Homozygous DBF4 mutation as a cause for severe congenital neutropenia

Background Severe congenital neutropenia presents with recurrent infections early in life due to arrested granulopoiesis. Multiple genetic defects are known to block granulocyte differentiation, however a genetic cause remains unknown in approximately 40% of cases. Objective We aimed to characterize a patient with severe congenital neutropenia and syndromic features without a genetic diagnosis. METHODS: Whole exome sequencing results were validated using flow cytometry, Western blotting, co-immunoprecipitation, quantitative PCR, cell cycle and proliferation analysis of lymphocytes and fibroblasts and granulocytic differentiation of primary CD34+ and HL-60 cells. Results We identified a homozygous missense mutation in DBF4 in a patient with mild extra-uterine growth retardation, facial dysmorphism and severe congenital neutropenia. DBF4 is the regulatory subunit of the CDC7 kinase, together known as DBF4-dependent kinase (DDK), the complex essential for DNA replication initiation. The variant allele demonstrated impaired ability to bind CDC7, resulting in decreased DDK-mediated phosphorylation, defective S phase entry and progression and impaired differentiation of granulocytes associated with activation of the p53-p21 pathway. The introduction of WT DBF4 into patient CD34+ cells rescued the promyelocyte differentiation arrest. Conclusion Hypomorphic DBF4 mutation causes autosomal recessive severe congenital neutropenia with syndromic features

Human OTULIN haploinsufficiency impairs cell-intrinsic immunity to staphylococcal alpha-toxin

The molecular basis of interindividual clinical variability upon infection with Staphylococcus aureus is unclear. We describe patients with haploinsufficiency for the linear deubiquitinase OTULIN, encoded by a gene on chromosome 5p. Patients suffer from episodes of life-threatening necrosis, typically triggered by S. aureus infection. The disorder is phenocopied in patients with the 5p- (Cri-du-Chat) chromosomal deletion syndrome. OTULIN haploinsufficiency causes an accumulation of linear ubiquitin in dermal fibroblasts, but tumor necrosis factor receptor-mediated nuclear factor kappa B signaling remains intact. Blood leukocyte subsets are unaffected. The OTULIN-dependent accumulation of caveolin-1 in dermal fibroblasts, but not leukocytes, facilitates the cytotoxic damage inflicted by the staphylococcal virulence factor alpha-toxin. Naturally elicited antibodies against alpha-toxin contribute to incomplete clinical penetrance. Human OTULIN haploinsufficiency underlies life-threatening staphylococcal disease by disrupting cell-intrinsic immunity to alpha-toxin in nonleukocytic cells.Peer reviewe

Gene Discovery in Inborn Errors of Immunity

The field of IEI is evolving at a rapid pace with the introduction of next generation sequencing (NGS) in 2010, close collaboration between clinicians and researchers, availability of new tools and model organisms and identification of novel clinical phenotypes. The historical view on IEI limited to a predisposition to infection has been abandoned for a comprehensive view on disease with classification based on the primary immune subset disturbed and increased recognition of autoimmunity and immune dysregulation. In the first chapter we reported on a novel mutation in IKZF1, encoding a hematopoietic transcription factor, in a patient with a phenotype predominated by systemic lupus erythematosus. There were 4 prior reports on loss-of-function IKZF1 mutations in patients with CVID and autoimmunity and the clinical spectrum attributed to mutant IKAROS has expanded since to include ALL and early onset CID. Our group demonstrated mutant IKAROS B cells are reprogrammed to a hyperresponsive state with depressed CD22 expression and reduced IKAROS DNA binding to the CD22 locus, contributing to a lower threshold for activation. Collectively these patient studies have revealed novel biological functions of IKZF1, including B cell development and signaling, T cell development and activation, and dendritic cell development and function. However, a thorough understanding of the mechanism leading to autoimmunity is still lacking with both arguments for defects in central tolerance studied in mice and peripheral tolerance as demonstrated by our group. In the second chapter, we expand the spectrum of SEC61A1-related Mendelian disease to include severe congenital neutropenia (SCN) through identification of a novel de novo mutation in SEC61A1. The Sec61 complex is responsible for transport of newly synthesized proteins into the ER and passive leakage of calcium. There were only 2 prior reports on SEC61A1 mutations, including AD tubulo-interstitial kidney disease and primary antibody deficiency. Our study provided definitive proof of causality through mutation-specific induction of the UPR leading to cellular arrest of myeloid precursors. However, protein instability, dysregulated calcium homeostasis and defective protein translocation with induction of ER stress have been implicated in the pathogenesis of all five SEC61A1 mutations, regardless of the phenotype, indicating there are genotype-specific effects that remain to be elucidated. The third and final chapter reveals novel clinical phenotypes attributable to mutations in OTULIN. Two consecutive papers reported on hypomorphic homozygous OTULIN mutations in patients with a potentially life-threatening, very early-onset autoinflammatory condition coined OTULIN-related autoinflammatory syndrome (ORAS). Here, we presented a patient harboring a private heterozygous variant with incomplete penetrance and a clearly distinct clinical phenotype, with episodic inflammation and recurrent sterile neutrophilic abscesses in his lungs in the absence of any skin manifestations. While his asymptomatic mother harbors the same pathogenic mutation, monocytes from mother and patient are equally hyperresponsive in vitro. This is another example of how unidentified environmental triggers or genetic modifiers may influence the penetrance of a particular allele.status: publishe

A Swollen, Red Areola in a Young Boy

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Bone involvement in monogenic autoinflammatory syndromes

Until recently the most common autoinflammatory diseases (AIDs) associated with bone disease in childhood included a few genetically complex (chronic non-bacterial osteomyelitis, synovitis, acne, pustulosis, hyperostosis and osteitis syndrome) and monogenic (Majeed syndrome, deficiency of IL-1 receptor antagonist, cherubism) AIDs. However, the spectrum of monogenic AIDs associated with bone manifestations has markedly expanded to include both recently identified diseases such as the type I interferonopathies and also newly recognized bone dysplasias in already established AIDs. In addition, we propose that some known bone dysplasia syndromes, especially those presenting with hyperostosis and associated systemic inflammation, be classified as AIDs. Collectively, we provide an overview of the diverse bone manifestations identified in the genetically defined AIDs, discuss the hypotheses of the underlying pathophysiological mechanisms and highlight potential novel therapeutic strategies.status: publishe

Whole exome sequencing in inborn errors of immunity: use the power but mind the limits

Next-generation sequencing, especially whole exome sequencing (WES), has revolutionized the molecular diagnosis of inborn errors of immunity. This review summarizes the generation and analysis of next-generation sequencing data.status: publishe

Machine learning identifies the immunological signature of Juvenile Idiopathic Arthritis

Juvenile idiopathic arthritis (JIA) is the most common childhood rheumatic disease, with a strongly debated pathophysiological origin. Both adaptive and innate immune processes have been proposed as primary drivers, which may account for the observed clinical heterogeneity, but few high-depth studies have been performed. Here we profiled the adaptive immune system of 85 JIA patients and 43 age-matched controls, identifying immunological changes unique to JIA and others common across a broad spectrum of childhood inflammatory diseases. The JIA immune signature was shared between clinically distinct subsets, but was accentuated in the systemic JIA patients and those patients with active disease. Despite the extensive overlap in the immunological spectrum exhibited by healthy children and JIA patients, machine learning analysis of the dataset proved capable of diagnosis of JIA patients with ~90% accuracy. These results pave the way for large-scale longitudinal studies of JIA, where machine learning could be used to predict immune signatures that correspond to treatment response group.status: publishe

Machine learning identifies an immunological pattern associated with multiple juvenile idiopathic arthritis subtypes

OBJECTIVES: Juvenile idiopathic arthritis (JIA) is the most common class of childhood rheumatic diseases, with distinct disease subsets that may have diverging pathophysiological origins. Both adaptive and innate immune processes have been proposed as primary drivers, which may account for the observed clinical heterogeneity, but few high-depth studies have been performed. METHODS: Here we profiled the adaptive immune system of 85 patients with JIA and 43 age-matched controls with indepth flow cytometry and machine learning approaches. RESULTS: Immune profiling identified immunological changes in patients with JIA. This immune signature was shared across a broad spectrum of childhood inflammatory diseases. The immune signature was identified in clinically distinct subsets of JIA, but was accentuated in patients with systemic JIA and those patients with active disease. Despite the extensive overlap in the immunological spectrum exhibited by healthy children and patients with JIA, machine learning analysis of the data set proved capable of discriminating patients with JIA from healthy controls with ~90% accuracy. CONCLUSIONS: These results pave the way for large-scale immune phenotyping longitudinal studies of JIA. The ability to discriminate between patients with JIA and healthy individuals provides proof of principle for the use of machine learning to identify immune signatures that are predictive to treatment response group.status: publishe

A kindred with mutant IKAROS and autoimmunity

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Enhanced cGAS-STING-dependent interferon signaling associated with mutations in ATAD3A

Mitochondrial DNA (mtDNA) has been suggested to drive immune system activation, but the induction of interferon signaling by mtDNA has not been demonstrated in a Mendelian mitochondrial disease. We initially ascertained two patients, one with a purely neurological phenotype and one with features suggestive of systemic sclerosis in a syndromic context, and found them both to demonstrate enhanced interferon-stimulated gene (ISG) expression in blood. We determined each to harbor a previously described de novo dominant-negative heterozygous mutation in ATAD3A, encoding ATPase family AAA domain-containing protein 3A (ATAD3A). We identified five further patients with mutations in ATAD3A and recorded up regulated ISG expression and interferon alpha protein in four of them. Knockdown of ATAD3A in THP-1 cells resulted in increased interferon signaling, mediated by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Enhanced interferon signaling was abrogated in THP-1 cells and patient fibroblasts depleted of mtDNA. Thus, mutations in the mitochondrial membrane protein ATAD3A define a novel type I interferonopathy.Peer reviewe