IKAROS-how many feathers have you lost: mild and severe phenotypes in IKZF1 deficiency.

Heterozygous germline variants in human IKZF1 encoding for IKAROS define an inborn error of immunity with immunodeficiency, immune dysregulation and risk of malignancy with a broad phenotypic spectrum. Growing evidence of underlying pathophysiological genotype-phenotype correlations helps to improve our understanding of IKAROS-associated diseases. We describe 6 patients from 4 kindreds with two novel IKZF1 variants leading to haploinsufficiency from 3 centers in Germany. We also provide an overview of first symptoms to a final diagnosis including data from the literature.

Multiple-breath washout to detect lung disease in patients with inborn errors of immunity.

Background: Pulmonary manifestations are the major cause of morbidity and mortality in patients with inborn errors of immunity (IEI). New and more sensitive diagnostic methods can potentially lead to earlier recognition and treatment of IEI lung disease and improve outcome. The aim of this study was to compare multiple-breath washout (MBW) and spirometry in patients with IEI and cystic fibrosis (CF) as well as healthy controls (HC) and to evaluate the sensitivity of lung clearance index (LCI) to assess lung disease in IEI. Methods: IEI patients (n=114) were recruited from our paediatric and adult immunodeficiency outpatient clinics and compared to age-matched CF patients (n=114) and HC (n=114). MBW measurements and spirometry were performed in the study participants, and MBW testing was repeated after 63-707 days in IEI patients (n=70). Results: The LCI was significantly higher in IEI patients than in HC (p<0.001) and significantly lower than in CF patients (p<0.001). The forced expiratory volume in 1 s (FEV1) z-score was significantly lower in IEI patients than in HC (p<0.01) and significantly higher than in CF patients (p<0.01). LCI and FEV1 z-score correlated moderately negatively in the total cohort, the IEI group and the CF group. Nineteen (20.7%) of 92 IEI patients and 35 (33.3%) of 105 CF patients had an elevated LCI but a normal FEV1 z-score. After a median of 364 days, the median LCI of 70 IEI patients increased significantly by 0.2. Conclusion: MBW is useful to detect lung disease in IEI and is more sensitive than spirometry.

Activated PI3Kδ syndrome - reviewing challenges in diagnosis and treatment.

Activated PI3Kδ syndrome (APDS) is a rare inborn error of immunity (IEI) characterized primarily by frequent infections, lymphoproliferation and autoimmunity. Since its initial description in 2013, APDS has become part of the growing group of nearly 500 IEIs affecting various components of the immune system. The two subtypes of APDS - APDS1 and APDS2 - are caused by variants in the PIK3CD and PIK3R1 genes, respectively. Due to the rarity of the disease and the heterogeneous clinical picture, many patients are not diagnosed until years after symptom onset. Another challenge is the large number of PIK3CD and PIK3R1 variants whose functional significance for developing APDS is inconclusive. Treatment of APDS has so far been mostly symptom-oriented with immunoglobulin replacement therapy, immunosuppressive therapies and antibiotic or antiviral prophylaxes. Additionally, allogeneic stem cell transplantation as well as new targeted therapies are options targeting the root cause that may improve patients' quality of life and life expectancy. However, the clinical course of the disease is difficult to predict which complicates the choice of appropriate therapies. This review article discusses diagnostic procedures and current and future treatment options, and highlights the difficulties that physicians, patients and their caretakers face in managing this complex disease. This article is based on cohort studies, the German and US guidelines on the management of primary immunodeficiencies as well as on published experience with diagnosis and compiled treatment experience for APDS.

Screening Newborns for Low T Cell Receptor Excision Circles (TRECs) Fails to Detect Immunodeficiency, Centromeric Instability, and Facial Anomalies Syndrome.

