A novel single nucleotide polymorphism of human thyrotropin receptor does not have a major effect on development of Graves disease
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《中华医药杂志》英文版
1 Shandong Provincial Hospital, Shandong University,Jinan 250021， China
2 The Center (4th) Hospital of Xuzhou,Xuzhou,Jiangsu Province 221009， China
3 Ruijin Hospital, Shanghai Institute of Endocrinology, the State Key Laboratory of Medical Genomics of Shanghai Second Medical University,Shanghai 200025， China
*These authors contributed equally to this work.
Correspondence to SONG Huai-dong，huaidong_s1966@163.com or ZHAO Jia-jun，jjzhao@medmail.com.cn
[Abstract] Objective Graves disease (GD) is induced by an interplay of genetic factors and environmental triggers. It is well recognized that the thyroid stimulating hormone receptor/ thyrotropin (TSH-R) functions as a B-cell autoantigen in autoimmune thyroid diseases and therefore may be a potential candidate gene contributing to the development of GD or influencing the clinical course of the disease. However, it remains unclear whether the TSH-R gene is directly involved in the initiation of GD. Previous reports have been contrary results due to racial differences, selection bias, or population stratification. In order to obtain whether the single nucleotide polymorphisms (SNPs) concerning of the TSH-R is associated with GD in Chinese Han Pedigrees.Methods In the present study,we assessed all 10 exons and some introns of the TSHR gene using case-control analysis. Results We found a novel SNP in exon 8 and other 7 SNPs previously published in introns 1,4,5,6 and exon 7. The novel SNP showed two genotypes: A/A, A/C and the frequency of allele C was 14.74% in patients and 16.67% in controls respectively. No statistically significant differences in allele or genotype frequencies were observed between GD and healthy control subjects for any of the eight SNPs studied. Conclusion Our findings suggested that neither the novel SNP nor the other 7 SNPs of the TSHR gene may be responsible for GD.
[Key words] TSHR gene;Graves disease;polymorphism
INTRODUCTION
Graves disease(GD) is a common organ-specific autoimmune disease. The etiology of GD is believed to involve a complex interaction between susceptibility genes and environmental insults. Ten years after the first description of activating mutations in the TSH-R gene in sporadic autonomous hyperfunctioning thyroid adenomas[1], there is general agreement in assigning a major pathogenic role for this genetic abnormality, acting via the constitutive activation of the cAMP pathway, in both the growth and functional characteristics of these tumours. In GD, thyroid-stimulating autoantibodies can mimic TSH action and stimulate thyroid cells leading to hyperthyroidism and abnormal overproduction of thyroid hormone. Mechanism of TSHR-autoantibodies production is more or less clear but a susceptibility gene, which is linked to their production, is still unknown. There have been provided some candidate genes for GD, such as GD-1,GD-2,CTLA-4,TG,TPO,NIS and so on[2], but several reports related to the association of TSHR gene and GD genetic studies show no linkage between the TSHR gene and GD. Among three common polymorphisms in the TSHR gene, only the D727E germline polymorphism in the cytoplasmic tail of the receptor tested an association with the disease, and this association is weak[3]. However, other investigators did not confirm this association due to different subjects involved of stripes and gender[4].That suggest interpopulation heterogeneity of genetic and environment affect disease exist, reflecting the important matching of cases and control individuals within ethnic group[5]. In order to elucidate whether polymorphisms of TSHR was associated with GD in Chinese Han Pedigrees, in the present study we identified all 10 exons and some introns of the TSHR gene through direct sequencing using a case-control dataset.
METHODS
Subjects
Seventy-eight patients with GD of Han Nationality of Shandong Province in China who were divided into two groups: thirty unrelated patients with a familial hereditary history of GD and forty-eight sporadic GD. Fifteen male and sixty-three female patients, mean age was 44.83±14.51 years. Ninety-six normal healthy donors were age- and sex-matched from Han Nationality of Shandong Province in China.
Diagnosis of GD was based on the evidence of clinical hyperthyroidism associated with elevated serum free thyroxine (FT4) and free triiodothyronine (FT3) levels and suppressed serum TSH concentrations. A typical diffuse goiter with increased vascularization was detected by ultrasound. All the patients owed an increased thyroidal iodine uptake in thyroid scintiscans and had high titers of thyroperoxidase antibodies (TPOAb), thyroglobulin antibodies (TgAb) and TRAb measured by a radioreceptor assay.
Normal subjects were specifically selected as a control group. In these subjects there was no evidence of thyroid disease. All controls were clinically and biochemically euthyroid and were negative for thyroid antibodies
Ethical approval and informed consent for genetic analysis of these samples had also been obtained.
Sequencing of TSHR Gene
Total genomic DNA was extracted from peripheral lymphocytes of whole blood using standard phenol-chloroform procedures[6]. The target DNA were amplified by polymerase chain reactions (PCR) with primers designed using sequence information from GenBank ( LocusID: 7253) (Table 1). PCR was performed in a 20-μl reaction mixture containing 50 ng of genomic DNA, 2 mM MgCl2,5 mM dNTP, 5 U polymerase, 2μl 10×buffer under the following conditions: pre-denaturation at 95 ℃ for 5 min, denaturation at 94 ℃ for 30 s and annealing for 40 s and extension at 72 ℃ for 45 s, for 35 cycles. Each PCR was purified with 1 U shrimp alkaline phosphatase and 0.2 U Exonuclease 1 (Amersham Pharmacia Biotech, Little Chalfont, UK), for 1 h at 37 ℃ followed by 80°C for 15 min. Then, the treated products were sequenced with ABI PrismR BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit [Applied Biosystems, Inc. (ABI), Perkin-Elmer, Norwalk, CT].
Table 1 Primers of the TSHR Gene and the Annealing Temperatures of PCR
Statistical Analysis
Alleles and genotypes frequencies were compared between groups using the Chi-square (χ2) test (SPSS 11.0). A P value <0.05 was considered significant.
RESULTS
8 SNPs were found at introns 1,4,5,6 and exons 7,8 of the TSH-R gene respectively ( Figure 1 and Table 2，3 ). A novel SNP at 40 bp of exon 8( E8+ 40) was synonymous mutation AGA(Arg)→CGA(Arg), which had never been reported in SNPs bank (Figure 2). It had two genotypes: A/A, A/C. The frequency of allele C was 14.74% in patients, 15% in familial GD, 14.58% in sporadic GD and 16.67% in controls. Others were I1- 81 (SNP in intron 1 at 81 bp upstream of exon 2), I4-135 (SNP in intron 4 at 135 bp upstream of exon 5)，I4-365 (SNP in intron 4 at 365 bp upstream of exon 5)，I5-69 ( SNP in intron 5 at 69 bp upstream of exon 6 )，I6+13 ( SNP in intron 6 at 13 bp downstream of exon 6 )，I6-187 ( SNP in intron 6 at 187 bp upstream of exon 7 ), E7+16 ( SNP in 16 bp of exon 7 ), also synonymous mutation AAT(Asn)→AAC(Asn). After detecting genotypes and allele frequencies of these eight SNPs respectively, it showed that there were no statistically significant differences in SNP frequencies between patients and healthy control individuals (Table 4). Also, no statistically significant differences in allele or genotype frequencies were observed between familial GD and sporadic GD.
