belgian thyroid club

Belgian Thyroid ClubBelgian Thyroid Club3030thth Meeting, April 21, 2007Meeting, April 21, 2007

Increasing our understanding of thyroid function Increasing our understanding of thyroid function and dysfunction by studying twinsand dysfunction by studying twins

from physiology to overt thyroid diseasefrom physiology to overt thyroid disease

Thomas H. Brix, MD, ph.d.Department of Endocrinology and Metabolism

Odense University HospitalOdense, Denmark

TwinTwin studies and the thyroidstudies and the thyroid

• 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 pleiotropybetween 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

Take home messageTake home message

AgendaAgenda

• 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

Twin methodology Twin methodology –– basic conceptsbasic concepts

Monozygotic

Dizygotic

Twin methodology Twin methodology –– basic concepts & assumptionsbasic concepts & assumptions

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

TwinsMZ

DZ

Individuals within each pair carry identical genes

Individuals within each pair share half of their genes

Twin methodology Twin methodology –– measuring degree of similaritymeasuring degree of similarity

• Concordance rates• Pairwise • Casewise• Probandwise

• Correlations• Pearson’s

• Heritability• Biometric modelling

Twin methodology Twin methodology –– the twin casethe twin case--control study 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

Twin methodology Twin methodology -- summarysummary

Thyroid Thyroid phenotypephenotype

Genetic Genetic factorsfactors

Environmental Environmental factorsfactors

Classic twin study

Twin case-control studySpecific environmental and genetic factors

Smoking

Birth weight

X chromosome inactivation

Classical twin studies in overt thyroid disease Classical twin studies in overt thyroid disease concordance ratesconcordance rates

0.00113 (6-24)42 (26-59)103GoiterBrix et all, 1999

0.00562 (52-72)83 (74-93)157Goiter

0.06757 (31-83)82 (69-95)68Goiter

0.0100 (0.0-25)55 (23-83)24HTBrix et al, 2000

0.0014 (0.5-13)29 (16-46)99GDRingold et al, 2002

0.0013 (0.5-12)35 (21-50)105GDBrix et al, 2001

0.0120 (0.0-11)36 (17-59)55GDBrix et al, 1998

P valueDZMZNumberPhenotypeStudy

Probandwise concordance rate in % (95% CI)

Greig et all, 1967Malamos et all, 1967

MModelling genetic and environmental factors odelling genetic and environmental factors in Goiter and Gravesin Goiter and Graves’’ diseasedisease

Proportion of phenotypic variance due to additive genetic factorsProportion of phenotypic variance due to environmental factors

Data adopted from Greig et al 1967, Brix et al 1999, 2001 Data adopted from Greig et al 1967, Brix et al 1999, 2001

100%

20%

40%

60%

80%

Scotland Denmark Denmark

NGNG GDGD

Interim conclusions on classic twin studiesInterim 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

How to investigate this?How to investigate this?

• 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

Investigating environmental factors of possible Investigating environmental factors of possible influence in thyroid disease by means of twinsinfluence 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

Twin caseTwin case--control study control study -- smoking and thyroid diseasesmoking and thyroid disease

Brix et al. Arch. Int. Med. 2000Brix et al. Arch. Int. Med. 2000

1

2

3

4

5

Odd

s ra

tio

0.004P- value

3.0OR

MZ + DZZygosity

Phenotype Thyroid dis

0.03

2.7

MZ + DZ

GoiterGD

MZ + DZ

3.5

0.02

In all phenotypes, the effect of smoking was more pronounced in MZ than in DZ pairs

-8

-4

0

4

8

Pack

yea

r dif f

e ren

c e

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

Twin caseTwin case--control studiescontrol studiesCigarette smoking in the etiology of overt thyroid diseaseCigarette smoking in the etiology of overt thyroid disease

Brix et al, 2000

Is low birth weight a risk factor forIs low birth weight a risk factor forthyroid disease?thyroid disease?

