research article functional analysis of thyroid peroxidase gene...
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Research ArticleFunctional Analysis ofThyroid Peroxidase Gene Mutations Detected in Patientswith Thyroid Dyshormonogenesis
Srikanta Guria1 Biswabandhu Bankura2 Nisha Balmiki2
Arup Kumar Pattanayak2 Tapas Kumar Das3 Anirban Sinha4 Sudipta Chakrabarti5
Subhankar Chowdhury3 and Madhusudan Das2
1 Post Graduate Department of Zoology Barasat Government College Barasat Kolkata 700 124 India2Department of Zoology University of Calcutta 35 Ballygunge Circular Road Kolkata West Bengal 700 019 India3 Bagnan Rural Hospital Bagnan Howrah West Bengal 711 303 India4Department of Endocrinology Institute of Post GraduateMedical Educationamp Research 244 A J C Bose Road Kolkata 700 020 India5 Institute of Life Sciences Nalco Square Chandra Sekharpur Bhubaneswar 751 023 India
Correspondence should be addressed to Madhusudan Das madhuzooyahoocom
Received 5 October 2013 Accepted 20 February 2014 Published 13 April 2014
Academic Editor Małgorzata Kotula-Balak
Copyright copy 2014 Srikanta Guria et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Thyroid peroxidase (TPO) is the key enzyme in the biosynthesis of thyroid hormones We aimed to identify the spectrum ofmutations in the TPO gene leading to hypothyroidism in the population of West Bengal to establish the genetic etiology of thedisease 200 hypothyroid patients (case) and their corresponding sex and age matched 200 normal individuals (control) werescreened depending on their clinical manifestations Genomic DNA was isolated from peripheral blood samples and TPO gene(Exon 7 to Exon 14) was amplified by PCRThe PCR products were subjected to sequencing to identify mutations Single nucleotidechanges such as Glu 641 Lys Asp 668 AsnThr 725 Pro Asp 620 Asn Ser 398Thr and Ala 373 Ser were found Changes in the TPOwere assayed in vitro to compare mutant and wild-type activities Five mutants were enzymatically inactive in the guaiacol andiodide assaysThis is a strong indication that the mutations are present at crucial positions of the TPO gene resulting in inactivatedTPOThe results of this study may help to develop a genetic screening protocol for goiter and hypothyroidism in the population ofWest Bengal
1 Introduction
TPO is a membrane-bound glycoprotein (102 kDa) foundas a dimer [1] Each monomer consists of 933 amino acidresidues and contains a peroxidase domain three additionalextracellular domains a transmembrane helix and a shortC-terminal intracellular tail [2] The human TPO geneis located on chromosome 2p25 and spans approximately150Kb containing 17 exons [3] Mutations in TPO gene(particularly nonsynonymous cSNPs) can lead to severedefects in thyroid hormone production due to total iodideorganification defects (TIOD) or partial iodide organificationdefects (PIOD) TPO mutations are inherited as autosomal
recessive traits [4] Iodination of salt is the most effective andsustainable long-term public health measure for the preven-tion and control of iodide organification defects (IDD) Butthe iodination programme will not be effective if the genemutations are the cause of dyshormonogenesis Thereforeit is important to screen the percentage of people havinggene mutations and iodine deficiency among the clinicallyidentified thyroid patients
Screening and identification of mutations in the TPOgene of patients with evidence of TIOD and PIOD have beendone by several groups in different countries of the world likeArgentina [4] Netherlands [6] Japan [7] Portugal [8] andChina [9]The present investigation is aimed at screening the
Hindawi Publishing CorporationInternational Journal of EndocrinologyVolume 2014 Article ID 390121 8 pageshttpdxdoiorg1011552014390121
2 International Journal of Endocrinology
Table 1 Primer sequence used to screen the different exons of TPO gene
Exons Primers Sequence Amplicon size (bp)
Exon 7 Forward 51015840-CTGGAGCTCTGTGAACAAGAA-31015840 433Reverse 51015840-CCCTGGGAATAGGACAAAGAAA-31015840
Exon 8 Forward 51015840-CCCTACGTAACAAACCTGCAC-31015840 474Reverse 51015840-GGCTGTCAAGGAAGATGCTC-31015840
Exon 9 Forward 51015840-CGTTGCTTAGAAGGCCTCAG-31015840 444Reverse 51015840-CTTGCAGTGAGCTGAGATCG-31015840
Exon 10 Forward 51015840-ACAACCTGACCAGGCTTACG-31015840 485Reverse 51015840-CAGGACTCTGCCCTGCTG-31015840
Exon 11 Forward 51015840-CTGCCCTGAGGGTGTAAGG-31015840 446Reverse 51015840-GAGAGGCTGGCAGCACACAG-31015840
Exon 12 Forward 51015840-CTATCCCCAGATTGCTCCTG-31015840 449Reverse 51015840-GCTCAGTGAGTGACCACAGC-31015840
Exon 13 Forward 51015840-GTGTGCTTCGAGGGTCTCTG-31015840 485Reverse 51015840-CCCTAGACCAGGTGGGATG-31015840
Exon 14 Forward 51015840-CCATGTCCAGAGGAAAGGAG-31015840 238Reverse 51015840-CAGACTCAGGCAGGACAACC-31015840
mutationspolymorphisms in TPO gene and their effects onthe function of TPO gene leading to hypothyroidism in thepopulation ofWest Bengal to establish the genetic etiology ofthe disease
2 Materials and Methods
200 hypothyroid patients (case) and their correspondingsex and age matched 200 normal individuals (control) werescreened depending on their clinical manifestations anddetailed familial history from the Institute of Post GraduateMedical Education amp Research (IPGME amp R) KolkataPeripheral blood samples were collected on the basis ofprior consent given by patientsnormal individuals familiesand parents on behalf of minor children To the extentpossible complete pedigree samples were obtained whichincluded the proband siblings parents and other bloodrelatives Age and sex matched subjects with no goitre noclinical evidence of hypothyroidism and normal levels ofserum T
3 free T
4(FT4) TSH and anti-TPO antibody were
enrolled as normal (control) The experimental protocol wasapproved by the institutional ethics committee of IPGMEamp R Kolkata Clinical information includes complains likelethargy cold intolerance loss of memory constipationand weight gain Personal history includes menarche lastchild birth abortion cycle duration and periodicity Delayedmilestones for children like sitting without support speechand walking were also documented
Quantitative sandwich immunoassay kit (Siemens India)was used to assay serum TSH level Serum T
3or FT
4con-
centrations were determined by radioimmunoassay (RIA)Quantitative measurement of serum TPO antibody wasperformed using ELISA method TPO antibody value
gt60 IUmL was considered as positive [10] Radiolabelediodine was administered to patients and control Emittanceof radioactivity was measured over the thyroid Potassiumperchlorate (a competitive inhibitor of iodide transport intothe thyroid) was administered Emittance of radioactivity wasmeasured over the thyroid and compared to initial result
The iodine in the urine is measured by a modification ofthe traditional colorimetric method of Sandell and Kolthoff(1937) [11] This was done using the ammonium persulfatemethod as described by Pino et al (1996) [12] Urine wasdigested with ammonium persulfate The iodine in the urinesamples catalyses the reduction of ceric ammonium sulphate(yellow colour) to the cerous form (colourless) in the presenceof arsenious acid The degree of reduction in colour intensityof the yellow ceric ammonium sulphate is proportional to theiodine content in the urine sample
Peripheral blood samples were collected from the caseand control individuals Genomic DNA was isolated fromthe blood leucocytes by using QIAamp Blood Kit (QIAGENHilden Germany)The humanTPO gene (Exon 7 to Exon 14)was amplified by PCR PCRwas performed in a thermocycler(Applied Biosystems Model number 9902) using specificprimers for each of the exons (Table 1) The reaction mix-ture (25 120583L) contained 40ndash100 ng of genomic DNA 15mMMgCl
2 100 120583M of each dNTP 04 120583M of each primer and
05 unit of Taq DNA polymerase (Applied Biosystems)Denaturation at 95∘C for 30 seconds annealing at 55ndash60∘Cfor 30 seconds and extension at 72∘C for 30 seconds times 44cycles were performedThe PCR product was analyzed in 2agarose gel electrophoresis for verifying the size of the PCRproduct PCR fragments were purified from agarose gel usingGel Extraction kit (Genei Bangalore)
The PCR products were sequenced by using Big Dye Ter-minator kit v31 (Applied Biosystems) in ABI Prism 377DNA
International Journal of Endocrinology 3
Sequencer (PE Applied Biosystems) Purified amplicons wereused for sequencing reaction and the reactionmixture (10120583L)contained 150 ng DNA 5 120583M primer (either forward orreverse) 05 120583L BD and 19 120583L 5 times BD buffer Denaturationat 96∘C for 10 seconds annealing at 50ndash55∘C for 10 secondsand extension at 60∘C for 4min times 25 cycles were performed10 120583LPCRmixwas addedwith solution I (10120583LmqH
2O 2 120583L
125mM EDTA and pH 80) and solution II (50 120583L ehanol2 120583L Na-acetate and pH 46) The mixture was kept in darkfor 25min and centrifuged at room temperature (13000 rpmfor 30min) and sample was dried in speed vac Then 12120583LHi-Di (formamide) was added in sample and stored at darkfor 15min and incubated at 96∘C dry bath and we allowed thesample for snap chill in ice Then the sample was loaded inDNA Sequencer
Sequences alignment between sequences of case andcontrol individuals was performed to find the best matchingpiecewise (local) or global alignments of two query sequencesusing clustalW programme
Polymorphism in the TPO gene identified in this studyincluding nonsynonymous SNPs in the coding region dele-tions and insertions have been assayed in vitro We usedInvitrogen GeneArt Site-Directed Mutagenesis System forthis purpose for example Glu 641 Lys Asp 668Asn Thr725 Pro Asp 620Asn Ser 398Thr and Ala 373 Ser thesechangeswere incorporated intowild TPO cDNA through thismethod
We used pcDNA31 (vector) for TPO cDNA cloningDouble restriction enzyme (EcoRI KpnI) digestions wereperformed by incubating TPO cDNA and vector moleculeswith an appropriate amount of restriction enzyme in itsrespective buffer as recommended by the supplier and at theoptimal temperature The cut DNA fragment and vector arecovalently joined together by DNA ligase
Transformation is the method of introducing foreignDNA into bacterial cells The recombinant plasmids (TPOcDNA + plasmid) were amplified in DH5 alpha competentcells The uptake of recombinant plasmids by DH5 alphacell was carried out in ice-cold CaCl
2(0ndash5∘C) and sub-
sequent heat shock (42∘C for about 90 sec) Selection ofDH5 alpha competent cells containing recombinant plasmids(TPO cDNA + plasmid) was done by ampicillin treatmentPlasmid was purified by use of the plasmid isolation kit(PureLinkQuick PlasmidMiniprepKit Invitrogen)We usedInvitrogen Lipofectamine Reagent for transfection COS 7cells were grown in 38 cm dishes in DMEM supplementedwith 50mLL bovine calf serum and penicillin-streptomycinin 5 CO
2atmosphere at 37∘C When the cells reached
sim90 confluence they were transfected with 1 120583g of recom-binant plasmid DNA (wild-type and mutant) per 38 cmdish with Lipofectamine After incubation for 48 h the cellswere harvested for activity study Cells were harvested withtrypsin EDTA treatment and protein concentration wasdetermined on a 100 120583L aliquot using the Bio-Rad proteinassay (BioRad Munchen Germany) The cells were pelletedand subsequently suspended in 01 deoxycholate containing1 aprotinin and incubated for 10 min at 4∘C The extractwas microcentrifuged for 5min and the supernatant wasremoved to measure enzymatic activity using the guaiacol
Table 2 Study population
Case (119899 = 200) Control (119899 = 200) 119875 valueSex
Male 29 (145) 32 (16) 088Female 171 (855) 168 (84) 080
Age (Mean plusmn SD) 2985 plusmn 1951 3038 plusmn 1290
and I3
minus assays For the guaiacol assay 50 120583Lof the supernatantwas assayed in a final volume of 750120583L containing 35mMguaiacol and 05mM H
2O2in 01M Tris-HCl pH 86
The absorbance at 470 nm was followed and activity wasexpressed as ΔA sdot minminus1 sdot mg proteinminus1 For the I
3
minus assay25 120583L of the supernatant was used in a final volume of750120583L containing 01M potassium phosphate pH 75 50mMpotassium iodide and 025mM H
2O2 The absorbance at
353 nmwas followed and activitywas expressed asΔAsdotminminus1sdotmg proteinminus1 Spectrophotometric analysis was done on aShimadzu UV-200
Thirty micrograms of the deoxycholate extracted mem-brane protein fraction containing recombinant TPO andnormal control TPO were electrophoresed on a 75 SDS-polyacrylamide gel The gel was blotted onto nitrocelluloseusing a Bio-Rad Mini Protean 2 system (Bio-Rad LabsRichmond CA) followed by incubation with rabbit anti-human TPO antibody (abcam EPR5379) TPO protein wasvisualized using ECL Western blotting detection reagent(abcam)
21 Statistical Methods 1205942 and Fisher exact tests were usedto test the allelic and genotypic associations of each SNPStudentrsquos t-test was used to calculate any statistically signif-icant difference of continuous independent variables like ageTSH and FT4 within the control and patient groups All testswere done using GraphPad InStat software (GraphPad InStatsoftware San Diego CA) Odds ratio and 95 confidentialintervals were also calculated using the same software ABonferroni correction was applied for multiple testing
3 Results
The case and control groups were well balanced in terms ofage and gender There is no significant difference betweentwo groups (Table 2) Case individuals exhibited varied clin-ical manifestations The associated clinical manifestations ofhypothyroidism were goiter (655) lethargy (61) musclecramp (55) loss of memory (51) hoarseness of voice(405) weight loss (8 in male and 30 in female) con-stipation (55) cold intolerance (60) weight gain (45)etc(Table 3)
The TSH level was significantly high and FT4and T
3
levels were significantly low in case population In controlpopulation average serumTSH level was 251plusmn093 120583UmL ascompared to 3514plusmn462 120583UmL in case population SimilarlyserumFT
