Genetic control of serum IgE levels and asthma: linkage and linkage disequilibrium studies in an isolated population
Genetic control of serum IgE levels and asthma: linkage and linkage disequilibrium studies in an isolated populationTarja Laitinen1,2, Paula Kauppi2, Jaakko Ignatius3, Tarja Ruotsalainen2, Mark J. Daly4, Helena Kääriäinen3, Leonid Kruglyak4, Hannu Laitinen5, Albert de la Chapelle1, Eric S. Lander4,6, Lauri A. Laitinen2 and Juha Kere1,*
1Department of Medical Genetics, Haartman Institute, Haartmaninkatu 3, 00014 University of Helsinki, Helsinki, Finland, 2Division of Pulmonary Medicine and Clinical Physiology, Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland, 3Department of Medical Genetics, the Family Federation of Finland, Helsinki, Finland, 4Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA, 5Department of Pulmonary Medicine, Kainuu Central Hospital, Kajaani, Finland and 6Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Received June 9, 1997;Revised and Accepted August 29, 1997
Immunoglobulin E (IgE) concentration in serum is elevated in atopic diseases such as asthma. A large genomic region on chromosome 5 has previously been implicated in the control of IgE levels and bronchial hyperreactivity and may, therefore, harbor genes predisposing to asthma. In an effort to confirm this linkage and to delimit the critical region, we took advantage of an isolated founder subpopulation in Finland to study genetic linkage and haplotype associations. Sixteen polymorphic markers, including the Interleukin-4 and -9 genes (IL4, IL9), were physically ordered and genotyped in 157 nuclear families. Genetic linkage studies involving sib- and cousin-pair analyses found no evidence of genetic linkage between markers in 5q and either serum IgE levels or asthma. Haplotype association studies were also performed. Although initial inspection suggested the possibility of linkage disequilibrium in the region of IL9, we developed a rigorous permutation test for assessing association and determined that the association was no greater than would be expected by chance. Sequence analysis of the IL9 gene in three patients sharing a possibly conserved haplotype revealed a T113M coding polymorphism, but this variant showed no association with either serum IgE levels or asthma. We conclude that allelic variation at chromosome 5q31 is not likely to contribute to inheritance of serum IgE levels or the development of asthma in this Finnish subpopulation.
Several recent studies have suggested that allelic variation in the region containing the interleukin gene cluster on chromosome 5q31-q33 may play a role in the inheritance of IgE levels and asthma. These phenotypes are related: asthma is often associated with atopy, a condition that is defined by elevated IgE response to common allergens and often shows high non-specific IgE production. Twin studies have shown that both serum IgE levels and asthma are largely regulated by genetic factors, although the estimates of heritability vary widely (1 ,2 ). Marsh et al. (3 ) reported that human chromosome 5q31-q33 carries one or more genes regulating serum IgE levels, based on quantitative trait analysis in 119 sibs from 20 nuclear families. Meyers et al. (4 ) subsequently reported linkage to chromosome 5q31-q33 based on both sib-pair and lod score analysis in 55 families. In addition, Postma et al. (5 ) reported evidence that the same region contains a gene predisposing to bronchial hyperresponsiveness, which is an important characteristic of asthma. The identity of the causative gene(s) on chromosome 5 remains unknown, but the Interleukin gene cluster contains many plausible candidates. One of them is IL4, which is essential in IgE production and in the development of peribronchial inflammation (6 ,7 ). However, no trait-causing mutations or variants have been reported to date.
Interestingly, some studies have failed to detect any evidence of linkage to chromosome 5q31-q33. In a genome wide search among 80 families including 172 sib pairs from Western Australia, Daniels et al. (8 ) found six potential linkages to phenotypes associated with asthma, but could not confirm linkage to IgE or bronchial hyperreactivity with 5q markers. In another study, 131 British families were typed for an IL9 polymorphism and no linkage to IgE or bronchial hyperreactivity was found. However, one of the IL9 alleles showed significant association with serum IgE (9 ).
We sought to study the role of chromosome 5q31-q33 in a genetically isolated population (10 ). In the hope of maximizing genetic homogeneity, we studied the Kainuu region in the eastern central part of Finland with a current population of 95 000. The population structure and regional history of Kainuu have been extensively studied (11 ,12 ). According to census records, the region had only 200 households with a total of ~2000 individuals in the mid 16th to mid 17th centuries. Most of these individuals had recently migrated from the south, and many were likely to have been related. Thus, the number of founding chromosomes may well have been in the neighborhood of 100. In agreement with this historical information, disease-gene studies in the Kainuu subpopulation have shown remarkable linkage disequilibrium consistent with founder effects (13 -16 ). Long ancestral haplotypes of up to 12 cM have been maintained (14 ,16 ).