Table 2 Genotype and Case Distribution of the Polymorphisms of the TSHR Gene
Every polymorphism has three genotypes; “n” is the number of subjects
Table 3 Alleles Frequencies and Case Distribution of the SNPs, of the TSHR Gene
Table 4 The P value of Allele and Genotype Frequency of SNPs between Patients and Controls
Take I1-81 for example, in thirty familial GD, there were 49 T allele and 11 A allele. By comparing with controls which had 155 T allele and 37 A allele, the P value 0.8718 (top). Also, it had three genotypes: T/T, T/A, A/A, 21 T/T genotype, 9 T/A and A/A genotype. By comparing with controls which had 64 T/T genotype, 32 T/A and A/A genotype, the P value 0.7338 (bottom). It was the same as other polymorphisms.P value <0.05 was considered significant
Table 5 Meta-analysis Combining the Results of Three previous Studies[16]
Values are the number of subjects, with the corresponding percentage in parentheses.RR, relative risk; NS, not significant
Figure 1 Schematic representation of TSHR
E:exon; I:intron; I1- 81: SNP in intron 1 at 81 bp upstream of exon 2; I4-135:SNP in intron 4 at 135 bp upstream of exon 5; I4-365:SNP in intron 4 at 365 bp upstream of exon 5; I5-69:SNP in intron 5 at 69 bp upstream of exon 6; I6+13:SNP in intron 6 at 13 bp downstream of exon 6; I6-187:SNP in intron 6 at 187 bp upstream of exon 7; E7 or E8 with frame:there is polymorphism in exon 7 or 8
Figure 2 The mutant allel in exon 8 of TSH-R
Underline indicate a synonymous mutation AGA(Arg)→CGA(Arg)DISCUSSION
The identification of genetic loci conferring susceptibility to common complex diseases such as GD has proved problematic. Although there have been provided some candidate genes for GD, whether these genes are directly involved in the initiation of the pathologic process remains unclear. The thyroid-stimulating hormone/thyrotropin (TSH) is the most relevant hormone in the control of thyroid gland physiology. TSH effects on the thyroid gland are mediated by the interaction with a specific TSH receptor (TSHR). Therefore, the TSHR gene is a likely candidate gene for thyroid diseases. The TSHR gene as a disease susceptibility locus has been studied under the hypothesis that a modified antigen may have novel immunogenic properties.
Since Parma first described the presence of activating mutations in the TSHR gene in sporadic autonomous hyperfunctioning thyroid adenomas in 1993[2], an impressive number of different mutations have been identified, totaling 65 SNPs by the year 2003[7~11].These mutations have been identified in the context of different thyroid pathologies including: toxic thyroid nodules[1]; hereditary nonautoimmune hyperthyroidism (activating germline mutations)[12]; congenital euthyroid or hypothyroid TSH resistance (inactivating germline mutations)[13]; hot thyroid cancers[14]; AITD (autoimmune thyroid disease)[15].
These mutations were heterozygous and transmitted in an autosomal dominant fashion in the familial cases, suggesting casual involvement in the pathogenesis of such a variety of thyroid diseases. From the beginning, however, the pathophysiologic and clinical relevance of somatic TSHR mutations have been debated[16,17]. Strong arguments still exist against a fully causative role for these mutations in any of these diseases[15,16].
For this reason, we recently screened all 10 exons and some introns of the TSHR gene to assess whether TSHR was associated with GD. 8 SNPs were found at introns 1,4,5,6 and exons 7,8 of TSH-R respectively. Both of the SNPs at the exons were synonymous mutation: one was AAT(Asn)→AAC(Asn)at exon 7, the other was AGA(Arg)→CGA(Arg)at exon 8. The SNP at exon 8 was a novel finding, which had never been reported in SNPs bank. Since the effect of heredity is predominant in a family which has more than two members suffering from thyroid disease, we divided all patients into two groups to determine whether these SNPs were related to GD. Our results showed that there were no statistically significant differences of 8 SNPs between patients and controls. These findings suggested that the SNPs of the TSHR gene may not be responsible for GD in Chinese Han Pedigrees.
Also, we found that there were racial differences in the distribution of SNPs of the TSHR gene. Two common germline SNPS of the TSHR were described: a proline to threonine substitution at position 52 ( P52T) and a substitution of glutamic acid for aspartic acid at position 727(D727E)[15]. Both were negative in our study, while in our study, there was a novel SNP, synonymous mutation AGA(Arg)→CGA(Arg), in exon 8. The frequency of allele C was 17.29% and 20% in patients and controls respectively. The difference between these findings could be the results of racial differences, selection bias, or population stratification[4].
Our study, showing no association between SNPs of TSHR and GD, could not rule out weak associations. Take D727E for example[4] (Table 5): Ban and colleagues also could not get an association between D727E SNP and GD in their study. They, therefore, performed a meta-analysis combining their data with other data reported in the previous two studies showing no association between D727E SNP and GD. The results of the meta-analysis suggested a weak association between the D727E SNP E allele and GD (P value=0.03, RR=1.6)[4]. Also, case-control studies have their own limits as a result of a chance event or random variation, especially in small data sets. Therefore, larger samples are needed in our study to detect weaker associations. In addition, other approaches can be used in the genetic analysis to make up the potential short cut of case-control study, such as linkage analysis and intrafamilial linkage disequilibrium.
In conclusion, our dataset did not show a direct association between SNPs of TSHR and GD. The SNPs of the TSHR gene may not be responsible for GD. There are racial differences in the distribution of SNPs of the TSHR gene. Larger samples and a combination of other approaches to genetic analysis are needed in further study to detect weaker associations.
ACKNOWLEDGEMENTS
This work was supported by the National Natural Science Foundation of China ( 30470815); the sustentation fund of 2003 Technology Development Program of Shandong Province ( Z2003C02 ); Jiangsu Province Department of Health (H200651); Xuzhou Board of Health(XW2005017).
We are grateful to State Key Laboratory of Medical Genomics, Ruijin Hospital Affiliated to Shanghai Second Medical University for their cooperation.
REFERENCES
1. Parma J, Duprez L, Van Sande J, et al. Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature,1993，365:649-651.
2. Tomer Y, Davies TF, et al.Searching for the autoimmune thyroid disease susceptibility genes: from gene mapping to gene function. Endocr Rev, 2003，24(5):694-717.
3. Chistiakov DA, et al.Thyroid-stimulating hormone receptor and its role in Graves disease. Mol Genet Metab,2003，80(4):377-88.
4. Yoshiyuki Ban,et al.A germline single nucleotide polymorphism at the intracellular domain of the human thyrotropin receptor does not have a major effect on the development of Graves disease. Thyroid, 2002，12:1079-1083.
5. J.E. Collins, et al.Lack of association of the vitamin D receptor gene with Graves disease in UK Caucasians. Clin Endocrinol (Oxf),2004，60(5):618-24.
6. Tonacchera M, Van Sande J, Cetani F, et al. Functional characteristics of three new germline mutations of the thyrotropin gene causing autosomal dominant toxic thyroid hyperplasia. J Clin Endocrinol Metab,1996，81:547-554.
7. Paschke R, Ludgate ME.The thyrotropin receptor in thyroid disease. N Engl J Med, 1997,337:1675-1681.
8. Fuhrer D,Paschke R.Thyroid-stimulating hormone receptor mutations: update and clinical implications.Curr Opin Endocrinol Diabetes,2000,7:288-294.
9. Corvilain B, Van Sande J,Dumont JE, Vassart G.Somatic and germline mutation of the TSH receptor and thyroid diseases.Clin Endocrinol (oxf),2001,55:143-158.
10. Wonerow P, Neumann S, Gudermann T,Paschke R.Thyrotropin receptor mutations as a tool to understand Thyrotropin receptor action. J Mol Med,2001,79:707-721.
11. Rodien P,Ho SC, Vlaeminck V,Vassart G, Costagliola S.Activating mutations of TSH receptor. ANN endocrinol,2003,64:12-16.
12. Duprez L, Parma J, Van Sande J, Allgeier A, Leclere J, Schvartz C, Delisle MJ, Decoulx M, Orgiazzi J, Dumont J, Vassart G.Germline mutations in the thyrotropin receptor gene cause non-autoimmune autosomal dominant hyperthyroidism. Nat Genet,1994,7:396-401.
13. Sunthorn
A novel single nucleotide polymorphism of human thyrotropin receptor does not have a major effect on development of Graves disease
LIANG Jun1,2*, GAO Ling1*,ZHANG Li1*, ZHU Rui-hua2,DOU Lian-jun2,ZHANG Tong2,WU Qing-qiang2, LI Yu-su2,HENG Hao2,XIA Zhong1, ZHAO Jia-jun1, SONG Huai-dong3
1 Shandong Provincial Hospital, Shandong University,Jinan 250021， China
2 The Center (4th) Hospital of Xuzhou,Xuzhou,Jiangsu Province 221009， China
3 Ruijin Hospital, Shanghai Institute of Endocrinology, the State Key Laboratory of Medical Genomics of Shanghai Second Medical University,Shanghai 200025， China
*These authors contributed equally to this work.
