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in the third article, bADAM33: A Newly Identified Gene in the Pathogenesisof Asthma.Q The fourth article, bHLA-G: An Asthma Gene on Chromosome 6pQin further understanding of the underlying pathophysiology of this disease. In
this issue of the Immunology and Allergy Clinics of North America, the various
approaches to these studies are discussed with presentation of current results.
The first article, bPhenotype Definition, Age, and Gender in the Genetics ofAsthma and AtopyQ by Drs. Bottema, Reijmerink, Koppelman, Kerkhof, andPostma, discusses the critical role of phenotype and phenotype definition. Fre-
quency and expression of these diseases are different in males versus females,
which is also discussed in this article in relationship to the role of genetics and
how sex differences may affect genetic studies and results.
The second article, bFamily Studies and Positional Cloning of Genes forAsthma and Related PhenotypesQ by Drs. Smith and Meyers, presents an over-view of family studies and positionally cloned genes for asthma and related
phenotypes. This article is followed by two articles that provide more detail
on two different positionally cloned genes. The evolving story on the role of
ADAM33 in asthma is presented by Drs. Holgate, Davies, Powell, and HollowayPreface
Genetics in Asthma and Allergy
Deborah A. Meyers, PhD
Guest Editor
Major advances have occurred in the genetics of asthma and allergy, resulting
Immunol Allergy Clin N Am
25 (2005) ixxby Dr. Ober, demonstrates the power of family studies in understanding the
genetics of asthma and allergy.Family studies are one approach to genetic studies; the other major approach
is casecontrol studies (which may also be family-based), which are discussed in
0889-8561/05/$ see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.iac.2005.09.005 immunology.theclinics.com
the fifth article, bCandidate Gene Association Studies and Evidence for Gene-by-Gene InteractionsQ by Michael Kabesch. Clearly, there are multiple suscep-tibility genes for common diseases such as asthma and allergy; therefore, it
is important to test for gene-by-gene interactions as further discussed in this
article. Just as there are multiple genes involved in common diseases, there are
also strong environmental influences that need to be considered in genetics
studies, which is addressed by Dr. Martinez in the sixth article, bGeneEnvironment Interactions in Asthma and Allergy: A New Paradigm to Under-
stand Disease Causation.QAn exciting and important area of genetics is pharmacogenetics (also a gene
environment [therapy] interaction), which is reviewed in the seventh article,
bAsthma PharmacogenomicsQ by Drs. Hawkins, Weiss, and Bleecker. The finalarticle, bNew Approaches to Understanding the Genetics of AsthmaQ byDr. Meyers, discusses several new approaches that are being applied to studies
on common diseases and are beginning to be applied to asthma and allergy.
prefacexTogether, these articles provide an overview on the important aspects of genetic
studies on asthma and allergy and discussion of current results.
Deborah A. Meyers, PhD
Center for Human Genomics
Wake Forest University School of Medicine
Medical Center Boulevard
Winston-Salem, NC 27157, USA
E-mail address: dmeyers@wfubmc.edu
involved in the development of complex diseases such as asthma and atopy. The
Immunol Allergy Clin N Am
25 (2005) 621639results of genome screens in 11 different populations identified at least 18 regionsUniversity Medical Center Groningen, Hanzeplein 1, Groningen 9700 RB, The NetherlandsdDepartment of Epidemiology, University Medical Center Groningen, Hanzeplein 1,
Groningen 9700 RB, The Netherlands
Asthma and atopy are complex genetic diseases. The development of dis-
ease results from an interplay between multiple genes and environmental factors.
The application of genetics has its origin in the tremendous progress in genetic
research over the last decades. This started in 1953 with the seminal paper of
Watson and Crick on the structure of DNA [1]. It was in 1956 that the correct
chromosome number in humans was announced in Copenhagen at the First
World Congress of Human Genetics: a diploid number of 46, not 48 as previously
thought [2]. It was not merely getting the count right that was of importance; it
showed that human chromosomes could be investigated with relative facility.
This led to the discovery of genetic origins of many human diseases with
Mendelian genetic disorders and proceeded with the publication of the working
draft of the sequence of the human genome in 2001 [3,4]. Since then, large steps
have been made in the hunt for genes and in understanding the complexitybDepartment of Pediatrics, Beatrix Childrens Hospital, University Medical Center Groningen,
Hanzeplein 1, Groningen 9700 RB, The NetherlandsPhenotype Definition, Age, and Gender in the
Genetics of Asthma and Atopy
R.W.B. Bottema, MDa,T, N.E. Reijmerink, MDb,G.H. Koppelman, MD, PhDc, M. Kerkhof, MD, PhDd,
D.S. Postma, MD, PhDa
aDepartment of Pulmonology, University Medical Center Groningen, University of Groningen,
Hanzeplein 1, Groningen 9700 RB, The Netherlands
cDepartment of Pediatric Pulmonology, Beatrix Childrens Hospital,This work was supported by a grant from Zon-Mw, The Netherlands Organisation for Health0889-8561/05/$ see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.iac.2005.07.002 immunology.theclinics.com
Research and Development.
T Corresponding author.E-mail address: r.w.b.bottema@int.umcg.nl (R.W.B. Bottema).
Another equally important reason for the contradicting results are the wide-spread differences in phenotype definitions. For example, when asthma was
defined as a doctors diagnosis it was shown to reflect less severe asthma than
when it was also confirmed with a measurement of airway hyper-responsiveness
(AHR) [7]. When studying atopy, one can imagine that atopic individuals defined
by self-reported atopy constitute a heterogeneous group compared with an atopic
population defined by a positive reaction to skin-prick testing.
Finally, genetic studies may have different results because important deter-
minants of the asthma phenotype, such as age and gender of the studied popu-
lations, differ. Because asthma and atopy are heterogeneous diseases that show
large differences between age groups in disease incidence and prevalence, the
influence of certain genes may be different in childhood compared with adult-
hood. Also, different genetic mechanisms or geneenvironment interactions may
be involved in the development of asthma and atopy in men or women. Findings
of genetic studies could thus be dependent on the window of time in which the
study subjects are examined and on the gender of the subjects studied. Few
genetic studies deal with age and gender issues, and frequently studies do not
even describe the age and gender of the study population.
This article discusses the importance and influence of phenotype definition.
The influence of age and gender on asthma and atopy phenotypes is addressed as
an epidemiologic issue. Finally, the influence of age and gender on the results of
genetic studies is discussed, and examples from the literature are provided.
