The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 8 2784-2787
Copyright © 1999 by The Endocrine Society
Mutation Analysis Reveals Novel Sequence Variants in NTRK1 in Sporadic Human Medullary Thyroid Carcinoma1
Oliver Gimm2,
Angela Greco,
Cuong Hoang-Vu,
Henning Dralle,
Marco A. Pierotti and
Charis Eng
Clinical Cancer Genetics and Human Cancer Genetics Programs (O.G.,
C.E.), Comprehensive Cancer Center, Ohio State University, Columbus,
Ohio 43210; Division of Experimental Oncology A (A.G., M.A.P.),
Istituto Nazionale Tumori, Via G. Venetion, 20133 Milan, Italy;
Department of General Surgery (C.H.-V., H.D.), Martin Luther University
of Halle-Wittenberg, 06097 Halle/Saale, Germany; Cancer Research
Campaign Human Cancer Genetics Research Group (C.E.), University of
Cambridge, Cambridge CB2 2QQ, United Kingdom
Address all correspondence and requests for reprints to: Charis Eng, M.D., Ph.D., Human Cancer Genetics Program, Comprehensive Cancer Center, Ohio State University, 420 West 12th Avenue, Room 690C MRF, Columbus, Ohio 43210. E-mail: eng-1{at}medctr.osu.edu
 |
Abstract
|
|---|
Tyrosine kinase NTRK1 is expressed in neural and nonneuronal
tissues. Like RET, NTRK1 is often
activated by rearrangements that involve one of at least five other
genes in papillary thyroid carcinoma (PTC). Because of similarities in
involvement of the two tyrosine kinases RET (rearranged
during transfection) and NTRK1 in the pathogenesis
of PTC, the obvious parallels between RET and
NTRK1 and between PTC and medullary thyroid
carcinoma (MTC), NTRK1 seemed to be an excellent
candidate gene to play a role in the genesis of MTC. Single-strand
conformational polymorphism analysis of 16 exons of
NTRK1, from 31 sporadic MTC, revealed variants in five
exons (exons 4 and 14–17). Sequence analysis demonstrated one sequence
variant each in exons 4, 14, 16, and 17, and four different variants in
exon 15. Differential restriction enzyme digestion specific for each
variant confirmed the sequencing results. All variants were also
present in the corresponding germline DNA. Interestingly, the sequence
variants at codon 604 (c1810C>T) and codon 613 (c1838G>T) of exon 15
always occurred together and might represent linkage disequilibrium.
The frequencies of the sequence variants in germline DNA from patients
with sporadic MTC did not differ significantly from those in a
race-matched control group. Although we did not find any somatic
mutations of NTRK1 in sporadic MTC, the single-strand
conformational polymorphism conditions reported here, together with the
knowledge of the frequency of various sequence variants, may help in
future mutation analyses of DNA from other neural and nonneural
tissues.
|
Introduction
|
|---|
MOST THYROID carcinomas derive from thyroid
follicular cells, among which papillary thyroid carcinoma (PTC) is, by
far, the most common type (60–80%). The diagnosis is based on a
constellation of features, such as papillary architecture, the presence
of psammoma bodies, ground-glass nuclei, and indentations of the
nuclear membrane (grooved nuclei), not all of which may be present in a
single tumor. Although PTC metastasizes relatively early, usually by
the lymphatic system, hematogenous metastasis is infrequent. The
prognosis is generally considered to be good. In contrast, medullary
thyroid carcinoma (MTC) derives from the parafollicular C cells and is
less common (5–10%). The histopathological pattern may vary
considerably (such as classic, papillary, amyloid-rich, insular,
trabecular, small-cell variants). Lymphogenous and hematogenous
metastases are common clinical presentations, and the overall prognosis
is worse, compared with patients having PTC. Surprisingly, despite
these obvious differences, PTC and MTC have one gene in common that
somehow contributes to their distinct carcinogenesis: the protooncogene
RET.
