Late Onset of Primary Hemophagocytic Lymphohistiocytosis (HLH) with a Novel Constellation of Compound Heterozygosity Involving Two Missense Variants in the PRF1 Gene.

Hemophagocytic lymphohistiocytosis (HLH) is a rare but in most cases life-threatening immune-mediated disease of the hematopoietic system frequently associated with hematologic neoplasms. Here, we report on a case in which we detected a novel constellation of two missense variants affecting the PRF1 gene, leading to de novo primary HLH. Diagnostics included a comprehensive clinical work-up and standard methods of hematopathology as well as extended molecular genomics based on polymerase chain reaction (PCR) reactions and the calculation of three-dimensional molecule reconstructions of PRF1. Subsequently, a comprehensive review of the literature was performed, which showed that this compound heterozygosity has not been previously described. The patient was a 20-year-old female. Molecular diagnostics revealed two heterozygous missense variants in the PRF1 gene (A91V and R104C) on exon 2. Apart from the finding of two inconclusive genetic variants, all clinical criteria defined by the HLH study group of Histiocyte Society were met at initial presentation. The final diagnosis was made in cooperation with the Consortium of German HLH-reference centers. Here, chemotherapy did not lead to sufficient sustained disease control. Therefore, the decision for allogenic hematopoietic stem cell transplantation (alloHSCT) was made. Hitherto, the duration of response was 6 months. Due to severe and unmanageable hepatic graft-versus-host disease (GvHD), the patient died. We report on a novel constellation of a compound heterozygosity containing two missense variants on exon 2 of the PRF1 gene. To the authors' best knowledge, this is the first presentation of a primary HLH case harboring this genomic constellation with late-onset clinical manifestation.

Abnormal biomarkers predict complex FAS or FADD defects missed by exome sequencing.

BACKGROUND: Elevated TCRαß+CD4-CD8- double-negative T cells (DNT) and serum biomarkers help identify FAS mutant patients with autoimmune lymphoproliferative syndrome (ALPS). However, in some patients with clinical features and biomarkers consistent with ALPS, germline or somatic FAS mutations cannot be identified on standard exon sequencing (ALPS-undetermined: ALPS-U). OBJECTIVE: We sought to explore whether complex genetic alterations in the FAS gene escaping standard sequencing or mutations in other FAS pathway-related genes could explain these cases. METHODS: Genetic analysis included whole FAS gene sequencing, copy number variation analysis, and sequencing of FAS cDNA and other FAS pathway-related genes. It was guided by FAS expression analysis on CD57+DNT, which can predict somatic loss of heterozygosity (sLOH). RESULTS: Nine of 16 patients with ALPS-U lacked FAS expression on CD57+DNT predicting heterozygous "loss-of-expression" FAS mutations plus acquired somatic second hits in the FAS gene, enriched in DNT. Indeed, 7 of 9 analyzed patients carried deep intronic mutations or large deletions in the FAS gene combined with sLOH detectable in DNT; 1 patient showed a FAS exon duplication. Three patients had reduced FAS expression, and 2 of them harbored mutations in the FAS promoter, which reduced FAS expression in reporter assays. Three of the 4 ALPS-U patients with normal FAS expression carried heterozygous FADD mutations with sLOH. CONCLUSION: A combination of serum biomarkers and DNT phenotyping is an accurate means to identify patients with ALPS who are missed by routine exome sequencing.

Revisiting autoimmune lymphoproliferative syndrome caused by Fas ligand mutations.

