Consuelo Anzilotti

Mutations in PIGY: expanding the phenotype of inherited glycosylphosphatidylinositol deficiencies

Glycosylphosphatidylinositol (GPI)-anchored proteins are ubiquitously expressed in the human body and are important for various functions at the cell surface. Mutations in many GPI biosynthesis genes have been described to date in patients with multi-system disease and together these constitute a subtype of congenital disorders of glycosylation. We used whole exome sequencing in two families to investigate the genetic basis of disease and used RNA and cellular studies to investigate the functional consequences of sequence variants in the PIGY gene. Two families with different phenotypes had homozygous recessive sequence variants in the GPI biosynthesis gene PIGY. Two sisters with c.137T>C (p.Leu46Pro) PIGY variants had multi-system disease including dysmorphism, seizures, severe developmental delay, cataracts and early death. There were significantly reduced levels of GPI-anchored proteins (CD55 and CD59) on the surface of patient-derived skin fibroblasts (∼20–50% compared with controls). In a second, consanguineous family, two siblings had moderate development delay and microcephaly. A homozygous PIGY promoter variant (c.-540G>A) was detected within a 7.7 Mb region of autozygosity. This variant was predicted to disrupt a SP1 consensus binding site and was shown to be associated with reduced gene expression. Mutations in PIGY can occur in coding and non-coding regions of the gene and cause variable phenotypes. This article contributes to understanding of the range of disease phenotypes and disease genes associated with deficiencies of the GPI-anchor biosynthesis pathway and also serves to highlight the potential importance of analysing variants detected in 5′-UTR regions despite their typically low coverage in exome data.

Chronic mucocutaneous candidiasis: characterization of a family with STAT‐1 gain‐of‐function and development of an ex‐vivo assay for Th17 deficiency of diagnostic utility

Chronic mucocutaneous candidiasis (CMC) is characterized by recurrent and persistent superficial infections, with Candida albicans affecting the mucous membranes, skin and nails. It can be acquired or caused by primary immune deficiencies, particularly those that impair interleukin (IL)−17 and IL‐22 immunity. We describe a single kindred with CMC and the identification of a STAT1 GOF mutation by whole exome sequencing (WES). We show how detailed clinical and immunological phenotyping of this family in the context of WES has enabled revision of disease status and clinical management. Together with analysis of other CMC cases within our cohort of patients, we used knowledge arising from the characterization of this family to develop a rapid ex‐vivo screening assay for the detection of T helper type 17 (Th17) deficiency better suited to the routine diagnostic setting than established in‐vitro techniques, such as intracellular cytokine staining and enzyme‐linked immunosorbent assay (ELISA) using cell culture supernatants. We demonstrate that cell surface staining of unstimulated whole blood for CCR6+CXCR3–CCR4+CD161+ T helper cells generates results that correlate with intracellular cytokine staining for IL‐17A, and is able to discriminate between patients with molecularly defined CMC and healthy controls with 100% sensitivity and specificity within the cohort tested. Furthermore, removal of CCR4 and CD161 from the antibody staining panel did not affect assay performance, suggesting that the enumeration of CCR6+CXCR3–CD4+ T cells is sufficient for screening for Th17 deficiency in patients with CMC and could be used to guide further investigation aimed at identifying the underlying molecular cause.

Analysis of exome data for 4293 trios suggests GPI-anchor biogenesis defects are a rare cause of developmental disorders

Over 150 different proteins attach to the plasma membrane using glycosylphosphatidylinositol (GPI) anchors. Mutations in 18 genes that encode components of GPI-anchor biogenesis result in a phenotypic spectrum that includes learning disability, epilepsy, microcephaly, congenital malformations and mild dysmorphic features. To determine the incidence of GPI-anchor defects, we analysed the exome data from 4293 parent–child trios recruited to the Deciphering Developmental Disorders (DDD) study. All probands recruited had a neurodevelopmental disorder. We searched for variants in 31 genes linked to GPI-anchor biogenesis and detected rare biallelic variants in PGAP3, PIGN, PIGT (n=2), PIGO and PIGL, providing a likely diagnosis for six families. In five families, the variants were in a compound heterozygous configuration while in a consanguineous Afghani kindred, a homozygous c.709G>C; p.(E237Q) variant in PIGT was identified within 10–12u2009Mb of autozygosity. Validation and segregation analysis was performed using Sanger sequencing. Across the six families, five siblings were available for testing and in all cases variants co-segregated consistent with them being causative. In four families, abnormal alkaline phosphatase results were observed in the direction expected. FACS analysis of knockout HEK293 cells that had been transfected with wild-type or mutant cDNA constructs demonstrated that the variants in PIGN, PIGT and PIGO all led to reduced activity. Splicing assays, performed using leucocyte RNA, showed that a c.336-2A>G variant in PIGL resulted in exon skipping and p.D113fs*2. Our results strengthen recently reported disease associations, suggest that defective GPI-anchor biogenesis may explain ~0.15% of individuals with developmental disorders and highlight the benefits of data sharing.

Mutation of Fnip1 is associated with B-cell deficiency, cardiomyopathy, and elevated AMPK activity

Significance Cellular metabolism is tightly regulated by AMP-activated protein kinase (AMPK): the function of which is influenced by folliculin (FLCN), folliculin-interacting protein (FNIP)1, and FNIP2. FLCN is a known tumor-suppressor protein that is mutated in Birt–Hogg–Dubé syndrome, whereas FNIP1 and FNIP2 are binding partners of FLCN. Previous reports have suggested that the FLCN/FNIP1/FNIP2 complex acts a positive regulator of AMPK, whereas other reports suggest the opposite. Using a new mouse model of FNIP1 deficiency, our findings support the latter: we found that mutation of Fnip1 leads to B-cell deficiency and the development of a cardiomyopathy similar to mice and humans with gain-of-function mutations in AMPK. Folliculin (FLCN) is a tumor-suppressor protein mutated in the Birt–Hogg–Dubé (BHD) syndrome, which associates with two paralogous proteins, folliculin-interacting protein (FNIP)1 and FNIP2, forming a complex that interacts with the AMP-activated protein kinase (AMPK). Although it is clear that this complex influences AMPK and other metabolic regulators, reports of its effects have been inconsistent. To address this issue, we created a recessive loss-of-function variant of Fnip1. Homozygous FNIP1 deficiency resulted in profound B-cell deficiency, partially restored by overexpression of the antiapoptotic protein BCL2, whereas heterozygous deficiency caused a loss of marginal zone B cells. FNIP1-deficient mice developed cardiomyopathy characterized by left ventricular hypertrophy and glycogen accumulation, with close parallels to mice and humans bearing gain-of-function mutations in the γ2 subunit of AMPK. Concordantly, γ2-specific AMPK activity was elevated in neonatal FNIP1-deficient myocardium, whereas AMPK-dependent unc-51–like autophagy activating kinase 1 (ULK1) phosphorylation and autophagy were increased in FNIP1-deficient B-cell progenitors. These data support a role for FNIP1 as a negative regulator of AMPK.

Capturing resting T cells: the perils of PLL

Supported by a Royal Society University Research Fellowship (UF120277 to S.F.L.) and Research Professorship (RP150066 to D.K.); the EPSRC (EP/L027631/1 to A.P.,); the Wellcome Trust (098274/Z/12/Z to S.J.D., and WT101609MA to R.A.F.); PA Cephalosporin Fund (C.E.); the Wolfson Imaging Centre Oxford (funded by the Wolfson Foundation and Wellcome Trust; 104924/14/Z/14); the Micron Advanced BioImaging Unit (Wellcome Trust Strategic Award 091911); the Medical Research Council (MC_UU_12010/Unit Programmes G0902418 and MC_UU_12025); an MRC/BBSRC/EPSRC award (MR/K01577X/1); and a Marie Sklodowska-Curie Intra-European grant (707348 to I.U.).

53BP1 cooperation with the REV7–shieldin complex underpins DNA structure-specific NHEJ

Abstract53BP1 governs a specialized, context-specific branch of the classical non-homologous end joining DNA double-strand break repair pathway. Mice lacking 53bp1 (also known as Trp53bp1) are immunodeficient owing to a complete loss of immunoglobulin class-switch recombination1,2, and reduced fidelity of long-range V(D)J recombination3. The 53BP1-dependent pathway is also responsible for pathological joining events at dysfunctional telomeres4, and its unrestricted activity in Brca1-deficient cellular and tumour models causes genomic instability and oncogenesis5–7. Cells that lack core non-homologous end joining proteins are profoundly radiosensitive8, unlike 53BP1-deficient cells9,10, which suggests that 53BP1 and its co-factors act on specific DNA substrates. Here we show that 53BP1 cooperates with its downstream effector protein REV7 to promote non-homologous end joining during class-switch recombination, but REV7 is not required for 53BP1-dependent V(D)J recombination. We identify shieldin—a four-subunit putative single-stranded DNA-binding complex comprising REV7, c20orf196 (SHLD1), FAM35A (SHLD2) and FLJ26957 (SHLD3)—as the factor that explains this specificity. Shieldin is essential for REV7-dependent DNA end-protection and non-homologous end joining during class-switch recombination, and supports toxic non-homologous end joining in Brca1-deficient cells, yet is dispensable for REV7-dependent interstrand cross-link repair. The 53BP1 pathway therefore comprises distinct double-strand break repair activities within chromatin and single-stranded DNA compartments, which explains both the immunological differences between 53bp1- and Rev7- deficient mice and the context specificity of the pathway.The specificity of 53BP1 and its co-factors for particular DNA substrates during non-homologous end joining (NHEJ) derives from REV7–shieldin, a four-subunit DNA-binding complex that is required for REV7-dependent NHEJ but not for REV7-dependent DNA interstrand cross-link repair.

Themis2 lowers the threshold for B cell activation during positive selection

The positive and negative selection of lymphocytes by antigen is central to adaptive immunity and self-tolerance, yet how this is determined by different antigens is not completely understood. We found that thymocyte-selection-associated family member 2 (Themis2) increased the positive selection of B1 cells and germinal center B cells by self and foreign antigens. Themis2 lowered the threshold for B–cell activation by low-avidity, but not high-avidity, antigens. Themis2 constitutively bound the adaptor protein Grb2, src-kinase Lyn and signal transducer phospholipase γ2 (PLC-γ2), and increased activation of PLC-γ2 and its downstream pathways following B cell receptor stimulation. Our findings identify a unique function for Themis2 in differential signaling and provide insight into how B cells discriminate between antigens of different quantity and quality.

A homozygous variant disrupting the PIGH start-codon is associated with developmental delay, epilepsy, and microcephaly

Defective glycosylphosphatidylinositol (GPI)‐anchor biogenesis can cause a spectrum of predominantly neurological problems. For eight genes critical to this biological process, disease associations are not yet reported. Scanning exomes from 7,833 parent–child trios and 1,792 singletons from the DDD study for biallelic variants in this gene‐set uncovered a rare PIGH variant in a boy with epilepsy, microcephaly, and behavioral difficulties. Although only 2/2 reads harbored this c.1A > T transversion, the presence of ∼25 Mb autozygosity at this locus implied homozygosity, which was confirmed using Sanger sequencing. A similarly‐affected sister was also homozygous. FACS analysis of PIGH‐deficient CHO cells indicated that cDNAs with c.1A > T could not efficiently restore expression of GPI‐APs. Truncation of PIGH protein was consistent with the utilization of an in‐frame start‐site at codon 63. In summary, we describe siblings harboring a homozygous c.1A > T variant resulting in defective GPI‐anchor biogenesis and highlight the importance of exploring low‐coverage variants within autozygous regions.

Immune Checkpoints as Therapeutic Targets in Autoimmunity

Antibodies that block the immune checkpoint receptors PD1 and CTLA4 have revolutionized the treatment of melanoma and several other cancers, but in the process, a new class of drug side effect has emerged—immune related adverse events. The observation that therapeutic blockade of these inhibitory receptors is sufficient to break self-tolerance, highlights their crucial role in the physiological modulation of immune responses. Here, we discuss the rationale for targeting immune checkpoint receptors with agonistic agents in autoimmunity, to restore tolerance when it is lost. We review progress that has been made to date, using Fc-fusion proteins, monoclonal antibodies or other novel constructs to induce immunosuppressive signaling through these pathways. Finally, we explore potential mechanisms by which these receptors trigger and modulate immune cell function, and how understanding these processes might shape the design of more effective therapeutic agents in future.

