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To evaluate the relationship between knowledge of genetic diagnosis before HSCT and outcome, we reviewed all HSCTs for primary immune deficiencies (PID) performed at UCSF from 2007 through 2018. SCID, a distinct entity identified since 2010 in California by newborn screening and treated early, was considered separately. The underlying genetic condition was known at the time of HSCT in 85% of cases. Graft failure was less frequent in patients with a genetic diagnosis (19% with a genetic diagnosis versus 47% without, p = 0.020). Furthermore, event-free survival and overall survival (OS) at 5 years were better for those with a genetic diagnosis (78% with versus 44% without, p = 0.006; and 93% versus 60% without, p = 0.0002, respectively). OS at 5 years was superior for known-genotype patients with both SCID (p = 0.010) and non-SCID PID (p = 0.010). There was no difference in OS between HSCT done in 2007-2010 compared to more recently (p = 0.19). These data suggest that outcomes of HSCT for PID with known genotype may reflect specific experience and literature, or that a substantial proportion of patients with PID of undetermined genotype may have had underlying conditions for which HSCT may carry greater risk. The higher rate of graft failure in PID with unknown genotype may be in part explained by insufficient conditioning, which in turn could be dictated by compromised organ function in patients undergoing HSCT late in the course. Widespread availability of PID gene sequencing as standard care can provide genetic diagnoses for most patients with PID prior to HSCT, permitting optimization of transplant approach.

BackgroundThe DNA-repair enzyme Artemis is essential for rearrangement of T- and B-cell receptors. Mutations in DCLRE1C, which encodes Artemis, cause Artemis-deficient severe combined immunodeficiency (ART-SCID), which is poorly responsive to allogeneic hematopoietic-cell transplantation.MethodsWe carried out a phase 1-2 clinical study of the transfusion of autologous CD34+ cells, transfected with a lentiviral vector containing DCLRE1C, in 10 infants with newly diagnosed ART-SCID. We followed them for a median of 31.2 months.ResultsMarrow harvest, busulfan conditioning, and lentiviral-transduced CD34+ cell infusion produced the expected grade 3 or 4 adverse events. All the procedures met prespecified criteria for feasibility at 42 days after infusion. Gene-marked T cells were detected at 6 to 16 weeks after infusion in all the patients. Five of 6 patients who were followed for at least 24 months had T-cell immune reconstitution at a median of 12 months. The diversity of T-cell receptor β chains normalized by 6 to 12 months. Four patients who were followed for at least 24 months had sufficient B-cell numbers, IgM concentration, or IgM isohemagglutinin titers to permit discontinuation of IgG infusions. Three of these 4 patients had normal immunization responses, and the fourth has started immunizations. Vector insertion sites showed no evidence of clonal expansion. One patient who presented with cytomegalovirus infection received a second infusion of gene-corrected cells to achieve T-cell immunity sufficient for viral clearance. Autoimmune hemolytic anemia developed in 4 patients 4 to 11 months after infusion; this condition resolved after reconstitution of T-cell immunity. All 10 patients were healthy at the time of this report.ConclusionsInfusion of lentiviral gene-corrected autologous CD34+ cells, preceded by pharmacologically targeted low-exposure busulfan, in infants with newly diagnosed ART-SCID resulted in genetically corrected and functional T and B cells. (Funded by the California Institute for Regenerative Medicine and the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT03538899.).

We report the updated classification of inborn errors of immunity, compiled by the International Union of Immunological Societies Expert Committee. This report documents the key clinical and laboratory features of 55 novel monogenic gene defects, and 1 phenocopy due to autoantibodies, that have either been discovered since the previous update (published January 2020) or were characterized earlier but have since been confirmed or expanded in subsequent studies. While variants in additional genes associated with immune diseases have been reported in the literature, this update includes only those that the committee assessed that reached the necessary threshold to represent novel inborn errors of immunity. There are now a total of 485 inborn errors of immunity. These advances in discovering the genetic causes of human immune diseases continue to significantly further our understanding of molecular, cellular, and immunological mechanisms of disease pathogenesis, thereby simultaneously enhancing immunological knowledge and improving patient diagnosis and management. This report is designed to serve as a resource for immunologists and geneticists pursuing the molecular diagnosis of individuals with heritable immunological disorders and for the scientific dissection of cellular and molecular mechanisms underlying monogenic and related human immune diseases.

BackgroundDevelopment of a diverse T-cell receptor β (TRB) repertoire is associated with immune recovery following hematopoietic cell transplantation (HCT) for severe combined immunodeficiency (SCID). High-throughput sequencing of the TRB repertoire allows evaluation of clonotype dynamics during immune reconstitution.ObjectivesWe investigated whether longitudinal analysis of the TRB repertoire would accurately describe T-cell receptor diversity and illustrate the quality of T-cell reconstitution following HCT or gene therapy for SCID.MethodsWe used high-throughput sequencing to study composition and diversity of the TRB repertoire in 27 infants with SCID at 3, 6, and 12 months and yearly posttreatment(s). Total RNA from peripheral blood was used as template to amplify TRB rearrangements.ResultsTRB sequence analysis showed poor diversity at 3 months, followed by significant improvement by 6 months after cellular therapies. Kinetics of development of TRB diversity were similar in patients with a range of underlying gene defects. However, in patients with RAG and DCLRE1C defects, HCT with no conditioning or immune suppression only resulted in lower diversity than did HCT with conditioning. HCT from a matched donor correlated with higher diversity than did HCT from a mismatched donor. Naive CD4+ T-cell count at 6 months post-HCT correlated with higher TRB diversity. A Shannon index of diversity of 5.2 or lower 3 months after HCT predicted a need for a second intervention.ConclusionsTRB repertoire after hematopoietic cell therapies for SCID provides a quantitative and qualitative measure of diversity of T-cell reconstitution and permits early identification of patients who may require a second intervention.

RationaleEosinophilic gastrointestinal disorders (EGID), including eosinophilic esophagitis (EoE), are inflammatory disorders of the gastrointestinal mucosa mediated by complex immune mechanisms. Although there have been initial reports of EGID in patients with inborn errors of immunity (IEI), little is known about the presentation of EGID in immunodeficient individuals.MethodsWe queried the U.S. Immunodeficiency Network (USIDNET) for patient records including the terms eosinophilic esophagitis, gastritis, enteritis, or colitis. We analyzed 74 patient records from the database, including diagnoses, demographics, infectious history, laboratory findings, genetic studies, therapeutic interventions, and clinical outcomes.ResultsWe examined 74 patient records. A total of 61 patients had isolated EoE, and 13 had distal gastrointestinal involvement consistent with EGID. The most common IEI were common variable immunodeficiency (43.2%), some form of combined immunodeficiency (21.6%), chronic granulomatous disease (8.1%), hyper-IgE syndrome (6.8%), and autoimmune lymphoproliferative syndrome (6.8%). The median age at presentation with IEI was 0.5 years (IQR 1.725, max 39 years) and 56.76% were male. Approximately 20% of the patients in the cohort received a hematopoietic stem cell transplantation for treatment of IEI, but the timing of the HSCT in relationship to the EGID diagnosis was unknown.ConclusionsHere, we report EGID in a diverse cohort of IEI patients, suggesting that both non-EoE EGID and EoE can be seen as comorbid conditions with a variety of IEI. Our data suggests that EGID may be more common in patients with IEI than would be expected based on estimates of EGID in the general population.