In 1985, Takahashi et al. found this new transforming gene,
activated by DNA rearrangement during transfection (RET) of NIH 3T3
cells (1). RET encodes a transmembrane tyrosine kinase
receptor that is expressed in neural and neuroendocrine tissues (2). As
of today, at least five types of RET rearrangements
(translocations and inversions) have been found in PTC (RET/PTC1–5)
(3, 4, 5, 6, 7). The subsequent activation of RET, in this manner, seems to be
restricted to PTCs (8), although one study reports RET
rearrangement in a thyroid adenoma (9).
In 1993, RET was also found to be involved in the
pathogenesis of MTC. Germline mutations of RET have been
found to be associated with familial forms of MTC (FMTC, MEN 2A, MEN
2B; for review see Ref. 10) (11, 12, 13, 14, 15). Of interest, 30–70% of sporadic
MTC harbor a somatic RET mutation (16). In contrast to PTC,
mutations found in MTC are almost exclusively missense mutations.
Another tyrosine kinase, NTRK1 (also known as TrkA), has also been
found to be widely expressed in neural and nonneuronal tissues (17, 18), and we have shown that NTRK1 is expressed in MTC (Gimm and Eng,
unpublished data). NTRK1, encoding one of the receptors for
nerve growth factor, consists of at least 17 exons and is located on
chromosome subband 1q21–22 (19). Furthermore, like RET,
NTRK1 is often activated in PTC (20). Similar to
RET, the activation is also caused by rearrangements that
involve one of at least three (TFG, TPR, TPM3) genes (TRK-T1–3)
(21, 22, 23). Because of similarities in involvement of the two tyrosine
kinases RET and NTRK1 in the pathogenesis of PTC, the obvious parallels
between RET and NTRK1, and between PTC and MTC,
NTRK1 seemed to be an excellent candidate gene to play a
role in the genesis of MTC. Here, we report the results of mutation
analysis of NTRK1 in sporadic MTC.
|
Materials and Methods
|
|---|
Tumor and peripheral blood leucocyte DNA was obtained from 31
patients who underwent surgery for MTC at the Department of General
Surgery, University of Halle, Germany. All samples were obtained with
informed consent. Patients were classified as having sporadic MTC in
the absence of any MEN 2-specific RET mutation in the
germline, i.e. no mutation in exons 10, 11, 13, 14, 15, or
16. DNA extraction was performed using the QIAamp tissue kit
(Qiagen, Santa Clarita, CA) according to the
manufacturer’s instruction.
PCR amplifications were carried out in 1x PCR buffer
(Perkin-Elmer Corp., Norwalk, CT) containing 200 µmol/L
deoxynucleotide triphosphate, 1 µmol/L of each primer (see Table 1
), 2.5 U Taq polymerase
(Perkin-Elmer Corp.), and 100–200 ng of genomic DNA
template in a 50-µL vol. PCR conditions were: 40 cycles of 1 min at
95 C, 1 min at 60–62 C (see Table 1), 1 min at 72 C followed by 10 min
at 72 C. Exon 1 could not be amplified, and exons 8 and 17 were divided
into two (a and b) because of their large sizes. Some PCR products (see
Table 1) were subjected to further digestion to get smaller
fragments that are believed to be more likely to show alternate banding
patterns using single-strand conformational polymorphism (SSCP) (24).
The digestion was performed according to the manufacturer’s
recommendation (New England Biolabs, Inc., Beverly,
MA).
Before SSCP, 2 µL of the resulting PCR/digestion product were added
to 3 µL of formamide buffer, then heated to 95 C for 10 min, and
subsequently cooled on dry ice. Immediately before SSCP, the samples
were quickly thawed and then run on a 10%
polyacrylamide/1xTris-Borate-EDTA gel, except exon 14, which
was electrophoresed through a 10%
polyacrylamide/1xTris-Borate-EDTA/10% glycerol gel. Gels were
run either at 100 V for 14 h at room temperature (exons 2, 4–7,
9–16, and 17a) or at 150 V for 16 h at 4 C (exons 3, 8a, 8b, and
17b). Subsequent silver staining was performed as follows: 10 min in
0.5% acetic acid and 10% ethanol (fixation), 10 min in 0.1% silver
nitrate (staining), 20 min in 1.5% sodium hydroxide and 0.01% sodium
borohydride and 0.05% formaldehyde (development), and 10 min in 0.75%
sodium carbonate (fixation).