4level was 125plusmn010 ngdL in control population as
compared to 061 plusmn 027 ngdL in case population Serum T3
4 International Journal of Endocrinology
Table 3 Clinical manifestations of case at initial presentation
Clinical manifestations Case(119899 = 200) Control
(119899 = 200) 119875 valuelowastFamily history
Present 93 465 6 3lt00001
Absent 107 535 194 97lowastGoiter
Present 131 655 mdash mdashlowastWeight gain
Yes 90 45 67 34 0024No 110 55 133 66
Loss of memoryYes 102 51 90 45 027No 98 49 110 55
lowastLethargyYes 122 61 91 46 0002No 78 39 109 54
lowastMuscle crampYes 110 55 88 44 003No 90 45 112 56
lowastCold intoleranceYes 120 60 113 57 054No 80 40 87 43
lowastConstipationYes 109 55 80 40 0005No 91 45 120 60
lowastat the time of diagnosis
level was 164plusmn008 ngdL in control population as comparedto 065 plusmn 022 ngdL in case population 44 patients showedpositive TPO antibody level (Figure 1)
The 123I uptake as shown by 83 patients at 3 hours was481 plusmn790 and 753 plusmn590 of 123I was discharged at 60minafter the oral administration of KClO
4at a dose of 04 g in the
perchlorate discharge test confirming iodide organificationdefect in the thyroid gland In our patient population wefound that 192 patients were within the level of 10ndash20120583gdLIn overall 96 of the patients had UIE levels in the ranges ofoptimal iodine nutrition (10ndash20120583gdL) (WHOUNICEF andICCIDD 2001)
Mutation screening of TPO gene in both case and controlgroups showed six nonsynonymous changes c 1117 GgtTAla 373 Ser c 1193GgtC Ser 398 Thr c 1858 GgtA Asp 620Asn c 1921 GgtA Glu 641 Lys c 2002GgtA Asp668Asnand c 2173AgtC Thr725Pro (Figure 2) After the Bonferronicorrection for multiple comparisons a strong association wasobserved between the Asp 620 Asn Glu 641 Lys andThr 725Pro SNPs and hypothyroidism (Table 4)
To measure wild-type and mutant enzyme activity boththe Iminus and guaiacol assays were carried out Wild-typerecombinant TPO showed enzymatic activity in both assays
Mutant TPO showed relatively nonenzymatic reaction rate(Table 5)
All mutants expressed reduced amount of TPO on theWestern blot In Figure 3(a) Western blot showed expressionof wild-type and mutated recombinant TPO Lane 1 showedwild-type recombinant TPO Lanes 2ndash4 showed recombinantTPO containing (2-Glu 641 Lys 3-Asp 668Asn and 4-Thr 725Pro) changes In Figure 3(b)Western blot showed expressionof wild-type and mutated recombinant TPO Lane 1 showedwild-type recombinant TPO Lanes 2ndash4 showed recombinantTPO Containing (2-Asp 620 Asn 3-Ser 398 Thr and 4-Ala373 Ser) changes
4 Discussion
The present study reveals clinical analysis of hypothyroidismin screened population Hypothyroid patients exhibited sig-nificant increase in the level of serum TSH than controlAs expected patients showed lower level of T
3and FT
4as
compared to control population 83 patients showed positiveresult in the perchlorate discharge test confirming iodideorganification defect in the thyroid glandThe urinary iodineexcretion (UIE) of 96 patients was within the levels of 10ndash20120583gdL which were in the range of optimal iodine nutrition
International Journal of Endocrinology 5
Table4AlleleandGenotyped
istrib
utionof
TPOgene
polymorph
ismsinthes
tudy
SNP
Allele
Allelefre
quency
Odd
sratio
(95
CI)119875value
Genotype
Case
(119899=200)
Con
trol(119899=200)
Odd
sratio
(95
CI)
119875value
Case
Con
trol
c1117
GgtT
G054
059
123(093ndash16
2)017
GG
34
35
Reference
Ala373Ser
T046
041
GT
40
485
GGversus
GT
085
(054ndash
133)
054
TT26
165
GGversus
TT162(094ndash
281)
011
GG+GTversus
TT178(109ndash
290)
0028
c119
3GgtC
C073
068
079
(058ndash10
7)014
CC575
49
Reference
Ser3
98Th
rG
027
032
GC
305
375
CCversus
GC
069
(045ndash10
7)012
GG
12
135
CCversus
GG076
(041ndash14
0)046
c1858GgtA
G090
096
267
(147ndash485)
0001
GG
850
930
Reference
Asp620As
nA
010
004
GA
105
650
GGversus
GA177(086ndash
364
)011
AA
45
050
GGversus
AA985(123ndash7854)
0008
GGversus
GA+AA234(120ndash
457)
0011
c1921
GgtA
G087
094
234
(141ndash388)
0001
GG
775
89
Reference
Glu64
1Lys
A013
006
GA
185
11
GGversus
GA193(109ndash
341)
0022
AA
40
00
GGversus
GA+AA235
(135
ndash409)
0002
c2002
GgtA
G093
094
118(067ndash207)
056
GG
865
885
Reference
Asp66
8As
nA
007
006
GA
125
115
GGversus
GA111
(061ndash203)
073
AA
10
00
c2173
AgtC
A054
063
145(109ndash
192)
001
AA
35
410
Reference
Thr7
25Pro
C046
037
AC375
440
AAversus
AC100(064ndash
156)
099
CC275
150
AAversus
CC215
(124ndash
371)
000
6AA+AC
versus
CC215
(131ndash353
)0002
Chi-squ
aretestw
asused
tocompare
theg
enotypea
ndallelefre
quencies
betweencasesa
ndcontrols
119875valuelt
005
isconsidered
tobe
statisticallysig
nificant
6 International Journal of Endocrinology
0
02
04
06
08
1
12
14
16
18
2
Patient Control
P lt 00001
T 3(N
gm
L)
065 plusmn 022
164 plusmn 008
(a)
0
02
04
06
08
1
12
14
16
Patient Control
P lt 00001
FT4
(Ng
dL)
061 plusmn 027
125 plusmn 010
(b)
0
5
10
15
20
25
30
35
40
45
Patient Control
TSH
(mcU
mL)
P lt 00001
3514 plusmn 462
251 plusmn 093
(c)
44
156
0
20
40
60
80
100
120
140
160N
umbe
r of p
atie
nts
Anti-TPO antibody level (IUmL)gt60 IUmL lt60 IUmL
(d)
Figure 1 T3and FT
4level of patient and control (normal) populations TSH and anti-TPO antibody level of patient and control (normal)
populations
(10ndash20120583gdL) (WHO UNICEF and ICCIDD 2001) There-fore UIE levels indicate that iodine deficiency is currently nota public health problem in our screened population
Our study identified six different SNPs among these threeSNPs (Asp 620 Asn Glu 641 Lys and Thr 725 Pro) aresignificantly associated with hypothyroidism We also try tofind out the association between TPO gene polymorphismsand Anti-TPO level in our study subjects and we did not findany association (data not shown) However several groups indifferent population like Tehranian population [13] and Ira-nian population [14] showed association between TPO genepolymorphisms and anti-TPO level Our study needs muchlarger sample size which may help in better understanding of
association between TPO gene polymorphisms and anti-TPOlevel
Based on the literature Exons 7ndash11 encode the catalyticcenter of the TPO protein (heme binding region) whichis crucial for the enzymatic activity [15] On the otherhand previous findings also indicate that Exons 7ndash14 harbormutational hot spots [15] Thus mutations in these regionsare expected to have major effects on TPO activity resultingin severe organification defect and severe hypothyroidismIn Glu 641 Lys negative charged amino acid is replaced bypositive charged amino acid As we know Threonine is thephosphorylation site of the protein which is important for theactivation of the protein [16 17] Therefore mutations in this
International Journal of Endocrinology 7
A C T
A T C A C T G G C
C C T
A T C C C T G G C
Normal Patient
(a)
A G CCC G C G C C A G C G A G G T
A C CC C G C G C C A C C G A G G T
Normal Patient
(b)
G C GG C G C G C C T G C G G C C T G T G C
T C GG C G C G C C T T C G G C C T G T G C
Normal Patient
(c)
Figure 2 Nucleotide polymorphism in TPO gene study population (a) Nucleotide polymorphism in patient ID-147 Exon11 and ACT gtCCT(Thr gt Pro) (b) Nucleotide polymorphism in patient ID-6 Exon7 and AGC gt ACC (Ser gtThr) (c) Nucleotide polymorphism in patientID-183 Exon7 and GCG gt TCG (Ala gt Ser)
1 2 3 4
GAPDH
(a)
1 2 3 4
GAPDH
(b)
Figure 3 Western blot showing expression of wild-type and mutated recombinant TPO (a) Lane 1 wild-type recombinant TPO Lanes 2ndash4recombinant TPO containing (2-Glu 641 Lys 3-Asp 668Asn and 4-Thr 725 Pro) changes In (b)Western blot showing expression of wild-typeandmutated recombinant TPO Lane 1 wild-type recombinant TPO Lanes 2ndash4 recombinant TPO containing (2-Asp 620Asn 3-Ser 398Thrand 4-Ala 373 Ser) changes
amino acid may change the activity of TPO enzyme whichmay ultimately reduce the functional efficacy of the enzyme
We established the effect of these polymorphisms throughin vitro assay Some polymorphisms (Glu 641 Lys Thr 725Pro andAsp 620Asn)were enzymatically inactive in both theguaiacol and iodide assaysThey also showed reduced amountof TPO on the Western blot This is a strong indication that
the changes are present at crucial positions of the TPO generesulting in inactivated TPO
5 Conclusions
Thus the above changes of amino acids may exert its effecton the structure and functional activity of TPO Our study
8 International Journal of Endocrinology
Table 5 Guaiacol and iodide oxidation activity of expressed humanTPO protein
Mutation Guaiacol oxidation Iodide oxidationWild type 076 plusmn 007 090 plusmn 005
Glu 641 Lys ND lowast
Asp 668 Asn 063 plusmn 007 069 plusmn 027
Thr 725 Pro ND lowast
Asp 620 Asn ND lowast
Ser 398Thr 070 plusmn 027 082 plusmn 029
Ala 373 Ser 062 plusmn 009 lowast
Enzyme activity was activity expressed as Δ119860sdotminminus1sdotmg proteinminus1lowastComparable with nonenzymatic reaction rate (051 plusmn 024) ND nodetectable activity (lt10 of wild-type expressed TPO) [5]
tried to detect the genetic etiology of this disease which mayfurther help us to risk categorize for hypothyroidism Thisstudy also helps to develop a genetic screening protocol forhypothyroidism specifically for the Indian population
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Srikanta Guria and Biswabandhu Bankura contributedequally to this paper
Acknowledgments
This work was supported by Grant from the Depart-ment of Biotechnology Government of India (no BTPR9342Med123462007) The authors would like to thankthe families who gave their consent and collaborated in thisstudy
References
[1] J R Baker Jr P Arscott and J Johnson ldquoAn analysis of thestructure and antigenicity of different forms of human thyroidperoxidaserdquoThyroid vol 4 no 2 pp 173ndash178 1994
[2] J P Banga D Mahadevan G J Barton et al ldquoPrediction ofdomain organisation and secondary structure of thyroid perox-idase a human autoantigen involved in destructive thyroiditisrdquoFEBS Letters vol 266 no 1-2 pp 133ndash141 1990
[3] S Kimura Y-S Hong T Kotani S Ohtaki and F KikkawaldquoStructure of the human thyroid peroxidase gene comparisonand relationship to the humanmyeloperoxidase generdquoBiochem-istry vol 28 no 10 pp 4481ndash4489 1989
[4] 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
[5] H Bikker F Baas and J J De Vijlder ldquoMolecular analy-sis of mutated thyroid peroxidase detected in patients with
total iodide organification defectsrdquo The Journal of ClinicalEndocrinology andMetabolism vol 82 no 2 pp 649ndash653 1997
[6] B Bakker H Bikker T Vulsma J S E De Randamie B MWiedijk and J J M De Vijlder ldquoTwo decades of screeningfor congenital hypothyroidism in the Netherlands TPO genemutations in total iodide organification defects (an update)rdquoJournal of Clinical Endocrinology and Metabolism vol 85 no10 pp 3708ndash3712 2000
[7] T Kotani K Umeki J-I Kawano et al ldquoPartial iodide organ-ification defect caused by a novel mutation of the thyroidperoxidase gene in three siblingsrdquo Clinical Endocrinology vol59 no 2 pp 198ndash206 2003
[8] C Rodrigues P Jorge J Pires Soares et al ldquoMutation screeningof the thyroid peroxidase gene in a cohort of 55 Portuguesepatients with congenital hypothyroidismrdquo European Journal ofEndocrinology vol 152 no 2 pp 193ndash198 2005
[9] 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
[10] A M Maskari and A Alnaqdy ldquoFrequency of thyroid micro-somal and thyroid peroxidase antibody levels in a selectedgroup of Omani patients with Gravesrsquo Diseaserdquo Kuwait MedicalJournal vol 38 no 1 pp 10ndash13 2006
[11] E B Sandell and I M Kolthoff ldquoMicro determination of iodineby a catalytic methodrdquoMikrochimica Acta vol 1 no 1 pp 9ndash251937
[12] S Pino S-L Fang and L E Braverman ldquoAmmonium persul-fate a safe alternative oxidizing reagent for measuring urinaryiodinerdquo Clinical Chemistry vol 42 no 2 pp 239ndash243 1996
[13] B FaamM SDaneshpour F AziziM Salehi andMHedayatildquoAssociation between TPO gene polymorphisms and Anti-TPOlevel in Tehranian population TLGSrdquo Gene vol 498 no 1 pp116ndash119 2012
[14] M Hedayati M S Jahromi M Z Yeganeh M S DaneshpourLH Rad andFAzizi ldquoAssociation between serum level of anti-TPO titer and polymorphisms G1193C Exon 8 and C2145TExon 12 of thyroid peroxidase gene in an Iranian populationrdquoInternational Journal of Endocrinology and Metabolism vol 8no 2 pp 64ndash67 2010
[15] H Bikker T Vulsma F Baas and J J M De Vijlder ldquoIdentifica-tion of five novel inactivating mutations in the human thyroidperoxidase gene by denaturing gradient gel electrophoresisrdquoHuman Mutation vol 6 no 1 pp 9ndash16 1995
[16] M Huse and J Kuriyan ldquoThe conformational plasticity ofprotein kinasesrdquo Cell vol 109 no 3 pp 275ndash282 2002
[17] A Krupa G Preethi and N Srinivasan ldquoStructural modesof stabilization of permissive phosphorylation sites in proteinkinases distinct strategies in SerThr and Tyr kinasesrdquo Journalof Molecular Biology vol 339 no 5 pp 1025ndash1039 2004
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
2 International Journal of Endocrinology
Table 1 Primer sequence used to screen the different exons of TPO gene
Exons Primers Sequence Amplicon size (bp)
Exon 7 Forward 51015840-CTGGAGCTCTGTGAACAAGAA-31015840 433Reverse 51015840-CCCTGGGAATAGGACAAAGAAA-31015840
Exon 8 Forward 51015840-CCCTACGTAACAAACCTGCAC-31015840 474Reverse 51015840-GGCTGTCAAGGAAGATGCTC-31015840
Exon 9 Forward 51015840-CGTTGCTTAGAAGGCCTCAG-31015840 444Reverse 51015840-CTTGCAGTGAGCTGAGATCG-31015840
Exon 10 Forward 51015840-ACAACCTGACCAGGCTTACG-31015840 485Reverse 51015840-CAGGACTCTGCCCTGCTG-31015840
Exon 11 Forward 51015840-CTGCCCTGAGGGTGTAAGG-31015840 446Reverse 51015840-GAGAGGCTGGCAGCACACAG-31015840
Exon 12 Forward 51015840-CTATCCCCAGATTGCTCCTG-31015840 449Reverse 51015840-GCTCAGTGAGTGACCACAGC-31015840
Exon 13 Forward 51015840-GTGTGCTTCGAGGGTCTCTG-31015840 485Reverse 51015840-CCCTAGACCAGGTGGGATG-31015840
