Welcome to the ESID 2022 Meeting Interactive Programme

Background and Aims

Rare missense variants are frequently detected by next-generation DNA sequencing but the deleterious functional consequence of these is often difficult or impossible to establish with computational means.

Methods

We utilize a database of regulatory post-translational modifications, accumulated from thousands of scientific papers, to search missense variants that affect post-translational modification sites known to regulate the protein activity.

Results

Using this approach, we identified three patients, monochorionic triamniotic identical triplets, with a homozygous variant, p.Y371C, in CBL. The patients suffer from recurrent pneumonia and emphysema associated with an excess of transitional B cells and moncytosis. The variant is loss-of-function affecting the phosphorylation site required for CBL function . The triplets have inherited the CBL mutation from their father by segmental uniparental disomy (UPD) mosaicism. Surprisingly, the mosaic distribution of the mutant allele is exactly identical between the three siblings, affecting bone marrow and peripheral blood, oral mucosa but not hair and fingernails. The homozygosity rate in whole blood is above 95%, explaining the homogeneity of the immunological phenotype between the three siblings.

Conclusions

In summary we discovered and described a novel inborn error of immunity (IEI): autosomal recessive, yet mosaic CBL deficiency, causing recurrent pneumonia, myeloproliferation and a B-cell defect. To date, this report represents the first IEI in monozygotic triplets.

Background and Aims

Three patients from a multigeneration family suffer since early childhood from recurrent respiratory tract infections associated to hypogammaglobulinemia and requiring Ig substitution. Skin manifestations and early hair greying were reported for all patients. Whole exome sequencing identified a novel heterozygous missense IRF4 mutation located in the sequence coding for the interferon activation domain. Our study aimed to characterize functional consequences of this mutation for the immune system.

Methods

Time-of-flight mass cytometry was performed to characterize the immune phenotype. Molecular analysis included luciferase reporter assays, transcriptomic and proteomic approaches. Ectopic expression and functional analysis were performed in naive T and B lymphoblastoid cell lines.

Results

Blood immunophenotyping of all patients indicated a striking absence of plasma cells. Lower proportion of naïve and elevated proportion of terminal effector T lymphocytes were observed in both CD4 and CD8 cell subsets. Luciferase reporter assays (containing three main IRF4 binding elements) revealed variably modified transcriptional activity for mutant IRF4 protein, depending on the analyzed element. Although chromatin immunoprecipitation experiments showed similar binding of wild type and mutated IRF4 to these elements, rapid immunoprecipitation mass spectrometry of endogenous proteins identified unexpected cofactors bound to DNA IRF4 complexes.

Ectopic expression of mutant IRF4 in naïve T cells was associated with a rapid loss of naïve markers. Experiments performed in lymphoblastoid B cell lines correlated the presence of the mutant IRF4 protein to a loss of pro-plasmablast gene expression.

Conclusions

We report the phenotypic and functional investigation of a novel dominant immune deficiency caused by an IRF4 missense heterozygous mutation.

Background and Aims

The NF-κB family of transcription factors are essential for immune function. Mutations in NFKB1 and NFKB2 are the most prevalent monogenic causes of Common Variable Immunodeficiency (CVID). NF-κB activity is regulated by the IκB family of inhibitor proteins, including 3 atypical nuclear IκBs (Bcl-3, IκBζ, and IκBNS) that bind to NF-κB dimers at target gene promoters to provide additional regulation. Disruption of any one of these interactions may thus have significant impact on immune responses.

We identified biallelic loss-of-function mutations in NFKBID (encoding IκBNS) in 3 unrelated patients with antibody deficiency complicated by significant immune dysregulation. Two adult patients were identified (Royal Melbourne Hospital, Australia; Vall d’Hebron Hospital, Barcelona, Spain) and one child (Royal Children’s Hospital, Australia).

Aims:

1. Characterise the clinical spectrum of NFKBID-deficiency

2. Determine the cellular and regulatory consequences of NFKBID-deficiency

Methods

Comprehensive clinical and immunological characterisation of 3 patients was performed. IκBNS null leukocyte cell lines were generated (Jurkat, THP-1, Bjab) by CRISPR/Cas9 editing, representing key immune cell lineages with specific point mutations introduced, and NF-κB activity assessed.

Results

Both adult IκBNS-deficient patients developed a progressive dysregulatory immune phenotype with shared clinical features of severe dermatitis, autoimmune cytopenia and recurrent respiratory infections We identified defects in NFκB signalling both upstream and downstream of IκBNS, suggesting atypical IκB inhibitors also regulate expression and function of classical IκBs and underlie severe disease in patients.

Conclusions

Mutations in NFKBID represent a new inborn error of immunity due to aberrant NF-κB signalling and disruption of classical IκB inhibiror proteins.

Background and Aims

CWF19L2 is a part of the post-mRNA release spliceosomal complex and is responsible for pre-mRNA splicing into its mature form. Here we present a compound heterozygous mutations (c.2200C>T, p.Gln734Ter; c.2251T>C, p.Cys751Arg) in a family with microcephaly, autism and immunodeficiency.

Methods

WES was used to identify biallelic CW19L2 variants. Immunological assessment included immunophenotyping, functional T and B cell assessment and neutrophil assays. Knockdown experiments using HEK293 cells were used to study cell division.

Results

Affected sibling pair presented with early onset bronchiectasis and recurrent sinopulmonary infections. Immunoglobulin profile showed reduction in IgM, but normal IgG and IgA levels. Lymphocyte populations and T cell proliferation in the probands were normal with the exception of a reduction in naïve T-cells. T-cell recall response to range of viral antigens was detectable. Patient peripheral blood B cells were able to differentiate in vitro and secrete IgM, IgA and IgG in the culture, following T dependent stimulation. Neutrophil burst test was normal but neutrophil migration was severely impaired in the probands. RNA sequencing from the proband identified increased expression of genes associated with the adaptive immune response and T cell receptor signalling, and a decrease in genes associated with the inflammatory response, chemokine-mediated signalling and actin cytoskeleton. CWF19L2 knockdown in HEK293 resulted in reduced cellular proliferation, increased asymmetric cell division, increased time required to complete mitosis and increased number of cells failing to complete mitosis.

Conclusions

These results suggest that biallelic loss of function variants in CW19L2 result in a novel primary immunodeficiency characterised by cytoskeleton defects and impaired neutrophil migration.

Background and Aims

MAP4K1 encodes for hematopoietic progenitor kinase 1 (HPK1) which negatively regulates TCR and BCR signaling in lymphocytes. MAP4K1-knockout mice show increased T cell proliferation, secretion of proinflammatory cytokines, and susceptibility to autoimmunity. In humans, decreased levels of HPK1 have been found in patients with psoriatic arthritis and systemic lupus erythematosus but evidence of direct causality between HPK1 and human immune disorders is lacking.

Methods

We employed genotyping and exome sequencing to identify the underlying genetic defect in a multigenerational pedigree with nine affected individuals exhibiting dominantly inherited autoimmunity/inflammation. RT-PCR, RNA-sequencing, flow cytometry and western blotting were utilized for patient characterization and analysis of candidate variant effects.

Results

Symptoms in the studied pedigree included prolonged fevers of unknown cause and recurrent joint inflammation, as well as untypical Still's disease, Kawasaki disease and urticaria in one patient, and HLH in another. Genetic linkage analysis indicated highest probability of disease variant at the genomic location chr19:35079781-45414451 (hg19, maximum LOD-score 2.067), where exome sequencing identified a novel splice-site variant in MAP4K1 (NM_007181:exon23:c.1778+2T>G). RT-PCR and western blotting confirmed altered splicing of MAP4K1 and loss of HPK1 protein production from the variant allele in patient-derived PBMCs. The patients had normal immune cell composition but exhibited increased levels of proinflammatory cytokines in their plasma. Gene Set Enrichment Analysis of RNA-sequencing data indicated increased expression of genes involved in NF-κB-mediated TNFα signaling and inflammatory responses in patient PBMCs. Further functional tests are pending.

Conclusions

Heterozygous loss of MAP4K1/HPK1 is a possible novel cause of autoinflammation and autoimmunity.

Background and Aims

Staphylococcus aureus is a major bacterial pathogen with a global impact on human health. The molecular basis of interindividual clinical variability upon exposure to and infection with S. aureus is unclear. Most cases of severe staphylococcal disease remain unexplained.

Methods

We aimed to discover human genetic and immunological determinants of severe staphylococcal disease in a genome-wide manner. We tested for genetic homogeneity in a cohort of patients with unexplained life-threatening staphylococcal disease in the exomes of 105 cases and 1,274 controls.

Results

We found enrichment for rare heterozygous OTULIN variants in patients with severe staphylococcal disease. OTULIN is a linear deubiquitinase and negative regulator of NF-κB-signaling encoded by a gene on chromosome 5p. Probands heterozygous for deleterious OTULIN variants suffered from episodes of life-threatening skin or pulmonary necrosis. Their disease was typically triggered by S. aureus infections. Haploinsufficiency was the mechanism of dominance and was both biochemically and clinically phenocopied in patients with the more common 5p- (Cri-du-Chat) chromosomal deletion syndrome. Blood leukocyte subsets were developmentally and functionally unaffected. In dermal fibroblasts, OTULIN haploinsufficiency increased the levels of linear ubiquitin, but TNF-receptor NF-κB-signaling remained intact. The OTULIN-dependent accumulation of caveolin-1 in dermal fibroblasts — but not leukocytes — facilitated the cytotoxic damage inflicted by the staphylococcal virulence factor α-toxin. Naturally elicited antibodies against α-toxin contributed to incomplete clinical penetrance.

Conclusions

By disrupting cell-intrinsic immunity to α-toxin in fibroblasts, human OTULIN haploinsufficiency underlies life-threatening staphylococcal disease of the skin and lungs.

Background and Aims

The DNA polymerase δ complex, comprising catalytic subunit POLD1, and accessory subunits POLD2, POLD3 and POLD4, is essential for DNA synthesis and central to genome integrity (Loeb and Monnat, 2008).

Biallelic loss-of-function mutations in POLD1 and POLD2, leading to reduced functionality of the polymerase δ complex, were reported previously (Conde et al. 2019, Cui et al. 2020, Nichols-Vinueza et al. 2021). The phenotype of polymerase δ deficient patients combines immunodeficiency, developmental abnormalities, and replicative stress and has implicated the DNA polymerase δ complex in T and B cell development.

The aim of this study is to validate a mutation in POLD3 as a novel cause of inborn error of immunity.

Methods

We performed molecular and functional analysis of the mutant DNA polymerase δ complex, assessed cell cycle progression as well as replication-associated DNA damage by means of imaging/standard flow cytometry.

Results

We identified a homozygous missense variant (c.1118CA>C; p.K373T) in POLD3 in a patient with severe combined immunodeficiency. The patient exhibited decreased numbers of naïve T cells associated with a restricted T-cell receptor repertoire and a defect in the early stages of T-cell receptor recombination. Protein expression of POLD1, POLD2 and POLD3 tends to be decreased in patient fibroblasts, associated with a marked defect in S-phase entry and an enhanced numbers of double-strand DNA break foci. Rescue of the phenotype will be presented. The patient received hematopoietic stem cell transplantation and is now two years old.

Conclusions

We describe a homozygous mutation in POLD3 as a novel cause of severe combined immunodeficiency.