Biology of Human Tumors Clinical

Cancer
Research
Biochemical, Molecular, and Clinical
Characterization of Succinate Dehydrogenase
Subunit A Variants of Unknown Significance
Amber E. Bannon1, Jason Kent1, Isaac Forquer2, Ajia Town3, Lillian R. Klug4,
Kelly McCann5, Carol Beadling6, Oliver Harismendy7, Jason K. Sicklick8,
Christopher Corless9, Ujwal Shinde10, and Michael C. Heinrich11

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Abstract
Purpose: Patients who inherit a pathogenic loss-of-function gathered SDHA VUS from two primary sources: The OHSU Knight
genetic variant involving one of the four succinate dehydrogenase Diagnostics Laboratory and the literature. We used a yeast model
(SDH) subunit genes have up to an 86% chance of developing one to identify the functional effect of a VUS on mitochondrial
or more cancers by the age of 50. If tumors are identified and function with a variety of biochemical assays. The computational
removed early in these high-risk patients, they have a higher model was used to visualize variants' effect on protein structure.
potential for cure. Unfortunately, many alterations identified in Results: We were able to draw conclusions on functional effects
these genes are variants of unknown significance (VUS), con- of variants using our three-prong approach to understanding VUS.
founding the identification of high-risk patients. If we could We determined that 16 (73%) of the alterations are actually
identify misclassified SDH VUS as benign or pathogenic SDH pathogenic, causing loss of SDH function, and six (27%) have
mutations, we could better select patients for cancer screening no effect upon SDH function.
procedures and remove tumors at earlier stages. Conclusions: We thus report the reclassification of the majority
Experimental Design: In this study, we combine data from of the VUS tested as pathogenic, and highlight the need for more
clinical observations, a functional yeast model, and a computa- thorough functional assessment of inherited SDH variants.
tional model to determine the pathogenicity of 22 SDHA VUS. We Clin Cancer Res; 1–11.
2017 AACR.

Introduction encoded by nuclear genes (SDHA, SDHB, SDHC, and SDHD,

collectively referred to as SDHx). The assembled SDH complex
SDH, succinate dehydrogenase, also known as complex II of the
localizes to the inner membrane of the mitochondria and links the
electron transport chain (ETC), is a four-subunit complex
tricarboxylic acid (TCA) cycle to the ETC, making SDH function
critical for aerobic respiration (1).
1
Department of Cell and Developmental Biology, Heinrich Lab, Oregon Health
Loss-of-function mutations affecting the SDH complex pre-
and Science University, Portland, Oregon. 2Portland VA Medical Center and dispose patients to develop multiple cancers, including gastro-
Oregon Health and Science University, Portland, Oregon. 3Heinrich Lab, Oregon intestinal stromal tumor (GIST), paraganglioma, pheochromo-
Health and Science University, Portland, Oregon. 4Department of Cancer Biol- cytoma, renal cell carcinoma, Hodgkin lymphoma, chronic
ogy, Heinrich Lab, Oregon Health and Science University, Portland, Oregon. lymphocytic leukemia, thyroid cancer, pituitary adenomas, and
5
Division of Hematology-Oncology, Department of Medicine, David Geffen
neuroendocrine tumors of the pancreas (2–6). Tumor forma-
School of Medicine at UCLA, Los Angeles, California. 6Department of Pathology,
Oregon Health and Science University, Portland, Oregon. 7Division of Biomedical
tion due to SDH-deficiency requires the complete loss of
Informatics, Department of Medicine, Moores UCSD Cancer Center, University of function of at least one SDHx subunit (e.g., A, B, C, or D),
California San Diego, La Jolla, California. 8Division of Surgical Oncology, Depart- causing destabilization and loss of enzymatic function of the
ment of Surgery, Moores UCSD Cancer Center, University of California San entire SDH complex (7). There are several genetic mechanisms
Diego, La Jolla, California. 9Department of Pathology, Knight Cancer Institute, that can lead to SDH-deficiency. Typically, loss of function of
Oregon Health and Science University, Portland, Oregon. 10Department of
an SDHx subunit is the result of a combination of an inactivat-
Biochemistry and Molecular Biology, Oregon Health and Science University,
Portland, Oregon. 11Departments of Medicine and Cell, Developmental, and
ing germline mutation (first hit) with a somatic LOH or other
Cancer Biology, Portland VA Health Care System and OHSU Knight Cancer inactivating mutation affecting the other allele (second hit).
Institute, Portland, Oregon. Less commonly, loss of SDH complex occurs due to somatic
Note: Supplementary data for this article are available at Clinical Cancer inactivation of both alleles of a given complex subunit or SDH
Research Online (http://clincancerres.aacrjournals.org/). assembly factor. Finally, SDH-deficiency can be caused by an
Corresponding Author: A.E. Bannon, Portland VA Medical Center and Oregon
SDHC epimutation, defined as hypermethylation of the SDHC
Health and Science University, R&D 19, Building 103, Room E223, 3710 SW US promoter, which leads to repression of SDHC transcription and
Veteran's Hospital Road, Portland, OR 97239. Phone: 503-220-8262; E-mail: depletion of SDHC protein levels, without a known underlying
[email protected] heritable cause (8).
doi: 10.1158/1078-0432.CCR-17-1397 Importantly, germline loss-of-function genetic SDHx variants
are associated with a high lifetime risk of developing the

2017 American Association for Cancer Research.

www.aacrjournals.org OF1
 Bannon et al.