Themis2: setting the threshold for B-cell selection

The strength of signal downstream of the B-cell receptor (BCR) determines the positive and negative selection of B cells. Expression of a functional BCR is required for the selection of immature B cells into the peripheral repertoire in the absence of antigen; but selection is also modulated by avidity and affinity-dependent interactions with self and foreign antigens. These antigendependent events shape the B-cell repertoire during ontogeny, development and the response to pathogens, and generation of B-cell memory; however, the mechanisms underlying these processes are not very well understood. During early ontogeny, interactions with intracellular or low abundance self-antigens allow positive selection of innate-like B1 B cells.1 In contrast, the presence of abundant soluble or lowavidity self-antigen leads to B-cell anergy, whereas stronger signals from highly abundant or multivalent antigens trigger receptor editing and/or negative selection of immature B cells through central tolerance.2 These processes must be determined by the state and environment of the B cells, defined in part by the presence of co-stimulation or other signals. Later in the periphery, antigens of similar affinity or avidity elicit B-cell activation, leading to rapid proliferation, plasma cell differentiation and antibody production. During an immune response to pathogens, the majority of B-cell activation involves interactions with lower avidity and/or low-affinity antigens in a soluble, potentially tolerogenic form or coupled via complement or Fc receptors, notably arrayed on the membranes of macrophages, B cells and follicular dendritic cells.3,4 However, despite the importance of antigen quality and quantity in modulating B-cell responses, very little is understood about the process that defines the signalling threshold between positive and negative selection, allowing B cells to discriminate between antigens of varying strength and form. In a recent paper published in Nature Immunology, Cheng et al.5 show how Themis2, a member of the newly described Themis-family of proteins, modulates the positive selection of B cells in response to self and foreign antigen. Themis2 determines the threshold for activation of B cells by lowaffinity and low-avidity ligands via PLCγ2 activation and ERK1/2-dependent pathways (Figure 1). These effects were detected using the immunoglobulin transgenic MD4 mouse model and the cognate antigen ‘hen egg lysozyme’ (HEL). Themis2 has no known catalytic domain and potentially functions as an adaptor protein by strengthening the interactions between PLCγ2 and the upstream kinase Lyn. Loss of Themis2 raises the threshold for the antigeninduced upregulation of CD69, which is required for the retention and proliferation of activated B cells in the lymph node, and reduces the expression of CD86, which is required for receiving ‘co-stimulatory’ T-cell help. Therefore, Themis2 may play an important part in the initial immune response, by increasing the sensitivity of naive B cells to rare and low-affinity antigens. The Themis-family of proteins is evolutionarily conserved in metazoans from mammals to cnidarians and is characterized by one or more copies of a novel cysteine containing globular CABIT domain.6 Themis2 has two tandem CABIT domains and has two close mammalian homologues: Themis1, expressed primarily in T cells, and Themis3, expressed in the intestine. In addition, mammals express two family members of unknown function, with single copies of the CABIT domain and a proline-rich region, Fam59A (Garem) and FAM59B. Like Themis2, Themis1 is proposed to be an analogue-to-digital convertor, which translates low-affinity signals into a selection event during the positive and negative selection of CD4+ CD8+ thymocytes. In the absence of Themis1, T-cell development is blocked at the CD4+CD8lo positive selection point.6–9 Themis1 is constitutively associated with Grb2 via its proline-rich domain and is rapidly phosphorylated by Lck and ZAP70 and recruited to LAT upon T-cell activation. Although research to elucidate the function of Themis1 demonstrates our MRC Human Immunology Unit, Weatherall Institute for Molecular Medicine, Nuffield Department of Medicine, University of Oxford, Oxford OX3 9DS, UK Correspondence: Dr RJ Cornall, MD, PhD, MRC Human Immunology Unit, Weatherall Institute for Molecular Medicine, Nuffield Department of Medicine, University of Oxford, OX3 9DS, UK E-mail: richard.cornall@ndm.ox.ac.uk Received: 12 April 2017; Accepted: 21 April 2017 Cellular & Molecular Immunology (2017) 14, 643–645 & 2017 CSI and USTC All rights reserved 2042-0226/17