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Int. J. Cancer (Pred. Oncol.): 69,312-316 (1996)
0 1996 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de I’llnion Internationale Contre le Cancer
DISTINCTION BETWEEN SPORADIC AND HEREDITARY MEDULLARY
THYROID CARCINOMA (MTC) BY MUTATION ANALYSIS OF THE RET
PROTO-ONCOGENE
Monika FINK’,Andreas WEINHAUSEL’,
Bruno NIEDERLE~
and Oskar A. HA AS',^ for the “Study Group Multiple Endocrine
Neoplasia Austria (SMENA)”
‘CCRI, St. Anna Children ’s Hospital, Vienna; and 2SSurgicalUniversity Clinic, Vienna,Austria.
Germline and somatic mutations of the RET proto-oncogene
are important pathogenetic factors in hereditary and sporadic
forms of medullary thyroid carcinoma (MTC). We have therefore analysed exons 10, I I. 13, 14 and 16 of this gene in 85
individuals from 16 Austrian families who, according to clinical
criteria, were at risk of suffering from hereditary forms of MTC.
We found mutations (codons 620,634 and 804) in the germline
of 3 families with familial medullary thyroid carcinoma (FMTC),
of 5 with multiple endocrine neoplasia type 2A (MEN 2A; codon
634) and of 2 with multiple endocrine neoplasia type 2B (MEN
2 B codon 9 18). The codon 804 mutation in one FMTC family led
to the substitution of Val (GTG) for Met (ATG) and has not been
reported previously. Within these I0 families, 32 carriers and 32
non-carriers were identified. Somatic mutations in the tumors
of 3 other families suggested a sporadic origin of the neoplasms.
In the remaining 3 families, no mutations were identified.
Fifty-nine individuals with an apparently sporadic MTC lacked
germline mutations in the RET gene, whereas 7 of 24 available
tumors (29%) contained a somatic mutation in codon 9 18. Our
findings provide further evidence that molecular genetic evaluation of hereditary and sporadic forms of MTC is a necessary
prerequisite for counselling and management of patients and
their families.
91996
Wiley-Liss,Inc.
Multiple endocrine neoplasia type 2A (MEN 2A), multiple
endocrine neoplasia type 2B (MEN 2B) and familial medullary
thyroid carcinoma (FMTC) are autosomal dominant inherited
disorders which predispose to the development of medullary
thyroid carcinoma (Donis-Keller et al., 1993; Hofstra et nl.,
1994; Mulligan et al., 1994a, 1995). Moreover, approximately
50% of MEN 2A and MEN 2B patients suffer from pheochromocytomas and approximately 5% of MEN 2A patients from
hyperplasia of the parathyroid glands (Donis-Keller et al.,
1993; Hofstra et al., 1994; Mulligan et al., 1994a, 1995). In
contrast to MEN 2B patients who can be easily recognized by
their distinct phenotype-a marfanoid habitus, mucosal neuromas, ganglioneuromas of the gastrointestinal tract and ophthalmic abnormalities-the diagnosis of FMTC is based solely on
the familial occurrence of MTC and/or CCH. With the recent
rccognition of specific mutations in the RET proto-oncogene,
which is located in the centromeric region of chromosome 10,
unequivocal determination of the carrier status became possible (Donis-Keller et a/., 1993; Mulligan et al., 1993). More
than 90% of MEN 2A and FMTC cases are due to constitutional mutations in 1 of 5 cysteine residues that are encoded by
exons 10 and 11 of the RET gene (Mulligan et a/., 1995). In
MEN 2A, a particular mutation at codon 634 that replaces a
cysteine with an arginine has been reported to specifically
increase the risk of parathyroid disease (Mulligan et al.,
1 9 9 4 ~ )In
. addition, exon 13 (codon 768) and exon 14 (codon
804) mutations have been reported recently in the germline of
3 and 2 families, respectively (Bolino et al., 1995; Eng et a/.,
199%). They affect the intracellular first tyrosine kinase
domain by substituting glutamic acid (codon 768 G A G ) for
aspartic acid (GAC) and valine (codon 804 GTG) for leucine
(TTG). respectively. The pathogenetic significance of the
mutation Glu768 > Asp was further confirmed by its somatic
occurrence in 4 of 10 sporadic MTCs (Eng et al., 1995b).