Exon 14 Forward 51015840-CCATGTCCAGAGGAAAGGAG-31015840 238Reverse 51015840-CAGACTCAGGCAGGACAACC-31015840
mutationspolymorphisms in TPO gene and their effects onthe function of TPO gene leading to hypothyroidism in thepopulation ofWest Bengal to establish the genetic etiology ofthe disease
2 Materials and Methods
200 hypothyroid patients (case) and their correspondingsex and age matched 200 normal individuals (control) werescreened depending on their clinical manifestations anddetailed familial history from the Institute of Post GraduateMedical Education amp Research (IPGME amp R) KolkataPeripheral blood samples were collected on the basis ofprior consent given by patientsnormal individuals familiesand parents on behalf of minor children To the extentpossible complete pedigree samples were obtained whichincluded the proband siblings parents and other bloodrelatives Age and sex matched subjects with no goitre noclinical evidence of hypothyroidism and normal levels ofserum T
3 free T
4(FT4) TSH and anti-TPO antibody were
enrolled as normal (control) The experimental protocol wasapproved by the institutional ethics committee of IPGMEamp R Kolkata Clinical information includes complains likelethargy cold intolerance loss of memory constipationand weight gain Personal history includes menarche lastchild birth abortion cycle duration and periodicity Delayedmilestones for children like sitting without support speechand walking were also documented
Quantitative sandwich immunoassay kit (Siemens India)was used to assay serum TSH level Serum T
3or FT
4con-
centrations were determined by radioimmunoassay (RIA)Quantitative measurement of serum TPO antibody wasperformed using ELISA method TPO antibody value
gt60 IUmL was considered as positive [10] Radiolabelediodine was administered to patients and control Emittanceof radioactivity was measured over the thyroid Potassiumperchlorate (a competitive inhibitor of iodide transport intothe thyroid) was administered Emittance of radioactivity wasmeasured over the thyroid and compared to initial result
The iodine in the urine is measured by a modification ofthe traditional colorimetric method of Sandell and Kolthoff(1937) [11] This was done using the ammonium persulfatemethod as described by Pino et al (1996) [12] Urine wasdigested with ammonium persulfate The iodine in the urinesamples catalyses the reduction of ceric ammonium sulphate(yellow colour) to the cerous form (colourless) in the presenceof arsenious acid The degree of reduction in colour intensityof the yellow ceric ammonium sulphate is proportional to theiodine content in the urine sample
Peripheral blood samples were collected from the caseand control individuals Genomic DNA was isolated fromthe blood leucocytes by using QIAamp Blood Kit (QIAGENHilden Germany)The humanTPO gene (Exon 7 to Exon 14)was amplified by PCR PCRwas performed in a thermocycler(Applied Biosystems Model number 9902) using specificprimers for each of the exons (Table 1) The reaction mix-ture (25 120583L) contained 40ndash100 ng of genomic DNA 15mMMgCl
2 100 120583M of each dNTP 04 120583M of each primer and
05 unit of Taq DNA polymerase (Applied Biosystems)Denaturation at 95∘C for 30 seconds annealing at 55ndash60∘Cfor 30 seconds and extension at 72∘C for 30 seconds times 44cycles were performedThe PCR product was analyzed in 2agarose gel electrophoresis for verifying the size of the PCRproduct PCR fragments were purified from agarose gel usingGel Extraction kit (Genei Bangalore)
The PCR products were sequenced by using Big Dye Ter-minator kit v31 (Applied Biosystems) in ABI Prism 377DNA
International Journal of Endocrinology 3
Sequencer (PE Applied Biosystems) Purified amplicons wereused for sequencing reaction and the reactionmixture (10120583L)contained 150 ng DNA 5 120583M primer (either forward orreverse) 05 120583L BD and 19 120583L 5 times BD buffer Denaturationat 96∘C for 10 seconds annealing at 50ndash55∘C for 10 secondsand extension at 60∘C for 4min times 25 cycles were performed10 120583LPCRmixwas addedwith solution I (10120583LmqH
2O 2 120583L
125mM EDTA and pH 80) and solution II (50 120583L ehanol2 120583L Na-acetate and pH 46) The mixture was kept in darkfor 25min and centrifuged at room temperature (13000 rpmfor 30min) and sample was dried in speed vac Then 12120583LHi-Di (formamide) was added in sample and stored at darkfor 15min and incubated at 96∘C dry bath and we allowed thesample for snap chill in ice Then the sample was loaded inDNA Sequencer
Sequences alignment between sequences of case andcontrol individuals was performed to find the best matchingpiecewise (local) or global alignments of two query sequencesusing clustalW programme
Polymorphism in the TPO gene identified in this studyincluding nonsynonymous SNPs in the coding region dele-tions and insertions have been assayed in vitro We usedInvitrogen GeneArt Site-Directed Mutagenesis System forthis purpose for example Glu 641 Lys Asp 668Asn Thr725 Pro Asp 620Asn Ser 398Thr and Ala 373 Ser thesechangeswere incorporated intowild TPO cDNA through thismethod
We used pcDNA31 (vector) for TPO cDNA cloningDouble restriction enzyme (EcoRI KpnI) digestions wereperformed by incubating TPO cDNA and vector moleculeswith an appropriate amount of restriction enzyme in itsrespective buffer as recommended by the supplier and at theoptimal temperature The cut DNA fragment and vector arecovalently joined together by DNA ligase
Transformation is the method of introducing foreignDNA into bacterial cells The recombinant plasmids (TPOcDNA + plasmid) were amplified in DH5 alpha competentcells The uptake of recombinant plasmids by DH5 alphacell was carried out in ice-cold CaCl
2(0ndash5∘C) and sub-
sequent heat shock (42∘C for about 90 sec) Selection ofDH5 alpha competent cells containing recombinant plasmids(TPO cDNA + plasmid) was done by ampicillin treatmentPlasmid was purified by use of the plasmid isolation kit(PureLinkQuick PlasmidMiniprepKit Invitrogen)We usedInvitrogen Lipofectamine Reagent for transfection COS 7cells were grown in 38 cm dishes in DMEM supplementedwith 50mLL bovine calf serum and penicillin-streptomycinin 5 CO
2atmosphere at 37∘C When the cells reached
sim90 confluence they were transfected with 1 120583g of recom-binant plasmid DNA (wild-type and mutant) per 38 cmdish with Lipofectamine After incubation for 48 h the cellswere harvested for activity study Cells were harvested withtrypsin EDTA treatment and protein concentration wasdetermined on a 100 120583L aliquot using the Bio-Rad proteinassay (BioRad Munchen Germany) The cells were pelletedand subsequently suspended in 01 deoxycholate containing1 aprotinin and incubated for 10 min at 4∘C The extractwas microcentrifuged for 5min and the supernatant wasremoved to measure enzymatic activity using the guaiacol
Table 2 Study population
Case (119899 = 200) Control (119899 = 200) 119875 valueSex
Male 29 (145) 32 (16) 088Female 171 (855) 168 (84) 080
Age (Mean plusmn SD) 2985 plusmn 1951 3038 plusmn 1290
and I3
minus assays For the guaiacol assay 50 120583Lof the supernatantwas assayed in a final volume of 750120583L containing 35mMguaiacol and 05mM H
2O2in 01M Tris-HCl pH 86
The absorbance at 470 nm was followed and activity wasexpressed as ΔA sdot minminus1 sdot mg proteinminus1 For the I
3
minus assay25 120583L of the supernatant was used in a final volume of750120583L containing 01M potassium phosphate pH 75 50mMpotassium iodide and 025mM H
2O2 The absorbance at
353 nmwas followed and activitywas expressed asΔAsdotminminus1sdotmg proteinminus1 Spectrophotometric analysis was done on aShimadzu UV-200
Thirty micrograms of the deoxycholate extracted mem-brane protein fraction containing recombinant TPO andnormal control TPO were electrophoresed on a 75 SDS-polyacrylamide gel The gel was blotted onto nitrocelluloseusing a Bio-Rad Mini Protean 2 system (Bio-Rad LabsRichmond CA) followed by incubation with rabbit anti-human TPO antibody (abcam EPR5379) TPO protein wasvisualized using ECL Western blotting detection reagent(abcam)
21 Statistical Methods 1205942 and Fisher exact tests were usedto test the allelic and genotypic associations of each SNPStudentrsquos t-test was used to calculate any statistically signif-icant difference of continuous independent variables like ageTSH and FT4 within the control and patient groups All testswere done using GraphPad InStat software (GraphPad InStatsoftware San Diego CA) Odds ratio and 95 confidentialintervals were also calculated using the same software ABonferroni correction was applied for multiple testing
3 Results
The case and control groups were well balanced in terms ofage and gender There is no significant difference betweentwo groups (Table 2) Case individuals exhibited varied clin-ical manifestations The associated clinical manifestations ofhypothyroidism were goiter (655) lethargy (61) musclecramp (55) loss of memory (51) hoarseness of voice(405) weight loss (8 in male and 30 in female) con-stipation (55) cold intolerance (60) weight gain (45)etc(Table 3)
The TSH level was significantly high and FT4and T
3
levels were significantly low in case population In controlpopulation average serumTSH level was 251plusmn093 120583UmL ascompared to 3514plusmn462 120583UmL in case population SimilarlyserumFT
4level was 125plusmn010 ngdL in control population as
compared to 061 plusmn 027 ngdL in case population Serum T3
4 International Journal of Endocrinology
Table 3 Clinical manifestations of case at initial presentation
Clinical manifestations Case(119899 = 200) Control
(119899 = 200) 119875 valuelowastFamily history
Present 93 465 6 3lt00001
Absent 107 535 194 97lowastGoiter
Present 131 655 mdash mdashlowastWeight gain
Yes 90 45 67 34 0024No 110 55 133 66
Loss of memoryYes 102 51 90 45 027No 98 49 110 55
lowastLethargyYes 122 61 91 46 0002No 78 39 109 54
lowastMuscle crampYes 110 55 88 44 003No 90 45 112 56
lowastCold intoleranceYes 120 60 113 57 054No 80 40 87 43
lowastConstipationYes 109 55 80 40 0005No 91 45 120 60
lowastat the time of diagnosis
level was 164plusmn008 ngdL in control population as comparedto 065 plusmn 022 ngdL in case population 44 patients showedpositive TPO antibody level (Figure 1)
The 123I uptake as shown by 83 patients at 3 hours was481 plusmn790 and 753 plusmn590 of 123I was discharged at 60minafter the oral administration of KClO
4at a dose of 04 g in the
perchlorate discharge test confirming iodide organificationdefect in the thyroid gland In our patient population wefound that 192 patients were within the level of 10ndash20120583gdLIn overall 96 of the patients had UIE levels in the ranges ofoptimal iodine nutrition (10ndash20120583gdL) (WHOUNICEF andICCIDD 2001)
Mutation screening of TPO gene in both case and controlgroups showed six nonsynonymous changes c 1117 GgtTAla 373 Ser c 1193GgtC Ser 398 Thr c 1858 GgtA Asp 620Asn c 1921 GgtA Glu 641 Lys c 2002GgtA Asp668Asnand c 2173AgtC Thr725Pro (Figure 2) After the Bonferronicorrection for multiple comparisons a strong association wasobserved between the Asp 620 Asn Glu 641 Lys andThr 725Pro SNPs and hypothyroidism (Table 4)
To measure wild-type and mutant enzyme activity boththe Iminus and guaiacol assays were carried out Wild-typerecombinant TPO showed enzymatic activity in both assays
Mutant TPO showed relatively nonenzymatic reaction rate(Table 5)
All mutants expressed reduced amount of TPO on theWestern blot In Figure 3(a) Western blot showed expressionof wild-type and mutated recombinant TPO Lane 1 showedwild-type recombinant TPO Lanes 2ndash4 showed recombinantTPO containing (2-Glu 641 Lys 3-Asp 668Asn and 4-Thr 725Pro) changes In Figure 3(b)Western blot showed expressionof wild-type and mutated recombinant TPO Lane 1 showedwild-type recombinant TPO Lanes 2ndash4 showed recombinantTPO Containing (2-Asp 620 Asn 3-Ser 398 Thr and 4-Ala373 Ser) changes
4 Discussion
The present study reveals clinical analysis of hypothyroidismin screened population Hypothyroid patients exhibited sig-nificant increase in the level of serum TSH than controlAs expected patients showed lower level of T
3and FT
4as
compared to control population 83 patients showed positiveresult in the perchlorate discharge test confirming iodideorganification defect in the thyroid glandThe urinary iodineexcretion (UIE) of 96 patients was within the levels of 10ndash20120583gdL which were in the range of optimal iodine nutrition
International Journal of Endocrinology 5
Table4AlleleandGenotyped
istrib
utionof
TPOgene
polymorph
ismsinthes
tudy
SNP
Allele
Allelefre
quency
Odd
sratio
(95
CI)119875value
Genotype
Case
(119899=200)
Con
trol(119899=200)
Odd
sratio
(95
CI)
119875value
Case
Con
trol
c1117
GgtT
G054
059
123(093ndash16
2)017
GG
34
35
Reference
Ala373Ser
T046
041
GT
40
485
GGversus
GT
085
(054ndash
133)
054
TT26
165
GGversus
TT162(094ndash
281)
011
GG+GTversus
TT178(109ndash
290)
0028
c119
3GgtC
C073
068
079
(058ndash10
7)014
CC575
49
Reference
Ser3
98Th
rG
027
032
GC
305
375
CCversus
GC
069
(045ndash10
7)012
GG
12
135
CCversus
GG076
(041ndash14
0)046
c1858GgtA
G090
096
267
(147ndash485)
0001
GG
850
930
Reference
Asp620As
nA
010
004
GA
105
650
GGversus
GA177(086ndash
364
)011
AA
45
050
GGversus
AA985(123ndash7854)
0008
GGversus
GA+AA234(120ndash
457)
0011
c1921
GgtA
G087
094
234
(141ndash388)
0001
GG
775
89
Reference
Glu64
1Lys
A013
006
GA
185
11
GGversus
GA193(109ndash
341)
0022
AA
40
00
GGversus
GA+AA235
(135
ndash409)
0002
c2002
GgtA
G093
094
118(067ndash207)
056
GG
865
885
Reference
Asp66
8As
nA
007
006
GA
125
115
GGversus
GA111
(061ndash203)
073
AA
10
00
c2173
AgtC
A054
063
145(109ndash
192)
001
AA
35
410
Reference
Thr7
25Pro
C046
037
AC375
440
AAversus
AC100(064ndash
156)
099
CC275
150
AAversus
CC215
(124ndash
371)
000
6AA+AC
versus
CC215
(131ndash353
)0002
Chi-squ
aretestw
asused
tocompare
theg
enotypea
ndallelefre
quencies
betweencasesa
ndcontrols
119875valuelt
005
isconsidered
tobe
statisticallysig
nificant
