Severe Combined Immunodeficiency Research Articles

Delayed-onset adenosine deaminase deficiency with a novel synonymous mutation and a case series from China.

Adenosine deaminase (ADA) is a key enzyme in the purine salvage pathway. Genetic defects of the ADA gene can cause a subtype of severe combined immunodeficiency. To date, few Chinese cases have been reported. We retrospectively reviewed the medical records of patients diagnosed with ADA deficiency in Beijing Children's Hospital and summarized the previously published ADA deficiency cases from China in the literature. Nine patients were identified with two novel mutations (W272X and Q202 =). Early-onset infection, thymic abnormalities and failure to thrive were the most common manifestations of Chinese ADA-deficient patients. The ADA genotype has a major effect on the clinical phenotype. Notably, a novel synonymous mutation (c.606G>A, p.Q202=) was identified in a delayed-onset patient, which affected pre-mRNA splicing leading to a frameshift and premature truncation of the protein. Furthermore, the patient showed γδT cells expansion with an increased effect or phenotype, which may be associated with the delayed onset of disease. In addition, we reported cerebral aneurysm and intracranial artery stenosis for the first time in ADA deficiency. Five patients died with a median age of four months, while two patients received stem cell transplantation and are alive. This study described the first case series of Chinese ADA-deficient patients. Early-onset infection, thymic abnormalities and failure to thrive were the most common manifestations in our patients. We identified a synonymous mutation that affected pre-mRNA splicing in the ADA gene, which had never been reported in ADA deficiency. Furthermore, we reported cerebral aneurysm in a delayed-onset patient for the first time. Further study is warranted to investigate the underlying mechanisms.

Quality of Life Evaluation in Saudi Arabian Pediatric Patients with Primary Immunodeficiency Diseases Receiving 20% Subcutaneous IgG Infusions at Home.

Subcutaneous immunoglobulin (SCIG) home infusion is widely used as an alternative to intravenous immunoglobulin (IVIG). This study aimed to determine the quality of life (QoL) of patients with primary immunodeficiency (PID) after switching to home-based SCIG. In this prospective open-label single-center study, QoL was determined using the validated Arabic version of the Child Health Questionnaire at baseline and 3 and 6 months after switching from IVIG to SCIG. Twenty-four patients were recruited from July 2018 to August 2021, including 14 females and 10 males. The median age of the patients was 5 years (range, 0-14 years). The patients' diagnoses included severe combined immunodeficiency, combined immunodeficiency, agammaglobulinemia, Omenn syndrome, immunodysregulation, hyper-IgE syndrome, common variable immunodeficiency, and bare lymphocyte syndrome. The median duration on IVIG before inclusion was 40 months (range, 5-125 months). The QoL score showed a significant improvement in the patients' global health at 3 and 6 months compared with those at baseline and a significant improvement in the patients' general health at 3 and 6 months compared with that at baseline. The mean baseline serum IgG trough level was 8.8 ± 2.1 g/L. The mean serum IgG level was significantly higher on SCIG at both 3 and 6 months (11.7 ± 2.3 and 11.7 ± 2.5 g/L, respectively). This is the first study involving an Arab population to show improvement in the QoL of patients with PID after switching from hospital-based IVIG to home-based 20% SCIG.

Understanding of Severe Combined Immunodeficiency and Carrier Status in the U.S.-Based Irish Traveller Population

IntroductionThe Irish Travellers are an ethnically distinct minority community originating from Ireland, with substantial immigration to the U. S. resulting in a current subpopulation estimated around 20,000. Given the history of cultural endogamy in this community, there are known increased rates of severe combined immunodeficiency (SCID), with two recombinase activating gene 1 pathogenic variants (RAG1; p.Arg897*and p.Ser259Alafs*5) and one novel non-homologous end-joining 1 gene variant (NHEJ1; p.Gln224Argfs*27) detected in the US community. However, knowledge of rare genetic disorders, including how they are passed and the value of carrier testing, is limited among some members of this population. MethodsA multi-institutional collaboration developed an outreach project for SCID patients and their relatives in the U.S.-based Irish Traveller community. SCID patients were identified through newborn screening and pedigree interviews. Genetic testing for SCID variants was offered to kindred members, prioritizing members of child-bearing age. An online educational program was designed to identify knowledge gaps and disseminate information on SCID and implications of carrier status. Results and DiscussionBaseline knowledge and attitude assessments prior to educational intervention were obtained from 28 kindred members, all of whom were female and the majority (n = 23) were of child bearing age. Similar to their counterparts in Ireland, the majority (n = 21) had less than a high school diploma. 12 respondents identified three or more family members diagnosed with SCID. The majority of respondents (n ≥17) strongly agreed on the importance of knowing what diseases run in their family. However, only a minority (n ≤8) were able to correctly identify how autosomal dominant, autosomal recessive, and X-linked diseases were passed down. Open ended discussions with key members of this kindred also helped identify religious and cultural barriers which contributed to hesitancy around genetic testing in this underserved community. Early diagnosis of affected members, enabled by educational sessions and carrier screening within this high-risk population, is vital to increase preparedness for the birth of a child with SCID. This is especially important in the case of SCID caused by NHEJ1, as the radiosensitivity caused by this mutation necessitates treatment and conditioning protocols which differ from that of SCID caused by RAG1 mutations.