BACKGROUND: Fas ligand (FasL) is expressed by activated T cells and induces death in target cells upon binding to Fas. Loss-of-function FAS or FASLG mutations cause autoimmune-lymphoproliferative syndrome (ALPS) characterized by expanded double-negative T cells (DNT) and elevated serum biomarkers. While most ALPS patients carry heterozygous FAS mutations, FASLG mutations are rare and usually biallelic. Only 2 heterozygous variants were reported, associated with an atypical clinical phenotype. OBJECTIVE: We revisited the significance of heterozygous FASLG mutations as a cause of ALPS. METHODS: Clinical features and biomarkers were analyzed in 24 individuals with homozygous or heterozygous FASLG variants predicted to be deleterious. Cytotoxicity assays were performed with patient T cells and biochemical assays with recombinant FasL. RESULTS: Homozygous FASLG variants abrogated cytotoxicity and resulted in early-onset severe ALPS with elevated DNT, raised vitamin B12, and usually no soluble FasL. In contrast, heterozygous variants affected FasL function by reducing expression, impairing trimerization, or preventing Fas binding. However, they were not associated with elevated DNT and vitamin B12, and they did not affect FasL-mediated cytotoxicity. The dominant-negative effects of previously published variants could not be confirmed. Even Y166C, causing loss of Fas binding with a dominant-negative effect in biochemical assays, did not impair cellular cytotoxicity or cause vitamin B12 and DNT elevation. CONCLUSION: Heterozygous loss-of-function mutations are better tolerated for FASLG than for FAS, which may explain the low frequency of ALPS-FASLG.

MAP kinase activating death domain deficiency is a novel cause of impaired lymphocyte cytotoxicity.

Most hereditary forms of hemophagocytic lymphohistiocytosis (HLH) are caused by defects of cytotoxicity, including the vesicle trafficking disorder Griscelli syndrome type 2 (GS2, RAB27A deficiency). Deficiency of the mitogen-activated protein kinase activating death domain protein (MADD) results in a protean syndrome with neurological and endocrinological involvement. MADD acts as a guanine nucleotide exchange factor for small guanosine triphosphatases, including RAB27A. A homozygous splice site mutation in MADD was identified in a female infant with syndromic features, secretory diarrhea, and features of HLH. Aberrant splicing caused by this mutation leads to an in-frame deletion of 30 base pairs and favors other aberrant variants. Patient natural killer (NK) cells and cytotoxic T cells showed a severe degranulation defect leading to absent perforin-mediated cytotoxicity. Platelets displayed defective adenosine triphosphate secretion, similar to that in GS2. To prove causality, we introduced a CRISPR/Cas9-based MADD knockout in the NK cell line NK-92mi. MADD-deficient NK-92mi cells showed a degranulation defect and impaired cytotoxicity similar to that of the patient. The defect of cytotoxicity was confirmed in another patient with MADD deficiency. In conclusion, RAB27A-interacting MADD is involved in vesicle release by cytotoxic cells and platelets. MADD deficiency causes a degranulation defect and represents a novel disease predisposing to an HLH phenotype.

Variant in the PLCG2 Gene May Cause a Phenotypic Overlap of APLAID/PLAID: Case Series and Literature Review.

Background: Variants in the phospholipase C gamma 2 (PLCG2) gene can cause PLCG2-associated antibody deficiency and immune dysregulation (PLAID)/autoinflammation and PLCG2-associated antibody deficiency and immune dysregulation (APLAID) syndrome. Linking the clinical phenotype with the genotype is relevant in making the final diagnosis. Methods: This is a single center case series of five related patients (4−44 years), with a history of autoinflammation and immune dysregulation. Clinical and laboratory characteristics were recorded and a literature review of APLAID/PLAID was performed. Results: All patients had recurrent fevers, conjunctivitis, lymphadenopathy, headaches, myalgia, abdominal pain, cold-induced urticaria and recurrent airway infections. Hearing loss was detected in two patients. Inflammatory parameters were slightly elevated during flares. Unswitched B-cells were decreased. Naïve IgD+CD27− B-cells and unswitched IgD+CD27+ B-cells were decreased; switched IgD-CD27+ B-cells were slightly increased. T-cell function was normal. Genetic testing revealed a heterozygous missense variant (c.77C>T, p.Thr26Met) in the PLCG2 gene in all patients. Genotype and phenotype characteristics were similar to previously published PLAID (cold-induced urticaria) and APLAID (eye inflammation, musculoskeletal complaints, no circulating antibodies) patients. Furthermore, they displayed characteristics for both PLAID and APLAID (recurrent infections, abdominal pain/diarrhea) with normal T-cell function. Conclusion: The heterozygous missense PLCG2 gene variant (c.77C>T, p.Thr26Met) might cause phenotypical overlap of PLAID and APLAID patterns.

