Increased susceptibility to Mycobacterium avium complex infection in miniature Schnauzer dogs caused by a codon deletion in CARD9.

Mammals are generally resistant to Mycobacterium avium complex (MAC) infections. We report here on a primary immunodeficiency disorder causing increased susceptibility to MAC infections in a canine breed. Adult Miniature Schnauzers developing progressive systemic MAC infections were related to a common founder, and pedigree analysis was consistent with an autosomal recessive trait. A genome-wide association study and homozygosity mapping using 8 infected, 9 non-infected relatives, and 160 control Miniature Schnauzers detected an associated region on chromosome 9. Whole genome sequencing of 2 MAC-infected dogs identified a codon deletion in the CARD9 gene (c.493_495del; p.Lys165del). Genotyping of Miniature Schnauzers revealed the presence of this mutant CARD9 allele worldwide, and all tested MAC-infected dogs were homozygous mutants. Peripheral blood mononuclear cells from a dog homozygous for the CARD9 variant exhibited a dysfunctional CARD9 protein with impaired TNF-α production upon stimulation with the fungal polysaccharide ß-glucan that activates the CARD9-coupled C-type lectin receptor, Dectin-1. While CARD9-deficient knockout mice are susceptible to experimental challenges by fungi and mycobacteria, Miniature Schnauzer dogs with systemic MAC susceptibility represent the first spontaneous animal model of CARD9 deficiency, which will help to further elucidate host defense mechanisms against mycobacteria and fungi and assess potential therapies for animals and humans.

IL-15 Priming Alters IFN-γ Regulation in Murine NK Cells.

NK effector functions can be triggered by inflammatory cytokines and engagement of activating receptors. NK cell production of IFN-γ, an important immunoregulatory cytokine, exhibits activation-specific IFN-γ regulation. Resting murine NK cells exhibit activation-specific metabolic requirements for IFN-γ production, which are reversed for activating receptor-mediated stimulation following IL-15 priming. Although both cytokine and activating receptor stimulation leads to similar IFN-γ protein production, only cytokine stimulation upregulates Ifng transcript, suggesting that protein production is translationally regulated after receptor stimulation. Based on these differences in IFN-γ regulation, we hypothesized that ex vivo IL-15 priming of murine NK cells allows a switch to IFN-γ transcription upon activating receptor engagement. Transcriptional analysis of primed NK cells compared with naive cells or cells cultured with low-dose IL-15 demonstrated that primed cells strongly upregulated Ifng transcript following activating receptor stimulation. This was not due to chromatin accessibility changes in the Ifng locus or changes in ITAM signaling, but was associated with a distinct transcriptional signature induced by ITAM stimulation of primed compared with naive NK cells. Transcriptional analyses identified a common signature of c-Myc (Myc) targets associated with Ifng transcription. Although Myc marked NK cells capable of Ifng transcription, Myc itself was not required for Ifng transcription using a genetic model of Myc deletion. This work highlights altered regulatory networks in IL-15-primed cells, resulting in distinct gene expression patterns and IFN-γ regulation in response to activating receptor stimulation.

IL-15 priming alters IFN-γ regulation in murine NK cells.

Natural killer (NK) effector functions can be triggered by inflammatory cytokines and engagement of activating receptors. NK cell production of IFN-γ, an important immunoregulatory cytokine, exhibits activation-specific IFN-γ regulation. Resting murine NK cells exhibit activation-specific metabolic requirements for IFN-γ production, which are reversed for activating receptor-mediated stimulation following IL-15 priming. While both cytokine and activating receptor stimulation leads to similar IFN-γ protein production, only cytokine stimulation upregulates Ifng transcript, suggesting that protein production is translationally regulated after receptor stimulation. Based on these differences in IFN-γ regulation, we hypothesized that ex vivo IL-15 priming of murine NK cells allows a switch to IFN-γ transcription upon activating receptor engagement. Transcriptional analysis of primed NK cells compared to naïve cells or cells cultured with low-dose IL-15 demonstrated that primed cells strongly upregulated Ifng transcript following activating receptor stimulation. This was not due to chromatin accessibility changes in the Ifng locus or changes in ITAM signaling, but was associated with a distinct transcriptional signature induced by ITAM stimulation of primed compared to naïve NK cells. Transcriptional analyses identified a common signature of c-Myc (Myc) targets associated with Ifng transcription. While Myc marked NK cells capable of Ifng transcription, Myc itself was not required for Ifng transcription using a genetic model of Myc deletion. This work highlights altered regulatory networks in IL-15 primed cells, resulting in distinct gene expression patterns and IFN-γ regulation in response to activating receptor stimulation.