conclusions on functional effect from clinical data alone. Our

Translational Relevance study gathered these VUS from two primary sources: OHSU and
Molecular testing plays an important role in the clinical the literature (12, 13). We then combine data from clinical
management of gastrointestinal stromal tumors, including observations, a functional yeast model, and a computational
decision making about the most appropriate medical or sur- model to understand the effects of SDHA VUS identified in GIST
gical therapy. As the routine use of multi-gene sequencing specimens on SDH complex function. Historically, yeasts have
panels has expanded, there has also been an increase in been a robust system for identifying the assembly and enzymatic
reported variants of unknown significance (VUS) of the SDHA activity of SDH (14–16). In addition, yeasts have proven to be an
gene. In many cases, these SDHA VUS are present in the ideal model to study the functional effect of SDHB/C/D variants
germline and are therefore potentially heritable by other on SDH complex activity (17–20). Yeasts are able to survive

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family members. In order to understand the functional con- without functional mitochondria (e.g., lacking SDH complex
sequences of these variants, we combined clinical observa- activity) if they are provided a fermentable carbon source; thus,
tions, data from a functional yeast model, and computational they provide a unique model system to study the biochemical
modeling to classify these SDHA VUS as having no effect or effects of SDHA VUS (21).
causing loss of function. These results will be helpful for Based on our findings, we discriminated between SDHA VUS
appropriate genetic counseling of individuals with these germ- that affected or did not affect SDH complex activity, and thus,
line variants. In addition, our data highlight the limitations of their potential for pathogenicity. These data will aid clinicians'
SDHA immunohistochemistry in clinical testing of tumors ability to provide genetic counseling and tumor surveillance to
with SDHA VUS. patients with germline inheritance of these specific SDHA
variants.

Materials and Methods

aforementioned malignancies. For example, the chance of a Yeast strains and vectors
patient with germline loss-of-function SDHD variant of develop- All Saccharomyces cerevisiae strains used in this study were
ing one or more primary tumors by the age of 50 was reported to derivatives of BY4741 (MATa his3D1 leu2D0 met15D0 ura3D0).
be 86% (9, 10). Therefore, if we could identify high-risk patients The SDH1 (yeast homolog to SDHA) deletion strain (sdh1D) was
through genetic testing and follow them serially with specialized purchased from the ATCC (catalog #4004998). The sdh1D was
screening tests, early tumor detection may lead to curative surgical constructed as part of the Saccharomyces Genome Deletion Project
resection before the tumors are metastatic/incurable. Early detec- by homologous recombination using the KanMX4 cassette (22).
tion is crucial because there are no effective medical treatments for We verified the deletion of SDH1 using PCR mapping of the SDH1
patients with advanced SDH-deficient cancers. locus with primer pairs recommended by the Saccharomyces
Currently, an SDH-deficient tumor is identified by measuring Genome Deletion Project.
SDHB protein abundance using immunohistochemistry (IHC); An amplicon containing the wild-type (WT) SDH1 [including
absence of SDHB protein is indicative of loss of SDH function. the native SDH1 promoter and 30 untranslated region (UTR)] was
However, it can be challenging to determine the underlying generated from WT BY4741 and cloned into the pRS416 plasmid
cause of SDH-deficiency in a tumor lacking SDHB expression. (ref. 23; ATCC) and expressed in the sdh1D strain. The SDH1 point
Clinical sequencing panels may turn up missense mutations in mutations were introduced by the QuikChange mutagenesis PCR
SDHx genes, but many of these are variants of unknown system (Agilent Technology; cat #200521). All mutations were
significance (VUS). In addition, such panels can miss large confirmed by Sanger sequencing. Yeast strains were transformed
intragenic deletions and are not designed to identify epigenetic using Frozen EZ Yeast Transformation II (Zymo Research; catalog
silencing of the SDHC promoter. Some SDHB, C, and D VUS # T2001). Strains were grown in synthetic complete medium
have been functionally characterized to determine their effect lacking uracil to maintain plasmid selection with either 2%
on function, and thus their pathogenicity in tumors like para- glucose or 3% glycerol as the carbon source.
ganglioma and pheochromocytoma. However, the study of the
functional consequences of SDHA VUS has lagged behind that Alignment of multiple species' succinate dehydrogenase
of other SDHx subunits (7). flavoprotein subunit
GIST is a heterogeneous group of tumors that arise from the Cluster Omega was used to align the protein sequences of SDH
interstitial cells of Cajal. However, there are several different driver flavoproteins including Escherichia coli (E. coli; P0AC41), yeast
genes that when mutated give rise to GIST (11). The molecular (Q00711), human (P31040), and pig (Q0QF01).
classification of GIST is especially important because of the
treatment implications of the different genetic drivers. The major- Immunoblotting
ity of GISTs have an activating receptor tyrosine kinase (RTK) Intact mitochondria were isolated using a previously described
mutation, but about 13% of GISTs lack RTK mutations (RTK-WT). method (24).Steady-state levels of mitochondrial proteins were
Most RTK-WT GISTs are SDH-deficient as assessed by IHC for resolved on SDS-PAGE, transferred to nitrocellulose membrane,
SDHB. SDHA pathogenic variants are found in 47% of SDH- probed using the indicated primary antibodies, and visualized
deficient GIST, and the majority of these SDHA mutations are using Amersham-enhanced chemiluminescence Western Blotting
germline, and thus heritable, variants (12). However, some the Detection Reagent (GE Life Sciences; catalog #RPN2106) with
SDHA variants we find in GIST are VUS. A universal problem in horseradish peroxidase–conjugated secondary antibodies
the field is that these variants are rarely seen with a complete (BioRad; catalog #1662408EDU). We used previously described
clinical and pathogenic annotation, making it difficult to draw polyclonal rabbit antibodies for immunodetection of Sdh1 and

