d11dab8c2b722912d4dd81a30bd50894 282328.pdf 82a048f1df09d901ef13456474cb16be1180c8dc 282328.pdf 0b43145042478b8cac8ef25c90803d5175702efef38a59c83cb1091c379ac03c 282328.pdf Title: An Integrative Analysis of DNA Methylation Pattern in Myotonic Dystrophy Type 1 Samples Reveals a Distinct DNA Methylation Profile between Tissues and a Novel Muscle-Associated Epigenetic Dysregulation Subject: Myotonic dystrophy type 1 (DM1) is a progressive, non-treatable, multi-systemic disorder. To investigate the contribution of epigenetics to the complexity of DM1, we compared DNA methylation profiles of four annotated CpG islands (CpGis) in the DMPK locus and neighbouring genes, in distinct DM1 tissues and derived cells, representing six DM1 subtypes, by bisulphite sequencing. In blood, we found no differences in CpGi 74, 43 and 36 in DNA methylation profile. In contrast, a CTCF1 DNA methylation gradient was found with 100% methylation in congenital cases, 50% in childhood cases and 13% in juvenile cases. CTCF1 methylation correlated to disease severity and CTG expansion size. Notably, 50% of CTCF1 methylated cases showed methylation in the CTCF2 regions. Additionally, methylation was associated with maternal transmission. Interestingly, the evaluation of seven families showed that unmethylated mothers passed on an expansion of the CTG repeat, whereas the methylated mothers transmitted a contraction. The analysis of patient-derived cells showed that DNA methylation profiles were highly preserved, validating their use as faithful DM1 cellular models. Importantly, the comparison of DNA methylation levels of distinct DM1 tissues revealed a novel muscle-specific epigenetic signature with methylation of the CTCF1 region accompanied by demethylation of CpGi 43, a region containing an alternative DMPK promoter, which may decrease the canonical promoter activity. Altogether, our results showed a distinct DNA methylation profile across DM1 tissues and uncovered a novel and dual epigenetic signature in DM1 muscle samples, providing novel insights into the epigenetic changes associated with DM1. Keywords: myotonic dystrophy; CpG islands; DNA methylation; epigenetics; phenotype severity; DM1 biopsies; cellular models Author: Emma Koehorst, Renato Odria, Júlia Capó, Judit Núñez-Manchón, Andrea Arbex, Miriam Almendrote, Ian Linares-Pardo, Daniel Natera-de Benito, Verónica Saez, Andrés Nascimento, Carlos Ortez, Miguel Ángel Rubio, Jordi Díaz-Manera, Jorge Alonso-Pérez, Giuseppe Lucente, Agustín Rodriguez-Palmero, Alba Ramos-Fransi, Alicia Martínez-Piñeiro, Gisela Nogales-Gadea and Mònica Suelves Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Jun 10 11:14:58 2022 CEST ModDate: Fri Jun 10 11:19:56 2022 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 21 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3186246 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- OOYTGH+VnURWPalladioL Type 1 Custom yes yes no 10 0 TDQWTO+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 DJDCSE+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 BLXYCR+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 XWMKTY+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 EBFBTV+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 46 0 BBRGFZ+CMSY10 Type 1 Builtin yes yes no 71 0 URYLRZ+URWPalladioL-BoldItal Type 1 Custom yes yes no 84 0 KGFFBP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 97 0 KGFFCB+PalatinoLinotype TrueType WinAnsi yes yes no 100 0 KGFFCD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 103 0 KGFFBP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 120 0 KGFFCB+PalatinoLinotype TrueType WinAnsi yes yes no 123 0 KGFFCD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 126 0 KGFFBP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 139 0 KGFFCB+PalatinoLinotype TrueType WinAnsi yes yes no 142 0 KGFFCD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 145 0 KGFFBP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 156 0 KGFFCB+PalatinoLinotype TrueType WinAnsi yes yes no 159 0 KGFFCD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 162 0 KGFFBP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 173 0 KGFFCB+PalatinoLinotype TrueType WinAnsi yes yes no 176 0 KGFFCD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 179 0 KGFFBP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 189 0 KGFFCB+PalatinoLinotype TrueType WinAnsi yes yes no 192 0 KGFFCD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 195 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-10-17 02:22:25 CEST RepresentationInformation: 282328.