Asthma and atopy phenotype definitions
To find the genes contributing to the asthma and atopy phenotypes, it is
important to have a clear definition of the phenotype. Atopy can be defined as
a hereditary predisposition to produce IgE antibodies against environmental
allergens that is associated with one or more atopic diseases such as bronchial
asthma, urticaria, eczema and allergic rhinitis [8]. Asthma has been defined by
the Global Initiative for Asthma [9] as a chronic inflammatory disorder of
the airways in which many cells and cellular elements play a role. The chronicof potential linkage to asthma and atopy [5]. Hundreds of genetic association
studies on asthma and atopy have been conducted, and these have reported
variants in more than 64 candidate genes that may contribute to the development
of asthma and atopy [6]. Despite the successes of genetic research, there are great
difficulties in replicating findings between study groups. Various reasons for the
discrepant results of studies have been described, such as (1) the different ethnic
backgrounds of the populations studied, (2) the variable influence of environ-
mental factors in different countries with different lifestyles of the studied
populations, and (3) insufficient power of studies to detect minor genetic effects.
bottema et al622inflammation causes an associated increase in AHR that leads to recurrent
episodes of wheezing, breathlessness, chest tightness and coughing, particularly
at night or in the early morning. These episodes are usually associated with
Box 1. Possible definitions of asthma and atopy in genetic studies
Asthma
Questionnaire data
Symptoms occurring once or several times at follow-up(wheeze, dyspnea, cough, nocturnal symptoms)
Self-reported (doctor-diagnosed) asthma Use of asthma treatment Video questionnaire Doctor diagnosis
Intermediate phenotypes of asthma
Airway hyper-responsivenessDirect (methacholine, histamine)Indirect (exercise, mannitol, AMP, cold-air challenge)
Reversibility on C2-agonist Variability of peak expiratory flow Lung function (eg, FEV1, VC, micro Rint)
Combination of questionnaires and intermediate phenotypes
Asthma score Asthma algorithm
Atopy
Questionnaire data
Symptoms of atopic asthma, rhinitis, and dermatitis Self-reported (doctor-diagnosed) atopic asthma, rhinitis,or dermatitis
Use of atopy treatment Doctor diagnosis
Intermediate phenotypes of atopy
Serum total IgE Serum allergen-specific IgE Allergy skin tests Number of eosinophils in peripheral blood
phenotype definition, age, and gender 623
but these methods have not been standardized and validated, and a gold standardis lacking at that age. Moreover, the methods differ from the standard methods
used in older children and adults. Therefore, comparison of these methods
for small children with the standard methods cannot be justified. One more
example is the difficulty to distinguish asthma from chronic obstructive pul-
monary disease in elderly populations because both groups include subjects with
respiratory symptoms, bronchial hyper-responsiveness, and airway obstruction
that is partly reversible. To assess the importance and suitability of an intermediate
phenotype in genetic research of complex diseases, the following criteria can be
used: (1) a high genetic component, (2) regulation by major genes, (3) objective
measurement, (4) quantification, (5) feasibility in every individual, (6) diagnostic
value for asthma or atopy (sensitive and specific), and (7) enabling replication
between studies.
This article focuses on the following phenotypes: asthma assessed by doctors
diagnosis or symptoms, total serum IgE, and sensitization defined as a positive
reaction to skin prick tests or the measurement of specific IgE, and AHR. These
phenotypes have proven their usefulness in genetic studies, and the influence of
age and gender on their expression has been extensively studied [10].
The influence of age and gender: epidemiology
Asthma and age
Asthma is a heterogeneous condition with variability between patients and
within each patient over time. There is a wide variation in how the disease
presents itself and how it is being diagnosed. As a consequence, several different
asthma phenotypes have been described.
Childhood asthma
According to Bel [11], childhood asthma can be divided into four differentwidespread but variable airway obstruction that is often reversible either spon-
taneously or with treatment. These definitions show that atopy and asthma have
many features and that there is not a single measurement that confirms or
excludes either disease. To circumvent this problem in studies on the genetics
of asthma and atopy, several strategies have been used (Box 1): use of interme-
diate phenotypes, an asthma algorithm or asthma score, and (video) question-
naires with questions regarding doctors diagnosis and symptoms. The success
of these strategies depends greatly on the population under study. For example,
in a child under 6 years of age, it is not possible to perform a spirometry and a
bronchial provocation with methacholine. Other methods to measure lung func-
tion and bronchial hyper-responsiveness in small children have been developed,
bottema et al624phenotypes based on age of onset, remission, (genetic) risk factors, pathogenetic
mechanisms, prognosis, and treatment approaches (Fig. 1). The first description
of different asthma phenotypes in childhood was provided by Martinez and
colleagues [12] based on the results of a large prospective study of 1246 newborns
Fig. 1. Clinical phenotypes of asthma in childhood and adulthood. (Data from Bel EH. Clinical
phenotypes of asthma. Curr Opin Pulm Med 2004;10:4450.)
phenotype definition, age, and gender 625in Arizona (the Tucson Childrens Respiratory Study) [13]: (1) the transient infant
wheezers includes children who show nonatopic wheezing until 3 years of age
and have a favorable prognosis. (2) The nonatopic wheezing toddlers include
children who continue to wheeze beyond the third year of life and seem to de-
velop airway obstruction in relation to viral infection. These two wheezing
phenotypes in childhood are self-limiting and do not reflect asthma. (3) The per-
sistent IgE-mediated wheezers develop persistent, chronic asthma. (4) A fourth
childhood asthma phenotype was derived from a retrospective study by De Marco
and colleagues [14]. This asthma phenotype occurs during or after puberty, af-
fects mainly girls, and has a low remission rate. Most children with mild inter-
mittent asthma outgrow their asthma as adults, but the more troublesome their
asthma in childhood, the less likely they are to outgrow the disease [15].
Adult asthma
Asthma starting in adulthood seems to be different from childhood asthma.
Adult-onset asthma can be subdivided in several subtypes with distinct under-
lying pathophysiologic mechanisms, such as aspirin-induced asthma or severe
asthma [16] and steroid-resistant asthma [17].
Asthma and gender
Notwithstanding the variation in phenotype definition used to assess asthma,
epidemiologic studies of questionnaire defined asthma find apparent gender
differences in the development and outcome of asthma. During childhood and
adolescence, boys are nearly twice as likely as girls to develop asthma. The
higher male incidence [1821] and male prevalence of asthma continues until
16 years of age [2225]. This is reflected by the hospitalization rates from asthma
in early childhood. For instance, the hospital admission rate at 1 year of age is
5.3/1000 for boys and 2.9/1000 for girls in Finland [26]. The higher incidence
and prevalence of asthma in boys reverses around the age of 16 years. In young
adulthood, female gender becomes an important risk factor for the development
of asthma [27], and throughout adulthood incidence and prevalence of asthma are
greater in women [20,23,2832]. Additionally, the subtype severe asthma seems
to affect mainly adult women [16].
Total IgE and age
At birth, IgE concentrations in cord blood are generally low. Johnson and
colleagues [33] found a geometric mean IgE of 0.20 IU/mL in 538 healthy
newborns recruited from a general population. IgE levels seem to rise during
childhood and reach a peak value between 8 and 12 years of age [3339]. During
adolescence, the mean total IgE level of asthmatic and general populations
declines steeply and continues to decline at a slower pace after 35 years of age
[35,38,39] (Fig. 2A). Epidemiologic studies have shown that IgE levels of an
individual track with age; thus, high IgE levels in infancy are highly correlated to
high IgE levels later in life [35,40]. These data are largely based on cross-
sectional studies and thus may be biased by the rising incidence of allergy
observed in the last decades, which specifically affects younger individuals,
thereby suggesting a reduction with age. This does not seem to offer the full
explanation of the cross-sectional observations because one longitudinal study
finds a pattern of IgE changes with age similar to the description above [39].
