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Evidence for Linkage of Chromosome 12q15–q24.1 Markers to High Total Serum IgE Concentrations in Children of the German Multicenter Allergy Study
SHORT COMMUNICATION Evidence for Linkage of Chromosome 12q15–q24.1 Markers to High Total Serum IgE Concentrations in Children of the German Multicenter Allergy Study Renate Nickel,*,† Ulrich Wahn,† Nobuyuki Hizawa,* Nancy Maestri,‡ David L. Duffy,‡ Kathleen C. Barnes,* Kirsten Beyer,*,† Johannes Forster,§ Renate Bergmann,† Fred Zepp,Ø Volker Wahn,\ and David G. Marsh*,1 *Division of Clinical Immunology, Department of Medicine, Johns Hopkins University, Baltimore, Maryland 21224; †Department of Pediatrics, Humboldt University, Berlin, Germany; ‡Department of Epidemiology, The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21224; and Department of Pediatrics, §University Hospital Freiburg, Ø University Hospital Mainz, and \University Hospital Du¨sseldorf, Germany Received February 24, 1997; accepted September 4, 1997
Linkage of asthma and high total serum IgE levels to chromosome 12q15–q24.1 has been recently described. To evaluate this region further in regard to total IgE responsiveness, we genotyped 52 unrelated German children with persistently ‘‘high’’ total serum IgE (selected from a noninterventional prospective multicenter cohort study) and their parents. We carefully defined a most extreme IgE phenotype and analyzed it as a dichotomous trait. We tested for linkage between high total IgE concentrations and nine polymorphic microsatellite markers on chromosome 12q15–q24.1 using the transmission/disequilibrium test. Evidence for linkage and allelic association for high total IgE was observed for four markers in this region. This study demonstrates the value of using extreme phenotypes in genetic analysis of a complex quantitative trait. q 1997 Academic Press
Elevated total IgE concentrations are characteristic of the atopic diseases. A clear association between elevated IgE levels and asthma has been established (4). Twin and numerous family studies have demonstrated that genetic factors influence total IgE concentrations (9, 11, 16, 20). However, studies on the genetics of total IgE responsiveness are complicated by various environmental factors, including infectious diseases and variable allergen exposure (23). Using the candidate gene approach, linkage of asthma and/or asthma-associated phenotypes to chromosome 6p21 (17), 11q13 (25, 26), 5q31.1–q33 (18, 21, 24), and 14q11.2 (22) has been described. The most recent region of interest is chromosome 12q, where evidence for linkage to asthma and/or asthma-associated traits has been shown (1, 5): Barnes et al. (1996) ob1 To whom all correspondence should be addressed at Johns Hopkins Asthma & Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, Maryland 21224. Telephone: (410) 550-2001. Fax: (410) 5502527. E-mail: [email protected].
served evidence for linkage of both asthma and elevated total IgE concentrations to markers on chromosome 12q15–q24.1 in an Afro-Caribbean population; evidence for linkage of log[total IgE] and elevated IgE levels was also described in an inbred Caucasian population (1). The statistical approaches in this study were the transmission/disequilibrium test (TDT), affected sib-pair analysis, and multipoint analysis. Two genome-wide searches on asthma and asthma-associated traits have been published (5, 6). Using multipoint analysis in affected sib-pairs of different racial backgrounds, chromosome 12q was one region that showed evidence for linkage to asthma in Caucasian and Hispanic sib-pairs in the Collaborative Study on the Genetics of Asthma (CSGA). No other atopy-associated phenotypes were analyzed (5). Daniels et al. (1996) observed no evidence for linkage of asthma-associated traits to chromosome 12q in two Caucasian populations (United Kingdom and Australia) by analyzing affected sib-pairs (6). The objective of this study was to conduct a TDT analysis of chromosome 12q markers in longitudinally characterized unrelated children, using very strict inclusion criteria for a high IgE phenotype to analyze it as a dichotomous trait. Children of bilateral German ethnicity