Journal: Nature Genetics

Article Title: Selection for somatic escape variants in SERPINA1 in the liver of patients with alpha-1 antitrypsin deficiency

doi: 10.1038/s41588-025-02125-1

Figure Lengend Snippet: ( a ) Genomic and clinicopathological features of patients with alpha-1 anti-trypsin (A1AT) deficiency (purple header bar, n = 5) and haemochromatosis (green header bar, n = 5) included in the study. Germline variants were called from the exome or whole genome sequencing and reported for genes previously associated with either A1AT-deficiency or haemochromatosis. ( b ) Example photomicrographs of serial liver sections stained for enhanced picosirius red (EPSR, upper panels), diastase-Periodic Acid Schiff (dPAS, middle panels) or Perls’ stain (lower panels) from three patients included in the study. Numbers on the panels are the dPAS granules / globules score (0–4) or hepatocellular iron deposition score (0–4) from global assessment of liver histology. Scale bars 1 mm.

Article Snippet: The human A1AT polymer-specific mAb 2C1 antibody (HM2289, Hycult Biotechnology) was used for native PAGE detection of polymers.

Techniques: Sequencing, Staining

Journal: Nature Genetics

Article Title: Selection for somatic escape variants in SERPINA1 in the liver of patients with alpha-1 antitrypsin deficiency

doi: 10.1038/s41588-025-02125-1

Figure Lengend Snippet: a , Overview of the hierarchical experimental design: ten livers from patients with A1AT deficiency and hemochromatosis were sampled. These samples underwent LCM to generate 306 individual microdissections; isolated DNA was then sequenced using WGS or WES approaches. b , Top panel, mean sequencing coverage in each microdissection. Donors (M, male; F, female) are ordered by disease and age. The number of SNVs and indels detected per microdissection within the coding exome across WGS ( n = 77) and WES ( n = 229) data (for five samples for which dual WGS and WES were performed, only WGS calls are shown). Variants are colored by type. c , Genes under positive selection in A1AT deficiency and hemochromatosis cohorts. Manhattan plots showing the distribution of P values testing for gene-level non-synonymous variants in A1AT deficiency (left panel) and hemochromatosis (right panel). Genes found to be under positive selection in A1AT deficiency, hemochromatosis or, previously, in SLD are labeled. Genes are ordered by genomic position, and those with significant q values (<0.1) are highlighted in bold.

Article Snippet: The human A1AT polymer-specific mAb 2C1 antibody (HM2289, Hycult Biotechnology) was used for native PAGE detection of polymers.

Techniques: Isolation, Sequencing, Laser Capture Microdissection, Selection, Labeling

Journal: Nature Genetics

Article Title: Selection for somatic escape variants in SERPINA1 in the liver of patients with alpha-1 antitrypsin deficiency

doi: 10.1038/s41588-025-02125-1

Figure Lengend Snippet: ( a ) Upper panel: burden of single-nucleotide variants (SNVs) per exome, corrected by sensitivity of mutation detection. Each box plot represents a patient (n = 10 patients; 305 microdissections) and each dot represents one laser-capture microdissected sample. The grey-to-black intensity of the points reflects the median variant allele fraction (VAF) of mutations in each microdissection. Boxes in the box plots indicate median and interquartile range; whiskers denote range. Lower panel: burden of indel variants per exome (n = 10 patients; 305 microdissections). ( b ) Relative proportional contributions of the six most abundant SBS signatures across A1AT-deficiency (purple-headed columns) and haemochromatosis (green-headed columns) donors. Each bar represents a laser capture microdissection, in ascending order of the number of coding mutations. ( c ) Chromothripsis involving chromosomes 5 and 10 (upper panel) and chromosomes 7 and 10 (lower panel), observed in patient with haemochromatosis (PD51607). Black points represent corrected read depth along the chromosome. Lines and arcs represent structural variants, coloured by the orientation of the joined ends (purple, tail-to-tail inverted; brown, head-to-head inverted; turquoise, tandem-duplication-type orientation; green, deletion-type orientation).

Article Snippet: The human A1AT polymer-specific mAb 2C1 antibody (HM2289, Hycult Biotechnology) was used for native PAGE detection of polymers.

Techniques: Mutagenesis, Variant Assay, Laser Capture Microdissection

Journal: Nature Genetics

Article Title: Selection for somatic escape variants in SERPINA1 in the liver of patients with alpha-1 antitrypsin deficiency

doi: 10.1038/s41588-025-02125-1

Figure Lengend Snippet: a , Phylogenetic tree of clonal structure from liver sample PD51606, with colored branches showing independently acquired SERPINA1 mutations. b , Spatial mapping of the clones from the phylogenetic tree onto an H&E-stained photomicrograph of the patient’s liver biopsy, with SERPINA1 mutant clones colored to match the tree shown in a . Each dot signifies a microdissection, with solid black dots indicating LCM microdissections harboring a SERPINA1 mutation. For WGS samples, clonal cell fraction is depicted, with a fully filled disk signifying all clones detected in the LCM sample and SERPINA1 mutant cell fraction depicted by the degree of disk filling. Dashed lines indicate SERPINA1 mutations identified exclusively in WES samples. Nodules are color-highlighted, whereby all microdissections within the nodule contain the same SERPINA1 mutant clone. c , Spatial location of clones with SERPINA1 variants in the H&E-stained liver of the remaining four patients with A1AT deficiency (colored area), in addition to SERPINA1 wild-type clones (gray circles).

Article Snippet: The human A1AT polymer-specific mAb 2C1 antibody (HM2289, Hycult Biotechnology) was used for native PAGE detection of polymers.

Techniques: Clone Assay, Staining, Mutagenesis, Laser Capture Microdissection

Journal: Nature Genetics

Article Title: Selection for somatic escape variants in SERPINA1 in the liver of patients with alpha-1 antitrypsin deficiency

doi: 10.1038/s41588-025-02125-1

Figure Lengend Snippet: a , Distribution of non-synonymous variants detected in SERPINA1 in liver samples from patients with A1AT deficiency ( n = 5) with gene-level q value corrected for multiple hypothesis testing reported. The coding sequence of the gene is represented on the x axis, with exons in pink and protein domains in purple. Position of the germline PiZ variant (E366K) shown in magenta; RCL, reactive center loop. b , Effect of C-terminal variants on Z-A1AT protein sequence; sequence alignment of Z-A1AT amino acids 361–418. The reactive center loop is highlighted in blue; amino acid sequence modified by frameshift in orange; E366K (PiZ) variant indicated in magenta; stop codon denoted by *. c , Left, cartoon representing the crystal structure of native A1AT derived from Protein Data Bank (PDB 1QLP ) . The position of residues K367 (light purple) and E387 (blue) is shown by colored spheres, with downstream residues colored similarly. A linear schematic of the protein shows relative mutation positioning using the same color code (upper). Middle, the crystal structure of two protomers in a Z-A1AT polymer from PDB 3T1P , showing the insertion of donor protomer residues downstream of K367 (light purple sphere) into the acceptor protomer. Right, structural model of two protomers in a polymer (PDB 3T1P ) highlighting the incompatibility of Z-K367* and Z-E387* C-terminal donation to a polymer.

Article Snippet: The human A1AT polymer-specific mAb 2C1 antibody (HM2289, Hycult Biotechnology) was used for native PAGE detection of polymers.

Techniques: Sequencing, Variant Assay, Modification, Derivative Assay, Mutagenesis, Polymer

Journal: Nature Genetics

Article Title: Selection for somatic escape variants in SERPINA1 in the liver of patients with alpha-1 antitrypsin deficiency

doi: 10.1038/s41588-025-02125-1

Figure Lengend Snippet: ( a ) Left: Cartoon of the primary sequence of the somatic A1AT mutant Z-M398delFS mapped as a portion of the structure of native full length A1AT derived from Protein Data Bank (PDB):1QLP17. Magenta and blue regions reflect portions of the protein sequence lost in the somatic mutants K367* and E387* respectively (mutation sites modeled as spheres), as shown in Fig. . Centre: Zoomed section of A1AT beta-sheet C showing the missense F396Y and L397D residues of Z-M398delFS in black with amino acid side chains rendered. Right: A comparative zoom of full length A1AT showing the side chains of native F396 and L397, with structure downstream of the Z-M398delFS premature STOP codon rendered opaque. ( b ) Cartoon of the primary sequence of A1AT Z-M398delFS mapped onto the crystal structure of a Z-A1AT polymer from PDB:3T1P18, where Z-M398delFS is displayed as the donor protomer and the acceptor Z-protomer is grey. Residues downstream of K367 and E387 are shown in magenta and blue respectively. F396Y and L397D are shown in black. ( c ) Left panel: Zoomed polymer interface shown from ( b ) showing contacts between protomers as dashed lines (polar contacts are green, others are grey). Right panel: A corresponding map of inter-protomer contacts between two molecules of full-length Z-A1AT.

Article Snippet: The human A1AT polymer-specific mAb 2C1 antibody (HM2289, Hycult Biotechnology) was used for native PAGE detection of polymers.

Techniques: Sequencing, Mutagenesis, Derivative Assay, Polymer

Journal: Nature Genetics

Article Title: Selection for somatic escape variants in SERPINA1 in the liver of patients with alpha-1 antitrypsin deficiency

doi: 10.1038/s41588-025-02125-1

Figure Lengend Snippet: a , Fluorescence micrographs of CHO cells expressing indicated HaloTagged variants of A1AT (magenta) with the ER marker protein (moxGFP-KDEL (green)). Dashed line boxes indicate the area expanded in the zoomed-in images. Scale bar, 10 μm. b , ER inclusions (characterized by a region of fragmented tubular ER network) in CHO cells as in a ; Z versus Z-K367*, P = 0.0283; Z versus Z-K387*, P = 0.0248. c , Levels of secreted A1AT from CHO cells expressing indicated A1AT variants; Z versus Z-K367*, P = 0.0053; Z versus Z-K387*, P = 0.017. d , SDS–PAGE and native PAGE immunoblotting of soluble and insoluble cell lysate fractions from an equivalent cell mass of CHO cells expressing indicated A1AT variants. Native PAGE immunoblotting was performed using antibodies recognizing total (a0409) and polymerized (mAB 2C1 ) A1AT. SDS–PAGE immunoblotting was performed using an N-terminal directed antibody against A1AT present in all variants (MA5-15521). e , Left, exemplar photomicrograph of liver tissue immunohistochemistry for Z-A1AT polymers. Circles indicate regions with microdissected hepatocyte clones analyzed by WES or WGS (as in Fig. ) in an adjacent section with the indicated SERPINA1 genotype; right, exemplar Z-A1AT polymer immunohistochemistry from regions with the indicated SERPINA1 genotype across the A1AT-deficiency cohort. f , Quantification of Z-A1AT polymer staining intensity (as in e ) of 91 microdissected areas from five patients with A1AT deficiency; Z-A1AT versus non-truncating variant, P = 0.202; Z-A1AT versus truncating variant, P < 0.0001; ns, not significant. g , Pulse–chase time course of CHO cells expressing indicated HaloTagged A1AT variants using the HaloTag ligand. Quantitation of HaloTag-band intensity is plotted. * and # denote the significance of Z versus Z-K367* ( P = 0.012, 0.010 and 0.005 at 3, 6 and 12 h, respectively) or Z versus Z-E387* ( P = 0.805, 0.020 and 0.022 at 3, 6 and 12 h, respectively), respectively. h , Pulse–chase of HaloTagged Z-K367* in the presence of dimethylsulfoxide (vehicle), lactacystin (proteosome inhibitor, 5 µM), bafilomycin (lysosomal inhibitor, 100 nM) or both. * and # denote significance of vehicle versus lactacystin ( P = 0.597, 0.002 and <0.0001 at 1.5, 3 and 6 h, respectively) or vehicle versus both inhibitors ( P = 0.172, 0.002 and <0.0001 at 1.5, 3 and 6 h, respectively), respectively. i , Proportion of CHO cells displaying ER inclusions when co-expressing HaloTagged Z-A1AT with untagged variants of A1AT (Z + M versus Z + Z, P = 0.027; Z + Z versus Z + Z-K367*, P = 0.012; Z + Z versus Z + Z-K387*, P = 0.002; Z + M versus Z + Z-K387*, P = 0.033); n = 4 ( a , b and i ) or n = 3 ( c , d , g and h ) biological replicates for all conditions. Columns ( b , c , f and i ) and points ( g and h ) represent means ± s.e.m. Analyses by one-way ( b , c , f and i ) or two-way ( g and h ) ANOVA with Šidák’s correction for multiple hypothesis testing; ns, not significant; * P ≤ 0.05; ** P ≤ 0.01; **** P ≤ 0.0001.

Article Snippet: The human A1AT polymer-specific mAb 2C1 antibody (HM2289, Hycult Biotechnology) was used for native PAGE detection of polymers.