Total IgE and gender
Although boys and girls have similar IgE levels measured from cord blood
at birth [33,41] and IgE levels increase with age in both genders, IgE rises more
rapidly in boys. Serum IgE levels are consistently found to be significantly higher
in boys compared with girls above the age of 6 months, and the levels remain to
be higher in boys throughout childhood [33,37,42,43]. In adulthood, men seem
to have a higher total IgE than women in a general and an asthmatic population
[4447]. This observation remains significant when corrected for smoking habits.
It is intriguing that men have higher IgE levels than women because in this age
group asthma prevalence and incidencewhich is known to be highly correlated
to the atopic status of an individualare higher among women.
Sensitization and age
bottema et al626Sensitization is defined as one or more positive skin prick test(s) or the
presence of specific IgE antibodies in serum. During childhood, the time course
Fig. 2. Hypothetical figures on the relationship between age, gender, and phenotype based on data
from the literature. (A) The geometric mean total IgE level and age in men and women in Caucasian
populations. (B) Sensitization to inhalant allergens and age (no gender difference). (C) Bronchial
hyper-responsiveness and age in men and women.
phenotype definition, age, and gender 627
Sensitization and genderStudies on sensitization in relation to gender do not show consistent results.
The results may vary with age of the population studied. The number and species
of the allergens tested also seems to be of importance. Some studies mention a
difference in the type of sensitization between males and females in childhood
and in adulthood [33,44,52,55]. This may reflect a difference in exposure to
environmental influences between males and females. However, in infancy this
explanation is not plausible.
Airway hyper-responsiveness and age
AHR is defined as an exaggerated response to contractile, nonallergic stim-
uli such as methacholine, histamine, or hypertonic saline [61]. Measurement of
AHR in early childhood is not feasible due to the cooperation and coordination
needed from the child to perform accurate lung function measurements. There is
no standardized measurement available for children younger than 6 years of age.
Because of these difficulties, studies of AHR in childhood are few and have
relatively small numbers of subjects. Furthermore, accurate dosing of the
challenging agents in children continues to be a matter of debate. As a result,
differences in study methods make comparison of studies for AHR in earlydiseases generally observed during the last decades. In one longitudinal study
[39], skin prick tests were performed at baseline and after a follow-up of 8 years
in 1333 subjects 3 to 65 years of age at baseline. This study shows an increase in
sensitization among all age groups, which has been confirmed by Broadfield
and colleagues [60]. The greatest increase in prevalence occurred among children
and teenagers, with only minimal increases above the age of 65 years. The
strength of the reaction, estimated by skin test index, peaked between 25 and
35 years of age.of sensitization to food and sensitization to inhalant allergens differs. IgE
synthesis starts during fetal life, and specific IgE to food and inhalant allergens
can be measured early in life in cord blood at birth, albeit in low concentrations
[48,49]. Sensitization to food allergens occurs mainly in infancy and tends to be
transient, whereas sensitization to aeroallergens increases throughout childhood
and tends to be persistent [50,51]. In general, sensitization seems to increase
during childhood and adolescence [36,5055] and reaches a peak in the third
decade of life. The prevalence of sensitization and the size of skin prick test
reactions decline in persons older than 30 years of age [35,44,5659] (Fig. 2B).
This time course in sensitization to common allergens was observed in cross-
sectional studies and may reflect a rising incidence and prevalence of allergic
bottema et al628childhood difficult to perform. Despite these difficulties, studies in children
above 5 years of age have shown that AHR changes with age (Fig. 2C). In
childhood, airway responsiveness seems to be relatively high. There is a decrease
larger airway lability [41]. A higher prevalence of AHR in women compared with
This could be exemplified by asthma-related genes on the sex chromosomes.3. Different environmental effects depending on age and gender, for exam-
ple, the use of oral contraceptives (gene-by-environment interaction)
4. Epigenetic effects that may be dependent on age and gender
Few genetic studies have addressed the influence of age and gender onmen [63,65,69,7173] has been identified in large epidemiologic studies in adult
populations. Several explanations for this gender difference at adult age have
been proposed. Correction for smaller airway size, lung volume, or baseline lung
function parameters in women does not explain the gender difference in some
studies [63,69,72,73], but it does in others [69,71]. These discrepancies may be
explained by differences in statistical analysis (eg, the use of a quantitative
measure [slope of dose-response curve] or an absolute measure of AHR [AHR:
yes or no]). Another explanation mentioned for the gender difference is a greater
susceptibility to the effects of smoking in females.
The influence of age and gender: genetics
Theoretically, age and gender effects could be explained by the follow-
ing characteristics:
1. Similar disease susceptibility genes but different disease expression
modified by age or gender (modifying effect)
2. Different susceptibility genes in childhood asthma compared with adult-
onset asthma and in males compared with females (genetic heterogeneity).in AHR during adolescence, and this stabilizes in adulthood [6268]. Some
studies mention an increase of airway responsiveness above approximately
55 years of age [63,65,69]. These changes in airway responsiveness with age occur
in symptomatic and in asymptomatic individuals.
Airway hyper-responsiveness and gender
Despite difficulties in comparing studies of AHR at very early age, studies
show evidence for a gender difference in AHR that is similar to the pattern of
incidence and prevalence of asthma. In childhood, le Souef and colleagues [70]
and Paoletti and colleagues [63] describe a greater responsiveness in boys than in
girls, a finding that reverts during adolescence, where girls have a higher
prevalence of hyper-responsiveness. Boys exposed to environmental tobacco
smoke show significantly greater peak expiratory flow variability, reflecting
phenotype definition, age, and gender 629the development of atopy and asthma. Notwithstanding the fact that the available
studies are small, they do provide the first evidence for age- and gender-related
effects in the genetics of asthma and atopy.
Age-related genetic studies
One of the first attempts to identify whether genetic associations vary with
age was performed by ODonell and colleagues [74]. They performed a genetic
association study on longitudinal data on atopy and a polymorphism of CD14,
a membrane receptor, involved in binding of lipopolysaccharide and thereby
possibly influencing the postnatal Th2/Th1 shift. Failure in this immune system
shift may be associated with atopic development, which makes it plausible that
polymorphisms in the CD14 gene are especially associated with early atopic
development. The CD14/159 polymorphism has been associated with alteredsoluble CD14 and IgE serum levels in several cross-sectional studies. In an
attempt to identify a possible age effect of CD14/159 polymorphism, ODonelland colleagues collected longitudinal data from age 8 to 25 years on atopy and
AHR from 305 subjects and genotyped these individuals. For atopy, AHR, and
wheeze, these individuals were classified as having early persistent (present at
age 8 or 10 years, then consistently present up to age 18 or 25 years), early
remittent (present at age 8 or 10 years and at the next visit but then consistently
absent up to age 18 or 25 years), late onset (absent at age 8 or 10 years, then
present on at least two subsequent visits, with no visits after the age of 10 years
showing absent), or no disease during follow-up. They discovered that
individuals with 159CC were at higher risk for developing early-onset atopy
and early-onset AHR (OR 2.2 and 2.6) compared with individuals carrying
159CT and 159TT. Cross-sectional analysis showed that the CD14/159 CCgenotype was associated with atopy and AHR in childhood but not in adulthood.
