Identification of germline monoallelic mutations in IKZF2 in patients with immune dysregulation

Helios, encoded by IKZF2, is a member of the Ikaros family of transcription factors with pivotal roles in T-follicular helper, NK- and T-regulatory cell physiology. Somatic IKZF2 mutations are frequently found in lymphoid malignancies. Although germline mutations in IKZF1 and IKZF3 encoding Ikaros and Aiolos have recently been identified in patients with phenotypically similar immunodeficiency syndromes, the effect of germline mutations in IKZF2 on human hematopoiesis and immunity remains enigmatic. We identified germline IKZF2 mutations (one nonsense (p.R291X)- and 4 distinct missense variants) in six patients with systemic lupus erythematosus, immune thrombocytopenia or EBV-associated hemophagocytic lymphohistiocytosis. Patients exhibited hypogammaglobulinemia, decreased number of T-follicular helper and NK cells. Single-cell RNA sequencing of PBMCs from the patient carrying the R291X variant revealed upregulation of proinflammatory genes associated with T-cell receptor activation and T-cell exhaustion. Functional assays revealed the inability of HeliosR291X to homodimerize and bind target DNA as dimers. Moreover, proteomic analysis by proximity-dependent Biotin Identification revealed aberrant interaction of 3/5 Helios mutants with core components of the NuRD complex conveying HELIOS-mediated epigenetic and transcriptional dysregulation.Peer reviewe

Severe congenital neutropenia due to G6PC3 deficiency: early and delayed phenotype of a patient

Abstract Background Severe Congenital Neutropenia type 4 (SCN4), is a rare autosomal recessive condition, due to mutations in the G6PC3 gene. The phenotype comprises neutropenia of variable severity and accompanying anomalies. Case presentation We report a male patient with confirmed G6PC3 deficiency presented with recurrent bacterial infections and multi-systemic complications. Our case was the first with a novel homozygous frameshift mutation in G6PC3. The patient demonstrated large platelets on his peripheral blood smear which is a rare presentation of this disease. Conclusion As SCN4 patients could be easily missed, it is recommended to consider G6PC3 mutation for any case of congenital, unexplained neutropenia

Novel Frameshift Autosomal Recessive Loss-of-Function Mutation inSMARCD2Encoding a Chromatin Remodeling Factor Mediates Granulopoiesis

Purpose Recently, a new form of congenital neutropenia that is caused by germline biallelic loss-of-function mutations in theSMARCD2gene was described in four patients. Given the rarity of the condition, the clinical spectrum of the disease has remained elusive. We here report a new patient with a novel frameshift mutation and compare our patient with the previously reportedSMARCD2-mutant patients, aiming to provide a more comprehensive understanding of the natural course of the disease. Methods Clinical and laboratory findings of all reported patients were reviewed. Next-generation sequencing was performed to identify the causative genetic defect. Data on the hematopoietic stem cell transplantation including stem cell sources, conditioning regimen, engraftment, graft-versus-host disease, and infections were also collected. Results An 11-year-old female patient had a variety of infections including sepsis, deep tissue abscesses, otitis, pneumonia, gingivitis, and diarrhea since infancy. A novel homozygous mutation inSMARCD2(c.93delG, p.Ala32Argfs*80) was detected. Bone marrow examination showed hypocellularity and decreased neutrophils with diminished granules and myeloid dysplasia, but no blast excess as in previously reported patients. The neutropenia was non-responsive even to higher doses of granulocyte colony-stimulating factor (G-CSF); therefore, the patient was transplanted at 10 years of age from a HLA-A allele-mismatched unrelated donor using a reduced toxicity conditioning regimen and recovered successfully. Compared with the previous four cases, our patient showed longer survival before transplantation without blastic transformation. Conclusion Distinctive myeloid features and long-term follow-up including therapy options are presented for the newly described case of SMARCD2 deficiency. This disorder is apparent at infancy and requires early transplantation due to the unrelenting disease course despite conventional therapy

Immune Repertoire Profiling Reveals that Clonally Expanded B and T Cells Infiltrating Diseased Human Kidneys Can Also Be Tracked in Blood

<div><p>Recent advances in high-throughput sequencing allow for the competitive analysis of the human B and T cell immune repertoire. In this study we compared Immunoglobulin and T cell receptor repertoires of lymphocytes found in kidney and blood samples of 10 patients with various renal diseases based on next-generation sequencing data. We used Biomed-2 primer panels and ImmunExplorer software to sequence, analyze and compare complementarity determining regions and V-(D)-J elements. While generally an individual’s renal receptor repertoire is different from the repertoire present in blood, 94% (30/32) of the lymphocytes with clonal expansion in kidney can also be traced in blood however, not all of these clonotypes are equally abundant. Summarizing the data of all analyzed patients, 68% of highly expanded T cell clonotypes and 30% of the highly expanded B cell clonotypes that have infiltrated the kidney can be found amongst the five most abundant clonotypes in blood. In addition, complementarity determining region 3 sequences of the immunoglobulin heavy chains are on average more diverse than T cell receptor beta chains. Immune repertoire analysis of tissue infiltrating B and T cells adds new approaches to the assessment of adaptive immune response in kidney diseases. Our data suggest that expanded clonotypes in the tissues might be traceable in blood samples in the course of treatment or the natural history of the disease.</p></div

Quantification of element frequency (%) in highly expanded clonotypes.

<p>(A) VH, DH and JH element distribution of expanded B cell clonotypes and (B) Vß, Dß and Jß element distribution of expanded T cell clonotypes. We would appreciate the creation of a publicly available database with common V-(D)-J element frequency distribution in human populations to identify potential shifts in the course of diseases as to our knowledge this has not been provided to the community until this date.</p

Average percentage of blood and kidney lymphocyte subpopulations in all patients.

<p>T cells were separately analyzed for CD4⁺ and CD8⁺ subpopulations. NKT cells were gated using CD3⁺/CD56⁺ and NK cells, by CD3^-/CD56⁺. Cells presenting CD19⁺ and/or CD20⁺ surface markers were categorized as B cells. Significant differences were marked by a connecting line and the corresponding P-value (paired two-sided t-test).</p

CDR3 sequences, V-(D)-J elements and functionality of highly expanded clonotypes in kidney and blood of all patients.

<p>Percent values for expanded clonotypes in kidney and blood samples are highlighted in bold. Expanded clonotypes that could not be detected in the other compartment were marked as not detected, n.d. Clonotypes are also categorized according to primer sets regarding Biomed-2 primer panel [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143125#pone.0143125.ref015" target="_blank">15</a>]. B cell results from patient 6 were discarded due to low amount of DNA input leading to inaccurate clonotype proportions. T cell results from blood sample of patient 10 were sorted in CD4⁺/CD8^- and CD4^-/CD8⁺ subpopulations. The T helper cell subpopulation showed no expansions and is therefore not listed (we found a very low abundance of the expanded clonotype in the kidney in this sample and we assume that these are traces from the CD4^-/CD8⁺ population and therefore this sample was discarded). Functionality of the rearranged chain is marked P for productive and UP for unproductive.</p><p>CDR3 sequences, V-(D)-J elements and functionality of highly expanded clonotypes in kidney and blood of all patients.</p

Inverse Simpson’s diversity index and cell input.

<p>(a) IGH and TRB diversity of the average of all patients and healthy volunteers based on the inverse Simpson’s diversity index. Independent two-sided t test were performed with a level of significance of 0.05. (b) Average cell input of all patients and healthy volunteers for B and T cells. Paired two-sided t test were performed with a level of significance of 0.05. Error bars represent the standard deviation between samples of the same group.</p