Functional characterization of the MTC-associated germline RET-K666E mutation: evidence of oncogenic potential enhanced by the G691S polymorphism ...
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Endocrine-Related Cancer (2011) 18 519–527
Functional characterization of the MTC-
associated germline RET-K666E mutation:
evidence of oncogenic potential enhanced
by the G691S polymorphism
Maria Grazia Borrello1†, Antonella Aiello2†, Bernard Peissel 3,
Maria Grazia Rizzetti1, Piera Mondellini1, Debora Degl’Innocenti1,
Veronica Catalano1, Morena Gobbo2, Paola Collini 2, Italia Bongarzone1,
Marco A Pierotti 4, Angela Greco1‡ and Ettore Seregni 5‡
1
Operative Unit Molecular Mechanisms, Department of Experimental Oncology, 2Pathology and Laboratory Medicine, 3Unit of
Medical Genetics, Department of Preventive and Predictive Medicine, 4Scientific Directorate and 5Nuclear Medicine, Therapy and
Endocrinology, IRCCS Istituto Nazionale dei Tumori Foundation, Milan, Italy
(Correspondence should be addressed to M G Borrello; Email: mariagrazia.borrello@istitutotumori.mi.it)
†
(M G Borrello and A Aiello contributed equally to this work)
‡
(A Greco and E Seregni share senior authorship)
Abstract
Activating mutations of RET, a gene encoding two isoforms of a tyrosine kinase receptor
physiologically expressed in several neural crest-derived cell lineages, are associated with
the inherited forms of medullary thyroid carcinoma (MTC). The identification and character-
ization of novel RET mutations involved in MTC is valuable, as RET gene testing plays a crucial
role in the management of these patients. In an MTC patient, we have identified a germline
c.1996AOG transition in heterozygosis leading to K666E substitution. In addition, the
conservative S904S (c.2712COG) and the non-conservative functional G691S (c.2071GOA)
polymorphisms have been identified. Through functional studies, we demonstrate for the first
time that K666E is a gain-of-function mutation with oncogenic potential, based on its ability to
transform NIH3T3 cells. It was not possible to define whether K666E is a de novo or inherited
RET variant in the patient, as the family history was negative for MTC, and the carrier status of
family members could not be tested. Our results, together with a recent report of co-segregation
of the mutation in three MTC families, suggest that K666E is a causative MTC mutation. As we
have shown that the same patient allele carries both K666E and G691S variants, the latter
known to increase downstream RET signaling, a possible role for the G691S polymorphism has
also been investigated. We have demonstrated that, although RET-G691S is not oncogenic
per se, it enhances the transforming activity of the RET-K666E mutant, thus suggesting a
modifier role for this functional polymorphism.
Endocrine-Related Cancer (2011) 18 519–527
Introduction (RET51) and a shorter protein of 1072 residues
The RET (rearranged during transfection) gene displaying nine unrelated C-terminal-specific amino
encodes a tyrosine kinase receptor with a crucial role acids (RET9), as shown in Fig. 1A. The RET receptor
in development. RET comprises 21 exons and system also includes the GFRa1–4 alternative
generates a transcript subjected to alternative splicing co-receptors and GDNF family ligands, including
leading to two main isoforms: a protein of 1114 GDNF, neurturin, persephin, and artemin (reviewed
residues displaying 51 C-terminal-specific amino acids in Arighi et al. (2005)).
