KLRG1-expressing CD8+ T cells are exhausted and polyfunctional in patients with chronic hepatitis B.

Killer cell lectin-like receptor G1 (KLRG1) has traditionally been regarded as an inhibitory receptor of T cell exhaustion in chronic infection and inflammation. However, its exact role in hepatitis B virus (HBV) infection remains elusive. CD8+ T cells from 190 patients with chronic hepatitis B were analyzed ex vivo for checkpoint and apoptosis markers, transcription factors, cytokines and subtypes in 190 patients with chronic hepatitis B. KLRG1+ and KLRG1- CD8+ T cells were sorted for transcriptome analysis. The impact of the KLRG1-E-cadherin pathway on the suppression of HBV replication mediated by virus-specific T cells was validated in vitro. As expected, HBV-specific CD8+ T cells expressed higher levels of KLRG1 and showed an exhausted molecular phenotype and function. However, despite being enriched for the inhibitory molecules, thymocyte selection-associated high mobility group box protein (TOX), eomesodermin (EOMES), and Helios, CD8+ T cells expressing KLRG1 produced significant levels of tumour necrosis factor (TNF)-α, interferon (IFN)-γ, perforin, and granzyme B, demonstrating not exhausted but active function. Consistent with the in vitro phenotypic assay results, RNA sequencing (RNA-seq) data showed that signature effector T cell and exhausted T cell genes were enriched in KLRG1+ CD8+ T cells. Furthermore, in vitro testing confirmed that KLRG1-E-cadherin binding inhibits the antiviral efficacy of HBV-specific CD8+ T cells. Based on these findings, we concluded that KLRG1+ CD8+ T cells are not only a terminally exhausted subgroup but also exhibit functional diversity, despite inhibitory signs in HBV infection.

Identification and functional analysis of perforin 1 from largemouth bass (Micropterus salmoides).

In this study, we present the first cloning and identification of perforin (MsPRF1) in largemouth bass (Micropterus salmoides). The full-length cDNA of MsPRF1 spans 1572 base pairs, encoding a 58.88 kDa protein consisting of 523 amino acids. Notably, the protein contains MACPF and C2 structural domains. To evaluate the expression levels of MsPRF1 in various healthy largemouth bass tissues, real-time quantitative PCR was employed, revealing the highest expression in the liver and gut. After the largemouth bass were infected by Nocardia seriolae, the mRNA levels of MsPRF1 generally increased within 48 h. Remarkably, the recombinant protein MsPRF1 exhibits inhibitory effects against both Gram-negative and Gram-positive bacteria. Additionally, the largemouth bass showed a higher survival rate in the N. seriolae challenge following the intraperitoneal injection of rMsPRF1, with observed reductions in the tissue bacterial loads. Moreover, rMsPRF1 demonstrated a significant impact on the phagocytic and bactericidal activities of largemouth bass MO/MΦ cells, concurrently upregulating the expression of pro-inflammatory factors. These results demonstrate that MsPRF1 has a potential role in the immune response of largemouth bass against N. seriolae infection.

Disruption of IFNγ, GZMB, PRF1, or LYST Results in Reduced Suppressive Function in Human CD8+ T Cells.

An imbalance between proinflammatory and regulatory processes underlies autoimmune disease pathogenesis. We have shown that acute relapses of multiple sclerosis are characterized by a deficit in the immune suppressive ability of CD8+ T cells. These cells play an important immune regulatory role, mediated in part through cytotoxicity (perforin [PRF]/granzyme [GZM]) and IFNγ secretion. In this study, we further investigated the importance of IFNγ-, GZMB-, PRF1-, and LYST-associated pathways in CD8+ T cell-mediated suppression. Using the CRISPR-Cas9 ribonucleoprotein transfection system, we first optimized efficient gene knockout while maintaining high viability in primary bulk human CD8+ T cells. Knockout was confirmed through quantitative real-time PCR assays in all cases, combined with flow cytometry where appropriate, as well as confirmation of insertions and/or deletions at genomic target sites. We observed that the knockout of IFNγ, GZMB, PRF1, or LYST, but not the knockout of IL4 or IL5, resulted in significantly diminished in vitro suppressive ability in these cells. Collectively, these results reveal a pivotal role for these pathways in CD8+ T cell-mediated immune suppression and provide important insights into the biology of human CD8+ T cell-mediated suppression that could be targeted for immunotherapeutic intervention.