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-10-16 12:31:58 CEST Size: 3186246 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 417 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Children: Item: Title: Patient Registry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Tissue and Cell Culture Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: DNA Isolation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: CTG Expansion Size Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Bisulphite Treatment and Sanger Sequencing of Four CpG Islands Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Children: Item: Title: A Study Cohort Encompassing All Clinical Subtypes of DM1 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: DNA Methylation Profiles across the DMPK Locus in Blood Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Aberrant DNA Methylation Profiles of CTCF1 Associated with Higher Disease Severity in Childhood Cases Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: A Higher Chance of Methylation in CTCF1 with Increasing CTG Expansion Size Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Increased, but Not Exclusive, Maternal Transmission in CTCF1 Methylated Cases Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Methylation Status Is Not Inheritable and Associated with the Transmission of CTG Repeat Contractions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: DNA Methylation Profiles Are Preserved in Blood-Derived Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: DM1 Is Associated with Hypomethylation of CpGi 43 in Muscle Tissue and Muscle-Derived Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: DM1 Is Associated with Hypermethylation in CTCF1 in Muscle Tissue and Muscle Derived Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Info: Title: An Integrative Analysis of DNA Methylation Pattern in Myotonic Dystrophy Type 1 Samples Reveals a Distinct DNA Methylation Profile between Tissues and a Novel Muscle-Associated Epigenetic Dysregulation Author: Emma Koehorst, Renato Odria, Júlia Capó, Judit Núñez-Manchón, Andrea Arbex, Miriam Almendrote, Ian Linares-Pardo, Daniel Natera-de Benito, Verónica Saez, Andrés Nascimento, Carlos Ortez, Miguel Ángel Rubio, Jordi Díaz-Manera, Jorge Alonso-Pérez, Giuseppe Lucente, Agustín Rodriguez-Palmero, Alba Ramos-Fransi, Alicia Martínez-Piñeiro, Gisela Nogales-Gadea and Mònica Suelves Subject: Myotonic dystrophy type 1 (DM1) is a progressive, non-treatable, multi-systemic disorder. To investigate the contribution of epigenetics to the complexity of DM1, we compared DNA methylation profiles of four annotated CpG islands (CpGis) in the DMPK locus and neighbouring genes, in distinct DM1 tissues and derived cells, representing six DM1 subtypes, by bisulphite sequencing. In blood, we found no differences in CpGi 74, 43 and 36 in DNA methylation profile. In contrast, a CTCF1 DNA methylation gradient was found with 100% methylation in congenital cases, 50% in childhood cases and 13% in juvenile cases. CTCF1 methylation correlated to disease severity and CTG expansion size. Notably, 50% of CTCF1 methylated cases showed methylation in the CTCF2 regions. Additionally, methylation was associated with maternal transmission. Interestingly, the evaluation of seven families showed that unmethylated mothers passed on an expansion of the CTG repeat, whereas the methylated mothers transmitted a contraction. The analysis of patient-derived cells showed that DNA methylation profiles were highly preserved, validating their use as faithful DM1 cellular models. Importantly, the comparison of DNA methylation levels of distinct DM1 tissues