Endocrine-Related Cancer (2011) 18 519–527 Downloaded from Bioscientifica.com at 09/25/2020 03:50:51PM
DOI: 10.1530/ERC-10-0306
via free access
1351–0088/11/018–519 q 2011 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.orgM G Borrello et al.: Germline RET K666E and G691S mutations
A B Lys666Glu C RET9
150 160 170
1S -
69 E
G 666
8T
R
6E
1S
k
34
K
91
oc
66
69
C6
t
M
M
CDL
w
K
G
RET
pERK1/2
C634 CR
ERK1/2
TM
K666
G691 JM Gly691Ser
230 240
TK
M918
RET9
RET51
Figure 1 (A) Schematic representation of RET protein. The codons mutated in the patient (K666 and G691) and the codons
corresponding to MEN2A RET-C634R and MEN2B RET-M918T mutations are indicated. CL, cadherin-like domain; CR, Cys-rich
domain; TM, transmembrane domain; JM, juxtamembrane domain; TK, tyrosine kinase domain. (B) Sequence electropherogram for
missense mutations found in exon 11. The mutations are marked by arrows. (C) Western blot analysis of RET protein and associated
ERK signaling of transiently transfected RET gene variants RET-K666E, RET-G691S, and RET-K666E–G691S. Lysates from
HEK293T cells transiently transfected with recombinant plasmids of the indicated RET mutants were analyzed by western blot with
anti-RET, anti p-ERK1/2, and anti-ERK1/2 antibodies.
RET is involved in thyroid neoplasias through largely exploited to optimize the diagnostic and
different gain-of-function alterations. Somatic clinical management of hereditary MTC (Chiefari
rearrangements of RET are present in about one-third et al. 1998, Marx 2005, Gagel & Marx 2008). The
of papillary thyroid carcinomas (reviewed in Greco presence of a known MEN2-associated RET mutation
et al. (2009)), whereas specific germline activating in the germline of an MTC patient identifies hereditary
mutations of RET are causally involved in hereditary MTC disease, thus allowing preclinical identification
medullary thyroid carcinoma (MTC), including of family members at risk of developing MTC, as well
multiple endocrine neoplasia type 2A (MEN2A, as providing information about the risk of the proband
OMIM 171400), type 2B (MEN2B, OMIM 162300), developing other tumors associated with MEN2
and familial MTC (FMTC; OMIM 155240) syndromes syndromes. Moreover, the RET mutation type, assessed
(Lodish & Stratakis 2008). Activating RET mutations by RET genetic testing, guides clinical decisions, as
involved in hereditary MTC forms are also present in a different RET mutants have been associated with
fraction of sporadic MTCs. different risk profiles. Consequently, prophylactic
MTC, which accounts for around 5% of thyroid thyroidectomy for asymptomatic mutation carriers is
tumors, is a neuroendocrine neoplasia arising from the recommended at early ages (from the first months of
thyroid parafollicular C cells. Physiologically, RET, life to 5 years) for higher risk mutations (codons
GFRa4, and persephin appear to be necessary for 883/918 and 634 respectively), whereas for milder
migration of neural crest calcitonin producing cells mutations surgery in carriers may be delayed
into the developing thyroid gland: markedly dimin- (Frank-Raue et al. 2010). Moreover, the identification
ished parafollicular C cells are found in RETK/K mice and functional characterization of novel activating
(Lindahl et al. 2000, Lindfors et al. 2006). RET mutations responsible for the disease contributes
MTC is relatively unresponsive to radiotherapy and to the knowledge of genetic–phenotypic relationships
conventional chemotherapy: thyroidectomy is the in hereditary MTC.
standard treatment and the only curative therapy In this study, we report the identification, in a
(Wells & Santoro 2009). RET mutation analysis is 48-year-old woman carrying unicentric MTC with
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520 www.endocrinology-journals.orgEndocrine-Related Cancer (2011) 18 519–527
C-cell hyperplasia, of the RET-K666E germline variant tumors/disorders, or pheochromocytomas in her
as well as two polymorphisms: the G691S non- family. Her father died at the age of 66 years from
synonymous variant and the conservative S904S liver cirrhosis. Her mother was healthy at the age of 82
(TCC/TCG) variant. K666E and G691S RET years and her brother at the age of 52 years. Her sister
mutations occur in the juxtamembrane domain, a developed breast cancer at the age of 50 years. The
region not frequently affected by mutations. In this patient had no offspring. The patient and her sister
study, the co-segregation of the K666E mutation with provided a written informed consent for RET mutation
MTC in our patient’s family was not verifiable. analysis and for research purposes approved by the
However, K666E has previously been reported in local ethical committee. To date, no other family
three unrelated families and it is thought to be a members have consented to testing.