OF2 Clin Cancer Res; 2017 Clinical Cancer Research

 Biochemical, Molecular, and Clinical Characteristics of SDHA VUS

Sdh2 (25). Anti-porin was purchased from ThermoFisher Scien- Results

tific (catalog # 459500).
Clinical analysis
All the known clinical information on the SDHA VUS identified
Analysis of Sdh1-bound flavin adenine dinucleotide and total in this study, including SDHA/B IHC, tumor-mutant allele frac-
mitochondrial flavin adenine dinucleotide tion, other disease references, population data from the ExAC
Levels of flavin adenine dinucleotide (FAD) covalently bound database (29), and ClinVar clinical significance (30), is listed
to Sdh1 were analyzed as previously described (14, 26). Briefly, in Table 1. Unfortunately, there were few variants with complete
mitochondrial proteins were resolved on SDS-PAGE, and the gel clinical and pathologic annotation, emphasizing the challenge of
was placed in a 10% acetic acid solution for 20 minutes to oxidize trying to understand the functional effects of these variants from

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flavin. FAD was visualized upon exposure to UV light using a Bio- available clinical data alone. Currently, SDHx subunit testing
Rad ChemiDoc MP Imaging System. remains uncommon for GIST, and only a limited number of
reference laboratories offer this testing. However, these laborato-
Oxygen consumption assay ries usually do not have access to full clinical annotation and/or
Yeast strains were grown to confluency in glucose-based media prior SDHB IHC testing results. This limitation applies to the
and then switched to glycerol-based media for 12 hours. Two published cases as well as the cases from the Knight Diagnostic
million cells were plated onto commercially available microplates laboratory. We listed all available additional clinical information
with oxygen sensors (Oxoplate; PreSens catalog #OP96U; ref. 27). for tumors with VUS in Supplementary Table S2.
Kinetic reading of oxygen consumption was measured using a
spectrofluorometer. A Student t test was used to determine sta- Functional studies in yeast
tistical significance between a variant of interest and yeast com- To understand the functional consequences of VUS in human
plemented with WT Sdh1. SDHA, we used complementation studies in a yeast strain lacking
Sdh1 (sdh1D). The flavoprotein subunit of the SDH complex is
Computational modeling of Sdh1 variants highly conserved across all species, including yeast Sdh1 (ySdh1)
A model of yeast Sdh1 (Q00711) from the Swiss Model and human SDHA (hSDHA; Supplementary Fig. S1). For sim-
repository was refined using Yasara Homology Modelling to plicity, we use yeast nomenclature throughout this study and
include the liganded FAD interactions with the peptide chain, reference the corresponding human variant in all of the tables.
followed by optimization of the loop and side-chains interac- As a positive control, we generated a WT SDH1 amplicon from
tions. Side-chain rotamers were fine-tuned considering electro- BY4741 that included the promoter and the 30 UTR which was
static and knowledge-based packing interactions as well as sol- cloned into a pRS416 plasmid and expressed in sdh1D yeast. We
vation effects. An unrestrained high-resolution refinement with used sdh1D as our negative control. Sdh1D do not have a functional
explicit solvent molecules was run, using YAMBER, a second- SDH complex and were unable to utilize their mitochondria for
generation self-parameterizing force field derived from the canonical TCA or ETC pathways, allowing growth on a ferment-
AMBER force field. Clinically identified variants were introduced able carbon source (glucose), but preventing growth with a
in the homology model, and the structures were minimized as nonfermentable carbon source such as glycerol (Fig. 1). Comple-
described earlier. The variants were compared with the WT model mentation with WT SDH1 (þSdh1) restored growth on glycerol
of E. coli or yeast Sdh1 using PYMOL. (Fig. 1). Using this strategy, we tested the ability of plasmids
encoding individual VUS to complement the sdh1D strain and
restore a normal growth phenotype. The two loss-of-function
FoldX analysis controls (yR19X and yH90S), as well as 16 of the variants shown
FoldX was used to measure the effect of point mutations on the in Fig. 1 (yG97R, yT134M, yR179W, yG251R, yH287Y, yR303C,
stability of the Sdh1 yeast model (28). Except as noted, we used yY399C, yG410R, yC431F, yG432E, yR444C, yR444H, yA447E,
the default software settings. The move neighbors setting was yR458W, yR582G, yH601Y), were unable to grow on glycerol,
turned off, and the average DDG was calculated after three runs. indicating that these variants result in loss of oxidative phosphor-
ylation (Fig. 1A). In contrast, six of the variants (yN109S, yR162H,
Mutation analysis yR186W, yT501I, yW605FF, and yV633I) were able to grow on
We gathered SDHA VUS from two primary sources: The glycerol, indicating these variants had no effect on the Sdh1
OHSU Knight Diagnostics Laboratory (Portland, OR) and the function as their phenotype was the same as WT Sdh1 (Fig. 1B).
literature (12, 13). The majority of the variants pulled from the As a secondary measure of SDH complex activity, we measured
literature were found in a review highlighting the need for a oxygen consumption to assess oxidative phosphorylation, a sur-
functional model to characterize SDHx VUS (12). The remain- rogate for measuring the ability of the SDH complex to transfer
ing literature variants are from an article identifying novel electrons to the ETC. In concordance with our previous results, the
causes of GIST that were WT for any known oncogenic driver same 18 variants that were unable to grow on glycerol consumed
of GIST (13). A collaboration with the OHSU Knight Diagnos- significantly less (P < 0.0001) oxygen than WT Sdh1 (Fig. 1C).
tics Laboratory, which offers a targeted exome panel to identify Variants that grew on glycerol had similar oxygen consumption to
genetic drivers in GIST which includes all four SDHx subunits WT Sdh1, confirming that these variants do not disrupt the ETC
(https://www.ohsu.edu/custom/knight-diagnostic-labs/home/ (Fig. 1D). To ensure oxygen consumption was due to the ETC,
test-details?id¼GeneTrailsGISTGenotypingPanel), leads to azide was added to each cell strain, and this promptly abolished
identification of several novel variants. All of the variants came oxygen consumption for all controls and variants.
from tumor samples that lacked any known oncogenic driver of To better understand how each variant affects Sdh1 protein
GIST (e.g., no KIT mutations). function, we measured the covalent attachment of FAD to Sdh1, a

