Omenn Syndrome in Two Infants with Different Hypomorphic Variants in Janus Kinase 3.

Biallelic null or hypomorphic variants in JAK3 cause SCID and less frequently Omenn syndrome. We investigated homozygous hypomorphic JAK3 mutations in two patients, and expression and function of a novel JAK3R431P variant in Omenn syndrome. Immunophenotyping of PBMC from the patient with the novel JAK3R431P variant was undertaken, by flow cytometry and Phosflow after stimulation with IL-2, IL-7, and IL-15. JAK3 expression was investigated by Western blotting. We report two patients with homozygous hypomorphic JAK3 variants and clinical features of Omenn syndrome. One patient had a previously described JAK3R775H variant, and the second had a novel JAK3R431P variant. One patient with a novel JAK3R431P variant had normal expression of JAK3 in immortalised EBV-LCL cells but reduced phosphorylation of STAT5 after stimulation with IL-2, IL-7, and IL-15 consistent with impaired kinase activity. These results suggest the JAK3R431P variant to be hypomorphic. Both patients are alive and well after allogeneic haematopoietic stem cell transplantation. They have full donor chimerism, restitution of thymopoiesis and development of appropriate antibody responses following vaccination. We expand the phenotype of hypomorphic JAK3 deficiency and demonstrate the importance of functional testing of novel variants in disease-causing genes.

A case of T-cell acute lymphoblastic leukemia in retroviral gene therapy for ADA-SCID.

Hematopoietic stem cell gene therapy (GT) using a γ-retroviral vector (γ-RV) is an effective treatment for Severe Combined Immunodeficiency due to Adenosine Deaminase deficiency. Here, we describe a case of GT-related T-cell acute lymphoblastic leukemia (T-ALL) that developed 4.7 years after treatment. The patient underwent chemotherapy and haploidentical transplantation and is currently in remission. Blast cells contain a single vector insertion activating the LIM-only protein 2 (LMO2) proto-oncogene, confirmed by physical interaction, and low Adenosine Deaminase (ADA) activity resulting from methylation of viral promoter. The insertion is detected years before T-ALL in multiple lineages, suggesting that further hits occurred in a thymic progenitor. Blast cells contain known and novel somatic mutations as well as germline mutations which may have contributed to transformation. Before T-ALL onset, the insertion profile is similar to those of other ADA-deficient patients. The limited incidence of vector-related adverse events in ADA-deficiency compared to other γ-RV GT trials could be explained by differences in transgenes, background disease and patient's specific factors.

Novel Presentation of Major Histocompatibility Complex Class II Deficiency with Hemophagocytic Lymphohistiocytosis.

PURPOSE: Major histocompatibility complex (MHC) class II deficiency is one of the combined immune deficiency disorders caused by defects in the MHC class II regulatory genes leading to abnormal T cells development and function. Therefore, patients mainly present with increased susceptibility to infections, diarrhea, and failure to thrive. In this report, we present one MHC class II deficient patient with a novel presentation with Hemophagocytic Lymphohistiocytosis (HLH). METHODS: Immunophenotyping of lymphocyte subpopulations and HLA-DR expression was assess by flow cytometry. Gene mutational analysis was performed by whole exome and Sanger sequencing. RESULTS: We reported a 7-year-old girl, who was diagnosed at age of 2 years with MHC class II deficiency by genetic testing and flow cytometry. Two years later, she developed disseminated BCGitis which was treated with proper antimicrobial agents. At the age of 7 years, she presented with clinical features fulfilling 6 diagnostic criteria of HLH including evidence of hemophagocytic activity in bone marrow aspiration. Accordingly, the diagnosis of HLH was established and the patient was started on IV Dexamethasone, Anakinra and IVIG. Eventually, patient started to improve and was discharged in good condition. Few months later, the patient was readmitted with severe pneumonia and sepsis leading to death. CONCLUSION: Patients with MHC class II deficiency might present with disseminated BCGitis especially if the patient has severe T cell lymphopenia. Additionally, this immune defect might be added to the list of inborn errors of immunity that can be complicated with HLH.

Long-term and real-world safety and efficacy of retroviral gene therapy for adenosine deaminase deficiency.