If variant SSCP banding patterns were seen, the remaining PCR aliquot
was subjected to purification and semiautomated sequencing using the
above primers and dye terminator technology, as previously described
(25, 26). If sequencing revealed a sequence variant, the corresponding
leucocyte DNA was examined in the same manner to determine whether the
sequence variant is somatic or germline. Further, we analyzed whether
the nucleotide change would create or remove the recognition site of a
restriction enzyme to prove, by three different means, the existence of
this variant. The frequencies of these sequence variants, in patients
with MTC and in a race-matched control group, were determined.
|
Results
|
|---|
SSCP analysis of all 16 exons of NTRK1 from 31 sporadic
MTC revealed variants in five exons (exons 4, 14–16, and 17a; Fig. 1). Sequencing revealed 1 sequence
variant each in exons 4, 14, 16, and 17a, and 4 different variants in
exon 15 (Table 2). Differential
restriction enzyme digestion, specific for each variant, confirmed the
sequencing results. Corresponding germline DNA was examined for the
presence of each of these variants, and all were also present in the
germline. Interestingly, the sequence variants at codon 604 (c1810C>T)
and codon 613 (c1838G>T) of exon 15 always occurred together. The
frequencies of the sequence variants in DNA from patients with sporadic
MTC did not differ significantly from those in a race-matched control
group (Table 2).
View this table:
[in this window]
[in a new window]
|
Table 2. Frequency of polymorphic sequence variants in
patients with sporadic medullary thyroid carcinoma and race-matched
controls
|
|
One previously described variant (27) was not detected (c1767T>C) in
this study either by SSCP or restriction analysis (Table 2
).
|
Discussion
|
|---|
In the present study, we detected 3 previously reported
polymorphisms (27) and 5 novel sequence variants in NTRK1,
and we determined their frequencies in DNA from patients with sporadic
MTC and a race-matched control group. One previously reported sequence
variant (27) in exon 14 (c1767T>C) could not be detected by SSCP and
restriction analysis of 126 total alleles (60 MTC alleles, 66 control
alleles).
We did not detect any somatic mutations of NTRK1 in tumor
DNA from 31 independent patients with sporadic MTC. The absence of any
somatic sequence variant of NTRK1 is somewhat surprising.
Though it is obvious that high-penetrance mutations of
NTRK1 are not associated with medullary thyroid
tumorigenesis, it is becoming more and more evident that development of
a cancer can result from an interplay of either a few high penetrance
mutations in key genes or from several, or many, sequence variants of
unknown significance (28). For example, overrepresentation of a rare
sequence variant of RET has been observed in patients with
sporadic MTC (29); this variant, together with variants in other genes,
may somehow predispose to MTC in a low penetrance fashion.
Although we did not find any somatic mutations of NTRK1 in
sporadic MTC, nor did we find an association with sequence variants,
the SSCP conditions reported here, together with the knowledge of the
frequency of various sequence variants, may help in future
mutation analyses of DNA from other neural and nonneural tissues, such
as squamous cell carcinoma of the esophagus, squamous cell carcinoma of
the uterus, and ductal carcinoma of the breast (18).
|
Acknowledgments
|
|---|
The authors thank K. Hammje, M. Sitte, I. Schwarz, and Drs.
Patricia L. M. Dahia, Debbie J. Marsh, and Sig Verselis for
technical assistance.
|
Footnotes
|
|---|
1 This study was supported partially by P30CA16058 from the National
Cancer Institute (Comprehensive Cancer Center), and generous donations
from the Brown family and the Abrams family (to C.E.). 
2 Recipient of a fellowship from the Deutsche
Forschungsgemeinschaft.
Received March 11, 1999.
Revised April 5, 1999.
