THYROID CANCER AND NODULES
THYROID
Volume 00, Number 00, 2011
ª Mary Ann Liebert, Inc.
DOI: 10.1089/thy.2010.0267
Molecular Analysis of the RET Proto-Oncogene Key Exons
in Patients with Medullary Thyroid Carcinoma:
A Comprehensive Study of the Iranian Population
Ehsan Alvandi,1 Seyed Mohammad Akrami,1 Mohsen Chiani,2,* Mehdi Hedayati,3 Babak Noori Nayer,2,{
Mohammad Reza Mohajeri Tehrani,4 Manouchehr Nakhjavani,5 and Mehrdad Pedram1
Background: Germ-line mutations of RET proto-oncogene are the known cause of hereditary medullary thyroid
carcinoma (MTC), which account for approximately 25% of all MTC cases and occur as multiple endocrine
neoplasia type 2 syndromes. Here, we present the first comprehensive genetic screening and analysis of MTC
among Iranian families.
Methods: A total of 55 patients with MTC (male to female ratio ¼ 1:1.6; average age of disease onset ¼ 33 13
years) from 53 independent families participated in this study. All of the patients had undergone total thyroidectomy between 1999 and 2006, and 51 of them were clinically characterized as apparently sporadic cases.
Genomic DNA samples were obtained and following highly-specific polymerase chain reaction amplification of
the 6 RET key exons (10, 11, 13, 14, 15, and 16) were subjected to direct DNA sequencing without a requirement
for a purification step.
Results: Sequence analysis revealed that 9 (17.6%) of the apparently sporadic cases (from 8 kindreds) carried an RET
germ-line mutation. Of the seven different mutations identified among all of the families studied, five were in the
cysteine codons, with Cys634Arg having the highest prevalence (45.5%) among the afflicted families. Mutation
carriers have an earlier age of onset (21 6) versus the sporadic cases (37 12).
Conclusions: This is the first comprehensive genetic screening and analysis of MTC among Iranian families. The
results further confirm the need and advantages of DNA sequencing for identification of hereditary MTC cases.
There does not seem to be a meaningful correlation between single nucleotide polymorphism patterns and the
average age of disease onset. Geographical distribution of the sporadic cases, however, shows a significant
concentration toward the Northern regions of the country, noticeably the provinces situated directly to the south
of the Caspian Sea.
MEN2B, which is the most aggressive type (characterized
by MTC and pheochromocytoma along with developmental disorders such as marfanoid habitus, mucosal and
intestinal ganglioneuromatosis) but accounts for only 5%
of cases; MEN2A, which accounts for the vast majority
(85%) of cases and is characterized by MTC, pheochromocytoma, and hyperparathyroidism; and familial MTC
(FMTC) that could be considered as a ‘‘clinical variant’’ or
an extremely mild version of MEN2A characterized by
MTC alone (1–3).
Introduction
M
edullary thyroid carcinoma (MTC), which accounts for 4%–10% of all thyroid cancers, stems from
parafollicular calcitonin-producing (C) cells. About 25% of
all MTC cases are hereditary and occur as multiple endocrine neoplasia type 2 (MEN2) syndromes with an autosomal dominant mode of inheritance (1,2). Based on the
involvement of the type of tissues and clinical symptoms,
MEN2 syndromes are classified into three major variants:
1
Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences (TUMS), Tehran, Iran.
Research Center for Gastroenterology and Liver Diseases and 3Research Institute for Endocrine Sciences, Shahid Beheshti University
of Medical Sciences, Tehran, Iran.
4
Endocrinology and Metabolism Research Center, TUMS, Tehran, Iran.
5
Endocrinology and Metabolism Research Center, Vali-Asr Hospital, Tehran, Iran.
*Present address: Pilot Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran.
{
Present address: Department of Gastrology and Hepatology, Mehrad General Hospital, Tehran, Iran.