6 International Journal of Endocrinology
0
02
04
06
08
1
12
14
16
18
2
Patient Control
P lt 00001
T 3(N
gm
L)
065 plusmn 022
164 plusmn 008
(a)
0
02
04
06
08
1
12
14
16
Patient Control
P lt 00001
FT4
(Ng
dL)
061 plusmn 027
125 plusmn 010
(b)
0
5
10
15
20
25
30
35
40
45
Patient Control
TSH
(mcU
mL)
P lt 00001
3514 plusmn 462
251 plusmn 093
(c)
44
156
0
20
40
60
80
100
120
140
160N
umbe
r of p
atie
nts
Anti-TPO antibody level (IUmL)gt60 IUmL lt60 IUmL
(d)
Figure 1 T3and FT
4level of patient and control (normal) populations TSH and anti-TPO antibody level of patient and control (normal)
populations
(10ndash20120583gdL) (WHO UNICEF and ICCIDD 2001) There-fore UIE levels indicate that iodine deficiency is currently nota public health problem in our screened population
Our study identified six different SNPs among these threeSNPs (Asp 620 Asn Glu 641 Lys and Thr 725 Pro) aresignificantly associated with hypothyroidism We also try tofind out the association between TPO gene polymorphismsand Anti-TPO level in our study subjects and we did not findany association (data not shown) However several groups indifferent population like Tehranian population [13] and Ira-nian population [14] showed association between TPO genepolymorphisms and anti-TPO level Our study needs muchlarger sample size which may help in better understanding of
association between TPO gene polymorphisms and anti-TPOlevel
Based on the literature Exons 7ndash11 encode the catalyticcenter of the TPO protein (heme binding region) whichis crucial for the enzymatic activity [15] On the otherhand previous findings also indicate that Exons 7ndash14 harbormutational hot spots [15] Thus mutations in these regionsare expected to have major effects on TPO activity resultingin severe organification defect and severe hypothyroidismIn Glu 641 Lys negative charged amino acid is replaced bypositive charged amino acid As we know Threonine is thephosphorylation site of the protein which is important for theactivation of the protein [16 17] Therefore mutations in this
International Journal of Endocrinology 7
A C T
A T C A C T G G C
C C T
A T C C C T G G C
Normal Patient
(a)
A G CCC G C G C C A G C G A G G T
A C CC C G C G C C A C C G A G G T
Normal Patient
(b)
G C GG C G C G C C T G C G G C C T G T G C
T C GG C G C G C C T T C G G C C T G T G C
Normal Patient
(c)
Figure 2 Nucleotide polymorphism in TPO gene study population (a) Nucleotide polymorphism in patient ID-147 Exon11 and ACT gtCCT(Thr gt Pro) (b) Nucleotide polymorphism in patient ID-6 Exon7 and AGC gt ACC (Ser gtThr) (c) Nucleotide polymorphism in patientID-183 Exon7 and GCG gt TCG (Ala gt Ser)
1 2 3 4
GAPDH
(a)
1 2 3 4
GAPDH
(b)
Figure 3 Western blot showing expression of wild-type and mutated recombinant TPO (a) Lane 1 wild-type recombinant TPO Lanes 2ndash4recombinant TPO containing (2-Glu 641 Lys 3-Asp 668Asn and 4-Thr 725 Pro) changes In (b)Western blot showing expression of wild-typeandmutated recombinant TPO Lane 1 wild-type recombinant TPO Lanes 2ndash4 recombinant TPO containing (2-Asp 620Asn 3-Ser 398Thrand 4-Ala 373 Ser) changes
amino acid may change the activity of TPO enzyme whichmay ultimately reduce the functional efficacy of the enzyme
We established the effect of these polymorphisms throughin vitro assay Some polymorphisms (Glu 641 Lys Thr 725Pro andAsp 620Asn)were enzymatically inactive in both theguaiacol and iodide assaysThey also showed reduced amountof TPO on the Western blot This is a strong indication that
the changes are present at crucial positions of the TPO generesulting in inactivated TPO
5 Conclusions
Thus the above changes of amino acids may exert its effecton the structure and functional activity of TPO Our study
8 International Journal of Endocrinology
Table 5 Guaiacol and iodide oxidation activity of expressed humanTPO protein
Mutation Guaiacol oxidation Iodide oxidationWild type 076 plusmn 007 090 plusmn 005
Glu 641 Lys ND lowast
Asp 668 Asn 063 plusmn 007 069 plusmn 027
Thr 725 Pro ND lowast
Asp 620 Asn ND lowast
Ser 398Thr 070 plusmn 027 082 plusmn 029
Ala 373 Ser 062 plusmn 009 lowast
Enzyme activity was activity expressed as Δ119860sdotminminus1sdotmg proteinminus1lowastComparable with nonenzymatic reaction rate (051 plusmn 024) ND nodetectable activity (lt10 of wild-type expressed TPO) [5]
tried to detect the genetic etiology of this disease which mayfurther help us to risk categorize for hypothyroidism Thisstudy also helps to develop a genetic screening protocol forhypothyroidism specifically for the Indian population
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Srikanta Guria and Biswabandhu Bankura contributedequally to this paper
Acknowledgments
This work was supported by Grant from the Depart-ment of Biotechnology Government of India (no BTPR9342Med123462007) The authors would like to thankthe families who gave their consent and collaborated in thisstudy
References
[1] J R Baker Jr P Arscott and J Johnson ldquoAn analysis of thestructure and antigenicity of different forms of human thyroidperoxidaserdquoThyroid vol 4 no 2 pp 173ndash178 1994
[2] J P Banga D Mahadevan G J Barton et al ldquoPrediction ofdomain organisation and secondary structure of thyroid perox-idase a human autoantigen involved in destructive thyroiditisrdquoFEBS Letters vol 266 no 1-2 pp 133ndash141 1990
[3] S Kimura Y-S Hong T Kotani S Ohtaki and F KikkawaldquoStructure of the human thyroid peroxidase gene comparisonand relationship to the humanmyeloperoxidase generdquoBiochem-istry vol 28 no 10 pp 4481ndash4489 1989
[4] 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
[5] H Bikker F Baas and J J De Vijlder ldquoMolecular analy-sis of mutated thyroid peroxidase detected in patients with
total iodide organification defectsrdquo The Journal of ClinicalEndocrinology andMetabolism vol 82 no 2 pp 649ndash653 1997
[6] B Bakker H Bikker T Vulsma J S E De Randamie B MWiedijk and J J M De Vijlder ldquoTwo decades of screeningfor congenital hypothyroidism in the Netherlands TPO genemutations in total iodide organification defects (an update)rdquoJournal of Clinical Endocrinology and Metabolism vol 85 no10 pp 3708ndash3712 2000
[7] T Kotani K Umeki J-I Kawano et al ldquoPartial iodide organ-ification defect caused by a novel mutation of the thyroidperoxidase gene in three siblingsrdquo Clinical Endocrinology vol59 no 2 pp 198ndash206 2003
[8] C Rodrigues P Jorge J Pires Soares et al ldquoMutation screeningof the thyroid peroxidase gene in a cohort of 55 Portuguesepatients with congenital hypothyroidismrdquo European Journal ofEndocrinology vol 152 no 2 pp 193ndash198 2005
[9] 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
[10] A M Maskari and A Alnaqdy ldquoFrequency of thyroid micro-somal and thyroid peroxidase antibody levels in a selectedgroup of Omani patients with Gravesrsquo Diseaserdquo Kuwait MedicalJournal vol 38 no 1 pp 10ndash13 2006
[11] E B Sandell and I M Kolthoff ldquoMicro determination of iodineby a catalytic methodrdquoMikrochimica Acta vol 1 no 1 pp 9ndash251937
[12] S Pino S-L Fang and L E Braverman ldquoAmmonium persul-fate a safe alternative oxidizing reagent for measuring urinaryiodinerdquo Clinical Chemistry vol 42 no 2 pp 239ndash243 1996
[13] B FaamM SDaneshpour F AziziM Salehi andMHedayatildquoAssociation between TPO gene polymorphisms and Anti-TPOlevel in Tehranian population TLGSrdquo Gene vol 498 no 1 pp116ndash119 2012
[14] M Hedayati M S Jahromi M Z Yeganeh M S DaneshpourLH Rad andFAzizi ldquoAssociation between serum level of anti-TPO titer and polymorphisms G1193C Exon 8 and C2145TExon 12 of thyroid peroxidase gene in an Iranian populationrdquoInternational Journal of Endocrinology and Metabolism vol 8no 2 pp 64ndash67 2010
[15] H Bikker T Vulsma F Baas and J J M De Vijlder ldquoIdentifica-tion of five novel inactivating mutations in the human thyroidperoxidase gene by denaturing gradient gel electrophoresisrdquoHuman Mutation vol 6 no 1 pp 9ndash16 1995
[16] M Huse and J Kuriyan ldquoThe conformational plasticity ofprotein kinasesrdquo Cell vol 109 no 3 pp 275ndash282 2002
[17] A Krupa G Preethi and N Srinivasan ldquoStructural modesof stabilization of permissive phosphorylation sites in proteinkinases distinct strategies in SerThr and Tyr kinasesrdquo Journalof Molecular Biology vol 339 no 5 pp 1025ndash1039 2004
Submit your manuscripts athttpwwwhindawicom
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of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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EndocrinologyInternational Journal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
International Journal of Endocrinology 3
Sequencer (PE Applied Biosystems) Purified amplicons wereused for sequencing reaction and the reactionmixture (10120583L)contained 150 ng DNA 5 120583M primer (either forward orreverse) 05 120583L BD and 19 120583L 5 times BD buffer Denaturationat 96∘C for 10 seconds annealing at 50ndash55∘C for 10 secondsand extension at 60∘C for 4min times 25 cycles were performed10 120583LPCRmixwas addedwith solution I (10120583LmqH
2O 2 120583L
125mM EDTA and pH 80) and solution II (50 120583L ehanol2 120583L Na-acetate and pH 46) The mixture was kept in darkfor 25min and centrifuged at room temperature (13000 rpmfor 30min) and sample was dried in speed vac Then 12120583LHi-Di (formamide) was added in sample and stored at darkfor 15min and incubated at 96∘C dry bath and we allowed thesample for snap chill in ice Then the sample was loaded inDNA Sequencer
Sequences alignment between sequences of case andcontrol individuals was performed to find the best matchingpiecewise (local) or global alignments of two query sequencesusing clustalW programme
Polymorphism in the TPO gene identified in this studyincluding nonsynonymous SNPs in the coding region dele-tions and insertions have been assayed in vitro We usedInvitrogen GeneArt Site-Directed Mutagenesis System forthis purpose for example Glu 641 Lys Asp 668Asn Thr725 Pro Asp 620Asn Ser 398Thr and Ala 373 Ser thesechangeswere incorporated intowild TPO cDNA through thismethod
We used pcDNA31 (vector) for TPO cDNA cloningDouble restriction enzyme (EcoRI KpnI) digestions wereperformed by incubating TPO cDNA and vector moleculeswith an appropriate amount of restriction enzyme in itsrespective buffer as recommended by the supplier and at theoptimal temperature The cut DNA fragment and vector arecovalently joined together by DNA ligase
Transformation is the method of introducing foreignDNA into bacterial cells The recombinant plasmids (TPOcDNA + plasmid) were amplified in DH5 alpha competentcells The uptake of recombinant plasmids by DH5 alphacell was carried out in ice-cold CaCl
2(0ndash5∘C) and sub-
sequent heat shock (42∘C for about 90 sec) Selection ofDH5 alpha competent cells containing recombinant plasmids(TPO cDNA + plasmid) was done by ampicillin treatmentPlasmid was purified by use of the plasmid isolation kit(PureLinkQuick PlasmidMiniprepKit Invitrogen)We usedInvitrogen Lipofectamine Reagent for transfection COS 7cells were grown in 38 cm dishes in DMEM supplementedwith 50mLL bovine calf serum and penicillin-streptomycinin 5 CO
2atmosphere at 37∘C When the cells reached
sim90 confluence they were transfected with 1 120583g of recom-binant plasmid DNA (wild-type and mutant) per 38 cmdish with Lipofectamine After incubation for 48 h the cellswere harvested for activity study Cells were harvested withtrypsin EDTA treatment and protein concentration wasdetermined on a 100 120583L aliquot using the Bio-Rad proteinassay (BioRad Munchen Germany) The cells were pelletedand subsequently suspended in 01 deoxycholate containing1 aprotinin and incubated for 10 min at 4∘C The extractwas microcentrifuged for 5min and the supernatant wasremoved to measure enzymatic activity using the guaiacol
Table 2 Study population
Case (119899 = 200) Control (119899 = 200) 119875 valueSex
Male 29 (145) 32 (16) 088Female 171 (855) 168 (84) 080
Age (Mean plusmn SD) 2985 plusmn 1951 3038 plusmn 1290
and I3
minus assays For the guaiacol assay 50 120583Lof the supernatantwas assayed in a final volume of 750120583L containing 35mMguaiacol and 05mM H
2O2in 01M Tris-HCl pH 86
The absorbance at 470 nm was followed and activity wasexpressed as ΔA sdot minminus1 sdot mg proteinminus1 For the I
3
minus assay25 120583L of the supernatant was used in a final volume of750120583L containing 01M potassium phosphate pH 75 50mMpotassium iodide and 025mM H
2O2 The absorbance at
353 nmwas followed and activitywas expressed asΔAsdotminminus1sdotmg proteinminus1 Spectrophotometric analysis was done on aShimadzu UV-200
Thirty micrograms of the deoxycholate extracted mem-brane protein fraction containing recombinant TPO andnormal control TPO were electrophoresed on a 75 SDS-polyacrylamide gel The gel was blotted onto nitrocelluloseusing a Bio-Rad Mini Protean 2 system (Bio-Rad LabsRichmond CA) followed by incubation with rabbit anti-human TPO antibody (abcam EPR5379) TPO protein wasvisualized using ECL Western blotting detection reagent(abcam)
21 Statistical Methods 1205942 and Fisher exact tests were usedto test the allelic and genotypic associations of each SNPStudentrsquos t-test was used to calculate any statistically signif-icant difference of continuous independent variables like ageTSH and FT4 within the control and patient groups All testswere done using GraphPad InStat software (GraphPad InStatsoftware San Diego CA) Odds ratio and 95 confidentialintervals were also calculated using the same software ABonferroni correction was applied for multiple testing
3 Results
The case and control groups were well balanced in terms ofage and gender There is no significant difference betweentwo groups (Table 2) Case individuals exhibited varied clin-ical manifestations The associated clinical manifestations ofhypothyroidism were goiter (655) lethargy (61) musclecramp (55) loss of memory (51) hoarseness of voice(405) weight loss (8 in male and 30 in female) con-stipation (55) cold intolerance (60) weight gain (45)etc(Table 3)
The TSH level was significantly high and FT4and T
3
levels were significantly low in case population In controlpopulation average serumTSH level was 251plusmn093 120583UmL ascompared to 3514plusmn462 120583UmL in case population SimilarlyserumFT
4level was 125plusmn010 ngdL in control population as
compared to 061 plusmn 027 ngdL in case population Serum T3
4 International Journal of Endocrinology
Table 3 Clinical manifestations of case at initial presentation
Clinical manifestations Case(119899 = 200) Control
(119899 = 200) 119875 valuelowastFamily history
Present 93 465 6 3lt00001
Absent 107 535 194 97lowastGoiter
Present 131 655 mdash mdashlowastWeight gain
Yes 90 45 67 34 0024No 110 55 133 66
Loss of memoryYes 102 51 90 45 027No 98 49 110 55
lowastLethargyYes 122 61 91 46 0002No 78 39 109 54
lowastMuscle crampYes 110 55 88 44 003No 90 45 112 56
lowastCold intoleranceYes 120 60 113 57 054No 80 40 87 43
lowastConstipationYes 109 55 80 40 0005No 91 45 120 60
lowastat the time of diagnosis
level was 164plusmn008 ngdL in control population as comparedto 065 plusmn 022 ngdL in case population 44 patients showedpositive TPO antibody level (Figure 1)
The 123I uptake as shown by 83 patients at 3 hours was481 plusmn790 and 753 plusmn590 of 123I was discharged at 60minafter the oral administration of KClO
4at a dose of 04 g in the
perchlorate discharge test confirming iodide organificationdefect in the thyroid gland In our patient population wefound that 192 patients were within the level of 10ndash20120583gdLIn overall 96 of the patients had UIE levels in the ranges ofoptimal iodine nutrition (10ndash20120583gdL) (WHOUNICEF andICCIDD 2001)
Mutation screening of TPO gene in both case and controlgroups showed six nonsynonymous changes c 1117 GgtTAla 373 Ser c 1193GgtC Ser 398 Thr c 1858 GgtA Asp 620Asn c 1921 GgtA Glu 641 Lys c 2002GgtA Asp668Asnand c 2173AgtC Thr725Pro (Figure 2) After the Bonferronicorrection for multiple comparisons a strong association wasobserved between the Asp 620 Asn Glu 641 Lys andThr 725Pro SNPs and hypothyroidism (Table 4)
To measure wild-type and mutant enzyme activity boththe Iminus and guaiacol assays were carried out Wild-typerecombinant TPO showed enzymatic activity in both assays
Mutant TPO showed relatively nonenzymatic reaction rate(Table 5)
All mutants expressed reduced amount of TPO on theWestern blot In Figure 3(a) Western blot showed expressionof wild-type and mutated recombinant TPO Lane 1 showedwild-type recombinant TPO Lanes 2ndash4 showed recombinantTPO containing (2-Glu 641 Lys 3-Asp 668Asn and 4-Thr 725Pro) changes In Figure 3(b)Western blot showed expressionof wild-type and mutated recombinant TPO Lane 1 showedwild-type recombinant TPO Lanes 2ndash4 showed recombinantTPO Containing (2-Asp 620 Asn 3-Ser 398 Thr and 4-Ala373 Ser) changes
4 Discussion
The present study reveals clinical analysis of hypothyroidismin screened population Hypothyroid patients exhibited sig-nificant increase in the level of serum TSH than controlAs expected patients showed lower level of T
3and FT
4as
compared to control population 83 patients showed positiveresult in the perchlorate discharge test confirming iodideorganification defect in the thyroid glandThe urinary iodineexcretion (UIE) of 96 patients was within the levels of 10ndash20120583gdL which were in the range of optimal iodine nutrition
International Journal of Endocrinology 5
Table4AlleleandGenotyped
istrib
utionof
TPOgene
polymorph
ismsinthes
tudy
SNP
Allele
Allelefre
quency
Odd
sratio
(95
CI)119875value
Genotype
Case
(119899=200)
Con
trol(119899=200)
Odd
sratio
(95
CI)
119875value
Case
Con
trol
c1117
GgtT
G054
059
123(093ndash16
2)017
GG
34
35
Reference
Ala373Ser
T046
041
GT
40
485
GGversus
GT
085
(054ndash
133)
054
TT26
165
GGversus
TT162(094ndash
281)
011
GG+GTversus
TT178(109ndash
290)
0028
c119
3GgtC
C073
068
079
(058ndash10
7)014
CC575
49
Reference
Ser3
98Th
rG
027
032
GC
305
375
CCversus
GC
069
(045ndash10
7)012
GG
12
135
CCversus
GG076
(041ndash14
0)046
c1858GgtA
G090
096
267
(147ndash485)
0001
GG
850
930
Reference
Asp620As
nA
010
004
GA
105
650
GGversus
GA177(086ndash
364
)011
AA
45
050
GGversus
AA985(123ndash7854)
0008
GGversus
GA+AA234(120ndash
457)
0011
c1921
GgtA
G087
094
234
(141ndash388)
0001
GG
775
89
Reference
Glu64
1Lys
A013
006
GA
185
11
GGversus
GA193(109ndash
341)
0022
AA
40
00
GGversus
GA+AA235
(135
ndash409)
0002
c2002
GgtA
G093
094
118(067ndash207)
056
GG
865
885
Reference
Asp66
8As
nA
007
006
GA
125
115
GGversus
GA111
(061ndash203)
073
AA
10
00
c2173
AgtC
A054
063
145(109ndash
192)
001
AA
35
410
Reference
Thr7
25Pro
C046
037
AC375
440
AAversus
AC100(064ndash
156)
099
CC275
150
AAversus
CC215
(124ndash
371)
000
6AA+AC
versus
CC215
(131ndash353
)0002
Chi-squ
aretestw
asused
tocompare
theg
enotypea
ndallelefre
quencies
betweencasesa
ndcontrols
119875valuelt
005
isconsidered
tobe
statisticallysig
nificant
6 International Journal of Endocrinology
0
02
04
06
08
1
12
14
16
18
2
Patient Control
P lt 00001
T 3(N
gm
L)
065 plusmn 022
164 plusmn 008
(a)
0
02
04
06
08
1
12
14
16
Patient Control
P lt 00001
FT4
(Ng
dL)
061 plusmn 027
125 plusmn 010
(b)
0
5
10
15
20
25
30
35
40
45
Patient Control
TSH
(mcU
mL)
P lt 00001
3514 plusmn 462
251 plusmn 093
(c)
44
156
0
20
40
60
80
100
120
140
160N
umbe
r of p
atie
nts
Anti-TPO antibody level (IUmL)gt60 IUmL lt60 IUmL
(d)
Figure 1 T3and FT
4level of patient and control (normal) populations TSH and anti-TPO antibody level of patient and control (normal)
populations
(10ndash20120583gdL) (WHO UNICEF and ICCIDD 2001) There-fore UIE levels indicate that iodine deficiency is currently nota public health problem in our screened population
Our study identified six different SNPs among these threeSNPs (Asp 620 Asn Glu 641 Lys and Thr 725 Pro) aresignificantly associated with hypothyroidism We also try tofind out the association between TPO gene polymorphismsand Anti-TPO level in our study subjects and we did not findany association (data not shown) However several groups indifferent population like Tehranian population [13] and Ira-nian population [14] showed association between TPO genepolymorphisms and anti-TPO level Our study needs muchlarger sample size which may help in better understanding of
association between TPO gene polymorphisms and anti-TPOlevel
Based on the literature Exons 7ndash11 encode the catalyticcenter of the TPO protein (heme binding region) whichis crucial for the enzymatic activity [15] On the otherhand previous findings also indicate that Exons 7ndash14 harbormutational hot spots [15] Thus mutations in these regionsare expected to have major effects on TPO activity resultingin severe organification defect and severe hypothyroidismIn Glu 641 Lys negative charged amino acid is replaced bypositive charged amino acid As we know Threonine is thephosphorylation site of the protein which is important for theactivation of the protein [16 17] Therefore mutations in this
International Journal of Endocrinology 7
A C T
A T C A C T G G C
C C T
A T C C C T G G C
Normal Patient
(a)
A G CCC G C G C C A G C G A G G T
A C CC C G C G C C A C C G A G G T
Normal Patient
(b)
G C GG C G C G C C T G C G G C C T G T G C
T C GG C G C G C C T T C G G C C T G T G C
Normal Patient
(c)
Figure 2 Nucleotide polymorphism in TPO gene study population (a) Nucleotide polymorphism in patient ID-147 Exon11 and ACT gtCCT(Thr gt Pro) (b) Nucleotide polymorphism in patient ID-6 Exon7 and AGC gt ACC (Ser gtThr) (c) Nucleotide polymorphism in patientID-183 Exon7 and GCG gt TCG (Ala gt Ser)
1 2 3 4
GAPDH
(a)
1 2 3 4
GAPDH
(b)
Figure 3 Western blot showing expression of wild-type and mutated recombinant TPO (a) Lane 1 wild-type recombinant TPO Lanes 2ndash4recombinant TPO containing (2-Glu 641 Lys 3-Asp 668Asn and 4-Thr 725 Pro) changes In (b)Western blot showing expression of wild-typeandmutated recombinant TPO Lane 1 wild-type recombinant TPO Lanes 2ndash4 recombinant TPO containing (2-Asp 620Asn 3-Ser 398Thrand 4-Ala 373 Ser) changes
amino acid may change the activity of TPO enzyme whichmay ultimately reduce the functional efficacy of the enzyme
We established the effect of these polymorphisms throughin vitro assay Some polymorphisms (Glu 641 Lys Thr 725Pro andAsp 620Asn)were enzymatically inactive in both theguaiacol and iodide assaysThey also showed reduced amountof TPO on the Western blot This is a strong indication that
the changes are present at crucial positions of the TPO generesulting in inactivated TPO
5 Conclusions
Thus the above changes of amino acids may exert its effecton the structure and functional activity of TPO Our study
8 International Journal of Endocrinology
Table 5 Guaiacol and iodide oxidation activity of expressed humanTPO protein
Mutation Guaiacol oxidation Iodide oxidationWild type 076 plusmn 007 090 plusmn 005
Glu 641 Lys ND lowast
Asp 668 Asn 063 plusmn 007 069 plusmn 027
Thr 725 Pro ND lowast
Asp 620 Asn ND lowast
Ser 398Thr 070 plusmn 027 082 plusmn 029
Ala 373 Ser 062 plusmn 009 lowast
Enzyme activity was activity expressed as Δ119860sdotminminus1sdotmg proteinminus1lowastComparable with nonenzymatic reaction rate (051 plusmn 024) ND nodetectable activity (lt10 of wild-type expressed TPO) [5]
tried to detect the genetic etiology of this disease which mayfurther help us to risk categorize for hypothyroidism Thisstudy also helps to develop a genetic screening protocol forhypothyroidism specifically for the Indian population
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Srikanta Guria and Biswabandhu Bankura contributedequally to this paper
Acknowledgments
This work was supported by Grant from the Depart-ment of Biotechnology Government of India (no BTPR9342Med123462007) The authors would like to thankthe families who gave their consent and collaborated in thisstudy
References
[1] J R Baker Jr P Arscott and J Johnson ldquoAn analysis of thestructure and antigenicity of different forms of human thyroidperoxidaserdquoThyroid vol 4 no 2 pp 173ndash178 1994
[2] J P Banga D Mahadevan G J Barton et al ldquoPrediction ofdomain organisation and secondary structure of thyroid perox-idase a human autoantigen involved in destructive thyroiditisrdquoFEBS Letters vol 266 no 1-2 pp 133ndash141 1990
[3] S Kimura Y-S Hong T Kotani S Ohtaki and F KikkawaldquoStructure of the human thyroid peroxidase gene comparisonand relationship to the humanmyeloperoxidase generdquoBiochem-istry vol 28 no 10 pp 4481ndash4489 1989
[4] 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
[5] H Bikker F Baas and J J De Vijlder ldquoMolecular analy-sis of mutated thyroid peroxidase detected in patients with
total iodide organification defectsrdquo The Journal of ClinicalEndocrinology andMetabolism vol 82 no 2 pp 649ndash653 1997
[6] B Bakker H Bikker T Vulsma J S E De Randamie B MWiedijk and J J M De Vijlder ldquoTwo decades of screeningfor congenital hypothyroidism in the Netherlands TPO genemutations in total iodide organification defects (an update)rdquoJournal of Clinical Endocrinology and Metabolism vol 85 no10 pp 3708ndash3712 2000
[7] T Kotani K Umeki J-I Kawano et al ldquoPartial iodide organ-ification defect caused by a novel mutation of the thyroidperoxidase gene in three siblingsrdquo Clinical Endocrinology vol59 no 2 pp 198ndash206 2003
[8] C Rodrigues P Jorge J Pires Soares et al ldquoMutation screeningof the thyroid peroxidase gene in a cohort of 55 Portuguesepatients with congenital hypothyroidismrdquo European Journal ofEndocrinology vol 152 no 2 pp 193ndash198 2005
[9] 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
[10] A M Maskari and A Alnaqdy ldquoFrequency of thyroid micro-somal and thyroid peroxidase antibody levels in a selectedgroup of Omani patients with Gravesrsquo Diseaserdquo Kuwait MedicalJournal vol 38 no 1 pp 10ndash13 2006
[11] E B Sandell and I M Kolthoff ldquoMicro determination of iodineby a catalytic methodrdquoMikrochimica Acta vol 1 no 1 pp 9ndash251937
[12] S Pino S-L Fang and L E Braverman ldquoAmmonium persul-fate a safe alternative oxidizing reagent for measuring urinaryiodinerdquo Clinical Chemistry vol 42 no 2 pp 239ndash243 1996
[13] B FaamM SDaneshpour F AziziM Salehi andMHedayatildquoAssociation between TPO gene polymorphisms and Anti-TPOlevel in Tehranian population TLGSrdquo Gene vol 498 no 1 pp116ndash119 2012
[14] M Hedayati M S Jahromi M Z Yeganeh M S DaneshpourLH Rad andFAzizi ldquoAssociation between serum level of anti-TPO titer and polymorphisms G1193C Exon 8 and C2145TExon 12 of thyroid peroxidase gene in an Iranian populationrdquoInternational Journal of Endocrinology and Metabolism vol 8no 2 pp 64ndash67 2010
[15] H Bikker T Vulsma F Baas and J J M De Vijlder ldquoIdentifica-tion of five novel inactivating mutations in the human thyroidperoxidase gene by denaturing gradient gel electrophoresisrdquoHuman Mutation vol 6 no 1 pp 9ndash16 1995
[16] M Huse and J Kuriyan ldquoThe conformational plasticity ofprotein kinasesrdquo Cell vol 109 no 3 pp 275ndash282 2002
[17] A Krupa G Preethi and N Srinivasan ldquoStructural modesof stabilization of permissive phosphorylation sites in proteinkinases distinct strategies in SerThr and Tyr kinasesrdquo Journalof Molecular Biology vol 339 no 5 pp 1025ndash1039 2004
Submit your manuscripts athttpwwwhindawicom
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Research and TreatmentAIDS
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Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
4 International Journal of Endocrinology
Table 3 Clinical manifestations of case at initial presentation
Clinical manifestations Case(119899 = 200) Control
(119899 = 200) 119875 valuelowastFamily history
Present 93 465 6 3lt00001
Absent 107 535 194 97lowastGoiter
Present 131 655 mdash mdashlowastWeight gain
Yes 90 45 67 34 0024No 110 55 133 66
Loss of memoryYes 102 51 90 45 027No 98 49 110 55
lowastLethargyYes 122 61 91 46 0002No 78 39 109 54
lowastMuscle crampYes 110 55 88 44 003No 90 45 112 56
lowastCold intoleranceYes 120 60 113 57 054No 80 40 87 43
lowastConstipationYes 109 55 80 40 0005No 91 45 120 60
lowastat the time of diagnosis
level was 164plusmn008 ngdL in control population as comparedto 065 plusmn 022 ngdL in case population 44 patients showedpositive TPO antibody level (Figure 1)
The 123I uptake as shown by 83 patients at 3 hours was481 plusmn790 and 753 plusmn590 of 123I was discharged at 60minafter the oral administration of KClO
4at a dose of 04 g in the
perchlorate discharge test confirming iodide organificationdefect in the thyroid gland In our patient population wefound that 192 patients were within the level of 10ndash20120583gdLIn overall 96 of the patients had UIE levels in the ranges ofoptimal iodine nutrition (10ndash20120583gdL) (WHOUNICEF andICCIDD 2001)
Mutation screening of TPO gene in both case and controlgroups showed six nonsynonymous changes c 1117 GgtTAla 373 Ser c 1193GgtC Ser 398 Thr c 1858 GgtA Asp 620Asn c 1921 GgtA Glu 641 Lys c 2002GgtA Asp668Asnand c 2173AgtC Thr725Pro (Figure 2) After the Bonferronicorrection for multiple comparisons a strong association wasobserved between the Asp 620 Asn Glu 641 Lys andThr 725Pro SNPs and hypothyroidism (Table 4)
To measure wild-type and mutant enzyme activity boththe Iminus and guaiacol assays were carried out Wild-typerecombinant TPO showed enzymatic activity in both assays
Mutant TPO showed relatively nonenzymatic reaction rate(Table 5)
All mutants expressed reduced amount of TPO on theWestern blot In Figure 3(a) Western blot showed expressionof wild-type and mutated recombinant TPO Lane 1 showedwild-type recombinant TPO Lanes 2ndash4 showed recombinantTPO containing (2-Glu 641 Lys 3-Asp 668Asn and 4-Thr 725Pro) changes In Figure 3(b)Western blot showed expressionof wild-type and mutated recombinant TPO Lane 1 showedwild-type recombinant TPO Lanes 2ndash4 showed recombinantTPO Containing (2-Asp 620 Asn 3-Ser 398 Thr and 4-Ala373 Ser) changes
4 Discussion
The present study reveals clinical analysis of hypothyroidismin screened population Hypothyroid patients exhibited sig-nificant increase in the level of serum TSH than controlAs expected patients showed lower level of T
3and FT
4as
compared to control population 83 patients showed positiveresult in the perchlorate discharge test confirming iodideorganification defect in the thyroid glandThe urinary iodineexcretion (UIE) of 96 patients was within the levels of 10ndash20120583gdL which were in the range of optimal iodine nutrition
International Journal of Endocrinology 5
Table4AlleleandGenotyped
istrib
utionof
TPOgene
polymorph
ismsinthes
tudy
SNP
Allele
Allelefre
quency
Odd
sratio
(95
CI)119875value
Genotype
Case
(119899=200)
Con
trol(119899=200)
Odd
sratio
(95
CI)
119875value
Case
Con
trol
c1117
GgtT
G054
059
123(093ndash16
2)017
GG
34
35
Reference
Ala373Ser
T046
041
GT
40
485
GGversus
GT
085
(054ndash
133)
054
TT26
165
GGversus
TT162(094ndash
281)
011
GG+GTversus
TT178(109ndash
290)
0028
c119
3GgtC
C073
068
079
(058ndash10
7)014
CC575
49
Reference
Ser3
98Th
rG
027
032
GC
305
375
CCversus
GC
069
(045ndash10
7)012
GG
12
135
CCversus
GG076
(041ndash14
0)046
c1858GgtA
G090
096
267
(147ndash485)
0001
GG
850
930
Reference
Asp620As
nA
010
004
GA
105
650
GGversus
GA177(086ndash
364
)011
AA
45
050
GGversus
AA985(123ndash7854)
0008
GGversus
GA+AA234(120ndash
457)
0011
c1921
GgtA
G087
094
234
(141ndash388)
0001
GG
775
89
Reference
Glu64
1Lys
A013
006
GA
185
11
GGversus
GA193(109ndash
341)
0022
AA
40
00
GGversus
GA+AA235
(135
ndash409)
0002
c2002
GgtA
G093
094
118(067ndash207)
056
GG
865
885
Reference
Asp66
8As
nA
007
006
GA
125
115
GGversus
GA111
(061ndash203)
073
AA
10
00
c2173
AgtC
A054
063
145(109ndash
192)
001
AA
35
410
Reference
Thr7
25Pro
C046
037
AC375
440
AAversus
AC100(064ndash
156)
099
CC275
150
AAversus
CC215
(124ndash
371)
000
6AA+AC
versus
CC215
(131ndash353
)0002
Chi-squ
aretestw
asused
tocompare
theg
enotypea
ndallelefre
quencies
betweencasesa
ndcontrols
119875valuelt
005
isconsidered
tobe
statisticallysig
nificant
6 International Journal of Endocrinology
0
02
04
06
08
1
12
14
16
18
2
Patient Control
P lt 00001
T 3(N
gm
L)
065 plusmn 022
164 plusmn 008
(a)
0
02
04
06
08
1
12
14
16
Patient Control
P lt 00001
FT4
(Ng
dL)
061 plusmn 027
125 plusmn 010
(b)
0
5
10
15
20
25
30
35
40
45
Patient Control
TSH
(mcU
mL)
P lt 00001
3514 plusmn 462
251 plusmn 093
(c)
44
156
0
20
40
60
80
100
120
140
160N
umbe
r of p
atie
nts
Anti-TPO antibody level (IUmL)gt60 IUmL lt60 IUmL
(d)
Figure 1 T3and FT
4level of patient and control (normal) populations TSH and anti-TPO antibody level of patient and control (normal)
populations
(10ndash20120583gdL) (WHO UNICEF and ICCIDD 2001) There-fore UIE levels indicate that iodine deficiency is currently nota public health problem in our screened population
Our study identified six different SNPs among these threeSNPs (Asp 620 Asn Glu 641 Lys and Thr 725 Pro) aresignificantly associated with hypothyroidism We also try tofind out the association between TPO gene polymorphismsand Anti-TPO level in our study subjects and we did not findany association (data not shown) However several groups indifferent population like Tehranian population [13] and Ira-nian population [14] showed association between TPO genepolymorphisms and anti-TPO level Our study needs muchlarger sample size which may help in better understanding of
association between TPO gene polymorphisms and anti-TPOlevel
Based on the literature Exons 7ndash11 encode the catalyticcenter of the TPO protein (heme binding region) whichis crucial for the enzymatic activity [15] On the otherhand previous findings also indicate that Exons 7ndash14 harbormutational hot spots [15] Thus mutations in these regionsare expected to have major effects on TPO activity resultingin severe organification defect and severe hypothyroidismIn Glu 641 Lys negative charged amino acid is replaced bypositive charged amino acid As we know Threonine is thephosphorylation site of the protein which is important for theactivation of the protein [16 17] Therefore mutations in this
International Journal of Endocrinology 7
A C T
A T C A C T G G C
C C T
A T C C C T G G C
Normal Patient
(a)
A G CCC G C G C C A G C G A G G T
A C CC C G C G C C A C C G A G G T
Normal Patient
(b)
G C GG C G C G C C T G C G G C C T G T G C
T C GG C G C G C C T T C G G C C T G T G C
Normal Patient
(c)
Figure 2 Nucleotide polymorphism in TPO gene study population (a) Nucleotide polymorphism in patient ID-147 Exon11 and ACT gtCCT(Thr gt Pro) (b) Nucleotide polymorphism in patient ID-6 Exon7 and AGC gt ACC (Ser gtThr) (c) Nucleotide polymorphism in patientID-183 Exon7 and GCG gt TCG (Ala gt Ser)
1 2 3 4
GAPDH
(a)
1 2 3 4
GAPDH
(b)
Figure 3 Western blot showing expression of wild-type and mutated recombinant TPO (a) Lane 1 wild-type recombinant TPO Lanes 2ndash4recombinant TPO containing (2-Glu 641 Lys 3-Asp 668Asn and 4-Thr 725 Pro) changes In (b)Western blot showing expression of wild-typeandmutated recombinant TPO Lane 1 wild-type recombinant TPO Lanes 2ndash4 recombinant TPO containing (2-Asp 620Asn 3-Ser 398Thrand 4-Ala 373 Ser) changes
amino acid may change the activity of TPO enzyme whichmay ultimately reduce the functional efficacy of the enzyme
We established the effect of these polymorphisms throughin vitro assay Some polymorphisms (Glu 641 Lys Thr 725Pro andAsp 620Asn)were enzymatically inactive in both theguaiacol and iodide assaysThey also showed reduced amountof TPO on the Western blot This is a strong indication that
the changes are present at crucial positions of the TPO generesulting in inactivated TPO
5 Conclusions
Thus the above changes of amino acids may exert its effecton the structure and functional activity of TPO Our study
8 International Journal of Endocrinology
Table 5 Guaiacol and iodide oxidation activity of expressed humanTPO protein
Mutation Guaiacol oxidation Iodide oxidationWild type 076 plusmn 007 090 plusmn 005
Glu 641 Lys ND lowast
Asp 668 Asn 063 plusmn 007 069 plusmn 027
Thr 725 Pro ND lowast
Asp 620 Asn ND lowast
Ser 398Thr 070 plusmn 027 082 plusmn 029
Ala 373 Ser 062 plusmn 009 lowast
Enzyme activity was activity expressed as Δ119860sdotminminus1sdotmg proteinminus1lowastComparable with nonenzymatic reaction rate (051 plusmn 024) ND nodetectable activity (lt10 of wild-type expressed TPO) [5]
tried to detect the genetic etiology of this disease which mayfurther help us to risk categorize for hypothyroidism Thisstudy also helps to develop a genetic screening protocol forhypothyroidism specifically for the Indian population
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Srikanta Guria and Biswabandhu Bankura contributedequally to this paper
Acknowledgments
This work was supported by Grant from the Depart-ment of Biotechnology Government of India (no BTPR9342Med123462007) The authors would like to thankthe families who gave their consent and collaborated in thisstudy
References
[1] J R Baker Jr P Arscott and J Johnson ldquoAn analysis of thestructure and antigenicity of different forms of human thyroidperoxidaserdquoThyroid vol 4 no 2 pp 173ndash178 1994
[2] J P Banga D Mahadevan G J Barton et al ldquoPrediction ofdomain organisation and secondary structure of thyroid perox-idase a human autoantigen involved in destructive thyroiditisrdquoFEBS Letters vol 266 no 1-2 pp 133ndash141 1990
[3] S Kimura Y-S Hong T Kotani S Ohtaki and F KikkawaldquoStructure of the human thyroid peroxidase gene comparisonand relationship to the humanmyeloperoxidase generdquoBiochem-istry vol 28 no 10 pp 4481ndash4489 1989
[4] 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
[5] H Bikker F Baas and J J De Vijlder ldquoMolecular analy-sis of mutated thyroid peroxidase detected in patients with
total iodide organification defectsrdquo The Journal of ClinicalEndocrinology andMetabolism vol 82 no 2 pp 649ndash653 1997
[6] B Bakker H Bikker T Vulsma J S E De Randamie B MWiedijk and J J M De Vijlder ldquoTwo decades of screeningfor congenital hypothyroidism in the Netherlands TPO genemutations in total iodide organification defects (an update)rdquoJournal of Clinical Endocrinology and Metabolism vol 85 no10 pp 3708ndash3712 2000
[7] T Kotani K Umeki J-I Kawano et al ldquoPartial iodide organ-ification defect caused by a novel mutation of the thyroidperoxidase gene in three siblingsrdquo Clinical Endocrinology vol59 no 2 pp 198ndash206 2003
[8] C Rodrigues P Jorge J Pires Soares et al ldquoMutation screeningof the thyroid peroxidase gene in a cohort of 55 Portuguesepatients with congenital hypothyroidismrdquo European Journal ofEndocrinology vol 152 no 2 pp 193ndash198 2005
[9] 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
[10] A M Maskari and A Alnaqdy ldquoFrequency of thyroid micro-somal and thyroid peroxidase antibody levels in a selectedgroup of Omani patients with Gravesrsquo Diseaserdquo Kuwait MedicalJournal vol 38 no 1 pp 10ndash13 2006
[11] E B Sandell and I M Kolthoff ldquoMicro determination of iodineby a catalytic methodrdquoMikrochimica Acta vol 1 no 1 pp 9ndash251937
[12] S Pino S-L Fang and L E Braverman ldquoAmmonium persul-fate a safe alternative oxidizing reagent for measuring urinaryiodinerdquo Clinical Chemistry vol 42 no 2 pp 239ndash243 1996
[13] B FaamM SDaneshpour F AziziM Salehi andMHedayatildquoAssociation between TPO gene polymorphisms and Anti-TPOlevel in Tehranian population TLGSrdquo Gene vol 498 no 1 pp116ndash119 2012
[14] M Hedayati M S Jahromi M Z Yeganeh M S DaneshpourLH Rad andFAzizi ldquoAssociation between serum level of anti-TPO titer and polymorphisms G1193C Exon 8 and C2145TExon 12 of thyroid peroxidase gene in an Iranian populationrdquoInternational Journal of Endocrinology and Metabolism vol 8no 2 pp 64ndash67 2010
[15] H Bikker T Vulsma F Baas and J J M De Vijlder ldquoIdentifica-tion of five novel inactivating mutations in the human thyroidperoxidase gene by denaturing gradient gel electrophoresisrdquoHuman Mutation vol 6 no 1 pp 9ndash16 1995
[16] M Huse and J Kuriyan ldquoThe conformational plasticity ofprotein kinasesrdquo Cell vol 109 no 3 pp 275ndash282 2002
[17] A Krupa G Preethi and N Srinivasan ldquoStructural modesof stabilization of permissive phosphorylation sites in proteinkinases distinct strategies in SerThr and Tyr kinasesrdquo Journalof Molecular Biology vol 339 no 5 pp 1025ndash1039 2004
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
International Journal of Endocrinology 5
Table4AlleleandGenotyped
istrib
utionof
TPOgene
polymorph
ismsinthes
tudy
SNP
Allele
Allelefre
quency
Odd
sratio
(95
CI)119875value
Genotype
Case
(119899=200)
Con
trol(119899=200)
Odd
sratio
(95
CI)
119875value
Case
Con
trol
c1117
GgtT
G054
059
123(093ndash16
2)017
GG
34
35
Reference
Ala373Ser
T046
041
GT
40
485
GGversus
GT
085
(054ndash
133)
054
TT26
165
GGversus
TT162(094ndash
281)
011
GG+GTversus
TT178(109ndash
290)
0028
c119
3GgtC
C073
068
079
(058ndash10
7)014
CC575
49
Reference
Ser3
98Th
rG
027
032
GC
305
375
CCversus
GC
069
(045ndash10
7)012
GG
12
135
CCversus
GG076
(041ndash14
0)046
c1858GgtA
G090
096
267
(147ndash485)
0001
GG
850
930
Reference
Asp620As
nA
010
004
GA
105
650
GGversus
GA177(086ndash
364
)011
AA
45
050
GGversus
AA985(123ndash7854)
0008
GGversus
GA+AA234(120ndash
457)
0011
c1921
GgtA
G087
094
234
(141ndash388)
0001
GG
775
89
Reference
Glu64
1Lys
A013
006
GA
185
11
GGversus
GA193(109ndash
341)
0022
AA
40
00
GGversus
GA+AA235
(135
ndash409)
0002
c2002
GgtA
G093
094
118(067ndash207)
056
GG
865
885
Reference
Asp66
8As
nA
007
006
GA
125
115
GGversus
GA111
(061ndash203)
073
AA
10
00
c2173
AgtC
A054
063
145(109ndash
192)
001
AA
35
410
Reference
Thr7
25Pro
C046
037
AC375
440
AAversus
AC100(064ndash
156)
099
CC275
150
AAversus
CC215
(124ndash
371)
000
6AA+AC
versus
CC215
(131ndash353
)0002
Chi-squ
aretestw
asused
tocompare
theg
