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The Impact of the Human Genome on Endocrinology: Original Articles |
Department of Internal Medicine I (M.A.P., K.R., K.-H.U., K.B.), University Hospital, Frankfurt am Main, Germany; Department of Pediatrics (M.S., A.M.P.), Endocrinology Unit, University "La Sapienza," Rome, Italy; and Department of Medicine (S.H., M.H.), Division of Endocrinology, University Hospital Göttingen, Göttingen, Germany
Address all correspondence and requests for reprints to: Klaus Badenhoop, M.D., Department of Internal Medicine I, University Hospital Frankfurt am Main, Theodor-Stern-Kai 7, D-60596 Frankfurt am Main, Germany. E-mail: . badenhoop{at}em.uni-frankfurt.de
Abstract
Graves’ disease and Hashimoto’s thyroiditis are common autoimmune thyroid disorders. Experimentally, 1,25(OH)2 D3 prevents Hashimoto’s thyroiditis. Vitamin D serum levels in Graves’ disease were found to be significantly lower than in nonautoimmune hyperthyroidism. The polymorphic vitamin D-binding protein (DBP) greatly facilitates vitamin D actions, and DBP alleles differ regarding their affinity for 1,25(OH)2 D3. Therefore, we investigated polymorphisms of the DBP gene for an association with thyroid autoimmunity. Families with an offspring affected by Graves’ disease (95 pedigrees) or by Hashimoto’s thyroiditis (92 pedigrees) encompassing 561 individuals of Caucasian origin were genotyped for three DBP polymorphisms [(TAAA)N in intron 8; StyI; and HaeIII in exon 11]. Indirect haplotyping and (extended) transmission disequilibrium testing were performed. There was a significant transmission disequilibrium of the intron 8 polymorphism in patients with Graves’ disease (P < 0.03) but not of the exon 11 polymorphism. In contrast, neither the intron 8 nor the exon 11 polymorphism was associated with Hashimoto’s thyroiditis. Maternal and paternal transmission as well as allele frequencies in DQ2+ and DQ2- patients did not differ in either disease. Therefore, allelic variants of the DBP gene confer susceptibility to Graves’ disease but not to Hashimoto’s thyroiditis in our population. These findings support a role of the vitamin D endocrine system in thyroid autoimmunity.
GRAVES’ DISEASE AND Hashimoto’s thyroiditis are the most common autoimmune thyroid disorders. Both environmental and genetic factors are involved in their etiology. Susceptibility to thyroid autoimmunity is conferred by the human leukocyte antigen (HLA) class II genes (1). Recently, X chromosomal factors were shown to be linked to thyroid autoimmunity (2) as well as type 1 diabetes (3). However, the majority of genetic risk factors for Graves’ disease and Hashimoto’s thyroiditis remains to be identified.
There is evidence for a role of the vitamin D endocrine system in the pathogenesis of thyroid autoimmunity. 1,25(OH)2 D3 prevents autoimmune thyroiditis in animal models (4). In humans, serum levels of 1,25(OH)2 D3 were found to be significantly lower in autoimmune than in nonautoimmune hyperthyroidism (5). 1,25(OH)2 D3 exerts its immunomodulatory effects by down-regulating the expression of HLA class II molecules on thyrocytes (6) and inhibiting lymphocyte proliferation as well as secretion of inflammatory cytokines (7).
The vitamin D-binding protein (DBP) is the main systemic transporter of 1,25(OH)2 D3 and is essential for its cellular endocytosis (8). Serum levels of 1,25(OH)2 D3 were shown to correlate with those of DBP (9).
The DBP gene on chromosome 4q contains three polymorphisms: a variable (TAAA)N repeat is located in intron 8 (10); a T
G substitution in exon 11 replaces aspartic acid by glutamic acid in codon 416; and a CG substitution in codon 420 leads to an exchange of threonine for lysine (11). These DBP variants lead to differences in the affinity for 1,25(OH)2 D3 (12). An association of the DBP exon 11 polymorphisms has been reported with rheumatoid arthritis (13), whereas similar studies in type 1 diabetes have led to controversial findings (14, 15, 16, 17). Furthermore, DBP polymorphisms have been correlated with glucose homeostasis in three ethnic groups (18, 19, 20, 21) and male osteoporosis (22). The immunomodulatory effects of the vitamin D endocrine system and the importance of DBP for systemic 1,25(OH)2 D3 levels led us to investigate genetic DBP variants in Graves’ disease and Hashimoto’s thyroiditis.
