Asthma families show transmission disequilibrium of gene variants in the vitamin D metabolism and signalling pathway

The vitamin D prophylaxis of rickets in pregnant women and newborns may play a role in early allergic sensitization. We now asked if an already diseased population may have inherited genetic variants in the vitamin D turnover or signalling pathway. Serum levels of calcidiol (25-OH-D3) and calcitriol (1,25-(OH)2-D3) were retrospectively assessed in 872 partipants of the German Asthma Family Study. 96 DNA single base variants in 13 different genes were genotyped with MALDI-TOF and a bead array system. At least one positive SNP with a TDT of p < 0.05 for asthma or total IgE and calcidiol or calcitriol was seen in IL10, GC, IL12B, CYP2R1, IL4R, and CYP24A1. Consistent strong genotypic association could not be observed. Haplotype association were found only for CYP24A1, the main calcidiol degrading enzyme, where a frequent 5-point-haplotype was associated with asthma (p = 0,00063), total IgE (p = 0,0014), calcidiol (p = 0,0043) and calcitriol (p = 0,0046). Genetic analysis of biological pathways seem to be a promising approach where this may be a first entry point into effects of a polygenic inherited vitamin D sensitivity that may affect also other metabolic, immunological and cancerous diseases.


Background
Asthma is a chronic inflammatory condition of the airways, variable airway obstruction and elevated serum IgE levels of unclear pathogenesis [1]. A hypothesis relating early vitamin D supplementation and induction of later allergy has initially been postulated as the main cholecalciferol metabolite calcitriol may suppresses dendritic cell maturation and consecutive development of Th1 cells [2] which is now supported by in vitro, animal and human studies [3,4].
Exposure studies in humans, however, are difficult as nearly all newborns in Western countries are now being exposed in utero or during the first year of life to vitamin supplements [5,6]. We now asked if there are DNA sequence variants that are associated with higher or lower levels of vitamin D metabolites. As it is unlikely that any complex disease is determined by variants in a single gene we tested the main genes that code for enzymes in the metabolic pathway of vitamin D conversion (Figure 1).

Study population
The German Asthma Family Study collected affected sib pairs in 26 paediatric centres in Germany and Sweden for a two-stage genome-wide linkage scan [7,8]. In these families at least two children were required with confirmed clinical asthma, while prematurity or low birth weight of the children were excluded, along with any other severe pulmonary disease. All affected children over age 3 had a history of at least 3 years of recurrent wheezing and with no other airway disease diagnosed. Unaffected siblings were also sampled if they were at least 6 years old and eligible for pulmonary function testing. Each study participant signed a consent form. All study methods were approved by the ethics commission of "Ärztekammer Nordrhein-Westfalen".
A complete pedigree of the family was drawn and information collected in a questionnaire. Participants were examined for several closely associated phenotypes. Pulmonary function tests were performed by forced flow volume tests and bronchial challenge was done by methacholine (discontinued in the second stage of the study) as reported earlier [7,8].
The current analysis differs from previous publications [7,8]. We excluded here all families with at least one member of non white skin colour (families 2, 14, 16, 19 to 21 and 27 to 32) as these individuals had considerable lower levels of 25-OH-D 3 (data not shown) compared to all other participants ( Figure 2).
Total IgE was determined with an ELISA (Pharmacia Diagnostics, Uppsala, Sweden). 25-OH-D 3 was determined with an enzymatic immunoassay (OCTEIA 25-Hydroxy Vitamin D kit, Immunodiagnostic Systems IDS, Frankfurt, Germany) that has a working range of 6-360 nmol/L, an intra-assay of 8% and inter-assay variation of 10% with a 100% specificity for 25-OH-D 3 and 75% specificity for 25-OH-D 2 according to the manufacturer. 1,25-OH2-D 3 was determined by immunoextraction followed by an enzyme-immunoassay (OCTEIA 1,25-Hydroxy Vitamin D kit, Immunodiagnostic Systems IDS, Frankfurt, Germany) that has a working range of 6-500 pmol/L, a 100% specificity for 1,25-OH 2 -D 3 and 0,009% specificity for 25-OH-D 2 . 25-OH-D 3 values reported are the mean of a duplicate analysis while due to limited serum availability only single assays have been performed for 1,25-OH 2 -D 3 .

Control population
191 anonymized DNAs were selected randomly from the ECRHS II study [9] to fill in remaining slots on the genotyping plates. These DNA samples served as populationbased controls to test if the parents of the famillies had different allele spectrum.

DNA preparation and genotyping
DNA was isolated from peripheral white blood cells using Qiamp (Qiagen, Germany) or Puregene isolation kits (Gentra Systems, Minneapolis, MN, USA).
Genes were selected as coding either for key enzymes in the vitamin D conversion pathway or being regulated by vitamin D metabolites [10]. SNPs were being picked more or less randomly either for tagging haplotypes or being functional relevant [11]. Most SNPs were genotyped using MALDI-TOF mass spectrometry of allele-specific primer extension products generated from amplified DNA Pathway diagram of genes tested for association Figure 1 Pathway diagram of genes tested for association.    (CYP24A1), rs4809958 (CYP24A1), rs2585427 (CYP24A1), rs2248359 (CYP24A1) and rs2426496 (CYP24A1).