Except for 2 novel dinucleotide changes in codons 620 and
634/635 (Landsvater et al., 1996), all mutations were due to
single nucleotide substitutions (Mulligan et al., 1995). In M E N
2B only one specific mutation has been identified so far,
namely a threonine for methionine substitution in codon 918 in
the intracellular second tyrosine kinase domain (Carlson et al.,
1994b; Hofstra et al., 1994). The same codon is also altered in
approximately 23% of sporadic MTCs (Blaugrund et al., 1994;
Hofstra et al., 1994; Zedenius et aL, 1994; Eng et al., 1995~;
Komminoth et al., 1995). In accordance with the more severe
lapse of the disease in MEN 2B patients, sporadic tumors
harboring this mutation seem also to behave more aggressively
than tumors with MEN 2A or FMTC mutations (Zedenius et
a/., 1994).
To substantiate the clinical suspicion of hereditary tumor
predisposition, we analysed material from individuals of 16
Austrian families by molecular genetic means as well as from
59 patients with apparently sporadic MTCs.
MATERIAL AND METHODS
Samples
DNA was obtained from the peripheral blood (PB) of 44
individuals. Eighty-five individuals (designated A ) were considered to be members of FMTC, MEN 2A and MEN 2B
kindreds or families in which inherited disease was suspected.
Fifty-nine patients (designated T) suffered from apparently
sporadic MTC. In addition, tumor tissue from 24 patients was
analysed; 5 tumors from individuals with suspected familial
predisposition and 19 tumors from apparently sporadic cases.
The mutation analysis of 2 of these tumors was published
previously (Eng et al., 1 9 9 5 ~ ) .
Patients
The patients or families were classified according to the
criteria proposed by the “International RET mutation consortium” (Mulligan et al., 1995):
MEN 2A (1)-MTC, pheochromocytoma and histologically
confirmed parathyroid hyperplasia o r adenoma;
MEN 2A (2)-MTC and pheochromocytoma, but without
parathyroid disease;
MEN 2A (3)-MTC without pheochromocytoma, but with
histologically confirmed parathyroid disease;
FMTC-at least 4 confirmed MTC without pheochromocytomas o r parathyroid disease;
Other-any
family fulfilling the above criteria in which
screening was not performed, or FMTC families with less
than 4 cases of MTC;
MEN 2B-MTC with or without pheochromocytoma together with the typical facies, mucosal neuromas and ganglioneuromatosis of the gut.
whom correspondence and reprint requests should be sent, at
the CCRI, St. Anna Children’s Hospital, KinderspItalgasse 6, A-1090
Vienna. Austria. Fax: 43 1 40170 481. email: o.a.haas(@magnet.at
Received: February 26, 1996 and in revised form April 15, 1996.
313
FINK ETAL.
TABLE I - SEQUENCES OF PRIMERS USED FOR PCR AND DNA SEQUENCING TOGETHER WITH THE
EXPECTED LENGTH OF PRODUCTS AND ANNEALING TEMPERATURE IN THE PCR
Exon
Orientation
Primerisequence
10
Sense
Anti-sense
Sequencing
Sense
Anti-sense
Sequencing
Sense
Anti-sense
Sequencing
Sense
Anti-sense
Sequencing
Sense
Anti-sense
Sequencing
5’-gcg ccc cag gag gct gag tg;3’
5’-cgt ggt ggt ccc ggc cgc C-3
S’-agg agg ctg agt ggg c-3’
5‘-cct ctg cgg tgc caa gcc tc-3’
5’-gac agc agc acc gag acg atg-3’
5’-ctg cgg tgc caa gcc tca-3’
5’-gca ggc ctc tct gtc tga act t-:’
5’-gga gaa cag ggc tgt atg ga-3
5‘-gcc tct ctg tct gaa ctt ggg-3‘
5’-aag acc caa gct gcc tga c-3’
S‘-gtg gtg ggt cag ggt gtg g-3’
5’-ccc aag ctg cct gac cc-3’
5’-agg gat agg gcc tgg gct tc-3’
5’-taa cct cca ccc caa gag ag-3’
5’-ata ggg cct ggg ctt ctc-3’
11
13
14
16
Preparation of samples and isolation of gnomic DNA
Mononucleated cells from peripheral blood samples of
patients and their relatives were obtained by gradient density
centrifugation on Lymphoprep (Nycomed, Oslo, Norway).
Fresh tumor was cut into small pieces.
Formaldehyde-fixed paraffin-embedded tumor material cut
into sections of 5-10 p m was provided by the study centers.