Subjects and Methods
Subjects
A total of 187 nuclear families of Caucasian origin with an offspring affected by Graves’ disease (95 pedigrees) or Hashimoto’s thyroiditis (92 pedigrees) encompassing 561 individuals were genotyped. Samples were collected in the outpatient clinics of the University Hospital Frankfurt am Main (Germany), the University Hospital Göttingen (Germany), and the Department of Pediatrics, University "La Sapienza," Rome (Italy). All patients recruited in Rome had a prepubertal and all others a postpubertal disease onset. Eighty-five individuals (69 females and 16 males) had a postpubertal and 10 a prepubertal (9 females and 1 male) onset of Graves’ disease. The onset of Hashimoto’s thyroiditis was prepubertal in 87 patients (67 females and 20 males) and postpubertal in five patients (all females). Informed consent was obtained. The diagnosis of Graves’ disease rested on autoimmune hyperthyroidism with TSH receptor antibodies and/or opthalmopathy. Hashimoto’s thyroiditis was diagnosed by positive Tg and/or thyroid peroxidase antibodies, reduced echogenicity on thyroid ultrasound, and normal or elevated TSH level. The male:female ratio was 1:5.6 for Graves’ disease and 1:4.6 for Hashimoto’s thyroiditis, respectively.
Genotype analysis
DNA was extracted from whole blood using standard protocols. Previously published primers were used to amplify exon 11 (11). Primers to amplify intron 8 containing the (TAAA)N repeat polymorphism were designed according to the DBP gene sequence (16) (GenBank accession no. L10641 and L10642).
PCRs were performed in a total volume of 25 µl for each pair of primers containing 50 mM KCl, 1.5 mM MgCl2, 10 mM Tris HCl (pH 8.3), 0.25 mM of each dNTP, 1.2 µM of each primer, and 0.25 U Taq polymerase (Promega Corp., Madison, WI) using 100 ng template DNA. Intron 8 PCR products were separated on 8% polyacrylamide gels and silver stained. An appropriate molecular weight marker was included on each gel. Intron 8 alleles were assigned according to the number of (TAAA)N repeats: 6 (187 bp), 8 (195 bp), 10 (203 bp), and 11 (207 bp).
Exon 11 PCR products (606 bp) were digested with HaeIII (New England Biolabs, Inc., Beverly, MA) and with StyI (New England Biolabs, Inc.) at 37 C for 3 h. If the respective restriction sites are present, digestion with HaeIII results in two fragments of 231 and 375 bp and after digestion with StyI two fragments of 240 and 266 bp can be visualized on a 2% agarose gel stained with ethidium bromide.
If both exon 11 restriction sites are absent, the respective haplotype is referred to as WT (formerly designated as 1f). Presence of only the HaeIII site characterizes the allele D416E (formerly named 1s) and presence of the StyI site marks the allele T420K (previously designated 2). The existence of both restriction sites on a single haplotype has not yet been described. Indirect haplotyping of the intron 8 and the exon 11 polymorphisms was performed as previously reported (23). HLA DQ genotyping was performed using a nested PCR approach with sequence-specific primers (1).
Statistical analysis
Pair-wise linkage disequilibrium between the intron 8 and exon 11 polymorphisms was calculated considering all parental genotypes (24). Transmission disequilibrium testing (TDT), a test for association in the presence of linkage, was used to detect preferential transmission of DBP alleles to affected offspring (25). To assess the overall transmission distortion for the multilocus polymorphisms, we performed genotype-wise extended transmission disequilibrium testing (ETDT) (26) by logistic regression analysis using LRTDT (available at ftp.gene.ucl.ac.uk). TDT results are given uncorrected, whereas ETDT results have been corrected according to the respective degree of freedom. Subsets stratified for HLA-DQ haplotypes, parental origin of DBP alleles, gender, and age of disease onset were compared using 
2 testing. A P less than 0.05 was considered significant.
Results
Altogether, 561 individuals were typed for the intron 8 (TAAA)N repeat and the exon 11 polymorphisms. The observed DBP allele frequencies (see Table 3
) were in accordance to previously reported data from Caucasians (10, 16). Pair-wise linkage disequilibrium data [D(intron 8_exon 11) ± SD] were as follows: D(10_WT) = 0.0954 ± 0.0132; D(10_D416E) = -0.0564 ± 0.0109; D(8_WT) = -0.0939 ± 0.0133 and D(8_D416E) = 0.0554 ± 0.0119. These values indicate the studied polymorphisms to be in weak but significant linkage disequilibrium as expected in the presence of genotype-phenotype associations (27).
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Allele 10 of the intron 8 (TAAA)N repeat polymorphism was significantly less often transmitted than expected to offspring with Graves’ disease (32 transmitted vs. 54 not transmitted; TDT: P = 0.0244). Allele 8, on the other hand, was significantly more often inherited to patients (54 transmitted vs. 33 not transmitted; TDT: P = 0.0177). The observed frequency of allele 6 was too low for a statistical evaluation (Table 1). Overall, we observed a significant transmission disequilibrium for the intron 8 (TAAA)N repeat polymorphism (ETDT: P = 0.0296).
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Forty-one patients with Graves’ disease were typed DQ2+ (DQ2: DQA1*0501-DQB1*0201) and the DQ2 haplotype was significantly more often transmitted to affected offspring (38 transmitted vs. 17 not transmitted, TDT: P = 0.0046) than expected. Transmission frequencies for DBP alleles did not differ between the subsets of HLA-DQ2+ and -DQ2- patients. Similarly, no differential transmission was observed between maternal and paternal DBP alleles (data not shown).
