Identification of a mutation in the coding sequence of the human thyroid peroxidase gene causing congenital goiter

Identification of a mutation in the coding

sequence of the human thyroid peroxidase

gene causing congenital goiter.

M J Abramowicz, … , H A Chester, G Vassart

J Clin Invest.

1992;

90(4)

:1200-1204.

https://doi.org/10.1172/JCI115981

.

Thyroid peroxidase (TPO) is the key enzyme in the synthesis of thyroid hormones, and the

TPO defects are believed to be the most prevalent causes of the inborn errors of thyroid

metabolism. We investigated an adopted boy with iodide organification defect, who

presented with florid hypothyroidism at the age of 4 mo, poorly complied with thyroxine

treatment, and developed a compressive goiter necessitating partial resection at the age of

12 yr. Biochemical studies revealed the absence of TPO activity in the resected tissue.

Genomic DNA studies identified a 4 base-pair insertion in the eighth exon of the TPO gene,

and showed that the patient was homozygous for this frameshift mutation. The direct genetic

diagnosis of this mutation can be made by digestion of polymerase chain reaction products

with NaeI restriction enzyme. This will help assessing its prevalence among the

heterogenous genetic group of TPO defects.

Research Article

Find the latest version:

Identification

of

a

Mutation in the Coding Sequence

of the Human Thyroid Peroxidase Gene Causing Congenital Goiter

Marc J. Abramowicz,* Hector M.Targovnik,** _{Viviana Varela,t Pascale}

_Cochaux,I

_{Leon Krawiec,11 MarioA. Pisarev,11}

Francisco V. E. Propato,t Guillermo Juvenal,11 Hugo A. Chester,11andGilbert Vassart*

*Institut deRechercheInterdisciplinaireenBiologie HumaineetNucleaire, Free University of Brussels, 1070Brussels,Belgium; tCatedra de GeneticayBiologia Molecular,Facultadde FarmaciayBioquimica, Universidadde Buenos Aires, 1113 Buenos Aires,

Argentina;

IlDivision

Bioquimica Nuclear, Gerencia de Aplicaciones, Comission Nacional de Energia Atomica, 1429Buenos Aires,Argentina;and §Service deGenetique, HopitalErasme, 1070 Brussels, Belgium

Abstract

Thyroidperoxidase (TPO) is the key enzyme in the synthesis ofthyroid hormones, and the TPO defects are believed to be the mostprevalent causes of the inborn errors of thyroid metabo-lism. Weinvestigated an adopted boy with iodide organification defect, who presented with florid hypothyroidism at the age of 4 mo, poorlycomplied with thyroxine treatment, and developed a compressive goiter necessitating partial resection at the age of 12yr.

Biochemicalstudies revealed the absence of TPO activity in the resectedtissue. Genomic DNA studies identified a 4

base-pair

insertion in theeighth exon of the TPO gene, and showed that the

patient

was homozygous for this frameshift mutation. The direct genetic diagnosis of this mutation can be made by

digestion

of

polymerase

chain reaction products with

NaeI

re-striction enzyme. This will help assessing its prevalence among the heterogenous genetic group of TPO defects. (J. Clin. In-vest. 1992.

90:1200-1204.)

Key words: alternative

splicing.

inborn errors of metabolism * molecular diagnosis * organifica-tion defect *

thyroid

hormone

biosynthesis

Introduction

Congenital hypothyroidismis apublichealth concernaffecting 1/4,000 births. Althoughmost cases result from dysembryo-genesisof thethyroid gland,aninterestingminorityare due to

inborn errors of

thyroid

hormones metabolism(1).

Congenital

goiters

duetodefectivesynthesisofthyroid prohormone

thyro-globulin (TG)' have been investigatedin man and in animal

models(

1-4),

and a pathogenic mutation has recently been

identified

in the human (5); however, themost

prevalent

cause

of

inherited

defects in

thyroid

metabolism is believed to bethe

thyroid peroxidase (TPO) deficiency (6).