We recruited a total of 157 asthma families from Kainuu, phenotyped the individuals for IgE levels and asthma, genotyped them for 16 polymorphic markers in 5q31-q33, and performed linkage and association analyses. Our results indicate that allelic variation in the Interleukin gene cluster is unlikely to play a significant role in the regulation of IgE levels or asthma in this isolated and homogeneous population.
Since comprehensive maps were not available, we began by constructing a dense genetic and physical map of the 30 cM region of chromosome 5q containing the Interleukin gene cluster, using as starting points the Genethon and CHLC genetic maps (17 -19 ) and the Whitehead physical map (20 ). To integrate the various genes and markers (Fig. 1 A) into a single physically ordered map, the entire set of loci was typed in two radiation hybrid panels with different levels of resolution. An integrated physical map based on these results is shown in Figure 1 B. The map is particularly dense in the 4 Mb region surrounding the Interleukin gene region.
Families were collected from the Kainuu province in eastern central Finland. Families were selected through a proband (parent or child) with self-reported asthma. In total, we ascertained 51 multiplex families and 106 uniplex families. Some of the nuclear families were merged by genealogical information to yield 54 pedigrees. These pedigrees were used in linkage analyses and included altogether 73 sibpairs, 41 first cousin and 21 second cousin pairs, in most cases with complete sets of parents. The uniplex families (used only for association analyses) consisted of an affected individual together with two relatives needed to determine linkage phase: either an affected child with both parents or an affected parent with spouse and offspring.
The clinical characteristics of the study subjects are shown in Table 1 . Uniform national criteria for asthma are used in Finland, based on the recommendations of the American Thoracic Society (21). The criteria include a documented history of asthmatic symptoms (wheezing, dyspnoea in rest or in exercise, excluding other causes for dyspnoea); wheezing by auscultation; and significant reversible obstruction of the airways and/or bronchial hyperreactivity as evidenced by histamine or methacholine challenge (22-26). Additional examinations included eosinophilic leukocyte counts in blood and sputum, and skin prick testing. In childhood, the diagnosis was based mainly on medical history verified by a pediatrician. Asthma diagnoses were independently verified by two pulmonary physicians. Based on all available hospital documentation, 87% (n = 185) of the self-reported asthmatics had been carefully examined and fulfilled the criteria above. A more detailed clinical analysis will be published elsewhere (Kauppi et al., in preparation).
For qualitative linkage and haplotype analyses, subjects were classified according to two phenotypes: serum IgE levels and asthma. For serum IgE values, subjects were divided into two groups based on their measured serum IgE values, high (IgE>100 kU/l) and low (IgE <= 100 kU/l) (4 ). The subgroups showed expected differences in screening results for airway allergies and in the prevalence of asthma (Table 1 ). The high IgE group was designated as `affected' in linkage and haplotype analyses. For asthma, only those patients whose diagnosis could be verified retrospectively (n = 185) were designated `affected'. The remaining 27 patients and four of 275 family members who reported asthma symptoms but had not been rigorously examined, were designated as `phenotype unknown'.
To verify the origin of families, we traced the birthplaces of parents and grandparents using population registries. The results are plotted for groups with high IgE (25%) and with low IgE (75%) in Figure 2 . The proportion originating in Kainuu proper was similar in the two groups, being 82% in the high IgE group and 77% in the low IgE group. Most of the remaining grandparents originated in neighboring, often southern municipalities (Fig. 2 ). Only a single grandparent was non-Finnish. We concluded that population stratification or admixture is unlikely.
To determine the inheritance pattern at chromosome 5q31-q33, we genotyped 313 individuals in multiplex families for 16 genetic markers in the 30 cM region spanning D5S404-D5S413. Multipoint linkage analysis was then performed to detect linkage to serum IgE levels and asthma, using the GENEHUNTER and MAPMAKER/SIBS computer packages. Specifically, we analyzed serum IgE levels as a quantitative trait and `high' serum IgE levels as a qualitative trait (Table 2 ). No suggestive evidence for linkage was found. Similarly, we analyzed asthma as a qualitative trait and found no evidence for linkage. The affected relative pair analysis with MAPMAKER/SIBS (measuring the frequency of allele sharing) yielded no LOD score greater than 0.56 in the multipoint analysis and the non-parametric linkage analysis with GENEHUNTER yielded NPL scores that were negative across the entire region. The P-values fall far below the level of statistical significance, even for a single-point test. MAPMAKER/SIBS was used to perform exclusion mapping on the asthma phenotype. We could exclude the presence of a locus with [lambda]s>5 at a LOD score of -2 and a locus with [lambda]s>3 at a LOD score of -1. Loci of smaller effects could not be excluded.
