Whole Exome Sequencing Identified a Pathogenic IL2RG Variant in Monozygotic Twins with Severe Combined Immunodeficiency

Whole Exome Sequencing Identified a Pathogenic IL2RG Variant in Monozygotic Twins with Severe Combined Immunodeficiency | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Whole Exome Sequencing Identified a Pathogenic IL2RG Variant in Monozygotic Twins with Severe Combined Immunodeficiency Seyed Pooria Tadayon Nabavi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6234637/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose This study aimed to determine the genetic cause of severe combined immunodeficiency (SCID) in monozygotic twin male infants who presented with recurrent severe infections and disseminated BCG-related complications. Methods Whole Exome Sequencing (WES) was performed on one twin to identify candidate pathogenic variants. The detected IL2RG variant was validated through PCR and Sanger sequencing, and family segregation analysis was conducted. Comprehensive immunological assessments, including T-cell receptor excision circle (TREC) assays and lymphocyte profiling, were employed. High-resolution HLA typing was also carried out to evaluate donor compatibility for hematopoietic stem cell transplantation (HSCT). Results WES revealed a hemizygous c.670C > T (p.Arg224Trp) variant in the IL2RG gene, a change absent from population databases and predicted to be deleterious by in silico tools. Sanger sequencing confirmed the variant in both twins, and maternal heterozygosity was identified, supporting an X-linked recessive inheritance pattern. Clinically, the twins exhibited profound lymphopenia and undetectable TRECs, consistent with a T⁻B⁺NK⁻ immunophenotype. HLA typing demonstrated only partial haplotype matches with the parents, indicating the necessity for a haploidentical transplant approach. Conclusion Integrating genomic analysis with immunological profiling effectively pinpointed a pathogenic IL2RG mutation as the cause of SCID in these twins. Early genetic diagnosis using WES is critical for guiding timely therapeutic interventions such as HSCT or gene therapy and underscores the need for implementing newborn screening and carrier testing programs, particularly in resource-limited settings. Severe combined immunodeficiency IL2RG Whole Exome Sequencing Genetic diagnosis Hematopoietic stem cell transplantation Figures Figure 1 Figure 2 Figure 3 Introduction Severe Combined Immunodeficiency (SCID) comprises a group of rare, monogenic disorders characterized by a block in T lymphocyte development, leading to life-threatening infections in early infancy​ [ 1 , 2 ]. Affected infants often inflict with opportunistic fungal, bacterial, or viral infections within the first months of life and, without immune reconstitution, succumb within the first year​ [ 2 ]. SCID is considered a pediatric medical emergency, given that timely diagnosis and intervention are critical for survival​. The estimated incidence of SCID is approximately 1 in 50,000–100,000 live births​. Particularly, in regions with high rates of consanguinity, the incidence can be markedly higher, due to the increased prevalence of autosomal recessive forms of SCID​. However, in many developing countries lacking newborn screening programs, SCID cases are frequently underdiagnosed or diagnosed late. Indeed, while several countries have implemented routine newborn screening for SCID using T-cell receptor excision circle (TREC) assays​, most low-income settings still rely on clinical suspicion that results in delays until severe infections manifest. SCID is genetically heterogeneous, with mutations in at least 20–, and as of recent classifications, up to 18 distinct genes known to cause a SCID phenotype​. These defects span various molecular pathways, including impaired V(D)J recombination (e.g. RAG1 , RAG2 , DCLRE1C ), cytokine signaling abnormalities (e.g. IL2RG , JAK3 , IL7R ), and metabolic defects (e.g. ADA ) ​ [ 3 – 5 ]. Among these, X-linked SCID caused by mutations in the IL2RG gene (encoding the interleukin-2 receptor common gamma chain, γc) is the single most common subtype. A historical cohort study indicated that IL2RG mutations account for ~ 40–50% of all SCID cases​ [ 6 ], making this the most prevalent form of the disease. Accordingly, in an analysis of 108 SCID infants, 49 had X-linked IL2RG mutations (~ 45% of cases), far exceeding any other genetic subtype​ [ 6 ]. IL2RG is located on Xq13.1 and encodes the shared γc subunit of at least six cytokine receptors, including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21​ [ 7 ]. This common γ-chain is critical for lymphocyte development and function. Consequently, IL2RG mutations abrogate multiple cytokine signaling pathways, leading to a characteristic SCID immunophenotype of absent T cells and NK cells with non-functional B cells (T − B + NK – SCID)​ [ 7 ]​. Basically, X-linked SCID is a disease of defective interleukin signaling, explaining its severe combined loss of cellular immunity​. Especially, autosomal recessive forms like JAK3 deficiency phenocopy X-linked SCID because JAK3 is the kinase that associates with γc, underscoring the central role of the IL2RG signaling axis in normal immune development​. Therapeutic option for SCID is available in the form of immune reconstitution, most commonly through hematopoietic stem cell transplantation (HSCT). Outcomes for HSCT in SCID have improved dramatically over time, and survival rates now exceed 90% for infants who receive a transplant from an HLA-identical sibling donor in the first few months of life​ [ 8 ]. Even with partially matched donors, early transplantation (before the onset of irreversible infections) significantly enhances survival, highlighting the importance of prompt diagnosis​. Experimental gene therapy has also shown success in certain SCID subsets (including IL2RG deficiency), further expanding treatment options​ [ 1 ]. The key to these favorable outcomes is early identification of affected infants, ideally at birth, before infections occur​ ​[ 8 , 9 ]. This has driven the adoption of newborn screening for SCID in many high-income countries, using the TREC assay to detect T-cell lymphopenia in dried blood spots​ [ 10 ]. TREC screening is highly sensitive for SCID and related T-cell defects​, allowing presymptomatic diagnosis. However, newborn screening by itself does not reveal the underlying genetic cause – it can flag an infant as likely SCID, but cannot distinguish IL2RG mutation from other etiologies​. Moreover, some atypical or "leaky" SCID cases (with milder T-cell deficits) might initially pass newborn screening, only to present later with immunodeficiency​ [ 11 ]. These limitations underscore the need for comprehensive genetic diagnostics following an abnormal screen or in patients with clinical SCID features in settings without screening. Advances in genomic technology, particularly Whole Exome Sequencing (WES), have revolutionized the diagnostic approach to SCID and other primary immunodeficiencies. WES enables rapid, parallel analysis of all protein-coding genes, allowing clinicians to pinpoint pathogenic variants in SCID patients within a single test​ [ 12 ]. This has proven invaluable for confirming the diagnosis and guiding therapy, especially in cases with atypical presentation or in families where the specific genetic defect is not evident. In this regard, using next-generation sequencing methods, over 90% of infants with SCID in contemporary North American cohorts is genetically characterized​ [ 13 ]. By contrast, traditional diagnostic approaches often required serial candidate gene testing guided by immunophenotype – a time-consuming process that may be unfeasible when a patient’s condition is rapidly deteriorating. Studies have demonstrated that WES can detect novel or unexpected mutations in SCID patients who had initially eluded diagnosis by routine tests​. Furthermore, genetic sequencing is crucial for distinguishing between different genetic forms of SCID that may present with similar clinical and immunologic features (like RAG1/2 mutations causing Omenn syndrome vs. other SCID forms)​ [ 2 ]. Knowing the exact molecular defect has practical implications. It informs family counseling and can influence treatment decisions, such as eligibility for gene therapy trials or the urgency and conditioning regimen for HSCT [ 1 , 8 ]​. That said, the widespread use of WES has also introduced new challenges, such as the interpretation of variants of uncertain significance (VUS). In some SCID cases, WES uncovers novel missense changes whose functional impact is not immediately clear, necessitating supplementary studies. As a finding, functional assays have been used to confirm the pathogenicity of ambiguous RAG1 variants discovered by sequencing as a definitive diagnosis​. Diagnosing and managing SCID poses particular challenges in resource-limited settings. Many low- and middle-income countries lack routine newborn screening programs for SCID, and awareness among clinicians may be limited, leading to missed or delayed diagnoses. Infants in these settings often present only after developing severe infections or failure to thrive, at which point opportunistic pathogens (e.g. Bacille Calmette-Guérin (BCG) vaccine strain, given as newborn tuberculosis prophylaxis in many countries) may have already caused disseminated disease​. The delay not only increases immediate mortality risk but can also compromise the success of curative therapy since active infections and organ damage at transplant are associated with worse outcomes​. A further obstacle is the limited availability of advanced diagnostic tools. In the absence of in-country genomic facilities, confirming a suspected SCID diagnosis genetically may rely on sending samples abroad or not be done at all, leaving the genetic subtype unknown​. This lack of definitive diagnosis can impede optimal treatment – for example, distinguishing IL2RG deficiency (X-linked) has implications for family screening and donor search, and identifying ADA deficiency might allow enzyme replacement therapy as a bridge to transplant. Moreover, access to HSCT itself is variable in resource-limited regions; even when a genetic diagnosis is made, specialized transplant centers and suitable donors may not be readily accessible. Paradoxically, the regions with higher SCID incidence due to consanguinity are often those with the scarcest resources for early detection and treatment​. This disparity highlights an urgent need for international collaboration and capacity-building, training healthcare providers to recognize SCID, and