PLCG2, A Regulator of Lung Adenocarcinoma Proliferation and Migration Associated with Immune Infiltration.

BACKGROUND: Results from the TCGA database showed that phosphatidylinositol-specific phospholipase Cγ2 (PLCG2) expression level in Lung Adenocarcinoma (LUAD) was notably decreased compared to adjacent tissues, so we unveiled its role of LUAD. OBJECTIVE: This study aims to explore the expression and clinical significance of Phosphatidyl-inositol-specific phospholipase Cγ2 (PLCG2) in lung adenocarcinoma (LUAD) cells and its role in cell proliferation and metastasis. METHODS: Differential PLCG2 mRNA and protein levels between LUAD tissues and adjacent tissues were analyzed from the TCGA database, TIMER, and UALCAN database. Differentially expressed genes were screened for patients in the high and low PLCG2 mRNA expression groups by the R package as well as GSEA. The expression level of PLCG2 in LUAD cells was detected using qRT-PCR and CCK8, clone formation, Transwell, and Western blot assays. RESULTS: PLCG2 was lowly expressed in LUAD and did not significantly correlate with the prognosis of LUAD. PLCG2 expression levels varied significantly in terms of patients' gender, age, T, N, and pathological stage. GO/KEGG enrichment analysis showed that co-expression of PLCG2 was mainly associated with the immune response- regulating cell-surface receptors, and so on. GSEA analysis showed enrichment pathways of PLCG2-related differential gees were primarily associated with the olfactory transduction pathway, ribosome, etc. R software analysis revealed a significant correlation between PLCG2 expression and six types of immune-infiltrat-ing cells, positively correlated with immune checkpoint-related genes and negatively regulated by tumor mutational load. Overexpressing PLCG2 showed reduced LUAD cell proliferation, clone formation, cell migration and invasion, and epithelial-mesenchymal transition-associated proteins, compared with the control group. CONCLUSION: PLCG2 is lowly expressed in LUAD tissues and is involved in immune infiltration of LUAD, inhibiting LUAD cell proliferation and metastasis.

Methyltransferase DNMT3B promotes colorectal cancer cell proliferation by inhibiting PLCG2.

Aberrant DNA methylation patterns in the promoter region of PLCG2 are associated with dysregulated signaling pathways and cellular functions. Its role in colorectal cancer cells is still unknown. In this study, qRT-PCR is used to measure DNMT3B expression in colorectal cancer. Western blot analysis and immunohistochemistry are used to analyze DNMT3B and PLCG2 protein levels in colorectal tissues and cell lines. Cell Counting Kit-8 (CCK-8) and colony formation assays are used to assess the proliferation of colorectal cancer cells. Methylation-specific PCR (MSP) and bisulfite-sequencing PCR (BSP) are used to measure DNA methylation level. Our results show that DNMT3B is overexpressed in colorectal cells in the TCGA datasets according to Kaplan-Meier plots. DNMT3B is significantly overexpressed in tumor tissues compared to that in adjacent nontumor tissues. Western blot analysis results demonstrate high expression of DNMT3B in tumor tissues. Compared to normal colonic epithelial cells, colorectal cancer cell lines exhibit elevated level of PLCG2 methylation. Overexpression of PLCG2 effectively prevents the growth of colorectal cancer xenograft tumors in vivo. PLCG2 is identified as a key downstream regulatory protein of DNMT3B in colorectal cancer. DNMT3B inhibits PLCG2 transcription through methylation of the PLCG2 promoter region. DNMT3B controls colorectal cancer cell proliferation through PLCG2, which is useful for developing therapeutic approaches that target PLCG2 expression for the treatment of colorectal cancer.

Differential NEUROD1, ASCL1, and POU2F3 Expression Defines Molecular Subsets of Bladder Small Cell/Neuroendocrine Carcinoma With Prognostic Implications.