Thus, it seems possible that the influence of the CD14/159 polymorphism maycause an effect during childhood that fades away in adulthood. This suggests that
other genetic or environmental factors play a role in the atopic development in
late childhood or early adulthood. Because these findings do not explain why in
other studies on CD14 associations are being found in adulthood [75,76], further
studies need to confirm their observations.
Child and colleagues [77] studied glutathione S-transferase 1 (GSTP-1) be-
cause it may be of importance in the development of AHR. The enzyme is
involved in detoxification of many environmental toxins, drugs, and by-products
of oxidative stress that may otherwise cause inflammation of the airways [78].
Furthermore, variants of the GSTP-1 gene have shown association with risk of
AHR and atopy in adults [79,80]. They indicated that it might be important to
adjust for age in genetic association studies using the phenotype AHR. The
interpretation of the measurement of AHR at young age is difficult because body
size, breathing pattern, and baseline lung function may influence the measure-
ment. In an attempt to solve these issues, they describe a method to correct dose-
response slopes and PC20 values for the baseline parameters age, lung function,
atopy, and height in 145 children 7 to 18 years of age [77]. The corrections
bottema et al630made in this study had a marked effect on the AHR status in 70 of 122 children,
5 of 122 to a more severe and 40 of 122 to a less severe PC20 category. The
corrections also resulted in a significant reduction of the mean dose-response
slope. The authors showed that this correction influenced the result of a genetic
association study in these children. A previously unidentified association between
the GSTP-1 genotype and AHR was found. This association would not have been
found in this population if uncorrected AHR was tested for association with the
GSTP-1 genotype [77].
One of the most extensively studied candidate genes for asthma and atopy
is interleukin (IL)-13. IL-13, a cytokine primarily produced by T-helper type
2 (Th2) cells, may be important in the development of asthma and atopy because
it promotes B-cell differentiation and is capable of inducing isotype class-
switching of B-cells to produce IgE and IgG4 [81]. In mice, IL-13 contributes to
AHR by inducing contractions of smooth muscle cells and stimulating overpro-
duction of mucus [82]. Many studies describe one or more associations between
various polymorphisms of the IL-13 gene with asthma and atopy phenotypes. We
took a closer look at the results of studies investigating IL-13 polymorphisms to
evaluate age and gender of the populations studied. To avoid bias by differences
in ethnicity, we considered only white and Caucasian populations. Table 1 shows
that there seems to be a difference between the associations found in childhood
and associations found in adulthood. In childhood, consistent associations of
single-nucleotide polymorphisms (SNPs) in the IL-13 gene with total serum IgE
are found [8387]. Given the difficulty to establish asthma in young children, in
this age group the asthma phenotype was evaluated in only a few studies, and
no associations with IL-13 SNPs were found [83,88]. In contrast, the studies
performed in adulthood do find associations with asthma or AHR [8991], but
they do not find association with total serum IgE [89,90,92,93]. It is not clear
why IL-13 is associated with IgE in childhood and not in adulthood. Oryszczyn
and colleagues [94] showed that IgE levels seem to be stable in mid-adulthood,
which suggests that in adulthood the environment may have a small effect on
IgE level. In contrast, environmental factors in childhood may have a larger
influence, which may bring about a geneenvironment interaction that is not
apparent in adulthood. Moreover, IgE levels are somewhat lower at adult age,
compared with more variable and higher levels at childhood age, which may
affect the outcome as well.
Finally, Ruse and colleagues [95] showed that serum IgE levels, but not
the high-affinity IgE receptor polymorphisms, were associated with late-onset
airway obstruction. They interpreted their findings as follows: interaction
between environmental and genetic factors control serum IgE levels and disease
pathogenesis may differ between early- and late-onset airway obstruction
phenotypes. Thus, IgE may have different roles in childhood asthma and late-
onset asthma.
Animal studies provide evidence for the existence of age-related genetics.
Garret and colleagues [96] conducted time-course genetic analysis in selectively
bred rats to evaluate the genetic causes of albuminuria and proteinuria as early
phenotype definition, age, and gender 631markers of renal disease. Genome scans performed at 8, 12, and 16 weeks of age
identified quantitative trait loci (QTL) on nine rat chromosomes. The QTL
identified were variable with time and age of the rats. A locus for proteinuria on
Table
1
AssociationstudiesonIL-13polymorphismsin
whiteand/orCaucasian
populations
Author
Number
of
subjects
Age
Association
IL-13SNPs
tested
aTotalIgE
Sensitization
Asthma/BHR
Hoffjan[85]
207
1yr
P=.0026
n.a.
n.t.
Arg130Gln
He[88]
329
2yr
n.t.
OR2.5,P=.014
n.a.
Arg130Gln
1111C/T
Liu
[86,87]
482
Cohort;17yr
OR2.38,95%CI
1.354.21,P=.003
OR3.49,95%CI
1.528.02(foodallergens)/
OR2.27,95%CI1.044.94
(outdoorallergens)
n.t.
Arg130Gln
1111C/T
Graves
[84]
1399
911yr
P=.000002
n.t.
n.t.
Arg130Gln
1111C/T
1512A/C
DeM
eo[83]
666
Mean8.16,SD
2.11yr
P=.04
P=.0069
n.a.
Arg130Gln
Hummelshoj[92]
342
Mean27(allergic
subjects);
31(controls);range1766yr
n.t.
OR2.1,P=.0053
n.t.
1111C/T
Heinzm
ann[89]
300
Youngadults;nofurther
inform
ation
n.a.
n.a.
OR1.83,95%CI
1.132.99,P=.014
Arg130Gln
Howard[90]
368
Mean52,range3476yr
n.a.
P=.02
P=.008,P=.007
Arg130Gln
1111C/T
3Vuntranslated
regionG/A
Van
der
Pouw
Kraan
[91]
208
Notmentioned
n.a.
n.a.
OR7.8,P=.002
1111C/T
Recruitmentfrom
outpatient
departm
entofpulm
onology;
thus,probably
adults.
Nieters
[93]
640
3565yr;nofurther
inform
ation
n.a.
n.a.
n.t.