Newborn screening has improved the survival of infants with severe combined immunodeficiency (SCID) – a Primary Immune Deficiency Treatment Consortium (PIDTC) study

BackgroundProspective/retrospective protocols of the PIDTC have collected detailed data from children undergoing allogeneic hematopoietic cell transplantation (HCT) for SCID at 33 North American centers since 1982. We previously reported that from 1982 to 2009, overall survival (OS) after HCT was unchanged. Newborn screening (NBS) for SCID, initiated in 2010, was hypothesized to improve SCID OS following HCT. MethodsChildren born and receiving HCT during 4 time intervals (1982–89; 1990–99; 2000–09; and 2010–18) were analyzed for categorial variables (chi-square test), continuous outcomes (Kruskal-Wallis test), and OS (Kaplan-Meier method). Results902 children with typical (n = 747) and atypical (n = 155) SCID were included. Unlike years 1982–2009 when 5-year OS after HCT was stagnant (72–73%), improvement was seen for the first time beginning in 2010, when 5-year OS increased to 87% (p < 0.001). To determine what contributed to this finding, multiple factors were evaluated to identify differences between children transplanted between 2010–18 compared to prior time intervals. Significant differences (all p < 0.001) were found in the trigger for diagnosis being NBS or family history as opposed to clinical illness, age at HCT, active infection at HCT, and transplant characteristics including conditioning intensity, stem cell source, and donor source. On multivariable analysis, which excluded matched sibling donors due to their consistently high rates of OS (≥92%) in all time intervals, active infection (HR 2.41, 95% CI 1.53–3.72; p < 0.001), age ≥3.5 months at HCT (HR 2.21, 95% CI 1.38–3.24; p = 0.001), certain genotypes (ADA, DCLRE1C/LIG4/NHEJ, and other rare genotypes) (HR 2.22–3.67, 95% CI 1.23–7.45; p < 0.001), and Black/African American race (HR 2.33, 95% CI 1.56–3.54; p < 0.001) were associated with lower survival rates. A subgroup multivariable analysis examining the effect of trigger for diagnosis in the era of NBS, adjusting for genotype and race, confirmed that improved OS with NBS was related to earlier age and fewer infections at time of HCT. ConclusionThis study confirmed for the first time the direct benefit of population-based SCID NBS in improving OS after HCT. Our findings support adoption of SCID NBS worldwide, but also point out that persistent racial disparities in survival despite universal availability of screening must be addressed.

Early immune reconstitution in an infant with adenosine deaminase deficient severe combined immunodeficiency started on enzyme replacement therapy

Severe Combined Immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency is an inborn error of immunity resulting in the accumulation of adenosine and deoxyadenosine leading to a T-B-NK-phenotype. Enzyme Replacement Therapy with Elapegademase-IvIr is typically used as a bridge prior to undergoing definitive treatment with HSCT or HSC-GT.We report a full-term female infant born to Caucasian parents. Immediate newborn course was unremarkable. She had two borderline newborn screens (NBS) for SCID. There was no consanguinity or family history of immunodeficiency. Physical exam was normal except for absent tonsils. She had profound lymphopenia (Absolute Lymphocyte Count of 210 cells/mL), and initial lymphocyte enumeration showed low CD3 cells (169/mL), decreased CD19 cells (6/mL) and CD16/56+ cells (26/mL) consistent with a T-B-NK- SCID. There was no maternal engraftment. Immunoglobulin studies showed normal IgG (325) with low IgM and IgA. Mitogen proliferation to phytohemagglutinin (PHA) was significantly low (10,200 CPM).A SCID genetics panel revealed two pathogenic variants in ADA (p.Glu319fs and p.Arg149Trp). Biochemical testing confirmed absent ADA activity and elevated total deoxyadenosine nucleotides (dAXP) of 40.3% in RBCs (normally undetectable), consistent with the diagnosis of ADA-SCID. She was promptly started on ERT twice weekly and developed full immune reconstitution of her T-cells, B-cells and NK-cells after 21 days. At that time, her dAXP remained elevated at 10%. Her T-cell function also normalized (PHA 239,562 CPM).We report an infant with ADA-SCID who had two borderline NBS results and developed full immune reconstitution 3 weeks after starting ERT. This is unusual since thymopoiesis typically requires 60–90 days for T-cell reconstitution. While B and NK immune reconstitution typically occurs early, early T-cell numbers lag behind, especially while dAXP levels remain measurable. Both genetic variants in this patient are severe, but somatic mosaicism with reversion of the Arg149Trp point mutation might account for rapid T-cell recovery; further T-cell analysis is planned to assess this possibility. This child is the first case of SCID diagnosed in North Carolina following a borderline NBS, as opposed to abnormal, reinforcing the utility of highly-sensitive TREC thresholds for identification of at-risk infants. [Display omitted] [Display omitted]

Racial and Ethnic Disparities in Outcomes following Hematopoietic Stem Cell Transplant (HCT) in Patients with Severe Combined Immunodeficiency (SCID)