BackgroundMonogenic conditions that disrupt proper development and/or function of the immune system are termed inborn errors of immunity (IEIs), also known as primary immunodeficiencies. Patients with IEIs often suffer from other manifestations in addition to infection, and allergic inflammation is an increasingly recognized feature of these conditions.MethodsWe performed a retrospective analysis of IEIs presenting with allergic inflammation as reported in the USIDNET registry. Our inclusion criteria comprised of patients with a reported monogenic cause for IEI where reported lab eosinophil and/or IgE values were available for the patient prior to them receiving potentially curative therapy. Patients were excluded if we were unable to determine the defective gene underlying their IEI. Patients were classified as having eosinophilia or elevated IgE when their record included at least 1 eosinophil count or IgE value that was greater than the age stratified upper limit of normal. We compared the proportion of patients with eosinophilia or elevated IgE with the proportion of samples in a reference population that fall above the upper limit of normal (2.5%).ResultsThe query submitted to the USIDNET registry identified 1409 patients meeting inclusion criteria with a monogenic cause for their IEI diagnosis, of which 975 had eosinophil counts and 645 had IgE levels obtained prior to transplantation or gene therapy that were available for analysis. Overall, 18.8% (183/975) of the patients evaluated from the USIDNET registry had eosinophilia and 20.9% (135/645) had an elevated IgE. IEIs caused by defects in 32 genes were found to be significantly associated with eosinophilia and/or an elevated IgE level, spanning 7 of the 10 IEI categories according to the International Union of Immunological Societies classification.ConclusionType 2 inflammation manifesting as eosinophilia or elevated IgE is found in a broad range of IEIs in the USIDNET registry. Our findings suggest that allergic immune dysregulation may be more widespread in IEIs than previously reported.

X-linked Severe Combined Immunodeficiency (SCID-X1) due to IL2RG mutations is potentially fatal in infancy where 'emergency' life-saving stem cell transplant may only achieve incomplete immune reconstitution following transplant. Salvage therapy SCID-X1 patients over 2 years old (NCT01306019) is a non-randomized, open-label, phase I/II clinical trial for administration of lentiviral-transduced autologous hematopoietic stem cells following busulfan (6 mg/kg total) conditioning. The primary and secondary objectives assess efficacy in restoring immunity and safety by vector insertion site analysis (VISA). In this ongoing study (19 patients treated), we report VISA in blood lineages from first eight treated patients with longer follow up found a > 60-fold increase in frequency of forward-orientated VIS within intron 3 of the High Mobility Group AT-hook 2 gene. All eight patients demonstrated emergence of dominant HMGA2 VIS clones in progenitor and myeloid lineages, but without disturbance of hematopoiesis. Our molecular analysis demonstrated a cryptic splice site within the chicken β-globin hypersensitivity 4 insulator element in the vector generating truncated mRNA transcripts from many transcriptionally active gene containing forward-oriented intronic vector insert. A two base-pair change at the splice site within the lentiviral vector eliminated splicing activity while retaining vector functional capability. This highlights the importance of functional analysis of lentivectors for cryptic splicing for preclinical safety assessment and a redesign of clinical vectors to improve safety.

Newborn screening for severe combined immunodeficiency (SCID) has not only accelerated diagnosis and improved treatment for affected infants, but also led to identification of novel genes required for human T cell development. A male proband had SCID newborn screening showing very low T cell receptor excision circles (TRECs), a biomarker for thymic output of nascent T cells. He had persistent profound T lymphopenia, but normal numbers of B and natural killer (NK) cells. Despite an allogeneic hematopoietic stem cell transplant from his brother, he failed to develop normal T cells. Targeted resequencing excluded known SCID genes; however, whole exome sequencing (WES) of the proband and parents revealed a maternally inherited X-linked missense mutation in MED14 (MED14V763A), a component of the mediator complex. Morpholino (MO)-mediated loss of MED14 function attenuated T cell development in zebrafish. Moreover, this arrest was rescued by ectopic expression of cDNA encoding the wild type human MED14 ortholog, but not by MED14V763A , suggesting that the variant impaired MED14 function. Modeling of the equivalent mutation in mouse (Med14V769A) did not disrupt T cell development at baseline. However, repopulation of peripheral T cells upon competitive bone marrow transplantation was compromised, consistent with the incomplete T cell reconstitution experienced by the proband upon transplantation with bone marrow from his healthy male sibling, who was found to have the same MED14V763A variant. Suspecting that the variable phenotypic expression between the siblings was influenced by further mutation(s), we sought to identify genetic variants present only in the affected proband. Indeed, WES revealed a mutation in the L1 cell adhesion molecule (L1CAMQ498H); however, introducing that mutation in vivo in mice did not disrupt T cell development. Consequently, immunodeficiency in the proband may depend upon additional, unidentified gene variants.

The pioneer transcription factor (TF) PU.1 controls hematopoietic cell fate by decompacting stem cell heterochromatin and allowing nonpioneer TFs to enter otherwise inaccessible genomic sites. PU.1 deficiency fatally arrests lymphopoiesis and myelopoiesis in mice, but human congenital PU.1 disorders have not previously been described. We studied six unrelated agammaglobulinemic patients, each harboring a heterozygous mutation (four de novo, two unphased) of SPI1, the gene encoding PU.1. Affected patients lacked circulating B cells and possessed few conventional dendritic cells. Introducing disease-similar SPI1 mutations into human hematopoietic stem and progenitor cells impaired early in vitro B cell and myeloid cell differentiation. Patient SPI1 mutations encoded destabilized PU.1 proteins unable to nuclear localize or bind target DNA. In PU.1-haploinsufficient pro-B cell lines, euchromatin was less accessible to nonpioneer TFs critical for B cell development, and gene expression patterns associated with the pro- to pre-B cell transition were undermined. Our findings molecularly describe a novel form of agammaglobulinemia and underscore PU.1's critical, dose-dependent role as a hematopoietic euchromatin gatekeeper.

The most recent updated classification of inborn errors of immunity/primary immunodeficiencies, compiled by the International Union of Immunological Societies Expert Committee, was published in January 2020. Within days of completing this report, it was already out of date, evidenced by the frequent publication of genetic variants proposed to cause novel inborn errors of immunity. As the next formal report from the IUIS Expert Committee will not be published until 2022, we felt it important to provide the community with a brief update of recent contributions to the field of inborn errors of immunity. Herein, we highlight studies that have identified 26 additional monogenic gene defects that reach the threshold to represent novel causes of immune defects.