Correspondence to SONG Huai-dong，huaidong_s1966@163.com or ZHAO Jia-jun，jjzhao@medmail.com.cn
[Abstract] Objective Graves disease (GD) is induced by an interplay of genetic factors and environmental triggers. It is well recognized that the thyroid stimulating hormone receptor/ thyrotropin (TSH-R) functions as a B-cell autoantigen in autoimmune thyroid diseases and therefore may be a potential candidate gene contributing to the development of GD or influencing the clinical course of the disease. However, it remains unclear whether the TSH-R gene is directly involved in the initiation of GD. Previous reports have been contrary results due to racial differences, selection bias, or population stratification. In order to obtain whether the single nucleotide polymorphisms (SNPs) concerning of the TSH-R is associated with GD in Chinese Han Pedigrees.Methods In the present study,we assessed all 10 exons and some introns of the TSHR gene using case-control analysis. Results We found a novel SNP in exon 8 and other 7 SNPs previously published in introns 1,4,5,6 and exon 7. The novel SNP showed two genotypes: A/A, A/C and the frequency of allele C was 14.74% in patients and 16.67% in controls respectively. No statistically significant differences in allele or genotype frequencies were observed between GD and healthy control subjects for any of the eight SNPs studied. Conclusion Our findings suggested that neither the novel SNP nor the other 7 SNPs of the TSHR gene may be responsible for GD.
[Key words] TSHR gene;Graves disease;polymorphism
INTRODUCTION
Graves disease(GD) is a common organ-specific autoimmune disease. The etiology of GD is believed to involve a complex interaction between susceptibility genes and environmental insults. Ten years after the first description of activating mutations in the TSH-R gene in sporadic autonomous hyperfunctioning thyroid adenomas[1], there is general agreement in assigning a major pathogenic role for this genetic abnormality, acting via the constitutive activation of the cAMP pathway, in both the growth and functional characteristics of these tumours. In GD, thyroid-stimulating autoantibodies can mimic TSH action and stimulate thyroid cells leading to hyperthyroidism and abnormal overproduction of thyroid hormone. Mechanism of TSHR-autoantibodies production is more or less clear but a susceptibility gene, which is linked to their production, is still unknown. There have been provided some candidate genes for GD, such as GD-1,GD-2,CTLA-4,TG,TPO,NIS and so on[2], but several reports related to the association of TSHR gene and GD genetic studies show no linkage between the TSHR gene and GD. Among three common polymorphisms in the TSHR gene, only the D727E germline polymorphism in the cytoplasmic tail of the receptor tested an association with the disease, and this association is weak[3]. However, other investigators did not confirm this association due to different subjects involved of stripes and gender[4].That suggest interpopulation heterogeneity of genetic and environment affect disease exist, reflecting the important matching of cases and control individuals within ethnic group[5]. In order to elucidate whether polymorphisms of TSHR was associated with GD in Chinese Han Pedigrees, in the present study we identified all 10 exons and some introns of the TSHR gene through direct sequencing using a case-control dataset.
METHODS
Subjects
Seventy-eight patients with GD of Han Nationality of Shandong Province in China who were divided into two groups: thirty unrelated patients with a familial hereditary history of GD and forty-eight sporadic GD. Fifteen male and sixty-three female patients, mean age was 44.83±14.51 years. Ninety-six normal healthy donors were age- and sex-matched from Han Nationality of Shandong Province in China.
Diagnosis of GD was based on the evidence of clinical hyperthyroidism associated with elevated serum free thyroxine (FT4) and free triiodothyronine (FT3) levels and suppressed serum TSH concentrations. A typical diffuse goiter with increased vascularization was detected by ultrasound. All the patients owed an increased thyroidal iodine uptake in thyroid scintiscans and had high titers of thyroperoxidase antibodies (TPOAb), thyroglobulin antibodies (TgAb) and TRAb measured by a radioreceptor assay.
Normal subjects were specifically selected as a control group. In these subjects there was no evidence of thyroid disease. All controls were clinically and biochemically euthyroid and were negative for thyroid antibodies
Ethical approval and informed consent for genetic analysis of these samples had also been obtained.
Sequencing of TSHR Gene
Total genomic DNA was extracted from peripheral lymphocytes of whole blood using standard phenol-chloroform procedures[6]. The target DNA were amplified by polymerase chain reactions (PCR) with primers designed using sequence information from GenBank ( LocusID: 7253) (Table 1). PCR was performed in a 20-μl reaction mixture containing 50 ng of genomic DNA, 2 mM MgCl2,5 mM dNTP, 5 U polymerase, 2μl 10×buffer under the following conditions: pre-denaturation at 95 ℃ for 5 min, denaturation at 94 ℃ for 30 s and annealing for 40 s and extension at 72 ℃ for 45 s, for 35 cycles. Each PCR was purified with 1 U shrimp alkaline phosphatase and 0.2 U Exonuclease 1 (Amersham Pharmacia Biotech, Little Chalfont, UK), for 1 h at 37 ℃ followed by 80°C for 15 min. Then, the treated products were sequenced with ABI PrismR BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit [Applied Biosystems, Inc. (ABI), Perkin-Elmer, Norwalk, CT].
Table 1 Primers of the TSHR Gene and the Annealing Temperatures of PCR
TP1
Statistical Analysis
Alleles and genotypes frequencies were compared between groups using the Chi-square (χ2) test (SPSS 11.0). A P value <0.05 was considered significant.
RESULTS
8 SNPs were found at introns 1,4,5,6 and exons 7,8 of the TSH-R gene respectively ( Figure 1 and Table 2，3 ). A novel SNP at 40 bp of exon 8( E8+ 40) was synonymous mutation AGA(Arg)→CGA(Arg), which had never been reported in SNPs bank (Figure 2). It had two genotypes: A/A, A/C. The frequency of allele C was 14.74% in patients, 15% in familial GD, 14.58% in sporadic GD and 16.67% in controls. Others were I1- 81 (SNP in intron 1 at 81 bp upstream of exon 2), I4-135 (SNP in intron 4 at 135 bp upstream of exon 5)，I4-365 (SNP in intron 4 at 365 bp upstream of exon 5)，I5-69 ( SNP in intron 5 at 69 bp upstream of exon 6 )，I6+13 ( SNP in intron 6 at 13 bp downstream of exon 6 )，I6-187 ( SNP in intron 6 at 187 bp upstream of exon 7 ), E7+16 ( SNP in 16 bp of exon 7 ), also synonymous mutation AAT(Asn)→AAC(Asn). After detecting genotypes and allele frequencies of these eight SNPs respectively, it showed that there were no statistically significant differences in SNP frequencies between patients and healthy control individuals (Table 4). Also, no statistically significant differences in allele or genotype frequencies were observed between familial GD and sporadic GD.
Table 2 Genotype and Case Distribution of the Polymorphisms of the TSHR Gene
TP2
Every polymorphism has three genotypes; “n” is the number of subjects
Table 3 Alleles Frequencies and Case Distribution of the SNPs, of the TSHR Gene
TP3
Table 4 The P value of Allele and Genotype Frequency of SNPs between Patients and Controls
TP4
Take I1-81 for example, in thirty familial GD, there were 49 T allele and 11 A allele. By comparing with controls which had 155 T allele and 37 A allele, the P value 0.8718 (top). Also, it had three genotypes: T/T, T/A, A/A, 21 T/T genotype, 9 T/A and A/A genotype. By comparing with controls which had 64 T/T genotype, 32 T/A and A/A genotype, the P value 0.7338 (bottom). It was the same as other polymorphisms.P value <0.05 was considered significant
Table 5 Meta-analysis Combining the Results of Three previous Studies[16]
TP5
Values are the number of subjects, with the corresponding percentage in parentheses.RR, relative risk; NS, not significant
TP6
Figure 1 Schematic representation of TSHR
E:exon; I:intron; I1- 81: SNP in intron 1 at 81 bp upstream of exon 2; I4-135:SNP in intron 4 at 135 bp upstream of exon 5; I4-365:SNP in intron 4 at 365 bp upstream of exon 5; I5-69:SNP in intron 5 at 69 bp upstream of exon 6; I6+13:SNP in intron 6 at 13 bp downstream of exon 6; I6-187:SNP in intron 6 at 187 bp upstream of exon 7; E7 or E8 with frame:there is polymorphism in exon 7 or 8
TP7
Figure 2 The mutant allel in exon 8 of TSH-R
Underline indicate a synonymous mutation AGA(Arg)→CGA(Arg)DISCUSSION
The identification of genetic loci conferring susceptibility to common complex diseases such as GD has proved problematic. Although there have been provided some candidate genes for GD, whether these genes are directly involved in the initiation of the pathologic process remains unclear. The thyroid-stimulating hormone/thyrotropin (TSH) is the most relevant hormone in the control of thyroid gland physiology. TSH effects on the thyroid gland are mediated by the interaction with a specific TSH receptor (TSHR). Therefore, the TSHR gene is a likely candidate gene for thyroid diseases. The TSHR gene as a disease susceptibility locus has been studied under the hypothesis that a modified antigen may have novel immunogenic properties.
