Primary immunodeficiencies (PIDs) are a heterogeneous group of inherited diseases of the immune system. The definite diagnosis of PID is ascertained by genetic analysis; however, this takes time and ...is costly. Flow cytometry provides a rapid and highly sensitive tool for diagnosis of PIDs.
Flow cytometry can evaluate specific cell populations and subpopulations, cell surface, intracellular and intranuclear proteins, biologic effects associated with specific immune defects, and certain functional immune characteristics, each being useful for the diagnosis and evaluation of PIDs. Flow cytometry effectively identifies major forms of PIDs, including severe combined immunodeficiency, X-linked agammaglobulinemia, hyper IgM syndromes, Wiskott-Aldrich syndrome, X-linked lymphoproliferative syndrome, familial hemophagocytic lymphohistiocytosis, autoimmune lymphoproliferative syndrome, IPEX syndrome, CTLA 4 haploinsufficiency and LRBA deficiency, IRAK4 and MyD88 deficiencies, Mendelian susceptibility to mycobacterial disease, chronic mucocuneous candidiasis, and chronic granulomatous disease. While genetic analysis is the definitive approach to establish specific diagnoses of PIDs, flow cytometry provides a tool to effectively evaluate patients with PIDs at relatively low cost.
Abstract
Autoinflammatory disease is an ‘inborn error of immunity’, resulting in systemic inflammation. Cryopyrin-associated periodic syndrome (CAPS) is a prototypical autoinflammatory disease caused ...by gain-of-function mutations in the NLRP3 (NLR family pyrin domain containing 3) gene; these mutations activate the NLRP3 inflammasome, resulting in overproduction of IL-1β. The first case of CAPS caused by somatic NLRP3 mosaicism was reported in 2005 after identification of variant small peaks by Sanger sequencing. An international collaborative study revealed that the majority of mutation-negative CAPS cases are due to low-level NLRP3 mosaicism, suggesting that central nervous system involvement in somatic mosaicism patients is milder than in genotype-matched heterozygous patients. Recent advances in next-generation sequencing have expanded the number of NLRP3 somatic mosaicism cases and identified a new entity called ‘late-onset CAPS with myeloid-specific NLRP3 mosaicism’; however, no mosaic-specific clinical features have been identified/confirmed yet. With respect to NLRP3 mosaicism in CAPS, a prospective longitudinal study on the variant genotype, its allele frequency and its tissue distribution (along with a comprehensive clinical phenotype) would provide better understanding of NLRP3 mosaicism, resulting in more appropriate patient care and genetic counseling.
Mosaicism in CAPS
Purpose
Pathogenic
MEFV
variants cause pyrin-associated autoinflammatory diseases (PAADs), which include familial Mediterranean fever (FMF), FMF-like disease, and pyrin-associated autoinflammation ...with neutrophilic dermatosis (PAAND). The diagnosis of PAADs is established by clinical phenotypic and genetic analyses. However, the pathogenicity of most
MEFV
variants remains controversial, as they have not been functionally evaluated. This study aimed to establish and validate a new functional assay to evaluate the pathogenicity of
MEFV
variants.
Methods
We transfected THP-1 monocytes with 32
MEFV
variants and analyzed their effects on cell death with or without stimulation with
Clostridium difficile
toxin A (TcdA) or UCN-01. These variants were classified using hierarchical cluster analysis. Macrophages were obtained from three healthy controls and two patients with a novel homozygous
MEFV
P257L
variant, for comparison of IL-1β secretion using a cell-based assay and a novel THP-1-based assay.
Results
Disease-associated
MEFV
variants induced variable degrees of spontaneous or TcdA/UCN-01-induced cell death in THP-1. Cell death was caspase-1 dependent and was accompanied by ASC speck formation and IL-1β secretion, indicating that pathogenic
MEFV
variants induced abnormal pyrin inflammasome activation and subsequent pyroptotic cell deaths in this assay. The
MEFV
variants (
n
= 32) exhibiting distinct response signatures were classified into 6 clusters, which showed a good correlation with the clinical phenotypes. Regarding the pathogenicity of
MEFV
P257L
variants, the results were consistent between the cell-based assay and the THP-1-based assay.
Conclusion
Our assay facilitates a rapid and comprehensive assessment of the pathogenicity of
MEFV
variants and contributes to a refined definition of PAAD subtypes.
Objective
Coatomer subunit alpha (COPA) syndrome, also known as autoinflammatory interstitial lung, joint, and kidney disease, is caused by heterozygous mutations in COPA. We identified a novel COPA ...variant in 4 patients in one family. We undertook this study to elucidate whether and how the variant causes manifestations of COPA syndrome by studying these 4 patients and by analyzing results from a gene‐targeted mouse model.
Methods
We performed whole‐exome sequencing in 7 family members and measured the type I interferon (IFN) signature of the peripheral blood cells. We analyzed the effects of COPA variants in in vitro experiments and in Copa mutant mice that were generated.
Results
We identified a heterozygous variant of COPA (c.725T>G, p.Val242Gly) in the 4 affected members of the family. The IFN score was high in the members carrying the variant. In vitro analysis revealed that COPA V242G, as well as the previously reported disease‐causing variants, augmented stimulator of interferon genes (STING)–induced type I IFN promoter activities. CopaV242G/+ mice manifested interstitial lung disease and STING‐dependent elevation of IFN‐stimulated gene expression. In CopaV242G/+ dendritic cells, the STING pathway was not constitutively activated but was hyperactivated upon stimulation, leading to increased type I IFN production.
Conclusion
V242G, a novel COPA variant, was found in 4 patients from one family. In gene‐targeted mice with the V242G variant, interstitial lung disease was recapitulated and augmented responses of the STING pathway, leading to an increase in type I IFN production, were demonstrated.
Dried blood spots (DBS) are widely used for screening biomolecular profiles, including enzymatic activities. However, detection of minor proteins in DBS by liquid chromatography–mass spectrometry ...(LC-MS/MS) without pre-enrichment remains challenging because of the coexistence of large quantities of hydrophilic proteins. In this study, we address this problem by developing a simple method using sodium carbonate precipitation (SCP). SCP enriches hydrophobic proteins from DBS, allowing substantial removal of soluble proteins. In combination with SCP, we used quantitative LC-MS/MS proteome analysis in a data-independent acquisition mode (DIA) to enhance the sensitivity and quantification limits of proteome analysis. As a result, identification of 1977 proteins in DBS is possible, including 585 disease-related proteins listed in the Online Mendelian Inheritance in Man.
Next, the IL-1β secretion from macrophages derived from monocytes in vitro (peripheral blood–derived macrophages PB-MPs) was evaluated. Because TcdA stimulation alone did not induce IL-1β secretion ...from PB-MPs (data not shown), PB-MPs were primed with LPS before TcdA stimulation. ...by examining both cell types from the same patients, we revealed the overall picture of the cytokine responses of monocytes and macrophages from patients with FMF. Induced pluripotent stem cell (iPSC) technology provides the opportunity to analyze the effect of genetic variants free from the influence of medication or differences in genetic background. ...we evaluated whether macrophages derived from patients' iPSCs (iPSC-derived macrophages iPS-MPs) recapitulated the phenotype of PB-MPs from patients with FMF. iPSC lines from 3 patients with FMF with the M694I and E148Q MEFV variants were established (patients 6-8; see Fig E2 and Table E1 in this article's Online Repository at www.jacionline.org) and differentiated into iPS-MPs (see Fig E3 in this article's Online Repository at www.jacionline.org). iPS-MPs with the M694I mutation recapitulated the enhanced pyrin inflammasome activation of PB-MPs, leading to increased IL-1β secretion (Fig 2, A), ASC speck formation (Fig 2, B), and cell death, which was dependent on MEFV expression (see Fig E4 in this article's Online Repository at www.jacionline.org). ...we applied our newly established method to 2 additional MEFV variants, T577N and N679H, which were identified in 2 families in which autoinflammatory disease with dominant inheritance was suspected.
Monocytic lineage cells (monocytes, macrophages and dendritic cells) play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells ...from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3 × 10(6) ± 0.3 × 10(6) floating monocytes from approximately 30 clusters of ESCs/iPSCs 5-6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery.
X-linked dominant incontinentia pigmenti (IP) and X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) are caused by loss-of-function and hypomorphic IKBKG (also known as ...NEMO) mutations, respectively. We describe a European mother with mild IP and a Japanese mother without IP, whose 3 boys with EDA-ID died from ID. We identify the same private variant in an intron of IKBKG, IVS4+866 C>T, which was inherited from and occurred de novo in the European mother and Japanese mother, respectively. This mutation creates a new splicing donor site, giving rise to a 44-nucleotide pseudoexon (PE) generating a frameshift. Its leakiness accounts for NF-κB activation being impaired but not abolished in the boys' cells. However, aberrant splicing rates differ between cell types, with WT NEMO mRNA and protein levels ranging from barely detectable in leukocytes to residual amounts in induced pluripotent stem cell-derived (iPSC-derived) macrophages, and higher levels in fibroblasts and iPSC-derived neuronal precursor cells. Finally, SRSF6 binds to the PE, facilitating its inclusion. Moreover, SRSF6 knockdown or CLK inhibition restores WT NEMO expression and function in mutant cells. A recurrent deep intronic splicing mutation in IKBKG underlies a purely quantitative NEMO defect in males that is most severe in leukocytes and can be rescued by the inhibition of SRSF6 or CLK.
Familial hemophagocytic lymphohistiocytosis type 3 is a fatal inborn error of immunity due to abnormal cytotoxic activity of T and NK cells and is caused by variants in UNC13D, which encodes ...Munc13–4. One published case was reported to carry a tandem duplication of UNC13D exons 7–12, and we here present another case with the exact same duplication breakpoints. The patient carried the tandem duplication from maternal origin, and a c.2346_2349 variant on the paternal allele. Single nucleotide polymorphism analysis around UNC13D revealed that the allele with tandem duplication was most likely a founder allele. Transposable element analysis showed that the breakpoints occurred within Alu elements in introns 12 and 6. Multiple sequence alignment revealed that Alu elements containing the truncated points are highly homologous. Sequence homology was thought to be a factor predisposing to the tandem duplication variant.
•Two distinct patients with familial hemophagocytic lymphohititocytosis type 3 had the same tandem duplication of UNC13D.•Transposable elements analysis showed the truncated point within the Alu elements of intron 12 and intron 6.•Alu elements containing the truncated points are highly homologous to each other among intron 12 and intron 6 Alu elements.
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