www.aacrjournals.org Clin Cancer Res; 2017 OF3

 Bannon et al.

Table 1. Clinical variants of human SDHA with unknown significance

Tumor-
hSDHA ySdh1 SDHB/ mutant allele Other disease Allele frequency from ClinVar clinical Conclusions
Mutation Mutation Source SDHA IHC fraction references EXAC database significance drawn
R31X R19X Control Neg/neg N/A GIST (38, 39) 1.647  104 Pathogenic/likely LOF (Control)
(38) Paraganglioma (40) pathogenic
H99S H90S Control N/A N/A None N/A N/A (H99Y Likely LOF (Control)
(14) pathogenic)
G106R G97R OHSU N/A 92% Novel N/A N/A LOF
N118S N109S OHSU N/A 50.7% Novel N/A N/A No effect
T143M T134M Literature N/A 22% Novel N/A (T143R reported Uncertain significance LOF

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(13) 8.31  106)
R171H R162H OHSU N/A 34% Novel 8.24  106 Uncertain significance No effect
R171H R162H OHSU N/A 40.3% Novel 8.24  106 Uncertain significance No effect
R188W R179W Literature Neg/pos N/A None N/A (R188Q reported N/A LOF
(12) [30] 1.65  105)
R195W R186W OHSU Neg/ND 66.7% GIST (37) N/A (R195Q reported N/A No effect
8.23  106)
G260R G251R Literature Pos/pos N/A None N/A Uncertain significance LOF
(12) [32]
H296Y H287Y Literature N/A 87% Novel N/A N/A LOF
(13)
R312C R303C OHSU N/A 34.5% Novel N/A N/A (R312P Uncertain LOF
significance)
R408C Y399C OHSU N/A 52.8% GIST (41); Late onset N/A N/A LOF
neurodegenerative
disease (42)
G419R G410R OHSU N/A 77% GIST (43) N/A N/A LOF
C438F C431F OHSU N/A 48.5% Novel N/A N/A LOF
G439E G432E OHSU N/A 87.6% Novel N/A N/A LOF
R451C R444C Literature N/A 18% Complex II deficiency N/A (R451H reported N/A LOF
(13) (42) 8.23  106)
R451H R444H OHSU N/A 47.5% Novel 8.23  106 N/A LOF
R451H R444H OHSU N/A 39.5% Novel 8.23  106 N/A LOF
A454E A447E Literature Neg/pos N/A None N/A N/A (A454T LOF
(12) [31] Uncertain
significance)
R465W R458W OHSU N/A 50% Novel 8.23  106 Uncertain significance LOF
T508I T501I OHSU N/A 50.6% Cardiomyopathy and 7.60  104 Conflicting No effect
leukodystrophy (44) interpretations of
pathogenicity
R589G R582G OHSU N/A 40.2% Paraganglioma (45) 8.29  106 Uncertain significance LOF
H625W H601Y Literature Neg/neg N/A Pituitary adenoma and N/A N/A LOF
(12) [39] pheochromocytoma/
paraganglioma
Y629F W605F OHSU N/A 99.6% 1.52  101 Benign/likely benign No effect
V657I V633I OHSU N/A 95.1% Not pathogenic (46) 1.30  101 Likely benign No effect
NOTE: All of the genetic variants were found in GIST, except for hG260R, which was found in a paraganglioma. A summary of available clinical data for each variant is
listed, including source of the variant, clinical IHC results for SDHB/SDHA, frequency of the variant in tumor, other SDHA variants found in the same tumor, information
on functional effect of other variants in the tumor, other disease references to the variant of interest in the literature, population allele frequency, and results from our
yeast model.
Abbreviations: LOF, loss of function; N/A, not available; neg, negative- SDHx absent; ND, not done; pos, positive- SDHx expression.