Accepted April 15, 1999.
|
References
|
|---|
-
Takahashi M, Ritz J, Cooper GM. 1985 Activation of a novel human transforming gene, ret, by DNA
rearrangement. Cell. 42:581–588.[CrossRef][Medline]
-
Takahashi M, Cooper GM. 1987 ret
transforming gene encodes a fusion protein homologous to tyrosine
kinases. Mol Cell Biol. 7:1378–1385.[Abstract/Free Full Text]
-
Grieco M, Santoro M, Berlingieri MT, et al. 1990 PTC is a novel rearranged form of the ret
proto-oncogene and is frequently detected in vivo in human
thyroid papillary carcinomas. Cell. 60:557–563.[CrossRef][Medline]
-
Sozzi G, Bongarzone I, Miozzo M, et al. 1994 A
t(10;17) translocation creates the RET/PTC2 chimeric
transforming sequence in papillary thyroid carcinoma. Genes Chromosom
Cancer. 9:244–250.[Medline]
-
Santoro M, Dathan NA, Berlingieri MT, et al. 1994 Molecular characterization of RET/PTC3; a novel rearranged
version of the RET proto-oncogene in a human thyroid
papillary carcinoma. Oncogene. 9:509–516.[Medline]
-
Fugazzola L, Pierotti MA, Vigano E, Pacini F,
Vorontsova TV, Bongarzone I. 1996 Molecular and biochemical
analysis of RET/PTC4, a novel oncogenic rearrangement
between RET and ELE1 genes, in a post-Chernobyl
papillary thyroid cancer. Oncogene. 13:1093–1097.[Medline]
-
Klugbauer S, Demidchik EP, Lengfelder E, Rabes HM. 1998 Detection of a novel type of RET rearrangement (PTC5)
in thyroid carcinomas after Chernobyl and analysis of the involved
RET-fused gene RFG5. Cancer Res. 58:198–203.[Abstract/Free Full Text]
-
Santoro M, Carlomagno F, Hay ID, et al. 1992 Ret oncogene activation in human thyroid neoplasms is
restricted to the papillary cancer subtype. J Clin Invest. 89:1517–1522.
-
Ishizaka Y, Kobayashi S, Ushijima T, Hirohashi S,
Sugimura T, Nagao M. 1991 Detection of retTPC/PTC transcripts in
thyroid adenomas and adenomatous goiter by an RT-PCR method. Oncogene. 6:1667–1672.[Medline]
-
Eng C. 1999 RET proto-oncogene in the
development of human cancer. J Clin Oncol. 17:380–393.[Abstract/Free Full Text]
-
Mulligan LM, Kwok JB, Healey CS, et al. 1993 Germ-line mutations of the RET proto-oncogene in multiple
endocrine neoplasia type 2A. Nature. 363:458–460.[CrossRef][Medline]
-
Donis-Keller H, Dou S, Chi D, et al. 1993 Mutations
in the RET proto-oncogene are associated with MEN 2A and
FMTC. Hum Mol Genet. 2:851–856.[Abstract/Free Full Text]
-
Eng C, Smith DP, Mulligan LM, et al. 1994 Point
mutation within the tyrosine kinase domain of the RET
proto-oncogene in multiple endocrine neoplasia type 2B and related
sporadic tumours. Hum Mol Genet. 3:237–241.[Abstract/Free Full Text]
-
Hofstra RM, Landsvater RM, Ceccherini I, et al. 1994 A mutation in the RET proto-oncogene associated with
multiple endocrine neoplasia type 2B and sporadic medullary thyroid
carcinoma. Nature. 367:375–376.[CrossRef][Medline]
-
Carlson KM, Dou S, Chi D, et al. 1994 Single
missense mutation in the tyrosine kinase catalytic domain of the
RET protooncogene is associated with multiple endocrine
neoplasia type 2B. Proc Natl Acad Sci USA. 91:1579–1583.[Abstract/Free Full Text]
-
Eng C, Mulligan LM. 1997 Mutations of the
RET proto-oncogene in the multiple endocrine neoplasia type
2 syndromes, related sporadic tumours, and hirschsprung disease. Hum
Mutat. 9:97–109.[CrossRef][Medline]
-
Lomen-Hoerth C, Shooter EM. 1995 Widespread
neurotrophin receptor expression in the immune system and other
nonneuronal rat tissues. J Neurochem. 64:1780–1789.[Medline]
-
Koizumi H, Morita M, Mikami S, Shibayama E, Uchikoshi
T. 1998 Immunohistochemical analysis of TrkA neurotrophin receptor
expression in human non-neuronal carcinomas. Pathol Int. 48:93–101.[Medline]
-
Greco A, Villa R, Pierotti MA. 1996 Genomic
organization of the human NTRK1 gene. Oncogene. 13:2463–2466.[Medline]
-
Bongarzone I, Pierotti MA, Monzini N, et al. 1989 High frequency of activation of tyrosine kinase oncogenes in human
papillary thyroid carcinoma. Oncogene. 4:1457–1462.[Medline]
-
Sozzi G, Bongarzone I, Miozzo M, et al. 1992 Cytogenetic and molecular genetic characterization of papillary thyroid
carcinomas. Genes Chromosom Cancer. 5:212–218.[Medline]
-
Greco A, Pierotti MA, Bongarzone I, Pagliardini S, Lanzi
C, Della Porta G. 1992 TRK-T1 is a novel oncogene
formed by the fusion of TPR and TRK genes in
human papillary thyroid carcinomas. Oncogene. 7:237–242.[Medline]
-
Greco A, Mariani C, Miranda C, et al. 1995 The DNA
rearrangement that generates the TRK-T3 oncogene involves a
novel gene on chromosome 3 whose product has a potential coiled-coil
domain. Mol Cell Biol. 15:6118–6127.[Abstract]
-
Jordanova A, Kalaydjieva L, Savov A, et al. 1997 SSCP analysis: a blind sensitivity trial. Hum Mutat. 10:65–70.[CrossRef][Medline]
-
Gimm O, Marsh DJ, Andrew SD, et al. 1997 Germline
dinucleotide mutation in codon 883 of the RET proto-oncogene
in multiple endocrine neoplasia type 2B without codon 918 mutation. J Clin Endocrinol Metab. 82:3902–3904.[Abstract/Free Full Text]
-
Liaw D, Marsh DJ, Li J, et al. 1997 Germline
mutations of the PTEN gene in Cowden disease, an inherited
breast and thyroid cancer syndrome. Nat Genet. 16:64–67.[CrossRef][Medline]
-
George DJ, Suzuki H, Bova GS, Isaacs JT. 1998 Mutational analysis of the TrkA gene in prostate cancer. Prostate. 36:172–180.[CrossRef][Medline]
-
Storey A, Thomas M, Kalita A, et al. 1998 Role of a
p53 polymorphism in the development of human
papillomavirus-associated cancer. Nature. 393:229–234.[CrossRef][Medline]
-
Gimm O, Neuberg DS, Marsh DJ, et al. 1999 Over-representation of a germline RET sequence variant in
patients with sporadic medullary thyroid carcinoma and somatic
RET codon 918 mutation. Oncogene. 18:1369–1373.[CrossRef][Medline]
This article has been cited by other articles:
|
|
|
|
 
A. Cerrato, V. De Falco, and M. Santoro
Molecular genetics of medullary thyroid carcinoma: the quest for novel therapeutic targets
J. Mol. Endocrinol.,
October 1, 2009;
43(4):
143 - 155.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. J. Morrison and A. B. Atkinson
Genetic Aspects of Familial Thyroid Cancer
Oncologist,
June 1, 2009;
14(6):
571 - 577.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Beckers and A. F Daly
The clinical, pathological, and genetic features of familial isolated pituitary adenomas
Eur. J. Endocrinol.,
October 1, 2007;
157(4):
371 - 382.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Y. Lau and D. I. Chasman
Functional classification of proteins and protein variants
PNAS,
April 27, 2004;
101(17):
6576 - 6581.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
O. Gimm, H. Chi, P. L. M. Dahia, A. Perren, R. Hinze, P. Komminoth, H. Dralle, P. R. Reynolds, and C. Eng
Somatic Mutation and Germline Variants of MINPP1, a Phosphatase Gene Located in Proximity to PTEN on 10q23.3, in Follicular Thyroid Carcinomas
J. Clin. Endocrinol. Metab.,
April 1, 2001;
86(4):
1801 - 1805.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
S. Mardy, Y. Miura, F. Endo, I. Matsuda, and Y. Indo
Congenital insensitivity to pain with anhidrosis (CIPA): effect of TRKA (NTRK1) missense mutations on autophosphorylation of the receptor tyrosine kinase for nerve growth factor
Hum. Mol. Genet.,
February 1, 2001;
10(3):
179 - 188.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
