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OTOXXX10.1177/0194599815569709Otola
Otolaryngology
Head and Neck Surgery
2015, V
ol. 152(4) 650654
American Academy of
OtolaryngologyHead and Neck
Surgery Foundation 2015
Reprints and permission:
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DOI: 10.1177/0194599815569709
http://otojournal.org
Abstract
Objective. The American Thyroid Association (ATA) medullary thyroid cancer (MTC) guidelines group RET variants, in
the setting of familial medullary thyroid cancer and multiple
endocrine neoplasia type 2, into 4 classes of severity based on
epidemiological data. The aim of this study was to determine if
genotype correlates with phenotype in RET missense mutations.
Study Design. In silico mutational tolerance prediction.
Setting. Academic research hospital.
Subjects and Methods. We analyzed all RET variants currently
listed in the ATA guidelines for the management of MTC using
2 computer-based (in silico) mutation tolerance prediction
approaches: PolyPhen-2 HumVar and PolyPhen-2 HumDiv.
Our analysis also included 27 different RET single-nucleotide
polymorphisms resulting in missense variants.
Results. There was a statistically significant difference in the
overall HumDiv score between ATA groups A and B (P = .025)
and a statistically significant different HumVar score between
benign polymorphisms and ATA group A (P = .023). Overall,
RET variants associated with a less aggressive clinical phenotype generally had a lower Hum Div/Var score.
Conclusions. Polyphen-2 Hum Div/Var may provide additional
clinical data to help distinguish benign from MEN2/familial
medullary thyroid carcinomacausing RET variants as well
as less aggressive phenotypes (ATA A) from more aggressive
ones (ATA B-C). In silico genetic analyses, with proper validation, may predict the phenotypic severity of RET variants, providing clinicians with a tool to aid clinical decision making in
cases in which the RET variant is currently unknown or little
epidemiological data are available.
Keywords
medullary thyroid cancer, multiple endocrine neoplasia, thyroidectomy, in silico analysis, RET gene
651
Heineman et al
Level D represents MEN2B and has the earliest need for prophylactic thyroidectomy.
Recently, several computer-modeling algorithms have
been developed to analyze the disruptive effect of a given
variant on protein function, termed in silico tolerance prediction, as they calculate whether a protein could tolerate a particular missense mutation.5 As any amino acid change can be
either a benign polymorphism or a disease-causing mutation,
in silico tolerance prediction attempts to predict which amino
acid substitutions will more severely disrupt protein function
by a multifaceted analysis. The programs assess pathogenicity
through 2 major criteria. The first is the evolutionary conservation of the mutated protein region, which assumes that
highly conserved regions between species are of more functional importance than lesser conserved regions. The second is
the change in amino acid side-chain encoded by the missense
mutation, since amino acids have a wide range of physical
properties, which may affect protein folding and function.
These properties include variability in amino acid side chain
size and polarity. This methodology is often used to rank
mutations observed in a genome by predicted effect size but
had not been systematically applied to the specific study of
genotype-phenotype associations for the RET gene.
This is in contrast to in vitro studies that required introducing
a particular variant into cell lines and observing the change in
cellular phenotype.6 In silico analyses of a subset of RET variants
were correlated with in vitro studies by Cosci et al.7 They analyzed 6 known and 7 unknown variants and found a direct relationship between the transforming ability of a particular variant
in cell lines with the Align-GVGD score. Furthermore, this work
proved the validity of in silico analysis as a valid alternative to in
vitro studies. Nakao et al8 used an in silico method to analyze two
MEN 2B variants, p.(Q781R) and p.(V804M). The p.(V804M)
variant was also examined in the context of 6 different tandem
variants. To our knowledge, these 2 studies are the only in silico
analyses of the RET proto-oncogene, but they both examine only
a fraction of the known MTC-causing RET variants. This is the
first attempt to our knowledge to analyze of all the ATA-classified
MEN-causing variants and compare them with known benign
polymorphisms of the RET gene.
Statistical Analysis
Statistical significance was calculated using SPSS (version
18, SPSS Inc, Chicago, Illinois). A 2-tailed Fisher exact test
was used with a significance threshold of P < .05, with the in
silico results binned into lower risk (<0.9) and higher risk
(>0.9).
