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Supplementary data
Supplementary Material and Methods
Sequencing
Genomic DNA was extracted from peripheral blood and purified on a spin column (Qiagen).
Screening for the VHL mutations (three coding exons and exon-intron junctions) and the eight
Single Nucleotide Polymorphisms (SNP) described in Liu et al., 2004 (1) was performed by
direct sequencing using BigDye Terminator v3.1 Kit (Applied Biosystems) and an ABI 3730
Genetic Analyzer (Applied Biosystems). We aligned Sequence data using Seqscape (Applied
Biosystems) or Sequencher (Gene Codes Corporation) software. Sequences of all primers
used in this study are available upon request.
Computational Methods
Molecular dynamics (MD) simulations were performed with the CHARMM 27(2) force field
using the NAMD program (3). The starting models of pVHL were constructed from the
crystal structure solved by Min and co-workers (2002) (PDB ID code 1lm8) (4). Mutant
models were generated from the wild-type model by in silico substitutions of the asparagines
in positions 161 and 200, with a glutamine (Q) and a tryptophan (W) respectively
(MODELLER 9v8) (5, 6). Each model was solvated in a TIP3P water box with a minimum
distance of 10 Å from the edge of the box to any protein atom. The charges of the system
were neutralized by adding counterions (Na+ or Cl-). To eliminate clashes between the solvent
and the protein, the solvated systems were first minimized for 3 000 steps with the protein
atoms restrained followed by another 3 000 steps of minimization with all atoms allowed to
1
move. The systems were then heated up to 300 K during 100 ps while constraining protein
backbone atoms to allow the relaxation of solvent molecules. The systems were then
equilibrated for 100 ps under constant volume and temperature (NVT) conditions constraining
protein backbone atoms followed by 500 ps equilibrium run under constant pressure and
temperature (NPT) conditions without any constraints. Production simulations were
performed for 20 ns with the NPT ensemble at 300 K and room pressure. Temperature and
pressure were controlled using a Langevin thermostat and Nose-Hoover Langevin piston
barostat (7) as implemented in NAMD (3). Short-range interactions employed a switch
function with a 12 Å cut-off and 10 Å switch distance, and the long range electrostatic
interactions were calculated with the Particle Mesh Ewald protocol (8). During production
simulations the time step was 2 fs, with a SHAKE constraint on all bonds containing
hydrogen atoms. Structures were saved every 1 ps. To reduce the results dependence on the
initial conditions, two replicas of the MD simulation were produced for each protein. The
trajectories were analyzed with the ptraj program of AMBER Tools 1.5. Structural alignments
and figure rendering were performed by VMD 1.8.7 (9). The angle rotation analysis during
simulation was performed using Hingefind (10).
In vitro analysis of HIF-1/pVHL binding
The HA-tagged pVHL protein was synthesized in vitro and incubated with a hydroxylatedbiotinylated peptide corresponding to the HIF-1 oxygen dependant domain (HIF-OH,
100ng). Site directed mutagenesis was performed on pRc-CMV-HA-VHL wild-type vector
(11) using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The pVHL protein
was synthesized in an in vitro transcription-translation system (T7 TNT quick coupled
transcription/translation kit, Promega). Proteins were then incubated with a hydroxylatedbiotinylated peptide (8-residue peptide: free-AP*YIPMDD-cooh, P*=hydroxyproline)
2
corresponding to the HIF-1 Oxygen Dependent degradation Domain (HIF-OH). HApVHL/HIF complexes were immunoprecipitated with streptavidin beads and analyzed by NuPage migration (Invitrogen). The hydroxylated peptides bound to HA-pVHL were revealed by
immunoblotting using an HA specific antibody (Santa Cruz Biotechnology).
