Clinical Chemistry 43: 453-457, 1997;
(Clinical Chemistry. 1997;43:453-457.)
© 1997 American Association for Clinical Chemistry, Inc.
Rapid, nonradioactive screening for mutations in exons 10, 11, and 16 of the RET protooncogene associated with inherited medullary thyroid carcinoma
Mark Siegelman1,
Ajay Mohabeer1,
Thomas J. Fahey, III2,
Gail Tomlinson3,
Chris Mayambala1,
Sepideh Jafari1,
Walter W. Noll4,
Stephen N. Thibodeau5 and
D. Brian Dawson1,a
1
Departments of Pathology,
2
Surgery, and
3
Pediatrics, UT Southwestern Medical Center of Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235-9072.
4
Department of Pathology, Dartmouth-Hitchcock
Medical Center, Lebanon, NH.
5
Department of Laboratory Medicine, Mayo Clinic,
Rochester, MN.
a Author for correspondence. Fax 214-648-4070; e-mail Dawson01{at}utsw.swmed.edu
 |
Abstract
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Germline mutations in exons 10, 11, and 16 of the RET
protooncogene are associated with the heritable cancer syndromes
multiple endocrine neoplasia (MEN) type 2A, familial medullary thyroid
carcinoma (FMTC), and MEN type 2B. Nonradioactive mutation analysis
with nondenaturing Phastgels® and the Phast
SystemTM was performed on DNA amplified by the polymerase
chain reaction from exons 10, 11, and 16 of the RET
protooncogene from patients with MEN 2A, MEN 2B, or FMTC. The analysis
requires ~45–90 min for electrophoresis and 35 min for staining.
This assay detected 20 of 21 different mutations that represented
~90% of all known mutations associated with these lesions. A rare
silent polymorphism within exon 10 was also detected. This form of
mutation analysis provides simple, rapid, and highly sensitive
nonradioactive detection of mutations known to be associated with MEN
2A, FMTC, and MEN 2B.
Key Words: indexing terms: cancer • thyroid disease • heritable disorders • polymerase chain reaction • single-strand conformation polymorphism
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Introduction
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About 20% of the cases of medullary thyroid carcinoma (MTC) are
inherited in an autosomal dominant fashion as one of three syndromes:
multiple endocrine neoplasia (MEN) type 2A or 2B, or familial medullary
thyroid carcinoma (FMTC).1
The inherited form of the disease has been linked to germline
point mutations within the RET protooncogene. Linkage
analysis has localized to chromosome 10 a gene associated with MEN
2A (1)(2). The RET protooncogene
was subsequently localized to this same region of chromosome 10
(3). Thorough investigation of the RET
protooncogene demonstrated that mutations causing MEN 2A and FMTC
appear to be mainly confined to codons for cysteine residues in the
extracellular domain within exons 10 and 11 of the gene
(4)(5). Individuals in MEN 2A families
carrying the defective gene may have a near-100% risk of medullary
thyroid cancer, the penetrance being age-related (12).
Case reports have recently shown that rare germline mutations
implicated in FMTC can also occur in exons 13 and 14
(6)(7). A single-point mutation in exon 16 has
been shown to cause MEN 2B in ~93% of patients
(8)(9)(10)(16).
Before recognition of the gene encoding the defect in these cancer
syndromes, diagnosis commonly relied on pentagastrin-induced calcitonin
release tests. The advent of direct DNA analysis has provided a highly
specific, reliable, and less-intrusive alternative to aid diagnosis
(11)(12)(13)(14)(15). DNA sequencing, currently the most definitive
method used to detect mutations in exons 10 and 11, is, however,
cumbersome, time-consuming, and expensive. Restriction endonuclease
digestion is useful for the single-point mutation in exon 16 but its
utility for analyzing exons 10 and 11 is limited because of the
multiplicity of point mutations (>20) described for this region
(16). Single nucleotide changes can also be detected by
using methods that analyze for conformation polymorphisms, e.g.,
single-stranded conformation polymorphisms (SSCP). PCR amplicons
between 50 and 250 bp are denatured into single strands, snap-cooled,
and electrophoresed in a nondenaturing gel. The electrophoresis
conditions favor the formation of intrastrand base-pairing.
