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Novel thyroid specific transcripts identified by SAGE: implication for congenital
hypothyroidism
Moreno Navarro, J.C.
Publication date
2003
Link to publication
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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CHAPTERR 6
6.11 Preface.
Duringg the last two decades, part of the genetic basis of congenital hypothyroidism (CH)) has been elucidated by the identification of molecular defects in pituitary- and thyroid-specificc proteins in patients with various types of hypothyroidism. However. soo far identified molecular defects only explain a small proportion of the cases with hypothyroidism.. Hence, novel research strategies are required to fill in the gaps in thee molecular events necessary for the formation of a mature, fully-functional hypothalamus-pituitary-thyroidd axis and to identify novel disease-related genes. Thiss thesis deals with the application of novel technologies in genomics to the identificationn of genes functionally relevant for thyroid physiology. The first step was thee development of a computational method that overcomes the difficulty of selectingg tissue-specific tags out of the overwhelming amount of data derived from thee analysis of a thyroid SAGE library. The best validation of the efficacy of the TPE substractionn approach has been the demonstration of important functions (or features}} for the three proteins selected and later characterized. Two of the selected tagss correspond to proteins whose functions ( H20 ^ generation for NM-56 and
dehalogenationn of iodotyrosines for NM159) were for long known to exist in the thyroidd but whose molecular nature remained elusive. The third tag (NM41) correspondss to a novel type of proteins that shares features with others known to actt as morphogens in development and tissue morphostasis.
Possiblee implications of the present work in the frame of current (and historical) thyroidologyy are discussed next.
6.22 Cloning of " t i s s u e - s p e c i f i c " genes: m e t h o d o l o g i c a l
considerations. .
Tissue-specificityy has been classically approached in terms of relative abundance of transcriptss among tissues. Categories have been proposed for tissue-specific geness based on their moderate (> 2 fold), strong (> 5 fold} or very strong (>10 fold) preferentiall expression in a given ceil type with respect to a range of other tissues [1],, The need to categorize implies that only a minimal number of transcripts fit the strictt definition of being tissue-specific, namely being exclusively expressed in a singlee tissue. Most proteins encoded by so-called 'tissue-specific1' genes should trulyy be termed "tissue-restricted" since they are expressed in a reduced and characteristicc pattern of tissues. Proteins with essential functions for thyroid metabolismm and development that are generally termed thyroid specific, are not expressedd exclusively in thyroid. The sodium iodide symporter is expressed in
thyroidd and a range of other tissues like sahvary ana mammary glands, gastrointestinall mucosa or placenta Pendnn mactivation results primarily in deafnesss and only affects thyrod function in a limited number of cases. The "thyroid"" transcription factor TTF1 (now NKX2.1 i is also important for development off lung and ventral forebram. and TTF2 (now FKHL15) and PAX8 show rather a thyroid-restrictedd than a thyroid-specific pattern of expression
Identificationn of novel functionally relevant genes and proteins using their specific./ restrictedd pattern of expression has been a goal for decades in Molecular Biology. Subtractivee hybridization and the PCR-based rnRNA differential display are proceduress which have been successful in isolating some relevant genes [2. 3]. However,, they only detect gene expression differences between a limited number of eel'' Wpes (usually two). The public!" available expressed sequence tag (EST) librariess of different human tissues opened a new tool for gene discovery. EST librariess can be mined and compared in search of subsets of mRNAs differentially expressedd among tissues or pathological conditions like cancer [1]. Drawback of the ESTT approacn is that, very mjch depending on library size. EST sequences frequentlyy correspond to RNAs with a relatively high expression level. Microarrays cann contain many thousands of transcripts and ESTs. and are used to elucidate correlationn of expression with metabolic pathways or disease [4. 5]. Since microarrrayss show the expression profile of transcripts selected and spotted on the array,, they by definition do not offer "new genes" .