Results
652
A (65%) using HumVar (P = .025). There was also a statistically significant difference using HumDiv between ATA
groups A (78%) and B (96%; P = .025). The data used for this
analysis are given in Table 2. Overall, RET variants associated with a less aggressive clinical phenotype generally had a
lower Hum Div/Var score.
In addition to MTC risk levels, we mined the ATA genotype-phenotype data for other presentations of a RET variant,
among the most studied of which is primary hyperparathyroidism. In variants known to have a higher predisposition to
primary hyperparathyroidism, 24 of 26 (92%) had the highest
disruption score. This is in comparison to 9 of 24 (38%) variants having the highest score with lesser predispositions to
primary hyperparathyroidism. There are insufficient data to
analyze the risk of pheochromocytoma and other manifestations of RET variants.
When proposing a diagnostic test, a clinician needs to
know the sensitivity and specificity of the test and positive
(PPV) and negative predictive value (NPV) of the test result if
presented with a patient having an unknown RET variant. The
sensitivity and specificity of HumVar in distinguishing
disease-causing (MEN2/FMTC) from nondisease-causing
(benign) variants were 74.0% and 78.6%, respectively (PPV =
86.1%, NPV = 62.9%). The sensitivity and specificity of
HumDiv in the same analysis were 88.0% and 57.1%, respectively (PPV = 78.6%, NPV = 72.7%). When analyzing the
tests ability to distinguish less severe phenotypes (ATA A)
from more severe (ATA B, C), HumVar had a sensitivity and
specificity of 81.8% and 41.2% (PPV = 73.0%, NPV = 53.9%)
compared with HumDiv with a sensitivity and specificity of
97.0% and 28.4% (PPV = 72.7%, NPV = 83.3%).
Discussion
A variant in the RET proto-oncogene may result in a spectrum
of disease, from no symptoms, to any combination of MTC,
primary hyperparathyroidism, pheochromocytoma, Hirschsprung
disease, and cutaneous lichen amyloidosis.4 The ATA classification of genotype-phenotype correlations is the result of
several hundred studies of families worldwide with a spectrum of variants. While the better understood variants may
involve many kindred, a significant percentage of variants
involve only a small number of families, making a consensus
on the pathogenicity of the variant extremely challenging.
The traditional method in analyzing the oncogenic potential of a RET variant was the in vitro focus formation assay, in
which the variant was introduced into cell lines to study
growth curves. This method is time-consuming, expensive
and does not always correlate with disease severity.14 An alternative to in vitro analysis is in silico analysis.
Overall, our computer-based analysis of RET variants generally reproduced the ATA classification. The in silico data
appeared to be binary, in that the scores were largely either
very low (<0.5) or very large (>0.95).
There was no statistically significant difference between
the ATA levels B and C; however, there was a large population
of high RET disruption in group B. Similarly, the ATA
HumDiv
HumVar
0.01
0.00
0.86
0.15
0.21
0.96
0.00
0.03
0.99
0.04
1.00
0.00
0.00
0.99
0.76
0.00
0.40
0.51
0.99
1.00
0.01
1.00
1.00
1.00
1.00
0.98
1.00
0.004
0.004
0.207
0.035
0.023
0.736
0.002
0.015
0.503
0.015
0.878
0.003
0.002
0.81
0.319
0.002
0.063
0.067
0.888
1
0.026
0.973
1
0.998
0.998
0.662
0.997
0.61
1.00
0.01
0.49
0.99
1.00
0.99
0.99
0.33
1.00
0.56
1.00
1.00
1.00
1.00
0.97
1.00
1.00
0.97
1.00
1.00
0.97
1.00
0.13
1.00
0.00
0.12
0.60
1.00
0.80
0.86
0.15
0.99
0.47
1.00
1.00
1.00
1.00
0.94
1.00
0.99
0.94
1.00
0.99
0.94
1.00
(continued)
653
Heineman et al
distinction between risk levels B and C is not entirely clear.
The current recommendation for age of prophylactic surgery
for level C is before age 5 years, while the recommendation
for level is to consider surgery before age 5 but may be delayed
if the patient has a normal annual basal serum calcitonin, normal annual neck ultrasound, less aggressive MTC family history, and family preference.4 This highlights the clinical
heterogeneity within the ATA risk levels and may indicate the
need for a more advanced classification scheme. Perhaps,
instead of comparing the in silico analysis to the current gold
standard, the ATA risk levels, both systems could be reconciled into a unified classification.