Since Parma first described the presence of activating mutations in the TSHR gene in sporadic autonomous hyperfunctioning thyroid adenomas in 1993[2], an impressive number of different mutations have been identified, totaling 65 SNPs by the year 2003[7~11].These mutations have been identified in the context of different thyroid pathologies including: toxic thyroid nodules[1]; hereditary nonautoimmune hyperthyroidism (activating germline mutations)[12]; congenital euthyroid or hypothyroid TSH resistance (inactivating germline mutations)[13]; hot thyroid cancers[14]; AITD (autoimmune thyroid disease)[15].
These mutations were heterozygous and transmitted in an autosomal dominant fashion in the familial cases, suggesting casual involvement in the pathogenesis of such a variety of thyroid diseases. From the beginning, however, the pathophysiologic and clinical relevance of somatic TSHR mutations have been debated[16,17]. Strong arguments still exist against a fully causative role for these mutations in any of these diseases[15,16].
For this reason, we recently screened all 10 exons and some introns of the TSHR gene to assess whether TSHR was associated with GD. 8 SNPs were found at introns 1,4,5,6 and exons 7,8 of TSH-R respectively. Both of the SNPs at the exons were synonymous mutation: one was AAT(Asn)→AAC(Asn)at exon 7, the other was AGA(Arg)→CGA(Arg)at exon 8. The SNP at exon 8 was a novel finding, which had never been reported in SNPs bank. Since the effect of heredity is predominant in a family which has more than two members suffering from thyroid disease, we divided all patients into two groups to determine whether these SNPs were related to GD. Our results showed that there were no statistically significant differences of 8 SNPs between patients and controls. These findings suggested that the SNPs of the TSHR gene may not be responsible for GD in Chinese Han Pedigrees.
Also, we found that there were racial differences in the distribution of SNPs of the TSHR gene. Two common germline SNPS of the TSHR were described: a proline to threonine substitution at position 52 ( P52T) and a substitution of glutamic acid for aspartic acid at position 727(D727E)[15]. Both were negative in our study, while in our study, there was a novel SNP, synonymous mutation AGA(Arg)→CGA(Arg), in exon 8. The frequency of allele C was 17.29% and 20% in patients and controls respectively. The difference between these findings could be the results of racial differences, selection bias, or population stratification[4].
Our study, showing no association between SNPs of TSHR and GD, could not rule out weak associations. Take D727E for example[4] (Table 5): Ban and colleagues also could not get an association between D727E SNP and GD in their study. They, therefore, performed a meta-analysis combining their data with other data reported in the previous two studies showing no association between D727E SNP and GD. The results of the meta-analysis suggested a weak association between the D727E SNP E allele and GD (P value=0.03, RR=1.6)[4]. Also, case-control studies have their own limits as a result of a chance event or random variation, especially in small data sets. Therefore, larger samples are needed in our study to detect weaker associations. In addition, other approaches can be used in the genetic analysis to make up the potential short cut of case-control study, such as linkage analysis and intrafamilial linkage disequilibrium.
In conclusion, our dataset did not show a direct association between SNPs of TSHR and GD. The SNPs of the TSHR gene may not be responsible for GD. There are racial differences in the distribution of SNPs of the TSHR gene. Larger samples and a combination of other approaches to genetic analysis are needed in further study to detect weaker associations.
ACKNOWLEDGEMENTS
This work was supported by the National Natural Science Foundation of China ( 30470815); the sustentation fund of 2003 Technology Development Program of Shandong Province ( Z2003C02 ); Jiangsu Province Department of Health (H200651); Xuzhou Board of Health(XW2005017).
We are grateful to State Key Laboratory of Medical Genomics, Ruijin Hospital Affiliated to Shanghai Second Medical University for their cooperation.
REFERENCES
1. Parma J, Duprez L, Van Sande J, et al. Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature,1993，365:649-651.
2. Tomer Y, Davies TF, et al.Searching for the autoimmune thyroid disease susceptibility genes: from gene mapping to gene function. Endocr Rev, 2003，24(5):694-717.
3. Chistiakov DA, et al.Thyroid-stimulating hormone receptor and its role in Graves disease. Mol Genet Metab,2003，80(4):377-88.
4. Yoshiyuki Ban,et al.A germline single nucleotide polymorphism at the intracellular domain of the human thyrotropin receptor does not have a major effect on the development of Graves disease. Thyroid, 2002，12:1079-1083.
5. J.E. Collins, et al.Lack of association of the vitamin D receptor gene with Graves disease in UK Caucasians. Clin Endocrinol (Oxf),2004，60(5):618-24.
6. Tonacchera M, Van Sande J, Cetani F, et al. Functional characteristics of three new germline mutations of the thyrotropin gene causing autosomal dominant toxic thyroid hyperplasia. J Clin Endocrinol Metab,1996，81:547-554.
7. Paschke R, Ludgate ME.The thyrotropin receptor in thyroid disease. N Engl J Med, 1997,337:1675-1681.
8. Fuhrer D,Paschke R.Thyroid-stimulating hormone receptor mutations: update and clinical implications.Curr Opin Endocrinol Diabetes,2000,7:288-294.
9. Corvilain B, Van Sande J,Dumont JE, Vassart G.Somatic and germline mutation of the TSH receptor and thyroid diseases.Clin Endocrinol (oxf),2001,55:143-158.
10. Wonerow P, Neumann S, Gudermann T,Paschke R.Thyrotropin receptor mutations as a tool to understand Thyrotropin receptor action. J Mol Med,2001,79:707-721.
11. Rodien P,Ho SC, Vlaeminck V,Vassart G, Costagliola S.Activating mutations of TSH receptor. ANN endocrinol,2003,64:12-16.
12. Duprez L, Parma J, Van Sande J, Allgeier A, Leclere J, Schvartz C, Delisle MJ, Decoulx M, Orgiazzi J, Dumont J, Vassart G.Germline mutations in the thyrotropin receptor gene cause non-autoimmune autosomal dominant hyperthyroidism. Nat Genet,1994,7:396-401.
13. Sunthornthepvarakul T, Gottschalk ME, Hayashi Y, Refetoff S.Brief report: resistance to thyrotropin caused by mutations in the thyrotropin-receptor gene. N Engl J Med,1995,332:155-160.
14. Russo D, Arturi F, Schlumberger M, Caillou B, Monier R, Filetti S, Suarez HG.Activating mutations of the TSH receptor in differentiated thyroid carcinomas, Oncogene,1995,11:1907-1911.
15. Tonacchera M, Pinchera V.Thyrotropin receptor polymorphisms and thyroid disease.J Clin Endocrinol Metab,2000,85:2637-2639.
16. Derwahl M.Editorial: TSH Receptor and Gs-α gene mutations in the pathogenesis of toxic thyroid adenomas: a note caution. J Clin Endocrinnol Metab,1996,81:2783-2785.
17. Derwahl M, Hamancher C,Russo D,Broecker M, Manole D, Schatz H,Kopp P, Filetti S..Constitutive activation of the Gs-alpha protein-Adenylate cyclase pathway may not be sufficient to generate toxic adenimas.J Clin Endocrinol Metab,1996,81:1898-1904.
(Editor Anne)thepvarakul T, Gottschalk ME, Hayashi Y, Refetoff S.Brief report: resistance to thyrotropin caused by mutations in the thyrotropin-receptor gene. N Engl J Med,1995,332:155-160.
14. Russo D, Arturi F, Schlumberger M, Caillou B, Monier R, Filetti S, Suarez HG.Activating mutations of the TSH receptor in differentiated thyroid carcinomas, Oncogene,1995,11:1907-1911.
15. Tonacchera M, Pinchera V.Thyrotropin receptor polymorphisms and thyroid disease.J Clin Endocrinol Metab,2000,85:2637-2639.