implementing cost-effective genetic testing (such as targeted sequencing panels or exome sequencing via regional centers) to facilitate prompt diagnosis. Within this context, IL2RG mutations remain a focal point because of their relative frequency and clear therapeutic implications. X-linked SCID cases can be identified by family history or by carrier testing in mothers once a mutation is known, which emphasizes the value of molecular diagnosis. Published reports from the Middle East and Asia have begun to catalog the spectrum of IL2RG mutations in their SCID populations​. However, data on SCID in certain regions, such as sub-Saharan Africa and parts of South Asia, remain very scarce. Even in countries like Iran, where consanguinity is observed and autosomal recessive SCID (e.g. RAG deficiencies) might be expected to predominate, X-linked IL2RG mutations still account for a substantial fraction of cases​. Every new case study adds to the collective knowledge needed to improve outcomes. Accordingly, the present study reports SCID in monozygotic twins, a rare but especially informative, providing a controlled look at genotype-phenotype correlation and the impact of environmental factors on disease course​. Such finding can improve our understanding of SCID pathogenesis and inheritance patterns. In this study, we present the case of monozygotic twin infants from a resource-limited setting who were diagnosed with SCID due to a pathogenic IL2RG mutation, identified through whole exome sequencing. We describe the clinical presentation (including severe recurrent infections and BCGiosis), the immunological findings, and the genetic analysis confirming an X-linked IL2RG variant, which was verified by family segregation. This report highlights the utility of WES in reaching a definitive diagnosis in the absence of newborn screening and shows the challenges of managing SCID in a setting with limited resources. By integrating genomic data with clinical and immunological evaluation, the critical role of IL2RG in immune development and advocate for broader implementation of genomic newborn screening and early referral for curative therapy is highlighted. Also, the findings contribute to the growing global registry of SCID mutations and support efforts to ensure that life-saving interventions for SCID, like timely HSCT or gene therapy, become accessible to all patients, regardless of geographic location or resource availability. Methods Study Participants and Sample Collection This study involved monozygotic twin male infants (5 months old) presenting with clinical features of SCID, including recurrent bacterial infections and failure to thrive. They had a history of disseminated Bacille Calmette–Guérin (BCG) infection following newborn vaccination. The infants were referred to a specialized immunology-genetics clinic in Iran after SCID was suspected. Peripheral blood samples (~ 2.5 mL) were collected in EDTA tubes from each twin and their parents (after informed consent). Genomic DNA was extracted from leukocytes using the QIAamp DNA Blood Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. DNA quality and concentration were assessed by agarose gel electrophoresis and spectrophotometry (NanoDrop 2000, Thermo Fisher Scientific) to ensure high-molecular-weight DNA suitable for sequencing. Whole Exome Sequencing (WES) To identify the genetic cause of immunodeficiency, whole exome sequencing was performed on one twin (the proband). An Illumina NovaSeq 6000 platform (Macrogen, South Korea) was used for high-throughput sequencing. Exome enrichment was done with the SureSelect Human All Exon V7 kit (Agilent Technologies), and a sequencing depth of ~ 200× was achieved. Raw reads were aligned to the human reference genome (GRCh38) using the Burrows-Wheeler aligner, and variant calling was performed with CLC Genomics Workbench v7.7 (QIAGEN). Variant annotation and filtering prioritized rare, protein-altering variants consistent with an X-linked inheritance pattern. Public databases including ClinVar, gnomAD, and dbSNP were consulted to exclude known benign variants and identify novel or pathogenic candidates. PCR and Sanger Sequencing Candidate variant validation and family segregation analysis were carried out by PCR amplification and Sanger sequencing of the IL2RG gene. Custom primers flanking the putative mutation site were designed (Primer3Plus/NCBI Primer-BLAST) with the following sequences: IL2RG exon 5 forward 5′-TACTCTTCCTGATACCAGATAG-3′ and reverse 5′-CTACTCTAACACACCCCAAC-3′. PCR was performed in a 40 µL reaction containing 20–50 ng genomic DNA, 0.8 µM of each primer, 2.5 µL of 10× PCR buffer, 1 µL of each dNTP (10 mM), 2 µL of MgCl₂ (25 mM), 0.1 µL of Taq DNA polymerase (5 U/µL), and nuclease-free water to volume. Thermocycling conditions were: initial denaturation at 95°C for 5 min; followed by 36 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 30 s; and a final extension at 72°C for 5 min. PCR products were confirmed on a 1.5% agarose gel and then purified for Sanger sequencing (performed by Macrogen, Korea). Chromatogram analysis (using CLC Workbench) enabled confirmation of the WES-identified variant in the twins and the determination of parental carrier status. Clinical and Immunological Assessments Comprehensive clinical evaluations were conducted. Key features recorded included the frequency and type of infections (e.g. pneumonia, sepsis with Staphylococcus haemolyticus), vaccination history (notably the BCG-related complications), and growth parameters. Laboratory tests revealed profound lymphopenia on complete blood counts (absolute lymphocyte count < 300 cells/µL, far below age-matched normals). Flow cytometry of lymphocyte subsets (if available) would show markedly low T cells and NK cells with non-functional B cells, consistent with SCID. A T-cell receptor excision circle (TREC) assay was used as a surrogate for thymic output; both patients had undetectable TRECs in peripheral blood, consistent with absent new T-cell production. In preparation for potential treatment, HLA typing was performed for the twins and their parents at HLA class I (A, B, C) and class II (DRB1, DQB1) loci using high-resolution sequence-based methods. HLA results were used to evaluate the feasibility of hematopoietic stem cell transplantation (HSCT) from family donors. Results Genetic Analysis WES of the proband identified a hemizygous variant in IL2RG, located on chromosome Xq13.1, that perfectly matched the clinical suspicion of X-linked SCID. The variant was a single nucleotide change c.670C > T in the IL2RG coding sequence, predicting a missense substitution p.Arg224Trp in the common gamma chain protein. This was the only rare, protein-altering variant on the X chromosome that could explain the phenotype. The variant was absent from population databases (gnomAD frequency 0.0%), and in silico predictors (PolyPhen-2, SIFT) indicated it is deleterious. Table 1 summarizes the genomic findings. Sanger sequencing confirmed the presence of the IL2RG c.670C > T mutation in both twins, and additionally showed that their mother is heterozygous for this variant (carrier), while the father’s sequence is wild-type. These results are consistent with X-linked recessive inheritance of the disease-causing mutation. The IL2RG p.Arg224Trp variant has previously been classified as pathogenic in X-SCID patients and was not found in healthy individuals. Table 1 WES results of the proband. Key variant identified by whole exome sequencing in the proband Gene Genomic Coordinate (GRCh38) Nucleotide Change Amino Acid Change Zygosity Predicted Pathogenicity IL2RG X:70,329,165 c.670C > T p.Arg224Trp Hemizygous (male) Damaging (PolyPhen-2, SIFT) Note : No other candidate variants explaining the immunodeficiency were found on autosomal genes (e.g., RAG1/2, JAK3, ADA ), consistent with IL2RG being the sole causative mutation. HLA Typing and Familial Segregation Family HLA typing revealed only partial haplotype matches between the twins and their parents, and no fully matched sibling donor was available (the twins have no other siblings). Table 2 shows the HLA alleles for the proband, mother, and father. The twins shared one HLA haplotype with the mother and one with the father, as expected for an X-linked condition inherited from a carrier mother. For example, the proband is HLA-DRB1*01:01/01:01 (inherited one DRB101 allele from each parent) and HLA-DQB105:01/*05:01 (one DQB105:01 from each parent), indicating haploidentical matches with both parents. The mother and father each share half of their HLA alleles with the affected infants, but neither parent is a full HLA match at all loci. These results indicate that an alternative donor source (such as a haploidentical parental transplant with T-cell depletion or an unrelated donor) would be required for HSCT, as no HLA-identical sibling donor exists. Table 2 HLA compatibility results. HLA genotyping of the family. The proband’s HLA alleles are compared to those of the mother and father. Family Member HLA-A HLA-B HLA-C HLA-DRB1 HLA-DQB1 Proband A11, A?