Newborn screening efforts focusing on the quantification of T cell receptor excision circles (TRECs), as a biomarker for abnormal thymic production of T cells, have allowed for the identification and definitive treatment of severe combined immunodeficiency (SCID) in asymptomatic neonates. With the adoption of TREC quantification in Guthrie cards across the USA and abroad, typical, and atypical SCID constitutes only ~ 10% of cases identified with abnormal TRECs associated with T cell lymphopenia. Several other non-SCID-related conditions may be identified by newborn screening in a term infant. Thus, it is important for physicians to recognize that other factors, such as prematurity, are often associated with low TRECs initially, but often improve with age. This paper focuses on a challenge that immunologists face: the diagnostic evaluation and management of cases in which abnormal TRECs are associated with variants of T cell lymphopenia in the absence of a genetically defined form of typical or atypical SCID. Various syndromes associated with T cell impairment, secondary forms of T cell lymphopenia, and idiopathic T cell lymphopenia are identified using this screening approach. Yet there is no consensus or guidelines to assist in the evaluation and management of these newborns, despite representing 90% of the patients identified, resulting in significant work for the clinical teams until a diagnosis is made. Using a case-based approach, we review pearls relevant to the evaluation of these newborns, as well as the management dilemmas for the families and team related to the resolution of genetic ambiguities.

Public health newborn screening (NBS) programs provide population-scale ascertainment of rare, treatable conditions that require urgent intervention. Tandem mass spectrometry (MS/MS) is currently used to screen newborns for a panel of rare inborn errors of metabolism (IEMs)1-4. The NBSeq project evaluated whole-exome sequencing (WES) as an innovative methodology for NBS. We obtained archived residual dried blood spots and data for nearly all IEM cases from the 4.5 million infants born in California between mid-2005 and 2013 and from some infants who screened positive by MS/MS, but were unaffected upon follow-up testing. WES had an overall sensitivity of 88% and specificity of 98.4%, compared to 99.0% and 99.8%, respectively for MS/MS, although effectiveness varied among individual IEMs. Thus, WES alone was insufficiently sensitive or specific to be a primary screen for most NBS IEMs. However, as a secondary test for infants with abnormal MS/MS screens, WES could reduce false-positive results, facilitate timely case resolution and in some instances even suggest more appropriate or specific diagnosis than that initially obtained. This study represents the largest, to date, sequencing effort of an entire population of IEM-affected cases, allowing unbiased assessment of current capabilities of WES as a tool for population screening.

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is a rare autosomal recessive disorder of β-oxidation caused by pathogenic variants in the ACADS gene. Analyte testing for SCADD in blood and urine, including newborn screening (NBS) using tandem mass spectrometry (MS/MS) on dried blood spots (DBSs), is complicated by the presence of two relatively common ACADS variants (c.625G>A and c.511C>T). Individuals homozygous for these variants or compound heterozygous do not have clinical disease but do have reduced short-chain acyl-CoA dehydrogenase (SCAD) activity, resulting in elevated blood and urine metabolites. As part of a larger study of the potential role of exome sequencing in NBS in California, we reviewed ACADS sequence and MS/MS data from DBSs from a cohort of 74 patients identified to have SCADD. Of this cohort, approximately 60% had one or more of the common variants and did not have the two rare variants, and thus would need no further testing. Retrospective analysis of ethylmalonic acid, glutaric acid, 2-hydroxyglutaric acid, 3-hydroxyglutaric acid, and methylsuccinic acid demonstrated that second-tier testing applied before the release of the newborn screening result could reduce referrals by over 50% and improve the positive predictive value for SCADD to above 75%.

Since 2013, the International Union of Immunological Societies (IUIS) expert committee (EC) on Inborn Errors of Immunity (IEI) has published an updated phenotypic classification of IEI, which accompanies and complements their genotypic classification into ten tables. This phenotypic classification is user-friendly and serves as a resource for clinicians at the bedside. There are now 430 single-gene IEI underlying phenotypes as diverse as infection, malignancy, allergy, autoimmunity, and autoinflammation. We herein report the 2019 phenotypic classification, including the 65 new conditions. The diagnostic algorithms are based on clinical and laboratory phenotypes for each of the ten broad categories of IEI.

Primary Immune Regulatory Disorders (PIRD) are an expanding group of diseases caused by gene defects in several different immune pathways, such as regulatory T cell function. Patients with PIRD develop clinical manifestations associated with diminished and exaggerated immune responses. Management of these patients is complicated; oftentimes immunosuppressive therapies are insufficient, and patients may require hematopoietic cell transplant (HCT) for treatment. Analysis of HCT data in PIRD patients have previously focused on a single gene defect. This study surveyed transplanted patients with a phenotypic clinical picture consistent with PIRD treated in 33 Primary Immune Deficiency Treatment Consortium centers and European centers. Our data showed that PIRD patients often had immunodeficient and autoimmune features affecting multiple organ systems. Transplantation resulted in resolution of disease manifestations in more than half of the patients with an overall 5-years survival of 67%. This study, the first to encompass disorders across the PIRD spectrum, highlights the need for further research in PIRD management.

We report the updated classification of Inborn Errors of Immunity/Primary Immunodeficiencies, compiled by the International Union of Immunological Societies Expert Committee. This report documents the key clinical and laboratory features of 430 inborn errors of immunity, including 64 gene defects that have either been discovered in the past 2 years since the previous update (published January 2018) or were characterized earlier but have since been confirmed or expanded upon in subsequent studies. The application of next-generation sequencing continues to expedite the rapid identification of novel gene defects, rare or common; broaden the immunological and clinical phenotypes of conditions arising from known gene defects and even known variants; and implement gene-specific therapies. These advances are contributing to greater understanding of the molecular, cellular, and immunological mechanisms of disease, thereby enhancing immunological knowledge while improving the management of patients and their families. This report serves as a valuable resource for the molecular diagnosis of individuals with heritable immunological disorders and also for the scientific dissection of cellular and molecular mechanisms underlying inborn errors of immunity and related human diseases.

Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies1-5. Here we report two improvements that increase the efficiency of CRISPR-Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTSs) added at the ends of the homology-directed repair (HDR) template interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template to the nucleus, enhancing HDR efficiency approximately two- to fourfold. Furthermore, stabilizing Cas9 RNPs into nanoparticles with polyglutamic acid further improves editing efficiency by approximately twofold, reduces toxicity, and enables lyophilized storage without loss of activity. Combining the two improvements increases gene targeting efficiency even at reduced HDR template doses, yielding approximately two to six times as many viable edited cells across multiple genomic loci in diverse cell types, such as bulk (CD3+) T cells, CD8+ T cells, CD4+ T cells, regulatory T cells (Tregs), γδ T cells, B cells, natural killer cells, and primary and induced pluripotent stem cell-derived6 hematopoietic stem progenitor cells (HSPCs).