[L-DEP regimen salvage therapy for refractory primary hemophagocytic lymphohistiocytosis triggered by Epstein-Barr virus infection in 4 children].

Objective: To analyze the efficacy and safety of the L-DEP regimen (asparaginase, liposome doxorubicin, etoposide and methylprednisolone) as a salvage therapy for the refractory primary hemophagocytic lymphohistocytosis triggered by Epstein-Barr virus infection (EBV-pHLH) in children. Methods: In this retrospective case study, clinical and laboratory data before and after L-DEP regimen of 4 children diagnosed with EBV-pHLH in Beijing Children's hospital between January 2016 and June 2022 were collected, and the efficacy and safety of L-DEP regimen for the treatment of EBV-pHLH were analyzed. Results: Among 4 patients, there were 3 females and 1 male with the age ranged from 0.8 to 7.0 years. Two of them showed compound heterozygous mutations of PRF1, one with a heterozygous mutation of UNC13D, one homozygous mutation of ITK. Before the L-DEP therapy, all of them had anemia and a soaring level of soluble CD25, 3 patients had neutropenia and thrombopenia, 3 patients had a high level of ferritin, 3 patients had hypofibrinogenemia and 1 patient had hypertriglyceridemia. After receiving 1 or 2 cycles of L-DEP treatment, three achieved remission, including complete remission (1 case) and partial remission (2 cases), and the other one had no remission. The levels of blood cell counts, soluble CD25, triglyceride, fibrinogen and albumin were recovered gradually in 3 patients who got remission. All four patients underwent hematopoietic stem cell transplantation (HSCT) after L-DEP regimen, and three survived. All patients had no severe chemotherapy related complications. The main side effects were bone marrow suppression, infection and pancreatitis, which recovered after appropriate treatments, apart from one who died from severe infection after urgent HSCT. Conclusion: L-DEP regimen could be served as an effective and safe salvage treatment for refractory pediatric EBV-pHLH, and also provide an opportunity for patients to receive HSCT.

Sea Anemone Membrane Attack Complex/Perforin Superfamily Demonstrates an Evolutionary Transitional State between Venomous and Developmental Functions.

Gene duplication is a major force driving evolutionary innovation. A classic example is generating new animal toxins via duplication of physiological protein-encoding genes and recruitment into venom. While this process drives the innovation of many animal venoms, reverse recruitment of toxins into nonvenomous cells remains unresolved. Using comparative genomics, we find members of the Membrane Attack Complex and Perforin Family (MAC) have been recruited into venom-injecting cells (cnidocytes), in soft and stony corals and sea anemones, suggesting that the ancestral MAC was a cnidocyte expressed toxin. Further investigation into the model sea anemone Nematostella vectensis reveals that three members have undergone Nematostella-specific duplications leading to their reverse recruitment into endomesodermal cells. Furthermore, simultaneous knockdown of all three endomesodermally expressed MACs leads to mis-development, supporting that these paralogs have nonvenomous function. By resolving the evolutionary history and function of MACs in Nematostella, we provide the first proof for reverse recruitment from venom to organismal development.

Early hematopoietic cell transplantation for familial hemophagocytic lymphohistiocytosis in a regional treatment network in Japan.