* (No exact match) B35, B35 (homozygous) C04, C15 DRB1 01:01, DRB1 01:01 (homozygous) DQB1 05:05, DQB1 05:05 (homozygous) Mother A30, A32 B35, B35 (homozygous) C04, C04 (homozygous) DRB1 01:01, DRB1 03:01 DQB1 02:01, DQB1 05:05 Father A11, A26 B35, B51 C04, C15 DRB1 01:01, DRB1 01:01 (homozygous) DQB1 05:05, DQB1 05:05 (homozygous) Note : The proband’s HLA-A alleles (likely A11 and A30) each match one parent (A11 from father, A30 from mother), but there is no overlap between parental A alleles; hence, no exact HLA-A match is shared by both parents. Clinical Findings Both twins manifested severe clinical symptoms of immunodeficiency early in life. They suffered recurrent, hard-to-treat infections. Notably, each developed pneumonia and sepsis with organisms such as Staphylococcus haemolyticus, and both had disseminated BCG infection (BCG-osis) following administration of the live attenuated BCG vaccine at birth. The disseminated mycobacterial infection was confirmed by finding granulomas on biopsy and acid-fast bacilli in a bone marrow aspirate. Immunologically, the infants were profoundly lymphopenic: total lymphocyte counts were 4,000/µL). There was an absence of functional T cells (CD3 + lymphocytes extremely low) and NK cells, whereas B cells were present (T − B + NK − phenotype). However, the B cells were non-functional, evidenced by extremely low immunoglobulin levels and lack of specific antibody production despite infection, consistent with the need for T-cell help. The TREC assay was undetectable in both patients, indicating virtually no new T cells emerging from the thymus. These findings confirmed a diagnosis of X-linked SCID (T⁻B⁺NK⁻ subtype). Both infants required isolation and prophylactic antimicrobial interventions while a definitive therapy was planned. Figure 1 . Sanger Sequencing Chromatogram of the IL2RG c.670C > T Variant. Chromatogram demonstrating the hemizygous c.670C > T mutation in the IL2RG gene in the proband. The arrow highlights the nucleotide substitution that results in the p.Arg224Trp amino acid change Fig. 2 . Pedigree Diagram Illustrating X-Linked Inheritance. Family pedigree showing the X-linked recessive transmission of the IL2RG mutation. Affected individuals (filled symbols) include the monozygotic twin boys, while the mother (half-filled symbol) is an asymptomatic carrier and the father (open symbol) is unaffected Fig. 3 . Schematic Overview of HLA Typing for HSCT Donor Selection Diagram outlining the high-resolution HLA typing workflow used to assess donor compatibility. The figure displays the HLA-A, -B, -C, -DRB1, and -DQB1 allele distributions among the proband and his parents, emphasizing the partial haplotype matches critical for selecting a suitable donor for hematopoietic stem cell transplantation Discussion This report provides a detailed genetic and clinical investigation of X-SCID in identical twin brothers. As a results, a hemizygous c.670C > T (p.Arg224Trp) mutation was identified in IL2RG as the cause of their condition. The IL2RG gene encodes the common γc, a crucial subunit shared by multiple cytokine receptors, including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 that regulate lymphocyte development [ 14 ]. Disruption of the common γc due to IL2RG mutations impairs signaling through all these cytokines, arresting the maturation of T cells and NK cells, while B cells develop but cannot function properly without T-cell help​. This immunologic mechanism explains the classic SCID phenotype observed in the patients, and aligns with the fact that mutations in IL2RG are the single most common cause of SCID globally [ 15 ]. Large cohort studies have shown that X-linked IL2RG deficiency accounts for roughly 40–50% of all SCID cases​ [ 15 ]. The specific IL2RG variant in this twins (p.Arg224Trp) is a known pathogenic mutation associated with X-SCID. It lies in the extracellular domain of the γc protein, and the substitution of a positively charged arginine with a bulky tryptophan is predicted to disrupt the receptor’s structure and its interaction with interleukins. Indeed, this variant has been reported in multiple unrelated SCID patients and is not found in healthy population databases [ 15 – 17 ]. These findings are consistent with previous reports that IL2RG missense mutations abolish common γc receptor function, leading to the absence of T and NK cells and nonfunctional B cells​[ 14 ]. By confirming the mutation in both twins and the carrier state in their mother, a genetic evidence of X-linked inheritance us provided, which is valuable information for family counseling such as a risk to future male children. Clinically, the twins’ presentation was characteristic of SCID. They experienced life-threatening infections within months of birth and did not respond normally to vaccines and developed disseminated disease from an attenuated vaccine strain. If untreated, infants with SCID typically succumb to severe infections within the first year of life​ [ 3 , 18 , 19 ], reflecting the absolute dependence of the adaptive immune system on functional T cells. In this twins, the absence of TRECs was a key laboratory finding. TRECs are circular DNA fragments excised during T-cell receptor gene rearrangement in the thymus, and they serve as a biomarker for recent thymic emigrants​ [ 10 , 20 , 21 ]. The twins’ undetectable TREC levels indicate almost complete failure of thymic T-cell production, consistent with X-SCID. These results mirror what is seen in newborn screening programs for SCID: babies with absent or extremely low TRECs are flagged for urgent evaluation and often found to have SCID​ [ 22 , 23 ]. Thanks to newborn screening, many SCID cases in developed programs are now identified at birth before infections occur​ [ 24 ]. Unfortunately, no such screening was in place for our patients in the time of this study. Their diagnosis was made after clinical illness had begun, illustrating the pediatric emergency nature of SCID – early diagnosis is crucial to prevent fatal infections​. The definitive treatment for SCID is allogeneic HSCT, which can reconstitute a functional immune system in the patient [ 1 ]. HSCT outcomes are vastly improved when performed in the first months of life, ideally before the infant has acquired severe infections​ [ 25 ]. Historical data and recent multicenter studies concur that infants transplanted at ≤ 3.5 months of age have survival rates > 90% [ 25 , 26 ]. In the case of the twins, the HLA typing results indicated that neither had an HLA-identical sibling donor. The best available options would likely be a haploidentical parental transplant (using the father or mother as a donor, with T-cell depletion to prevent graft-versus-host disease) or searching for a well-matched, unrelated donor. It is well documented that any delay in performing HSCT for SCID correlates with worse survival, largely due to infections acquired while waiting for a transplant​. Therefore, upon genetic confirmation of SCID, an expeditious referral for HSCT is recommended even if only a haploidentical donor is available, as outcomes with parental donors can be life-saving when done early in life​ [ 25 , 26 ]. The present study also highlights the disparities in healthcare infrastructure. Regions without newborn screening programs or easy access to transplant centers face significant challenges in managing SCID. In countries like Iran (where our patients reside), SCID diagnoses often rely on clinical recognition of infections and failure to thrive, which may occur late in the disease course​. This delay leads to higher infection-related morbidity and mortality. Moreover, areas with a high rate of consanguineous marriages see a greater prevalence of autosomal recessive forms of SCID and other primary immunodeficiencies, further increasing the burden of undiagnosed cases​. These findings emphasize the urgent need for implementing population-wide SCID newborn screening in such regions. Even a simple TREC-based DNA test on Guthrie cards can identify SCID in the first days of life, enabling prompt protective measures and timely curative therapy [ 24 ]. Alongside screening, genetic counseling and carrier testing in families with known X-SCID is essential. In the case of the twins, identification of the carrier mother allows informed reproductive choices and early testing of future male infants in the extended family. Another important aspect of this case is the demonstration of how genomic technologies complement immunological tests for a precise diagnosis. Traditional diagnostics for suspected SCID include lymphocyte phenotyping and functional assays like mitogen proliferation tests, which can confirm an immunodeficiency but not its genetic cause. Here, WES was decisive in pinpointing IL2RG as the mutated gene, which not only clinched the diagnosis of X-SCID but also guided the family workup, identifying the carrier mother. The power of WES to reveal the molecular basis of rare diseases has revolutionized medical genetics​ [ 12 ]. It is especially valuable in genetically heterogeneous conditions like SCID, which can result from mutations in over a dozen different genes​ [ 1 ]. Recent studies from Iran and other countries with limited resources have shown that applying WES can significantly improve the diagnostic yield for patients with primary immunodeficiencies, uncovering mutations that targeted gene panels might miss​ [ 4 ]. Our report adds to this body of evidence, illustrating that even in a resource-constrained setting, integrating WES into the diagnostic workflow for infants with severe immune syndromes is feasible and impactful. By identifying a specific genetic defect, we can tailor the management and provide accurate genetic counseling. In cases where WES identifies variants of uncertain significance, further functional studies by measuring cytokine signaling in patient T cells or targeted gene assays may be needed​. Looking forward, advancements in therapy, such as gene therapy, offer hope to patients with X-SCID. Early trials demonstrated that integrating a correct copy of IL2RG into patient hematopoietic stem cells could reconstitute T-cell immunity, but some patients developed leukemia due to retroviral vector insertion near oncogenes [ 27 , 28 ]. In recent years, improved vectors (such as self-inactivating lentiviral vectors) have made gene therapy for SCID much safer, and new trials have shown successful immune reconstitution without unexpected cancers​ [ 1 ]. Gene therapy has thus become an alternative for patients who lack a suitable HSCT donor or who have contraindications to transplantation​ [ 1 ]. In the context of the presented case, gene therapy could be considered, although at present HSCT from a haploidentical donor is the standard approach. Long-term follow-up of SCID patients treated with HSCT or gene therapy is an important need​. Issues such as chronic graft-versus-host disease, incomplete immune reconstitution like poor B-cell function requiring lifelong immunoglobulin replacement, or late complications like secondary malignancies need to be monitored in survivor cohorts. Fortunately, outcomes for X-SCID have dramatically improved over the past few decades with > 90% of infants now surviving if treated early​ [ 26 ]. This study of cases with X-SCID contributes to the literature by expanding the known mutation spectrum of IL2RG by adding p.Arg224Trp as a recurrent pathogenic variant and by emphasizing the multi-faceted approach required – from genomics to clinical care – to achieve the best outcomes for SCID patients. Conclusions This study highlights the critical importance of early genetic diagnosis in infants with SCID and the life-saving potential of early intervention. The identification of the IL2RG c.670C > T (p.Arg224Trp) mutation in these twins enabled a timely, definitive diagnosis and informed treatment planning. Here, the routine use of whole exome sequencing or comprehensive genetic panels in any infant suspected of SCID was advocated, especially in settings without newborn screening. Such an approach can rapidly confirm the diagnosis, guiding urgent treatment like HSCT, which offers the chance for a normal life​. This study also supports efforts to expand newborn screening programs globally and to improve access to transplantation and emerging gene therapies for patients in all regions​​. Through early detection and intervention, the historically dismal prognosis of SCID, the “bubble boy” disease, can be converted into excellent long-term survival and quality of life for affected children. Declarations Acknowledgements Not applicable. Authorship contributions The author contributed substantially to the conception, design, data acquisition, analysis, and interpretation of the study. Seyed Pooria Tadayon Nabavi led the project, coordinated the study design, and drafted the manuscript. The coauthor critically revised the manuscript for intellectual content, approved the final version, and agree to be accountable for all aspects of the work. Conflicts of interest/Competing interests The author has no relevant financial or non-financial interests to disclose. Funding This study was self-founded, and the author has provided all supports to perform the study. Data availability The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. All relevant data supporting the findings of this study are included in the manuscript. Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. 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Available from: https://pubmed.ncbi.nlm.nih.gov/15696101/ Currier R, Puck JM. SCID newborn screening: What we’ve learned. J Allergy Clin Immunol [Internet]. 2021 [cited 2025 Mar 13];147:417–26. Available from: https://pubmed.ncbi.nlm.nih.gov/33551023/ Rabbani B, Tekin M, Mahdieh N. The promise of whole-exome sequencing in medical genetics. J Hum Genet [Internet]. 2014 [cited 2025 Mar 13];59:5–15. Available from: https://pubmed.ncbi.nlm.nih.gov/24196381/ Dvorak CC, Haddad E, Buckley RH, Cowan MJ, Logan B, Griffith LM et al. The genetic landscape of severe combined immunodeficiency in the United States and Canada in the current era (2010–2018). J Allergy Clin Immunol [Internet]. 2019 [cited 2025 Mar 13];143:405–7. Available from: https://pubmed.ncbi.nlm.nih.gov/30193840/ hematology Uribe L. KW-S in, 1998 undefined. X-linked SCID and other defects of cytokine pathways. europepmc.orgL Uribe, KI WeinbergSeminars in hematology, 1998•europepmc.org [Internet]. [cited 2025 Mar 13]; Available from: https://europepmc.org/article/med/9801259 Puck J, Pepper A, Blood PH-, Journal T. 1997 undefined. Mutation analysis of IL2RG in human X-linked severe combined immunodeficiency. ashpublications.orgJM Puck, AE Pepper, PS Henthorn, F Candotti, J Isakov, T Whitwam, ME Conley, RE FischerBlood, The Journal of the American Society of Hematology, 1997•ashpublications.org [Internet]. [cited 2025 Mar 13]; Available from: https://ashpublications.org/blood/article-abstract/89/6/1968/139070 Lee PPW, Chan KW, Chen TX, Jiang LP, Wang XC, Zeng HS et al. Molecular diagnosis of severe combined immunodeficiency–identification of IL2RG, JAK3, IL7R, DCLRE1C, RAG1, and RAG2 mutations in a cohort of Chinese and Southeast Asian children. J Clin Immunol [Internet]. 2011 [cited 2025 Mar 13];31:281–96. Available from: https://pubmed.ncbi.nlm.nih.gov/21184155/ O’Marcaigh AS, Puck JM, Pepper AE, De Santes K, Cowan MJ. Maternal mosaicism for a novel interleukin-2 receptor gamma-chain mutation causing X-linked severe combined immunodeficiency in a Navajo kindred. J Clin Immunol [Internet]. 1997 [cited 2025 Mar 13];17:29–33. Available from: https://pubmed.ncbi.nlm.nih.gov/9049783/ Notarangelo LD, Fischer A, Geha RS, Casanova JL, Chapel H, Conley ME et al. Primary immunodeficiencies: 2009 update: The International Union of Immunological Societies (IUIS) Primary Immunodeficiencies (PID) Expert Committee. J Allergy Clin Immunol [Internet]. 2009 [cited 2025 Mar 13];124:1161. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC2797319/ Wekell P, Hertting O, Holmgren D, Fasth A. Fifteen-minute consultation: Recognising primary immune deficiencies in children. Arch Dis Child Educ Pract Ed [Internet]. 2019 [cited 2025 Mar 13];104:235–43. Available from: https://pubmed.ncbi.nlm.nih.gov/30733240/ Morinishi Y, Imai K, Nakagawa N, Sato H, Horiuchi K, Ohtsuka Y et al. Identification of severe combined immunodeficiency by T-cell receptor excision circles quantification using neonatal guthrie cards. J Pediatr [Internet]. 2009 [cited 2025 Mar 13];155:829–33. Available from: https://pubmed.ncbi.nlm.nih.gov/19628217/ Kwan A, Church JA, Cowan MJ, Agarwal R, Kapoor N, Kohn DB et al. Newborn Screening for SCID and T Cell Lymphopenia in California: Results of the First Two Years. J Allergy Clin Immunol [Internet]. 2013 [cited 2025 Mar 13];132:140. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC3759317/ Puck JM. Laboratory technology for population-based screening for severe combined immunodeficiency in neonates: the winner is T-cell receptor excision circles. J Allergy Clin Immunol [Internet]. 2012 [cited 2025 Mar 13];129:607–16. Available from: https://pubmed.ncbi.nlm.nih.gov/22285280/ Kwan A, Church JA, Cowan MJ, Agarwal R, Kapoor N, Kohn DB et al. Newborn Screening for SCID and T Cell Lymphopenia in California: Results of the First Two Years. J Allergy Clin Immunol [Internet]. 2013 [cited 2025 Mar 13];132:140. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC3759317/ Kwan A, Church JA, Cowan MJ, Agarwal R, Kapoor N, Kohn DB et al. Newborn screening for severe combined immunodeficiency and T-cell lymphopenia in California: results of the first 2 years. J Allergy Clin Immunol [Internet]. 2013 [cited 2025 Mar 13];132. Available from: https://pubmed.ncbi.nlm.nih.gov/23810098/ Buckley RH. Advances in the understanding and treatment of human severe combined immunodeficiency. Immunol Res [Internet]. 2000 [cited 2025 Mar 13];22:237–51. Available from: https://pubmed.ncbi.nlm.nih.gov/11339359/ Pai S-Y, Logan BR, Griffith LM, Buckley RH, Parrott RE, Dvorak CC et al. Transplantation outcomes for severe combined immunodeficiency, 2000–2009. N Engl J Med [Internet]. 2014 [cited 2025 Mar 13];371:434–46. Available from: https://pubmed.ncbi.nlm.nih.gov/25075835/ Pavel-Dinu M, Wiebking V, Dejene BT, Srifa W, Mantri S, Nicolas CE et al. Gene correction for SCID-X1 in long-term hematopoietic stem cells. Nature Communications 2019 10:1 [Internet]. 2019 [cited 2025 Mar 14];10:1–15. Available from: https://www.nature.com/articles/s41467-019-09614-y Blanco E, Izotova N, Booth C, Thrasher AJ. Immune Reconstitution After Gene Therapy Approaches in Patients With X-Linked Severe Combined Immunodeficiency Disease. Front Immunol [Internet]. 2020 [cited 2025 Mar 14];11:608653. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC7729079/ Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6234637","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":433454037,"identity":"b058fb63-995a-4741-8068-d46e840f6866","order_by":0,"name":"Seyed Pooria Tadayon Nabavi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYBAC+wYgkQBiSYAYFQwMBoS0GBxA0XKGWC0MMC2MbcRoOd587MODGoY8/tnNzyQezjssb87efIDhR8U2nFrse44lz0g4xlAsceeYmUTitsOGO3uOJTD2nLmN2xaJHGOGBDaGxIYbCcYGQC2MG27kGDAzthHS8o8hcf6N9M8GiXMO2xOnJbGNIRGo0vBBYsPhRMJaeI4lMyT2SSRuvHOm8EHCsfTkDWeOJRzE6xf25sOMP77ZJM673b7h4I8aa9sNx5sPPvhRgVsLFEjAGM1g8gAh9cigjhTFo2AUjIJRMEIAAI32XweCWpEAAAAAAElFTkSuQmCC","orcid":"","institution":"Islamic Azad University","correspondingAuthor":true,"prefix":"","firstName":"Seyed","middleName":"Pooria Tadayon","lastName":"Nabavi","suffix":""}],"badges":[],"createdAt":"2025-03-15 20:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6234637/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6234637/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79573001,"identity":"b6f7534f-9f24-4416-9a3e-f7c80bf90f0f","added_by":"auto","created_at":"2025-03-31 11:04:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2004600,"visible":true,"origin":"","legend":"\u003cp\u003eSanger sequencing chromatograms demonstrating the IL2RG c.670C\u0026gt;T (p.Arg224Trp) variant in the family. The top panel shows the father’s wild-type sequence (A), the middle panel depicts the mother’s heterozygous state (B), and the bottom panels (C and D) confirm the hemizygous mutation in both monozygotic twins. The arrow highlights the nucleotide substitution (C→T), which leads to the pathogenic p.Arg224Trp change associated with X-linked SCID.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6234637/v1/7c8c055a56aac103a51a9dd5.jpg"},{"id":79573002,"identity":"39ffe952-fc06-4297-999d-da0b7ea94eef","added_by":"auto","created_at":"2025-03-31 11:04:06","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":24548,"visible":true,"origin":"","legend":"\u003cp\u003eFamilial segregation of the \u003cem\u003eIL2RG\u003c/em\u003e variant. Pedigree showing X-linked inheritance. Squares: males; circles: females; filled symbols: affected; half-filled: carriers\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6234637/v1/0cd08503f396022281f8642b.jpg"},{"id":79576242,"identity":"31eceb92-ea83-4487-800f-806f0c099f43","added_by":"auto","created_at":"2025-03-31 11:20:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3183907,"visible":true,"origin":"","legend":"\u003cp\u003eHLA typing workflow.\u003cstrong\u003e \u003c/strong\u003eSchematic of HLA compatibility assessment for HSCT\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6234637/v1/f2d724130132ce0c6b04d687.jpg"},{"id":82375490,"identity":"c0b8b06c-30f6-420d-885e-d0ef67597711","added_by":"auto","created_at":"2025-05-09 14:31:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5782078,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6234637/v1/1c598fdd-8b2e-46f5-b5bb-ff7f04ecba50.