2
1
2
Germ-line missense mutations of RET proto-oncogene are
the known cause of all hereditary MEN2 variants (3,4). RET
gene (RET), which is located on chromosome 10 near the
centromere (10q11.2), encodes a transmemebrane receptor
tyrosine kinase protein that is primarily expressed in neural
crest and urogenital precursor cells. RET molecule contains an
extracelluar, a transmembrane, and two intracellular tyrosine
kinase domains. Traditionally, elevated serum calcitonin
levels, both basal and following pentagastrin stimulation,
were used as an indicator for C-cell hyperfunction and
MEN2 carrier status. However, molecular genetic studies
have provided a reliable method for identification of RET
mutations and established a strong correlation between the
genotypes and MEN2 phenotypes (2,5–7). Mutations in exons 10 and 11 (e.g., Cys620Tyr and Cys634Arg), affecting the
cysteine-residue encoding codons within RET extra-cellular
domain, play a major role in causing MEN2A. These mutations often lead to a gain-of-function in RET tyrosine kinase
signaling as a result of auto-dimerization of two RET molecules. Some other mutations in exons 10 and 11 as well as
mutations in exons 13, 14, and 15 (which encode the RET
intra-cellular domain) could lead to FMTC. By contrast,
MEN2B is almost exclusively related to Met918Thr mutation
in exon 16 that also leads to a gain-of-function as a result of
substrate alteration (8–13). Therefore, analysis of the 6 RET
key exons (no. 10, 11, 13, 14, 15, and 16) is a very reliable
method in order to differentiate hereditary MTC cases from
sporadic ones (14).
Before the present work, the available published data on
the rate, distribution, and mutation analysis of MTC among
the Iranian population were very limited. In a retrospective
study, Larijani and colleagues analyzed the medical records of
five major tertiary referral centers in the Iranian capital,
Tehran. The statistical analysis of 1,177 thyroid carcinoma
cases, from a 14-year period (1980–1994), showed that only 42
patients (3.6%) were diagnosed as MTC, with a female to male
ratio of 1:1.1 (15). The molecular study of patients with MTC
in Iran has been limited to a report by Hedayati et al., in which
examination of RET exons 10 and 11 in 57 MTC cases (with a
female to male ratio of 1.2:1) by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) led to
identification of only four germ-line mutation carriers (16).
Here, we present the first comprehensive molecular analysis of patients with MTC among the Iranian population.
After a thorough genetic counseling phase, a total of 55 patients with MTC from 53 independent kindreds were examined by direct DNA sequencing of all 6 RET key exons:10, 11,
13, 14, 15, and 16. This led to a reliable strategy for identification of MTC germ-line mutation carriers, exact genotyping
of all index patients, and a prelude to molecular screening of
their first-degree relatives. Molecular characterization of the
asymptomatic germ-line mutation carriers provided a highly
significant preventive opportunity by their timely referral for
prophylactic thyroidectomy.
Materials and Methods
Patients
Fifty-five patients with MTC from 53 independent families
were referred from three main specialized medical centers in
the capital city, Tehran (TUMS Endocrinology and Metabolism Research Center, EMRC, at Shariati Hospital; Shahid
ALVANDI ET AL.
Beheshti University’s Endocrine Research Center at Taleghani
Hospital; and Endocrinology and Metabolism Research
Center at Vali-Asr Hospital), which deal with most MTC cases
in Iran. These cases were diagnosed and treated during an
eight-year period from 1999 to 2006.
MTC or C-cell hyperplasia was initially detected by elevated serum-calcitonin levels, followed by fine needle aspiration. After surgical removal of the thyroid gland, the diagnosis
of MTC was further confirmed by histopathology. The initial
MEN2 variant type assignments were made based on clinical
manifestations, medical records, including biochemical data
and histopathological features, and family pedigrees.
Genetic counseling
All of the index patients were subjected to genetic counseling, during which background of the disease, the aim of the
study, and the potential risk for other family members were
discussed. An informed consent form was obtained from each
patient and approved by TUMS ethics committee in accordance with institutional guidelines and national regulations.
After molecular analysis, a formal report containing the
results was prepared for each family/patient, by addressing
respective endocrinologists. After identification of the families with RET germ-line mutation carrier/s, a second genetic
counseling session was held, during which arrangements
were made for collecting blood samples from the first-degree
relatives of the index patients. In case of detecting an
asymptomatic germ-line carrier during the screening process,
he/she was referred for prophylactic thyroidectomy.
DNA extraction
Using a modified salting-out technique, genomic DNA was
extracted from peripheral blood lymphocytes of ethylenediaminetetraacetic acid-anticogulated samples. Briefly, 500 ml of
each blood sample was mixed with 1 mL of RBC-lysis buffer
(0.32 M sucrose, 10 mM Tris-HCl, pH 8.2, 5 mM MgCl2, and 1%
v/v Triton X-100), incubated on ice for 15 min, and centrifuged
at 10,000 rpm for 2 min. After discarding the supernatant, the
same procedure was repeated twice before addition of 300 ml
of WBC-lysis buffer (0.45 M NaCl, 10 mM Tris-HCl, pH 8.2,
and 2 mM ethylenediaminetetraacetic acid), 20 ml of 10% sodium dodecyl sulfate, and 10 ml of proteinase K (20 mg/mL),
followed by incubation at 558C for 2 h. gDNA was precipitated
by addition of 100 ml of 5M NaCl and isopropanol (1v/v),
fished out using a thin glass rod, washed with 70% EtOH, and
dissolved in 1TE buffer. The quality of gDNA samples and
their quantification were determined by agarose gel electrophoresis and UV spectrophotometry (A260/A280).
