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Frans P.M. Cremers, PhD Department of Human Genetics Radboud University Nijmegen Medical Centre

N. HUMA. GE. N. ETICS. GE. N. NIJME. 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 Genetics Radboud University Nijmegen Medical Centre

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Frans P.M. Cremers, PhD Department of Human Genetics Radboud University Nijmegen Medical Centre

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  1. N HUMA GE N ETICS GE N NIJME 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 Genetics Radboud University Nijmegen Medical Centre Nijmegen, The Netherlands HGM2006 Helsinki; 2-6-2006

  2. Retinal detachment Photo- Receptors: rods & cones Neural retina Retinal pigment epithelium Choroid Human Genetics Nijmegen

  3. 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

  4. Wagner disease Normal 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. Human Genetics Nijmegen

  5. Erosive vitreoretinopathy • Invariably: • “empty” vitreous • night blindness • RPE erosion (progressive) • visual field loss • Variable: • pigmentations • retinal detachment Courtesy: C.E. van Nouhuys Brown et al. Ophthalmology. 101:694-704, 1994. Human Genetics Nijmegen

  6. XRCC4 CTRL1 CSPG2 EDIL3 Wagner disease/ERVR linkage interval Chromosome 5q13-q14 D5S650 D5S626 5’CTRL1 D5S107 D5S2103 D5S618 81,5 83,0 83,6 Mbp 89,0 90,0 72,0 Brown et al. ‘95 Zech et al. ‘99 Perveen et al. ‘99 Black et al. ‘99 G. Black and coworkers: sequence analysis of ORFs: no variants identified!! Human Genetics Nijmegen

  7. 310 135 134 254 310 135 134 254 310 135 134 254 310 135 134 154 ? ? Wagner disease - linkage W04-153 D5S1347 286 310 D5S2094 129 135 D5S107 154 134 D5S428 246 254 306 129 152 244 306 135 144 248 306 135 148 244 314 131 150 244 306 135 144 248 306 135 148 244 310 135 148 250 310 135 148 250 310 131 154 252 310 131 154 252 310 131 154 252 310 131 154 252 Human Genetics Nijmegen

  8. 310 135 134 254 310 135 134 254 Wagner Disease W04-153 310 135 134 254 310 135 134 254 310 135 134 254 310 135 134 254 310 135 134 254 310 135 134 254 ? ? ? Erosive vitreoretinopathy - linkage W95-124 D5S1347 310 310 D5S2094 135 135 D5S107 148 134 D5S428 248 254 286 129 144 244 310 129 148 254 314 135 150 250 310 135 148 248 302 135 156 250 310 135 148 248 286 129 144 244 286 129 144 244 286 129 148 254 290 129 144 254 290 129 144 254 290 129 144 254 298 135 ? 244 Human Genetics Nijmegen

  9. Wagner disease: founder haplotype in the NL W95-023 W95-131 W04-153 W05-088 W95-038 Mbp D5S424 122 ? ? ? 130 136 v 2.3 D5S1501 96 110 ? 110 106 96 v 2.9 D5S2029 149 149 149 149 137 129 v 0.2 D5S626 ? ? ? ? 1 ? v 0.4 D5S641 267 267 267 v 267 267 269 0.3 D5S1347 310 310 310 310 310 v 302 0.1 D5S2094 135 135 135 135 135 135 v 0.5 5’CRTL1 230 230 230 230 230 224 v 0.6 D5S107 148 134 134 150 134 152 v 1.8 D5S428 254 254 ? 244 254 252 v 0.7 D5S617 196 188 ? 188 188 196 v 2.3 D5S1722 227 ? ? ? 231 223 v 1.8 D5S1463 169 ? ? ? 193 177 Human Genetics Nijmegen