Consequently, the sense and anti-sense single-stranded DNA fold into
different conformations, as dictated by their unique primary nucleotide
sequence. A single nucleotide change alters these conformations, which
can be detected by gel electrophoresis. Generally described methods for
SSCP require addition of formamide/KOH/sodium dodecyl sulfate to
denature the DNA into single strands; large sequencing gels with or
without glycerol to be run in a cold room; 32P-labeling of
the DNA for detection; and 24–76 h to complete the electrophoresis and
subsequent autoradiography (17)(18).
A rapid, nonradioactive technique for detecting conformation
polymorphisms has been previously described by one of us (A.M.)
for detecting point mutations within the p53 gene
(19). Additional recent studies have also shown the
utility of Phastgels® and the Phast
SystemTM (both from Pharmacia BioTech, Piscataway, NJ) for
SSCP analysis (20)(21)(22). We have adapted this technique to
detect mutations in exons 10, 11, and 16 associated with inherited MTC
(MEN 2A, MEN 2B, or FMTC). To determine the sensitivity of the system,
we examined DNA from 21 patients with distinct mutations in codons 609,
611, 618, 620, 634, and 918.
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Materials and Methods
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Patients.
Whole-blood samples were collected into EDTA
or acid–citrate–dextrose preservative from 21 patients. All of the
patients had inherited MTC (MEN 2A, MEN 2B, or FMTC) and had known
genomic RET mutations previously analyzed by sequence
analysis (by S.N.T. and W.W.N.). DNA was prepared from the peripheral
blood leukocytes by standard phenol/chloroform extraction techniques
followed by ethanol precipitation.
PCR.
Exons 10, 11, and 16 of the RET
protooncogene were amplified by use of the following primer pairs:
exon 10, forward: 5' GGG-GCA-GCA-TTG-TTG-GGG-GAC 3';
reverse: 5' CGT-GGT-GGT-CCC-GGC-CGC-C 3'
exon 11, forward: 5' CCT-CTG-CGG-TGC-CAA-GCC-TC 3';
reverse: 5' GAA-GAG-GAC-AGC-GGC-TGC-GAT 3'
exon 16, forward: 5' AGG-GAT-AGG-GCC-TGG-GCT-T 3';
reverse: 5' TAA-CCT-CCA-CCC-CAA-GAG 3'
The PCR reaction mixture consisted of 50 ng of template DNA,
0.5 µmol/L of each primer, 2.5 U of Taq polymerase
(Perkin-Elmer, Norwalk, CT), 1x PCR buffer N for exons 10 and 11 (2.0
mmol/L MgCl2, pH 10.0; Invitrogen, San Diego, CA) or PCR
buffer M for exon 16 (1.5 mmol/L MgCl2, pH 10.0;
Invitrogen), and 200 µmol/L dNTPs in a final total volume of 25 µL.
The reaction mixture and genomic DNA were mixed in thin-walled
microfuge tubes and placed in a Model 9600 Thermocycler (Perkin-Elmer).
The DNA was initially denatured at 95 °C for 2 min. The program for
exons 10 and 11 consisted of 40 cycles of 95 °C for 60 s,
65 °C for 60 s, and 72 °C for 60 s, with a final
elongation step of 72 °C for 10 min. The program for exon 16
consisted of 40 cycles of 95 °C for 30 s, 60 °C for 30
s, and 72 °C for 45 s, with a final elongation step of 72 °C
for 10 min.
Mutation analysis.
The PCR product (5 µL) was diluted
10-fold in water, heat-denatured at 95 °C for 5 min, and placed
without delay on ice for ~15 min. From the diluted product, 4 µL
was subjected to electrophoresis at 4 °C (for exons 10 and 16) and
4, 10, and 15 °C (for exon 11) on the Phast System, with use of a
20% precast, nondenaturing polyacrylamide gel (Phastgel). The gel was
prerun at 400 V, 5.0 mA, 1.0 W, and 100 Vh. Sample was automatically
applied at 25 V, 5.0 mA, 1.0 W, and 2 Vh. Electrophoresis was performed
at 400 V, 5.0 mA, 1.0 W, and 350–700 Vh (45–90 min, determined
empirically). The DNA products were detected by using the automated
Phast System staining apparatus and silver stain kit (35 min).