Notably, tolerance predictors were able to successfully
determine pathogenic MEN2/FMTC-causing variants from
benign polymorphisms. This may provide important evidence
to determine whether polymorphisms of undetermined significance are potentially pathologic. With the rapidly decreasing
cost and increasing availability of DNA sequencing, the ability to interpret and make meaningful predictions of sequencing data is becoming ever more important.
In silico tolerance predictors have the potential to fill this void
but are not yet ready to make perfect RET variant predictions. We
postulate that the inability of these methods to detect perfect differences within our analyzed variants was several fold.
Firstly, genotype-phenotype variation may be influenced
by epigenetic factors. The ability to regulate the expression of
various genes through the covalent modification of DNA and
histones, known as epigenetics, has been shown to play an
important role in the activation of proto-oncogenes, thereby
driving the development of cancer.15 These changes are not
detectable with standard DNA sequencing.
Another potential contributor to the discordance between
in silico tolerance predictions is the complex genetic makeup
of every patient as well as the theoretical nature of the in
silico analyses, both tolerance predictions and structural
analysis. The correlation between clinical presentation and
the in silico analyses were not perfect. Clearly, each case of
MEN2/FMTC will have a unique genetic milieu with a combination of local, nonpathologic polymorphisms as well as
other genes that may retard or accelerate tumorigenesis. The
mutation analysis programs may also miss important regions
of the protein that are not conserved or would have a larger
biological role than would be expected, resulting in erroneous predictions. In subsequent studies, we would like to
examine more RET missense variants to further elucidate
which mutation prediction methodologies have the best
prognostic value.
We do not advocate that computer algorithms replace
patient preference, clinical acumen, and past epidemiological
and in vitro studies; rather, we propose it be added to the discourse and decision making on an individual patient basis.
Patients and families with diseases on the MEN spectrum
require care at tertiary care hospitals that can deliver integrated care with surgeons, endocrinologists, and geneticists.
Lastly, in silico mutation predictors have the capability of
analyzing any genetic variant and may be applied to many different genetic problems facing otolaryngologists.
Table 1. (continued)
RET Variant
ATA B
p.(C609F)
p.(C609G)
p.(C609R)
p.(C609S)
p.(C609Y)
p.(C611F)
p.(C611G)
p.(C611R)
p.(C611S)
p.(C611W)
p.(C611Y)
p.(C618F)
p.(C618G)
p.(C618R)
p.(C618S)
p.(C618Y)
p.(C620F)
p.(C620G)
p.(C620R)
p.(C620S)
p.(C620W)
p.(C620Y)
p.(C630F)
p.(C630R)
p.(C630S)
p.(C630Y)
ATA C
p.(C634F)
p.(C634G)
p.(C634R)
p.(C634S)
p.(C634W)
p.(C634Y)
HumDiv
HumVar
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.96
0.96
0.96
0.02
0.96
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.97
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.82
0.69
0.69
0.02
0.69
0.95
0.97
0.98
0.95
0.96
1.00
0.96
0.98
0.78
0.98
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
Benign
ATA A
ATA B
16
22
28
5
7
17
1
6
27
654
Conclusions
We have shown that PolyPhen-2 has the potential to distinguish benign from disease causing RET variants as well as
ATA group A and B from one another on the basis of the variant alone with a moderate degree of sensitivity and specificity.
We acknowledge the current limitations of this analysis; however, we propose that that the accuracy of in silico genetic
analytics will only improve with increasing computing power
in the setting of the widespread availability of sequencing.
The use of computer-based protein modeling to predict clinical outcomes for rare, genetic diseases, such as the timing of
prophylactic thyroidectomy in MEN2, may add crucial evidence to aid in decision making. Furthermore, if a previously
unknown variant (non-ATA classified) is discovered in a
patient, in silico analysis may help determine the potential
aggressiveness and predict the ideal age for prophylactic thyroidectomy for that patients kindred.
Author Contributions
Thomas E. Heineman, acquisition, analysis, drafting, approval,
accountable; Rohan Joshi, analysis, drafting, approval, accountable;
Marc A. Cohen, data interpretation, drafting, approval, accountable;
William I. Kuhel, data interpretation, drafting, approval, accountable; David I. Kutler, analysis, drafting, approval, accountable.
Disclosures
Competing interests: None.
Sponsorships: None.
Funding source: None.
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