Familial hemophagocytic lymphohistiocytosis (FHLH) is a fatal hyperinflammation syndrome arising from the genetic defect of perforin-mediated cytolysis. Curative hematopoietic cell transplantation (HCT) is needed before development of central nervous system (CNS) disease. We studied treatment outcomes of 13 patients (FHLH2 n = 11, FHLH3 n = 2) consecutively diagnosed from 2011 to 2022 by flow cytometric screening for non-myeloablative HCT in a regional treatment network in Kyushu, Japan. One patient with a novel PRF1 variant escaped screening, but all patients with FHLH2 reached diagnosis and 8 of them received HCT until 3 and 9 months of age, respectively. The earliest HCT was conducted 65 days after birth. Three pretransplant deaths occurred in newborns with liver failure at diagnosis. Ten posttransplant patients have remained disease-free, 7 of whom had no neurological involvement. Time from first etoposide infusion to HCT was shorter in patients without CNS disease or bleeding than in patients with those factors (median [range] days: 62 [50-81] vs. 122 [89-209], p = 0.016). Six of 9 unrelated patients had a PRF1 c.1090_1091delCT variant. These results suggest that the critical times to start etoposide and HCT are within 3 months after birth and during etoposide control, respectively. Newborn screening may increase the percentage of disease-free survivors without complications.

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.

Cell softness renders cytotoxic T lymphocytes and T leukemic cells resistant to perforin-mediated killing.

Mechanical force contributes to perforin pore formation at immune synapses, thus facilitating the cytotoxic T lymphocytes (CTL)-mediated killing of tumor cells in a unidirectional fashion. How such mechanical cues affect CTL evasion of perforin-mediated autolysis remains unclear. Here we show that activated CTLs use their softness to evade perforin-mediated autolysis, which, however, is shared by T leukemic cells to evade CTL killing. Downregulation of filamin A is identified to induce softness via ZAP70-mediated YAP Y357 phosphorylation and activation. Despite the requirements of YAP in both cell types for softness induction, CTLs are more resistant to YAP inhibitors than malignant T cells, potentially due to the higher expression of the drug-resistant transporter, MDR1, in CTLs. As a result, moderate inhibition of YAP stiffens malignant T cells but spares CTLs, thus allowing CTLs to cytolyze malignant cells without autolysis. Our findings thus hint a mechanical force-based immunotherapeutic strategy against T cell leukemia.

ERG mediates the inhibition of NK cell cytotoxicity through the HLX/STAT4/Perforin signaling pathway, thereby promoting the progression of myocardial infarction.

This study aimed to investigate the role of ERG in the HLX/STAT4/Perforin signaling axis, impacting natural killer (NK) cell cytotoxicity and myocardial infarction (MI) progression. NK cell cytotoxicity was assessed via co-culture and 51Cr release assays. Datasets GSE34198 and GSE97320 identified common differentially expressed genes in MI. NK cell gene expression was analyzed in MI patients and healthy individuals using qRT-PCR and Western blotting. ERG's regulation of HLX and STAT4's regulation of perforin were studied through computational tools (MEM) and ChIP experiments. HLX's influence on STAT4 was explored with the MG132 proteasome inhibitor. Findings were validated in a mouse MI model.ERG, a commonly upregulated gene, was identified in NK cells from MI patients and mice. ERG upregulated HLX, leading to STAT4 proteasomal degradation and reduced Perforin expression. Consequently, NK cell cytotoxicity decreased, promoting MI progression. ERG mediates the HLX/STAT4/Perforin axis to inhibit NK cell cytotoxicity, fostering MI progression. These results provide vital insights into MI's molecular mechanisms.

Upregulation of CX3CR1 expression in circulating T cells of systemic lupus erythematosus patients as a reflection of autoimmune status through characterization of cytotoxic capacity.