BACKGROUND: Assessment of T-cell receptor excision circles (TRECs) in dried blood spots of newborns allows the detection of severe combined immunodeficiency (SCID) (T cells <300/µL at birth) with a presumed sensitivity of 100%. TREC screening also identifies patients with selected combined immunodeficiency (CID) (T cells >300/µL, yet <1500/µL at birth). Nevertheless, relevant CIDs that would benefit from early recognition and curative treatment pass undetected. OBJECTIVE: We hypothesized that TREC screening at birth cannot identify CIDs that develop with age. METHODS: We analyzed the number of TRECs in dried blood spots in archived Guthrie cards of 22 children who had been born in the Berlin-Brandenburg area between January 2006 and November 2018 and who had undergone hematopoietic stem-cell transplantation (HSCT) for inborn errors of immunity. RESULTS: All patients with SCID would have been identified by TREC screening, but only 4 of 6 with CID. One of these patients had immunodeficiency, centromeric instability, and facial anomalies syndrome type 2 (ICF2). Two of 3 patients with ICF whom we have been following up at our institution had TREC numbers above the cutoff value suggestive of SCID at birth. Yet all patients with ICF had a severe clinical course that would have justified earlier HSCT. CONCLUSIONS: In ICF, naïve T cells may be present at birth, yet they decline with age. Therefore, TREC screening cannot identify these patients. Early recognition is nevertheless crucial, as patients with ICF benefit from HSCT early in life.

Prospective Newborn Screening for SCID in Germany: A First Analysis by the Pediatric Immunology Working Group (API).

BACKGR OUND: T-cell receptor excision circle (TREC)-based newborn screening (NBS) for severe combined immunodeficiencies (SCID) was introduced in Germany in August 2019. METHODS: Children with abnormal TREC-NBS were referred to a newly established network of Combined Immunodeficiency (CID) Clinics and Centers. The Working Group for Pediatric Immunology (API) and German Society for Newborn Screening (DGNS) performed 6-monthly surveys to assess the TREC-NBS process after 2.5 years. RESULTS: Among 1.9 million screened newborns, 88 patients with congenital T-cell lymphocytopenia were identified (25 SCID, 17 leaky SCID/Omenn syndrome (OS)/idiopathic T-cell lymphocytopenia, and 46 syndromic disorders). A genetic diagnosis was established in 88%. Twenty-six patients underwent hematopoietic stem cell transplantation (HSCT), 23/26 within 4 months of life. Of these, 25/26 (96%) were alive at last follow-up. Two patients presented with in utero onset OS and died after birth. Five patients with syndromic disorders underwent thymus transplantation. Eight syndromic patients deceased, all from non-immunological complications. TREC-NBS missed one patient, who later presented clinically, and one tracking failure occurred after an inconclusive screening result. CONCLUSION: The German TREC-NBS represents the largest European SCID screening at this point. The incidence of SCID/leaky SCID/OS in Germany is approximately 1:54,000, very similar to previous observations from North American and European regions and countries where TREC-NBS was implemented. The newly founded API-CID network facilitates tracking and treatment of identified patients. Short-term HSCT outcome was excellent, but NBS and transplant registries will remain essential to evaluate the long-term outcome and to compare results across the rising numbers of TREC-NBS programs across Europe.

Case Report: Rubella Virus-Induced Cutaneous Granulomas in Two Pediatric Patients With DNA Double Strand Breakage Repair Disorders - Outcome After Hematopoietic Stem Cell Transplantation.

We report two patients with DNA repair disorders (Artemis deficiency, Ataxia telangiectasia) with destructive skin granulomas, presumably triggered by live-attenuated rubella vaccinations. Both patients showed reduced naïve T cells. Rapid resolution of skin lesions was observed following hematopoietic stem cell transplantation. However, the patient with AT died due to complications of severe hepatic veno-occlusive disease 6 month after HSCT. Dried blood spots obtained after birth were available from this patient and showed absent T-cell receptor excision circles (TRECs). Therefore, newborn screening may help to prevent patients with moderate T-cell deficiency from receiving live-attenuated rubella vaccine potentially causing granulomas.

Genetic Analysis of a Cohort of 275 Patients with Hyper-IgE Syndromes and/or Chronic Mucocutaneous Candidiasis.