revealed a novel muscle-specific epigenetic signature with methylation of the CTCF1 region accompanied by demethylation of CpGi 43, a region containing an alternative DMPK promoter, which may decrease the canonical promoter activity. Altogether, our results showed a distinct DNA methylation profile across DM1 tissues and uncovered a novel and dual epigenetic signature in DM1 muscle samples, providing novel insights into the epigenetic changes associated with DM1. Keywords: myotonic dystrophy; CpG islands; DNA methylation; epigenetics; phenotype severity; DM1 biopsies; cellular models Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Jun 10 11:14:58 CEST 2022 ModDate: Fri Jun 10 11:19:56 CEST 2022 ID: 0xb1333dcbe77402b20443b2bbed66bc71, 0xb1333dcbe77402b20443b2bbed66bc71 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: DJDCSE+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: DJDCSE+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: URYLRZ+URWPalladioL-BoldItal FontSubset: true FirstChar: 110 LastChar: 110 FontDescriptor: FontName: URYLRZ+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: BBRGFZ+CMSY10 FontSubset: true FirstChar: 6 LastChar: 48 FontDescriptor: FontName: BBRGFZ+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: BLXYCR+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 122 FontDescriptor: FontName: BLXYCR+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: OOYTGH+VnURWPalladioL FontSubset: true FirstChar: 129 LastChar: 243 FontDescriptor: FontName: OOYTGH+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: XWMKTY+VnURWPalladioL-Bold FontSubset: true FirstChar: 129 LastChar: 243 FontDescriptor: FontName: XWMKTY+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: TDQWTO+URWPalladioL-Bold FontSubset: true FirstChar: 2 LastChar: 241 FontDescriptor: FontName: TDQWTO+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, 313, 105, 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, 197, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 246, 623, 257, 636 Annotation: Subtype: Link Rect: 355, 611, 366, 624 Annotation: Subtype: Link Rect: 406, 598, 417, 611 Annotation: Subtype: Link Rect: 517, 598, 528, 611 Annotation: Subtype: Link Rect: 175, 585, 186, 599 Annotation: Subtype: Link Rect: 536, 585, 547, 599 Annotation: Subtype: Link Rect: 130, 17, 302, 28 Annotation: Subtype: Link Rect: 389, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 480, 87, 499 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 549, 644, 556, 653 ActionDest: 208 Annotation: Subtype: Link Rect: 346, 594, 353, 603 ActionDest: 208 Annotation: Subtype: Link Rect: 430, 566, 437, 578 ActionDest: 65 Annotation: Subtype: Link Rect: 445, 569, 452, 578 ActionDest: 208 Annotation: Subtype: Link Rect: 455, 569, 467, 578 ActionDest: 208 Annotation: Subtype: Link Rect: 276, 141, 288, 150 ActionDest: 208 Annotation: Subtype: Link Rect: 437, 104, 449, 113 ActionDest: 208 Annotation: Subtype: Link Rect: 452, 104, 464, 113 ActionDest: 208 Annotation: Subtype: Link Rect: 402, 66, 414, 75 ActionDest: 208 Annotation: Subtype: Link Rect: 414, 66, 426, 75 ActionDest: 211 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 493, 723, 505, 732 ActionDest: 211 Annotation: Subtype: Link Rect: 449, 622, 461, 631 ActionDest: 211 Annotation: Subtype: Link Rect: 276, 597, 288, 606 ActionDest: 211 Annotation: Subtype: Link Rect: 291, 597, 303, 606 ActionDest: 211 Annotation: Subtype: Link Rect: 212, 535, 224, 544 ActionDest: 211 Annotation: Subtype: Link Rect: 413, 434, 425, 443 ActionDest: 211 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 411, 497, 423, 506 ActionDest: 211 Annotation: Subtype: Link Rect: 523, 484, 535, 493 ActionDest: 211 Annotation: Subtype: Link Rect: 276, 408, 287, 417 ActionDest: 211 Annotation: Subtype: Link Rect: 426, 396, 438, 405 ActionDest: 211 Annotation: Subtype: Link Rect: 350, 180, 452, 192 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 334, 635, 346, 644 ActionDest: 211 Annotation: Subtype: Link Rect: 252, 484, 264, 493 ActionDest: 211 Annotation: Subtype: Link Rect: 302, 472, 314, 481 ActionDest: 211 Annotation: Subtype: Link Rect: 512, 204, 519, 216 ActionDest: 81 Page: Sequence: 6 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 198, 662, 205, 674 ActionDest: 91 Annotation: Subtype: Link Rect: 226, 625, 233, 637 ActionDest: 91 Annotation: Subtype: Link Rect: 310, 612, 317, 624 ActionDest: 91 Annotation: Subtype: Link Rect: 413, 575, 419, 587 ActionDest: 91 Annotation: Subtype: Link Rect: 199, 499, 206, 511 ActionDest: 91 Annotation: Subtype: Link Rect: 549, 313, 556, 325 ActionDest: 81 Page: Sequence: 8 Page: Sequence: 9 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 310, 556, 317, 568 ActionDest: 91 Annotation: Subtype: Link Rect: 259, 382, 266, 394 ActionDest: 114 Annotation: Subtype: Link Rect: 504, 193, 511, 205 ActionDest: 131 Annotation: Subtype: Link Rect: 288, 168, 295, 180 ActionDest: 131 Annotation: Subtype: Link Rect: 414, 130, 421, 142 ActionDest: 131 Annotation: Subtype: Link Rect: 437, 93, 444, 105 ActionDest: 131 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 532, 289, 539, 301 ActionDest: 150 Annotation: Subtype: Link Rect: 544, 239, 551, 251 ActionDest: 150 Annotation: Subtype: Link Rect: 467, 201, 474, 213 ActionDest: 150 Annotation: Subtype: Link Rect: 347, 176, 353, 188 ActionDest: 150 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 312, 291, 319, 303 ActionDest: 167 Annotation: Subtype: Link Rect: 418, 191, 425, 203 ActionDest: 167 Annotation: Subtype: Link Rect: 372, 103, 379, 115 ActionDest: 167 Annotation: Subtype: Link Rect: 522, 91, 529, 103 ActionDest: 167 Page: Sequence: 13 Page: Sequence: 14 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 511, 685, 518, 697 ActionDest: 183 Annotation: Subtype: Link Rect: 486, 282, 559, 293 Annotation: Subtype: Link Rect: 357, 212, 369, 221 ActionDest: 211 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 410, 712, 422, 721 ActionDest: 211 Annotation: Subtype: Link Rect: 422, 712, 434, 721 ActionDest: 211 Annotation: Subtype: Link Rect: 434, 712, 446, 721 ActionDest: 211 Annotation: Subtype: Link Rect: 446, 712, 458, 721 ActionDest: 211 Annotation: Subtype: Link Rect: 230, 687, 242, 696 ActionDest: 211 Annotation: Subtype: Link Rect: 242, 687, 254, 696 ActionDest: 211 Annotation: Subtype: Link Rect: 254, 687, 266, 696 ActionDest: 211 Annotation: Subtype: Link Rect: 518, 675, 530, 684 ActionDest: 211 Annotation: Subtype: Link Rect: 531, 675, 543, 684 ActionDest: 211 Annotation: Subtype: Link Rect: 543, 675, 555, 684 ActionDest: 211 Annotation: Subtype: Link Rect: 526, 574, 538, 583 ActionDest: 211 Annotation: Subtype: Link Rect: 541, 574, 553, 583 ActionDest: 211 Annotation: Subtype: Link Rect: 418, 474, 430, 483 ActionDest: 211 Annotation: Subtype: Link Rect: 469, 461, 481, 470 ActionDest: 211 Annotation: Subtype: Link Rect: 464, 436, 476, 445 ActionDest: 211 Annotation: Subtype: Link Rect: 476, 436, 488, 445 ActionDest: 211 Annotation: Subtype: Link Rect: 491, 436, 503, 445 ActionDest: 211 Annotation: Subtype: Link Rect: 299, 361, 311, 370 ActionDest: 211 Annotation: Subtype: Link Rect: 312, 361, 324, 370 ActionDest: 211 Annotation: Subtype: Link Rect: 422, 323, 434, 332 ActionDest: 211 Annotation: Subtype: Link Rect: 519, 323, 531, 332 ActionDest: 211 Annotation: Subtype: Link Rect: 531, 323, 543, 332 ActionDest: 211 Annotation: Subtype: Link Rect: 544, 323, 556, 332 ActionDest: 211 Annotation: Subtype: Link Rect: 288, 298, 300, 307 ActionDest: 211 Annotation: Subtype: Link Rect: 301, 298, 313, 307 ActionDest: 211 Annotation: Subtype: Link Rect: 390, 248, 402, 257 ActionDest: 211 Annotation: Subtype: Link Rect: 402, 248, 414, 257 ActionDest: 211 Annotation: Subtype: Link Rect: 352, 172, 364, 182 ActionDest: 211 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 202, 738, 214, 746 ActionDest: 211 Annotation: Subtype: Link Rect: 193, 650, 205, 659 ActionDest: 211 Annotation: Subtype: Link Rect: 210, 625, 222, 633 ActionDest: 211 Annotation: Subtype: Link Rect: 456, 612, 463, 621 ActionDest: 208 Annotation: Subtype: Link Rect: 268, 537, 280, 546 ActionDest: 211 Annotation: Subtype: Link Rect: 271, 486, 283, 495 ActionDest: 211 Annotation: Subtype: Link Rect: 323, 449, 335, 458 