Inn chapter 2 of this thesis, a new approach to identify novel genes preferentially expressedd in tissues is proposed, based on Serial Analysis of Gene Expression (SAGE}} combined with the selection of tags by a computational algorithm. SAGE is currentlyy the only technique that allows determining the complete and quantitative expressionn profile of a tissue. Indeed, the approximately 300,000 rnRNA molecules containedd in human cells can be fully determined by a SAGE library of approximatelyy 300.000 tags. A i'brary of this size includes transcripts (83% of the total)) present at levels as low as one copy per cell [6]. This unprecedented power in genee expression profiling is responsible for the fact that SAGE libraries so far made invariablyy contain a percentage of SAGE tags (near to 50%) representing completelyy unknown mRNAs. These tags are named no-match (NM) tags, since thesee sequences do not match any identified qene in the GenBank database Thus, thesee thousands of no-match tags constitute a bunch of novel genes waiting to be characterized.. Since both putative housekeeping genes as well as tissue-specific geness will be among them, the main problem that had to be faced was how to isolatee an interesting group of tags in the absence of any functional or additional genomicc information to just a 10-basepair cDNA sequence.
Thee SAGE technique has been usee to gam insight into the pathogenesis of differentt diseases through the comparison of gene expression profiles between normall and pathological tissues [7-9], To date, this line of research has lead to the identificationn of subsets of sequences, most of them ESTs. putatively involved in pathogenesis.. In rare reports, a novel cDNA or a group of ESTs with tissue-restrictedd expression was isolated, but not functionally characterized [10. 11]. Thus. untill now no truly novel and fully functionally characterized gene with a restricted patternn of expression has been identified by SAGE.
Byy in silico analysis of SAGE libraries, each no-match tag can be graded based on itss expression level and tissue abundance in other SAGE libraries, using the tissue preferentiall expression (TPE) algorithm [12]. In this way we identified 3 novel cDNAss with a thyroid-restricted pattern of expression, showing that the cloning of tissuee specific genes is attainable upon the construction of only one. medium-sized SAGEE library and the use of publicly available SAGE databases. Further research onn these cDNAs led to the full functional characterization of DEHAL1 (our no-match tagg 159) that encodes an enzyme that catalyses the dehalogenation of lodotyrosiness in the thyroid. No-match 56 turned out to be the thyroid oxidase and thee inactivating mutations we identified in the THOX2 gene of patients with congenitall hypothyroidism are the strongest available evidence of the involvement off this oxidase in H202 production in thyroid. With respect to the function of a third
cDNA.. NM41. currently of a more elusive nature, experiments are ongoing.
Thee recent mapping of the human genome has opened the way to further uses and applicationn of SAGE in the identification of novel genes. The demonstration that SAGEE tags can actually be mapped along the human genome, defining unexpected chromosomall regions of increased gene expression (RIDGES) [13], which have beenn linked to the clustering of housekeeping genes [14], opens an attractive model off gene order in the human genome. Finally, the recent development of LongSAGE. aa SAGE method that generates longer tags that due to their size can be more easily mappedd to the genome, seems to have the potential to reveal undiscovered genes knownn to be contained within specific genomic regions [15],
6.33 TH0X2 defects in CH patients: implications for t h y r o i d
basicc research.
Fromm biochemical studies, nydroger peroxide iH;.0:-. i generation was expected to
existt in the thyroid follicle, since t is required for lodination of thyroglobulin that is catalyzedd by thyroperoxidase [16J However, evidence for thyroidal H^O^ production wass only demonstrated m the nmeteer eighties [17. 18]. At that time, also the first patientt with an e orgamficat on defect due to defective h^O- generation was reportedd [19]. The corresponding e-vymatic activity was padially punfied in 1996 [20]. .
Thee first report on the cloning of components of the thyroidal HL-O- generating systemm came after the purifica:>oc of a flavoprotem with NADPH- and C az" -dependentt activity from pig thyroid piasma membranes that was foliowed by peptide sequencingg and cDNA cloning of a 138 kDa protein named p138i : : x [21]. Subsequently,, two cDNAs. nam-eu T h O X I anc TnOX2 (5-compiete version of p138'Ü Xii encoding NADPH oxidases were cloned from thyroid based on the hypothesiss that H202 generation in thyroid and phagocytes would be functionally
similarr [22]. The same transcripts were cloned under the name DUOX1 and DUOX2 [23]] and THOX2 (internally designated NM56.I was also identified by our group usingg the SAGE-TPE algorithm approach [12].
