1. MOLECULAR ANALYSIS OF BRAF AND RAS FAMILY GENES IN
THYROID CARCINOMA IN THE GREEK POPULATION
Papadopoulou Alexandra1, Loukou Kalliopi1, Argyropoulou Marilena1, Papoutsidakis George1,
Athanassiou-Kostoglou Ifigenia2, Vilaras George3, Karameris Αndreas3, Liadaki Kalliopi1
1 University of Thessaly, Department of Biochemistry and Biotechnology, Ploutonos 26 & Aeolou Str., Larissa 41221, Greece
2 Red Cross Hospital, Department of Endocrinology, 7 Korinthias Str., Athens 11526, Greece
3 417 Veterans Administration Hospital (Ν.Ι.Μ.Τ.S.), Department of Pathology, Monis Petraki 12, Athens 11521, Greece
INTRODUCTION
Thyroid cancer is one of the most common malignancies of the endocrine system with a
steadily increasing incidence worldwide (1). It originates from follicular thyroid epithelial
cells or calcitonin-secreting parafollicular C cells. Based on histopathological
characteristics, thyroid tumors are classified into four major types: papillary, follicular,
medullary and anaplastic. Additional variants exist among these major types, with the most
common being the follicular subtype of papillary carcinoma (2).
The understanding of the molecular pathogenesis and the identification of molecular
markers, which will be used for diagnosis and prognosis of thyroid cancer, is of high clinical
significance. Previous studies have identified genetic mutations, especially ones resulting in
the activation of the MAP kinase signaling pathway, which contribute to the pathogenesis of
thyroid cancer. These include mutations of the BRAF gene, mutations of the RAS (HRAS,
NRAS and KRAS) genes, as well as RET/PTC and PAX8/PPARγ genetic rearrangements
(3, 4).
BRAF is a serine-threonine kinase that belongs to the family of RAF proteins. BRAF
activation is triggered by RAS binding and results in phosphorylation and activation of
downstream targets along the MAPK cascade. Point mutations in the BRAF gene (codon
600), which lead to the constitutive activation of the kinase, have been reported to be
tumorigenic for thyroid cells (5). The family of human RAS genes includes HRAS, NRAS
and KRAS. They encode highly related G proteins which propagate signals arising from cell
membrane receptors along the MAPK and other signaling pathways. Activating point
mutations in discrete domains of the RAS genes (codons 12, 13 and 61) are common in
different types of human cancers, including certain types of thyroid carcinomas (3).
The aim of the present study was to investigate the association of BRAF and RAS
(HRAS, NRAS, KRAS) point mutations with different types of thyroid carcinoma in the
Greek population.
MATERIALS AND METHODS
The study involved 33 patient samples obtained from the NIMITS Hospital of Athens. The
samples include different types of thyroid carcinomas with a prevalence that is
representative of their prevalence reported in the general population (Table 1). Following the
isolation of genomic DNA from paraffin-embedded tissue biopsies, Polymerase Chain
Reaction and sequencing were performed for the identification of mutations in codon 600 of
the BRAF gene and mutations in codons 12, 13 and 61 of the RAS genes (HRAS, KRAS
and NRAS) (Table 2).
RESULTS AND DISCUSSION
Thyroid carcinoma types Sample number
Prevalence in
current study (%)
Prevalence in
population (%) (6)
Papillary 11 33
75
Follicular subtype of papillary
carcinoma
13 39
Follicular 7 21 16
Medullary 2 6 5
Gene Primer Sequences (5΄ → 3΄) Mutation Amino acid(s)
Product Size
(bp)
BRAF
F: CATAATGCTTGCTCTGATAGGAA
R: AGTAACTCAGCAGCATCTCAG
codon 600
(GTG)
Valine 244
HRAS
F: CAGGAGACCCTGTAGGAG
R: TATCCTGGCTGTGTCCTG
codons 12-13
(GGC-GGT)
Glycine-
Glycine
225
HRAS
F: TGTCCTCCTGCAGGATTC
R: GTACTGGTGGATGTCCTC
codon 61
(CAG)
Glutamine 190
NRAS
F: AAAGTACTGTAGATGTGGCTC
R: GTGAGAGACAGGATCAGG
codons 12-13
(GGT-GGT)
Glycine-
Glycine
224
NRAS
F: GATTCTTACAGAAAACAAGTG
R: ATGACTTGCTATTATTGATGG
codon 61
(CAA)
Glutamine 157
KRAS
F: AACCTTATGTGTGACATGTTC
R: TCCTGCACCAGTAATATGC
codons 12-13
(GGT-GGC)
Glycine -
Glycine
216
KRAS
F: AATCCAGACTGTGTTTCTCC
R: TTAAACCCACCTATAATGGTG
codon 61
(CAA)
Glutamine 217
Thyroid carcinoma type Prevalence of BRAF mutations Prevalence of RAS mutations
Papillary 40-45% (8, 9, 10) / 27.3% (7) 0% (3, 4) / 54.5% (7)
Follicular subtype of papillary
carcinoma
12% (11, 12) 10-20% (4, 13, 14)
Follicular 0% (3, 5) 18-50% (15, 16, 17, 18)
Medullary 0% (3, 20, 21, 22) / 68.2% (7) 0-7.6% (19, 20, 21, 22) / 40.9% (7)
Table 1. Types of thyroid carcinoma and their prevalence in this study and in the general population.