pathogenic mutation (Ahmed et al. 2005). In this study,
we have demonstrated the in vitro oncogenic potential Mutation analysis
associated with the RET-K666E mutant, based on its
DNA was extracted from peripheral venous blood using
ability to transform NIH3T3 cells, thus suggesting that
a commercial kit (Qiagen Blood Extraction Kit) as
this RET variant is indeed responsible for hereditary
recommended by the manufacturer. The screening for
MTC. Moreover, our results, showing that the
germline RET gene mutations was carried out in exons
oncogenic activity of RET-K666E is enhanced by the
8, 10, 11, 13, 14, 15, and 16, where most known
presence of the polymorphism G691S, suggest a
mutations take place. RET exons 10, 11, 13, and 14 were
possible modifier role for this known non-synonymous
analyzed by double-gradient denaturing gradient gel
RET polymorphism.
electrophoresis (DG-DGGE) with primers and con-
ditions previously described (Hofstra et al. 1996). RET
exons 8, 15, and 16 (together with exon 11 that showed a
Materials and methods DG-DGGE migration pattern suggestive of base
Patient presentation and analysis substitution) were amplified with exon flanking primers
and analyzed by automated sequencing (ABIprism 377;
The patient, a 48-year-old woman, underwent neck
Applied Biosystems, Foster City, CA, USA). The
ultrasound after clinical demonstration of cervical
amplified exon 11 PCR product was cloned in pCRII-
lymphadenomegaly. At the inferior region of the left
TOPO vector (Invitrogen) according to the manufac-
thyroid lobe, a 6!5!5 mm nodule with irregular
turer’s instructions, and single clones were sequenced.
margins was detected. A fine needle aspiration biopsy
gave a cytological diagnosis of follicular proliferation
Construction of the RET mutants
with oxyphilic metaplasia, and for this reason, a left
hemithyroidectomy was performed. The histological Recombinant plasmids carrying RET9-WT (the short
examination, however, showed that the patient dis- isoform of the proto-RET gene), RET51-WT (the long
played a unicentric MTC of 5 mm, associated with isoform of the proto-RET gene), RET9-C634R
C-cell hyperplasia, and vascular invasion. Before (containing an MEN2A causing mutation), and
completing the thyroidectomy, abdomen computed RET9-M918T (containing the main MEN2B causing
tomography (CT) and 24 h urinary catecholamine mutation) are described elsewhere (Arighi et al. 2004).
determination were performed in order to exclude the Site-directed mutagenesis was performed, using an
presence of pheochromocytoma. In addition, C-cell in vitro oligonucleotide mutagenesis system (Quik-
hyperplasia and lymphocytic chronic thyroiditis were Change XL site-directed mutagenesis; Stratagene, La
histologically detected in the right lobe. The patient is Jolla, CA, USA), on RET9-WT and RET51-WT
currently in active follow-up, which includes laboratory cDNAs cloned in the eukaryotic expression vector
tests (e.g. s-calcitonin; carcinoembryonic antigen pCDNA3 (Invitrogen), to obtain RET9-K666E,
(s-CEA), s-chromogranin A; s-calcium; s-parathyroid RET51-K666E, RET9-G691S and RET51-G691S
hormone (s-PTH), s-25(OH)D, u-catecholamines) and constructs. The K666E single mutants were subjected
instrumental examination (e.g. neck and abdomen to a second cycle of site-directed mutagenesis to obtain
ultrasound; chest and abdomen CT). At present, 3 the double mutants RET9-K666E–G691S and RET51-
years after surgery, these diagnostic procedures provide K666E–G691S. The mutant clones were identified by
no evidence of clinical recurrence or metastases. direct sequencing and then entirely sequenced to
The patient attended genetic counseling and family exclude possible additional mutations. Plasmid DNA
history data were collected. The clinical investiga- was extracted using the MAXI PREP Kit (Qiagen) as
tion did not reveal any history of MTC, thyroid suggested by the supplier.
