8ab3ad4a839c5702009caf3afa5278f2 275207.pdf 13621f85310573d989cfd26cb0daa2be34d2061c 275207.pdf b4684ae651e7552d9c2634d40ba420de7ed900fb3cfb2e48d9a2d4f112dccf4c 275207.pdf Title: Deep Molecular Characterization of Milder Spinal Muscular Atrophy Patients Carrying the c.859G>C Variant in SMN2 Subject: Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by biallelic loss or pathogenic variants in the SMN1 gene. Copy number and modifier intragenic variants in SMN2, an almost identical paralog gene of SMN1, are known to influence the amount of complete SMN proteins. Therefore, SMN2 is considered the main phenotypic modifier of SMA, although genotype–phenotype correlation is not absolute. We present eleven unrelated SMA patients with milder phenotypes carrying the c.859G>C-positive modifier variant in SMN2. All were studied by a specific NGS method to allow a deep characterization of the entire SMN region. Analysis of two homozygous cases for the variant allowed us to identify a specific haplotype, Smn2-859C.1, in association with c.859G>C. Two other cases with the c.859G>C variant in their two SMN2 copies showed a second haplotype, Smn2-859C.2, in cis with Smn2-859C.1, assembling a more complex allele. We also identified a previously unreported variant in intron 2a exclusively linked to the Smn2-859C.1 haplotype (c.154-1141G>A), further suggesting that this region has been ancestrally conserved. The deep molecular characterization of SMN2 in our cohort highlights the importance of testing c.859G>C, as well as accurately assessing the SMN2 region in SMA patients to gain insight into the complex genotype–phenotype correlations and improve prognostic outcomes. Keywords: spinal muscular atrophy; SMN2 copies; phenotype–genotype correlations; positive modifiers; next-generation sequencing Author: Laura Blasco-Pérez, Mar Costa-Roger, Jordi Leno-Colorado, Sara Bernal, Laura Alias, Marta Codina-Solà, Desirée Martínez-Cruz, Claudia Castiglioni, Enrico Bertini, Lorena Travaglini, José M. Millán, Elena Aller, Javier Sotoca, Raúl Juntas, Christina Engel Hoei-Hansen, Antonio Moreno-Escribano, Encarna Guillén-Navarro, Laura Costa-Comellas, Francina Munell, Susana Boronat, Ricardo Rojas-García, Mónica Povedano, Ivon Cuscó and Eduardo F. Tizzano Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Aug 1 12:29:54 2022 CEST ModDate: Mon Aug 1 12:33:15 2022 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1307167 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- YLPNQQ+VnURWPalladioL Type 1 Custom yes yes no 10 0 DVIXYK+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 NTAUGU+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 ODZUED+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 BTDXFZ+URWPalladioL-BoldItal Type 1 Custom yes yes no 28 0 UQZHSH+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 50 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 53 0 MPUDFL+PazoMath Type 1 Builtin yes yes no 75 0 AOPHAZ+CMSY10 Type 1 Builtin yes yes no 85 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 100 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 104 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 108 0 BCDIEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 112 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 128 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 132 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 136 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 152 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 156 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 160 0 BCDIEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 164 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-07-04 02:31:29 CEST RepresentationInformation: 275207.