16. Derwahl M.Editorial: TSH Receptor and Gs-α gene mutations in the pathogenesis of toxic thyroid adenomas: a note caution. J Clin Endocrinnol Metab,1996,81:2783-2785.
17. Derwahl M, Hamancher C,Russo D,Broecker M, Manole D, Schatz H,Kopp P, Filetti S..Constitutive activation of the Gs-alpha protein-Adenylate cyclase pathway may not be sufficient to generate toxic adenimas.J Clin Endocrinol Metab,1996,81:1898-1904.
(Editor Anne)(LIANG Jun1,2*, GAO Ling1*)
2 The Center (4th) Hospital of Xuzhou,Xuzhou,Jiangsu Province 221009， China
3 Ruijin Hospital, Shanghai Institute of Endocrinology, the State Key Laboratory of Medical Genomics of Shanghai Second Medical University,Shanghai 200025， China
*These authors contributed equally to this work.
Correspondence to SONG Huai-dong，huaidong_s1966@163.com or ZHAO Jia-jun，jjzhao@medmail.com.cn
[Abstract] Objective Graves disease (GD) is induced by an interplay of genetic factors and environmental triggers. It is well recognized that the thyroid stimulating hormone receptor/ thyrotropin (TSH-R) functions as a B-cell autoantigen in autoimmune thyroid diseases and therefore may be a potential candidate gene contributing to the development of GD or influencing the clinical course of the disease. However, it remains unclear whether the TSH-R gene is directly involved in the initiation of GD. Previous reports have been contrary results due to racial differences, selection bias, or population stratification. In order to obtain whether the single nucleotide polymorphisms (SNPs) concerning of the TSH-R is associated with GD in Chinese Han Pedigrees.Methods In the present study,we assessed all 10 exons and some introns of the TSHR gene using case-control analysis. Results We found a novel SNP in exon 8 and other 7 SNPs previously published in introns 1,4,5,6 and exon 7. The novel SNP showed two genotypes: A/A, A/C and the frequency of allele C was 14.74% in patients and 16.67% in controls respectively. No statistically significant differences in allele or genotype frequencies were observed between GD and healthy control subjects for any of the eight SNPs studied. Conclusion Our findings suggested that neither the novel SNP nor the other 7 SNPs of the TSHR gene may be responsible for GD.
[Key words] TSHR gene;Graves disease;polymorphism
INTRODUCTION
Graves disease(GD) is a common organ-specific autoimmune disease. The etiology of GD is believed to involve a complex interaction between susceptibility genes and environmental insults. Ten years after the first description of activating mutations in the TSH-R gene in sporadic autonomous hyperfunctioning thyroid adenomas[1], there is general agreement in assigning a major pathogenic role for this genetic abnormality, acting via the constitutive activation of the cAMP pathway, in both the growth and functional characteristics of these tumours. In GD, thyroid-stimulating autoantibodies can mimic TSH action and stimulate thyroid cells leading to hyperthyroidism and abnormal overproduction of thyroid hormone. Mechanism of TSHR-autoantibodies production is more or less clear but a susceptibility gene, which is linked to their production, is still unknown. There have been provided some candidate genes for GD, such as GD-1,GD-2,CTLA-4,TG,TPO,NIS and so on[2], but several reports related to the association of TSHR gene and GD genetic studies show no linkage between the TSHR gene and GD. Among three common polymorphisms in the TSHR gene, only the D727E germline polymorphism in the cytoplasmic tail of the receptor tested an association with the disease, and this association is weak[3]. However, other investigators did not confirm this association due to different subjects involved of stripes and gender[4].That suggest interpopulation heterogeneity of genetic and environment affect disease exist, reflecting the important matching of cases and control individuals within ethnic group[5]. In order to elucidate whether polymorphisms of TSHR was associated with GD in Chinese Han Pedigrees, in the present study we identified all 10 exons and some introns of the TSHR gene through direct sequencing using a case-control dataset.
METHODS
Subjects
Seventy-eight patients with GD of Han Nationality of Shandong Province in China who were divided into two groups: thirty unrelated patients with a familial hereditary history of GD and forty-eight sporadic GD. Fifteen male and sixty-three female patients, mean age was 44.83±14.51 years. Ninety-six normal healthy donors were age- and sex-matched from Han Nationality of Shandong Province in China.
Diagnosis of GD was based on the evidence of clinical hyperthyroidism associated with elevated serum free thyroxine (FT4) and free triiodothyronine (FT3) levels and suppressed serum TSH concentrations. A typical diffuse goiter with increased vascularization was detected by ultrasound. All the patients owed an increased thyroidal iodine uptake in thyroid scintiscans and had high titers of thyroperoxidase antibodies (TPOAb), thyroglobulin antibodies (TgAb) and TRAb measured by a radioreceptor assay.
Normal subjects were specifically selected as a control group. In these subjects there was no evidence of thyroid disease. All controls were clinically and biochemically euthyroid and were negative for thyroid antibodies
Ethical approval and informed consent for genetic analysis of these samples had also been obtained.
Sequencing of TSHR Gene
Total genomic DNA was extracted from peripheral lymphocytes of whole blood using standard phenol-chloroform procedures[6]. The target DNA were amplified by polymerase chain reactions (PCR) with primers designed using sequence information from GenBank ( LocusID: 7253) (Table 1). PCR was performed in a 20-μl reaction mixture containing 50 ng of genomic DNA, 2 mM MgCl2,5 mM dNTP, 5 U polymerase, 2μl 10×buffer under the following conditions: pre-denaturation at 95 ℃ for 5 min, denaturation at 94 ℃ for 30 s and annealing for 40 s and extension at 72 ℃ for 45 s, for 35 cycles. Each PCR was purified with 1 U shrimp alkaline phosphatase and 0.2 U Exonuclease 1 (Amersham Pharmacia Biotech, Little Chalfont, UK), for 1 h at 37 ℃ followed by 80°C for 15 min. Then, the treated products were sequenced with ABI PrismR BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit [Applied Biosystems, Inc. (ABI), Perkin-Elmer, Norwalk, CT].
Table 1 Primers of the TSHR Gene and the Annealing Temperatures of PCR
Statistical Analysis
Alleles and genotypes frequencies were compared between groups using the Chi-square (χ2) test (SPSS 11.0). A P value <0.05 was considered significant.
RESULTS
8 SNPs were found at introns 1,4,5,6 and exons 7,8 of the TSH-R gene respectively ( Figure 1 and Table 2，3 ). A novel SNP at 40 bp of exon 8( E8+ 40) was synonymous mutation AGA(Arg)→CGA(Arg), which had never been reported in SNPs bank (Figure 2). It had two genotypes: A/A, A/C. The frequency of allele C was 14.74% in patients, 15% in familial GD, 14.58% in sporadic GD and 16.67% in controls. Others were I1- 81 (SNP in intron 1 at 81 bp upstream of exon 2), I4-135 (SNP in intron 4 at 135 bp upstream of exon 5)，I4-365 (SNP in intron 4 at 365 bp upstream of exon 5)，I5-69 ( SNP in intron 5 at 69 bp upstream of exon 6 )，I6+13 ( SNP in intron 6 at 13 bp downstream of exon 6 )，I6-187 ( SNP in intron 6 at 187 bp upstream of exon 7 ), E7+16 ( SNP in 16 bp of exon 7 ), also synonymous mutation AAT(Asn)→AAC(Asn). After detecting genotypes and allele frequencies of these eight SNPs respectively, it showed that there were no statistically significant differences in SNP frequencies between patients and healthy control individuals (Table 4). Also, no statistically significant differences in allele or genotype frequencies were observed between familial GD and sporadic GD.