Whilee these novel THOX proteins contain the consensus sequences for FAD- and NADPH-bindingg sites that are essential for H/02 generation in the phagocyte
oxidasee system (gp91:':':::>\) [24]. tie experimental proof that THOX1 and THOX2. aree capable of H;.02 generation is currently lacking [25]. Chapter 3 of this thesis
describess in vivo evidence of the involvement of THOX2 in thyroid physiology in the formm of a biallelic inactivating THOX2 mutation that prematurely truncates the THOX22 protein in a patient with complete lacK of thyroid hormone production that causess severe CH [26]. In the absence of THOX1 mutations in our cohort of CH patientss with iodide organification defects, the role of THOX1 in thyroid physiology remainss presently unclear.
Thee proposed model for THOX proteins in the apical membrane of the thyroid cells. withh Liie amino leiminai pari of the protein located in the foiiicuiar lumen, does not containn any suggestions with resoect to possible netero- or nomo-dimerization of THOX11 and THOX2. neither on oligomenzation of the thyroid oxidases with other co-factors.. The C. elegans homologue of THOX1 is capable to catalyze cross linkingg of tyrosine residues involved in the stabilization of its cuticular extracellular matrixx [23] and this raises the tempting speculation that THOX might have a similar
Generall discussion
rolee in the coupling of (lodinated) tyrosine residues in thyroglobulin to form thyroid hormone,, partially overlapping with the organification and coupling properties attributedd to TPO.
Ourr study also describes 3 patients with PIOD and moderate and transient CH that havee monoallelic mutations in the THOX2 gene, This suggests hapioinsufficiency as thee mechanism for this transient disorder. THOX2 hapioinsufficiency represents an examplee of dominantly inherited disease (although with transient biochemical expression),, unprecedented in the field of thyroid dyshormonogenesis. It is currently nott clear why one normal allele of THOX2 gene is not enough to overcome hypothyroidism,, while this is the case for most other genes involved in dyshormonogenicc hypothyroidism. A possible clue to this question is that, at least in thee rat model, chronic TSH stimulation of THOX genes does not lead to overexpressionn of the THOX proteins [27].
6.44 THOX2 defects in CH patients: clinical implications.
Inn Chapter 3, one patient with permanent and severe CH due to a total iodide organificationn defect (TIOD) was found to carry a homozygous stopcodon that prematurelyy truncates the THOX2 protein, deleting all functional domains. This findingg demonstrates that CH due to a TIOD is a genetically heterogeneous entity. Inn 34 out of 35 families, TIOD is caused by inactivating TPO mutations [28], while in 11 family inactivating mutations in THOX2 were causally related to the disease. Inn the same chapter, 3 patients with transient CH are shown to have monoallelic inactivatingg mutations in the THOX2 gene. They had a moderate hypothyroidism, detectablee by neonatal screening, associated with partial perchlorate discharges (PIOD).. These findings represent the first evidence of a genetic origin for transient congenitall hypothyroidism, previously thought to be exclusively caused by environmental,, immune or iatrogenic factors. It strongly suggests that the 10-25% of "idiopathic"" transient CH cases where the classical etiologies can be excluded correspondd to genetically inherited forms of mild dyshormonogenesis.
Thiss has practical implications for the screening of CH and the clinical management off transient CH. The incidence of CH is almost exclusively presented in relation to permanentt primary CH [29] and the incidence of transient CH is not systematically reportedd in literature. From the Dutch screening program we know that the incidencee of transient CH (1:2200) is higher than that of permanent CH (1:3100) [30],, This is probably because it is generally accepted that etiologies of transient CH cannott recur later in life and that patients will be safely free of disease during
Chapterr 6
childhoodd and adult life. A concept that now has to be revised. We have described a subsett of patients with transient CH that is due to a genetic (and obviously permanent)) defect. In literature, there is one report of a patient with transient neonatall hypothyroidism attributed to ""probable immaturity of thyroidal iodine organification''' [31], and another on 2 patients with severe dyshormonogenic hypothyroidismm (positive perchlorate test) detected after normal neonatal TSH screeningg [32]. No follow-up has been reported of these patients during childhood andd adult life, but these cases might overlap the phenotype we describe for monoallelicc THOX2 mutations. Since there is a chance of recurrence of subclinical/ overtt hypothyroidism or goiter development during pregnancy, monoallelic THOX2 mutationss currently represent a crossroad between permanent and transient CH. Ann accurate prevalence of these mild transient dyshormonogenic defects is difficult too estimate, since the suspicion is high that cases are missed by the current T4-basedd screening for CH. As shown in chapter 3. the sib in family 3 with a monoallelicc mutation in the THOX2 gene was not detected by screening because he hadd a thyroxine bloodspot value of -0.6 S.D. of the mean vaiue for the thyroxine distributionn curve of that particular day. while cut-off levels for TSH determination in thee Dutch screening are established at -0.8 S.D.