STAT3-confusion-of-function: Beyond the loss and gain dualism.

BACKGROUND: Germline mutations of signal transducer and activator of transcription 3 (STAT3) are responsible for 2 distinct human diseases: autosomal-dominant hyper-IgE syndrome (AD-HIES) caused by STAT3 loss-of-function mutations and STAT3 gain-of-function disease. So far, these entities have been regarded as antithetic, with AD-HIES mainly associated with characteristic infections and a connective tissue phenotype and STAT3 gain-of-function characterized by lymphoproliferation and poly-autoimmunity. The R335W substitution in the DNA-binding domain of STAT3 was initially described in 2 patients with typical AD-HIES, but paradoxically, recent functional analysis demonstrated a gain-of-function effect of this variant. OBJECTIVES: A patient with Sjögren syndrome and features of AD-HIES with this mutation is described and the molecular consequences are further characterized. METHODS: This study provides a clinical and immunological description of the patient. STAT phosphorylation in primary patient cells was studied and A4 cells transfected with the patient allele were used to study phosphorylation kinetics, transcriptional activity, and target-gene induction. RESULTS: The hybrid clinical features of the patient were associated with normal TH17 cells. Enhanced and prolonged STAT3 phosphorylation, an increased STAT3 driven luciferase reporter activity upon IL-6 stimulation, but reduced IL-6-induced SOCS3 production were all observed. CONCLUSIONS: The germline R335W-STAT3 variant displays a mixed behavior in vitro that mainly shows gain-of-function, but also loss-of-function features. This is matched by an ambiguous clinical and immunological phenotype that dismantles the classical antithetic dualism of gain- versus loss-of-function. Germline STAT3 mutation-related disease represents a pathological spectrum with the p.R335W associated phenotype locating between the 2 recognized clinical disease patterns.

Stable isotopes (δ13C, δ15N) and biomarkers as indicators of the hydrological regime of fens in a European east-west transect.

Peatland degradation is tightly connected to hydrological changes and microbial metabolism. To better understand these metabolism processes, more information is needed on how microbial communities and substrate cycling are affected by changing hydrological regimes. These activities should be imprinted in stable isotope bulk values (δ 15N, δ 13C) due to specific isotopic fractionation by different microbial communities, their metabolic pathways and nutrient sources. We hypothesize that stable isotope values and microbial abundance are correlated and act as indicators of different hydrological regimes. We sampled an East-West transect across European fens in 14 areas and conducted a stable isotope (δ 13C, δ 15N) and membrane fatty acid (mFA) analysis. Within each area an undrained, drained and rewetted site was selected. Rewetted sites were separated based on when rewetting occurred. We found differences in the upper layers of all sites in microbial-derived mFAs and stable isotope values corresponding to hydrological regimes. The highest and lowest quantities of microbial-derived mFAs were measured in undrained and drained sites, respectively. Fungal-derived mFAs were especially lower in drained sites. Simultaneously, δ15N stable isotope values were highest in drained sites. In addition, stable isotope values and microbial-derived mFAs showed distinct depth trends. In undrained sites stable isotopes values slightly increased with depth. In drained sites, δ15N values decreased downwards, whereas δ13C values increased. Overall microbial-derived mFAs decreased with depth. These patterns presumably result from anoxic conditions and high peat recalcitrance in the deeper layers. In sites with short time of rewetting, the microbial-derived mFAs and stable isotope values were similar to values of drained sites, while with increasing rewetting time values shifted to those of undrained sites. We conclude that biomarkers indicate that stable isotope values reflect specific microbial metabolic processes, which differ with hydrological regimes, and thus could indicate both drainage and rewetting in fens.

Rubella vaccine-induced granulomas are a novel phenotype with incomplete penetrance of genetic defects in cytotoxicity.