Arg130Gln
Abbreviations:
BHR,bronchialhyperresponsiveness;CI,confidence
interval;n.a.,notassociated;n.t.,nottested;OR,oddsratio;SNP,single-nucleotidepolymorphism.
aSynonymsofSNPs:Arg130Gln=Arg110Gln=Gln110Arg;1
111C/T
=C-1112T=1
055C/T
=1
024C/T
=1
112C/T.
bottema et al632
chromosome 10 was present at age 12 weeks and not at age 8 and 16 weeks. This
suggests that the genes underlying these disease loci have a variable influence on
the phenotype that changes with age. This indicates that the complex genetic
mechanisms causing renal disease differ between early onset and late onset or
progression of the disease. Future studies have to elucidate whether this is also
the case for atopy or asthma.
Gender-related genetic studies
The cytotoxic T lymphocyte-associated 4 receptor (CTLA-4) may be im-
portant in the development of atopy and asthma because it is involved in a
costimulatory pathway regulating T-cell activation and subsequent IgE produc-
tion. Chang and colleagues [97] found an association between the CTLA-4
genotype and cord blood IgE in a population of 644 Chinese newborns. This asso-
ciation with the CTLA-4 (+49 A/G) polymorphism was found only in females. In
an adult Chinese population, Yang and colleagues [98] found an association of
the CTLA-4 (+49 A/G) genotype and serum IgE levels, again only in females.
It had been previously shown that these same CTLA-4 polymorphisms are asso-
ciated with atopy or asthma [99,100]. However, these studies did not stratify for
gender in their analysis. Thus, the results on a putative role of CTLA-4 SNPs in
the development of atopy and the specific gender effects have been replicated in a
second population. Furthermore, the CTLA-4 (+49 A/G) polymorphism has been
shown to alter T-cell activation in human cells [101]. To our knowledge, there is
no biologic mechanism described to explain the gender differences observed in
this genetic association.
Szczeklik and colleagues [102] have described a polymorphism that shows
a genetic association in women with asthma, but not in men: the prostaglandin
endoperoxide H synthase COX-2 (165 G/C). This COX-2 enzyme may beinvolved in asthma development as a mediator of bronchial inflammation. The
COX-2 (165 CC) homozygotes were over-represented in female but not inmale asthmatics (odds ratio 3.08, 95% confidence interval 1.356.63, P = .01).
A functional effect of this polymorphism was confirmed by investigating pros-
taglandin production by peripheral blood monocytes in vitro as related to the
genotype of patients. Peripheral blood monocytes obtained from CC homozygous
women produced increased quantities of prostaglandins compared with mono-
cytes from female GG homozygotes. The researchers investigated only mono-
cytes from female patients; thus the question of whether this functional effect was
present only in female monocytes remains unanswered by the authors [102]. An
explanation for the gender difference in this association study is not available.
COX-2 has been studied extensively in relation to heart disease, and this may
provide a clue to the gender differences. One COX-2 inhibitor (Rofecoxib) was
recently discovered to enhance cardiovascular risks and was taken off the market.
phenotype definition, age, and gender 633This effect on cardiovascular risks was seen particularly in females and especially
in younger women who produce estrogen [103]. COX-2 is known to produce
prostacyclin, and production of this fatty acid is blocked by the COX-2 inhibitors.
Prostacyclin acts through the prostacyclin receptor. Egan and colleagues [104]
studied mice lacking this prostacyclin receptor. These mice were highly
inhibitors enhance cardiovascular risks by extinguishing the beneficial effect of
estrogen in premenopausal women. The presumed estrogen dependent biosyn-thesis of COX-2 also explains why the genetic association of the COX-2
(165 G/C) polymorphism with asthma before was only found in females. Inmales, who have low levels of estrogen, a polymorphism in the COX-2 gene has
small effects.
A polymorphism of the proinflammatory cytokine IL-1b was found to beassociated with asthma only in men in a cohort of 245 asthma patients and
405 controls [105]. The difference in genotype between asthmatics and controls
was seen comparing heterozygote men with homozygote men, irrespective of the
alleles. This is difficult to explain biologically, and the authors could not solve
this problem. There was also no biologic explanation for the gender difference
observed. These results need to be replicated before conclusions are drawn.
Summary
When studying genetics of complex diseases it is important to have a clearly
described and objective phenotype. When drawing conclusions in association
studies, age and gender of the population studied should be considered. Until we
know what causes phenotypic differences between males and females and
between children and adults, we should try to study longitudinal cohorts with
phenotype assessment at different time points and stratify our analyses for gender.
To acquire sufficient power for these types of analyses, international collabo-
ration may be the only way to elucidate the intricate geneenvironmental
interactions in atopy and asthma in an age- and gender-dependent manor.susceptible to atherosclerosis. In these mice, there was no gender gap in heart
diseasea divergence long observed in people and in mice in which younger
males are at higher risk for heart disease than younger females. Female mice
lacking this prostacyclin receptor were highly susceptible to atherosclerosis
through susceptibility to oxidative stress from free radicals, which boost plaque
formation in arteries. The authors studied the effect of estrogen in relation
to prostacyclin production and found that in mice, estrogen increased prostacyclin
biosynthesis and depressed oxidative stress. This suggests that in premenopausal
females, the relative protection for atherosclerosis may be induced by their
endogenous estrogen that, acting through one of its receptors, stimulates COX-2
production and prostacyclin production. This also explains why COX-2bottema et al634References
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$11.3 billion in 1998, of which $7.8 billion were direct medical expenses.
Therefore, genetic studies have sought to identify the genes that influence thedevelopment or progression of asthma with the hope of contributing to the un-polymorphism. A threshold may also be present at which a certain number of
variants in contributing genes are required before a disease is expressed [1].
Both the incidence of asthma and the cost of treatment have been steadily
increasing for decades. Approximately 6.7 million Americans were affected with
asthma in 1980 compared with 17.3 million in 1998 [2]. Also in 1998, there were
2 million asthma-related visits to emergency departments and 5438 reported
deaths [3]. Asthma was a contributing cause of death in another 6850 indi-
viduals, increasing the number of asthma-related deaths to 12,288 for that year
[4]. Total annual costs of asthma in the United States were estimated to beenvironmental stimulus. Interaction of multiple genes may also be required to
confer susceptibility or to increase severity. Therefore, the phenotypic effects of aFamily Studies and Positional Cloning of Genes
for Asthma and Related Phenotypes
Alicia K. Smith, PhDa, Deborah A. Meyers, PhDb,TaCenters for Disease Control and Prevention, Atlanta, GA, USA
bCenter for Human Genomics, Wake Forest University School of Medicine,
Medical Center Boulevard, Winston-Salem, NC 27157, USA
Asthma is characterized by atopy, bronchial hyperresponsiveness (BHR),
inflammation, and intermittent airway obstruction, all of which occur as a result of
the interaction between individual susceptibility and environmental exposures.