Paraffin was removed by 2 10-min treatments with xylene. The
tissue was washed twice with absolute ethanol and dried
briefly. Samples were digested with 0.1 mg/ml proteinase K in
extraction buffer (100 mM NaC1, 50 mM Tris-HC1, p H 7.5, 1
mM EDTA, 0.5% SDS) at 55°C overnight when D N A was
purified by phenol-chloroform extraction. Otherwise a rapid
DNA-extraction procedure with DNAzol (ViennaLab, Vienna, Austria) was used to purify high-quality D N A from
whole blood or protease digests of tumor tissue.
Polymerase chain reaction (PCR)
Primers used to amplify exons 10, 11, 13, 14 and 16 and the
respective PCR conditions are shown in Table I (Donis-Keller
et al., 1993; Mulligan et al., 1993; Eng et al., 1994, 19956;
Hofstra et al., 1994; Ceccherini et al., 1994).
Single-strand conformationpolymorphism (SSCP)
PCR product (6 pI) was mixed with 3 p1 formamide loading
buffer (included in fmol Sequencing System, Promega, Madison, WI). heat-denatured and loaded on a non-denaturing
polyacrylamide gel (12% polyacryalmide, 3% glycerol, 0.5 x
TBE). Gels were run in an Easy 111-4 electrophoresis cell
(KemEnTech, Copenhagen, Denmark) at 360 to 400 V at 4°C
for 4 hr. Gels were stained in ethidium bromide (0.5 pg/ml) for
5 min and destained in running tap water for 30 sec.
Restriction digest
Codon 918 RET gene mutations in MEN 2B patients and in
sporadic MTCs can be detected by Fok I digestion of PCR
products of exon 16 (Hofstra et al., 1994) and the codon 768
mutation by Alu I digestion of PCR products of exon 13 (Eng et
a/., 19956). Mutations eliminate the restriction sites and leave
the mutant allele uncut. All samples were loaded on 2% agarose
gels. Restriction enzymes were purchased from Promega.
Several mutations in exon 11, codon 634, generate new
restriction sites. The T G C to CGC transition generates a
restriction site for Cfo I, the mutated sequence T G C to TGG is
cleaved by Hae 11, TAC by Rsa I and G G C by Hae 111. As an
alternative to SSCP analysis, MEN2A carrier status was
therefore determined by restriction digestion in families A02
(Hae 11), A03 (Cfo I), A11 and A12 (Rsa I) and A10 (Hae 111)
which harbored the respective mutations. Without further
Product [bp]
Annealing temp. (“C)
186
69
118
68
295
63
327
68
192
64
purification, 15 pl of the PCR products were mixed with 5 p1
digestion mix (containing the appropriate buffer, enzyme and
water), incubated at 37°C for at least 2 hr and loaded on 3%
agarose gels.
Screening strategy and sequence analysis
Initially, we used SSCP and restriction analysis for screening
exons 10, 11, 13 and 16 in all tissues. Mutations which were
identified through this approach were then further verified by
cycle sequencing of PCR products.
Before sequencing, we purified PCR-amplicons with the
Talent clean mix kit (Talent, Trieste, Italy). For cycle sequencing we used a LI-Cor Model 4000L (Li-Cor, Lincoln, NE)
DNA sequencer with 5‘-IRD-41 labeled nested forward
(5‘ -+ 3’) primers and the Thermo Sequenase cycle sequencing
kit (Amersham, Aylesbury, UK) (Table I).
PB and tumor tissue (if available) from all families and
patients with apparently sporadic MTCs in whom no mutation
was detected in exons 10, 11, 13 and 16 were subjected to
screening for exon-14 mutations by automated sequencing.
