Conversion of monoclonal IgG to dimeric and secretory IgA restores neutralizing ability and prevents infection of Omicron lineages.

The emergence of Omicron lineages and descendent subvariants continues to present a severe threat to the effectiveness of vaccines and therapeutic antibodies. We have previously suggested that an insufficient mucosal immunoglobulin A (IgA) response induced by the mRNA vaccines is associated with a surge in breakthrough infections. Here, we further show that the intramuscular mRNA and/or inactivated vaccines cannot sufficiently boost the mucosal secretory IgA response in uninfected individuals, particularly against the Omicron variant. We thus engineered and characterized recombinant monomeric, dimeric, and secretory IgA1 antibodies derived from four neutralizing IgG monoclonal antibodies (mAbs 01A05, rmAb23, DXP-604, and XG014) targeting the receptor-binding domain of the spike protein. Compared to their parental IgG antibodies, dimeric and secretory IgA1 antibodies showed a higher neutralizing activity against different variants of concern (VOCs), in part due to an increased avidity. Importantly, the dimeric or secretory IgA1 form of the DXP-604 antibody significantly outperformed its parental IgG antibody, and neutralized the Omicron lineages BA.1, BA.2, and BA.4/5 with a 25- to 75-fold increase in potency. In human angiotensin converting enzyme 2 (ACE2) transgenic mice, a single intranasal dose of the dimeric IgA DXP-604 conferred prophylactic and therapeutic protection against Omicron BA.5. Thus, dimeric or secretory IgA delivered by nasal administration may potentially be exploited for the treatment and prevention of Omicron infection, thereby providing an alternative tool for combating immune evasion by the current circulating subvariants and, potentially, future VOCs.

Metagenomic next-generation sequencing as a diagnostic tool in the clinical routine of an infectious diseases department: a retrospective cohort study.

BACKGROUND: Metagenomic next-generation sequencing (mNGS) of circulating cell-free DNA from plasma is a hypothesis-independent broadband diagnostic method for identification of potential pathogens. So far, it has only been investigated in special risk populations (e.g. patients with neutropenic fever). PURPOSE: To investigate the extent to which mNGS (DISQVER® platform) can be used in routine clinical practice. METHODS: We collected whole blood specimens for mNGS testing, blood cultures (BC), and pathogen-specific PCR diagnostics. Clinical data and pathogen diagnostics were retrospectively reviewed by an infectious disease expert panel regarding the adjustment of anti-infective therapy. RESULTS: In 55 selected patients (median age 53 years, 67% male) with heterogeneous diagnoses, a total of 66 different microorganisms and viruses were detected using mNGS (51% viruses, 38% bacteria, 8% fungi, 3% parasites). The overall positivity rate of mNGS was 53% (29/55). Fifty-two out of 66 (79%) potential pathogens detected by mNGS were found in patients with primary or secondary immunodeficiency. The concordance rates of BC and pathogen-specific PCR diagnostics with mNGS testing were 14% (4/28) and 36% (10/28), respectively (p < 0.001). An additional bacterial pathogen (Streptococcus agalactiae) could only be detected by BC. Therapeutic consequences regarding anti-infective therapy were drawn from 23 pathogens (35% of detections), with 18 of these detections occurring in patients with immunodeficiency. CONCLUSIONS: We conclude that mNGS is a useful diagnostic tool, but should only be performed selectively in addition to routine diagnostics of infectious diseases. The limited number of patients and the retrospective study design do not allow any further conclusions.

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.

Long-Term Follow-Up of Newborns with 22q11 Deletion Syndrome and Low TRECs.

