Gardie PresentationHoustonOct4 3 2018def - VHL Alliance
Transcript of Gardie PresentationHoustonOct4 3 2018def - VHL Alliance
11/10/2018
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L’unité de recherche de l’institut du thoraxInserm UMR 1087 / CNRS UMR 6291Nantes, France
Oct 4, 2018, Houston
New lessons from an old gene: complex splicing and mutations in a novel cryptic exon in VHL gene cause
erythrocytosis and VHL disease
Betty Gardie,
Maître de Conférences de l’Ecole Pratique des hautes Etudes
Institut du Thorax, UMR INSERM1087/CNRS 6291
NANTES, FRANCE
ErythrocytosisHealthy von Hippel‐Lindau DiseaseType 2BType 2C
R200W/wt R200W/R200W L188V/wt
type2
Type 2A
Y98H/wt
type2
R167Q/wt
Type 1
C162F/wt
Genotype (VHL):
Phenotype:
Representativegenotypes:
VHL Genotype/Phenotype correlations
ProjectProject
Molecular mechanisms at the origin of the different phenotypes?
ChuvashPolycythemia
ErythrocytosisHealthy von Hippel‐Lindau DiseaseType 2BType 2C
R200W/wt R200W/R200W L188V/wt
type2
Type 2A
Y98H/wt
type2
R167Q/wt
Type 1
C162F/wt
Genotype (VHL):
Phenotype:
Representativegenotypes:
VHL Genotype/Phenotype correlations
P138P/wtD143D/D143D
R200W/wtD143D/wtG144R/wtQ164H/wt
wt/wt
Unsolved cases:
ErythrocytosisHealthy von Hippel‐Lindau DiseaseType 2BType 2C
R200W/wt R200W/R200W L188V/wt
type2
Type 2A
Y98H/wt
type2
R167Q/wt
Type 1
C162F/wt
Genotype (VHL):
Phenotype:
Representativegenotypes:
VHL Genotype/Phenotype correlations
P138P/wtD143D/D143DUnsolved cases:
Synonymous mutations induce splicing alteration:visit POSTER Marion Lenglet
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ErythrocytosisHealthy von Hippel‐Lindau DiseaseType 2BType 2C
R200W/wt R200W/R200W L188V/wt
type2
Type 2A
Y98H/wt
type2
R167Q/wt
Type 1
C162F/wt
Genotype (VHL):
Phenotype:
Representativegenotypes:
VHL Genotype/Phenotype correlations
R200W/wtD143D/wtG144R/wtQ164H/wtwt/wt
wt/wt
Unsolved cases:
E1 E2 E3
E1 E2 E3
STOP
E1 E3
ATG STOP
mRNA
VHL structure
VHL gene
ProteinspVHL213/p30pVHL160/p19
pVHL172/ pVHLΔE2
Latif et al., Science 1993
ATGATG
E1 E2 E3
E1 E3
Lymphoblastoid Cell Line (LCL) cDNA
Cloning and sequencing of all VHL transcripts
Erythrocytosis
ATG STOP
E1 E2 E3E1’VHL gene
Identification of new VHL cryptic exon
E1 E1’ E2 E3
E1
Protein pVHLX1 :114 aa (E1) + 79 aa (E1’)
New VHL isoforms
?