with persistently high total IgE (n Å 44) and their parents were recruited for genotyping from the German Multicenter Atopy Study (MAS ’90). The MAS Cohort initially comprised 1314 children that were followed from birth (1990) to the age of 5 years (2). At 1, 2, 3, and 5 years total serum IgE levels were determined with CAP RAST FEIA (Pharmacia, Freiburg, Germany). Log[total IgE] concentrations were sex-adjusted according to a linear regression model (SYSTAT). The cut points for high IgE were 27.6, 108.0, 155.0, and 214.0 kU/liter, respectively, according to the 85th percentile of the tested MAS population. The persistently high total IgE phenotype was defined as high total IgE concentrations GENOMICS
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TABLE 1 Global TDTs for Persistently High Total IgE and Chromosome 12q Markers in 52 Unrelated German Children Locus
Distance (cM)a
Het.b
No. of allelesc
nd
Global TDTe P value
D12S335 D12S1052 D12S326 D12S379 D12S1064 D12S351 D12S311 D12S95 PAH
0.0 13.2 17.7 22.7 26.5 29.2 32.7 36.1 50.4
0.80 0.72 0.79 0.72 0.83 0.76 0.64 0.76 0.79
12 6 12 6 10 7 7 9 9
51 35 44 40 45 37 34 46 42
0.46 0.72 0.39 0.001 0.11 0.0009 0.95 0.83 0.23
a
Distances according to Ref. 15 and adjusted by Cri-Map (Ref. 10) on a heterogeneous Afro-Caribbean population. Heterozygosity rate. c Number of alleles observed for each marker. d Number of informative probands. e Ewens allelic TDT x2 test. b
that were documented at ¢2 timepoints. An additional eight children (born 1988–1993) who were not participants of the MAS ’90 were included in the study. They had been followed as outpatients for various atopic diseases for at least 2 years. Total serum IgE levels had been determined in the same laboratory at least twice (and at least 1 year apart) between the ages of 1 and 5 years and clearly exceeded the described cut-points calculated from the MAS ’90 cohort (292 to 64.200 kU/ liter). Thirty of the genotyped children were male, and 22 were female. Structured interviews did not indicate parasitic disease in any of the genotyped children. Nine markers on chromosome 12q15–q24.1 (Table 1) were analyzed for linkage and allelic association. All primers were supplied by Research Genetics (Huntsville, AL). Microsatellite markers were analyzed as described previously (18). The program SIB-PAIR (7) was used to perform multiallelic forms of the TDT in 52 children with the high IgE phenotype. In the absence of linkage and allelic association between the chosen marker locus and the trait, one expects each allele of a heterozygous parental genotype to be transmitted equally likely to the proband. We present results from two TDT procedures. The first is a multiallelic TDT testing for symmetry in the full N 1 N transmission table, where N is the number of alleles at the locus (3, 14). The null hypothesis is tested with Ewens x2. We will call this the ‘‘global (symmetry)’’ TDT. The second is the TDT performed using each allele in turn (the comparison group being all other alleles at that locus). This we will denote an ‘‘allele-by-allele’’ TDT. We expect that if multiple independently arising mutant alleles are present at the trait locus, or if association is generated by population admixture/stratification, then the global test might be more powerful. We observed significant evidence for linkage of high total IgE levels to four polymorphic markers in chromosome 12q15–q24.1 in 52 unrelated German children with longitudinally stable high IgE phenotypes. Re-
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sults from global allelic TDTs are summarized in Table 1. Alleles associated with the high IgE phenotype are shown in Table 2. We attempted to estimate linkage disequilibrium between two alleles positively associated with the high IgE trait (D12S351, allele 161 and D12S379, allele 201). We assigned a D12S351(161)/ D12S379(201) parental haplotype and tested for linkage disequilibrium in 12 informative probands. The ratio of transmitted/not transmitted D12S351(161)/ D12S379(201) haplotypes was 13/2 (Põ0.05). Because we did not study additional siblings or extended families, the number of affected individuals whereby transmission of this