BACKGROUND: Population-based neonatal screening using T-cell receptor excision circles (TRECs) identifies infants with profound T lymphopenia, as seen in cases of severe combined immunodeficiency, and in a subgroup of infants with 22q11 deletion syndrome (22q11DS). PURPOSE: To investigate the long-term prognostic value of low levels of TRECs in newborns with 22q11DS. METHODS: Subjects with 22q11DS and low TRECs at birth (22q11Low, N=10), matched subjects with 22q11DS and normal TRECs (22q11Normal, N=10), and matched healthy controls (HC, N=10) were identified. At follow-up (median age 16 years), clinical and immunological characterizations, covering lymphocyte subsets, immunoglobulins, TRECs, T-cell receptor repertoires, and relative telomere length (RTL) measurements were performed. RESULTS: At follow-up, the 22q11Low group had lower numbers of naïve T-helper cells, naïve T-regulatory cells, naïve cytotoxic T cells, and persistently lower TRECs compared to healthy controls. Receptor repertoires showed skewed V-gene usage for naïve T-helper cells, whereas for naïve cytotoxic T cells, shorter RTL and a trend towards higher clonality were found. Multivariate discriminant analysis revealed a clear distinction between the three groups and a skewing towards Th17 differentiation of T-helper cells, particularly in the 22q11Low individuals. Perturbations of B-cell subsets were found in both the 22q11Low and 22q11Normal group compared to the HC group, with larger proportions of naïve B cells and lower levels of memory B cells, including switched memory B cells. CONCLUSIONS: This long-term follow-up study shows that 22q11Low individuals have persistent immunologic aberrations and increased risk for immune dysregulation, indicating the necessity of lifelong monitoring. CLINICAL IMPLICATIONS: This study elucidates the natural history of childhood immune function in newborns with 22q11DS and low TRECs, which may facilitate the development of programs for long-term monitoring and therapeutic choices.

Gut Microbiota Perturbation in IgA Deficiency Is Influenced by IgA-Autoantibody Status.

BACKGROUND & AIMS: IgA exerts its primary function at mucosal surfaces, where it binds microbial antigens to regulate bacterial growth and epithelial attachment. One third of individuals with IgA deficiency (IgAD) suffers from recurrent mucosal infections, possibly related to an altered microbiota. We aimed to delineate the impact of IgAD and the IgA-autoantibody status on the composition and functional capacity of the gut microbiota. METHODS: We performed a paired, lifestyle-balanced analysis of the effect of IgA on the gut microbiota composition and functionality based on fecal samples from individuals with IgAD and IgA-sufficient household members (n = 100), involving quantitative shotgun metagenomics, species-centric functional annotation of gut bacteria, and strain-level analyses. We supplemented the data set with 32 individuals with IgAD and examined the influence of IgA-autoantibody status on the composition and functionality of the gut microbiota. RESULTS: The gut microbiota of individuals with IgAD exhibited decreased richness and diversity and was enriched for bacterial species encoding pathogen-related functions including multidrug and antimicrobial peptide resistance, virulence factors, and type III and VI secretion systems. These functional changes were largely attributed to Escherichia coli but were independent of E coli strain variations and most prominent in individuals with IgAD with IgA-specific autoreactive antibodies. CONCLUSIONS: The microbiota of individuals with IgAD is enriched for species holding increased proinflammatory potential, thereby potentially decreasing the resistance to gut barrier-perturbing events. This phenotype is especially pronounced in individuals with IgAD with IgA-specific autoreactive antibodies, thus warranting a screening for IgA-specific autoreactive antibodies in IgAD to identify patients with IgAD with increased risk for gastrointestinal implications.

Sensitive and specific serological ELISA for the detection of SARS-CoV-2 infections.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered the worldwide coronavirus disease 2019 (COVID-19) pandemic. Serological assays for the detection of SARS-CoV-2 infections are important to understand the immune response in patients and to obtain epidemiological data about the number of infected people, especially to identify asymptomatic persons not aware of a past infection. METHODS: We recombinantly produced SARS-CoV-2 nucleocapsid (N)-protein in Escherichia coli. We used the purified protein to develop an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV-2 specific antibodies. This ELISA method was optimized and validated with serum samples collected from 113 patients with RT-PCR-confirmed SARS-CoV-2 infections including hospitalized COVID-19 patients and 1500 control sera mostly collected before 2015 with different clinical background. RESULTS: The optimized N-protein-ELISA provided a sensitivity of 89.7% (n = 68) for samples collected from patients with confirmed SARS-CoV-2 infections and mild to severe symptoms more than 14 days after symptom onset or a positive PCR test. The antibody levels remained low for serum samples collected in the first six days (n = 23) and increased in the second week (n = 22) post symptom onset or PCR confirmation. At this early phase, the ELISA provided a sensitivity of 39.1% and 86.4%, respectively, reflecting the time of an IgG immune response against pathogens. The assay specificity was 99.3% (n = 1500; 95% CI 0.995-0.999). Serum samples from persons with confirmed antibody titers against human immunodeficiency viruses 1/2, parvovirus B19, hepatitis A/B virus, cytomegalovirus, Epstein Barr virus, and herpes simplex virus were tested negative. CONCLUSIONS: We conclude that the N-protein-based ELISA developed here is well suited for the sensitive and specific serological detection of SARS-CoV-2 specific IgG antibodies in human serum for symptomatic infections. It may also prove useful to identify previous SARS-CoV-2 infections in vaccinated people, as all currently approved vaccines rely on the SARS-CoV-2 spike (S-) protein.

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.

Study of an extended family with CTLA-4 deficiency suggests a CD28/CTLA-4 independent mechanism responsible for differences in disease manifestations and severity.

The CTLA-4 checkpoint regulates the activation of T cells. Individuals with heterozygous mutations in CTLA-4 have a complex phenotype typically characterized by antibody deficiency alongside variable autoimmunity. Despite severe disease in some individuals, others remain largely unaffected with reasons for this variation unknown. We studied a large family carrying a single point mutation in CTLA-4 leading to an amino acid change R75W and compared both unaffected with affected individuals. We measured a variety of features pertaining to T cell and CTLA-4 biology and observed that at the cellular level there was complete penetrance of CTLA-4 mutations. Accordingly, unaffected individuals were indistinguishable from those with disease in terms of level of CTLA-4 expression, percentage of Treg, upregulation of CTLA-4 upon stimulation and proliferation of CD4 T cells. We conclude that the wide variation in disease phenotype is influenced by immune variation outside of CTLA-4 biology.

Newborn screening using TREC/KREC assay for severe T and B cell lymphopenia in Iran.

T-cell receptor excision circles (TRECs) and κ-deleting recombination excision circles (KRECs) are recently used for detection of T or B cell lymphopenia in neonates based on region-specific cutoff levels. Here, we report cutoffs for TREC and KREC copies useful for newborn screening and/or diagnosis of primary immunodeficiency diseases (PID) in Iran. DNA was extracted from a single 3.2 mm punch of dried blood spots collected from 2160 anonymized newborns referred to two major referral health centers between 2014 and 2016. For refinement of the cutoffs, 51 patients with a definite diagnosis of severe combined immunodeficiency, X-linked agammaglobulinaemia and combined immunodeficiency, including ataxia telangiectasia, human phosphoglucomutase 3 and Janus kinase-3 deficiency, as well as 47 healthy controls were included. Samples from patients with an X-linked hyper-IgM-syndrome, Wiskott-Aldrich syndrome and DNA ligase 4 deficiency were considered as disease controls. Triplex-quantitative real-time PCR was used. Cutoffs were calculated as TRECs < 11 and KRECs < 6 copies with an ACTB > 700 copies with sensitivity of 100% for TREC and 97% for KREC. Among thirty anonymized newborn samples (1.5%) with abnormal results for TREC and/or KREC, only twenty one available cases were retested and shown to be in the normal range except for three samples (0.15%). All of the patients with a definitive diagnosis were correctly identified based on our established TREC/KREC copy numbers. Determining cutoffs for TREC/KREC is essential for correctly identifying children with PID in newborn screening. Early diagnosis of PID patients enables appropriate measures and therapies like stem cell transplantation. This article is protected by copyright. All rights reserved.

Newborn Screening for Severe Primary Immunodeficiency Diseases in Sweden-a 2-Year Pilot TREC and KREC Screening Study.

Newborn screening for severe primary immunodeficiencies (PID), characterized by T and/or B cell lymphopenia, was carried out in a pilot program in the Stockholm County, Sweden, over a 2-year period, encompassing 58,834 children. T cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) were measured simultaneously using a quantitative PCR-based method on DNA extracted from dried blood spots (DBS), with beta-actin serving as a quality control for DNA quantity. Diagnostic cutoff levels enabling identification of newborns with milder and reversible T and/or B cell lymphopenia were also evaluated. Sixty-four children were recalled for follow-up due to low TREC and/or KREC levels, and three patients with immunodeficiency (Artemis-SCID, ATM, and an as yet unclassified T cell lymphopenia/hypogammaglobulinemia) were identified. Of the positive samples, 24 were associated with prematurity. Thirteen children born to mothers treated with immunosuppressive agents during pregnancy (azathioprine (n = 9), mercaptopurine (n = 1), azathioprine and tacrolimus (n = 3)) showed low KREC levels at birth, which spontaneously normalized. Twenty-nine newborns had no apparent cause identified for their abnormal results, but normalized with time. Children with trisomy 21 (n = 43) showed a lower median number of both TREC (104 vs. 174 copies/µL blood) and KREC (45 vs. 100 copies/3.2 mm blood spot), but only one, born prematurely, fell below the cutoff level. Two children diagnosed with DiGeorge syndrome were found to have low TREC levels, but these were still above the cutoff level. This is the first large-scale screening study with a simultaneous detection of both TREC and KREC, allowing identification of newborns with both T and B cell defects.

The Extended Clinical Phenotype of 26 Patients with Chronic Mucocutaneous Candidiasis due to Gain-of-Function Mutations in STAT1.

PURPOSE: Gain-of-function (GOF) mutations in the signal transducer and activator of transcription 1 (STAT1) result in unbalanced STAT signaling and cause immune dysregulation and immunodeficiency. The latter is often characterized by the susceptibility to recurrent Candida infections, resulting in the clinical picture of chronic mucocutaneous candidiasis (CMC). This study aims to assess the frequency of GOF STAT1 mutations in a large international cohort of CMC patients. METHODS: STAT1 was sequenced in genomic DNA from 57 CMC patients and 35 healthy family members. The functional relevance of nine different STAT1 variants was shown by flow cytometric analysis of STAT1 phosphorylation in patients' peripheral blood cells (PBMC) after stimulation with interferon (IFN)-α, IFN-γ or interleukin-27 respectively. Extended clinical data sets were collected and summarized for 26 patients. RESULTS: Heterozygous mutations within STAT1 were identified in 35 of 57 CMC patients (61%). Out of 39 familial cases from 11 families, 26 patients (67%) from 9 families and out of 18 sporadic cases, 9 patients (50%) were shown to have heterozygous mutations within STAT1. Thirteen distinct STAT1 mutations are reported in this paper. Eight of these mutations are known to cause CMC (p.M202V, p.A267V, p.R274W, p.R274Q, p.T385M, p.K388E, p.N397D, and p.F404Y). However, five STAT1 variants (p.F172L, p.Y287D, p.P293S, p.T385K and p.S466R) have not been reported before in CMC patients. CONCLUSION: STAT1 mutations are frequently observed in patients suffering from CMC. Thus, sequence analysis of STAT1 in CMC patients is advised. Measurement of IFN- or IL-induced STAT1 phosphorylation in PBMC provides a fast and reliable diagnostic tool and should be carried out in addition to genetic testing.

Deficiency of innate and acquired immunity caused by an IKBKB mutation.

BACKGROUND: Severe combined immunodeficiency (SCID) comprises a heterogeneous group of heritable deficiencies of humoral and cell-mediated immunity. Many patients with SCID have lymphocyte-activation defects that remain uncharacterized. METHODS: We performed genetic studies in four patients, from four families of Northern Cree ancestry, who had clinical characteristics of SCID, including early onset of severe viral, bacterial, and fungal infections despite normal B-cell and T-cell counts. Genomewide homozygosity mapping was used to identify a candidate region, which was found on chromosome 8; all genes within this interval were sequenced. Immune-cell populations, signal transduction on activation, and effector functions were studied. RESULTS: The patients had hypogammaglobulinemia or agammaglobulinemia, and their peripheral-blood B cells and T cells were almost exclusively of naive phenotype. Regulatory T cells and γδ T cells were absent. All patients carried a homozygous duplication--c.1292dupG in exon 13 of IKBKB, which encodes IκB kinase 2 (IKK2, also known as IKKß)--leading to loss of expression of IKK2, a component of the IKK-nuclear factor κB (NF-κB) pathway. Immune cells from the patients had impaired responses to stimulation through T-cell receptors, B-cell receptors, toll-like receptors, inflammatory cytokine receptors, and mitogens. CONCLUSIONS: A form of human SCID is characterized by normal lymphocyte development despite a loss of IKK2 function. IKK2 deficiency results in an impaired response to activation stimuli in a variety of immune cells, leading to clinically relevant impairment of adaptive and innate immunity. Although Ikk2 deficiency is lethal in mouse embryos, our observations suggest a more restricted, unique role of IKK2-NF-κB signaling in humans. (Funded by the German Federal Ministry of Education and Research and others.).

Prospective neonatal screening for severe T- and B-lymphocyte deficiencies in Seville.

BACKGROUND: Early diagnosis of primary immunodeficiency such as severe combined immunodeficiency (SCID) and X-linked agammaglobulinemia (XLA) improves outcome of affected children. T-cell-receptor-excision circles (TRECs) and kappa-deleting-recombination-excision circles (KRECs) determination from dried blood spots (DBS) identify neonates with severe T- and/or B-lymphopenia. No prospective data exist of the impact of gestational age (GA) and birth weight (BW) on TRECs and KRECs values. METHODS: TRECs and KRECs determination using triplex RT-PCR (TRECS-KRECS-ß-actin-Assay) from prospectively collected DBS between 02/2014 and 02/2015 in three hospitals in Seville, Spain. Cut-off levels were TRECs < 6/punch, KRECs < 4/punch and -ß-actin>700/punch. Internal (SCID, XLA, ataxia telangiectasia) and external controls (NBS quality assurance program, CDC) were included. RESULTS: A total of 5160 DBS were tested. Re-punch was needed in 77 samples (1.5%) due to insufficient ß-actin (<700 copies/punch). Pre-term neonates (GA<37 weeks) and neonates with a BW<2500 g showed significantly lower TRECs and KRECs levels (p < 0.001). Due to repeat positive results five neonates were re-called (<0.1%): Fatal chromosomopathy (n = 1; TRECs 1/KRECs 4); extreme pre-maturity (n = 2; TRECs 0/KRECs 0 and TRECs 1/KRECs 20 copies/punch); neonates born to mothers receiving azathioprine during pregnancy (n = 2; TRECs 92/KRECs 1 and TRECs 154/KRECs 3 copies/punch). All internal and external controls were correctly identified. CONCLUSIONS: TRECS-KRECS-ß-actin-Assay correctly identifies T- and B-cell lymphopenias. Pre-maturity and low BW is associated with lower TREC and KREC levels. Extreme pre-maturity and maternal immune suppressive therapy may be a cause for false positive results of TRECs and KRECs values, respectively. To reduce the rate of insufficient samples, DBS extraction and storage need to be improved.

Novel mutations in TNFRSF7/CD27: Clinical, immunologic, and genetic characterization of human CD27 deficiency.

BACKGROUND: The clinical and immunologic features of CD27 deficiency remain obscure because only a few patients have been identified to date. OBJECTIVE: We sought to identify novel mutations in TNFRSF7/CD27 and to provide an overview of clinical, immunologic, and laboratory phenotypes in patients with CD27 deficiency. METHODS: Review of the medical records and molecular, genetic, and flow cytometric analyses of the patients and family members were performed. Treatment outcomes of previously described patients were followed up. RESULTS: In addition to the previously reported homozygous mutations c.G24A/p.W8X (n = 2) and c.G158A/p.C53Y (n = 8), 4 novel mutations were identified: homozygous missense c.G287A/p.C96Y (n = 4), homozygous missense c.C232T/p.R78W (n = 1), heterozygous nonsense c.C30A/p.C10X (n = 1), and compound heterozygous c.C319T/p.R107C-c.G24A/p.W8X (n = 1). EBV-associated lymphoproliferative disease/hemophagocytic lymphohistiocytosis, Hodgkin lymphoma, uveitis, and recurrent infections were the predominant clinical features. Expression of cell-surface and soluble CD27 was significantly reduced in patients and heterozygous family members. Immunoglobulin substitution therapy was administered in 5 of the newly diagnosed cases. CONCLUSION: CD27 deficiency is potentially fatal and should be excluded in all cases of severe EBV infections to minimize diagnostic delay. Flow cytometric immunophenotyping offers a reliable initial test for CD27 deficiency. Determining the precise role of CD27 in immunity against EBV might provide a framework for new therapeutic concepts.

SYK expression endows human ZAP70-deficient CD8 T cells with residual TCR signaling.

Autosomal recessive human ZAP70 deficiency is a rare cause of combined immunodeficiency (CID) characterized by defective CD4 T cells and profound CD8 T cell lymphopenia. Herein, we report two novel patients that extend the molecular genetics, the clinical and functional phenotypes associated with the ZAP70 deficiency. The patients presented as infant-onset CID with severe infections caused by varicella zoster virus and live vaccines. Retrospective TCR excision circle newborn screening was normal in both patients. One patient carried a novel non-sense mutation (p.A495fsX75); the other a previously described misense mutation (p.A507V). In contrast to CD4 T cells, the majority of the few CD8 T cells showed expression of the ZAP70-related tyrosine kinase SYK that correlated with residual TCR signaling including calcium flux and degranulation. Our findings highlight the differential requirements of ZAP70 and SYK during thymic development, peripheral homeostasis as well as effector functions of CD4 and CD8 T cells.

Diagnosis of immunodeficiency caused by a purine nucleoside phosphorylase defect by using tandem mass spectrometry on dried blood spots.

BACKGROUND: Purine nucleoside phosphorylase (PNP) deficiency is a rare form of autosomal recessive combined primary immunodeficiency caused by a enzyme defect leading to the accumulation of inosine, 2'-deoxy-inosine (dIno), guanosine, and 2'-deoxy-guanosine (dGuo) in all cells, especially lymphocytes. Treatments are available and curative for PNP deficiency, but their efficacy depends on the early approach. PNP-combined immunodeficiency complies with the criteria for inclusion in a newborn screening program. OBJECTIVE: This study evaluate whether mass spectrometry can identify metabolite abnormalities in dried blood spots (DBSs) from affected patients, with the final goal of individuating the disease at birth during routine newborn screening. METHODS: DBS samples from 9 patients with genetically confirmed PNP-combined immunodeficiency, 10,000 DBS samples from healthy newborns, and 240 DBSs from healthy donors of different age ranges were examined. Inosine, dIno, guanosine, and dGuo were tested by using tandem mass spectrometry (TMS). T-cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) levels were evaluated by using quantitative RT-PCR only for the 2 patients (patients 8 and 9) whose neonatal DBSs were available. RESULTS: Mean levels of guanosine, inosine, dGuo, and dIno were 4.4, 133.3, 3.6, and 3.8 µmol/L, respectively, in affected patients. No indeterminate or false-positive results were found. In patient 8 TREC levels were borderline and KREC levels were abnormal; in patient 9 TRECs were undetectable, whereas KREC levels were normal. CONCLUSION: TMS is a valid method for diagnosis of PNP deficiency on DBSs of affected patients at a negligible cost. TMS identifies newborns with PNP deficiency, whereas TREC or KREC measurement alone can fail.

Clinical picture and treatment of 2212 patients with common variable immunodeficiency.

BACKGROUND: Common variable immunodeficiency (CVID) is an antibody deficiency with an equal sex distribution and a high variability in clinical presentation. The main features include respiratory tract infections and their associated complications, enteropathy, autoimmunity, and lymphoproliferative disorders. OBJECTIVE: This study analyzes the clinical presentation, association between clinical features, and differences and effects of immunoglobulin treatment in Europe. METHODS: Data on 2212 patients with CVID from 28 medical centers contributing to the European Society for Immunodeficiencies Database were analyzed retrospectively. RESULTS: Early disease onset (<10 years) was very frequent in our cohort (33.7%), especially in male subjects (39.8%). Male subjects with early-onset CVID were more prone to pneumonia and less prone to other complications suggesting a distinct disease entity. The diagnostic delay of CVID ranges between 4 and 5 years in many countries and is particularly high in subjects with early-onset CVID. Enteropathy, autoimmunity, granulomas, and splenomegaly formed a set of interrelated features, whereas bronchiectasis was not associated with any other clinical feature. Patient survival in this cohort was associated with age at onset and age at diagnosis only. There were different treatment strategies in Europe, with considerable differences in immunoglobulin dosing, ranging from 130 up to 750 mg/kg/mo. Patients with very low trough levels of less than 4 g/L had poor clinical outcomes, whereas higher trough levels were associated with a reduced frequency of serious bacterial infections. CONCLUSION: Patients with CVID are being managed differently throughout Europe, affecting various outcome measures. Clinically, CVID is a truly variable antibody deficiency syndrome.

