Tim Niehues

Hotspot Mutations in H3F3A and IDH1 Define Distinct Epigenetic and Biological Subgroups of Glioblastoma

Glioblastoma (GBM) is a brain tumor that carries a dismal prognosis and displays considerable heterogeneity. We have recently identified recurrent H3F3A mutations affecting two critical amino acids (K27 and G34) of histone H3.3 in one-third of pediatric GBM. Here, we show that each H3F3A mutation defines an epigenetic subgroup of GBM with a distinct global methylation pattern, and that they are mutually exclusive with IDH1 mutations, which characterize a third mutation-defined subgroup. Three further epigenetic subgroups were enriched for hallmark genetic events of adult GBM and/or established transcriptomic signatures. We also demonstrate that the two H3F3A mutations give rise to GBMs in separate anatomic compartments, with differential regulation of transcription factors OLIG1, OLIG2, and FOXG1, possibly reflecting different cellular origins.

Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations

The protein cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of immune responses, and its loss causes fatal autoimmunity in mice. We studied a large family in which five individuals presented with a complex, autosomal dominant immune dysregulation syndrome characterized by hypogammaglobulinemia, recurrent infections and multiple autoimmune clinical features. We identified a heterozygous nonsense mutation in exon 1 of CTLA4. Screening of 71 unrelated patients with comparable clinical phenotypes identified five additional families (nine individuals) with previously undescribed splice site and missense mutations in CTLA4. Clinical penetrance was incomplete (eight adults of a total of 19 genetically proven CTLA4 mutation carriers were considered unaffected). However, CTLA-4 protein expression was decreased in regulatory T cells (Treg cells) in both patients and carriers with CTLA4 mutations. Whereas Treg cells were generally present at elevated numbers in these individuals, their suppressive function, CTLA-4 ligand binding and transendocytosis of CD80 were impaired. Mutations in CTLA4 were also associated with decreased circulating B cell numbers. Taken together, mutations in CTLA4 resulting in CTLA-4 haploinsufficiency or impaired ligand binding result in disrupted T and B cell homeostasis and a complex immune dysregulation syndrome.

Girls homozygous for an IL-2–inducible T cell kinase mutation that leads to protein deficiency develop fatal EBV-associated lymphoproliferation

The fatal immune dysregulation that sometimes follows EBV infection in boys has been linked to mutations in two X chromosome-encoded genes, SLAM-associated protein (SAP) and X-linked inhibitor of apoptosis (XIAP). In this study we describe 2 girls from a consanguineous Turkish family who died after developing severe immune dysregulation and therapy-resistant EBV-positive B cell proliferation following EBV infection. SNP array-based genome-wide linkage analysis revealed IL-2-inducible T cell kinase (ITK) as a candidate gene for this immunodeficiency syndrome. Both girls harbored a homozygous missense mutation that led to substitution of a highly conserved residue (R335W) in the SH2 domain of ITK. Characteristics of ITK deficiency in mouse models, such as absence of NKT cells and high levels of eomesodermin in CD8+ cells, were seen in either one or both of the girls. Two lines of evidence suggested that R335W caused instability of the ITK protein. First, in silico modeling of the mutant protein predicted destabilization of the SH2 domain. Additionally, Western blot analysis revealed that, unlike wild-type ITK, the R335W mutant was nearly undetectable when expressed in 293 T cells. Our results suggest that ITK deficiency causes what we believe to be a novel immunodeficiency syndrome that leads to a fatal inadequate immune response to EBV. Because ITK deficiency resembles EBV-associated lymphoproliferative disorders in boys, we suggest that this molecular cause should be considered during diagnosis and treatment.

Colchicine use in children and adolescents with familial Mediterranean fever: literature review and consensus statement.

The daily application of colchicine is the standard therapy for prophylaxis of attacks and amyloid deposition in familial Mediterranean fever. However, because of many issues (eg, dosage, time of introduction, etc), no standardized treatment recommendations have been established. In this work we review the available literature on colchicine use with respect to its indication, efficacy, mode of application, and safety in children and adolescents with familial Mediterranean fever. On the basis of this analysis, a consensus statement on the application of colchicine in children and adolescents with familial Mediterranean fever was developed by caregivers from Germany, Austria, and Turkey.

Mutations in STAT3 and diagnostic guidelines for hyper-IgE syndrome

BACKGROUND The hyper-IgE syndrome (HIES) is a primary immunodeficiency characterized by infections of the lung and skin, elevated serum IgE, and involvement of the soft and bony tissues. Recently, HIES has been associated with heterozygous dominant-negative mutations in the signal transducer and activator of transcription 3 (STAT3) and severe reductions of T(H)17 cells. OBJECTIVE To determine whether there is a correlation between the genotype and the phenotype of patients with HIES and to establish diagnostic criteria to distinguish between STAT3 mutated and STAT3 wild-type patients. METHODS We collected clinical data, determined T(H)17 cell numbers, and sequenced STAT3 in 100 patients with a strong clinical suspicion of HIES and serum IgE >1000 IU/mL. We explored diagnostic criteria by using a machine-learning approach to identify which features best predict a STAT3 mutation. RESULTS In 64 patients, we identified 31 different STAT3 mutations, 18 of which were novel. These included mutations at splice sites and outside the previously implicated DNA-binding and Src homology 2 domains. A combination of 5 clinical features predicted STAT3 mutations with 85% accuracy. T(H)17 cells were profoundly reduced in patients harboring STAT3 mutations, whereas 10 of 13 patients without mutations had low (<1%) T(H)17 cells but were distinct by markedly reduced IFN-gamma-producing CD4(+)T cells. CONCLUSION We propose the following diagnostic guidelines for STAT3-deficient HIES. Possible: IgE >1000IU/mL plus a weighted score of clinical features >30 based on recurrent pneumonia, newborn rash, pathologic bone fractures, characteristic face, and high palate. Probable: These characteristics plus lack of T(H)17 cells or a family history for definitive HIES. Definitive: These characteristics plus a dominant-negative heterozygous mutation in STAT3.