PCR amplification of RET key exons
Forward (F) and reverse (R) primers for RET exons 10, 11, 13,
14, 15, and 16 were designed based on analysis of DNA sequences as well as some previous studies (17–20): exon 10,
RET10-F2 (50 -CTATGCTTGCGACACCAGTTG-30 ) and RET10R2 (50 -CTCCTGGGTGGAGTAACAGAG-30 ); exon 11, RET11F2 (50 -CAGAGCATACGCAGCCTGTAC-30 ) and RET11-R1
(50 -GCCTCGTCTGCCCAGCGTTG-30 ); exon 13, RET13-F1 (50 AGAAGCCTCAAGCAGCATCGTC-30 ) and RET13-R1 (50 AGGAGCAGTAGGGAAAGGGAGA-30 ); exon 14, RET14-F1
and
RET14-R1
(50 -GAAGACCCAAGCTGCCTGAC-30 )
GENETIC ANALYSIS OF MTC IN IRAN
3
(50 -GGCTAGAGTGTGGCATGGTG-30 ); exon 15, RET15-F1
(50 -GGTCTCACCAGGCCGCTAC-30 ) and RET15-R2 (50 -TCGG
TATCTTTCCTAGGCTTC-30 ); exon 16, RET16-F1 (50 -AGGGATAGGGCCTGGGCTTC-30 ) and RET16-R1 (50 -CCAGCCATTT
GCCTCACGAAC-30 ).
PCRs were performed in 25-mL mixtures containing
50–100 ng of template gDNA, 0.2–0.4 mM of each primer,
1PCR buffer with 1–2 mM of Mg2þ, and 0.625 U of Taq
polymerase (Roche Diagnostics). The concentrations of Mg2þ
(CinnaGen Inc.), dNTPs (CinnaGen Inc.), and template gDNA
was specifically worked out for each exon. PCR amplification
was performed with a pre-heating cycle of 948C for 3 min,
followed by 35 cycles of denaturation at 948C for 30 s, annealing at 608C–628C for 30 s, extension at 728C for 40 s, and a final
extension cycle at 728C for 7 min. Afterward, 5 ml of each PCR
was analyzed by gel electrophoresis on 1% agarose, and a
portion of each reaction was used for direct DNA sequencing.
by another round of PCR and DNA sequencing using the respective reverse primers for further assurance. The ABI chromatograms were each carefully examined using Sequence
Scanner (Applied Biosystems) before subjecting them to alignment analysis with Lasergene MegAlign software (DNASTAR).
Restriction fragment length polymorphism
In order to detect the Cys634Arg mutation (TGC ? CGC)
by a restriction endonuclease, the PCR products of RET exon
11 (454 bp) were first purified and quantified, as mentioned
earlier, and were then digested by Hha I (CinnaGen Inc.) following the manufacturer’s instructions. Digested fragments
along with nondigested ones (as negative controls) were analyzed on 2% agarose gels.
Results
MTC patients characterization and demographics
DNA sequence analysis
DNA sequencing reactions were performed by BigDye Terminator Cycle Sequencing kit (Applied Biosystems) in a 20 ml
final volume, using 10 ng of each PCR product as template and
3.2 pmol of the respective primer per reaction. The reactions
were run on an automated ABI Sequence Analyzer 3130xl
(Applied Biosystems). The PCRs for each sample were initially
sequenced using the forward primers. In the case of germ-line
mutation carriers, however, the results were also re-confirmed
A total of 55 patients with MTC, with a female to male ratio
of 1.6:1, from 53 kindreds (i.e., unrelated families) were identified by reviewing the medical records from major referral
centers in the Iranian capital city, Tehran, and enrolled for this
study (Table 1). The average age of disease onset was 33 (13)
years. All of these cases had undergone total thyroidectomy
and were still alive during the course of study. Based on reviewing the clinical records and information obtained during
the genetic counseling sessions, only four patients from four
Table 1. Distribution of Patients/Kindreds with Medullary Thyroid Carcinoma
Indexed by Age of Disease Onset, Gender, and Province
Family/patient no. MTC age of onset Gender
1
2a
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26b
27
28
32
26
29
45
10
31
13
47
50
48
27
25
40
37
28
39
61
32
49
34
15
34
57
65
43
26
46
57
F
F
M
M
M
F
F
F
F
F
F
F
F
F
F
F
M
M
M
F
F
F
F
F
M
M
M
M
Province
Markazi
E. Azarbayejan
Markazi
Kermanshah
Khorasan
Khorasan
Tehran
Esfahan
Lorestan
Gilan
Gilan
Khorasan
Gilan
Mazandaran
Mazandaran
E. Azarbayejan
Semnan
Kerman
Mazandaran
Gilan
E. Azarbayejan
Gilan
Mazandaran
Tehran
Zanjan
Tehran
Markazi
Mazandaran
Family/patient No. MTC age of onset Gender
29
30
31/1
31/2a
31/3
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53c
38
48
21
21
21
29
37
20
38
36
45
43
20
38
33
45
42
17
25
32
21
22
33
13
22
23
14
Index patients’ phenotypes: aMEN2A; bFMTC; cMEN2B.