Small cell carcinomas (SMC) of the lung are now molecularly classified based on the expression of transcriptional regulators (NEUROD1, ASCL1, POU2F3, and YAP1) and DLL3, which has emerged as an investigational therapeutic target. PLCG2 has been shown to identify a distinct subpopulation of lung SMC with stem cell-like and prometastasis features and poor prognosis. We analyzed the expression of these novel neuroendocrine markers and their association with traditional neuroendocrine markers and patient outcomes in a cohort of bladder neuroendocrine carcinoma (NEC) consisting of 103 SMC and 19 large cell NEC (LCNEC) assembled in tissue microarrays. Coexpression patterns were assessed and integrated with detailed clinical annotation including overall (OS) and recurrence-free survival (RFS) and response to neoadjuvant/adjuvant chemotherapy. We identified 5 distinct molecular subtypes in bladder SMC based on the expression of ASCL1, NEUROD1, and POU2F3: ASCL1+/NEUROD1- (n = 33; 34%), ASCL1- /NEUROD1+ (n = 21; 21%), ASCL1+/NEUROD1+ (n = 17; 17%), POU2F3+ (n = 22, 22%), and ASCL1- /NEUROD1- /POU2F3- (n = 5, 5%). POU2F3+ tumors were mutually exclusive with those expressing ASCL1 and NEUROD1 and exhibited lower expression of traditional neuroendocrine markers. PLCG2 expression was noted in 33 tumors (32%) and was highly correlated with POU2F3 expression (P < .001). DLL3 expression was high in both SMC (n = 72, 82%) and LCNEC (n = 11, 85%). YAP1 expression was enriched in nonneuroendocrine components and negatively correlated with all neuroendocrine markers. In patients without metastatic disease who underwent radical cystectomy, PLCG2+ or POU2F3+ tumors had shorter RFS and OS (P < .05), but their expression was not associated with metastasis status or response to neoadjuvant/adjuvant chemotherapy. In conclusion, the NEC of the bladder can be divided into distinct molecular subtypes based on the expression of ASCL1, NEUROD1, and POU2F3. POU2F3-expressing tumors represent an ASCL1/NEUROD1-negative subset of bladder NEC characterized by lower expression of traditional neuroendocrine markers. Marker expression patterns were similar in SMC and LCNEC. Expression of PLCG2 and POU2F3 was associated with shorter RFS and OS. DLL3 was expressed at high levels in both SMC and LCNEC of the bladder, nominating it as a potential therapeutic target.

In vivo validation of late-onset Alzheimer's disease genetic risk factors.

INTRODUCTION: Genome-wide association studies have identified over 70 genetic loci associated with late-onset Alzheimer's disease (LOAD), but few candidate polymorphisms have been functionally assessed for disease relevance and mechanism of action. METHODS: Candidate genetic risk variants were informatically prioritized and individually engineered into a LOAD-sensitized mouse model that carries the AD risk variants APOE ε4/ε4 and Trem2*R47H. The potential disease relevance of each model was assessed by comparing brain transcriptomes measured with the Nanostring Mouse AD Panel at 4 and 12 months of age with human study cohorts. RESULTS: We created new models for 11 coding and loss-of-function risk variants. Transcriptomic effects from multiple genetic variants recapitulated a variety of human gene expression patterns observed in LOAD study cohorts. Specific models matched to emerging molecular LOAD subtypes. DISCUSSION: These results provide an initial functionalization of 11 candidate risk variants and identify potential preclinical models for testing targeted therapeutics. HIGHLIGHTS: A novel approach to validate genetic risk factors for late-onset AD (LOAD) is presented. LOAD risk variants were knocked in to conserved mouse loci. Variant effects were assayed by transcriptional analysis. Risk variants in Abca7, Mthfr, Plcg2, and Sorl1 loci modeled molecular signatures of clinical disease. This approach should generate more translationally relevant animal models.

Human PLCG2 haploinsufficiency results in a novel natural killer cell immunodeficiency.

