Prognostic value of anti-IFI16 autoantibodies in pulmonary arterial hypertension and mortality in patients with systemic sclerosis / Valor pronóstico de los autoanticuerpos anti-IFI16 en la hipertensión arterial pulmonar y en la mortalidad en los pacientes con esclerodermia sistémica

Objectives: To determine the diagnostic value of anti-interferon gamma inducible protein 16 (IFI16) autoantibodies in systemic sclerosis (SSc) patients negative for all tested SSc-specific autoantibodies (SSc-seronegative patients) and to evaluate the clinical significance of these autoantibodies, whether isolated or in the presence of anti-centromere autoantibodies (ACA). Methods: Overall, 58 SSc-seronegative and 66 ACA-positive patients were included in the study. All patients were tested for anti-IFI16 autoantibodies by an in-house direct ELISA. Associations between clinical parameters and anti-IFI16 autoantibodies were analysed. Results: Overall, 17.2% of SSc-seronegative and 39.4% of ACA-positive patients were positive for anti-IFI16 autoantibodies. Anti-IFI16 autoantibodies were found only in patients within the limited cutaneous SSc (lcSSc) subset. A positive association between anti-IFI16 positivity and isolated pulmonary arterial hypertension (PAH) was found (odds ratio [OR]=5.07; p=0.014) even after adjusting for ACA status (OR=4.99; p=0.019). Anti-IFI16-positive patients were found to have poorer overall survival than negative patients (p=0.032). Cumulative survival rates at 10, 20 and 30 years were 96.9%, 92.5% and 68.7% for anti-IFI16-positive patients vs. 98.8%, 97.0% and 90.3% for anti-IFI16-negative-patients, respectively. Anti-IFI16-positive patients also had worse overall survival than anti-IFI16-negative patients after adjusting for ACA status in the multivariate Cox analysis (hazard ratio [HR]=3.21; p=0.043). Conclusion: Anti-IFI16 autoantibodies were associated with isolated PAH and poorer overall survival. Anti-IFI16 autoantibodies could be used as a supplementary marker of lcSSc in SSc-seronegative patients and for identifying ACA-positive patients with worse clinical outcome. (AU)

Objetivos: Determinar el valor diagnóstico de los autoanticuerpos anti-interferon gamma inducible protein 16 (IFI16) en los pacientes con esclerodermia sistémica (SSc) negativos para todos los autoanticuerpos específicos de SSc (pacientes SSc seronegativos) y evaluar el significado clínico de estos autoanticuerpos, aislados o en combinación con autoanticuerpos anticentrómero (ACA). Métodos: Se incluyeron 58 pacientes SSc seronegativos y 66 pacientes ACA positivos. Todos los pacientes se testaron para los autoanticuerpos anti-IFI16 mediante un ELISA directo «in-house». Las asociaciones entre parámetros clínicos y los autoanticuerpos anti-IFI16 fueron analizadas. Resultados: En total, el 17,2% de los pacientes SSc seronegativos y el 39,4% de los pacientes ACA positivos fueron positivos para anti-IFI16. Los autoanticuerpos anti-IFI16 se detectaron solamente en los pacientes con la forma limitada cutánea de SSc (lcSSc). Se encontró una asociación entre la positividad de anti-IFI16 y la hipertensión arterial pulmonar (HAP) aislada (odds ratio [OR]: 5,07; p=0,014), incluso cuando se ajustó el análisis a la presencia o ausencia de ACA (OR: 4,99; p=0,019). Los pacientes anti-IFI16 positivos mostraron una peor supervivencia general que los pacientes negativos (p=0,032). Las ratios de supervivencia acumulada a 10, 20 y 30 años fueron respectivamente del 96,9, 92,5 y 68,7% para los pacientes anti-IFI16 positivos frente al 98,8, 97,0 y 90,3% para los anti-IFI16 negativos. Los pacientes anti-IFI16 positivos también tenían una supervivencia general menor que los pacientes anti-IFI16 negativos tras ajustar para la presencia o ausencia de ACA mediante análisis multivariado de Cox (hazard ratio [HR]: 3,21; p=0,043)... (AU)

Prognostic value of anti-IFI16 autoantibodies in pulmonary arterial hypertension and mortality in patients with systemic sclerosis.

