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Primary immune deficiency (PID) disorders are clinically and molecularly heterogeneous diseases. T cell receptor excision circles (TRECs) and κ (kappa)-deleting excision circles (KRECs) are markers of T and B cell development, respectively. They are useful tools to assess T and B cell function and immune reconstitution and have been used for newborn screening for severe combined immunodeficiency disease (SCID) and agammaglobulinemia, respectively. Their profiles in several genetically confirmed PIDs are still lacking. The objective of this study was to determine TREC and KREC genomic profiling among various molecularly confirmed PIDs. We used real-time-quantitative polymerase chain reaction (RT-qPCR)-based triplex analysis of TRECs, KRECs and β-actin (ACTB) in whole blood genomic DNA isolated from 108 patients with molecularly confirmed PIDs. All agammaglobulinemia patients had low KREC counts. All SCIDs and Omenn syndrome patients secondary to mutations in RAG1, RAG2, DCLRE1C and NHEJ1 had low TREC and KREC counts. JAK3-deficient patients had normal KREC and the TREC count was influenced by the type of mutation. Early-onset ADA patients had low TREC and KREC counts. Four patients with zeta-chain-associated protein kinase 70 (ZAP70) had low TREC. All purine nucleoside phosphorylase (PNP) patients had low TREC. Combined immunodeficiency (CID) patients secondary to AK2, PTPRC, CD247, DCLREC1 and STAT1 had normal TREC and KREC counts. Most patients with ataxia-telangiectasia (AT) patients had low TREC and KREC, while most DOCK8-deficient patients had low TRECs only. Two of five patients with Wiskott-Aldrich syndrome (WAS) had low TREC counts as well as one patient each with bare lymphocyte syndrome (BLS) and chronic granulomatous disease. All patients with Griscelli disease, Chediak-Higashi syndrome, hyper-immunoglobulin (Ig)M syndrome and IFNGR2 had normal TREC and KREC counts. These data suggest that, in addition to classical SCID and agammaglobulinemia, TREC/KREC assay may identify ZAP70 patients and secondary target PIDs, including dedicator of cytokinesis 8 (DOCK8) deficiency, AT and some individuals with WAS and BLS., (© 2020 British Society for Immunology.)

Severe combined immunodeficiency disease (SCID) is the most severe form of primary immunodeficiency disorders (PID). T-cell receptor excision circle (TREC) copy number analysis is an efficient tool for population-based newborn screening (NBS) for SCID and other T cell lymphopenias. We sought to assess the incidence of SCID among Saudi newborn population and examine the feasibility of using targeted next generation sequencing PID gene panel (T-NGS PID) on DNA isolated from dried blood spots (DBSs) in routine NBS programs as a mutation screening tool for samples with low TREC count. Punches from 8,718 DBS collected on Guthrie cards were processed anonymously for the TREC assay. DNA was extracted from samples with confirmed low TREC count, then screened for 22q11.2 deletion syndrome by real-time polymerase chain reaction and for mutations in PID-related genes by T-NGS PID panel. Detected mutations were confirmed by Sanger sequencing. Sixteen out of the 8,718 samples were confirmed to have low TREC copy number. Autosomal recessive mutations in AK2, JAK3, and MTHFD1 were confirmed in three samples. Two additional samples were positive for the 22q11.2 deletion syndrome. In this study, we provide evidence for high incidence of SCID among Saudi population (1/2,906 live births) and demonstrate the feasibility of using T-NGS PID panel on DNA extracted from DBSs as a new reliable, rapid, and cost-effective mutation screening method for newborns with low TREC assay, which can be implemented as part of NBS programs for SCID.