Newborn screening for severe T and B cell lymphopenia identifies a fraction of patients with Wiskott-Aldrich syndrome.

The lack or marked reduction of recently formed T and B cells provides a basis for neonatal screening for severe combined immunodeficiencies (SCID) and X-linked agammaglobulinemia (XLA). Newborns with other conditions are also identified if a severe T or B cell lymphopenia is present at birth. We retrospectively analyzed Guthrie card samples from 11 children with Wiskott-Aldrich syndrome (WAS), a rare disease that requires early diagnosis and treatment, to determine whether combined T-cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) screening could identify these patients. 4 of 11 patients showed markedly reduced TREC or KREC copy numbers in their DBS as compared to storage-time matched controls and prospectively screened Swedish and German newborns. No correlation was observed between the WAS gene mutations, the clinical severity/course and the result of the screening assay. A diagnosis of WAS should thus be considered in newborns with positive TREC or KREC screening results.

Novel NLRP12 mutations associated with intestinal amyloidosis in a patient diagnosed with common variable immunodeficiency.

Heterozygous mutations in the NLRP12 gene have been found in patients with systemic auto-inflammatory diseases. However, the NLRP12-associated periodic fever syndromes show a wide clinical spectrum, including patients without classical diagnostic symptoms. Here, we report on a 20-year-old female patient diagnosed with common variable immunodeficiency (CVID), who developed intestinal amyloidosis and carried novel compound heterozygous mutations in NLRP12, identified by whole exome and transcriptome sequencing. CVID is a primary immunodeficiency characterized by low serum immunoglobulins, recurrent bacterial infections and development of malignancy, but it also presents with a magnitude of autoimmune features. Because of the unspecific heterogeneous clinical features of the disease, a delay in diagnosis is common. Secondary, inflammatory (AA type) amyloidosis has infrequently been observed in CVID patients. Based on our case observation and a critical review of the literature, we suggest that NLRP12 mutations might account for a small fraction of CVID patients with severe auto-inflammatory complications.

Retrospective analysis of TREC based newborn screening results and clinical phenotypes in infants with the 22q11 deletion syndrome.

PURPOSE: Population-based newborn screening using T-cell receptor excision circles (TREC) identifies infants with severe T-lymphopenia, seen in severe combined immunodeficiencies (SCID), but also infants with the 22q11 deletion syndrome (22q11DS). Methods for analysis of kappa-deleting recombination excision circles (KREC) help identifying infants with B-lymphopenia. We aimed to evaluate the occurrence of abnormal TREC or KREC newborn screening results in 22q11DS patients and assessed the clinical relevance of abnormal screening reports. METHODS: Simultaneous TREC and KREC analysis was performed on stored original Guthrie cards. Patients with abnormal screening reports were compared to patients with normal reports, regarding lymphocyte counts and clinical severity, obtained by retrospective analysis of medical charts. RESULTS: Of 48 included patients, nine (19 %) had abnormal TREC copy numbers. All 22q11DS patients with abnormal TRECs had CD3+ T-lymphopenia at the time of diagnosis, but only one patient had the complete DiGeorge syndrome. Identified 22q11DS patients with abnormal TREC copy numbers showed significantly lower CD8+ T-lymphocytes at time-of-diagnosis and were significantly more prone to viral infections, compared to 22q11DS patients with normal TREC copy numbers. All 22q11DS patients showed KREC copies within the normal range. CONCLUSIONS: In this retrospective study a high proportion of 22q11DS patients were identified by TREC-based newborn screening. Although only one of them had the complete DiGeorge syndrome with no T-lymphocytes, all of them had T-lymphopenia and most of them had recurrent viral infections, as well as other medical problems, warranting early recognition of the syndrome.