Because the relevant environmental exposures may vary, the genes involved may
be different even within a phenotypically similar group of people because of the
complexity and redundancy of many biochemical pathways. Expression of a
functional variant may not be apparent without the presence of a specific
functional variant in one gene may not be apparent without the presence of another
Immunol Allergy Clin N Am
25 (2005) 641654derstanding of asthma pathogenesis, clinical diagnoses, and treatments.0889-8561/05/$ see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.iac.2005.09.003 immunology.theclinics.com
T Corresponding author. Centers for Disease Control and Prevention, Atlanta, GA.E-mail address: dmeyers@wfubmc.edu (D.A. Meyers).
as families, are useful to address this question. The rate of phenotypic dis-
cordance is compared between monozygotic twins, who share both genetic andenvironmental backgrounds, and dizygotic twins, who share approximately 50%
of their genetic background and share their environment. Significantly higher
disease concordance in monozygotic twins suggests a genetic component of
the disease.
Asthma has been shown to aggregate in families [5], and heritability is esti-
mated from 36% [6] to 75% [7]. The associated phenotype of total serum IgE
levels have been shown to aggregate in families and are correlated with asthma
risk [8]. Family studies have also shown that hyperresponsiveness aggregates in
families with heritability estimated between 22% and 66% [911]. Twin studies
support these observations as well [12]. Twin studies also indicate that genetic
influences regulate components of asthma such as total serum IgE levels and
bronchial hyperresponsiveness [9].
Genome-wide screening
After a genetic component has been suggested, the complexity of the cell
types and signaling molecules involved makes identification of the contribut-
ing variants difficult. Candidate gene analysis is based on knowledge about a
gene and its contribution to the pathology of a disease. In a disease where the
etiology is well known and when there are a limited number of genes which may
be involved, this is a reasonable approach. However, if a disease involves
multiple biochemical pathways or if the etiology is not well characterized, ge-
nome scans can be used to identify genes for study based solely on position
within the genome.
To perform a genome scan, related individuals are used to determine if regions
of the genome are cotransmitted with a disease or phenotype. Genetic markers areDetermination of genetic component
Initially, studies are performed to examine whether or not a particular con-
dition has a genetic component. This often includes family heritability studies
which relate the proportion of total phenotypic variance into genetic and en-
vironmental components as described by:
H Vg=Vp
where H represents heritability, Vg represents the proportion of genetic vari-
ance, and Vp represents the phenotypic variance due to both genetic and envi-
ronmental components. Studies of monozygotic versus dizygotic twins, as well
smith & meyers642genotyped in families at evenly spaced intervals throughout the genome. Although
not all family members express the phenotype being studied, it is possible to
determine which markers cosegregate with the disease or a component of it. The
degree of linkage in a family is represented by a lod score which corresponds to
the log10 of the odds for linkage and is represented by the following formula:
z x log10L pedigree given x L pedigree given 0:5
where z(x) is the two-point log of odds (LOD) score (the linkage between a
marker locus and another marker or phenotype locus) and L is the likelihood of
observing a particular configuration of a phenotype and a marker locus in a family.
q is the fraction of recombinant offspring verses nonrecombinant offspring in afamily, and ranges from 0 for loci that are completely linked to 0.5 for loci that
assort independently. A LOD score of 3.3 would indicate a likelihood ratio of over
1000:1 in favor of linkage and is considered significant evidence of linkage
[13]. However, in common diseases, regions with LOD scores greater than one
may be chosen for further study, especially if the evidence for linkage has been
replicated in multiple populations. Further addition of markers, known as fine
mapping, may be used to refine a linked region or to potentially eliminate false
positive results.
Specifying a model in the LOD score analysis (parametric or model depen-
dent analysis) can often increase the power to detect linkage. In this case, a model
is built that includes estimates of mode of inheritance such as dominant or
recessive and estimated degree of penetrance and phenocopy rate. However,
because the mode of inheritance is often not known in common diseases, a
nonparametric or model independent analysis is usually performed. Analysis of
both qualitative traits (eg, asthma or not) and quantitative traits (eg, log total
serum IgE levels) are often used in genome-wide screens, and quantitative trait
loci analyses are usually more powerful in detecting linkage.
Although multiple regions of the genome have been identified by genome-wide
screens, linked regions are not always replicated between studies. Families often
have different environmental exposures or genetic backgrounds, making detection
of genes with small to moderate effects much more difficult [1]. Other reasons
for the discrepancies may include differences in parameters used in each LOD
score analysis or variations in marker density or information content. To increase
the power of a study, families with a more homogeneous genetic background
are often helpful because there may be fewer disease genes contributing to the
phenotype, and these genes may be easier to identify. However, candidate genes
identified in a more isolated group may not be relevant outside of that population.
In recent years, linkage studies and positional cloning have been used to
successfully identify genes that contribute to asthma and atopy. Genome-wide
linkage studies for asthma or related phenotypes have been completed in at least
10 populations (Table 1). Several of these linked regions have been reported in
positional cloning of genes for asthma 643multiple populations and with related phenotypes including 1p31-pter, 2q32-q34
5q23-q31, 6p21-p24, 7q11-q22, 9p21-p23, 11p13-p15, 12q13-q21, 12q23-q25,
13q14-q31, and 19q13. Fine mapping and evaluation of candidate genes within
Table 1
Linkages reported for asthma and related phenotypes
Chromosome Asthma Atopy
Pulmonary
function
1p31-pter French Dutch Chinese
Danish German
1p21-22 Japanese
Danish
1q41 Japanese
2pter German German German
2p25 Chinese
2q14 Hutterite
2q21 German
2q24 Dutch
2q32-34 United States Hispanics Dutch Dutch
German
Hutterites
3p23-26 Japanese Hutterite
3q22 Danish Danish
3q29-qter Danish Dutch
Danish
4p13 Danish
4q23 Finnish Chinese
4q32 Japanese Danish
Danish
4q34-35 Japanese German Australian
Danish
5p13-15 African Americans Hutterite
5q15 Hutterite Dutch
5q23-31 United States Caucasians Dutch
Hutterite Danish
Japanese
Danish
5q33-35 Japanese Danish
6p21-24 United States Caucasians Dutch
German German
Japanese Australian
Danish Danish
7p14-15 Finnish Finnish
7q11-22 Japanese Dutch Australian
German
Australian
7q32 Finnish
8q13 Hutterite
9p13 German
9p21-23 Hutterite Hutterite
German German
Japanese
9q31-32 German German
10p14 Chinese
11p13-15 United States Caucasians French
Australian
(continued on next page)
smith & meyers644
Chromosome Asthma Atopy
Pulmonary
function11q13 Australian
French
Danish
11q25 GermanTable 1 (continued)
positional cloning of genes for asthma 645these areas has led to the identification of several potential susceptibility or
severity genes (Box 1). Some of these regions and genes identified therein are
reviewed below.