OBJECTIVE: This study investigated CX3CR1 expression in human peripheral blood T lymphocytes and their subsets, exploring changes in SLE patients and its diagnostic potential. METHODS: Peripheral blood samples from 31 healthy controls and 50 SLE patients were collected. RNA-Seq data from SLE patient PBMCs were used to analyze CX3CR1 expression in T cells. Flow cytometry determined CX3CR1-expressing T lymphocyte subset proportions in SLE patients and healthy controls. Subset composition and presence of GZMB, GPR56, and perforin in CX3CR1+ T lymphocytes were analyzed. T cell-clinical indicator correlations were assessed. ROC curves explored CX3CR1's diagnostic potential for SLE. RESULTS: CX3CR1+CD8+ T cells exhibited higher GPR56, perforin, and GZMB expression than other T cell subsets. The proportion of CX3CR1+ was higher in TEMRA and lower in Tn and TCM. PMA activation reduced CX3CR1+ T cell proportions. Both RNA-Seq and flow cytometry revealed elevated CX3CR1+ T cell proportions in SLE patients. Significantly lower perforin+ and GPR56+ proportions were observed in CX3CR1+CD8+ T cells in SLE patients. CX3CR1+ T cells correlated with clinical indicators. CONCLUSION: CX3CR1+ T cells display cytotoxic features, with heightened expression in CD8+ T cells, particularly in adult SLE patients. Increased CX3CR1 expression in SLE patient T cells suggests its potential as an adjunctive diagnostic marker for SLE.

Regulation and distribution of European sea bass perforins point to their role in the adaptive cytotoxic response against NNV.

Cell-mediated cytotoxicity is a complex immune mechanism that involves the release of several killing molecules, being perforin (PRF) one of the most important effector players. Perforin is synthesized by T lymphocytes and natural killer cells in mammals and responsible for the formation of pores on the target cell membrane during the killing process. Although perforin has been extensively studied in higher vertebrates, this knowledge is very limited in fish. Therefore, in this study we have identified four prf genes in European sea bass (Dicentrarchus labrax) and evaluated their mRNA levels. All sea bass prf genes showed the typical and conserved domains of its human orthologue and were closely clustered by the phylogenetic analysis. In addition, all genes showed constitutive and ubiquitous tissular expression, being prf1.9 gene the most highly expressed in immune tissues. Subsequently, in vitro stimulation of head-kidney (HK) cells with phytohemagglutinin, a T-cell activator, showed an increase of all prf gene levels, except for prf1.3 gene. European sea bass HK cells increased the transcription of prf1.2 and prf1.9 during the innate cell-mediated cytotoxic activity against xenogeneic target cells. In addition, sea bass infected with nodavirus (NNV) showed a similar expression pattern of all prf in HK and brain at 15 days post-infection, except for prf1.3 gene and in the gonad. Finally, the use of a polyclonal antibody against PRF1.9 showed an increase of positive cells in HK, brain and gonad from NNV-infected fish. Taken together, the data seem to indicate that all prf genes, except prf1.3, appear to be involved in the European sea bass immunity, and probably in the cell-mediated cytotoxic response, with PRF1.9 playing the most important role against nodavirus. The involvement of the PRFs and the CMC activity in the vertical transmission success of the virus is also discussed.

Concomitant assessment of PD-1 and CD56 expression identifies subsets of resting cord blood Vδ2 T cells with disparate cytotoxic potential.

Vγ9Vδ2 T lymphocytes are programmed for broad antimicrobial responses with rapid production of Th1 cytokines even before birth, and thus thought to play key roles against pathogens in infants. The process regulating Vδ2 cell acquisition of cytotoxic potential shortly after birth remains understudied. We observed that perforin production in cord blood Vδ2 cells correlates with phenotypes defined by the concomitant assessment of PD-1 and CD56. Bulk RNA sequencing of sorted Vδ2 cell fractions indicated that transcripts related to cytotoxic activity and NK function are enriched in the subset with the highest proportion of perforin+ cells. Among differentially expressed transcripts, IRF8, previously linked to CD8 T cell effector differentiation and NK maturation, has the potential to mediate Vδ2 cell differentiation towards cytotoxic effectors. Our current and past results support the hypothesis that distinct mechanisms regulate Vδ2 cell cytotoxic function before and after birth, possibly linked to different levels of microbial exposure.

Case Report: Chronic inflammatory demyelinating polyradiculoneuropathy rather than hemophagocytic lymphohistiocytosis-the initial phenotype of PRF1 gene mutation.