BACKGROUND: Although most individuals effectively control herpesvirus infections, some suffer from severe and/or recurrent infections. A subset of these patients possess defects in natural killer (NK) cells, lymphocytes that recognize and lyse herpesvirus-infected cells; however, the genetic etiology is rarely diagnosed. PLCG2 encodes a signaling protein in NK-cell and B-cell signaling. Dominant-negative or gain-of-function variants in PLCG2 cause cold urticaria, antibody deficiency, and autoinflammation. However, loss-of-function variants and haploinsufficiency have not been reported to date. OBJECTIVES: The investigators aimed to identify the genetic cause of NK-cell immunodeficiency in 2 families and herein describe the functional consequences of 2 novel loss-of-function variants in PLCG2. METHODS: The investigators employed whole-exome sequencing in conjunction with mass cytometry, microscopy, functional assays, and a mouse model of PLCG2 haploinsufficiency to investigate 2 families with NK-cell immunodeficiency. RESULTS: The investigators identified novel heterozygous variants in PLCG2 in 2 families with severe and/or recurrent herpesvirus infections. In vitro studies demonstrated that these variants were loss of function due to haploinsufficiency with impaired NK-cell calcium flux and cytotoxicity. In contrast to previous PLCG2 variants, B-cell function remained intact. Plcg2+/- mice also displayed impaired NK-cell function with preserved B-cell function, phenocopying human disease. CONCLUSIONS: PLCG2 haploinsufficiency represents a distinct syndrome from previous variants characterized by NK-cell immunodeficiency with herpesvirus susceptibility, expanding the spectrum of PLCG2-related disease.

Multi-omics analyses reveal novel effects of PLCγ2 deficiency in the mouse brain.

Phospholipase C gamma-2 (PLCγ2) catalyzes the hydrolysis of the membrane phosphatidylinositol-4,5-bisphosphate (PIP2) to form diacylglycerol (DAG) and inositol trisphosphate (IP3), which subsequently feed into numerous downstream signaling pathways. PLCG2 polymorphisms are associated with both reduced and increased risk of Alzheimer's disease (AD) and with longevity. In the brain, PLCG2 is highly expressed in microglia, where it is proposed to regulate phagocytosis, secretion of cytokines/chemokines, cell survival and proliferation. We analyzed the brains of three-month-old PLCγ2 knockout (KO), heterozygous (HET), and wild-type (WT) mice using multiomics approaches, including shotgun lipidomics, proteomics, and gene expression profiling, and immunofluorescence. Lipidomic analyses revealed sex-specific losses of total cerebrum PIP2 and decreasing trends of DAG content in KOs. In addition, PLCγ2 depletion led to significant losses of myelin-specific lipids and decreasing trends of myelin-enriched lipids. Consistent with our lipidomics results, RNA profiling revealed sex-specific changes in the expression levels of several myelin-related genes. Further, consistent with the available literature, gene expression profiling revealed subtle changes on microglia phenotype in mature adult KOs under baseline conditions, suggestive of reduced microglia reactivity. Immunohistochemistry confirmed subtle differences in density of microglia and oligodendrocytes in KOs. Exploratory proteomic pathway analyses revealed changes in KO and HET females compared to WTs, with over-abundant proteins pointing to mTOR signaling, and under-abundant proteins to oligodendrocytes. Overall, our data indicate that loss of PLCγ2 has subtle effects on brain homeostasis that may underlie enhanced vulnerability to AD pathology and aging via novel mechanisms in addition to regulation of microglia function.

Cold Urticaria Syndromes: Diagnosis and Management.

Cold urticaria is a chronic condition causing episodic symptoms of cold-induced wheals or angioedema in response to direct or indirect exposure to cold temperatures. Whereas symptoms of cold urticaria are typically benign and self-limiting, severe systemic anaphylactic reactions are possible. Acquired, atypical, and hereditary forms have been described, each with variable triggers, symptoms, and responses to therapy. Clinical testing, including response to cold stimulation, helps define disease subtypes. More recently, monogenic disorders characterized by atypical forms of cold urticaria have been described. Here, we review the different forms of cold-induced urticaria and related syndromes and propose a diagnostic algorithm to aid clinicians in making a timely diagnosis for the appropriate management of these patients.

Carriers of the p.P522R variant in PLCγ2 have a slightly more responsive immune system.