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Whole Exome Sequencing Identified a Pathogenic IL2RG Variant in Monozygotic Twins with Severe Combined Immunodeficiency","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSevere Combined Immunodeficiency (SCID) comprises a group of rare, monogenic disorders characterized by a block in T lymphocyte development, leading to life-threatening infections in early infancy​ [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Affected infants often inflict with opportunistic fungal, bacterial, or viral infections within the first months of life and, without immune reconstitution, succumb within the first year​ [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. SCID is considered a pediatric medical emergency, given that timely diagnosis and intervention are critical for survival​. The estimated incidence of SCID is approximately 1 in 50,000\u0026ndash;100,000 live births​. Particularly, in regions with high rates of consanguinity, the incidence can be markedly higher, due to the increased prevalence of autosomal recessive forms of SCID​. However, in many developing countries lacking newborn screening programs, SCID cases are frequently underdiagnosed or diagnosed late. Indeed, while several countries have implemented routine newborn screening for SCID using T-cell receptor excision circle (TREC) assays​, most low-income settings still rely on clinical suspicion that results in delays until severe infections manifest. SCID is genetically heterogeneous, with mutations in at least 20\u0026ndash;, and as of recent classifications, up to 18 distinct genes known to cause a SCID phenotype​. These defects span various molecular pathways, including impaired V(D)J recombination (e.g. \u003cem\u003eRAG1\u003c/em\u003e, \u003cem\u003eRAG2\u003c/em\u003e, \u003cem\u003eDCLRE1C\u003c/em\u003e), cytokine signaling abnormalities (e.g. \u003cem\u003eIL2RG\u003c/em\u003e, \u003cem\u003eJAK3\u003c/em\u003e, \u003cem\u003eIL7R\u003c/em\u003e), and metabolic defects (e.g. \u003cem\u003eADA\u003c/em\u003e) ​ [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Among these, X-linked SCID caused by mutations in the \u003cem\u003eIL2RG\u003c/em\u003e gene (encoding the interleukin-2 receptor common gamma chain, γc) is the single most common subtype. A historical cohort study indicated that \u003cem\u003eIL2RG\u003c/em\u003e mutations account for ~\u0026thinsp;40\u0026ndash;50% of all SCID cases​ [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], making this the most prevalent form of the disease. Accordingly, in an analysis of 108 SCID infants, 49 had X-linked \u003cem\u003eIL2RG\u003c/em\u003e mutations (~\u0026thinsp;45% of cases), far exceeding any other genetic subtype​ [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. IL2RG is located on Xq13.1 and encodes the shared γc subunit of at least six cytokine receptors, including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21​ [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This common γ-chain is critical for lymphocyte development and function. Consequently, \u003cem\u003eIL2RG\u003c/em\u003e mutations abrogate multiple cytokine signaling pathways, leading to a characteristic SCID immunophenotype of absent T cells and NK cells with non-functional B cells (T\u003csup\u003e\u0026minus;\u003c/sup\u003e B\u003csup\u003e+\u003c/sup\u003e NK\u003csup\u003e\u0026ndash;\u003c/sup\u003e SCID)​ [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]​. Basically, X-linked SCID is a disease of defective interleukin signaling, explaining its severe combined loss of cellular immunity​. Especially, autosomal recessive forms like \u003cem\u003eJAK3\u003c/em\u003e deficiency phenocopy X-linked SCID because JAK3 is the kinase that associates with γc, underscoring the central role of the IL2RG signaling axis in normal immune development​.\u003c/p\u003e \u003cp\u003eTherapeutic option for SCID is available in the form of immune reconstitution, most commonly through hematopoietic stem cell transplantation (HSCT). Outcomes for HSCT in SCID have improved dramatically over time, and survival rates now exceed 90% for infants who receive a transplant from an HLA-identical sibling donor in the first few months of life​ [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Even with partially matched donors, early transplantation (before the onset of irreversible infections) significantly enhances survival, highlighting the importance of prompt diagnosis​. Experimental gene therapy has also shown success in certain SCID subsets (including \u003cem\u003eIL2RG\u003c/em\u003e deficiency), further expanding treatment options​ [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The key to these favorable outcomes is early identification of affected infants, ideally at birth, before infections occur​ ​[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This has driven the adoption of newborn screening for SCID in many high-income countries, using the TREC assay to detect T-cell lymphopenia in dried blood spots​ [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. TREC screening is highly sensitive for SCID and related T-cell defects​, allowing presymptomatic diagnosis. However, newborn screening by itself does not reveal the underlying genetic cause \u0026ndash; it can flag an infant as likely SCID, but cannot distinguish \u003cem\u003eIL2RG\u003c/em\u003e mutation from other etiologies​. Moreover, some atypical or \"leaky\" SCID cases (with milder T-cell deficits) might initially pass newborn screening, only to present later with immunodeficiency​ [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These limitations underscore the need for comprehensive genetic diagnostics following an abnormal screen or in patients with clinical SCID features in settings without screening.\u003c/p\u003e \u003cp\u003eAdvances in genomic technology, particularly Whole Exome Sequencing (WES), have revolutionized the diagnostic approach to SCID and other primary immunodeficiencies. WES enables rapid, parallel analysis of all protein-coding genes, allowing clinicians to pinpoint pathogenic variants in SCID patients within a single test​ [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This has proven invaluable for confirming the diagnosis and guiding therapy, especially in cases with atypical presentation or in families where the specific genetic defect is not evident. In this regard, using next-generation sequencing methods, over 90% of infants with SCID in contemporary North American cohorts is genetically characterized​ [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. By contrast, traditional diagnostic approaches often required serial candidate gene testing guided by immunophenotype \u0026ndash; a time-consuming process that may be unfeasible when a patient\u0026rsquo;s condition is rapidly deteriorating. Studies have demonstrated that WES can detect novel or unexpected mutations in SCID patients who had initially eluded diagnosis by routine tests​. Furthermore, genetic sequencing is crucial for distinguishing between different genetic forms of SCID that may present with similar clinical and immunologic features (like \u003cem\u003eRAG1/2\u003c/em\u003e mutations causing Omenn syndrome vs. other SCID forms)​ [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Knowing the exact molecular defect has practical implications. It informs family counseling and can influence treatment decisions, such as eligibility for gene therapy trials or the urgency and conditioning regimen for HSCT [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]​. That said, the widespread use of WES has also introduced new challenges, such as the interpretation of variants of uncertain significance (VUS). In some SCID cases, WES uncovers novel missense changes whose functional impact is not immediately clear, necessitating supplementary studies. As a finding, functional assays have been used to confirm the pathogenicity of ambiguous \u003cem\u003eRAG1\u003c/em\u003e variants discovered by sequencing as a definitive diagnosis​.\u003c/p\u003e \u003cp\u003eDiagnosing and managing SCID poses particular challenges in resource-limited settings. Many low- and middle-income countries lack routine newborn screening programs for SCID, and awareness among clinicians may be limited, leading to missed or delayed diagnoses. Infants in these settings often present only after developing severe infections or failure to thrive, at which point opportunistic pathogens (e.g. Bacille Calmette-Gu\u0026eacute;rin (BCG) vaccine strain, given as newborn tuberculosis prophylaxis in many countries) may have already caused disseminated disease​. The delay not only increases immediate mortality risk but can also compromise the success of curative therapy since active infections and organ damage at transplant are associated with worse outcomes​. A further obstacle is the limited availability of advanced diagnostic tools. In the absence of in-country genomic facilities, confirming a suspected SCID diagnosis genetically may rely on sending samples abroad or not be done at all, leaving the genetic subtype unknown​. This lack of definitive diagnosis can impede optimal treatment \u0026ndash; for example, distinguishing \u003cem\u003eIL2RG\u003c/em\u003e deficiency (X-linked) has implications for family screening and donor search, and identifying ADA deficiency might allow enzyme replacement therapy as a bridge to transplant. Moreover, access to HSCT itself is variable in resource-limited regions; even when a genetic diagnosis is made, specialized transplant centers and suitable donors may not be readily accessible. Paradoxically, the regions with higher SCID incidence due to consanguinity are often those with the scarcest resources for early detection and treatment​. This disparity highlights an urgent need for international collaboration and capacity-building, training healthcare providers to recognize SCID, and implementing cost-effective genetic testing (such as targeted sequencing panels or exome sequencing via regional centers) to facilitate prompt diagnosis.\u003c/p\u003e \u003cp\u003eWithin this context, \u003cem\u003eIL2RG\u003c/em\u003e mutations remain a focal point because of their relative frequency and clear therapeutic implications. X-linked SCID cases can be identified by family history or by carrier testing in mothers once a mutation is known, which emphasizes the value of molecular diagnosis. Published reports from the Middle East and Asia have begun to catalog the spectrum of \u003cem\u003eIL2RG\u003c/em\u003e mutations in their SCID populations​. However, data on SCID in certain regions, such as sub-Saharan Africa and parts of South Asia, remain very scarce. Even in countries like Iran, where consanguinity is observed and autosomal recessive SCID (e.g. \u003cem\u003eRAG\u003c/em\u003e deficiencies) might be expected to predominate, X-linked \u003cem\u003eIL2RG\u003c/em\u003e mutations still account for a substantial fraction of cases​. Every new case study adds to the collective knowledge needed to improve outcomes. Accordingly, the present study reports SCID in monozygotic twins, a rare but especially informative, providing a controlled look at genotype-phenotype correlation and the impact of environmental factors on disease course​. Such finding can improve our understanding of SCID pathogenesis and inheritance patterns.