VHL mutants construction
To avoid cell death during reintroduction of VHL in the VHL-defective 786.O cell line (12),
we used an tetracycline (or doxycycline) inducible system to study the VHL mutants. The
ViraPower T-REX Lentiviral Expression System (Invitrogen) was used to construct inducible
lenti-vectors encoding non-tagged VHL. The VHL coding sequences (wild-type or mutants)
were cloned into a pENTR vector and transferred into the pLenti4/TO (Tet Operator)/V5/Dest
vector using GATEWAY cloning technology (Invitrogen) (13). The vectors were stably
transfected into 786.O cells a VHL-defective sporadic ccRCC cell line mutated for VHL that
overexpresses HIF-2. The 786.O cells are a gift from the laboratory of Pr Kaelin (Boston).
They have been genotyped by direct sequencing for the VHL gene (mutation c.311delG;
p.G104fs*55) after reception in our laboratory. The cells were transduced with the
pLenti6/TR (Tet Repressor) vector, selected with blasticidin (Invitrogen) and cloned. The
expression of the Tetracycline Repressor was tested and the selected clone was transduced
with the pLenti4/TO/VHL constructions and selected with zeocin (Invitrogen). Clones were
grown in DMEM medium (Gibco) supplemented with 5% tetracycline free foetal calf serum
(PAA), Zeocin 0.2 mg/mL, blacticidin 3 g/mL and sodium pyruvate 1 mM (Gibco) and
were tested for VHL expression after induction with doxycyclin 1 g/mL (Sigma) that
releases the Tet Repressor from the Tet Operator and causes induction of VHL expression. We
selected clones with equivalent VHL mRNA expression for further studies.
3
Transcriptomic study
RNA was extracted using Trizol reagent (Sigma) and purified on Qiagen columns. 500 ng of
total RNA from each RNA sample was amplified and labelled with two fluorescent dyes (Cy5
and Cy3) using the Quick Amp Labeling Two-Color kit (Agilent Technologies, Palo Alto,
CA, USA). Labelled cRNA were hybridized to the Agilent Human Whole Genome Oligo
Microarray format 4x44K (Agilent Technologies, G4112F), prior to washing and scanning.
All hybridizations were performed in duplicate with dye swap to eliminate possible dye bias.
Data were extracted from scanned images using Feature Extraction software (v 10.5.1.1
Agilent) with default settings. Data from all hybridizations were analysed with Rosetta
Resolver software. Unsupervised hierarchical clustering was generated using cosine
correlation as similarity metrics and average link of both for genes and samples. Genes were
considered significant when their p-values were below 10-5 with more than a 2-fold change of
expression. The microarray data related to this paper have been submitted to the Array
Express
data
repository
at
the
European
Bioinformatics
Institute
(http://www.ebi.ac.uk/arrayexpress/) under the accession number E-MTAB-1269.
Statistical calculation:
The presence of an increasing gradient in gene expression profiles (datas from TaqMan) was
tested by dose-response statistical analysis as suggested by Pramana et al., 2010 (14). We
considered six increasing doses (wild type (wt), R200W, R161Q, R200W+R161Q, C162F,
empty) and tested the null hypothesis of homogeneity of means (no dose effect) against
ordered alternatives. We performed isotonic regression of the observed means (15, 16) and
used the modified M (M') statistics for an increasing dose response (14). Due to the small
sample size, the significance was evaluated using a Monte-Carlo simulation where H0
4
samples was generated using permutation of the dose ordering. N=10,000 simulations was
performed for each gene to produce an empirical p-value with 90% confidence intervals. All
computations were performed using the R software (version 2.15.1) and the R package
IsoGene (version 1.0).
Expression profiles of the 30 identified genes in clear cell renal cell carcinoma
The GSM14994 dataset (17), was used to analyse the expression of the 30 genes identified as
direct target of the pVHL/HIF-2 pathway. The Affymetrix HT Human Genome U133A
Arrays [[HT_HG-U133A] (that do not include LncRNAs, micro RNA, and other small RNA
changes, as well as aberrantly processed mRNAs) was normalized with the justRMA
procedure in BrB Array Tools v. 4.4.0 (http://linus.nci.nih.gov/BRB-ArrayTools.html). The
signature corresponding to the comparaison of 52 clear cell renal cell carcinoma (VHL
mutated) versus 11 normal kidney tissues was performed with the Class Comparison
procedure on a restrictive list of 26 genes (probes for KCNIP3, TMEM141, ARRDC3,
ERRFI1 were absent of the dataset (17)).
Legends of supplementary Tables and Figures:
Table S1: Phenotypes associated with R161Q and R200W VHL germline mutations.
Summary of the R200W and R161Q VHL carriers and associated phenotypes described in the
literature. Pheo: pheochromocytoma, RCC: renal cell carcinoma, Hb: hemangioblastoma,
NET: pancreatic neuroendocrine tumor, CP: Chuvash Polycythemia. Ref: references. A : (1,
18-25), B : (21, 24, 25),(26), C : (22, 23, 27, 28), D : (29),(30), E : (27, 31-40), F : (41).
Genotype-phenotype analysis of families harboring R161Q mutations reveals a strong
5
correlation between the R161Q/wt genotype and predisposition to pheochromocytomas,
excepted for a single Japanese family in which very few R161Q mutation carriers had
pheochromocytomas, thus suggesting that the penetrance of this specific mutation is under the
control of modifier genes operating in the Japanese genetic context (41). The occurence of
renal cancer in the R161Q carriers is low (frequency : 0.26 calculated from our database), and
the mutant has been classified as a VHL Type-A (in comparison, the frequency of renal
tumors in patients carring the typical Type-2B mutation R167W is 0.38 in our database). The
family carrying the double mutation R200W+R161Q was firstly described as carrier of a
unique R200W mutation (30, 40) and was cited as a puzzling case (23, 24, 26, 42-51). NA:
data Not Available.
Table S2: VHL mutations described on patients with polycythemia. The table lists the
VHL mutations described in the literature in patients with a polycythemia. The genetic status
of the mutations (homozygous, compound heterozygous or heterozygous) is indicated in the
third column. The fourth column indicates the references ((19-23, 28, 52-58)). VHL mutations
in patients with erythrocytosis are always missenses mutations excepted for one truncating
mutation, VHL-E10X (personal data, (53)). However, this particular mutation is located
between the two translation initiation codons and has the capacity to produce a pVHL19
isoform still able to regulate HIF. The same VHL mutations have already been described in
some patients with a VHL disease (references in the fifth column (27, 30, 40, 59-61)) or have
never been described in such cases (ND: not described). None of these mutations as been
described in patients with a severe VHL disease, excepted the VHL-V130L that has been
described in one family with renal carcinoma (a same family described three times). The
genotyping of this family has been performed on the same time as the present described
6
family (with the misdiagnosed VHL-R200W mutation, Olschwang et al., 1996), therefore, it
should be retested before final conclusion.
Table S3: Hematological data for the family carring the VHL-R200W+R161Q
mutations. RBC: Red Blood Cells, N: normal values; f: females; Hb: Hemoglobin; Ht:
Hematocrit; EPO: Erythropoietin; NP: not performed (EPO dosage was not performed on
patient III-2 as he was not alive at the time of our study); NA: data Not Available that
corresponds to old data that were not kept because they were normal.
Table S4: Hydrogen bonds recorded during molecular dynamics (MD) simulation of
mutants. The H-bonds occupancies are given in percentages of the simulation time, averaged
over two MD trajectories. Top: H-bonds formed within the α-domain between H1, H2 and H3
in wild-type (wt) pVHL and the mutants R161Q and R200W+R161Q. The R161Q mutation
indirectly causes the weakening of crucial stabilizing interactions involving primarily Arg
167. Bottom: H-bonds formed within the β-domain between H4 and the seven-stranded βsandwich in wild type (wt) pVHL and the mutants R200W and R200W+R161Q. The R200W
mutation induces a partial disappearance of stabilizing H-bonds. Local intra-domain effects
observed in the single mutants are increased in the double mutant R200W+R161Q.
