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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