Hirokazu Kanegane

Epstein–Barr virus (EBV) primarily infects B cells but can occasionally target T and/or NK cells, leading to chronic active EBV disease or EBV‐associated hemophagocytic lymphohistiocytosis, which are often difficult to treat. EBV‐associated diseases exhibit a wide range of phenotypes. EBV‐infected cells play a major role in pathogenesis, making their identification essential for diagnosis. EBV‐encoded small RNA (EBER) flow fluorescence in situ hybridization (FISH), combined with multicolor flow cytometry, allows highly sensitive detection of EBV‐infected cells, even from limited samples. However, EBER flow FISH is labor‐intensive and requires technical expertise, limiting its use in standard facilities. We investigated whether the technique used in our department could be successfully implemented in other facilities. We performed EBER flow FISH at our department (Facility A) on cryopreserved peripheral blood mononuclear cell samples from seven patients diagnosed with EBV‐associated diseases. The same assay was then conducted at Facility B, and results were compared for cell populations and the frequency of EBER‐positive cells within each population. Significant correlations were observed between facilities for both cell fraction proportions and the proportion of EBER‐positive cells. Among data points where both facilities detected ≥5% EBER‐positive cells, 6 of 8 data points (75%) were within the acceptance interval of ±20%. Although one patient with very low proportions of EBER‐positive cells showed inconsistent findings, overall results did not differ significantly between facilities. Although further validation in larger multicenter studies is required, these findings suggest preliminary interlaboratory comparability and support the potential transferability of EBER flow FISH across institutions.

Clinical targeted sequencing identified low-VAF KRAS variants in two of 26 children with childhood-onset systemic lupus erythematosus (SLE). Both had severe inflammatory manifestations but lacked Noonan syndrome features, and neither developed persistent lymphoproliferation suggestive of RAS-associated autoimmune lymphoproliferative disease during follow-up. These findings suggest that KRAS mosaicism may contribute to the development or severity of childhood-onset SLE.

A cluster of cases of IRAK4 deficiency has been identified in Okinawa Prefecture, Japan. All affected families carry the homozygous c.123_124insA variant, suggesting a founder effect and supporting the implementation of targeted newborn screening of IRAK4 deficiency in Okinawa.

Multidisciplinary genomic evaluation is increasingly recognized for its diagnostic and therapeutic implications in adults with suspected inborn errors of immunity (IEI) presenting with rheumatic and musculoskeletal disease (RMD) phenotypes. We retrospectively analyzed 50 adults with suspected IEI who underwent genetic testing and were pre-classified into immunodeficiency (ID), autoinflammatory disorders (AID), and non-ID/AID groups. Genetic findings, clinical classification, treatment modifications, and exploratory machine learning analyses were evaluated. A final genetic diagnosis consistent with IEI was identified in 15 patients (30.0%), with the highest yield in the non-ID/AID group (44.4%). Variants were most frequently associated with autoinflammatory diseases (40.0%). Four patients, all initially classified as non-ID/AID, were reclassified based on genetic findings and IUIS classification. Treatment was modified in 12 patients, including eight genetically diagnosed patients and three genotype-driven interventions. Two patients died from disease-related complications. Machine learning analyses provided heterogeneous feature contributions across groups. These findings highlight the utility of multidisciplinary genomic evaluation for refining diagnoses in challenging adult patients.

Activated phosphoinositide 3-kinase delta syndrome (APDS) is a form of common variable immunodeficiency (CVID) caused by gain-of-function variants in PIK3CD (APDS1) or loss-of-function variants in PIK3R1 (APDS2), resulting in hyperactivation of the PI3Kδ pathway. Clinically, it is characterized by recurrent respiratory infections, lymphadenopathy, splenomegaly, and progressive airway destruction, often leading to bronchiectasis. Enteropathy, cytopenia, and immune dysregulation have also been observed in several patients. Some patients develop malignant lymphomas, which can be fatal. Prophylactic antimicrobial therapy and regular immunoglobulin replacement therapy are administered in a manner similar to that used for CVID. However, since excessive activation of the class IA phosphoinositide 3-kinase signaling pathway is the primary pathophysiology of APDS, mammalian target of rapamycin inhibitors and selective p110δ inhibitors are expected to be licensed therapies. Early diagnosis based on diagnostic clues specific to APDS, followed by prompt treatment, is expected to improve patient prognosis.

X-linked hyper-IgM syndrome (XHIGM) is an inborn error of immunity caused by variants in the CD40LG gene, leading to disrupted T-cell function and impaired immunoglobulin class-switch recombination. Supportive care alone is linked to unfavorable outcomes, necessitating allogeneic hematopoietic cell transplantation (HCT) as a curative option. Although myeloablative conditioning is recommended for HCT, concerns remain regarding its toxicity. We reviewed ten patients with XHIGM who underwent HCT using reduced-toxicity conditioning (RTC) at our institution between 2010 and 2024. All patients underwent HCT by 5 years of age (range: 2–5 years; median: 3 years). Donor sources included unrelated bone marrow, unrelated cord blood, and haploidentical-related donors. Conditioning primarily consisted of fludarabine and busulfan (target area under the curve: 60–65 mg/L·h), with low-dose total body irradiation (TBI) administered in five patients. Two patients experienced graft failure, requiring a second transplantation, and one died from post-transplant complications. The remaining nine patients survived. The 5-year overall survival rate was 88.9%; the 5-year graft-versus-host disease-free, relapse-free, and second transplantation-free survival rate was 67.5%. The addition of low-dose TBI to fludarabine/busulfan was associated with favorable long-term donor chimerism, although this finding should be considered hypothesis-generating. RTC may represent a feasible conditioning strategy for XHIGM.

Griscelli syndrome type 2 (GS2), caused by biallelic RAB27A variants, is classically associated with hypopigmentation and life-threatening hemophagocytic lymphohistiocytosis (HLH). However, a few patients exhibit normal pigmentation, termed GS2 sine albinism. We identified two unrelated Japanese patients with this presentation caused by compound heterozygous RAB27A variants, representing the first functionally characterized cases among Japanese patients. Functional studies were performed using RAB27A-null cell systems to evaluate the two allele pairs: p.Trp73Arg/p.Val143Ala (patient 1) and p.Ser106PhefsTer18/p.Ser115Arg (patient 2). The truncating allele p.Ser106PhefsTer18 and the missense allele p.Trp73Arg failed to restore peripheral melanosome distribution and lost binding to both melanophilin (MLPH) and MUNC13-4. In contrast, p.Val143Ala and p.Ser115Arg preserved MLPH interaction but showed reduced binding to MUNC13-4. These findings demonstrate that RAB27A variants can dissociate melanosome transport from immune effector function in an allele-dependent manner. Importantly, the absence of hypopigmentation does not exclude GS2. Combined genetic and functional analyses are essential for precise variant interpretation and for guiding timely curative intervention in GS2.