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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%