Detection of the off-target effects of base editors is important for identifying their safety risks but current methods for understanding their global activities have limitations in terms of sensitivity or bias by computationally selecting a subset of sites for experimental analysis. We present CHANGE-seq-BE, a method to assess the guide RNA-dependent off-target profile of both adenine and cytosine base editors that is simultaneously sensitive and unbiased. CHANGE-seq-BE relies on selective sequencing of base-editor-modified genomic DNA in vitro and provides comprehensive identification of genome-wide off-target mutations. We found that 98.8% of validated off-target sites were unique to ABE8e adenine base editors compared to Cas9 nuclease, suggesting substantially higher off-target activity of the former. We further applied CHANGE-seq-BE to support genotoxicity studies in an emergency investigational new drug application for customized adenine base editor treatment for a person with CD40L-deficient X-linked hyper IgM syndrome. Our results emphasize the importance of using a base-editor-specific method for identifying off-target activity.

Talaromyces marneffei (TM), a temperature-dependent dimorphic fungus and opportunistic pathogen, poses a significant threat to immunocompromised individuals, particularly in Southeast Asian regions such as China and India. This case report details an 8-month-old HIV negative Chinese infant with recurrent cough and fever, who was diagnosed with TM infection through blood culture and metagenomic next-generation sequencing (mNGS). Additionally, whole exome sequencing identified a point mutation (c.346+1G>T) in the child's CD40LG gene, primary immunodeficiency calized to chromosome position chrX:135736590, leading to X-linked Hyper IgM Syndrome (XHIGM). The patient was managed with intravenous immunoglobulin (IVIG) and a 12-day course of amphotericin B and itraconazole, which led to significant clinical improvement and discharge on a quarterly IVIG regimen. However, he required readmission for recurrent TM pneumonia at 9 and 40 months post-discharge. This case highlights the diagnostic challenge and management complexity of TM infection in the context of primary immunodeficiency. Primary immunodeficiencies are a heterogeneous group of inherited disorders affecting the immune system, with more than 450 distinct genetic defects identified to date. Patients with an X-linked immunodeficiency typically present with frequent and recurrent infections alongside immune dysregulation manifesting as autoimmunity, lymphoproliferation, granuloma formation, chronic inflammatory diseases, and increased susceptibility to malignancies. Among this group of disorders, 7 major X-linked immunodeficiency disorders have been well-characterized: X-linked chronic granulomatous disease (X-CGD), X-linked hyper-IgM syndrome, X-linked lymphoproliferative syndrome (XLP), Wiskott-Aldrich syndrome (WAS), X-linked severe combined immunodeficiency (X-SCID), X-linked agammaglobulinemia (XLA), and IPEX (Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked) syndrome. These conditions result in cellular or humoral immune deficiency, leading to serious infections and increased morbidity and mortality from early life. Identifying carriers remains challenging in the absence of family history, despite advances in genetic testing. CD40 ligand deficiency, a disorder of abnormal T- and B-cell function, is characterized by low serum concentrations of immunoglobulin (Ig) G, IgA, and IgE with normal or elevated serum concentrations of IgM. Mitogen proliferation may be normal, but NK- and T-cell cytotoxicity can be impaired. Antigen-specific responses are usually decreased or absent. Total numbers of B cells are normal but there is a marked reduction of class-switched memory B cells. Defective oxidative burst of both neutrophils and macrophages has been reported. The range of clinical findings varies, even within the same family. More than 50% of males with CD40 ligand deficiency develop symptoms by age one year, and more than 90% are symptomatic by age four years. CD40 ligand deficiency usually presents in infancy with recurrent upper- and lower-respiratory tract bacterial infections, opportunistic infections including Pneumocystis jirovecii pneumonia, and recurrent or protracted diarrhea that can be infectious or noninfectious and is associated with faltering growth. Neutropenia is common; thrombocytopenia and anemia are less commonly seen. Autoimmune and/or inflammatory disorders (such as sclerosing cholangitis) as well as increased risk for neoplasms have been reported as medical complications of this disorder. Significant neurologic complications, often the result of a central nervous system infection, are seen in 5%-15% of affected males. Liver disease, a serious complication of CD40 ligand deficiency once observed in more than 80% of affected males by age 20 years, may be decreasing with adequate screening and treatment of Cryptosporidium infection. The diagnosis of CD40 ligand deficiency is established in a male proband with typical clinical and laboratory findings and a hemizygous pathogenic variant in CD40LG identified by molecular genetic testing. Targeted therapy: Hematopoietic stem cell transplantation (the only curative treatment currently available) is ideally performed before age ten years or prior to evidence of organ dysfunction. Treatment of manifestations: Ig replacement therapy (either intravenous or subcutaneous); appropriate antimicrobial therapy for acute infections; antimicrobial prophylaxis for opportunistic infection against Pneumocysitis jirovecii pneumonia; recombinant granulocyte colony-stimulating factor for chronic neutropenia; immunosuppressants for autoimmune disorders. Agents/circumstances to avoid: Areas that place the affected individual at risk of contracting Cryptosporidium including pools, lakes, ponds, or certain water sources; drinking unpurified or unfiltered water; live vaccines such as rotavirus, MMR, varicella, live attenuated polio, and BCG. Surveillance: At least annually, complete blood count with differential to monitor for cytopenias, testing of IgG levels and lymphocyte subpopulations, and pulmonary function tests after age seven years. Regular assessment of liver function, with consideration of abdominal imaging, and polymerase chain reaction-based testing for the presence of enteric pathogens including Cryptosporidium. Monitor growth and general health with a low threshold for lymph node biopsy given elevated oncologic risk. Evaluation of relatives at risk: It is appropriate to clarify the genetic status of newborn at-risk male relatives of an affected individual to allow early diagnosis and prompt initiation of treatment and prevention of infections. CD40 ligand deficiency is inherited in an X-linked manner. The risk to sibs of a male proband depends on the genetic status of the mother. If the mother of the proband has a pathogenic variant in CD40LG, the chance of the mother transmitting it in each pregnancy is 50%: males who inherit the pathogenic variant will be affected; females who inherit the pathogenic variant will be heterozygotes. Heterozygous females are typically asymptomatic but may have a range of clinical manifestations depending on X-chromosome inactivation. Once the CD40LG pathogenic variant has been identified in an affected family member, heterozygote testing for at-risk female relatives and prenatal/preimplantation genetic testing for CD40 ligand deficiency are possible.

