research article functional analyses of c.2268dup in...

Research ArticleFunctional Analyses of c2268dup in Thyroid Peroxidase GeneAssociated with Goitrous Congenital Hypothyroidism

Ching Chin Lee1 Fatimah Harun2 Muhammad Yazid Jalaludin2 Chor Yin Lim1

Khoon Leong Ng3 and Sarni Mat Junit1

1 Department of Molecular Medicine Faculty of Medicine University of Malaya 50603 Lembah Pantai Kuala Lumpur Malaysia2 Department of Paediatrics Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia3 Department of Surgery Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia

Correspondence should be addressed to Sarni Mat Junit sarniumedumy

Received 3 September 2013 Revised 23 January 2014 Accepted 6 February 2014 Published 17 March 2014

Academic Editor Markus Schuelke

Copyright copy 2014 Ching Chin Lee et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The c2268dup mutation in thyroid peroxidase (TPO) gene was reported to be a founder mutation in Taiwanese patientswith dyshormonogenetic congenital hypothyroidism (CH) The functional impact of the mutation is not well documented Inthis study homozygous c2268dup mutation was detected in two Malaysian-Chinese sisters with goitrous CH Normal andalternatively spliced TPO mRNA transcripts were present in thyroid tissues of the two sisters The abnormal transcript contained34 nucleotides originating from intron 12 The c2268dup is predicted to generate a premature termination codon (PTC) atposition 757 (pGlu757X) Instead of restoring the normal reading frame the alternatively spliced transcript has led to anotherstop codon at position 740 (pAsp739ValfsX740) The two PTCs are located at 116 and 201 nucleotides upstream of the exons 1314junction fulfilling the requirement for a nonsense-mediated mRNA decay (NMD) Quantitative RT-PCR revealed an abundanceof unidentified transcripts believed to be associated with the NMD TPO enzyme activity was not detected in both patients eventhough a faint TPO band of about 80 kD was present In conclusion the c2268dup mutation leads to the formation of normal andalternatively spliced TPO mRNA transcripts with a consequential loss of TPO enzymatic activity in Malaysian-Chinese patientswith goitrous CH

1 Introduction

Abnormalities in thyroid hormone synthesis or thyroiddyshormonogenesis accounts for 10 to 20 cases of congeni-tal hypothyroidism (CH) a common endocrine disorderwithan incidence of 1 in 2370 to 6000 live births worldwide [1ndash3]The remaining 80ndash85 cases of CH are due to thyroid dysge-nesis [4] Thyroid dyshormonogenesis is linked to defects ingenes encoding proteins that are involved in thyroid hormonesynthesis secretion or recycling [5 6] The candidate genesinclude the thyroid peroxidase (TPO) [7 8] dual oxidase2 (DUOX2) [9] solute carrier family 5 or sodium iodidesymporter (SLC5ANIS) [10] and thyroglobulin (Tg) [11] Insome cases CH patients with dyshormonogenesis developdiffuse or nodular goiter at birth [8 12 13] or later in theirlives

TPO gene abnormality is the most common cause ofcongenital dyshormonogenetic hypothyroidism [14] To datemore than 60 mutations that affect the TPO activity to vary-ing extents have been described [15] Among the mutationsthe c2268dup (c2268 insT) in exon 13 of the TPO geneis a common cause of thyroid dyshormonogenesis amongpatients in Taiwan and China [16ndash18] Ancestors of thestudied subjects were believed to have originated from thesouthern China [16]

Human TPO is a 110 kDA membrane-bound glycosy-lated heme-containing protein that catalyzes the iodinationof thyroglobulin and the coupling of iodotyrosyl residues togenerate functionally active thyroid hormones T3 and T4The single gene encoding TPO is located on chromosome2p25 which spans at least 150 kb and contains 17 exons [19]The full length of theTPOmRNA transcript (TPO1) is 3152 bp

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 370538 11 pageshttpdxdoiorg1011552014370538

2 BioMed Research International

(GenBank accession numberNM 0005475) in size and codesfor a protein consisting of 933 amino acids [20ndash23]

Apart from TPO1 seven different TPO transcripts whichare generated through alternative splicing had also beendiscovered Differential splicing generates shorter transcriptsTPO2 TPO3 TPO4 and TPO5 lacking exons 10 16 14 and8 respectively [24ndash26] In addition three other multisplicedtranscripts such as TPO23 (lacking exons 10 and 16) TPO24(lacking exon 10 and 14) and TPO6 that lacks exons 10 12ndash14 and 16 had also been described [26] Among these eighttypes of TPO transcripts only TPO1 TPO3 and TPO4 areexpected to produce enzymatically active TPO protein andcan therefore be expected to play important roles in thyroidhormone synthesis [26 27] TPO2 which is lacking in exon10 leads to a production of TPO protein which does not haveany enzymatic activity and is rapidly degraded after synthesis[24] TPO5 lacks exon 8 which has been suggested to code fora site that participates in catalytic mechanism The proteintranslated from TPO5 is also unable to acquire a properthree dimensional configuration and reach the cell surfaceIn contrast TPO4 which is lacking in exon 14 is able to foldcorrectly and reach the cell surface However this isoformhasa shorter half-life as compared to TPO1 [26]

The c2268dup mutation was predicted to cause a frameshift that leads to a stop signal after the insertion point[16] The sequence is then hypothesized to translate into atruncated protein of 756 amino acids that lack the entiretransmembrane and intracellular domain which are encodedby exon 15 to exon 17 causing impairment to the expressionat the apical membrane [7 28] The actual effects of themutation on the TPO mRNA transcript and the subsequentprotein function remain unclear In this study we charac-terized the c2268dup mutation in two Malaysian-Chinesesisters with goitrous congenital hypothyroidismThe possibleeffects of the mutation to the TPO mRNA splicing activityprotein function and expression were also investigated

2 Subjects and Methods

21 Subjects and Family Members Two sisters of Malaysian-Chinese origin were diagnosed with CH during infancyThe proband III-2 was detected to have CH by neonatalscreening and the diagnosis was confirmed at 10 days afterbirth with serum TSH of 1200120583IUmL (Normal range03ndash50 120583IUmL) and free T

4of 50 pmolL (Normal range

20ndash68 pmolL) Her elder sister III-1 was also detectedto have CH (but not by neonatal TSH screening as itwas not available) at 3 weeks of life with serum TSHof 965120583IUmL and free T

4of 20 pmolL when she pre-

sented with prolonged jaundice Both sisters received thy-roid replacement with L-thyroxine Technetium-99m thyroidscintigraphy was performed when they were 3 years oldwhen L-thyroxine was temporarily stopped for 6 weeks andthe thyroid function test was repeated The thyroid scanrevealed the presence of thyroid glands despite having a risein TSH and low free T

4 In their late teenage years they

developed goiterThyroid ultrasonography revealed that bothof them had multinodular goiter (MNG) Both sisters had

elevated levels of thyroglobulin as the goiters progressedIII-2rsquos thyroglobulin (hTG) level measured when she was20 years old was 193 ngmL (reference range 0ndash55 ngmL)whilst her elder sisterrsquos was much higher at 2288 ngmLThe proband and her sister (III-1) subsequently underwenttotal thyroidectomy at the age of 20 years and 245 yearsrespectively Histology of their thyroid glands revealed thepresence of multiple macro- and microfollicular adenomataarising in a background of dyshormonogenetic goiter Theelder sister had a small ventricular septal defect while theproband had no other congenital anomalies and both ofthemhad normal growth and developmentTheir parents (II-1 and II-2) who are biologically unrelated were all healthyand euthyroid with no congenital anomalies However theirmaternal grandmother (I-4) was reported to have goiterand had subsequently undergone thyroidectomy with thehistology results unknown Written informed consent wasobtained from the parents for their blood and their childrenrsquos(III-1 and III-2) blood and thyroid tissues for TPO geneanalysisThis study was approved by the University ofMalayaMedical Centre (UMMC) Ethical Committee (InstitutionalReview Board) in accordance with the ICH-GCP guidelineand the Declaration of Helsinki (Reference number 65416)

22 Screening for c2268dup Mutation and Other PotentialNucleotide Alterations in the TPO Gene in the Proband HerSister and the FamilyMembers GenomicDNAwas extractedfrom peripheral venous blood from the proband the affectedsister and their healthy family members using QIAampDNA Blood Mini Kit (Qiagen Germany) according to themanufacturerrsquos protocol The TPO gene was PCR-amplifiedwith flanking intronic primers covering all the 17 exons [1729] A PCR reaction mixture (50 120583L) containing 100ndash250 ngwas prepared in the presence of a final concentration of 1XTaq buffer with KCl 25mM of MgCl 200120583M of dNTP 8dimethyl sulfoxide (DMSO) 1 unit of Taq DNA polymerase(Fermentas USA) and 20 pmol of each forward and reverseprimers Thirty-five cycles of amplification were carried outwith standard PCR protocol at annealing temperature of 55∘Cfor all coding exons for 30 seconds The PCR products werepurified using QIAquick PCR purification kit (Qiagen Ger-many) and then sequenced using ABI PrismGene SequencerModel 3100 Version 37 (Research Biolabs Singapore)

23 In Silico Analysis of the Potential Impact of the c2268dupon Splicing Activity Using HSF Algorithm TheHuman Splic-ing Finder (httpwwwumdbeHSF) [30] was used toanalyze the existence of alternative splice sites in exon 13 ofthe TPO gene as a consequence of the c2268dup mutationThe analysis was done on the whole of exon 13 (171 bp) 50nucleotides upstream of exon 13 (31015840 end of intron 12) and 50nucleotides downstream of exon 13 (51015840 end of intron 13)

24 Detection of Alternatively Spliced TPO mRNA TranscriptAssociated with the c2268dup Mutation Following thy-roidectomy tissue samples were immediately kept frozen indry ice and then stored atminus80∘CA piece of the pathologically

BioMed Research International 3

normal tissue was taken near the colloid nodules and theabnormal tissue was taken from the thyroid lesion Tumorswere diagnosed histopathologically andno evidence ofmalig-nancy was detected Each piece of tissue sample of about30mg was homogenized and total cellular RNA (tcRNA)was then extracted from the normal and lesionic areas ofthe thyroid tissues using RNeasy Mini (Qiagen Germany)according to the manufacturerrsquos instruction The extractedtcRNA was treated with DNase (Ambion UK) and thenreverse transcribed to complimentary DNA (cDNA) usingthe High Capacity RNA-to-cDNA Kit (Applied BiosystemsUSA)The cDNAwas then used as a template to PCR-amplifythe coding exons (exons 2ndash17) using overlapping primersthat cover the exon-exon junctions (see SupplementaryTable 1 (S1) in the Supplementary Material available onlineat httpdxdoiorg1011552014370538) PCR amplificationand the subsequent PCRproduct purification and sequencingwere performed as described earlier except that cDNA wasused as a template instead of genomic DNA ComplementaryDNA samples prepared from thyroid tissue taken from threeindividuals without the c2268dup mutation were used aspositive controls

25 Quantitation of Normal and Abnormally Spliced TPOmRNA Transcripts in Thyroid Tissues from the Proband andHer Sister Quantitative real time PCR (qRT-PCR) was usedto analyze TPO gene expression in the normal and lesionareas of the thyroid tissues of the proband and her sisterComparison of the TPO gene expression in thyroid tissuesof the proband and her sister was also made The expressionof the TPO gene was normalized to that of a housekeepinggene the TATA box-binding protein (TBP) In addition theratio of TPO mRNA variants in c2268dup mutation-freeindividuals was also calculated and compared to the twosisters Total TPO mRNA transcripts were analyzed usingE4F and E5R primer pair covering a region from exons 4to 5 Primer pairs spanning exons 12 to 13 (E1213F andE13R) were designed to target normally spliced transcripts(TPO1 TPO2 TPO3 TPO4 TPO5 TPO23 and TPO24)Meanwhile the E12I12F and E13R primer pair was usedto target alternatively spliced-derived transcripts with aninsertion of 34 bp between exon 12 and exon 13 TPO6 thatdoes not contain exon 12 and exon 13 was amplified using E11and E1115 primers All qRT-PCR oligonucleotide sequencesare listed in Supplementary Table 2 (S2)The qRT-PCR exper-iments were performed on a StepOnePlus system (Perkin-ElmerApplied Biosystems) The amplification reaction wascarried out using 1X SYBR Green PCR Mastermix (AppliedBiosystems USA) containing 10 ng of cDNA and 200 nMof each primer The sample was initially denatured at 95∘Cfor 5min followed by 40 cycles of denaturation at 95∘Cfor 15 sec and subsequent annealing and elongation step at59∘C for 1min Melting curves were performed to check thespecificity of the amplicons Each experiment was carriedout in triplicate Comparison of total or target gene mRNAexpression was done by calculating the differences in thethreshold cycles

The fold change in expression between tissue samples wascalculated by

Fold Change = 2minusΔΔCt (1)

where ΔΔCt = (Ct targeted TPO mRNA minus Ct TBP) lesionic area minus(Ct targeted TPO mRNA minus Ct TBP) normal area

The fold change in expression between patients wascalculated by


where ΔΔCt = (Ct targeted TPO mRNA minus Ct TBP) proband minus(Ct targeted TPO mRNA minus Ct TBP) probandrsquos sister

Total 10 ng of cDNA sample from normal and lesionicareas of thyroid tissue (5 ng template from each area) of eachpatient was used as the template

Meanwhile the ratio of TPOmRNAvariants to total TPOmRNA in each tissue types was determined by the followingcalculation

For normal area of thyroid tissue

Targeted TPO mRNA speciesnormal areatotal TPO mRNAnormal area

= 2minusΔCt (3)

For lesionic area of thyroid tissue

Targeted TPO mRNA specieslesion areatotal TPO mRNAlesion area

= 2minusΔCt (4)

26 Microsomal Protein Isolation from Thyroid TissuesMicrosomal protein was isolated from the frozen thyroid tis-sue specimens Thirty milligram of tissue was homogenizedin ice-cold phosphate buffered saline (PBS) containing PierceProtease Inhibitors Cocktail (Thermo Scientific USA) andsubsequently centrifuged for 15min at 3000timesg at 4∘C toremove nuclei and cytoskeletal componentsThe supernatantwas then ultracentrifuged in the Optima L-100k (BeckmanCoulter USA) at 100000timesg for 2 h at 4∘C The pellet wascollected and then dissolved in lysis buffer (50mMTris-HClpH 73 150mMNaCl 5mMEDTA 10 glycerol (wv) 1Triton X-100 (wv) protease inhibitors) [31]Themixture wasultrasonicated using Power Sonic 405 (Hwashin TechnologyKorea) to improve the yield of the extracted protein

27 Immunodetection of TPO Protein Immunodetectionanalysis was performed using mouse monoclonal MoAb47primary antibody (Abcam UK) and WesternDot 625 West-ern blot kits (Invitrogen USA) to detect the presence ofTPO protein in the tissue specimens from both patientsTwenty 120583g ofmicrosomal fraction extract for each sample and300 ng of a positive control (recombinant fragment of humanthyroid peroxidase fromAbcam product code ab73765 UK)were used in this analysis The expression of TPO in normaland lesion areas of thyroid tissue from both patients werequantitated by densitometry scanning using Image-J software(httprsbwebnihgovij) The expression of TPO in normaland lesionic areas of the thyroid tissues was normalized to theexpression of beta actin The total expression of TPO proteinin the proband was also compared to that of her sister


NA NANA

1 2 3 4

1 2

1 2

(I)

(II)

(III)

Male

Female

c 2268dup homozygous

c2268dup heterozygous

Goiter

NA Not available

TSH 1200 120583IUmLTSH 965 120583IUmLFree 50pmolL

lowast

lowast

lowastlowast

Free 20pmolL T4T4

Figure 1 Family pedigree of the index patient A family pedigree of twoMalaysian sisters with congenital hypothyroidism and multinodulargoiter The proband is indicated by the arrow

28 Guaiacol Oxidation Assay Microsomal fraction extractsprepared from patientsrsquo thyroid gland tissues were immedi-ately used for enzymatic assay Microsomes prepared fromthyroid tissues from rats were extracted using the samemethod and were then used as a positive control Twenty 120583gof each microsomal fraction were added to 1mL of reactionmixture containing 40mM guaiacol (Sigma-Aldrich USA)and 67mM sodium phosphate buffer pH 75 The enzy-matic reaction was initiated by adding final concentrationof 025mMH

2O2 The absorbance was measured at 470 nm

(OD470

) at every second for 3min at room temperature

3 Results

31 Mutational Screening of the TPO Gene in the Proband andHer Family Members PCR amplification followed by DNAsequencing of the TPO gene in the proband III-2 revealed ahomozygous c2268dup mutation in exon 13The mutation ispredicted to create a premature termination codon at position757 (pGlu757x) Further DNA sequencing analysis revealedthat her affected sister III-1 was also homozygous for thesame mutation On the other hand both parents (II-1 and II-2) and the probandrsquos maternal grandmother (I-4) were foundto be heterozygous for the mutation A family tree of thepatient showing the mode of inheritance of the mutation isshown in Figure 1

32 HSF Analysis of the Potential Impact of the c2268dupMutation on Splicing Activity in Exon 13 HSF analysis

predicted that apart from the natural acceptor and donorsplice sites there are an additional 18 potential acceptor splicesites and 7 potential donor splice sites in exon 13 and theadjacent introns 12 and 13 (50 nucleotides each) of the TPOgene (data not shown) The TPO exon 13 has a strong donorsplice site with a consensus value of 9402 and a weakeracceptor site with a consensus value of 7612 (Table 1(a))A potential cryptic acceptor splice site exists at nucleotideposition minus46 with a stronger score than that of the wild type(8399 versus 7612) The mutation which is located closer tothe acceptor site than to the donor site is predicted by the ESEfinder software to disrupt binding sites for three regulatory SRproteins SRp40 SF2ASF and SRp55 at c2267 c2268 andc2269 respectively (Table 1(b)) In addition ESE motif fromHSF algorithm suggested that the c2268dupmutation createsa new 9G8 protein binding sites at c2268 (Table 1(c))

33 Detection of Alternatively Spliced TPO mRNA TranscriptAssociated with the c2268dup Mutation RT-PCR analysisof the TPO mRNA transcripts in thyroid tissue of theproband showed two amplicons for exons 9 to 12 (499 bpand 328 bp) exons 12 to 13 (300 bp and 350 bp) exons13 to 15 (250 bp and 118 bp) and exons 15 to 17 (261 bpand 131 bp) (Figures 2(a) and 2(b)) Sequencing analysisof these RT-PCR fragments confirmed that the extra banddetected in exons 9 to 12 exons 13 to 15 and exons 15 to17 correspond to the TPO2 TPO4 and TPO5 transcriptsrespectively (data not shown) Meanwhile the extra banddetected in exons 12 to 13 amplicons contained 34 nucleotides


Table 1 (a) HSF analyses showing a summary of HSF matrices for potential splice sites (b) ESE finder matrices for SRp40 SC35 SF2ASFand SRp55 proteins (c) ESE finder matrices for ESE proteins

(a)

Nucleotide position Splice site type Motif New potential splice site Consensus value (0ndash100)minus46 Acceptor cttttctcgtagtt cttttctcgtagTT 8399 (CT)minus12 Acceptor cacatttcatagAC cacatttcatagAC 7612 (WT)169 Donor AAGgtcagt AAGgtcagt 9402 (WT)CT cryptic site WT wild type

(b)

Nucleotide position cDNA position Linked SR protein Reference motif Variationlowast

(0ndash100) (0ndash100)

51 c2267 SRp40 TGTGAGG Site broken(7850) (minus100)

52 c2268 SF2ASF GTGAGGA Site broken(7927) (minus100)

53 c2269 SRp55 TGAGGA Site broken(7482) (minus100)

lowastVariation expresses the difference between reference and mutant values Wild-type value is taken as a reference

(c)

Nucleotide position cDNA position Linked ESE protein Reference motif Variationlowast


52 c2268 9G8 GTTGAG New site(6349) (100)


that originate from intron 12 Further analysis showed thatthis unknown TPO variant was detected in all thyroid issuespecimens from both patients (Figure 2(c)) but not in thethree c2268dup mutation-free individuals (Figure 2(d))Thesequence of this novel transcript matched with that of thecryptic acceptor splice (cttttctcgtagTT) in exon 13 (Table 1(a))that was predicted earlier from the HSF analysis Apart fromthe c2268dup no other nucleotide alterations were detectedin intron 12 and exon 13 of the proband (III-2) and her sister(III-1)

A schematic diagram shows the position of the c2268dupin genomicDNA (Figure 3(a)) and the possible consequencesof the mutation on mRNA splicing (Figures 3(b) and 3(c))and translation (Figures 3(d) and 3(e)) As presented by thedotted line in Figure 3(a) the c2268dup is located at +52nucleotides (nt) from the exon 12exon 13 junction and 116 ntupstream of the exon 13exon 14 junctionThe c2268dup canbe predicted to induce a frameshift in the normally splicedproduct and the appearance of a premature terminationcodon (PTC) at position 757 pGlu757X (Figure 3(d)) On theother hand alternative splicing generating inclusion of 34 ntof intron 12 at the beginning of exon 13 can also be predictedto lead to a frameshift and appearance of PTC at position740 (pAsp739ValfsX740) In silico analysis using PolyPhen-2 revealed that aspartate at codon 739 was not well conserved(data not shown)

34 TPOGene Expression Differences between the Normal andLesion Areas of Thyroid Tissue of the Proband and Her SisterThe expression of ldquoall TPO mRNA transcriptsrdquo in the lesionarea of thyroid tissue of the proband was found to be higherthan the normal area of the thyroid tissue The expressionof normally spliced TPO mRNA transcripts was concurrentwith this result The level of the alternatively spliced TPOmRNA transcripts was also higher in the lesion compared tothe normal area in thyroid tissue of the proband Comparedto the proband the expression of ldquoall TPOmRNA transcriptsrdquoin the lesionic area of thyroid tissue of her affected sister wasfound to be only slightly higher (lt15 fold) than that of thenormal area In addition the total amount of the normallyspliced TPO mRNA transcripts and the alternatively splicedtranscript was lower than the normal area of the thyroidtissue The amount of TPO6 in both patients was not ableto be determined due to a low yield of PCR product Therelative expression level of TPO mRNA transcripts betweenthe normal and lesionic areas of thyroid tissue of proband andher affected sister is shown in Figure 4(a)

35 TPO Gene Expression Difference between the Probandand Her Sister The expression of the TPO gene in III-1 wasfound to be lower than that of the proband regardless of thetype of the TPO transcripts produced However the degreeof downregulation of the known TPO transcripts including


(bp)

(bp)

1000

500

100

1 2 3 4 5 6

803671499

328

300350

(a)

1000

500

100

1 2 3 4 5 6

(bp)

(bp)437

488

118131

261290

(b)

(bp)

(bp)

1000

500

100

1 2 3 4 5 6

300350

(c)

(bp)

(bp)

1000

500

100

1 2 3 4

300

(d)

Figure 2 Agarose gel electrophoresis of reverse-transcription PCR (RT-PCR) products (a)TheRT-PCR products of exons 2 to 13 that includethe exon-exon junctions Abnormal spliced products were not detected in exons 2 to 12 based on the expected PCR products present (lanes 3to 5) However an unknown product of approximately 350 bp was detected for the exons 1213 fragment in addition to the expected productof 300 bp Lane 1 GeneRuler 100 bp DNA Ladder lane 2 negative control lane 3 exons 2 to 7 671 bp lane 4 exons 7 to 9 803 bp lane 5 exons9 to 12 499 bp and 328 bp lane 6 exons 12 to 13 300 bp and an unknown product of 350 bp (b) The RT-PCR products of exons 7 to 9 andexons 13 to 17 that include the exon-exon junctions Abnormal spliced products were not detected in exons 7 to 9 and exons 13 to 17 basedon the expected PCR products present (lanes 3 to 6) Lane 1 GeneRuler 100 bp DNA Ladder lane 2 negative control lane 3 exons 13 to 15250 bp and 118 bp lane 4 exons 15 to 17 261 bp and 131 bp lane 5 exons 7 to 8 488 bp lane 6 exons 8 to 9 437 bp (c) The RT-PCR productsof exons 12 to 13 that include the exon-exon junction An unknown product of approximately 350 bp and an expected product of 300 bp weredetected in all thyroid tissue specimens from proband and III-1 with c2268dup mutation Lane 1 GeneRuler 100 bp DNA Ladder lane 2normal area of thyroid tissue of III-1 lane 3 lesion area of thyroid tissue of III-1 lane 4 normal area of thyroid tissue of proband lane 5 lesionarea of thyroid tissue of proband lane 6 negative control (d)The RT-PCR products of exons 12 to 13 that include the exon-exon junction Anexpected product of 300 bp was detected in all c2268dup-mutation free individuals Lane 1 GeneRuler 100 bp DNA Ladder lane 2 ControlI lane 3 Control II lane 4 Control III

TPO1 TPO2 TPO3 TPO4 TPO5 TPO23 and TPO24with (minus424 plusmn 041) or without normal splicing (minus394 plusmn031) is greater than that of the ldquoall TPO mRNA transcriptsrdquo(minus289plusmn011)The relative expression level of the TPOmRNAtranscripts between the proband and her sister is shown inFigure 4(b)

36 Quantification of TPO mRNA Variants The expressionratio of TPO1 TPO2 TPO3 TPO4 TPO5 TPO23 andTPO24 with or without the 34 nt insertion between exon12 and exon 13 to the ldquoall TPO mRNA transcriptsrdquo in bothnormal and lesionic areas of thyroid tissue of the probandwas similar The percentage of TPO1 TPO2 TPO3 TPO4TPO5 TPO23 and TPO24 in normal and lesionic areas was4033 plusmn 341 and 3994 plusmn 273 respectively Meanwhile

the percentage of the alternatively spliced transcript with theinsertion of 34 nt was 396 plusmn 074 in the normal area and377 plusmn 021 in the area of lesion Overall the expressionof all known TPO mRNA transcripts with or without the34 nt insertion in normal area was slightly higher than thatof the lesionic area About 5571 and 5629 of TPOmRNAtranscript in both normal and lesion areas of thyroid tissueof proband was unidentified (unknown TPO transcript)The normally spliced mutant TPO mRNA transcript gavehigher level of expression than the alternatively spliced TPOtranscript in the ratio of 99 1 in normal area and 95 1 inlesionic area

Compared to the proband the expression ratio of TPO1TPO2 TPO3 TPO4 TPO5 TPO23 and TPO24 with orwithout insertion of 34 bp between exon 12 and exon 13 to


Intron 12 Exon 12 Exon 13 Intron 13

116nt52 nt

c2268dup (pGlu756X)

(a)

Exon 13Exon 12

(b)

Exon 12 Exon 13

34nt

(c)

c2268dup

Exon 13 (c2216)Exon 12 (c2215)

XQ D H C

756aa

middot middot middotmiddot middot middot

CUCAAG AC CACUGUUGA

(d)

c2268dup

739aa

Exon 12 (c2215)

XQ V

Exon 13 (c2216)

pAsp739ValfsX740

201 nt

Intron 12 (c2216-34)

c2216-1 CUCAAG

middot middot middot

AU AC UUGA uuugacuacaugucaaccuguccacauuucauag

(e)

Figure 3 The location and the predicted effects of the c2268dup mutation A schematic diagram shows the position of the c2268dup ingenomic DNA (a) and the possible consequences of the mutation on mRNA splicing (b c) and translation (d e) Exon sequences are inuppercase intron sequences are in lower case letters Deduced protein sequence is shown in one letter code First in-frame stop codon isrepresented by a capital letter ldquoXrdquo Source httpwwwncbinlmnihgovnuccoreNM 0005475 (NCBI Reference Sequence NM 0005475)

total TPO transcripts in III-1 normal area of thyroid tissuewas higher than lesion area of the thyroid tissueThe percent-age of normally spliced TPO1 TPO2 TPO3 TPO4 TPO5TPO23 and TPO24 in the normal area was 4345 plusmn 1059whereas in lesionic area it was only 3041plusmn538Meanwhilethe percentage of alternative spliced transcripts in normal andlesionic area was 410 plusmn 068 and 244 plusmn 028 respectivelyFifty-two percent and 67 of TPO transcripts in the normaland lesionic areas of thyroid tissue respectively belonged tothe unknown TPO transcript The normally spliced mutantTPO transcripts gave higher level of expression than thealternatively spliced TPO transcript in the ratio of 106 1 innormal area of thyroid tissue and 125 1 in lesionic area ofthyroid tissue

In contrast the ldquoall TPO mRNA transcriptsrdquo in thec2268dup mutation-free individuals comprised at least729 of TPO1 TPO2 TPO3 TPO4 TPO5 TPO23 andTPO24 TPO6 and alternative spliced transcripts with inser-tion of 34 bpbetween exon 12 and exon 13were either detectedin small amount (lt01) or not detected The amount ofunknown TPO transcripts in the mutation free individualswas estimated in the range of 145 to 27 The sum () ofall examined TPO variants in c2268dupmutation in patientsand mutation-free individuals is shown in Figure 4(c)

37 Western Blotting and Guaiacol Oxidation Assay of theMutant TPO Protein (pGlu757X) Western blot analysisusing theMoAb47 primary antibody revealed that themicro-somal protein extract from thyroid tissues of both patientscontained a faint band of about 80 kDa (Figure 5 lanes 2 to 7)The recombinant fragment of human TPO (positive control)gave a band of 101 kDa (Figure 5 lane 8) Densitometricevaluation of the blots using Image-J algorithm showed thatthere was a lower expression of TPO protein in the lesionarea compared to the normal area of the thyroid tissues Theexpression level of the TPO protein found in the lesion area

of thyroid tissue of the proband and her sister (III-1) was136-fold and 22-fold lower compared to the normal area oftheir own thyroid tissue Besides the amount of TPO proteinfound in the thyroid tissue of III-1 was significantly lower(675 fold) if compared to that of the proband No peroxidaseactivity was detected in microsomal protein extracted fromthyroid tissues of both patients (data not shown)

4 Discussion

TPO abnormality is the most common cause of congenitaldyshormonogenetic hypothyroidism [14] where more than60mutations in theTPOgene that affect TPOactivity to vary-ing extents have been described [15] Among the mutationsthe c2268dup nonsense mutation in exon 13 of the TPO genehad been reported to be common amongst dyshormono-genetic Taiwanese patients with CH with evidence of afounder effect [16 17] The Taiwanese CH patients originatedfrom the same area of Southern Mainland China [17] Inthis present study we identified a homozygous c2268dupmutation in the TPO gene in two sisters from Malaysian-Chinese family with CH with development of goiters laterin their lives Earlier mutational screening excluded thyroidsynthesis-related genes the TSHR and FOXE1 to be the causeof CH and goiter in these two patients [32 33] Both parentsand the maternal grandmother are all heterozygous for thesame mutation

Studies have suggested that mutations that generate aPTC can disrupt the reading frame and thus result inthe production of alternatively spliced mRNAs through anonsense-associated altered splicing (NAS) mechanism [34]Other studies have shown that abnormal splicing mechanismcan also be triggered as a result of disruption of ESE-binding site sequence by a mutation [35 36] In this studythe alternatively spliced TPO transcript with an insertionof 34 bp between exon 12 and exon 13 is postulated to


1 1 1 1 11

005

115

225

3

Normal areaLesion area

All TPO transcripts

Proband

Fold

chan

ge

Alternatively spliced transcripts with

insertion of 34 bpbetween exon 12

and exon 13

Normally spliced transcripts including

TPO4 TPO5 TPO23TPO1 TPO2 TPO3

and TPO24

III-1 Proband III-1 Proband III-1

minus25

minus05

minus15minus2

minus1

218 plusmn 013

141 plusmn 012172 plusmn 023 172 plusmn 023

minus181 plusmn 028minus174 plusmn 024

(a)

All TPO transcripts

Normally spliced

transcripts including

TPO4 TPO5 TPO23

TPO1 TPO2 TPO3

and TPO24

100 100 100

ProbandIII-1

Alternatively spliced transcripts with insertion of 34 bp between

exon 12 and exon 13

minus289 plusmn 011

minus424 plusmn 041minus394 plusmn 031minus5

minus4

minus3

minus2

minus1

0

1

2

Fold

chan

ge

(b)

Control 3 lesion area



III-1 lesion area

III-1 normal area

Proband lesion area

Proband normal area

0 20()

40 60 80 100

TPO1 TPO2 TPO3 TPO4 TPO5 TPO23

TPO6

Novel alternative splicing derived TPO transcript

Others

and TPO24 transcripts

(c)

Figure 4 Quantitative real time PCR analysis Comparison of the TPO gene expression level between the (a) normal and lesion areas ofthyroid tissue of proband and III-1 (b) TPO gene expression between proband and III-1 and (c) sum () of examined TPO variants inthyroid tissue of proband III-1 and controls I II and III

be generated as a result of the interruption of the ESE-binding site sequence in exon 13 of the TPO gene and hasabolished the binding motif of three SR proteins SRp40SF2ASF and SRp55 which are required to strengthen theuse of the wild-type (WT) splice site The alternative splicingmechanism triggered by the c2268dup has unmasked oneof the potential acceptor splice sites by the substitution ofthe natural acceptor splice site with a sequence motif ofcacatttcatagAC located at minus12 with an alternative acceptorsplice site with a motif of cttttctcgtagTT located at positionminus46 generating a sequence of 51015840 sdot sdot sdotAAGTTsdot sdot sdot 31015840 insteadof 51015840 sdot sdot sdotAAGACsdot sdot sdot 31015840 at the spliced junction Sometimesthe alternative splicing mechanism might help to restore

the normal reading frame disrupted by a nonsense mutation[12 37] However the alternatively spliced mutant TPOtranscript that was generated in this study did not restorethe normal reading frame but indeed has led to another stopcodon after the 739th amino acid Despite the activation ofa potential splice site in exon 13 the natural acceptor siteremains favorable for splicing activity

In accordance with the mRNA surveillance mecha-nism the majority of nonsense-associated transcripts will bedegraded via endogenous nonsense-mediated mRNA decay(NMD) However studies showed that only PTCs that arelocated at 50ndash55 nucleotides upstream of an exon-exonjunction mediate the mRNA decay The activation of NMD

BioMed Research International 9(k

Da)

170130

95725543

34

26

17

TPO 101 kDaTPO 80kDa

81 2 3 4 5 6 7

120573-actin 42kDa

Figure 5 Western blot and Image-J densitometric analyses Micro-somal protein samples were first tested with anti-TPO antibody fol-lowed by anti-beta actin antibody (loading control) Equal amountof microsomal protein (20 120583g) for each sample (100 ng for thepositive control) was used Lane 1 PageRuler prestained proteinladder lane 2 normal area of thyroid tissue of probandrsquos sister (III-1) lane 3 lesion area of thyroid tissue of probandrsquos sister (III-1) lane4 normal area of thyroid tissue of proband lane 5 lesion area ofthyroid tissue of proband lane 6 normal and lesion areas (10 120583geach) of thyroid tissue of probandrsquos sister (III-1) lane 7 normaland lesion areas (10 120583g each) of thyroid tissue of proband (III-2)lane 8 recombinant fragment of human thyroid peroxidase (positivecontrol)

is believed to limit the expression of abnormal transcriptand thus protect the cell from possible harmful effect ofthe encoded truncated protein [38ndash42] In this study thetwo PTCs caused by the c2268dup mutation fulfill therequirement for the mRNA decay where they are locatedat 116 and 201 nucleotides away from the exon 13exon14 junction Theoretically the expression of normally andalternatively spliced mutant transcripts which was reflectedby the total amount of PCR amplicon of exons 1213 shouldbe similar or close to the expression of the total TPOtranscripts that was represented by the amplicon of exons 45However in this study it was found that the TPO transcriptsthat contained PTCs due to the c2268dup mutation werefound to be far less abundant compared to the total TPOtranscripts Further analysis showed no evidence of theremaining unidentified TPO transcripts belonged to TPO6which does not contain exons 10 12 13 14 and 16 To the bestof our knowledge there is no other similar TPO transcriptthat has been reported so farTherefore the abundant amountof unidentified transcripts is believed to be due to (1) partiallydegraded PTC-containing TPO transcripts or (2) the increasein shorter alternatively spliced TPO isoforms Under bothcircumstances the reduction of functional TPO transcriptscould subsequently lead to insufficient TPO protein transla-tion

In general the amount of the unidentified TPO tran-scripts in the lesion area of thyroid tissues of both patientswas higher than that of the normal area The expression ofTPO protein in the lesion area of thyroid tissues of bothpatients was significantly lower than that of the normal areasuggesting that the abundant unidentified TPO transcriptswere probably degraded and did not get translated consistent

with the hypothesized mRNA surveillance mechanism Thedownregulation of TPO expression in lesion area of thyroidtissue and disruption of TPO mRNA maturation has beensuggested to be associated with malignant neoplasm [43]Interestingly when comparison ismade between the probandand her elder sister III-1 the expression of all known TPOtranscripts and the corresponding protein was significantlyhigher in the former than the latter suggesting that theseverity of NMD increases with age The lower expressionof TPO in these two patients may increase the risk of theirbenign lesion developing into a carcinoma if the thyroidgland is not removed

In the early part of this study a smaller size TPO proteinin native form was successfully detected in both patientsusingMoAb47 antibodies which recognizes an epitope that islocated in-between amino acid sequence 713ndash721 [44] How-ever the source of the TPO protein remained unclear sincesimilar size protein can be translated from both normallyspliced and alternatively spliced TPO transcriptsThemutantTPO detected in thyroid cells of proband and her sister didnot show any enzymatic activity despitethe retention of itsimportant functional sites suggesting that the absent regionis not only important as a hinge for insertion to themembranebutmay also play an important role in proper protein folding

5 Conclusion

Thec2268dupmutation leads to the formation of normal andalternatively spliced TPO transcripts with a consequentialloss of TPO protein enzymatic activity in the dyshormono-genetic Malaysian-Chinese patients with CH

Conflict of Interests

The authors declare that no conflict of interests exists

Acknowledgments

This project was financially supported by Grants from theMinistry of Higher Education Malaysia (FP0502010B)University of Malaya Research Grant (RG42012HTM) andPostgraduate Research Fund University of Malaya (PV116-2012A)

References

[1] J-E Toublanc ldquoComparison of epidemiological data on con-genital hypothyroidism in Europe with those of other parts inthe worldrdquo Hormone Research vol 38 no 5-6 pp 230ndash2351992

[2] K B Harris and K A Pass ldquoIncrease in congenital hypothy-roidism in New York State and in the United StatesrdquoMolecularGenetics and Metabolism vol 91 no 3 pp 268ndash277 2007

[3] L LWu B S Sazali N Adeeb and B A K Khalid ldquoCongenitalhypothyroid screening using cord blood TSHrdquo Singapore Med-ical Journal vol 40 no 1 pp 23ndash26 1999

[4] M Castanet M Polak C Bonaıti-Pellie S Lyonnet P Czer-nichow and J Leger ldquoNineteen years of national screeningfor congenital hypothyroidism familial cases with thyroid


dysgenesis suggest the involvement of genetic factorsrdquo Journalof Clinical Endocrinology and Metabolism vol 86 no 5 pp2009ndash2014 2001

[5] J J M de Vijlder C Ris-Stalpers and T Vulsma ldquoInborn errorsof thyroid hormone biosynthesisrdquo Experimental and ClinicalEndocrinology and Diabetes vol 105 supplement 4 pp 32ndash371997

[6] P Kopp ldquoPerspective genetic defects in the etiology of congen-ital hypothyroidismrdquo Endocrinology vol 143 no 6 pp 2019ndash2024 2002

[7] J Deladoey N Pfarr J-M Vuissoz et al ldquoPseudodominantinheritance of goitrous congenital hypothyroidism caused byTPO mutations molecular and in silico studiesrdquo Journal ofClinical Endocrinology and Metabolism vol 93 no 2 pp 627ndash633 2008

[8] C C Lee F Harun M Y Jalaludin C H Heh R Othman andS Mat Junit ldquoA novel homozygous c 1502TgtG (pVal501Gly)mutation in the thyroid peroxidase gene in Malaysian sisterswith congenital hypothyroidismandmultinodular goiterrdquo Inter-national Journal of Endocrinology vol 2013 Article ID 9871867 pages 2013

[9] V Varela C M Rivolta S A Esperante L Gruneiro-Papendieck A Chiesa and H M Targovnik ldquoThree muta-tions (pQ36H pG418fsX482 and gIVS19-2AgtC) in the dualoxidase 2 gene responsible for congenital goiter and iodideorganification defectrdquoClinical Chemistry vol 52 no 2 pp 182ndash191 2006

[10] J Pohlenz and S Refetoff ldquoMutations in the sodiumiodidesymporter (NIS) gene as a cause for iodide transport defects andcongenital hypothyroidismrdquo Biochimie vol 81 no 5 pp 469ndash476 1999

[11] T Ieiri P Cochaux H M Targovnik et al ldquoA 31015840 splice sitemutation in the thyroglobulin gene responsible for congenitalgoiter with hypothyroidismrdquo Journal of Clinical Investigationvol 88 no 6 pp 1901ndash1905 1991

[12] M J Abramowicz H M Targovnik V Varela et al ldquoIdentifica-tion of a mutation in the coding sequence of the human thyroidperoxidase gene causing congenital goiterrdquo Journal of ClinicalInvestigation vol 90 no 4 pp 1200ndash1204 1992

[13] G Medeiros-Neto H M Targovnik and G Vassart ldquoDefec-tive thyroglobulin synthesis and secretion causing goiter andhypothyroidismrdquo Endocrine Reviews vol 14 no 2 pp 165ndash1831993

[14] M Avbelj H Tahirovic M Debeljak et al ldquoHigh prevelanceif thyroid peroxidase gene mutations in patients with thyroiddyshormonogenesisrdquo European Journal of Endocrinology vol156 no 5 pp 511ndash519 2007

[15] C Ris-Stalpers and H Bikker ldquoGenetics and phenomics ofhypothyroidism and goiter due to TPO mutationsrdquo Molecularand Cellular Endocrinology vol 322 no 1-2 pp 38ndash43 2010

[16] D-M Niu B Hwang Y-K Chu C-J Liao P-L Wang andC-Y Lin ldquoHigh prevalence of a novel mutation (2268 insT)of the thyroid peroxidase gene in Taiwanese patients with totaliodide organification defect and evidence for a founder effectrdquoJournal of Clinical Endocrinology and Metabolism vol 87 no 9pp 4208ndash4212 2002

[17] J-Y Wu S-G Shu C-F Yang C-C Lee and F-J TsaildquoMutation analysis of thyroid peroxidase gene in Chinesepatients with total iodide organification defect identification offive novel mutationsrdquo Journal of Endocrinology vol 172 no 3pp 627ndash635 2002

[18] H Li Y Liu W Wu and S Li ldquoMutation analysis of thyroidperoxidase gene in a Chinese family with congenital hypothy-roidismrdquo Chinese Journal of Birth Health amp Heredity no 5 pp36ndash37 2011

[19] Y Endo S Onogi K Umeki et al ldquoRegional localization of thegene for thyroid peroxidase to human chromosome 2p25 andmouse chromosome 12Crdquo Genomics vol 25 no 3 pp 760ndash7611995

[20] S Kimura T Kotani and O W McBride ldquoHuman thyroid per-oxidase complete cDNA and protein sequence chromosomemapping and identification of two alternately spliced mRNAsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 84 no 16 pp 5555ndash5559 1987

[21] F Libert J Ruel M Ludgate et al ldquoThyroperoxidase anauto-antigen with a mosaic structure made of nuclear andmitochondrial gene modulesrdquoThe EMBO Journal vol 6 no 13pp 4193ndash4196 1987

[22] P Seto H Hiray R O Magnusson et al ldquoIsolation of acomplementary DNA clone for thyroid microsomal antigenHomology with the gene for thyroid peroxidaserdquo Journal ofClinical Investigation vol 80 no 4 pp 1205ndash1208 1987

[23] R P Magnusson G D Chazenbalk J Gestautas et al ldquoMolec-ular cloning of the complementary deoxyribonucleic acid forhuman thyroid peroxidaserdquoMolecular Endocrinology vol 1 no11 pp 856ndash861 1987

[24] P Niccoli L Fayadat V Panneels J Lanet and J-L FrancldquoHuman thyroperoxidase in its alternatively spliced form(TPO2) is enzymatically inactive and exhibits changes inintracellular processing and traffickingrdquo Journal of BiologicalChemistry vol 272 no 47 pp 29487ndash29492 1997

[25] E Zanelli M Henry B Charvet and Y Malthiery ldquoEvidencefor an alternative splicing in the thyroperoxidase messengerfrompatients withGravesrsquo diseaserdquoBiochemical and BiophysicalResearch Communications vol 170 no 2 pp 735ndash741 1990

[26] M Ferrand V le Fourn and J-L Franc ldquoIncreasing diversityof human thyroperoxidase generated by alternative splicingcharacterization by molecular cloning of new transcripts withsingle- and multispliced mRNAsrdquo Journal of Biological Chem-istry vol 278 no 6 pp 3793ndash3800 2003

[27] P Niccoli-Sire L Fayadat S Siffroi-Fernandez Y Malthierryand J L Franc ldquoAlternatively spliced form of human thyroper-oxidase TPOzanelli activity intracellular trafficking and rolein hormonogenesisrdquo Biochemistry vol 40 no 8 pp 2572ndash25792001

[28] R Kuliawat J Ramos-Castaneda Y Liu and P Arvan ldquoIntra-cellular trafficking of thyroid peroxidase to the cell surfacerdquoJournal of Biological Chemistry vol 280 no 30 pp 27713ndash27718 2005

[29] C M Rivolta S A Esperante L Gruneiro-Papendieck et alldquoFive novel inactivating mutations in the thyroid peroxidasegene responsible for congenital goiter and iodide organificationdefectrdquo Human Mutation vol 22 no 3 p 259 2003

[30] F-O Desmet D Hamroun M Lalande G Collod-Beroud MClaustres and C Beroud ldquoHuman Splicing Finder an onlinebioinformatics tool to predict splicing signalsrdquo Nucleic AcidsResearch vol 37 no 9 article e67 2009

[31] D O McDonald and S H S Pearce ldquoThyroid peroxi-dase forms thionamide-sensitive homodimers relevance forimmunomodulation of thyroid autoimmunityrdquo Journal ofMolecular Medicine vol 87 no 10 pp 971ndash980 2009


[32] M Musa F Harun and S M Junit ldquoMolecular analysis of TSHreceptor gene in unrelated patients with congenital hypothy-roidismrdquo Malaysian Journal of Biochemistry and MolecularBiology vol 15 no 1 pp 8ndash18 2007

[33] I-N Kang M Musa F Harun and S M Junit ldquoCharacteri-zation of mutations in the FOXE1 gene in a cohort of unrelatedmalaysian patients with congenital hypothyroidism and thyroiddysgenesisrdquo Biochemical Genetics vol 48 no 1-2 pp 141ndash1512010

[34] P A Frischmeyer andH C Dietz ldquoNonsense-mediatedmRNAdecay in health and diseaserdquo Human Molecular Genetics vol 8no 10 pp 1893ndash1900 1999

[35] L Cartegni S L Chew and A R Krainer ldquoListening to silenceand understanding nonsense exonic mutations that affectsplicingrdquo Nature Reviews Genetics vol 3 no 4 pp 285ndash2982002

[36] C R Valentine ldquoThe association of nonsense codons with exonskippingrdquo Mutation ResearchmdashReviews in Mutation Researchvol 411 no 2 pp 87ndash117 1998

[37] M H Ricketts M J Simons and J Parma ldquoA nonsensemutation causes hereditary goitre in the Afrikander cattleand unmasks alternative splicing of thyroglobulin transcriptsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 84 no 10 pp 3181ndash3184 1987

[38] Y-F Chang J S Imam and M F Wilkinson ldquoThe Nonsense-mediated decay RNA surveillance pathwayrdquo Annual Review ofBiochemistry vol 76 pp 51ndash74 2007

[39] J Weischenfeldt J Lykke-Andersen and B Porse ldquoMessengerRNA surveillance neutralizing natural nonsenserdquoCurrent Biol-ogy vol 15 no 14 pp R559ndashR562 2005

[40] L E Maquat ldquoNonsense-mediated mRNA decay in mammalsrdquoJournal of Cell Science vol 118 part 9 pp 1773ndash1776 2005

[41] S Brogna and J Wen ldquoNonsense-mediated mRNA decay(NMD) mechanismsrdquoNature Structural and Molecular Biologyvol 16 no 2 pp 107ndash113 2009

[42] X Sun and L E Maquat ldquomRNA surveillance in mammaliancells the relationship between introns and translation termina-tionrdquo RNA vol 6 no 1 pp 1ndash8 2000

[43] J di Cristofaro M Silvy A Lanteaume M Marcy P Carayonand C de Micco ldquoExpression of tpo mRNA in thyroid tumorsquantitiative PCR analysis and correlation with alterations ofret Braf ras and pax8 genesrdquo Endocrine-Related Cancer vol 13no 2 pp 485ndash495 2006

[44] R Finke P Seto J Ruf P Carayon and B Rapoport ldquoDeter-mination at the molecular level of a B-cell epitope on thyroidperoxidase likely to be associated with autoimmune thyroiddiseaserdquo Journal of Clinical Endocrinology and Metabolism vol73 no 4 pp 919ndash921 1991

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


(GenBank accession numberNM 0005475) in size and codesfor a protein consisting of 933 amino acids [20ndash23]

Apart from TPO1 seven different TPO transcripts whichare generated through alternative splicing had also beendiscovered Differential splicing generates shorter transcriptsTPO2 TPO3 TPO4 and TPO5 lacking exons 10 16 14 and8 respectively [24ndash26] In addition three other multisplicedtranscripts such as TPO23 (lacking exons 10 and 16) TPO24(lacking exon 10 and 14) and TPO6 that lacks exons 10 12ndash14 and 16 had also been described [26] Among these eighttypes of TPO transcripts only TPO1 TPO3 and TPO4 areexpected to produce enzymatically active TPO protein andcan therefore be expected to play important roles in thyroidhormone synthesis [26 27] TPO2 which is lacking in exon10 leads to a production of TPO protein which does not haveany enzymatic activity and is rapidly degraded after synthesis[24] TPO5 lacks exon 8 which has been suggested to code fora site that participates in catalytic mechanism The proteintranslated from TPO5 is also unable to acquire a properthree dimensional configuration and reach the cell surfaceIn contrast TPO4 which is lacking in exon 14 is able to foldcorrectly and reach the cell surface However this isoformhasa shorter half-life as compared to TPO1 [26]

The c2268dup mutation was predicted to cause a frameshift that leads to a stop signal after the insertion point[16] The sequence is then hypothesized to translate into atruncated protein of 756 amino acids that lack the entiretransmembrane and intracellular domain which are encodedby exon 15 to exon 17 causing impairment to the expressionat the apical membrane [7 28] The actual effects of themutation on the TPO mRNA transcript and the subsequentprotein function remain unclear In this study we charac-terized the c2268dup mutation in two Malaysian-Chinesesisters with goitrous congenital hypothyroidismThe possibleeffects of the mutation to the TPO mRNA splicing activityprotein function and expression were also investigated

2 Subjects and Methods

21 Subjects and Family Members Two sisters of Malaysian-Chinese origin were diagnosed with CH during infancyThe proband III-2 was detected to have CH by neonatalscreening and the diagnosis was confirmed at 10 days afterbirth with serum TSH of 1200120583IUmL (Normal range03ndash50 120583IUmL) and free T

4of 50 pmolL (Normal range

20ndash68 pmolL) Her elder sister III-1 was also detectedto have CH (but not by neonatal TSH screening as itwas not available) at 3 weeks of life with serum TSHof 965120583IUmL and free T

4of 20 pmolL when she pre-

sented with prolonged jaundice Both sisters received thy-roid replacement with L-thyroxine Technetium-99m thyroidscintigraphy was performed when they were 3 years oldwhen L-thyroxine was temporarily stopped for 6 weeks andthe thyroid function test was repeated The thyroid scanrevealed the presence of thyroid glands despite having a risein TSH and low free T

4 In their late teenage years they

developed goiterThyroid ultrasonography revealed that bothof them had multinodular goiter (MNG) Both sisters had

elevated levels of thyroglobulin as the goiters progressedIII-2rsquos thyroglobulin (hTG) level measured when she was20 years old was 193 ngmL (reference range 0ndash55 ngmL)whilst her elder sisterrsquos was much higher at 2288 ngmLThe proband and her sister (III-1) subsequently underwenttotal thyroidectomy at the age of 20 years and 245 yearsrespectively Histology of their thyroid glands revealed thepresence of multiple macro- and microfollicular adenomataarising in a background of dyshormonogenetic goiter Theelder sister had a small ventricular septal defect while theproband had no other congenital anomalies and both ofthemhad normal growth and developmentTheir parents (II-1 and II-2) who are biologically unrelated were all healthyand euthyroid with no congenital anomalies However theirmaternal grandmother (I-4) was reported to have goiterand had subsequently undergone thyroidectomy with thehistology results unknown Written informed consent wasobtained from the parents for their blood and their childrenrsquos(III-1 and III-2) blood and thyroid tissues for TPO geneanalysisThis study was approved by the University ofMalayaMedical Centre (UMMC) Ethical Committee (InstitutionalReview Board) in accordance with the ICH-GCP guidelineand the Declaration of Helsinki (Reference number 65416)

22 Screening for c2268dup Mutation and Other PotentialNucleotide Alterations in the TPO Gene in the Proband HerSister and the FamilyMembers GenomicDNAwas extractedfrom peripheral venous blood from the proband the affectedsister and their healthy family members using QIAampDNA Blood Mini Kit (Qiagen Germany) according to themanufacturerrsquos protocol The TPO gene was PCR-amplifiedwith flanking intronic primers covering all the 17 exons [1729] A PCR reaction mixture (50 120583L) containing 100ndash250 ngwas prepared in the presence of a final concentration of 1XTaq buffer with KCl 25mM of MgCl 200120583M of dNTP 8dimethyl sulfoxide (DMSO) 1 unit of Taq DNA polymerase(Fermentas USA) and 20 pmol of each forward and reverseprimers Thirty-five cycles of amplification were carried outwith standard PCR protocol at annealing temperature of 55∘Cfor all coding exons for 30 seconds The PCR products werepurified using QIAquick PCR purification kit (Qiagen Ger-many) and then sequenced using ABI PrismGene SequencerModel 3100 Version 37 (Research Biolabs Singapore)

23 In Silico Analysis of the Potential Impact of the c2268dupon Splicing Activity Using HSF Algorithm TheHuman Splic-ing Finder (httpwwwumdbeHSF) [30] was used toanalyze the existence of alternative splice sites in exon 13 ofthe TPO gene as a consequence of the c2268dup mutationThe analysis was done on the whole of exon 13 (171 bp) 50nucleotides upstream of exon 13 (31015840 end of intron 12) and 50nucleotides downstream of exon 13 (51015840 end of intron 13)

24 Detection of Alternatively Spliced TPO mRNA TranscriptAssociated with the c2268dup Mutation Following thy-roidectomy tissue samples were immediately kept frozen indry ice and then stored atminus80∘CA piece of the pathologically














= 2minusΔCt (3)



= 2minusΔCt (4)




NA NANA

1 2 3 4

1 2

1 2

(I)

(II)

(III)

Male

Female



Goiter

NA Not available


lowast

lowast

lowastlowast

Free 20pmolL T4T4




(OD470


3 Results







(a)


(b)







(c)










(bp)

(bp)

1000

500

100

1 2 3 4 5 6

803671499

328

300350

(a)

1000

500

100

1 2 3 4 5 6

(bp)

(bp)437

488

118131

261290

(b)

(bp)

(bp)

1000

500

100

1 2 3 4 5 6

300350

(c)

(bp)

(bp)

1000

500

100

1 2 3 4

300

(d)








116nt52 nt

c2268dup (pGlu756X)

(a)

Exon 13Exon 12

(b)

Exon 12 Exon 13

34nt

(c)

c2268dup

Exon 13 (c2216)Exon 12 (c2215)

XQ D H C

756aa


CUCAAG AC CACUGUUGA

(d)

c2268dup

739aa

Exon 12 (c2215)

XQ V

Exon 13 (c2216)

pAsp739ValfsX740

201 nt

Intron 12 (c2216-34)

c2216-1 CUCAAG



(e)






4 Discussion




1 1 1 1 11

005

115

225

3


All TPO transcripts

Proband

Fold

chan

ge



and exon 13



and TPO24


minus25

minus05

minus15minus2

minus1

218 plusmn 013



(a)

All TPO transcripts

Normally spliced


TPO4 TPO5 TPO23

TPO1 TPO2 TPO3

and TPO24

100 100 100

ProbandIII-1


exon 12 and exon 13

minus289 plusmn 011


minus4

minus3

minus2

minus1

0

1

2

Fold

chan

ge

(b)




III-1 lesion area

III-1 normal area

Proband lesion area

Proband normal area

0 20()

40 60 80 100


TPO6


Others


(c)






Da)

170130

95725543

34

26

17


81 2 3 4 5 6 7

120573-actin 42kDa






5 Conclusion




Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























= 2minusΔCt (3)



= 2minusΔCt (4)




NA NANA

1 2 3 4

1 2

1 2

(I)

(II)

(III)

Male

Female



Goiter

NA Not available


lowast

lowast

lowastlowast

Free 20pmolL T4T4




(OD470


3 Results







(a)


(b)







(c)










(bp)

(bp)

1000

500

100

1 2 3 4 5 6

803671499

328

300350

(a)

1000

500

100

1 2 3 4 5 6

(bp)

(bp)437

488

118131

261290

(b)

(bp)

(bp)

1000

500

100

1 2 3 4 5 6

300350

(c)

(bp)

(bp)

1000

500

100

1 2 3 4

300

(d)








116nt52 nt

c2268dup (pGlu756X)

(a)

Exon 13Exon 12

(b)

Exon 12 Exon 13

34nt

(c)

c2268dup

Exon 13 (c2216)Exon 12 (c2215)

XQ D H C

756aa


CUCAAG AC CACUGUUGA

(d)

c2268dup

739aa

Exon 12 (c2215)

XQ V

Exon 13 (c2216)

pAsp739ValfsX740

201 nt

Intron 12 (c2216-34)

c2216-1 CUCAAG



(e)






4 Discussion




1 1 1 1 11

005

115

225

3


All TPO transcripts

Proband

Fold

chan

ge



and exon 13



and TPO24


minus25

minus05

minus15minus2

minus1

218 plusmn 013



(a)

All TPO transcripts

Normally spliced


TPO4 TPO5 TPO23

TPO1 TPO2 TPO3

and TPO24

100 100 100

ProbandIII-1


exon 12 and exon 13

minus289 plusmn 011


minus4

minus3

minus2

minus1

0

1

2

Fold

chan

ge

(b)




III-1 lesion area

III-1 normal area

Proband lesion area

Proband normal area

0 20()

40 60 80 100


TPO6


Others


(c)






Da)

170130

95725543

34

26

17


81 2 3 4 5 6 7

120573-actin 42kDa






5 Conclusion




Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NA NANA

1 2 3 4

1 2

1 2

(I)

(II)

(III)

Male

Female



Goiter

NA Not available


lowast

lowast

lowastlowast

Free 20pmolL T4T4




(OD470


3 Results







(a)


(b)







(c)










(bp)

(bp)

1000

500

100

1 2 3 4 5 6

803671499

328

300350

(a)

1000

500

100

1 2 3 4 5 6

(bp)

(bp)437

488

118131

261290

(b)

(bp)

(bp)

1000

500

100

1 2 3 4 5 6

300350

(c)

(bp)

(bp)

1000

500

100

1 2 3 4

300

(d)








116nt52 nt

c2268dup (pGlu756X)

(a)

Exon 13Exon 12

(b)

Exon 12 Exon 13

34nt

(c)

c2268dup

Exon 13 (c2216)Exon 12 (c2215)

XQ D H C

756aa


CUCAAG AC CACUGUUGA

(d)

c2268dup

739aa

Exon 12 (c2215)

XQ V

Exon 13 (c2216)

pAsp739ValfsX740

201 nt

Intron 12 (c2216-34)

c2216-1 CUCAAG



(e)






4 Discussion




1 1 1 1 11

005

115

225

3


All TPO transcripts

Proband

Fold

chan

ge



and exon 13



and TPO24


minus25

minus05

minus15minus2

minus1

218 plusmn 013



(a)

All TPO transcripts

Normally spliced


TPO4 TPO5 TPO23

TPO1 TPO2 TPO3

and TPO24

100 100 100

ProbandIII-1


exon 12 and exon 13

minus289 plusmn 011


minus4

minus3

minus2

minus1

0

1

2

Fold

chan

ge

(b)




III-1 lesion area

III-1 normal area

Proband lesion area

Proband normal area

0 20()

40 60 80 100


TPO6


Others


(c)






Da)

170130

95725543

34

26

17


81 2 3 4 5 6 7

120573-actin 42kDa






5 Conclusion




Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)


(b)







(c)










(bp)

(bp)

1000

500

100

1 2 3 4 5 6

803671499

328

300350

(a)

1000

500

100

1 2 3 4 5 6

(bp)

(bp)437

488

118131

261290

(b)

(bp)

(bp)

1000

500

100

1 2 3 4 5 6

300350

(c)

(bp)

(bp)

1000

500

100

1 2 3 4

300

(d)








116nt52 nt

c2268dup (pGlu756X)

(a)

Exon 13Exon 12

(b)

Exon 12 Exon 13

34nt

(c)

c2268dup

Exon 13 (c2216)Exon 12 (c2215)

XQ D H C

756aa


CUCAAG AC CACUGUUGA

(d)

c2268dup

739aa

Exon 12 (c2215)

XQ V

Exon 13 (c2216)

pAsp739ValfsX740

201 nt

Intron 12 (c2216-34)

c2216-1 CUCAAG



(e)






4 Discussion




1 1 1 1 11

005

115

225

3


All TPO transcripts

Proband

Fold

chan

ge



and exon 13



and TPO24


minus25

minus05

minus15minus2

minus1

218 plusmn 013



(a)

All TPO transcripts

Normally spliced


TPO4 TPO5 TPO23

TPO1 TPO2 TPO3

and TPO24

100 100 100

ProbandIII-1


exon 12 and exon 13

minus289 plusmn 011


minus4

minus3

minus2

minus1

0

1

2

Fold

chan

ge

(b)




III-1 lesion area

III-1 normal area

Proband lesion area

Proband normal area

0 20()

40 60 80 100


TPO6


Others


(c)






Da)

170130

95725543

34

26

17


81 2 3 4 5 6 7

120573-actin 42kDa






5 Conclusion




Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(bp)

(bp)

1000

500

100

1 2 3 4 5 6

803671499

328

300350

(a)

1000

500

100

1 2 3 4 5 6

(bp)

(bp)437

488

118131

261290

(b)

(bp)

(bp)

1000

500

100

1 2 3 4 5 6

300350

(c)

(bp)

(bp)

1000

500

100

1 2 3 4

300

(d)








116nt52 nt

c2268dup (pGlu756X)

(a)

Exon 13Exon 12

(b)

Exon 12 Exon 13

34nt

(c)

c2268dup

Exon 13 (c2216)Exon 12 (c2215)

XQ D H C

756aa


CUCAAG AC CACUGUUGA

(d)

c2268dup

739aa

Exon 12 (c2215)

XQ V

Exon 13 (c2216)

pAsp739ValfsX740

201 nt

Intron 12 (c2216-34)

c2216-1 CUCAAG



(e)






4 Discussion




1 1 1 1 11

005

115

225

3


All TPO transcripts

Proband

Fold

chan

ge



and exon 13



and TPO24


minus25

minus05

minus15minus2

minus1

218 plusmn 013



(a)

All TPO transcripts

Normally spliced


TPO4 TPO5 TPO23

TPO1 TPO2 TPO3

and TPO24

100 100 100

ProbandIII-1


exon 12 and exon 13

minus289 plusmn 011


minus4

minus3

minus2

minus1

0

1

2

Fold

chan

ge

(b)




III-1 lesion area

III-1 normal area

Proband lesion area

Proband normal area

0 20()

40 60 80 100


TPO6


Others


(c)






Da)

170130

95725543

34

26

17


81 2 3 4 5 6 7

120573-actin 42kDa






5 Conclusion




Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















116nt52 nt

c2268dup (pGlu756X)

(a)

Exon 13Exon 12

(b)

Exon 12 Exon 13

34nt

(c)

c2268dup

Exon 13 (c2216)Exon 12 (c2215)

XQ D H C

756aa


CUCAAG AC CACUGUUGA

(d)

c2268dup

739aa

Exon 12 (c2215)

XQ V

Exon 13 (c2216)

pAsp739ValfsX740

201 nt

Intron 12 (c2216-34)

c2216-1 CUCAAG



(e)






4 Discussion




1 1 1 1 11

005

115

225

3


All TPO transcripts

Proband

Fold

chan

ge



and exon 13



and TPO24


minus25

minus05

minus15minus2

minus1

218 plusmn 013



(a)

All TPO transcripts

Normally spliced


TPO4 TPO5 TPO23

TPO1 TPO2 TPO3

and TPO24

100 100 100

ProbandIII-1


exon 12 and exon 13

minus289 plusmn 011


minus4

minus3

minus2

minus1

0

1

2

Fold

chan

ge

(b)




III-1 lesion area

III-1 normal area

Proband lesion area

Proband normal area

0 20()

40 60 80 100


TPO6


Others


(c)






Da)

170130

95725543

34

26

17


81 2 3 4 5 6 7

120573-actin 42kDa






5 Conclusion




Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1 1 1 1 11

005

115

225

3


All TPO transcripts

Proband

Fold

chan

ge



and exon 13



and TPO24


minus25

minus05

minus15minus2

minus1

218 plusmn 013



(a)

All TPO transcripts

Normally spliced


TPO4 TPO5 TPO23

TPO1 TPO2 TPO3

and TPO24

100 100 100

ProbandIII-1


exon 12 and exon 13

minus289 plusmn 011


minus4

minus3

minus2

minus1

0

1

2

Fold

chan

ge

(b)




III-1 lesion area

III-1 normal area

Proband lesion area

Proband normal area

0 20()

40 60 80 100


TPO6


Others


(c)






Da)

170130

95725543

34

26

17


81 2 3 4 5 6 7

120573-actin 42kDa






5 Conclusion




Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Da)

170130

95725543

34

26

17


81 2 3 4 5 6 7

120573-actin 42kDa






5 Conclusion




Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article functional analyses of c.2268dup in...

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