Thiss case illustrates that patients with so-called "borderline hypothyroidism" can actuallyy be missed by CH screening programs, in the light of our own data, supportedd by recent reports showing that most of these patients maintain disturbed thyroidd function until 3 years of age [33] and that newborns classified false positive att CH screening have a high risk of subclinical hypothyroidism in infancy and early childhoodd [34]. it is important to establish if this borderline hypothyroidism hampers development.. If these data change the cost/benefit ratio of the screening procedure, thenn cut-off levels used in both TSH- and T4-based screening methods may have to bee revised.
Thee need for treatment of subclinical or "compensated" hypothyroidism (TSH elevationn with normal T4 levels) has long been a matter of debate in children and adultt patient care. In the nineteen eighties, neonates with "transient infantile hyperthyrotropinemia"" were described and followed without treatment, showing in somee cases relapse of TSH elevation and development of hypothyroidism or goiter duringg infancy and childhood [35], In this publication, mental development was reportedd normal, as assessed by the Tsumon-lnage or the Tsumon-lsobi developmentall scales (usually used in Japan to evaluate psychomotor development)) and sometimes the IQ Wechsler scale. Contradicting these results, a recentt study showed a lower intelligence quotient at the age of 7-8 years in children whoo suffered transient congenital hypothyroidism and hyperthyrotropinemia [36].
Generall discission
Furthermore,, classical studies have shown that newborns with TSH levels higher thann 7.0 mU/ml and normal T4 have a low basal metabolic rate, reversible after thyroxinee treatment [37]. Thus, on the basis of evidence that abnormalities of thyroid functionn at birth are associated with impaired intellectual development and hypometabolicc state in children, we advise to treat patients with transient CH and hyperthyrotropinemiaa as soon as possible after birth. We agree with the current vieww that, irrespective to the age of patients, subclinical hypothyroidism should be consideredd a mild thyroid failure, and should consequently be treated [38]
6.55 DEHAL1: a novel gene for an "old" phenotype.
Ass early as in 1954, the phenotype of iodotyrosine dehalogenase deficiency was recognizedd as an inborn error of thyroid metabolism [39-41], The clinical presentationn was originally described in an inbred family with non-endemic goitrous cretinismm that seemed subjected to hereditary transmission. Study of additional pedigreess was suggestive of autosomal recessive inheritance with heterozygote carrierss showing milder signs of disease [41.42], In vitro assays on thyroid tissue fromm hypothyroid goitrous patients showed diminished capacity to deiodinate either MIT,, DIT or both and, in some cases, also impaired deiodination of intravenously injectedd radiolabeled iodotyrosines was demonstrated [42-44], Whether these variablee data reflect the characteristics of this enzymatic activity or were due to technicall variations is at the present moment difficult to evaluate. In all cases, pathophysiologyy of the disease was established as the impaired ability for deiodinationn of mono- and di-iodotyrosmes in the thyroid and other tissues. This resultss in continuous urinary loss of iodine causing hypothyroidism due to simple iodinee depletion.
AA rat model for a dehalogenase defect was generated by the administration of 3-nitro-L-tyrosine,, an inhibitor of dehalogenase activity [45], but the characterization of thee enzyme remained elusive. In 1979, a partial purification of a flavoprotein with iodotyrosinee deiodinase activity was reported from bovine thyroid tissue [46], The enzymee contained flavin mononucleotide (FMN) and consisted of two subunits. The molecularr nature of this enzymatic system has to date remained unknown.
