A truncating TPO mutation (Y55X) in patients with hypothyroidism and total iodide organification defect

A truncating TPO mutation (Y55X) in patients with hypothyroidism and total iodide organification defect
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Transcript 0743-5800 (print), 1532-4206 (electronic) Endocr Res, Early Online: 1–5 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/07435800.2014.967354 ORIGINAL ARTICLE A truncating TPO mutation (Y55X) in patients with hypothyroidism andtotal iodide organification defect Hakan Cangul 1 , Feyza Darendeliler 2 , Yaman Saglam 3 , Banu Kucukemre 2 , Michaela Kendall 4 , Kristien Boelaert 5 ,Timothy G. Barrett 5 , and Eamonn R Maher 6 1 Department of Medical Genetics, Bahcesehir University School of Medicine, Istanbul, Turkey,  2 Pediatric Endocrinology Unit, Istanbul Faculty of Medicine, Istanbul University, Turkey,  3 Centre for Genetic Diagnosis, Medical Park Goztepe Hospital, Istanbul, Turkey,  4 Department of Child Health,Division of Clinical and Experimental Sciences, Faculty of Medicine, Southampton, UK,  5 Centre for Diseases and Personalised Medicine, School of Clinical and Experimental Medicine, University of Birmingham, Birmingham, UK, and   6  Academic Department of Medical Genetics, University of Cambridge Clinical School, Cambridge, UK  Absract Purpose : Mutations in the  TPO  gene have been reported to cause congenital hypothyroidism(CH), and our aim in this study was to determine the genetic basis of congenitalhypothyroidism in two affected children coming from a consanguineous family. Methods : Since CH is usually inherited in autosomal recessive manner in consanguineous/multicase-families, we adopted a two-stage strategy of genetic linkage studies and targetedsequencing of the candidate genes. First we investigated the potential genetic linkage of thefamily to any known CH locus using microsatellite markers and then screened for mutations inlinked-gene by Sanger sequencing.  Results : The family showed potential linkage to the  TPO gene and we detected a non-sense mutation (Y55X) in both cases that had total iododeorganification defect (TIOD). The mutation segregated with disease status in the family. Y55X isthe only truncating mutation in the exon 2 of the  TPO  gene reported in the literature andresults in the earliest stop codon known in the gene to date.  Conclusions : This study confirmsthe pathogenicity of Y55X mutation and demonstrates that a nonsense mutation in the amino-terminal coding region of the  TPO  gene could totally abolish the function of the TPO enzymeleading to TIOD. Thus it helps to establish a strong genotype/phenotype correlation associatedwith this mutation. It also highlights the importance of molecular genetic studies in thedefinitive diagnosis and accurate classification of CH. Keywords Congenital hypothyroidism, genetics,molecular, mutation, thyroiddyshormonogenesis,  TPO  gene History Received 24 April 2014Revised 15 September 2014Accepted 15 September 2014Published online 20 October 2014 Introduction Congenital hypothyroidism is the most common neonatalendocrine disorder with an incidence of 1/3500 live births andcauses mental retardation and growth delay unless a timelyand proper treatment is introduced (1). About 2% of CH isfamilial and to date 11 causative genes have been describedfor the pathogenesis of inherited CH (2). Table 1 shows the details of all these loci and associated clinical phenotypes.Some of these genes are associated with primary thyroiddysgenesis phenotype (CHNG, OMIM#274400) (3,4) while some are with thyroid dyshormonogenesis (TDH,OMIM#275200) (5). Currently there are seven genes knownto cause congenital TDH which encode for proteins involvedin thyroid hormone biosynthesis (6). Major steps in thyroidhormone synthesis include iodide transfer from blood tothyrocytes and then into the follicular lumen, oxidation andcovalent linkage to tyrosine residues of thyroglobulin and,upon their hydrolysis, eventual coupling of iodinated tyrosylresidues into iodothyronines (T 4  and T 3 ) (7).Oxidation and organification of iodide are achieved bythyroid peroxidase (TPO) enzyme which requires hydrogenperoxide (H 2 O 2 ) as the final electron acceptor (8,9). Therefore the generation of hydrogen peroxide is a critical step in thesynthesis of thyroid hormones (10). Previous reports havefound that a defect in the system that generates