BACKGROUND: The rs72824905 single-nucleotide polymorphism in the PLCG2 gene, encoding the p.P522R residue change in Phospholipase C gamma 2 (PLCγ2), associates with protection against several dementia subtypes and with increased likelihood of longevity. Cell lines and animal models indicated that p.P522R is a functional hypermorph. We aimed to confirm this in human circulating peripheral immune cells. METHODS: We compared effects of p.P522R on immune system function between carriers and non-carriers (aged 59-103y), using in-depth immunophenotyping, functional B-cell and myeloid cell assays, and in vivo SARS-CoV-2 vaccination. RESULTS: In line with expectations, p.P522R impacts immune cell function only slightly, but it does so across a wide array of immune cell types. Upon B-cell stimulation, we observed increased PLCγ2 phosphorylation and calcium release, suggesting increased B-cell sensitivity upon antigen recognition. Further, p.P522R-carriers had higher numbers of CD20++CD21-CD24+ naive B cells and IgG1+ memory B cells. In myeloid cells, normalized ROS production was higher upon PLCγ2-dependent stimulation. On classical monocytes, CD33 levels were elevated. Furthermore, carriers expressed lower levels of allergy-related FcεRI on several immune cell subsets. Nevertheless, carriers and non-carriers had similar serological responses to SARS-CoV-2 vaccination. CONCLUSION: The immune system from p.P522R-carriers is slightly more responsive to stimulation than in non-carriers.

A novel fluorogenic reporter substrate for 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2 (PLCγ2): Application to high-throughput screening for activators to treat Alzheimer's disease.

A rare coding variant in PLCγ2 (P522R) expressed in microglia induces a mild activation of enzymatic activity when compared to wild-type. This mutation is reported to be protective against the cognitive decline associated with late-onset Alzheimer's disease (LOAD) and therefore, activation of wild-type PLCγ2 has been suggested as a potential therapeutic target for the prevention and treatment of LOAD. Additionally, PLCγ2 has been associated with other diseases such as cancer and some autoimmune disorders where mutations with much greater increases in PLCγ2 activity have been identified. Here, pharmacological inhibition may provide a therapeutic effect. In order to facilitate our investigation of the activity of PLCγ2, we developed an optimized fluorogenic substrate to monitor enzymatic activity in aqueous solution. This was accomplished by first exploring the spectral properties of various "turn-on" fluorophores. The most promising turn-on fluorophore was incorporated into a water-soluble PLCγ2 reporter substrate, which we named C8CF3-coumarin. The ability of PLCγ2 to enzymatically process C8CF3-coumarin was confirmed, and the kinetics of the reaction were determined. Reaction conditions were optimized to identify small molecule activators, and a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed with the goal of identifying small molecule activators of PLCγ2. The optimized screening conditions allowed identification of potential PLCγ2 activators and inhibitors, thus demonstrating the feasibility of this approach for high-throughput screening.

Clinical and immunological features of an APLAID patient caused by a novel mutation in PLCG2.

Background: The APLAID syndrome is a rare primary immunodeficiency caused by gain-of-function mutations in the PLCG2 gene. We present a 7-year-old APLAID patient who has recurrent blistering skin lesions, skin infections in the perineum, a rectal perineal fistula, and inflammatory bowel disease. Methods: To determine the genetic cause of our patient, WES and bioinformatics analysis were performed. Flow cytometry was used for phenotyping immune cell populations in peripheral blood. Cytokines released into plasma were analyzed using protein chip technology. The PBMCs of patient and a healthy child were subjected to single-cell RNA-sequencing analysis. Results: The patient carried a novel de novo missense mutation c.2534T>C in exon 24 of the PLCG2 gene that causes a leucine to serine amino acid substitution (p.Leu845Ser). Bioinformatics analysis revealed that this mutation had a negative impact on the structure of the PLCγ2 protein, which is highly conserved in many other species. Immunophenotyping by flow cytometry revealed that in addition to the typical decrease in circulating memory B cells, the levels of myeloid dendritic cells (mDCs) in the children's peripheral blood were significantly lower, as were the CD4+ effector T cells induced by their activation. Single-cell sequencing revealed that the proportion of different types of cells in the peripheral blood of the APLAID patient changed. Conclusions: We present the first case of APLAID with severely reduced myeloid dendritic cells carrying a novel PLCG2 mutation, and conducted a comprehensive analysis of immunological features in the ALPAID patient, which has not been mentioned in previous reports. This study expands the spectrum of APLAID-associated immunophenotype and genotype. The detailed immune analyses in this patient may provide a basis for the development of targeted therapies for this severe autoinflammatory disease.

In vivo validation of late-onset Alzheimer's disease genetic risk factors.