E1’ E3
Lenglet, Robriquet et al., 2018
ATG STOP
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ATG
ATG
E1 E2 E3E1’VHL gene
Identification of new VHL cryptic exon
E1 E1’ E2 E3
STOP
E1
New VHL isoforms
E1’ E3
STOP
NCBI: E1’ E2 E3NON CODING ENST00000477538.1
pVHLX1
Erythrocytosis
E1VHL gene E2 E3
c.340+574A>T
c.340+694_711dup
c.340+770T>Cc.340+816A>C
E1’
Identification of mutations in the new E1’‐VHL exon
Lenglet, Robriquet et al., 2018
* Whole genomesequencingJenny TaylorCarme Camps
* *
*
von Hippel‐Lindau disease
c.340+617C>Gc.340+648T>C (SNP)
E1 E1’Gène VHL E2 E3
Identification of mutations in the new E1’‐VHL exon
Lenglet, Robriquet et al., 2018
Stéphane Richard
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RNAseq
Upregulation of isoforms containing E1’-VHL exon
Lenglet, Robriquet et al., 2018
Pheo F8 I.2X1-L128V+L138P/WT
Pheo F8 II.1X1-L128V+L138P/WT
Pheo controlVHL WT
LCL ControlWT
LCL F8 II.6X1-L128V+L138P/WT
VHL disease
Exon 1 Exon 1’ Exon 2 Exon 3VHL
291 reads
137 reads
172 reads
121 reads
98 reads
VHL
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Upregulation of isoforms containing E1’-VHL exon
RT‐qPCR TaqMan
Lenglet, Robriquet et al., 2018
***
ATG STOP
E1 E1’ E2 E3
E1 E1’ E3
ATG STOP
probe 14
Reporter splicing assay:
Minigene experiments
VHL – E1’
Differential effect of VHL mutations on Exon1’ retention
Erythrocytosis
Lenglet, Robriquet et al., 2018
pCAS2
15
Minigene experiments
VHL – E1’
Differential effect of VHL mutations on Exon1’ retention
Erythrocytosis
Lenglet, Robriquet et al., 2018
pCAS2
16
Western Blot
Downregulation of expression of all VHL protein isoforms
pVHLX1
Yannick Arlot, Franck ChesnelAnne Couturier
pVHL213 pVHL160
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17
Expression of new VHL isoforms
E1 E2 E3E1’VHL gene
ENST00000477538.1
Probe E1‐E1’
Probe Upstream E1’
Tissues Cell lines
Lenglet, Robriquet et al., 2018
E1 E1’ E2 E3
E1 E1’ E3
E1’ E2 E3
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MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPRPVLRSVNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPYPTLPPGTGRRIHSYRVLMTPVGQFCVVPALVENTFLLGRLTDAKTGTSQGHVGAGRADRVWRGKLTYLPAGRWRGCGCVVSVKEHFPEKEESRME
pVHLX1
pVHLX1 contains 16/17 aa involved in HIF binding
Lenglet, Robriquet et al., 2018
ATG STOP
E1 E1’ E2 E3
E1 E1’ E3
ATG STOP
No pVHLX1 activity on HIF pathway
Hypoxia Responsive Element Reporter Assay in 786.O cells
Lenglet, Robriquet et al., 2018 20
Conclusion- Identification of a new VHL exon
- Mutations in patients with erythrocytosis or von Hippel-Lindau disease that alter splicing
- Unknown physiological role of the new VHL isoforms(non coding RNA?)
- Screen unsolved cases for this new exonGenomic data (Whole genome sequencing)
- Study the role of the new isoformsExpression (RNAsequencing)?....
Perspectives
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AcknowledgmentsAcknowledgments
Marion Lenglet,Florence Robriquet,Stéphane Richard,Anne‐Paule Gimenez‐Roqueplo,Yannick Arlot‐Bonnemains,Holger CarioKlaus Schwarz,Carme Camps,Anne Couturier,David Hoogewijs,Alexandre Buffet,Nelly Burnichon,Samantha JL. Knight,Sophie Gad,Sophie Couvé,Franck Chesnel,Pierre Lindenbaum,
Thomas Besnard,Sophie Deveaux,Sophie Ferlicot,Fabrice Airaud,Céline Garrec,Richard Redon,Stéphane Bezieau,Brigitte Bressac‐de Paillerets,François Girodon,Maria‐Luigia Randi,Vincent Bours,Joachim R. Göthert,Antonis Kattamis,Nicolas Janin,Celeste Bento,Jenny C. Taylor.