Hyper-IgE syndromes and chronic mucocutaneous candidiasis constitute rare primary immunodeficiency syndromes with an overlapping clinical phenotype. In recent years, a growing number of underlying genetic defects have been identified. To characterize the underlying genetic defects in a large international cohort of 275 patients, of whom 211 had been clinically diagnosed with hyper-IgE syndrome and 64 with chronic mucocutaneous candidiasis, targeted panel sequencing was performed, relying on Agilent HaloPlex and Illumina MiSeq technologies. The targeted panel sequencing approach allowed us to identify 87 (32 novel and 55 previously described) mutations in 78 patients, which generated a diagnostic success rate of 28.4%. Specifically, mutations in DOCK8 (26 patients), STAT3 (21), STAT1 (15), CARD9 (6), AIRE (3), IL17RA (2), SPINK5 (3), ZNF341 (2), CARMIL2/RLTPR (1), IL12RB1 (1), and WAS (1) have been detected. The most common clinical findings in this cohort were elevated IgE (81.5%), eczema (71.7%), and eosinophilia (62.9%). Regarding infections, 54.7% of patients had a history of radiologically proven pneumonia, and 28.3% have had other serious infections. History of fungal infection was noted in 53% of cases and skin abscesses in 52.9%. Skeletal or dental abnormalities were observed in 46.2% of patients with a characteristic face being the most commonly reported feature (23.1%), followed by retained primary teeth in 18.9% of patients. Targeted panel sequencing provides a cost-effective first-line genetic screening method which allows for the identification of mutations also in patients with atypical clinical presentations and should be routinely implemented in referral centers.

A Pathogenic Missense Variant in NFKB1 Causes Common Variable Immunodeficiency Due to Detrimental Protein Damage.

In common variable immunodeficiency (CVID), heterozygous damaging NFKB1 variants represent the most frequent monogenic cause. NFKB1 encodes the precursor p105, which undergoes proteasomal processing to generate the mature NF-κB transcription factor subunit p50. The majority of NFKB1 sequence changes comprises missense variants of uncertain significance (VUS), each requiring functional evaluation to assess causality, particularly in families with multiple affected members presenting with different phenotypes. In four affected members of a German family, all diagnosed with CVID, we identified a previously uncharacterized heterozygous NFKB1 missense variant (c.1049A>G; p.Tyr350Cys). The clinical phenotypes varied markedly regarding onset, frequency and severity of infections. Consistent immunologic findings were hypogammaglobulinemia with normal specific antibody response to protein- and polysaccharide-based vaccinations, reduced switched memory B cells and decreased lymphocyte proliferation upon stimulation with the B cell mitogen SAC. To assess the pathogenicity of the NFKB1 missense variant, we employed immunophenotyping and functional analyses in a routine in vitro cell culture model. Following site-directed mutagenesis to introduce the variant into overexpression vectors encoding EGFP-fused p105 or p50, we analyzed transiently transfected HEK293T cells by confocal imaging and Western blotting. The cytoplasmic p105-Tyr350Cys precursor gained only weak expression levels indicating accelerated decay. The missense change disabled processing of the precursor to prevent the generation of mutant p50. Unlike the wildtype p50, the overexpressed mutant p50-Tyr350Cys was also not sustainable and showed a conspicuous subnuclear mislocalization with accumulation in dense aggregates instead of a homogenous distribution. Electrophoretic mobility shift assays, fluorescence-based reporter gene analyses and co-transfection experiments however demonstrated, that the DNA-binding activity of p50-Tyr350Cys and the interaction with RelA(p65), IκBα and wildtype p50 were preserved. Mutation carriers had reduced p105 and p50 levels, indicating insufficient protein amounts as the most likely primary defect. In conclusion, the missense variant c.1049A>G caused a detrimental defect, preventing the persistent expression of both, the p105-Tyr350Cys precursor and the mature p50-Tyr350Cys. The variable clinical phenotypes among affected family members sharing an identical pathogenic NFKB1 variant support a disease mechanism provoked by a p105/p50 (haplo)insufficient condition.