Introduction: Genome-wide association studies have identified over 70 genetic loci associated with late-onset Alzheimer's disease (LOAD), but few candidate polymorphisms have been functionally assessed for disease relevance and mechanism of action. Methods: Candidate genetic risk variants were informatically prioritized and individually engineered into a LOAD-sensitized mouse model that carries the AD risk variants APOE4 and Trem2*R47H. Potential disease relevance of each model was assessed by comparing brain transcriptomes measured with the Nanostring Mouse AD Panel at 4 and 12 months of age with human study cohorts. Results: We created new models for 11 coding and loss-of-function risk variants. Transcriptomic effects from multiple genetic variants recapitulated a variety of human gene expression patterns observed in LOAD study cohorts. Specific models matched to emerging molecular LOAD subtypes. Discussion: These results provide an initial functionalization of 11 candidate risk variants and identify potential preclinical models for testing targeted therapeutics.

Multiple gene variants linked to Alzheimer's-type clinical dementia via GWAS are also associated with non-Alzheimer's neuropathologic entities.

The classic pathologic hallmarks of Alzheimer's disease (AD) are amyloid plaques and neurofibrillary tangles (AD neuropathologic changes, or ADNC). However, brains from individuals clinically diagnosed with "AD-type" (amnestic) dementia usually harbor heterogeneous neuropathologies in addition to, or other than, ADNC. We hypothesized that some AD-type dementia associated genetic single nucleotide variants (SNVs) identified from large genomewide association studies (GWAS) were associated with non-ADNC neuropathologies. To test this hypothesis, we analyzed data from multiple studies with available genotype and neuropathologic phenotype information. Clinical AD/dementia risk alleles of interest were derived from the very large GWAS by Bellenguez et al. (2022) who reported 83 clinical AD/dementia-linked SNVs in addition to the APOE risk alleles. To query the pathologic phenotypes associated with variation of those SNVs, National Alzheimer's disease Coordinating Center (NACC) neuropathologic data were linked to AD Sequencing Project (ADSP) and AD Genomics Consortium (ADGC) data. Separate data were obtained from the harmonized Religious Orders Study and the Rush Memory and Aging Project (ROSMAP). A total of 4811 European participants had at least ADNC neuropathology data and also genotype data available; data were meta-analyzed across cohorts. As expected, a subset of dementia-associated SNVs were associated with ADNC risk in Europeans-e.g., BIN1, PICALM, CR1, MME, and COX7C. Other gene variants linked to (clinical) AD dementia were associated with non-ADNC pathologies. For example, the associations of GRN and TMEM106B SNVs with limbic-predominant age-related TDP-43 neuropathologic changes (LATE-NC) were replicated. In addition, SNVs in TNIP1 and WNT3 previously reported as AD-related were instead associated with hippocampal sclerosis pathology. Some genotype/neuropathology association trends were not statistically significant at P < 0.05 after correcting for multiple testing, but were intriguing. For example, variants in SORL1 and TPCN1 showed trends for association with LATE-NC whereas Lewy body pathology trended toward association with USP6NL and BIN1 gene variants. A smaller cohort of non-European subjects (n = 273, approximately one-half of whom were African-Americans) provided the basis for additional exploratory analyses. Overall, these findings were consistent with the hypothesis that some genetic variants linked to AD dementia risk exert their affect by influencing non-ADNC neuropathologies.

Cardio-omentopexy requires a cardioprotective innate immune response to promote myocardial angiogenesis in mice.