process known as flavination. We also measured the protein marked decrease in Sdh1 protein abundance (R179W, G251R,
abundance of Sdh1 and Sdh2 (Fig. 2). Sdh1 flavination is critical H287Y, R303C, Y399C, G410R, C431F, G432E, R444H, A447E,
for the catalytic activity of Sdh1; without covalent attachment of R458W, and R582G). All of the loss-of-function variants resulted
flavin, the Sdh1 protein is unable to oxidize succinate (14). As a in a decrease in Sdh2 protein abundance, consistent with loss of a
positive control for a variant that inhibited insertion of FAD into functional SDH complex as described previously (25). In a subset
Sdh1, we used the previously described Sdh1 H90S variant (14). of no effect and loss-of-function variants, we confirmed that Sdh1
Variants that complemented sdh1D yeast in our functional assays mRNA expression was not significantly different from WT.
had similar levels of flavinated Sdh1, total Sdh1, and total Sdh2 as Based on all of the above results, we characterized 16 variants
yeast complemented with WT Sdh1 (Fig. 2D). However, variants (73%) as causing loss of function and six variants (27%) as having
that were unable to perform oxidative phosphorylation had no effect on SDH function (Table 2). All the loss-of-function
differential effects on the levels of flavinated Sdh1 and total Sdh1 variants were unable to grow on glycerol, had decreased oxygen
protein (Fig. 2A–C). Some loss-of-function variants inhibited consumption, and showed decreased Sdh2 protein abundance. In
flavination without affecting the abundance of Sdh1 (H90S, contrast, the no effect group had similar results to cells comple-
G97R, T134M, R444C, and H601Y), whereas others caused a mented with WT Sdh1 protein in these assays.

OF4 Clin Cancer Res; 2017 Clinical Cancer Research

 Biochemical, Molecular, and Clinical Characteristics of SDHA VUS

A GLUCOSE GLYCEROL
B GLUCOSE GLYCEROL

WT WT

-SDH1 -SDH1

+SDH1 +SDH1

R19X N109S

H90S R162H

G97R R186W

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T134M T501I

R179W W605F
Figure 1.
ySdh1 variants causing loss of function G251R V633I
are unable to use glycerol as a sole H287Y
carbon source or consume oxygen.
R303C
Yeasts were grown to a confluency
of 2  107/mL and then serially diluted Y399C
and plated onto either glucose or
G410R
glycerol media plates (left to right:
highest to lowest). All variants, C431F
regardless of SDH complex function,
G432E
can grow on glucose, but only those
variants that complement sdh1D and R444C
restore SDH complex activity are able
R444H
to grow on glycerol and consume
oxygen. A, Loss-of-function variants A447E
that are unable to grow with glycerol R458W
as their sole carbon source. B, Variants
with no effect are able to grow with R582W
glycerol as their sole carbon source. H601Y
C, Sdh1 variants that consume
significantly less oxygen than cells
complemented with WT Sdh1 (þSdh1
C D
1.5 1.5
control). D, Variants that consume
oxygen at a similar rate to the þSdh1

Relative oxygen consumption

Relative oxygen consumption

control.     , P < 0.0001.

1.0 1.0

0.5 0.5

0.0 0.0

T D 1 X S R R Y C C R F E C H E G Y T D 1 S H I F I
W h1 dh 19 90 97 34M79W51 87 03 99 10 31 32 44 44 47 58W 82 01 W h1 dh 09 62 86W 501 05 633
4 1 6
Sd +S R H G T1 R1 G2 H2 R3 Y3 G4 C4 G4 R4 R4 A R4 R5 H6 Sd +S N R1 R1 T W V

Computational modeling function for most of these amino acids (31). In addition to the
We next performed computational modeling to predict the amino acids directly interacting with the flavin-binding pocket, it
potential structural implications of each variant. Using the two has been previously shown that the C-terminal domain of Sdh1 is
groups (loss-of-function and no effect) identified in our yeast crucial for the flavination of Sdh1 (25). Two variants located in the
model, we further characterized each variant by visualizing the C-terminal domain of Sdh1, yR582G and yH601Y, both inhibited
location in the Sdh1 protein. Both groups, loss-of-function and no flavination of Sdh1. To visualize the substrate (succinate) and
effect, were located throughout the four domains of Sdh1, sug- cofactor (FAD), which were not visualized using the yeast struc-
gesting that there are not "hotspot" areas or domains for path- ture, these Sdh1 variants were modeled using the E. Coli crystal
ogenic variants, unlike the situation for some proteins. Within the structure of Sdh1.
loss-of-function group, 12 variants [yH90S (loss-of-function con- The other six loss-of-function variants [yR19X (control—not
trol), yG97R, yG251R, yH287Y, yR303C, yY399C, yG432E, pictured in computational models because it is an early truncation
yR444C, yR444H, yA447E, yR582G, yH601Y] were identified as of the protein), yT134M, yR179W, yG410R, yG431F, and
being involved in cofactor (FAD) or substrate (succinate) binding yR458W] are not located in the active site of Sdh1. Changes in
in the Sdh1-active site (Fig. 3, Table 2, and Supplementary Video protein structure based on each of these variants are visualized
S1). Structural studies from the homologous protein quinol: in Fig. 4A and Supplementary Video S1. Further, FoldX analyses of
fumarate reductase in E. coli suggests an important catalytic the variants compared with WT provided insight into how the

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 Bannon et al.