Twin caseTwin case--control studiescontrol studiesImpact of birth weight in clinically overt thyroid diseaseImpact of birth weight in clinically overt thyroid disease

-1000

-500

0

500

1000 Autoimmune thyroid disease

Birt

h w

eigh

t diff

eren

ce (g

)

Proband vs co-twin, 49 pairs, p = 0.78

GD, n = 35 pairs, p = 0.74. HT, n = 14 pairs, p = 0.95

-1000

-500

0

500

1000Non-autoimmune thyroid disease

Birt

h w

eigh

t diff

eren

ce (g

)

proband vs co-twin, 82 pairs, p = 0.55

Brix et al. Clin. Endocrinol. 2000Brix et al. Clin. Endocrinol. 2000

Twin caseTwin case--control studiescontrol studiesOdds ratio of different birth characteristics in Odds ratio of different birth characteristics in AITDAITD and NGand NG

Autoimmune aa Non-autoimmune bb

Risk factor odds ratio (95 % limits) odds ratio (95 % limits)

Birth weight / 100 g 0.98 ( 0.92 - 1.08 ) 1.01 ( 0.95 - 1.08 )

Birth length per cm 0.97 ( 0.83 - 1.14 ) 1.00 ( 0.88 - 1.15 )

Birth order: 1. vs 2. 0.56 ( 0.25 - 1.25 ) 0.82 ( 0.45 - 1.52 )

Term vs premature 1.10 ( 0.44 - 2.23 ) 0.92 ( 0.54 - 1.86 )

a Covers both Graves’ disease and Hashimotos’ thyroiditisb Covers both simple goiter and toxic nodular goiter

Brix et al. Clin. Endocrinol. 2000Brix et al. Clin. Endocrinol. 2000

Investigating epigenetic factors of possible Investigating epigenetic factors of possible influence in AITD by means of twinsinfluence in AITD by means of twins

• Matched case-control study of same sex twin pairs discordant for AITD

Exemplified by:- X chromosome inactivation

The phenomenon of X chromosome inactivationThe 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

X chromosome inactivationX chromosome inactivationMatched twin caseMatched twin case--control studycontrol study

AITD twins versus co-twins, p = 0.003

20

40

60

80

100

Co-twins

Random Skewed

AITD twins

prevalence of random and skewed X-inactivation

66

89

34

11

Brix et al. JCEM 2005Brix et al. JCEM 2005

• 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

Conclusions Conclusions –– overt thyroid diseaseovert thyroid disease

• However, despite intensive research, it has not been possible to identify genes or genetic polymorphisms with major impact

How to overcome this?How to overcome this?

• Clinically overt thyroid diseases are complex and multidetermined phenotypes with numerous genes and environmental triggers each making small contributions to the overall clinical presentation

Endophenotypes Endophenotypes –– Why study healthy twins?Why study healthy twins?

Genes

Low levelphenotype

Intermediatephenotype

Clinical EndpointThyroid Disease

Thyroid Volume

Thyroid Morphology

Presence of thyroid antibodies

TSH

FreeT4FreeT3

Thyroid Function

Immunesystem

Regulation of blood glucose

Lipidmetabolism

Regulation ofgrowth hormone

Environmental contribution

ln TSHln TSH

lnTS

H in

Tw

in 2

lnTSH in Twin 1-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5

-2.5

-2

-1.5

-1

-.5

0

.5

1

1.5

2

2.5

lnTS

H in

Tw

in 2

lnTSH in Twin 1-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5

-2.5

-2

-1.5

-1

-.5

0

.5

1

1.5

2

2.5rMZ=0.64 (CI 0.56-0.70) rDZ=0.29 (CI 0.18-0.39)

MZn=284 pairs

DZn=285 pairs

p-value<0.00005 Hansen PS et al. JCEM 2004Hansen PS et al. JCEM 2004

Thyroid function Thyroid function –– euthyroid subjectseuthyroid subjects

Free T4Free T4

Free

T4

in T

win

2

Free T4 in Twin 18 10 12 14 16 18 20 22

8

10

12

14

16

18

20

22

Free

T4

in T

win

2

Free T4 in Twin 18 10 12 14 16 18 20 22

8

10

12

14

16

18

20

22

MZn=284 pairs

DZn=285 pairs

p-value<0.00005

rMZ=0.63 (CI 0.56-0.70) rDZ=0.30 (CI 0.19-0.40)

Hansen PS et al. JCEM 2004Hansen PS et al. JCEM 2004


Free

T3

in T

win

2

Free T3 in Twin 13 4 5 6 7 8 9 10 11 12

3

4

5

6

7

8

9

10

11

12

Free

T3

in T

win

2

Free T3 in Twin 13 4 5 6 7 8 9 10 11 12

3

4

5

6

7

8

9

10

11

12

MZn=284 pairs

DZn=285 pairs

p-value<0.00005

rMZ=0.59 (CI 0.51-0.66) rDZ=0.37 (CI 0.27-0.47)

Free T3Free T3



6436 35 366564

0

20

40

60

80

100

lnTSH Free T4 Free T3

Genetic effects Environmental effects

Per

cent


Thyroid function Thyroid function –– euthyroid subjectseuthyroid subjectsBiometric modellingBiometric modelling

Hypothalamus

Paraventricular TRH-neuron

somatostatin-producing neuron

GRF-synthesizing cells

SST

Deiodinase I

ppTRH ppTRH TRH

TRHRTSHα

Thyrotroph cell

TSHβ

Somatotroph cell

GH

TSHα TSHβ

Apical membrane

TSH

TSHR activated

TPO

NIS

TSHRIGF1

Deiodinase I

TG

TGF β1

EGF

T4T3

TG

Follicular cell

Proteolysis ofTG

Deiodinase I

leptincold exposure

Pituitary

ThyroidGHRH

TRH degradingenzyme

Liver

rT3

Suprachiasmatic nucleus

Per1 Per2

light

Deiodinase III

Skin

Deiodinase III

Deiodinase II Deiodinase II

IGF1

FGF β1

Insulin

Iodine

ThyroidThyroid functionfunction regulation regulation -- complexcomplex

Hansen PS et al. Am J Physiol Endocrinol Metab 2007

Thyroid function Thyroid function –– euthyroid subjectseuthyroid subjectsGenetic pleiotropy?Genetic pleiotropy?

•• 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

Thyroid function Thyroid function –– summarysummary

• 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 T3and serum free T4 levels

Thyroid volumen and morphologyThyroid volumen and morphology

-1.5 -1 -0.5 0 0.5 1 1.5 2

-1.5

-1

-0.5

0

0.5

1

1.5

2

-1.5 -1 -0.5 0 0.5 1 1.5 2

-1.5

-1

-0.5

0

0.5

1

1.5

2rMZ=0.71 (0.60-0.79) rDZ=0.18 (-0.01-0.36)

lnVo

lum

e in

Tw

in 2

lnVo

lum

e in

Tw

in 2

lnVolume in Twin 1 lnVolume in Twin 1

a

b

MZn=104 pairs

DZn=107 pairs

p-value < 0.00005

a+b, outliers. Excluded from the correlation

Thyroid size, total cohortThyroid size, total cohort

Results Results -- thyroid sizethyroid size


rDZ=0.10 (-0.17-0.35)

-1.5 -1 -0.5 0 0.5 1 1.5 2

-1.5

-1

-0.5

0

0.5

1

1.5

2

aln

Volu

me

in T

win

2

rMZ=0.80 (0.68-0.87)

-1.5 -1 -0.5 0 0.5 1 1.5 2

-1.5

-1

-0.5

0

0.5

1

1.5

2

lnVo

lum

e in

Tw

in 2


MZ, malesn = 58 pairs

DZ, malesn = 57 pairs

p-value < 0.00005

a, outlier. Excluded from the correlation

Thyroid size, malesThyroid size, males



Thyroid size, femalesThyroid size, females

-1.5 -1 -0.5 0 0.5 1 1.5 2

-1.5

-1

-0.5

0

0.5

1

1.5

2 rMZ=0.48 (0.21-0.68)

-1.5 -1 -0.5 0 0.5 1 1.5 2

rDZ=0.13 (-0.16-0.40)

-1.5

-1

-0.5

0

0.5

1

1.5

2

bln

Volu

me

in T

win

2

lnVo

lum

e in

Tw

in 2


MZ, femalesn = 46 pairs

DZ, femalesn = 48 pairs

p-value = 0.035

b, outlier. Excluded from the correlation



5029 21 5079710

20

40

60

80

100

lnVolume Males Females

Per

cent



Thyroid size Thyroid size –– biometric modellingbiometric modellingTheThe heritabilityheritability estimate was not estimate was not significantly different between males significantly different between males and femalesand females

Results Results –– thyroid nodularitythyroid nodularity

0.6780.30 (0.09 – 0.53)0.22 (0.00 – 0.53)Solitary nodules

0.0390.17 (0.00 – 0.53)0.59 (0.25 – 0.84)Multiple nodules

0.0740.36 (0.17 – 0.56)0.57 (0.36 – 0.76)Nodules,overall

MZ vs DZDZMZ

p-valueProbandwise concordance

Hansen PS et al. Clin Endocrinol 2005Hansen PS et al. Clin Endocrinol 2005

3033 23 7077670

20

40

60

80

100


Per

cent

Nodules, overall Multiple nodules Solitary nodules

Hansen PS et al. Clin Endocrinol 2005Hansen PS et al. Clin Endocrinol 2005

Thyroid nodularity Thyroid nodularity –– biometric modellingbiometric modelling

TheThe heritabilityheritability estimate was estimate was significantly different betweensignificantly different betweenmultiple and solitary nodulesmultiple and solitary nodules

Thyroid size and morphology Thyroid size and morphology -- summarysummary

• 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

Thyroid autoantibodiesThyroid autoantibodies

0.15

0.45

0

0.20

0.40

0.60

0.80

1.00

MZ twins DZ twins

p=0.022

Probandwise concordance according to zygosity

Thyroid antibody statusThyroid antibody status

Hansen PS et al. EJE 2006Hansen PS et al. EJE 2006

ln TPOabln TPOab

-2 -1 0 1 2 3 4 5

-2

-1

0

1

2

3

4

5

ln T

POab

in T

win

2

ln TPOab in Twin 1

rDZ=0.13 (CI 0.02-0.25)

MZn=284 pairs

DZn=285 pairs

p-value<0.0001

-2 -1 0 1 2 3 4 5

-2

-1

0

1

2

3

4

5

ln T

POab

in T

win

2

ln TPOab in Twin 1

rMZ=0.65 (CI 0.57-0.71)


Classical twin studies in AITD Classical twin studies in AITD -- correlationscorrelations

-2 -1 0 1 2 3 4 5

-2

-1

0

1

2

3

4

5

-2 -1 0 1 2 3 4 5

-2

-1

0

1

2

3

4

5

ln T

gab

in T

win

2

ln T

gab

in T

win

2

ln Tgab in Twin 1 ln Tgab in Twin 1

rMZ=0.48 (CI 0.39-0.57) rDZ=0.18 (CI 0.06-0.29)

MZn=284 pairs

DZn=285 pairs

p-value<0.0001

ln Tgabln Tgab


Classical twin studies in AITD Classical twin studies in AITD -- correlationscorrelations

6227 33 3867730

20

40

60

80

100

Thyroid antibody status serum TPOab concentration serum Tgab concentration


Per

cent

Thyroid antibodies Thyroid antibodies –– biometric modellingbiometric modelling


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

Investigating environmental factors of possible Investigating environmental factors of possible influence on the level of thyroid antibodies by influence on the level of thyroid antibodies by

means of twinsmeans of twins

• Matched case-control study of same sex twin pairs discordant for AITD

Exemplified by:- Smoking habits- Iodine intake- Stress- Infections- Fetal conditions

Twin caseTwin case--control studiescontrol studiesIs infection with Yersinia enterocolitica a risk factor for thyrIs infection with Yersinia enterocolitica a risk factor for thyroid oid

autoantibodies?autoantibodies?

89 euthyroid twin pairs discordant for thyroid autoantibodies

1.35 (0.72 – 2.53)YOP IgG-ab

0.94 (0.49 – 1.83)YOP IgA-ab

Odds ratio (95% CI)

Hansen et al, Clin Exp Immunol 2006

Is Is there a link between there a link between low birth low birth weight weight and and thyroithyroid autoantibodiesd autoantibodies??

TPOab (14 pairs), p = 0.01 Tgab (16 pairs), p = 0.15

Phillips et al., 2002

Twin caseTwin case--control studiescontrol studiesBirth weight and the level of thyroid autoantibodiesBirth weight and the level of thyroid autoantibodies

Within pair difference in birth weight (100 gram) -15 -10 -5 0 5 10 15

-5

0

5

p = 0.75

n = 512 pairs

Within pair difference in birth weight (100 gram) -15 -10 -5 0 5 10 15

-5

0

5

p = 0.59

Brix et al, JCEM 2006

• 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

Thyroid antibodies Thyroid antibodies -- summarysummary

TwinTwin studies and the thyroidstudies and the thyroid

• 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 pleiotropybetween 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

Take home messageTake home message

TwinTwin studies and the thyroidstudies and the thyroidResearch pointersResearch 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

AcknowledgmentsAcknowledgments

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 fromThe Agnes & Knut Mørks FoundationThe Dagmar Marshalls FoundationThe Novo Nordisk Foundation CommitteThe A.P. Møller & Hustru Chastine Mc-Kinney Møllers FoundationThe Clinical Research Institute, University of Southern Denmark

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