FULL PAPER Preferential transmission and association of the 403 G-A promoter RANTES polymorphism with atopic asthma

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1 (2005) 6, & 2005 Nature Publishing Group All rights reserved /05 $ FULL PAPER Preferential transmission and association of the 403 G-A promoter RANTES polymorphism with atopic asthma SA Al-Abdulhadi, PJ Helms, M Main, O Smith and G Christie Department of Child Health, University of Aberdeen, Royal Aberdeen Children Hospital, Westburn Road, Aberdeen, Scotland, UK Asthma is a complex inherited disease. The study was undertaken to identify the association of RANTES promoter polymorphisms with atopy and asthma using family-based association tests (FBATs) and generation-specific case control analyses. We identified 154 nuclear families (453 individuals) in whom we established RANTES promoter status using the RFLP-PCR method. Of the two known promoter polymorphisms 403G/A and 28C/G, only the former appeared with a clinically relevant frequency. A total of 61 families were eligible for assessment of transmission of the allele with asthma and atopy by the pedigree disequilibrium test (PDT). Overall, allele frequency for 403A was 38.3% and 84 of 89 (94.3%) alleles were transmitted with physician diagnosed asthma (PDA) (P ¼ 0.001). All 89 children with atopy received the mutant allele, which was more than expected following Mendelian Laws of transmission (P ¼ ). In 303 unrelated parents, significant associations of the mutant allele were for atopy with or without asthma (P ¼ 0.001). In 150 unrelated children, significant associations were for atopy alone (P ¼ 0.001) and asthma (P ¼ 0.001). No associations were found for bronchial hyperresponsiveness (BHR). The 403 G-A is transmitted with atopy and atopic asthma, although its contribution appears to relate more to atopy than asthma and BHR. (2005) 6, doi: /sj.gene Published online 9 December 2004 Keywords: RANTES promoter; polymorphism; PDT; atopy; asthma; association Introduction Asthma is a common respiratory disease with a high morbidity and significant mortality, 1 and the majority of affected individuals first present before the age 9 years. 2 Asthma, particularly in children and young adults, is closely related to atopy in that the majority of those affected also have positive skin prick tests (SPTs) to common inhaled allergens. 3 Eosinophils are the major inflammatory cells involved and for which RANTES, a member of the Beta-Chemokines subfamily, is a potent chemoattractant. 4,5 Elevated plasma levels of RANTES have been observed during acute episodes of asthma, suggesting a role in the pathogenesis of the disease. 6 Studies in the mouse model have confirmed that CCR5 and its major ligand RANTES/CCL5 are key contributors to the development of chronic fungal asthma, that RANTES can remain functional in the absence of its receptor and that neutralization of RANTES reduces the hallmarks of asthma in this model. 7 RANTES is also one of the major HIV-1-suppressive factors produced by CD8 þ cells. In this context, RANTES functions as one of the natural ligands for the chemokine receptor CCR5, Correspondence: Dr SA Al-Abdulhadi, Department of Child Health, University of Aberdeen, Royal Aberdeen Children Hospital, Westburn Road, Aberdeen AB25 2ZG, Scotland,UK. saa@iahs.abdn.ac.uk Received 01 September 2004; accepted 06 October 2004; published online 9 December 2004 thereby suppressing in vitro replication of the R5 strains of HIV-1, which use CCR5 as a coreceptor. 8 The gene that regulates this chemokine (SCYA5, CCL5) is one of several CC cytokine genes clustered on the q-arm of chromosome 17 and is one of several regions that have been shown to be in linkage with asthma. 9 A mutation in position 403 of the promoter region of RANTES gene has been associated with atopic dermatitis, polymyalgia rheumatica and rheumatoid arthritis. 10 Two polymorphisms, a C to G substitution at position 28 and a G to A substitution at position 403, have been identified by sequencing a 1031 bp PCR fragment of the upstream noncoding region of the RANTES gene in a case control HIV-1 population. 11 Three haplotypes have been found in a Japanese population with allele frequencies of for the 28C/G and for the 403G/A polymorphism, 12 although in Caucasian populations the distribution of these two polymorphisms has been found to be different with a lower allele frequency of and heterozygosity of for the 28C/G polymorphism 13 and an overall frequency of and heterozygosity of for the 403 G/A polymorphism. 14 The 403G/A point mutation results in a new consensus element for the GATA transcription factor family, 15 which suggests a role in the differential regulation of gene expression. The 403G/A single-nucleotide polymorphism has been found to be independently associated with both atopy and asthma suggesting that this chemokine may have a role both in the development of airway obstruction and