Early-Onset Parkinson's Disease: Creating the Right Environment for a Genetic Disorder.

Parkinson's disease (PD) by its common understanding is a late-onset sporadic movement disorder. However, there is a need to recognize not only the fact that PD pathogenesis expands beyond (or perhaps to) the brain but also that many early-onset patients develop motor signs before the age of 50 years. Indeed, studies have shown that it is likely the protein aggregation observed in the brains of patients with PD precedes the motor symptoms by perhaps a decade. Studies on early-onset forms of PD have shown it to be a heterogeneous disease with multiple genetic and environmental factors determining risk of different forms of disease. Genetic and neuropathological evidence suggests that there are α-synuclein centric forms (e.g., SNCA genomic triplication), and forms that are driven by a breakdown in mitochondrial function and specifically in the process of mitophagy and clearance of damaged mitochondria (e.g., PARKIN and PINK1 recessive loss-of-function mutations). Aligning genetic forms with recognized environmental influences will help better define patients, aid prognosis, and hopefully lead to more accurately targeted clinical trial design. Work is now needed to understand the cross-talk between these two pathomechanisms and determine a sense of independence, it is noted that autopsies studies for both have shown the presence or absence of α-synuclein aggregation. The integration of genetic and environmental data is critical to understand the etiology of early-onset forms of PD and determine how the different pathomechanisms crosstalk.

A human STAT3 gain-of-function variant confers T cell dysregulation without predominant Treg dysfunction in mice.

Primary immune regulatory disorders (PIRD) represent a group of disorders characterized by immune dysregulation, presenting with a wide range of clinical disease, including autoimmunity, autoinflammation, or lymphoproliferation. Autosomal dominant germline gain-of-function (GOF) variants in STAT3 result in a PIRD with a broad clinical spectrum. Studies in patients have documented a decreased frequency of FOXP3+ Tregs and an increased frequency of Th17 cells in some patients with active disease. However, the mechanisms of disease pathogenesis in STAT3 GOF syndrome remain largely unknown, and treatment is challenging. We developed a knock-in mouse model harboring a de novo pathogenic human STAT3 variant (p.G421R) and found these mice developed T cell dysregulation, lymphoproliferation, and CD4+ Th1 cell skewing. Surprisingly, Treg numbers, phenotype, and function remained largely intact; however, mice had a selective deficiency in the generation of iTregs. In parallel, we performed single-cell RNA-Seq on T cells from STAT3 GOF patients. We demonstrate only minor changes in the Treg transcriptional signature and an expanded, effector CD8+ T cell population. Together, these findings suggest that Tregs are not the primary driver of disease and highlight the importance of preclinical models in the study of disease mechanisms in rare PIRD.

Systematic Functional Analysis of PINK1 and PRKN Coding Variants.

Loss of either PINK1 or PRKN causes an early onset Parkinson's disease (PD) phenotype. Functionally, PINK1 and PRKN work together to mediate stress-activated mitochondrial quality control. Upon mitochondrial damage, PINK1, a ubiquitin kinase and PRKN, a ubiquitin ligase, decorate damaged organelles with phosphorylated ubiquitin for sequestration and degradation in lysosomes, a process known as mitophagy. While several genetic mutations are established to result in loss of mitophagy function, many others have not been extensively characterized and are of unknown significance. Here, we analyzed a set of twenty variants, ten in each gene, focusing on understudied variants mostly from the Parkinson's progressive marker initiative, with sensitive assays to define potential functional deficits. Our results nominate specific rare genetic PINK1 and PRKN variants that cause loss of enzymatic function in line with a potential causative role for PD. Additionally, we identify several variants with intermediate phenotypes and follow up on two of them by gene editing midbrain-derived neuronal precursor cells. Thereof derived isogenic neurons show a stability defect of the rare PINK1 D525N mutation, while the common PINK1 Q115L substitution results in reduced kinase activity. Our strategy to analyze variants with sensitive functional readouts will help aid diagnostics and disease treatment in line with current genomic and therapeutic advances.

Immunological Findings and Clinical Outcomes of Infants With Positive Newborn Screening for Severe Combined Immunodeficiency From a Tertiary Care Center in the U.S.

The implementation of severe combined immunodeficiency (SCID) newborn screening has played a pivotal role in identifying these patients early in life as well as detecting various milder forms of T cell lymphopenia (TCL). In this study we reviewed the diagnostic and clinical outcomes, and interesting immunology findings of term infants referred to a tertiary care center with abnormal newborn SCID screens over a 6-year period. Key findings included a 33% incidence of non-SCID TCL including infants with novel variants in FOXN1, TBX1, MYSM1, POLD1, and CD3E; 57% positivity rate of newborn SCID screening among infants with DiGeorge syndrome; and earlier diagnosis and improved transplant outcomes for SCID in infants diagnosed after compared to before implementation of routine screening. Our study is unique in terms of the extensive laboratory workup of abnormal SCID screens including lymphocyte subsets, measurement of thymic output (TREC and CD4TE), and lymphocyte proliferation to mitogens in nearly all infants. These data allowed us to observe a stronger positive correlation of the absolute CD3 count with CD4RTE than with TREC copies, and a weak positive correlation between CD4RTE and TREC copies. Finally, we did not observe a correlation between risk of TCL and history of prenatal or perinatal complications or low birth weight. Our study demonstrated SCID newborn screening improves disease outcomes, particularly in typical SCID, and allows early detection and discovery of novel variants of certain TCL-associated genetic conditions.

Reliance on Cox10 and oxidative metabolism for antigen-specific NK cell expansion.

Natural killer (NK) cell effector functions are dependent on metabolic regulation of cellular function; however, less is known about in vivo metabolic pathways required for NK cell antiviral function. Mice with an inducible NK-specific deletion of Cox10, which encodes a component of electron transport chain complex IV, were generated to investigate the role of oxidative phosphorylation in NK cells during murine cytomegalovirus (MCMV) infection. Ncr1-Cox10Δ/Δ mice had normal numbers of NK cells but impaired expansion of antigen-specific Ly49H+ NK cells and impaired NK cell memory formation. Proliferation in vitro and homeostatic expansion were intact, indicating a specific metabolic requirement for antigen-driven proliferation. Cox10-deficient NK cells upregulated glycolysis, associated with increased AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) activation, although this was insufficient to protect the host. These data demonstrate that oxidative metabolism is required for NK cell antiviral responses in vivo.

Fine-mapping of the non-coding variation driving the Caucasian LRRK2 GWAS signal in Parkinson's disease.

INTRODUCTION: Genome-wide association studies (GWAS) have confirmed the leucine-rich repeat kinase 2 (LRRK2) gene as a susceptibility locus for idiopathic Parkinson's disease (PD) in Caucasians. Though the rs1491942 and rs76904798 variants have shown the strongest associations, the causal variant(s) remains unresolved. Therefore, the aim of this study was to identify variants that may be driving the LRRK2 GWAS signal by sequencing the entire LRRK2 gene in Caucasian PD patients and controls. METHODS: A discovery series (287 PD patients, 294 controls) and replication series (362 PD patients, 168 controls) were included. The entire LRRK2 gene as well as 10 Kb upstream/downstream was sequenced. Candidate potential causal variants were considered to be those that (a) were in at least weak linkage disequilibrium with the two GWAS-nominated variants (rs1491942 and rs76904798), and (b) displayed an association odds ratio (OR) that is stronger than the two GWAS variants. RESULTS: Thirty-four candidate variants (all intronic/intergenic) that may drive the LRRK2 PD GWAS signal were identified in the discovery series. However, examination of the replication series for these variants did not reveal any with a consistently stronger OR than both PD GWAS variants. Evaluation of public databases to determine which candidate variants are most likely to have a direct functional effect on LRRK2 expression was inconclusive. CONCLUSION: Though our findings provide novel insights into the LRRK2 GWAS association, a clear causal variant was not identified. The identified candidate variants can form the basis for future experiments and functional studies that can more definitively assess causal LRRK2 variants.

CLEC3B p.S106G Mutant in a Caucasian Population of Successful Neurological Aging.

A number of efforts are underway to better understand the role of genetic variation in successful aging and longevity. However, to date, only two genes have been consistently associated with longevity in humans: APOE and FOXO3, with the APOE ɛ2 allele also protective against dementia. Recently, using an exome-wide SNP array approach, a missense variant CLEC3B c.316G>A (rs13963 p.S106G) was reported to associate with longevity in two independent cohorts of Japanese and Chinese participants. Interestingly, CLEC3B p.S106G is more frequent in Caucasian populations. Herein, we examined the frequency of CLEC3B p.S106G in a Caucasian series of 1,483 neurologically healthy individuals with a specific subset >80 years of age. Although our findings do not support an association between CLEC3B p.S106G and aging without neurological disease (p = .89), we confirmed the association between the APOE ε2 allele and better survival without neurological disease (p = .001). Further assessment of healthy aged cohorts that retain intact neurological function will be critical to understand the etiology of neurodegenerative disease and the role of age at risk.

A homozygous PIGN missense mutation in Soft-Coated Wheaten Terriers with a canine paroxysmal dyskinesia.

Hereditary paroxysmal dyskinesias (PxD) are a heterogeneous group of movement disorders classified by frequency, duration, and triggers of the episodes. A young-adult onset canine PxD has segregated as an autosomal recessive trait in Soft-Coated Wheaten Terriers. The medical records and videos of episodes from 25 affected dogs were reviewed. The episodes of hyperkinesia and dystonia lasted from several minutes to several hours and could occur as often as >10/day. They were not associated with strenuous exercise or fasting but were sometimes triggered by excitement. The canine PxD phenotype most closely resembled paroxysmal non-kinesigenic dyskinesia (PNKD) of humans. Whole genome sequences were generated with DNA from 2 affected dogs and analyzed in comparison to 100 control canid whole genome sequences. The two whole genome sequences from dogs with PxD had a rare homozygous PIGN:c.398C > T transition, which predicted the substitution of an isoleucine for a highly conserved threonine in the encoded enzyme. All 25 PxD-affected dogs were PIGN:c.398T allele homozygotes, whereas there were no c.398T homozygotes among 1185 genotyped dogs without known histories of PxD. PIGN encodes an enzyme involved in the biosynthesis of glycosylphosphatidylinositol (GPI), which anchors a variety of proteins including CD59 to the cell surface. Flow cytometry of PIGN-knockout HEK239 cells expressing recombinant human PIGN with the c.398T variant showed reduced CD59 expression. Mutations in human PIGN have been associated with multiple congenital anomalies-hypotonia-seizures syndrome-1 (MCAHS1). Movement disorders can be a part of MCAHS1, but this is the first PxD associated with altered GPI anchor function.

Variants within the SP110 nuclear body protein modify risk of canine degenerative myelopathy.

Canine degenerative myelopathy (DM) is a naturally occurring neurodegenerative disease with similarities to some forms of amyotrophic lateral sclerosis (ALS). Most dogs that develop DM are homozygous for a common superoxide dismutase 1 gene (SOD1) mutation. However, not all dogs homozygous for this mutation develop disease. We performed a genome-wide association analysis in the Pembroke Welsh Corgi (PWC) breed comparing DM-affected and -unaffected dogs homozygous for the SOD1 mutation. The analysis revealed a modifier locus on canine chromosome 25. A haplotype within the SP110 nuclear body protein (SP110) was present in 40% of affected compared with 4% of unaffected dogs (P = 1.5 × 10(-5)), and was associated with increased probability of developing DM (P = 4.8 × 10(-6)) and earlier onset of disease (P = 1.7 × 10(-5)). SP110 is a nuclear body protein involved in the regulation of gene transcription. Our findings suggest that variations in SP110-mediated gene transcription may underlie, at least in part, the variability in risk for developing DM among PWCs that are homozygous for the disease-related SOD1 mutation. Further studies are warranted to clarify the effect of this modifier across dog breeds.