SUBJECTS and METHODS

One hundred and ninety-two children of bilateral German ethnicity diagnosed with AD were recruited from the MAS'90 cohort along with 59 non-atopic children. Genomic DNA was collected from one or both parents in 77 of the 192 children with the diagnosis of AD. The MAS'90 cohort initially comprised 1314 unrelated, healthy, mature newborns born in 1990 (^{Bergman et al. 1994}). Parents participating in the study gave their written informed consent as approved by the Ethics Committee (Berlin, Germany). Yearly follow-up visits from birth until present were conducted. the diagnosis of AD was defined by the criteria of MAS'90 (^{Bergmann et al. 1997}): a combination of ''dry skin'' and at least three of nine typical signs (e.g., itching, erythema, red papules) at more than three of 26 typical anatomic sites (e.g., cheeks, retroauricular, extensor and flexural areas).

In addition, 40 Swedish families in which at least one member had AD were recruited from the International Study of Asthma and Allergy in Children (ISAAC) cohort (3537 children, aged 13–14 y) in Linköping. Recruited families had at most one atopic parent and at least three children, at least one of whom had a history of allergy as defined by the criteria in the ISAAC (^{Asher et al. 1995}). The ISAAC questionnaire included five questions related to skin symptoms, including AD (^{Williams et al. 1999}). A diagnosis of AD was based on the criteria suggested by^{Hanifin & Rajka, (1980)}.

Genomic DNA was purified from blood (Qiagen Blood DNA Kit) or jugal epithelial cells (^{Aron et al. 1994}). Microsatellite markers were analyzed as described before (^{Marsh et al. 1994}). Markers were analyzed for linkage and allelic association on the following chromosomes: 5q31–33 (D5S642, D5S666, D5S 1972, D5S643, D5S436, D5S207, D5S210, D5S2090, CSF1R), 6p21.3 (D6S291, DQCAR, and D6S273), 12q15–24.1 (D12S1052, D12S379, D12S1064, D12S351, D12S95, PAH), 13q12–31 (D13S219, D13S894, D13S1491, D13S218, D13S1288, D13S1248, D13S263, D13S291, D13S328, D13S270, D13S284, D13S318), 14q11.2, and 14q32.1–32.3 (TCRD, D14S34, D14S260, D14S542). All primers were supplied by Research Genetics (Huntsville, AL).

To test for association in the German population the frequencies of various alleles were compared among children affected with AD and unaffected controls using a ² test for a 2N (alleles) contingency table (GAS, Version 2.0, routine ASSCOMPARE). Aditionally, a ² test for an allele of interest is performed by collapsing the 2N table into a 22 table comparing a given allele of interest with all others. In the 77 German trios and 40 Swedish nuclear families, parental transmission of marker alleles was analyzed using results from two transmission disequilibrium test (TDT) procedures (SIB-PAIR, David L.Duffy, Version 0.93, 1995/1996;^{Duffy 1996}). The first is a multiallalic test for complete symmetry in the full NN transmission table, where N is the number of alleles at the locus (^{Duffy et al. 1990}). The null hypothesis is tested with a Pearson ² statistic proposed by^{Ewens & Spielman 1995}), the ''global (symmetry)'' TDT. The second analysis is performed using each allele in turn with the comparison group being all other alleles, an ''allele-by-allele'' TDT (^{Spielman et al. 1993}). Affected sib pairs from the Swedish nuclear families were used in an affected sib-pair analysis using the program SIBPAL 2.7 included in the SAGE 2.2A suite (^{SAGE, 1994}).

Results

AD is associated with chromosome 13q12–14 and chromo- some 5q31–33 markers in the German population

In the German population comparison of 192 unrelated AD children with the unaffected control group showed a significant association between AD and D13S218 on chromosome 13q12–14 using both the global and allele-by-allele statistics (Table 1). Using the TDT, parental transmission of marker alleles showed significant evidence for linkage to the same marker (Table 1). On chromosome 5q31–33 evidence for association for the markers D5S436 and D5S643 (1 cM upstream of D5S436) were also found by both analysis (Table 2).

Table 1 - Association and linkage to a polymorphic marker (D13S218) on chromosome 13q12–14 in the German population.

Full table

Table 2 - Association and linkage to two adjacent markers (D5S436, D5S643) on chromosome 5q31–33 in the German population.

