Belgian Thyroid Club

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1 Belgian Thyroid Club 30 th Meeting, April 21, 2007 Increasing our understanding of thyroid function and dysfunction by studying twins from physiology to overt thyroid disease Thomas H. Brix, MD, ph.d. Department of Endocrinology and Metabolism Odense University Hospital Odense, Denmark

2 Twin studies and the thyroid Take home message The comparison of concordance between MZ and DZ twins provides irrefutable evidence of a genetic component in the etiology of GD, HT and goiter But also a clue to the etiology of thyroid function, morphology and autoimmunity in healthy euthyroid subjects Biometric modelling showes that 60-80% and 20-40% of the phenotypic variance is attributable to genetic and environmental effects, respectively Biometric modelling suggests the presence of discrete genetic pleiotropy between the serum levels of free T4 and free T3 Despite the well known gender difference in thyroid autoimmunity, our analyses indicate that the same set of genes operate in males and females Investigating discordant twin pairs showed that smoking and skewed XCI is, while birth characteristics and infection with Yersinia are not, associated with an increased risk of thyroid disease / autoimmunity

3 Agenda Basic concepts of twin methodology» The classical twin study» Twin case-control study Twin studies in overt thyroid disease» Goiter» Graves disease» Hashimoto s thyroiditis Twin studies dealing with euthyroid subjects» Thyroid function» Thyroid size» Thyroid morphology» Thyroid antibodies Conclusion and perspectives

4 Twin methodology basic concepts Monozygotic Dizygotic

5 Twin methodology basic concepts & assumptions Twins MZ DZ Individuals within each pair carry identical genes Individuals within each pair share half of their genes Concepts Greater phenotypic similarity among MZ than DZ pairs must be due to the greater genetic similarity Discordance in MZ must be due to environmental effects Assumptions Equal intra-pair environment in MZ and DZ twin pairs Twins are representative of the general population Correct classification of zygosity Well defined phenotypes No gene-gene interaction (epistasis) No gene-environment interaction

6 Twin methodology measuring degree of similarity Concordance rates Pairwise Casewise Probandwise Correlations Pearson s Heritability Biometric modelling

7 Twin methodology the twin case-control control study Matched case-control study of twin pairs discordant for thyroid disease / dysfunction In other words, when studying discordant twin pairs, the healthy co-twin serves as a control for the affected twin Because the twin pairs are matched for genetic, intrauterine and early childhood environment, this design controls for confounding by these factors

8 Twin methodology - summary Classic twin study Genetic factors Thyroid phenotype Environmental factors Twin case-control study Specific environmental and genetic factors Smoking Birth weight X chromosome inactivation

9 Study Classical twin studies in overt thyroid disease concordance rates Brix et al, 1998 Brix et al, 2001 Ringold et al, 2002 Brix et al, 2000 Greig et all, 1967 Malamos et all, 1967 Brix et all, 1999 Phenotype GD GD GD HT Goiter Goiter Goiter Number Probandwise concordance rate in % (95% CI) MZ DZ 36 (17-59) 35 (21-50) 29 (16-46) 55 (23-83) 82 (69-95) 83 (74-93) 42 (26-59) 0 (0.0-11) 3 (0.5-12) 4 (0.5-13) 0 (0.0-25) 57 (31-83) 62 (52-72) 13 (6-24) P value

10 Modelling genetic and environmental factors in Goiter and Graves disease 100% 80% 60% 40% 20% NG GD Scotland Denmark Denmark Proportion of phenotypic variance due to additive genetic factors Proportion of phenotypic variance due to environmental factors Data adopted from Greig et al 1967, Brix et al 1999, 2001

11 Interim conclusions on classic twin studies Higher concordance rates in MZ than in DZ twins gives evidence of a genetic component in the etiology of GD, HT, and goiter Biometric modelling showes that 40-80%of the phenotypic variance is attributable to genetic effects Concordance rates < 1 suggests environmental factors to be of importance Biometric modelling showes that 20-60%of the phenotypic variance is attributable to environmental effects How to investigate this?