haplotype could be followed unambiguously is restricted. Therefore, we cannot exclude that these alleles were transmitted independently. Because we chose children with IgE concentrations above the 85th percentile from an atopy-enriched study population (MAS ’90), our definition of a high IgE phenotype is presumably even more extreme in regard to a nonselected population. The careful definition of most extreme IgE phenotypes in a longitudinally characterized study population is in contrast with the design of other genetic studies on IgE or other atopy-associated phenotypes. A clear association between asthma and elevated total IgE concentrations has been demonstrated (4). The data we presented in this study could therefore reflect evidence for linkage to asthma rather than high IgE. Because of the young age of our study subjects pulmonary function tests and bronchial challenges have not yet been performed. In addition, wheezing in early childhood does not necessarily result in asthma (19). The analysis of atopy-related phenotypes other than high IgE will therefore be performed when an asthmatic phenotype can be reliably assigned in the MAS cohort. The results of our study confirm the observations of Barnes et al. (1996), who reported linkage of asthma and high total IgE concentration to chromosome 12q15–q24.1 markers in an Afro-Caribbean outbred population and linkage to log[total IgE] and elevated
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TABLE 2 Allelic Associations between High IgE and Four Chromosome 12q Markers in 52 Unrelated German Children Allele
Transmitted (n)a
Not transmitted (n)b
P, allele-by-allele TDT
D12S379
197 201 213
1 27 0
12 14 10
0.0023 0.042 0.0016
D12S1064
181 189
11 3
3 11
0.032 0.032
D12S351
161 165
31 6
7 19
0.0001 0.0093
PAH
239 243
21 11
10 23
0.048 0.04
Locus
a b
Number of times specific allele was transmitted from heterozygous parents to high IgE offspring. Number of times specific allele was not transmitted from heterozygous parents to high IgE offspring.
total IgE concentrations to markers in this region in a Caucasian inbred population (1). Studies of autosomal markers showed that linkage disequilibrium in general extends over approximately 500 kb (12). Therefore, our results showing positive TDTs over 6 cM are surprising. Ewens and Spielman (1995) demonstrated that the TDT is a valid test for linkage when the association is being generated by admixture rather than linkage disequilibrium (8). The allelic association generated by such a mechanism could in theory be genome-wide. This is one mechanism that could lead to allelic association over a broad region. A second mechanism is that the presence of multiple disease gene mutations at different times in the same population can also lead to disequilibrium over larger regions, suggested as the cause of linkage disequilibrium over a 3-cM region with Huntington disease (13). Interestingly, Barnes et al. (1996) also observed positive TDTs for asthma and high IgE over a large region (ú10 cM) on chromosome 12q (1). Our results emphasize the value of carefully selecting individuals with consistent expression of an extreme phenotype from longitudinally well-characterized study populations. More closely spaced marker analysis in chromosome 12q in the described population should further restrict the region of interest. Eventually, large-scale sequencing must be performed to identify the gene(s) in this region involved in the regulation of total IgE responsiveness. ACKNOWLEDGMENTS We thank the families in Germany for their generous participation in this study and the physicians, research nurses, and technicians in Germany for their essential contributions to the German Multicenter Allergy Study. This work was funded by the German Ministry of Research and Technology (BMFT) Grant 01EE9406 and NIH Grant AI20059. Renate Nickel was supported by BMFT Grant 01EE9606 and by a postdoctoral stipend from Genome Therapeutics Corporation.
REFERENCES 1. Barnes, K. C., Neely, J. D., Duffy, D. L., Freidhoff, L. R., Breazeale, D. R., Schou, C., Naidu, R. P., Levett, P. N., Renault, B.,