NC, not confirmed; MTC, medullary thyroid carcinoma; MEN2A, multiple endocrine neoplasia type 2.
F
F
F
F
M
F
M
F
F
F
F
M
M
F
M
F
M
F
M
M
M
F
M
F
M
F
F
Province
Gilan
Tehran
Markazi
Markazi
Markazi
NC
NC
Ardebil
NC
NC
NC
Mazandaran
NC
Kerman
NC
NC
Khuzestan
Semnan
Esfahan
Gilan
Markazi
Esfahan
E. Azarbayejan
Gilan
Markazi
NC
Khuzestan
4
ALVANDI ET AL.
Table 2. Polymerase Chain Reaction Conditions and Oligonucleotide Primers Used for Efficient
Amplification of RET Key Exons for the Purpose of Direct DNA Sequencing
Exon
10
11
13
14
15
16
Primer
sequence
Size of PCR
product
Mg2þ
(mM)
dNTPs
(mM)a
DNA
(ng)
Annealing
temperature (8C)
10-F2: 50 -CTATGCTTGCGACACCAGTTG-30
10-R2: 50 -CTCCTGGGTGGAGTAACAGAG-30
11-F2: 50 -CAGAGCATACGCAGCCTGTAC-30
11-R1: 50 -GCCTCGTCTGCCCAGCGTTG-30
13-F1: 50 -AGAAGCCTCAAGCAGCATCGTC-30
13-R1: 50 -AGGAGCAGTAGGGAAAGGGAGA-30
14-F1: 50 -GAAGACCCAAGCTGCCTGAC-30
14-R1: 50 -GGCTAGAGTGTGGCATGGTG-30
15-F1: 50 -GGTCTCACCAGGCCGCTAC-30
15-R2: 50 -TCGGTATCTTTCCTAGGCTTC-30
16-F1: 50 -AGGGATAGGGCCTGGGCTTC-30
16-R1: 50 -CCAGCCATTTGCCTCACGAAC-30
392 bp
1.5
0.4
100
60
454 bp
2
0.4
100
60
348 bp
1.5
0.4
100
62
385 bp
1.5
0.4
50
60
332 bp
2
0.2
100
60
351 bp
1
0.2
100
60
Reaction volume: 50 ml.
a
Concentration of each of dNTP.
F, forward primer; R, reverse primer; PCR, polymerase chain reaction.
families were characterized with MEN2 phenotypes: no. 2 and
31/2, MEN2A; no. 26, FMTC; and no. 53, MEN2B. The remaining 51 patients, representing 49 independent families,
were considered as apparently sporadic cases.
Molecular analysis of the RET proto-oncogene
key exons
PCR set-ups. One of the goals of the present work was to
devise an efficient, time-saving, and cost-effective strategy for
amplification and sequencing of the 6 RET key exons (10, 11,
13, 14, 15, and 16). This was achieved by working out a detailed PCR-setup for highly specific amplification of each
exon, as well as efficient primer usage, without the need for
either a gel- or PCR-purification step before DNA sequencing
(Table 2). An example of this approach, how the specific
conditions were worked out for RET exon 11, is shown in
Figure 1 (also see Materials and Methods for details on
gDNAs quality and sequencing reactions). It should be noted
that except for Lane 1, which was the first amplification
FIG. 1. RET exon 11 polymerase chain reaction (PCR) set-up. (A) The PCR conditions for various reactions are listed below
the lanes. Each lane is a representative of a series of reactions aimed at optimization of the final product. Lane 7 shows the
final set-up for exon-11 amplification, with no visible non-specific products and an efficient primer usage. (B) Application of
PCR conditions listed for lane 7 in panel A to various gDNA samples. A portion of each reaction, without going through a
purification step, was sent out for direct DNA sequencing. Lanes: M, 100-bp DNA ladder; 1–7, representatives of various PCR
experiments; 33–42, index patients; C, no-template negative control. Arrow indicates the 454-bp specific band for exon 11.