Adenosine deaminase (ADA) deficiency leads to severe combined immunodeficiency (SCID). Previous clinical trials showed that autologous CD34+ cell gene therapy (GT) following busulfan reduced-intensity conditioning is a promising therapeutic approach for ADA-SCID, but long-term data are warranted. Here we report an analysis on long-term safety and efficacy data of 43 patients with ADA-SCID who received retroviral ex vivo bone marrow-derived hematopoietic stem cell GT. Twenty-two individuals (median follow-up 15.4 years) were treated in the context of clinical development or named patient program. Nineteen patients were treated post-marketing authorization (median follow-up 3.2 years), and two additional patients received mobilized peripheral blood CD34+ cell GT. At data cutoff, all 43 patients were alive, with a median follow-up of 5.0 years (interquartile range 2.4-15.4) and 2 years intervention-free survival (no need for long-term enzyme replacement therapy or allogeneic hematopoietic stem cell transplantation) of 88% (95% confidence interval 78.7-98.4%). Most adverse events/reactions were related to disease background, busulfan conditioning or immune reconstitution; the safety profile of the real world experience was in line with premarketing cohort. One patient from the named patient program developed a T cell leukemia related to treatment 4.7 years after GT and is currently in remission. Long-term persistence of multilineage gene-corrected cells, metabolic detoxification, immune reconstitution and decreased infection rates were observed. Estimated mixed-effects models showed that higher dose of CD34+ cells infused and younger age at GT affected positively the plateau of CD3+ transduced cells, lymphocytes and CD4+ CD45RA+ naive T cells, whereas the cell dose positively influenced the final plateau of CD15+ transduced cells. These long-term data suggest that the risk-benefit of GT in ADA remains favorable and warrant for continuing long-term safety monitoring. Clinical trial registration: NCT00598481 , NCT03478670 .

From variant of uncertain significance to likely pathogenic in two siblings with atypical RAG2 Deficiency: a case report and review of the literature.

BACKGROUND: Severe combined immunodeficiencies (SCIDs) are hereditary disorders characterized by impaired T and B cell function, resulting in significant immune system dysfunction. Recombination-activating gene (RAG) mutations account for a substantial proportion of SCID cases. Here, we present two sibling cases of SCID caused by a novel RAG2 gene mutation. CASE PRESENTATION: The index case was an 8-year-old boy who had a history of recurring infections. After a comprehensive immunological workup, the initial diagnosis of agammaglobulinemia was revised to combined immunodeficiency (CID). The patient underwent hematopoietic stem cell transplantation (HSCT) but succumbed to cytomegalovirus (CMV) infection. His brother, a 4-month-old boy, presented with CMV chorioretinitis. Leaky SCID was diagnosed based on genetic tests and immunological findings. The patient received appropriate treatment and was considered for HSCT. Both siblings had a homozygous RAG2 gene variant, with the first case classified as a variant of uncertain significance (VUS). The presence of the same mutation in the second brother, and the clinical phenotype, supports considering the mutation as likely pathogenic. CONCLUSIONS: This case report highlights a novel RAG2 gene mutation associated with CID. The classification of a VUS may evolve with accumulating evidence, and additional studies are warranted to establish its pathogenicity. Proper communication between genetic counselors and immunologists, accurate documentation of patient information, increased public awareness, and precise utilization of genetic techniques are essential for optimal patient management.

CRISPR/Cas9-Based Disease Modeling and Functional Correction of Interleukin 7 Receptor Alpha Severe Combined Immunodeficiency in T-Lymphocytes and Hematopoietic Stem Cells.

Interleukin 7 Receptor alpha Severe Combined Immunodeficiency (IL7R-SCID) is a life-threatening disorder caused by homozygous mutations in the IL7RA gene. Defective IL7R expression in humans hampers T cell precursors' proliferation and differentiation during lymphopoiesis resulting in the absence of T cells in newborns, who succumb to severe infections and death early after birth. Previous attempts to tackle IL7R-SCID by viral gene therapy have shown that unregulated IL7R expression predisposes to leukemia, suggesting the application of targeted gene editing to insert a correct copy of the IL7RA gene in its genomic locus and mediate its physiological expression as a more feasible therapeutic approach. To this aim, we have first developed a CRISPR/Cas9-based IL7R-SCID disease modeling system that recapitulates the disease phenotype in primary human T cells and hematopoietic stem and progenitor cells (HSPCs). Then, we have designed a knockin strategy that targets IL7RA exon 1 and introduces through homology-directed repair a corrective, promoterless IL7RA cDNA followed by a reporter cassette through AAV6 transduction. Targeted integration of the corrective cassette in primary T cells restored IL7R expression and rescued functional downstream IL7R signaling. When applied to HSPCs further induced to differentiate into T cells in an Artificial Thymic Organoid system, our gene editing strategy overcame the T cell developmental block observed in IL7R-SCID patients, while promoting full maturation of T cells with physiological and developmentally regulated IL7R expression. Finally, genotoxicity assessment of the CRISPR/Cas9 platform in HSPCs using biased and unbiased technologies confirmed the safety of the strategy, paving the way for a new, efficient, and safe therapeutic option for IL7R-SCID patients.