  10. Wagner disease & ERVR: samefounder haplotype ERVR W95-023 W95-131 W04-153 W05-088 W95-124 W95-038 Mbp D5S424 122 ? ? 122 ? 130 136 v 2.3 D5S1501 96 110 ? 96 110 106 96 v 2.9 D5S2029 149 149 149 149 149 137 129 v 0.2 D5S626 ? ? ? ? ? 1 ? v 0.4 D5S641 267 267 267 267 v 267 267 269 0.3 D5S1347 310 310 310 310 310 310 v 302 0.1 D5S2094 135 135 135 135 135 135 135 v 0.5 5’CRTL1 230 230 230 230 230 230 224 v 0.6 D5S107 148 134 134 134 150 134 152 v 1.8 D5S428 254 254 ? 254 244 254 252 v 0.7 D5S617 196 188 ? 188 188 188 196 v 2.3 D5S1722 227 ? ? 231 ? 231 223 v 1.8 D5S1463 169 ? ? 181 ? 193 177 Human Genetics Nijmegen

  11. Nijmegen Wagner disease & ERVR: common ancestor W95-131 W05-088 W95-023 W04-153 W95-124 (ERVR) Human Genetics Nijmegen

  12. Wagner disease/ERVR linkage interval Chromosome 5q13-q14 D5S650 D5S626 5’CTRL1 D5S107 D5S2103 D5S618 81,5 83,0 83,6 Mbp 89,0 90,0 72,0 XRCC4 CTRL1 CSPG2 EDIL3 Human Genetics Nijmegen

  13. Miyamoto et al. IOVS, 46:2726, 2005 CSPG2/Versican 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ATG TGA c.4004-2A>G AG GT 8 15 1 Wild type 7 GG GT 8 15 1 7 Mutant Human Genetics Nijmegen

  14. Miyamoto et al. IOVS, 46:2726, 2005 CSPG2/Versican 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ATG TGA c.4004-2A>G AG GT 8 15 1 Wild type 7 GT GG AG 8 15 1 7 Mutant In-frame 39 nt deletion Human Genetics Nijmegen

  15. CSPG2/Versican splice variants V0 1 6 7 8 9 15 V1 1 6 7 8 9 15 V2 1 6 7 8 9 15 V3 1 6 7 8 9 15 Human Genetics Nijmegen

  16. CSPG2/Versican 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 α β Hyaluronan binding region Glycosaminoglycan (GAG) attachment domains EGF domains Lectin-C binding domain Sushi domain • 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. Human Genetics Nijmegen

  17. CSPG2/Versican: intron 7 variants 7 8 -2 aaaactctgtttttttcagGTCGAATGAGTG Miyamoto et al. IOVS, 46:2726, 2005 Human Genetics Nijmegen

  18. Binding Capacity: 64% 14% 0% CSPG2/Versican: intron 7 variants 7 8 aaaactctgtttttttcagGTCGAATGAGTG -5 -1 c.4004-5T>C c.4004-5T>A c.4004-1G>A Human Genetics Nijmegen

  19. CSPG2/Versican c.4004-5T>Csegregates with Wagner disease Family W95-131 T/C T/T T/C T/C T/T T/T T/T T/C T/C T/C T/T T/C T/C T/T T/C T/C T/T T/C T/C Human Genetics Nijmegen

  20. c.4004-5T>A c.4004-5T>C CSPG2 C C C C C C A Wagner disease & ERVR: same founder variant ERVR W95-038 W95-023 W95-131 W04-153 W05-088 W95-124 136 D5S424 122 ? ? 122 ? 130 96 D5S1501 96 110 ? 96 110 106 129 D5S2029 149 149 149 149 149 137 ? D5S626 ? ? ? ? ? 1 269 D5S641 267 267 267 267 267 267 302 D5S1347 310 310 310 310 310 310 135 D5S2094 135 135 135 135 135 135 224 5’CRTL1 230 230 230 230 230 230 152 D5S107 148 134 134 134 150 134 252 D5S428 254 254 ? 254 244 254 196 D5S617 196 188 ? 188 188 188 223 D5S1722 227 ? ? 231 ? 231 177 D5S1463 169 ? ? 181 ? 193 Human Genetics Nijmegen