12 Investigating environmental factors of possible influence in thyroid disease by means of twins Matched case-control study of same sex twin pairs discordant for thyroid disease Exemplified by: - Smoking habits - Fetal conditions - Skewed X chromosome inactivation

13 Twin case-control control study - smoking and thyroid disease 5 4 In all phenotypes, the effect of smoking was more pronounced in MZ than in DZ pairs Odds ratio Phenotype Thyroid dis GD Goiter Zygosity MZ + DZ MZ + DZ MZ + DZ OR P- value Brix et al. Arch. Int. Med. 2000

14 Twin case-control control studies Cigarette smoking in the etiology of overt thyroid disease 8 Pack year difference Thyroid disease: proband vs co-twin, n = 51 pairs, p = 0.02 AITD: proband vs co-twin, n = 17 pairs, p = 0.03 Goiter: proband vs co-twin, n = 34 pairs, p = 0.20 Brix et al, 2000

15 Is low birth weight a risk factor for thyroid disease?

16 Twin case-control control studies Impact of birth weight in clinically overt thyroid disease Birth weight difference (g) 1000 Autoimmune thyroid disease Proband vs co-twin, 49 pairs, p = GD, n = 35 pairs, p = HT, n = 14 pairs, p = 0.95 Birth weight difference (g) Non-autoimmune thyroid disease proband vs co-twin, 82 pairs, p = 0.55

17 Twin case-control control studies Odds ratio of different birth characteristics in AITD and NG Autoimmune a Non-autoimmune b Risk factor odds ratio (95 % limits) odds ratio (95 % limits) Birth weight / 100 g 0.98 ( ) 1.01 ( ) Birth length per cm 0.97 ( ) 1.00 ( ) Birth order: 1. vs ( ) 0.82 ( ) Term vs premature 1.10 ( ) 0.92 ( ) a Covers both Graves disease and Hashimotos thyroiditis b Covers both simple goiter and toxic nodular goiter Brix et al. Clin. Endocrinol. 2000

18 Investigating epigenetic factors of possible influence in AITD by means of twins Matched case-control study of same sex twin pairs discordant for AITD Exemplified by: - X chromosome inactivation

19 The phenomenon of X chromosome inactivation In female mammalian cells one of the two X chromosomes is inactivated in early embryonic life Thus, females are mosaics for two cell lines, maternel vs paternal X Most females have a 50:50 distribution of these two cell lines Skewed X chromosome inactivation is a marked deviation from the 50:50 distribution Skewed X chromosome inactivation offers a potential mechanism whereby X linked self antigenes may escape presentation in the thymus

20 X chromosome inactivation Matched twin case-control control study prevalence of random and skewed X-inactivation Random 66 Skewed AITD twins Co-twins AITD twins versus co-twins, p = Brix et al. JCEM 2005

21 Conclusions overt thyroid disease Genetic factors play a major role in GD, HT and NG Environmental factors are also of importance. Best documented for iodine intake and smoking habits However, despite intensive research, it has not been possible to identify genes or genetic polymorphisms with major impact Clinically overt thyroid diseases are complex and multidetermined phenotypes with numerous genes and environmental triggers each making small contributions to the overall clinical presentation How to overcome this?

24 Endophenotypes Why study healthy twins? Thyroid Disease Clinical Endpoint Regulation of growth hormone Lipid metabolism Regulation of blood glucose Thyroid Volume Thyroid Morphology TSH Thyroid Function Presence of thyroid antibodies Immune system Intermediate phenotype Low level phenotype Environmental contribution FreeT3 FreeT4 Genes

25 Thyroid function euthyroid subjects p-value< Hansen PS et al. JCEM 2004 MZ n=284 pairs ln TSH DZ n=285 pairs r MZ =0.64 (CI ) 2.5 r DZ =0.29 (CI ) lntsh in Twin lntsh in Twin lntsh in Twin lntsh in Twin 1

26 Thyroid function euthyroid subjects MZ n=284 pairs Free T4 DZ n=285 pairs 22 r MZ =0.63 (CI ) r DZ =0.30 (CI ) Free T4 in Twin Free T4 in Twin Free T4 in Twin Free T4 in Twin 1 p-value< Hansen PS et al. JCEM 2004

27 Thyroid function euthyroid subjects MZ n=284 pairs Free T3 DZ n=285 pairs r MZ =0.59 (CI ) r DZ =0.37 (CI ) Free T3 in Twin Free T3 in Twin Free T3 in Twin Free T3 in Twin 1 p-value< Hansen PS et al. JCEM 2004

28 Hansen PS et al. JCEM 2004 Thyroid function euthyroid subjects Biometric modelling Percent lntsh Free T4 Free T3 Genetic effects Environmental effects