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www.endocrinology-journals.org 521M G Borrello et al.: Germline RET K666E and G691S mutations
Cell culture, transfections, focus formation Results
assay, and anchorage-independent assay
Patient’s and family’s RET mutation analysis
Human HEK293T cells were maintained in DMEM
DG-DGGE analysis and/or direct DNA sequencing
with 10% FCS. The recombinant plasmids were
after PCR amplification of RET exons 8, 10, 11, 13, 14,
transfected using Lipofectamine 2000 (Invitrogen)
15, and 16, from the patient’s peripheral blood, has
according to the manufacturer’s instructions.
shown three variations in the RET gene sequence: a
NIH3T3 cells were cultured in DMEM supplemented
c.1996AOG transition in heterozygosis, leading to the
with 10% FCS. Stable transfection of NIH3T3 cells was
missense K666E mutation, a c.2071GOA transversion
performed using the CaPO4 method (Bongarzone et al.
in heterozygosis, leading to a missense mutation
1989) and using 250 ng plasmid DNA together with
corresponding to the known non-conservative functional
30 mg NIH3T3-derived DNA carrier. Transfected cells
G691S polymorphism (SNIP rs1799939), both in exon
were grown in DMEM with 10% FCS and selected in
11 (Fig. 1B), and a c.2712COG transversion, a silent
the presence of G418 antibiotic (650 mg/ml) to
mutation leading to the conservative S904S polymorphic
determine the transfection efficiency; transforming
variant (SNIP rs1800863) in exon 15 (not shown). The
activity was determined in medium containing 5%
patient’s sister, the only family member who gave
serum in the presence or absence of 10 ng/ml GDNF
informed consent, carried the G691S polymorphism
(AlomoneLabs, Jerusalem, Israel). Both G418-resistant
but not the K666E genetic variant (not shown).
colonies and transformed foci were fixed and counted.
Most mutations associated with hereditary MTC map
For anchorage-independent assay, cell lines NIH3T3
in the Cys-rich region, such as the classical MEN2A-
expressing RET mutants were suspended in DMEMC
associated C634R mutation, or in the kinase domain,
10% FCS and plated at 1000–10 000 cells per 60 mm
such as the prevalent MEN2B-associated mutation
dish in the same medium 0.33% final agar. After
M918T (Fig. 1A); at variance, the RET-K666E variant
2 weeks, the colonies were stained with p-iodonitrote-
maps in the juxtamembrane region of the receptor.
trazolium violet, photographed, and counted.
To assess whether the putative MTC-associated
K666E mutation and the polymorphic G691S mutation
Western blot analysis carried by the proband were on the same allele, the
Cells were serum-starved for 24 h, then lysed in amplified exon 11 PCR product was cloned in pCR2.1-
ice-cold RIPA buffer (20 mM Tris, pH 7.4, 137 mM TOPO vector. A total of 11 clones were sequenced: six
NaCl, 10% glycerol, 0.1% SDS, 0.5% sodium deox- presented both wt codons and five presented both
ycholate, 1% Triton X-100, 2 mM EDTA, pH 8.0) mutated codons, thus indicating that the two mutations
supplemented with protease/phosphatase inhibitors. are on the same allele.
