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We present a case of X-linked thrombocytopenia (XLT) with a novel WAS gene variant expressing a normal amount of Wiskott-Aldrich syndrome protein (WASp) in lymphocytes. XLT usually decreases WASp expression not only in platelets, but also in lymphocytes. However, there were cases, such as the present one, in which WASp was expressed normally in lymphocytes and absent only in platelets. Our finding suggests that it is of greater diagnostic sensitivity to perform an expression analysis of WASp in both platelets and lymphocytes when XLT is suspected.

Key Clinical Message. A 15‐year‐old girl developed inherited cardiomyopathy and macrothrombocytopenia revealing pathogenic variants of both MYH7 and MYH9 genes. This underlies the importance of repeated genetic testing in diagnosing and managing inherited disorders. Abstract The MYH7 and MYH9 genes encode for distinct myosin heavy chain proteins. Our case features a 15‐year‐old girl, presenting with inherited cardiomyopathy and macrothrombocytopenia, revealing distinct pathogenic variants of both MYH7 and MYH9 genes. This underlines the relevance of genetic testing and personalized medicine in diagnosing and managing inherited disorders.

Abstract Introduction Adenosine deaminase (ADA) deficiency is an ultrarare inherited purine metabolism disorder characterized by severe combined immunodeficiency. Elapegademase‐lvlr is a new pegylated recombinant bovine ADA used in enzyme‐replacement therapy (ERT) for ADA deficiency. Therefore, replacement with the new drug may eliminate the infectious risks associated with the currently used bovine intestinal‐derived product, pegademase. Methods We conducted a multicenter, single‐arm, open‐label, phase 3, and postmarketing clinical study of elapegademase for patients with ADA deficiency. The following biochemical markers were monitored to determine an appropriate dose of elapegademase: the trough deoxyadenosine nucleotide (dAXP) level ≤0.02 μmol/mL in erythrocytes or whole blood and the trough serum ADA activity ≥1100 U/L (equivalent to plasma levels ≥15 μmol/h/mL) indicated sufficient enzyme activity and detoxification as efficacy endpoints and monitored adverse events during the study as safety endpoints. Results A total of four patients (aged 0–25 years) were enrolled. One infant patient died of pneumonia caused by cytomegalovirus infection whereas the other three completed the study and have been observed in the study period over 3 years. The infant patient had received elapegademase at 0.4 mg/kg/week until decease and the others received elapegademase at maximum doses of 0.3 mg/kg/week for 164–169 weeks. As a result, all four patients achieved undetectable levels of dAXPs together with sufficient enzyme activity, increased T and B cell numbers, and slightly elevated and maintained IgM and IgA immunoglobulin levels. Serious adverse events occurred in three patients, all of which were assessed as unrelated to elapegademase. Conclusions This study showed that elapegademase had comparable safety and efficacy to pegademase as ERT for ADA deficiency by demonstrating stable maintenance of sufficient ADA activity and lowering dAXP to undetectable levels, while no drug‐related adverse events were reported (Trial registration: JapicCTI‐163204).

Two patients with adenosine deaminase (ADA)-deficient severe combined immunodeficiency (ADA-SCID) received stem cell-based gene therapy (SCGT) using GCsapM-ADA retroviral vectors without preconditioning in 2003 and 2004. The first patient (Pt1) was treated at 4.7 years old, and the second patient (Pt2), who had previously received T cell gene therapy (TCGT), was treated at 13 years old. More than 10 years after SCGT, T cells showed a higher vector copy number (VCN) than other lineages. Moreover, the VCN increased with differentiation toward memory T and B cells. The distribution of vector-marked cells reflected variable levels of ADA requirements in hematopoietic subpopulations. Although neither patient developed leukemia, clonal expansion of SCGT-derived clones was observed in both patients. The use of retroviral vectors yielded clonal dominance of vector-marked clones, irrespective of the lack of leukemic changes. Vector integration sites common to all hematopoietic lineages suggested the engraftment of gene-marked progenitors in Pt1, who showed severe osteoblast (OB) insufficiency compared to Pt2, which might cause a reduction in the stem/progenitor cells in the bone marrow (BM). The impaired BM microenvironment due to metabolic abnormalities may create space for the engraftment of vector-marked cells in ADA-SCID, despite the lack of preconditioning.

