2e453eae49f07ec6a192442c60deab33 pmc_33260630.pdf 2b46104e6ee58e6f7ecb077d2d54142f99fd46a3 pmc_33260630.pdf 52056659dda0023114b7b347cb5cfb944966c5fc424be43f439227f824307ff2 pmc_33260630.pdf Title: TYK2 Variants in B-Acute Lymphoblastic Leukaemia Subject: B-cell precursor acute lymphoblastic leukaemia (B-ALL) is a malignancy of lymphoid progenitor cells with altered genes including the Janus kinase (JAK) gene family. Among them, tyrosine kinase 2 (TYK2) is involved in signal transduction of cytokines such as interferon (IFN) / through IFN-/ receptor alpha chain (IFNAR1). To search for disease-associated TYK2 variants, bone marrow samples from 62 B-ALL patients at diagnosis were analysed by next-generation sequencing. TYK2 variants were found in 16 patients (25.8%): one patient had a novel mutation at the four-point-one, ezrin, radixin, moesin (FERM) domain (S431G) and two patients had the rare variants rs150601734 or rs55882956 (R425H or R832W). To functionally characterise them, they were generated by direct mutagenesis, cloned in expression vectors, and transfected in TYK2-deficient cells. Under high-IFN doses, the three variants were competent to phosphorylate STAT1/2. While R425H and R832W induced STAT1/2-target genes measured by qPCR, S431G behaved as the kinase-dead form of the protein. None of these variants phosphorylated STAT3 in in vitro kinase assays. Molecular dynamics simulation showed that TYK2/IFNAR1 interaction is not affected by these variants. Finally, qPCR analysis revealed diminished expression of TYK2 in B-ALL patients at diagnosis compared to that in healthy donors, further stressing the tumour immune surveillance role of TYK2. Keywords: B-cell precursor acute lymphoblastic leukaemia; TYK2 variants; IFN; IFN/ receptor alpha chain (IFNAR1); next-generation sequencing; molecular dynamics; TYK2 expression; immune surveillance Author: Edgar Turrubiartes-Martínez, Irene Bodega-Mayor, Pablo Delgado-Wicke, Francisca Molina-Jiménez, Diana Casique-Aguirre, Martín González-Andrade, Inmaculada Rapado, Mireia Camós, Cristina Díaz-de-Heredia, Eva Barragán, Manuel Ramírez-Orellana, Beatriz Aguado, Ángela Figuera, Joaquín Martínez-López and Elena Fernández-Ruiz Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Wed Dec 2 12:39:01 2020 CET ModDate: Wed Dec 2 13:08:28 2020 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2487236 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- 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(2019-04-17) LastModified: 2020-12-28 03:47:07 CET Size: 2487236 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 548 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4923ab24 Item: Title: Material and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Children: Item: Title: Patients and Data Collection Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: Ethics Statement Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: DNA Isolation and Next-Generation Sequencing Assay (NGS) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Site-Direct Mutagenesis, Plasmids, Cell Culture, and Transfection Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Western Blot and Immunoprecipitation Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: In Vitro Kinase Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Expression Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Structural Analysis of R425H and S431G TYK2 Variants Interacting with IFNAR1 and Molecular Dynamics Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Children: Item: Title: TYK2 Non-Synonymous Variants Found in B-ALL Patients Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: TYK2 Variants Present Differences in IFN Signalling Response and in STAT-Target Genes Induction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: In Vitro Kinase Activity of TYK2 Variants Is Impaired Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Molecular Dynamics of the TYK2 R425H  and S431G IFNAR1 Complex Interaction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: TYK2 Expression Analysis in B-ALL Patients 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: TYK2 Variants in B-Acute Lymphoblastic Leukaemia Author: Edgar Turrubiartes-Martínez, Irene Bodega-Mayor, Pablo Delgado-Wicke, Francisca Molina-Jiménez, Diana Casique-Aguirre, Martín González-Andrade, Inmaculada Rapado, Mireia Camós, Cristina Díaz-de-Heredia, Eva Barragán, Manuel Ramírez-Orellana, Beatriz Aguado, Ángela Figuera, Joaquín Martínez-López and Elena Fernández-Ruiz Subject: B-cell precursor acute lymphoblastic leukaemia (B-ALL) is a malignancy of lymphoid progenitor cells with altered genes including the Janus kinase (JAK) gene family. Among them, tyrosine kinase 2 (TYK2) is involved in signal transduction of cytokines such as interferon (IFN) / through IFN-/ receptor alpha chain (IFNAR1). To search for disease-associated TYK2 variants, bone marrow samples from 62 B-ALL patients at diagnosis were analysed by next-generation sequencing. TYK2 variants were found in 16 patients (25.8%): one patient had a novel mutation at the four-point-one, ezrin, radixin, moesin (FERM) domain (S431G) and two patients had the rare variants rs150601734 or rs55882956 (R425H or R832W). To functionally characterise them, they were generated by direct mutagenesis, cloned in expression vectors, and transfected in TYK2-deficient cells. Under high-IFN doses, the three variants were competent to phosphorylate STAT1/2. While R425H and R832W induced STAT1/2-target genes measured by qPCR, S431G behaved as the kinase-dead form of the protein. None of these variants phosphorylated STAT3 in in vitro kinase assays. Molecular dynamics simulation showed that TYK2/IFNAR1 interaction is not affected by these variants. Finally, qPCR analysis revealed diminished expression of TYK2 in B-ALL patients at diagnosis compared to that in healthy donors, further stressing the tumour immune surveillance role of TYK2. Keywords: B-cell precursor acute lymphoblastic leukaemia; TYK2 variants; IFN; IFN/ receptor alpha chain (IFNAR1); next-generation sequencing; molecular dynamics; TYK2 expression; immune surveillance Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Wed Dec 02 12:39:01 CET 2020 ModDate: Wed Dec 02 13:08:28 CET 2020 ID: 0xd59ead0598190e165abe2e5c88475abf, 0xd59ead0598190e165abe2e5c88475abf Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: OFLXAT+Rpxr FontSubset: true FirstChar: 11 LastChar: 62 FontDescriptor: FontName: OFLXAT+Rpxr Flags: Symbolic FontBBox: -40, -283, 904, 728 FontFile: true Font: BaseFont: IZXEMB+EURM10 FontSubset: true FirstChar: 11 LastChar: 31 FontDescriptor: FontName: IZXEMB+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: 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: 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92 Checksum: a5102d72 Type: CRC32 Checksum: 2e453eae49f07ec6a192442c60deab33 Type: MD5 Checksum: 2b46104e6ee58e6f7ecb077d2d54142f99fd46a3 Type: SHA-1