Perforin is essentially involved in the granule-dependent killing activities of cytotoxic T lymphocytes and NK cells. Monoallelic PRF1 mutation increases the risk of autoimmune diseases, and biallelic PRF1 mutation causes familial hemophagocytic lymphohistiocytosis-2. Here, we report a case of a 12-year-old girl with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), followed by a rapidly progressive onset of hemophagocytic lymphohistiocytosis (HLH) 9 months later, alongside manifestations of demyelinating encephalopathy. Genetic sequencing revealed a heterozygous nonsense mutation in the PRF1 gene (c.984G>A; p.W328*) and a heterozygous missense mutation in the PRF1 gene (c.1349C>T; p.T450M). Eventually, she died because of no suitable allogeneic hematopoietic stem cell available in time. Our observations suggest that CIPD might represent the initial phenotype of biallelic PRF1 mutation and could serve as an early sign of subsequent HLH. A comprehensive understanding of this condition is paramount for timely diagnosis, treatment, and ultimately improved patient outcomes.

Genome-Wide Analysis of the Membrane Attack Complex and Perforin Genes and Their Expression Pattern under Stress in the Solanaceae.

The Membrane Attack Complex and Perforin (MACPF) proteins play a crucial role in plant development and adaptation to environmental stresses. Heretofore, few MACPF genes have been functionally identified, leaving gaps in our understanding of MACPF genes in other plants, particularly in the Solanaceae family, which includes economically and culturally significant species, such as tomato, potato, and pepper. In this study, we have identified 26 MACPF genes in three Solanaceae species and in the water lily, which serves as the base group for angiosperms. Phylogenetic analysis indicates that angiosperm MACPF genes could be categorized into three distinct groups, with another moss and spikemoss lineage-specific group, which is further supported by the examination of gene structures and domain or motif organizations. Through inter-genome collinearity analysis, it is determined that there are 12 orthologous SolMACPF gene pairs. The expansion of SolMACPF genes is primarily attributed to dispersed duplications, with purifying selection identified as the principal driving force in their evolutionary process, as indicated by the ω values. Furthermore, the analysis of expression patterns revealed that Solanaceae genes are preferentially expressed in reproductive tissues and regulated by various environmental stimuli, particularly induced by submergence. Taken together, these findings offer valuable insights into and a fresh perspective on the evolution and function of SolMACPF genes, thereby establishing a foundation for further investigations into their phenotypic and functional characteristics.

Complementary HLH susceptibility factors converge on CD8 T-cell hyperactivation.

Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperinflammatory syndromes. Familial HLH is caused by genetic impairment of granule-mediated cytotoxicity (eg, perforin deficiency). MAS is linked to excess activity of the inflammasome-activated cytokine interleukin-18 (IL-18). Though individually tolerated, mice with dual susceptibility (Prf1⁻/⁻Il18tg; DS) succumb to spontaneous, lethal hyperinflammation. We hypothesized that understanding how these susceptibility factors synergize would uncover key pathomechanisms in the activation, function, and persistence of hyperactivated CD8 T cells. In IL-18 transgenic (Il18tg) mice, IL-18 effects on CD8 T cells drove MAS after a viral (lymphocytic choriomeningitis virus), but not innate (toll like receptor 9), trigger. In vitro, CD8 T cells also required T-cell receptor (TCR) stimulation to fully respond to IL-18. IL-18 induced but perforin deficiency impaired immunoregulatory restimulation-induced cell death (RICD). Paralleling hyperinflammation, DS mice displayed massive postthymic oligoclonal CD8 T-cell hyperactivation in their spleens, livers, and bone marrow as early as 3 weeks. These cells increased proliferation and interferon gamma production, which contrasted with increased expression of receptors and transcription factors associated with exhaustion. Broad-spectrum antibiotics and antiretrovirals failed to ameliorate the disease. Attempting to genetically "fix" TCR antigen-specificity instead demonstrated the persistence of spontaneous HLH and hyperactivation, chiefly on T cells that had evaded TCR fixation. Thus, drivers of HLH may preferentially act on CD8 T cells: IL-18 amplifies activation and demand for RICD, whereas perforin supplies critical immunoregulation. Together, these factors promote a terminal CD8 T-cell activation state, combining features of exhaustion and effector function. Therefore, susceptibility to hyperinflammation may converge on a unique, unrelenting, and antigen-dependent state of CD8 T-cell hyperactivation.