Severe combined immunodeficiency diagnosis and genetic defects.

Severe combined immunodeficiency (SCID) is a rare and life-threatening genetic disorder that severely impairs the immune system's ability to defend the body against infections. Often referred to as the "bubble boy" disease, SCID gained widespread recognition due to the case of David Vetter, a young boy who lived in a sterile plastic bubble to protect him from germs. SCID is typically present at birth, and it results from genetic mutations that affect the development and function of immune cells, particularly T cells and B cells. These immune cells are essential for identifying and fighting off infections caused by viruses, bacteria, and fungi. In SCID patients, the immune system is virtually non-existent, leaving them highly susceptible to recurrent, severe infections. There are several forms of SCID, with varying degrees of severity, but all share common features. Newborns with SCID often exhibit symptoms such as chronic diarrhea, thrush, skin rashes, and persistent infections that do not respond to standard treatments. Without prompt diagnosis and intervention, SCID can lead to life-threatening complications and a high risk of mortality. There are over 20 possible affected genes. Treatment options for SCID primarily involve immune reconstitution, with the most well-known approach being hematopoietic stem cell transplantation (HSCT). Alternatively, gene therapy is also available for some forms of SCID. Once treated successfully, SCID patients can lead relatively normal lives, but they may still require vigilant infection control measures and lifelong medical follow-up to manage potential complications. In conclusion, severe combined immunodeficiency is a rare but life-threatening genetic disorder that severely compromises the immune system's function, rendering affected individuals highly vulnerable to infections. Early diagnosis and appropriate treatment are fundamental. With this respect, newborn screening is progressively and dramatically improving the prognosis of SCID.

What a Clinician Needs to Know About Genome Editing: Status and Opportunities for Inborn Errors of Immunity.

During the past 20 years, gene editing has emerged as a novel form of gene therapy. Since the publication of the first potentially therapeutic gene editing platform for genetic disorders, increasingly sophisticated editing technologies have been developed. As with viral vector-mediated gene addition, inborn errors of immunity are excellent candidate diseases for a corrective autologous hematopoietic stem cell gene editing strategy. Research on gene editing for inborn errors of immunity is still entirely preclinical, with no trials yet underway. However, with editing techniques maturing, scientists are investigating this novel form of gene therapy in context of an increasing number of inborn errors of immunity. Here, we present an overview of these studies and the recent progress moving these technologies closer to clinical benefit.

Heterogeneity in RAG1 and RAG2 deficiency: 35 cases from a single-centre.

Recombination activating genes (RAG)1 and RAG2 deficiency leads to combined T/B-cell deficiency with varying clinical presentations. This study aimed to define the clinical/laboratory spectrum of RAG1 and RAG2 deficiency. We retrospectively reviewed the clinical/laboratory data of 35 patients, grouped them as severe combined immunodeficiency (SCID), Omenn syndrome (OS), and delayed-onset combined immunodeficiency (CID) and reported nine novel mutations. The male/female ratio was 23/12. Median age of clinical manifestations was 1 months (mo) (0.5-2), 2 mo (1.25-5), and 14 mo (3.63-27), age at diagnosis was 4 mo (3-6), 4.5 mo (2.5-9.75), and 27 mo (14.5-70) in SCID (n = 25; 71.4%), OS (n = 5; 14.3%), and CID (n = 5; 14.3%) patients, respectively. Common clinical manifestations were recurrent sinopulmonary infections 82.9%, oral moniliasis 62.9%, diarrhea 51.4%, and eczema/dermatitis 42.9%. Autoimmune features were present in 31.4% of the patients; 80% were in CID patients. Lymphopenia was present in 92% of SCID, 80% of OS, and 80% of CID patients. All SCID and CID patients had low T (CD3, CD4, and CD8), low B, and increased NK cell numbers. Twenty-eight patients underwent hematopoietic stem cell transplantation (HSCT), whereas seven patients died before HSCT. Median age at HSCT was 7 mo (4-13.5). Survival differed in groups; maximum in SCID patients who had an HLA-matched family donor, minimum in OS. Totally 19 (54.3%) patients survived. Early molecular genetic studies will give both individualized therapy options, and a survival advantage because of timely diagnosis and treatment. Further improvement in therapeutic outcomes will be possible if clinicians gain time for HSCT.