22
1 1’ 2 3
ATG STOP STOP
1 1’ 3
ATG STOP STOP
MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPRPVLRSVNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPYPTLPPGTGRRIHSYRVLMTPVGQFCVVPALVENTFLLGRLTDAKTGTSQGHVGAGRADRVWRGKLTYLPAGRWRGCGCVVSVKEHFPEKEESRME
pVHLX1
pVHLX1 conservation: only in higher primates
Lenglet, Robriquet et al., 2018
brain placenta kidney lung heart liver pancreas sk muscle0
1
2
3 E1E1'
brain placenta kidney lung heart liver pancreas sk muscle0
1
2
3
4
5 E1E2
brain placenta kidney lung heart liver pancreas sk muscle0
2
4
6
8 E1E3
Supplemental Figure 1
0
0.5
1
1.5
2 E1E1'
LCL C1 HK2 UT7 RC1 Hep3B Hela 786-O HEK 293T
0
0.5
1
1.5 E1E2
0
0.5
1
1.5 E1E3
LCL C1 HK2 UT7 RC1 Hep3B Hela 786-O HEK 293T
LCL C1 HK2 UT7 RC1 Hep3B Hela 786-O HEK 293T
Rel
ativ
e ge
ne e
xpre
ssio
n no
rmal
ized
to k
idne
y
Rel
ativ
e ge
ne e
xpre
ssio
n no
rmal
ized
to H
ep3B
Rel
ativ
e ge
ne e
xpre
ssio
n no
rmal
ized
to k
idne
yR
elat
ive
gene
exp
ress
ion
norm
aliz
ed to
kid
ney
Rel
ativ
e ge
ne e
xpre
ssio
n no
rmal
ized
to H
ep3B
Rel
ativ
e ge
ne e
xpre
ssio
n no
rmal
ized
to H
ep3B
0
0.5
1.0
1.5
2.0
2.5Upstream E1'
Rel
ativ
e ge
ne e
xpre
ssio
n no
rmal
ized
to H
ep3B
brain placenta kidney lung heart liver pancreas sk muscle LCL C1 HK2 UT7 RC1 Hep3B Hela 786-O HEK 293T
Rel
ativ
e ge
ne e
xpre
ssio
n no
rmal
ized
to k
idne
y
A
0
5
10
15
20Upstream E1'
Supplemental Figure 1
B E1E2 E1E3 Upstream E1' E1E1' RPLP0brain 26,76 32,31 28,16 32,90 21,425
placenta 25,54 30,90 26,125 30,25 25,2kidney 25,10 31,10 26,595 30,38 22,03lung 25,31 31,42 26,85 30,54 21,51heart 26,76 31,96 26,74 30,39 19,155liver 24,12 29,09 24,745 28,94 24,025
pancreas 23,98 29,57 24,045 29,47 21,44skeletal muscle 29,48 36,34 30,775 34,41 19,105
E1E2 E1E3 Upstream E1' E1E1' RPLP0LLB C1 22,90 28,44 25,12 27,47 18,6HK2 22,73 28,93 25,58 27,68 18,4UT7 22,37 26,75 24,62 26,99 18,2RC1 22,70 28,00 25,77 28,67 21,3Hep3B 21,10 26,49 24,32 27,31 19,1Hep3B 21,52 27,01 24,495 27,80 18,9Hela 22,93 28,99 25,225 27,66 19,27786‐O 22,77 28,64 25,74 26,92 19,065HEK 293 22,25 27,83 24,99 26,54 18,385
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Human :Chimp :Gorilla :Orangutan :Olive_baboon :Macaque :Rhesus :Gibbon :Marmoset : Squirrel_monkey :
Human :Chimp :Gorilla :Orangutan :Olive_baboon :Macaque :Rhesus :Gibbon :Marmoset : Squirrel_monkey :
Supplemental Figure 2
Ac.340+617C>G c.340+648T>C
c.340+694_711dup c.340+770T>G
c.340+574A>T
c.340+816A>C
B
Human :
Chimp :
Gorilla :
Orangutan :
Olive_baboon :
Macaque :
Rhesus :
Gibbon :
Marmoset :
Squirrel_monkey :
Human : Chimp : Gorilla : Orangutan : Olive_baboon : Macaque : Rhesus : Gibbon : Marmoset : Squirrel_monkey :