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Kucherlapati, R., Iozzino, S., Ehrlich, E., Beaty, T. H., and Marsh, D. G. (1996). Linkage of Asthma and total serum IgE concentrations to markers on chromosome 12q: Evidence from Afro-Caribbean and Caucasian populations. Genomics 37: 41– 50. 2. Bergmann, R. L., Bergmann, K. E., Lau-Schadendorf, S., Luck, W., Dannemann, A., Bauer, C. P., Dorsch, W., Forster, J., Schmidt, E., Schulz, J., and Wahn, U. (1994). Atopic disease in infancy. The German Multicenter Atopy Study (MAS-90). Pediatr. Allergy Immunol. 5(Suppl 1): 19–25. 3. Bickeboller, H., and Clerget-Darpoux, F. (1995). Statistical properties of the allelic and genotypic transmission/disequilibrium test for multiallelic markers. Genet. Epidemiol. 12: 865– 870. 4. Burrows, B., Martinez, F. D., Halonen, M., Barbee, R. A., and Cline, M. G. (1989). Association of asthma with serum IgE levels and skin-test reactivity to allergens. N. Engl. J. Med. 320: 271– 277. 5. The Collaborative Study on the Genetics of Asthma (CSGA) (1997). A genome-wide search for asthma susceptibility loci in ethnically diverse populations. Nature Genet. 15: 389–392. 6. Daniels, S. E., Bhattacharrya, S., James, A., Leaves, N. I., Young, A., Hill, M. R., Faux, J. A., Ryan, G. F., le Souef, P. N., Lathrop, G. M., Musk, A. W., and Cookson, W. O. C. M. (1996). A genome wide search for quantitative trait loci underlying asthma. Nature 383: 247–250. 7. Duffy, D. L. (1996). SIB-PAIR, Version 0.93. Queensland Institute of Medical Research, Brisbane. Available at: http://www. qimr.edu.au/davidd.html. [Computer program] 8. Ewens, W. J., and Spielman, R. S. (1995). The transmission/ disequilibrium test: History, subdivision, and admixture. Am. J. Hum. Genet. 57: 455–464. 9. Gerrard, J. W., Rao, D. C., and Morton, N. E. (1978). A genetic study of immunoglobulin E. Am. J. Hum. Genet. 30: 46–58. 10. Green, P., Falls, K., and Crooks, S. (1990). Cri-Map, Version 2.4. [Computer program] 11. Hanson, B., McGue, M., Roitman-Johnson, B., Segal, N. L., Bouchard, T. J., and Blumenthal, M. N. (1991). Atopic disease and immunoglobulin E in twins reared apart and together. Am. J. Hum. Genet. 48: 873–879. 12. Jorde, L. B., Watkins, W. S., Carlson, M., Groden, J., Albertsen, H., Thliveris, A., and Leppert, M. (1994). Linkage disequilibrium predicts physical distance in the adenomatous polyposis coli region. Am. J. Hum. Genet. 54: 884–898. 13. Kaplan, N. L., Hill, W. G., and Weir, B. S. (1995). Likelihood methods for locating disease genes in nonequilibrium populations. Am. J. Hum. Genet. 56: 18–32.
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14. Kaplan, N. L., Martin, N. R., and Weir, B. S. (1997). Power studies for the transmission/disequilibrium tests with multiple alleles. Am. J. Hum. Genet. 60: 691–702. 15. Krauter, K., Montgomery, K., Yoon, S.-J., LeBlanc-Straceski, J., Renault, B., Marondel, I., Herdman, V., Cupelli, L., Banks, A., Lieman, J., Menninger, J., Bray-Ward, P., Nadkarni, P., Weissenbach, J., Le Paslier, D., Rigault, P., Chumakov, I., and Cohen, D. (1995). A second-generation YAC contig map of human chromosome 12. Nature 377: 321–333. 16. Marsh, D. G., Bias, W. B., and Ishizaka, K. (1974). Genetic control of basal serum immunoglobulin E level and its effect on specific reaginic sensitivity. Proc. Natl. Acad. Sci. USA 71: 3588–3592. 17. Marsh, D. G., Hsu, S. H., Roebber, M., Ehrlich-Kautzky, E., Freidhoff, L. R., Meyers, D. A., Pollard, M. K., and Bias, W. B. (1982). HLA-Dw2: A genetic marker for human immune response to short ragweed pollen allergen Ra5. I. Response resulting primarily from natural antigenic exposure. J. Exp. Med. 155: 1439–1451. 18. Marsh, D. G., Neely, J. D., Breazeale, D. R., Gosh, B., Freidhoff, L. R., Ehrlich, K. E., Schou, C., Krishnaswamy, G., and Beaty, T. H. (1994). Linkage analysis of Il4 and other chromosome 5q31.1 markers and total serum immunoglobulin E concentrations. Science 264: 1152–1156. 19. Martinez, F. D., Wright, A. L., Taussig, L. M., Holberg, C. J., Halonen, M., and Morgan, W. J., (1995). Asthma and wheezing in the first six years of life. The Group Health Medical Associates. N. Engl. J. Med. 332: 133–138. 20. Meyers, D. A., Beaty, T. H., Freidhoff, L. R., and Marsh, D. G.
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21.