RESULTS
Molecular genetic analysis revealed characteristic point
mutations in the germline of 10 Austrian families and thus
confirmed the clinical diagnosis of either FMTC, MEN 2A or
MEN 2B (Fig. 1, Table 11). The types of the respective
mutations in the RET proto-oncogene in our patients were in
accordance with those reported previously (Table 11) (DonisKeller et al., 1993; Blaugrund et al., 1994; Carlson et al., 19946;
Hofstra et al., 1994; Maruyama et al., 1994; Mulligan et al.,
1994a; Schuffenecker et al., 1994; Komminoth et al., 1995;
Landsvater et al., 1996). In all 5 MEN 2A families and in 1
FMTC family the mutation was located in codon 634. Interestingly, the most common mutation (TGC > CGC) which accounts for 52% of all codon 634 mutations (Mulligan et al.,
1995) was found in one family only. The mutation 634 T G C >
TAC that occurs at a frequency of 25% was detected in 2
families. Two rare mutations in this codon (TGC > TGG,
6.6%, and T G C > AGC, 0.8%) were found in families A02
and A04, respectively. Three of the carriers are still asymptomatic (in family A02 and family A04). They are under close
surveillance by the clinicians. One individual in family A04 was
thyroidectomized because of a positive pentagastrin stimulation test. He suffered from CCH, but molecular genetic
analysis proved that he as well as his mother and sister had no
germline mutation as yet identified in MEN 2A, 2B or FMTC
families. Two FMTC families had codon 620, one had codon
634 and another had codon 804 mutations, respectively. This
314
RET MUTATIONS IN SPORADIC AND HEREDITARY MTC
MTC with germline mutation
I MTC with somatic mutation
E MTC without mutation
m
M
14
13
0 CCH with gennlme mutation
healthy with germline mutation
CCH without mutation
0 healthy without mutation
cd
3
2*
3
.CI
4-(
0
3E
a
s:
familv:
A01 A02 A03 A04 A05’A06 A07 A08 A09 A10 A l l A12 A13 A14 A15 A16’
codoiaffected: 620 634 634 634 918 - 804 918 - 634 9 1 8 634 634 918 918 FIGURE1- Disease and mutation patterns of 16 Austrian families who, according to clinical criteria, had a suspected genetic
predisposition to development of MTC.
Exon
Case
FMTC
A01
A07
A10
MEN 2A’
A02 (1)
A03 1
A04 2{
A12 12
A13
MEN 2B
A05
A15
sMTC*
A08
All
A14
1)
Codon
Nucleolide
Amino acid
chanee
change
10
14
11
620
804
634
TGC > TAC
GTG > ATG
TGC > GGC
Cys > Tyr
Val > Met
Cys > Gly
11
11
634
634
634
634
634
TGC
TGC
TGC
TGC
TGC
> TGG
> CGC
> AGC
> TAC
> TAC
Cys > Trp
Cys > Arg
Cys > Ser
Cys > Tyr
Cys > Tyr
16
16
918
918
ATG > ACG
ATG > ACG
Met > Thr
Met > Thr
16
16
16
918
918
918
ATG > TTG
ATG > ACG
ATG > ACG
Met > Leu
Met > Thr
Met > Thr
11
11
11
‘(1)and (2). classification according to the criteria proposed by
the “International RET mutation consortium” (Mulligan et aZ.,
199.5)(see text).’Somatic mutations restricted to the tumor-tissue.
last mutation differs from the one reported previously in this
codon (GTG > TTG) (Bolino et al., 1995). It changes the
nucleotide sequence from GTG to ATG and thereby replaces
the normal valine with a methionine (Table 11, Fig. 2).
Two patients with the characteristic symptoms of MEN 2B
carried the codon 918 mutation in exon 16. This mutation was
found in 93.8% of all MEN 2B patients analysed so far
(Mulligan et a/., 1995). In one of the patients (A15) the
mutation occurred de noc’o as both parents and a brother were
non-carriers. Non-paternity was excluded by analysis with
polymorphic PCR markers on chromosomes 8,16 and 21 (data
not shown). The phenotypically unaffected father of the
second MEN 2B patient (A05) was not available for the study.
The mother of the patient is a non-carrier.
In 6 families (A06, A08, A09, A l l , A14 and A16) with 1or 2
MTCs and/or CCH, no germline mutations were detected in
the analysed exons (Fig. 1). Paraffin-embedded tumor was
available from 5 patients of 4 families (A08, A l l , A14 and
A16) who suffered from MTC. Digestion of the PCR product
of exon 16 with Fok I revealed a somatic mutation in codon 918
in 4 of these patients.
Analysis of the respective exons in 59 patients with MTC
with no apparent familial predisposition did not reveal any
new germline mutations. However, the detection of somatic
codon 918 mutations in 3 of 19 available tumors confirmed
their sporadic origin. In the remaining 16 tumors we also
searched for the glutamic acid to aspartic acid transition at
codon 768 in exon 13 by AluI digestion and for the mis-sense
mutation at codon 804 in exon 14 by direct sequencing of the
PCR products. Despite a reported incidence of approximately
40% in codon 918 mutation-negative tumors (Eng et al.,
19956), we could not identify a codon 768 mutation by Alu I
digestion in our tumors. Table 111 summarizes the clinical
outcome and results of mutation analyses of the tumor tissues
from 24 patients.