2 atopy. 16 In view of the plausible role of the 403G/A polymorphism in promoting allergic responses and its high alleleic frequency in Caucasian populations, we sought to confirm or refute the previous reported case control study 16 using a family-based population in order to account for the inherent problems associated with case control studies such as linkage disequilibrium, population admixture and sampling error. We also sought to confirm the allelic frequencies of both 403G/A and 28C/G in our population and, to our knowledge and for the first time, assess whether the mutant alleles were preferentially transmitted with physician diagnosed asthma (PDA) atopy and/or other associated phenotypes including elevated serum IgE, baseline lung function and nonspecific bronchial hyper-responsiveness (BHR). Results Of the 308 parents (age years (median 43)), 303 and 150 of the154 unrelated children (age 8 24 years (median 14)) had complete data and 138 were defined as atopic asthmatics, 24 as nonatopic asthmatics, 157 as atopic without asthma and 134 as neither atopic nor asthmatic (Table 1). The overall allele frequency for the mutant 403G-A polymorphism was 38.3%. The mutant 403 G-A polymorphism was in Hardy Weinburg equilibrium in the whole population although not in all the population subcategories. Functional binding site within 403 RANTES promoter The Matlnd program found the matrix number of the inserted sequence as V$GATA/GATA3.03. This was then applied in MatInspector to identify the functional binding site of that matrix by screening the bp length human genomic library for matches with matrix similarities of greater than Of the 153 matches found in the vertebrate group, 21 functional binding sites matched the sequence with core similarities of between 0.92 and Only one of these binding site sequences corresponded to the IUPAC string aggagataagatc of the RANTES promoter 403G-A polymorphism. This polymorphism had a core similarity of 1.00 and a matrix similarity of Family-based association tests (FBATs) Informative child parent trios were assembled from the 150 pedigrees with complete data and with at least one heterozygous parent for the mutant polymorphism. In these informative families, the mutant (G-A allele) was preferentially transmitted at high levels of significance to children with PDA (affected ¼ 89 vs nonaffected ¼ 33, P ¼ 0.001) and to children with positive SPT (affected ¼ 117 vs nonaffected ¼ 23, P ¼ ). Borderline levels of transmission were noted for elevated IgE (affected ¼ 47 vs nonaffected ¼ 47, P ¼ 0.046) and low baseline FEV1 (affected ¼ 67 vs nonaffected ¼ 41, P ¼ 0.021), but with no suggestion of significance for BHR (affected ¼ 88 vs nonaffected ¼ 40, P ¼ 0.194) (Table 2). Transmission of the mutant allele to affected children was higher than expected following Mendelian laws of transmission. As atopy (assessed by þ ve SPT) exhibited the strongest evidence for transmission with the mutant allele, we explored whether there was any suggestion of preferential transmission from mothers or fathers in those families in which only one parent carried the mutant allele. For atopy, there was no such suggestion in that 42 alleles were transmitted from mothers and 38 from fathers. Table 1 children Population characteristics of unrelated parents and Unrelated parents (n ¼ 303) Unrelated children (n ¼ 150) Gender Male 149 (49.1%) 84 (56.0%) Female 154 (50.9%) 66 (44.0%) Cigarette smokers Yes 122 (40.3%) 12 (8.0%) No 181 (59.7%) 138 (92.0%) Asthma status (PDA) Yes 93 (30.7%) 83 (55.3%) No 210 (69.3%) 67 (44.7%) Atopy status Yes (SPT Zwheal 3 mm) 210 (69.3%) 112 (74.7%) No (SPT wheal 0 2 mm) 93 (30.7%) 38 (25.3%) IgE r100 IU/ml 207 (68.3%) 41 (27.3%) IgE 4100 IU/ml 96 (31.7%) 109 (72.7%) Diagnostic categories Atopic/asthmatic 68 (22.5%) 70 (46.7%) Not atopic/asthmatic 11 (3.6%) 13 (8.7%) Atopic/not asthmatic 128 (42.2%) 29 (19.3%) Not atopic/not asthmatic 96 (31.7%) 38 (25.3%) 25 Table 2 Results of PDT analyses for 403A allele by phenotype Phenotype Informative pedigrees Parent genotype Children affected Allele transmission Z-score P pdt AG/AA GG A (%) G (%) PDA SPT s-ige FEV BHR The table evaluates only those alleles transmitted from heterozygous parents (G/A) to affected children. PDA ¼ physician diagnosed asthma, SPT ¼skin prick test, s-ige ¼ serum IgE 4120 IU/ml, FEV1r83.5% predicted, BHR ¼ PC 20o8 mg/ml methacholine.