Full table

AD is associated with chromosome 13q12–14 markers in the Swedish population

In the Swedish families, evidence for association and linkage was also found for D13S218 using the global and allelic TDT and the affected sib-pair analysis (Table 3). The only other evidence for association was found with an allele at marker D5S436 (allelic TDT=4.0, p=0.046 for allele 250 where four were transmitted and 12 were not transmitted to the affected child; Ewens TDT=13.5 (12 df), Ewens p=NS). None of the investigated markers on chromosome 6p, 12q, and 14q showed significant evidence for association with AD in either study population (data not shown).

Table 3 - Association and linkage to a polymorphic marker (D13S218) on chromosome 13q12–14 in the Swedish population.

Full table

Discussion

Using the candidate gene approach, linkage of bronchial hyper- responsiveness and high total serum IgE to chromosome 5q31.1–q33.3 has been described (^{Marsh et al. 1994;Meyers et al. 1994;Postma et al. 1995}). Interestingly,^{Meyers et al. (1994)} and^{Postma et al. (1995)} found their most significant finding for the marker D5S436, the same marker showing evidence for association with AD in this study. Chromosome 5q31.1–q33.3 is a region of special interest as several candidate genes for atopy and atopy-related phenotypes map to this region: interleukin (IL)-13, IL-4, IL-5, interferon regulatory factor 1, IL-3, granulocyte–macrophage colony-stimulating factor, IL-9, and granulocyte–macrophage colony-stimulating factor 1 receptor (^{Marquet et al. 1996}). Interestingly, D5S436 maps in the region centromeric of CSF1R and telomeric of CSF2 (^{Marquet et al. 1996}), where the glucocorticoid receptor is located (^{Huebner et al. 1990}). Abnormalities in glucocorticoid receptor binding have been observed in patients with steroid-resistant asthma (^{Sher et al. 1994}) and in patients with AD (^{Clayton et al. 1995}).

One candidate gene that maps in the region of chromosome 13q is the IgE-dependent histamine-releasing factor (^{MacDonald et al. 1999}). One nearby marker yielding evidence of linkage to atopy in two distinct populations (Australian, UK) (D13S270) was also used in our analysis (Daniels et al. 1996). This marker showed no evidence for linkage to AD in our two study populations. Recently, a genome-wide scan described evidence for linkage to asthma in Caucasian sib-pairs in 13q21.3-qter using multipoint analysis (The Collaborative Study on the Genetics of Asthma, 1997); however, we did not observe evidence for linkage nor significant association to AD with marker D13S318. Few known candidate genes for AD map in region of D13S218, e.g., the high-mobility group protein 1. High-mobility group protein 1 plays a part in gene regulation as a transactivator or quasi-transcription factor (^{Aizawa et al. 1994}). High-mobility group protein 1 binds to conserved lymphokine element-0 in the IL-5 promoter (^{Marrugo et al. 1996}). It also binds to the conserved lymphokine element 0 of IL-4, granulocyte–macrophage colony-stimulating factor and granulocyte colony-stimulating factor genes. Polymorphisms in the high-mobility group protein 1 gene may lead to interference in the transcription of IL-4 and IL-5.

Many AD patients will develop asthma or allergic rhinitis later in life. It seems that similar genetic and immunologic mechanisms are involved in these associated atopic diseases. So it is likely that a region on chromosome 5q31–33 is associated with and linked to bronchial hyperresponsiveness, total IgE, and AD. Asthma, atopy, and elevated IgE levels are unlikely to be caused by single gene alterations but rather by a complex interaction of both major and minor disease susceptibility genes and environmental factors. Although there is an overlap in these phenotypes, differences do occur. We hypothesize that there are both common and distinct areas on chromosomes that are associated with one of these phenotypes. Unlike chromosome 5, on chromosome 13 it seems that the different phenotypes are linked not to the same, but to different regions; also, on chromosome 12, evidence for linkage was shown to both, asthma and total IgE (^{Barnes et al. 1996;Nickel et al. 1997}), but not to AD.

In conclusion, we have identified two chromosomal regions showing evidence for both association and linkage with AD. Further linkage studies using a genome-wide screen would provide further insight in the genetic basis of AD.

References

References

Acknowledgments

We thank all families for their generous participation in this study and the physicians, research nurses and technicians for their essential contribution. We especially thank Xie Lun Xue, Xiu Liu, and Eva Ehrlich for excellent technical support. This study was funded by the German Ministry of Research and Technology (BMFT) grant no. 01EE9406, NIH grant AI20059, and by the Vardal Foundation and the National Association for the Prevention of Asthma and Allergies. Kirsten Beyer was supported by BMFT grant no. 01EE9606.