DISCUSSION
We identified germline mutations in 10 of 16 Austrian
families with a clinically suspected, heritable predisposition to
315
FINKETAL.
TABLE 111-CHARACTERISTICS OF 24 PATIENTS WITH MTC FROM WHOM
TUMOR TISSUE WAS ANALYSED. SEVEN OF 24 (29%) TUMORS HAD
MUTATIONS IN CODON 918 OF THE RET GENE
Case
Sex
Age at
diagnosis
(vears)
FIGURE
2 - Sequence pattern of the novel germline RET mutation of codon 804 in exon 14 (GTG > ATG) in an FMTC family.
development of MTC. This enabled us to unequivocally
determine the carrier status of all family members. The
demonstration of somatic mutations in the tumors of another 4
patients, on the other hand, provided strong evidence against a
specific genetic predisposition in these families with multiple
thyroid diseases. The absence of known MEN 2A-associated
germline mutations in at least 2 of the remaining families (A08
and A14), however, was still surprising. We cannot exclude the
presence of as yet unidentified germline mutations in other
parts of the RET gene, but it also remains possible that
neoplastic diseases in these families resulted from an increased mutagenic exposure. The index patient in family A08
had bilateral MTC and CCH. Despite the fact that several
sporadic cases with bilateral MTC have been reported previously, such a multifocal onset of the disease is nevertheless
generally considered a definite feature of FMTC and MEN 2A
(Franc et aL, 1987). In addition, the patient's brother was
thyroidectomized because of CCH. With an incidence of
approximately S%, CCH is quite a common feature in the
normal population (Lips et al., 1987). CCH may reflect a
physiological or a preneoplastic condition that may also be
more common in one family than in another. Moreover,
proliferating cells in CCH are probably more prone to acquire
mutations. The index patient from family A14 developed MTC
at a young age. His father had an elevated calcitonin level after
stimulation with pentagastrin and was therefore also thyroidectomized. The paternal grandmother and a cousin of the patient
suffered from primary localized cutaneous amyloidosis (PLCA)
from the age of 18. Similar pigmented interscapular skin
lesions, which were also seen in families A02 and A13, were
previously reported in several FMTC and MEN 2A kindreds
associated with a 634 mutation and seem to arise prior to the
onset of thyroid disease (Ferrer et al., 1991). The existence of
an apparently sporadic MTC in a young individual in the
context of such a particular heritable skin disease therefore
raises the question whether this association represents a mere
coincidence or rather has a common genetic basis other than
mutations of the RET proto-oncogene. The families in which
neither constitutional nor somatic mutations were found
remain under close clinical surveillance and biochemical
screening is being continued.
The detection of a new FMTC or MEN 2A case can be of
great significance to both patients and offspring alike. Indeed,
A08
A1 1
A1 1
A14
A16
TO1
TO2
TO3
TO4
TO5
TO6
TO7
TO8
TO9
T10
T11
T12
T13
T14
T15
T16
T17
T18
T19
M
M
M
M
F
M
M
F
F
F
F
M
F
F
F
F
F
M
F
M
M
F
F
M
33
61
30
22
69
45
51
48
47
74
50
50
49
61
75
49
53
54
41
28
53
49
56
36
Clinical outcome
Mutation
(tumorlgermline)
Persist. MTC
Dead of disease
n.a.'
Persist. MTC
n.a.
Free of disease
Persist. MTC
n.a.
n.a.
n.a.
Dead of disease
Free of disease
Free of disease
Free of disease
n.a.
Persist. MTC
Dead of disease
Dead of disease
n.a.
Free of disease
n.a.
n.a.
n.a.
n.a.
Codon 918/neg.
Codon 918ineg.
Codon 918/neg.
Codon 918/neg.
Neg./neg.
Neg./neg.
Codon 918ineg.
NegJneg.
Neg./neg.
NegJneg.
Neg./neg.
Neg./neg.
NegJneg.
Neg./neg.
Neg./neg.
Codon 918ineg.
Codon 918/neg.
NegJneg.
Neg./neg.
Neg./neg.
Neg./neg.
Neg./neg.
NegJneg.
Nedneg.