Linkage analysis between markers on the chromosome 5q and serum IgE levels using Haseman-Elston QTL regression analysis in computer package MAPMAKER/SIBS and non-parametric (NPL) Z-score in computer package GENEHUNTER
Although the Kainuu families showed no evidence of linkage of genetic markers to either IgE levels or asthma, they could still reveal evidence of allelic association. Studies of late-onset Alzheimer's disease (AD) provide an example: the chromosomal region containing the ApoE locus shows only weak evidence of linkage in families, but the ApoE4 allele shows striking allelic association with AD (27 -29 ).
If the population contains only a limited number of disease-predisposing ancestral alleles, then affected individuals should show an excess of those ancestral haplotypes. To search for such association, we examined each chromosome in the multiplex and uniplex families and determined haplotypes for the 16 genetic markers studied (by using relatives to assign phase). For each family, a chromosome was classified as `trait-associated' if it occurred in any affected family member and as `control' if it occurred only in unaffected family members; each chromosome was counted only once per family. Depending on the mode of action of the putative predisposing gene(s), the set of trait-associated chromosomes will also contain normal chromosomes but, nonetheless, should still be enriched for disease-predisposing alleles if chromosome 5q31-q33 plays a role in the trait.
A computer program was used to enumerate all possible haplotypes of adjacent markers, considering all haplotype lengths (1, 2, 3, ...16 markers). For each haplotype, we counted the frequency of its occurrence among trait-associated (for the qualitative traits of IgE levels and asthma) and control chromosomes, and observed all instances in which the distribution showed any evidence of skewing (P<0.05 by Fisher's test). Such haplotypes are shown in Figure 3 . Visual inspection suggested a clustering of apparently identical segments between distinct haplotypes in the region of IL9, possibly reminiscent of a conserved founder haplotype.
A simple permutation test was developed to test whether the occurrence of skewed haplotypes was truly greater than would be expected simply by chance. For the test, the haplotypes were treated as fixed but the classification of the chromosomes (as trait-associated or control) was randomly permuted. We performed 100 such permutations, noting the number of haplotypes with P<0.05 and P<0.01 as well as the smallest P-value observed. Virtually every permutation gave results similar to those seen for the actual data. In fact, 67 of 100 permutations showed a larger number of significant associations at the P<0.05 level than the actual data set. Moreover, apparent clustering of haplotypes was also seen in most permutations, suggesting that it is a result of visually imposing order on the data, rather than of true clustering. Based on these statistical tests, we concluded that there was no statistically significant evidence for haplotype association with either IgE levels or asthma.
Having found no evidence of association, we developed a test to determine the power to detect the presence of an ancestral chromosome with haplotype h (without recombinational or mutational change) carried at frequency F among affected chromosomes (see Materials and Methods). The power to detect association was very high (98%) for F = 0.20; high (83%) for F = 0.15; moderate (51%) for F = 0.10, and low (10%) for F = 0.05. We could exclude association with F = 0.20 (P = 0.001) and with F = 0.15 (P = 0.03), but not with F = 0.10 (P = 0.20).
Based on this analysis, it is most likely that any ancestral chromosome contributes to only a small proportion of chromosomes in asthma patients (<15%, and probably <10%). Alternatively, but more unlikely given the young age of the population (10 -16 ), a susceptibility haplotype might have eroded by recombination and mutation to become unrecognizable.
In spite of the lack of statistically significant allelic association, we decided to sequence the IL9 gene (near the center of the region that had initially showed suggestive evidence of a haplotype). The gene was sequenced in four subjects having different combinations of three of the most strongly skewed haplotypes to search for a variant and one control to verify the published sequence. Associated with one haplotype, we found a C -> T nucleotide substitution at position 338 in exon 5 of IL9, encoding the predicted amino acid change T113M. The nucleotide change creates a new restriction site for NcoI, providing a convenient assay that was used to type all subjects. The T113M variant occurred in 15% of the chromosomes (80/542), but showed no association with high serum IgE (38/253) or asthma (43/323). We concluded that the IL9 variant does not predispose to or protect from asthma or influence serum IgE.