H 2 O 2  causescongenital hypothyroidism (11–13). Two dual oxidases(DUOX1 and 2) have recently been identified as thecomponents of thyroid H 2 O 2  generating system (14, 15). TPO is a thyroid-specific heme peroxidase localized in theapical membrane of thyrocytes and plays a central role inthyroid hormone biosynthesis by catalysing (i) oxidation of iodide; (ii) its organification (binding to thyrosine residuesof thyroglobulin); and also (iii) coupling reactions of inertmonoiodothyrosine and diiodothyrosines to form the activethyroid hormones T 4  and T 3  (2). As for the other genesinvolved in the thyroid hormone synthesis, mutations in  TPO Correspondence: Dr Hakan Cangul, Department of Medical Genetics,Bahcesehir University School of Medicine, Istanbul, Turkey .  Tel: +90533 7449433. Fax: +90 216 4684567. E-mail:     E  n   d  o  c  r   R  e  s   D  o  w  n   l  o  a   d  e   d   f  r  o  m    i  n   f  o  r  m  a   h  e  a   l   t   h  c  a  r  e .  c  o  m    b  y   S   i  m  o  n  e   S   k  e  e  n  o  n   1   1   /   0   7   /   1   4   F  o  r  p  e  r  s  o  n  a   l  u  s  e  o  n   l  y .  causing permanent CH are mostly inherited in an autosomalrecessive fashion and to date more than 60 distinct mutationshave been described in this gene (8,9). To investigate genetic background of CH we developed atwo-tier strategy combining genetic linkage studies and fullsequencing of candidate genes in familial cases and identifiedseveral mutations to date in different CH genes (16–29). Inthe current study we aimed to determine genetic cause of CHin a consanguineous family with two affected siblings. Herewe report detailed genetic analyses and associated clinicalphenotypes of these cases. Molecular genetic analysesfacilitate definitive diagnosis and accurate classification of CH and might also describe patient-specific targets foralternative treatment of the disease. Materials and methods Subjects Two cases born to a consanguineous Turkish family wereascertained through our studies on the genetics of congenitalhypothyroidism. The older sister was first diagnosed at fivemonths of age with hormone values of TSH 120mIU/L,T4 5 4.5 m g/dL (normal 4.5–12.5) and T3 5 80ng/dL (normal80–120). Thyroid ultrasonoghraphy and scintigraphy showedthyroid hyperplasia, and perchlorate discharge test indicatedTIOD (total iodide organification defect). In her last follow-up at age of 18, she was on 100 m g/day L-thyroxine and herhormone levels were TSH 1.71mIU/L and fT4 1.40ng/dl.Her weight was 5 3rd centile and height was at 25th centile.Her younger brother was born at term and first diagnosedat 40 days of age through investigations for prolonged jaundice. Hormone values at diagnosis were TSH 180mIU/L,T4 1.5 m g/dL (normal 4.5–12.5) and T3 20ng/dL (normal80–120). Thyroid ultrasonography performed at 2 months of age showed diffuse hyperplasia while a scintighraphy at age 5demonstrated bilateral adenomatous hyperplasia with homo-geneous dissemination of activity. Perchlorate discharge testindicated TIOD. At the age of 15, he has mild mentalretardation and his weight is under 10th centile and height at3rd centile. Their parents were healthy and free of any signsor symptoms of hypothyroidism. Informed consent wasobtained from the family and venous blood samples werecollected from all family members. All procedures performedwere in accordance with the Declaration of Helsinki and thestudy was approved by relevant IRBs/Ethics Committees.DNA was extracted by using standard methods and storedat   20  C until analysed. Potential linkage analysis First, we performed linkage analysis to all 11 known CH lociin all family members with the use of microsatellite markers.Four primer pairs surrounding each locus were selected(Table 1). Fluorescent labelling of one oligonucleotide of eachprimer pair enabled the sizing of PCR products in a capillaryelectrophoresis machine by using GeneMapper v4.0 softwaresuite (Applied Biosystems, Warrington, UK). By combininggenotypes for microsatellite markers we constructed haplo-type tables for each family member. As autosomal recessiveinheritance was assumed in consanguineous families, homo-zygosity of a particular haplotype for a locus in casesaccompanied by heterozygosity of the same haplotype in bothparents was taken as suggestive of linkage to that locus. Direct sequence analysis of the  TPO  gene The DNA template of the  TPO  gene was