Objective: The pedicled greater omentum, when applied onto stressed hearts using omentopexy, has been shown to be protective in humans and animals. The mechanisms underlying cardioprotection using omentopexy remain elusive. This study examined whether macrophage-mediated angiogenesis accounts for the cardioprotective effect of omentopexy in mice. Methods: C57BL/6 mice were subjected to minimally invasive transverse aortic constriction for 6 weeks and subsequent cardio-omentopexy for 8 weeks. Control mice underwent the same surgical procedures without aortic constriction or cardio-omentopexy. Results: Transverse aortic constriction led to left ventricular concentric hypertrophy, reduced mitral E/A ratio, increased cardiomyocyte size, and myocardial fibrosis in the mice that underwent sham cardio-omentopexy surgery. The negative effects of transverse aortic constriction were prevented by cardio-omentopexy. Myocardial microvessel density was elevated in the mice that underwent aortic constriction and sham cardio-omentopexy surgery, and cardio-omentopexy further enhanced angiogenesis. Nanostring gene array analysis uncovered the activation of angiogenesis gene networks by cardio-omentopexy. Flow cytometric analysis revealed that cardio-omentopexy triggered the accumulation of cardiac MHCIIloLyve1+TimD4+ (Major histocompatibility complex class IIlow lymphatic vessel endothelial hyaluronan receptor 1+ T cell immunoglobulin and mucin domain conataining 4+) resident macrophages at the omental-cardiac interface. Intriguingly, the depletion of macrophages with clodronate-liposome resulted in the failure of cardio-omentopexy to protect the heart and promote angiogenesis. Conclusions: Cardio-omentopexy protects the heart from pressure overload-elicited left ventricular hypertrophy and dysfunction by promoting myocardial angiogenesis. Cardiac MHCIIloLyve1+TimD4+ resident macrophages play a critical role in the cardioprotective effect and angiogenesis of cardio-omentopexy.

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.

Heterozygous expression of the Alzheimer's disease-protective PLCγ2 P522R variant enhances Aß clearance while preserving synapses.

BACKGROUND: A rare coding variant, P522R, in the phospholipase C gamma 2 (PLCG2) gene has been identified as protective against late-onset Alzheimer's disease (AD), but the mechanism is unknown. PLCG2 is exclusively expressed in microglia within the central nervous system, and altered microglial function has been implicated in the progression of AD. METHODS: Healthy control hiPSCs were CRISPR edited to generate cells heterozygous and homozygous for the PLCG2P522R variant. Microglia derived from these hiPSC's were used to investigate the impact of PLCγ2P522R on disease relevant processes, specifically microglial capacity to take up amyloid beta (Aß) and synapses. Targeted qPCR assessment was conducted to explore expression changes in core AD linked and microglial genes, and mitochondrial function was assessed using an Agilent Seahorse assay. RESULTS: Heterozygous expression of the P522R variant resulted in increased microglial clearance of Aß, while preserving synapses. This was associated with the upregulation of a number of genes, including the anti-inflammatory cytokine Il-10, and the synapse-linked CX3CR1, as well as alterations in mitochondrial function, and increased cellular motility. The protective capacity of PLCγ2P522R appeared crucially dependent on (gene) 'dose', as cells homozygous for the variant showed reduced synapse preservation, and a differential gene expression profile relative to heterozygous cells. CONCLUSION: These findings suggest that PLCγ2P522R may result in increased surveillance by microglia, and prime them towards an anti-inflammatory state, with an increased capacity to respond to increasing energy demands, but highlights the delicate balance of this system, with increasing PLCγ2P522R 'dose' resulting in reduced beneficial impacts.

The P522R protective variant of PLCG2 promotes the expression of antigen presentation genes by human microglia in an Alzheimer's disease mouse model.

The P522R variant of PLCG2, expressed by microglia, is associated with reduced risk of Alzheimer's disease (AD). Yet, the impact of this protective mutation on microglial responses to AD pathology remains unknown. Chimeric AD and wild-type mice were generated by transplanting PLCG2-P522R or isogenic wild-type human induced pluripotent stem cell microglia. At 7 months of age, single-cell and bulk RNA sequencing, and histological analyses were performed. The PLCG2-P522R variant induced a significant increase in microglial human leukocyte antigen (HLA) expression and the induction of antigen presentation, chemokine signaling, and T cell proliferation pathways. Examination of immune-intact AD mice further demonstrated that the PLCG2-P522R variant promotes the recruitment of CD8+ T cells to the brain. These data provide the first evidence that the PLCG2-P522R variant increases the capacity of microglia to recruit T cells and present antigens, promoting a microglial transcriptional state that has recently been shown to be reduced in AD patient brains.