Techniques: Fluorescence, Expressing, Marker, SDS Page, Clear Native PAGE, Western Blot, Immunohistochemistry, Clone Assay, Polymer, Staining, Variant Assay, Pulse Chase, Quantitation Assay

Journal: Nature Genetics

Article Title: Selection for somatic escape variants in SERPINA1 in the liver of patients with alpha-1 antitrypsin deficiency

doi: 10.1038/s41588-025-02125-1

Figure Lengend Snippet: ( a ) Western blot analysis of A1AT secretion by immunoblotting of media conditioned for 24 h by CHO cells transfected with the indicated A1AT-variant constructs. Immunoblotting was performed using a monoclonal antibody raised against an N-terminal fragment of human α1-antitrypsin (MA5-15521). Images representative of n = 3 biologically independent experiments. ( b ) Micrographs of the ER marker protein mEmerald-KDEL expressed in COS7 cells (left images) with accompanying single-particle tracks of HaloTagged variants of A1AT labelled with PAJF646, colour coded by mean velocity (right images). Frequency distribution histograms show mean velocity of tracks analysed. A dashed line denotes the median velocity of M-A1AT. For each condition over 10,000 particle tracks were analysed across a minimum of 17 cells from 3 repeats. ( c ) CHO cells conditionally-expressing untagged Tetracycline-inducible (Tet-ON) Z-A1AT were transfected with expression vectors encoding HaloTagged variants of A1AT, 2 h prior to 1 µg/mL doxycycline treatment. Input and Halo-affinity purified samples were then separated by SDS-PAGE prior to immunoblotting with antibody recognising total (MA5-15521) A1AT. Images representative of n = 3 biologically independent experiments.

Article Snippet: The human A1AT polymer-specific mAb 2C1 antibody (HM2289, Hycult Biotechnology) was used for native PAGE detection of polymers.

Techniques: Western Blot, Transfection, Variant Assay, Construct, Marker, Single Particle, Expressing, Affinity Purification, SDS Page

Journal: Nature Genetics

Article Title: Selection for somatic escape variants in SERPINA1 in the liver of patients with alpha-1 antitrypsin deficiency

doi: 10.1038/s41588-025-02125-1

Figure Lengend Snippet: ( a ) Left: Homozygous MM- SERPINA1 leads to expression, translation and secretion of functional A1AT protein; middle: Homozygous ZZ- SERPINA1 leads to translation and subsequent polymerisation of Z-A1AT, leading to ER disruption. Right: Acquisition of a somatic escape variant in a single Z- SERPINA1 allele leading to missense or truncation of the C-terminus reduces Z-A1AT polymer accumulation and ER disruption, providing a selective advantage compared to neighbouring hepatocytes with ZZ- SERPINA1 .

Article Snippet: The human A1AT polymer-specific mAb 2C1 antibody (HM2289, Hycult Biotechnology) was used for native PAGE detection of polymers.

Techniques: Expressing, Functional Assay, Disruption, Variant Assay, Polymer

Journal: bioRxiv

Article Title: Organobodies: A robust and size-controllable system for generating scalable hiPSC-derived liver organoids for drug toxicity screening

doi: 10.1101/2025.10.20.683433

Figure Lengend Snippet: A) Hematoxylin and eosin (H&E) staining of hepatic OBs 13 days after encapsulation of day 8 hepatic progenitors derived from the HC3X line, showing morphology, cellular distribution, and structural organization of HLCs. B) Immunohistochemical staining of OB sections 13 days post-encapsulation, demonstrating expression of key hepatocyte-specific markers: HNF4α, HNF6, CK18, PCK2, CYP3A4, ALB, ASGR1, A1AT, NTCP, and MRP2. The cholangiocyte marker CK7 was also detected. C) Immunostaining for ECM components laminin α5 and collagen I, and structural protein β-actin, revealed organized HLC clustering within the SAP hydrogel, indicative of matrix remodelling and ECM deposition. Staining for activated caspase-3 (aCASP3) confirmed minimal apoptosis within OBs. Nuclei were counterstained with DAPI. All images are representative of at least three independent experiments.

Article Snippet: Albumin secretion was measured using the Human Albumin DuoSet ELISA Kit, and alpha-1 antitrypsin (A1AT) levels were measured using the Human A1AT DuoSet ELISA Kit (both from R&D Systems), following the manufacturer’s instructions.

Techniques: Staining, Encapsulation, Derivative Assay, Immunohistochemical staining, Expressing, Marker, Immunostaining

Journal: bioRxiv

Article Title: Organobodies: A robust and size-controllable system for generating scalable hiPSC-derived liver organoids for drug toxicity screening

doi: 10.1101/2025.10.20.683433

Figure Lengend Snippet: A) Comparative RT-qPCR analysis between 2D HLC cultures ( N = 1-3 independent differentiations, n = 3-9 technical replicates) and 3D hepatic OBs ( N = 3-7 independent differentiation, n = 9-21 technical replicates) generated from HC3X line for 11 key markers at early (day 20) and late (days 37 – 40) stages of differentiation in comparison with 2D HepG2 cells and freshly isolated PHHs (from 2-3 individual batch). RPL19 was used as housekeeping gene to normalise gene expression. Fold change has been calculated against the expression of the same genes in undifferentiated HC3X hiPSC. To avoid overcrowded graphs, hiPSC and HepG2 cells have been excluded from statistical analysis. B) Albumin secretion levels in OBs versus 2D HLCs at day 40 of differentiation, measured by ELISA (N = 3; n = 9–12). C) A1AT secretion in the culture medium by hepatic OBs and 2D HLC cultures at days 20, 30, 40, 50, and 60 of differentiation measured by ELISA. The secretion level is significantly higher for OB cultures at days 40 and 50. ( N = 3, n = 9-12). D) qPCR analysis showing significant increased expression of CYP3A4 in 3D hepatic OBs following induction with 10 μM Rifampicin compared to vehicle (DMSO) and untreated control ( N = 2, n = 6). Statistical significance was determined at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 . Data are presented as mean ± SD.

Article Snippet: Albumin secretion was measured using the Human Albumin DuoSet ELISA Kit, and alpha-1 antitrypsin (A1AT) levels were measured using the Human A1AT DuoSet ELISA Kit (both from R&D Systems), following the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Generated, Comparison, Isolation, Gene Expression, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Nature Communications

Article Title: Stereo-random oligonucleotides enable efficient recruitment of ADAR in vitro and in vivo

doi: 10.1038/s41467-025-64434-7

Figure Lengend Snippet: A Repetitive 2´ modification of large stretches of nucleosides of an ON targeting a GAPDH ORF site. B Partial 2´ modification, only at pyrimidine nucleosides. C Representative assessment of ON stability with an ON degradation assay in 100% FBS (Comprehensive, uncropped images are in Source data file). D Editing performance of partially stabilized ON in RPE, NHA and NHBE cells. GAPDH ORF target. Interferon was only applied with the v25.1 ON. E Gymnotic uptake of ON v117.28 (1 µM and 0.2 µM) in HeLa cells. GAPDH ORF target. F Repair of the SERPINA1 E342K mutation causing α-1-antitrypsin (A1AT) deficiency in a HeLa cell model stably overexpressing SERPINA1 E342K cDNA. Editing yields and normalized total A1AT levels in the supernatant of SERPINA1 E342K-piggyBac HeLa cells after treatment with partially modified ON designed to repair the SERPINA1 E342K mutation. The mock ON was the GAPDH ORF-targeting ON v117.19 (see Supplementary Table ). Data in A, B, D-F is shown as the mean ± s.d., N = 3 independent experiments. Experiments in A , B , E and F were done in HeLa cells. All editing experiments were performed with 50 nM ON, each datapoint represents a biological replicate.

Article Snippet: Goat anti-human A1AT antibody-HRP conjugate (Bethyl Laboratories, A80-122P) was applied at 0.5 μg/mL in PBS (100 μL) and incubated at room temperature for 1 h with gentle shaking (50 rpm).

Techniques: Modification, Degradation Assay, Mutagenesis, Stable Transfection

Journal: Nature Communications

Article Title: Stereo-random oligonucleotides enable efficient recruitment of ADAR in vitro and in vivo

doi: 10.1038/s41467-025-64434-7

Figure Lengend Snippet: A Treatment scheme of the PiZZ (SERPINA1 E342K) A1AT deficiency mouse model. Mice were treated with a single dose of 0–5 mg/kg LNP-encapsulated SERPINA1 E342K-targeting ON v117.82 (40 nt, 5’-29-1-10 symmetry, 5’-C A A) via tail vein injection. LNP formulation was based on the FDA-approved siRNA drug Onpattro. Mice were sacrificed 3 days post injection. B Sanger editing yields from hepatocytes (blue) and serum A1AT (human, wildtype) concentrations (yellow) clearly show a dose dependent correction at RNA and protein level. The considered therapeutic threshold of 11 µM serum A1AT (ref. ) is depicted as the dotted horizontal line. C Correlation of in vivo editing yields and resulting wildtype A1AT serum concentration. Data in B is shown as the mean ± s.d., N = 2–5 independent animals per condition. Figure 5A contains an icon created in BioRender (Stafforst, T. (2025), https://BioRender.com/zfhahgx .

Article Snippet: Goat anti-human A1AT antibody-HRP conjugate (Bethyl Laboratories, A80-122P) was applied at 0.5 μg/mL in PBS (100 μL) and incubated at room temperature for 1 h with gentle shaking (50 rpm).

Techniques: Injection, Formulation, In Vivo, Concentration Assay