IntroductionBlack race and Hispanic ethnicity are associated with higher mortality among patients with SCID who undergo HCT. We hypothesized that Black and Hispanic patients have a higher incidence of infection pre-transplant and greater age at treatment, and that newborn screening (NBS) could substantially narrow these disparities. MethodsPatients with SCID who received HCT between 1982–2020 at one of 33 North American institutions in the Primary Immune Deficiency Treatment Consortium (PIDTC) were included. The probability of survival was estimated using the Kaplan-Meier method. Cox proportional hazards regression models were used for multivariable analysis. The effect of NBS on disparities was evaluated by performing stratified analyses based on the trigger for diagnosis. Changes in racial/ethnic disparities were assessed over time. ResultsOf 925 patients included, 52% were non-Hispanic White, 22% were Hispanic, and 10% were Black. Genotypes varied by race and ethnicity as shown in Table 1. Overall survival (OS) among Black patients who received a transplant from donors other than matched siblings was significantly lower compared to non-Hispanic White patients (aHR 2.43, 95%CI 1.62, 3.65) after accounting for age, conditioning, donor, genotype, infection status, and year of HCT. OS among Hispanic patients was also lower (aHR 1.34, 95%CI 0.94, 1.90), but did not reach statistical significance (Figure 1). [Display omitted] Thirty-nine percent of both Black and Hispanic patients had an active infection at HCT compared to 34% of non-Hispanic White patients (p = 0.52). There were no racial/ethnic differences in age at treatment or baseline infection among those diagnosed by NBS, whereas disparities were noted among those diagnosed by family history (White: 57 days, Hispanic: 95 days, Black: 99 days; p = 0.02). When evaluated by treatment era, racial/ethnic disparities in age at treatment and baseline infection resolved in the modern era (after 2010).Table 1Frequency of SCID genotypes by Race and Ethnicity.GenotypeTotal N = 925NH White N = 477Hispanic N = 201Black N = 96Asian/PI N = 43Nat. American N = 42ADA55 (8.5)33 (10.0)9 (6.5)7 (10.6)0 (0.0)2 (6.9)CD3 delta10 (1.6)7 (2.1)0 (0.0)0 (0.0)0 (0.0)0 (0.0)DCLRE1C39 (6.1)11 (3.3)5 (3.6)0 (0.0)3 (8.6)16 (55.2)IL2RG274 (42.5)157 (47.4)41 (29.5)41 (62.1)21 (60.0)4 (13.8)IL7R67 (10.4)26 (7.9)31 (22.3)3 (4.5)1 (2.9)1 (3.4)JAK340 (6.2)19 (5.7)10 (7.2)8 (12.1)2 (5.7)0 (0.0)RAG180 (12.4)41 (12.4)17 (12.2)6 (9.1)3 (8.6)6 (20.7)RAG235 (5.4)10 (3.0)16 (11.5)0 (0.0)2 (5.7)0 (0.0)RMRP (CHH)13 (2.0)8 (2.4)2 (1.4)0 (0.0)1 (2.9)0 (0.0)Other/Unknown2811466230813 ConclusionBlack patients with SCID had a more than two-fold hazard of death compared to non-Hispanic White patients, even after accounting for age and infection status. Hispanic patients also demonstrated higher mortality, but this difference was not statistically significant. Disparities across all races and ethnicities in age at treatment and baseline infection were not noted among those diagnosed in the modern era.

RAC2 mutations and immune deficiency – functional spectrum of an international cohort

Mutations in RAC2 were first identified in neonates carrying RAC2 dominant negative p.D57N, who presented with severe neutrophil defects and T-cell lymphopenia. Subsequently, dominant-activating mutations were reported in patients with combined immunodeficiency/immunodysregulation (CIID) or severe combined immunodeficiency (SCID). We assembled an international cohort of 48 published and unpublished patients with RAC2 mutations to characterize the spectrum of disease.Clinical and immunologic phenotypes of 48 germline mutant RAC2 patients were obtained from referring physicians and literature reports. All patients had significant T lymphopenia with several patients detected by newborn screening, most patients exhibited B and NK lymphopenia as well. Respiratory infections were common, affecting 90% of patients while 56% had significant viral infections and 15% had malignancies. Heterologous RAC2 expression assays including superoxide production, PAK1 binding, and AKT activation were used to characterize RAC2 variant effects on downstream signaling. Protein stability was determined by Western blot. Patient presentation varied by mutation: five patients presented as SCID with reticular dysgenesis, five as neonatal neutrophil deficiency (D57N), and 38 as CIID. Patients with dominant, active RAC2 variants with high protein stability (Q61R, Q61K) presented in the first days of life with SCID and absent lymphocytes. Activating variants with lower stability (C18W, G30R, E62K, N92T, R174) presented as CIID with lymphopenia, detectable by newborn screening but presenting later with recurrent sinopulmonary and viral infections. A third class of mutations with unstable transcript or protein were phenotypic only in homozygosity (W56*, R68W). Consistent with the role of RAC2 in the NADPH oxidase complex, patients with mutations causing reduced superoxide production exhibited bacterial infections. Severity of phenotype and initial clinical presentation were correlated with protein stability such that high stability led to neonatal presentation whereas dominant mutations with decreased stability had later clinical onset.Clinical presentation of RAC2 mutation-bearing patients is determined by a combination of protein activity and stability. Heterologous expression together with RAC2 functional analysis is useful for deciphering the molecular basis of the clinical phenotype related to these immunodeficiencies.