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Results
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DNA was obtained from 21 individuals with inherited MTC. The
germline RET mutations had been previously characterized by
DNA sequence analysis, and samples were selected to represent different
point mutations within one of five cysteine codons in exons 10 and 11
of the RET protooncogene or the T
C transition mutation in
exon 16 (see Table 1
). PCR primers chosen on the basis of relevant literature
(14) and available sequence data were used to amplify
exons 10, 11, and 16 of the RET protooncogene. The Phast
System was used for mutation analysis because of the convenience of
precast polyacrylamide gels; accurate, flexible temperature control;
nonradioactive silver staining; and rapid electrophoresis time. The
protocol was further simplified by diluting the PCR products 1:10 in
water and using heat denaturation with quick placement of the products
on ice rather than denaturing with formamide/KOH/sodium dodecyl
sulfate.
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Table 1. SSCP analysis of RET protooncogene exons 10,
11, and 16 on samples previously characterized by sequence analysis at
a reference laboratory.
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Using this method of mutation analysis, we were able to identify 20 of
21 mutations in exons 10, 11, and 16 of the RET
protooncogene, obtaining an overall sensitivity of 95%. Moreover, the
banding patterns for each of these mutations were reproducible over
repeated runs. All 14 mutations in RET exon 10 were easily
identified in the same electrophoretic conditions, performed at 4 °C
(Fig. 1
). In general, the mutant bands were as intense as wild-type
bands and were well separated from them. Five of six mutations in exon
11 were identified (Fig. 2
); however, not all of these were separated at 4 °C. Four of
the five mutations were detected by electrophoresis at 10 °C,
although one of these (C634Y) was more distinct at 4 °C. The fifth
mutation (C634R) was detected at 15 °C. Additionally, we detected a
rare silent polymorphism in codon 623 of exon 10 in one individual
(mutation analysis performed at 4 °C). The single known exon 16
mutation associated with MEN 2B and sporadic MTC was identified by the
Phastgel system at an electrophoretic temperature of 4 °C (Fig. 3
). The total time for mutation analysis of each exon was <2 h,
including electrophoresis and silver staining.
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Figure 1. Mutation analysis of RET protooncogene exon 10.
DNA from samples previously characterized by sequence analysis was
amplified by PCR, diluted 1:10 with distilled water, and denatured.
Electrophoresis was performed with the Phast System and a 20%
nondenaturing polyacrylamide Phastgel at 4 °C (see text). DNA
fragments were visualized with silver stain in the automated Phast
System. NL, normal control demonstrating wild-type pattern;
611, DNA with a mutant base in codon 611; 618,
DNA with a mutant base in codon 618.
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Figure 2. Mutation analysis of RET protooncogene exon 11.
Assay details as in Fig. 1
. Electrophoresis was performed at 4 °C,
10 °C, and 15 °C. NL, normal control; 634,
DNA with a mutant base in codon 634.
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Figure 3. Mutation analysis of RET protooncogene exon 16.
Assay details as in Fig. 1
. NL, normal control;
918, DNA with a mutant base in codon 918.
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Discussion
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The genetic mutations within the RET protooncogene
predisposing to MTC provide a dramatic example of the impact molecular
analysis can have on patient management and diagnosis. Unlike
biochemical assays, methodologies based on DNA analysis require a
single blood test with only the mild side effects of venipuncture.
Also, unlike provocative biochemical assays, the test can provide
diagnostic information before development of C-cell hyperplasia or foci
of MTC. Because a mutation in the RET protooncogene remains
consistent within a family, once a mutation is identified, at-risk
members need be screened only once, as opposed to annually for
biochemical screening. Family members with a mutation would benefit
from total thyroidectomy. Family members without a mutation (and their
descendants) require no further testing.
Despite their high degree of clinical utility, DNA analyses frequently
can be laborious, time intensive, and expensive and can require
considerable technical skill. The rapid, nonradioactive screening
technique presented here, performed on the Phast System, eliminates
many of these impediments to common clinical utility, and can detect
germline mutations in the RET protooncogene with a
sensitivity of ~90%. The use of dilution in water and boiling to
denature the PCR products before electrophoresis produced a banding
pattern that was generally very easy to interpret. Whether this gentle
denaturing actually produces single-stranded DNA or
heteroduplex/homoduplex formation is unknown at present. In our
experience, the mutant bands detected in exons 10 and 16 were generally
quite well-resolved and distinct compared with those in exon 11, and
all of the former yielded to analysis under our standard conditions at
4 °C. Analysis of exon 11 frequently required the additional
maneuver of altered temperature. Using three different downstream
primers for exon 11 did nothing to resolve the bands more distinctly or
to detect the one mutation that our standard conditions did not detect.