Our study was designed to examine the role of the region on chromosome 5q31-q33 which has been implicated in the control of IgE levels and asthma in previous studies (3 ,4 ). The genetic marker set included a total of 16 loci spanning a total of 30 cM, and the physical order of these markers was determined in order to allow reliable haplotype construction for association study. The results refine the maps in comparison to those used in previous studies; for example, the [beta]2 adrenergic receptor gene was shown to lie ~13 Mb distal to the cytokine cluster. We focused our study especially on the Interleukin gene cluster and typed 11 markers within 4 Mb of genomic DNA. The information content of markers in our study was greater than in previous studies. Thus, our study covers the longest genomic distance and has the highest number and density of markers so far applied to linkage and association studies of the 5q31-q33 region with IgE levels, bronchial hyperreactivity or asthma.
We studied genetic linkage, because earlier studies reported positive results in similarly sized samples. Our negative findings are not surprising because the sample has fewer affected pairs and thus lower power to detect loci with weak or moderate effects. Specifically, the sample has reasonable power to detect a locus with [lambda]s>5 for the qualitative phenotype of asthma and a locus explaining >80% of the variance in IgE levels. Our data thus cannot distinguish among the hypotheses of false positive results in earlier studies, a false negative result (failure to detect a weak locus) in our study, and genuine heterogeneity between different studies (8 ,9 ,30 ).
We also searched for evidence of haplotype association. Visual inspection of haplotypes suggested possible founder segments. However, a more rigorous analysis involving a permutation test indicated that the skewing was no greater than expected by chance. The permutation test, developed for this study, should provide a useful general tool for assessing association. In addition, we found a polymorphism in the coding region of IL9, but determined that it was not associated with IgE levels or asthma.
Our sample has a high likelihood for detecting association provided that a common susceptibility allele is present in at least 15% of the patients' chromosomes. Our failure to detect association therefore indicates that either 5q31-q33 does not play an important role in determining IgE levels or asthma in Kainuu, or that the allelic complexity of the susceptibility gene(s) is quite high (effective allele number >6-7) even in this young, isolated population.
Published PCR assays were used for the genes IL4, IRF1 and IL9 (3 ), and for markers D5S642, D5S666, D5S1995, D5S458, D5S2115, D5S393, D5S399, D5S816, D5S396, D5S500, D5S436, D5S413 and D5S434 (17 ,18 ). Primers were designed based on published sequences for the genes FGFA (CGC CAC AAG CAG CAG CTG and ACA AAT TCA GGC TCT GTG GG), CD14 (GCC GCT GTG TAG AAA GAA GC and CCA AGG CAG TTT GAG TCC AT) and ADB2 (AGT TCC CCT AAA GTC CTG TG and CTG CAC ACT CAG CTT GTC). DNA samples from two radiation hybrid panels (GeneBridge 4 and Stanford panel, Research Genetics, Inc., USA) were amplified by PCR and the presence or absence of each gene or marker was scored. The Whitehead Institute network server was used in the analysis of the results with the GeneBridge 4 panel. Physical distances were estimated using the conversion of 1 cR corresponding to 300 kb in the GeneBridge 4 panel.
Local radio and newspaper advertisements were used to recruit families with one or more asthmatics in the Kainuu province (Fig. 2 ). Blood samples for serum IgE and allergen measurements and DNA extraction were collected from members of nuclear families (proband, father and mother; or proband, spouse and at least one child; or proband, one parent and at least one sibling), in order to establish haplotypes unambiguously. Additional family members (uncles, aunts, grandparents, further sibs, cousins) were included when available.
Serum total IgE (kU/l) was determined by solid phase immunoassay (Diagnostics CAP FEIA, Kabi Pharmacia, Sweden), and IgE specific antibodies for the most common airway allergens in Finland (birch, mugwort and timothy pollen, horse, cat and dog dander, home dust mite and Cladosporium herbarum mould) were determined by Phadiatop CAP FEIA (Kabi Pharmacia, Sweden).
DNA was extracted from blood samples by a standard non-enzymatic method. PCR amplifications were carried out in 20 [mu]l reactions containing 50 ng of genomic DNA, 0.2 mM of each primer, 50 mM KCl, 1.5 M MgCl2,10 mM Tris-HCl (pH 8.8 at 20oC), 0.2 mM of each dNTP and 0.3 U thermostable DNA polymerase (Dynazyme, Finnzymes, Finland). The samples were denatured for 10 min at 93oC, followed by 24-30 cycles each of 15-30 s at 93oC, 15-30 s at 55-60oC, 15-30 s at 72oC, and finally elongated for 8 min at 72oC. The samples were electrophoresed on denaturing 7 M urea/6% polyacrylamide gels and the alleles were visualized by silver staining.