Table 2 Genotype and Case Distribution of the Polymorphisms of the TSHR Gene
Every polymorphism has three genotypes; “n” is the number of subjects
Table 3 Alleles Frequencies and Case Distribution of the SNPs, of the TSHR Gene
Table 4 The P value of Allele and Genotype Frequency of SNPs between Patients and Controls
Take I1-81 for example, in thirty familial GD, there were 49 T allele and 11 A allele. By comparing with controls which had 155 T allele and 37 A allele, the P value 0.8718 (top). Also, it had three genotypes: T/T, T/A, A/A, 21 T/T genotype, 9 T/A and A/A genotype. By comparing with controls which had 64 T/T genotype, 32 T/A and A/A genotype, the P value 0.7338 (bottom). It was the same as other polymorphisms.P value <0.05 was considered significant
Table 5 Meta-analysis Combining the Results of Three previous Studies[16]
Values are the number of subjects, with the corresponding percentage in parentheses.RR, relative risk; NS, not significant
Figure 1 Schematic representation of TSHR
E:exon; I:intron; I1- 81: SNP in intron 1 at 81 bp upstream of exon 2; I4-135:SNP in intron 4 at 135 bp upstream of exon 5; I4-365:SNP in intron 4 at 365 bp upstream of exon 5; I5-69:SNP in intron 5 at 69 bp upstream of exon 6; I6+13:SNP in intron 6 at 13 bp downstream of exon 6; I6-187:SNP in intron 6 at 187 bp upstream of exon 7; E7 or E8 with frame:there is polymorphism in exon 7 or 8
Figure 2 The mutant allel in exon 8 of TSH-R
Underline indicate a synonymous mutation AGA(Arg)→CGA(Arg)DISCUSSION
The identification of genetic loci conferring susceptibility to common complex diseases such as GD has proved problematic. Although there have been provided some candidate genes for GD, whether these genes are directly involved in the initiation of the pathologic process remains unclear. The thyroid-stimulating hormone/thyrotropin (TSH) is the most relevant hormone in the control of thyroid gland physiology. TSH effects on the thyroid gland are mediated by the interaction with a specific TSH receptor (TSHR). Therefore, the TSHR gene is a likely candidate gene for thyroid diseases. The TSHR gene as a disease susceptibility locus has been studied under the hypothesis that a modified antigen may have novel immunogenic properties.
Since Parma first described the presence of activating mutations in the TSHR gene in sporadic autonomous hyperfunctioning thyroid adenomas in 1993[2], an impressive number of different mutations have been identified, totaling 65 SNPs by the year 2003[7~11].These mutations have been identified in the context of different thyroid pathologies including: toxic thyroid nodules[1]; hereditary nonautoimmune hyperthyroidism (activating germline mutations)[12]; congenital euthyroid or hypothyroid TSH resistance (inactivating germline mutations)[13]; hot thyroid cancers[14]; AITD (autoimmune thyroid disease)[15].
These mutations were heterozygous and transmitted in an autosomal dominant fashion in the familial cases, suggesting casual involvement in the pathogenesis of such a variety of thyroid diseases. From the beginning, however, the pathophysiologic and clinical relevance of somatic TSHR mutations have been debated[16,17]. Strong arguments still exist against a fully causative role for these mutations in any of these diseases[15,16].
For this reason, we recently screened all 10 exons and some introns of the TSHR gene to assess whether TSHR was associated with GD. 8 SNPs were found at introns 1,4,5,6 and exons 7,8 of TSH-R respectively. Both of the SNPs at the exons were synonymous mutation: one was AAT(Asn)→AAC(Asn)at exon 7, the other was AGA(Arg)→CGA(Arg)at exon 8. The SNP at exon 8 was a novel finding, which had never been reported in SNPs bank. Since the effect of heredity is predominant in a family which has more than two members suffering from thyroid disease, we divided all patients into two groups to determine whether these SNPs were related to GD. Our results showed that there were no statistically significant differences of 8 SNPs between patients and controls. These findings suggested that the SNPs of the TSHR gene may not be responsible for GD in Chinese Han Pedigrees.
Also, we found that there were racial differences in the distribution of SNPs of the TSHR gene. Two common germline SNPS of the TSHR were described: a proline to threonine substitution at position 52 ( P52T) and a substitution of glutamic acid for aspartic acid at position 727(D727E)[15]. Both were negative in our study, while in our study, there was a novel SNP, synonymous mutation AGA(Arg)→CGA(Arg), in exon 8. The frequency of allele C was 17.29% and 20% in patients and controls respectively. The difference between these findings could be the results of racial differences, selection bias, or population stratification[4].
Our study, showing no association between SNPs of TSHR and GD, could not rule out weak associations. Take D727E for example[4] (Table 5): Ban and colleagues also could not get an association between D727E SNP and GD in their study. They, therefore, performed a meta-analysis combining their data with other data reported in the previous two studies showing no association between D727E SNP and GD. The results of the meta-analysis suggested a weak association between the D727E SNP E allele and GD (P value=0.03, RR=1.6)[4]. Also, case-control studies have their own limits as a result of a chance event or random variation, especially in small data sets. Therefore, larger samples are needed in our study to detect weaker associations. In addition, other approaches can be used in the genetic analysis to make up the potential short cut of case-control study, such as linkage analysis and intrafamilial linkage disequilibrium.
In conclusion, our dataset did not show a direct association between SNPs of TSHR and GD. The SNPs of the TSHR gene may not be responsible for GD. There are racial differences in the distribution of SNPs of the TSHR gene. Larger samples and a combination of other approaches to genetic analysis are needed in further study to detect weaker associations.
ACKNOWLEDGEMENTS
This work was supported by the National Natural Science Foundation of China ( 30470815); the sustentation fund of 2003 Technology Development Program of Shandong Province ( Z2003C02 ); Jiangsu Province Department of Health (H200651); Xuzhou Board of Health(XW2005017).
We are grateful to State Key Laboratory of Medical Genomics, Ruijin Hospital Affiliated to Shanghai Second Medical University for their cooperation.
REFERENCES
1. Parma J, Duprez L, Van Sande J, et al. Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature,1993，365:649-651.
2. Tomer Y, Davies TF, et al.Searching for the autoimmune thyroid disease susceptibility genes: from gene mapping to gene function. Endocr Rev, 2003，24(5):694-717.
3. Chistiakov DA, et al.Thyroid-stimulating hormone receptor and its role in Graves disease. Mol Genet Metab,2003，80(4):377-88.
4. Yoshiyuki Ban,et al.A germline single nucleotide polymorphism at the intracellular domain of the human thyrotropin receptor does not have a major effect on the development of Graves disease. Thyroid, 2002，12:1079-1083.
5. J.E. Collins, et al.Lack of association of the vitamin D receptor gene with Graves disease in UK Caucasians. Clin Endocrinol (Oxf),2004，60(5):618-24.
6. Tonacchera M, Van Sande J, Cetani F, et al. Functional characteristics of three new germline mutations of the thyrotropin gene causing autosomal dominant toxic thyroid hyperplasia. J Clin Endocrinol Metab,1996，81:547-554.
7. Paschke R, Ludgate ME.The thyrotropin receptor in thyroid disease. N Engl J Med, 1997,337:1675-1681.
8. Fuhrer D,Paschke R.Thyroid-stimulating hormone receptor mutations: update and clinical implications.Curr Opin Endocrinol Diabetes,2000,7:288-294.
9. Corvilain B, Van Sande J,Dumont JE, Vassart G.Somatic and germline mutation of the TSH receptor and thyroid diseases.Clin Endocrinol (oxf),2001,55:143-158.
10. Wonerow P, Neumann S, Gudermann T,Paschke R.Thyrotropin receptor mutations as a tool to understand Thyrotropin receptor action. J Mol Med,2001,79:707-721.
11. Rodien P,Ho SC, Vlaeminck V,Vassart G, Costagliola S.Activating mutations of TSH receptor. ANN endocrinol,2003,64:12-16.
12. Duprez L, Parma J, Van Sande J, Allgeier A, Leclere J, Schvartz C, Delisle MJ, Decoulx M, Orgiazzi J, Dumont J, Vassart G.Germline mutations in the thyrotropin receptor gene cause non-autoimmune autosomal dominant hyperthyroidism. Nat Genet,1994,7:396-401.
13. Sunthorn
A novel single nucleotide polymorphism of human thyrotropin receptor does not have a major effect on development of Graves disease
LIANG Jun1,2*, GAO Ling1*,ZHANG Li1*, ZHU Rui-hua2,DOU Lian-jun2,ZHANG Tong2,WU Qing-qiang2, LI Yu-su2,HENG Hao2,XIA Zhong1, ZHAO Jia-jun1, SONG Huai-dong3
1 Shandong Provincial Hospital, Shandong University,Jinan 250021， China
2 The Center (4th) Hospital of Xuzhou,Xuzhou,Jiangsu Province 221009， China
3 Ruijin Hospital, Shanghai Institute of Endocrinology, the State Key Laboratory of Medical Genomics of Shanghai Second Medical University,Shanghai 200025， China
*These authors contributed equally to this work.