271
265
265
259
166
249
250
256
235
234
97%
271
265
259 259
164 164
249 249
250 250
256 256 256
235237 237
235 235 234
97%
97%
271
95%
95%
95%
270
167
251
252
61%
60%
61%
60%
180
178
179
166
154
155
90%
90%
90%
90%
90%
90%
91% 91%
64%
275
274
255
239
236
65%
99%
275
256
240
237
93%
93%
93%
93%
60%
92%
93%
275
243
243
85%
86%
86%
85%
56%
86%
87%
88%
274
248
85%
85%
85%
85%
56%
85%
86%
90%
88%
274
Human :Chimp :Gorilla :Orangutan :Olive_baboon :Macaque :Rhesus :Gibbon :Marmoset : Squirrel_monkey :
Human :Chimp :Gorilla :Orangutan :Olive_baboon :Macaque :Rhesus :Gibbon :Marmoset : Squirrel_monkey :
Supplemental Figure 2
C c.340+617C>G c.340+648T>C
c.340+694_711dup c.340+770T>G
Cat :Horse :White_rhinoceros :Microbat :Elephant :Sheep :Pig :Squirrel :
Cat :Horse :White_rhinoceros :Microbat :Elephant :Sheep :Pig :Squirrel :
c.340+574A>T
c.340+816A>C
Human :
Chimp :
Gorilla :
Orangutan :
OliveBaboon :
Macaque :
Marmoset :
Gibbon :
Greenmonkey :
D
c.340+617C>GX1, c.382C>G,
pLeu128Val
c.340+648T>CSNP X1, c.413T>C
p.Leu138Pro
c.340+770C>GX1, c.535T>Cp.Ser179Pro
c.340+816A>CX1, c.583A>Cp.*Serext*24
****
**
***
Supplemental Figure 3
Supplemental Figure 4
F1
I.1
I.2
II.1
VHL c.429C>T, p.Asp143Asp E1’, c.340+770T>C X1, c.535T>C, p.Ser179Pro
F3
VHL c.598C>T, p.Arg200Trp
53
92
E1’, c.340+770T>C X1, c.535T>C, p.Ser179Pro
F4
II.1
I.1
VHL c.598C>T, p.Arg200TrpE1’, c.340+694_711dup
X1 c.458_476dup, p.Trp159X
F2 II.1
E1’, c.340+770T>C X1, c.535T>C, p.Ser179Pro
VHL c.598C>T, p.Arg200Trp
II.1
II.2
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Supplemental Figure 4
VHL c.430G>A, p.Gly144Arg E1’, c.340+694_711dup X1 c.458_476dup, p.Trp159X
I.2
I.1
II.1
II.2
II.3
F5
VHL c.492G>C, p.Gln164His c.340+574A>T
F6
I.1
I.2
II.1
Germline DNA
WT
F8 I.2
Tumor DNA
Pheo F8 I.2
Pheo F8 II.1
RCC F8 I.2
c.340+617C>G c.340+648T>C
F8
Supplemental Figure 5
ATGCCCCGGAGGGCGGAGAACTGGGACGAGGCCGAGGTAGGCGCGGAGGAGGCAGGCGTCGAAGAGTACGGCCCTGAAGAAGACGGCGGGGAGGAGTCGGGCGCCGAGGAGTCCGGCCCGGAAGAGTCCGGCCCGGAGGAACTGGGCGCCGAGGAGGAGATGGAGGCCGGGCGGCCGCGGCCCGTGCTGCGCTCGGTGAACTCGCGCGAGCCCTCCCAGGTCATCTTCTGCAATCGCAGTCCGCGCGTCGTGCTGCCCGTATGGCTCAACTTCGACGGCGAGCCGCAGCCCTACCCAACGCTGCCGCCTGGCACGGGCCGCCGCATCCACAGCTACCGAGgtacgggcccggcgcttaggcccgacccagcagggacgatagcacggtctgaagcccctctaccgccccggggtccattttgcagacggggaactgaggccccttgaggcaggacacatccagggtgacgctgctcgtaagcgtcagagcattcttttttttttttttttttttctgagacggagtctcgctctgtcgcccaggctggagtgcagtggcgcgatctcgactcactgcagcctccgcctcccgggttcaagcgattctcctgcctcagcctcctgagtagctgggattacaggcgtgcgccaccgcgcccggctgatttttatatttttagtagagacggggtttcaccatgttggtcaggctggtctcgaactgctgacctcgtgatccgcccgcctcggcctcccaaagtgctgggcttatgggcatgagcctccgcgcccggcccagagcattctttataaggccgaatagtttgcatttgaaggtggctccccccagtcccccaccccacgtgtattttcccctcaaagaaaagctgcatccttaacaccccatctgttcagtcctcatgactccagtgggccagttctgcgtagtccctgccctcgtggagaacacattcctcctggggagactgacagatgcaaagacaggaacaagccagggtcatgttggcgccggaagagccgaccgtgtgtggcgtgggaaattgacttacctgcctgctgggagatggaggggttgcggttgtgtggtttcagttaaggagcacttcccggagaaggaagagagcaggatggagtaggaactagccaaccctaggtaagaggttctagacatgcgtgcgttgagacctggagtcttgggagaggatgcttaaaaggtgattttacccctaggaatatgggggcactgaaatttttttttttttttgagacgggagtcttgctctgcaagctggagtgcagtggcccacgctagaatgcagtggcgcgattgcggctcattgcaacatctgccacctggggtcaagtggttctcttgcctcagcctcccgaggagcggggattacaggcgtgcgccaccactcctggctaattttttttttagtagagacgggggtttcgtcattttggctaggctggtctcgaactcctgacctcagatgatccacccgccttggcctcccaaagtgctgagattacaggtgtaagccactgcgcccagccctttgaaagtttttcagtatttatgtatatatatttttgagttggagtctggatctgtcgccagactggagtgctgttgcac
340+770T>C
340+1150T>CChuvash SNP
Exon 1
Exon 1’
SNP Chuvash (Liu et al., 2004) : rs779808 (Chuvash Allele = C)[Homo sapiens] gagacgggggtttcgtcattttggc[C/T]aggctggtctcgaactcctgacctc
340+770T>C 340+1150T>C
Allele 1(CHUVASH)
Allele 2
Patient F2
A.2) Downstream filtering for patients F2 II.1, F3 II.1 and F3 II.2 (all heterozygous for the VHL p.R200W mutation)
High confidence variants which are rare in general population 543,445 variants
A.2.1) Variants within VHL gene +/- 5Kb
Genomic change Gene Region
Transcript Variant
Protein Variant
GenotypeCADD 1000G
FreqESP Freq
ExACFreq
WGS500FreqF2 II.1 F3 II.1 F3 II.2
chr3:10184641T>C Intronic NM_000551.3:c.340+770T>C Het Het Het 12.26 NR NR NR NR
chr3:10191605C>T Exonic NM_000551.3:c.598C>T p.R200W Het Het Het 35 NR 0.015 0.023 NR
1078variants
Variants within ERY gene list: genes in erythrocytosis gene panel and/or IVA gene list for erythrocytosis/ polycythaemia (120 genes)Freq in WGS500 < 3%
Same variant in all 3 patients (same genotype required for all)
102variants
Genes with variants in all 3 patients (same variant and genotype required for
brothers)
46 genes
Predicted deleterious 3 variants Variants in all 3 patients predicted deleterious 0 genes
Genomic change Gene Gene Region Transcript Variant Protein
Variant
GenotypeCADD 1000G
FreqESP Freq
ExACFreq
WGS500FreqF2 II.1 F3 II.1 F3 II.2
chr3:10191605C>T VHL Exonic NM_000551.3:
c.598C>T p.R200W Het Het Het 35 NR 0.015 0.023 NR
chr9:135853324T>C GFI1B Promoter
Intronic
NM_004188.6:c.‐925T>C; c.‐355‐570T>C
Het Het Het 7.34 0.26 NR NR 0.55
chr11:33758819delCACG CD59 Promoter NM_203331.2:
c.‐958_‐955delCGTG; Hom Hom Hom NA NR NR NR NR
Supplementary Figure 6
Filtering stepNumber of variants