Table S5: Rotation angle calculated in the MD simulation of wild-type (wt) pVHL,
R200W and R161Q single mutants and R161+R200W double mutant. The angle
maximum (Max) and mean (Mean) values along with standard deviations (Sd) are given in
degrees for each MD trajectory (Sim 1, 2) of each protein.
7
Table S6: Statistical calculation of the graded dysregulation of the VHL mutants. The
presence of an increasing gradient in gene expression measured by RT-quantitative PCR has
been tested using a dose-response statistic suggested by Pramana and collaborators (14). We
considered six increasing doses (with this order: wild type (wt), R200W, R161Q,
R200W+R161Q, C162F, empty) and tested the null hypothesis of homogeneity of means (no
dose effect) against ordered alternatives. Following (15, 16), we performed an isotonic
regression of the observed means and used the modified M (M') statistics for increasing dose
response (see Pramana et al, 2010) (14). Due to the small samples, the significance was
evaluated using Monte-Carlo simulations where H0 samples was generated using permutation
of the dose ordering. N=10,000 was performed for each genes in order to produce an
empirical p-value with 90% confidence intervals. All computations were performed using the
R software (version 2.15.1) and the R package IsoGene (version 1.0). The results are
expressed, for each gene, in p-values (%) for an ascending gradient of VHL dysfunction.
Confidence intervals are indicated in square brackets. NA: Not Applicable. Overall, a gradient
for all these genes is highly significant (overall p-value<1e-10 under the null hypothesis of
homogeneity of means).
Table S7: pVHL/HIF-2 target genes which are differentially expressed among normal
kidney versus ccRCC. The list of the 30 genes regulated by the direct pVHL/HIF-2
pathway has been analysed using the Dataset GSM14994 (17). The genes significant at
0.001 level of the univariate test are listed in the table. Genes are Sorted by p-value of the
univariate test. Class 1: N, normal kidney tissues; Class 2: T, ccRCC with mutated VHL. FDR:
8
False Discovery Rate. Function of the genes have been determined from data in the litterature
(62-80).
Figure S1: Genotype/phenotype correlations associated with VHL germline mutations
described in the literature. On the top, chromosome 3 is represented. The VHL inherited
germline mutation is indicated with a red circle for predisposition to Chuvash polycythemia
or with a red cross for predisposition to von Hippel-Lindau disease. Below, polycythemia is
represented by a red contour, VHL disease is characterized by the development of tumors
(circles): CNS hemangioblastomas that can occur anywhere along the brain/spine areas (red),
retinal hemangioblastomas (blue), pheochromocytomas (black) and clear cell renal cell
carcinomas (ccRCC) (yellow) with a low (small circle) or high risk (large circle), pancreatic
cysts and neuroendocrine tumors (purple) and epididymal cystadenomas (brown). A
representative VHL mutation of each von Hippel-Lindau disease type is indicated below. The
purpose of this study is to determine the discriminating effect between germline VHL
mutation that predispose heterozygous carriers to develop tumors or not.
Figure S2: Structure and interaction networks of pVHL altered by the R161Q or
R200W mutations. (A): Crystal structure of pVHL (PDB ID code: 1lm8) displayed as a
cartoon. The α-helices and the loops are labelled. The asparagines in positions 161 and 200
are shown using stick and surface representations. (B): Interaction network between the H4
helix and the seven-stranded β-sandwich, in wild-type pVHL (in blue) and in the R200W
mutant (in cyan). (C): Interaction network between the H1 helix and the H2 and H3 helices,
in the wild-type pVHL (in blue) and in the R161Q mutant (in yellow). Interacting residues are
displayed in sticks and the residues in positions 161 and 200 subject to mutations are
9
highlighted by a thicker stick radius. Oxygen, nitrogen and hydrogen atoms are colored in red,
blue and white respectively and H-bonds are drawn as dashed red lines.
Figure S3: VHL expression in 786.O cells transduced by different VHL inducible
lentiviral constructs. TaqMan real-time quantitative RT-PCR analysis of the VHL transcripts
after induction with doxycyclin (DOX).
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