Protein samples were boiled in NuPAGE LDS sample
buffer (Invitrogen), separated by 7% NuPAGE Novex Transforming activity of RET-K666E, RET-G691S,
Bis–Tris Gels with the appropriate running buffer and RET-K666E/G691S gene variants
(Invitrogen), then transferred onto nitrocellulose filters,
and immunoblotted with the indicated antibodies. For Site-directed mutagenesis was performed on pCDNA3
densitometric analyses, blots were analyzed using the eukaryotic expression vector carrying the two RET
Image Quant 5.2 Software (Molecular Dynamics, GE isoforms (RET9 or RET51) to obtain the following
Healthcare, CA, USA). The following antibodies were mutants: RET9-K666E, RET51-K666E, RET9-G691S,
used in blotting experiments: anti-RET (C-19) from RET51-G691S, RET9-K666E–G691S, and RET51-
Santa Cruz Biotechnology (Santa Cruz, CA, USA), K666E–G691S.
anti-phospho-RET (Tyr905) from Cell Signaling Tech- To assess the quality and efficiency of recombinant
nologies (Beverly, MA, USA), anti-phospho-Tyr 4G10 plasmids, all the constructs were transiently transfected
from Upstate Biotechnology (Lake Placid, NY, USA) into HEK293T cells to analyze RET protein
anti-MAP kinase (ERK1/2), and anti-MAP kinase expression, as well as their associated downstream
activated (pERK1/2) from Sigma–Aldrich. signaling on ERK1/2, as read-out of RET activation, in
comparison with RET-wt, and the MEN2A- and
MEN2B-associated RET-C634R and RET-M918T
Statistical analysis
mutants (Fig. 1C). All the constructs express proteins
Excel software was used to analyze all the data. of the expected molecular weight. RET protein
Significance of differences was determined by two- displaying K666E amino acid variant shows a stronger
tailed Student’s t-test with unequal variance. ERK phosphorylation than RET-wt. The RET protein
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522 www.endocrinology-journals.orgEndocrine-Related Cancer (2011) 18 519–527
encoded by the double mutants, RET K666E–G691S, significantly lower than RET carrying the classical
shows an even stronger ERK phosphorylation, similar C634R mutation (P!0.01). At variance, no transform-
to mutant proteins encoded by RET-C634R and RET- ing activity was associated with the non-synonymous
M918T controls. The respective long isoforms behave polymorphic variant G691S carried by either RET
similarly (data not shown). This preliminary analysis isoforms. Interestingly, RET gene isoforms carrying
prompted us to analyze the biological activity of either both variants RET9- and RET51-K666E–G691S,
single- or double-mutated RET forms. mimicking the mutated allele of the patient, proved
To define the oncogenic potential of the K666E to be significantly more efficient (P!0.01) in focus
mutation, the RET-K666E constructs, together with formation compared with the respective single mutants
vectors expressing the short and the long isoforms of (Fig. 2A). Moreover, RET9-K666E–G691S generates
RET-wt and RET-C634R, were stably transfected in foci larger than single mutant RET9-K666E and more
NIH3T3 cells for a focus formation assay in the similar to those obtained with C634R mutation, as
presence or absence of chronic stimulation with the shown in representative plates for each RET mutant
RET ligand GDNF. G418 selection was also performed (Fig. 2B). Similar data, but with lower foci numbers
to normalize the results. Both isoforms of RET were (about 1/4), were obtained without GDNF ligand
tested, as we have demonstrated that long isoforms of stimulation (data not shown).
oncogenic RETs display a stronger transforming Several NIH3T3-transformed cell lines carrying
activity compared with the short ones (Borrello et al. RET-K666E or RET-K666E–G691S mutants were
2002), thus enhancing the possibility of identifying isolated and analyzed. At variance with RET-wt
mild activating mutations. The results of three expressing cells, all the NIH3T3 cells expressing
transfections, performed in triplicate and normalized RET mutants are able to grow in soft agar. The
on the number of NIH3T3 G418-resistant colonies, are RET-K666E–G691S agar clones were generally
shown in Fig. 2. Both RET isoforms carrying the bigger than the RET-K666E ones and comparable to
K666E mutation show transforming activity, although RET-C634R clones (Fig. 2C).
*
A 4.5 *
*PM G Borrello et al.: Germline RET K666E and G691S mutations
K666E K666E–G691S Discussion
Mock 1 2 3 4 5 6 7 8 A complete classification and functional character-
RET ization of all RET mutations involved in inherited
MTC is essential for an effective genetic analysis in
pTyr-905
clinical practice. Timely thyroidectomy in family
pERK1/2 members carrying an inherited mutated RET allele
prevents MTC, the most common cause of death in
ERK1/2 MEN2 syndromes (Modigliani et al. 1998).
*P=0.02 In this study, we report the identification of three
1.2 RET variants in an MTC patient: the K666E mutation,
1 the non-synonymous polymorphism G691S, and
pERK/ERK
0.8 the conservative polymorphism S904S. Through
0.6 functional analysis of the corresponding RET mutants,
0.4 we have demonstrated the in vitro oncogenic potential
0.2 associated with RET-K666E, based on its ability to
0
transform NIH3T3 cells, thus suggesting that this RET
variant is indeed responsible for hereditary MTC.
Figure 3 Western blot analysis of RET protein and associated
signaling in NIH3T3 cells stably expressing RET gene variants Moreover, we have shown that the oncogenic activity
RET9-K666E and RET9-K666E–G691S. Lysates from of RET-K666E is enhanced by the presence of G691S,
NIH3T3 cell lines were analyzed by western blot with anti-RET, suggesting a possible modifier role of this non-
anti-p-RET (Tyr905), anti-ERK1/2, and anti-p-ERK1/2
antibodies. *Statistically significant result. synonymous RET polymorphism.
Sequence variations in the intracellular juxtamem-
brane domain of RET have been infrequently reported
The same NIH3T3-derived cell lines were analyzed and affect codons 665 and 666 (Ahmed et al. 2005,
for RET protein expression and Tyr905 phospho- Cordella et al. 2006, Muzza et al. 2010). Both K666N
rylation, as well as for ERK1/2 phosphorylation, as and the complex K666NinsS mutations have been
read-out of RET receptor activation (Fig. 3). All the shown to display oncogenic potential (Cordella et al.
analyzed transformed cell lines, either expressing 2006, Muzza et al. 2010). In particular, K666N and
RET-K666E–G691S or RET-K666E, show different K666E mutants show a comparable transforming
levels of RET protein and are able to elicit a stronger activity; in fact, in both cases, their transforming activity
ERK1/2 phosphorylation than the mock-transfected is one half compared to that of the C634R mutant
NIH3T3 cells. The cell lines expressing RET-K666E– (Muzza et al. 2010). Moreover, biochemical analysis of
G691S receptor are significantly more efficient in K666E shows higher amounts of fully glycosylated
ERK1/2 phosphorylation (PZ0.02) than those expres- mutant receptor compared to RET-wt and other mutants.
sing RET-K666E receptor, in keeping with the This feature was also previously displayed by the
described biological effect of the respective RET K666NinsS mutant (Cordella et al. 2006).
mutants. RET-K666E clones show a certain correlation The K666E variant has already been described in
between RET expression and phosphorylation (eval- three unrelated families (Ahmed et al. 2005) and it is
uated either as total Tyr- (data not shown) or as Tyr905- reported as a pathogenic mutation in the web-based
phosphorylation) and ERK1/2 activation levels. ARUP online Scientific Resource RET database http://
At variance, ERK1/2 activation is high in all the RET- www.arup.utah.edu/database/MEN2/MEN2_search.
K666E–G691S expressing clones, despite the different php (Margraf et al. 2009). Pathogenicity was deduced
levels of RET protein and autophosphorylation, thus from clinical and segregation analysis, although
highlighting the capability of double mutant to strongly penetrance was incomplete and one of the MEN2A/
activate ERK1/2, even at low expression level, as FMTC families had one discordant member displaying
shown by clone 5. no mutation and a positive pentagastrin stimulation. A
Overall, our in vitro analyses suggest that K666E functional analysis for the RET-K666E variant has not
is a gain-of-function alteration of RET receptor kinase been performed earlier. We have now demonstrated for
with a weaker oncogenic potential than the MEN2A- the first time that K666E is a gain-of-function mutation
associated C634R mutation; however, its oncogenic with oncogenic potential, as it displays transforming
ability is enhanced by the presence of the G691S activity by NIH3T3 focus formation assay. It is not
polymorphism. known whether K666E is a de novo or inherited RET
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524 www.endocrinology-journals.orgEndocrine-Related Cancer (2011) 18 519–527
variation in the presented patient, as the family history The RET-G691S polymorphism has been suggested
was negative for MTC, and the carrier status of family as a genetic modifier in MEN2A (Gil et al. 2002),
members could be tested only for the patient’s sister, but this issue is controversial (Lesueur et al. 2006).
who proved negative for the mutation. Conflicting results are also reported with regard to a
However, the RET-K666E variant has a clear-cut possible role for this polymorphism in susceptibility to
transforming activity, displayed by short and long sporadic MTC ((Fugazzola et al. 2008) and references
isoforms of RET and in the presence/absence of the herein). Interestingly, the G691S polymorphism has
RET ligand GDNF. This is consistent with the fact that been suggested to play a role in a specific mutation
the presence of lysine residue at codon 666 is highly affecting K666 residue. In fact, a complex in frame
conserved among species, and with a recent report insertion–deletion mutation (insTTGTdelG) at codon
showing the presence of the wt codon for Lys in a series 666 (resulting in replacement of the lysine by
of 400 normal control alleles (Muzza et al. 2010). asparagine and serine insertion) was detected in the
Our in vitro studies show a milder transforming germline of a 12-year-old boy displaying MTC, and in
activity of the K666E mutation compared with the his unaffected mother and maternal grandfather.
classical MEN2A-associated C634R mutation. The Interestingly, the MTC-affected boy, but not the
clinical data obtained from three families (Ahmed et al. unaffected family members, displayed the G691S
2005), along with the patient described in this study, polymorphic variant allele inherited from his father
showed that among 15 carriers of the K666E variant, (Vandenbosch et al. 2005). Thus, the G691S poly-
five developed MTC (35 to 64 years old), one morphism seems to enhance RET oncogenicity of
presented pheochromocytoma and elevated calcitonin distinct mutations affecting RET codon 666, by
at 35 years old, two developed C cell hyperplasia at 35 increasing its penetrance in the clinical onset
and 71 years old, and seven remained asymptomatic (Vandenbosch et al. 2005), and its transforming
individuals (2–66 years old). Altogether, this varia- activity in in vitro assay (our study). Overall, these
bility suggests that K666E could be a mild mutation data strengthen the potential role of the RET-G691S
with variable expressivity and penetrance. The polymorphism in MTC. This is the first demonstration
presence of modifier genes or of an undetected of an in vitro effect of the G691S variant on the
segregating mutation could explain the segregation transforming activity of a RET mutant. This effect may
pattern observed in these families. RET polymorphic be related to the capacity of the polymorphic variant
variants might be possible modifiers contributing to the G691S to enhance ERK1/2 activation triggered by
variable penetrance and expressivity associated with RET-K666E mutant receptor, demonstrated by our
K666E mutation (see below). biochemical analysis of NIH3T3 cell lines expressing
The patient in this study presents the non- RET-K666E or RET-K666E–G691S, and previously
synonymous polymorphism RET-G691S in the same shown for RET-wt (Sawai et al. 2005, Narita et al. 2009).
allele carrying the K666E mutation. RET-G691S is It remains to be investigated whether the effect of
a common polymorphic variant with a reported G691S on in vitro transforming activity is relevant for
allele frequency of 11–33% (ARUP database). It was all the gain-of-function RET mutations causing
shown that RET-G691S is able to increase downstream hereditary MTC. Different mutations might be differ-
signaling compared with RET-wt (Sawai et al. 2005, entially affected by the polymorphism, depending on
Narita et al. 2009). However, this variant is not their ‘strength’. Alternatively, as the variation at 691
oncogenic in focus formation assays, as previously residue has been hypothesized to modify the protein
shown by Fugazzola et al. (2008) for the RET9 structure (Lesueur et al. 2006), it might differentially
isoform and now confirmed in this paper for both synergize with specific gain-of-function mutations.