GENETIC ANALYSIS OF MTC IN IRAN
5
Table 3. Characterization of RET Germ-Line Mutation Carriers
RET mutation
Family/patient No.
2
5
6
7
11
21
26
31/1
31/2
31/3
39
51
53
MTC age of onset
26
10
31
13
27
15
26
21
21
21
20
22
14
Gender
F
M
F
F
F
F
M
F
F
M
M
M
F
Exon
Codon
Nucleotide/amino acid
Phenotype
11
14
10
11
10
11
11
11
11
11
11
11
16
634
804
611
634
618
634
630
634
634
634
634
634
918
TGC ? TAC (Cys634Tyr)
GTG ? ATG (Val804Met)
TGC ? TAC (Cys611Tyr)
TGC ? CGC (Cys634Arg)
TGC ? CGC (Cys618Arg)
TGC ? CGC (Cys634Arg)
TGC ? CGC (Cys630Arg)
TGC ? CGC (Cys634Arg)
TGC ? CGC (Cys634Arg)
TGC ? CGC (Cys634Arg)
TGC ? CGC (Cys634Arg)
TGC ? CGC (Cys634Arg)
ATG ? ACG (Met918Thr)
MEN2A
Sporadic
Sporadic
Sporadic
Sporadic
Sporadic
FMTC
Sporadic (?)
MEN2A
Sporadic (?)
Sporadic
Sporadic
MEN2B
Notice that phenotypes assignments were done before molecular characterization. Question marks for patients 31/1 and 31/3 indicate
suspicion for being a mutation carrier based on their family history.
FMTC, familial medullary thyroid carcinoma.
reaction from the 6-exon PCR set, each lane is a representative
of a set of reactions focusing on a specific PCR condition, such
as the amount of template gDNA, annealing temperature, and
Mg2þ or dNTPs concentrations (Fig. 1).
Characterization and distributions of RET mutations. Among the 53 kindreds studied here, 11 (20.7%)
were identified as carrying RET germ-line mutation carriers
(Table 3). The 4 families (no. 2, 26, 31, and 53) with earlier
MEN2 phenotype assignments matched their predictive genotypes. There were 13 patients who were index mutationcarriers with an average age of disease onset of 20.5 years old
(6), ranging from 10 to 31 years. Each of these identified
mutation carriers harbored only a single germ-line missense
point-mutation. Although one cannot rule out the possibility
of changes within other RET exons, we did not find any evidence of double mutations or deletion/insertions within the 6
FIG. 2. Distribution of RET germ-line mutations. The
brackets show the affected codons and resulting amino acid
changes, followed by the number of kindreds identified. Color
images available online at www.liebertonline.com/thy.
key exons examined here. Overall, a total of 7 different heterozygote missense point-mutations were identified in RET
exons 10, 11, 14, and 16 (Fig. 2). There were no mutations in
exons 13 and 15. The most affected part of the RET molecule
came from exons 11 (seven families, 63.6%) and 10 (two
families, 18.2%), which encode the cysteine-rich regions of the
extra-cellular domain, followed by exons 14 and 16 (one
family each, 9.1%), which encode the tyrosine kinase regions
of the intra-cellular domain. In fact, 9 of the 11 afflicted families carry germ-line mutations in cysteine codons.
The most prevalent mutation, Cys634Arg (TGC ? CGC),
which is indicative of MEN2A, was seen in 7 carriers
(53.9%) from 5 (45.5%) kindreds. In fact, apart from cases
with assigned MEN2 phenotypes, the Cys634Arg mutation
was found in 4 out of 49 (i.e., 8.2%) apparently sporadic
MTC cases. The remaining six mutations (Cys611Tyr,
Cys618Arg, Cys630Arg, Cys634Tyr, Val804Met, and
Met918Thr) each represented a single index patient from
one (9.1%) mutation-carrier kindred. Surprisingly, the
mutation carrier with the youngest age of disease onset
(patient no. 5, 10 years) was genotyped as Val804Met
(GTG ? ATG), which is typically classified as level 1 (i.e.,
the lowest) risk level.
Single nucleotide polymorphism mapping and geographical distribution of MTC cases. In addition to characterization of germ-line mutations, DNA sequencing provided a
complete and detailed genotype for each index MTC patient.
