Tomohito Yagi

Characteristic perforin gene mutations of haemophagocytic lymphohistiocytosis patients in Japan

Summary. Perforin gene (PRF1) mutations appear to occur in about 30% of patients with haemophagocytic lymphohistiocytosis (HLH). We tested perforin expression and gene mutations in 14 HLH patients and six patients with Epstein–Barr virus‐associated HLH (EBV‐HLH) in Japan. Five of the 14 HLH patients had perforin abnormalities. The presence of PRF1 genetic abnormality correlated well with the lack of perforin expression as determined by flow cytometry. Sequencing showed that four patients had a compound heterozygous mutation while the fifth patient had a homozygous mutation. Three of the mutations we detected were novel. In contrast, none of the six EBV‐HLH patients showed perforin abnormalities. Our data, combined with the PRF1 mutations in three previously reported Japanese patients, suggest that the 1090–1091delCT and 207delC mutations of the perforin gene are frequently present in Japanese HLH patients (62·5% and 37·5% respectively). Examination of the geographical origins of the ancestors in the perforin‐mutant HLH patients revealed that they mostly came from the Western part of Japan, suggesting that the present‐day cases may largely derive from a common ancestor.

Three susceptible loci associated with primary open-angle glaucoma identified by genome-wide association study in a Japanese population

Primary open-angle glaucoma (POAG) is the major type of glaucoma. To discover genetic markers associated with POAG, we examined a total of 1,575 Japanese subjects in a genome-wide association study (stage 1) and a subsequent study (stage 2). Both studies were carried out at a single institution. In the stage 1 association study, we compared SNPs between 418 POAG patients and 300 control subjects. First, low-quality data were eliminated by a stringent filter, and 331,838 autosomal SNPs were selected for analysis. Poorly clustered SNPs were eliminated by a visual assessment, leaving 255 that showed a significant deviation (P < 0.001) in the allele frequency comparison. In the stage 2 analysis, we tested these 255 SNPs for association in DNA samples from a separate group of 409 POAG and 448 control subjects. High-quality genotype data were selected and used to calculate the combined P values of stages 1 and 2 by the Mantel–Haenszel test. These analyses yielded 6 SNPs with P < 0.0001. All 6 SNPs showed a significant association (P < 0.05) in stage 2, demonstrating a confirmed association with POAG. Although we could not link the SNPs to the annotated gene(s), it turned out that we have identified 3 genetic loci probably associated with POAG. These findings would provide the foundation for future studies to build on, such as for the metaanalysis, to reveal the molecular mechanism of the POAG pathogenesis.

Quantitative Analysis of Cell-free Epstein-Barr Virus Genome Copy Number in Patients with EBV-associated Hemophagocytic Lymphohistiocytosis

To determine whether the EBV genome content in serum or plasma reflects clinical features and outcome in EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), we quantified the cell-free EBV genome copy number by real-time PCR in 38 patients with EBV-HLH, and compared this to the values from 15 patients with infectious mononucleosis (IM). The median (range) cell-free EBV genome copy number at diagnosis was 3.0   ×   10 3 (undetectable m 5.5   ×   10 7 ) copies/ml in EBV-HLH, which was significantly higher than the 6.6   ×   10 1 (undetectable m 1.0   ×   10 3 ) copies/ml in IM (P =0.0008). We serially analyzed cell-free EBV genome copy number in 10 cases of EBV-HLH up to 4 months from diagnosis. In four patients who achieved remission, the EBV genome became undetectable soon after starting therapy. In the remaining six patients who responded poorly to therapy, the EBV genome copy number in the serum or plasma remained at high levels except for one case. In addition, we confirmed that the EBV genome became undetectable after hematopoietic stem cell transplantation in 4 EBV-HLH cases. These results suggest that the quantitative analysis of cell-free EBV genome copy number is useful for evaluating disease activity and for predicting the response to therapy in EBV-HLH.