Lanadelumab Efficacy, Safety, and Injection Interval Extension in HAE: A Real-Life Study.

BACKGROUND: Lanadelumab has been available in Germany for the prophylactic treatment of hereditary angioedema since February 2019. OBJECTIVE: To investigate real-life treatment outcome of lanadelumab and gain practical experience in adapting the therapy to individual patients. METHODS: The study included 34 patients. In 24 patients with hereditary angioedema and 4 patients with angioedema due to acquired C1-inhibitor deficiency, the previous treatment was switched to lanadelumab. In 6 patients with hereditary angioedema, lanadelumab from the open-label Hereditary Angioedema Long-term Prophylaxis study was continued in regular care. During the transition, patients were monitored using the angioedema control test and the angioedema quality of life questionnaire. At the time at which patients became symptom-free, the dosage interval was increased gradually (+3 days). RESULTS: On average, the angioedema control test values improved from 7.5 (poorly controlled disease) to 14.9 (well-controlled disease), and all patients showed adequate disease control. All treated patients, except 1 outlier, scored angioedema quality of life questionnaire values representing only a slight reduction in quality of life (mean, 14 points). At the time point of data collection, 9 patients used an average fixed injection interval of 30 days. Twenty-two patients were symptom-free from the beginning of the treatment phase and intended to extend their injection interval from 30 to 32.5 days (median). We recommended reducing the initial dosing interval from 24 to 21 days (median) to 3 patients because of intermediately occurring symptoms. CONCLUSIONS: Gradual extension of injection intervals of lanadelumab presented in this study can minimize the burden of therapy without losing efficacy.

CD70 Deficiency Associated With Chronic Epstein-Barr Virus Infection, Recurrent Airway Infections and Severe Gingivitis in a 24-Year-Old Woman.

Most of the few patients with homozygous CD70 deficiency described to date suffered from EBV-related malignancies in early childhood. We present a woman with CD70 deficiency diagnosed in adulthood. She presented in childhood with recurrent airway infections due to encapsulated bacteria, herpes zoster and a fulminant EBV infection followed by chronic EBV infection with mild lymphoproliferation and severe gingivitis/periodontal disease with high EBV viral load in saliva and gingival plaques as an adult. Up to the age of 24 years she developed no malignancy despite constant EBV viremia since primary EBV infection 15 years previously. Immunologic evaluation in childhood showed hypogammaglobulinemia with impaired polysaccharide responsiveness. She has been stable on immunoglobulin substitution with no further severe viral infections and no bacterial airway infections in adulthood. Targeted panel sequencing at the age of 20 years revealed a homozygous CD70 missense mutation (ENST00000245903.3:c.2T>C). CD70 deficiency was confirmed by absent CD70 expression of B cells and activated T cell blasts. The patient finished high school, persues an academic career and has rarely sick days at college. The clinical course of our patient may help to counsel parents of CD70-deficient patients with regard to prognosis and therapeutic options including haematopoetic stem cell transplantation.

Hereditary angioedema in children and adolescents - A consensus update on therapeutic strategies for German-speaking countries.

BACKGROUND/METHODS: At a consensus meeting in August 2018, pediatricians and dermatologists from German-speaking countries discussed the therapeutic strategy for the treatment of pediatric patients with type I and II hereditary angioedema due to C1 inhibitor deficiency (HAE-C1-INH) for Germany, Austria, and Switzerland, taking into account the current marketing approval status. HAE-C1-INH is a rare disease that usually presents during childhood or adolescence with intermittent episodes of potentially life-threatening angioedema. Diagnosis as early as possible and an optimal management of the disease are important to avoid ineffective therapies and to properly treat swelling attacks. This article provides recommendations for developing appropriate treatment strategies in the management of HAE-C1-INH in pediatric patients in German-speaking countries. An overview of available drugs in this age-group is provided, together with their approval status, and study results obtained in adults and pediatric patients. RESULTS/CONCLUSION: Currently, plasma-derived C1 inhibitor concentrates have the broadest approval status and are considered the best available option for on-demand treatment of HAE-C1-INH attacks and for short- and long-term prophylaxis across all pediatric age-groups in German-speaking countries. For on-demand treatment of children aged 2 years and older, recombinant C1-INH and bradykinin-receptor antagonist icatibant are alternatives. For long-term prophylaxis in adolescents, the parenteral kallikrein inhibitor lanadelumab has recently been approved and can be recommended due to proven efficacy and safety.