\u003c/p\u003e \u003cp\u003eIn this study, we present the case of monozygotic twin infants from a resource-limited setting who were diagnosed with SCID due to a pathogenic \u003cem\u003eIL2RG\u003c/em\u003e mutation, identified through whole exome sequencing. We describe the clinical presentation (including severe recurrent infections and BCGiosis), the immunological findings, and the genetic analysis confirming an X-linked \u003cem\u003eIL2RG\u003c/em\u003e variant, which was verified by family segregation. This report highlights the utility of WES in reaching a definitive diagnosis in the absence of newborn screening and shows the challenges of managing SCID in a setting with limited resources. By integrating genomic data with clinical and immunological evaluation, the critical role of \u003cem\u003eIL2RG\u003c/em\u003e in immune development and advocate for broader implementation of genomic newborn screening and early referral for curative therapy is highlighted. Also, the findings contribute to the growing global registry of SCID mutations and support efforts to ensure that life-saving interventions for SCID, like timely HSCT or gene therapy, become accessible to all patients, regardless of geographic location or resource availability.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Participants and Sample Collection\u003c/h2\u003e \u003cp\u003eThis study involved monozygotic twin male infants (5 months old) presenting with clinical features of SCID, including recurrent bacterial infections and failure to thrive. They had a history of disseminated Bacille Calmette\u0026ndash;Gu\u0026eacute;rin (BCG) infection following newborn vaccination. The infants were referred to a specialized immunology-genetics clinic in Iran after SCID was suspected. Peripheral blood samples (~\u0026thinsp;2.5 mL) were collected in EDTA tubes from each twin and their parents (after informed consent). Genomic DNA was extracted from leukocytes using the QIAamp DNA Blood Mini Kit (Qiagen, Germany) according to the manufacturer\u0026rsquo;s instructions. DNA quality and concentration were assessed by agarose gel electrophoresis and spectrophotometry (NanoDrop 2000, Thermo Fisher Scientific) to ensure high-molecular-weight DNA suitable for sequencing.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWhole Exome Sequencing (WES)\u003c/h3\u003e\n\u003cp\u003eTo identify the genetic cause of immunodeficiency, whole exome sequencing was performed on one twin (the proband). An Illumina NovaSeq 6000 platform (Macrogen, South Korea) was used for high-throughput sequencing. Exome enrichment was done with the SureSelect Human All Exon V7 kit (Agilent Technologies), and a sequencing depth of ~\u0026thinsp;200\u0026times; was achieved. Raw reads were aligned to the human reference genome (GRCh38) using the Burrows-Wheeler aligner, and variant calling was performed with CLC Genomics Workbench v7.7 (QIAGEN). Variant annotation and filtering prioritized rare, protein-altering variants consistent with an X-linked inheritance pattern. Public databases including ClinVar, gnomAD, and dbSNP were consulted to exclude known benign variants and identify novel or pathogenic candidates.\u003c/p\u003e\n\u003ch3\u003ePCR and Sanger Sequencing\u003c/h3\u003e\n\u003cp\u003eCandidate variant validation and family segregation analysis were carried out by PCR amplification and Sanger sequencing of the IL2RG gene. Custom primers flanking the putative mutation site were designed (Primer3Plus/NCBI Primer-BLAST) with the following sequences: IL2RG exon 5 forward 5\u0026prime;-TACTCTTCCTGATACCAGATAG-3\u0026prime; and reverse 5\u0026prime;-CTACTCTAACACACCCCAAC-3\u0026prime;. PCR was performed in a 40 \u0026micro;L reaction containing 20\u0026ndash;50 ng genomic DNA, 0.8 \u0026micro;M of each primer, 2.5 \u0026micro;L of 10\u0026times; PCR buffer, 1 \u0026micro;L of each dNTP (10 mM), 2 \u0026micro;L of MgCl₂ (25 mM), 0.1 \u0026micro;L of Taq DNA polymerase (5 U/\u0026micro;L), and nuclease-free water to volume. Thermocycling conditions were: initial denaturation at 95\u0026deg;C for 5 min; followed by 36 cycles of 95\u0026deg;C for 30 s, 58\u0026deg;C for 30 s, 72\u0026deg;C for 30 s; and a final extension at 72\u0026deg;C for 5 min. PCR products were confirmed on a 1.5% agarose gel and then purified for Sanger sequencing (performed by Macrogen, Korea). Chromatogram analysis (using CLC Workbench) enabled confirmation of the WES-identified variant in the twins and the determination of parental carrier status.\u003c/p\u003e\n\u003ch3\u003eClinical and Immunological Assessments\u003c/h3\u003e\n\u003cp\u003eComprehensive clinical evaluations were conducted. Key features recorded included the frequency and type of infections (e.g. pneumonia, sepsis with Staphylococcus haemolyticus), vaccination history (notably the BCG-related complications), and growth parameters. Laboratory tests revealed profound lymphopenia on complete blood counts (absolute lymphocyte count\u0026thinsp;\u0026lt;\u0026thinsp;300 cells/\u0026micro;L, far below age-matched normals). Flow cytometry of lymphocyte subsets (if available) would show markedly low T cells and NK cells with non-functional B cells, consistent with SCID. A T-cell receptor excision circle (TREC) assay was used as a surrogate for thymic output; both patients had undetectable TRECs in peripheral blood, consistent with absent new T-cell production. In preparation for potential treatment, HLA typing was performed for the twins and their parents at HLA class I (A, B, C) and class II (DRB1, DQB1) loci using high-resolution sequence-based methods. HLA results were used to evaluate the feasibility of hematopoietic stem cell transplantation (HSCT) from family donors.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGenetic Analysis\u003c/h2\u003e \u003cp\u003eWES of the proband identified a hemizygous variant in IL2RG, located on chromosome Xq13.1, that perfectly matched the clinical suspicion of X-linked SCID. The variant was a single nucleotide change c.670C\u0026thinsp;\u0026gt;\u0026thinsp;T in the IL2RG coding sequence, predicting a missense substitution p.Arg224Trp in the common gamma chain protein. This was the only rare, protein-altering variant on the X chromosome that could explain the phenotype. The variant was absent from population databases (gnomAD frequency 0.0%), and in silico predictors (PolyPhen-2, SIFT) indicated it is deleterious. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the genomic findings. Sanger sequencing confirmed the presence of the IL2RG c.670C\u0026thinsp;\u0026gt;\u0026thinsp;T mutation in both twins, and additionally showed that their mother is heterozygous for this variant (carrier), while the father\u0026rsquo;s sequence is wild-type. These results are consistent with X-linked recessive inheritance of the disease-causing mutation. The IL2RG p.Arg224Trp variant has previously been classified as pathogenic in X-SCID patients and was not found in healthy individuals.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eWES results of the proband. \u003cem\u003eKey variant identified by whole exome sequencing in the proband\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenomic Coordinate (GRCh38)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNucleotide Change\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAmino Acid Change\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eZygosity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePredicted Pathogenicity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIL2RG\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX:70,329,165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.670C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep.Arg224Trp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHemizygous (male)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDamaging (PolyPhen-2, SIFT)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNote\u003c/em\u003e: No other candidate variants explaining the immunodeficiency were found on autosomal genes (e.g., \u003cem\u003eRAG1/2, JAK3, ADA\u003c/em\u003e), consistent with \u003cem\u003eIL2RG\u003c/em\u003e being the sole causative mutation.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHLA Typing and Familial Segregation\u003c/h3\u003e\n\u003cp\u003eFamily HLA typing revealed only partial haplotype matches between the twins and their parents, and no fully matched sibling donor was available (the twins have no other siblings). Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the HLA alleles for the proband, mother, and father. The twins shared one HLA haplotype with the mother and one with the father, as expected for an X-linked condition inherited from a carrier mother. For example, the proband is HLA-DRB1*01:01/01:01 (inherited one DRB101 allele from each parent) and HLA-DQB105:01/*05:01 (one DQB105:01 from each parent), indicating haploidentical matches with both parents. The mother and father each share half of their HLA alleles with the affected infants, but neither parent is a full HLA match at all loci. These results indicate that an alternative donor source (such as a haploidentical parental transplant with T-cell depletion or an unrelated donor) would be required for HSCT, as no HLA-identical sibling donor exists.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHLA compatibility results. \u003cem\u003eHLA genotyping of the family. The proband\u0026rsquo;s HLA alleles are compared to those of the mother and father.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFamily Member\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHLA-A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHLA-B\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHLA-C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHLA-DRB1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHLA-DQB1\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProband\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA11, A?