29 Hypothalamus TRH degrading enzyme pptrh pptrh Paraventricular TRH-neuron GHRH Thyroid function regulation - complex GRF-synthesizing cells TRH cold exposure leptin Pituitary TRHR TSHα TSHα TSHβ TSHβ Thyroid TSH TSHR activated Insulin Follicular ce SST somatostatin-producing neuron Per1 Liver Per2 Suprachiasmatic nucleus light Thyrotroph cell GH Somatotroph cell Deiodinase II Deiodinase II NIS TPO Deiodinase I TSHR TG IGF1 EGF TGF β1 FGF β1 IGF1 Deiodinase I Skin Deiodinase III rt3 T3 T4 Deiodinase I Proteolysis of TG TG Iodine Apical membrane Deiodinase III

30 Thyroid function euthyroid subjects Genetic pleiotropy? Hansen PS et al. Am J Physiol Endocrinol Metab 2007 Discrete genetic pleiotropy between serum free T3 and serum free T4 levels only 7% of the genetic component of T3 levels is shared with T4 Serum TSH and thyroid hormones did not share any genetic influences

31 Thyroid function summary A much larger phenotypic similarity in MZ twins as compared to DZ twins was found in serum TSH, serum Free T4 and Free T3 levels Using structural equation model fitting it was estimated that about 65% of the variation was due to genetic effects Discrete genetic pleiotropy between serum free T3 and serum free T4 levels

32 Thyroid volumen and morphology

33 Results - thyroid size Hansen PS et al. JCEM MZ n=104 pairs Thyroid size, total cohort DZ n=107 pairs r MZ =0.71 ( ) r 2 DZ =0.18 ( ) lnvolume in Twin lnvolume in Twin a b lnvolume in Twin 1 lnvolume in Twin 1 a+b, outliers. Excluded from the correlation

34 Results - thyroid size Hansen PS et al. JCEM MZ, males n = 58 pairs r MZ =0.80 ( ) Thyroid size, males DZ, males n = 57 pairs r DZ =0.10 ( ) lnvolume in Twin lnvolume in Twin a lnvolume in Twin 1 lnvolume in Twin 1 a, outlier. Excluded from the correlation

35 Results - thyroid size Hansen PS et al. JCEM MZ, females n = 46 pairs r MZ =0.48 ( ) Thyroid size, females DZ, females n = 48 pairs r DZ =0.13 ( ) lnvolume in Twin lnvolume in Twin b lnvolume in Twin 1 lnvolume in Twin 1 b, outlier. Excluded from the correlation

36 Thyroid size biometric modelling 100 The heritability estimate was not significantly different between males and females 80 Percent lnvolume Males Females Genetic effects Environmental effects Hansen PS et al. JCEM 2004

37 Results thyroid nodularity Probandwise concordance p-value MZ DZ MZ vs DZ Nodules, overall 0.57 ( ) 0.36 ( ) Multiple nodules 0.59 ( ) 0.17 ( ) Solitary nodules 0.22 ( ) 0.30 ( ) Hansen PS et al. Clin Endocrinol 200

38 Thyroid nodularity biometric modelling 100 The heritability estimate was significantly different between multiple and solitary nodules 80 Percent Nodules, overall Multiple nodules Solitary nodules Genetic effects Environmental effects

39 Thyroid size and morphology - summary A much larger phenotypic similarity in MZ twins as compared to DZ twins was found Using structural equation model fitting, we found that about 70% of the variation was due to genetic effects Model fitting suggests that the genetic contribution to thyroid nodularity depends on the subtype uni or multinodularity

40 Thyroid autoantibodies

41 Thyroid antibody status p= MZ twins DZ twins Probandwise concordance according to zygosity Hansen PS et al. EJE 2006

42 Classical twin studies in AITD - correlations MZ n=284 pairs ln TPOab DZ n=285 pairs rmz=0.65 (CI ) rdz=0.13 (CI ) ln TPOab in Twin ln TPOab in Twin ln TPOab in Twin ln TPOab in Twin 1 p-value< Hansen PS et al. EJE 2006

43 Classical twin studies in AITD - correlations MZ n=284 pairs ln Tgab DZ n=285 pairs rmz=0.48 (CI ) rdz=0.18 (CI ) ln Tgab in Twin ln Tgab in Twin ln Tgab in Twin 1 ln Tgab in Twin 1 p-value< Hansen PS et al. EJE 2006

44 Thyroid antibodies biometric modelling 100 Adding OS pairs to the biometric modelling did not significantly change the estimates, indicating that it is the same set of genes that operate in males and females 80 Percent Thyroid antibody status serum TPOab concentration serum Tgab concentration Genetic effects Environmental effects Hansen PS et al. EJE 2006