Correspondence to SONG Huai-dong，huaidong_s1966@163.com or ZHAO Jia-jun，jjzhao@medmail.com.cn
[Abstract] Objective Graves disease (GD) is induced by an interplay of genetic factors and environmental triggers. It is well recognized that the thyroid stimulating hormone receptor/ thyrotropin (TSH-R) functions as a B-cell autoantigen in autoimmune thyroid diseases and therefore may be a potential candidate gene contributing to the development of GD or influencing the clinical course of the disease. However, it remains unclear whether the TSH-R gene is directly involved in the initiation of GD. Previous reports have been contrary results due to racial differences, selection bias, or population stratification. In order to obtain whether the single nucleotide polymorphisms (SNPs) concerning of the TSH-R is associated with GD in Chinese Han Pedigrees.Methods In the present study,we assessed all 10 exons and some introns of the TSHR gene using case-control analysis. Results We found a novel SNP in exon 8 and other 7 SNPs previously published in introns 1,4,5,6 and exon 7. The novel SNP showed two genotypes: A/A, A/C and the frequency of allele C was 14.74% in patients and 16.67% in controls respectively. No statistically significant differences in allele or genotype frequencies were observed between GD and healthy control subjects for any of the eight SNPs studied. Conclusion Our findings suggested that neither the novel SNP nor the other 7 SNPs of the TSHR gene may be responsible for GD.
[Key words] TSHR gene;Graves disease;polymorphism
INTRODUCTION
Graves disease(GD) is a common organ-specific autoimmune disease. The etiology of GD is believed to involve a complex interaction between susceptibility genes and environmental insults. Ten years after the first description of activating mutations in the TSH-R gene in sporadic autonomous hyperfunctioning thyroid adenomas[1], there is general agreement in assigning a major pathogenic role for this genetic abnormality, acting via the constitutive activation of the cAMP pathway, in both the growth and functional characteristics of these tumours. In GD, thyroid-stimulating autoantibodies can mimic TSH action and stimulate thyroid cells leading to hyperthyroidism and abnormal overproduction of thyroid hormone. Mechanism of TSHR-autoantibodies production is more or less clear but a susceptibility gene, which is linked to their production, is still unknown. There have been provided some candidate genes for GD, such as GD-1,GD-2,CTLA-4,TG,TPO,NIS and so on[2], but several reports related to the association of TSHR gene and GD genetic studies show no linkage between the TSHR gene and GD. Among three common polymorphisms in the TSHR gene, only the D727E germline polymorphism in the cytoplasmic tail of the receptor tested an association with the disease, and this association is weak[3]. However, other investigators did not confirm this association due to different subjects involved of stripes and gender[4].That suggest interpopulation heterogeneity of genetic and environment affect disease exist, reflecting the important matching of cases and control individuals within ethnic group[5]. In order to elucidate whether polymorphisms of TSHR was associated with GD in Chinese Han Pedigrees, in the present study we identified all 10 exons and some introns of the TSHR gene through direct sequencing using a case-control dataset.
METHODS
Subjects
Seventy-eight patients with GD of Han Nationality of Shandong Province in China who were divided into two groups: thirty unrelated patients with a familial hereditary history of GD and forty-eight sporadic GD. Fifteen male and sixty-three female patients, mean age was 44.83±14.51 years. Ninety-six normal healthy donors were age- and sex-matched from Han Nationality of Shandong Province in China.
Diagnosis of GD was based on the evidence of clinical hyperthyroidism associated with elevated serum free thyroxine (FT4) and free triiodothyronine (FT3) levels and suppressed serum TSH concentrations. A typical diffuse goiter with increased vascularization was detected by ultrasound. All the patients owed an increased thyroidal iodine uptake in thyroid scintiscans and had high titers of thyroperoxidase antibodies (TPOAb), thyroglobulin antibodies (TgAb) and TRAb measured by a radioreceptor assay.
Normal subjects were specifically selected as a control group. In these subjects there was no evidence of thyroid disease. All controls were clinically and biochemically euthyroid and were negative for thyroid antibodies
Ethical approval and informed consent for genetic analysis of these samples had also been obtained.
Sequencing of TSHR Gene
Total genomic DNA was extracted from peripheral lymphocytes of whole blood using standard phenol-chloroform procedures[6]. The target DNA were amplified by polymerase chain reactions (PCR) with primers designed using sequence information from GenBank ( LocusID: 7253) (Table 1). PCR was performed in a 20-μl reaction mixture containing 50 ng of genomic DNA, 2 mM MgCl2,5 mM dNTP, 5 U polymerase, 2μl 10×buffer under the following conditions: pre-denaturation at 95 ℃ for 5 min, denaturation at 94 ℃ for 30 s and annealing for 40 s and extension at 72 ℃ for 45 s, for 35 cycles. Each PCR was purified with 1 U shrimp alkaline phosphatase and 0.2 U Exonuclease 1 (Amersham Pharmacia Biotech, Little Chalfont, UK), for 1 h at 37 ℃ followed by 80°C for 15 min. Then, the treated products were sequenced with ABI PrismR BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit [Applied Biosystems, Inc. (ABI), Perkin-Elmer, Norwalk, CT].
Table 1 Primers of the TSHR Gene and the Annealing Temperatures of PCR
TP1
Statistical Analysis
Alleles and genotypes frequencies were compared between groups using the Chi-square (χ2) test (SPSS 11.0). A P value <0.05 was considered significant.
RESULTS
8 SNPs were found at introns 1,4,5,6 and exons 7,8 of the TSH-R gene respectively ( Figure 1 and Table 2，3 ). A novel SNP at 40 bp of exon 8( E8+ 40) was synonymous mutation AGA(Arg)→CGA(Arg), which had never been reported in SNPs bank (Figure 2). It had two genotypes: A/A, A/C. The frequency of allele C was 14.74% in patients, 15% in familial GD, 14.58% in sporadic GD and 16.67% in controls. Others were I1- 81 (SNP in intron 1 at 81 bp upstream of exon 2), I4-135 (SNP in intron 4 at 135 bp upstream of exon 5)，I4-365 (SNP in intron 4 at 365 bp upstream of exon 5)，I5-69 ( SNP in intron 5 at 69 bp upstream of exon 6 )，I6+13 ( SNP in intron 6 at 13 bp downstream of exon 6 )，I6-187 ( SNP in intron 6 at 187 bp upstream of exon 7 ), E7+16 ( SNP in 16 bp of exon 7 ), also synonymous mutation AAT(Asn)→AAC(Asn). After detecting genotypes and allele frequencies of these eight SNPs respectively, it showed that there were no statistically significant differences in SNP frequencies between patients and healthy control individuals (Table 4). Also, no statistically significant differences in allele or genotype frequencies were observed between familial GD and sporadic GD.
Table 2 Genotype and Case Distribution of the Polymorphisms of the TSHR Gene
TP2
Every polymorphism has three genotypes; “n” is the number of subjects
Table 3 Alleles Frequencies and Case Distribution of the SNPs, of the TSHR Gene
TP3
Table 4 The P value of Allele and Genotype Frequency of SNPs between Patients and Controls
TP4
Take I1-81 for example, in thirty familial GD, there were 49 T allele and 11 A allele. By comparing with controls which had 155 T allele and 37 A allele, the P value 0.8718 (top). Also, it had three genotypes: T/T, T/A, A/A, 21 T/T genotype, 9 T/A and A/A genotype. By comparing with controls which had 64 T/T genotype, 32 T/A and A/A genotype, the P value 0.7338 (bottom). It was the same as other polymorphisms.P value <0.05 was considered significant
Table 5 Meta-analysis Combining the Results of Three previous Studies[16]
TP5
Values are the number of subjects, with the corresponding percentage in parentheses.RR, relative risk; NS, not significant
TP6
Figure 1 Schematic representation of TSHR
E:exon; I:intron; I1- 81: SNP in intron 1 at 81 bp upstream of exon 2; I4-135:SNP in intron 4 at 135 bp upstream of exon 5; I4-365:SNP in intron 4 at 365 bp upstream of exon 5; I5-69:SNP in intron 5 at 69 bp upstream of exon 6; I6+13:SNP in intron 6 at 13 bp downstream of exon 6; I6-187:SNP in intron 6 at 187 bp upstream of exon 7; E7 or E8 with frame:there is polymorphism in exon 7 or 8
TP7
Figure 2 The mutant allel in exon 8 of TSH-R
Underline indicate a synonymous mutation AGA(Arg)→CGA(Arg)DISCUSSION
The identification of genetic loci conferring susceptibility to common complex diseases such as GD has proved problematic. Although there have been provided some candidate genes for GD, whether these genes are directly involved in the initiation of the pathologic process remains unclear. The thyroid-stimulating hormone/thyrotropin (TSH) is the most relevant hormone in the control of thyroid gland physiology. TSH effects on the thyroid gland are mediated by the interaction with a specific TSH receptor (TSHR). Therefore, the TSHR gene is a likely candidate gene for thyroid diseases. The TSHR gene as a disease susceptibility locus has been studied under the hypothesis that a modified antigen may have novel immunogenic properties.