OBJECTIVES: To determine the diagnostic value of anti-interferon gamma inducible protein 16 (IFI16) autoantibodies in systemic sclerosis (SSc) patients negative for all tested SSc-specific autoantibodies (SSc-seronegative patients) and to evaluate the clinical significance of these autoantibodies, whether isolated or in the presence of anti-centromere autoantibodies (ACA). METHODS: Overall, 58 SSc-seronegative and 66 ACA-positive patients were included in the study. All patients were tested for anti-IFI16 autoantibodies by an in-house direct ELISA. Associations between clinical parameters and anti-IFI16 autoantibodies were analysed. RESULTS: Overall, 17.2% of SSc-seronegative and 39.4% of ACA-positive patients were positive for anti-IFI16 autoantibodies. Anti-IFI16 autoantibodies were found only in patients within the limited cutaneous SSc (lcSSc) subset. A positive association between anti-IFI16 positivity and isolated pulmonary arterial hypertension (PAH) was found (odds ratio [OR]=5.07; p=0.014) even after adjusting for ACA status (OR=4.99; p=0.019). Anti-IFI16-positive patients were found to have poorer overall survival than negative patients (p=0.032). Cumulative survival rates at 10, 20 and 30 years were 96.9%, 92.5% and 68.7% for anti-IFI16-positive patients vs. 98.8%, 97.0% and 90.3% for anti-IFI16-negative-patients, respectively. Anti-IFI16-positive patients also had worse overall survival than anti-IFI16-negative patients after adjusting for ACA status in the multivariate Cox analysis (hazard ratio [HR]=3.21; p=0.043). CONCLUSION: Anti-IFI16 autoantibodies were associated with isolated PAH and poorer overall survival. Anti-IFI16 autoantibodies could be used as a supplementary marker of lcSSc in SSc-seronegative patients and for identifying ACA-positive patients with worse clinical outcome.

Role of Skewed X-Chromosome Inactivation in Common Variable Immunodeficiency.

The term common variable immunodeficiency (CVID) encompasses a clinically diverse group of disorders, mainly characterized by hypogammaglobulinemia, insufficient specific antibody production, and recurrent infections. The genetics of CVID is complex, and monogenic defects account for only a portion of cases, typically <30%. Other proposed mechanisms include digenic, oligogenic, or polygenic inheritance and epigenetic dysregulation. In this study, we aimed to assess the role of skewed X-chromosome inactivation (XCI) in CVID. Within our cohort of 131 genetically analyzed CVID patients, we selected female patients with rare variants in CVID-associated genes located on the X-chromosome. Four patients harboring heterozygous variants in BTK (n = 2), CD40LG (n = 1), and IKBKG (n = 1) were included in the study. We assessed XCI status using the HUMARA assay and an NGS-based method to quantify the expression of the 2 alleles in mRNA. Three of the 4 patients (75%) exhibited skewed XCI, and the mutated allele was predominantly expressed in all cases. Patient 1 harbored a hypomorphic variant in BTK (p.Tyr418His), patient 3 had a pathogenic variant in CD40LG (c.288+1G>A), and patient 4 had a hypomorphic variant in IKBKG (p.Glu57Lys) and a heterozygous splice variant in TNFRSF13B (TACI) (c.61+2T>A). Overall, the analysis of our cohort suggests that CVID in a small proportion of females (1.6% in our cohort) is caused by skewed XCI and highly penetrant gene variants on the X-chromosome. Additionally, skewed XCI may contribute to polygenic effects (3.3% in our cohort). These results indicate that skewed XCI may represent another piece in the complex puzzle of CVID genetics.

Novel frameshift variants expand the map of the genetic defects in IRF2BP2.