BACKGROUND: Rubella virus-induced granulomas have been described in patients with various inborn errors of immunity. Most defects impair T-cell immunity, suggesting a critical role of T cells in rubella elimination. However, the molecular mechanism of virus control remains elusive. OBJECTIVE: This study sought to understand the defective effector mechanism allowing rubella vaccine virus persistence in granulomas. METHODS: Starting from an index case with Griscelli syndrome type 2 and rubella skin granulomas, this study combined an international survey with a literature search to identify patients with cytotoxicity defects and granuloma. The investigators performed rubella virus immunohistochemistry and PCR and T-cell migration assays. RESULTS: This study identified 21 patients with various genetically confirmed cytotoxicity defects, who presented with skin and visceral granulomas. Rubella virus was demonstrated in all 12 accessible biopsies. Granuloma onset was typically before 2 years of age and lesions persisted from months to years. Granulomas were particularly frequent in MUNC13-4 and RAB27A deficiency, where 50% of patients at risk were affected. Although these proteins have also been implicated in lymphocyte migration, 3-dimensional migration assays revealed no evidence of impaired migration of patient T cells. Notably, patients showed no evidence of reduced control of concomitantly given measles, mumps, or varicella live-attenuated vaccine or severe infections with other viruses. CONCLUSIONS: This study identified lymphocyte cytotoxicity as a key effector mechanism for control of rubella vaccine virus, without evidence for its need in control of live measles, mumps, or varicella vaccines. Rubella vaccine-induced granulomas are a novel phenotype with incomplete penetrance of genetic disorders of cytotoxicity.

A distinct CD38+CD45RA+ population of CD4+, CD8+, and double-negative T cells is controlled by FAS.

The identification and characterization of rare immune cell populations in humans can be facilitated by their growth advantage in the context of specific genetic diseases. Here, we use autoimmune lymphoproliferative syndrome to identify a population of FAS-controlled TCRαß+ T cells. They include CD4+, CD8+, and double-negative T cells and can be defined by a CD38+CD45RA+T-BET- expression pattern. These unconventional T cells are present in healthy individuals, are generated before birth, are enriched in lymphoid tissue, and do not expand during acute viral infection. They are characterized by a unique molecular signature that is unambiguously different from other known T cell differentiation subsets and independent of CD4 or CD8 expression. Functionally, FAS-controlled T cells represent highly proliferative, noncytotoxic T cells with an IL-10 cytokine bias. Mechanistically, regulation of this physiological population is mediated by FAS and CTLA4 signaling, and its survival is enhanced by mTOR and STAT3 signals. Genetic alterations in these pathways result in expansion of FAS-controlled T cells, which can cause significant lymphoproliferative disease.

Griscelli Syndrome Type 2 Sine Albinism: Unraveling Differential RAB27A Effector Engagement.

Griscelli syndrome type 2 (GS-2) is an inborn error of immunity characterized by partial albinism and episodes of hemophagocytic lymphohistiocytosis (HLH). It is caused by RAB27A mutations that encode RAB27A, a member of the Rab GTPase family. RAB27A is expressed in many tissues and regulates vesicular transport and organelle dynamics. Occasionally, GS-2 patients with RAB27A mutation display normal pigmentation. The study of such variants provides the opportunity to map distinct binding sites for tissue-specific effectors on RAB27A. Here we present a new case of GS-2 without albinism (GS-2 sine albinism) caused by a novel missense mutation (Val143Ala) in the RAB27A and characterize its functional cellular consequences. Using pertinent animal cell lines, the Val143Ala mutation impairs both the RAB27A-SLP2-A interaction and RAB27A-MUNC13-4 interaction, but it does not affect the RAB27A-melanophilin (MLPH)/SLAC2-A interaction that is crucial for skin and hair pigmentation. We conclude that disruption of the RAB27A-MUNC13-4 interaction in cytotoxic lymphocytes leads to the HLH predisposition of the GS-2 patient with the Val143Ala mutation. Finally, we include a review of GS-2 sine albinism cases reported in the literature, summarizing their genetic and clinical characteristics.

NCKAP1L defects lead to a novel syndrome combining immunodeficiency, lymphoproliferation, and hyperinflammation.