3 26 Case control analyses Table 3a,b shows the RANTES genotype and allele frequencies in atopy subgroups. Genotype frequencies were not adequate, so allele frequencies were compared with nonatopic nonasthmatic group, in which the following was suggested. In 303 unrelated parents, the presence of the mutant allele was significantly associated with atopy alone (P ¼ 0.001, OR ¼ 5.4, 95% CI ¼ ) and with atopic asthma (P ¼ 0.001, OR ¼ 7.8, 95% CI ¼ ), but not with nonatopic asthma (P ¼ 0.29) (Table 3a). In unrelated children, atopy without asthma was significantly associated with the mutant allele (P ¼ 0.001, OR ¼ 19.2, 95% CI ¼ ), and with both atopic (P ¼ 0.001, OR ¼ 10.0, 95% CI ¼ ) and nonatopic asthma (P ¼ 0.001, OR ¼ 9.5, 95% CI ¼ ) (Table 3b). However, univariate analyses suggested that the association with atopy was independent of asthma in both parents and children. Table 4 shows the genotype and allele frequencies in asthma-related phenotypes. Genotype frequencies AG and AA were compared to GG in each phenotype group. However, AA genotype shows no significant association in which AG and AA combined genotypes were evaluated in Table 4. Also, allele frequencies were compared for each phenotype group. In both unrelated parents (P ¼ 0.001) and unrelated children (P ¼ 0.001), highly significant associations were found between the 403A allele and atopy, as defined by at least one positive skin test (Table 4). In unrelated parents, a higher frequency of the 403A allele was also seen with elevated serum IgE although this failed to achieve statistical significance (P ¼ 0.493). No such trend was apparent in unrelated children (Table 4). In the whole population, the median FEV1 was 83.5% predicted and when subjects were categorized as below this level, no association was observed with the allele in unrelated parents (P ¼ 0.217). However, a significant association with this index of disease severity was found in unrelated children (P ¼ 0.001) (Table 4). No significant associations between 403A allele were seen for BHR in either parents (P ¼ 0.730) or children (P ¼ 0.217) after accounting for age and gender (Table 4). Similar but not identical association was obtained when allele frequencies were compared for each phenotype group. Discussion The allelic frequencies of the two main RANTES promoter polymorphisms found in our population are consistent with findings in Caucasian populations. However, there are population differences in the allelic frequencies, and possible contributions to allergic disease, of the two identified promoter polymorphisms as seen in the very low rates for the 28C/G polymorphism in our population in comparison to Japanese and Chinese populations. 12,17 In addition to population differences in genetic and environmental contributions to disease expression, the inheritance of asthma and atopy is likely to be complex and to be confounded by relatively small contributions from a number of different genes. It is also likely to be confounded by partial penetrance, by asymptomatic carriers who have not yet been exposed to the relevant environmental hazard and by disease phenocopies. All of these factors are likely to be operating, thereby decreasing the power of linkage/ association analyses, which assume that asthma and atopy exhibits Mendelian inheritance. Despite these potential problems, we have shown that the RANTES promoter 403G- A polymorphism is not only transmitted preferentially to affected children but that in their unrelated parents the allele is associated with atopy alone and in combination with asthma, but not with nonatopic asthma, BHR or baseline FEV1. These observations would suggest that it is atopy rather than airway function per se that is influenced by the mutant allele and the likely associated upregulation of RANTES. In the children we studied, it was not possible to separate these features as there were relatively few asymptomatic siblings in our families and most of our asthmatic child probands were also atopic. Further examination of this issue would require a different study design and inclusion of