Table 4 shows the distribution of known single nucleotide
polymorphism (SNPs) among all 53 patients with MTC. Only
one of the observed SNPs, codon 691 from exon 11, results in
an amino-acid change: GGT ? AGT; Gly691Ser. In addition to
the SNPs listed on the table, we detected a potentially new
SNP (AAG ? AAA; Lys887Lys) at codon 887 from exon 15 in
four members of kindred number 31, including two index
patients: 31/1 and 31/2 (Fig. 3).
We found no apparent significant correlation between observed SNPs and patient gender or age of disease onset. By
contrast, verified geographical distribution of the patients
with MTC shows a significant concentration of sporadic cases
6
ALVANDI ET AL.
Table 4. Characterization and Distribution of Single Nucleotide Polymorphisms
among Patients with Medullary Thyroid Carcinoma
Exon 11
Exon 13
Exon 14
Exon 15
Exon 11
Exon 13
Exon 14
Exon 15
Patient GGT ? AGT CTT ? CTG GGT ? AGT CTT ? CTG Patient AGC ? AGT TCC ? TCG AGC ? AGT TCC ? TCG
no.
G691S
L769L
S836S
S904S
no.
G691S
L769L
S836S
S904S
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
P
PP
PP
P
P
P
P
—
P
—
P
P
—
P
P
—
P
PP
P
—
—
—
—
P
—
—
—
P
P
—
—
—
—
P
—
P
P
—
—
P
—
—
—
PP
P
—
P
PP
P
P
PP
—
—
—
P
P
P
—
—
—
—
—
—
P
P
—
—
P
—
—
—
—
—
—
P
—
PP
P
—
—
—
—
—
—
P
PP
PP
P
P
P
—
—
P
—
P
P
—
P
P
—
P
PP
P
—
—
—
—
P
—
—
—
P
29
30
31/1a
31/2a
31/3
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
P
PP
—
P
P
P
—
—
P
PP
PP
P
P
—
—
—
—
P
PP
P
—
P
—
—
PP
—
—
—
—
—
—
P
—
PP
—
—
—
P
P
P
P
—
—
PP
—
—
P
—
—
P
P
—
P
P
—
—
—
—
P
—
P
—
—
—
—
P
P
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
PP
P
P
P
P
—
—
P
—
—
P
P
—
—
—
—
P
PP
P
—
P
P
—
PP
—
—
Bold numbers (2, 5–7, 11, 21, 26 31/1–3, 39, 51, and 53) indicate the RET germ-line mutation carriers.
a
Also contained AAG ? AAA (K887K).
SNP, single nucleotide polymorphism; —, No SNP detected; P, heterozygote SNP; PP, homozygote SNP.
toward the North and Northwestern regions of the country
(Fig. 4).
Discussion
Considering the prevalence of MTC in Iran, based on the
available current information and two previous studies
spanning a 26-year period (1980–2005) (15,16), our sample
size of 53 independent families, from a recent 8-year period
(1999–2006), covers a very significant portion, if not the vast
majority, of MTC cases in the country. To our knowledge, this
is the first comprehensive genetic analysis and screening for
MTC among the Iranian population. Sequencing of the 6 RET
key exons resulted in identification of 13 germ-line mutation
carriers from 11 independent families. Mutation carriers had
an earlier age of MTC onset compared with the sporadic cases:
21 6 versus 37 12, respectively. Noticeably, both of these
numbers are indicative of earlier ages of onset for MTC in Iran
compared with the global averages [around the age of 30 for
hereditary vs. the 6th decade of life for sporadic cases (4,5)].
The initial screening of the patients with MTC, based on reviewing their medical records and information obtained during
the genetic counseling sessions, resulted in characterization of
only four patients, from four independent families, with MEN2
phenotypes: two cases of MEN2A and one case of MEN2B and
FMTC each, from families number 2, 31, 53, and 26, respectively
(Table 3). Later on, molecular analysis of RET germ-line mutations confirmed the initial phenotypic assignments. It should be
noted that none of the patients had exhibited the symptoms of
Hirschsprung’s disease. Two patients showed the characteristics of MEN2A: patient no. 2 carrying the Cys634Tyr mutation
had hyperparathyroidism, and no. 31/2 carrying Cys634Arg
mutation exhibited pheochromocytoma.
We also suspected two patients (31/1, and 31/3) as potential cases of MEN2A based on the assignment of MEN2A
phenotype to another member (31/2) of the same kindred
(Fig. 3A). However, these suspected cases lacked the proper
clinical manifestations for such a diagnosis. Most notably,
unlike patient no. 31/2, they had shown no evidence of
pheochromocytoma. Nonetheless, as shown in Figure 3B,
genetic screening revealed that both suspected patients carried the Cys634Arg germ-line mutation, which is the genetic
hallmark of MEN2A.