Since Parma first described the presence of activating mutations in the TSHR gene in sporadic autonomous hyperfunctioning thyroid adenomas in 1993[2], an impressive number of different mutations have been identified, totaling 65 SNPs by the year 2003[7~11].These mutations have been identified in the context of different thyroid pathologies including: toxic thyroid nodules[1]; hereditary nonautoimmune hyperthyroidism (activating germline mutations)[12]; congenital euthyroid or hypothyroid TSH resistance (inactivating germline mutations)[13]; hot thyroid cancers[14]; AITD (autoimmune thyroid disease)[15].
These mutations were heterozygous and transmitted in an autosomal dominant fashion in the familial cases, suggesting casual involvement in the pathogenesis of such a variety of thyroid diseases. From the beginning, however, the pathophysiologic and clinical relevance of somatic TSHR mutations have been debated[16,17]. Strong arguments still exist against a fully causative role for these mutations in any of these diseases[15,16].
For this reason, we recently screened all 10 exons and some introns of the TSHR gene to assess whether TSHR was associated with GD. 8 SNPs were found at introns 1,4,5,6 and exons 7,8 of TSH-R respectively. Both of the SNPs at the exons were synonymous mutation: one was AAT(Asn)→AAC(Asn)at exon 7, the other was AGA(Arg)→CGA(Arg)at exon 8. The SNP at exon 8 was a novel finding, which had never been reported in SNPs bank. Since the effect of heredity is predominant in a family which has more than two members suffering from thyroid disease, we divided all patients into two groups to determine whether these SNPs were related to GD. Our results showed that there were no statistically significant differences of 8 SNPs between patients and controls. These findings suggested that the SNPs of the TSHR gene may not be responsible for GD in Chinese Han Pedigrees.
Also, we found that there were racial differences in the distribution of SNPs of the TSHR gene. Two common germline SNPS of the TSHR were described: a proline to threonine substitution at position 52 ( P52T) and a substitution of glutamic acid for aspartic acid at position 727(D727E)[15]. Both were negative in our study, while in our study, there was a novel SNP, synonymous mutation AGA(Arg)→CGA(Arg), in exon 8. The frequency of allele C was 17.29% and 20% in patients and controls respectively. The difference between these findings could be the results of racial differences, selection bias, or population stratification[4].
Our study, showing no association between SNPs of TSHR and GD, could not rule out weak associations. Take D727E for example[4] (Table 5): Ban and colleagues also could not get an association between D727E SNP and GD in their study. They, therefore, performed a meta-analysis combining their data with other data reported in the previous two studies showing no association between D727E SNP and GD. The results of the meta-analysis suggested a weak association between the D727E SNP E allele and GD (P value=0.03, RR=1.6)[4]. Also, case-control studies have their own limits as a result of a chance event or random variation, especially in small data sets. Therefore, larger samples are needed in our study to detect weaker associations. In addition, other approaches can be used in the genetic analysis to make up the potential short cut of case-control study, such as linkage analysis and intrafamilial linkage disequilibrium.
In conclusion, our dataset did not show a direct association between SNPs of TSHR and GD. The SNPs of the TSHR gene may not be responsible for GD. There are racial differences in the distribution of SNPs of the TSHR gene. Larger samples and a combination of other approaches to genetic analysis are needed in further study to detect weaker associations.
ACKNOWLEDGEMENTS
This work was supported by the National Natural Science Foundation of China ( 30470815); the sustentation fund of 2003 Technology Development Program of Shandong Province ( Z2003C02 ); Jiangsu Province Department of Health (H200651); Xuzhou Board of Health(XW2005017).
We are grateful to State Key Laboratory of Medical Genomics, Ruijin Hospital Affiliated to Shanghai Second Medical University for their cooperation.
REFERENCES
1. Parma J, Duprez L, Van Sande J, et al. Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature,1993，365:649-651.
2. Tomer Y, Davies TF, et al.Searching for the autoimmune thyroid disease susceptibility genes: from gene mapping to gene function. Endocr Rev, 2003，24(5):694-717.
3. Chistiakov DA, et al.Thyroid-stimulating hormone receptor and its role in Graves disease. Mol Genet Metab,2003，80(4):377-88.
4. Yoshiyuki Ban,et al.A germline single nucleotide polymorphism at the intracellular domain of the human thyrotropin receptor does not have a major effect on the development of Graves disease. Thyroid, 2002，12:1079-1083.
5. J.E. Collins, et al.Lack of association of the vitamin D receptor gene with Graves disease in UK Caucasians. Clin Endocrinol (Oxf),2004，60(5):618-24.
6. Tonacchera M, Van Sande J, Cetani F, et al. Functional characteristics of three new germline mutations of the thyrotropin gene causing autosomal dominant toxic thyroid hyperplasia. J Clin Endocrinol Metab,1996，81:547-554.
7. Paschke R, Ludgate ME.The thyrotropin receptor in thyroid disease. N Engl J Med, 1997,337:1675-1681.
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13. Sunthornthepvarakul T, Gottschalk ME, Hayashi Y, Refetoff S.Brief report: resistance to thyrotropin caused by mutations in the thyrotropin-receptor gene. N Engl J Med,1995,332:155-160.
14. Russo D, Arturi F, Schlumberger M, Caillou B, Monier R, Filetti S, Suarez HG.Activating mutations of the TSH receptor in differentiated thyroid carcinomas, Oncogene,1995,11:1907-1911.
15. Tonacchera M, Pinchera V.Thyrotropin receptor polymorphisms and thyroid disease.J Clin Endocrinol Metab,2000,85:2637-2639.
16. Derwahl M.Editorial: TSH Receptor and Gs-α gene mutations in the pathogenesis of toxic thyroid adenomas: a note caution. J Clin Endocrinnol Metab,1996,81:2783-2785.
17. Derwahl M, Hamancher C,Russo D,Broecker M, Manole D, Schatz H,Kopp P, Filetti S..Constitutive activation of the Gs-alpha protein-Adenylate cyclase pathway may not be sufficient to generate toxic adenimas.J Clin Endocrinol Metab,1996,81:1898-1904.
(Editor Anne)thepvarakul T, Gottschalk ME, Hayashi Y, Refetoff S.Brief report: resistance to thyrotropin caused by mutations in the thyrotropin-receptor gene. N Engl J Med,1995,332:155-160.
14. Russo D, Arturi F, Schlumberger M, Caillou B, Monier R, Filetti S, Suarez HG.Activating mutations of the TSH receptor in differentiated thyroid carcinomas, Oncogene,1995,11:1907-1911.
15. Tonacchera M, Pinchera V.Thyrotropin receptor polymorphisms and thyroid disease.J Clin Endocrinol Metab,2000,85:2637-2639.
16. Derwahl M.Editorial: TSH Receptor and Gs-α gene mutations in the pathogenesis of toxic thyroid adenomas: a note caution. J Clin Endocrinnol Metab,1996,81:2783-2785.
17. Derwahl M, Hamancher C,Russo D,Broecker M, Manole D, Schatz H,Kopp P, Filetti S..Constitutive activation of the Gs-alpha protein-Adenylate cyclase pathway may not be sufficient to generate toxic adenimas.J Clin Endocrinol Metab,1996,81:1898-1904.
(Editor Anne)(LIANG Jun1,2*, GAO Ling1*)