Adenosine deaminase 2 deficiency in a Chinese patient: Report of one novel mutation and literature review.

OBJECTIVE: Through a case of deficiency of adenosine deaminase 2 (DADA2) to improve domestic clinicians' understanding of the disease, and to review the literature, promote dermatologists for clinical secondary primary lesion diagnosis. METHOD: Analysis of a case diagnosed with DADA2 deficiency of clinical manifestations, laboratory, imaging examination and treatment methods, and discussion through literature analysis. RESULTS: The child with recurrent fever, limbs nodular erythema, gradually in the limbs. CT of lower limb skin showed mild edema of the spinous layer, intact basal layer, dilated vascular congestion in the superficial dermis, visible RBC extravasation, and changes of telangiectasia ring purpura were considered. Cranial magnetic resonance imaging (MRI) showed a left choroidal cleft cyst. Genetic test was the CECR1 mutation. The treatment with adalimumab was effective. CONCLUSION: In this case, DADA2 is the seventh case in China, and the CECR1 mutation site (c.254A> T p.N85I,c.851G>T p. G284V) was a compound heterozygous mutation. Mastering the clinical characteristics is helpful for clinicians to diagnose this disease.

Gene therapy for adenosine deaminase severe combined immune deficiency-An unexpected journey of four decades.

Severe combined immune deficiency due to adenosine deaminase deficiency (ADA SCID) is an inborn error of immunity with pan-lymphopenia, due to accumulated cytotoxic adenine metabolites. ADA SCID has been treated using gene therapy with a normal human ADA gene added to autologous hematopoietic stem cells (HSC) for over 30 years. Iterative improvements in vector design, HSC processing methods, and clinical HSC transplant procedures have led nearly all ADA SCID gene therapy patients to achieve consistently beneficial immune restoration with stable engraftment of ADA gene-corrected HSC over the duration of observation (as long as 20 years). One gene therapy for ADA SCID is approved by the European Medicines Agency (EMA) in the European Union (EU) and another is being advanced to licensure in the U.S. and U.K. Despite the clear-cut benefits and safety of this curative gene and cell therapy, it remains challenging to achieve sustained availability and access, especially for rare disorders like ADA SCID.

Genetically corrected RAG2-SCID human hematopoietic stem cells restore V(D)J-recombinase and rescue lymphoid deficiency.

ABSTRACT: Recombination-activating genes (RAG1 and RAG2) are critical for lymphoid cell development and function by initiating the variable (V), diversity (D), and joining (J) (V(D)J)-recombination process to generate polyclonal lymphocytes with broad antigen specificity. The clinical manifestations of defective RAG1/2 genes range from immune dysregulation to severe combined immunodeficiencies (SCIDs), causing life-threatening infections and death early in life without hematopoietic cell transplantation (HCT). Despite improvements, haploidentical HCT without myeloablative conditioning carries a high risk of graft failure and incomplete immune reconstitution. The RAG complex is only expressed during the G0-G1 phase of the cell cycle in the early stages of T- and B-cell development, underscoring that a direct gene correction might capture the precise temporal expression of the endogenous gene. Here, we report a feasibility study using the CRISPR/Cas9-based "universal gene-correction" approach for the RAG2 locus in human hematopoietic stem/progenitor cells (HSPCs) from healthy donors and RAG2-SCID patient. V(D)J-recombinase activity was restored after gene correction of RAG2-SCID-derived HSPCs, resulting in the development of T-cell receptor (TCR) αß and γδ CD3+ cells and single-positive CD4+ and CD8+ lymphocytes. TCR repertoire analysis indicated a normal distribution of CDR3 length and preserved usage of the distal TRAV genes. We confirmed the in vivo rescue of B-cell development with normal immunoglobulin M surface expression and a significant decrease in CD56bright natural killer cells. Together, we provide specificity, toxicity, and efficacy data supporting the development of a gene-correction therapy to benefit RAG2-deficient patients.