TPO is thekey enzyme in

thyroid

hormones

formation;

it

catalyzes

both the iodination and the

coupling

of

hormono-genic tyrosyl

residues of TG

(7, 8).

TPO isa 110-kD

glycopro-M. J.Abramowicz and H.M. Targovnik contributed equally to the

study.

Address reprint requests to Dr. Abramowicz, IRIBHN, Campus ErasmebatC, 808 Route de Lennik, 1070 Brussels, Belgium.

Receivedfor publication 12February 1992 and in revisedform 4 May 1992.

1.Abbreviationsused in this paper: TG, thyroglobulin; TPO,thyroid peroxidase.

tein, using heme as a cofactor, whose primary structure has been deducedfromthe sequence of the cloned complementary DNA (cDNA) (9-1 1). Sequences involved in the enzymatic mechanismhave been postulated bycomparison with related peroxidase enzymes (12). The TPO gene, located on chro-mosome2 [2pter-2p24] (10, 13) consists of17exonsand 16 introns ( 14).

CongenitalTPOdefects, typically transmittedasautosomal recessive traits, result in hypothyroid goiters with failure to convert iodide intoorganic iodine (organification defect)

(8).

Genetic heterogeneityisexpectedinthissyndrome,asit may resultfrom several mechanisms ( 1), including total absence of TPO activity, inability of TPO to bind to the heme cofactor (15),inability to interact with the TG substrate, and abnormal subcellular localization

(8).

Recent

linkage

studies

supported

heterogeneity

of the mutations

involved,

and

suggested

that

onepathogenic TPO mutation might consist ofa notyet identi-fiedpartial genomicdeletion(6).

Inthe present study,wehaveidentifiedahuman mutation

causing

TPO

deficiency.

It consistsofa

duplication

ofa

tetran-ucleotide -GGCC- in exon8 of the TPO gene

occurring

in a

homozygous

subject presenting

with

congenital goiter

and

hy-pothyroidism. Theresultingframeshiftgenerates stopcodons

in exon 9, which would result in a

grossly

truncated

protein

with no

expected activity.

Methods

Patient.D.M.,a4-mo-oldadopted male,wasreferredtotheendocrine

service (CentrodeInvestigationesEndocrinologicas,CONICET,

Hos-pitaldeniftos"RicardoGutierrez,"BuenosAires,Argentina),wherea

diagnosis ofhypothyroidism was made. The serum thyroxine

(14)

levelwas0.1

tg/dl

(normal,5-13

Ag/dl)

and thepatientwas

immedi-atelygiventhyroidhormonetherapy.Atthe ageof5 yr thephysical

examination showed moderate to severe mental retardation with

speechdifficulties and stuntedgrowth.Themedicationwas discontin-ued andlaboratorytestingrevealed: lowserumtriiodothyronine(T3) (25 ng/dl,normal100-200ng/ml)andT4 (1.6,g/dl), elevated thyro-tropin (>80uUU/ml,normal<9

AU/ml)

andapositivethiocyanate

dischargetest(27% dischargeof labeled iodine,normal<10%).No

informationsareavailableregarding complianceofthepatientwith his

treatment. The subsequent evaluation atthe age of 10 yr showed:

serumT4, 0.5,lg/dl;serumT3,27ng/dl;

'31I-thyroid

uptakewas20%

at2and 24h(normal,30-60%at2 h and 15-55%at24h)and the

thyroidscanrevealedapattern ofdiffusegoiter.Microsomal antibodies

wereabsent.Thyroidectomywasperformedatthe ageof 12 yr 9mo

because ofcompressivesymptoms, and thepatientwassubsequently

treated withappropriate thyroidhormonereplacement therapy.

Histo-logicalexamination disclosedamultinodulargoiter.Thefollicular

epi-thelial cellsshowedhyperplasiawithpapillary projectionsin thecystic

follicles.

Biochemicaland molecular geneticstechniques.Allmolecular ge-netics manipulations involved standard procedures (16). Standard

J.Clin.Invest.