Data handling and statistical analysis
Clinical data and genotypes were all transferred to a SQL 2000 database and checked for completeness, paternity, and Hardy-Weinberg equilibrium. Further analyses were performed using R 2.0 statistical software [14]. Linkage disequilibrium was determined by Haploview [15] using the Gabriel method for block definition. TDT association for quantitative and qualitative traits was done with SIB-PAIR [16] using the TDT option for qualitative and the Haseman-Elston regression for quantitative traits. Familybased haplotype association analysis was performed by FBAT [17] using a dominant model.

Results
The There were no major differences in serum levels between children and parents. There was also no major influence by sex or age. An important factor, however, was found with month of examination representing seasonal sun exposure in mid Europe ( Figure 2). Even after serum storage of 10 years, the individual 25-OH-D 3 levels followed a clear time course with a major peak in August. The hormonal form 1,25-OH 2 -D 3 did not vary over the course of the year, as the conversion rate decreased with higher levels of 25-OH-D 3 (Figure 3).
The overall heritability index H 2 for 25-OH-D 3 was 80.3% while the H 2 for 1,25-OH 2 -D 3 was only 30.0% [27]. There was neither an association of 25-OH-D3 and total IgE nor an association of 1,25-OH 2 -D 3 and total IgE levels.

Discussion
We have shown that serum 25-OH-D 3 (calcidiol) levelsalthough highly influenced by environmental sunlight exposure-is a heritable trait in asthma families. In contrast, a major genetic influence on 1,25-OH 2 -D 3 (calcitriol) levels could not be found, a finding that requires replication in further family and population-based studies.
The reason for this discrepancy is not fully clear as the conversion of 25-OH-D 3 to 1,25-OH 2 -D 3 is closely regulated by a direct feedback loop. It is generally agreed, however, that 25-OH-D 3 reflects best the current vitamin D status [28]. Unfortunately standardized reference values for this age group are not available but values for 25-OH-D 3 in children seem to be in the upper normal range [29]. An explanation therefore could be that a delayed downstream metabolism is leading to an (unintended) afflux or -also possible-that an increased peripheral demand needs a larger reservoir.
We observed a number of positive associations with single nucleotide polymorphisms. Although the selection of   candidate genes was rather subjective, it turned out that some of the tested candidate genes are associated with both allergy and vitamin D metabolites. Statistical significance, however, was weak, and varied even with different analysis strategies and software packages (unpublished own observation). There was also no fully consistent pattern when comparing the family transmission and the case-control approach which makes it unlikely that any of the tested SNPs is already an important functional variant.
The new associations may instead indicate the effects of physically closely related variants in these genes (which is also supported by the haplotype results of CYP24A1).
The associated candidate genes are of particular interest. CYP24A1 is the major enzyme of the calcitriol degradation pathway that showed nearly 100-fold increase after vitamin D treatment of rats [30]. Previous studies also suggest that CYP24A1 null mice cannot clear calcitriol efficiently [31] which would support the above mentioned afflux hypothesis. Genomic organization of CYP24A1 gene, location of genotyped SNPs, linkage disequilibrium between SNPs (with R2 indicated by bullet size) and LD block structure (highlighted by red boxes; rs2248359 was excluded from LD calculations for not being in HWE) Figure 4 Genomic organization of CYP24A1 gene, location of genotyped SNPs, linkage disequilibrium between SNPs (with R2 indicated by bullet size) and LD block structure (highlighted by red boxes; rs2248359 was excluded from LD calculations for not being in HWE). SNPs indicated by ¶ were used to build haplotypes.
with the renal enzyme responsible for 1-α-hydroxylation being CYP27B1. A loss of function mutation in CYP2R1 has also been described [34] and deserves further testing.
Variants in CYP2R1, CYP27B1 and CYP24A1 or other genes in the metabolic pathway of vitamin D have not been tested so far with asthma or allergy but several of the VDR-controlled genes tested here already have been associated with asthma and allergy. The many positive but weak associations represent a common dilemma in complex disease. In asthma more than 75 genes have now been claimed to be associated [50] but none of them has been shown to contribute to risk in all populations studied [51]. Obviously there are only small genetic effects and a large heterogeneity; sometimes there is unidentified population stratification and there might be phenotyping and genotyping errors. Most likely, however, not the "center" SNPs have been choosen [11]. The current pathway based approach seems to be an alternative in particular when an environmental trait can be included. It is likely that some of the genes identified here are acting in concert to determine the overall vitamin D sensitivity.
Besides increasing sample size and testing additional populations, further work may concentrate on monitoring vitamin D supplementation by immunological readouts and the identification of contributing functional genetic elements. The present rediscovery of a genetic vitamin D sensitivity [52] may be an important step in allergy induction and also surmount many other diseases including type 1 diabetes, osteoporosis, tuberculosis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel diseases, and prostate cancer where adequate vitamin D support has been found to be beneficial.  Authors' contributions M.W. initiated the study, applied for funding, developed protocols, trained investigators, planned laboratory analysis, did statistical analysis and wrote the report. J.A. did the clinical survey, C.B. did the SNP analysis, M.B. built serum and DNA bank and did the vitamin D assays together with E.A. who supervised also laboratory work and did functional assays. T.F-K. participated in the data analysis. All authors critically revised the paper.

Conflicts of Interest
The author(s) declare that they have no competing interests.