families with larger numbers of unaffected children and/or a case control study in unrelated children. Although the whole population was in Table 3 RANTES genotypes and allele frequencies in (a) unrelated parents; (b) unrelated children Patient group Genotypes Alleles GG AG AA G A P a OR a 95% CI a (a) Nonatopic nonasthmatic 86 (89.6%) 6 (6.2%) 4 (4.2%) 178 (92.7%) 14 (7.3%) Nonatopic asthma 9(81.8%) 1 (9.1%) 1 (9.1%) 19 (86.3%) 3 (13.6%) Atopic alone (no asthma) 44 (34.1%) 70 (54.6%) 14 (10.8%) 159 (61.6%) 98(38.3%) Atopic asthma 8 (11.7%) 50 (73.5%) 10 (14.7%) 66 (48.5%) 70(51.5%) (b) Nonatopic nonasthmatic 35 (92.1%) 2 (5.3%) 1 (2.6%) 72 (94.7%) 4 (5.3%) Nonatopic asthma 6 (48.0%) 5 (42.0%) 2 (10.0%) 17 (65.4%) 9 (34.6%) Atopy alone (no asthma) 1 (3.44%) 26 (89.6%) 2 (6.9%) 28 (48.3%) 30(51.7%) Atopic asthma 24 (34.0%) 42 (60.0%) 4 (5.7%) 90 (64.3%) 50(35.7%) Cross-tabulation analysis. Reference category (nonatopic, nonasthmatic) a G vs A alleles.

4 Table 4 RANTES genotype and allele frequencies according to asthma-related phenotypes: (a) unrelated parents; (b) unrelated children 27 Genotypes Alleles GG AG AA P a OR a 95% CI a G A P b OR b 95% CI b (a) Skin test Negative 59 (63.4%) 28 (30.1%) 6 (6.5%) 146 (78.5%) 40 (21.5%) Positive 88 (41.9%) 99 (47.1%) 23 (11.0%) (65.5%) 145 (34.5%) IgE level IgE o100 IU/l 101 (48.8%) 87 (42.0%) 19 (9.2%) 289 (69.8%) 125 (30.2%) IgE 4100 IU/l 46 (47.9%) 40 (41.7%) 10 (10.4%) (68.7%) 60 (31.2%) BHR 48 mg/ml 84 (61.2%) 70 (41.2%) 16 (9.4%) 238 (70.0%) 102 (30.0%) r8 mg/ml 63 (47.4%) 57 (42.8%) 13 (9.7%) (68.8%) 83 (31.2%) FEV1 % 483% predicted 82 (50.9%) 64 (39.8%) 15 (9.3%) 228 (70.8%) 94 (29.2%) r83% predicted 65 (45.7%) 63 (44.5%) 14 (9.8%) (67.9%) 91 (32.1%) (b) Skin test Negative 28 (73.7%) 6 (15.8%) 4 (10.5%) 62 (81.6%) 14 (18.4%) Positive 38 (33.9%) 69 (61.5%) 5 (4.6%) (64.7%) 79 (35.3%) IgE level IgE o100 IU/l 18 (43.9%) 20 (48.8%) 3 (7.3%) 56 (68.3%) 26 (31.7%) IgE 4100 IU/l 48 (44.0%) 55 (50.4%) 6 (5.6%) (69.3%) 67 (30.7%) BHR 48 mg/ml 17 (36.2%) 25 (53.2%) 5 (10.6%) 59 (62.8%) 35 (37.3%) r8 mg/ml 49 (47.6%) 50 (48.5%) 4 (3.9%) (71.8%) 58 (28.2%) FEV1 % 483% predicted 36 (60.0%) 22 (36.7%) 2 (3.3%) 94 (78.3 %) 26 (21.7%) r83% predicted 30 (33.3%) 53 (58.9%) 7 (7.8%) (62.8%) 67 (37.2%) Cross-tabulation analysis. a AG + AA genotype compared with GG genotype. b A compared with G alleles. Gender and age accounted for in the model. Hardy Weinberg equilibrium, the lack of such equilibrium in some of the population subgroups was probably due to the small size of some of these subgroups. Although we could not demonstrate any preferential maternal transmission of atopy with the mutant allele, we have to concede that this negative finding would require confirmation in a larger number of families using more sophisticated techniques such as loglinear analysis and assessment of imprinting. That the frequency of individual genotypes and the 403A allele was strongly associated with positive SPT but not significance with serum IgE suggests that RANTES contributes to the recruitment of inflammatory cells such as basophils, mast cells and memory T cells in response to allergen exposure, 18 rather than in regulating IgE production. The same trends were observed by Fryer et al 16 in their population (24 46 years), using similar diagnostic criteria for atopy and asthma, although our population was larger and we were able to study the association in two generations (children aged 8 24 years and adults aged 34 61years) and the transmission of the allele with phenotypes. As the age range in association studies is often wide, 16 we also examined these associations separately in children and parents. These analyses suggested that the association was with atopy independent of asthma in parents in contrast to the association with both asthma and atopy in their children (Table 3). However, most of the younger generation with asthma we studied was also atopic, hence rendering the dual association insecure. In the younger generation we studied, we were able to confirm the suggestion by Fryer et al 16 that the mutant allele might