Expression of the Ikaros gene family in childhood acute lymphoblastic leukaemia

Summary. The Ikaros (Ik) gene family, which includes Ik, Aiolos (Ai), and Helios (He), is a primary regulator of lymphocyte differentiation, and is involved in the development of acute lymphoblastic leukaemia (ALL). We analysed the expression of the Ik gene family isoforms in 97 ALL cases, consisting of 64 childhood and 33 infant ALL cases, using reverse transcription‐polymerase chain reaction (RT‐PCR). Expression of Ik was detected in all cases, 87 of which expressed either Ik1 or Ik2, or both, five of which expressed Ik1/Ik2 and Ik6, and another five of which expressed only Ik6. Therefore, the dominant negative isoform of Ik6 was expressed in 10 of the 38 cases of childhood precursor B ALL, but was absent in other types of childhood ALL (26·3%, χ2‐test, P = 0·0001). In terms of Aiolos and Helios expression, 49 (65·3%) out of the 75 and 40 (50%) out of the 80 ALL cases tested showed non‐spliced Ai1 and He1 respectively. Only one case of T lineage ALL expressed a small‐sized isoform of Helios (designated as He6). It was also found that the expression of Ai1 and He1 was low in Ik6‐positive patients (Fishers exact test; Ai1 P = 0·005, Hel P = 0·035).

Association between prostaglandin E receptor 3 polymorphisms and Stevens-Johnson syndrome identified by means of a genome-wide association study

BACKGROUND Stevens-Johnson syndrome (SJS) and its severe variant, toxic epidermal necrolysis (TEN), are acute inflammatory vesiculobullous reactions of the skin and mucosa. They often affect the ocular surface and can result in permanent visual dysfunction. OBJECTIVES We sought to discover genetic markers for SJS/TEN susceptibility. METHODS We performed a genome-wide association study with 60 patients and 300 control subjects. We applied stringent filter and visual assessments for selecting single nucleotide polymorphisms (SNPs) and a high false discovery rate threshold. We fine-mapped the region where a candidate SNP was found and confirmed the results by means of sequencing. We evaluated the function of agonist-activated prostaglandin E receptor 3 (EP3), the gene for which contained several SNPs, in regulating cytokine production in human conjunctival epithelial (CE) cells. The expression levels of EP3 in the CE cells from patients and control subjects were also compared. RESULTS We identified 3 SNPs that passed the false discovery rate threshold. One (rs17131450) was close to the EP3 gene. Therefore we analyzed the EP3 region in detail and identified 5 other SNPs. We confirmed the association between SJS/TEN and all 6 SNPs. Activated EP3 was expressed in control CE cells, and it suppressed polyI:C-stimulated cytokine production, suggesting that EP3 might help prevent ocular surface inflammation. Concordantly, the EP3 levels were much lower in the CE cells of the patients than in those of the control subjects. CONCLUSION We demonstrated, using both genetic and functional analyses, that EP3 could be a key player in the pathogenesis of SJS/TEN accompanied by ocular complications.

Ikaros dominant negative isoform (Ik6) induces IL-3-independent survival of murine pro-B lymphocytes by activating JAK-STAT and up-regulating Bcl-xl levels

Ikaros is an essential regulator of lymphocyte differentiation. Mice transgenic for the Ikaros dominant negative (DN) mutation rapidly develop lymphoid malignancies. Various human leukemias have also been reported to express Ikaros DN isoforms, but its role in leukemogenesis is yet to be defined. We demonstrate that overexpressed Ikaros DN (Ik6) prolonged the survival of two different murine pro-B cell lines in cytokine deprived condition, and this was associated with increased expression of Bcl-xl. A survey of the upstream controller(s) of Bcl-xl expression revealed Ik6 overexpression enhanced the phosphorylation of JAK2 and STAT5. Interestingly, the Ik6 expressing cell lines showed reduced expression of B-cell differentiation surface marker CD45R (B220), which is also known as a JAK2 inhibitor. Although further evaluation with human clinical materials are required, these results propose a putative role of Ik6 in the development of B-lineage acute lymphoblastic leukemia, by activating the JAK2-STAT5 pathway and thus stimulating the production of Bcl-xl.

Defective p53 engagement after the induction of DNA damage in cells deficient in topoisomerase 3β

The type IA topoisomerases have been implicated in the repair of dsDNA breaks by homologous recombination and in the resolution of stalled or damaged DNA replication forks; thus, these proteins play important roles in the maintenance of genomic stability. We studied the functions of one of the two mammalian type IA enzymes, Top3β, using murine embryonic fibroblasts (MEFs) derived from top3β−/− embryos. top3β−/− MEFs proliferated more slowly than TOP3β+/+ control MEFs, demonstrated increased sensitivity to DNA-damaging agents such as ionizing and UV radiation, and had increased DNA double-strand breaks as manifested by increased γ-H2-AX phosphorylation. However, incomplete enforcement of the G1–S cell cycle checkpoint was observed in top3β−/− MEFs. Notably, ataxia-telangiectasia, mutated (ATM)/ATM and Rad3-related (ATR)-dependent substrate phosphorylation after UV-B and ionizing radiation was impaired in top3β−/− versus TOP3β+/+ control MEFs, and impaired up-regulation of total and Ser-18-phosphorylated p53 was observed in top3β−/− cells. Taken together, these results suggest an unanticipated role for Top3β beyond DNA repair in the activation of cellular responses to DNA damage.