[Clinical characteristics of primary hemophagocytic lymphohistiocytosis associated with perforin gene deficiency: a single-center retrospective study].

Objective: This study aimed to investigate the clinical characteristics of patients diagnosed with primary hemophagocytic lymphohistiocytosis (pHLH) associated with perforin gene deficiency. Methods: We retrospectively analyzed the clinical data of 16 pHLH patients associated with perforin gene deficiency at Beijing Friendship Hospital, Capital Medical University, from April 2014 to August 2021. The mutation sites, mutation types, family history, clinical characteristics, and prognosis of the patients were assessed. Results: A total of 16 patients, including ten males and six females, with a median onset age of 17.5 years (range: 4-42 years), were enrolled in this study. Sixteen different mutations were identified, consisting of 11 missense mutations, one nonsense mutation, two frameshift mutations, and two in-frame mutations. All patients harbored at least one deleterious missense mutation, with the most common mutation sites being c.1349C>T (p.T450M) and c.503G>A (p.S168N). Decreased natural killer (NK) cell activity was observed in 11 patients, reduced perforin protein expression in ten patients, concurrent Epstein-Barr virus (EBV) infection at onset in eight patients, a family history in two patients, and central nervous system involvement in four patients. Eleven cases underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT), with eight cases surviving. The median survival time of non-transplanted patients was eight months (range: 4-18 months), while that of transplanted patients was reported as "not reached". Conclusions: Emphasizing the diagnosis of pHLH in adults with perforin gene deficiency. In addition, it should be noted that EBV infection can potentially act as a triggering factor in such disease, and allo-HSCT exerts a substantial effect on the prognosis of patients.

Granzyme-A deficiency attenuates experimental osteoarthritis in mice, but perforin deficiency does not.

OBJECTIVES: This study aims to assess the development of osteoarthritis (OA) in granzyme A- (gzmA) and B- (gzmB) and perforin- (perf) knockout mice. MATERIALS AND METHODS: A total of 75 male and female C57BL/6 (eight to nine-week-old) mice were allocated to: gzmA-deficient (gzmA-/-) (11 females, 8 males), gzmB-deficient (gzmB-/-) (9 females, 8 males), perf-deficient (perf-/-) (10 females, 9 males), and control group (10 females, 10 males). Osteoarthritis was induced in the right knee by instability of the meniscus medial ligament. Sham surgery was practiced in the left knee. Knee samples obtained eight weeks after surgery were stained (Safranin-O) and blindly scored in lateral and medial femur and tibia using the Osteoarthritis Research Society International scale (OARSI) (from Grade 0, cartilage intact to 6, deformation), (five stages from 0, no OA to 4, >50% surface involvement); OARSI score (Grade x Stage); and a semi-quantitative scale from Grade 0 (normal) to 6 (cartilage erosion >80%). RESULTS: Significantly higher values in all scales in the right knees compared to the left knees in male and female mice were observed (p<0.05). Males of all strains showed in the right knee higher values than females on all scales. Deficiency of perforin did not modify OA severity in any sex. The gzmA-/- females presented less degenerative changes than the other groups. CONCLUSION: Our study results show that sex plays an important role in the development of experimental OA in mice. Deficiency of gzmA can protect from the development of OA in female mice.

Hemophagocytic lymphohistiocytosis with a hemizygous PRF1 c.674G>A mutation.

Hemophagocytic lymphohistiocytosis(HLH) is a rare highly-fatal disease presenting with fever, hepatosplenomegaly, and pancytopenia and has a poor prognosis. Homozygous or semi-zygous or complex heterozygous variants can cause familial HLH and heterozygous carriers are frequently seen in secondary HLH. A 42-year-old male patient was admitted to the hospital for persistent fever, fatigue, and splenomegaly. Investigations revealed hypertriglyceridemia, hyperlactatemia dehydrogenaseemia, hyperferritinemia, and elevated levels of soluble cluster of differentiation 25. We found a heterozygous mutation of PRF1: c.674G>A (p.R225Q) through next-generation sequencing technology of hemophagocytic-lymphohistiocytosis-related genes. After a brief remission with dexamethasone and etoposide-based therapy, the disease relapsed quickly, and an allogeneic hematopoietic stem cell transplant was performed to achieve complete remission. To date, the patient's condition was in complete remission. Our study detected a rare missense mutation in the PRF1 gene in a patient with HLH disease and the c.674G>A mutation may be rated as a possible pathogenic variant.