Inn chapter 4 of this thesis, we describe the identification, using SAGE and the computationall subtraction of tissue-preferentially expressed tags, of a novel gene expressedd mainly in thyroid, but also in liver and kidney. The gene encodes proteins belongingg to the family of nitroreductases, enzymes known to use FMN as cofactor. Thee putative use of FMN and the pattern of expression in tissues known to have
Oapterr 6
dehalogenatingg activity, resulted in the hypothesis that our NM 159 was the iodotyrosinee dehalogenase. A cell assay was developed and the transcript indeed encodess a protein that is able to dehalogenate iodotyrosines. The activity was completelyy inhibited by the specific substrate competitor 3-nitro-L-tyrosine. Computerr three dimensional modeling predicts that DEHAL1 forms a homodimer. bindingg 2 FMN molecules per dimer. Characterization of DEHAL1 makes it the candidatee gene for the human iodotyrosine dehalogenase deficiency.
Itt is exciting to envisage the detection of molecular defects in this novel gene in familiess with hypothyroidism and goiter. Identification of patients with this disorder wass mostly done in the pre-screemng era and it is an unsolved question if patients withh dehalogenase deficiency (a disorder that can have mild manifestations) are actuallyy detected by neonatal CH screening programs. Detectability is expected to dependd on the level of iodine intake and. thus, on geographical areas The cloning off DEHAL1 now opens the way to molecular testing of candidate patients with unaffiliatedd congenital hypothyroidism. However, the diagnosis of iodotyrosine dehalogenasee deficiency needs the development and adaptation of the radioactive proceduress into modern, non-radioactive diagnostic tools for the determination of MtT.. DIT and iodine in the urine of patients. The accurate determination of these compounds,, for example by mass spectrometry, would allow the detailed characterizationn of the disorder, making structure-function analysis of the human thyroidd dehalogenase-1 possible.
6.66 NM41\ a cystine-knotWke protein in the t h y r o i d .
Understandingg of the type of molecules that regulate patterning of the body came fromm large-scale genetic screens by embryologists [47]. From these studies, several importantt types of patterning molecules are known with high conservation between species.. These patterning molecules fall into two categories: intrinsic, autonomouss regulators of cell fate (that act at the level of the cell} and extrinsic, cell-nonautonomouss regulators of cell fate (that act between cells). Transcription factors aree the most important intrinsic regulators whereas morphogens and the Notch signallingg pathway have emerged the most important extrinsic regulators. Four majorr families of morphogens have been so far identified: the Hedgehog (Hh). Wnt. Fibroblastt Growth factor (FGF) and Transforming Growth Factor (TGF) \\ families. In orderr to serve as vehicles of information between cells, these molecules are typicallyy secreted to the extracellular milieu [48].
Genera-.Genera-. discuss:
cr
Molecularr players involved in embryogenesis of the thyroid are largely unknown. Whilee three transcription factors TTF1/NKX2.1. TTF2/FKHL15 and PAX8. have beenn shown to partly control developmental processes of the thyroid anlage like earlyy migration or survival and proliferation of cells, no effector or mediator of these eventss has so far been characterized. Notably, no extrinsic regulator of patterning eventss in the thyroid has been so far reported
Inn the gut (structure from which the thyroid is derived), several molecules known for theirr role as extrinsic morphogens have been shown to play a role during developmentt and in the maintenance of epithelial homeostasis ("morphostasisH) of thee adult gastrointestinal tract. One of them is TGF |.i. a molecule structurally belongingg to the family of cystine-knot proteins [49. 50].
Inn Chapter 5 of this thesis, the identification of a novel protein with preferential expressionn in the thyroid is presented. Its cleavable signal peptide and the immunohistochemicall localization of the protein in the thyroid follicle strongly suggestt that NM41 is a secretable protein. Partial conservation of the cysteine framee typical for cystine-knot proteins, a large family of extracellular molecules early expressedd in development [51]. is suggestive of a similar function for the NM41 protein.. The tissue pattern of expression, including the gut and embryologically relatedd structures, further supports the working hypothesis of an involvement of NM411 in developmental processes or morphostatic events that maintain microarchitecturee of thyroid and/or other epithelial tissues.
Furtherr characterization of the functional features of this protein in the future constitutess the basis for the possible identification of NM41 genetic defects responsiblee for human phenotypes of disease, including congenital hypothyroidism.
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