Abstract Purpose Mosaic loss of chromosome Y (mLOY) is a common feature in elderly men. If mLOY can also occur in young men, it may lead to spermatogenic failure due to loss of spermatogenic genes. Indeed, previous studies detected the 45,X/46,XY karyotype in a few young men with spermatogenic failure. The present study aimed to clarify the frequency of cryptic mLOY in reproductive‐aged men with spermatogenic failure. Methods We studied 198 men at ages 24‐55 years who presented with etiology‐unknown non‐obstructive azoospermia. Prior this study, these patients underwent G‐banding analysis for 20 leukocytes and were found to have 46,XY karyotype. We analyzed copy numbers of chromosome Y in blood cells by using semi‐quantitative multiplex PCR for AMELY/AMELX, array‐based comparative genomic hybridization (CGH) for the AMELY locus, and droplet digital PCR for SRY, USP9Y, and UTY. Results Multiplex PCR showed borderline low AMELY/AMELX ratios in three patients. However, for the three patients, CGH excluded deletion of the AMELY locus, and droplet digital PCR suggested preserved copy numbers of all tested loci. Conclusion This study highlights the rarity of leukocyte mLOY in reproductive‐aged men with spermatogenic failure. In addition, our data imply that standard karyotyping is sufficient to screen early onset mLOY.

Abstract Background Haploinsufficiency of A20 (HA20) is caused by loss-of-function TNFAIP3 variants. Phenotypic and genetic features of HA20 remain uncertain; therefore, the clinical distinction between HA20 and Behçet’s disease (BD) requires clarification. Methods We have collected 12 Japanese BD-like families. Probands of these families were analyzed by whole exome sequencing (WES) and subsequent Sanger sequencing. Clinical features were compared between 54 HA20 patients (including previously reported and new cases) and 520 Japanese BD patients. Results We identified c.1434C>A:p.(Cys478*) in one family and a 236 kb deletion at 6q23.3 containing TNFAIP3 in another family. Four HA20 patients in the two families presented with childhood-onset recurrent oral and genital ulcers and were initially diagnosed and treated as BD. Consistent with the clinical features of HA20, recurrent, refractory fever attacks (three of four patients), and digestive ulcers (two of the four patients) were observed. A comparison of clinical features between HA20 patients and cohorts of BD patients revealed several critical features specific to HA20. These were early-onset, familial occurrence, recurrent fever attacks, gastrointestinal involvement, and infrequent ocular involvement. Conclusions We identified a novel nonsense variant and deletion of the entire TNFAIP3 gene in two unrelated Japanese HA20 families. Genetic screening of TNFAIP3 should be considered for familial BD-like patients with early-onset recurrent fevers.

Clinical improvement in stem cell gene therapy (SCGT) for primary immunodeficiencies depends on the engraftment levels of genetically corrected cells, and tracing the transgene in each hematopoietic lineage is therefore extremely important in evaluating the efficacy of SCGT. We established a single cell-based droplet digital PCR (sc-ddPCR) method consisting of the encapsulation of a single cell into each droplet, followed by emulsion PCR with primers and probes specific for the transgene. A fluorescent signal in a droplet indicates the presence of a single cell carrying the target gene in its genome, and this system can clearly determine the ratio of transgene-positive cells in the entire population at the genomic level. Using sc-ddPCR, we analyzed the engraftment of vector-transduced cells in two patients with severe combined immunodeficiency (SCID) who were treated with SCGT. Sufficient engraftment of the transduced cells was limited to the T cell lineage in peripheral blood (PB), and a small percentage of CD34+ cells exhibited vector integration in bone marrow, indicating that the transgene-positive cells in PB might have differentiated from a small population of stem cells or lineage-restricted precursor cells. sc-ddPCR is a simplified and powerful tool for the detailed assessment of transgene-positive cell distribution in patients treated with SCGT.