Recommendations from the ClinGen SCID VCEP: Implementation of ACMG/AMP Variant Curation Guidelines for Severe Combined Immunodeficiency Disease

BackgroundThe Clinical Genome Resource (ClinGen) is a National Institutes of Health (NIH)-funded program that aims to improve our understanding of the relationship between genetic variations and human health. ClinGen has formed Variant Curation Expert Panels (VCEPs) that provide evidence-based interpretations of genomic variants associated with specific diseases. The ClinGen Severe Combined Immunodeficiency Disease (SCID) VCEP was created to develop specifications-rules for SCIDassociated genes because the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines from 2015 are not gene-specific. The panel has focused on IL2RG, JAK3, ADA, DCLRE1C, IL7R, RAG1, and RAG2 to aid in precise diagnosis, clinical management, and genetic counseling.Methods and Results: Of the 28 ACMG/AMP criteria, the SCID VCEP adapted 18 rules to the 7 selected genes and determined that 10 rules were not applicable (with slight differences between each gene). The key specifications of the rules included setting thresholds for minor allele frequency, developing a method for counting probands and segregations, reviewing functional assays, and determining phenotypic characteristics specific to each gene. Pilot testing of the rules modifications demonstrated appropriate performance as determined by the VCEP. ConclusionsThe VCEP will curate 5,460 variants using these gene-specific rules and categorize them into five groups: pathogenic, likely pathogenic, uncertain significance, likely benign, and benign. The pathogenicity classifications will be published in ClinVar with 3-star review status. This standardized and evidence-based interpretation of genetic variants associated with SCID is crucial to ensure accurate diagnosis, effective clinical management and informed genetic counseling for affected individuals and their families.

A novel IL2RG transmembrane mutation resulting in severe combined immunodeficiency and maternal T cell engraftment

IntroductionX-linked severe combined immunodeficiency (SCID) is commonly associated with mutations in IL2RG, an integral part of several receptors essential for lymphocyte development. In this case, our patient has a novel IL2RG missense mutation in the transmembrane domain leading to a T-B+NK+ phenotype. Case PresentationA 2-week-old boy born full-term from an uncomplicated, non-consanguineous pregnancy presents for absent T-cell receptor excision circles (TREC) on newborn screen. He had been growing well, no thrush/diarrhea/infections. Patient had an uncle who died at 1-year-old from pneumonia but had no other family history. On exam he was healthy appearing, no dysmorphic features, rash, or hepatosplenomegaly.CBC was notable for absolute lymphocyte count of 530. TBNK flow cytometry at 2-weeks-old revealed < 1% CD3+ cells/CD4+ helper cells/CD8+ cytotoxic T cells, B cells were normal at 410, NK cells were mildly low 148, CD45Ra was < 1%, and CD45Ro was 100% (Table 1). Mitogen stimulation was markedly decreased. Microarray was normal, without 22q11.2 deletion. Primary immunodeficiency panel revealed hemizygous variance at the IL2RG gene c.811G>A (p.Gly 271Arg) that is not seen in the general population.Patient was diagnosed with T-B+NK+ SCID. Mom was CMV+ and stopped breastfeeding. Patient was started on IVIG and prophylactic therapy: fluconazole, acyclovir, and bactrim. He was referred to BMTand enrolled in a gene therapy trial as he has no matched sibling donors. At 2-months-old, repeat TBNK was notable for increased CD3 and CD4 cells, raising the concern for maternal engraftment (Table 1). Subsequent HLA chimerism study of T cells demonstrated HLA from mother(CD3) 91% and HLA from patient(CD3) 9% indicating engraftment of maternal cells. Patient was asymptomatic, no diarrhea or rash.Table 1Labs2-weeks-old2-months-oldTBNKAbsolute CD3+ (%)1 (<1%)49 (3%)Absolute CD3+4+ (%)1 (<1%)47 (3%)Absolute CD3+CD8+(%)1 (<1%)1 (<1%)CD4CD8 Ratio233.67Absolute CD19+ (%)410 (72%)1611 (92%)Absolute CD56+16+ (%)148 (26%)79 (5%)CD45 Ra<1%CD45 Ro100%Mitogen StimulationPHA2988 cpmCon A3774 cpmPWM1654 cpm DiscussionThe IL2RG gene G271A mutation results in a missense mutation that changes the charge of the amino acid glycine(uncharged) to arginine(positively charged) at the transmembrane region of the gamma chain, which likely interferes with the receptor's localization on the cell surface. Interestingly, NK cells are preserved in this patient indicating some preservation of signaling by IL15. This case illustrates a novel mutation in the IL2RG transmembrane domain that results in T-B+NK+ phenotype with maternal engraftment.

