Gardie PresentationHoustonOct4 3 2018def - VHL Alliance

11/10/2018

1

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



type2

Type 2A

Y98H/wt

type2

R167Q/wt

Type 1

C162F/wt

Genotype (VHL):

Phenotype:



P138P/wtD143D/D143D

R200W/wtD143D/wtG144R/wtQ164H/wt

wt/wt

Unsolved cases:



type2

Type 2A

Y98H/wt

type2

R167Q/wt

Type 1

C162F/wt

Genotype (VHL):

Phenotype:



P138P/wtD143D/D143DUnsolved cases:

Synonymous mutations induce splicing alteration:visit POSTER Marion Lenglet

11/10/2018

2



type2

Type 2A

Y98H/wt

type2

R167Q/wt

Type 1

C162F/wt

Genotype (VHL):

Phenotype:



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

11/10/2018

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ATG

ATG


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


* 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


Stéphane Richard

12

RNAseq

Upregulation of isoforms containing E1’-VHL exon


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

11/10/2018

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Upregulation of isoforms containing E1’-VHL exon

RT‐qPCR TaqMan


***

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


pCAS2

15

Minigene experiments

VHL – E1’

Differential effect of VHL mutations on Exon1’ retention

Erythrocytosis


pCAS2

16

Western Blot

Downregulation of expression of all VHL protein isoforms

pVHLX1

Yannick Arlot, Franck ChesnelAnne Couturier

pVHL213 pVHL160

11/10/2018

5

17

Expression of new VHL isoforms


ENST00000477538.1

Probe E1‐E1’

Probe Upstream E1’

Tissues Cell lines


E1 E1’ E2 E3

E1 E1’ E3

E1’ E2 E3

18

MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPRPVLRSVNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPYPTLPPGTGRRIHSYRVLMTPVGQFCVVPALVENTFLLGRLTDAKTGTSQGHVGAGRADRVWRGKLTYLPAGRWRGCGCVVSVKEHFPEKEESRME

pVHLX1

pVHLX1 contains 16/17 aa involved in HIF binding


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

11/10/2018

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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


brain placenta kidney lung heart liver pancreas sk muscle0

1

2

3 E1E1'


1

2

3

4

5 E1E2


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



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'


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

11/10/2018

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Human :Chimp :Gorilla :Orangutan :Olive_baboon :Macaque :Rhesus :Gibbon :Marmoset : Squirrel_monkey :



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




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

****

**

***



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

11/10/2018

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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


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


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


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

11/10/2018

9

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)


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


11/10/2018

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


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

*


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


Documents

Gardie PresentationHoustonOct4 3 2018def - VHL Alliance