The Nck-associated protein 1-like (NCKAP1L) gene, alternatively called hematopoietic protein 1 (HEM-1), encodes a hematopoietic lineage-specific regulator of the actin cytoskeleton. Nckap1l-deficient mice have anomalies in lymphocyte development, phagocytosis, and neutrophil migration. Here we report, for the first time, NCKAP1L deficiency cases in humans. In two unrelated patients of Middle Eastern origin, recessive mutations in NCKAP1L abolishing protein expression led to immunodeficiency, lymphoproliferation, and hyperinflammation with features of hemophagocytic lymphohistiocytosis. Immunophenotyping showed an inverted CD4/CD8 ratio with a major shift of both CD4+ and CD8+ cells toward memory compartments, in line with combined RNA-seq/proteomics analyses revealing a T cell exhaustion signature. Consistent with the core function of NCKAP1L in the reorganization of the actin cytoskeleton, patients' T cells displayed impaired early activation, immune synapse morphology, and leading edge formation. Moreover, knockdown of nckap1l in zebrafish led to defects in neutrophil migration. Hence, NCKAP1L mutations lead to broad immune dysregulation in humans, which could be classified within actinopathies.

TIM-3 deficiency presenting with two clonally unrelated episodes of mesenteric and subcutaneous panniculitis-like T-cell lymphoma and hemophagocytic lymphohistiocytosis.

This report offers novel clinical and diagnostic aspects of the association between germline mutations in HAVCR2 and subcutaneous panniculitis-like T-cell lymphoma (SPTCL). The patient presented with panniculitis-like T-cell lymphoma involving mesenteric fatty tissue associated with hemophagocytic lymphohistiocytosis (HLH). Five years later, he developed a clonally unrelated SPTCL and underwent hematopoietic stem cell transplantation. Retrospectively, he was found to carry germline mutations in HAVCR2 associated with reduced T-cell immunoglobulin mucin-3 (TIM-3) expression. We show that mesenteric fatty tissue localization of SPTCL can be the presenting manifestation of TIM-3 deficiency, that this condition predisposes to recurrent lymphoma, and that flow cytometry is a possible screening tool.

Retroviral UNC13D Gene Transfer Restores Cytotoxic Activity of T Cells Derived from Familial Hemophagocytic Lymphohistiocytosis Type 3 Patients In Vitro.

Familial hemophagocytic lymphohistiocytosis (FHL) is a group of life-threatening, autosomal recessive disorders of severe hyperinflammation. FHL type 3 (FHL-3) accounts for about 30% of FHL cases. It is characterized by mutations in the UNC13D gene that give rise to functionally impaired or absent Munc13-4 protein, resulting in impaired secretion of lytic granules by cytotoxic lymphocytes. Etoposide-based therapy is currently used as the standard of care that results in around 60% 5-year survival, illustrating the need for novel treatment approaches. Key problems include treatment toxicity and failure to induce or maintain remission of the hyperinflammation. Instead of immunosuppression, transplantation of autologous gene-corrected T cells can be envisaged as an approach to restore the impaired immune reaction. This study established a protocol that enabled hyperactivated, FHL-3 patient-derived T cells to be cultured and a codon-optimized UNC13D expression cassette to be delivered by either alpha- or gamma-retroviral gene transfer. The data demonstrate that the established protocol can be applied to FHL-3 patient cells with various genetic backgrounds and that gamma-retroviral UNC13D transfer restored expression of functional Munc13-4, as well as degranulation capacity and cell-mediated cytotoxicity of those patient-derived CD8+ T cells. Furthermore, the study shows that the co-introduction of a truncated low-affinity nerve growth factor receptor coding sequence enabled the therapeutic effect to be optimized by enriching transduced cells in a Good Manufacturing Practice-compliant manner. In conclusion, this study lays the foundation for an adaptive immune cell therapy approach aiming at immunological stabilization of FHL-3 patients with autologous, immune-competent T cells prior to hematopoietic stem-cell transplantation.

Between Party, People, and Profession: The Many Faces of the 'Doctor' during the Cultural Revolution.