Background: At present, the knowledge about disease-causing mutations in IRF2BP2 is very limited because only a few patients affected by this condition have been reported. As previous studies have described, the haploinsufficiency of this interferon transcriptional corepressors leads to the development of CVID. Very recently, a more accurate phenotype produced by truncating variants in this gene has been defined, manifesting CVID with gastrointestinal inflammatory symptoms and autoimmune manifestations. Methods: We analyzed 5 index cases with suspected primary immunodeficiency by high throughput sequencing. They were submitted for a genetic test with a panel of genes associated with immune system diseases, including IRF2BP2. The screening of SNVs, indels and CNVs fulfilling the criteria with very low allelic frequency and high protein impact, revealed five novel variants in IRF2BP2. In addition, we isolated both wild-type and mutated allele of the cDNA from one of the families. Results: In this study, we report five novel loss-of-function (LoF) mutations in IRF2BP2 that likely cause primary immunodeficiency, with CVID as more frequent phenotype, variable expression of inflammatory gastrointestinal features, and one patient with predisposition of viral infection. All identified variants were frameshift changes, and one of them was a large deletion located on chromosome 1q42, which includes the whole sequence of IRF2BP2, among other genes. Both de novo and dominant modes of inheritance were observed in the families here presented, as well as incomplete penetrance. Conclusions: We describe novel variants in a delimited low-complex region, which may be considered a hotspot in IRF2BP2. Moreover, this is the first time that a large CNV in IRF2BP2 has been reported to cause CVID. The distinct mechanisms than LoF in IRF2BP2 could cause different phenotype compared with the mainly described. Further investigations are necessary to comprehend the regulatory mechanisms of IRF2BP2, which could be under variable expression of the disease.

Anti-nuclear valosin-containing protein-like autoantibody is associated with calcinosis and higher risk of cancer in systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc)-specific autoantibodies allow the diagnosis and predict the prognosis of SSc patients with different clinical characteristics. The aim of this study was to describe new SSc-related autoantibodies by a novel protein immunoprecipitation (IP) assay. METHODS: Serum samples and clinical data were collected from 307 SSc patients. Antinuclear autoantibodies were tested in all patients by indirect immunofluorescence (IIF) on HEp-2 cells. SSc-specific autoantibodies were evaluated with a commercial immunoblot and chemiluminescence immunoassay, and traditional RNA-IP. Patients negative for all these autoantibodies (n = 51) were further tested with a non-radioactive protein IP assay. Protein bands detected on SDS-PAGE were then analysed by mass spectrometry (MS) and confirmed by western blot (WB). Additional 56 patients with nucleolar pattern by IIF were tested by protein IP-WB. RESULTS: Five patients who underwent protein IP testing showed a 110-115kDa molecular weight band on SDS-PAGE and a homogeneous nucleolar pattern by IIF. MS identified the bands as nuclear valosin-containing protein-like (NVL). An additional positive patient was detected by IP-WB. As compared with the remaining 101 negative patients, anti-NVL positive patients showed a greater prevalence of calcinosis (100% vs 18.9%, p< 0.001), and cancer (66.7% vs 8.9%, p= 0.002), with a particular association with synchronous cancer (OR = 16.3; p= 0.024). CONCLUSION: We identified NVL as a new autoantibody target by a novel protein IP assay in SSc patients with a homogeneous nucleolar IIF pattern, testing negative for all known SSc-specific autoantibodies by commercial assays and RNA IP. Anti-NVL identifies a new clinical phenotype, characterized by calcinosis and cancer.

Molecular Challenges in the Diagnosis of X-Linked Chronic Granulomatous Disease: CNVs, Intronic Variants, Skewed X-Chromosome Inactivation, and Gonosomal Mosaicism.