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(1987). Inheritance of serum total IgE (basal levels) in man. Am. J. Hum. Genet. 41: 51–62. Meyers, D. A., Postma, D. S., Panhuysen, C. I. M., Xiu, J., Amelung, P. J., Levitt, R. C., and Bleecker, E. R. (1994). Evidence for a locus regulating total serum IgE levels mapping to chromosome 5. Genomics 23: 464–470. Moffatt, M. F., Hill, M. R., Corne´lis, F., Schou, C., Faux, J. A., Young, R. P., James, A. L., Ryan, G., le Souef, P., Musk, A. W., Hopkin, J. M., and Cookson, W. O. C. M. (1994). Genetic linkage of T-cell receptor a/d complex to specific IgE responses. Lancet 343: 1597–1600. Ownby, D. R. (1993). Clinical significance of IgE. In ‘‘Allergy, Principles and Practice’’ (E.Middleton, Jr., C. E. Reed, E. F. Ellis, N. F. Adkinson, Jr., J. W. Yuninger, and W. W. Busse, Eds.), Vol. 1, pp 1059–1076, Mosby-Year Book, St. Louis. Postma, D. S., Bleecker, E. R., Amelung, P. J., Holroyd, K. J., Xu, J., Panhuysen, C. I. M., and Meyers, D. (1995). Genetic susceptibility to asthma-bronchial hyperresponsiveness coinherited with a major gene for atopy. N. Engl. J. Med. 333: 894– 900. Sandford, A. J., Shirakawa, T., Moffatt, M. F., Daniels, S. E., Ra, C., Faux, J. A., Young, R. P., Nakamura, Y., Lathrop, G. M., Cookson, W. O.C. M., and Hopkin, J. M. (1993). Localisation of atopy and b subunit of high-affinity IgE receptor (FceRI) on chromosome 11q. Lancet 341: 332–334. Shirakawa, T., Li, A., Dubowitz, M., Dekker, J. W., Shaw, A. E., Faux, J. A., Ra, C., Cookson, W. O.C. M., and Hopkin, J. M. (1994). Association between atopy and the variants of the b subunit of the high-affinity immunoglobulin E receptor. Nature Genet. 7: 125–130.
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Linkage of asthma and high total serum IgE levels to chromosome 12q15–q24.1 has been recently described. To evaluate this region further in regard to total IgE responsiveness, we genotyped 52 unrelated German children with persistently ‘‘high’’ total serum IgE (selected from a noninterventional prospective multicenter cohort study) and their parents. We carefully defined a most extreme IgE phenotype and analyzed it as a dichotomous trait. We tested for linkage between high total IgE concentrations and nine polymorphic microsatellite markers on chromosome 12q15–q24.1 using the transmission/disequilibrium test. Evidence for linkage and allelic association for high total IgE was observed for four markers in this region. This study demonstrates the value of using extreme phenotypes in genetic analysis of a complex quantitative trait. q 1997 Academic Press
Elevated total IgE concentrations are characteristic of the atopic diseases. A clear association between elevated IgE levels and asthma has been established (4). Twin and numerous family studies have demonstrated that genetic factors influence total IgE concentrations (9, 11, 16, 20). However, studies on the genetics of total IgE responsiveness are complicated by various environmental factors, including infectious diseases and variable allergen exposure (23). Using the candidate gene approach, linkage of asthma and/or asthma-associated phenotypes to chromosome 6p21 (17), 11q13 (25, 26), 5q31.1–q33 (18, 21, 24), and 14q11.2 (22) has been described. The most recent region of interest is chromosome 12q, where evidence for linkage to asthma and/or asthma-associated traits has been shown (1, 5): Barnes et al. (1996) ob1 To whom all correspondence should be addressed at Johns Hopkins Asthma & Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, Maryland 21224. Telephone: (410) 550-2001. Fax: (410) 5502527. E-mail: [email protected].