During the Chinese Cultural Revolution (1966-76), Chairman Mao fundamentally reformed medicine so that rural people received medical care. His new medical model has been variously characterised as: revolutionary Maoist medicine, a revitalised form of Chinese medicine; and the final conquest by Western medicine. This paper finds that instead of Mao's vision of a new 'revolutionary medicine', there was a new medical synthesis that drew from the Maoist ideal and Western and Chinese traditions, but fundamentally differed from all of them. Maoist medicine's ultimate aim was doctors as peasant carers. However, rural people and local governments valued treatment expertise, causing divergence from this ideal. As a result, Western and elite Chinese medical doctors sent to the countryside for rehabilitation were preferable to barefoot doctors and received rural support. Initially Western-trained physicians belittled elite Chinese doctors, and both looked down on barefoot doctors and indigenous herbalists and acupuncturists. However, the levelling effect of terrible rural conditions made these diverse conceptions of the doctor closer during the Cultural Revolution. Thus, urban doctors and rural medical practitioners developed a symbiotic relationship: barefoot doctors provided political protection and local knowledge for urban doctors; urban doctors' provided expertise and a medical apprenticeship for barefoot doctors; and both counted on the local medical knowledge of indigenous healers. This fragile conceptual nexus had fallen apart by the end of the Maoist era (1976), but the evidence of new medical syntheses shows the diverse range of alliances that become possible under the rubric of 'revolutionary medicine'.

Heparanase 2 interacts with heparan sulfate with high affinity and inhibits heparanase activity.

Heparanase activity is highly implicated in cell dissemination associated with tumor metastasis, angiogenesis, and inflammation. Heparanase expression is induced in many hematological and solid tumors, associated with poor prognosis. Heparanase homolog, termed heparanase 2 (Hpa2), was cloned based on sequence homology. Detailed characterization of Hpa2 at the biochemical, cellular, and clinical levels has not been so far reported, and its role in normal physiology and pathological disorders is obscure. We provide evidence that unlike heparanase, Hpa2 is not subjected to proteolytic processing and exhibits no enzymatic activity typical of heparanase. Notably, the full-length Hpa2c protein inhibits heparanase enzymatic activity, likely due to its high affinity to heparin and heparan sulfate and its ability to associate physically with heparanase. Hpa2 expression was markedly elevated in head and neck carcinoma patients, correlating with prolonged time to disease recurrence (follow-up to failure; p = 0.006) and inversely correlating with tumor cell dissemination to regional lymph nodes (N-stage; p = 0.03). Hpa2 appears to restrain tumor metastasis, likely by attenuating heparanase enzymatic activity, conferring a favorable outcome of head and neck cancer patients.

Acetylcholine-induced seizure-like activity and modified cholinergic gene expression in chronically epileptic rats.

The entorhinal cortex (EC) plays an important role in temporal lobe epilepsy. Under normal conditions, the enriched cholinergic innervation of the EC modulates local synchronized oscillatory activity; however, its role in epilepsy is unknown. Enhanced neuronal activation has been shown to induce transcriptional changes of key cholinergic genes and thus alter cholinergic responses. To examine cholinergic modulations in epileptic tissue we studied molecular and electrophysiological cholinergic responses in the EC of chronically epileptic rats following exposure to pilocarpine or kainic acid. We confirmed that while the total activity of the acetylcholine (ACh)-hydrolysing enzyme, acetylcholinesterase (AChE) was not altered, epileptic rats showed alternative splicing of AChE pre-mRNA transcripts, accompanied by a shift from membrane-bound AChE tetramers to soluble monomers. This was associated with increased sensitivity to ACh application: thus, in control rats, ACh (10-100 microm) induced slow (< 1Hz), periodic events confined to the EC; however, in epileptic rats, ACh evoked seconds-long seizure-like events with initial appearance in the EC, and frequent propagation to neighbouring cortical regions. ACh-induced seizure-like events could be completely blocked by the non-specific muscarinic antagonist, atropine, and were partially blocked by the muscarinic-1 receptor antagonist, pirenzepine; but were not affected by the non-specific nicotinic antagonist, mecamylamine. Epileptic rats presented reduced transcript levels of muscarinic receptors with no evidence of mRNA editing or altered mRNA levels for nicotinic ACh receptors. Our findings suggest that altered cholinergic modulation may initiate seizure events in the epileptic temporal cortex.