enotypea
ndallelefre
quencies
betweencasesa
ndcontrols
119875valuelt
005
isconsidered
tobe
statisticallysig
nificant
6 International Journal of Endocrinology
0
02
04
06
08
1
12
14
16
18
2
Patient Control
P lt 00001
T 3(N
gm
L)
065 plusmn 022
164 plusmn 008
(a)
0
02
04
06
08
1
12
14
16
Patient Control
P lt 00001
FT4
(Ng
dL)
061 plusmn 027
125 plusmn 010
(b)
0
5
10
15
20
25
30
35
40
45
Patient Control
TSH
(mcU
mL)
P lt 00001
3514 plusmn 462
251 plusmn 093
(c)
44
156
0
20
40
60
80
100
120
140
160N
umbe
r of p
atie
nts
Anti-TPO antibody level (IUmL)gt60 IUmL lt60 IUmL
(d)
Figure 1 T3and FT
4level of patient and control (normal) populations TSH and anti-TPO antibody level of patient and control (normal)
populations
(10ndash20120583gdL) (WHO UNICEF and ICCIDD 2001) There-fore UIE levels indicate that iodine deficiency is currently nota public health problem in our screened population
Our study identified six different SNPs among these threeSNPs (Asp 620 Asn Glu 641 Lys and Thr 725 Pro) aresignificantly associated with hypothyroidism We also try tofind out the association between TPO gene polymorphismsand Anti-TPO level in our study subjects and we did not findany association (data not shown) However several groups indifferent population like Tehranian population [13] and Ira-nian population [14] showed association between TPO genepolymorphisms and anti-TPO level Our study needs muchlarger sample size which may help in better understanding of
association between TPO gene polymorphisms and anti-TPOlevel
Based on the literature Exons 7ndash11 encode the catalyticcenter of the TPO protein (heme binding region) whichis crucial for the enzymatic activity [15] On the otherhand previous findings also indicate that Exons 7ndash14 harbormutational hot spots [15] Thus mutations in these regionsare expected to have major effects on TPO activity resultingin severe organification defect and severe hypothyroidismIn Glu 641 Lys negative charged amino acid is replaced bypositive charged amino acid As we know Threonine is thephosphorylation site of the protein which is important for theactivation of the protein [16 17] Therefore mutations in this
International Journal of Endocrinology 7
A C T
A T C A C T G G C
C C T
A T C C C T G G C
Normal Patient
(a)
A G CCC G C G C C A G C G A G G T
A C CC C G C G C C A C C G A G G T
Normal Patient
(b)
G C GG C G C G C C T G C G G C C T G T G C
T C GG C G C G C C T T C G G C C T G T G C
Normal Patient
(c)
Figure 2 Nucleotide polymorphism in TPO gene study population (a) Nucleotide polymorphism in patient ID-147 Exon11 and ACT gtCCT(Thr gt Pro) (b) Nucleotide polymorphism in patient ID-6 Exon7 and AGC gt ACC (Ser gtThr) (c) Nucleotide polymorphism in patientID-183 Exon7 and GCG gt TCG (Ala gt Ser)
1 2 3 4
GAPDH
(a)
1 2 3 4
GAPDH
(b)
Figure 3 Western blot showing expression of wild-type and mutated recombinant TPO (a) Lane 1 wild-type recombinant TPO Lanes 2ndash4recombinant TPO containing (2-Glu 641 Lys 3-Asp 668Asn and 4-Thr 725 Pro) changes In (b)Western blot showing expression of wild-typeandmutated recombinant TPO Lane 1 wild-type recombinant TPO Lanes 2ndash4 recombinant TPO containing (2-Asp 620Asn 3-Ser 398Thrand 4-Ala 373 Ser) changes
amino acid may change the activity of TPO enzyme whichmay ultimately reduce the functional efficacy of the enzyme
We established the effect of these polymorphisms throughin vitro assay Some polymorphisms (Glu 641 Lys Thr 725Pro andAsp 620Asn)were enzymatically inactive in both theguaiacol and iodide assaysThey also showed reduced amountof TPO on the Western blot This is a strong indication that
the changes are present at crucial positions of the TPO generesulting in inactivated TPO
5 Conclusions
Thus the above changes of amino acids may exert its effecton the structure and functional activity of TPO Our study
8 International Journal of Endocrinology
Table 5 Guaiacol and iodide oxidation activity of expressed humanTPO protein
Mutation Guaiacol oxidation Iodide oxidationWild type 076 plusmn 007 090 plusmn 005
Glu 641 Lys ND lowast
Asp 668 Asn 063 plusmn 007 069 plusmn 027
Thr 725 Pro ND lowast
Asp 620 Asn ND lowast
Ser 398Thr 070 plusmn 027 082 plusmn 029
Ala 373 Ser 062 plusmn 009 lowast
Enzyme activity was activity expressed as Δ119860sdotminminus1sdotmg proteinminus1lowastComparable with nonenzymatic reaction rate (051 plusmn 024) ND nodetectable activity (lt10 of wild-type expressed TPO) [5]
tried to detect the genetic etiology of this disease which mayfurther help us to risk categorize for hypothyroidism Thisstudy also helps to develop a genetic screening protocol forhypothyroidism specifically for the Indian population
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Srikanta Guria and Biswabandhu Bankura contributedequally to this paper
Acknowledgments
This work was supported by Grant from the Depart-ment of Biotechnology Government of India (no BTPR9342Med123462007) The authors would like to thankthe families who gave their consent and collaborated in thisstudy
References
[1] J R Baker Jr P Arscott and J Johnson ldquoAn analysis of thestructure and antigenicity of different forms of human thyroidperoxidaserdquoThyroid vol 4 no 2 pp 173ndash178 1994
[2] J P Banga D Mahadevan G J Barton et al ldquoPrediction ofdomain organisation and secondary structure of thyroid perox-idase a human autoantigen involved in destructive thyroiditisrdquoFEBS Letters vol 266 no 1-2 pp 133ndash141 1990
[3] S Kimura Y-S Hong T Kotani S Ohtaki and F KikkawaldquoStructure of the human thyroid peroxidase gene comparisonand relationship to the humanmyeloperoxidase generdquoBiochem-istry vol 28 no 10 pp 4481ndash4489 1989
[4] 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
[5] H Bikker F Baas and J J De Vijlder ldquoMolecular analy-sis of mutated thyroid peroxidase detected in patients with
total iodide organification defectsrdquo The Journal of ClinicalEndocrinology andMetabolism vol 82 no 2 pp 649ndash653 1997
[6] B Bakker H Bikker T Vulsma J S E De Randamie B MWiedijk and J J M De Vijlder ldquoTwo decades of screeningfor congenital hypothyroidism in the Netherlands TPO genemutations in total iodide organification defects (an update)rdquoJournal of Clinical Endocrinology and Metabolism vol 85 no10 pp 3708ndash3712 2000
[7] T Kotani K Umeki J-I Kawano et al ldquoPartial iodide organ-ification defect caused by a novel mutation of the thyroidperoxidase gene in three siblingsrdquo Clinical Endocrinology vol59 no 2 pp 198ndash206 2003
[8] C Rodrigues P Jorge J Pires Soares et al ldquoMutation screeningof the thyroid peroxidase gene in a cohort of 55 Portuguesepatients with congenital hypothyroidismrdquo European Journal ofEndocrinology vol 152 no 2 pp 193ndash198 2005
[9] 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
[10] A M Maskari and A Alnaqdy ldquoFrequency of thyroid micro-somal and thyroid peroxidase antibody levels in a selectedgroup of Omani patients with Gravesrsquo Diseaserdquo Kuwait MedicalJournal vol 38 no 1 pp 10ndash13 2006
[11] E B Sandell and I M Kolthoff ldquoMicro determination of iodineby a catalytic methodrdquoMikrochimica Acta vol 1 no 1 pp 9ndash251937
[12] S Pino S-L Fang and L E Braverman ldquoAmmonium persul-fate a safe alternative oxidizing reagent for measuring urinaryiodinerdquo Clinical Chemistry vol 42 no 2 pp 239ndash243 1996
[13] B FaamM SDaneshpour F AziziM Salehi andMHedayatildquoAssociation between TPO gene polymorphisms and Anti-TPOlevel in Tehranian population TLGSrdquo Gene vol 498 no 1 pp116ndash119 2012
[14] M Hedayati M S Jahromi M Z Yeganeh M S DaneshpourLH Rad andFAzizi ldquoAssociation between serum level of anti-TPO titer and polymorphisms G1193C Exon 8 and C2145TExon 12 of thyroid peroxidase gene in an Iranian populationrdquoInternational Journal of Endocrinology and Metabolism vol 8no 2 pp 64ndash67 2010
[15] H Bikker T Vulsma F Baas and J J M De Vijlder ldquoIdentifica-tion of five novel inactivating mutations in the human thyroidperoxidase gene by denaturing gradient gel electrophoresisrdquoHuman Mutation vol 6 no 1 pp 9ndash16 1995
[16] M Huse and J Kuriyan ldquoThe conformational plasticity ofprotein kinasesrdquo Cell vol 109 no 3 pp 275ndash282 2002
[17] A Krupa G Preethi and N Srinivasan ldquoStructural modesof stabilization of permissive phosphorylation sites in proteinkinases distinct strategies in SerThr and Tyr kinasesrdquo Journalof Molecular Biology vol 339 no 5 pp 1025ndash1039 2004
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
6 International Journal of Endocrinology
0
02
04
06
08
1
12
14
16
18
2
Patient Control
P lt 00001
T 3(N
gm
L)
065 plusmn 022
164 plusmn 008
(a)
0
02
04
06
08
1
12
14
16
Patient Control
P lt 00001
FT4
(Ng
dL)
061 plusmn 027
125 plusmn 010
(b)
0
5
10
15
20
25
30
35
40
45
Patient Control
TSH
(mcU
mL)
P lt 00001
3514 plusmn 462
251 plusmn 093
(c)
44
156
0
20
40
60
80
100
120
140
160N
umbe
r of p
atie
nts
Anti-TPO antibody level (IUmL)gt60 IUmL lt60 IUmL
(d)
Figure 1 T3and FT
4level of patient and control (normal) populations TSH and anti-TPO antibody level of patient and control (normal)
populations
(10ndash20120583gdL) (WHO UNICEF and ICCIDD 2001) There-fore UIE levels indicate that iodine deficiency is currently nota public health problem in our screened population
Our study identified six different SNPs among these threeSNPs (Asp 620 Asn Glu 641 Lys and Thr 725 Pro) aresignificantly associated with hypothyroidism We also try tofind out the association between TPO gene polymorphismsand Anti-TPO level in our study subjects and we did not findany association (data not shown) However several groups indifferent population like Tehranian population [13] and Ira-nian population [14] showed association between TPO genepolymorphisms and anti-TPO level Our study needs muchlarger sample size which may help in better understanding of
association between TPO gene polymorphisms and anti-TPOlevel
Based on the literature Exons 7ndash11 encode the catalyticcenter of the TPO protein (heme binding region) whichis crucial for the enzymatic activity [15] On the otherhand previous findings also indicate that Exons 7ndash14 harbormutational hot spots [15] Thus mutations in these regionsare expected to have major effects on TPO activity resultingin severe organification defect and severe hypothyroidismIn Glu 641 Lys negative charged amino acid is replaced bypositive charged amino acid As we know Threonine is thephosphorylation site of the protein which is important for theactivation of the protein [16 17] Therefore mutations in this
International Journal of Endocrinology 7
A C T
A T C A C T G G C
C C T
A T C C C T G G C
Normal Patient
(a)
A G CCC G C G C C A G C G A G G T
A C CC C G C G C C A C C G A G G T
Normal Patient
(b)
G C GG C G C G C C T G C G G C C T G T G C
T C GG C G C G C C T T C G G C C T G T G C
Normal Patient
(c)
Figure 2 Nucleotide polymorphism in TPO gene study population (a) Nucleotide polymorphism in patient ID-147 Exon11 and ACT gtCCT(Thr gt Pro) (b) Nucleotide polymorphism in patient ID-6 Exon7 and AGC gt ACC (Ser gtThr) (c) Nucleotide polymorphism in patientID-183 Exon7 and GCG gt TCG (Ala gt Ser)
1 2 3 4
GAPDH
(a)
1 2 3 4
GAPDH
(b)
Figure 3 Western blot showing expression of wild-type and mutated recombinant TPO (a) Lane 1 wild-type recombinant TPO Lanes 2ndash4recombinant TPO containing (2-Glu 641 Lys 3-Asp 668Asn and 4-Thr 725 Pro) changes In (b)Western blot showing expression of wild-typeandmutated recombinant TPO Lane 1 wild-type recombinant TPO Lanes 2ndash4 recombinant TPO containing (2-Asp 620Asn 3-Ser 398Thrand 4-Ala 373 Ser) changes
amino acid may change the activity of TPO enzyme whichmay ultimately reduce the functional efficacy of the enzyme
We established the effect of these polymorphisms throughin vitro assay Some polymorphisms (Glu 641 Lys Thr 725Pro andAsp 620Asn)were enzymatically inactive in both theguaiacol and iodide assaysThey also showed reduced amountof TPO on the Western blot This is a strong indication that
the changes are present at crucial positions of the TPO generesulting in inactivated TPO
5 Conclusions
Thus the above changes of amino acids may exert its effecton the structure and functional activity of TPO Our study
8 International Journal of Endocrinology
Table 5 Guaiacol and iodide oxidation activity of expressed humanTPO protein
Mutation Guaiacol oxidation Iodide oxidationWild type 076 plusmn 007 090 plusmn 005
Glu 641 Lys ND lowast
Asp 668 Asn 063 plusmn 007 069 plusmn 027
Thr 725 Pro ND lowast
Asp 620 Asn ND lowast
Ser 398Thr 070 plusmn 027 082 plusmn 029
Ala 373 Ser 062 plusmn 009 lowast
Enzyme activity was activity expressed as Δ119860sdotminminus1sdotmg proteinminus1lowastComparable with nonenzymatic reaction rate (051 plusmn 024) ND nodetectable activity (lt10 of wild-type expressed TPO) [5]
tried to detect the genetic etiology of this disease which mayfurther help us to risk categorize for hypothyroidism Thisstudy also helps to develop a genetic screening protocol forhypothyroidism specifically for the Indian population
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Srikanta Guria and Biswabandhu Bankura contributedequally to this paper
Acknowledgments
This work was supported by Grant from the Depart-ment of Biotechnology Government of India (no BTPR9342Med123462007) The authors would like to thankthe families who gave their consent and collaborated in thisstudy
References
[1] J R Baker Jr P Arscott and J Johnson ldquoAn analysis of thestructure and antigenicity of different forms of human thyroidperoxidaserdquoThyroid vol 4 no 2 pp 173ndash178 1994
[2] J P Banga D Mahadevan G J Barton et al ldquoPrediction ofdomain organisation and secondary structure of thyroid perox-idase a human autoantigen involved in destructive thyroiditisrdquoFEBS Letters vol 266 no 1-2 pp 133ndash141 1990
[3] S Kimura Y-S Hong T Kotani S Ohtaki and F KikkawaldquoStructure of the human thyroid peroxidase gene comparisonand relationship to the humanmyeloperoxidase generdquoBiochem-istry vol 28 no 10 pp 4481ndash4489 1989