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-07-03 14:14:00 CEST Size: 1307167 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 364 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Clinical and Molecular Characterization of Patients Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Haplotype Characterization by Deep Sequencing of SMN2 Genes Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Children: Item: Title: Establishment of Two Haplotypes Associated with the c.859G>C Modifier Variant Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Difference between Haplotypes and Detection of a Novel Variant Exclusively Associated with the Smn2-859C.1 Haplotype Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Children: Item: Title: Study Participants Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: SMN2 Genotyping and Haplotype Characterization Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Info: Title: Deep Molecular Characterization of Milder Spinal Muscular Atrophy Patients Carrying the c.859G>C Variant in SMN2 Author: Laura Blasco-Pérez, Mar Costa-Roger, Jordi Leno-Colorado, Sara Bernal, Laura Alias, Marta Codina-Solà, Desirée Martínez-Cruz, Claudia Castiglioni, Enrico Bertini, Lorena Travaglini, José M. Millán, Elena Aller, Javier Sotoca, Raúl Juntas, Christina Engel Hoei-Hansen, Antonio Moreno-Escribano, Encarna Guillén-Navarro, Laura Costa-Comellas, Francina Munell, Susana Boronat, Ricardo Rojas-García, Mónica Povedano, Ivon Cuscó and Eduardo F. Tizzano Subject: Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by biallelic loss or pathogenic variants in the SMN1 gene. Copy number and modifier intragenic variants in SMN2, an almost identical paralog gene of SMN1, are known to influence the amount of complete SMN proteins. Therefore, SMN2 is considered the main phenotypic modifier of SMA, although genotype phenotype correlation is not absolute. We present eleven unrelated SMA patients with milder phenotypes carrying the c.859G>C-positive modifier variant in SMN2. All were studied by a specific NGS method to allow a deep characterization of the entire SMN region. Analysis of two homozygous cases for the variant allowed us to identify a specific haplotype, Smn2-859C.1, in association with c.859G>C. Two other cases with the c.859G>C variant in their two SMN2 copies showed a second haplotype, Smn2-859C.2, in cis with Smn2-859C.1, assembling a more complex allele. We also identified a previously unreported variant in intron 2a exclusively linked to the Smn2-859C.1 haplotype (c.154-1141G>A), further suggesting that this region has been ancestrally conserved. The deep molecular characterization of SMN2 in our cohort highlights the importance of testing c.859G>C, as well as accurately assessing the SMN2 region in SMA patients to gain insight into the complex genotype phenotype correlations and improve prognostic outcomes. Keywords: spinal muscular atrophy; SMN2 copies; phenotype genotype correlations; positive modifiers; next-generation sequencing Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Aug 01 12:29:54 CEST 2022 ModDate: Mon Aug 01 12:33:15 CEST 2022 ID: 0x16f0191445b6024677f0ef38bd51bbca, 0x16f0191445b6024677f0ef38bd51bbca Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: UQZHSH+VnURWPalladioL-Bold FontSubset: true FirstChar: 160 LastChar: 243 FontDescriptor: FontName: UQZHSH+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: NTAUGU+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: NTAUGU+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: AOPHAZ+CMSY10 FontSubset: true FirstChar: 48 LastChar: 48 FontDescriptor: FontName: AOPHAZ+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: ODZUED+URWPalladioL-Ital FontSubset: true FirstChar: 40 LastChar: 122 FontDescriptor: FontName: ODZUED+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: YLPNQQ+VnURWPalladioL FontSubset: true FirstChar: 129 LastChar: 226 FontDescriptor: FontName: YLPNQQ+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: MPUDFL+PazoMath FontSubset: true FirstChar: 68 LastChar: 68 FontDescriptor: FontName: MPUDFL+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true Font: BaseFont: BTDXFZ+URWPalladioL-BoldItal FontSubset: true FirstChar: 49 LastChar: 83 FontDescriptor: FontName: BTDXFZ+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: DVIXYK+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 122 FontDescriptor: FontName: DVIXYK+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 34, 323, 152, 332 Annotation: Subtype: Link Rect: 34, 311, 77, 320 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 126, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 147, 87 Annotation: Subtype: Link Rect: 34, 756, 180, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 134, 661, 145, 674 Annotation: Subtype: Link Rect: 238, 661, 250, 674 Annotation: Subtype: Link Rect: 430, 661, 441, 674 Annotation: Subtype: Link Rect: 134, 648, 145, 662 Annotation: Subtype: Link Rect: 366, 648, 377, 662 Annotation: Subtype: Link Rect: 115, 635, 126, 649 Annotation: Subtype: Link Rect: 264, 635, 275, 649 Annotation: Subtype: Link Rect: 306, 624, 317, 637 Annotation: Subtype: Link Rect: 408, 610, 419, 624 Annotation: Subtype: Link Rect: 139, 17, 278, 28 Annotation: Subtype: Link Rect: 422, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 444, 87, 463 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 532, 569, 539, 578 ActionDest: 175 Annotation: Subtype: Link Rect: 539, 569, 546, 578 ActionDest: 175 Annotation: Subtype: Link Rect: 200, 418, 207, 427 ActionDest: 175 Annotation: Subtype: Link Rect: 210, 418, 217, 427 ActionDest: 175 Annotation: Subtype: Link Rect: 249, 393, 256, 402 ActionDest: 175 Annotation: Subtype: Link Rect: 256, 393, 263, 402 ActionDest: 175 Annotation: Subtype: Link Rect: 438, 343, 445, 352 ActionDest: 175 Annotation: Subtype: Link Rect: 448, 343, 460, 352 ActionDest: 175 Annotation: Subtype: Link Rect: 403, 318, 415, 327 ActionDest: 175 Annotation: Subtype: Link Rect: 505, 280, 517, 289 ActionDest: 175 Annotation: Subtype: Link Rect: 517, 280, 529, 289 ActionDest: 175 Annotation: Subtype: Link Rect: 230, 230, 242, 239 ActionDest: 179 Annotation: Subtype: Link Rect: 284, 179, 296, 189 ActionDest: 175 Annotation: Subtype: Link Rect: 513, 167, 525, 176 ActionDest: 179 Annotation: Subtype: Link Rect: 208, 142, 220, 151 ActionDest: 179 Annotation: Subtype: Link Rect: 223, 142, 235, 151 ActionDest: 179 Annotation: Subtype: Link Rect: 495, 92, 507, 101 ActionDest: 179 Annotation: Subtype: Link Rect: 510, 92, 522, 101 ActionDest: 179 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 334, 735, 346, 744 ActionDest: 179 Annotation: Subtype: Link Rect: 347, 736, 359, 744 ActionDest: 179 Annotation: Subtype: Link Rect: 361, 685, 373, 694 ActionDest: 179 Annotation: Subtype: Link Rect: 376, 685, 388, 694 ActionDest: 179 Annotation: Subtype: Link Rect: 522, 623, 534, 631 ActionDest: 175 Annotation: Subtype: Link Rect: 255, 597, 267, 606 ActionDest: 179 Annotation: Subtype: Link Rect: 552, 393, 559, 405 ActionDest: 79 Annotation: Subtype: Link Rect: 404, 