  21. CSPG2/Versican RT-PCR analysis Δ39 bp  • 2 out of 3 known CSPG2/Versican mutations activate a cryptic splice site in exon 8 Human Genetics Nijmegen

  22. 39 nt/13 aa deletion: no effect on GAG binding V0 1 6 7 8 9 15 V1 1 6 7 8 9 15 V2 1 6 7 8 9 15 V3 1 6 7 8 9 15  Are their quantitative effects? Human Genetics Nijmegen

  23. Relative quantitation: Q PCR Reference gene: GUSB Gene of interest: CSPG2 splice variants Ct1 Ct2 threshold cDNA amount ΔCt = Ct1 - Ct2 20 30 PCR cycles Human Genetics Nijmegen

  24. Controls (8) 1.0 1.0 1.0 1.0 Relative quantities of CSPG2/Versican mRNA splice variants in patients vs controls CSPG2/Versican splice variant Sequence variant Individual V0 V1 V2 V3 W95-038P1 c.4004-5T>A 0.62 0.54 52.0* 13.9* W95-131P1 c.4004-5T>C 0.81 0.66 45.3* 12.1* W95-124P1 c.4004-5T>C 1.02 0.64 38.3* 12.8* W95-137P1 c.4004-1G>A 0.81 0.66 137.2* 22.6* W05-282P1 Unknown 0.87 0.66 477.7* 52.0* W95-124P1: erosive vitreoretinopathy *p<0.0001 Human Genetics Nijmegen

  25. CSPG2/Versican isoforms in eye Normal mutant / + + / + mutant / + 100 50 V1 V2 V3 V0 V1 V2 V3 V0 V0 V1 V2 V3 Adapted from: Zao & Russell, Mol. Vis.11:603 - 608, 2005. Human Genetics Nijmegen

  26. CSPG2/Versican isoforms in eye Wagner disease Normal + / + c.4004-1G>A / + 100 50 V0 V1 V2 V3 V0 V1 V2 V3 Human Genetics Nijmegen

  27. CSPG2/Versican isoforms in eye Wagner disease Normal + / + c.4004-1G>A / + 100 50 V0 V1 V2 V3 V0 V1 V2 V3 Total CSPG2 molecules not changed! Human Genetics Nijmegen

  28. CSPG2/Versican splice variants V0 1 6 7 8 9 15 V1 1 6 7 8 9 15 V2 1 6 7 8 9 15 V3 1 6 7 8 9 15 : glycosaminoglycan (GAG) attachment sites Human Genetics Nijmegen

  29. GAG-attachment sites: 100% 63% CSPG2-GAG attachment sites Wagner disease Normal + / + c.4004-1G>A / + 100 50 V0 V1 V2 V3 V0 V1 V2 V3 Human Genetics Nijmegen

  30. Wagner disease mechanism? • Total amount CSPG2 unaltered • Reduction in GAG attachment sites • Cause: V0/V1 or V2/V3 ? Human Genetics Nijmegen

  31. Wagner disease families with no V2/V3 upregulation! CSPG2/Versican splice variant Sequence variant Patient V0 V1 V2 V3 W95-038P1 c.4004-5T>A 0.62 0.54 52.0 13.9 W95-131P1 c.4004-5T>C 0.81 0.66 45.3 12.1 W95-124P1 c.4004-5T>C 1.02 0.64 38.3 12.8 W95-137P1 c.4004-1G>A 0.81 0.66 137.2 22.6 W05-282P1 Unknown 0.87 0.66 477.7 52.0 W95-061P1 Unknown 1.11 0.73 0.95 0.84 W95-152P1 Unknown 0.75 0.69 0.44 0.66 W05-542P1 Unknown 0.76 0.68 0.32 0.53 Human Genetics Nijmegen