served evidence for linkage of both asthma and elevated total IgE concentrations to markers on chromosome 12q15–q24.1 in an Afro-Caribbean population; evidence for linkage of log[total IgE] and elevated IgE levels was also described in an inbred Caucasian population (1). The statistical approaches in this study were the transmission/disequilibrium test (TDT), affected sib-pair analysis, and multipoint analysis. Two genome-wide searches on asthma and asthma-associated traits have been published (5, 6). Using multipoint analysis in affected sib-pairs of different racial backgrounds, chromosome 12q was one region that showed evidence for linkage to asthma in Caucasian and Hispanic sib-pairs in the Collaborative Study on the Genetics of Asthma (CSGA). No other atopy-associated phenotypes were analyzed (5). Daniels et al. (1996) observed no evidence for linkage of asthma-associated traits to chromosome 12q in two Caucasian populations (United Kingdom and Australia) by analyzing affected sib-pairs (6). The objective of this study was to conduct a TDT analysis of chromosome 12q markers in longitudinally characterized unrelated children, using very strict inclusion criteria for a high IgE phenotype to analyze it as a dichotomous trait. Children of bilateral German ethnicity with persistently high total IgE (n Å 44) and their parents were recruited for genotyping from the German Multicenter Atopy Study (MAS ’90). The MAS Cohort initially comprised 1314 children that were followed from birth (1990) to the age of 5 years (2). At 1, 2, 3, and 5 years total serum IgE levels were determined with CAP RAST FEIA (Pharmacia, Freiburg, Germany). Log[total IgE] concentrations were sex-adjusted according to a linear regression model (SYSTAT). The cut points for high IgE were 27.6, 108.0, 155.0, and 214.0 kU/liter, respectively, according to the 85th percentile of the tested MAS population. The persistently high total IgE phenotype was defined as high total IgE concentrations GENOMICS
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TABLE 1 Global TDTs for Persistently High Total IgE and Chromosome 12q Markers in 52 Unrelated German Children Locus
Distance (cM)a
Het.b
No. of allelesc
nd
Global TDTe P value
D12S335 D12S1052 D12S326 D12S379 D12S1064 D12S351 D12S311 D12S95 PAH
0.0 13.2 17.7 22.7 26.5 29.2 32.7 36.1 50.4
0.80 0.72 0.79 0.72 0.83 0.76 0.64 0.76 0.79
12 6 12 6 10 7 7 9 9
51 35 44 40 45 37 34 46 42
0.46 0.72 0.39 0.001 0.11 0.0009 0.95 0.83 0.23
a
Distances according to Ref. 15 and adjusted by Cri-Map (Ref. 10) on a heterogeneous Afro-Caribbean population. Heterozygosity rate. c Number of alleles observed for each marker. d Number of informative probands. e Ewens allelic TDT x2 test. b
that were documented at ¢2 timepoints. An additional eight children (born 1988–1993) who were not participants of the MAS ’90 were included in the study. They had been followed as outpatients for various atopic diseases for at least 2 years. Total serum IgE levels had been determined in the same laboratory at least twice (and at least 1 year apart) between the ages of 1 and 5 years and clearly exceeded the described cut-points calculated from the MAS ’90 cohort (292 to 64.200 kU/ liter). Thirty of the genotyped children were male, and 22 were female. Structured interviews did not indicate parasitic disease in any of the genotyped children. Nine markers on chromosome 12q15–q24.1 (Table 1) were analyzed for linkage and allelic association. All primers were supplied by Research Genetics (Huntsville, AL). Microsatellite markers were analyzed as described previously (18). The program SIB-PAIR (7) was used to perform multiallelic forms of the TDT in 52 children with the high IgE phenotype. In the absence of linkage and allelic association between the chosen marker locus and the trait, one expects each allele of a heterozygous parental genotype to be transmitted equally likely to the proband. We present results from two TDT procedures. The first is a multiallelic TDT testing for symmetry in the full N 1 N transmission table, where N is the number of alleles at the locus (3, 14). The null hypothesis is tested with Ewens x2. We will call this the ‘‘global (symmetry)’’ TDT. The second is the TDT performed using each allele in turn (the comparison group being all other alleles at that locus). This we will denote an ‘‘allele-by-allele’’ TDT. We expect that if multiple independently arising mutant alleles are present at the trait locus, or if association is generated by population admixture/stratification, then the global test might be more powerful. We observed significant evidence for linkage of high total IgE levels to four polymorphic markers in chromosome 12q15–q24.1 in 52 unrelated German children with longitudinally stable high IgE phenotypes. Re-