BackgroundIn 6.5 years of newborn screening for severe combined immunodeficiency in California, 3,252,156 infants had DNA from dried blood spots (DBSs) assayed for T-cell receptor excision circles. Infants with T-cell receptor excision circle values of less than a designated cutoff on a single DBS, 2 DBS samples with insufficient PCR amplification, or known genetic risk of immunodeficiency had peripheral blood complete blood counts and lymphocyte subsets assayed in a single flow cytometry laboratory. Cases in which immune defects were ruled out were available for analysis.ObjectiveWe sought to determine reference intervals for lymphocyte subsets in racially/ethnically diverse preterm and term newborns who proved to be unaffected by any T-lymphopenic immune disorder.MethodsEffective gestational age (GA) was defined as GA at birth plus postnatal age at the time of sample collection. After determining exclusion criteria, we analyzed demographic and clinical information, complete and differential white blood cell counts, and lymphocyte subsets for 301 infants, with serial measurements for 33 infants. Lymphocyte subset measurements included total T cells, helper and cytotoxic T-cell subsets, naive and memory phenotype of each T-cell subset, B cells, and natural killer cells.ResultsReference intervals were generated for absolute numbers and lymphocyte subsets from infants with effective GAs of 22 to 52 weeks. Sex and ethnicity were not significant determinants of lymphocyte subset counts in this population. Lymphocyte counts increased postnatally.ConclusionThis study provides a baseline for interpreting comprehensive lymphocyte data in preterm and term infants, aiding clinicians to determine which newborns require further evaluations for immunodeficiency.

BACKGROUND:Although autoimmunity and hyperinflammation secondary to recombination activating gene (RAG) deficiency have been associated with delayed diagnosis and even death, our current understanding is limited primarily to small case series. OBJECTIVE:Understand the frequency, severity, and treatment responsiveness of autoimmunity and hyperinflammation in RAG deficiency. METHODS:In reviewing the literature and our own database, we identified 85 patients with RAG deficiency, reported between 2001 and 2016, and compiled the largest case series to date of 63 patients with prominent autoimmune and/or hyperinflammatory pathology. RESULTS:Diagnosis of RAG deficiency was delayed a median of 5 years from the first clinical signs of immune dysregulation. Most patients (55.6%) presented with more than 1 autoimmune or hyperinflammatory complication, with the most common etiologies being cytopenias (84.1%), granulomas (23.8%), and inflammatory skin disorders (19.0%). Infections, including live viral vaccinations, closely preceded the onset of autoimmunity in 28.6% of cases. Autoimmune cytopenias had early onset (median, 1.9, 2.1, and 2.6 years for autoimmune hemolytic anemia, immune thrombocytopenia, and autoimmune neutropenia, respectively) and were refractory to intravenous immunoglobulin, steroids, and rituximab in most cases (64.7%, 73.7%, and 71.4% for autoimmune hemolytic anemia, immune thrombocytopenia, and autoimmune neutropenia, respectively). Evans syndrome specifically was associated with lack of response to first-line therapy. Treatment-refractory autoimmunity/hyperinflammation prompted hematopoietic stem cell transplantation in 20 patients. CONCLUSIONS:Autoimmunity/hyperinflammation can be a presenting sign of RAG deficiency and should prompt further evaluation. Multilineage cytopenias are often refractory to immunosuppressive treatment and may require hematopoietic cell transplantation for definitive management.

BackgroundAllogeneic hematopoietic stem-cell transplantation for X-linked severe combined immunodeficiency (SCID-X1) often fails to reconstitute immunity associated with T cells, B cells, and natural killer (NK) cells when matched sibling donors are unavailable unless high-dose chemotherapy is given. In previous studies, autologous gene therapy with γ-retroviral vectors failed to reconstitute B-cell and NK-cell immunity and was complicated by vector-related leukemia.MethodsWe performed a dual-center, phase 1-2 safety and efficacy study of a lentiviral vector to transfer IL2RG complementary DNA to bone marrow stem cells after low-exposure, targeted busulfan conditioning in eight infants with newly diagnosed SCID-X1.ResultsEight infants with SCID-X1 were followed for a median of 16.4 months. Bone marrow harvest, busulfan conditioning, and cell infusion had no unexpected side effects. In seven infants, the numbers of CD3+, CD4+, and naive CD4+ T cells and NK cells normalized by 3 to 4 months after infusion and were accompanied by vector marking in T cells, B cells, NK cells, myeloid cells, and bone marrow progenitors. The eighth infant had an insufficient T-cell count initially, but T cells developed in this infant after a boost of gene-corrected cells without busulfan conditioning. Previous infections cleared in all infants, and all continued to grow normally. IgM levels normalized in seven of the eight infants, of whom four discontinued intravenous immune globulin supplementation; three of these four infants had a response to vaccines. Vector insertion-site analysis was performed in seven infants and showed polyclonal patterns without clonal dominance in all seven.ConclusionsLentiviral vector gene therapy combined with low-exposure, targeted busulfan conditioning in infants with newly diagnosed SCID-X1 had low-grade acute toxic effects and resulted in multilineage engraftment of transduced cells, reconstitution of functional T cells and B cells, and normalization of NK-cell counts during a median follow-up of 16 months. (Funded by the American Lebanese Syrian Associated Charities and others; LVXSCID-ND ClinicalTrials.gov number, NCT01512888.).

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.

Newborn screening for severe combined immunodeficiency (SCID) was instituted in California in 2010. In the ensuing 6.5 years, 3 252 156 infants in the state had DNA from dried blood spots assayed for T-cell receptor excision circles (TRECs). Abnormal TREC results were followed-up with liquid blood testing for T-cell abnormalities. We report the performance of the SCID screening program and the outcomes of infants who were identified. Data that were reviewed and analyzed included demographics, nursery summaries, TREC and lymphocyte flow-cytometry values, and available follow-up, including clinical and genetic diagnoses, treatments, and outcomes. Infants with clinically significant T-cell lymphopenia (TCL) were successfully identified at a rate of 1 in 15 300 births. Of these, 50 cases of SCID, or 1 in 65 000 births (95% confidence interval 1 in 51 000-1 in 90 000) were found. Prompt treatment led to 94% survival. Infants with non-SCID TCL were also identified, diagnosed and managed, including 4 with complete DiGeorge syndrome who received thymus transplants. Although no cases of typical SCID are known to have been missed, 2 infants with delayed-onset leaky SCID had normal neonatal TREC screens but came to clinical attention at 7 and 23 months of age. Population-based TREC testing, although unable to detect immune defects in which T cells are present at birth, is effective for identifying SCID and clinically important TCL with high sensitivity and specificity. The experience in California supports the rapid, widespread adoption of SCID newborn screening.

