Array-based sequence capture and next-generation sequencing for the identification of primary immunodeficiencies.

Primary immunodeficiencies are genetic disorders in which components of immunological pathways are either missing or dysregulated. With the advent of next-generation sequencing, testing for genes in conditions with a heterogeneous genetic background seems more promising. We designed a custom microarray with 385K probe capacity to capture exons of 395 human genes, known or predicted to be associated with primary immunodeficiency and immune regulation. Enriched target DNA was sequenced using a GS FLX Titanium 454 platform. The patients selected were likely to have an underlying immunodeficiency. In one patient with hepatosplenomegaly, recurrent infections and an elevated IgM level, sequence analysis of the patient and his two unaffected parents identified ATM (ataxia telangiectasia mutated) as the underlying defect. In a second child with a clinical SCID phenotype, we detected a mutation in the ARTEMIS gene after focusing on SCID-associated genes. 454 sequencing yielded 152,000-397,000 high-quality reads per patient. 78-99% of the targeted nucleotides were covered at least one time, 76-82% at least five times. Array-based sequence capture expands our capacities to sequence large targeted DNA regions in a less laborious and time-consuming approach. Our array was capable to find the underlying genetic defect in two patients with suspected primary immunodeficiency. Upcoming whole-exome sequencing definitely will add more valuable data, but bioinformatical analysis and validation of variants already pose major challenges.

iNKT cell frequency in peripheral blood of Caucasian children and adolescent: the absolute iNKT cell count is stable from birth to adulthood.

Human invariant natural killer T cells (iNKT cells) are a unique population of T cells that express a semi-invariantly rearranged T cell receptor (TCR) and are involved in a variety of immunoregulatory processes. We assessed the frequency of peripheral blood iNKT cells in 64 healthy Caucasian children from 7 months to 18 years of age and five cord blood samples by flow cytometry. iNKT cells were measured as CD3(+) cells co-expressing TCRVα24 and TCRVß11 and using the monoclonal antibody 6B11, which recognizes specifically their invariant TCR rearrangement. The absolute number of iNKT cells ranged from 86 to 10,499 (CD3(+) /TCRVα24(+) / TCRVß11(+)) and 233 to 11,167 (CD3(+) /6B11(+)) iNKT cells per millilitre of blood. This range is stable from birth to adulthood. The relative iNKT cell count was found to be 0.003-0.71% (CD3(+) /TCRVα24/TCRVß11) and 0.019-0.776% (CD3/6B11) of peripheral blood T cells and shows only a slight increase with age.

MHC class II deficiency cured by unrelated mismatched umbilical cord blood transplantation: case report and review of 68 cases in the literature.

MHC class II deficiency is a rare and fatal form of primary combined immunodeficiency caused by a lack of T-cell-dependent humoral and cellular immune response to foreign antigens, which can only be cured by allogenic stem cell transplantation. In the literature search, we identified 68 cases of HSCT in MHC class II deficiency in the last 14 yr. Pre- and post-transplant MHC class II deficiency is complicated by overwhelming viral infections, a high incidence of GvHD, and graft failure with a poor overall survival rate below 50%. We report an eight-month-old boy presenting with severe respiratory infections and chronic diarrhea, whose sister died at the age of four yr from septicemia. MHC II deficiency was caused by an RFXANK-mutation and treated successfully by 4/6 mismatched unrelated CBT after a myeloablative conditioning regimen based on anti-thymocyte globulin, busulfane, fludarabine, and cyclophosphamide. At present, our patient is well with full immune reconstitution 3(4/12) yr after CBT. CB may represent an alternative source of stem cells for children with MHC class II deficiency without a suitable donor.

Terminally differentiated CD8 cells in HIV-infected children: HIV-GAG/POL specificity and IFN-γ production.

BACKGROUND: CD8 cells are key to antiviral immunity and can be divided by phenotype into early (CD28+ CD27+), intermediate (CD28-CD27+) and terminally differentiated subsets (CD28- CD27-). Despite effective HAART there is an unexplained expansion of CD8+CD28-CD27-T cells in HIV-infected children. The cytokine production and specificity of this terminally differentiated CD8 T cell subset in chronic virus infection is unclear. PATIENTS, METHODS & RESULTS: In a cohort of 26 HIV-infected children the cytokine production of terminally differentiated CD8 cells was analyzed by intracellular staining and FACS analysis and was compared to children with chronic hepatitis B infection and to healthy children. The specificity of CD8 subsets was analyzed by staining with Gag/Pol tetramers in a cohort of 13 patients. We show that an increased production of interferon-γ in terminally and early/intermediate differentiated CD8 cell subsets after stimulation is specific for HIV-infection. The expanded population of terminally differentiated CD8+CD28-CD27- T cells does include HIV Gag/Pol specific T cells in adults but not in children. CONCLUSION: The expansion of terminally differentiated CD8 cells might be important for immunomodulation but in children it does not appear to play a role in HIV Gag and Pol specific immunity.