Chronic granulomatous disease (CGD) is a prototypical inborn error of immunity affecting phagocytes, in which these cells are unable to produce reactive oxygen species. CGD is caused by defects in genes encoding subunits of the NADPH oxidase enzyme complex (CYBA, CYBB, CYBC1, NCF1, NCF2, NCF4); inflammatory responses are dysregulated, and patients are highly susceptible to recurrent severe bacterial and fungal infections. X-linked CGD (XL-CGD), caused by mutations in the CYBB gene, is the most common and severe form of CGD. In this study, we describe the analytical processes undertaken in 3 families affected with XL-CGD to illustrate several molecular challenges in the genetic diagnosis of this condition: in family 1, a girl with a heterozygous deletion of CYBB exon 13 and skewed X-chromosome inactivation (XCI); in family 2, a boy with a hemizygous deletion of CYBB exon 7, defining its consequences at the mRNA level; and in family 3, 2 boys with the same novel intronic variant in CYBB (c.1151 + 6 T > A). The variant affected the splicing process, although a small fraction of wild-type mRNA was produced. Their mother was a heterozygous carrier, while their maternal grandmother was a carrier in form of gonosomal mosaicism. In summary, using a variety of techniques, including an NGS-based targeted gene panel and deep amplicon sequencing, copy number variation calling strategies, microarray-based comparative genomic hybridization, and cDNA analysis to define splicing defects and skewed XCI, we show how to face and solve some uncommon genetic mechanisms in the diagnosis of XL-CGD.

Single cell transcriptomic analysis of Graves' disease thyroid glands reveals the broad immunoregulatory potential of thyroid follicular and stromal cells and implies a major re-interpretation of the role of aberrant HLA class II expression in autoimmunity.

The study of the immune response in thyroid autoimmunity has been mostly focused on the autoantibodies and lymphocytes, but there are indications that intrinsic features of thyroid tissue cells may play a role in disrupting tolerance that needs further investigation. The overexpression of HLA and adhesion molecules by thyroid follicular cells (TFC) and our recent demonstration that PD-L1 is also moderately expressed by TFCs in autoimmune thyroid indicates that TFCs they may activate but also inhibit the autoimmune response. Intriguingly, we have recently found that in vitro cultured TFCs are able to suppress the proliferation of autologous lymphocyte T in a contact-dependent manner which is independent of the PD-1/PD-L1 signaling pathway. To get a more comprehensive picture of TFC activating and inhibitory molecules/pathways driving the autoimmune response in the thyroid glands, preparations of TFCs and stromal cells from five Graves' disease (GD) and four control thyroid glands were compared by scRNA-seq. The results confirmed the previously described interferon type I and type II signatures in GD TFCs and showed unequivocally that they express the full array of genes that intervene in the processing and presentation of endogenous and exogeneous antigens. GD TFCs lack however expression of costimulatory molecules CD80 and CD86 required for priming T cells. A moderate overexpression of CD40 by TFCs was confirmed. GD Fibroblasts showed widespread upregulation of cytokine genes. The results from this first single transcriptomic profiling of TFC and thyroid stromal cells provides a more granular view of the events occurring in GD. The new data point at an important contribution of stromal cells and prompt a major re-interpretation of the role of MHC over-expression by TFC, from deleterious to protective. Most importantly this re-interpretation could also apply to other tissues, like pancreatic beta cells, where MHC over-expression has been detected in diabetic pancreas.

Rare predicted loss-of-function variants of type I IFN immunity genes are associated with life-threatening COVID-19.

BACKGROUND: We previously reported that impaired type I IFN activity, due to inborn errors of TLR3- and TLR7-dependent type I interferon (IFN) immunity or to autoantibodies against type I IFN, account for 15-20% of cases of life-threatening COVID-19 in unvaccinated patients. Therefore, the determinants of life-threatening COVID-19 remain to be identified in ~ 80% of cases. METHODS: We report here a genome-wide rare variant burden association analysis in 3269 unvaccinated patients with life-threatening COVID-19, and 1373 unvaccinated SARS-CoV-2-infected individuals without pneumonia. Among the 928 patients tested for autoantibodies against type I IFN, a quarter (234) were positive and were excluded. RESULTS: No gene reached genome-wide significance. Under a recessive model, the most significant gene with at-risk variants was TLR7, with an OR of 27.68 (95%CI 1.5-528.7, P = 1.1 × 10-4) for biochemically loss-of-function (bLOF) variants. We replicated the enrichment in rare predicted LOF (pLOF) variants at 13 influenza susceptibility loci involved in TLR3-dependent type I IFN immunity (OR = 3.70[95%CI 1.3-8.2], P = 2.1 × 10-4). This enrichment was further strengthened by (1) adding the recently reported TYK2 and TLR7 COVID-19 loci, particularly under a recessive model (OR = 19.65[95%CI 2.1-2635.4], P = 3.4 × 10-3), and (2) considering as pLOF branchpoint variants with potentially strong impacts on splicing among the 15 loci (OR = 4.40[9%CI 2.3-8.4], P = 7.7 × 10-8). Finally, the patients with pLOF/bLOF variants at these 15 loci were significantly younger (mean age [SD] = 43.3 [20.3] years) than the other patients (56.0 [17.3] years; P = 1.68 × 10-5). CONCLUSIONS: Rare variants of TLR3- and TLR7-dependent type I IFN immunity genes can underlie life-threatening COVID-19, particularly with recessive inheritance, in patients under 60 years old.