© The AmericanSocietyfor Clinical

Investigation,

Inc.

0021-9738/92/10/1200/05 $2.00

precautions were taken to avoidpolymerasechainreaction(PCR) con-taminations.

The presence ofTGin thethyroid tissue from patientDM was

investigatedby agarose gelelectrophoresis (indenatured and reduced conditions), gel filtration onSephacryl S-300column,and immuno-electrophoresis. TPOactivitywasmeasuredbythetyrosine iodinase assay(17).Organificationof'1'I(givenas a25-uCitracerdosebefore

surgery) was studied bySephadex G-200 elution pattern ofthyroid

homogenate.RNA and DNA samples were preparedfrom goitertissue frozen in liquid nitrogen at the time of surgery.GenomicDNA from controlsubjectswasextractedfrom circulating leukocytes.The goiter

genomicDNAfrom patientD.M. was analyzedby Southern blotting

using BglII, EcoRI,PstI, and PvuII restrictionenzymes andthe TG cDNAprobepbTgM3 (18).

Amplificationofoverlapping portions ofthegoiterTG messenger RNA (mRNA), encompassing thewhole lengthofthe mRNA, was

performed by reversetranscription of totalgoiterRNAaliquots fol-lowed by PCRamplificationexactly asdescribedinIerietal. (5). By

usingthe samemethod, couples of primersweredesignedtoamplify

the[6-1066], [744-1703], [1524-2400],and[2221-3024]segmentsof

the3-kb TPO cDNA(19). To facilitate cloning,acoupleofnested PCRprimers containing restriction sitesin 5'overhangs,wereusedto

reamplifythe1019-1558segmentfromthe[744-1703]fragment

(for-ward primer complementary to exon 8 (1019F): 5'tagaattccaccgtg-tatggcagctc3'; reverse primer complementary to exon 9 (1558R):

5'tggaagcttgcgtccagcctcctcacc3'). Standard PCR conditionswere the

following:1minat930C,2 minat550C,3 minat720C,X35 cycles,in

100-,ul reactionvolumeswith 1.5 mMMgCl2and 10% DMSO. The PCR productwas visualized by electrophoresisin a 2% agarose gel

followed byethidium bromide staining,andaliquotswerecloned in M13mpl8andmpl9and sequencedonboth strands (model 370A DNA sequencer,Applied Biosystems,Inc., FosterCity,CA).

A460-bpgenomic DNAfragment containingthe exon8/intron 8

junction wasamplifiedunderidentical conditions from goiterDNA

usingthesameforward primer complementaryto exon8(1019F)and

areverseprimer complementarytointron8

(IN8R:5'ataagcttggagagag-aagccacgatgc3'). Digestion ofthePCR product with SstII(Bethesda

Research Laboratories, Gaithersburg, MD) or NaeI (Boehringer Mannheim GmbH, Mannheim, FRG)wasperformedasdescribedby themanufacturers.NaeIrecognizesthe sequence5'GCCGGC3'. The PCR product was extractedwith phenol, CHC13,then

ethanol-precipi-tatedbefore digestion,andstained withethidiumbromideafter electro-phoresis in a 3% agarose gel. Aliquots were cloned in Ml3 and se-quencedonboth strands. Additional amplificationswereperformed usingIN8R anda more5'exonicforward primer (951 F:5'ttgaattctttggg-aacctgtccacg3'); SstIIandNaeI digestion,aswell as M 13sequencing of thisPCRproductyieldedthesameresults. A350-bp fragment

contain-ingtheintron8 / exon 9junctionwasamplifiedusingaforwardprimer complementarytothe3'region ofintron8

(IN8F:5'tagaattcgcaagaaggc-atttctgg3')andthereverseprimer complementaryto exon9(1558R).

The PCR productwascloned inM13 and sequenced on both strands.

An additional amplification product spanningthe intron 8/exon 9

junctionwas obtainedusing 1558R anda more3'intronic forward primer(IN8Fd:5'atgaattcgaggcgaccctcctctgg3');itwascloned inMl3 and sequenced on both strands, andyieldedthe same results.