R179W

G410R
R303C

G432E
G251R

Sdh1D

H287Y

Y399C
+Sdh1
T134M

C431F
Sdh1D

+Sdh1
B

G97R
R19X

H90S
A

WT
WT
FAD-Sdh1 (72 kDa) FAD-Sdh1 (72 kDa)

Sdh1 (72 kDa) Sdh1 (72 kDa)

Sdh2 (30 kDa) Sdh2 (30 kDa)

Porin (30 kDa) Porin (30 kDa)

C D

R458W

R582G
R444C

R444H

R186W
Sdh1D

A447E

H601Y
+Sdh1

W605F
R162H
Sdh1D

N109S
+Sdh1

V633I
T501I
WT

WT

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FAD-Sdh1 (72 kDa) FAD-Sdh1 (72 kDa)

Sdh1 (72 kDa) Sdh1 (72 kDa)

Sdh2 (30 kDa) Sdh2 (30 kDa)

Porin (30 kDa) Porin (30 kDa)

E 1.4
F 1.2
1.2

Normalized Sdh1/Sdh2
1
1
Normalized Sdh2

0.8
0.8
0.6
0.6

0.4 0.4

0.2 0.2

0 0
-Sdh1D
WT

Sdh1
R19X
H90S
G97R
T134M
R179W
G251R
H287Y
R303C
Y399C
G410R
C431F
G432E
R444C
R444H
A447E
R458W
R582G
H601Y
N109S
R162H
R186W
T501I
W605F
V633I

-Sdh1D
WT

Sdh1

N109S
R162H

R186W

T501I

W605F

V633I
Figure 2.
Abundance of flavinated Sdh1, total Sdh1, and total Sdh2 in mitochondrial lysates from yeast expressing each variant. Western blotting of mitochondrial
lysates from each variant is shown. Positive and negative control lanes are included on each Western blot (WT, SDH1delta, þSDH1). WT and þSdh1 show
bands at expected size for each protein. Sdh1D does not show bands for Sdh1-FAD, Sdh1, or Sdh2, but does have normal expression of the mitochondrial
marker, porin. A–C, Loss-of-function variants are shown in numerical order by altered amino acid residue. D, No effect variants are shown in numerical
order by altered amino acid residue and have with no reduction in flavin, Sdh1, or Sdh2 expression compared with WT controls. E, Sdh2 protein abundance
was calculated relative to Sdh1 and then normalized to porin (control for mitochondrial protein loading). The no-effect variants are labeled in black, whereas
the loss-of-function variants are in gray. No-effect variants have Sdh2 protein abundance more similar to complemented Sdh1, and loss-of-function
variants have decreased Sdh2 similar to sdh1D. F, Ratio of Sdh1/Sdh2 protein abundance in no effect variants.

variants affect stability (Supplementary Table S2; ref. 28). problem is exacerbated in GIST as loss of SDHA protein is the
yT134M, yR179W, yG410R, and yG431F all have positive DDG most common cause of SDH deficiency in GIST, but SDHA
(change in free energy), indicating these changes are highly variants have not been extensively characterized at the func-
destabilizing. Some of these also were associated with decreased tional level. Finally, many SDHA VUS are also found in the
Sdh1 protein abundance (Fig. 2). germline, meaning that they can be inherited. Knowing a
Interestingly, all the variants with no functional effects were patient has a loss-of-function SDHA variant in a tumor dra-
localized to the surface of the protein (Fig. 4B; Supplementary matically changes clinical decision making concerning screen-
Video S1) where it would be predicted that they would be less ing recommendations for tumor syndromes, which affects both
likely to affect protein structure and function. Notably, these the patient and other family members. Because there are no
mutations did not map into any of the known critical domains effective medical treatments for advanced SDH-deficient
of Sdh1 or predicted Sdh2 interaction sites. tumors, early detection of tumors allowing curative surgical
resection is crucial.
A major problem in the field has been the lack of a validated
Discussion model system to assess the biochemical function of SDHA VUS.
Molecular testing plays an important role in the clinical Currently, there are no human SDHA-deficient cell lines, and
management of GIST, including decision making about appro- assessing SDH complex activity in tumor samples is difficult.
priate medical and surgical therapy. SDH deficiency is a poten- Previously, it has been shown that a yeast model can be used to
tial cause of GIST in tumors lacking known oncogenic drivers evaluate the pathogenic significance of SDHB mutations
such as gain-of-function KIT mutations. To attempt to identify (17, 18). Extending these studies, we report the successful use
the cause of SDH deficiency in such GIST, it is necessary to use of a yeast model to characterize SDHA variants found in GIST
multi-gene sequencing panels to search for SDHx pathogenic tumors as either causing loss of function or having no bio-
mutations; however, as the routine use of multi-gene sequenc- chemical consequence. Together with computational model-
ing panels has expanded, there has also been an increase in ing, structural homology, and patient data, we have drawn
reported VUS. Due to the multiple potential causes of SDH- conclusions on how and why the SDHA variants we studied
deficiency, it is difficult to determine if a newly discovered VUS affect SDH function, providing clinicians with information to
is responsible for the defect, or if a different genetic mechanism guide genetic counseling of patients and family members who
is responsible for the loss of SDH complex activity. This harbor one of these germline VUS.