The following polymorphic microsatellites were typed: D5S404, D5S622, D5S490, D5S642, D5S1995, D5S2115, D5S393, D5S399, D5S816, D5S500, D5S436, D5S413, D5S434 and intronic polymorphisms within the IL4, IRF1 and IL9 genes. Typing results were read independently by two persons who were blind to the clinical data. In cases of ambiguity, the typing was repeated. To get an estimate of the rate of typing errors, IL9 typing was done twice (second primer pair: AAA GAG TCC CCA GAA AAG GC and ACA GAG CAA ATA GGT GGG CA). Both primer pairs amplified the same CA-repeat and gave results that were scored identically in all cases.
The IL9 gene was sequenced in selected individuals using the following primers: GTG GCC CCA ACT TAC AGA GA and CCT GCA AGG GAA ATT TCA GA (exons 1 and 2), AAT ATG TAT GAG GAT GAA AAA ACT CG and TTC AAG ATC GTA GTT TTA AAA GGG (exon 3), AAG TCT CCT CCA GGG GAT GT and ATC AAC CTT GAA CTA CCA ATT CC (exon 4), ACT CTG GCT CTT GGC AGG TA and CCT ATG AGC CTG AGG GTC TG (exon 5). These amplicons covered all exons and 40% of the whole genomic length of the IL9 gene. The unsequenced segments were in the middle of introns. Screening for the T113M variant was carried out using NcoI digestion. The length of the PCR product of the fifth exon when electrophoresed on agarose gel was 462 bp in the wild type (T) and the length of two NcoI digested fragments in the presence of the variant (M) were 321 and 141 bp.
For comparisons of clinical parameters, a two-tailed Student's t-test was used. For linkage analysis, we studied the qualitative phenotypes of high IgE level (>100 kU/l) and asthma by performing multipoint affected relative pair analysis with the computer package MAPMAKER/SIBS (31 ) and non-parametric multipoint linkage analysis with the computer package GENEHUNTER (32 ). We also studied IgE levels as a quantitative trait, using MAPMAKER/SIBS to perform the Haseman-Elston test.
For allele and haplotype association analysis, we used Fisher's exact test to compare the number of occurrences of each haplotype in phenotypic categories (high IgE versus low IgE and asthma versus no asthma). Since many tests were performed (every haplotype for every set of consecutive markers was examined), we developed a permutation test to estimate the significance of the overall observations as follows: taking the observed haplotypes as fixed, we randomly permuted the IgE and asthma status among the individuals and repeated the complete analysis as before on the permuted data. The apparent associations resulting from 100 such permutations were then compared with the actual results, both in the number of significant results at significance levels of 0.05 and 0.01 and in the significance levels of the most extreme results.
We performed simulations to assess the power of the sample to detect the presence of an ancestral chromosome carrying susceptibility to a trait. Specifically, we considered the hypothesis that there was an ancestral chromosome with three-locus haplotype h present (without recombinational or mutational change to the haplotype) at frequency F among current affected chromosomes. The haplotype H was chosen at random for each replicate according to its frequency among control chromosomes. For each value of F = 5%, 10%, 15%, 20%, we generated 10 000 replicates in which affected chromosomes were chosen with probability F to be h and with probability 1-F from the control distribution. We used Fisher's exact test to compare the resulting distribution with that observed on control chromosomes. The power to detect association was measured by the fraction of replicates in which the resulting P-value exceeded the corrected 0.05 significance level (obtained from the observed data by performing a permutation test on the phenotypes). The confidence to exclude association was measured by the fraction of replicates yielding P-values less extreme than the most extreme value observed in our actual data set.
Ms Liisa Rajasalo, Ms Päivikki Pajunen and their staff of the Kainuu Central Hospital are gratefully acknowledged for help in organizing the sample collection drive. We thank Ms Siv Knaappila, Ms Merja Nissinen and Ms Hanna Lampinen for their careful and skilled genotyping work. This study was supported by the Academy of Finland, Helsinki University Central Hospital Research Funds, Association for the Pulmonary Disabled, Sigrid Juselius Foundation, Ida Montin Foundation, Finnish Anti-Tuberculosis Association Foundation, Finnish Cancer Organization, Whitehead Institute for Biomedical Research and US National Institutes of Health. Part of the study was done at Folkhälsan Institute of Genetics.
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*To whom correspondence should be addressed. Tel: +358 9 1912 6538; Fax: +358 9 1912 6677; Email: juha.kere@helsinki.fi
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