downloaded fromthe Ensembl database (ENSG00000115705). All alternativetranscripts (17 in total) were included to ensure that primerswere designed to cover all coding exons and intron/exonboundaries. Intronic primers flanking the coding sequencewere designed for PCR amplification using ExonPrimer andPrimer3. Primer sequences and PCR conditions are availableupon request. PCR products were size-checked on 1%horizontal agarose gels and cleaned up using MicroCLEAN(Microzone, Haywards Heath, UK) or gel-extracted usingQIAquickTM Gel Extraction kit (Qiagen, Crawley, UK). Thepurified PCR products were sequenced in both forward andreverse directions using the ABI BigDye Terminator v3.1Cycle Sequencing kits on an ABI Prism 3730 DNA Analyzer(Applied Biosystems, Warrington, UK). Analysed sequenceswere then downloaded using Chromas software and assessedfor the presence of alterations. Results and discussion Haplotype tables were constructed for each family member bycombining the scores for each marker to observe thesegregation of the genotype along with the disease status.The linkage analysis using these tables indicated a potentiallinkage to the  TPO  locus in the family, i.e. both CH caseswere homozygous for a disease associated haplotype whileboth parents were heterozygous for the same haplotype(Figure 1). The family showed no such linkage to other 10 locitested, i.e. cases did not have homozygosity for the micro-satellite markers designed for these loci. These results Table 1. Genes causing congenital hypothyroidism, associatedphenotypes and microsatellite markers used for their linkage analysis.Gene Ch locus Phenotype* Microsatellite markersNIS 19p13.2 TDH1 D19S566, D19S593 D19S103,D19S898TPO 2p25 TDH2A D2S2980, D2S323 D2S1780,D2S2245PDS 7q31 TDH2B D7S2459, D7S692 D7S2456,D7S799TG 8q24 TDH3 D8S1740, D8S256 D8S1746,D8S558DEHAL1 6q25.1 TDH4 D6S1654, D6S440 D6S1687,D6S960THOXA2 15q21.1 TDH5 D15S100, D15S123 D15S132,D15S977THOX2 15q21.1 TDH6 D15S100, D15S123 D15S132,D15S978TSHR 14q31 CHNG1 D14S1433, D14S606 D14S1008,D14S610PAX8 2q12–q14 CHNG2 D2S2269, D2S160, D2S410,D2S1893TSHB 1p13 CHNG4 D1S2756, D1S2881, D1S2852,D1S189NKX2-5 5q34 CHNG5 D5S400, D5S2075, D5S211,ATA52D02*As described in OMIM database: TDH, Thyroid dyshormonogenesis;CHNG, Congenital non-goitrous hypothyroidism; Ch: Choromosomal. 2  H. Cangul et al.  Endocr Res, Early Online: 1–5    E  n   d  o  c  r   R  e  s   D  o  w  n   l  o  a   d  e   d   f  r  o  m    i  n   f  o  r  m  a   h  e  a   l   t   h  c  a  r  e .  c  o  m    b  y   S   i  m  o  n  e   S   k  e  e  n  o  n   1   1   /   0   7   /   1   4   F  o  r  p  e  r  s  o  n  a   l  u  s  e  o  n   l  y .  suggested that the disease associated haplotype segregatedwith the disease status in the family assuming autosomalrecessive inheritance model which is the most likely pattern inconsanguineous families. Therefore we proceeded tosequence the coding region (and flanking sequences) of the TPO  gene in all family members.Direct sequencing analysis revealed a homozygous C to Atransversion in the exon 2 of the  TPO  gene (c.165C 4 A) inboth affected siblings which results in a stop codon early inthe molecule (p.Y55X). Both parents carried the mutation atheterozygous state (Figure 2) which was consistent with thelinkage results. TPO protein is comprised of 933 amino acidresidues and the truncation of the molecule at 55th residue bythis mutation will result in lack of all catalytic domains in theenzyme (Figure 3). Therefore Y55X mutation is most likely torender TPO protein completely non-functional in our patients. D2S2980D2S323D2S2245D2S1780171239248311171239242331171239260319171239260319171239260319171239260319171239260319171239260319D2S2980D2S323D2S2245D2S1780 Figure 1. The scores of microsatellite marker analysis surrounding the TPO  locus in family members. The markers used are listed on the leftand the disease associated haplotype is shaded in grey.Figure 2. Sequence traces from exon 2 of the  TPO  gene showing the mutation Y55X in the case (c.165C 4 A, in grey rectangle, numbering according toEnsembl transcript ENST00000329066 as ATG transcript start site). Note the homozygosity of the mutation in the cases (middle panel) andheterozygosity in the parents (bottom panel) compared to the control (top panel). DOI: 10.3109/07435800.2014.967354  TPO mutation  3    E  n   d  o  c  r   R  e  s   D  o  w  n   l  o  a   d  e   d   f  r  o  m    i  n   f  o  r  m  a   h  e  a   l   t   h  c  a  r  e .  c  o  m    b  y   S   i  m  o  n  e   S   k  e  e  n  o  n   1   1   /   0   7   /   1   4   F  o  r  p  e  r  s  o  n  a   l  u  s  e  o  n   l  y .  The  TPO  gene is located on chromosome 2p25 and coversabout 150Kb of DNA (1,19). It is comprised of 17 coding exons with a 3048 nucleotide-full-length transcript whichencodes 933-amino acid TPO enzyme. The  TPO  genealteration detected in this study (Y55X) is the only nonsenseexon 2 mutation reported in the literature. If expressed at all,the resulting protein truncated at the 55th residue will lack remaining 878 amino acids which include all catalyticdomains of the enzyme molecule. On the other hand, non-sense mutations located before the last exon of any gene isexpected to trigger non-sense mediated decay of transcriptsresulting with no protein expression (30). Therefore Y55Xmutation located in exon 2 of the  TPO  gene is most likely toresult in total lack of enzyme activity in the thyroid tissue of our patients. However further functional studies are needed tounequivocally establish the pathogenicity of this mutation.Towards this purpose, mRNA and protein-expression analysesand functional assays of the TPO activity are indicated forfollow-up studies.In terms of phenotypic presentation, both siblings in ourstudy showed TIOD further confirming that TPO activitymust totally be lost in them. In their comprehensive review(8), Ris-Stalpers and Bikker stated that homozygous orcompound heterozygous inactivating  TPO  mutations areassociated with goitre and TIOD. As some missense muta-tions might result in partial loss function (LOF) in the TPOenzyme with some residual activity, they may be manifestedwith partial iodide organification defect (PIOD). Clinicalpresentation in cases with these mutations could be furthermodified with iodine status. Therefore the phenotype in thesecases is more likely to be variable. In contrast, total LOFmutations like Y55X invariably lead to TIOD and thus allowthe establishment of more consistent genotype/phenotypecorrelations. Accordingly it is plausible to suggest that Y55Xmutation detected in this study is phenotypically associatedwith severe TDH and TIOD.While only 15% of sporadic CH is caused by defects inthyroid hormone synthesis, most of the familial cases resultfrom thyroid dyshormonogenesis inherited autosomal reces-sively. We previously reported  TPO  mutations as the mostcommon cause of familial TDH (24) and stated that  TPO mutation analysis should take a major place in determiningthe aetiology the disease in such cases. Since mutations inother TDH genes such as  DUOX2, DUOXA2, TG, IYG  aremore likely to be associated with PIOD, screening for  TPO mutations should be the first line of investigation especially incases with TIOD. A prior linkage analysis to the  TPO  locus infamilial cases would further ensure if mutation analysis wouldbe feasible.In summary, with this study we showed that TDH in ourcases was likely to be caused by Y55X mutation. Thismutation is the only exon 2 non-sense mutation reported in theliterature and causes TIOD. Therefore our results make animportant contribution to the understanding of clinicalconsequences associated with  TPO  mutations. It also high-lights the role of genetic investigations in determining theputative aetiology of CH. Acknowledgements We are grateful to the family who agreed to participate inthis study. Declaration of interest This study was funded by Birmingham Children HospitalResearch Foundation. References 1. Medeiros-Neto G, Knobel M, DeGroot LJ. Genetic disorders of the thyroid hormone system. In: Baxter JD, ed. Genetics inendocrinology. Philadelphia: Lippincott Williams & Wilkins;2002:375–402.2. Park SM, Chatterjee VKK. Genetics of congenital hypothyroidism. J Med Genet 2005;42:379–89.3. Castanet M, Polak M, Bonaiti-Pellie C, et al. Nineteen years of national screening for congenital hypothyroidism: familial caseswith thyroid dysgenesis suggest the involvement of genetic factors. J Clin Endocrinol Metab 2001;84:2502–6.4. Kopp P. Genetic defects in the etiology of congenital hypothyroid-ism. Endocrinology 2001;143:2019–24.5. Caputo M, Rivolta CM, Esperante SA, et al. Congenital hypothy-roidism with goitre caused by new mutations in the thyroglobulingene. Clin Endocrinol (Oxf) 2007;67:351–7.6. 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