Chromosome 1p
Multiple family studies including those in French [14] and Danish [15]
populations have reported that asthma susceptibility genes map to a region on
chromosome 1p31. Other studies link this region to atopy in Dutch [16,17] and
12q13-21 United States Caucasians German
United States Hispanics
Hutterite
German
12q23-25 Japanese French
Danish Dutch
German
13q12-13 Japanese Dutch Hutterite
13q14-31 Japanese Australian
French
Dutch
13q32-qter United States Caucasians Dutch
14q11-13 United States Caucasians
15q11 Dutch
15q22 German
16p12 Chinese
16q21 Danish
16q24 Hutterite Australian
17p12-q12 African American
17q12-q21 French French
17q25 Japanese Dutch
19q13 United States Caucasian Hutterite
Hutterite French
21q21-22 United States Hispanics Hutterite
Hutterite
22q12-13 Danish Chinese
Xp11 Danish Danish
Xq25-26 German
The following populations are represented: Australian [39], US populationsAfrican American,
Caucasian, and Hispanic [22], Hutterite [23,64], German [18], French [14], Japanese [26], Dutch
[16,17], Finnish [60], Chinese [19], and Danish [15]. Studies involving total serum IgE levels, skin
testing, or peripheral blood eosinophils are included under Atopy. Studies involving BHR, FEV1,
FVC, or FEV1/FVC are included under Pulmonary Function.
mouse model of asthma showed that gob-5 is involved in mucus secretion and is
Studies of candidate genes within this region have identified CTLA4 which is
expressed exclusively on activated T cells and acts as a negative regulator ofT cell activation. Association studies have reported the association of CTLA4
polymorphisms with asthma susceptibility, total serum IgE levels, and bronchial
hyperresponsiveness in a Dutch population [24] and with total serum IgE levels
in a Japanese population [25].
Chromosome 5qupregulated in sensitized mice [20]. Studies of its human homologchloride
channel calcium-activated 1in a Japanese population consisting of adults and
children with asthma reported association of single nucleotide polymorphism
(SNP)s with asthma as well as the identification of risk haplotypes [21].
Chromosome 2q
Evidence for linkage of markers within chromosome 2q32-q34 to asthma has
been observed in a Hispanic population in the United States [22] and to atopic
phenotypes in Hutterite [23], German [18], and Dutch populations [16,17].German [18] populations and to pulmonary function in the Chinese [19]. A
Box 1. Fine mapping and candidate gene analysis
Genotype additional markers and narrow region of linkage Test for association and linkage disequilibrium to further nar-row the region of interest
Genotype polymorphisms in candidate genes to test for as-sociation with the disease trait (multiple populations)
smith & meyers646Multiple family studies have demonstrated that asthma and atopy suscepti-
bility genes map to a region on chromosome 5q23-q35 including those in Dutch
[16,17], American [22], Hutterite [23], Japanese [26], Danish [15], and British
[27,28] populations. A cluster of cytokines important in immune regulation are
located within the region of linkage that has been genetically and functionally
implicated in asthma and atopy. Polymorphisms in interleukin (IL)-4 have been
associated with asthma, increased total serum IgE levels, and pulmonary function
[2932]. Polymorphisms in IL-13 have also been associated with asthma, total
serum IgE levels, and BHR [3335]. Both IL-13 and IL-4 are capable of inducing
isotype class-switching of B cells to produce IgE after allergen exposure, and
binding of either IL13 or IL4 to the IL4 receptor (IL4R) promotes the initial
response for Th2 lymphocyte polarization often observed in asthma.
Danish [15] populations and to atopic phenotypes in Dutch [16,17], German [18],
Australian [39], and Danish [15] populations. Candidate genes in this region
lations [22] as well as in the Hutterites [23] and Germans [18]. Pulmonary
function phenotypes have mapped to this region in a German [18] population aswell. Signal transducer and activator of transcription 6 (STAT6) is located on
chromosome 12q13. It is a transcription factor involved in both IL-4 and IL-13
mediated responses and exhibits increased expression in bronchial biopsies of
patients with asthma versus controls [46]. Tamura and colleagues published
the first association study with STAT6 and suggested its association with pre-
disposition to allergic diseases in Japanese children [47]. Additional studies sup-include human leukocyte antigen DRB1, a class II molecules of the major his-
tocompatibility complex, which has been associated with both total serum IgE
levels and specific IgE titres to common allergens in 1004 individuals from
230 families from the rural Australian town of Busselton [40]. Moffatt and col-
leagues also reported a relationship between human leukocyte antigen DRB1
variants and total and specific IgE levels in Australian Aborigines suffering from
endemic hookworm infection [41].
Another well-characterized candidate gene in this region is tumor necrosis
factor a, a multifunctional proinflammatory cytokine. Associations of the tumornecrosis factor a308 polymorphism has been reported in subjects with mild/moderate and those with fatal/near fatal asthma versus those without asthma in a
Canadian population [42]. Associations have also been reported with atopy in a
Spanish population [43], self-reported childhood asthma in a UK/Irish population
[44], and atopic asthma in Taiwanese children [45].
Chromosome 12q
Linkage analyses have demonstrated that chromosome 12q13-q21 is likely to
contain genes related to asthma in United States Caucasian and Hispanic popu-Monocyte differentiation antigen CD14 is also located on chromosome 5q31.
It functions as a receptor for bacterial cell wall components, including endotoxin
and lipopolysaccharide, and may be involved in the polarization of T lym-
phocytes. Polymorphisms in CD14 have been associated with asthma and total
serum IgE levels in Dutch [36], Indian [37], and Taiwanese [38] populations.
Chromosome 6p
Evidence for linkage of markers within chromosome 6p21-p24 to asthma has
been observed in United States Caucasian [22], German [18], Japanese [26], and
positional cloning of genes for asthma 647port the association of STAT6 variants to atopic phenotypes including total serum
IgE levels and eosinophilia [4850]. Associations of STAT6 SNPs and haplotypes
with asthma were recently reported in an Indian population [51].
Positional cloning
Advances in genetic mapping and technology have yielded more power-
ful approaches to identifying disease genes. Analysis of single markers can be
enhanced by considering the structure of linkage disequilibrium (LD) blocks
within a region. LD is the nonrandom association of alleles at adjacent loci. In
general, the closer two SNPs are to each other, the more likely they are to be in a
region that is inherited as a block. A study using a SNP within an LD block
can provide information about other markers within that block and has led to
the concept of haplotype tagging SNPs. Use of this technique can reduce the
number of markers needed as well as the cost of experiments. This, combined
with advances in high throughput genotyping, has made genome-wide studies
more accessible and allowed the study of complex diseases to evolve.
The next step in family-based genetic studies, known as positional cloning,
also uses a systematic process of locating genetic regions that are coinherited with
a disease (Fig. 1). Construction of a high-density SNP map is used to identify
common variants in the region. Association studies using these markers can
further narrow the region of interest. Replication in one or more additional popu-
lations is often used to focus the associations to the most relevant areas. Positional
cloning can identify disease-related genes based solely on position within the
smith & meyers648genome, even if the function of that gene is not yet known, and it requires no
A
B
a
b
c C
SNP 1:
SNP 2:
SNP 3:
Chromosomefrom Mother
d dSNP 3:
The high-risk variantof the disease gene is
located here
Chromosomefrom Father
A
B
a
b
C
d d
a
b
A
B
C
d d
c
c
Crossoverevent
The closer together that 2 loci are, the lesslikely a recombination event will occur
between them and they tend to segregatetogether through generations.