A Case of X-Linked Moesin-Associated Immune Deficiency in a Pediatric Patient

BackgroundX-Linked Moesin-Associated Immune Deficiency (X-MAID) is a rare severe combined immunodeficiency (SCID) subtype that can present at any age due to its variability. Depending on severity, patients demonstrate failure to thrive, recurrent bacterial and viral infections, and increased susceptibility to varicella zoster. It has been characterized by marked lymphopenia with hypogammaglobulinemia and impaired T-cell migration and proliferation. Case PresentationThis is a report of a Cuban 7-year-old male with poor weight gain and facial dysmorphia. He had a history of recurrent bacterial gastrointestinal infections and pneumonia beginning at 4 months of age. He additionally had 4–6 upper respiratory tract and ear infections annually. While still living in Cuba, he was admitted for a profound EBV infection in the setting of significant leukopenia. A bone marrow biopsy confirmed no malignancy. After he moved to the United States, his laboratory work-up revealed marked leukopenia with low absolute neutrophil and lymphocyte count with low T and B cells, very low immunoglobulin levels IgG, IgA, and IgM, and poor vaccination responses to streptococcus pneumonia, varicella zoster, and SARS-CoV-2. Genetic testing revealed a missense pathogenic variant c.511C>T (p.Arg171Trp) in the moesin (MSN) gene associated with X-MAID. He was managed with Bactrim and acyclovir prophylaxis, and immunoglobulin replacement therapy, and considered for hematopoietic stem cell transplantation. DiscussionDiagnosis of X-MAID should be considered in patients with recurrent infections and profound lymphopenia. As with SCID, early diagnosis and intervention is of utmost importance to prevent morbidity and mortality. This case demonstrates the importance of genetic testing in identifying this disease as it may prompt an immunologist to consider HSCT if conservative management is suboptimal. In the current literature, HSCT appears promising, but the long-term outcomes have yet to be described.

Diagnostic significance of TREC and KRE Cevaluation in case of primary immunodeficiencies

IntroductionTREC and KREC are products of V(D)J recombination of Tand B-lymphocyte receptors in thymus and bone marrow, respectively. Low levels of TREC/KREC in peripheral blood are connected to T- and/or B-cell lymphopenia Materials and methodsWe have studied DNA of healthy children (n = 98) aged 0.0 (0–15.0) years, patients with primary immunodeficiency (n = 84) (SCID (n = 13), AT (n = 15), NBS (n = 17), X-linked agammaglobulinemia (n = 15), diseases of immune dysregulation (n = 14), Wicksott-Aldrich syndrome (n = 10)) aged 7.0 (0.1–15.0) years by multiplex RQ-PCR.The analysis of diagnostic significance has been performed by tracing of characteristic ROC curves and defining the area under the AUC curve. ResultsThe AUC(TREC) and KREC for the SCID diagnostics has been equal to 1.00 ± 0.0 (p < 0.0001) with a diagnostic sensitivity and specificity of 100%.The AUC(TREC) for the X-linked agammaglobulemia diagnosis has made 0.62 ± 0.07 (p = 0.16) with diagnostic sensitivity and specificity of 14% and 93.6%, respectively. The AUC(KREC) has been 1.00 ± 0.0 (p < 0.0001) with diagnostic sensitivity and specificity of 100%.AUC(TREC) for patients with syndromes of chromosomal instability has been equal to 0.99 ± 0.007 (p < 0.0001) with diagnostic sensitivity of 93.4% and specificity of – 97.4%. The AUC(KREC) has been 0.98 ±0.015 (p < 0.0001)) with diagnostic sensitivity of 93.5% and specificity of 98.7%.In case of diseases of immune dysregulation, AUC(TREC) has been equal to 0.99 ± 0.007 (p < 0.0001) with diagnostic sensitivity of 93.3% and specificity of 96.3%. The AUC(KREC) has been 0.87 ± 0.06 (p < 0.0001) with diagnostic sensitivity of 86.6% and specificity of 94.6%.The AUC(TREC) for Wiskott-Aldrich syndrome diagnostics using analysis has made 0.63 ± 0.1 (p = 0.16), with sensitivity and specificity of 30% and 78.1%, respectively. AUC(KREC) has been 0.61 ± 0.09 (p = 0.23) with sensitivity of 20.0% and specificity of 92.35%. ConclusionEvaluation of TREC/KREC is diagnostically significant during the primary diagnosis of diseases, associated with severe combined immune deficiency, agammaglobulinemia, syndromes of chromosomal instability, immune dysregulation. Evaluation of TREC/KREC has low information content and, thus, has no diagnostic significance for Wiskott-Aldrich syndrome.

Analysis of novel RAC2 variants associated with neutrophil dysfunction, lymphopenia, and primary immunodeficiency

Abstract RAC2 is a member of Rho-family GTPases, abundant in neutrophils serving roles in NADPH oxidase activation and cytoskeleton dynamics. Patients with dominant, activating mutations in RAC2 (E62K, N92T, Q61R) are associated with neutrophil dysfunction, lymphopenia and immunodeficiency. We now characterize additional disease-related RAC2 variants with in-vitro analysis of RAC2 effector functions including NOX2-based superoxide production, regulation of PAK1-PDB binding, activation of AKT, protein stability, subcellular localization, and F-actin formation. We identified both active and inactive variants associated with lymphopenia and immunodeficiency. Patients with dominant, active RAC2 variants with high protein stability (Q61R, Q61K) presented in the first days of life with severe combined immunodeficiency and absent lymphocytes. Activating variants with lower protein stability (I21S, E62K, N92K, N92S) presented as combined variable immunodeficiency with lymphopenia, detectable by newborn screening but clinically presenting later with recurrent sinopulmonary infections, reduced B-cells and hypogammaglobulinemia. A third class of mutations with unstable transcript or protein were phenotypic only in homozygosity (W56*, R68W). Severity of phenotype and initial clinical presentation was correlated with protein stability, with increased stability leading to neonatal presentation and decreased stability of dominant mutations having later clinical onset. Thus, heterologous expression and analysis of RAC2 variants is useful for deciphering the genetic and molecular mechanistic basis for the common underlying clinical phenotype related to lymphopenia and immunodeficiency.