T Cell Impairment Is Predictive for a Severe Clinical Course in NEMO Deficiency.

PURPOSE: NEMO-deficient patients present with variable degrees of immunodeficiency. Accordingly, treatment ranges from antibiotic prophylaxis and/or IgG-substitution to allogenic hematopoietic stem cell transplantation (HSCT). The correct estimation of the immunodeficiency is essential to avoid over- as well as under-treatment. We compare the immunological phenotype of a NEMO-deficient patient with a newly-described splice site mutation that causes truncation of the NEMO zinc-finger (ZF) domain and a severe clinical course with the immunological phenotype of three NEMO-deficient patients with missense mutations and milder clinical courses and all previously published patients. METHODS: Lymphocyte subsets, proliferation, and intracellular NEMO-expression were assessed by FACS. NF-κB signal transduction was determined by measuring IκBα-degradation and the production of cytokines upon stimulation with TNF-α, IL-1ß, and TLR-agonists in immortalized fibroblasts and whole blood, respectively. RESULTS: The patient with truncated ZF-domain of NEMO showed low levels of IgM and IgG, reduced class-switched memory B cells, almost complete skewing towards naïve CD45RA+ T cells, impaired T cell proliferation as well as cytokine production upon stimulation with TNF-α, IL-1ß, and TLR-agonists. He suffered from severe infections (sepsis, pneumonia, osteomyelitis) during infancy. In contrast, three patients with missense mutations in IKBKG presented neither skewing of T cells towards naïvety nor impaired T cell proliferation. They are stable on prophylactic IgG-substitution or even off any prophylactic treatment. CONCLUSION: The loss of the ZF-domain and the impaired T cell proliferation accompanied by almost complete persistence of naïve T cells despite severe infections are suggestive for a profound immunodeficiency. Allogenic HSCT should be considered early for these patients before chronic sequelae occur.

Impaired polysaccharide responsiveness without agammaglobulinaemia in three patients with hypomorphic mutations in Bruton Tyrosine Kinase-No detection by newborn screening for primary immunodeficiencies.

Hypomorphic mutations in the gene encoding Bruton tyrosine kinase (BTK) may result in milder phenotypes and delayed diagnosis of B-cell related immunodeficiencies due to residual BTK function. Newborn screening for kappa-deleting-recombination-excision circles (KRECs) reliably identifies classical X-linked agammaglobulinaemia (XLA) patients with profound B-cell lymphopenia at birth but has not been evaluated in patients with residual BTK function. We aimed to evaluate clinical findings, BTK function and KREC copy numbers in three patients with BTK mutations presenting with impaired polysaccharide responsiveness without agammaglobulinaemia. One patient had an invasive pneumococcal infection at the age of 4 years. All three patients (two brothers) had visible tonsils, normal to slightly decreased immunoglobulin G levels, undetectable pneumococcal antibodies despite pneumococcal conjugate vaccinations, no antibody response after a diagnostic polysaccharide vaccination as well as profound B-cell lymphopenia with residual B-cell differentiation. BTK mutations were identified by Sanger sequencing. BTK staining and phosphorylation assays were performed on peripheral B cells. KREC copy numbers were determined from dried blood spots obtained within the first week of life as well as once at the age of 8, 6 and 3 years, respectively. BTK staining showed residual protein expression. Also, residual BTK activity could be demonstrated. KREC copy numbers from dried blood spots were above the threshold set for detection of patients with profound B-cell lymphopenia. Male patients with impaired polysaccharide responsiveness should be evaluated for B-cell lymphopenia followed by BTK analyses irrespective of immunoglobulin levels or tonsil size.