Optimized expression and purification of a human adenosine deaminase in E. coli and characterization of its Asp8Asn variant.

Homo sapiens adenosine deaminase isoform 1 (HsADA1) hydrolyzes adenosine and 2-deoxyadenosine as a key step in the purine nucleoside salvage pathway. Some HsADA1 mutations have severe deleterious effects, as is the case in a severe combined immunodeficiency resulting from loss of enzyme activity (ADA-SCID). Other mutations that reduce enzyme activity, for instance the Asp8Asn (D8N) variant, do not cause ADA-SCID but are correlated with other consequences to health. To ease further study of HsADA1 and its variants, we optimized an inexpensive, recombinant expression process in an Escherichia coli host through multiplexed parameter testing enabled by a lysate-based microtiter plate assay. We demonstrate the importance of gene codon usage, induction time and temperature, and alcohol supplementation towards improving enzyme yield to a final titer of 5 mg per liter of culture. We further show that use of a double-histidine-tag (his-tag) system greatly improves purity. We then utilize our expression and purification framework to produce the HsADA1 D8N variant, which had previously not been purified to homogeneity. We confirm that the D8N variant is ∼30% less active than the wildtype HsADA1 and show that it better retains its activity in human serum. Additionally, we show that both HsADA1 and the D8N variant have heightened activity in serum, driven in part by a previously undescribed phenomenon involving albumin. Therefore, this work presents a valuable process to produce HsADA1 that allows for insights into it and its variants' behavior. We also confirm the utility of lysate-based activity assays towards finding optimal E. coli expression conditions for enzymes and show how fusing his-tags in tandem can enhance product purity.

Human Autosomal Recessive DNA Polymerase Delta 3 Deficiency Presenting as Omenn Syndrome.

The DNA polymerase δ complex (PolD), comprising catalytic subunit POLD1 and accessory subunits POLD2, POLD3, and POLD4, is essential for DNA synthesis and is central to genome integrity. We identified, by whole exome sequencing, a homozygous missense mutation (c.1118A > C; p.K373T) in POLD3 in a patient with Omenn syndrome. The patient exhibited severely decreased numbers of naïve T cells associated with a restricted T-cell receptor repertoire and a defect in the early stages of TCR recombination. The patient received hematopoietic stem cell transplantation at age 6 months. He manifested progressive neurological regression and ultimately died at age 4 years. We performed molecular and functional analysis of the mutant POLD3 and assessed cell cycle progression as well as replication-associated DNA damage. Patient fibroblasts showed a marked defect in S-phase entry and an enhanced number of double-stranded DNA break-associated foci despite normal expression levels of PolD components. The cell cycle defect was rescued by transduction with WT POLD3. This study validates autosomal recessive POLD3 deficiency as a novel cause of profound T-cell deficiency and Omenn syndrome.

Novel Compound Heterozygous ZAP70 R37G A507T Mutations in Infant with Severe Immunodeficiency.

Zeta-chain associated protein kinase 70 kDa (ZAP70) combined immunodeficiency (CID) is an autosomal recessive severe immunodeficiency that is characterized by abnormal T-cell receptor signaling. Children with the disorder typically present during the first year of life with diarrhea, failure to thrive, and recurrent bacterial, viral, or opportunistic infections. To date, the only potential cure is hematopoietic stem cell transplant (HSCT). The majority of described mutations causing disease occur in the homozygous state, though heterozygotes are reported without a clear understanding as to how the individual mutations interact to cause disease. This case describes an infant with novel ZAP-70 deficiency mutations involving the SH2 and kinase domains cured with allogeneic HSCT utilizing a reduced-intensity conditioning regimen and graft manipulation. We then were able to further elucidate the molecular signaling alterations imparted by these mutations that lead to altered immune function. In order to examine the effect of these novel compound ZAP70 heterozygous mutations on T cells, Jurkat CD4+ T cells were transfected with either wild type, or with individual ZAP70 R37G and A507T mutant constructs. Downstream TCR signaling events and protein localization results link these novel mutations to the expected immunological outcome as seen in the patient's primary cells. This study further characterizes mutations in the ZAP70 gene as combined immunodeficiency and the clinical phenotype.

Partial correction of immunodeficiency by lentiviral vector gene therapy in mouse models carrying Rag1 hypomorphic mutations.