A novel infant acute lymphoblastic leukemia cell line with MLL-AF5q31 fusion transcript.

Infant acute lymphoblastic leukemia (ALL) is characterized by the presence of the proB phenotype (CD10−/CD19+), poor prognosis and frequent rearrangement of the mixed lineage leukemia (MLL) gene. The most frequent rearrangement is t(4;11)(q21;q23), the role of whose product, the MLL-AF4 fusion transcript, has been extensively studied in leukemogenesis. In a cell line of infant leukemia with MLL rearrangement denoted KP-L-RY, panhandle PCR amplification of cDNA revealed the presence of a fusion transcript, MLL-AF5q31, indicating that AF5q31 is also a partner gene of MLL. In this fusion transcript the MLL exon 6 is fused in frame to the 5′ side of the putative transactivation domain of AF5q31. The AF5q31 protein is a member of the AF4/LAF4/FMR2-related family of proteins, which have been suggested to play a role in hematopoietic cell growth and differentiation. The MLL-AF5q31 fusion transcript, although probably rare, appears to be associated with the pathogenesis of infant ALL like MLL-AF4. Co-expression of HoxA9 and Meis1 genes in the KP-L-RY cell line indicated possible functional similarity between MLL-AF4 and MLL-AF5q31. Further understanding of the function of AF5q31 as well as the specific leukemogenic mechanism of MLL-AF5q31 awaits future studies.

Molecular Analysis of Latent Membrane Protein 1 in Patients with Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis in Japan

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is considered to be an oncoprotein because it is crucial for B-lymphocyte transformation. Since a 30 base pair (bp) deletion in the carboxy-terminal portion of the LMP1 gene was found in a CAO cell line derived from nasopharyngeal carcinoma containing EBV, an association between EB viral genetic alteration and tumorigenicity has been postulated. In this study we have analyzed LMP1 DNA isolated from 10 Japanese patients with EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH). In all HLH patients, we found the 30bp deletion and 4-8-tandem repeats of the sequence DNGPQDPDNTD in the LMP1 gene. Furthermore, detailed amino acid (aa) sequence analysis revealed that 7 aa substitutions identical to those found in CAO-LMP1 but not in B95.8 cell line-LMPl were found in all the HLH cases. NF-κB assay revealed that HLH-LMP1 activated NF-κB significantly more than that of B95.8-LMP1 (p=0.032). We conclude that EBV from all of our HLH cases shared common genetic characteristics with EBV obtained from the CAO cell line, which is distinct from the wild-type EBV isolated from the B95.8 cell line. These data suggest that the mutational changes of the LMP1 gene may play an important role in the pathogenesis of these fatal EBV-related disorders.

Cytokine storm arising on the ocular surface in a patient with Stevens–Johnson syndrome

Stevens–Johnson syndrome (SJS) and its more extreme variant, toxic epidermal necrolysis (TEN), are acute, adverse systemic reactions that can affect anyone who takes medications. SJS/TEN predominantly affects the skin and mucosal membranes and predisposes patients to life-threatening complications such as sepsis, respiratory dysfunction and multi-organ failure. Even when a patient does survive this disease, serious ocular discomfort and morbidity often persists life long.1 2 In May 2008, a 59-year-old female inpatient had a case of red eyes, and 2 days later she presented with a sudden onset of high fever and eruption and erosion in the mucocutaneous regions including the mouth, paronychia and bilateral conjunctivitis. Slit-lamp examination revealed a large epithelial defect of the conjunctiva with severe hyperaemia in both eyes (figure 1A,B). There was no viral or bacterial infection, and skin biopsy specimens of the erythematous macules revealed necrotic keratinocytes and liquefaction, compatible with the diagnosis of SJS. Steroid pulse therapy and intensive topical betamethasone (0.1%, 10 times daily) were …