Detection and evolutionary dynamics of somatic FAS variants in autoimmune lymphoproliferative syndrome: Diagnostic implications.

Autoimmune lymphoproliferative syndrome (ALPS) is a rare primary immune disorder characterized by impaired apoptotic homeostasis. The clinical characteristics include lymphoproliferation, autoimmunity (mainly cytopenia), and an increased risk of lymphoma. A distinctive biological feature is accumulation (>2.5%) of an abnormal cell subset composed of TCRαß+ CD4-CD8- T cells (DNTs). The most common genetic causes of ALPS are monoallelic pathogenic variants in the FAS gene followed by somatic FAS variants, mainly restricted to DNTs. Identification of somatic FAS variants has been typically addressed by Sanger sequencing in isolated DNTs. However, this approach can be costly and technically challenging, and may not be successful in patients with normal DNT counts receiving immunosuppressive treatment. In this study, we identified a novel somatic mutation in FAS (c.718_719insGTCG) by Sanger sequencing on purified CD3+ cells. We then followed the evolutionary dynamics of the variant along time with an NGS-based approach involving deep amplicon sequencing (DAS) at high coverage (20,000-30,000x). Over five years of clinical follow-up, we obtained six blood samples for molecular study from the pre-treatment (DNTs>7%) and treatment (DNTs<2%) periods. DAS enabled detection of the somatic variant in all samples, even the one obtained after five years of immunosuppressive treatment (DNTs: 0.89%). The variant allele frequency (VAF) range was 4%-5% in pre-treatment samples and <1.5% in treatment samples, and there was a strong positive correlation between DNT counts and VAF (Pearson's R: 0.98, p=0.0003). We then explored whether the same approach could be used in a discovery setting. In the last follow-up sample (DNT: 0.89%) we performed somatic variant calling on the FAS exon 9 DAS data from whole blood and purified CD3+ cells using VarScan 2. The c.718_719insGTCG variant was identified in both samples and showed the highest VAF (0.67% blood, 1.58% CD3+ cells) among >400 variants called. In summary, our study illustrates the evolutionary dynamics of a somatic FAS mutation before and during immunosuppressive treatment. The results show that pathogenic somatic FAS variants can be identified with the use of DAS in whole blood of ALPS patients regardless of their DNT counts.

Rare predicted loss-of-function variants of type I IFN immunity genes are associated with life-threatening COVID-19.