*\u003c/p\u003e \u003cp\u003e(No exact match)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB35, B35\u003c/p\u003e \u003cp\u003e(homozygous)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC04, C15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDRB1\u003cem\u003e01:01, DRB1\u003c/em\u003e01:01\u003c/p\u003e \u003cp\u003e(homozygous)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDQB1\u003cem\u003e05:05, DQB1\u003c/em\u003e05:05\u003c/p\u003e \u003cp\u003e(homozygous)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMother\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA30, A32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB35, B35\u003c/p\u003e \u003cp\u003e(homozygous)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC04, C04\u003c/p\u003e \u003cp\u003e(homozygous)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDRB1\u003cem\u003e01:01, DRB1\u003c/em\u003e03:01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDQB1\u003cem\u003e02:01, DQB1\u003c/em\u003e05:05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFather\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA11, A26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB35, B51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC04, C15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDRB1\u003cem\u003e01:01, DRB1\u003c/em\u003e01:01\u003c/p\u003e \u003cp\u003e(homozygous)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDQB1\u003cem\u003e05:05, DQB1\u003c/em\u003e05:05\u003c/p\u003e \u003cp\u003e(homozygous)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNote\u003c/b\u003e: The proband\u0026rsquo;s HLA-A alleles (likely A11 and A30) each match one parent (A11 from father, A30 from mother), but there is no overlap between parental A alleles; hence, no exact HLA-A match is shared by both parents.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eClinical Findings\u003c/h3\u003e\n\u003cp\u003eBoth twins manifested severe clinical symptoms of immunodeficiency early in life. They suffered recurrent, hard-to-treat infections. Notably, each developed pneumonia and sepsis with organisms such as Staphylococcus haemolyticus, and both had disseminated BCG infection (BCG-osis) following administration of the live attenuated BCG vaccine at birth. The disseminated mycobacterial infection was confirmed by finding granulomas on biopsy and acid-fast bacilli in a bone marrow aspirate. Immunologically, the infants were profoundly lymphopenic: total lymphocyte counts were \u0026lt;\u0026thinsp;300 cells/\u0026micro;L (normal for age is \u0026gt;\u0026thinsp;4,000/\u0026micro;L). There was an absence of functional T cells (CD3\u003csup\u003e+\u003c/sup\u003e lymphocytes extremely low) and NK cells, whereas B cells were present (T\u003csup\u003e\u0026minus;\u003c/sup\u003e B\u003csup\u003e+\u003c/sup\u003e NK\u003csup\u003e\u0026minus;\u003c/sup\u003e phenotype). However, the B cells were non-functional, evidenced by extremely low immunoglobulin levels and lack of specific antibody production despite infection, consistent with the need for T-cell help. The TREC assay was undetectable in both patients, indicating virtually no new T cells emerging from the thymus. These findings confirmed a diagnosis of X-linked SCID (T⁻B⁺NK⁻ subtype). Both infants required isolation and prophylactic antimicrobial interventions while a definitive therapy was planned.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Sanger Sequencing Chromatogram of the IL2RG c.670C\u0026thinsp;\u0026gt;\u0026thinsp;T Variant. Chromatogram demonstrating the hemizygous c.670C\u0026thinsp;\u0026gt;\u0026thinsp;T mutation in the IL2RG gene in the proband. The arrow highlights the nucleotide substitution that results in the p.Arg224Trp amino acid change Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Pedigree Diagram Illustrating X-Linked Inheritance. Family pedigree showing the X-linked recessive transmission of the IL2RG mutation. Affected individuals (filled symbols) include the monozygotic twin boys, while the mother (half-filled symbol) is an asymptomatic carrier and the father (open symbol) is unaffected Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Schematic Overview of HLA Typing for HSCT Donor Selection\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDiagram outlining the high-resolution HLA typing workflow used to assess donor compatibility. The figure displays the HLA-A, -B, -C, -DRB1, and -DQB1 allele distributions among the proband and his parents, emphasizing the partial haplotype matches critical for selecting a suitable donor for hematopoietic stem cell transplantation\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis report provides a detailed genetic and clinical investigation of X-SCID in identical twin brothers. As a results, a hemizygous c.670C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Arg224Trp) mutation was identified in \u003cem\u003eIL2RG\u003c/em\u003e as the cause of their condition. The \u003cem\u003eIL2RG\u003c/em\u003e gene encodes the common γc, a crucial subunit shared by multiple cytokine receptors, including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 that regulate lymphocyte development [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Disruption of the common γc due to \u003cem\u003eIL2RG\u003c/em\u003e mutations impairs signaling through all these cytokines, arresting the maturation of T cells and NK cells, while B cells develop but cannot function properly without T-cell help​. This immunologic mechanism explains the classic SCID phenotype observed in the patients, and aligns with the fact that mutations in \u003cem\u003eIL2RG\u003c/em\u003e are the single most common cause of SCID globally [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Large cohort studies have shown that X-linked \u003cem\u003eIL2RG\u003c/em\u003e deficiency accounts for roughly 40\u0026ndash;50% of all SCID cases​ [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe specific \u003cem\u003eIL2RG\u003c/em\u003e variant in this twins (p.Arg224Trp) is a known pathogenic mutation associated with X-SCID. It lies in the extracellular domain of the γc protein, and the substitution of a positively charged arginine with a bulky tryptophan is predicted to disrupt the receptor\u0026rsquo;s structure and its interaction with interleukins. Indeed, this variant has been reported in multiple unrelated SCID patients and is not found in healthy population databases [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These findings are consistent with previous reports that \u003cem\u003eIL2RG\u003c/em\u003e missense mutations abolish common γc receptor function, leading to the absence of T and NK cells and nonfunctional B cells​[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. By confirming the mutation in both twins and the carrier state in their mother, a genetic evidence of X-linked inheritance us provided, which is valuable information for family counseling such as a risk to future male children.\u003c/p\u003e \u003cp\u003eClinically, the twins\u0026rsquo; presentation was characteristic of SCID. They experienced life-threatening infections within months of birth and did not respond normally to vaccines and developed disseminated disease from an attenuated vaccine strain. If untreated, infants with SCID typically succumb to severe infections within the first year of life​ [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], reflecting the absolute dependence of the adaptive immune system on functional T cells. In this twins, the absence of TRECs was a key laboratory finding. TRECs are circular DNA fragments excised during T-cell receptor gene rearrangement in the thymus, and they serve as a biomarker for recent thymic emigrants​ [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The twins\u0026rsquo; undetectable TREC levels indicate almost complete failure of thymic T-cell production, consistent with X-SCID. These results mirror what is seen in newborn screening programs for SCID: babies with absent or extremely low TRECs are flagged for urgent evaluation and often found to have SCID​ [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Thanks to newborn screening, many SCID cases in developed programs are now identified at birth \u003cem\u003ebefore\u003c/em\u003e infections occur​ [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Unfortunately, no such screening was in place for our patients in the time of this study. Their diagnosis was made after clinical illness had begun, illustrating the pediatric emergency nature of SCID \u0026ndash; early diagnosis is crucial to prevent fatal infections​.\u003c/p\u003e \u003cp\u003eThe definitive treatment for SCID is allogeneic HSCT, which can reconstitute a functional immune system in the patient [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. HSCT outcomes are vastly improved when performed in the first months of life, ideally before the infant has acquired severe infections​ [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Historical data and recent multicenter studies concur that infants transplanted at \u0026le;\u0026thinsp;3.5 months of age have survival rates\u0026thinsp;\u0026gt;\u0026thinsp;90% [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the case of the twins, the HLA typing results indicated that neither had an HLA-identical sibling donor. The best available options would likely be a haploidentical parental transplant (using the father or mother as a donor, with T-cell depletion to prevent graft-versus-host disease) or searching for a well-matched, unrelated donor. It is well documented that any delay in performing HSCT for SCID correlates with worse survival, largely due to infections acquired while waiting for a transplant​. Therefore, upon genetic confirmation of SCID, an expeditious referral for HSCT is recommended even if only a haploidentical donor is available, as outcomes with parental donors can be life-saving when done early in life​ [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The present study also highlights the disparities in healthcare infrastructure. Regions without newborn screening programs or easy access to transplant centers face significant challenges in managing SCID. In countries like Iran (where our patients reside), SCID diagnoses often rely on clinical recognition of infections and failure to thrive, which may occur late in the disease course​. This delay leads to higher infection-related morbidity and mortality. Moreover, areas with a high rate of consanguineous marriages see a greater prevalence of autosomal recessive forms of SCID and other primary immunodeficiencies, further increasing the burden of undiagnosed cases​. These findings emphasize the urgent need for implementing population-wide SCID newborn screening in such regions. Even a simple TREC-based DNA test on Guthrie cards can identify SCID in the first days of life, enabling prompt protective measures and timely curative therapy [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Alongside screening, genetic counseling and carrier testing in families with known X-SCID is essential. In the case of the twins, identification of the carrier mother allows informed reproductive choices and early testing of future male infants in the extended family.