PLCG2 is associated with the inflammatory response and is induced by amyloid plaques in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is characterized by robust microgliosis and phenotypic changes that accompany disease pathogenesis. Accumulating evidence from genetic studies suggests the importance of phospholipase C γ 2 (PLCG2) in late-onset AD (LOAD) pathophysiology. However, the role of PLCG2 in AD is still poorly understood. METHODS: Using bulk RNA-Seq (N=1249) data from the Accelerating Medicines Partnership-Alzheimer's Disease Consortium (AMP-AD), we investigated whether PLCG2 expression increased in the brains of LOAD patients. We also evaluated the relationship between PLCG2 expression levels, amyloid plaque density, and expression levels of microglia specific markers (AIF1 and TMEM119). Finally, we investigated the longitudinal changes of PLCG2 expression in the 5xFAD mouse model of AD. To further understand the role of PLCG2 in different signaling pathways, differential gene expression and co-expression network analyses were performed using bulk RNA-Seq and microglial single-cell RNA-Seq data. To substantiate the human analyses, we performed differential gene expression analysis on wild-type (WT) and inactivated Plcg2 mice and used immunostaining to determine if the differentially expressed genes/pathways were altered by microglial cell coverage or morphology. RESULTS: We observed significant upregulation of PLCG2 expression in three brain regions of LOAD patients and significant positive correlation of PLCG2 expression with amyloid plaque density. These findings in the human brain were validated in the 5xFAD amyloid mouse model, which showed disease progression-dependent increases in Plcg2 expression associated with amyloid pathology. Of note, increased Plcg2 expression levels in 5xFAD mice were abolished by reducing microglia. Furthermore, using bulk RNA-Seq data, we performed differential expression analysis by comparing cognitively normal older adults (CN) with 75th percentile (high) and 25th percentile (low) PLCG2 gene expression levels to identify pathways related to inflammation and the inflammatory response. The findings in the human brain were validated by differential expression analyses between WT and plcg2 inactivated mice. PLCG2 co-expression network analysis of microglial single-cell RNA-Seq data identified pathways related to the inflammatory response including regulation of I-kappaB/NF-kappa B signaling and response to lipopolysaccharide. CONCLUSIONS: Our results provide further evidence that PLCG2 plays an important role in AD pathophysiology and may be a potential target for microglia-targeted AD therapies.

A novel likely pathogenic PLCG2 variant in a patient with a recurrent skin blistering disease and B-cell lymphopenia.

Pathogenic variants of PLCG2 encoding phospholipase C gamma 2 (PLCγ2) were first reported in 2012 and their clinical manifestations vary widely. PLCG2-associated antibody deficiency and immune dysregulation (PLAID) and autoinflammation and PLCγ2-associated antibody deficiency and immune dysregulation (APLAID) are representative examples of PLCG2 pathogenic variants. In this report, we describe a 17-year-old male with recurrent blistering skin lesions, B-cell lymphopenia, and asthma. Distinct from the patients in previous reports, this patient had the heterozygous de novo c.2119T > C missense variant (NM_002661.4) resulting in a serine to proline amino acid substitution (p.Ser707Pro). The variant located to the PLCγ2 C-terminal Src homology 2 (cSH2) domain, which is a critical site for the restriction of intrinsic enzyme activity. This variant could be classified as "likely pathogenic" according to American College of Medical Genetics and Genomics guidelines. Laboratory results showed a reduction in circulating B cells without a decrease of serum IgG and IgA. Our findings expand the variety of clinical phenotypes for PLCG2 missense variants.

Signatures of plasticity, metastasis, and immunosuppression in an atlas of human small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive malignancy that includes subtypes defined by differential expression of ASCL1, NEUROD1, and POU2F3 (SCLC-A, -N, and -P, respectively). To define the heterogeneity of tumors and their associated microenvironments across subtypes, we sequenced 155,098 transcriptomes from 21 human biospecimens, including 54,523 SCLC transcriptomes. We observe greater tumor diversity in SCLC than lung adenocarcinoma, driven by canonical, intermediate, and admixed subtypes. We discover a PLCG2-high SCLC phenotype with stem-like, pro-metastatic features that recurs across subtypes and predicts worse overall survival. SCLC exhibits greater immune sequestration and less immune infiltration than lung adenocarcinoma, and SCLC-N shows less immune infiltrate and greater T cell dysfunction than SCLC-A. We identify a profibrotic, immunosuppressive monocyte/macrophage population in SCLC tumors that is particularly associated with the recurrent, PLCG2-high subpopulation.