Table 2. Primer sequences used for the screening of the specific mutations of the BRAF and RAS genes.
Table 3. Average prevalence of the specific mutations having reported in the most major thyroid carcinoma types.
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13 Zhu Z et al, Am J Clin Pathol, 2003, 120(1): 71
14 Adeniran AJ et al, Am J Surg Pathol, 2006, 30: 216
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17 Motoi N et al, Pathol Res Pract, 2000, 196(1): 1
18 Basolo et al, Thyroid, 2000, 10: 19
19 Schulten HJ et al, Anticancer Res, 2011, 31: 4179
20 Bockhorn M et al, Exp Clin Endocrinol Diabetes, 2000, 108: 49
21 Cerrato A et al, J Mol Endocrinol. 2009, 43(4): 143
22 Cho U et al, J K Med Sci, 2014, 29: 1054
REFERENCES
Funding from: Postgraduate Program “Applications of Molecular Biology-Genetics-Diagnostic Markers”,
Department of Biochemistry and Biotechnology, School of Health Sciences, University of Thessaly.
*F: Forward Primer/ R: Reverse Primer
Μ : Molecular Weight Marker
A-G: PCR products
A: BRAF
Β: HRAS 12/13
C: HRAS 61
D: NRAS 12/13
E: NRAS 61
F: KRAS 12/13
G: KRAS 61
H-K: Sequence chromatograms
H: BRAF
I: KRAS 12/13
K: KRAS 61
H I
M A
250 →
200 →
←244bp
M B
250→
200→ ←225bp
M C
250 →
200 → ←190bp
M D
250 →
200 → ←224bp
M E
200 →
150 →
←157bp
M F
250 →
200 → ←216bp
M G
250 →
200 →
←217bp
K
The low prevalence of mutations in specific carcinoma types in combination with the fact
that genetic mutations in thyroid carcinoma are reported to be mutually exclusive require that
the results of this study are confirmed in a larger sample population and expanded to include
the identification of RET/PTC and PAX8/PPARγ genetic rearrangements.
We did not identify mutations at codon 600 of the BRAF gene and at codons 12, 13 and 61
of the HRAS, NRAS and KRAS genes in the examined thyroid carcinoma samples. Although
the patient sample used in this study is representative of the prevalence of the different types
of thyroid carcinoma in the Greek population, our results should be interpreted with caution due
to small size of the population.
A previous study (7) analyzed BRAF (codon 600) and KRAS (codon 12) mutations in a
Greek papillary and medullary carcinoma cohort and demonstrated a high frequency of these
mutations compared to the reported literature, which is summarized in Table 3. It should be
mentioned that the prevalence of molecular alterations differs depending on the type of
carcinoma, even between different variants of the same type. Pure papillary carcinoma is
reported to harbor a high frequency (~45%) of BRAF mutations and no RAS mutations,
whereas papillary carcinoma of follicular subtype harbors lower frequency of BRAF mutations,
and higher (up to 20%) frequency of RAS mutations. The previous study (7) did not distinguish
between papillary carcinoma and its variant. Furthermore, we did not identify any mutations in
the limited number of medullary carcinoma samples, in contrast to the high frequency reported
previously in the Greek population (7), as opposed to other studies (Table 3).