be related to disease severity, assessed in our case as baseline FEV1, although even in this population it was not possible to separate the influence of atopy as few of the younger population with PDA were nonatopic. Our data appear to confirm the association of the 403A allele with atopy (defined as specific allergen sensitization) but are less supportive of the view that this allele contributes directly to asthma, to airway function and to disease severity independently of its contribution to atopy. Matching of the promoter sequence confirmed that it belongs to the GATA-binding factor 3 family, which is consistent with it being a T-cell antigen receptor. 19 However, the matching technique we used may find false positives by scanning large genomic sequences and the sensitivity of the method remains to be established by

5 28 comparison with experimental data. 30 Despite these limitations, the close matching we observed suggests that the 403G-A RANTES promoter polymorphism is functional and that it is likely to have a role in upregulating an important component of the allergic response. Further confirmation for the functional role of the RANTES 403G-A polymorphism will require verification along the lines already established for the 28C/G polymorphism. 20 The known biological activity and contribution of the chemokine pathway to allergic inflammation supports an important role for RANTES in the expression of atopy and atopic asthma. 5 Increased production of RANTES would be expected to increase the level of inflammatory cell recruitment, as it is one of the major chemoattractant proteins for eosinophils 4 and others (basophils, mast cells and T lymphocytes) into asthmatic airways after allergen challenge. 21,22 The role of this particular pathway to immune regulation is also supported by observations that the GATA transcription motif in position 403 of the chemokine RANTES gene is associated with a number of inflammatory-mediated disease including rheumatoid arthritis 10 in delaying the progression of HIV infection, 23 and in the expression of childhood atopic dermatitis. 24 We have strengthened the evidence that this association is real by demonstrating familial transmission with asthma and atopy and by confirming that the association is predominantly toward atopy and atopic asthma. Family-based studies enable robust methods such as PDT for confirming or refuting the contribution of genetic candidates to complex diseases such as asthma and atopy as they are less subject to the confounding errors associated with case control analyses. PDT has advantages over TDT as it is able to increase power by including more than one trio per family and may better account for mixed genetic models. In Caucasian populations, the contribution of RANTES 403G-A promoter polymorphism to asthma would appear to be modulated through the mechanism of airway sensitization to allergen rather than to mechanisms contributing directly to airway remodeling and BHR. Materials and methods Subjects We identified 154 unrelated nuclear families (598 individuals, including children and parents) from our local Grampian population. A total of 105 of these families were identified from a local network of general practices as families containing at least two children and young adults between 8 and 24 years with a physician diagnosis of asthma (PDA) and current symptoms (wheeze in the past 12 months) and 49 families identified through parent probands who had been studied as children in our community in the mid 1960s. 25,26 All 156 families were white Caucasian and were drawn from a population in which the UK 2001 census showed that 84% were born in Scotland, 12% in other parts of the UK and only 4% out of the UK. Atopy was defined as at least one positive SPT with a wheal size of Z3 mm among five inhalant allergens (cat, dog, house dust mite, grass and alternaria) referenced to negative control and IgE status defined as above or below 120 IU/ml. BHR was assessed by methacholine challenge 27 only omitting this test in subjects with a baseline FEV1 below 70% predicted. Predicted FEV1 