Newborn screening (NBS) for severe combined immunodeficiency (SCID) utilizing T-cell receptor excision circles (TRECs) has been implemented in all 50 states as of December 2018 and has been transformative for the clinical care of SCID patients. Though having high sensitivity for SCID, NBS-SCID has low specificity, therefore is able to detect other causes of lymphopenia in newborns including many inborn errors of immunity (IEIs). In a recent study, three of six newborns later diagnosed with Warts, Hypogammaglobulinemia, Infections, and Myelokathexis (WHIM) syndrome were found to have a low TRECs and lymphopenia at birth. This presents an opportunity to increase the detection and diagnosis of WHIM syndrome by NBS-SCID with immunological follow-up along with a combination of flow cytometry for immune cell subsets, absolute neutrophil count, and genetic testing, extending beyond the conventional bone marrow studies. Coupled with emerging technologies such as next-generation sequencing, transcriptomics and proteomics, dried blood spots used in NBS-SCID will promote earlier detection, diagnosis, and therefore treatment of IEIs such as WHIM syndrome., (© 2022 Wiley Periodicals LLC.)

Purpose: The measurement of T-cell receptor excision circle (TREC) is used for newborn screening (NBS) in dried blood spot (DBS) samples from Guthrie card for severe combined immunodeficiency (SCID). Here, we report the results of first newborn screening pilot program for SCID conducted in Türkiye. Methods: The study was carried out together with Ankara University School of Medicine and The Ministry of Health, Public Health General Directorate, Pediatric and Adolescent Health Department. TREC measurements were performed in randomly selected Guthrie card samples obtained from 20253 babies born between October 2018 and October 2020. The TREC analyses were performed together with beta Actin (b-Actin) via RT-PCR (Real Time Polymerase Chain Reaction). Results: TRECs found to be normal (≥15 copies/µl) in 98,6% of the newborns (n: 19975) but low (<15 copies/µl) in 1.4% (n:278) at the initial analyses. TRECs were retested in 278 suspected infants and found to be normal in 160 (0.8%) while low in 118 (0.58%). New DBS were obtained from the babies with low TRECs (new sample test). TRECs were normal in 108 (0.53%) of the new sample tests and low in 10 (0.049%). Two among 10 babies who had abnormal (undetectable) TRECs were diagnosed as SCID; ADA (P1) and RAG1 (P2) defects were confirmed respectively. They both received curative treatments [gene therapy (P1) and HSCT (P2)]. The remaining 6 of 8 newborns with abnormal TRECs were found normal after clinical and laboratory immune work-up, while medical records of other two revealed early postnatal death due to extreme prematurity. Conclusion: In the light of this study the incidence of SCID was detected at least 1/10000 live births in Türkiye. This study shows the feasibility and usefulness of initiating SCID screening in Türkiye [ABSTRACT FROM AUTHOR]

Severe combined immunodeficiency (SCID) is the most severe heterogeneous group of inherited disorders characterized by profound abnormalities such as humoral and cell-mediated immunity defects and hindered natural killer cell development and function. The knowledge of the molecular basis of SCID is essential for precise diagnosis and early treatment. In recent years, new genetic defects that cause SCID have been discovered, and the molecular and immunological mechanisms of SCID have been better understood. SCID symptoms include candidiasis, chronic diarrhea, failure to grow, and oral thrush. Hematopoietic stem cell transplantation, enzyme replacement therapy, and gene therapy are used to treat SCID. The prevalence of SCID varies worldwide. More than 80% of SCID infants have no family history of the condition. However, the development of a newborn screening test has enabled SCID detection before symptoms appear, ensuring that affected infants receive life-saving treatments. Countries that organize newborn screening programs for SCID can detect patients in their early stages of life and treat them accordingly. This review will serve as a source of up-to-date information on the identification of various genetic disorders that cause SCID, as well as their clinical characteristics, treatments, and diagnosis options, potentially saving the lives of many infants before pathogenic infections occur. [ABSTRACT FROM AUTHOR]

