, Dr.
Steve Kesten, Dr. Kathy Siminovitch, and Dr. Susan Tarlo; in China, Dr.
Shui-Wang Zhang, Dr. Jixui Zhang, and Dr. Yongming Zhou; in Montreal,
Dr. Jean-Luc Malo; in Paris, Dr. Alain Lockhart; and in Tristan da Cunha, Dr.
Peter Sandell and all of the Islanders who participated with tremendous enthusiasm.
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