ARDS as presenting symptom in an infant with CD40L deficiency (Hyper-IgM syndrome Type 1).

We report on a 4 month old male infant with respiratory syncytial virus (RSV) infection leading to acute respiratory distress syndrome (ARDS). A diagnostic algorithm including extended infectiological and immunological work-up revealed absence of CD40-ligand. ARDS was treated successfully with a complex respiratory therapy plus intravenous immunoglobulin substitution. Molecular analysis detected mutations in the CD40L gene (Hyper-IgM syndrome Type 1). The case underlines the importance of an extended diagnostic work-up in an uncommonly severe course of respiratory infection in early infancy.

Description of the mutations in 15 subjects with variant forms of maple syrup urine disease.

BACKGROUND: In maple syrup urine disease (MSUD), disease-causing mutations can affect the BCKDHA, BCKDHB or DBT genes encoding for the E1 alpha, E1 beta and E2 subunits of the multienzyme branched-chain 2-keto acid dehydrogenase (BCKD) complex. AIM: The aim of this study was to screen DNA samples of 15 subjects with distinct well-characterized variant MSUD phenotypes for mutations in the three genes in order to demonstrate a potential correlation between specific nucleotide changes and particular variant phenotypes. METHODS: The exonic coding sequences of all three genes were studied using genomic DNA and cellular RNA derived from peripheral blood leukocytes. RESULTS: In 37% of the cases (total 30 alleles), disease-causing mutations were located in the BCKDHA, in 46% in the BCKDHB, and in 13% in the DBT gene. Novel mutations occurring homozygously were p.Ala328Thr in the BCKDHA gene and p.Gly249_Lys257del in the DBT gene. Both are associated with a mild MSUD variant. The same holds true for the novel mutations p.Pro200Ala in BCKDHB and p.Phe307Ser in DBT which were identified in heterozygous fashion. Among the known mutant alleles, p.Gly278Ser in the BCKDHB gene was relatively frequent and also associated with a mild MSUD variant. CONCLUSION: The results of this study indicate that genotyping may be predictive of clinical severity of variant MSUD phenotypes and might be of prognostic value particularly in subjects with variant MSUD identified in newborn screening in whom early treatment fortunately slows the natural course of the disease.

[Concepts on the pathogenesis of juvenile idiopathic arthritis]. / Vorstellungen zur Pathogenese der juvenilen idiopathischen Arthritis.

There are various explanations for the development of juvenile idiopathic arthritis (JIA).Gene changes in the immune system can predispose to JIA and regulation of the immune system is crucial in the pathogenesis. The adaptive, acquired immune system probably plays a central role. Thus, in the case of JIA a conspicuous population of highly activated T-cells can be found in the synovia. B-cells are also involved, as indicated by positive ANA titers in JIA patients. Regulatory T-cells (Tregs) attempt to prevent the expansion of autoreactive T-cells.However, the natural or the innate immune system also plays a role. Thus a disorder of the inflammasome could underlie the cause of JIA with systemic onset. The interaction between congenital and adaptive immune system shows that a distinct spatial and temporal separation between the two immune systems is becoming increasingly difficult. An infection- and virus-related immune reaction could also be the cause of JIA. Proinflammatory cytokines are of proven significance in pathogenesis in terms of how they are released under stress, for example. New genomic and proteomic techniques are able to produce individualized profiles for each patient and allow for increasingly fine separation between subtypes, thus improving therapeutic possibilities.

Impaired CD95-mediated apoptosis in autoimmunity and occurrence of a p22 Caspase-8 cleavage product in JIA.