values for adults Z18 years were from Crapo et al and for children o18 yrs from Wang et al Baseline FEV1 was categorized as above or below the median whole population % predicted value. DNA was isolated from EDTA anticoagulated whole blood using the phenol chloroform method. Ethical approval for all studies was awarded by the Grampian Research Ethics Committee. Functional binding site within 403 RANTES promoter The online bioinformatics application, MatInspector, 30 available at was used to identify transcription factor consensus binding sites in the putative promoter sequence. Matlnd carries out alignment and thus allows identification of subsets of binding sites according to their space length. MatInspector identifies binding sites with variable spaces provided that sufficient sequences for the generation of multiple matrices are available. 30 DNA Pooling and Allelic Quantification (AQ) Diluted genomic DNA samples were pooled (pre-pcr pools) separately for cases and controls. Six pre-pcr pools were produced by randomly sampling 200 individuals from cases and controls with pools of 100 random even and odd numbered individuals. Each pre- PCR pool was amplified by PCR for both RANTES candidate SNPs in triplicate using the following PCR conditions: 50 ml volumes containing 100 ng of genomic DNA; 10 PCR Buffer; 25 mm MgCl 2 ; 10 mm dntps; 10 pmol/ml of each primer. SNPs used were 403A 5 0 -Biotin-TGTGAGTGTGCTCACCTCCT-3 0, 5 0 -TGTGAG TCTTCAAAGTTCCTGCT-3 0 ; SNP-28G 5 0 -Biotin- TGCTT GGTTGCTATTTTGGA-3 0, 5 0 -CCACGTGCTGTCTTGAT CCTC-3 0. For each run, 5 U of Taq DNA polymerase was used with PCR conditions of 40 cycles of 951C for 45 s, 601C for 45 s and 721C for 1.5 min. The PCR products (post-pcr pools) were inspected for clearly scored product using 1.5% agarose gel electrophoresis stained with ethidium bromide. In total, 30 ml of each post-pcr pool was then used to determine the allelic frequencies using Pyrosequencingt technology (PSQ). 31 The PCR products were prepared for PSQ using a PSQ96 Sample Prep tool and PSQ reactions were performed using the PSQt96 SNP reagent kit according to the manufacturer s instructions with two different sequencing primers for each SNP. Primers used were 5 0 -CCTGCTTATTCATTA CAGAT-3 0 for 403A and 5 0 -CCCTTTATAGGGCCA-3 0 for 28G and each run was performed in triplicate. Each PSQ96 plate contained one negative control (no template) and one positive control per genotype. The program PSQt96 evaluation AQ software was used to obtain the ratio of each separate allele peak height to the sum of height of both allele peaks. To allow the conversion of this peak height ratio to allele frequency for the DNA pools, a standard curve based on individual samples was established and the ratios from pooled samples were plotted against individual sample frequency. The equation of the linear regression best-fit line was then determined for each SNP and used to convert the allele peak height ratios to allele frequencies in the DNA pools.

6 Using this pooling technique, the allelic frequencies for the 28G SNP was so low in both cases and controls ( and 0.007, respectively) that further analyses for this SNP were not pursued in our population. This was in contrast to the 403A SNP, which had a high allelic frequency of 0.36 in cases and a low frequency of in controls. Genotyping and sequencing The 403 G-A SNP was identified in individuals by RFLP-PCR using the primers 5 0 -GCCTCAATTTA CAGTGTG-3 0 and 5 0 -TGCTTATTCATTACAGATCGTA 3 0. PCR was performed in 50 ml final volumes, containing 1 NH 3 buffer (Bioline), 1.5 mm MgCl 2, 0.2 mm dntps (Bioline), 6.3 pmol of each primer and 1 U of Taq polymerase (Bioline). PCR cycles were 951C for 2 min followed by 35 cycles of 951C, 501C and 721C each for 40 s with a final 5 min extension step at 721C. In the presence of the 403G allele, the RsaI enzyme cuts the PCR product giving two bands of 115 and 20 bp, while in the presence of the mutant allele the PCR product remains undigested. The RANTES genotype was visualized on 4% agarose gels stained with ethidium bromide. PCR products were then