Results

Organification

defect

andnormal expression

of

the TG gene. Southern

blotting

analysis ofthe TG locus in patient D.M.

showedanormal band pattern.

Similarly,

all

fragments

of the

goiter

TG mRNA

displayed

normal sizes after reverse

tran-scription and PCR analysis (see Methods). The protein was detected in thetissue

by

agarose

gel

electrophoresis,

gel

filtra-tion assay, and

immunoelectrophoresis;

those three tests showed a normal size ofthe TG

protein (data

not

shown).

Consistent with the

_{preoperative thiocyanate discharge}

test in-dicative ofaniodide

organification defect,

TPO

activity

in the resected

goiter

tissue

wasverylowasmeasured

by

the

tyrosine

iodinaseassay

(19 U,

with 323 U inacontrol froma

_patient

with multinodular

goiter ( 17),

and the

sephadex

G-200 elution

pattern

oflabeled

_{thyroid proteins}

showed concentration of

_3"I

in the

free iodine

peak,

withnodetectable

iodination

of

high-molecular-weight proteins

eluted from the column.

Deletion

in

goiter

TPO mRNA.

By using

thesame

method-ology

as for

TG,

PCR

amplification

of four

overlapping

seg-mentsofthe reverse-transcribed

goiter

TPO mRNA

(see

Meth-ods),

each±

1 kb in

length,

identifieda±

I00-bp

deletion in the

1000-1550

region

of

the normal TPO

_{cDNA; Fig. 1,}

lane

3,

shows the size of this deleted segment

(420 bp)

as

_compared

with

a control from normal

thyroid

RNA

(540

bp,

lane

2),

wherea

fainter

_420-bp

band is also

observed;

this

420-bp

band

is

not

observed

in the

amplification

product

from a normal TPO cDNA molecule cloned ina

plasmid

vectorshown in lane 1.Asmaller PCR

product

observed in the

goiter

is also found in the

background

of

amplification generated

in the absence of DNA

template (H20 control,

lane

4).

The

420-bp

segment

from

the

goiter

was cloned and

_sequenced.

The deleted

se-quence

(see below)

corresponds

tothe first 124

bp

ofexon

_9,

i.e., fragment

[1379-1502]

of the normal

(3 kb)

TPO mRNA

(10, 19).

Inspection

of the

goiter

TPO cDNA sequence upstream of the deletion

readily

identified

a

4-bp

insertion

at

position

1227 inexon

8,

duetothe

duplication

ofaGGCC tetranucleotide.

By

its

expected

deleterious

effect,

this frameshift mutation mustbeconsidered

responsible

for the

_phenotype.

Homozygous

mutation ingoiter genomicDNA. In orderto confirm the presence of the

insertion

in

genomic

DNA andto

determine whether the

patient

was

homozygous

for the

muta-tion,

the

corresponding region

of

genomic

DNAwas

amplified,

cloned,

and

sequenced. Duplication

ofthe GGCC tetranucleo-tidewasidentified inexon8 of the TPO gene, 152

bp

upstream

from the

junction

with

intron

8

(Fig.

2

A).

In

addition,

two

point

mutationswerefound

by

comparison

with

published

se-quences

( 10,

11,

14, 19) (Fig.

2

A;

coordinatesasin reference

19).

All sequences from the total PCR

product

showed identi-cal mutations

(five

clones

sequenced). Homozygosity

was fur-ther

demonstrated

by

the

complete digestion

of the uncloned PCR

product

with NaeI

(Fig.

2

B).

This enzyme

recognizes

specifically

the mutated sequence. The specificity of the

major

PCR

amplification

product

is

indicated by

theSstII

digestion

that generates the

expected

restriction bands. Also shown in

Fig.