F2 II.1, F3 II.1, F3 II.2 Family 7 trioImport of multi‐vcf file into IVA(PASS variants with freq ≤ 3% in 1000G, NHLBI ESP6500 and ExAC )
694,445 661,545
High confidence variants(Quality ≥ 20; Read depth ≥ 10; Allele freq ≥ 0.05)
684,034 653,857
Rare variants in general population(Freq ≤ 1% in 1000G, NHLBI ESP, ExAC and gnomAD)
543,445 419,479
A.1) Initial filtering steps common to both WGS datasets (patients F2 II.1, F3 II.1, F3 II.2; family 7 trio)
A.2.2) Variants in genes related or suspected to be related to erythrocytosis
A) Analysis of single nucleotide variants and small insertions/ deletions
Literature search does not support candidacy for any of these variants
A.3) Downstream filtering for family 7 (trio: proband affected, parents unaffected)
419,479 variants
Predicted deleterious by IVA
Genetic analysisFreq WGS500 < 3% and inspection on IGV to eliminate
artefactsMode of Inheritance N. Variants N. Variants in ERY gene list
de novo 1 0
Autosomal recessive 3 0
X‐linked 12 0
Compound heterozygous 30 2 (SLC4A1, FOXP1)
2990 variants
High confident variants which are rare in general population
A.3.1) Analysis of single nucleotide variants and small insertions/ deletions
Same variant in all 3 patients (same genotype required for all)
2 variants
Genes with variants in all 3 patients (same variant and genotype required for
brothers)
1 gene
320 variants
A.2.3) Variants predicted deleterious with high functional impact
Predicted deleterious and CADD score > 20Freq in WGS500 < 3%
Genomic change Gene Gene Region Transcript Variant Protein
Variant
GenotypeCADD 1000G
FreqESP Freq
ExACFreq
WGS500FreqF2 II.1 F3 II.1 F3 II.2
chr3:10191605C>T VHL Exonic NM_000551.3:
c.598C>T p.R200W Het Het Het 35 NR 0.015 0.023 NR
chr19:925968T>G ARID3A Promoter NM_005224.2:
c.‐359T>G NA Het Het Het 21.9 NR NR NR NR
chr7:148963937G>A ZNF783 Exonic NM_001195220.1:
c.448G>A p.V150M Het HomRef
HomRef 26.2 NR 0.046 0.056 0.18
chr7:148979220G>A ZNF783 Exonic NM_001195220.1:
c.1427G>A p.R476H HomRef Het Het 29.5 NR NR NR NR
Literature search does not support candidacy for any of these variants
Genomic change Gene Gene Region Transcript Variant
GenotypeCADD 1000G
FreqESP Freq
ExACFreq
WGS500FreqProband Father Mother
chr3:71114069G>T FOXP1 Intronic5’UTR
NM_032682.5:c.283‐11145C>ANM_001244813.1:c.‐113C>A
Het HomRef Het 17.6 NR NR NR NR
chr3:71448771G>A FOXP1 Intronic NM_032682.5:c.‐167‐40376C>T Het Het Hom
Ref 1.4 0.04 NR NR NR
chr17:42343875C>T SLC4A1 Intronic NM_000342.3:c.‐69+1547G>A Het Het Hom
Ref 9.5 0.08 NR NR NR
chr17:42344297A>G SLC4A1 Intronic NM_000342.3:c.‐69+1125T>C Het Hom
Ref Het 4.9 0.99 NR NR 0.36
Literature search does not support candidacy for any of the variants obtained in the genetic analysis
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No relevant copy number variants were identified
A.3.2) Variants within VHL new cryptic exon
B) Analysis of copy number variants
chrX:47829755-47834837 : Hemizygous loss in F3 II.1 and F3 II.2.• In ZNF182 3’UTR. This region has highly conserved POLR2A TFBS.• Literature search on this gene does not support candidacy for this variant
Genomic change Gene Region Transcript Variant
GenotypeCADD 1000G
FreqESP Freq
ExACFreq
WGS500FreqProband Father Mother
chr3:10184687A>C Intronic NM_000551.3:c.340+816A>C Hom Het Het 9.95 NR NR NR NR
B.1) Patients F2 II.1, F3 II.1 and F3 II.2 (all heterozygous for the VHL p.R200W mutation)
B.2) Family 7 (trio: proband affected, parents unaffected)
Supplemental Figure 8
APosition on Chromosome 3 (hg19) :Junction E1 on 3’ : 10183872Junction E1‘ on 5’ : 10184447Junction E1‘ on 3’ : 10184706Junction E2 on 5’ : 10188198Junction E2 on 3’ : 10188321Junction E3 on 5’ : 10191471
B
ControlF11 II.6
E1E2 (%) E2E3 (%) E1E3 (%) E1E1’ (%) E1’E2 (%) E1’E3 (%)47.8 33.9 2.8 2 13.5 026.8 20.3 5.9 13.7 33.3 0
LCL
Total reads251153
Pheochromocytoma
ControlF11 II.1
E1E2 (%) E2E3 (%) E1E3 (%) E1E1’ (%) E1’E2 (%) E1’E3 (%)41 48.2 7.2 1.2 1.2 1.2
22.8 17.2 5 11.7 42.2 1.1
Total reads83
180F11 I.2 28.6 30.6 0 16.3 20.4 4.1 98
C
Pheo F11 I.2
Pheo F11 II.1
LCL F11 II.6
c.340+617C>G c.340+648T>C
C G T C
26%
26%
28%
74%
74%
72%
25%
26%
31%
75%
74%
69%
E1E2 E1E3 E1E1' RPLP0LCL C1 23,15 28,70 27,60 19,37LCL C2 23,46 29,07 27,98 19,15
LCL F1 I.2 24,39 27,51 27,73 19,37LCL F1 I.1 23,75 29,50 26,30 19,56LCL F1 II.1 26,05 27,28 26,17 19,45LCL F2 II.1 23,22 29,08 25,75 18,93LCL F8 II.6 23,88 29,33 25,69 19,32LCL C1 22,25 27,83 23,76 19,71LCL C2 21,85 27,28 24,54 19,22
LCL F1 I.2 23,20 27,32 25,66 19,73LCL F1 I.1 22,63 28,71 22,99 19,71LCL F1 II.1 25,35 26,41 23,14 20,05LCL F2 II.1 22,32 28,73 22,84 19,61LCL F8 II.6 23,50 29,03 23,49 20,05
‐ puro
+ puro
A
Erythrocytosis compound heterozygous
VHL disease with heterozygous mutation in E1’
Healthy with heterozygous mutation in VHL
Control Wild Type
Rel
ativ
e ge
ne e
xpre
ssio
n
Healthy with heterozygous mutation in E1’
0
2
4
6
8
10 E1E3
B
C1 C2 F1I.2
F1II.1
F1I.1
F8II.6
F2II.1
C1 C2 F1I.2
F1II.1
F1I.1
F8II.6
F2II.1
Without Puromycin With Puromycin
Supplemental Figure 9
11/10/2018
10
Supplemental Figure 10
Exon 1
Exon 2
Exon 3
Exon 1 Exon 2
Exon 3
Exon 1’ F6
Exon 1
Deletion : use thenext AG splicing acceptor site
Exon 1’ F6
