INTRODUCTION

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Asthma is a common inflammatory disorder characterized by respiratory symptoms such as wheeze, cough, shortness of breath, and presence of airways hyperresponsiveness. It is associated with other clinical conditions such as atopy, and total serum elevated IgE (1). Asthma, hay fever, and eczema are associated under the general term atopic hypersensitivity (OMIM*147050). However, atopy is quite common in nonasthmatic subjects. Atopic asthma is a complex disease with multiple genetic and environmental determinants.

Several reports have indicated multiple regions of the genome that are likely to contain susceptibility genes for asthma and related phenotypes in different racial groups (2). Different markers on chromosome 12 have been described to be linked to atopic asthma phenotypes in different populations: in the U.S. whites and Hispanics (3), in the British (6), in the Hutterites (4), in Minnesota families (7), in Afro-Caribbean and in Amish kindreds (8), and in the Germans (5, 9).

The purpose of this study was to investigate linkage of chromosome 12 markers with allergic asthma phenotypes in a large and well-characterized family sample of the Italian population.

	METHODS

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Families and Phenotypes

During a period of 3 yr (1995-1998), 116 families were ascertained through an affected allergic asthmatic child, at the Paediatrics Clinic of the University of Verona or at the Hospital of Bolzano, both located in northeastern Italy. Each family includes at least two affected children. A panel of 560 individuals (308 males; 252 females) was phenotyped for asthma through a physician diagnosis according to the definition of the American Thoracic Society (ATS) (1). All the patients defined as asthmatics had recurrent symptoms, clinical improvement from inhaled steroids, and reversibility of the bronchial obstruction after administration of ₂-agonists with an increase in FEV₁ greater than 12% (10). Moreover, determination of total serum IgE, skin-prick test (SPT) reactivity against common allergens, and bronchial hyperresponsiveness (BHR) to methacholine were performed as previously described (11, 12). The protocol was approved by the local ethical committee. High IgE was defined as a qualitative trait with serum concentration greater than 200 kilounits/liter [kU/L] for the adults, and an age-adjusted value for children younger than 10 yr of age. A subject was defined as positive to SPT when he or she was positive against at least one of the allergens tested (11). Atopy was defined as a qualitative trait with high IgE and/or SPT positivity. BHR phenotype was defined as positivity to a methacholine challenge test defined as a decrease in baseline FEV₁ by  20% (PC₂₀) at a concentration < 25 mg/ml methacholine. Table 1 shows the distribution of the intermediate phenotypes relative to clinical asthma status. The geometric mean of IgE in asthmatics was 407.2 kU/L (range, 4.34 to 8,125 kU/L) and 78.8 kU/L in nonasthmatics (range, 3.29 to 7,720 kU/ L). In subjects with PC₂₀ < 25 mg/ml (PC₂₀ was not measured for values > 25 mg/ml) the geometric mean of PC₂₀ was 2.88 mg/ml in asthmatics (range, 0.08 to 24 mg/ml) and 4.98 mg/ml in nonasthmatics (range: 0.24 to 22 mg/ml). The 116 families, which include some multigeneration pedigrees, were split into 134 nuclear two-generation families made up of at least one affected sib pair (ASP) and parents. Therefore, some individuals have been considered twice, once as parent and once as child. Thus, the number of families used in the affected sib pair analysis was: 50 families for the clinical asthma phenotype (69 ASPs), 110 families for atopy (204 ASPs), 61 families for BHR (74 ASPs), 53 families for IgE (83 ASPs), and 103 families for SPT (178 ASPs).

Genotyping

Genomic DNA was prepared from whole blood by standard methods. Genotypes were determined on a ABI377 Nucleic Acid Analyzer (PE Applied Biosystems, Foster City, CA) after polymerase chain reaction (PCR) with fluorescent-labeled primers. DNA markers used were the following: D12S87, D12S2080, D12 S1301, D12S2196, D12S390, D12S398, D12S1056, D12S1294, D12S375, D12S1052, D12S1071, D12S1064, D12S360 (Research Genetics Inc., Huntsville, AL). All individuals were typed with all markers except 44 families that were not genotyped for D12S360.

Statistical Analyses

Statistical analyses were performed using the following computer programs: ASPEX version 2.3 for linkage analysis (13, 14) and Simulate (15, 16). ASPEX computes a multipoint parametric logarithm of odds (LOD) score (mlod) maximized over the parameter lambda(s) (the ratio of recurrence risk in a sibling with an affected sib and the prevalence of the disorder in the population). All the analyses were performed under a multiplicative model. The sib_phase module (ASPEX) was used to estimate genotypes of missing parents (26 individuals), to compute the probabilities of each identity by descent (IBD) state, and to calculate the maximum likelihood sharing at each marker locus. Marker frequencies were estimated from the genotypes of family founder members of the data set. For the simulation, genotypes of the family members for the map of linked markers (unlinked to the affection locus) were constructed with the Simulate computer program. The simulation studies were performed on asthma phenotype and gene frequencies where estimated from the family founder members. Crimap 2.4 was used for construction of the reference map (17).