'No information available.
germline mutations were reported in 1 of 15 (Blaugrund et al.,
1994), in 1 of 10 (Zedenius et al., 1994) in 1 of 67 (Eng et al.,
1995a) and in 3 of 16 (Komminoth et al., 1995) randomly
chosen patients with MTC who lacked a positive family history
(on average 6% of apparently sporadic MTC). We did not
identify any germline mutations in the 59 patients analysed,
whereas 3 of 19 tumors harbored a somatic mutation. Because
somatic and germline mutations can both be detected in the
tumor, the neoplastic tissue should be the first choice for
mutation screening. Moreover, somatic mutations may also
turn out to be of prognostic value, since at least some of them
seem to indicate a less favorable prognosis (Zedenius et al.,
1994). The correlation between the presence of a codon 918
mutation and an aggressive course of the disease is also evident
in our study, since all patients with this particular tumorrestricted mutation suffered from recurrence of the tumor. It is
likely that other factors and/or mutations can also determine
the severity of the disease because, in several cases with fatal
outcome in the group, we were unable to identify any particular mutation (Table 111). Much to our surprise, we could not
detect any tumor-restricted or germline mutation at codon
768, although it has been suggested that this particular
alteration may be present in a considerable proportion of
sporadic tumors (Eng et al., 199%).
In contrast to MEN 2A, approximately SO% of the MEN 2B
cases are due to de nuvu germline mutations (Carlson et al.,
1994~).It was recently shown that the affected allele is always
of paternal origin and that two-thirds of the de nuvo cases are
females (Carlson et al., 1994~).The implication of this parentof-origin bias and the uneven sex distribution among the
offspring remains an enigma (Carlson et aZ., 1994~).One
possible explanation is a selective disadvantage for male
embryos with a codon 918 mutation in a certain stage of
development. In this context it is also noteworthy that 2 of the
4 de nuvo mutated RET alleles in MEN 2A patients reported so
far originated from the father and that both patients are
females (Mulligan et nl., 1994b; Zedenius et al., 1994). Since, in
the 2 other cases with a putative de novu mutation, neither
parents nor siblings of the patient were examined, parental
transmission of the RET mutation in de nuvu MEN2A cases
cannot be excluded (Blaugrund et al., 1994).
316
RET MUTATIONS IN SPORADIC AND HEREDITARY MTC
Conventional biochemical screening in individuals of families with hereditary MTC can give false-positive results because of physiological conditions unconnected with the disease, whereas molecular genetic analysis unambiguously reveals
the carrier status. Our approach of directly sequencing the
PCR products not only circumvents the laborious optimization
of SSCP conditions, which have to be defined stringently for
each particular mutation, but also avoids the risk of falsenegative results. Moreover, it immediately provides complete
information about additional mutations and/or polymorphisms. The detection of non-carriers among family members
with CCH in our and other studies confirms that thyroidectomy should not be performed before the carrier status has
been established by molecular genetic means (Lips et nl.,
1994). On the other hand, the detection of a germline mutation
in asymptomatic relatives serves as an alert for a more
intensive clinical surveillance, allows earlier detection of neoplastic lesions and enhances the chance of curing by preventive
thyroidectomy. Analysis of tumor material and blood is extremely helpful in cases in which the diagnosis of FMTC is
uncertain, particularly in families in which only 1 or 2 members
are affected. Furthermore, the specific codon 918 mutation
might be of prognostic value, as tumors containing this
mutation seem to behave more aggressively.
Biochemical examination of the families of affected persons
is painful, raises anxiety and demands the active cooperation
of patients, relatives and physicians over years. In contrast,
only one blood sample per individual is required for molecular
genetic screening. This simplicity and the high accuracy of
analysis contribute to the high compliance of individuals at risk
of developing familial MTC.
ACKNOWLEDGEMENTS
We thank the patients and their families involved in this
study and the clinicians in the Study Group Multiple Endocrine
Neoplasia Austria: Drs. A. Behmel, R. Pfragner, K. Dam,
M. Manzl, G. Galvan, M. Stockhammer, W. Teufl, A. Haidenthaler, A. Brandl, S. Lax, H. Vierhapper, J. Flores, M. Weissel
and B. Markt. We are grateful to Miss Margit M. Konig and
Dr. 0. Chevchtik for technical assistance. DNA sequencing
was performed by Dr. R. Felber at the Biozentrum, University,
Vienna. This work was supported by the “Osterreichische
Kinderkrebshilfe”.
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