Apart from the six cases discussed earlier, the remaining 49
independent patients with MTC had neither the clinical
manifestations nor a family history indicative or pointing at
MEN2 subtypes. Therefore, they were characterized as apparently sporadic cases. However, molecular analysis of the 6
key RET exons by DNA sequencing revealed that seven of
these patients harbored single germ-line mutations.
Among the 53 families studied here, RET germ-line mutations were detected in 11 families (20.7%). The index patients
GENETIC ANALYSIS OF MTC IN IRAN
7
FIG. 3. Pedigree analysis and sequencing results for kindred no. 31. (A) As the pedigree shows, three members of the family
(II-2, 4, and 7) had been diagnosed with medullary thyroid carcinoma (MTC), but only patient II-4 (indicated by the
arrow) exhibited the clinical manifestation of multiple endocrine neoplasia type 2 phenotype. (B) DNA sequencing chromatograms. Molecular analysis of the three patients with MTC revealed that they all carried the genetic hallmark of multiple
endocrine neoplasia type 2 (i.e., Cys634Arg; TGC ? CGC). Later on, screening of asymptomatic first-degree relatives also
revealed that members I-2 and III-2 were RET germ-line mutation carriers as well. The rectangle on each sequencing diagram,
except for the last one (codon 887), indicates the codon 634. M shows the position of the heterozygote point mutation. The last
diagram shows a potentially new single nucleotide polymorphisms (SNP) (AAG ? AAA) identified in codon 887 of four
family members. Black circles and squares in panel A indicate patients with MTC or the proband. The gray squares show the
member identified as mutation carriers. The strikethrough circle marks a deceased female member. Color images available
online at www.liebertonline.com/thy.
in six (54.5%) of these families harbored a mutation in codon
634, with Cys634Arg (TGC ? CGC) being the most frequent
one. The Cys634Arg is the most frequent, and exclusive,
mutation associated with MEN2A. There is also generally a
strong correlation between mutations of codon 634 and the
occurrence of pheochromocytoma and/or hyperparathyroidism as well as an increasing probability of developing
these phenotypes with age (1–3). Therefore, we concluded
and recommended that all of the Cys634Arg carriers go
through regular clinical follow-ups.
Despite the exact criteria of International RET Mutation
Consortium for classification of FMTC (i.e., families with
more than 10 carriers and affected members over the age of
50) (14), we classified one family (no. 26) as FMTC. Based on
the clinical data, only four members of this family had developed MTC, and they all lacked other MEN2-associated
manifestations. However, this is in line with a less rigid definition of FMTC and the recent trend advocated by the
American Thyroid Association (2). Moreover, random genetic
screening of two of the four members revealed that they both
8
FIG. 4. Geographical distribution of patients with MTC in
Iran. The locations of the families harboring RET germ-line
mutation carriers are shown with black circles, whereas those
with sporadic cases are indicated by gray circles. The high
concentration of sporadic cases in the two Northern provinces
of Gilan and Mazandaran (labeled on the map by numbers 3
and 4, respectively) might be because of localized environmental factors. Numbers indicate various provinces as confirmed origins of patients with MTC: 1, East Azarbayejan; 2,
Ardebil; 3, Gilan; 4, Mazandaran; 5, Golestan; 6, Zanjan; 7,
Tehran; 8, Markazi; 9, Semnan; 10, Khorasan; 11, Kermanshah;
12, Lorestan; 13, Esfahan; 14, Khuzestan; and 15, Kerman.
carried the rare germ-line mutation TGC ? CGC (Cys630Arg)
in exon 11, which is in agreement with FMTC characterization
(21,22).
We detected a Val804Met mutation in RET exon 14 of a 10year-old patient (family no. 5). In some reports, mutations in
codon 804 have been reported to be associated with late age of
MTC onset and low tumor aggressiveness (4,5,23). By contrast, another study reported a Val804Met carrier showing the
FIG. 5. Restriction fragment
length polymorphism (RFLP)
analysis of Cys634Arg. Restriction
digestion of 454-bp amplified
product of RET exon 11, containing
Cys634Arg, by Hha I results in
formation of 2 DNA fragments
(358 and 96 bp). Due to the lack of
mutated allele, the PCR product
from sample 1 does not get cut.
By contrast, samples 7, 21, 39,
and 51 from respective patients
all contain the heterozygote
mutation. Lanes: M, 100-bp DNA
ladder; þ and , with and
without the restriction enzyme,
respectively.
