b76166e4a719781ea341bf3cd4e4be3c pmc_9139148.pdf ec55750e941d6ef357e38ff50bcde95ba88aeb84 pmc_9139148.pdf 7ffb6a97c935005946beee55af31e28abfa6fb28e417d8f3e3c5fb531f0a1c6e pmc_9139148.pdf Title: Hepatitis B Virus Variants with Multiple Insertions and/or Deletions in the X Open Reading Frame 3' End: Common Members of Viral Quasispecies in Chronic Hepatitis B Patients Subject: Deletions in the 3' end region of the hepatitis B virus (HBV) X open reading frame (HBX) may affect the core promoter (Cp) and have been frequently associated with hepatocellular carcinoma (HCC). The aim of this study was to investigate the presence of variants with deletions and/or insertions (Indels) in this region in the quasispecies of 50 chronic hepatitis B (CHB) patients without HCC. We identified 103 different Indels in 47 (94%) patients, in a median of 3.4% of their reads (IQR, 1.3–8.4%), and 25% (IQR, 13.1–40.7%) of unique sequences identified in each quasispecies (haplotypes). Of those Indels, 101 (98.1%) caused 44 different altered stop codons, the most commonly observed were at positions 128, 129, 135, and 362 (putative position). Moreover, 39 (37.9%) Indels altered the TATA-like box (TA) sequences of Cp; the most commonly observed caused TA2 + TA3 fusion, creating a new putative canonical TATA box. Four (8%) patients developed negative clinical outcomes after a median follow-up of 9.4 (8.7–12) years. In conclusion, we observed variants with Indels in the HBX 3' end in the vast majority of our CHB patients, some of them encoding alternative versions of HBx with potential functional roles, and/or alterations in the regulation of transcription. Keywords: hepatitis B virus; hepatitis B X open reading frame; HBX 3' end region; insertions; deletions; quasispecies; next-generation sequencing Author: Selene García-García, Andrea Caballero-Garralda, David Tabernero, Maria Francesca Cortese, Josep Gregori, Francisco Rodriguez-Algarra, Josep Quer, Mar Riveiro-Barciela, Maria Homs, Ariadna Rando-Segura, Beatriz Pacin-Ruiz, Marta Vila, Roser Ferrer-Costa, Tomas Pumarola, Maria Buti and Francisco Rodriguez-Frias Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sat May 21 11:44:42 2022 CEST ModDate: Sat May 21 11:48:22 2022 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 18 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1053191 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- WLENKM+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 CIQLLF+VnURWPalladioL Type 1 Custom yes yes no 15 0 SZZOJW+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 OALVSQ+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 IAFOCY+CMSY10 Type 1 Builtin yes yes no 28 0 ARMLRS+URWPalladioL-Ital Type 1 Custom yes yes no 32 0 PXEKZD+CMBSY10 Type 1 Builtin yes yes no 36 0 EBFBTV+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 54 0 ZXDKIP+EURM10 Type 1 Builtin yes yes no 79 0 EVMTFM+URWPalladioL-BoldItal Type 1 Custom yes yes no 89 0 FNTSBS+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 98 0 FNTSBS+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 104 0 FNTSBS+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 110 0 FNTSBS+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 126 0 FNTSBS+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 132 0 FNTSBS+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 138 0 FNTSBS+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 157 0 FNTSBS+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 163 0 FNTSBS+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 169 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-05-04 02:38:01 CEST RepresentationInformation: pmc_9139148.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-06-17 07:12:18 CEST Size: 1053191 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 412 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: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Patients and Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Serological and Virological Determinations Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Amplification of HBV Genome Region Analyzed and Next-Generation Sequencing Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Next-Generation Sequencing Data Treatment Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Cloning to Confirm NGS Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: RNA Structural Modelling Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Children: Item: Title: Patients and Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Overview of Next-Generation Data Analyses Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Alternative HBX Stop Codons Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Indels Affecting the TATA-Like Box Region Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Confirmation of Indel Variants by Cloning Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Clinical Outcome of Patients Studied Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Info: Title: Hepatitis B Virus Variants with Multiple Insertions and/or Deletions in the X Open Reading Frame 3' End: Common Members of Viral Quasispecies in Chronic Hepatitis B Patients Author: Selene García-García, Andrea Caballero-Garralda, David Tabernero, Maria Francesca Cortese, Josep Gregori, Francisco Rodriguez-Algarra, Josep Quer, Mar Riveiro-Barciela, Maria Homs, Ariadna Rando-Segura, Beatriz Pacin-Ruiz, Marta Vila, Roser Ferrer-Costa, Tomas Pumarola, Maria Buti and Francisco Rodriguez-Frias Subject: Deletions in the 3' end region of the hepatitis B virus (HBV) X open reading frame (HBX) may affect the core promoter (Cp) and have been frequently associated with hepatocellular carcinoma (HCC). The aim of this study was to investigate the presence of variants with deletions and/or insertions (Indels) in this region in the quasispecies of 50 chronic hepatitis B (CHB) patients without HCC. We identified 103 different Indels in 47 (94%) patients, in a median of 3.4% of their reads (IQR, 1.3 8.4%), and 25% (IQR, 13.1 40.7%) of unique sequences identified in each quasispecies (haplotypes). Of those Indels, 101 (98.1%) caused 44 different altered stop codons, the most commonly observed were at positions 128, 129, 135, and 362 (putative position). Moreover, 39 (37.9%) Indels altered the TATA-like box (TA) sequences of Cp; the most commonly observed caused TA2 + TA3 fusion, creating a new putative canonical TATA box. Four (8%) patients developed negative clinical outcomes after a median follow-up of 9.4 (8.7 12) years. In conclusion, we observed variants with Indels in the HBX 3' end in the vast majority of our CHB patients, some of them encoding alternative versions of HBx with potential functional roles, and/or alterations in the regulation of transcription. Keywords: hepatitis B virus; hepatitis B X open reading frame; HBX 3' end region; insertions; deletions; quasispecies; next-generation sequencing Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sat May 21 11:44:42 CEST 2022 ModDate: Sat May 21 11:48:22 CEST 2022 ID: 0x16e6997925d6abaefa1fbcd64e0d82b7, 0x16e6997925d6abaefa1fbcd64e0d82b7 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: ARMLRS+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 122 FontDescriptor: FontName: ARMLRS+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: SZZOJW+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 134 FontDescriptor: FontName: SZZOJW+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: PXEKZD+CMBSY10 FontSubset: true FirstChar: 48 LastChar: 48 FontDescriptor: FontName: PXEKZD+CMBSY10 Flags: Symbolic FontBBox: -26, -940, 1332, 825 FontFile: true Font: BaseFont: OALVSQ+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: OALVSQ+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: EVMTFM+URWPalladioL-BoldItal FontSubset: true FirstChar: 66 LastChar: 88 FontDescriptor: FontName: EVMTFM+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: WLENKM+VnURWPalladioL-Bold FontSubset: true FirstChar: 189 LastChar: 189 FontDescriptor: FontName: WLENKM+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: IAFOCY+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: IAFOCY+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: CIQLLF+VnURWPalladioL FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: CIQLLF+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: ZXDKIP+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: ZXDKIP+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: 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: 157, 641, 168, 654 Annotation: Subtype: Link Rect: 418, 641, 429, 654 Annotation: Subtype: Link Rect: 165, 630, 176, 641 Annotation: Subtype: Link Rect: 255, 628, 266, 641 Annotation: Subtype: Link Rect: 406, 628, 417, 641 Annotation: Subtype: Link Rect: 491, 628, 502, 641 Annotation: Subtype: Link Rect: 218, 615, 229, 629 Annotation: Subtype: Link Rect: 353, 615, 364, 629 Annotation: Subtype: Link Rect: 125, 603, 136, 616 Annotation: Subtype: Link Rect: 460, 603, 471, 616 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, 695, 556, 704 ActionDest: 191 Annotation: Subtype: Link Rect: 548, 657, 555, 666 ActionDest: 191 Annotation: Subtype: Link Rect: 417, 619, 424, 629 ActionDest: 191 Annotation: Subtype: Link Rect: 425, 620, 432, 629 ActionDest: 191 Annotation: Subtype: Link Rect: 489, 582, 496, 591 ActionDest: 191 Annotation: Subtype: Link Rect: 296, 494, 303, 503 ActionDest: 191 Annotation: Subtype: Link Rect: 304, 494, 311, 503 ActionDest: 191 Annotation: Subtype: Link Rect: 296, 469, 303, 478 ActionDest: 191 Annotation: Subtype: Link Rect: 187, 431, 194, 440 ActionDest: 191 Annotation: Subtype: Link Rect: 193, 393, 200, 403 ActionDest: 194 Annotation: Subtype: Link Rect: 287, 381, 299, 390 ActionDest: 194 Annotation: Subtype: Link Rect: 462, 356, 474, 365 ActionDest: 194 Annotation: Subtype: Link Rect: 529, 318, 541, 327 ActionDest: 194 Annotation: Subtype: Link Rect: 225, 243, 236, 252 ActionDest: 194 Annotation: Subtype: Link Rect: 532, 230, 544, 239 ActionDest: 194 Annotation: Subtype: Link Rect: 544, 230, 556, 239 ActionDest: 194 Annotation: Subtype: Link Rect: 419, 218, 431, 227 ActionDest: 194 Annotation: Subtype: Link Rect: 432, 218, 444, 227 ActionDest: 194 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 252, 392, 264, 401 ActionDest: 194 Annotation: Subtype: Link Rect: 444, 141, 456, 150 ActionDest: 194 Annotation: Subtype: Link Rect: 545, 141, 556, 150 ActionDest: 194 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 491, 685, 503, 694 ActionDest: 194 Annotation: Subtype: Link Rect: 515, 623, 527, 631 ActionDest: 194 Annotation: Subtype: Link Rect: 376, 610, 388, 619 ActionDest: 194 Annotation: Subtype: Link Rect: 475, 610, 487, 619 ActionDest: 194 Annotation: Subtype: Link Rect: 217, 305, 502, 317 Annotation: Subtype: Link Rect: 390, 295, 402, 305 ActionDest: 194 Annotation: Subtype: Link Rect: 393, 220, 405, 229 ActionDest: 194 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 202, 723, 209, 732 ActionDest: 86 Annotation: Subtype: Link Rect: 471, 410, 478, 422 ActionDest: 93 Annotation: Subtype: Link Rect: 530, 134, 538, 146 ActionDest: 93 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 303, 397, 314, 405 ActionDest: 194 Annotation: Subtype: Link Rect: 309, 358, 316, 367 ActionDest: 191 Annotation: Subtype: Link Rect: 366, 346, 372, 354 ActionDest: 191 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 425, 720, 432, 732 ActionDest: 118 Annotation: Subtype: Link Rect: 444, 346, 451, 358 ActionDest: 118 Annotation: Subtype: Link Rect: 514, 295, 521, 307 ActionDest: 93 Annotation: Subtype: Link Rect: 289, 270, 304, 282 ActionDest: 121 Annotation: Subtype: Link Rect: 472, 120, 479, 132 ActionDest: 118 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 393, 733, 400, 745 ActionDest: 93 Annotation: Subtype: Link Rect: 266, 547, 278, 556 ActionDest: 194 Annotation: Subtype: Link Rect: 281, 547, 293, 556 ActionDest: 194 Annotation: Subtype: Link Rect: 426, 509, 438, 519 ActionDest: 194 Annotation: Subtype: Link Rect: 476, 507, 482, 519 ActionDest: 121 Annotation: Subtype: Link Rect: 194, 482, 201, 494 ActionDest: 93 Annotation: Subtype: Link Rect: 449, 482, 456, 494 ActionDest: 118 Annotation: Subtype: Link Rect: 549, 97, 556, 109 ActionDest: 93 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 260, 723, 267, 732 ActionDest: 146 Annotation: Subtype: Link Rect: 246, 171, 253, 183 ActionDest: 118 Annotation: Subtype: Link Rect: 391, 83, 398, 95 ActionDest: 93 Annotation: Subtype: Link Rect: 452, 73, 459, 83 ActionDest: 191 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 325, 736, 337, 744 ActionDest: 194 Annotation: Subtype: Link Rect: 544, 735, 556, 744 ActionDest: 194 Annotation: Subtype: Link Rect: 216, 647, 223, 657 ActionDest: 191 Annotation: Subtype: Link Rect: 223, 647, 235, 657 ActionDest: 194 Annotation: Subtype: Link Rect: 444, 635, 456, 644 ActionDest: 194 Annotation: Subtype: Link Rect: 288, 595, 295, 607 ActionDest: 146 Annotation: Subtype: Link Rect: 364, 570, 371, 582 ActionDest: 93 Annotation: Subtype: Link Rect: 322, 547, 334, 556 ActionDest: 197 Annotation: Subtype: Link Rect: 549, 519, 556, 531 ActionDest: 152 Annotation: Subtype: Link Rect: 191, 356, 198, 368 ActionDest: 152 Annotation: Subtype: Link Rect: 288, 269, 295, 280 ActionDest: 152 Page: Sequence: 11 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 194, 554, 201, 566 ActionDest: 118 Annotation: Subtype: Link Rect: 288, 491, 295, 503 ActionDest: 118 Annotation: Subtype: Link Rect: 258, 393, 270, 402 ActionDest: 194 Annotation: Subtype: Link Rect: 270, 393, 282, 402 ActionDest: 194 Annotation: Subtype: Link Rect: 399, 355, 411, 365 ActionDest: 194 Annotation: Subtype: Link Rect: 412, 356, 424, 365 ActionDest: 194 Annotation: Subtype: Link Rect: 475, 355, 486, 365 ActionDest: 197 Annotation: Subtype: Link Rect: 412, 343, 424, 352 ActionDest: 194 Annotation: Subtype: Link Rect: 393, 318, 405, 327 ActionDest: 194 Annotation: Subtype: Link Rect: 347, 280, 359, 289 ActionDest: 197 Annotation: Subtype: Link Rect: 278, 180, 289, 189 ActionDest: 194 Annotation: Subtype: Link Rect: 290, 180, 302, 189 ActionDest: 194 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 421, 723, 433, 732 ActionDest: 197 Annotation: Subtype: Link Rect: 534, 660, 541, 669 ActionDest: 194 Annotation: Subtype: Link Rect: 203, 635, 215, 644 ActionDest: 194 Annotation: Subtype: Link Rect: 191, 497, 203, 506 ActionDest: 197 Annotation: Subtype: Link Rect: 406, 434, 418, 443 ActionDest: 197 Annotation: Subtype: Link Rect: 214, 409, 226, 418 ActionDest: 197 Annotation: Subtype: Link Rect: 443, 384, 455, 393 ActionDest: 197 Annotation: Subtype: Link Rect: 433, 283, 445, 293 ActionDest: 194 Annotation: Subtype: Link Rect: 445, 284, 457, 293 ActionDest: 194 Annotation: Subtype: Link Rect: 324, 108, 331, 117 ActionDest: 191 Annotation: Subtype: Link Rect: 532, 83, 544, 92 ActionDest: 197 Annotation: Subtype: Link Rect: 545, 83, 557, 92 ActionDest: 197 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 204, 720, 211, 732 ActionDest: 146 Annotation: Subtype: Link Rect: 359, 635, 371, 644 ActionDest: 197 Annotation: Subtype: Link Rect: 194, 585, 201, 594 ActionDest: 191 Annotation: Subtype: Link Rect: 544, 560, 556, 569 ActionDest: 197 Annotation: Subtype: Link Rect: 291, 535, 303, 544 ActionDest: 194 Annotation: Subtype: Link Rect: 343, 484, 355, 493 ActionDest: 194 Annotation: Subtype: Link Rect: 337, 409, 344, 418 ActionDest: 191 Annotation: Subtype: Link Rect: 345, 409, 357, 418 ActionDest: 194 Annotation: Subtype: Link Rect: 511, 397, 523, 406 ActionDest: 194 Annotation: Subtype: Link Rect: 196, 359, 208, 368 ActionDest: 197 Annotation: Subtype: Link Rect: 397, 334, 409, 343 ActionDest: 194 Annotation: Subtype: Link Rect: 313, 246, 325, 255 ActionDest: 194 Annotation: Subtype: Link Rect: 326, 246, 338, 255 ActionDest: 194 Annotation: Subtype: Link Rect: 470, 108, 482, 117 ActionDest: 194 Annotation: Subtype: Link Rect: 483, 108, 495, 117 ActionDest: 197 Annotation: Subtype: Link Rect: 495, 108, 507, 117 ActionDest: 197 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 289, 422, 301, 431 ActionDest: 197 Annotation: Subtype: Link Rect: 317, 296, 329, 305 ActionDest: 197 Annotation: Subtype: Link Rect: 191, 271, 203, 280 ActionDest: 197 Annotation: Subtype: Link Rect: 307, 233, 319, 242 ActionDest: 197 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 525, 646, 560, 657 Annotation: Subtype: Link Rect: 165, 634, 401, 645 Annotation: Subtype: Link Rect: 291, 217, 560, 228 Annotation: Subtype: Link Rect: 59, 184, 96, 194 Annotation: Subtype: Link Rect: 59, 161, 96, 171 Annotation: Subtype: Link Rect: 133, 138, 170, 148 Annotation: Subtype: Link Rect: 350, 114, 388, 125 Annotation: Subtype: Link Rect: 95, 92, 133, 102 Annotation: Subtype: Link Rect: 438, 79, 475, 90 Annotation: Subtype: Link Rect: 520, 68, 557, 79 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 296, 722, 334, 733 Annotation: Subtype: Link Rect: 346, 699, 384, 710 Annotation: Subtype: Link Rect: 507, 676, 544, 687 Annotation: Subtype: Link Rect: 431, 652, 469, 664 Annotation: Subtype: Link Rect: 319, 629, 357, 640 Annotation: Subtype: Link Rect: 140, 596, 177, 606 Annotation: Subtype: Link Rect: 183, 596, 219, 606 Annotation: Subtype: Link Rect: 265, 560, 303, 571 Annotation: Subtype: Link Rect: 184, 527, 221, 537 Annotation: Subtype: Link Rect: 109, 481, 146, 491 Annotation: Subtype: Link Rect: 112, 446, 150, 456 Annotation: Subtype: Link Rect: 336, 411, 374, 422 Annotation: Subtype: Link Rect: 99, 377, 137, 387 Annotation: Subtype: Link Rect: 180, 354, 218, 364 Annotation: Subtype: Link Rect: 147, 330, 560, 341 Annotation: Subtype: Link Rect: 56, 319, 101, 330 Annotation: Subtype: Link Rect: 59, 274, 96, 284 Annotation: Subtype: Link Rect: 102, 274, 138, 284 Annotation: Subtype: Link Rect: 59, 251, 96, 261 Annotation: Subtype: Link Rect: 102, 251, 138, 261 Annotation: Subtype: Link Rect: 125, 227, 393, 238 Annotation: Subtype: Link Rect: 519, 204, 557, 215 Annotation: Subtype: Link Rect: 59, 193, 95, 203 Annotation: Subtype: Link Rect: 455, 181, 492, 192 Annotation: Subtype: Link Rect: 498, 181, 534, 192 Annotation: Subtype: Link Rect: 104, 147, 141, 157 Annotation: Subtype: Link Rect: 147, 147, 183, 157 Annotation: Subtype: Link Rect: 519, 123, 557, 134 Annotation: Subtype: Link Rect: 59, 113, 95, 123 Annotation: Subtype: Link Rect: 392, 89, 429, 100 Annotation: Subtype: Link Rect: 519, 66, 557, 77 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 456, 722, 493, 733 Annotation: Subtype: Link Rect: 458, 699, 496, 710 Annotation: Subtype: Link Rect: 108, 665, 146, 675 Annotation: Subtype: Link Rect: 152, 665, 188, 675 Annotation: Subtype: Link Rect: 478, 641, 515, 652 Annotation: Subtype: Link Rect: 521, 641, 557, 652 Annotation: Subtype: Link Rect: 122, 607, 160, 617 Annotation: Subtype: Link Rect: 166, 607, 202, 617 Annotation: Subtype: Link Rect: 475, 595, 513, 606 Annotation: Subtype: Link Rect: 174, 573, 211, 583 Annotation: Subtype: Link Rect: 509, 526, 546, 537 Annotation: Subtype: Link Rect: 404, 503, 441, 514 Annotation: Subtype: Link Rect: 279, 480, 316, 491 Annotation: Subtype: Link Rect: 456, 457, 494, 468 Annotation: Subtype: Link Rect: 140, 423, 176, 433 Annotation: Subtype: Link Rect: 211, 400, 249, 410 Annotation: Subtype: Link Rect: 255, 400, 291, 410 Annotation: Subtype: Link Rect: 344, 377, 381, 387 Annotation: Subtype: Link Rect: 388, 377, 424, 387 Annotation: Subtype: Link Rect: 367, 342, 404, 353 Annotation: Subtype: Link Rect: 410, 342, 447, 353 Checksum: 67245774 Type: CRC32 Checksum: b76166e4a719781ea341bf3cd4e4be3c Type: MD5 Checksum: ec55750e941d6ef357e38ff50bcde95ba88aeb84 Type: SHA-1