355, 411, 367 ActionDest: 79 Annotation: Subtype: Link Rect: 103, 205, 112, 212 ActionDest: 179 Annotation: Subtype: Link Rect: 147, 205, 156, 212 ActionDest: 179 Annotation: Subtype: Link Rect: 358, 206, 367, 212 ActionDest: 175 Annotation: Subtype: Link Rect: 401, 205, 410, 212 ActionDest: 179 Annotation: Subtype: Link Rect: 532, 205, 541, 212 ActionDest: 179 Annotation: Subtype: Link Rect: 510, 68, 516, 78 ActionDest: 92 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 191, 579, 199, 591 ActionDest: 89 Annotation: Subtype: Link Rect: 194, 530, 201, 541 ActionDest: 89 Annotation: Subtype: Link Rect: 228, 428, 235, 440 ActionDest: 89 Annotation: Subtype: Link Rect: 299, 340, 306, 352 ActionDest: 89 Annotation: Subtype: Link Rect: 486, 315, 493, 327 ActionDest: 89 Annotation: Subtype: Link Rect: 428, 265, 435, 278 ActionDest: 92 Annotation: Subtype: Link Rect: 367, 215, 374, 227 ActionDest: 92 Annotation: Subtype: Link Rect: 319, 190, 327, 202 ActionDest: 92 Annotation: Subtype: Link Rect: 351, 152, 358, 164 ActionDest: 92 Annotation: Subtype: Link Rect: 243, 127, 250, 139 ActionDest: 92 Page: Sequence: 5 Page: Sequence: 6 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 350, 722, 357, 734 ActionDest: 89 Annotation: Subtype: Link Rect: 300, 675, 312, 684 ActionDest: 179 Annotation: Subtype: Link Rect: 315, 675, 327, 684 ActionDest: 179 Annotation: Subtype: Link Rect: 263, 662, 275, 671 ActionDest: 179 Annotation: Subtype: Link Rect: 203, 649, 215, 659 ActionDest: 179 Annotation: Subtype: Link Rect: 347, 499, 359, 508 ActionDest: 175 Annotation: Subtype: Link Rect: 402, 433, 409, 445 ActionDest: 120 Annotation: Subtype: Link Rect: 421, 125, 428, 137 ActionDest: 92 Annotation: Subtype: Link Rect: 311, 81, 323, 90 ActionDest: 179 Annotation: Subtype: Link Rect: 323, 81, 335, 90 ActionDest: 179 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 202, 737, 214, 746 ActionDest: 179 Annotation: Subtype: Link Rect: 214, 687, 226, 696 ActionDest: 179 Annotation: Subtype: Link Rect: 229, 687, 241, 696 ActionDest: 179 Annotation: Subtype: Link Rect: 241, 687, 253, 696 ActionDest: 179 Annotation: Subtype: Link Rect: 254, 687, 266, 696 ActionDest: 179 Annotation: Subtype: Link Rect: 269, 687, 281, 696 ActionDest: 179 Annotation: Subtype: Link Rect: 498, 675, 510, 684 ActionDest: 179 Annotation: Subtype: Link Rect: 496, 612, 508, 621 ActionDest: 179 Annotation: Subtype: Link Rect: 338, 574, 350, 583 ActionDest: 179 Annotation: Subtype: Link Rect: 405, 524, 417, 533 ActionDest: 179 Annotation: Subtype: Link Rect: 549, 509, 556, 521 ActionDest: 79 Annotation: Subtype: Link Rect: 351, 474, 363, 483 ActionDest: 179 Annotation: Subtype: Link Rect: 401, 471, 408, 483 ActionDest: 120 Annotation: Subtype: Link Rect: 266, 311, 278, 320 ActionDest: 179 Annotation: Subtype: Link Rect: 273, 145, 280, 158 ActionDest: 92 Annotation: Subtype: Link Rect: 483, 132, 490, 144 ActionDest: 89 Annotation: Subtype: Link Rect: 542, 132, 549, 144 ActionDest: 144 Annotation: Subtype: Link Rect: 462, 97, 474, 106 ActionDest: 179 Annotation: Subtype: Link Rect: 319, 82, 326, 94 ActionDest: 144 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 367, 697, 374, 709 ActionDest: 144 Annotation: Subtype: Link Rect: 429, 234, 435, 247 ActionDest: 89 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 375, 611, 387, 620 ActionDest: 179 Annotation: Subtype: Link Rect: 194, 584, 201, 595 ActionDest: 79 Annotation: Subtype: Link Rect: 302, 561, 313, 570 ActionDest: 175 Annotation: Subtype: Link Rect: 545, 548, 556, 557 ActionDest: 179 Annotation: Subtype: Link Rect: 228, 535, 240, 544 ActionDest: 