  32. Wagner disease families with noV2/V3 upregulation link to 5q13-q14 W95-061 W95-152 178 230 188 230 D5S2029 176 166 D5S641 234 230 D5S1347 334 338 D5S107 197 201 314 193 338 197 334 197 338 201 176 234 178 230 166 230 178 230 166 230 178 230 166 230 188 230 176 234 188 230 ? 338 197 334 197 314 193 334 197 338 199 318 197 W95-542 D5S1347 342 334 D5S107 199 205 334 197 338 197 ? 318 197 334 197 338 199 334 197 314 193 338 199 334 205 178 230 338 197 342 199 334 197 342 199 Human Genetics Nijmegen

  33. c.4004-5T>C, -5T>A, -1G>A Dutch c.4004-2A>G Japanese c.9265+1G>A Swiss* Observations & discussion 5.3 kb 14.7 kb 3.3 kb 7 8 9 Observations: • Only intronic mutations affecting splicing of exon 8 found in Wagner disease and ERVR • Exonic stopmutations are not found in 3396-aa ORF 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. *Kloeckener-Gruissem et al. Mol. Vis.12:250-255, 2006 Human Genetics Nijmegen

  34. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 8 CAGgtaagatc ctctgtttttttcagGTC Wagner disease/ERVR: an amazing quest Chromosome 5q13-q14 D5S626 D5S107 81,5 XRCC4 CTRL1 83,6 CSPG2 EDIL3 Human Genetics Nijmegen

  35. Acknowledgements N HUMA GE N ETICS GE N NIJME Dept. of Human Genetics Nijmegen, NL Canisius Wilhelmina Hospital, Nijmegen, NL Erik van Nouhuys Arijit Mukhopadhyay Konstantinos Nikopoulos Alessandra Maugeri Arjan de Brouwer Saskia van der Velde-Visser Marloes Wennemers Han Brunner Bartimeus Zeist, Zeist, NL Pieter van den Biesen Nienke Boonstra Manchester, UK Rahat Perveen Graeme Black Dept. of Ophthalmology Nijmegen, NL Camiel Boon Carel Hoyng Tuebingen, Germany Bernd Wissinger

  36. QUESTIONS

  37. CSPG2/Versican isoforms in eye Exon 8 stop Normal + / + mutation/ + 100 50 V0 V1 V2 V3 V0 V1 V2 V3 Total CSPG2 molecules: 100% 57% Human Genetics Nijmegen

  38. GAG-attachment sites: 100% 51% CSPG2-GAG attachment sites Exon 8 stop Normal + / + mutation/ + 100 50 V0 V1 V2 V3 V0 V1 V2 V3 Human Genetics Nijmegen

  39. CSPG2/Versican isoforms in eye Intron 6 splice Normal + / + mutation/ + 100 50 V0 V1 V2 V3 V0 V1 V2 V3 Total CSPG2 molecules unchanged Human Genetics Nijmegen

  40. GAG-attachment sites: 100% 95% CSPG2-GAG attachment sites Intron 6 splice Normal + / + mutation/ + 100 50 V0 V1 V2 V3 V0 V1 V2 V3 Human Genetics Nijmegen

  41. Intron 7 or 8 splice site mutations α β V0 1 6 7 8 9 15 V1 1 6 7 8 9 15 V2 1 6 7 8 9 15 V3 1 6 7 8 9 15 Predicted effect of total CSPG2total GAGs intron7/8mutations:100%63% (50% + 13%) Human Genetics Nijmegen

  42. Exon 8 stopmutations α β V0 1 6 7 8 9 15 V1 1 6 7 8 9 15 V2 1 6 7 8 9 15 V3 1 6 7 8 9 15 Predicted effect of total CSPG2total GAGs exon 8 stopmutations:57%51% (50% + 1%) Human Genetics Nijmegen

  43. Exon 8 stopmutations α β V0 1 6 7 8 9 15 V1 1 6 7 8 9 15 V2 1 6 7 8 9 15 V3 1 6 7 8 9 15 Predicted effect of total CSPG2total GAGs - exon 8 stopmutations:57%51% - intron7/8mutations:100%60% Human Genetics Nijmegen

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