ALVANDI ET AL.
symptoms of the disease at the age of six, who died 6 years
later due to widespread metastases (24). Finally, the presence
of pheochromocytoma and/or hyperparathyroidism in
Val804Met carriers have been reported in a number of studies
(25–27). Patient no. 5 did not exhibit any other endocrinerelated phenotypes. However, the presence of MTC at the age
of 10 supports the previously proposed wide range for the age
of disease onset among Val804Met carriers (28).
In approximately 95% of cases, MEN2B is associated with a
methionine-to-threonine substitution in the intra-cellular domain of RET as a result of mutation in codon 918 of exon 16
(Met918Thr) (10–12,29). We found this germ-line mutation in
patient no. 53 (age of MTC onset: 14) who had already been
characterized as MEN2B based on her medical records. Interestingly, both parents, who were over the age of 35, appeared healthy. Although we did not succeed in arranging for
genetic screening of the parents, considering their ages, it is
likely that the index patient is a de novo germ-line mutation
carrier. The presence of de novo germ-line Met918Thr mutation has been reported by other investigators as well and, in
fact, accounts for more than 50% of the cases (2,30,31).
In a previous study from Iran by Hedayati and colleagues,
the investigators looked at RET exons 10 and 11 of 57 patients
with MTC (collected within a 15 years period) by PCR-RFLP
analysis. The authors reported finding of 4 RET germ-line
mutation carriers (16). In a collaborative effort with Dr. Hedayati’s laboratory and careful cross-referencing of patients’
information, 22 of our MTC samples were from the same
patients as the previous study. Among these patients, using
the PCR-RFLP technique, Hedayati et al. had found only one
patient carrying a Cys620Arg mutation by digesting the
gDNA with BstU I. Surprisingly, by using DNA sequencing,
however, we did not detect any germ-line mutation in this
patient. Further, RET sequencing led to identification of 2
germ-line Cys634Arg carriers that had escaped detection by
PCR-RFLP in the previous study. We repeated the molecular
analysis of these patients by DNA sequencing of fresh samples, and the same results ensued. Further, subjecting our
gDNA samples to PCR-RFLP using the restriction enzyme
Hha I also provided the expected outcomes (Fig. 5). Nonetheless, such discrepancies confirm the reliability of DNA
GENETIC ANALYSIS OF MTC IN IRAN
sequencing and its preference over RFLP in detection of RET
germ-line mutations as also stated by the International RET
Mutation Consortium (14).
In addition to identifying the germ-line mutations in an
unbiased manner, another advantage of RET sequencing is
that it provides a clear genotype, which includes an SNP map
as well. We have obtained clear and complete genotypic profiles of the 6 RET key exons for our patients with MTC. Interestingly, we also detected a potentially new SNP, without
amino acid substitution, in exon 15 (AAG ? AAA; Lys887Lys)
of four members of one family (Fig. 3). Whether this nucleotide
change has any effect on MTC progression requires further
investigation. There have been some contradictory results in
different studies regarding the association of SNPs with a
germ-line mutation and/or mean age of the disease onset (32–
40). Given our limited sample size, we did not find a statistically significant correlation between the SNP maps and patient
gender and/or age of disease onset within our samples.
We found no specific geographical concentration of RET
germ-line mutation carriers in Iran. By contrast, geographical
distribution of sporadic cases shows a high concentration
within the Northern regions, especially in the provinces of
Gilan and Mazandaran, which are located by the Caspian Sea
(Fig. 4). Although these regions are densely populated, considering the fact that they make up only about 8% of the entire
population of the country (41), this might be suggestive of
involvement of some environmental factors.
This study is the first comprehensive survey of patients with
MTC in Iran. Despite the poor system of assorting data of patients with MTC, along with very low frequency of the disease,
through concerted effort and teamwork, we managed to collect
data from three major endocrinology and surgery departments
in the country. We also set up a reliable step-by-step strategy
for molecular analysis of RET proto-oncogene for patients with
MTC in Iran. During the course of this study, by screening the
first-degree relatives of identified hereditary cases among three
of the 11 families containing index germ-line carriers, we were
successful in detecting four asymptomatic germ-line mutation
carriers and referred them for prophylactic thyroidectomy.
Further follow-up of families carrying germ-line mutation is
needed to improve their health state.
Acknowledgment
This work was supported in part by TUMS grant number
3320.
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Mehrdad Pedram, Ph.D.
Department of Medical Genetics
School of Medicine
Tehran University of Medical Sciences
100 Poursina Ave.
Keshavarz Blvd.
Tehran 14176-1351
Iran
E-mail: [email protected]