Delayed onset ADA-SCID presenting as secondary HLH in a patient with normal newborn screening

Severe combined immunodeficiency (SCID) is a rare genetic syndrome caused by aberrant immune cell development or function and is fatal without early identification and treatment. The Centers for Disease Control estimates the annual incidence of SCID to be one case per 40,000 to 100,000 live births. Newborn screening using T-cell receptor excision circles (TREC) allows for early detection and timely treatment of SCID. Adenosine deaminase (ADA) is a vital enzyme in the purine salvage pathway, and its deficiency results in disruption of T, B and NK cell development, accounting for 10–20% of SCID cases. The timing of ADA-SCID presentation ranges from birth to delayed onset disease. Importantly, without early recognition and treatment, delayed onset ADA-SCID results in accumulation of toxic metabolites causing immune dysfunction, recurrent infections, and subsequent organ damage. Recently, some groups have questioned whether traditional TREC newborn screening adequately identifies ADA-SCID, especially those cases with delayed onset. We present the case of a 2-year-old male with normal newborn screening but a history of recurrent otitis media and respiratory infections who developed multisystem organ failure and septicemia with Escherichia coli, candida, adenovirus and enterovirus despite broad-spectrum antimicrobials. The patient also had features of a hyperinflammatory syndrome with fever, pancytopenia, hypertriglyceridemia, and hyperferritinemia and was treated for hemophagocytic lymphohistiocytosis (HLH) with dexamethasone and plasma exchange followed by anakinra, etoposide, and emapalumab. The patient was maximally supported with mechanical ventilation, extracorporeal membrane oxygenation, and renal replacement therapy but had progressive clinical deterioration and ultimately expired. Autopsy was notable for thymic aplasia and erythrophagocytosis in the bone marrow and lymph nodes. Genetic testing revealed a heterozygous missense pathogenic variant in exon 7 in the ADA gene as well as novel heterozygous intronic variant of unknown significance in the ADA gene. Postmortem data suggests this patient had ADA-SCID with multi-pathogen infections and secondary HLH. This case potentially identifies a novel pathogenic variant in ADA missed by traditional TREC newborn screening and highlights the need to retain a high clinical suspicion of SCID in patients with a history of recurrent infections despite normal TREC screening.

Characterization of expanded γδ T cells from patient with atypical X-linked severe combined immunodeficiency reveals preserved function and IL2RG-mediated signaling

Abnormally high γδ T cell numbers are common among atypical severe combined immunodeficiency (SCID) patients but detailed immunophenotyping and functional characterization of these expanded γδ T cells are limited. We have previously reported atypical SCID phenotype caused by hypomorphic IL2RG c.172C > T;p.(Pro58Ser) variant causing failure in IL2RG (CD132) plasma membrane targeting leading to impaired IL-2 responses in CD4 and CD8 T cells. Our aim was to investigate the phenotype and function of the index patient's abnormally large peripheral γδ T cell population.We found that in contrast to his αβ T cells, the patient's γδ T cells showed normal IL2RG cell surface expression and normal or enhanced IL2RG-mediated signaling. Vδ2+ population was proportionally increased with a preponderance of memory phenotypes and high overall tendency towards perforin expression. The patient's γδ T cells showed enhanced cytotoxicity towards A549 cancer cells. TCRvγ repertoire was versatile but sequencing of IL2RG revealed a novel c.534C>A; p.(Phe178Leu) somatic missense variant restricted to γδ T cells. Over time this variant became predominant in patient's γδ T cells and was enriched during in vitro expansion of patient's γδ T cells, indicating that the clones with this somatic variant may have growth or survival advantage. In silico analysis with DynaMut2 tool indicated that the overall effect of Phe178Leu substitution for IL2RG was predicted to be stabilizing and IL2RG-Pro58Ser/Phe178Leu to be more stable than IL2RG-Pro58Ser variant alone. Furthermore, IL2RG-Pro58Ser/Phe178Leu showed significantly increased cell surface expression when compared to IL2RG-Pro58Ser variant in stable HEK293 cell lines, suggesting that somatic IL2RG p.(Phe178Leu) variant may at least partially rescue the pathogenic effect of germline IL2RG p.(Pro58Ser) variant.In conclusion, our results indicate that expansion of γδ T cells associated with atypical SCID needs further studying and cannot exclusively be deemed as a homeostatic response to low numbers of conventional T cells. (Tuovinen et al., Journal of Clinical Immunology 2022)