ATGCCCCGGAGGGCGGAGAACTGGGACGAGGCCGAGGTAGGCGCGGAGGAGGCAGGCGTCGAAGAGTACGGCCCTGAAGAAGACGGCGGGGAGGAGTCGGGCGCCGAGGAGTCCGGCCCGGAAGAGTCCGGCCCGGAGGAACTGGGCGCCGAGGAGGAGATGGAGGCCGGGCGGCCGCGGCCCGTGCTGCGCTCGGTGAACTCGCGCGAGCCCTCCCAGGTCATCTTCTGCAATCGCAGTCCGCGCGTCGTGCTGCCCGTATGGCTCAACTTCGACGGCGAGCCGCAGCCCTACCCAACGCTGCCGCCTGGCACGGGCCGCCGCATCCACAGCTACCGAGGTCACCTTTGGCTCTTCAGAGATGCAGGGACACACGATGGGCTTCTGGTTAACCAAACTGAATTATTTGTGCCATCTCTCAATGTTGACGGACAGCCTATTTTTGCCAATATCACACTGCCAGTGTATACTCTGAAAGAGCGATGCCTCCAGGTTGTCCGGAGCCTAGTCAAGCCTGAGAATTACAGGAGACTGGACATCGTCAGGTCGCTCTACGAAGATCTGGAAGACCACCCAAATGTGCAGAAAGACCTGGAGCGGCTGACACAGGAGCGCATTGCACATCAACGGATGGGAGATTGA
ATGCCCCGGAGGGCGGAGAACTGGGACGAGGCCGAGGTAGGCGCGGAGGAGGCAGGCGTCGAAGAGTACGGCCCTGAAGAAGACGGCGGGGAGGAGTCGGGCGCCGAGGAGTCCGGCCCGGAAGAGTCCGGCCCGGAGGAACTGGGCGCCGAGGAGGAGATGGAGGCCGGGCGGCCGCGGCCCGTGCTGCGCTCGGTGAACTCGCGCGAGCCCTCCCAGGTCATCTTCTGCAATCGCAGTCCGCGCGTCGTGCTGCCCGTATGGCTCAACTTCGACGGCGAGCCGCAGCCCTACCCAACGCTGCCGCCTGGCACGGGCCGCCGCATCCACAGCTACCGAGGTACGGGCCCGGCGCTTAGGCCCGACCCAGCAGGGACGATAGCACGGTCTGAAGCCCCTCTACCGCCCCGGGGTCCATTTTGCAGACGGGGAACTGAGGCCCCTTGAGGCAGGACACATCCAGGGTGACGCTGCTCGTAAGCGTCAGAGCATTCTTTTTTTTTTTTTTTTTTTTCTGAGACGGAGTCTCGCTCTGTCGCCCAGGCTGGAGTGCAGTGGCGCGATCTCGACTCACTGCAGCCTCCGCCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGATTACAGGCGTGCGCCACCGCGCCCGGCTGATTTTTATATTTTTAGTAGAGACGGGGTTTCACCATGTTGGTCAGGCTGGTCTCGAACTGCTGACCTCGTGATCCGCCCGCCTCGGCCTCCCAAAGTGCTGGGCTTATGGGCATGAGCCTCCGCGCCCGGCCCAGAGCATTCTTTATAAGGCCGAATAGTTTGCATTTGAAGGTGGCTCCCCCCAGTCCCCCACCCCACGTGTATTTTCCCCTCAAAGAAAAGCTGCATCCTTAACACCCCATCTGTTCAGTCCTCATGACTCCAGTGGGCCAGTTCTGCGTAGTCCCTGCCCTCGTGGAGAACACATTCCTCCTGGGGAGACTGACAGATGCAAAGACAGGAACAAGCCAGGGTCATGTTGGCGCCGGAAGAGCCGACCGTGTGTGGCGTGGGAAATTGACTTACCTGCCTGCTGGGAGATGGAGGGGTTGCGGTTGTGTGGTTTCAGTTAAGGAGCACTTCCCGGAGAAGGAAGAGAGCAGGATGGAGTAGGAACTAGCCAACCCTAGGTAAGAGGTTCTAGACATGCGTGCGTTGAGACCTGGAGTCTTGGGAGAGGATGCTTAAAAGGTGATTTTACCCCTA
Supplemental Figure 12
Caco-2
293-T
E1’ WTH2O pcDNA pCas2 c.340
+617
C>G
A
A
B
B
1’
HK2
c.340
+617
C>G
+c.3
40+6
48T>
C
c.340
+648
T>C
*
Supplemental Figure 13
A
B
11/10/2018
11
ControlF9 II.1
E1E2 (%) E2E3 (%) E1E3 (%) E1E1’ (%) E1’E2 (%) E1’E3 (%)47.8 33.9 2.8 2 13.5 010.7 22.8 55.7 0.7 23 8.1
Total number and percentage of reads identified for specific VHL Exons splicing junctions
LCL
Total reads251149
Pheochromocytoma
ControlF11 III.1
41 48.2 7.2 1.2 1.2 1.213.2 11.3 73.6 1.9 0 0
Total reads8353
BGenesHK2
EGFREGLN3PRKCA
FLT1BCL2HGF
ANGPT2ENO1
PFKFB3TEK
SLC2A1TFRCPGK1LDHAHK3
GAPDHVEGFA
HK1PFKL
EGLN1
Fold change7.801136.386685.215864.678873.891763.753223.735893.683373.476013.448183.427053.341493.102832.958992.912982.327312.183492.171182.023621.9217
1.86894
E1E2 (%) E2E3 (%) E1E3 (%) E1E1’ (%) E1’E2 (%) E1’E3 (%)
A
Supplemental Figure 15