Therefore, evidence for linkagedisequilibrium will be obtained leading to
gene identificationFig. 1. Example of linkage disequilibrium. The location of the risk variant for the disease gene that
is being mapped is displayed on the chromosome inherited from the father. The alleles at the neigh-
boring SNPs 1 and 2 cosegregate. Identification of such a pattern leads to gene identification.
assumptions about the probable disease pathogenesis. Therefore, genes that
have been identified by positional cloning can provide additional insight into the
etiology of a disease. Asthma genes identified by this approach include PHF11
[52], GPRA [53], DPP10 [54], and ADAM33 [55] and human leukocyte antigen G
[56]; the latter two genes are discussed elsewhere in this issue.
Plant homeodomain zinc finger gene 11 (PHF11) is located on chromosome
13q14, where linkage for asthma [26] and atopy [14,17,39] has been reported.
Anderson and colleagues [57] had previously reported association in this region
with the novel microsatellite marker USAT24G1 and continued to explore the
region by developing a comprehensive SNP map for the surrounding region.
Fifty-four common variants were studied in 364 individuals from 80 Australian
nuclear families. Association of SNPs and haplotypes to serum IgE levels indi-
cated a 100-kb linkage disequilibrium block that contained SETDB2, PHF11, and
RCBTB1. Further analysis using a stepwise procedure including the most
significant SNP as a covariate until no association remained identified three PHF11
SNPs with independent effects. Six markers were then genotyped in a second set
of Australian atopic families, a British set of families with asthma, and a set of
European families with atopic dermatitis. Haplotype associations were observed
in each of the populations with asthma or total serum IgE levels and suggested a
more localized region influencing total serum IgE levels. Functional analysis
identified multiple transcript variants and noted uniform distribution among tissue
types and prominent expression in immune-related tissues. The function of this
gene is not yet fully known; however, based on structural motifs, it is predicted to
be involved in chromatin-mediated transcriptional regulation [58]. Although these
results have not yet been replicated in additional asthma populations, a family-
based association study reported variations in PHF11 were significantly associated
with childhood atopic dermatitis in an Australian cohort [59].
G-proteincoupled receptor for asthma (GPRA) is located in the chromosome
region 7p14-p15, which has been associated with asthma and atopy in a Finnish
Kainuu subpopulation [60]. Sequential rounds of fine mapping and haplotype
association analysis for serum IgE levels in the original Kainuu families and an
additional 103 trios were used to narrow the 20-cM linkage region. A 46-kb
haplotype was identified, and nonrepetitive DNA segments within this haplotype
were sequenced for SNP detection, revealing 72 novel SNPs and eight insertions
or deletions. An additional 131 trios with high IgE levels were genotyped, in-
dicating a conserved 133-kb region. Association analysis with a Canadian popu-
lation ascertained for asthma and a Finnish population ascertained for high IgE
levels confirmed the same 133-kb region. The two genes within this region were
GPRA and a series of untranslated, alternatively spliced transcripts (asthma-
associated, alternatively spliced 1 [AAA1]) which do not appear to code for a
protein. Bronchial biopsies suggested that one isoform of GPRA was differen-
tially expressed in bronchial epithelial cells and smooth muscle cells from in-
positional cloning of genes for asthma 649dividuals who have asthma when compared with healthy controls [53]. A study
of European children supports these findings [61]. Melen and colleagues [61]
examined the same haplotype blocks as the original study and reported asso-
ciation of single SNPs and haplotypes with allergic sensitization, asthma, and
allergic rhinoconjunctivitis in western European children. Associations with both
SNPs and haplotypes were also reported in a study of German children [62].
Box 2. Example of fine mapping
Association with D2S308 (allele 3) and asthma (P= .00001) Based on linkage disequilibrium, asthma gene located with100 kb of D2S308
Found evidence for significant association with asthma forSNPs in DPP10
Data from Allen M, Heinzmann A, Noguchi E, et al. Positional clon-ing of a novel gene influencing asthma from chromosome 2q14.Nat Genet 2003;35:25863.
smith & meyers650Both studies were able to replicate associations with some of the haplotypes
conferring risk and exerting protective effects as the original study by Laitinen
and colleagues [53].
Dipeptidyl peptidase 10 (DPP10) is a member of a protease family that can
cleave off terminal dipeptides from chemokines and cytokines. It is located at
2q14, where linkage to asthma has been reported [23]. Association of D2S308
with asthma was observed in a population of 244 families. Allen and colleaguesFig. 2. Example of the use of linkage disequilbirum to identify a disease gene. The degree of
disequilibrium between SNPs is shown in the square, and the degree of association with the phenotype
is shown on the y axis. (From Allen M, Heinzmann A, Noguchi E, et al. Positional cloning of a novel
gene influencing asthma from chromosome 2q14. Nat Genet 2003;35:25863; with permission.)
sequenced 462 kb of 2q14 surrounding this microsatellite and constructed a
high-density SNP LD map (Box 2). Association studies with 82 of 105 poly-
Although it is not yet known how many genes may contribute to the suscep-tibility or the severity of asthma and related phenotypes, genome-wide screens and
positional cloning techniques have been successful in identifying contributing
genes in multiple populations. The results of these studies provide additional
insight into the molecular mechanisms responsible for the development of a variety
of phenotypes. Replication with additional populationsparticularly in large-scale
studieshas been used to distinguish between false positive results or population-
specific effects or to further quantify the conferred risk. Even when individual
markers do not replicate in multiple population, association of the same region or
gene has been useful in directing future studies.
As further understanding of the linkage disequilibrium patterns within the
genome has allowed greater efficiency for genetic studies, advances in high-
throughput genotyping technology, genetic analysis methodologies, and a more
in-depth understanding of clinical phenotypes has made genome-wide studies
more accessible and cost-effective. In the future, identification of function vari-
ants with clinical relevance may be used to influence the diagnosis and treatment
of asthma.morphisms identified four islands of LD (AD), though associations with asthma
were limited to island B (Fig. 2). The most significant association was with
WTC122P, which alters the consensus binding site of CdxA, a DPP family pro-
moter element. Three SNPs were then examined in 1047 German school children.
The D2S308 allele associated in the family study demonstrated consistent
association with both asthma and atopy. Haplotypes such as WTC122P were
also significant, but it was not reported whether WTC122P was associated on its
own. Because no open reading frames had been reported in the area surround-
ing D2S308 and WTC122P, a cDNA library was screened. Further characteri-
zation including 5V and 3V RACE led to the identification of DPP10. D2S308and WTC122P were located near exon 2 of this gene. Functional analysis of
the locus indicated a complex pattern of transcript splicing with eight alternate
first exons and reported DPP10 expression in the trachea [54]. A study of
DPP10 function in the central nervous system suggested that it is a modulator of
Kv4-mediated A-type potassium channels [63]. However, its function in airway
physiology remains undetermined.
Summarypositional cloning of genes for asthma 651References
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