2A, is the absence of mutation in the region spanning the 3'

splice

site of intron 8. This

region

was

investigated

in search

1 2 3 4 MW Figure 1. Deletion in

goiter

bp

540-*

420-

I.-z.s

4*4W

TPO mRNA. Ethidium bromide

_staining

of the PCR productof the

[1019-1558]

region

ofTPO cDNA

amplified

from: normal TPO cDNA_template cloned inpBS plasmid

(lane

1);

reverse-tran-scribed thyroid RNA from

anormal control

subject

(lane 2);reverse-transcribed goiterRNA frompatient DM (lane3);

andH20control(lane 4). MW, molecular size markers (kbladder,

A

S T V Y G S S P A L E R Q L R N W

1019:TCCACCGTGTATGGCAGCTCCCCGGCCCTAGAGAGGCAGCTGCGGAACTG

T S A E G L L R V H A R L R D S

1069:GACCAGTGCCGAAGGGCTGCTCCGCGTCCACGCGCGCCTCCGGGACTCCG

G R A Y L P F V P P R A P

_IS

A C A

1119:GCCGCC'CCTACCTGCCCTTCGTGCCGCCACGCGCGCCTTCGGCCTGTGCG

P E P G I P G E T R G P C F L A G

1169:CCCGAGCCCGGCATCCCCGGAGAGACCCGCGGGCCCTGCTTCCTGGCCGG

D G R P R

QJ

R G P L P D G T A H

1219:AGACGGCCGGCCGCGCCACCGAGGTCCCCTCCCTGACGGCACTGCACACG

A V A A R A Q P P G R G A Q G P Q

1265:CTGTGGCTGCGCGAGCACAACCGCCTGGCCGCGGCGCTCAAGGCCCTCAA

C A L E R G R R V P G G A Q G R G

1315:_{TGCGCACTGGAGCGCGGACGCCGTGTACCAGGAGGCGCGCAAGGTCGTGG}

R S A P

1365:GCGCTCTGCACCAGgtgcgcggggtggtcctgggcgccctgggtggctgc

gggcaaagcggggggcgcctcgtgtgggtgcgcag----//---gaagtt

cagctgaggcccttattacaagacgagaagggtccagttccctggggctg tcaaggaagatgctcttccacactgccgctcgaggcgaccctcctctggt