Molecular profile of patients with inborn errors of immunity from Nepal

Background and AimsInborn errors of immunity (IEIs) are increasingly being diagnosed in various regions of the world. There are significant challenges in the diagnosis and treatment of IEIs in resource-limited countries. With the availability of one immunologist and increasing awareness, IEIs are diagnosed with increasing frequency and accuracy in Nepal. IEIs are also being evaluated in children presenting with autoimmune manifestations. We describe the profile of patients diagnosed with IEIs in Nepal during 2020–2022. MethodsRecords of all patients with IEIs who were diagnosed and treated at our tertiary care center in Nepal from August 2020 to April 2022 were analysed. Lead author (DB) has examined and diagnosed all cases. IEIs were diagnosed based on the European Society for Immunodeficiencies (ESID) diagnostic criteria and genetic analysis. ResultsThirty-two patients with IEI (19 boys; 13 girls) and 801 children with autoimmune disorders were diagnosed during the study period. Genetic analysis was done on 18 patients. The diagnostic profile of the patients includes patients with chronic granulomatous disease, X-linked agammaglobulinemia, severe combined immunodeficiency, Job syndrome, selective IgA deficiency, specific antibody deficiency, Wiskott-Aldrich Syndrome, CTLA4 deficiency, IPEX syndrome, IFN-IL12 axis defect, hereditary angioedema, MonoMac syndrome, ARPC1B deficiency, leukocyte adhesion defect, PAPA syndrome, autoimmune lymphoproliferative syndrome, ADA2 deficiency, early-complement deficiency lupus, familial cold auto-inflammatory syndrome, A20 haploinsufficiency, and Blau syndrome. Some patients succumbed before the scope of diagnosis. HSCT is planned for 3 patients. ConclusionsOur is the first report of genetically proven IEIs in Nepal. Logistic constraints coupled with a lack of awareness of IEIs among laity and pediatrician accounted for a missed diagnosis, late diagnosis, and poor outcomes in resource-limited settings. Antimicrobial prophylaxis reduced the incidence of breakthrough infections. The lack of HSCT facilities precludes definitive treatment of many IEIs.

Treatment of Omenn syndrome using multiple immunosuppressive strategies: a case series

Introduction: Omenn syndrome represents a therapeutic challenge as a state of florid immune dysregulation born out of autoreactive oligoclonal T cells due to hypomorphic variants in genes associated with severe combined immunodeficiency (SCID).Aims: To describe the therapeutic strategies used in the care of three patients with Omenn syndrome from 2013 to 2022. All had negative maternal engraftment studies and received immunosuppression to control autoreactive T cells. All patients ultimately received myeloablative conditioning with anti-thymocyte globulin (ATG), busulfan, and fludarabine for transplant. All patients are currently alive and well.Case 1: 2-month-old female with adenosine deaminase (ADA)-deficient SCID on pegylated ADA replacement developed respiratory distress, rash, and diarrhea in the setting of rising CD3+/CD4+/CD45RO+ cell count. Treated with high-dose systemic steroids. Due to continued CD3+ increase, systemic tacrolimus was added. She clinically improved and underwent matched unrelated donor cord blood transplant at 5 months of age.Case 2: A male with RAG1-SCID developed generalized erythroderma and rapidly rising CD3+/CD4+/CD45RO+ cell and absolute eosinophil counts soon after birth. Systemic steroids were given with clinical improvement. With taper of steroid, ATG 1 mg/kg was given. Initially, the absolute lymphocyte count (ALC) improved; however, within 5 days, his rash worsened and ALC rose. This necessitated re-initiation of high-dose steroids and addition of tacrolimus until matched related donor bone marrow transplant (MRD BMT) at 2 months old.Case 3: 9-day-old male with RAG1-SCID developed diffuse maculopapular rash, lymphadenopathy, and elevated memory T cells and eosinophils. Initiated high-dose systemic steroids and cyclosporine A (CSA) with quick clinical improvement. CSA discontinued due to toxicity and rising ALC and eosinophils. Given ATG 3 mg/kg/dose for 4 doses over 2 weeks with pattern of quick drop then rapid rise in ALC. Treated with steroids and tacrolimus until MRD BMT at 2 months old. ConclusionsWe describe consistent disease control with immunosuppression by systemic steroids and tacrolimus prior to transplant and suggest ATG as a potential adjunctive consideration in poorly-controlled disease. A growing body of knowledge and experience in optimal Omenn syndrome treatment strategies is imperative to improving outcomes.