\u003c/p\u003e \u003cp\u003eAnother important aspect of this case is the demonstration of how genomic technologies complement immunological tests for a precise diagnosis. Traditional diagnostics for suspected SCID include lymphocyte phenotyping and functional assays like mitogen proliferation tests, which can confirm an immunodeficiency but not its genetic cause. Here, WES was decisive in pinpointing \u003cem\u003eIL2RG\u003c/em\u003e as the mutated gene, which not only clinched the diagnosis of X-SCID but also guided the family workup, identifying the carrier mother. The power of WES to reveal the molecular basis of rare diseases has revolutionized medical genetics​ [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It is especially valuable in genetically heterogeneous conditions like SCID, which can result from mutations in over a dozen different genes​ [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Recent studies from Iran and other countries with limited resources have shown that applying WES can significantly improve the diagnostic yield for patients with primary immunodeficiencies, uncovering mutations that targeted gene panels might miss​ [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Our report adds to this body of evidence, illustrating that even in a resource-constrained setting, integrating WES into the diagnostic workflow for infants with severe immune syndromes is feasible and impactful. By identifying a specific genetic defect, we can tailor the management and provide accurate genetic counseling. In cases where WES identifies variants of uncertain significance, further functional studies by measuring cytokine signaling in patient T cells or targeted gene assays may be needed​.\u003c/p\u003e \u003cp\u003eLooking forward, advancements in therapy, such as gene therapy, offer hope to patients with X-SCID. Early trials demonstrated that integrating a correct copy of \u003cem\u003eIL2RG\u003c/em\u003e into patient hematopoietic stem cells could reconstitute T-cell immunity, but some patients developed leukemia due to retroviral vector insertion near oncogenes [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In recent years, improved vectors (such as self-inactivating lentiviral vectors) have made gene therapy for SCID much safer, and new trials have shown successful immune reconstitution without unexpected cancers​ [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Gene therapy has thus become an alternative for patients who lack a suitable HSCT donor or who have contraindications to transplantation​ [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In the context of the presented case, gene therapy could be considered, although at present HSCT from a haploidentical donor is the standard approach. Long-term follow-up of SCID patients treated with HSCT or gene therapy is an important need​. Issues such as chronic graft-versus-host disease, incomplete immune reconstitution like poor B-cell function requiring lifelong immunoglobulin replacement, or late complications like secondary malignancies need to be monitored in survivor cohorts. Fortunately, outcomes for X-SCID have dramatically improved over the past few decades with \u0026gt;\u0026thinsp;90% of infants now surviving if treated early​ [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This study of cases with X-SCID contributes to the literature by expanding the known mutation spectrum of \u003cem\u003eIL2RG\u003c/em\u003e by adding p.Arg224Trp as a recurrent pathogenic variant and by emphasizing the multi-faceted approach required \u0026ndash; from genomics to clinical care \u0026ndash; to achieve the best outcomes for SCID patients.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study highlights the critical importance of early genetic diagnosis in infants with SCID and the life-saving potential of early intervention. The identification of the \u003cem\u003eIL2RG\u003c/em\u003e c.670C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Arg224Trp) mutation in these twins enabled a timely, definitive diagnosis and informed treatment planning. Here, the routine use of whole exome sequencing or comprehensive genetic panels in any infant suspected of SCID was advocated, especially in settings without newborn screening. Such an approach can rapidly confirm the diagnosis, guiding urgent treatment like HSCT, which offers the chance for a normal life​. This study also supports efforts to expand newborn screening programs globally and to improve access to transplantation and emerging gene therapies for patients in all regions​​. Through early detection and intervention, the historically dismal prognosis of SCID, the \u0026ldquo;bubble boy\u0026rdquo; disease, can be converted into excellent long-term survival and quality of life for affected children.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eAuthorship contributions\u003c/p\u003e\n\u003cp\u003eThe author contributed substantially to the conception, design, data acquisition, analysis, and interpretation of the study. Seyed Pooria Tadayon Nabavi led the project, coordinated the study design, and drafted the manuscript. The coauthor critically revised the manuscript for intellectual content, approved the final version, and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003eConflicts of interest/Competing interests\u003c/p\u003e\n\u003cp\u003eThe author has no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was self-founded, and the author has provided all supports to perform the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. All relevant data supporting the findings of this study are included in the manuscript.\u003c/p\u003e\n\u003cp\u003eEthics approval\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Islamic Azad University, Damghan Branch, Dāmghān, Iran.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsent to participate\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from the parents.\u003c/p\u003e\n\u003cp\u003eConsent to publish\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFischer A, Notarangelo LD, Neven B, Cavazzana M, Puck JM. Severe combined immunodeficiencies and related disorders. Nat Rev Dis Primers [Internet]. 2015 [cited 2025 Mar 13];1. 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Advances in the understanding and treatment of human severe combined immunodeficiency. Immunol Res [Internet]. 2000 [cited 2025 Mar 13];22:237\u0026ndash;51. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmed.ncbi.nlm.nih.gov/11339359/\u003c/span\u003e\u003cspan address=\"https://pubmed.ncbi.nlm.nih.gov/11339359/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePai S-Y, Logan BR, Griffith LM, Buckley RH, Parrott RE, Dvorak CC et al. Transplantation outcomes for severe combined immunodeficiency, 2000\u0026ndash;2009. N Engl J Med [Internet]. 2014 [cited 2025 Mar 13];371:434\u0026ndash;46. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmed.ncbi.nlm.nih.gov/25075835/\u003c/span\u003e\u003cspan address=\"https://pubmed.ncbi.nlm.nih.gov/25075835/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePavel-Dinu M, Wiebking V, Dejene BT, Srifa W, Mantri S, Nicolas CE et al. Gene correction for SCID-X1 in long-term hematopoietic stem cells. Nature Communications 2019 10:1 [Internet]. 2019 [cited 2025 Mar 14];10:1\u0026ndash;15. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.nature.com/articles/s41467-019-09614-y\u003c/span\u003e\u003cspan address=\"https://www.nature.com/articles/s41467-019-09614-y\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlanco E, Izotova N, Booth C, Thrasher AJ. Immune Reconstitution After Gene Therapy Approaches in Patients With X-Linked Severe Combined Immunodeficiency Disease. Front Immunol [Internet]. 2020 [cited 2025 Mar 14];11:608653. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pmc.ncbi.nlm.nih.gov/articles/PMC7729079/\u003c/span\u003e\u003cspan address=\"https://pmc.ncbi.nlm.nih.gov/articles/PMC7729079/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Severe combined immunodeficiency, IL2RG, Whole Exome Sequencing, Genetic diagnosis, Hematopoietic stem cell transplantation","lastPublishedDoi":"10.21203/rs.3.rs-6234637/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6234637/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eThis study aimed to determine the genetic cause of severe combined immunodeficiency (SCID) in monozygotic twin male infants who presented with recurrent severe infections and disseminated BCG-related complications.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWhole Exome Sequencing (WES) was performed on one twin to identify candidate pathogenic variants. The detected IL2RG variant was validated through PCR and Sanger sequencing, and family segregation analysis was conducted. Comprehensive immunological assessments, including T-cell receptor excision circle (TREC) assays and lymphocyte profiling, were employed. High-resolution HLA typing was also carried out to evaluate donor compatibility for hematopoietic stem cell transplantation (HSCT).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWES revealed a hemizygous c.670C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Arg224Trp) variant in the IL2RG gene, a change absent from population databases and predicted to be deleterious by in silico tools. Sanger sequencing confirmed the variant in both twins, and maternal heterozygosity was identified, supporting an X-linked recessive inheritance pattern. Clinically, the twins exhibited profound lymphopenia and undetectable TRECs, consistent with a T⁻B⁺NK⁻ immunophenotype. HLA typing demonstrated only partial haplotype matches with the parents, indicating the necessity for a haploidentical transplant approach.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIntegrating genomic analysis with immunological profiling effectively pinpointed a pathogenic IL2RG mutation as the cause of SCID in these twins. Early genetic diagnosis using WES is critical for guiding timely therapeutic interventions such as HSCT or gene therapy and underscores the need for implementing newborn screening and carrier testing programs, particularly in resource-limited settings.\u003c/p\u003e","manuscriptTitle":"Whole Exome Sequencing Identified a Pathogenic IL2RG Variant in Monozygotic Twins with Severe Combined Immunodeficiency","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-31 11:04:01","doi":"10.21203/rs.3.rs-6234637/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"42c60ac7-71bf-477a-bc94-50544aa02f12","owner":[],"postedDate":"March 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-09T14:23:49+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-31 11:04:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6234637","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6234637","identity":"rs-6234637","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}