RET isoforms. Even in the presence of GDNF ligand, Future research on this issue could contribute toward
no valuable transforming activity different from clarifying the controversial role of G691S as a genetic
RET-wt background was appreciated. Regarding modifier. It is worth noting that strong modifying
S904S polymorphism, also detected in the presented effects of different RET variations on the oncogenic
patient, strong co-segregation with G691S poly- RET-V804M mutant have been reported. In particular,
morphism has already been demonstrated (Gil et al. the double mutants V804M–E805K, V804M–Y806C,
2002, Robledo et al. 2003). Both polymorphisms have and V804M–S940C, at variance with V804M
been shown to be under-represented in Hirschsprung’s causing FMTC, have been found in MEN2B patients
disease (HSCR, OMIM 142623) patients compared (Miyauchi et al. 1999, Menko et al. 2002, Cranston
with controls, thus suggesting that they might protect et al. 2006). Thus, the risk level associated with the
against the development of HSCR (Borrego et al. 1999). double mutants is significantly higher (Kloos et al.
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www.endocrinology-journals.org 525M G Borrello et al.: Germline RET K666E and G691S mutations
2009). The V804M–V778I double mutant has also Arighi E, Borrello MG & Sariola H 2005 RET tyrosine
been reported, and it has been classified at higher risk kinase signaling in development and cancer. Cytokine &
level than V804M (Kasprzak et al. 2001). However, at Growth Factor Reviews 16 441–467. (doi:10.1016/j.
variance with our report, in these cases, the mutations cytogfr.2005.05.010)
in tandem with V804M are classified as ‘uncertain’ Bongarzone I, Pierotti MA, Monzini N, Mondellini P,
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Declaration of interest sporadic medullary thyroid carcinoma. Journal of
The authors declare that there is no conflict of interest Endocrinological Investigation 21 358–364.
that could be perceived as prejudicing the impartiality of Cordella D, Muzza M, Alberti L, Colombo P, Travaglini P,
the research reported. Beck-Peccoz P, Fugazzola L & Persani L 2006 An
in-frame complex germline mutation in the juxtamem-
brane intracellular domain causing RET activation in
Funding familial medullary thyroid carcinoma. Endocrine-Related
This work was partially supported by grants from Associa- Cancer 13 945–953. (doi:10.1677/erc.1.01144)
zione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Cranston A, Carniti C, Oakhill K, Radzio-Andzelm E, Stone
Guido Berlucchi, Institutional Strategic Projects ‘contri- EA, McCallion AS, Hodgson S, Clarke S, Mondellini P,
bution 5 per mille’ Fondazione IRCCS Istituto Nazionale Leyland J et al. 2006 RET is constitutively activated by
Tumori. novel tandem mutations that alter active site resulting in
MEN2B. Cancer Research 66 10179–10187. (doi:10.
1158/0008-5472.CAN-06-0884)
Acknowledgements Frank-Raue K, Rondot S & Raue F 2010 Molecular genetics
We thank Silvia Grassi for secretarial assistance and and phenomics of RET mutations: impact on prognosis of
Dr Laura Fugazzola for helpful discussion. MTC. Molecular and Cellular Endocrinology 322 2–7.
(doi:10.1016/j.mce.2010.01.012)
Fugazzola L, Muzza M, Mian C, Cordella D, Barollo S,
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