Introduction: Recombination activating genes (RAG) 1 and 2 defects are the most frequent form of severe combined immunodeficiency (SCID). Patients with residual RAG activity have a spectrum of clinical manifestations ranging from Omenn syndrome to delayed-onset combined immunodeficiency, often associated with granulomas and/or autoimmunity (CID-G/AI). Lentiviral vector (LV) gene therapy (GT) has been proposed as an alternative treatment to the standard hematopoietic stem cell transplant and a clinical trial for RAG1 SCID patients recently started. However, GT in patients with hypomorphic RAG mutations poses additional risks, because of the residual endogenous RAG1 expression and the general state of immune dysregulation and associated inflammation. Methods: In this study, we assessed the efficacy of GT in 2 hypomorphic Rag1 murine models (Rag1F971L/F971L and Rag1R972Q/R972Q), exploiting the same LV used in the clinical trial encoding RAG1 under control of the MND promoter. Results and discussion: Starting 6 weeks after transplant, GT-treated mice showed a decrease in proportion of myeloid cells and a concomitant increase of B, T and total white blood cells. However, counts remained lower than in mice transplanted with WT Lin- cells. At euthanasia, we observed a general redistribution of immune subsets in tissues, with the appearance of mature recirculating B cells in the bone marrow. In the thymus, we demonstrated correction of the block at double negative stage, with a modest improvement in the cortical/medullary ratio. Analysis of antigenspecific IgM and IgG serum levels after in vivo challenge showed an amelioration of antibody responses, suggesting that the partial immune correction could confer a clinical benefit. Notably, no overt signs of autoimmunity were detected, with B-cell activating factor decreasing to normal levels and autoantibodies remaining stable after GT. On the other hand, thymic enlargement was frequently observed, although not due to vector integration and insertional mutagenesis. In conclusion, our work shows that GT could partially alleviate the combined immunodeficiency of hypomorphic RAG1 patients and that extensive efficacy and safety studies with alternative models are required before commencing RAG gene therapy in thesehighly complex patients.

Generation of inactivated IL2RG and RAG1 monkeys with severe combined immunodeficiency using base editing.

Severe combined immunodeficiency (SCID) encompasses a range of inherited disorders that lead to a profound deterioration of the immune system. Among the pivotal genes associated with SCID, RAG1 and IL2RG play crucial roles. IL2RG is essential for the development, differentiation, and functioning of T, B, and NK cells, while RAG1 critically contributes to adaptive immunity by facilitating V(D)J recombination during the maturation of lymphocytes. Animal models carrying mutations in these genes exhibit notable deficiencies in their immune systems. Non-human primates (NHPs) are exceptionally well-suited models for biomedical research due to their genetic and physiological similarities to humans. Cytosine base editors (CBEs) serve as powerful tools for precisely and effectively modifying single-base mutations in the genome. Their successful implementation has been demonstrated in human cells, mice, and crop species. This study outlines the creation of an immunodeficient monkey model by deactivating both the IL2RG and RAG1 genes using the CBE4max system. The base-edited monkeys exhibited a severely compromised immune system characterized by lymphopenia, atrophy of lymphoid organs, and a deficiency of mature T cells. Furthermore, these base-edited monkeys were capable of hosting and supporting the growth of human breast cancer cells, leading to tumor formation. In summary, we have successfully developed an immunodeficient monkey model with the ability to foster tumor growth using the CBE4max system. These immunodeficiency monkeys show tremendous potential as valuable tools for advancing biomedical and translational research.

Case report: IKZF1-related early-onset CID is expected to be missed in TREC-based SCID screening but can be identified by determination of KREC levels.

This report illustrates a case that would have been missed in the most common screening algorithms used worldwide in newborn screening (NBS) for severe combined immunodeficiency (SCID). Our patient presented with a clinical picture that suggested a severe inborn error of immunity (IEI). The 6-month-old baby had normal T-cell receptor excision circle (TREC) levels but no measurable level of kappa-deleting recombination excision circles (KRECs) in the NBS sample. A de novo IKZF1-mutation (c.476A>G, p.Asn159Ser) was found. The clinical picture, immunologic workup, and genetic result were consistent with IKZF1-related combined immunodeficiency (CID). Our patient had symptomatic treatment and underwent allogeneic hematopoietic cell transplantation (HCT). IKZF1-related CID is a rare, serious, and early-onset disease; this case provides further insights into the phenotype, including KREC status.