X-linked severe combined immunodeficiency (SCID-X1) is an inherited genetic immunodeficiency associated with mutations in the common cytokine receptor γ chain (γc) gene, and characterized by a complete defect of T and natural killer (NK) cells. Gene therapy for SCID-X1 using conventional retroviral (RV) vectors carrying the γc gene results in the successful reconstitution of T cell immunity. However, the high incidence of vector-mediated T cell leukemia, caused by vector insertion near or within cancer-related genes has been a serious problem. In this study, we established a gene therapy model of mouse SCID-X1 using a modified foamy virus (FV) vector expressing human γc. Analysis of vector integration in a human T cell line demonstrated that the FV vector integration sites were significantly less likely to be located within or near transcriptional start sites than RV vector integration sites. To evaluate the therapeutic efficacy, bone marrow cells from γc-knockout (γc-KO) mice were infected with the FV vector and transplanted into γc-KO mice. Transplantation of the FV-treated cells resulted in the successful reconstitution of functionally active T and B cells. These data suggest that FV vectors can be effective and may be safer than conventional RV vectors for gene therapy for SCID-X1.

Mixed-lineage-leukemia (MLL) fusion oncogenes are intimately involved in acute leukemia and secondary therapy-related acute leukemia. To understand MLL-rearranged leukemia, several murine models for this disease have been established. However, the mouse leukemia derived from mouse hematopoietic stem cells (HSCs) may not be fully comparable with human leukemia. Here we developed a humanized mouse model for human leukemia by transplanting human cord blood-derived HSCs transduced with an MLL-AF10 oncogene into a supra-immunodeficient mouse strain, NOD/Shi-scid, IL-2Rγ(-/-) (NOG) mice. Injection of the MLL-AF10-transduced HSCs into the liver of NOG mice enhanced multilineage hematopoiesis, but did not induce leukemia. Because active mutations in ras genes are often found in MLL-related leukemia, we next transduced the gene for a constitutively active form of K-ras along with the MLL-AF10 oncogene. Eight weeks after transplantation, all the recipient mice had developed acute monoblastic leukemia (the M5 phenotype in French-American-British classification). We thus successfully established a human MLL-rearranged leukemia that was derived in vivo from human HSCs. In addition, since the enforced expression of the mutant K-ras alone was insufficient to induce leukemia, the present model may also be a useful experimental platform for the multi-step leukemogenesis model of human leukemia.

DNA polymerase epsilon (Pol e), a component of the core replisome, is involved in DNA replication. Although genetic defects of Pol e have been reported to cause immunodeficiency syndromes, its role in haematopoiesis remains unknown. Here, we identified compound heterozygous variants (p.[Asp1131fs];[Thr1891del]) in POLE, encoding Pol e catalytic subunit A (POLE1), in siblings with a syndromic form of severe congenital transfusion-dependent anaemia. In contrast to Diamond-Blackfan anaemia, marked reticulocytopenia or marked erythroid hypoplasia was not found. Their bone marrow aspirates during infancy revealed erythroid dysplasia with strongly positive TP53 in immunostaining. Repetitive examinations demonstrated trilineage myelodysplasia within 2 years from birth. They had short stature and facial dysmorphism. HEK293 cell-based expression experiments and analyses of patient-derived induced pluripotent stem cells (iPSCs) disclosed a reduced mRNA level of Asp1131fs-POLE1 and defective nuclear translocation of Thr1891del-POLE1. Analysis of iPSCs showed compensatory mRNA upregulation of the other replisome components and increase of the TP53 protein, both suggesting dysfunction of the replisome. We created Pole-knockout medaka fish and found that heterozygous fishes were viable, but with decreased RBCs. Our observations expand the phenotypic spectrum of the Pol e defect in humans, additionally providing unique evidence linking Pol e to haematopoiesis. [ABSTRACT FROM AUTHOR]