[4] 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
[5] H Bikker F Baas and J J De Vijlder ldquoMolecular analy-sis of mutated thyroid peroxidase detected in patients with
total iodide organification defectsrdquo The Journal of ClinicalEndocrinology andMetabolism vol 82 no 2 pp 649ndash653 1997
[6] B Bakker H Bikker T Vulsma J S E De Randamie B MWiedijk and J J M De Vijlder ldquoTwo decades of screeningfor congenital hypothyroidism in the Netherlands TPO genemutations in total iodide organification defects (an update)rdquoJournal of Clinical Endocrinology and Metabolism vol 85 no10 pp 3708ndash3712 2000
[7] T Kotani K Umeki J-I Kawano et al ldquoPartial iodide organ-ification defect caused by a novel mutation of the thyroidperoxidase gene in three siblingsrdquo Clinical Endocrinology vol59 no 2 pp 198ndash206 2003
[8] C Rodrigues P Jorge J Pires Soares et al ldquoMutation screeningof the thyroid peroxidase gene in a cohort of 55 Portuguesepatients with congenital hypothyroidismrdquo European Journal ofEndocrinology vol 152 no 2 pp 193ndash198 2005
[9] 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
[10] A M Maskari and A Alnaqdy ldquoFrequency of thyroid micro-somal and thyroid peroxidase antibody levels in a selectedgroup of Omani patients with Gravesrsquo Diseaserdquo Kuwait MedicalJournal vol 38 no 1 pp 10ndash13 2006
[11] E B Sandell and I M Kolthoff ldquoMicro determination of iodineby a catalytic methodrdquoMikrochimica Acta vol 1 no 1 pp 9ndash251937
[12] S Pino S-L Fang and L E Braverman ldquoAmmonium persul-fate a safe alternative oxidizing reagent for measuring urinaryiodinerdquo Clinical Chemistry vol 42 no 2 pp 239ndash243 1996
[13] B FaamM SDaneshpour F AziziM Salehi andMHedayatildquoAssociation between TPO gene polymorphisms and Anti-TPOlevel in Tehranian population TLGSrdquo Gene vol 498 no 1 pp116ndash119 2012
[14] M Hedayati M S Jahromi M Z Yeganeh M S DaneshpourLH Rad andFAzizi ldquoAssociation between serum level of anti-TPO titer and polymorphisms G1193C Exon 8 and C2145TExon 12 of thyroid peroxidase gene in an Iranian populationrdquoInternational Journal of Endocrinology and Metabolism vol 8no 2 pp 64ndash67 2010
[15] H Bikker T Vulsma F Baas and J J M De Vijlder ldquoIdentifica-tion of five novel inactivating mutations in the human thyroidperoxidase gene by denaturing gradient gel electrophoresisrdquoHuman Mutation vol 6 no 1 pp 9ndash16 1995
[16] M Huse and J Kuriyan ldquoThe conformational plasticity ofprotein kinasesrdquo Cell vol 109 no 3 pp 275ndash282 2002
[17] A Krupa G Preethi and N Srinivasan ldquoStructural modesof stabilization of permissive phosphorylation sites in proteinkinases distinct strategies in SerThr and Tyr kinasesrdquo Journalof Molecular Biology vol 339 no 5 pp 1025ndash1039 2004
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
International Journal of Endocrinology 7
A C T
A T C A C T G G C
C C T
A T C C C T G G C
Normal Patient
(a)
A G CCC G C G C C A G C G A G G T
A C CC C G C G C C A C C G A G G T
Normal Patient
(b)
G C GG C G C G C C T G C G G C C T G T G C
T C GG C G C G C C T T C G G C C T G T G C
Normal Patient
(c)
Figure 2 Nucleotide polymorphism in TPO gene study population (a) Nucleotide polymorphism in patient ID-147 Exon11 and ACT gtCCT(Thr gt Pro) (b) Nucleotide polymorphism in patient ID-6 Exon7 and AGC gt ACC (Ser gtThr) (c) Nucleotide polymorphism in patientID-183 Exon7 and GCG gt TCG (Ala gt Ser)
1 2 3 4
GAPDH
(a)
1 2 3 4
GAPDH
(b)
Figure 3 Western blot showing expression of wild-type and mutated recombinant TPO (a) Lane 1 wild-type recombinant TPO Lanes 2ndash4recombinant TPO containing (2-Glu 641 Lys 3-Asp 668Asn and 4-Thr 725 Pro) changes In (b)Western blot showing expression of wild-typeandmutated recombinant TPO Lane 1 wild-type recombinant TPO Lanes 2ndash4 recombinant TPO containing (2-Asp 620Asn 3-Ser 398Thrand 4-Ala 373 Ser) changes
amino acid may change the activity of TPO enzyme whichmay ultimately reduce the functional efficacy of the enzyme
We established the effect of these polymorphisms throughin vitro assay Some polymorphisms (Glu 641 Lys Thr 725Pro andAsp 620Asn)were enzymatically inactive in both theguaiacol and iodide assaysThey also showed reduced amountof TPO on the Western blot This is a strong indication that
the changes are present at crucial positions of the TPO generesulting in inactivated TPO
5 Conclusions
Thus the above changes of amino acids may exert its effecton the structure and functional activity of TPO Our study
8 International Journal of Endocrinology
Table 5 Guaiacol and iodide oxidation activity of expressed humanTPO protein
Mutation Guaiacol oxidation Iodide oxidationWild type 076 plusmn 007 090 plusmn 005
Glu 641 Lys ND lowast
Asp 668 Asn 063 plusmn 007 069 plusmn 027
Thr 725 Pro ND lowast
Asp 620 Asn ND lowast
Ser 398Thr 070 plusmn 027 082 plusmn 029
Ala 373 Ser 062 plusmn 009 lowast
Enzyme activity was activity expressed as Δ119860sdotminminus1sdotmg proteinminus1lowastComparable with nonenzymatic reaction rate (051 plusmn 024) ND nodetectable activity (lt10 of wild-type expressed TPO) [5]
tried to detect the genetic etiology of this disease which mayfurther help us to risk categorize for hypothyroidism Thisstudy also helps to develop a genetic screening protocol forhypothyroidism specifically for the Indian population
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Srikanta Guria and Biswabandhu Bankura contributedequally to this paper
Acknowledgments
This work was supported by Grant from the Depart-ment of Biotechnology Government of India (no BTPR9342Med123462007) The authors would like to thankthe families who gave their consent and collaborated in thisstudy
References
[1] J R Baker Jr P Arscott and J Johnson ldquoAn analysis of thestructure and antigenicity of different forms of human thyroidperoxidaserdquoThyroid vol 4 no 2 pp 173ndash178 1994
[2] J P Banga D Mahadevan G J Barton et al ldquoPrediction ofdomain organisation and secondary structure of thyroid perox-idase a human autoantigen involved in destructive thyroiditisrdquoFEBS Letters vol 266 no 1-2 pp 133ndash141 1990
[3] S Kimura Y-S Hong T Kotani S Ohtaki and F KikkawaldquoStructure of the human thyroid peroxidase gene comparisonand relationship to the humanmyeloperoxidase generdquoBiochem-istry vol 28 no 10 pp 4481ndash4489 1989
[4] 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
[5] H Bikker F Baas and J J De Vijlder ldquoMolecular analy-sis of mutated thyroid peroxidase detected in patients with
total iodide organification defectsrdquo The Journal of ClinicalEndocrinology andMetabolism vol 82 no 2 pp 649ndash653 1997
[6] B Bakker H Bikker T Vulsma J S E De Randamie B MWiedijk and J J M De Vijlder ldquoTwo decades of screeningfor congenital hypothyroidism in the Netherlands TPO genemutations in total iodide organification defects (an update)rdquoJournal of Clinical Endocrinology and Metabolism vol 85 no10 pp 3708ndash3712 2000
[7] T Kotani K Umeki J-I Kawano et al ldquoPartial iodide organ-ification defect caused by a novel mutation of the thyroidperoxidase gene in three siblingsrdquo Clinical Endocrinology vol59 no 2 pp 198ndash206 2003
[8] C Rodrigues P Jorge J Pires Soares et al ldquoMutation screeningof the thyroid peroxidase gene in a cohort of 55 Portuguesepatients with congenital hypothyroidismrdquo European Journal ofEndocrinology vol 152 no 2 pp 193ndash198 2005
[9] 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
[10] A M Maskari and A Alnaqdy ldquoFrequency of thyroid micro-somal and thyroid peroxidase antibody levels in a selectedgroup of Omani patients with Gravesrsquo Diseaserdquo Kuwait MedicalJournal vol 38 no 1 pp 10ndash13 2006
[11] E B Sandell and I M Kolthoff ldquoMicro determination of iodineby a catalytic methodrdquoMikrochimica Acta vol 1 no 1 pp 9ndash251937
[12] S Pino S-L Fang and L E Braverman ldquoAmmonium persul-fate a safe alternative oxidizing reagent for measuring urinaryiodinerdquo Clinical Chemistry vol 42 no 2 pp 239ndash243 1996
[13] B FaamM SDaneshpour F AziziM Salehi andMHedayatildquoAssociation between TPO gene polymorphisms and Anti-TPOlevel in Tehranian population TLGSrdquo Gene vol 498 no 1 pp116ndash119 2012
[14] M Hedayati M S Jahromi M Z Yeganeh M S DaneshpourLH Rad andFAzizi ldquoAssociation between serum level of anti-TPO titer and polymorphisms G1193C Exon 8 and C2145TExon 12 of thyroid peroxidase gene in an Iranian populationrdquoInternational Journal of Endocrinology and Metabolism vol 8no 2 pp 64ndash67 2010
[15] H Bikker T Vulsma F Baas and J J M De Vijlder ldquoIdentifica-tion of five novel inactivating mutations in the human thyroidperoxidase gene by denaturing gradient gel electrophoresisrdquoHuman Mutation vol 6 no 1 pp 9ndash16 1995
[16] M Huse and J Kuriyan ldquoThe conformational plasticity ofprotein kinasesrdquo Cell vol 109 no 3 pp 275ndash282 2002
[17] A Krupa G Preethi and N Srinivasan ldquoStructural modesof stabilization of permissive phosphorylation sites in proteinkinases distinct strategies in SerThr and Tyr kinasesrdquo Journalof Molecular Biology vol 339 no 5 pp 1025ndash1039 2004
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
8 International Journal of Endocrinology
Table 5 Guaiacol and iodide oxidation activity of expressed humanTPO protein
Mutation Guaiacol oxidation Iodide oxidationWild type 076 plusmn 007 090 plusmn 005
Glu 641 Lys ND lowast
Asp 668 Asn 063 plusmn 007 069 plusmn 027
Thr 725 Pro ND lowast
Asp 620 Asn ND lowast
Ser 398Thr 070 plusmn 027 082 plusmn 029
Ala 373 Ser 062 plusmn 009 lowast
Enzyme activity was activity expressed as Δ119860sdotminminus1sdotmg proteinminus1lowastComparable with nonenzymatic reaction rate (051 plusmn 024) ND nodetectable activity (lt10 of wild-type expressed TPO) [5]
tried to detect the genetic etiology of this disease which mayfurther help us to risk categorize for hypothyroidism Thisstudy also helps to develop a genetic screening protocol forhypothyroidism specifically for the Indian population
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Srikanta Guria and Biswabandhu Bankura contributedequally to this paper
Acknowledgments
This work was supported by Grant from the Depart-ment of Biotechnology Government of India (no BTPR9342Med123462007) The authors would like to thankthe families who gave their consent and collaborated in thisstudy
References
[1] J R Baker Jr P Arscott and J Johnson ldquoAn analysis of thestructure and antigenicity of different forms of human thyroidperoxidaserdquoThyroid vol 4 no 2 pp 173ndash178 1994
[2] J P Banga D Mahadevan G J Barton et al ldquoPrediction ofdomain organisation and secondary structure of thyroid perox-idase a human autoantigen involved in destructive thyroiditisrdquoFEBS Letters vol 266 no 1-2 pp 133ndash141 1990
[3] S Kimura Y-S Hong T Kotani S Ohtaki and F KikkawaldquoStructure of the human thyroid peroxidase gene comparisonand relationship to the humanmyeloperoxidase generdquoBiochem-istry vol 28 no 10 pp 4481ndash4489 1989
[4] 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
[5] H Bikker F Baas and J J De Vijlder ldquoMolecular analy-sis of mutated thyroid peroxidase detected in patients with
total iodide organification defectsrdquo The Journal of ClinicalEndocrinology andMetabolism vol 82 no 2 pp 649ndash653 1997
[6] B Bakker H Bikker T Vulsma J S E De Randamie B MWiedijk and J J M De Vijlder ldquoTwo decades of screeningfor congenital hypothyroidism in the Netherlands TPO genemutations in total iodide organification defects (an update)rdquoJournal of Clinical Endocrinology and Metabolism vol 85 no10 pp 3708ndash3712 2000
[7] T Kotani K Umeki J-I Kawano et al ldquoPartial iodide organ-ification defect caused by a novel mutation of the thyroidperoxidase gene in three siblingsrdquo Clinical Endocrinology vol59 no 2 pp 198ndash206 2003
[8] C Rodrigues P Jorge J Pires Soares et al ldquoMutation screeningof the thyroid peroxidase gene in a cohort of 55 Portuguesepatients with congenital hypothyroidismrdquo European Journal ofEndocrinology vol 152 no 2 pp 193ndash198 2005
[9] 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
[10] A M Maskari and A Alnaqdy ldquoFrequency of thyroid micro-somal and thyroid peroxidase antibody levels in a selectedgroup of Omani patients with Gravesrsquo Diseaserdquo Kuwait MedicalJournal vol 38 no 1 pp 10ndash13 2006
[11] E B Sandell and I M Kolthoff ldquoMicro determination of iodineby a catalytic methodrdquoMikrochimica Acta vol 1 no 1 pp 9ndash251937
[12] S Pino S-L Fang and L E Braverman ldquoAmmonium persul-fate a safe alternative oxidizing reagent for measuring urinaryiodinerdquo Clinical Chemistry vol 42 no 2 pp 239ndash243 1996
[13] B FaamM SDaneshpour F AziziM Salehi andMHedayatildquoAssociation between TPO gene polymorphisms and Anti-TPOlevel in Tehranian population TLGSrdquo Gene vol 498 no 1 pp116ndash119 2012
[14] M Hedayati M S Jahromi M Z Yeganeh M S DaneshpourLH Rad andFAzizi ldquoAssociation between serum level of anti-TPO titer and polymorphisms G1193C Exon 8 and C2145TExon 12 of thyroid peroxidase gene in an Iranian populationrdquoInternational Journal of Endocrinology and Metabolism vol 8no 2 pp 64ndash67 2010
[15] H Bikker T Vulsma F Baas and J J M De Vijlder ldquoIdentifica-tion of five novel inactivating mutations in the human thyroidperoxidase gene by denaturing gradient gel electrophoresisrdquoHuman Mutation vol 6 no 1 pp 9ndash16 1995
[16] M Huse and J Kuriyan ldquoThe conformational plasticity ofprotein kinasesrdquo Cell vol 109 no 3 pp 275ndash282 2002
[17] A Krupa G Preethi and N Srinivasan ldquoStructural modesof stabilization of permissive phosphorylation sites in proteinkinases distinct strategies in SerThr and Tyr kinasesrdquo Journalof Molecular Biology vol 339 no 5 pp 1025ndash1039 2004
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom