69e106be3dd77acb3b19c5f64fe4355c pmc_9369323.pdf e1d2d9001825f4d75548335b1605b88d353c95ea pmc_9369323.pdf 20ab199df6f8e8093ef92d78fe0966894cdb0406986019068ad6e6d4537fddd7 pmc_9369323.pdf Title: Anatomical Laser Microdissection of the Ileum Reveals mtDNA Depletion Recovery in A Mitochondrial Neuro-Gastrointestinal Encephalomyopathy (MNGIE) Patient Receiving Liver Transplant Subject: mitochondrial neuro-gastrointestinal encephalomyopathy (MNGIE) is a rare genetic disorder characterized by thymidine phosphorylase (TP) enzyme defect. The absence of TP activity induces the imbalance of mitochondrial nucleotide pool, leading to impaired mitochondrial DNA (mtDNA) replication and depletion. Since mtDNA is required to ensure oxidative phosphorylation, metabolically active tissues may not achieve sufficient energy production. The only effective life-saving approach in MNGIE has been the permanent replacement of TP via allogeneic hematopoietic stem cell or liver transplantation. However, the follow-up of transplanted patients showed that gut tissue changes do not revert and fatal complications, such as massive gastrointestinal bleeding, can occur. The purpose of this study was to clarify whether the reintroduction of TP after transplant can recover mtDNA copy number in a normal range. Using laser capture microdissection and droplet-digital-PCR, we assessed the mtDNA copy number in each layer of full-thickness ileal samples of a naive MNGIE cohort vs. controls and in a patient pre- and post-TP replacement. The treatment led to a significant recovery of gut tissue mtDNA amount, thus showing its efficacy. Our results indicate that a timely TP replacement is needed to maximize therapeutic success before irreversible degenerative tissue changes occur in MNGIE. Keywords: gastrointestinal degeneration; microanatomical dissection; mitochondrial disorders; MNGIE; mtDNA depletion Author: Elisa Boschetti, Leonardo Caporali, Roberto D’Angelo, Carolina Malagelada, Anna Accarino, Maria Teresa Dotti, Roberta Costa, Giovanna Cenacchi, Loris Pironi, Rita Rinaldi, Vincenzo Stanghellini, Stefano Ratti, Lucia Manzoli, Valerio Carelli and Roberto De Giorgio Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Aug 8 08:35:25 2022 CEST ModDate: Mon Aug 8 08:56:08 2022 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 10 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2588327 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- IDEOOS+VnURWPalladioL Type 1 Custom yes yes no 10 0 KOCVHH+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 WDCRRF+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 NSUXGF+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 42 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 45 0 VYVZGZ+CMSY10 Type 1 Builtin yes yes no 70 0 HAAKNC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 77 0 HAAKNE+PalatinoLinotype TrueType WinAnsi yes yes no 80 0 HAAKOF+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 83 0 HAAKNC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 93 0 HAAKNE+PalatinoLinotype TrueType WinAnsi yes yes no 96 0 HAAKOF+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 99 0 OVNEXW+EURM10 Type 1 Builtin yes yes no 110 0 HAAKNC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 120 0 HAAKND+PalatinoLinotype CID TrueType Identity-H yes yes yes 123 0 HAAKNE+PalatinoLinotype TrueType WinAnsi yes yes no 129 0 HAAKOF+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 132 0 AWDAAW+VnURWPalladioL-Italic Type 1 Custom yes yes no 139 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-09-28 02:34:14 CEST RepresentationInformation: pmc_9369323.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-09-23 02:18:08 CEST Size: 2588327 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 258 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: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Children: Item: Title: mtDNA Assessment Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: mtDNA Assessment Pre- vs. Post-LT Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: mtDNA Assessment Post-AHSCT Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Children: Item: Title: Patients and Sampling Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Clinical History and Pre- and Post-LT Tissue Sampling of Patient #1 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: LCM of Ileal Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Total DNA Extraction and mtDNA Copy Number and Deletion Quantification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Statistic 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: Anatomical Laser Microdissection of the Ileum Reveals mtDNA Depletion Recovery in A Mitochondrial Neuro-Gastrointestinal Encephalomyopathy (MNGIE) Patient Receiving Liver Transplant Author: Elisa Boschetti, Leonardo Caporali, Roberto D’Angelo, Carolina Malagelada, Anna Accarino, Maria Teresa Dotti, Roberta Costa, Giovanna Cenacchi, Loris Pironi, Rita Rinaldi, Vincenzo Stanghellini, Stefano Ratti, Lucia Manzoli, Valerio Carelli and Roberto De Giorgio Subject: mitochondrial neuro-gastrointestinal encephalomyopathy (MNGIE) is a rare genetic disorder characterized by thymidine phosphorylase (TP) enzyme defect. The absence of TP activity induces the imbalance of mitochondrial nucleotide pool, leading to impaired mitochondrial DNA (mtDNA) replication and depletion. Since mtDNA is required to ensure oxidative phosphorylation, metabolically active tissues may not achieve sufficient energy production. The only effective life-saving approach in MNGIE has been the permanent replacement of TP via allogeneic hematopoietic stem cell or liver transplantation. However, the follow-up of transplanted patients showed that gut tissue changes do not revert and fatal complications, such as massive gastrointestinal bleeding, can occur. The purpose of this study was to clarify whether the reintroduction of TP after transplant can recover mtDNA copy number in a normal range. Using laser capture microdissection and droplet-digital-PCR, we assessed the mtDNA copy number in each layer of full-thickness ileal samples of a naive MNGIE cohort vs. controls and in a patient pre- and post-TP replacement. The treatment led to a significant recovery of gut tissue mtDNA amount, thus showing its efficacy. Our results indicate that a timely TP replacement is needed to maximize therapeutic success before irreversible degenerative tissue changes occur in MNGIE. 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