ActionDest: 211 Annotation: Subtype: Link Rect: 362, 336, 374, 345 ActionDest: 211 Annotation: Subtype: Link Rect: 217, 273, 229, 282 ActionDest: 211 Annotation: Subtype: Link Rect: 461, 135, 473, 144 ActionDest: 211 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 320, 738, 332, 746 ActionDest: 211 Annotation: Subtype: Link Rect: 278, 599, 290, 608 ActionDest: 208 Annotation: Subtype: Link Rect: 290, 599, 302, 608 ActionDest: 214 Annotation: Subtype: Link Rect: 303, 599, 314, 608 ActionDest: 214 Annotation: Subtype: Link Rect: 536, 447, 560, 458 Annotation: Subtype: Link Rect: 165, 435, 409, 446 Page: Sequence: 19 Annotations: Annotation: Subtype: Link Rect: 356, 427, 394, 438 Annotation: Subtype: Link Rect: 400, 427, 436, 438 Annotation: Subtype: Link Rect: 519, 415, 557, 426 Annotation: Subtype: Link Rect: 59, 405, 95, 415 Annotation: Subtype: Link Rect: 173, 382, 211, 392 Annotation: Subtype: Link Rect: 217, 382, 253, 392 Annotation: Subtype: Link Rect: 287, 358, 324, 369 Annotation: Subtype: Link Rect: 330, 358, 366, 369 Annotation: Subtype: Link Rect: 59, 324, 96, 334 Annotation: Subtype: Link Rect: 519, 312, 557, 323 Annotation: Subtype: Link Rect: 59, 301, 95, 311 Annotation: Subtype: Link Rect: 192, 278, 230, 288 Annotation: Subtype: Link Rect: 236, 278, 272, 288 Annotation: Subtype: Link Rect: 225, 254, 263, 265 Annotation: Subtype: Link Rect: 269, 254, 305, 265 Annotation: Subtype: Link Rect: 372, 231, 409, 242 Annotation: Subtype: Link Rect: 59, 174, 96, 185 Annotation: Subtype: Link Rect: 102, 174, 138, 185 Annotation: Subtype: Link Rect: 59, 151, 96, 162 Annotation: Subtype: Link Rect: 102, 151, 138, 162 Annotation: Subtype: Link Rect: 59, 128, 96, 139 Annotation: Subtype: Link Rect: 102, 128, 138, 139 Annotation: Subtype: Link Rect: 181, 94, 218, 104 Annotation: Subtype: Link Rect: 520, 82, 557, 93 Annotation: Subtype: Link Rect: 468, 70, 506, 81 Page: Sequence: 20 Annotations: Annotation: Subtype: Link Rect: 427, 735, 464, 746 Annotation: Subtype: Link Rect: 470, 735, 506, 746 Annotation: Subtype: Link Rect: 467, 724, 504, 735 Annotation: Subtype: Link Rect: 59, 690, 96, 700 Annotation: Subtype: Link Rect: 135, 655, 173, 665 Annotation: Subtype: Link Rect: 179, 655, 215, 665 Annotation: Subtype: Link Rect: 491, 631, 529, 642 Annotation: Subtype: Link Rect: 519, 608, 557, 619 Annotation: Subtype: Link Rect: 59, 598, 95, 608 Annotation: Subtype: Link Rect: 104, 563, 142, 573 Annotation: Subtype: Link Rect: 148, 563, 184, 573 Annotation: Subtype: Link Rect: 408, 539, 446, 550 Annotation: Subtype: Link Rect: 452, 539, 488, 550 Annotation: Subtype: Link Rect: 375, 505, 413, 516 Annotation: Subtype: Link Rect: 419, 505, 455, 516 Annotation: Subtype: Link Rect: 331, 470, 369, 481 Annotation: Subtype: Link Rect: 104, 425, 142, 435 Annotation: Subtype: Link Rect: 463, 401, 500, 412 Annotation: Subtype: Link Rect: 507, 401, 543, 412 Annotation: Subtype: Link Rect: 439, 367, 476, 378 Annotation: Subtype: Link Rect: 482, 367, 519, 378 Annotation: Subtype: Link Rect: 118, 345, 155, 355 Annotation: Subtype: Link Rect: 161, 345, 197, 355 Annotation: Subtype: Link Rect: 113, 310, 151, 320 Annotation: Subtype: Link Rect: 157, 310, 193, 320 Annotation: Subtype: Link Rect: 403, 275, 440, 286 Annotation: Subtype: Link Rect: 158, 253, 196, 263 Annotation: Subtype: Link Rect: 202, 253, 238, 263 Annotation: Subtype: Link Rect: 300, 229, 337, 240 Annotation: Subtype: Link Rect: 214, 207, 252, 217 Annotation: Subtype: Link Rect: 258, 207, 294, 217 Annotation: Subtype: Link Rect: 417, 183, 455, 194 Annotation: Subtype: Link Rect: 296, 160, 334, 171 Annotation: Subtype: Link Rect: 519, 137, 557, 148 Annotation: Subtype: Link Rect: 104, 115, 141, 125 Annotation: Subtype: Link Rect: 147, 115, 183, 125 Annotation: Subtype: Link Rect: 520, 91, 557, 102 Page: Sequence: 21 Annotations: Annotation: Subtype: Link Rect: 147, 724, 185, 735 Annotation: Subtype: Link Rect: 520, 701, 557, 712 Annotation: Subtype: Link Rect: 59, 690, 95, 700 Checksum: 54353400 Type: CRC32 Checksum: d11dab8c2b722912d4dd81a30bd50894 Type: MD5 Checksum: 82a048f1df09d901ef13456474cb16be1180c8dc Type: SHA-1