The development of a T cell receptor excision circle (TREC) assay utilizing dried blood spots (DBS) made possible universal newborn screening (NBS) for severe combined immunodeficiency (SCID) as a public health measure. Upon being flagged by an abnormal screening test in a SCID screening program, an infant can receive further diagnostic testing for SCID in the neonatal period, prior to onset of infectious complications, to permit immediate institution of protective measures and definitive, life-saving treatment to establish a functional immune system. SCID screening is now the accepted standard of care in state public health departments across the United States, and it is being adopted in many countries. It has proven effective, with infants having this otherwise inapparent but serious, rare disorder achieving survival and immune reconstitution. In addition to bringing to attention infants with the primary screening target diseases, typical SCID and leaky SCID (due to hypomorphic mutations in known SCID genes), the NBS assay for insufficient TRECs in DBS also reveals infants with non-SCID T lymphopenic conditions. Experience has accumulated regarding the range and limitations of diagnoses of newborns with low TRECs and low T cells. Previously unknown immune defects have been discovered, as well as conditions not formerly recognized to have low T cells in the neonatal period.

Now widely adopted, SCID newborn screening has proven effective for early identification and treatment of SCID. In addition, screening has improved our understanding of SCID and related disorders, which are more diverse than originally believed. Newborn screening for SCID illustrates how adding new disorders to newborn screening panels can be enormously beneficial if evidence-based guidelines are adhered to and if mechanisms are in place to track outcomes and learn along the way. These lessons should guide all additions to newborn screening, including those involving sequencing.

BackgroundPrimary antibody deficiencies (PADs) are the most frequent primary immunodeficiencies in human subjects. The genetic causes of PADs are largely unknown. Sec61 translocon alpha 1 subunit (SEC61A1) is the major subunit of the Sec61 complex, which is the main polypeptide-conducting channel in the endoplasmic reticulum membrane. SEC61A1 is a target gene of spliced X-box binding protein 1 and strongly induced during plasma cell (PC) differentiation.ObjectiveWe identified a novel genetic defect and studied its pathologic mechanism in 11 patients from 2 unrelated families with PADs.MethodsWhole-exome and targeted sequencing were conducted to identify novel genetic mutations. Functional studies were carried out ex vivo in primary cells of patients and in vitro in different cell lines to assess the effect of SEC61A1 mutations on B-cell differentiation and survival.ResultsWe investigated 2 families with patients with hypogammaglobulinemia, severe recurrent respiratory tract infections, and normal peripheral B- and T-cell subpopulations. On in vitro stimulation, B cells showed an intrinsic deficiency to develop into PCs. Genetic analysis and targeted sequencing identified novel heterozygous missense (c.254T>A, p.V85D) and nonsense (c.1325G>T, p.E381*) mutations in SEC61A1, segregating with the disease phenotype. SEC61A1-V85D was deficient in cotranslational protein translocation, and it disturbed the cellular calcium homeostasis in HeLa cells. Moreover, SEC61A1-V85D triggered the terminal unfolded protein response in multiple myeloma cell lines.ConclusionWe describe a monogenic defect leading to a specific PC deficiency in human subjects, expanding our knowledge about the pathogenesis of antibody deficiencies.

Since the 1990s, the International Union of Immunological Societies (IUIS) PID expert committee (EC), now called Inborn Errors of Immunity Committee, has published every other year a classification of the inborn errors of immunity. This complete catalog serves as a reference for immunologists and researchers worldwide. However, it was unadapted for clinicians at the bedside. For those, the IUIS PID EC is now publishing a phenotypical classification since 2013, which proved to be more user-friendly. There are now 320 single-gene inborn errors of immunity underlying phenotypes as diverse as infection, malignancy, allergy, auto-immunity, and auto-inflammation. We herein propose the revised 2017 phenotypic classification, based on the accompanying 2017 IUIS Inborn Errors of Immunity Committee classification.

Newborn screening (NBS) for rare conditions is performed in all 50 states in the USA. We have partnered with the California Department of Public Health Genetic Disease Laboratory to determine whether sufficient DNA can be extracted from archived dried blood spots (DBS) for next-generation sequencing in the hopes that next-generation sequencing can play a role in NBS. We optimized the DNA extraction and sequencing library preparation protocols for residual infant DBS archived over 20 years ago and successfully obtained acceptable whole exome and whole genome sequencing data. This sequencing study using DBS DNA without whole genome amplification prior to sequencing library preparation provides evidence that properly stored residual newborn DBS are a satisfactory source of DNA for genetic studies.

We studied three patients with severe skeletal dysplasia, T cell immunodeficiency, and developmental delay. Whole-exome sequencing revealed homozygous missense mutations affecting exostosin-like 3 (EXTL3), a glycosyltransferase involved in heparan sulfate (HS) biosynthesis. Patient-derived fibroblasts showed abnormal HS composition and altered fibroblast growth factor 2 signaling, which was rescued by overexpression of wild-type EXTL3 cDNA. Interleukin-2-mediated STAT5 phosphorylation in patients' lymphocytes was markedly reduced. Interbreeding of the extl3-mutant zebrafish (box) with Tg(rag2:green fluorescent protein) transgenic zebrafish revealed defective thymopoiesis, which was rescued by injection of wild-type human EXTL3 RNA. Targeted differentiation of patient-derived induced pluripotent stem cells showed a reduced expansion of lymphohematopoietic progenitor cells and defects of thymic epithelial progenitor cell differentiation. These data identify EXTL3 mutations as a novel cause of severe immune deficiency with skeletal dysplasia and developmental delay and underline a crucial role of HS in thymopoiesis and skeletal and brain development.

The rapid development of genomic sequencing technologies has decreased the cost of genetic analysis to the extent that it seems plausible that genome-scale sequencing could have widespread availability in pediatric care. Genomic sequencing provides a powerful diagnostic modality for patients who manifest symptoms of monogenic disease and an opportunity to detect health conditions before their development. However, many technical, clinical, ethical, and societal challenges should be addressed before such technology is widely deployed in pediatric practice. This article provides an overview of the Newborn Sequencing in Genomic Medicine and Public Health Consortium, which is investigating the application of genome-scale sequencing in newborns for both diagnosis and screening.

BackgroundSevere combined immunodeficiency (SCID) is characterized by arrested T-lymphocyte production and by B-lymphocyte dysfunction, which result in life-threatening infections. Early diagnosis of SCID through population-based screening of newborns can aid clinical management and help improve outcomes; it also permits the identification of previously unknown factors that are essential for lymphocyte development in humans.MethodsSCID was detected in a newborn before the onset of infections by means of screening of T-cell-receptor excision circles, a biomarker for thymic output. On confirmation of the condition, the affected infant was treated with allogeneic hematopoietic stem-cell transplantation. Exome sequencing in the patient and parents was followed by functional analysis of a prioritized candidate gene with the use of human hematopoietic stem cells and zebrafish embryos.ResultsThe infant had "leaky" SCID (i.e., a form of SCID in which a minimal degree of immune function is preserved), as well as craniofacial and dermal abnormalities and the absence of a corpus callosum; his immune deficit was fully corrected by hematopoietic stem-cell transplantation. Exome sequencing revealed a heterozygous de novo missense mutation, p.N441K, in BCL11B. The resulting BCL11B protein had dominant negative activity, which abrogated the ability of wild-type BCL11B to bind DNA, thereby arresting development of the T-cell lineage and disrupting hematopoietic stem-cell migration; this revealed a previously unknown function of BCL11B. The patient's abnormalities, when recapitulated in bcl11ba-deficient zebrafish, were reversed by ectopic expression of functionally intact human BCL11B but not mutant human BCL11B.ConclusionsNewborn screening facilitated the identification and treatment of a previously unknown cause of human SCID. Coupling exome sequencing with an evaluation of candidate genes in human hematopoietic stem cells and in zebrafish revealed that a constitutional BCL11B mutation caused human multisystem anomalies with SCID and also revealed a prethymic role for BCL11B in hematopoietic progenitors. (Funded by the National Institutes of Health and others.).

A brother and sister developed a previously undescribed constellation of autoimmune manifestations within their first year of life, with uncontrollable bullous pemphigoid, colitis, and proteinuria. The boy had hemophilia due to a factor VIII autoantibody and nephrotic syndrome. Both children required allogeneic hematopoietic cell transplantation (HCT), which resolved their autoimmunity. The early onset, severity, and distinctive findings suggested a single gene disorder underlying the phenotype. Whole-exome sequencing performed on five family members revealed the affected siblings to be compound heterozygous for two unique missense mutations in the 70-kD T cell receptor ζ-chain associated protein (ZAP-70). Healthy relatives were heterozygous mutation carriers. Although pre-HCT patient T cells were not available, mutation effects were determined using transfected cell lines and peripheral blood from carriers and controls. Mutation R192W in the C-SH2 domain exhibited reduced binding to phosphorylated ζ-chain, whereas mutation R360P in the N lobe of the catalytic domain disrupted an autoinhibitory mechanism, producing a weakly hyperactive ZAP-70 protein. Although human ZAP-70 deficiency can have dysregulated T cells, and autoreactive mouse thymocytes with weak Zap-70 signaling can escape tolerance, our patients' combination of hypomorphic and activating mutations suggested a new disease mechanism and produced previously undescribed human ZAP-70-associated autoimmune disease.

Background and objectiveThe potential application of whole-genome sequencing (WGS) to state-mandated standard newborn screening (NBS) challenges the traditional public health approach to NBS and raises ethical, policy, and clinical practice issues. This article examines the perspectives and values of diverse healthy pregnant women and parents of children diagnosed with a primary immunodeficiency disorder about traditional NBS and expanded NBS with the use of WGS.MethodsWe conducted 4 focus groups (3 in English and 1 in Spanish) with socioeconomically and ethnically diverse pregnant women (n = 26), and a comparison group with parents of children diagnosed with a primary immunodeficiency disorder (n = 5).ResultsPediatric policy-relevant themes that emerged from our analysis of the focus group data are presented within 4 categories: (1) perspectives on traditional NBS, (2) informed consent, (3) return of results, and (4) storage and retrieval of results. Analyses indicate that study participants desired greater inclusion in the NBS process. Despite an optimistic orientation to the potential benefits and limited harms likely to result from genomic applications of NBS, parents voiced concerns about privacy and control over test results. Limited trust in the medical system and the state-run NBS program informed these concerns.ConclusionsExpanded NBS with WGS for pediatricians may require management of more genetic conditions, including mutations that convey risk to both the child and parents for adult-onset disorders, and an informed-consent process to manage the genomic data and storage of blood spots. Attention to how these technologies are understood in diverse populations is needed for effective implementation.

Patients with mutations of the recombination-activating genes (RAG) present with diverse clinical phenotypes, including severe combined immune deficiency (SCID), autoimmunity, and inflammation. However, the incidence and extent of immune dysregulation in RAG-dependent immunodeficiency have not been studied in detail. Here, we have demonstrated that patients with hypomorphic RAG mutations, especially those with delayed-onset combined immune deficiency and granulomatous/autoimmune manifestations (CID-G/AI), produce a broad spectrum of autoantibodies. Neutralizing anti-IFN-α or anti-IFN-ω antibodies were present at detectable levels in patients with CID-G/AI who had a history of severe viral infections. As this autoantibody profile is not observed in a wide range of other primary immunodeficiencies, we hypothesized that recurrent or chronic viral infections may precipitate or aggravate immune dysregulation in RAG-deficient hosts. We repeatedly challenged Rag1S723C/S723C mice, which serve as a model of leaky SCID, with agonists of the virus-recognizing receptors TLR3/MDA5, TLR7/-8, and TLR9 and found that this treatment elicits autoantibody production. Altogether, our data demonstrate that immune dysregulation is an integral aspect of RAG-associated immunodeficiency and indicate that environmental triggers may modulate the phenotypic expression of autoimmune manifestations.

Severe combined immunodeficiency (SCID) is most frequently caused by mutations in the cytokine receptor common gamma chain, CD132, encoded by the X-linked gene, IL2RG. Most patients present in the first year of life with failure to thrive, severe, opportunistic infections and absence of CD3+ T cells. We present a patient with pediatric illness and a diagnosis of combined variable immune deficiency (CVID) who was diagnosed at age 23 with an inherited IL2RG mutation causing loss of signal transduction through CD132. His peripheral blood included CD3/CD4 and CD3/CD8 positive cells as well as low levels of CD19+ B cells containing a reversion to the wildtype IL2RG allele. The reversion, which was not present at birth, may account for his mild phenotype and late diagnosis.

Newborn screening (NBS) for severe combined immunodeficiency (SCID) identifies affected infants before the onset of life-threatening infections, permitting optimal treatment. Navajo Native Americans have a founder mutation in the DNA repair enzyme Artemis, resulting in frequent Artemis SCID (SCID-A). A pilot study at 2 Navajo hospitals assessed the feasibility of SCID NBS in this population. Dried blood spots from 1800 infants were assayed by PCR for T-cell receptor excision circles (TRECs), a biomarker for naïve T cells. Starting in February 2012, TREC testing transitioned to standard care throughout the Navajo Area Indian Health Service, and a total of 7900 infants were screened through July 2014. One infant had low TRECs and was diagnosed with non-SCID T lymphopenia, while 4 had undetectable TRECs due to SCID-A, all of whom were referred for hematopoietic cell transplantation. This report establishes the incidence of SCID-A and demonstrates effectiveness of TREC NBS in the Navajo.

The development of a T-cell receptor excision circle (TREC) assay utilizing dried blood spots in universal newborn screening has allowed the early detection of T-cell lymphopenia in newborns. Diagnosis of severe combined immunodeficiency (SCID) in affected infants in the neonatal period, while asymptomatic, permits early treatment and restoration of a functional immune system. SCID was the first immunodeficiency disease to be added to the Recommended Uniform Screening Panel of Core Conditions in the United States in 2010, and it is now implemented in 26 states in the U.S. This review covers the development of newborn screening for SCID, the biology of the TREC test, its current implementation in the U.S., new findings for SCID in the newborn screening era, and future directions.

A framework for open discourse on the use of CRISPR-Cas9 technology to manipulate the human genome is urgently needed

PurposeA male infant developed generalized rash, intestinal inflammation and severe infections including persistent cytomegalovirus. Family history was negative, T cell receptor excision circles were normal, and engraftment of maternal cells was absent. No defects were found in multiple genes associated with severe combined immunodeficiency. A 9/10 HLA matched unrelated hematopoietic cell transplant (HCT) led to mixed chimerism with clinical resolution. We sought an underlying cause for this patient's immune deficiency and dysregulation.MethodsClinical and laboratory features were reviewed. Whole exome sequencing and analysis of genomic DNA from the patient, parents and 2 unaffected siblings was performed, revealing 2 MALT1 variants. With a host-specific HLA-C antibody, we assessed MALT1 expression and function in the patient's post-HCT autologous and donor lymphocytes. Wild type MALT1 cDNA was added to transformed autologous patient B cells to assess functional correction.ResultsThe patient had compound heterozygous DNA variants affecting exon 10 of MALT1 (isoform a, NM_006785.3), a maternally inherited splice acceptor c.1019-2A > G, and a de novo deletion of c.1059C leading to a frameshift and premature termination. Autologous lymphocytes failed to express MALT1 and lacked NF-κB signaling dependent upon the CARMA1, BCL-10 and MALT1 signalosome. Transduction with wild type MALT1 cDNA corrected the observed defects.ConclusionsOur nonconsanguineous patient with early onset profound combined immunodeficiency and immune dysregulation due to compound heterozygous MALT1 mutations extends the clinical and immunologic phenotype reported in 2 prior families. Clinical cure was achieved with mixed chimerism after nonmyeloablative conditioning and HCT.

PurposeSevere combined immunodeficiency (SCID) encompasses a group of disorders characterized by reduced or absent T-cell number and function and identified by newborn screening utilizing T-cell receptor excision circles (TRECs). This screening has also identified infants with T lymphopenia who lack mutations in typical SCID genes. We report an infant with low TRECs and non-SCID T lymphopenia, who proved upon whole exome sequencing to have Nijmegen breakage syndrome (NBS).MethodsExome sequencing of DNA from the infant and his parents was performed. Genomic analysis revealed deleterious variants in the NBN gene. Confirmatory testing included Sanger sequencing and immunoblotting and radiosensitivity testing of patient lymphocytes.ResultsTwo novel nonsense mutations in NBN were identified in genomic DNA from the family. Immunoblotting showed absence of nibrin protein. A colony survival assay demonstrated radiosensitivity comparable to patients with ataxia telangiectasia.ConclusionsAlthough TREC screening was developed to identify newborns with SCID, it has also identified T lymphopenic disorders that may not otherwise be diagnosed until later in life. Timely identification of an infant with T lymphopenia allowed for prompt pursuit of underlying etiology, making possible a diagnosis of NBS, genetic counseling, and early intervention to minimize complications.

BackgroundThe recombination-activating gene (RAG) 1/2 proteins play a critical role in the development of T and B cells by initiating the VDJ recombination process that leads to generation of a broad T-cell receptor (TCR) and B-cell receptor repertoire. Pathogenic mutations in the RAG1/2 genes result in various forms of primary immunodeficiency, ranging from T(-)B(-) severe combined immune deficiency to delayed-onset disease with granuloma formation, autoimmunity, or both. It is not clear what contributes to such heterogeneity of phenotypes.ObjectiveWe sought to investigate the molecular basis for phenotypic diversity presented in patients with various RAG1 mutations.MethodsWe have developed a flow cytometry-based assay that allows analysis of RAG recombination activity based on green fluorescent protein expression and have assessed the induction of the Ighc locus rearrangements in mouse Rag1(-/-) pro-B cells reconstituted with wild-type or mutant human RAG1 (hRAG1) using deep sequencing technology.ResultsHere we demonstrate correlation between defective recombination activity of hRAG1 mutant proteins and severity of the clinical and immunologic phenotype and provide insights on the molecular mechanisms accounting for such phenotypic diversity.ConclusionsUsing a sensitive assay to measure the RAG1 activity level of 79 mutations in a physiologic setting, we demonstrate correlation between recombination activity of RAG1 mutants and the severity of clinical presentation and show that RAG1 mutants can induce specific abnormalities of the VDJ recombination process.

BackgroundThe approach to the diagnosis of severe combined immunodeficiency disease (SCID) and related disorders varies among institutions and countries.ObjectivesThe Primary Immune Deficiency Treatment Consortium attempted to develop a uniform set of criteria for diagnosing SCID and related disorders and has evaluated the results as part of a retrospective study of SCID in North America.MethodsClinical records from 2000 through 2009 at 27 centers in North America were collected on 332 children treated with hematopoietic stem cell transplantation (HCT), enzyme replacement therapy, or gene therapy for SCID and related disorders. Eligibility for inclusion in the study and classification into disease groups were established by using set criteria and applied by an expert review group.ResultsTwo hundred eighty-five (86%) of the patients were determined to be eligible, and 47 (14%) were not eligible. Of the 285 eligible patients, 84% were classified as having typical SCID; 13% were classified as having leaky SCID, Omenn syndrome, or reticular dysgenesis; and 3% had a history of enzyme replacement or gene therapy. Detection of a genotype predicting an SCID phenotype was accepted for eligibility. Reasons for noneligibility were failure to demonstrate either impaired lymphocyte proliferation or maternal T-cell engraftment. Overall (n = 332) rates of testing were as follows: proliferation to PHA, 77%; maternal engraftment, 35%; and genotype, 79% (mutation identified in 62%).ConclusionLack of complete laboratory evaluation of patients before HCT presents a significant barrier to definitive diagnosis of SCID and related disorders and prevented inclusion of subjects in our observational HCT study. This lesson is critical for patient care, as well as the design of future prospective treatment studies for such children because a well-defined and consistent study population is important for precision in outcomes analysis.

Autoimmune lymphoproliferative syndrome (ALPS) presents in childhood with nonmalignant lymphadenopathy and splenomegaly associated with a characteristic expansion of mature CD4 and CD8 negative or double negative T-cell receptor αβ(+) T lymphocytes. Patients often present with chronic multilineage cytopenias due to autoimmune peripheral destruction and/or splenic sequestration of blood cells and have an increased risk of B-cell lymphoma. Deleterious heterozygous mutations in the FAS gene are the most common cause of this condition, which is termed ALPS-FAS. We report the natural history and pathophysiology of 150 ALPS-FAS patients and 63 healthy mutation-positive relatives evaluated in our institution over the last 2 decades. Our principal findings are that FAS mutations have a clinical penetrance of

BackgroundAdenosine deaminase (ADA) deficiency causes severe cellular and humoral immune defects and dysregulation because of metabolic toxicity. Alterations in B-cell development and function have been poorly studied. Enzyme replacement therapy (ERT) and hematopoietic stem cell (HSC) gene therapy (GT) are therapeutic options for patients lacking a suitable bone marrow (BM) transplant donor.ObjectiveWe sought to study alterations in B-cell development in ADA-deficient patients and investigate the ability of ERT and HSC-GT to restore normal B-cell differentiation and function.MethodsFlow cytometry was used to characterize B-cell development in BM and the periphery. The percentage of gene-corrected B cells was measured by using quantitative PCR. B cells were assessed for their capacity to proliferate and release IgM after stimulation.ResultsDespite the severe peripheral B-cell lymphopenia, patients with ADA-deficient severe combined immunodeficiency showed a partial block in central BM development. Treatment with ERT or HSC-GT reverted most BM alterations, but ERT led to immature B-cell expansion. In the periphery transitional B cells accumulated under ERT, and the defect in maturation persisted long-term. HSC-GT led to a progressive improvement in B-cell numbers and development, along with increased levels of gene correction. The strongest selective advantage for ADA-transduced cells occurred at the transition from immature to naive cells. B-cell proliferative responses and differentiation to immunoglobulin secreting IgM after B-cell receptor and Toll-like receptor triggering were severely impaired after ERT and improved significantly after HSC-GT.ConclusionsADA-deficient patients show specific defects in B-cell development and functions that are differently corrected after ERT and HSC-GT.