There was a significant transmission disequilibrium for the intron 8 polymorphisms in the subset of female patients with postpubertal onset of Graves’ disease (69 individuals, ETDT: P = 0.0188) and the transmission pattern in the other subsets stratified for gender and age of disease onset did not differ significantly form the above group. No preferential transmission in any subset was observed for the exon 11 alleles (data not shown).
Hashimoto’s thyroiditis
No association with Hashimoto’s thyroiditis was found for the intron 8 (TAAA)N repeat polymorphism (ETDT: P = 0.4312). Transmission frequencies for allele 10 (25 transmitted vs. 34 not transmitted; TDT: P = 0.2413), allele 8 (35 transmitted vs. 25 not transmitted; TDT: P = 0.1967) and allele 6 (7 transmitted vs. 8 not transmitted; TDT: P = 0.7958) were in accordance to expected values (Table 2).
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Forty-two patients with Hashimoto’s thyroiditis were DQ2+ (DQ2: DQA1*0501-DQB1*0201) and the DQ2 haplotype was preferentially, although not significantly, transmitted to patients (37 transmitted vs. 23 not transmitted, TDT: P = 0.0707). Transmission of DBP alleles to HLA-DQ2+ did not differ from transmission to the subsets of HLA-DQ2- patients affected by Hashimoto’s thyroiditis. Similarly, there were no differences in the transmission of maternal and paternal DBP alleles (data not shown).
Neither the intron 8 (ETDT: P = 0.2393) nor the exon 11 polymorphisms were preferentially transmitted in the subset of female patients with prepubertal onset of Hashimoto’s thyroiditis (67 individuals), and the transmission pattern in the other subsets stratified for gender and age of disease onset did not differ significantly from the above group either (data not shown).
Discussion
In this first study investigating the role of three DBP polymorphisms in thyroid autoimmunity, we found the intron 8 (TAAA)N repeat polymorphism to be associated with Graves’ disease but not with Hashimoto’s thyroiditis. No association with either disease was observed for the exon 11 polymorphisms. In both conditions, no significant interaction between DBP genotypes and HLA haplotypes could be observed. There was no difference in transmission between maternal and paternal DBP alleles nor among the groups stratified for gender and age of disease onset excluding these as confounding factors in our analysis.
We observed a preferential transmission of HLA-DQ2 to offspring with Graves’ disease and Hashimoto’s thyroiditis, which is in accordance with previous findings in Caucasians (28, 29) and suggests that our population is representative for thyroid autoimmunity.
The association of the DBP intron 8 polymorphism with Graves’ disease may be owing to functional differences themselves or to linkage with a nearby gene. In a case-control study, the DBP intron 8 genotype 10/8 was associated with lower bone mineral density, increased fracture risk (22), and higher DBP protein serum concentrations than in individuals with genotype 10/10.
DBP is the major transporter of 1,25(OH)2 D3 and facilitates its peripheral bioavailability. DBP knockout mice have a markedly disturbed vitamin D metabolism (30). In humans the vitamin D-binding affinity differs in exon 11 variants (12), which might cause subtle alterations of the 1,25(OH)2 D3 concentrations and affect immune regulation. However, these exon 11 polymorphisms are not associated with Graves’ disease in our population.
Under physiological conditions, only 2% of DBP is saturated with vitamin D metabolites (31). Thus, slight alterations in its affinity toward 1,25(OH)2 D3 might not have any functional effect at all. Apart from transporting vitamin D, DBP has multifunctional properties including an important role in macrophage activation (32, 33, 34) DBP’s macrophage activating role is distinct from its vitamin D-binding ability. Variations of this property could affect adequate functioning of the immune system, as shown for DBP knockout mice that exhibited an impaired immune response to bacterial infections (35). The DBP intron 8 polymorphism is an Alu repeat polymorphism and might therefore affect DBP gene expression by a transcriptional mechanism, similar to the variation in enhancer activity demonstrated for an HRAS1 minisatellite polymorphism (36). The consensus sequence for the major Alu subfamily contains a functional retinoic acid response element, which was shown to interact with RAR in vitro (37).
To verify the observed association of the DBP intron 8 polymorphism with Graves’ disease, but not with Hashimoto’s thyroiditis, larger studies comprising families from different ethnic origins are needed. Functional analyses should address the differences of DBP intron 8 and 10 alleles with regard to DBP function, both in terms of its vitamin D binding ability as well as its macrophage activating properties.
Acknowledgments
Footnotes
This work was supported by the Deutsche Forschungsgemeinschaft (Grant Ba 976/8-1) and by the European Foundation for the Study of Diabetes.
Abbreviations: DBP, Vitamin D-binding protein; ETDT, extended transmission disequilibrium testing; HLA, human leukocyte antigen; TDT, transmission disequilibrium testing.
Received October 5, 2001.
Accepted February 26, 2002.
References
This article has been cited by other articles:
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  Y. Tomer and T. F. Davies Searching for the Autoimmune Thyroid Disease Susceptibility Genes: From Gene Mapping to Gene Function Endocr. Rev., October 1, 2003; 24(5): 694 - 717. [Abstract] [Full Text] [PDF]
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