Fig 2: Mutations in TOB2B cause peripheral B-cell immunodeficiency and dysmorphic features. a Pedigrees in three unrelated families with variants in TOP2B. Filled symbols, subjects with mutation; open symbols, unaffected subjects. Arrows indicate individuals subjected to whole genome sequencing. Three variants were identified: a missense mutation c.1448C > T, p.S483L, a de novo inframe deletion c.1761_1763AGAdel p.EE587E, a missense mutation c.1897G > A, p. G633S. b Modeling of the identified variants in protein domains. Linear modeling shows the approximate location of patient mutations in the TOPRIM domain. c 3D modeling with green and blue helices representing two domains of the homodimer, and black and white spheres representing the DNA helix in the binding site. Mutations are displayed in red in the structure of the TOPRIM domain

Fig 3: Heterozygous mutation in Top2b negatively affects B-cell development beyond haploinsufficiency. Expression of mutant Top2b (n = 7) results in reduced CD19+ B cells (a) in peripheral blood compared to wild-type (n = 6) or hemizygous mice (n = 6), while T cells (b) are unaffected.(shown as mean ± SEM, *p < 0.05, **p < 0.005, two-tailed Student’s t test). c qPCR analysis of kappa-deleting recombination excision circles demonstrates that B cells isolated from Top2b+/EE587E (n = 7) or Top2b+/- mice (n = 7) have a less robust replication history compared to wild-type mice (n = 8 mice), shown as mean ± SEM, while T-cell proliferation as measured by T-cell receptor excision circle (TREC) formation is unaffected (d), (n = 6 WT, 6 mutant and 4 hemizygous mice, performed with technical duplicates shown as mean ± SEM). *p < 0.05, **p < 0.005, one-way ANOVA. e–g Comet assay shows increased DNA strand breaks in B cells (e, f) isolated from Top2b+/EE587E mice compared to wildtype, expressed as comet tail length. T cells (g) are not affected (n = 3 mice per group, 10 images per mouse (X40); *p < 0.05; ****p < 0.0001, by Kruskal–Wallis test (f), or nonsignificant (n.s.) by Student’s t test (g)). h, i Expression of Top2b is elevated in wild-type B cells compared to wild-type T cells by RT-qPCR (h, n = 5 mice, performed in technical triplicates, shown as mean ± SEM, *p < 0.05, two-tailed Student’s t test), and immunoblot (i, protein isolated from B and T cells from three wild-type mice, representative of four experiments; source data are provided as a Source Data file)

Fig 4: B cell development is negatively affected whenTOP2B is mutated. a Spleen to body weight ratio for wild-type (n = 3), Top2b-/--mb1 cre (n = 3), and Top2b+/EE587E mice (n = 4) reveal smaller ratios in knockin and knockout mice compared to controls (shown as mean ± SEM). *p < 0.05 by Kruskal–Wallis test. b Total serum IgG is reduced in Top2b-/- mb1 cre mice (n = 6) and Top2b+/EE587E (n = 4) mice compared to controls (n = 7, shown as mean ± SEM). *p < 0.05 by Kruskal–Wallis test. c, d Heterozygous mutant mice fail to produce specific IgG antibody in response to vaccination with ovalbumin (OVA, n = 4, (c) or pneumovax-23 (d) (n = 3 mice per group, shown as mean ± SEM, *p < 0.05 by the Kruskal–Wallis test). Bone marrow (e) and spleen (f) cells were isolated from Top2b+/EE587E mice and littermate controls. Phenotypic analysis by flow cytometry demonstrates that the bone marrow of Top2b+/EE587E mice (n = 3) have fewer PrePro B cells (Fr. A, B220+, CD43-, BP1-, CD24-), Pre B cells (Fr. D, B220+, CD43-, IgD-, IgM-), and immature B cells (Fr. E, CD19+, B220+, CD43-, IgM+, IgD-) compared to control mice (e). In the spleen (f), there are overall fewer B cells with significant reductions in transitional (CD19+, B220+, IgDlo, IgM+, CD21/35int, CD23 variable), follicular (CD19+, B220+, IgMlo, IgDhi, CD38+) and activated (CD19+, B220+, IgDhi, IgMhi, Flt3+) B cells compared to control mice (n = 4, shown as mean ± SEM, absolute numbers calculated from whole tissue numbers). *p < 0.05; **p < 0.01, two-tailed Student’s t test. Expression of mutant Top2b results does not affect E2A (g, n = 6)) or Rag1 (h, n = 8) expression in common lymphoid progenitor cells (compared to WT n = 6), whereas B cell-specific transcription factors Pax5 (i) and Foxo1 (j) show reduced expression of compared to wild-type mice. (n = 4 mice per group, shown as mean ± SEM, *p < 0.05, two-tailed Student’s t test)

Fig 5: B cells are functionally impaired in the absence of Top2b, in vivo and in vitro. a Mice fail to produce specific IgG antibody in response to vaccination with ovalbumin (OVA) in the absence of Top2b in B cells (n = 9 mice per group, shown as mean ± SEM, ***p < 0.005 by the Kruskal–Wallis test). b–d Adult Top2b-/- mb1 cre and littermate mice were immunized with Pneumovax-23 (PPV23). At 6 days post-immunization (b), B cells produce PPV23-specific IgM in the absence of TOP2B, at similar levels to wild-type control mice. Four weeks after immunization with Pneumovax-23, anti-PPV23 IgG3 (c) and IgG2b (d) production is impaired in Top2b-/- mb1-cre mice compared to wild-type control animals (n = 6 WT or 5 Top2b-/- mb1 cre, shown as mean ± SEM, *p < 0.05 by the Kruskal–Wallis test). e In vitro, Top2b-/- mb1 cre mouse-derived B cells fail to proliferate, as assessed by CFSE dilution, in the presence of stimulation with LPS (blue histogram). Untreated, CFSE stained cells are shown in red. FACS plots are representative of 4 mice per group. Total number of CFSE positive cells is quantified in (f). g Top2b-/- mb1-cre mouse-derived B cells have increased cell death as measured by LDH release (n = 7 WT or 8 Top2b-/- mb1 cre mice per group, in duplicate, shown as mean ± SEM). *p < 0.05, two-tailed Student’s t test. h The B cells that do survive have reduced upregulation of the activation marker MHC II, as shown by mean fluorescence intensity (MFI). *p < 0.05, two-tailed Student’s t test (n = 4 mice per group)