45 Investigating environmental factors of possible influence on the level of thyroid antibodies by means of twins Matched case-control study of same sex twin pairs discordant for AITD Exemplified by: - Smoking habits - Iodine intake -Stress - Infections - Fetal conditions

46 Twin case-control control studies Is infection with Yersinia enterocolitica a risk factor for thyroid autoantibodies? 89 euthyroid twin pairs discordant for thyroid autoantibodies Odds ratio (95% CI) YOP IgA-ab YOP IgG-ab 0.94 ( ) 1.35 ( ) Hansen et al, Clin Exp Immunol 2006

47 Is there a link between low birth weight and thyroid d autoantibodies?

48 Twin case-control control studies Birth weight and the level of thyroid autoantibodies TPOab (14 pairs), p = 0.01 Tgab (16 pairs), p = 0.15 Phillips et al., p = p = n = 512 pairs Within pair difference in birth weight (100 gram) Within pair difference in birth weight (100 gram) Brix et al, JCEM 2006

49 Thyroid antibodies - summary A much larger phenotypic similarity in MZ twins as compared to DZ twins was found in TPOab and Tgab levels Using quantitative genetic model fitting, we found that about 70% of the variation was due to genetic effects Most likely it is the same set of genes that operate in males and females No link between the serum concentration of thyroid antibodies and low birth weight or infection with Yersinia

50 Twin studies and the thyroid Take home message The comparison of concordance between MZ and DZ twins provides irrefutable evidence of a genetic component in the aetiology of GD, HT and goiter But also a clue to the etiology of thyroid function, morphology and autoimmunity in healthy euthyroid subjects Biometric modelling showes that 60-80% and 20-40% of the phenotypic variance is attributable to genetic and environmental effects, respectively Biometric modelling suggests the presence of discrete genetic pleiotropy between the serum levels of free T4 and free T3 Despite the well known gender difference in thyroid autoimmunity, our analyses indicate that the same set of genes operate in males and females Investigating discordant twin pairs showed that smoking and skewed XCI is, while birth characteristics and infection with Yersinia are not, associated with an increased risk of thyroid disease / autoimmunity

51 Twin studies and the thyroid Research pointers Genetic pleiotropy? - Is the correlation between the circulating levels of TPOAb and TgAb or between GD and HT influenced by the same set of genes? To quantify the precise effect of specific genotypes (e.g. polymorphisms in HLA, CTLA-4,TSH-R and others) Using DZ twins, provides a means to enhance the power of conventional strategies to detect genetic influence via linkage and association Impact of epigenetic modifications comparison of MZ pairs is an ideal design for investigating the impact of epigenetic modifications such as DNA methylation

Acknowledgments Department of Endocrinology Laszlo Hegedüs, M.D., Professor Steen J. Bonnema, M.D., Ph.D. The Danish Twin Registry Pia S. Hansen, M.D., Ph.D. Kirsten O. Kyvik, M.D., Ph.D. The work has been supported by grants from The Agnes & Knut Mørks Foundation The Dagmar Marshalls Foundation The Novo Nordisk Foundation Committe The A.

52 Acknowledgments Department of Endocrinology Laszlo Hegedüs, M.D., Professor Steen J. Bonnema, M.D., Ph.D. The Danish Twin Registry Pia S. Hansen, M.D., Ph.D. Kirsten O. Kyvik, M.D., Ph.D. The work has been supported by grants from The Agnes & Knut Mørks Foundation The Dagmar Marshalls Foundation The Novo Nordisk Foundation Committe The A.P. Møller & Hustru Chastine Mc-Kinney Møllers Foundation The Clinical Research Institute, University of Southern Denmark

ORIGINAL INVESTIGATION. Thomas Heiberg Brix, MD; Pia Skov Hansen, MD; Kirsten Ohm Kyvik, MD, PhD; Laszlo Hegedüs, MD

ORIGINAL INVESTIGATION. Thomas Heiberg Brix, MD; Pia Skov Hansen, MD; Kirsten Ohm Kyvik, MD, PhD; Laszlo Hegedüs, MD ORIGINAL INVESTIGATION Cigarette Smoking and Risk of Clinically Overt Thyroid Disease A Population-Based Twin Case-Control Study Thomas Heiberg Brix, MD; Pia Skov Hansen, MD; Kirsten Ohm Kyvik, MD, PhD;