An Immunodeficiency Disease with RAG Mutations and Granulomas

We describe three unrelated girls who had an immunodeficiency disease with granulomas in the skin, mucous membranes, and internal organs. All three girls had severe complications after viral infections, including B-cell lymphoma associated with Epstein-Barr virus (EBV). Other findings were hypogammaglobulinemia, a diminished number of T and B cells, and sparse thymic tissue on ultrasonography. Molecular analysis revealed that the patients were compound heterozygotes for mutations in recombination activating gene 1 or 2 (RAG1 or RAG2). In each case, both parents were heterozygous carriers of a RAG mutation. The mutations were associated with reduced function of RAG in vitro (3 to 30% of normal activity). The parents and one sibling in the three families were healthy.

Factors affecting lymphocyte subset reconstitution after either related or unrelated cord blood transplantation in children - a Eurocord analysis

Immune recovery after cord blood transplantation (CBT) is of concern owing to the low number of lymphocytes transferred with the graft and their immaturity. Risk factors influencing lymphocyte subset reconstitution related to disease, patient, donor and transplant were studied in 63 children (< 16 years), given either related (n = 14) or unrelated (n = 49) CBT for malignant (n = 33) or non‐malignant diseases (n = 30). Only children with sustained myeloid engraftment were analysed. Absolute numbers of T (CD3+, CD4+, CD8+), B and natural killer (NK) cells were reported 2–3, 6, 9, 12 and 12–24 months after CBT. Median patient age was 4·0 years (0–15) and median follow‐up was 23 months (1·7–61·0). Twenty‐six patients received human leucocyte antigen (HLA)‐matched CBT and 37 received HLA‐mismatched CBT. The median number of nucleated cells (NCs) collected/recipient weight was 6·1 × 107/kg. In this selected population, the estimate 2 year survival was 85%. Lymphocyte reconstitution (defined as the median time to reach the normal value of age‐matched healthy children) was 3, 6 and 8 months for NK, B and CD8+ cells, while it was 11·7 months for both CD3+ and CD4+ lymphocytes. In the multivariate analysis, factors favouring T‐cell recovery were: related donor (P = 0·002); higher NCs/kg (P = 0·005) and recipient cytomegalovirus (CMV)‐positive serology (P = 0·04). Presence of acute graft‐versus‐host disease (GVHD) delayed T‐cell recovery (P = 0·04). To summarize, in children with sustained myeloid engraftment the concern that lymphocyte recovery after CBT could be delayed does not appear to be substantiated by our results.

More than just SCID—The phenotypic range of combined immunodeficiencies associated with mutations in the recombinase activating genes (RAG) 1 and 2

Combined immunodeficiencies with impaired numbers and function of T- and B-cells can be attributed to defects in the recombinase activating genes (RAG). The products of these genes, the RAG1 and 2 proteins, are key players in the V(D)J recombination process leading to the assembly of antigen receptor genes. Complete RAG deficiency (RAGD) with no V(D)J (<1% recombination activity of wild type) is associated with classical SCID and absence of T- and B-cells. In RAGD with residual V(D)J activity (>1% recombination activity of wild type), several clinical and immunological subtypes have been described: RAGD with skin inflammation and alphabeta T-cell expansion (classical Omenn syndrome), RAGD with skin inflammation and without T-cell expansion (incomplete Omenn syndrome), RAGD with gammadelta T-cell expansion and RAGD with granulomas. Engraftment of maternal T-cells can add to variation in phenotype. The potential role of epigenetic factors that influence the emergence of these phenotypes is discussed. Thorough assessment and interpretation of clinical and immunological findings will guide treatment modalities as intense as hematopoietic stem cell transplantation.

Evidence-based, interdisciplinary guidelines for anti-inflammatory treatment of uveitis associated with juvenile idiopathic arthritis

Uveitis in juvenile idiopathic arthritis (JIA) is frequently associated with the development of complications and visual loss. Topical corticosteroids are the first-choice therapy, and immunosuppression is commonly used. However, treatment has not been standardized. Representatives from the German Ophthalmological Society, Society for Childhood and Adolescent Rheumatology, and the German Society for Rheumatology reached consensus on a standardized treatment strategy according to disease severity in the individual patient. The recommendations were based on a systematic literature analysis in MEDLINE and consensus expert meetings. Evidence and recommendations were graded, and an algorithm for anti-inflammatory treatment and final statements confirmed in a Delphi method. An interdisciplinary, evidence-based treatment guideline for JIA uveitis is presented.

The NEMO mutation creating the most-upstream premature stop codon is hypomorphic because of a reinitiation of translation.

Amorphic mutations in the NF- kappa B essential modulator (NEMO) cause X-dominant incontinentia pigmenti, which is lethal in males in utero, whereas hypomorphic mutations cause X-recessive anhidrotic ectodermal dysplasia with immunodeficiency, a complex developmental disorder and life-threatening primary immunodeficiency. We characterized the NEMO mutation 110_111insC, which creates the most-upstream premature translation termination codon (at codon position 49) of any known NEMO mutation. Surprisingly, this mutation is associated with a pure immunodeficiency. We solve this paradox by showing that a Kozakian methionine codon located immediately downstream from the insertion allows the reinitiation of translation. The residual production of an NH(2)-truncated NEMO protein was sufficient for normal fetal development and for the subsequent normal development of skin appendages but was insufficient for the development of protective immune responses.