D H H P

atcctgggcctcactgagatgcttttcctatctgcacagATCATCACCCT

E G L H P Q D P G T R G L P A V R

1390:GAGGGATTACATCCCCAGGATCCTGGGACCCGAGGCCTTCCAGCAGTACG

G S L *

1440:TGGGTCCCTATGAAGGCTATGACTCCACCGCCAACCCCACTGTGTCCAAC

1490:GTGTTCTCCACAGCCGCCTTCCGCTTCGGCCATGCCACGATCCACCCGCT

1540:GGTGAGGAGGCTGGACGC

B

NC

Sstil

Nael

MW

N

G

N

G

N

G

bp

_w i t ^

~~~~~460

I!_.

....₂₅₀

*

-210

ofamutation

causing

a

splicing

error

(e.g.,

amutation affect- Discussion

ing

the 3'acceptor

splice

sitein intron

8)

that could have

ex-plained the

alternative

splicing

(20).

To the best of

Fig. 3 shows the expected effects ofthe mutation and of scribeinthisst

alternative

splicing

on the

primary

structureoftheTPOpro- TPO mutation

tein. Normal

splicing

ofthe mutated

transcript

wouldgenerate consistent with

a

polypeptide

lessthan halfthe

length

of normal

TPO,

with

activity

results

mutation of the proximal-and deletion of the

distal-puta-

thyroidism,

an( tive

heme-binding

histidine residues

( 12).

Interestingly,

alter-

Interestingl

nativesplicingasobserved inthe

goiter

TPOmRNArestores

disrupting

the I the normal

reading

frame. The result is a nearly

full-length

ducing

stop co protein with a 91-amino acid segmentat

position

[397-487]

cryptic

accepto

replacedbya5

1-residue

unrelated segmentwhich is readina

splice

siteactiv

different frame. This translation

product

is

expected

tohavean containsashoi

unchanged distal

putative

heme-binding His,

butto lack the cag,see

_Fig.

2 v

proximalputative

heme-binding

His. (21

).

The pres

Figure2. Homozygous mutationingoiter geno-micDNA. (A) Partial sequence of the goiterTPO

geneexon8,intron 8(lowercaseletters),and exon9. Theduplicated tetranucleotideis Dou-BLE-UNDERLINED. The 124-bp deleted from goiter TPO messengerRNA areunderlined. Deduced protein sequence is shown in the one letter code,

assumingnormalsplicing ofthetranscript.

Coor-dinates refertothe normal cDNA sequence

(ref-erence19).Theverticalarrowsindicate thepoint

mutations. *First in-frame stop codon. (B) Ethi-dium bromide staining ofa460-bp genomicDNA

fragment encompassingtheexon 8/intron 8 junc-tion, andcorresponding to the 5' part of the se-quence described inpanelA(from position 1019 in exon 8 through the interruption in intron 8), PCR-amplified from normal (N) or goiter (G)

ge-nomic DNA anddigestedasindicated by

restric-tion enzymes SstII or NaeI, which cleaves only if theGGCC duplication is present. NC, not cleaved. MW, molecular size markers (HaeIII fragments of

OXl74).

ourknowledge, the GGCC duplicationwe de-Ludy represents the first description ofahuman Lresultingincongenital goiter. Homozygosityis

ia recessive trait, and a nearabsence of TPO in iodideorganification defect, primary hypo-,d elevatedthyrotropin.

ly,besidesabolishingtheenzymatic activity by reading frame of the messenger RNA and

intro-dons, the GGCC duplication also unmasks a

)rsplicesite inexon 9. Consistent with cryptic

vation, the 3'

extremity

ofthe mRNAdeletion

rtpyrimidinetractand ends with AG (ttctcca-A4);it thus fits the acceptorsplicesiteconsensus sencein the normal thyroidcontrol ofa small

-1000

AA

a a a a

TM

I

I

I COOH

350 exon8 400

I

..z. . A: pp.

N :EGLLRVHARLRDSGRAYLPFVPPRAPAACAPEPGIPGETRGPCFLAGDGRASEVPSLTALHTLWLREHNRLAAALKALNAHWSADAVYQEARK Gns:EGLLRVHARLRDSGRAYLPFVPPRAPSACAPEPGIPGETRGPCFLAGDGRPRORGPLPDGTAHAVAARAOPPGRGAOGPOCALERGRRVPGGA

Gas:EGLLRVHARLRDSGRAYLPFVPPRAPSACAPEPGIPGETRGPCFLAGDGRPRQRGPLPDGTAHAVAARAOPPGRGAOGPOCALERGRRVPGGA

sna

:T* * *. . d. .

N :VVGALHQ IITLRDYIPRILGPEAFQQYVGPYEGYDSTANPTVSNVFSTAAFRFGHATIHPLVRRLDASFQEHPDLPGLWLHQAFFSPWTLLR

Gns:QGRGRSAP DHHPEGLHPQDPGTRGLPAVRGSL*

Gas:R2GBRSA ---AAFRFGHATIHPLVRRLDASFQEHPDLPGLWLHQAFFSPWTLLR

exon9

Figure 3. Effects of mutations and splicingonTPOproteinsequence.Primarystructuresoftheproteinsdeduced fromcDNAsequencesare

compared.N,normalgeneproduct; Gns, product of goitergeneand normalsplicing; Gas, productofgoitergeneandalternativesplicingas

ob-servedingoitermRNAfrompatient DM.pandd, putative proximaland distalheme-bindinghistidineresidues,respectively. TM,