Background: We previously reported inborn errors of TLR3- and TLR7-dependent type I interferon (IFN) immunity in 1-5% of unvaccinated patients with life-threatening COVID-19, and auto-antibodies against type I IFN in another 15-20% of cases. Methods: We report here a genome-wide rare variant burden association analysis in 3,269 unvaccinated patients with life-threatening COVID-19 (1,301 previously reported and 1,968 new patients), and 1,373 unvaccinated SARS-CoV-2-infected individuals without pneumonia. A quarter of the patients tested had antibodies against type I IFN (234 of 928) and were excluded from the analysis. Results: No gene reached genome-wide significance. Under a recessive model, the most significant gene with at-risk variants was TLR7 , with an OR of 27.68 (95%CI:1.5-528.7, P= 1.1×10 -4 ), in analyses restricted to biochemically loss-of-function (bLOF) variants. We replicated the enrichment in rare predicted LOF (pLOF) variants at 13 influenza susceptibility loci involved in TLR3-dependent type I IFN immunity (OR=3.70 [95%CI:1.3-8.2], P= 2.1×10 -4 ). Adding the recently reported TYK2 COVID-19 locus strengthened this enrichment, particularly under a recessive model (OR=19.65 [95%CI:2.1-2635.4]; P= 3.4×10 -3 ). When these 14 loci and TLR7 were considered, all individuals hemizygous ( n =20) or homozygous ( n =5) for pLOF or bLOF variants were patients (OR=39.19 [95%CI:5.2-5037.0], P =4.7×10 -7 ), who also showed an enrichment in heterozygous variants (OR=2.36 [95%CI:1.0-5.9], P =0.02). Finally, the patients with pLOF or bLOF variants at these 15 loci were significantly younger (mean age [SD]=43.3 [20.3] years) than the other patients (56.0 [17.3] years; P= 1.68×10 -5 ). Conclusions: Rare variants of TLR3- and TLR7-dependent type I IFN immunity genes can underlie life-threatening COVID-19, particularly with recessive inheritance, in patients under 60 years old.

Genome-wide detection of human variants that disrupt intronic branchpoints.

Pre-messenger RNA splicing is initiated with the recognition of a single-nucleotide intronic branchpoint (BP) within a BP motif by spliceosome elements. Forty-eight rare variants in 43 human genes have been reported to alter splicing and cause disease by disrupting BP. However, until now, no computational approach was available to efficiently detect such variants in massively parallel sequencing data. We established a comprehensive human genome-wide BP database by integrating existing BP data and generating new BP data from RNA sequencing of lariat debranching enzyme DBR1-mutated patients and from machine-learning predictions. We characterized multiple features of BP in major and minor introns and found that BP and BP-2 (two nucleotides upstream of BP) positions exhibit a lower rate of variation in human populations and higher evolutionary conservation than the intronic background, while being comparable to the exonic background. We developed BPHunter as a genome-wide computational approach to systematically and efficiently detect intronic variants that may disrupt BP recognition. BPHunter retrospectively identified 40 of the 48 known pathogenic BP variants, in which we summarized a strategy for prioritizing BP variant candidates. The remaining eight variants all create AG-dinucleotides between the BP and acceptor site, which is the likely reason for missplicing. We demonstrated the practical utility of BPHunter prospectively by using it to identify a novel germline heterozygous BP variant of STAT2 in a patient with critical COVID-19 pneumonia and a novel somatic intronic 59-nucleotide deletion of ITPKB in a lymphoma patient, both of which were validated experimentally. BPHunter is publicly available from https://hgidsoft.rockefeller.edu/BPHunter and https://github.com/casanova-lab/BPHunter.

Common Variable Immunodeficiency and Neurodevelopmental Delay Due to a 13Mb Deletion on Chromosome 4 Including the NFKB1 Gene: A Case Report.

Syndromic immunodeficiencies are a heterogeneous group of inborn errors of immunity that can affect the development of non-immune organs and systems. The genetic basis of these immunodeficiencies is highly diverse, ranging from monogenic defects to large chromosomal aberrations. Antibody deficiency is the most prevalent immunological abnormality in patients with syndromic immunodeficiencies caused by chromosomal rearrangements, and usually manifests as a common variable immunodeficiency (CVID)-like phenotype. Here we describe a patient with a complex phenotype, including neurodevelopmental delay, dysmorphic features, malformations, and CVID (hypogammaglobulinemia, reduced pre-switch and switch memory B cells, and impaired vaccine response). Microarray-based comparative genomic hybridization (aCGH) revealed a 13-Mb deletion on chromosome 4q22.2-q24 involving 53 genes, some of which were related to the developmental manifestations in our patient. Although initially none of the affected genes could be linked to his CVID phenotype, subsequent reanalysis identified NFKB1 haploinsufficiency as the cause. This study underscores the value of periodic reanalysis of unsolved genetic studies performed with high-throughput technologies (eg, next-generation sequencing and aCGH). This is important because of the ongoing incorporation of new data establishing the relationship between genes and diseases. In the present case, NFKB1 had not been associated with human disease at the time aCGH was performed. Eight years later, reanalysis of the genes included in the chromosome 4 deletion enabled us to identify NFKB1 haploinsufficiency as the genetic cause of our patient's CVID. In the future, other genes included in the deletion may be linked to human disease, allowing us to better define the molecular basis of our patient's complex clinical phenotype.