Introduction: Renal AA amyloidosis with X-linked hyper-IgM immunodeficiency is rare diseases, and their simultaneous presentation in the same patient is exceptional. Case Presentation: We present a case of renal AA amyloidosis in a 20-year-old man with nephrotic syndrome and reduced glomerular filtration rate (GFR). Clinically, serologically, histopathological, and genetically, we confirm renal amyloidosis in the presence of X-linked hyper-IgM syndrome; in turn, we detected a new hemizygous pathogenic variant in the CD40L gene (c.345delA). Conclusion: Our hypothesis suggests that these conditions predisposed the patient to a combined (cellular and humoral) immunodeficiency, leading to recurrent infectious episodes throughout his life, ultimately resulting in renal amyloidosis due to deposition of serum amyloid protein.

Immune dysregulation in children can lead to a variety of health issues, including infections, allergies and autoimmune diseases. However, the coexistence of autoimmune diseases and primary immunodeficiency disorders is extremely rare in clinical practice. A 4-year-old male patient was admitted in July 2017 with joint swelling and pain, alongside a history of recurrent respiratory infections and severe pneumonia. Physical examination revealed tenderness and swelling in multiple joints, and laboratory tests indicated elevated inflammatory markers. Imaging studies showed joint effusion and inflammatory lesions in the lungs. He was diagnosed with rheumatoid factor-negative polyarticular juvenile idiopathic arthritis (PJIA) and treatment was initiated with naproxen, methotrexate and etanercept, leading to significant symptom improvement. In July 2019, following a decline in immunoglobulin (Ig) M (IgM) levels (IgM 0.36 g/L) and recurrent infections, genetic testing was conducted, revealing a frameshift mutation in the CD40LG gene (c.621dup A, p.A208Sfs * 23), which confirmed the diagnosis of X-linked hyper IgM syndrome (XHIGM). The treatment regimen was adjusted to include monthly intravenous Ig infusions and prophylactic antibiotics, significantly reducing the frequency of respiratory infections. By January 2021, PJIA was in clinical remission, allowing for the discontinuation of immunosuppressive therapy, with follow-ups indicating continued recovery without discomfort. In conclusion, this case underscores the rare coexistence of XHIGM and PJIA in the field of pediatrics and identified a new pathogenic variant in CD40LG, enhancing our understanding of the clinical management of individuals with concurrent autoimmune and immunodeficiency disorders.

Hyper IgM syndrome (HIGM) is a rare immunodeficiency caused by impaired immunoglobulin class switching, leading to recurrent infections. The present report describes the case of an 18-year-old man initially diagnosed with common variable immunodeficiency at 3 years of age. Genetic analysis revealed a hemizygous CD40LG missense variant (p.Arg203Ile) associated with X-linked HIGM (XHIGM). Structural and flow cytometric analyses indicated normal CD40 ligand (CD40L) expression on activated CD4+ T-cells but impaired CD40 binding, indicating disrupted immune signaling. Notably, the patient experienced neither bacterial infections requiring hospitalization nor opportunistic infections during 15 years of immunoglobulin replacement therapy. These findings indicate that the p.Arg203Ile variant destabilizes CD40L-CD40 interactions without affecting CD40L expression, suggesting a hypomorphic phenotype. This report highlights the importance of combining genetic testing with functional analysis when evaluating atypical XHIGM presentations to predict clinical severity and provide a scientific basis for personalized treatment strategies. Additional studies are required to assess the long-term outcomes and potential curative therapies for similar cases.