	RESULTS

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The order and the relative genetic distance of the markers were determined using genotypes of 15 Centre d'Etude du Polymorphisme Humain families. They are indicated on the horizontal axis in Figure 1. The order of the markers is the same as in other published maps (Genetic Location DataBase[LDB]: cedar. ). Multipoint analysis displayed the highest mlod score estimation for the asthma phenotype with D12S390 DNA marker (mlod = 2.81; p = 0.003; lambda(s) = 2.25), as shown in Figure 1. The significance of the mlod statistics was determined through simulations. A total of 10,000 replicates were simulated for all markers in the reference map. A mlod greater than 2.81 was reached for 30 replicates.

View larger version (10K): [in this window] [in a new window]   Figure 1.   Maximal multipoint lod score (mlod) of clinical asthma against a sex-averaged genetic map of markers on chromosome 12. On the x-axis, relative marker distances (centiMorgans) organized from centromeric to telomeric (right) direction.

No other multipoint LOD score was greater than 1 except in one case, for the asthma phenotype again, around marker D12S1052 [mlod = 1.32; lambda(s) = 1.67]. BHR showed a maximum LOD score around marker D12S390 [mlod = 0.81, lambda(s) = 1.47]. A mlod = 0.95 [lambda(s) = 1.25] was associated with marker D12S1294 and atopy. SPT showed two peaks, one with a mlod = 0.71 [lambda(s) = 1.25] around marker D12S1294, and one with a mlod = 0.64 [lambda(s) = 1.25] around marker D12S390.

	DISCUSSION

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We indicate suggestive linkage for clinical asthma with marker D12S390 in Italian families. According to the guidelines of Lander and Kruglyak, significant linkage would have been demonstrated with a LOD value of 3.6 (18).

In our sample, 23.7% of asthmatic subjects do not have BHR (46 of 194, see Table 1). At the time of enrollment in the study, the majority of the patients were already receiving inhaled corticosteroids. Therefore, the treatment may have reduced their BHR (19). We do not believe that they represent misdiagnosis, as the diagnosis of asthma was always performed by the same persons (A.B. or L.P.), according to the definition of the ATS (1). All the patients defined as asthmatic has recurrent symptoms, clinical improvement from inhaled steroids, and reversibility of bronchial obstruction after administration of ₂-agonists. The treatment of the different asthmatic children was planned according to the International Guidelines (20), and specifically, children with infrequent episodic asthma received ₂-agonist when needed, and those with frequent episodic or perennial asthma were treated with inhaled corticosteroids.

Linkage of D12S390 with clinical asthma, and lack of linkage with BHR may suggest that genetic determinants located in the region may influence the appearance of symptoms and lung function, which were the parameters considered for the doctor's diagnosis of clinical asthma, but the effect of the therapeutic measures may also be involved.

In the D12S390 region, several possible candidate genes are located, e.g., natural resistance associated macrophage protein (NRAMP2), signal transducer and activator of transcription 6 (STAT6), and integrin ₇ (ITGB7) genes. No gene localized in this region has yet been linked to clinical asthma or any intermediate phenotype. More extensive mapping, and candidate gene studies will possibly identify the gene or genes involved in atopic asthma in this region of chromosome 12.

Linkage of allergic asthma associated phenotypes to chromosome 12 has been studied in several chromosome-specific searches and in genome-wide studies. These investigations were performed in different ethnic groups with a variety of DNA markers, statistical approaches, and phenotypes. Despite the differences, all the studies, except a genome scan for atopy in the British population (2), described linkage of some chromosome 12 markers with asthma or intermediate phenotypes, although in different chromosomal regions. With reference to the chromosomal region here described, we indicate the following literature data. In the Collaborative Study on the Genetics of Asthma (CSGA), a minor peak for asthma is linked to marker D12S398 and is located in the same region as here described (CSGA, see web site http://www.csga.org). Marker D12S398 in fact is the closest marker we have used distal to D12S390, around which we have detected peak linkage. In the CSGA the major peak is linked to marker D12S1064, for which marker we do not detect linkage (3). A recent study in the Barbados population indicated linkage to marker D12S313 region which includes interferon-gamma (IFN-) (21). Marker D12S1052, which showed linkage with clinical asthma in our sample (mlod = 1.32), is located approximately 6 cM from the D12S313 marker. The region for which we describe suggestive linkage to asthma coincides with the region linked to BHR slope in the German population (5, see the Asthma Gene Database web site http://cooke.gsf.de/), while another chromosome 12 region is linked with clinical asthma in that population.

These reports suggest that more than one region containing susceptibility genes for asthma or some of intermediate phenotype may exist on chromosome 12.