transmem-brane domain.x,GGCCduplication. *Stop codon.Aminoacidsarein theone-letter code. Themutatedregioniscross-hatched in theupper panelandunderlined in the lowerpanel.Arrowsindicate thepointmutations.The arrowhead indicates theexon8/exon9junction.Thedashed

line denotes deleted amino acids.

amount ofPCR-amplified transcript displaying thesame size asthegoiter transcript (Fig. 1, lane 2)suggeststhatalternative splicinginthegoiter mightrepresentthe exacerbationofa

natu-rally occurring phenomenon. Although deleterious in the nor-malallele, in the mutated allele bearing the GGCC duplication, the alternativesplicing mightrepresenta"salvage" mechanism becauseit restoresthe normal reading frame disrupted by the mutation andeliminates thestopcodons whichwouldtruncate

theproteininitscarboxy-terminal half. Although the physio-logical relevance of the alternative splicing remainstobe eluci-dated inthe case of this study, it is reminiscent ofasimilar situationdescribed inananimalmodel ofeuthyroid congenital goiter with TG defect (22), and of mutations of Becker

muscu-lardystrophy (23). Althoughnoenzymatic activitycanbe

ex-pected from the normal splicing product of the mutatedgene

(see Results and Fig. 3), the product of alternative splicing of the mutatedgene asobservedinthegoiter mRNA might have

someresidualTPOactivity inspite of mutation of the

proxi-malputative heme-binding His, as other His residues in the

peptide might possibly function in heme binding. This could

accountfor theverylowresidual TPO activity observed in the goiter (5% ofanormal control).

Intheabsenceof results fromNorthern blots of TPO

tran-scripts (duetolimitation of tissue availability), it is difficultto

estimate theamountof alternatively spliced transcripts inthe

goiterascomparedto the normalmessagepresent incontrol

tissue.

Absence

of

amplification

of normal-sized TPO cDNA

from

the

goiter

is

likely

to result from

destabilization of

the

mRNA

bearing

stopcodons in the

middle

of the coding se-quence

(24).

As nootherobservations

of

naturally occurring TPOgene

mutation

have been reported,

it is difficult

tospeculateonthe

nature

of

the

additional

point mutations found

inour

patient.

They may have accumulated because of lack of selective

pressure onthe abnormal gene.

Alternatively,

although they inducea

change

inamino acids

(11

157:G

-> T:Ala-- Ser and

1237:G -- C:Ser-- Thr in the

reading

frame of the normal

allele) they

may represent

polymorphisms

thatwerepresent

before the GGCC

duplication;

these

point

mutationsare

how-evernot

found

inany

of

thefour

independently published

se-quences

(

11,

1 14, 19).

Froma

practical viewpoint,

thepresenceof theNaeIsite in

the TPO alleles

amplified

by PCR as describedin Methods

providesarapidtestfor directgenetic diagnosis of this

muta-tion.

Considering

theexpected

genetic heterogeneity

of

organi-fication

defects, analysis

of furthercases will

provide

new

in-sight

on thestructure-function relationships of

thyroid

peroxi-dase.

Acknowledgments

This workwassupported bygrantsfrom theFonds de laRecherche

Scientifique MWdicale,and theBelgian Ministerede laPolitique

Scien-N

NH2 [

p

v

d

Gns:

Gas:

II

I

0

I

x

I

I

---x

d

tifique and the Loterie Nationale. Dr.AbramowiczisaResearch Assis-tant at the Fonds NationaldelaRecherche Scientifique, Belgium.Drs.

Targovnik, Krawiec, and Pisarevare established investigatorsofthe Argentine National Research Council (CONICET)and Dr.Targovnik

was avisiting investigatorattheInstitut deRecherche

Interdisciplin-aireenBiologie Humaineet Nucleaire, from 2 April to 2 June 1991,

supportedbyagrant from theUnited NationsEducational,Scientific

and CulturalOrganization(UNESCO) andthe ThirdWorld Academy ofSciences (TWAS). Thistextpresents research resultsof theBelgian ProgrammeonInter-University Polesof Attraction, initiatedby the

Belgian State, Prime Minister's Office, SciencePolicyprogramming.

Thescientificresponsibilityisassumed by theauthors.

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