BACKGROUND: Altered apoptosis can lead to autoimmune diseases such as systemic lupus erythematosus (SLE). Juvenile idiopathic arthritis (JIA) is another autoimmune disease characterized by autoinflammation. During this process activated T-cells accumulate in the synovial fluid. We hypothesized that resistance to CD95-mediated apoptosis could contribute to autoimmune phenotypes. PATIENTS/METHOD: We isolated highly activated T cells (CD45RO+ and CD95+) by magnetic separation from healthy controls, JIA patients and patients with other autoimmune diseases. In these purified cells, apoptosis was induced by stimulation with recombinant human soluble CD95 ligand (rhsCD95L) or dexamethasone and analyzed by flow cytometry. In addition, cleavage and expression of apoptosis mediators (Caspase-8 and -3) and regulators (FLIP, Bcl-2, Bcl-xL) were analyzed in mononuclear cells using immunoblot technique. RESULTS: Apoptosis upon CD95 stimulation, but not dexamethasone treatment, was reduced in JIA patients and patients with other autoimmune diseases compared to healthy controls. Additionally we observed a non-canonical cleavage pattern of Caspase-8 resulting in a p22 fragment and high expression of FLIP in SFMCs of patients with JIA. CONCLUSION: Expression and cleavage of proteins of the CD95 pathway is altered in JIA providing a possible explanation for resistance against death receptor-mediated apoptosis. Dexamethasone-induced apoptosis, however, is intact arguing against a general defect in apoptosis. The implications of the p22 fragment regarding apoptosis have to be further analyzed. The strong expression of FLIPShort in SFMCs may as well result from the highly activated status of the cells or be a feature of autoimmunity.

The BH3-only member Noxa causes apoptosis in melanoma cells by multiple pathways.

The molecular causes for resistance of melanoma to apoptosis are currently only partly understood. In the present study, we examined gene transfer and expression of the proapoptotic BH3-only protein Noxa as an alternative approach to chemotherapy and investigated the molecular mechanisms regulating Noxa-induced apoptosis. Noxa gene transfer caused dysregulation of both mitochondria and, as shown for the first time, also the endoplasmic reticulum, resulting in the accumulation of reactive oxygen species. Interestingly, expression of Noxa not only triggered the classical mitochondrial caspase cascade, but also resulted in the activation of apoptosis signal-regulating kinase1 and its downstream effectors c-Jun N-terminal kinase and p38. The activation of these kinases was abolished by antioxidants. Moreover, inhibition of the kinases by RNA interference or pharmacological inhibitors significantly attenuated Noxa-induced apoptosis. Thus, our data provide evidence for the involvement of multiple pathways in Noxa-induced apoptosis that are triggered at mitochondria and the endoplasmic reticulum, and suggest Noxa gene transfer as a complementary approach to chemotherapy.

Multiple eruptive myxoid dermatofibromas: report of first case and review of literature.

Multiple eruptive dermatofibromas are a rare presentation of dermatofibroma which are frequently associated with underlying diseases such as human immunodeficiency virus infection or lupus erythematosus. Eruptive dermatofibromas generally present a characteristic histology with a poorly circumscribed lesion showing hyperplasia of the epidermis, prominent bundles of collagen and a diffuse proliferation of fibrocytes. We report an unusual case of multiple eruptive dermatofibromas showing massive depositions of mucin within the dermis. A 20-year-old woman presented with nearly 100 red to yellowish papules and nodules distributed symmetrically all over the integument which developed over a period of 9 years. Comprehensive clinical and laboratory diagnostics showed no signs indicating any underlying disease. To our knowledge this is the first report of multiple eruptive myxoid dermatofibromas. We consider this case to be a unique presentation of multiple eruptive dermatofibroma showing massive deposition of mucin.

Long-term haematopoietic reconstitution and survival without interleukin-7 in a murine syngeneic bone marrow transplantation model.

We created a syngeneic mouse bone marrow transplantation (BMT) model to examine the effect of endogenous interleukin-7 (IL-7) on long-term (>or=140 days) haematopoietic reconstitution and survival after BMT. Wild-type (WT) IL-7(+/+) and knockout (KO) IL-7(-/-) mice were lethally irradiated and transplanted with bone marrow. Survival is best (85.7%) in the group WT grafts transplanted into WT recipients (WT-->WT) with a trend towards poorer survival in the other groups (WT-->KO: 60%, KO-->WT: 50%, KO-->KO: 45.5%, differences statistically not significant). If the recipient is deficient for IL-7-producing cells, T- and B-cell reconstitution remain incomplete. If the graft lacks IL-7-producing cells there is a significant delay in T- and NK-cell reconstitution. Interestingly, in the absence of IL-7, T-cell reconstitution is neither delayed nor incomplete because of an expansion of TCRalphabeta(+)/CD4(-)/CD8(-) double negative T cells. Long-term survival and lymphocyte reconstitution after syngeneic BMT can occur despite the absence of IL-7.