The undetected exon 11 mutation accounts for ~5% of the codon 634
mutations reported by the International RET Mutation
Consortium (16). The basis for the relative resistance to
this analysis of the exon 11 PCR product remains unclear. Nonetheless,
all but one of these mutations was resolved with this method.
Any patient with MTC, whether suspected to be inherited or sporadic,
should be screened for germline point mutations in both exon 10 and 11
of the RET protooncogene by one of the methods for direct
DNA analysis (11)(12)(13)(14)(15). In our mutation analysis method
(Fig. 4
), exon 11 would be screened initially for mutations at an
electrophoretic temperature of 10 °C, given that 87% of the
mutations associated with MEN 2A involve codon 634 in exon 11
(16). If no abnormal bands are detected, then
electrophoresis at 4 °C and 15 °C would be performed. Finally,
mutations within exon 10 would be analyzed at an electrophoretic
temperature of 4 °C. MEN 2B patients who need exon 16 analysis are
generally identified ahead of time by the unique clinical features of
this disease. A presumptive positive diagnosis can be made if any bands
of altered electrophoretic mobility are noted. Because this is a
screening technique, DNA sequence analysis of the initial proband is
needed to confirm that the mutation did indeed affect one of the five
cysteine codons. Once the presence of the mutation is confirmed, the
method could be used to screen the additional family members at
relatively low cost. If no abnormal bands are present, 20 of 21
mutations can be confidently ruled out. We hope to improve the assay to
allow us to detect all of the mutations associated with the disease.
Currently, for complete confidence, DNA sequence analysis is
recommended for all initial probands with a negative screen.
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Figure 4. Possible testing algorithm for families with MTC.
Probands in these studies were confirmed by sequencing.
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These results are similar to those of another study that successfully
used SSCP and the Phast System to detect 19 known
mutations/polymorphisms in the insulin receptor, GLUT-4, and
transthyretin genes (20). Several other authors describe
the utility of various SSCP techniques, both radioactive and
nonradioactive, for analysis of the RET protooncogene
(23)(24)(25)(26)(27). However, in only one of these papers do the
authors begin to address the sensitivity of their SSCP method, with
less-comprehensive analysis than that presented here (15 mutations in
these exons) for these syndromes (26). In addition, none
of the other methods described is as easy to perform as this mutation
analysis technique performed with the Phast System.
One limitation of any screening assay based on conformation
polymorphisms in predicting a disease phenotype is that point mutations
that are silent polymorphisms or polymorphisms having no functional
consequences for the molecule are also detected. However,
reports of silent polymorphisms within exons 10, 11, and 16 are
very rare (28). In addition, because banding patterns
by our methodology are so reproducible and so much less complex than
those in many radioactive-based SSCP techniques, we believe the
patterns generated by new mutations would probably be distinct and
could be selected for further characterization, with ultimate
confirmation by sequence analysis. In fact, using this SSCP method, we
successfully predicted (and later confirmed by sequence analysis)
previously uncharacterized mutations in at least three families.
In summary, this inexpensive, rapid, nonradioactive mutation
analysis has advantages over other DNA-based assays by being simple to
perform, requiring no radiolabeling or organic denaturants, and
providing rapid turnaround. The whole procedure, after PCR, can be
completed in <2 h. Mutation analysis with the Phast System provides a
simple, rapid technique that may have general applicability for
analyzing discrete point mutations within the human genome with better
sensitivity than other SSCP methods.
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Acknowledgments
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This work was supported in part by NIH Grant RO1 CA46806 (W.W.N.)
and the Norris Cotton Cancer Center Core Grant CA23108.
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Footnotes
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1 Nonstandard abbreviations: MEN 2A, multiple endocrine neoplasia type 2A; MEN 2B, multiple endocrine neoplasia type 2B; FMTC, familial medullary thyroid carcinoma; PCR, polymerase chain reaction; SSCP, single-strand conformation polymorphism; and MTC, medullary thyroid carcinoma. 
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Abstract
Introduction