OF6 Clin Cancer Res; 2017 Clinical Cancer Research

 Biochemical, Molecular, and Clinical Characteristics of SDHA VUS

Table 2. Consistent findings across multiple functional assays for each variant in our yeast model
Growth Oxygen consump- Sdh2 Protein
hSDHA ySdh1 phenotype on tion (% of (% of Structural implications of
Mutation Mutation glycerol complemented) complemented) variants Group name
R31X R19X Unable to grow Decreased (20%) Decreased (0%) Truncates mitochondrial Loss-of-function
(control) (control) targeting sequence (47) (control)
H99S H90S Unable to grow Decreased (2%) Decreased (15%) Inhibits covalent bond to Loss-of-function
(control) (control) FAD (14) (control)
G106R G97R Unable to grow Decreased (7%) Decreased (14%) Distorts the active site (31) Loss-of-function
N118S N109S Growth Similar (108%) Similar (83%) Surface of protein No effect
T143M T134M Unable to grow Decreased (3%) Decreased (35%) No obvious disturbances Loss-of-function

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but causes loss of
function
R171H R162H Growth Similar (84%) Similar (78%) Surface of protein No effect
R188W R179W Unable to grow Decreased (21%) Decreased (0%) Disrupts salt bridge with Loss-of-function
D108
R195W R186W Growth Similar (91%) Similar (82%) Surface of protein No effect
G260R G251R Unable to grow Decreased (27%) Decreased (0%) Obstructs flavin binding Loss-of-function
pocket
H296Y H287Y Unable to grow Decreased (4%) Decreased (11%) Inhibits succinate binding Loss-of-function
(31)
R312C R303C Unable to grow Decreased (4%) Decreased (3%) Contributes to proper Loss-of-function
orientation and
activation of the flavin
isoalloxazine ring to
facilitate formation of
the covalent FAD bond
and disrupts salt bridge
(48)
R408C Y399C Unable to grow Decreased (9%) Decreased (4%) In flavin binding site Loss-of-function
G419R G410R Unable to grow Decreased (19%) Decreased (2%) Distorts helix Loss-of-function
C438F C431F Unable to grow Decreased (19%) Decreased (2%) Bulky change disrupts Loss-of-function
helix
G439E G432E Unable to grow Decreased (-2%) Decreased (3%) Obstructs flavin binding Loss-of-function
pocket
R451C R444C Unable to grow Decreased (12%) Decreased (6%) Disrupts flavin binding, Loss-of-function
succinate binding and
proton shuttle
necessary for catalytic
activity (31)
R451H R444H Unable to grow Decreased (3%) Decreased (0%) Disrupts flavin binding, Loss-of-function
succinate binding, and
proton shuttle
necessary for catalytic
activity (31)
A454E A447E Unable to grow Decreased (11%) Decreased (0%) Lines succinate binding Loss-of-function
site (31)
R465W R458W Unable to grow Decreased (5%) Decreased (21%) Disrupts salt bridge with Loss-of-function
E136
T508I T501I Growth Similar (113%) Similar (75%) Surface of protein No effect
R589G R582G Unable to grow Decreased (12%) Decreased (0%) Inhibits C-terminal Loss-of-function
flavination (25)
H625W H601Y Unable to grow Decreased (18%) Decreased (2%) Inhibits C-terminal Loss-of-function
flavination
Y629F W605F Growth Similar (113%) Similar (101%) Surface of protein No effect
V657I V633I Growth Similar (108%) Similar (82%) Surface of protein No effect
NOTE: A summary of results, including the growth on glycerol, oxygen consumption, Sdh2 protein abundance, consequences of changing the WT amino acid using
computational modeling, and literature search for studies in other species' flavoprotein, is tabulated along with our classification of each variant based on our yeast
model. Numerical values for oxygen consumption were taken from Fig. 1C and D. Numerical values for Sdh2 protein were taken from Fig. 2E.

Using our yeast model, we characterized variants as either the loss-of-function variants were associated with a dramatic
causing loss of protein function or having no effect. All variants decrease in Sdh2 protein abundance, which is in concordance
in the loss-of-function group were unable to grow on a nonfer- with current clinical testing to determine SDH-deficiency in
mentable carbon source (glycerol). In addition, these variants also tumors—assessment of SDHB (human equivalent to ySdh2)
failed to consume oxygen, indicating defective oxidative phos- expression using IHC (2, 32–36). Our data provide further
phorylation. Interestingly, these variants had differential effects evidence supporting the role of SDHB IHC as the most reliable
on Sdh1 flavination and/or protein abundance. As a group, all of clinical test to identify SDH-deficiency in a tumor. Notably, we

www.aacrjournals.org Clin Cancer Res; 2017 OF7

 Bannon et al.

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Figure 3.
Variants involving the active site cause
loss of Sdh1 function. A ribbon
representation of Sdh1 (E. coli model
PBD file 2WP9) is shown with WT
protein carbons colored gray and the
variant carbons colored red. The FAD
is shown as teal-colored spheres,
whereas the dicarboxylic acid
substrate (succinate analog) is
depicted with yellow-colored spheres.
In each case, the view has been rotated
so that residues of interest are clearly
observed. References detailing
structural implications of each variant
can be found in Table 2.