175 Annotation: Subtype: Link Rect: 417, 422, 429, 431 ActionDest: 175 Annotation: Subtype: Link Rect: 430, 422, 442, 431 ActionDest: 179 Annotation: Subtype: Link Rect: 442, 422, 454, 431 ActionDest: 182 Annotation: Subtype: Link Rect: 205, 397, 217, 406 ActionDest: 175 Annotation: Subtype: Link Rect: 295, 359, 307, 368 ActionDest: 182 Annotation: Subtype: Link Rect: 206, 171, 218, 180 ActionDest: 179 Annotation: Subtype: Link Rect: 450, 171, 462, 180 ActionDest: 179 Annotation: Subtype: Link Rect: 536, 82, 560, 93 Annotation: Subtype: Link Rect: 165, 70, 377, 81 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 332, 414, 370, 425 Annotation: Subtype: Link Rect: 376, 414, 412, 425 Annotation: Subtype: Link Rect: 260, 380, 298, 391 Annotation: Subtype: Link Rect: 304, 380, 340, 391 Annotation: Subtype: Link Rect: 407, 357, 444, 368 Annotation: Subtype: Link Rect: 450, 357, 486, 368 Annotation: Subtype: Link Rect: 104, 335, 141, 345 Annotation: Subtype: Link Rect: 519, 311, 557, 322 Annotation: Subtype: Link Rect: 59, 300, 95, 310 Annotation: Subtype: Link Rect: 108, 277, 146, 287 Annotation: Subtype: Link Rect: 152, 277, 188, 287 Annotation: Subtype: Link Rect: 352, 253, 389, 264 Annotation: Subtype: Link Rect: 395, 253, 431, 264 Annotation: Subtype: Link Rect: 500, 230, 537, 241 Annotation: Subtype: Link Rect: 404, 173, 441, 184 Annotation: Subtype: Link Rect: 324, 150, 362, 161 Annotation: Subtype: Link Rect: 59, 116, 96, 126 Annotation: Subtype: Link Rect: 102, 116, 138, 126 Annotation: Subtype: Link Rect: 104, 82, 141, 92 Annotation: Subtype: Link Rect: 147, 82, 183, 92 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 211, 724, 248, 735 Annotation: Subtype: Link Rect: 254, 724, 290, 735 Annotation: Subtype: Link Rect: 519, 701, 557, 712 Annotation: Subtype: Link Rect: 59, 690, 95, 700 Annotation: Subtype: Link Rect: 469, 666, 507, 677 Annotation: Subtype: Link Rect: 513, 666, 549, 677 Annotation: Subtype: Link Rect: 152, 632, 190, 642 Annotation: Subtype: Link Rect: 196, 632, 232, 642 Annotation: Subtype: Link Rect: 347, 608, 385, 619 Annotation: Subtype: Link Rect: 357, 574, 395, 585 Annotation: Subtype: Link Rect: 378, 551, 415, 562 Annotation: Subtype: Link Rect: 357, 516, 394, 527 Annotation: Subtype: Link Rect: 393, 493, 430, 504 Annotation: Subtype: Link Rect: 436, 493, 472, 504 Annotation: Subtype: Link Rect: 268, 460, 305, 470 Annotation: Subtype: Link Rect: 311, 460, 347, 470 Annotation: Subtype: Link Rect: 285, 436, 323, 447 Annotation: Subtype: Link Rect: 329, 436, 365, 447 Annotation: Subtype: Link Rect: 393, 401, 431, 412 Annotation: Subtype: Link Rect: 437, 401, 473, 412 Annotation: Subtype: Link Rect: 59, 368, 96, 378 Annotation: Subtype: Link Rect: 140, 345, 177, 355 Annotation: Subtype: Link Rect: 381, 321, 561, 332 Annotation: Subtype: Link Rect: 494, 286, 531, 297 Annotation: Subtype: Link Rect: 291, 263, 329, 274 Annotation: Subtype: Link Rect: 59, 230, 96, 240 Annotation: Subtype: Link Rect: 125, 195, 162, 205 Annotation: Subtype: Link Rect: 278, 161, 315, 171 Annotation: Subtype: Link Rect: 321, 161, 357, 171 Annotation: Subtype: Link Rect: 122, 126, 159, 136 Annotation: Subtype: Link Rect: 165, 126, 201, 136 Annotation: Subtype: Link Rect: 519, 102, 557, 113 Annotation: Subtype: Link Rect: 59, 92, 95, 102 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 108, 701, 146, 712 Annotation: Subtype: Link Rect: 152, 701, 188, 712 Annotation: Subtype: Link Rect: 370, 678, 408, 689 Checksum: 77d22aba Type: CRC32 Checksum: 8ab3ad4a839c5702009caf3afa5278f2 Type: MD5 Checksum: 13621f85310573d989cfd26cb0daa2be34d2061c Type: SHA-1