Severe combined immunodeficiency (SCID) is a group of inborn errors of immunity (IEI) characterized by severe T- and/or B-lymphopenia. At birth, there are usually no clinical signs of the disease, but in the first year of life, often in the first months the disease manifests with severe infections. Timely diagnosis and treatment play a crucial role in patient survival. In Ukraine, the expansion of hemostatic stem cell transplantation and the development of a registry of bone marrow donors in the last few years have created opportunities for early correction of IEI and improving the quality and life expectancy of children with SCID. For the first time in Ukraine, we initiated a pilot study on newborn screening for severe combined immunodeficiency and T-cell lymphopenia by determining T cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs). The analysis of TREC and KREC was performed by real-time polymerase chain reaction (RT-PCR) followed by analysis of melting curves in neonatal dry blood spots (DBS). The DBS samples were collected between May 2020 and January 2022. In total, 10,350 newborns were screened. Sixty-five blood DNA samples were used for control: 25 from patients with ataxia-telangiectasia, 37 - from patients with Nijmegen breakage syndrome, 1 - with X-linked agammaglobulinemia, 2 - with SCID (JAK3 deficiency and DCLRE1C deficiency). Retest from the first DBS was provided in 5.8% of patients. New sample test was needed in 73 (0.7%) of newborns. Referral to confirm or rule out the diagnosis was used in 3 cases, including one urgent abnormal value. CID (TlowB+NK+) was confirmed in a patient with the urgent abnormal value. The results of a pilot study in Ukraine are compared to other studies (the referral rate 1: 3,450). Approbation of the method on DNA samples of children with ataxia-telangiectasia and Nijmegen syndrome showed a high sensitivity of TRECs (a total of 95.2% with cut-off 2000 copies per 106 cells) for the detection of these diseases. Thus, the tested method has shown its effectiveness for the detection of T- and B-lymphopenia and can be used for implementation of newborn screening for SCID in Ukraine. [ABSTRACT FROM AUTHOR]

Background: Inborn errors of immunity (IEIs) are being recognized as an important cause of morbidity and mortality in communities with a high frequency of consanguinity, such as Oman, and thus recessively inherited conditions. Various monogenic causes of IEI have been recently discovered; however, the disease phenotype may be variable and does not always include infection at presentation, leading to a delay in diagnosis and a poor outcome. It is now well recognized that immune dysregulation manifestations are observed in a significant proportion of patients with IEI and occasionally precede infection. Methods: Here, we retrospectively report the epidemiological, clinical, immunological, and molecular findings and outcomes from 239 patients with IEI who were diagnosed and managed at the Royal Hospital, Oman, from January 2010 to October 2021. Results: The estimated annual cumulative mean incidence of IEI was 25.5 per 100,000 Omani live births with a total prevalence of 15.5 per 100,000 Omani population. Both the high incidence and prevalence are attributed to the high rate of consanguinity (78.2%). Defects affecting cellular and humoral immunity including severe combined immunodeficiency (SCID), combined immunodeficiency (CID), and CID with syndromic features were the predominant defects in IEI (36%). Immune dysregulation was a prominent manifestation and occurred in approximately a third of all patients with IEI (32%), with a mean age of onset of 81 months and a mean diagnostic delay of 50.8 months. The largest percentage of patients who showed such clinical signs were in the category of diseases of immune dysregulation (41%), followed by predominantly antibody deficiency (18%). The overall mortality rate in our cohort was 25.1%, with higher death rates seen in CID including SCID and diseases of immune dysregulation. Conclusion: Immune dysregulation is a frequent manifestation of Omani patients with IEI. Early detection through raising awareness of signs of IEI including those of immune dysregulation and implementation of newborn screening programs will result in early intervention and improved overall outcome. [ABSTRACT FROM AUTHOR]

Next-generation sequencing (NGS) has been used to detect severe combined immunodeficiency (SCID) in patients, and some patients with DNA cross-link repair 1C (DCLRE1C) variants have been identified. Moreover, some compound variants, such as copy number variants (CNV) and single nucleotide variants (SNV), have been reported. The purpose of this study was to expand the genetic data related to patients with SCID carrying the compound DCLRE1C variant. Whole-exome sequencing (WES) was performed for genetic analysis, and variants were verified by performing Sanger sequencing or quantitative PCR. Moreover, we searched PubMed and summarized the data of the reported variants. Four SCID patients with DCLRE1C variants were identified in this study. WES revealed a homozygous deletion in the DCLRE1C gene from exons 1–5 in patient 1, exons 1–3 deletion and a novel rare variant (c.92T>C, p.L31P) in patient 2, exons 1–3 deletion and a novel rare variant (c.328C>G, p.L110V) in patient 3, and exons 1–4 deletion and a novel frameshift variant (c.449dup, p.His151Alafs*20) in patient 4. Based on literature review, exons 1–3 was recognized as a hotspot region for deletion variation. Moreover, we found that compound variations (CNV + SNV) accounted for approximately 7% variations in all variants. When patients are screened for T-cell receptor excision circles (TRECs), NGS can be used to expand genetic testing. Deletion of the DCLRE1C gene should not be ignored when a variant has been found in patients with SCID. [ABSTRACT FROM AUTHOR]

Introduction: Severe combined immunodeficiency (SCID) results from various monogenic defects that impair immune function and brings on early severe and life-threatening infections. The main stay of treatment for SCID is hematopoietic stem cell transplant (HSCT) with near normal survival at 5 years for an early transplant done at or before the age of 3.5 months of life and the patient is maintained free of infections. Although overall rare, it constitutes a major burden on affected children, their families and on the health system especially in communities with a high rate of consanguinity where incidence and prevalence of recessive inborn errors of immunity (IEI) are expected to be high. Method: Here, we report the clinical, immunological, and molecular findings in 36 children diagnosed with SCID from a single tertiary center in Oman for the last decade. Results: We observed a median annual incidence rate of 4.5 per 100,000 Omani live births, and 91.7% of affected children were born to consanguineous parents. Twenty-three children (63.9%) fulfilled the criteria for typical SCID. The median age at onset, diagnosis and diagnostic delay were 54, 135, and 68 days, respectively. The most common clinical manifestations were pneumonia, septicemia, and chronic diarrhea. Eleven children (30.6%) have received hematopoietic stem cell transplant (HSCT) with a survival rate of 73%. The most frequent genetic cause of SCID in this cohort (n = 36) was (RAG-1), encoding for recombination activating gene (n = 5, 13.9%). Similarly, Major histocompatibility complex type II deficiency accounted for (n = 5, 13.9%) of our cohort. Conclusion: Our report broadens the knowledge of clinical and molecular manifestations in children with SCID in the region and highlights the need to initiate newborn based screening program (NBS) program. [ABSTRACT FROM AUTHOR]

Severe combined immunodeficiency (SCID) and other T cell lymphopenias can be detected during newborn screening (NBS) by measuring T cell receptor excision circles (TRECs) in dried blood spot (DBS) DNA. Second tier next generation sequencing (NGS) with an amplicon based targeted gene panel using the same DBS DNA was introduced as part of our prospective pilot research project in 2015. With written parental consent, 21 000 newborns were TREC-tested in the pilot. Three newborns were identified with SCID, and disease-causing variants in IL2RG, RAG2 , and RMRP were confirmed by NGS on the initial DBS DNA. The molecular findings directed follow-up and therapy: the IL2RG -SCID underwent early hematopoietic stem cell transplantation (HSCT) without any complications; the leaky RAG2- SCID received prophylactic antibiotics, antifungals, and immunoglobulin infusions, and underwent HSCT at 1 year of age. The child with RMRP -SCID had complete Hirschsprung disease and died at 1 month of age. Since January 2018, all newborns in Norway have been offered NBS for SCID using 1st tier TRECs and 2nd tier gene panel NGS on DBS DNA. During the first 20 months of nationwide SCID screening an additional 88 000 newborns were TREC tested, and four new SCID cases were identified. Disease-causing variants in DCLRE1C, JAK3, NBN , and IL2RG were molecularly confirmed on day 8, 15, 8 and 6, respectively after birth, using the initial NBS blood spot. Targeted gene panel NGS integrated into the NBS algorithm rapidly delineated the specific molecular diagnoses and provided information useful for management, targeted therapy and follow-up i.e., X rays and CT scans were avoided in the radiosensitive SCID. Second tier targeted NGS on the same DBS DNA as the TREC test provided instant confirmation or exclusion of SCID, and made it possible to use a less stringent TREC cut-off value. This allowed for the detection of leaky SCIDs, and simultaneously reduced the number of control samples, recalls and false positives. Mothers were instructed to stop breastfeeding until maternal cytomegalovirus (CMV) status was determined. Our limited data suggest that shorter time-interval from birth to intervention, may prevent breast milk transmitted CMV infection in classical SCID. [ABSTRACT FROM AUTHOR]