identified four novel loss-of-function variants that did not affect The conclusions drawn from our yeast model are largely
Sdh1 protein abundance (yG97R, yT134M, yR444C, yH601Y). supported by our computational modeling results. For many
These variants prevent flavination of Sdh1, making Sdh1 dysfunc- variants, we could find confirmatory structural modeling evi-
tional, but still expressed at normal levels. Our findings are dence that the amino acids affected by these mutations played a
consistent with previous reports where SDHA expression was role in the catalytic site of Sdh1. The computational model and
retained in some SDHA-mutant tumors that lack SDHB expres- energy minimization also allowed us to explain why some
sion (37), confirming that not all dysfunctional SDHA (or Sdh1) variants could reduce Sdh1 protein abundance. Furthermore,
variants lead to decreased protein expression of this subunit. all the variants that did not affect protein function were on the
There are three clinical samples (R188W, G260R, A454E) that surface of the protein as visualized by our computational
we characterized as loss-of-function variants but are associated model, suggesting that they would not interact with the cata-
with normal sdh1 in our yeast model. Based on these results and lytic mechanism of Sdh1.
coupled with the fact the SDHA IHC is not universally available, Based on our biochemical data, there are still several tumors
we advocate for the use of SDHB IHC to identify tumors that are that have no known mechanism for loss of SDH activity
SDH-deficient. (Table 1). One of these tumors had the hR195W (yR186W)

OF8 Clin Cancer Res; 2017 Clinical Cancer Research

 Biochemical, Molecular, and Clinical Characteristics of SDHA VUS

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Figure 4.
Structural consequences of variants
visualized in a yeast model (yeast
model PBD file 1ORZ). A, Variants not
in the active site causing loss of
function. WT Sdh1 protein is
represented as a gray ribbon with the
amino acid of interest labeled as a gray
stick and the refolded variant Sdh1
protein as a red ribbon with the amino
acid of interest indicated by a red stick.
In each case, the view has been rotated
such that residues of interest are
clearly observed. Potential structural
implications of each variant can be
found in Table 2. B, Variants that do
not affect protein function are located
on the surface of Sdh1. The surface
area of WT Sdh1 is shown in gray, the
variant residue is labeled red, and the
surface area of Sdh2 is labeled in
green. In each case, the view has been
rotated such that residues of interest
are clearly observed.

variant with an allele frequency of 66.7% and negative SDHB sequencing panels (15, 16). Given the limitations of using
IHC. This tumor is likely driven by a different mechanism of deidentified results from clinical testing, we did not have access
SDH-deficiency. The rest of the unexplained tumors had no to residual tumor samples to perform additional IHC or geno-
IHC data available, so it is possible that these GISTs are not mic testing.
driven by SDH-deficiency and instead belong to a different We used a yeast model to characterize 22 SDHA VUS. These
subtype of GIST (11). Alternatively, these GISTs may be SDH- data revealed 16 (73%) of SDHA VUS as loss of function
deficient by a different genetic mechanism than the SDHA (and therefore pathogenic), highlighting the importance of
variant identified using targeted exome sequencing. There are understanding such variants to provide better clinical recommen-
several weaknesses of various targeted exome sequencing dations for genetic counselors concerning family screening
panels including potential lack of coverage for regions of and early detection protocols. However, our approach using a
certain SDHx genes, failure to detect large genomic deletions functional yeast model paired with computational modeling
involving SDHx subunits, as well as the inability to measure can distinguish between SDHA VUS that cause loss of
hypermethylation of the SDHC promoter. In addition, muta- function and those that have no biochemical effect, allowing
tions that inactivate newly identified SDH assembly factors us to discriminate between pathogenic and nonpathogenic
(SDHAF3, 4) are typically not included in clinical cancer gene variants.

www.aacrjournals.org Clin Cancer Res; 2017 OF9

 Bannon et al.

Disclosure of Potential Conflicts of Interest Acknowledgments

J.K. Sicklick reports receiving commercial research grants from Foundation Thank you to Arin McKinley, Diana Griffith, Janice Patterson, and Ashley
Medicine, Inc., and Novartis Pharmaceuticals. M.C. Heinrich is an employee of Young for their constant support during the completion of this project. The
MolecularMD. No potential conflicts of interest were disclosed by the other advice and feedback from Amanda McCullough, Maureen Hoatlin, and David
authors. Koeller were extremely beneficial for experimental design and interpretation of
results. Also, thank you to Dennis Winge for his generous gift of primary
Authors' Contributions antibodies for Sdh1 and Sdh2.
Conception and design: A.E. Bannon, L.R. Klug, K. McCann, M.C. Heinrich
Development of methodology: A.E. Bannon, I. Forquer, K. McCann, U. Shinde,
M.C. Heinrich Grant Support
A.E. Bannon was supported by the OHSUOCTRI TL1TR000129.

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Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): A.E. Bannon, I. Forquer, A. Town, C. Corless, O. Harismendy was supported by NIHR21CA177519 and U01CA196406.
U. Shinde, M.C. Heinrich J.K. Sicklick was supported by NIHK08CA168999, R21CA192072, and
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, P30CA023100. M.C. Heinrich was supported by GIST Cancer Research
computational analysis): A.E. Bannon, J. Kent, I. Forquer, L.R. Klug, K. McCann, Fund, Life Raft Group, and a Merit Review Grant from the Department of
O. Harismendy, U. Shinde, M.C. Heinrich Veterans Affairs (2I01BX000338-05).
Writing, review, and/or revision of the manuscript: A.E. Bannon, J. Kent, The costs of publication of this article were defrayed in part by the payment of
L.R. Klug, K. McCann, O. Harismendy, J.K. Sicklick, C. Corless, U. Shinde, page charges. This article must therefore be hereby marked advertisement in
M.C. Heinrich accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): A.E. Bannon, C. Beadling, M.C. Heinrich Received May 22, 2017; revised June 20, 2017; accepted July 14, 2017;
Study supervision: A.E. Bannon, I. Forquer, M.C. Heinrich published OnlineFirst July 19, 2017.

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