Epigenetic profiling linked to multisystem inflammatory syndrome in children (MIS-C): A multicenter, retrospective study.

Background: Most children and adolescents infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain asymptomatic or develop a mild coronavirus disease 2019 (COVID-19) that usually does not require medical intervention. However, a small proportion of pediatric patients develop a severe clinical condition, multisystem inflammatory syndrome in children (MIS-C). The involvement of epigenetics in the control of the immune response and viral activity prompted us to carry out an epigenomic study to uncover target loci regulated by DNA methylation that could be altered upon the appearance of MIS-C. Methods: Peripheral blood samples were recruited from 43 confirmed MIS-C patients. 69 non-COVID-19 pediatric samples and 15 COVID-19 pediatric samples without MIS-C were used as controls. The cases in the two groups were mixed and divided into discovery (MIS-C = 29 and non-MIS-C = 56) and validation (MIS-C = 14 and non-MIS-C = 28) cohorts, and balanced for age, gender and ethnic background. We interrogated 850,000 CpG sites of the human genome for DNA methylation variants. Findings: The DNA methylation content of 33 CpG loci was linked with the presence of MIS-C. Of these sites, 18 (54.5%) were located in described genes. The top candidate gene was the immune T-cell mediator ZEB2; and others highly ranked candidates included the regulator of natural killer cell functional competence SH2D1B; VWA8, which contains a domain of the Von Willebrand factor A involved in the pediatric hemostasis disease; and human leukocyte antigen complex member HLA-DRB1; in addition to pro-inflammatory genes such as CUL2 and AIM2. The identified loci were used to construct a DNA methylation profile (EPIMISC) that was associated with MIS-C in both cohorts. The EPIMISC signature was also overrepresented in Kawasaki disease patients, a childhood pathology with a possible viral trigger, that shares many of the clinical features of MIS-C. Interpretation: We have characterized DNA methylation loci that are associated with MIS-C diagnosis. The identified genes are likely contributors to the characteristic exaggerated host inflammatory response observed in these patients. The described epigenetic signature could also provide new targets for more specific therapies for the disorder. Funding: Unstoppable campaign of Josep Carreras Leukaemia Foundation, Fundació La Marató de TV3, Cellex Foundation and CERCA Programme/Generalitat de Catalunya.

Case Report: X-Linked SASH3 Deficiency Presenting as a Common Variable Immunodeficiency.

SASH3 is a lymphoid-specific adaptor protein. In a recent study, SASH3 deficiency was described as a novel X-linked combined immunodeficiency with immune dysregulation, associated with impaired TCR signaling and thymocyte survival in humans. The small number of patients reported to date showed recurrent sinopulmonary, cutaneous and mucosal infections, and autoimmune cytopenia. Here we describe an adult patient previously diagnosed with common variable immunodeficiency (CVID) due to low IgG and IgM levels and recurrent upper tract infections. Two separate, severe viral infections drew our attention and pointed to an underlying T cell defect: severe varicella zoster virus (VZV) infection at the age of 4 years and bilateral pneumonia due type A influenza infection at the age of 38. Genetic testing using an NGS-based custom-targeted gene panel revealed a novel hemizygous loss-of-function variant in the SASH3 gene (c.505C>T/p.Gln169*). The patient's immunological phenotype included marked B cell lymphopenia with reduced pre-switch and switch memory B cells, decreased CD4+ and CD8+ naïve T cells, elevated CD4+ and CD8+ TEMRA cells, and abnormal T cell activation and proliferation. The patient showed a suboptimal response to Streptococcus pneumoniae (polysaccharide) vaccine, and a normal response to Haemophilus influenzae type B (conjugate) vaccine and SARS-CoV-2 (RNA) vaccine. In summary, our patient has a combined immunodeficiency, although he presented with a phenotype resembling CVID. Two severe episodes of viral infection alerted us to a possible T-cell defect, and genetic testing led to SASH3 deficiency. Our patient displays a milder phenotype than has been reported previously in these patients, thus expanding the clinical spectrum of this recently identified inborn error of immunity.

