Schafiq Nabhani

Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation.

Mutations in LPS-responsive and beige-like anchor (LRBA) gene were recently described in patients with combined immunodeficiency, enteropathy and autoimmune cytopenia. Here, we extend the clinical and immunological phenotypic spectrum of LRBA associated disorders by reporting on three patients from two unrelated families who presented with splenomegaly and lymphadenopathy, cytopenia, elevated double negative T cells and raised serum Fas ligand levels resembling autoimmune lymphoproliferative syndrome (ALPS) and one asymptomatic patient. Homozygous loss of function mutations in LRBA were identified by whole exome analysis. Similar to ALPS patients, Fas mediated apoptosis was impaired in LRBA deficient patients, while apoptosis in response to stimuli of the intrinsic mitochondria mediated apoptotic pathway was even enhanced. This manuscript illustrates the phenotypic overlap of other primary immunodeficiencies with ALPS-like disorders and strongly underlines the necessity of genetic diagnosis in order to provide early correct diagnosis and subsequent care.

Early-onset Evans syndrome, immunodeficiency and premature immunosenescence associated with tripeptidyl-peptidase II deficiency

Autoimmune cytopenia is a frequent manifestation of primary immunodeficiencies. Two siblings presented with Evans syndrome, viral infections, and progressive leukopenia. DNA available from one patient showed a homozygous frameshift mutation in tripeptidyl peptidase II (TPP2) abolishing protein expression. TPP2 is a serine exopeptidase involved in extralysosomal peptide degradation. Its deficiency in mice activates cell death programs and premature senescence. Similar to cells from naïve, uninfected TPP2-deficient mice, patient cells showed increased major histocompatibility complex I expression and most CD8(+) T-cells had a senescent CCR7-CD127(-)CD28(-)CD57(+) phenotype with poor proliferative responses and enhanced staurosporine-induced apoptosis. T-cells showed increased expression of the effector molecules perforin and interferon-γ with high expression of the transcription factor T-bet. Age-associated B-cells with a CD21(-) CD11c(+) phenotype expressing T-bet were increased in humans and mice, combined with antinuclear antibodies. Moreover, markers of senescence were also present in human and murine TPP2-deficient fibroblasts. Telomere lengths were normal in patient fibroblasts and granulocytes, and low normal in lymphocytes, which were compatible with activation of stress-induced rather than replicative senescence programs. TPP2 deficiency is the first primary immunodeficiency linking premature immunosenescence to severe autoimmunity. Determination of senescent lymphocytes should be part of the diagnostic evaluation of children with refractory multilineage cytopenias.

De novo PIK3R1 gain-of-function with recurrent sinopulmonary infections, long-lasting chronic CMV-lymphadenitis and microcephaly.

PIK3R1 (phosphoinositide-3-kinase, regulatory subunit 1) gain-of-function has recently been described in patients with recurrent sinopulmonary infections, chronic CMV-/EBV-infections, lymphoproliferation, and hypogammaglobulinemia. Here we report a 15-year-old boy with treatment refractory CMV lymphadenitis, severe combined immunodeficiency, microcephaly and a severe developmental defect of Th17 cells. To avoid poor outcome, hematopoietic stem cell transplantation (HSCT) was performed. Subsequently, whole exome sequencing revealed a de novo heterozygous G-to-C mutation (chr5: 5:67,589,663: G>C) at the splice donor site of the PIK3R1 gene. Our data suggest that PIK3R1 gain-of-function leads to developmental defects in helper and regulatory T-cell subsets, the latter expanding the immunological features of PIK3R1 gain-of-function. T-cell subsets play a critical role in the regulation of immune response against infectious agents and of autoimmunity and thus may be particularly accountable for the clinical phenotype of affected patients.

A novel homozygous Fas ligand mutation leads to early protein truncation, abrogation of death receptor and reverse signaling and a severe form of the autoimmune lymphoproliferative syndrome.

We report a novel type of mutation in the death ligand FasL that was associated with a severe phenotype of the autoimmune lymphoproliferative syndrome in two patients. A frameshift mutation in the intracellular domain led to complete loss of FasL expression. Cell death signaling via its receptor and reverse signaling via its intracellular domain were completely abrogated. In vitro lymphocyte proliferation induced by weak T cell receptor stimulation could be blocked and cell death was induced by engagement of FasL in T cells derived from healthy individuals and a heterozygous carrier, but not in FasL-deficient patient derived cells. Expression of genes implicated in lymphocyte proliferation and activation (CCND1, NFATc1, NF-κB1) was increased in FasL-deficient T cells and could not be downregulated by FasL engagement as in healthy cells. Our data thus suggest, that deficiency in FasL reverse signaling may contribute to the clinical lymphoproliferative phenotype of ALPS.