Antibiotic Prophylaxis, Immunoglobulin Substitution and Supportive Measures Prevent Infections in MECP2 Duplication Syndrome.

Respiratory infections are the main cause of early death in patients with MECP2 duplication syndrome. We report on a 20-year-old patient with MECP2 duplication syndrome, IgG2/IgG4/IgA/IgM deficiency and polysaccharide-specific antibody deficiency, who had 46 episodes of pneumonia in his first 13 8/12 years of life. Immunoglobulin substitution, daily antibiotic prophylaxis with two agents and supportive measures reduced occurrence of pneumonia to four episodes in the following 6 2/12 years of life.

IgG subclass deficiencies in children: Facts and fiction.

The chance to analyse the four IgG subclasses arose with the publication of Terry and Fahey1 . Since then, a lot of new information on the role of subclasses and their deficiency states in humans has been obtained. This review tries to analyse critically our current knowledge of subclass deficiencies in children.

From immune substitution to immunomodulation.

Intravenous immunoglobulins (IVIGs) are currently used in many fields of medicine for replacement and immunomodulation. This review focuses on the milestones in the history of human immunoglobulins since the initial observation by Ogden C. Bruton who described replacement therapy in a boy with agammaglobulinemia. Since then, the preparations used for treatment have been markedly improved with respect to tolerability, clinical efficacy, and pathogen safety. Preparations and appropriate pumps for subcutaneous administration of IgG have been developed and offer an alternative mode of treatment for immunodeficient patients. Appropriate replacement today allows patients with humoral immunodeficiencies to reach adulthood and normal or near-normal quality of life. In 1981 a second fundamental discovery was made. Paul Imbach and coauthors in children with idiopathic thrombocytopenic purpura (ITP) showed that IVIG has immunomodulatory potential, offering a chance for affected children to receive effective treatment with little or no side effects compared to systemic corticosteroids. This new principle of treatment encouraged many researchers worldwide to exploit the potential of IVIG in many other immunopathological situations. As an example, Rhesus hemolytic disease in newborn babies is discussed.

Key findings to expedite the diagnosis of hyper-IgE syndromes in infants and young children.

BACKGROUND: Hyper-IgE syndromes (HIES) are primary immunodeficiency disorders characterized by elevated serum IgE, eczema, and recurrent infections. Despite the availability of confirmatory molecular diagnosis of several distinct HIES entities, the differentiation of HIES particularly from severe forms of atopic dermatitis remains a challenge. The two most common forms of HIES are caused by mutations in the genes STAT3 and DOCK8. METHODS: Here, we assess the clinical and immunologic phenotype of DOCK8- and STAT3-HIES patients including the cell activation, proliferation, and cytokine release after stimulation. RESULTS: Existing HIES scoring systems are helpful to identify HIES patients. However, those scores may fail in infants and young children due to the age-related lack of clinical symptoms. Furthermore, our long-term observations showed a striking variation of laboratory results over time in the individual patient. Reduced memory B-cell counts in concert with low specific antibody production are the most consistent findings likely contributing to the high susceptibility to bacterial and fungal infection. In DOCK8-HIES, T-cell lymphopenia and low IFN-gamma secretion after stimulation were common, likely promoting viral infections. In contrast to STAT3-HIES, DOCK8-HIES patients showed more severe inflammation with regard to allergic manifestations, elevated activation markers (HLA-DR, CD69, CD86, and CD154), and significantly increased inflammatory cytokines (IL1-beta, IL4, IL6, and IFN-gamma). CONCLUSION: Differentiating HIES from other diseases such as atopic dermatitis early in life is essential for patients because treatment modalities differ. To expedite the diagnosis process, we propose here a diagnostic workflow.