elucidation of the molecular basis of retinal detachment: intronic mutations in cspg2/ versican...

Elucidation of the molecular basis of retinal detachment: intronic mutations in CSPG2/

Versican cause Wagner disease and erosive vitreoretinopathy

Frans P.M. Cremers, PhD

Department of Human GeneticsRadboud University Nijmegen Medical Centre

Nijmegen, The Netherlands

HUMANGENETICS

NIJMEGENHGM2006 Helsinki; 2-6-2006

Human Genetics Nijmegen

Retinal detachment

Retinal pigment epithelium

Choroid

Neuralretina

Photo-Receptors:

rods & cones

Human Genetics Nijmegen

Non-syndromic retinal detachment

• Non-syndromic retinal detachment- Isolated – multifactorial (frequent; 1/125)- Autos. dominant (rare; COL2A1)

• Familial exudative vitreoretinopathy- Autos. dominant/recessive (rare; FZD4, LRP5)- X-linked (rare; NDP)

• Wagner disease / erosive vitreoretinopathy

- Autosomal dominant (rare; 5q13-q14)

Human Genetics Nijmegen

Wagner disease

Invariable: • “empty” vitreous• preretinal membrane

Variable:• abnormal vessel architecture• fovea shifted• cortical cataracts• chorioretinal atrophy• retinal detachment

Wagner H. Klin Mbl Augenheilk. 100:840-858, 1938.

Normal

Wagner disease

Human Genetics Nijmegen

Erosive vitreoretinopathy

Brown et al. Ophthalmology. 101:694-704, 1994.

Courtesy: C.E. van Nouhuys

Invariably: • “empty” vitreous• night blindness• RPE erosion (progressive)• visual field loss

Variable:• pigmentations• retinal detachment

XRCC4

CSPG2

CTRL1

EDIL3

Wagner disease/ERVR linkage interval

Human Genetics Nijmegen

83,0 90,072,0 Mbp81,5

Zech et al. ‘99

Perveen et al. ‘99

Black et al. ‘99

Chromosome 5q13-q14

D5S650 5’CTRL1D5S626 D5S618

Brown et al. ‘95

G. Black and coworkers: sequence analysis of ORFs: no variants identified!!

D5S2103

89,083,6

D5S107

Human Genetics Nijmegen

Wagner disease - linkage

W04-153

D5S1347 286 310D5S2094 129 135D5S107 154 134D5S428 246 254

310135148250

?310135134254

310135134254

310131154252

314131150244

310131154252

310135134154

306129152244

310131154252

310135148250

310131154252

306135144248

?310135134254

306135148244

306135148244

306135144248

Human Genetics Nijmegen

Erosive vitreoretinopathy - linkage

W95-124

D5S1347 310 310D5S2094 135 135D5S107 148 134D5S428 248 254

286129144244

310129148254

? ?

310135134254

314135150250

310135148248

302135156250

310135134254

286129144244

286129144244

310135148248

?

310135134254

286129148254

310135134254

310135134254

310135134254

310135134254

290129144254

290129144254

290129144254

298135

?244

310135134254

WagnerDiseaseW04-153

D5S424D5S1501

D5S626D5S641D5S1347D5S2094

5’CRTL1

D5S428D5S617D5S1722D5S1463

D5S2029

D5S107

W95-038

13696

?269302135

252196223177

224

129

152

W04-153

?110

?267310135

254188??

230

149

134

W95-023

12296

?267310135

254196227169

230

149

148

W05-088

??

?267310135

????

230

149

134

W95-131

130106

1267310135

254188231193

230

137

134

?110

?267310135

244188??

230

149

150

2.3

0.20.40.30.1

0.5

1.80.72.31.8

Mbp

2.9

0.6

vv

vv

vv

vv

vv

vv

Wagner disease: founder haplotype in the NL

Human Genetics Nijmegen

D5S424D5S1501

D5S626D5S641D5S1347D5S2094

5’CRTL1

D5S428D5S617D5S1722D5S1463

D5S2029

D5S107

W95-038

13696

?269302135

252196223177

224

129

152

W04-153

?110

?267310135

254188??

230

149

134

W95-023

12296

?267310135

254196227169

230

149

148

W05-088

??

?267310135

????

230

149

134

W95-131

130106

1267310135

254188231193

230

137

134

?110

?267310135

244188??

230

149

150

2.3

0.20.40.30.1

0.5

1.80.72.31.8

Mbp

2.9

0.6

vv

vv

vv

vv

vv

vv

Wagner disease & ERVR: samefounder haplotype

Human Genetics Nijmegen

W95-124

12296

?267310135

254188231181

230

149

134

ERVR

W95-131

W05-088

W95-023

W04-153

W95-124 (ERVR)

Human Genetics Nijmegen

Wagner disease & ERVR: common ancestor

Nijmegen

XRCC4

CSPG2

CTRL1

EDIL3

Wagner disease/ERVR linkage interval

Human Genetics Nijmegen

83,0 90,072,0 Mbp81,5

Chromosome 5q13-q14

D5S650 5’CTRL1D5S626 D5S618D5S2103

89,083,6

D5S107

Human Genetics Nijmegen

Miyamoto et al. IOVS, 46:2726, 2005

Wild type 1 157 8

Mutant 1 157 8

AGGT

GGGT

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

ATG TGAc.4004-2A>G

CSPG2/Versican

Human Genetics Nijmegen

Wild type 1 157 8

Mutant

AGGT

1 157 8GGGT

In-frame 39 nt deletion

AG

Miyamoto et al. IOVS, 46:2726, 2005

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

ATG TGAc.4004-2A>G

CSPG2/Versican

CSPG2/Versican splice variants

V0

V1

V2

V3

6 8

8

8

86

7

7

7

76

6

1

1

1

1

9 15

9 15

9 15

9 15

Human Genetics Nijmegen

• 3396-amino acid extracellular matrix protein

• Synthesised in virtually all human tissues: cell adhesion, proliferation, migration and extracellular matrix assembly.

• Maintains structure of the vitreous body in the human eye.

CSPG2/Versican

Hyaluronan binding region

α β

Glycosaminoglycan (GAG) attachment

domains

Lectin-C binding domain

Sushi domain

EGF domains

Human Genetics Nijmegen

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Human Genetics Nijmegen

-2

aaaactctgtttttttcagGTCGAATGAGTG

CSPG2/Versican: intron 7 variants

Miyamoto et al. IOVS, 46:2726, 2005

7 8

-5 -1

Human Genetics Nijmegen

CSPG2/Versican: intron 7 variants

c.4004-5T>C c.4004-5T>A c.4004-1G>A

7 8

BindingCapacity:

64% 14% 0%

aaaactctgtttttttcagGTCGAATGAGTG

Human Genetics Nijmegen

CSPG2/Versican c.4004-5T>Csegregates with Wagner disease

Family W95-131

T/CT/C

T/C T/C T/T T/C

T/T

T/CT/C T/T

T/C

T/C T/T T/C

T/TT/T

T/C

T/TT/C

D5S424D5S1501

D5S626D5S641D5S1347D5S2094

5’CRTL1

D5S428D5S617D5S1722D5S1463

D5S2029

D5S107

W04-153

?110

?267310135

254188??

230

149

134

W95-023

12296

?267310135

254196227169

230

149

148

W05-088

??

?267310135

????

230

149

134

W95-131

130106

1267310135

254188231193

230

137

134

?110

?267310135

244188??

230

149

150

Wagner disease & ERVR: same founder variant

Human Genetics Nijmegen

W95-124

12296

?267310135

254188231181

230

149

134

ERVRc.4004-5T>C

CSPG2 CCCCCC

W95-038

13696

?269302135

252196223177

224

129

152

c.4004-5T>A

A

Δ39 bp

Human Genetics Nijmegen

2 out of 3 known CSPG2/Versican mutations activate a cryptic splice sitein exon 8

CSPG2/Versican RT-PCR analysis

39 nt/13 aa deletion: no effect on GAG binding

Are their quantitative effects?

Human Genetics Nijmegen

V0

V1

V2

V3

6 8

8

8

86

7

7

7

76

6

1

1

1

1

9 15

9 15

9 15

9 15

20 30

PCR cycles

Ct1 Ct2

ΔCt = Ct1 - Ct2

Reference gene:GUSB

Gene of interest:CSPG2 splice variants

Human Genetics Nijmegen

Relative quantitation: Q PCR

cDNAamount

threshold

Relative quantities of CSPG2/Versican mRNA splice variants in patients vs controls

*p<0.0001

52.0* 477.7* 0.660.87UnknownW05-282P1

22.6* 137.2*0.660.81c.4004-1G>A W95-137P1

12.8* 38.3*0.641.02c.4004-5T>C W95-124P1

12.1* 45.3*0.660.81c.4004-5T>C W95-131P1

13.9*52.0*0.540.62c.4004-5T>A W95-038P1

V3V2V1 V0

CSPG2/Versican splice variantSequence variant

Individual

Human Genetics Nijmegen

W95-124P1: erosive vitreoretinopathy

1.01.01.01.0Controls (8)

Human Genetics Nijmegen

mutant / +

V0 V1 V2 V3V0 V1 V2 V3

100

50

+ / + mutant / +

V0 V1 V2 V3

CSPG2/Versican isoforms in eye

Normal

Adapted from: Zao & Russell, Mol. Vis. 11:603 - 608, 2005.

CSPG2/Versican isoforms in eye

Human Genetics Nijmegen

V0 V1 V2 V3

100

50

+ / + c.4004-1G>A / +

V0 V1 V2 V3

Normal Wagner disease

CSPG2/Versican isoforms in eye

Human Genetics Nijmegen

V0 V1 V2 V3

100

50

+ / + c.4004-1G>A / +

V0 V2 V3

Total CSPG2 molecules not changed!

Normal Wagner disease

V1

CSPG2/Versican splice variants

V0

V1

V2

V3

6 8

8

8

86

7

7

7

76

6

1

1

1

1

9 15

9 15

9 15

9 15

Human Genetics Nijmegen

: glycosaminoglycan (GAG) attachment sites

CSPG2-GAG attachment sites

Human Genetics Nijmegen

V0 V1 V2 V3

100

50

V0 V1 V2 V3

GAG-attachment sites: 100% 63%

+ / + c.4004-1G>A / +Normal Wagner disease

•Total amount CSPG2 unaltered

•Reduction in GAG attachment sites

•Cause: V0/V1 or V2/V3 ?

Wagner disease mechanism?

Human Genetics Nijmegen

52.0 477.7 0.660.87UnknownW05-282P1

22.6 137.20.660.81c.4004-1G>A W95-137P1

12.8 38.30.641.02c.4004-5T>C W95-124P1

12.1 45.30.660.81c.4004-5T>C W95-131P1

13.952.00.540.62c.4004-5T>A W95-038P1

V3V2V1 V0

CSPG2/Versican splice variantSequence variant

Patient

Human Genetics Nijmegen

Wagner disease families with no V2/V3 upregulation!

W95-061P1

W05-542P1

W95-152P1

Unknown

Unknown

Unknown

0.731.11

0.75

0.76

0.69

0.68

0.95

0.44

0.32

0.84

0.66

0.53

Wagner disease families with noV2/V3 upregulation link to 5q13-q14

Human Genetics Nijmegen

W95-061

D5S2029 176 166D5S641 234 230

176234

188230

176234

178230

188230

178230

188230

166230

178230

166230

178230

166230

W95-542

D5S1347 342 334D5S107 199 205

342199

334197

334205

178230

338197

334197

338197

342199

W95-152

D5S1347 334 338D5S107 197 201

334197

314193

314193

338197

334197

334197

338201

?338197

334197

338199

318197

?334197

338199

314193

318197

338199

Observations: Only intronic mutations affecting splicing of exon 8

found in Wagner disease and ERVR

Exonic stopmutations are not found in 3396-aa ORF

Observations & discussion

7 8 914.7 kb

5.3 kb3.3 kb

Human Genetics Nijmegen

c.4004-5T>C, -5T>A, -1G>ADutch

c.4004-2A>G Japanese

c.9265+1G>ASwiss*

*Kloeckener-Gruissem et al. Mol. Vis. 12:250-255, 2006

Hypotheses: Wagner disease / ERVR due to moderate reduction

of GAG attachment sites of CSPG2/Versican.

Haploinsufficiency of all CSPG2 isoforms is lethal.

Remaining mutations are located deep intronic.

XRCC4

CSPG2

CTRL1

EDIL3

Wagner disease/ERVR: an amazing quest

Human Genetics Nijmegen

81,5

Chromosome 5q13-q14

D5S626

83,6

D5S107

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

8

ctctgtttttttcagGTC CAGgtaagatc

Arijit MukhopadhyayKonstantinos NikopoulosAlessandra MaugeriArjan de BrouwerSaskia van der Velde-VisserMarloes WennemersHan Brunner

Dept. of Human GeneticsNijmegen, NL

Dept. of OphthalmologyNijmegen, NLCamiel BoonCarel Hoyng

Bartimeus Zeist, Zeist, NL

Canisius Wilhelmina Hospital,Nijmegen, NL

Pieter van den BiesenNienke Boonstra

Erik van Nouhuys

Manchester, UKRahat PerveenGraeme Black

Tuebingen, GermanyBernd Wissinger

Acknowledgements

HUMANGENETICS

NIJMEGEN

QUESTIONS

CSPG2/Versican isoforms in eye

Human Genetics Nijmegen

V0 V1 V2 V3

100

50

+ / + mutation / +

V0 V1 V2 V3

Normal Exon 8 stop

Total CSPG2 molecules: 100% 57%

CSPG2-GAG attachment sites

Human Genetics Nijmegen

V0 V1 V2 V3

100

50

V0 V1 V2 V3

GAG-attachment sites: 100% 51%

+ / +Normal

mutation / +Exon 8 stop

CSPG2/Versican isoforms in eye

Human Genetics Nijmegen

V0 V1 V2 V3

100

50

+ / + mutation / +

V0 V1 V2 V3

Normal Intron 6 splice

Total CSPG2 molecules unchanged

CSPG2-GAG attachment sites

Human Genetics Nijmegen

V0 V1 V2 V3

100

50

V0 V1 V2 V3

GAG-attachment sites: 100% 95%

+ / +Normal

mutation / +Intron 6 splice

Intron 7 or 8 splice site mutations

Human Genetics Nijmegen

α β

Predicted effect of total CSPG2 total GAGs

intron 7/8 mutations: 100% 63% (50% + 13%)

V0

V1

V2

V3

6 8

8

8

86

7

7

7

76

6

1

1

1

1

9 15

9 15

9 15

9 15

Exon 8 stopmutations

Human Genetics Nijmegen

α β

V0

V1

V2

V3

6 8

8

8

86

7

7

7

76

6

1

1

1

1

9 15

9 15

9 15

9 15

Predicted effect of total CSPG2 total GAGs

exon 8 stopmutations: 57% 51% (50% +

1%)

Exon 8 stopmutations

Human Genetics Nijmegen

α β

V0

V1

V2

V3

6 8

8

8

86

7

7

7

76

6

1

1

1

1

9 15

9 15

9 15

9 15

Predicted effect of total CSPG2 total GAGs

- exon 8 stopmutations: 57% 51%

- intron 7/8 mutations: 100% 60%