Specific antibody deficiency and autoinflammatory disease extend the clinical and immunological spectrum of heterozygous NFKB1 loss-of-function mutations in humans

The nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NF-κB1) is a master regulator of immune and inflammatory responses.[1][1],[2][2] NF-κB1 belongs to the NF-κB/Rel family of transcription factors that consists of five members in humans: NF-κB1 (p105/p50), NF-κB2 (p100/p52

The cell cycle regulator CCDC6 is a key target of RNA-binding protein EWS.

Genetic translocation of EWSR1 to ETS transcription factor coding region is considered as primary cause for Ewing sarcoma. Previous studies focused on the biology of chimeric transcription factors formed due to this translocation. However, the physiological consequences of heterozygous EWSR1 loss in these tumors have largely remained elusive. Previously, we have identified various mRNAs bound to EWS using PAR-CLIP. In this study, we demonstrate CCDC6, a known cell cycle regulator protein, as a novel target regulated by EWS. siRNA mediated down regulation of EWS caused an elevated apoptosis in cells in a CCDC6-dependant manner. This effect was rescued upon re-expression of CCDC6. This study provides evidence for a novel functional link through which wild-type EWS operates in a target-dependant manner in Ewing sarcoma.

STAT3 gain-of-function mutations associated with autoimmune lymphoproliferative syndrome like disease deregulate lymphocyte apoptosis and can be targeted by BH3 mimetic compounds

Autoimmune lymphoproliferative syndrome (ALPS) is typically caused by mutations in genes of the extrinsic FAS mediated apoptotic pathway, but for about 30% of ALPS-like patients the genetic diagnosis is lacking. We analyzed 30 children with ALPS-like disease of unknown cause and identified two dominant gain-of-function mutations of the Signal Transducer And Activator Of Transcription 3 (STAT3, p.R278H, p.M394T) leading to increased transcriptional activity. Hyperactivity of STAT3, a known repressor of FAS, was associated with decreased FAS-mediated apoptosis, mimicking ALPS caused by FAS mutations. Expression of BCL2 family proteins, further targets of STAT3 and regulators of the intrinsic apoptotic pathway, was disturbed. Cells with hyperactive STAT3 were consequently more resistant to intrinsic apoptotic stimuli and STAT3 inhibition alleviated this effect. Importantly, STAT3-mutant cells were more sensitive to death induced by the BCL2-inhibitor ABT-737 indicating a dependence on anti-apoptotic BCL2 proteins and potential novel therapeutic options.

Deregulation of Fas ligand expression as a novel cause of autoimmune lymphoproliferative syndrome-like disease.

Autoimmune lymphoproliferative syndrome is frequently caused by mutations in genes involved in the Fas death receptor pathway, but for 20–30% of patients the genetic defect is unknown. We observed that treatment of healthy T cells with interleukin-12 induces upregulation of Fas ligand and Fas ligand-dependent apoptosis. Consistently, interleukin-12 could not induce apoptosis in Fas ligand-deficient T cells from patients with autoimmune lymphoproliferative syndrome. We hypothesized that defects in the interleukin-12 signaling pathway may cause a similar phenotype as that caused by mutations of the Fas ligand gene. To test this, we analyzed 20 patients with autoimmune lymphoproliferative syndrome of unknown cause by whole-exome sequencing. We identified a homozygous nonsense mutation (c.698G>A, p.R212*) in the interleukin-12/interleukin-23 receptor-component IL12RB1 in one of these patients. The mutation led to IL12RB1 protein truncation and loss of cell surface expression. Interleukin-12 and -23 signaling was completely abrogated as demonstrated by deficient STAT4 phosphorylation and interferon γ production. Interleukin-12-mediated expression of membrane-bound and soluble Fas ligand was lacking and basal expression was much lower than in healthy controls. The patient presented with the classical symptoms of autoimmune lymphoproliferative syndrome: chronic non-malignant, non-infectious lymphadenopathy, splenomegaly, hepatomegaly, elevated numbers of double-negative T cells, autoimmune cytopenias, and increased levels of vitamin B12 and interleukin-10. Sanger sequencing and whole-exome sequencing excluded the presence of germline or somatic mutations in genes known to be associated with the autoimmune lymphoproliferative syndrome. Our data suggest that deficient regulation of Fas ligand expression by regulators such as the interleukin-12 signaling pathway may be an alternative cause of autoimmune lymphoproliferative syndrome-like disease.