cleaned using the ExoSAP ITTM kit (USB Corporation, Cleveland, OH, USA) and sequenced using the DYEnamic ET dye terminator cycle sequencing kit (Amersham Pharmacia Biotech UK Ltd, Buckinghamshire, UK). DNA sequencing was performed in 96-well plates, which were run on a MEGABACE-1000 automated DNA sequencer (Amersham Pharmacia Biotech UK Ltd, Buckinghamshire, UK). Direct sequencing was performed in random samples of each genotype and in all cases were identical to the genotypes inferred from PCR RFLP. Statistical analysis FBATs. In order to assess familial transmission of the alleles, the pedigree disequilibrium test (PDT) was used as this test has advantages over the classical transmission/disequilibrium test (TDT). 32,33 TDT is not a valid test of association if related nuclear families and/or siblings from larger pedigrees are used PDT overcomes these problems by treating triads (parent child trios) as independent entities in contrast to TDT, which treats the contribution from heterozygous parents as independent, 32,33 and which makes this test susceptible to confounding by phenocopies or heterogeneity. The PDT test is based on two alleles M 1 (the common allele) and M 2 (the affected allele), and pairs of these alleles ((M 1 M 2 ) or (M 2 M 2 )) transmitted from heterozygote parents to an affected child, and pairs of alleles (M 1 M 1 ) not transmitted with the disease or trait under investigation. Results are tested for significance, as in any family-based test, with the McNemar test. 34 PDT was applied separately for PDA, BHR, % predicted FEV-1 below the population median, atopy defined as at least one SPT at least 3 mm greater than negative control, and for serum IgE greater than 120 IU/ml. Case control study Of the 598 available subjects, 453 contributed to the case control study, as participants had to be unrelated to each other. None of the parents were related, thus providing 212 cases of PDA and 96 controls. For the case control study in the younger generation, we selected only the first identified child within each family, 83 cases and 67 controls. Allele frequencies for the 403G and the 403A alleles were compared in all diagnostic categories, atopic asthmatic, atopic not asthmatic and not atopic asthmatic referenced to not atopic not asthmatic for both parent and child generations. Genotype and allele frequencies among different groups were tested by w 2 and ORs and 95% CIs after adjustment for age and gender. As asthma and atopy are often coassociated, we performed univariate analyses for nonatopic asthma, atopic asthma, and atopy without asthma. Hardy Weinberg equilibrium was tested in a contingency table of observed genotype frequencies ( applets/kitest.htm). Association analyses were carried out using SPSS version 11 (SPSS Inc., Chicago, IL, USA) with w 2 tests applied to 2 2 genotype/phenotype tables and PDT with version 3.12 ( Option/pdt.html). Acknowledgements GlaxoSmithKline Research and development and the Scottish Heart Chest and Stroke Association supported the family collection. We thank Julie Black and Joanna Gordon for valuable assistant in family recruitment. References 1 Milos F, Snezana C. The role of eosinophils in asthma. Med Biol 2001; 8: Cantani A, Herrera RJ, Cameron C. Respiratory symptoms and lung function in relation to atopy in children. J Allergy Clin Immunol 1999; 6: Settipane RJ, Calvani M, Alessandri C et al. Asthma in childhood. Eur J Immunogenet 1994; 6: Teran LM, Noso N, Carroll M, Davies DE, Holgate S, Schroder JM. Eosinophil recruitment following allergen challenge is associated with the release of the chemokine RANTES into asthmatic airways. J Immunol 1996; 157: Conti P, Barbacane RC, Reale M. Chemokines in inflammatory states. Allergy Asthma Proc 1999; 20: Chihara J, Yasuba H, Tsuda A et al. Elevation of the plasma level of RANTES during asthma attacks. J Allergy Clin Immunol 1997; 100: S52 S55. 7 Schuh JM, Blease K, Hogaboam CM. The role of CC chemokine receptor 5 (CCR5) and RANTES/CCL5 during chronic fungal asthma in mice. FASEB J 2002; 16: Cocchi F, DeVico AL, Garzino-Demo A, Arya SK, Gallo RC, Lusso P. Identification of RANTES, MIP-1-alpha, and MIP- 1-beta as the major HIV suppressive factors produced by CD8(+) T cells. Science 1995; 270: Cookson WO. A new gene for asthma: would you ADAM and Eve it? Trends Genet 2003; 19: Makki RF, al Sharif F, Gonzalez-Gay MA, Garcia-Porrua C, Ollier WE, Hajeer AH. RANTES gene polymorphism in polymyalgia rheumatica, giant cell arteritis and rheumatoid arthritis. Clin Exp Rheumatol 2000; 18: Liu H, Chao D, Nakayama EE et al. Polymorphism in RANTES chemokine promoter affects HIV-1 disease progression. Proc Natl Acad Sci USA 1999; 96:

7 30 12 Hizawa N, Yamaguchi E, Konno S, Tanino Y, Jinushi E, Nishimura M. A functional polymorphism in the RANTES gene promoter is associated with the development of lateonset asthma. Am J Resp Crit Care Med 2002; 166: al Sharif F, Ollier WE, Hajeer AH. A rare polymorphism at position 28 in the human RANTES promoter. Eur J Immunogenet 1999; 26: Hajeer AH, al Sharif F, Ollier WE. A polymorphism at position 403 in the human RANTES promoter. Eur J Immunogenet 1999; 26: Weiss MJ, Orkin SH. GATA-transcription factors: key regulators of hematopoiesis. Exp Hematol 1995; 23: Fryer AA, Spiteri MA, Bianco A et al. The 403G-A promoter polymorphism in the RANTES gene is associated with atopy and asthma. Genes Immun 2000; 1: Yao TC, Kuo ML, See LC et al. The RANTES promoter polymorphism: a genetic risk factor for near-fatal asthma in Chinese children. J Allergy Clin Immunol 2003; 111: Schall TJ, Bacon K, Toy KJ, Goeddel DV. Selective attraction of monocytes and T lymphocytes of the memory phenotype by cytokine RANTES. Nature 1990; 347: Marine J, Winoto A. The human enhancer-binding protein Gata3 binds to several T-cell receptor regulatory elements. Proc Natl Acad Sci USA 1991; 88: Liu H, Chao D, Nakayama E et al. Polymorphism in RANTES chemokine promoter affects HIV-1 disease progression. Proc Natl Acad Sci USA 1999; 96: Gauvreau GM, Watson RM, O Byrne PM. Kinetics of allergen-induced airway eosinophilic cytokine production and airway inflammation. Am J Resp Crit Care Med 1999; 160: Kameyoshi Y, Dorschner A, Mallet AI, Christophers E, Schroder JM. Cytokine RANTES released by thrombinstimulated platelets is a potent attractant for human eosinophils. J Exp Med 1992; 176: Gonzalez E, Dhanda R, Bamshad M et al. Global survey of genetic variation in CCR5, RANTES, and MIP-1alpha: impact on the epidemiology of the HIV-1 pandemic. Proc Natl Acad Sci USA 2001; 98: Nickel RG, Casolaro V, Wahn U et al. Atopic dermatitis is associated with a functional mutation in the promoter of the C-C chemokine RANTES. J Immunol 2000; 164: Dawson B, Horobin G, Illsley R, Mitchell R. A survey of childhood asthma in Aberdeen. Lancet 1969; 1: Christie GL, Helms PJ, Godden DJ et al. Asthma, wheezy bronchitis, and atopy across two generations. Am J Resp Crit Care Med 1999; 159: Cockcroft DW, Killian DN, Mellon JJ, Hargreave FE. Bronchial reactivity to inhaled histamine: a method and clinical survey. Clin Allergy 1977; 7: Crapo RO, Morris AH, Gardner RM. Reference spirometric values using techniques and equipment that meets ATS recommendations. Am Rev Resp Dis 1981; 123: Wang XB, Dockery DW, Wypij D Fay ME, Ferris BG. Pulmonary function between 6 and 18 years of age. Pediatr Pulmonol 1993; 5: Quadt K, Frech K, Karas H, Wingender E, Werner T. Matlnd and Matinspector: new fast and versatile tools for detecting of consensus matches in nucleotide sequence data. Nucleic Acids Res 1995; 23: Eriksson S, Berg L, Wadelius M, Alderborn A. Cytochrome P450 genotyping by multiplexed real-time DNA sequencing with pyrosequencingt technology. Assay Drug Dev Technol 2002; 1: Martin ER, Monks SA, Warren LL, Kaplan NL. A test for linkage and association in general pedigrees: the pedigree disequilibrium test. Am J Hum Genet 2000; 67: Martin ER, Bass MP, Kaplan NL. Correcting for potential bias in the pedigree disequilibrium test. Am J Hum Genet 2001; 68: Spielman RS, McGinnis RE, Ewens WJ. Transmission test for linkage disequilibrium: the insulin gene region and insulindependent diabetes mellitus (IDDM). Am J Hum Genet 1993; 52: