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Novel thyroid specific transcripts identified by SAGE: implication for congenital
hypothyroidism
Moreno Navarro, J.C.
Publication date
2003
Document Version
Final published version
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Citation for published version (APA):
Moreno Navarro, J. C. (2003). Novel thyroid specific transcripts identified by SAGE:
implication for congenital hypothyroidism.
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Novell thyroid specific
transcriptss ^ ^ ^ ^ H I
identifiedd by SAGE: I
implicationss for congenita
NovelNovel thyroid specific transcripts
identifiedidentified by SAGE:
implicationsimplications for congenital hypothyroidism
Moreno,, José Carlos
"Novel"Novel thyroid specific transcripts identified by SAGE: implications for congenital hypothyroidism" hypothyroidism"
Thesiss University of Amsterdam with summary in Dutch, English and Spanish
©© J. C. Moreno, Amsterdam, The Netherlands. 2003.
Alll rights reserved. No part of this publication may be reproduced or transmitted in anyy form or by any means, electronic or mechanical, including photocopy, recording orr any information storage and retrieval system, without written permission of the author. .
Cover:: View of a three dimensional model for the thyroidal DEHAL1 enzyme. Design:: Eliane Beyer, based on an illustration included in Chapter 4 of this thesis. Printing:: PrintPartners Ipskamp B.V., Enschede, The Netherlands.
Financiall support from the Stichting Amsterdam Thyroid Club, the University of Amsterdam,, Emma Children's Hospital AMC. the Fund Pediatric Endocrinology AMC,, Novo Nordisk A/S. and Pharmacia is gratefully acknowledged.
NovelNovel thyroid specific transcripts identified by SAGE:
impiicationsimpiications for congenital hypothyroidism
Academischh Proefschrift
terr verkrijging van de graad van doctor aann de Universiteit van Amsterdam opp gezag van de Rector Magnificus
Prof.. mr. P.F. van der Heijden
tenn overstaan van een door het college voor promoties ingestelde commissie,, in het openbaar te verdedigen in de Aula der Universiteit
opp vrijdag 11 april 2003. te 12.00 uur
door r
Joséé Carlos Moreno Navarro
Promotiee commissie:
Promotorr Prof. dr. J.J.M. de Vijlder
Co-promotor:: Dr. C. Ris-Stalpers
Overigee leden: Prof. Dr. H. Heijmans Prof.. dr. D. Roos Dr.. P. Sannsteban Dr.. T. Vulsma
Prof.. Dr. W. Wiersinga
Universiteitt van Amsterdam Faculteitt Geneeskunde
Thee research described in this thesis was carried out in the Laboratory of Pediatrie Endocrinology.. Academic Medical Center. Amsterdam, The Netherlands.
"Desocupado"Desocupado lector, sin juramento me pod ras ere t't' que quisiera que este
tibro,tibro, eomo lü/'o de nu entendimiento, fuera el mds hermoso, el mdsgaflardo x
masmas discreto que pudiera imaginarse. Lero no he pod id o xo eontravenir a la
ordenorden de naturaleza, que en ella eada cosa engendra su semejaute '.
DonDon Q^ui/ote de La Mancha.
'Miguel'Miguel de (enautes.
may believe me without an oath, gentle reader that 1 (Vish this
bool{,bool{, LIS the child oj nix brain, nere the most beautiful, the most sprightly,
andand the most ingenious, that can be imagined. Hut I could not control the
orderorder of nature, alierehx each thing engenders its lif{e'.
Chiiehotte of -The 'Mancha.
de Cervantes
"Ledige"Ledige lezer, je zult zonder mijn eren oord best (villen getoi en hoe graag
il{.ditil{.dit boef{,, als spruit (an mijn brein, had zien uitgroeien tol het mooiste,
fierstefierste en verstandigste dat men zich r;qn deugen. :Maar ih k[on de nat nu ra et
nietniet breien die inhoudt dat alles zifusgelijke -voortbrengt".
Quichot -van La Manctta.
MiguelMiguel de ("ervautes.
AA mi padre,
queque me ensenó la religion del trabajo v, quizes
ineonscientemente,ineonscientemente, a pcrseguir lo imposible.
AA mi madre,
fuentefuente inagotable de pequenas v grander
ilusiones. ilusiones.
AA todo el que apareció en mi vida,
ensenandomeensenandome algo.
ToTo my father,
whowho taught me a religion called, work , and that
searchingsearching for the impossible has a meaning.
ToTo my mother,
aa never ending source of hopeful support.
ToTo anyone that appeared in my life, teaching
meme something.
Contents s
Chapterr 1 General introduction 11
1.11 Preface 13 1.22 Congenial hypothyroidism 17
1.33 Molecular basis of congenital hypothyroidism 19
1.44 Gaps and controversies 35 1.55 Strategies for CH-oriented research, the SAGE technique. 37
1.66 Scope of the Ihesis. 41
Chapterr 2 Cloning of tissue-specific genes using serial analysis of gene 53 expressionn and a novel computational substruction approach.
Chapterr 3 Inactivating mutations in the gene for thyroid oxidase 2 (THOX2) 73 andd congenital hypothyroidism.
Chapterr 4 Cloning and characterization of the human iodotyrosine 93 dehalogenase. .
Chapterr 5 Cloning of NM41, a novel cystine-knot like protein preferentially 111 expressedd in the thyroid.
Chapterr 6 General Discussion 129
66 1 Preface. 131 6.22 Cloning of 'tissue-specfic genes' methodological considerations. 131
6.33 THOX2 defects <n CH patients: implications f
or thyroid basic research 134
6.44 THOX2 defects in CH patients: clinical imp.ications. 135
6.55 DEHAL1: a novel gene for an " phenotype 137
6.66 NM41; a cystine-knot like protein ?n the thyroid 138
Chapterr 7 Summaries 145
Samenvattingg in het Nederlands 147
Summaryy in English 149 Resumenn en Espanol 151
Appendix:: nucleotide and amino acid sequence 155
Curriculumm Vitae 159 Publicationss 161 Abbreviationss 163 Acknowledgementss 167
CHAPTERR 1
Generall introduction
Partt of this chapter has been accepted for publication inn Trends in Endocrinology and Metabolism:
"Genetic"Genetic bases of hypothyroidism:
Genera!! introduction
1.11 Preface
ActionAction of thyroid hormone.
Thyroidd hormone exerts a broad range of effects on development, growth and metabolism.. The clinical manifestations of thyroid hormone deficiency and excess aree expressions of the multiple actions of thyroid hormone. Thyroxine (T4). the primaryy secretory product of the thyroid gland, is practically inactive until it is convertedd to the active hormone triiodothyronine (T3), a reaction catalyzed by iodothyroninee deiodmases. T4 can be considered a prohormone.
Thee actions of thyroid hormone are the result of the interaction of T3 with specific nuclearr receptors. The T3-receptor complexes bind to regulatory regions of target
genes;; modifying their expression [1], These receptors have been cloned [2], and
thee specific functions of the various T3-receptor complexes have experienced considerablee progress in recent years.
SynthesisSynthesis of thyroid hormone.
Thee synthesis of thyroid hormone takes place exclusively in the thyroid gland, which iss located in the lower part of the neck, ventral to the trachea. The functional unit of thee thyroid is the follicle, formed by a single layer of thyrocytes surrounding a lumen. Thee lumen mainly contains thyroglobulin (Tg) that serves as a matrix for the synthesiss of thyroid hormone, mainly T4 [3], which takes place at the apical border off thyrocytes.
Circulatingg plasma iodide is actively transported into the thyrocytes and is organified throughh a series of enzymatically catalyzed steps to form thyroid hormone. A numberr of thyroid-specific proteins are involved in the multi-step process of thyroid hormonogenesis.. First, the Na+/I" symporter traps iodide into the thyroid cell at the basall membrane. Once within the cell, iodide is transported through the apical membranee into the lumen by anion transporters, among which is pendrim an iodide/ chloridee transporter, iodide oxidation and binding to the tyrosine residues of Tg, as welll as the coupling of iodotyrosines to form T4 and T3, is catalyzed by
thyroperoxidasee (TPO) in the presence of hydrogen peroxide ( H202) . The H202
-generatingg system of the thyroid is a membrane system composed of at least two NADPHH oxidases, named THOX1 and THOX2. localized in the apical membrane [4].. Thyroid hormone secretion requires endocytosis of iodinated Tg from the lumen too the cytoplasm of the thyrocyte. where vesicles containing Tg fuse with lysosomes.. Proteases such as cathepsins lyse Tg. and subsequently T4 and T3 are released.. Mono- and di-iodotyrosines. the main iodinated side products of Tg
Chaoterr "
hydrolysis,, are also released anc are deiodinated by a dehalogenase whose molecularr nature is sofar unknown.
Thee thyroid stimulating hormone {TSH), also known as thyrotropin, is the major factorr regulating the synthesis and secretion of thyroid hormone. The thyrotropin receptorr (TSH-R) is a G protein-coupled receptor that acts via the PKA and PKC metabolicc pathways. Furthermore. 3 thyroidal transcription factors, NKX2.1. PAX8 andd FKHL15, are known to control the expression of thyroidal proteins necessary forr the synthesis of thyroid hormones. All these thyroid-specific proteins will be discussedd in more detail in connection with defects causing congenital hypothyroidism. .
RegulationRegulation of thyroid hormone synthesis.
Thee thyroidal secretory activity is regulated by the hypothalamic-pituitary unit throughh the negative feedback control mechanism via the plasma free T4 and free T33 concentrations (Fig. 1). The tripeptide hypothalamic hormone TRH (Thyrotropin-Releasingg Hormone) is synthesized in the paraventricular nuclei of the hypothalamuss and transported via the hypothalamic-pituitary stalk (portal vessels) too the anterior pituitary, where, through the activation of a specific receptor (TRH-R) willl make TSH released. This classical view of TSH control has been challenged (or completed)) by the demonstration of a functional TSH receptor in the pituitary folliculo-stellatee cells [5], suggesting a paracrine short-loop control of TSH in pituitary.. Further, the recent identification in pituitary tissue of thyrostimulin [6], a novell member of the glycoprotein hormones with capacity to stimulate the TSH-R. furtherr supports the existence of pituitary control mechanisms for TSH secretion. Finally.. TSH secreted in the bloodstream will reach the thyroid cells, stimulating the metabolismm of the gland via the TSH-R
EmbryologyEmbryology of the hypothalamus-pituitary-thyroid axis.
Thee thyroid gland develops as a ventral bulge of the endoderm at the position of the firstt and second branchial arches in the human embryo [7], In certain occasions, a remnantt of the median anlage' is recognizable as the foramen caecum of the tongue,, later in life (Fig. 2). About 17 days after conception, the human primordial
thwrf^i/jj Qgri Kg Hg+gQtgH close to the d e v e ! onmn heart and around dav 30 of
embryologicall life a bilobar structure is formed. During the next step both lobes fuse withh an ultimobranchial body, also referred to as lateral 'anlage', which develops fromm the fourth branchial pouches. These bodies contain the C-cells. in charge of thee secretion of calcitonin. Eight weeks after conception, the thyrocytes are organizedd in tubes and two weeks later intercellular follicles are formed and iodine
Generall introduction
cann be bound, indicating functional differentiation of thyrocytes. The number of follicless increases for some time by budding from the primary follicles; later in developmentt the size of the thyroid increases mainly by enlargement of the volume off existing follicles [8],
Thee pituitary gland is formed by fusion of an invagination of the floor of the third cerebrall ventricle and Rathke's pouch, which is an invagination of the oral ectoderm.. Subsequently there is differentiation of the five types of cells that produce thee different pituitary hormones: thyrotropes, somatotropes, lactotropes.
gonadotropes:: and corticotropes. During fetal development of the anterior pituitary
gland,, a number of sequential processes occur that regulate cell differentiation and proliferation. .
Moleculess expressed early in development of the thyroid gland and pituitary, mainly transcriptionn factors, are known to control the necessary steps that lead to the formationn of a mature thyroid-pituitary-hypothalamus axis. These molecular factors willl be addressed in connection to defects causing congenital hypothyroidism.
Chapterr 1
F i g .. 1 Schematic representation of the system regulating the
hypothalamic-pituitary-thyroidpituitary-thyroid function. Hypothalamic stimulation of thyrotropic function is balanced by thethe negative feed-back inhibition exerted by T3 and 14. +. stimulation: -. inhibition.
G e r e r ss .rtroducticn
1.22 Congenital h y p o t h y r o i d i s m
Hypothyroidismm is the most common inborn endocrine disease, overall affecting 1 in everyy 1200 newborns [7]. Neonatal screening programs allow its early detection andd treatment, thus preventing the cognitive and motor impairment caused by lack off thyroid hormone during the (early) postnatal phase of brain development [2], Dependingg on the screening method, only CH of thyroidal origin (using TSH screening)) or both thyroidal and central CH (using T4-based screening) will be detectedd in the first weeks of life. In North America. Japan. Australia and some countriess in Europe, screening is performed by the determination of total T4 in filter paper.. Positive cases are further screened by the determination of TSH and. as in thee case of The Netherlands, the additional determination of thyroxine-bmding globulinn (TBG) to trace relatively common cases of TBG deficiency. Most countries inn Europe use TSH determination as method of screening. Cut-off levels established forr these determinations slightly differ among countries, in order to optimalize the cost-benefitt ratio of mass screening based on variable geographical circumstances (iodinee deficiency) or conceptual reasons (biochemical definition of disease, lack of majorr psychomotor sequelae in children with early diagnosed and treated CH).
TypesTypes of CH.
Hypothyroidismm can be of a permanent or transient nature. Incidence of permanent CHH is estimated from international screening reports to be approximately 1:3500 newborns,, with considerable ethnic differences [7]. The etiology of permanent CH of primaryy origin has been linked to defects in proteins involved in thyroid hormone synthesis,, and transcription factors involved in development of the thyroid gland. Incidencee of transient CH is more difficult to estimate since, internationally, incidencee figures are almost exclusively related to permanent thyroidal CH. In The Netherlands,, an incidence as high as 1:2200 newborns was estimated from a follow-upp study of 288 CH patients born in 1981 and 1982. These figures vary amongg countries, depending mainly on the iodine intake of the population. Transient congenitall hypothyroidism can be caused by endemic iodine deficiency, exposure to iodinee excess at perinatal time (e.g. by the use of iodinated compounds) or fetal exposuree to maternally derived thyroid-blocking antibodies or antithyroid drugs in casee of women with thyroid autoimmune disease [10-13]. It can also be a consequencee of premature birth [14.15], or rare protein-loosing nephrosis [16]. However,, in about 20% of patients with transient disease the underlying etiology remainedd elusive ( Table 1) [17-20]. Recently, genetic defects have been identified inn this type of the disease, disclosing explanation for unaffiliated cases of transitory CHH (Chapter 2 of this thesis).
O a u t e rr '
Attendingg to the site where the abnormality resides, hypothyroidism can be classifiedd as thyroidal central (hypothalamic and pituitary) or peripheral. Resistance too thyroid hormone, a disorder in the oenpherai action of thyroid hormone, invariably impliess a hypothyroid state for seme tissues and will be considered in this introduction. .
Tablee 1 Prevalence and etiology of Transient Congenital Hypothyroidism (TCH).
DespiteDespite the different geographical a>eai. methods and cut-off levels used to screen for CH in differentdifferent countries (see references), a proportion of "idiopathic" cases of TCH is invariably found.found. Data include the cases of elevaieci TSH with low-normal or norm ai T4
S t u d yy period S c r e e n i n gg m e t h o d N e o n a t e ss s c r e e n e d P r e v a l e n c ee T C H E T I O L O G Y Y IodineIodine excess MH.MH. AID. TBA PrematurityPrematurity LBW T4T4 losses TnyioidTnyioid dysgeneses "Idiopathic" "Idiopathic" R e f e r e n c e e A:)::::reviH, .io", ii VIM IJnie ^ - . b M . i f c i .. •>:•>-: !<v-: : • ; • : af'.e'' ?ollüv".-up Arc rec--.fi
cü'i'iDine':!! figure for 2 r
A u s t r a l i a a 1977-1986 6 T 4 - T S H H 570.000 0 11 13.750 -: 588 0 88 5 -•*- --291 --- , [17] ] •-..-.. r ,v> : ^ ' .JratK.;r-- of CH •:.Ti^-r ifforontt causes of"
F r a n c e e ' 9 8 2 - 1 9 8 7 7 T S H H 2 0 22 930 11 2 7 - 2 599 , J.-.. J.-.. 11 0 :. 2 1 '' , PR] ] -TO- : vri :t, T ö '' .,r. n ^ . -•• '•' De-'o-stra'f:! "Ob. . Thee N e t h e r l a n d s 1 9 8 1 - 1 9 8 2 2 T 4 - T S H H 3400 355 '' 2.249 : 577 '• 33 5' -. 233 . 22 f)-. 11 4 •• ;; 1 91 i b tt -< ,:,--%r;:;,, ...rL;q. .-.rrr. .-: - . M M r. :» rvv o " o - o b -...c.:^:\fc\ r. USAA / C a n a d a 1972-1978 8 Diverse e '' 046. 362 '' 26.159 222 5 \ 2 0JJ •: [20] ] ; uAA .h. . .p, H M , , - , , ^ , . n - -- b ^ . „ , t ^ i i n o ^ o x ^ l . . . -
--1.33 Molecular basis of congenital h y p o t h y r o i d i s m
Overr the last 2 decades, the genetic basis of hypothyroidism has been unraveled throughh the analysis of animals with impaired thyroid function (Table 2). These findingss were occasionally linked to parallel defects in patients with distinct forms of hypothyroidism.. Following the classification by mechanism of disease, the etiology off the subtypes of hypothyroidism for which the genetic bases has been disclosed willl be introduced, including controversial aspects and possible guides to clarify them. .
1.3.11 Thyroidal h y p o t h y r o i d i s m
Hypothyroidismm originated from maldevelopment or dysfunction of the thyroid gland accountss for the majority of CH cases, as detected by current screening programs. Thyroidd dysgenesis, in its forms of agenesis, ectopy or hypoplasia, is the most prevalentt cause of primary CH (85-90%). while various defects in the synthesis of thyroidd hormone account for CH in the rest of cases Inversely to the prevalence, molecularr pathogenesis of thyroid dyshormonogenesis is more in-depth understood thann is dysgenesis.
D y s g e n e s i ss of t h e t h y r o i d g l a n d
AA unique combination of transcription factors controls the embryonal development off the thyroid gland [21]. Three major transcription factors. FKHL15. NKX2.1 and PAX8.. have been so far implicated in a small percentage of syndromic and nonsyndromicc forms of thyroid dysgenesis in man (Fig. 3). In animals, a whole net off novel proteins expressed at different stages of thyroid organogenesis is involved inn the sequence of molecular events required to generate a mature, fully-functional thyroidd (Table 2) [22-27], In many cases, the corresponding human molecular defect hass not yet been established.
ThyroidThyroid agenesis or hypoplasia: the FKHL15 gene.
Thee human FKHL15/FOXE1 gene, commonly referred to as TTF2, is a member of a familyy of proteins that bind DNA through a forkhead domain. During development, it iss expressed in thyroid. Rathke's pouch, pharyngeal structures and hair follicles [28].. Mice lacking the titf2 gene show cleft palate and either sublingual thyroid remnantss or agenesis of the gland [29], In humans, biallelic mutations have been foundd in rare cases of Bamforth's syndrome, defined by severe CH due to thyroid agenesis,, a constellation of midline defects as cleft palate, choanal atresia or bifid epiglottis,, and spiky hair [30], Recently, a less severe mutation has been described
Clatterr '
inn patients with incomplete clinical phenotype. suggesting partial preservation of FKHL155 function in vivo [31]. Phenotypsc variability in titf2 knockout mice (thyroid hypoplasiaa or agenesis) [29] suggests the involvement of "modifier" genes in early organogenesiss of the thyroid.
ThyroidThyroid ectopy or hypoplasia: the PAX8 gene.
PAX88 is a transcription factor that binds DNA via a conserved paired domain, and is expressedd in thyroid and kidney. Mice lacking the pax8 locus show hypoplastic thyroidss without follicular organization [32]. In humans, monoallelic PAX8 mutations havee been found in patients with sporadic and familial hypothyroidism due to thyroid dysgenesiss [33-35]. Haplomsufficiency in this case might be caused by monoallelic expression,, a feature reported for mammalian pax5 during development [36]. A characteristicc feature is the variable phenotype of the disorder, ranging from ectopy andd hypoplasia of the thyroid associated with severe CH to eutopic thyroid associatedd with mild hypothyroidism. It is currently unclear which molecular mechanismss underlie this phenomenon. A recent report snowing a polygenic backgroundd for thyroid dysgenesis in mice [37] suggests a role for paralogous pax geness or other co-factors on phenotypic modulation of this disorder [38],
Thyroidal,Thyroidal, pulmonary and neurological defects: the NKX2.1 gene.
NKX2.1.. also known as TTF1 and TEBP. is a transcriptional regulator that belongs too the family of homeodomain transcription factors. Sites for expression during embryogenesiss are thyroid, lung and specific areas of ventral forebrain. Titfl knockoutt mice die at birth because of severe lung hypoplasia and have complete absencee of thyroid and pituitary glands [39]. In Titfl homozygous null mutant embryos,, thyroid rudiments are initially formed but are eliminated through apoptosis [40].. In humans, only monoallelic deletions or inactivating mutations have been foundd responsible for a new syndrome defined by neurological, thyroidal and respiratoryy impairment [41-44], In published cases, thyroid phenotype varied from hypoplasiaa to mild TSH elevation. The attractive hypothesis that these mutations wouldd be responsible for the secretion of a less bioactive TSH from the pituitary [44],, would make NKX2.1 defects the first ones to cross the etiological border betweenn thyroidal and central hypothyroidism.
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T a b l ee 2 Animal models for hypothyroidism
S p e c i e ss G e n e G e n o t y p e ThyroidThyroid development M o u s ee t ' / 2 ' f o x e ' - - .K 0 . ; Mo..;soo oaxS - - :K 0 . M o u s ee titf 1 r K * 2 1 - - : K . 0 : M o u s ee f gfr? - - ' y ' ^ - . i b i ~ M o u s ee rar-i e • ra^"-/ - - i D . K 0 i o k u s ee "ex - - :K Ü . ; M o u s ee aoxa'5 -•- iK 0 ..• M o u s ee a v a l - - •:< 0 • Z e b r a h s hh r v j i ; paxz - - ' ' K . O . : ThyroidThyroid function A f r i k a n d e rr cattle tg N o n s e n s e r-,._ D L . ' C "" g o a l s tcj \ r ; n s e n s e n \ i M o u s ee i c c c / c o n : :y M i s s e n s o m.it Ratt ird-.vruvvj ;y M i s s o n s e m u l Catt :po U m d e - t r o d M o u s ee pes -.- ;K 0 ; M o u s ee ;f'y- h y t : tsh-" M i s s e n s e n u t M o u s ee sreo D N c r e ; ; ! T; ::: A b o r e v i a t o n s -'- h o m o z y g o u s mactivation * - . tie d o u b l ee k n o c k o u t m o u s e . T b a n s g e m o m o u s e ' O D '' R e f e r s mainly to features c a u s i n g or r e f e c t i n g the tiess a s s o c i a t e s s e v e r e d y s o i a s i a of h.ngs anc b a s a s:ss " a s s o c i a t e s hver a " d forebcain tiypof; as a d u e mo^.see e x p r e s s i n g a d o m m a n ' " e g a t i v e c e b p r o t e i n s nott stud-ed. P h e n o t y p ee "
"""'"•yroidd a g e n e s i s and c!e:
t oa a'.e T h y r oo d r y p o p asia a n d or or.topy T h y r o i dd and p i t u c ar y a g e n e s i s ii A g e n e s i s of thyro d anteno: " ptuita^y a n dd i u r g s . ': T " y o i dd n a l d e v e o p m e n t : T h y r o i dd d o v o l o o m e ' M a ^ e s t at the bucii s t a g e :; T h y r o i dd lobe a g e n e s i s a n d ' o r h y p o -p!! a s i a 11 hyrocj c b e h y p o p l a s i a Failuree o ' t n y o i d "oiLae f o r m a t i o n . :: Goiter and l u d i i - e - r o v c s i b i e i h y p O t - y r O I O I S a i i G o i t e rr and c o n g e n i t a h y p o t h y r o i d -ism m C o n g e n i t a !! h y o o t c y r o i d i s m . H v p o t h v T j i c i s mm a n d 1 0 D (loose y a n c h o r e dd TPO.... D e a ' n e s ss a n d vestibular d y s f u r c -:ion. . T h y r o i dd i p o s t r ' a t a h h v p o o l a s i a a n d h y p o t h y r o i d i s m m
Thyroidd h y p o p l a s i a ',vith poor different,, a h c o
o r o z y u o u ss :nachvation K 0 . . k n o c k o u t T O L l o d i c ee o r g a n i f i c a t i o n d e f e c ; e eva ted. -m p a r -m e n tt of thyroid p h y s i o l o g y a*~d -m a y o
g a n g l i aa of the brain " a s s o c i a t e s m u l h - c r y ; too if'icK of fusion of u t i m o b r a n o h i a l o o d l e s ' [)err ficaNy in ' h e t h y r o i d , ex s t e n c e of partia
Ref. . ; i o ] ] \"i'. \"i'. "201 1 i-d d
H H
[5| | :ej j j \ \ •S: : ; J 8 ] ]m m
: i f j ] ] [411 1 [48| | [55] ] (22 7 28] [33] ] .so.. D.K d e o r e a s s rr a b n o r m v-.v-. d y s g e T r a r s g c c M O DD •,' 3 3 ed d T l l i e --as sTablee 2 Animal models for hypothyroidism (cont.)
Speciess Gene Genotype Phenotype ' Ref.
ii Pituitary-hypothalamus development and function
PeripheralPeripheral action and intracellular availability of T3
r.. O..SO i-TH - . ' , . ; • r ; :.r^ ,ï e c =,ero,it •. :'.•. v- ~ :; . "
-• - - o - aa " $ h
11 S H . a m go t o
-b'o...b-- :!T.-i * th--;-: . . J K H . " ' J ' " S - ;,;.•.•- V'tl.itV
V o o s oo " ' • ; i - - 0 CetU'-Hi ' H f ^ ; r ; : , ; o.; " ' . : S H Vo.o-..-- !••'. f.-<r • • • > - ' . : ' ; ';eri:..!'(:ni ""•: r o s ^ o r : : ^ : ' " S H r i mm T S -:: A b b r e v a :ir,..,[:ir,..,[;;\,:\,: k r ; :: 4 p f f - r - . : •, !! f ; s n s s c s ii ' a s s o '"•"T.Hjprr o x f n o tt s b . c i i o : : : : n s .. - -. t.-.•;:.,.tt T . ; r i ' i ! - ,, ' f o i a t o ss ss ;; a ' . e s l i v ' • o s s m aa r i i O o n i O Z v a O L i BB i r a o t -vJ.-Hf-- " t - a r ^ . ; . : e i : . r t - a k . ^ ,, - a i S n, , . . , v e r ee d y s p l a s i a o f ?rr a m f o ' - p b r f v n b y ii d o a i n a - ; n e g a t o * v a tt o r * - b e t e r . l - C . . ; , - j .. ! C D 10 r r - : f | . - : . , - - ^ , - ,, - O ^ , v . n g ss ; • - - b n s r , o , : : o : N ^ r ii o u e t o .,, .-r r,.- p - n t f1 r- s [ > ' • a . r ! . - , . . i r x i l i a a ; i C kk n i . ' f i c a a ,ss n a o t •:.;ari:f::j j , , .. , .f , „ j ff l - e ü f u s i o n n vv • ' O " ' v a t i c c itt o r . ..-„.-< < "a!! r ?ff -_J i I t'Tv'"' ' -- K 0 k r o c k n u m ' o o t .. o l o . o o :''b,^'i.i.'.ij... ;•)!"••: -:: a s s o o a t e i , - n u l ' . ' O ' o b ' a n r . f i aa o o ; !! r! o / i s ' o n c . e T ' f tt m o u s e 3 K 0 :.:: . . : . i e o ; m ; o J - a . O -- ^ w : - ! , ; ; ,. o r g a aa a y = . : . ; e " e -<oss • ~ ' a m a o n ••;->".. m 1 0 D A a s
Generall int'ocïjctiar
D y s h o r m o n o g e n e s i ss of the t h y r o i d g l a n d
Thee origin of thyroid dyshormonogenesis can reside at virtually any step of the metabolicc pathways of follicular cells, especially in proteins directly involved in thyroidd hormone synthesis (Fig. 4).
HyporesponsivenessHyporesponsiveness to TSH: the TSHR and GNAS1 genes.
TSH-receptorTSH-receptor defects
Thee thyrotropin receptor (TSHR) is a predicted seven transmembrane glycoprotein that,, through binding to its ligand TSH. leads to activation of thyroid metabolism via GG proteins. Since the cloning of the TSHR gene in 1989, biallelic inactivating mutationss have been described in patients with total and partial insensitivity to TSH [45],, leading to hypoplasia of the thyroid with severe hypothyroidism or to diminishedd synthesis and secretion of thyroid hormone, respectively. A similar phenotypee is present in the hyt/hyt mice with a homozygous mutation in the homologouss murine tshr gene [46]. Recent reevaluation of these mice showed that thyroidd growth is not controlled by tshr during development, but exclusively during postnatall life [47].
Interestingly,, also a dominant mode of inheritance has been described for the partial resistancee to TSH, suggesting that monoallelic mutations which completely disrupt TSHRR function [48] can originate the same clinical phenotype as biallelic mutations withh a less severe impact on receptor function [49].
Gsi/.-subunitGsi/.-subunit defects.
GG proteins are a family of membrane-bound proteins that transmit the signal from stimulatedd seven-transmembrane receptors triggering cAMP transduction pathways.. Among the four Gu protein subtypes. Gsu and Gqa have been shown to mediatee TSHR signals. The Gs</. subunit is encoded by the GNAS1 gene, in which numerouss monoallelic inactivating mutations have been found in patients with pseudohypoparathyroidismm type la (PHP la). Manifestations of the disorder include mildd hypothyroidism due to resistance to TSH (and to other glycoprotein hormones) amongg a constellation of skeletal and developmental anomalies referred to as
Albright'sAlbright's hereditary osteodystrophy. The hormone unresponsiveness is the
consequencee of mutations in the maternal copy of the GNAS1 gene, in combination withh tissue-specific imprinting. If the mutation is transmitted on the paternal allele. thee phenotype is limited to the osteodystrophic features [50].
Chapterr 1
\wmL
JM\\M^ H f ^ ^
PAX8,, NKX2.1 ffrf&1* &1*
Hf f
Budding Budding Migration Migration SurvivalSurvival and ''iffei^ntiation ''iffei^ntiation Pi&nata) Pi&nata) FolHcte FolHcte formation formation G e n e e FKHL15 5 PAX8 8 NKX2.1 1 L o c u s s 9q22 2 2q13-14 4 14q12-q21 1 P r o t e i nn f u n c t i o n Transcriptionall regulation of geness involved in migration off the thyroid anlage and otherr midline developmental processes. .Transcriptionall regulation of geness involved in survival andd differentiation of migratingg thyroid cells.
Transcriptionall regulation of geness involved in survival andd differentiation of migrat-ingg thyroid cells.
P h e n o t y p e e
Agenesiss of the thyroid, cleftt palate and choanal atresia. .
Hypoplasiaa and/or ectopy off the thyroid, with severe CH. .
Choreoathetosis.. neona-tall respiratory distress andd thyroid hypoplasia and/orr TSH elevation. Inher. . A.R R A D . . A.D. . O M I M M 602617 7 167415 5 600635 5
Fig.. 3 Proteins involved in prenatal development and differentiation of the thyroid
gland,gland, as determined in mouse Factors responsible for the budding of the thyroid anlage fromfrom the primitive foregut and for the typical follicular organization of the mature thyroid are notnot yet identified. Gene localization, clinical phenotypes of congenital hypothyroidism and modemode of inheritance (Inher.) linked to genetic defects refer to human (A.R.: autosomal recessive:recessive: A.D.: autosomal dominant: OMIM: accession numbers in the Online Mendelian InheritanceInheritance in Man database). (™ p. 171)
Generall introduction Blood d circulation n ?? Follicular ?? lumen Gene e TSH-R R N l --THOX2 2
Locuss Protein function
14q311 Activation of thyroid-specific meta-bolicc pathways.
20q133 Signal transduction from GPCRs for stimulationn of adenilyl cyclase. 19p133 Basal transport of iodine from blood
streamm into the thyroid cell. 8q244 Matrix (pro-hormone) for synthesis
andd storage of thyroid hormones. 2p255 lodination of tyrosine residues of
thy-roglobulinn (organification) and cou-plingg of iodotyrosines to form T3+T4. 7q317q31 Transport of iodide from the cytoplasm
too the follicular lumen.
15q211 Generation of H202 in the thyroid
folli-cle. .
Dehalogenationn of MIT and DIT for iodinee recycling
Phenotypee Inhe Thyroidd hypoplasia and severe CH. A.R.
Euthyroidd hyperthyrotropinemia. A.R. Resistancee to TSH and/or Albright's heredi- A.D.
tarytary osteodystrophy.
Severee or moderate CH. A R. Euthyroidd goiter.
Goiterr and severe to moderate CH. A.R
Euthyroidd goiter. A.D. r. . AA D OMIM M 275200 0 103580 0 601843 3 600044 4 188450 0
Severee CH due to TIOD.
"Pendred"Pendred syndrome" deafness and goiter or A.R.
moderatee hypothyroidism due to PIOD. Permanentt and severe CH (TIOD). Transientt and moderate CH (PIOD) lodotyrosinee dehalogenase defect.
AA R AA D 274500 0 274600 0 607200 0 A.R.. A.D. 274800
Fig.. 4 Proteins involved in thyroid hormone synthesis. Except for the dehalogenase
activity,activity, most relevant steps in thyroid hormonogenesis have been identified at the molecular level.level. Gene localization, clinical phenotypes of congenital hypothyroidism and mode of inheritanceinheritance (Inher.) linked to genetic defects refer to the human (GPCR: G protein coupled receptor:receptor: MIT/DIT: mono-/di-iodotyrosine; TIOD/PIOD: Total/Partial Iodide Organification DefectDefect OMIM: accession numbers in the Online Mendelian Inheritance in Man database). PicturePicture modified from Van de Graaf et al. (ref. 109).(*> p. 172)
Finally,, resistance to TSH in some families is not associated with genetic defects in eitherr the TSHR or GNAS1 genes, implying that other genes involved in the TSH-T S H R - G s "" cascade are candidate genes for the disorder [51.52].
IodideIodide trapping defects: the NIS gene.
Iodidee is an essential component of thyroid hormones. Transport of iodide into the thyroidd is performed by NIS (Natrum/lodide Symporter). a 13 transrnembrane-domainn protein localized at the basal membrane of the thyroid cells [53], Numerous casess of CH due to an "iodide trapping defect" have been reported with the diagnosticc hallmark of a decreased saliva-serum radioiodide ratio. A subset of patientss has been studied at the molecular ievei. revealing homozygous or compoundd heterozygous inactivating mutations in the NIS coding region [53].
Mutationss result in the impairment of Na+- or l"-bindmg to NIS. disturbed ion
translocation,, the premature truncation of NIS or the defective trafficking of NIS mutantss with failure to reach the plasma membrane [54-56],
Strikingly,, some patients with biallelc (inactivating) mutations in the NIS gene only-showw partial impairment of iodide transport, clinically manifested as euthyroid goiter, whichh can be overcome by increased iodine intake. This suggests the existence of unidentifiedd additional transporters of iodide at the apical membrane of the thyrocyte. .
Pendred'sPendred's syndrome: the PDS gene.
Pendred'ss syndrome is an autosomal recessive disorder characterized by goiter. overtt or subclinical hypothyroidism and congenital hearing loss of variable degree. Typically,, the thyroid defect is characterized by a partial iodide organification defect (PIOD)) at the perchlorate test [57].
Pendred'ss syndrome is caused by mutations in the PDS gene [58], which encodes thee ion transporter pendnn. The function of this transmembrane protein, was recentlyy characterized as an iodide-specific apical porter in thyroid cells [59]. In the mouse,, pendrin inactivation causes early-onset deafness and vestibular dysfunction,, but no goiter or hypothyroidism [60],
Pendred'ss syndrome is caused by homozygous mutations in the PDS gene that prematurelyy truncate pendrin. as well as by missense mutations shown to disturb thee intracellular trafficking of pendrin towards the apical membrane [58.61].
AA major hallmark of Pendred's syndrome is the inter- and intra-familial variability of thee clinical phenotype [62]. The molecular explanation of this phenomenon remains unclear,, but different functional properties among different pendrin mutants have beenn proposed [63],
Thee typically incomplete organification defect of Pendred's syndrome could be explainedd by the existence of other apical iodide porters with (at least partially! overlappingg function with pendrin. Recently, a novel molecule was isolated basec onn NIS homology but localized in the apical membrane [64], representing a possible candidatee for unaffiliated cases of hypothyroidism due to PIODs.
Tola!Tola! iodide organification defects (TIODs): TPO defects.
Oxidationn and binding of iodide to tyrosine residues in Tg is commonly referred to as iodidee organification. This reaction takes place at the follicular side of the apical membranee of the thyrocyte and is catalyzed by the membrane-bound
thyroperoxidasee (TPO) in the presence of hydrogen peroxide ( H202) The four
factorss currently known to play a role in the organification reaction, including pendrin,, the transporter of iodide into the follicle, and Tg. acceptor of iodide, are depictedd in Fig. 5.
Patientss with TPO defects typically show severe biochemical hypothyroidism accompaniedd by a discharge of over 90% radioiodide after the administration of perchlorate.. defining a total iodide organification defect (TIOD). In a recent survey fromm The Netherlands, the molecular findings of 45 congenitally hypothyroid patientss with TIOD (in 35 families) were summarized [65]. Most cases were due to homozygouss or compound heterozygous mutations in the TPO gene. 1 patient had partiall maternal isodisomy of chromosome 2p (harboring an inactivating TPO mutation)) and. in one patient, no mutation could be found. This latter patient is discussedd in Chapter 3 of the thesis.
Recently,, two missense TPO mutations causing CH were investigated in vitro using immunofluorescencee and electron microscopy, showing retention of TPO mutants in thee ER [66], These findings are in line with early descriptions of abnormal cellular localizationn of TPO in a hypothyroid cat model [67],
PartialPartial iodide organification defects (PIODs).
Ass defined by the intravenous perchlorate discharge test, a PIOD is a partial impairmentt of iodide organification that originates a washout of 10 to 90% of the
i 2 3
l-radioiodidee taken up by the thyroid gland. CH patients with Pendred's syndrome,, caused by mutations in the PDS gene, typically show PIOD. But PIOD hass long been suspected to be a genetically heterogeneous entity. It remains controversiall whether TPO mutations that do not completely block enzyme activity [68)) might be responsible for certain cases of PIOD. Furthermore, the other player
Chapterr 1
off at least two oxidases [4], was always regarded as a molecular candidate for
PIOD.. The first proof of the involvement of H202-generating system in PIOD can be
foundd in chapter 3 of this thesis [69],
Fig.. 5 Proteins and enzymatic systems involved in the organification reaction that
takestakes place in the follicular lumen of the thyroid . (' p. 173)
ThyreoglobulinThyreoglobulin synthesis defects: the TG gene.
Thyroglobulin.. a homodimeric glycoprotein, is a key element in thyroid hormone synthesiss and storage. It is encoded by a very large gene (TG), mapped to human chromosomee 8q24, that spans more than 300 kb and contains 48 exons [70]. Thee first demonstration of the genetic basis of hypothyroidism came from studies on
tgtg in animal models with goiter and/or hypothyroidism. In the Afrikander cattle [71]
andd Dutch goats [72], homozygous stopcodons in the tg gene result in the synthesis off prematurely truncated thyroglobulin molecules that cause the disease. More recently,, the cog/cog mouse and rdw/rdw rat. which respectively develop goitrous andd non-goitrous hypothyroidism, were shown to synthesize full-length Tg moleculess with a defect in trafficking that are retained in the endoplasmic reticulum [73.74]. .
Generall '"•tro^j^.on
Afterr the identification of the human thyroglobuiin gene. CH could be linked to homozygouss defects in the TG coding region, including premature termination signalss and in-frame deletions of the TG mRNA [75], However, some TG defects cann be transmitted in an autosomal dominant manner, since monoallelic point mutationss and (partial) deletions in the TG gene have been described that cosegregatee with goiter development later in life [76.77]. Interestingly, these cases appearr among people living in iodine-deficient areas, suggesting that a combination off genetic and environmental factors might be required to cross the threshold for clinicall expression in the case of monoallelic defects with a less severe impact on hormonogenesis. .
Recently,, patients meeting the diagnostic criteria for a "Tg synthesis defect" (TSD) weree shown not to have molecular defects in the TG gene [78]. This implies that definitionn of TSD exclusively by clinical or biochemical criteria might not be appropriate.. Likely, as yet unidentified genes involved in intracellular trafficking and/ orr maturation of Tg are defective in patients who are now erroneously classified as sufferingg from TSD. Alternatively. TSD could be caused by transcriptional defects [79]. .
DehalogenaseDehalogenase defects.
Mono-- and di-iodotyrosine (MIT and DIT), the main iodinated side products of thyroidd hormonogenesis. are known to be deiodinated in the thyroid cell by a dehalogenasee [80]. In this way, the usually scarce iodine is recycled within the thyroidd for further synthesis of thyroid hormones.
Patientss with iodotyrosine dehalogenation defects have been extensively described inn literature. They show goiter and CH with the diagnostic hallmark of abnormal lossess of MIT and DIT in the urine [81-84], The molecular background for these defectss has not yet been elucidated, but a candidate gene for the thyroid dehalogenasee deficiency was recently reported encoding proteins with a conserved nitroreductasee domain and capacity for iodotyrosine dehalogenation [85], and Chapterr 4 of this thesis.
1.3.22 Central h y p o t h y r o i d i s m
Centrall hypothyroidism -.s the lack of stimulation of the thyroid giand by thyrotropin
(TSH).. due to maldevelopm.ent or dysfunction of the hypothalamus or the pituitary gland.. It occurs in approximately 1 in every 20.000 newborns \1). and. in general, hass a milder clinical and biochemical expression than thyroidal CH.
Developmentt of the various cell lineages of the anterior pituitary requires the transcriptionall activity of a net of factors that triggers the differentiation of GH-, PRL-.. TSH-. FSH- and LH-. ACTH- or MSH- producing cells in a temporally and spatiallyy regulated fashion (Fig 6i ^86]. Formation of TSH-secretmg cells requires thee functionality of. at least. 4 transcription factors. HESX1. LHX3. PROP1 and P O U 1 F 1 .. for which mutations have ceen described in familial cases of combined pituitaryy hormone deficiency ( C P H D J inducing hypothyroidism [87-90]. Defects in otherr proteins Known to co-operate in murine pituitary organogenesis, as pitx.1. pitx2 orr gata2. have not been reported in humans. Further, the combinatorial transcriptionall regulation leading to tne finai celi-type determination of the 2 dishnct populationss of pituitary thyrotropes still needs to be elucidated in detail [86],
Pituitaryy hypothyroidism of developmental origin is always presented m association withh other pituitary hormone deficiencies. In some syndromic cases, the clinical phenotypee includes malformation of various brain structures, the optic nerves, or thee cervical spine (Fig. 6). Inheritance of these defects has an autosomal recessive character,, with exception of some POU1F1 defects with a dom.riant negative effect [91]. .
Isolatedd impairment of TSH secretion has been hnked to biallelic inactivation of two genes:: the TSHB gene, encoding the ['.-chain of the i/ + ii TSH heterodimec and the TRH-receptorr gene 92.93]. In certain cases. TSHB mutations cause diminished TSHH bioactivity [92]. Besides, newly identified transcription factors. liKe the
Trap22G~subunitt of the TRAP coactivator complex, could play essential roles <n
TSHBB gene transcription, as suggested by the phenotype of pituitary
hypothyroidismm exhibited by the trap220 * ' mice [94], Finally, defects in the TRH genee and in the common i/ subunt of TSH have been modeled in mice but not yet describedd in humans (Table 2) [95.96].
Differentt pathogenic mechanisms to consider for central hypothyroidism are possiblee abnormalities of the regulatory system for TRH and TSH secretion. Recently,, a novel heterodimenc glycoprotein hormone, termed thyrostimulin. was shownn to be expressed in the pituitary and some peripheral tissues [6].
Generaa ' H f i j d u c t i o r
Thyrostimulinn has the capacity to specifically activate the TSH receptor and. based onn the reported presence of the TSHR in the anterior pituitary [5], it is tempting to speculatee on a possible role of thyrostimulin in (paracrine) regulation of pituitary thyrotropicc function. Alternatively, secretion of significant amounts of thyrostimulin to thee bloodstream would suggest redundant metabolic functions of thyrostimulin and TSH.. both acting through the thyroidal TSHR. This hypothesis could explain the typicall mildness of central hypothyroidism (as caused by classical TSH deficiency) comparedd to thyroidal hypothyroidism.
Chapterr 1 Gene e HESX1 1 LHX3 3 PROP1 1 POU1F1 1 TRH H TRH-R R TSHA A --L o c u s s ••'--• • 9q3.4 4 5q q :' -66 |2 :'
; ;
Proteinn functionGenee transcription regulation involved in differentiationn of all anterior pituitary cell lines and developmentt of optic nerve and bra.n Genee transcription regulation involved in differentiationn of all pituitary cell lines except for corticotropess and melanotropes.
Genee transcription involved in ontogenesis and differentiationn of all anterior pituitary coll lines, except thee corticotropes and melanotropes.
Genee transcription involved in dtffc thyrotropes.. lactotropes and somatotropes. Activationn of TRH-receptor.
Activationn of Signal transduction leading to secretion off TSH in the thyrotropes
Activationn of GPCRs (dimerization with [\ chains) Activationn of TSH-receptor (dimenzatón with TSH-<;)
Phenotype e
Seplo-oplicSeplo-oplic dysplasia: optic nerve hypoplasia.. CPHD and agenesis of midline brainn structures.
Deficiencyy of TSH. GH. FSH and LH, and =t>htyy to rotate the head.
CPHD. .
TSH.. GH and PRL deficiencies.
Nott reported
Isolatedd central hypothyroidism of late onsett and growth retardation Glycoproteinn hormones deficiency. Isolatedd hereditary TSH deficiency.
Inner. . ..
-:
AA 0 -- R .-.. K O M I M M 601802 2 600577 7 601538 8 173110 0 275120 0 188545 5 118850 0 188540 0Fig.. 6 Proteins involved in development and function of pituitary thyrotropic cells.
TheThe timely-ordered expression of a net of pituitary transcription factors govern the
differentiationdifferentiation of pituitary cell lineages. TSH secretion by the thyrotropes requires stimulation fromfrom hypothalamic peptide TRH ( thyrotropin releasing hormone) and proper expression of
thethe TRH-receptor. TSHA and TSHB genes. Gene localization, clinical phenotypes of congenitalcongenital hypothyroidism and mode of inheritance (Inher.) refer to human (A.R., autosomal recessive;recessive; A.D.. autosomal dominant: OMIM: accession nos. in database Online Mendelian InheritanceInheritance in Man). POU1F1 is the human homologue of the mouse Pit-1 gene, f' p. 174)
Gereraii nTod'JCl'on
1.3.33 Peripheral h y p o t h y r o i d i s m
ResistanceResistance to thyroid hormone.
Resistancee to thyroid hormones (RTH) defines a situation of intrinsic cellular hypothyroidism,, sometimes associated with biochemical euthyroidism or even mild hyperthyroidismm in blood. Actions of thyroid hormones are mediated mainly through fourr nuclear receptors (A1, A2, B1 and B2) generated by alternative splicing of the TRAA and TRB genes (Fig. 7). So far, only defects in the TRB gene were shown to causee decreased responsiveness to thyroid hormone in humans [97], TRs knockout andd transgenic mouse models have shown that these receptors serve varied non-redundantt physiological functions, but can. to some extent, cooperate or substitute forr each other in certain tissues (Table 2) [98], Interestingly, a subset of patients with clinicall RTH does not have mutations in the TR genes [99], suggesting alternative molecularr mechanisms accounting for cases of peripheral hypothyroidism not mediatedd by TRs. Reduced availability of thyroid hormones in peripheral tissues cann also be explained at the molecular level by abnormalities in other nuclear factors,, like RXRs, RARs or coactivators. which regulate thyroid hormone action in associationn with TRs (Table 3) [100-102].
ReducedReduced intracellular availability of thyroid hormone.
Att a pre-receptor level, two additional mechanisms are envisaged: impairment of putativee transporters of thyroid hormone in peripheral tissues [103], and defects in
thee system of deiodinases; especially DI01 and DI02. enzymes responsible for the
intracellularr conversion of T4 into the active thyroid hormone T3 [104] (See Fig. 7). Whilee the physiological relevance of different membrane proteins with capacity for thyroidd hormone transport has yet to be defined [103], to date, no molecular defects havee been described in the human deiodinase system or in the transcriptional partnerss of TRs, as identified in mice (Table 2) [105,106].
Chapterr 1 Gene e T R a a T R P P THH transporters DI01 1 DI02 2 DI03 3 Locus s 17q11 1 3p23 3 1p32 2 14q24 4 14q32 2 Proteinn function
Recruitmentt of nuclear T3 and transcriptionn of T3 target genes.
Recruitmentt of nuclear T3 and transcriptionn of T3 target genes. Transportt of T4/T3 into peripheral cells s
Deiodmationn of T4 into T3 in liver andd kidney.
Deiodmationn of T4 into T3 n pituitary,, thyroid, muscle and brain.
Deiodinationn of T3 into reverse T3 orr T2 in placenta, brain and vessels. .
Phenotype e
Nott identified in human.
Unresponsivenesss to thyroid hormone e
Nott described
Euthyroidd hyperthyroxinemia. Mutationss not identified in humans
Nott described (Pituitary TH resistance,, suspected). Severee hypothyroidism in giant hemangiomas.. No mutations identified. . Inher. . Not t reported d A.R.. A D Not t reported d
'..
reported d Not t reported d OMIM M 188450 0 190160 0--147892 2 601038 8
Fig.. 7 Proteins involved in intracellular availability and action of thyroid hormones
inin peripheral tissues. Gene localization, clinical phenotypes of congenital hypothyroidism andand mode of inheritance (Inher.) refer to human (A.R.. autosomal recessive; A.D.. autosomal dominant:dominant: OMIM: accession nos. in database Online Mendelian Inheritance in Man). (fp.(fp. 175)
G&"eraii intror.kjaiop
1.44 Gaps and controversies.
Manyy molecular aspects of thyroid development and thyroid metabolism still remain poorlyy understood. The identification of novel proteins relevant for thyroid physiologyy and the unraveling of the molecular basis of thyroid diseases is necessaryy to gain a more holistic view of thyroid biology and pathophysiology. In the currentt literature, there are numerous gaps and controversies that reflect both the presentt knowledge and the obscure (and challenging) pitfalls in thyroid research (Tablee 3) [64], [107-112].
T a b l ee 3 Gaps and controversies in thyroid patho-physiology and research. Processs in thyroid p h y s i o l o g yy and disease H.;, "•;^,, t'.v'OiC o e . e :.;.•""';!"•: T hh , rv.-:=:J f;/.;:;;-: :"•:•'"! •:•;(( t r « ( u a r ' , ' r . i n . i r e . U p I H * ' ;; uf •;,;•] f.]fc (rrjT- V e : ^ s i r a ! i y : r f : t N r MM ::ia-ci |r;rjj -t.* f a ^ n f - : . ; - ! - , • - ; i.. v'e :. v ' c s c i ':j-\ :l .. J1" : ; , ' ! ' KK "
Genee and protein identified forr the process
T K h ^ ' v :: P A . ^ \ K . X l 1 r- h.;:VH'--'' > " ^ ~ « * ' S H R R - A XX f :S '• -. SS..yy,, , . . .r.! r K J^! ; S . - ; - ; - I ^ ' M S Pc-r-rvv •-A p i ^ a '' o l i o r - ;r,v S I V - T ; ^ •-A. 1 .
Molecularr question marks
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1.55 Strategies for CH-oriented research: the SAGE technique.
AA requisite for molecular medicine is the identification of functionally specialized geness in the search of defects responsible for human disease.Theree are two mainstream approaches to identify such functionally relevant genes: Onee is based on the mapping of clinical phenotypes to chromosomal loci, the subsequentt linkage refinement and delineation of a (usually extensive) area that containss the gene responsible for the phenotype followed by the search for candidatee genes within the region. In this approach, the physiological relevance of thee gene and protein to be cloned is supported by the connection of putative genetic defectss to an already known human disease. Disadvantages are that the molecular basess of clinically poorly defined phenotypes cannot be studied. Practical hampers off this "phenotype to genotype" approach are the rarity of large family pedigrees thatt are an absolute requirement to obtain positive linkage and the time-consuming stepp of sequencing a high number of genes within the putative locus. A typical examplee of this approach in the thyroid (and auditory) field is the identification of PDS.. the gene encoding pendrin, responsible for Pendred's syndrome (58). More thann a century after the original description of the syndrome, in 1996 the phenotype wass mapped to human chromosome 7q22-31.1. followed by a further refinement of linkagee and the final identification of the gene upon the systematic sequencing of thee region during the Human Genome sequencing project.
Thee second strategy "genotype to phenotype" relies on the identification of functionallyy relevant genes that after elucidation of the function of the corresponding protein,, can be linked to clinical phenotypes unaffiliated at the molecular level. Advantagess of this approach are that the research is independent from the fact that thee disease might or might not be clinically well defined or even recognized, or not suspectedd to have a genetic origin. Furthermore, this line of research is not limited byy the fact that the disease might eventually have not been mapped to any human chromosomee or to syntenic chromosomes in animal models. Main difficulties are relatedd to the cloning strategy directed to find "physiologically relevant' genes. Tissuee specificity of expression can be a major clue to overcome some of these difficulties.. Tissue specificity has been approached classically in terms of comparativee abundance of transcripts among tissues. Cloning strategies like the cDNAA subtraction or the differential display use the basic principle of isolating mRNAss based on their different level of expression in different tissues or under differentt conditions.
Thee recent mapping of the human genome has opened the way to novel technologiess that identify new genes and proteins in a tissue- and cell-specific manner.. Microarrays and serial analysis of gene expression (SAGE) are powerful high-throughputt technigues to this respect [113]. The advantage of SAGE is that it hass the capacity not only to generate a complete and guantitative gene expression profile,, but also to identify transcripts not yet characterized [114]. A SAGE library consistss of 10-basepair sequence tags that, due to their location at the 3' end of the transcriptt can be linked to a specific mRNA. Abundance of mRNA transcripts is determinedd by the number of times that the corresponding tag is scored within the libraryy (Fig.8 and 9).
Applicationn of SAGE to the identification of novel tissue-specific genes poses the difficultyy of selection. TIssue-specric "no-match" tags (tags that do not match any knownn gene in the GenBank and putatively represent novel transcripts) have to be identifiedd from many thousands of tags present in a SAGE library. Recently, a computationall substraction method was developed (Chapter 2 of this thesis) to pinpointt tissue-specific SAGE tags using the "tissue preferential expression' (TPEj algorithm,, which was able to identify novel thyroid-specific tags [115], Cloning of the correspondingg full-length cDNAs and mutation screening of candidate patients with "idiopathic"" CH led to the molecular affiliation of 2 distinct clinical forms of hypothyroidismm [69]. Thus, high-throughput genomics represents a powerful alternativee to the classical identification of disease-related genes by genetic linkage. Biomformaticc searches for homologous sequences to physiologically relevant cDNAss or proteins [116], or the combined data-mining in databases containing diversee biomedical information [117] have also become essential tools to study geneticallyy inherited diseases.
Generall introduction •AAAAA A •AAAAA A •AAAAA A •AAAAA A •AAAAA A •AAAAA A •AAAAA A •AAAAA A
Isolatee SAGE tags
L i n kk tags together
Sequencee linked tags
Q u a n t i t a t ee tags and determine patternss of gene expression
Genee product
N o r m a l l
Genee p r o d u c t
Disease e
Fig.. 8 General flow chart of the SAGE technique. General steps to be taken when
performingperforming Serial Analysis of Gene Expression (SAGE) are: 1. mRNA isolation from the tissuetissue of interest: 2. Isolation of SAGE tags representing each one of the mRNA molecules: 3.
ConcatemerizationConcatemerization of tags: 4. Sequencing of linked tags, and 5. Quantification of tag abundanceabundance using specialized software. The technique allows the comparison of the abundanceabundance of individual tags between 2 (or more) SAGE libraries, as depicted in the picture. OverexpressionOverexpression or downregulation of tags (A to H in the scheme) in a SAGE library constructedconstructed from a pathological tissue with respect to the abundance in a normal tissue is the basisbasis to implicate the corresponding gene in the pathogenesis of the disease. (*& p. 176)
Chapterr 1
Fig.. 9 Detailed scheme of the SAGE technique. SAGE allows quantitative and
simultaneoussimultaneous analysis of the complete pool of mRNA transcripts present in a tissue. First, doubledouble stranded cDNA is synthesized from mRNA isolated from the tissue, using biotinylated oligoprimers.oligoprimers. The cDNA is then cleaved with a restriction enzyme (anchoring enzyme. AE) thatthat is expected to cleave every transcript at least once. Binding to streptavidin beads
isolatesisolates the 3' portion of the cleaved cDNA. The pool is divided in half and ligated through the AEAE restriction site to two different linkers (A and B) containing a type IIS restriction site (tagging(tagging enzyme. TE). This type of enzymes cut at a defined distance from their recognition sites.sites. Then, addition of TE results in the release of a short piece of DNA bound to the linker. TheThe two pools are then ligated together with their blunt ends (forming the so-called ditags). servingserving as a template for a PCR reaction with specific primers for each linker. The resulting productsproducts are digested with the AE. liberating the ditags which are subsequently ligated togethertogether (concatemerized). subcloned in appropriate vectors and sequenced in a serial fashion.fashion. Scheme from. Velculescu et al. Science 270: 484-487 (1995). (« p. 177)
1.66 Scope of the thesis.
Thee aim of this thesis was the identification of novel genes relevant for thyroid physiologyy involved in the pathogenesis of Congenital Hypothyroidism. The Serial Analysiss of Gene Expression (SAGE) was chosen as the cloning strategy.
Chapterr 2 deals with the development and experimental validation of a computationall substraction method that accurately discriminates between tissue-specificc tags and tags corresponding to housekeeping genes. From a thyroid SAGE libraryy containing 4260 "no-match" tags, the relative abundance of 80 tags were comparedd to their level of expression in 14 other SAGE libraries from 9 different humann tissues, using the "tissue preferential expression" (TPE) algorithm. This led too the identification of 4 novel thyroidal cDNAs.
Onee of the selected tags (internally designated NM56) corresponds to the THOX2 gene,, encoding one of the two oxidases putatively involved in hydrogen peroxide generationn in the thyroid follicle. In Chapter 3. genetic screening in the THOX2 genee showed that premature stopcodons cause CH. Biallelic mutations were associatedd with permanent and severe CH due to a total iodide organification defect (TIOD)) while, unexpectedly, monoallelic mutations cause a moderate and transient formm of the disease, due to a partial iodide organification defect (PIOD). In combinationn with the biochemical/etiological classification of these patients, this provedd the role of THOX2 in thyroid hormonogenesis.
Chapterr 4, describes the identification of DEHAL1 after detection of the correspondingg tag NM159 by the TPE algorithm. This novel gene is expressed in thyroid,, kidney and liver and encodes proteins with a conserved nitroreductase domain.. The pattern of expression and the FMN dependency of nitroreductases hallmarkedd the function of this enzyme. Subsequently it was proved to deiodinate mono-- and di-iodotyrosines (MIT and DIT), the main side products of thyroid hormonee synthesis. DEHAL1 represents the best candidate gene for the human dehalogenasee deficiency, known to cause goiter and urinary loss of MIT and DIT thatt will result in varying degrees of CH depending on iodine intake.
Chapterr 5 deals with the characterization of a novel thyroidal protein from another selectedd No-match tag (NM41). This protein has structural homologies with the familyy of cystine-knot proteins, exhibits a conserved glycoprotein signature (typical forr TSH, FSH, LH and CGH a. and [i subunits) and a signal peptide for secretion. Functionn of the protein is currently unknown.
References s
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