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sults from global allelic TDTs are summarized in Table 1. Alleles associated with the high IgE phenotype are shown in Table 2. We attempted to estimate linkage disequilibrium between two alleles positively associated with the high IgE trait (D12S351, allele 161 and D12S379, allele 201). We assigned a D12S351(161)/ D12S379(201) parental haplotype and tested for linkage disequilibrium in 12 informative probands. The ratio of transmitted/not transmitted D12S351(161)/ D12S379(201) haplotypes was 13/2 (Põ0.05). Because we did not study additional siblings or extended families, the number of affected individuals whereby transmission of this haplotype could be followed unambiguously is restricted. Therefore, we cannot exclude that these alleles were transmitted independently. Because we chose children with IgE concentrations above the 85th percentile from an atopy-enriched study population (MAS ’90), our definition of a high IgE phenotype is presumably even more extreme in regard to a nonselected population. The careful definition of most extreme IgE phenotypes in a longitudinally characterized study population is in contrast with the design of other genetic studies on IgE or other atopy-associated phenotypes. A clear association between asthma and elevated total IgE concentrations has been demonstrated (4). The data we presented in this study could therefore reflect evidence for linkage to asthma rather than high IgE. Because of the young age of our study subjects pulmonary function tests and bronchial challenges have not yet been performed. In addition, wheezing in early childhood does not necessarily result in asthma (19). The analysis of atopy-related phenotypes other than high IgE will therefore be performed when an asthmatic phenotype can be reliably assigned in the MAS cohort. The results of our study confirm the observations of Barnes et al. (1996), who reported linkage of asthma and high total IgE concentration to chromosome 12q15–q24.1 markers in an Afro-Caribbean outbred population and linkage to log[total IgE] and elevated
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TABLE 2 Allelic Associations between High IgE and Four Chromosome 12q Markers in 52 Unrelated German Children Allele
Transmitted (n)a
Not transmitted (n)b
P, allele-by-allele TDT
D12S379
197 201 213
1 27 0
12 14 10
0.0023 0.042 0.0016
D12S1064
181 189
11 3
3 11
0.032 0.032
D12S351
161 165
31 6
7 19
0.0001 0.0093
PAH
239 243
21 11
10 23
0.048 0.04
Locus
a b
Number of times specific allele was transmitted from heterozygous parents to high IgE offspring. Number of times specific allele was not transmitted from heterozygous parents to high IgE offspring.
total IgE concentrations to markers in this region in a Caucasian inbred population (1). Studies of autosomal markers showed that linkage disequilibrium in general extends over approximately 500 kb (12). Therefore, our results showing positive TDTs over 6 cM are surprising. Ewens and Spielman (1995) demonstrated that the TDT is a valid test for linkage when the association is being generated by admixture rather than linkage disequilibrium (8). The allelic association generated by such a mechanism could in theory be genome-wide. This is one mechanism that could lead to allelic association over a broad region. A second mechanism is that the presence of multiple disease gene mutations at different times in the same population can also lead to disequilibrium over larger regions, suggested as the cause of linkage disequilibrium over a 3-cM region with Huntington disease (13). Interestingly, Barnes et al. (1996) also observed positive TDTs for asthma and high IgE over a large region (ú10 cM) on chromosome 12q (1). Our results emphasize the value of carefully selecting individuals with consistent expression of an extreme phenotype from longitudinally well-characterized study populations. More closely spaced marker analysis in chromosome 12q in the described population should further restrict the region of interest. Eventually, large-scale sequencing must be performed to identify the gene(s) in this region involved in the regulation of total IgE responsiveness. ACKNOWLEDGMENTS We thank the families in Germany for their generous participation in this study and the physicians, research nurses, and technicians in Germany for their essential contributions to the German Multicenter Allergy Study. This work was funded by the German Ministry of Research and Technology (BMFT) Grant 01EE9406 and NIH Grant AI20059. Renate Nickel was supported by BMFT Grant 01EE9606 and by a postdoctoral stipend from Genome Therapeutics Corporation.
REFERENCES 1. Barnes, K. C., Neely, J. D., Duffy, D. L., Freidhoff, L. R., Breazeale, D. R., Schou, C., Naidu, R. P., Levett, P. N., Renault, B.,
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