Inborn errors of TLR3- or MDA5-dependent type I IFN immunity in children with enterovirus rhombencephalitis.

Enterovirus (EV) infection rarely results in life-threatening infection of the central nervous system. We report two unrelated children with EV30 and EV71 rhombencephalitis. One patient carries compound heterozygous TLR3 variants (loss-of-function F322fs2* and hypomorphic D280N), and the other is homozygous for an IFIH1 variant (loss-of-function c.1641+1G>C). Their fibroblasts respond poorly to extracellular (TLR3) or intracellular (MDA5) poly(I:C) stimulation. The baseline (TLR3) and EV-responsive (MDA5) levels of IFN-ß in the patients' fibroblasts are low. EV growth is enhanced at early and late time points of infection in TLR3- and MDA5-deficient fibroblasts, respectively. Treatment with exogenous IFN-α2b before infection renders both cell lines resistant to EV30 and EV71, whereas post-infection treatment with IFN-α2b rescues viral susceptibility fully only in MDA5-deficient fibroblasts. Finally, the poly(I:C) and viral phenotypes of fibroblasts are rescued by the expression of WT TLR3 or MDA5. Human TLR3 and MDA5 are critical for cell-intrinsic immunity to EV, via the control of baseline and virus-induced type I IFN production, respectively.

Case Report: Characterizing the Role of the STXBP2-R190C Monoallelic Mutation Found in a Patient With Hemophagocytic Syndrome and Langerhans Cell Histiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory disorder. HLH can be considered as a threshold disease depending on the trigger and the residual NK-cell cytotoxicity. In this study, we analyzed the molecular and functional impact of a novel monoallelic mutation found in a patient with two episodes of HLH. A 9-month-old child was diagnosed at 2 months of age with cutaneous Langerhans cell histiocytosis (LCH). After successful treatment, the patient developed an HLH episode. At 16 month of age, the patient went through an HSCT losing the engraftment 5 months later concomitant with an HLH relapse. The genetic study revealed a monoallelic mutation in the STXBP2 gene (.pArg190Cys). We transfected COS7 cells to analyze the STXBP2-R190C expression and to test the interaction with STX11. We used the RBL-2H3 cell line expressing STXBP2-WT-EGFP or R190C-EGFP for degranulation assays. Mutation STXBP2-R190C did not affect protein expression or interaction with syntaxin-11. However, we have demonstrated that STXBP2-R190C mutation diminishes degranulation in the RBL-2H3 cell line compared with the RBL-2H3 cell line transfected with STXBP2-WT or nontransfected. These results suggest that STXBP2-R190C mutation acts as a modifier of the degranulation process producing a decrease in degranulation. Therefore, under homeostatic conditions, the presence of one copy of STXBP2-R190 could generate sufficient degranulation capacity. However, it is likely that early in life when adaptive immune system functions are not sufficiently developed, an infection may not be resolved with this genetic background, leading to a hyperinflammation syndrome and eventually develop HLH. This analysis highlights the need for functional testing of new mutations to validate their role in genetic susceptibility and to establish the best possible treatment for these patients.

Early Diagnosis and Treatment of Purine Nucleoside Phosphorylase (PNP) Deficiency through TREC-Based Newborn Screening.

Purine nucleoside phosphorylase (PNP) deficiency is a rare inherited disorder, resulting in severe combined immunodeficiency. To date, PNP deficiency has been detected in newborn screening only through the use of liquid chromatography tandem mass spectrometry. We report the first case in which PNP deficiency was detected by TREC analysis.