Familial hemophagocytic phohistiocytosis induced by PRF1 mutation with neurologic manifestations as the initial clinical presentations: A case report.

BACKGROUND: To investigate the clinical characteristics of familial hemophagocytic phohistiocytosis (FHL) induced by PRF1 gene mutation and with central nervous injury as the initial presentation. CASE PRESENTATION: Herein, we presented 2 cases of a familial hemophagocytic syndrome caused by PRF1 gene mutation in 1 family with central nervous injury as the first symptom and searched relevant literature for clinical analysis of its pathogenic characteristics. Two children from 1 family were included in this study, both of whom had complex heterozygous mutations of C. 1189_1190dupTG (p.H398Afs*23) and C. 394G>A (p.G132R). Literature search further revealed 20 cases of PRF1 gene mutation-induced familial FHL with central nervous injury as the initial presentation. The main neurological symptoms included cranial nerve injury (81.8%), convulsion (77.3%), ataxia (63.6%), encephalopathy (59.1%), and limb paralysis (40.9%). Cranial imaging findings were dominated by the cerebral hemisphere (100%), cerebellar hemisphere (85%), brainstem (55%), and periventricular white matter (40%), and 73.7% of cases had elevated white blood cell count in CSF. Most cases were confirmed by differential diagnosis and gene sequencing, which suggested that C. 673C>T (P.r225W), C. 394G>A (P.G132r), C. 666C>A (p.H222Q), C. 1349C>T (p.T450M), C. 1349C>T (p.T450M), and C. 443C>C (p.A148G) could be focal mutations of this disease. CONCLUSION: Lesions involving the cerebellum and brainstem in children with ataxia and cranial nerve damage could be indicative of primary FHL; thus, the inherent immune test and gene test should be timely performed to help confirm the diagnosis, guide the treatment, and improve the prognosis.

First case of very late-onset FHL2 in Spain with two variants in the PRF1 gene.

Hemophagocytic lymphohistiocytosis (HLH) is a rare but fatal disorder characterized by the proliferation and infiltration of macrophages and hyperactivated T lymphocytes that escape from the physiological control pathways and favour the existence of an environment of excessive inflammation and tissue destruction. HLH has been classified into two types: a primary or familial autosomal recessive form, caused by mutations in genes encoding proteins involved in the granule-dependent cytotoxic pathway (familial hemophagocytic lymphohistiocytosis [FHL] types 1-5); and other secondary or acquired form, generally associated with infections, malignancy, autoimmune diseases, metabolic disorders or primary immunodeficiencies. Since the first familial hemophagocytic lymphohistiocytosis-2 (FHL2) causative mutation in the PRF1 gene was described in 1999, more than 200 mutations have been identified to date. Here, we report the first case of very late-onset FHL2 in a Spanish 72-year-old female with splenomegaly, hypertriglyceridemia, hypofibrinogenemia, pancytopenia and marrow hemophagocytosis harbouring in heterozygosity two PRF1 variants proposed as causative in this study. The heterozygous mutation c.445G>A (p.Gly149Ser) identified in the exon 2 results in a missense mutation previously described as a probable pathogenic variant associated with the development of FHL2. Affecting the same exon, c.272C>T (p.Ala91Val) is the most prevalent variant of this gene. Although it was initially classified as benign, recent studies support its potential pathogenic role, considering it a variant of uncertain significance associated with a risk of developing FHL2. The genetic confirmation of FHL made possible an adequate counselling to the patient and direct relatives and provided important information for her control and follow-up.