Somatic genetic reversion of large duplication event in IL2RG in siblings

BackgroundDeleterious mutations in the common gamma chain (γC, CD132) are associated with X-linked severe combined immunodeficiency (X-SCID) characterized by a T-B+NK- phenotype. Hypomorphic variants in the IL2RG gene have also been associated with a milder or atypical SCID phenotype. Milder phenotypes can also occur when somatic genetic rescue occurs in the setting of severe IL2RG mutations. Spontaneous somatic variants particularly in hematologic disorders can occur particularly in genes with known selection pressure including IL2RG. ResultsHere, we report the clinical and molecular findings from two brothers with atypical X-SCID phenotype including hypogammaglobulinemia, elevated IgM, T cell lymphopenia and extensive verruca plana. One brother required splenectomy secondary to a benign vascular tumor and had recurrent pancreatitis consistent with primary sclerosing cholangitis. Both brothers developed recurrent suppurative pneumonia resulting in early bronchiectasis in their first decade. Next generation sequencing initially was negative, yet reanalysis found a large duplication event in exon 3 of IL2RG gene with somatic genetic rescue events that occurred twice in two brothers. Functional studies revealed a unique mosaic pattern in each affected sibling when stimulating peripheral blood mononuclear cells with IL-2 or IL-21 resulting in bimodal phospho-STAT5 or phospho-STAT3 respectively. The genetic analysis revealed the maternal allele was heterozygous as expected, although the ratio of these events and clonal analysis revealed cell type skewing. ConclusionOur data demonstrate an atypical SCID phenotype in two brothers secondary to the combination of a large duplication event in the IL-2RG gene partially rescued by somatic genetic rescue. Although previous somatic reversion cases have been reported involving the IL2RG genetic locus, our cases demonstrate a large duplication reversion event with recurrence. Our case highlights the unique selection pressure to amplify rare genetic events at the IL2RG locus.

Hypomorphic splice site IL2RG variants associated with Cryptosporidium liver disease

BackgroundX-linked severe combined immunodeficiency (X-SCID) is caused by mutations in IL2RG encoding for the common gamma chain (γc). This disease is typically fatal within the first year without immune reconstitution. We present two patients with IL2RG variants leading to a phenotype of Cryptosporidium-related sclerosing cholangitis. MethodsMedical history and laboratories were reviewed for two patients with IL2RG variants at c.270G. For one patient, we examined cDNA, γc expression, TCR repertoire, cytokine signaling, in vitro B-cell plasmablast differentiation, and in vitro T-cell differentiation of peripheral blood CD34+ cells. ResultsTwo unrelated male patients (17 and 21 years old) were identified with liver disease, recurrent childhood pneumonia and resultant bronchiectasis. P1 has persistent warts. Elevated transaminases in P1 led to liver imaging and biopsy consistent with sclerosing cholangitis. Stool PCR was positive for Cryptosporidium. P2 was diagnosed at age 6 with cryptogenic cirrhosis. He underwent orthotopic liver transplant at age 19; diarrhea and cholestatic liver disease developed 4 months post-transplant, and cryptosporidium was found in stool and liver. Cryptococcus meningitis also developed on immunosuppression. Both patients were hemizygous for an IL2RG variant resulting in p.W90C (P1:c.270G>T; P2:c.270G>C). P1 had mildly reduced CD4+ lymphocytes, CD4+ and CD8+ central/ effector memory T-lymphocytes, memory B-lymphocytes, and normal NK counts. P1 cDNA revealed a leaky splicing defect with a mix of exons 2–4 skipping along with full-length transcript encoding p.W90C. Cells from heterozygous mother expressed wild-type sequence suggesting skewed X-inactivation. Surface protein expression and distribution of TCR V-Beta repertoire was largely normal. IL-2-mediated STAT5 phosphorylation was impaired in T- and NK-cells. P1’s B-lymphocytes proliferated in response to in vitro IL-21/CD40L co-stimulation but failed to differentiate into CD27+CD38+ plasmablasts (< 1%). In vitro T-cell differentiation showed impaired survival of developing thymocytes, despite capability of maturing intoTCRaB+ CD3+ T cells. ConclusionsWe present two patients hemizygous for IL2RG variants leading to milder clinical phenotype compared to classic X-SCID. Consistent with other diseases of impaired IL-21 signaling, there was significant Cryptosporidium-related liver disease in addition to clinical manifestations of hypomorphic IL-2 signaling defect. Further work is necessary to address restoring IL-21 signaling to improve liver disease.

Use of Monoclonal Antibodies in a Pediatric Patient With Severe Combined Immunodeficiency and Persistent SARS-CoV-2 Infection.

We describe the use of monoclonal antibodies for the treatment of persistent SARS-CoV-2 infection in a pediatric patient with severe combined immunodeficiency who required urgent stem cell transplantation to cure his disease.

Two patients with ZAP-70 deficiency in China present with a different genetic, immunological, and clinical phenotype

Zeta(ζ)-Chain Associated Protein Kinase 70 kDa (ZAP-70) deficiency is a rare autosomal recessive primary immunodeficiency disease. Little is known about this disease. In this study, we report two patients to extend the range of clinical phenotypes and immunophenotypes associated with ZAP-70 mutations. We describe the clinical, genetic, and immunological phenotypes of two patients with ZAP-70 deficiency in China, and the data are also compared with the literature. Case 1 presented with leaky severe combined immunodeficiency with low to the absence of CD8 + T cells, while case 2 suffered from a recurrent respiratory infection and had a past medical history of non-EBV-associated Hodgkin’s lymphoma. Sequencing revealed novel compound heterozygous mutations in ZAP-70 of these patients. Case 2 is the second ZAP-70 patient presenting a normal CD8 + T cell number. These two cases have been treated with hematopoietic stem cell transplantation. Selective CD8 + T cell loss is an essential feature of the immunophenotype of ZAP-70 deficiency patients, but there are exceptions. Hematopoietic stem cell transplantation can provide excellent long-term immune function and resolution of clinical problems.