We report the updated classification of primary immunodeficiencies (PIDs) compiled by the Expert Committee of the International Union of Immunological Societies. In comparison to the previous version, more than 30 new gene defects are reported in this updated version. In addition, we have added a table of acquired defects that are phenocopies of PIDs. For each disorder, the key clinical and laboratory features are provided. This classification is the most up-to-date catalog of all known PIDs and acts as a current reference of the knowledge of these conditions and is an important aid for the molecular diagnosis of patients with these rare diseases.

[This corrects the article on p. 162 in vol. 5, PMID: 24795713.].

The Primary Immune Deficiency Treatment Consortium (PIDTC) consists of 33 centers in North America. We hypothesized that the analysis of uniform data on patients with severe combined immunodeficiency (SCID) enrolled in a prospective protocol will identify variables that contribute to optimal outcomes following treatment. We report baseline clinical, immunologic, and genetic features of the first 50 patients enrolled, and the initial therapies administered, reflecting current practice in the diagnosis and treatment of both typical (n = 37) and atypical forms (n = 13) of SCID. From August 2010 to May 2012, patients with suspected SCID underwent evaluation and therapy per local center practices. Diagnostic information was reviewed by the PIDTC eligibility review panel, and hematopoietic cell transplantation (HCT) details were obtained from the Center for International Blood and Marrow Transplant Research. Most patients (92 %) had mutations in a known SCID gene. Half of the patients were diagnosed by newborn screening or family history, were younger than those diagnosed by clinical signs (median 15 vs. 181 days; P =

BackgroundAssay of T-cell receptor excision circles (TRECs) in dried blood spots obtained at birth permits population-based newborn screening (NBS) for severe combined immunodeficiency (SCID).ObjectiveWe sought to report the first 2 years of TREC NBS in California.MethodsSince August 2010, California has conducted SCID NBS. A high-throughput TREC quantitative PCR assay with DNA isolated from routine dried blood spots was developed. Samples with initial low TREC numbers had repeat DNA isolation with quantitative PCR for TRECs and a genomic control, and immunophenotyping was performed within the screening program for infants with incomplete or abnormal results. Outcomes were tracked.ResultsOf 993,724 infants screened, 50 (1/19,900 [0.005%]) had significant T-cell lymphopenia. Fifteen (1/66,250) required hematopoietic cell or thymus transplantation or gene therapy; these infants had typical SCID (n = 11), leaky SCID or Omenn syndrome (n = 3), or complete DiGeorge syndrome (n = 1). Survival to date in this group is 93%. Other T-cell lymphopenic infants had variant SCID or combined immunodeficiency (n = 6), genetic syndromes associated with T-cell impairment (n = 12), secondary T-cell lymphopenia (n = 9), or preterm birth (n = 8). All T-cell lymphopenic infants avoided live vaccines and received appropriate interventions to prevent infections. TREC test specificity was excellent: only 0.08% of infants required a second test, and 0.016% required lymphocyte phenotyping by using flow cytometry.ConclusionsTREC NBS in California has achieved early diagnosis of SCID and other conditions with T-cell lymphopenia, facilitating management and optimizing outcomes. Furthermore, NBS has revealed the incidence, causes, and follow-up of T-cell lymphopenia in a large diverse population.

T-cell receptor excision circles (TRECs) are circular DNA molecules formed during rearrangement of the T-cell receptor (TCR) genes during lymphocyte development. Copy number of the junctional portion of the δRec-ψJα TREC, assessed by quantitative PCR (qPCR) using DNA from dried blood spots (DBS), is a biomarker for newly formed T cells and absent or low numbers of TRECs indicate SCID (severe combined immunodeficiency) or T lymphocytopenia. No quantitation standard for TRECs exists. To permit comparison of TREC qPCR results with a reliable method for counting TRECs across different laboratories, we sought to construct a stable cell line containing a normal human chromosomal constitution and a single copy of the TREC junction sequence. A human EBV (Epstein Barr virus)-transformed B-cell line was transduced with a lentivirus encoding mCherry fluorescence, puromycin resistance and the δRec-ψJα TREC sequence. A TREC-EBV cell line, with each cell carrying a single lentiviral insertion was established, expanded and shown to have one TREC copy per diploid genome. Graded numbers of TREC-EBV cells added to aliquots of T lymphocyte depleted blood showed TREC copy number proportional to TREC-EBV cell number. TREC-EBV cells, therefore, constitute a reproducible cellular calibrator for TREC assays, useful for both population-based screening for severe combined immunodeficiency and evaluation of naïve T-cell production in clinical settings.

Absent T lymphocytes were unexpectedly found in homozygotes of a transgenic mouse from an unrelated project. T cell development did not progress beyond double-negative stage 1 thymocytes, resulting in a hypocellular, vestigial thymus. B cells were present, but NK cell number and B cell isotype switching were reduced. Transplantation of wild-type hematopoietic cells corrected the defect, which was traced to a deletion involving five contiguous genes at the transgene insertion site on chromosome 12C3. Complementation using bacterial artificial chromosome transgenesis implicated zinc finger BTB-POZ domain protein 1 (Zbtb1) in the immunodeficiency, confirming its role in T cell development and suggesting involvement in B and NK cell differentiation. Targeted disruption of Zbtb1 recapitulated the T(-)B(+)NK(-) SCID phenotype of the original transgenic animal. Knockouts for Zbtb1 had expanded populations of bone marrow hematopoietic stem cells and also multipotent and early lymphoid lineages, suggesting a differentiation bottleneck for common lymphoid progenitors. Expression of mRNA encoding Zbtb1, a predicted transcription repressor, was greatest in hematopoietic stem cells, thymocytes, and pre-B cells, highlighting its essential role in lymphoid development.

We report the updated classification of primary immunodeficiency diseases, compiled by the ad hoc Expert Committee of the International Union of Immunological Societies. As compared to the previous edition, more than 15 novel disease entities have been added in the updated version. For each disorders, the key clinical and laboratory features are provided. This updated classification is meant to help in the diagnostic approach to patients with these diseases.