Correction: Noguchi et al. PCR-Based Screening of Spinal Muscular Atrophy for Newborn Infants in Hyogo Prefecture, Japan. Genes 2022, 13, 2110.

The authors wish to make the following correction to this paper [...].

PCR-Based Screening of Spinal Muscular Atrophy for Newborn Infants in Hyogo Prefecture, Japan.

Spinal muscular atrophy (SMA) is a common devastating neuromuscular disorder, usually involving homozygous deletion of the SMN1 gene. Newly developed drugs can improve the motor functions of infants with SMA when treated in the early stage. To ensure early diagnosis, newborn screening for SMA (SMA-NBS) via PCR-based genetic testing with dried blood spots (DBSs) has been spreading throughout Japan. In Hyogo Prefecture, we performed a pilot study of SMA-NBS to assess newborn infants who underwent routine newborn metabolic screening between February 2021 and August 2022. Hyogo Prefecture has ~40,000 live births per year and the estimated incidence of SMA is 1 in 20,000-25,000 based on genetic testing of symptomatic patients with SMA. Here, we screened 8336 newborns and 12 screen-positive cases were detected by real-time PCR assay. Multiplex ligation-dependent probe amplification assay excluded ten false positives and identified two patients. These false positives might be related to the use of heparinized and/or diluted blood in the DBS sample. Both patients carried two copies of SMN2, one was asymptomatic and the other was symptomatic at the time of diagnosis. SMA-NBS enables us to prevent delayed diagnosis of SMA, even if it does not always allow treatment in the pre-symptomatic stage.

Large-scale serosurveillance of COVID-19 in Japan: Acquisition of neutralizing antibodies for Delta but not for Omicron and requirement of booster vaccination to overcome the Omicron's outbreak.

Continuous appearance of SARS-CoV-2 variants and mass vaccination have been intricately influencing on the COVID-19 situation. To elucidate the current status in Japan, we analyzed totally 2,000 sera in August (n = 1,000) and December (n = 1,000) 2021 collected from individuals who underwent a health check-up. The anti-N seropositive rate were 2.1% and 3.9% in August and December 2021, respectively, demonstrating a Delta variant endemic during that time; it was approximately twofold higher than the rate based on the PCR-based diagnosis. The anti-S seropositive rate was 38.7% in August and it reached 90.8% in December, in concordance with the vaccination rate in Japan. In the December cohort, 78.7% of the sera showed neutralizing activity against the Delta variant, whereas that against the Omicron was much lower at 36.6%. These analyses revealed that effective immunity against the Delta variant was established in December 2021, however, prompt three-dose vaccination is needed to overcome Omicron's outbreak.

Seroepidemiological Survey of the Antibody for Severe Acute Respiratory Syndrome Coronavirus 2 with Neutralizing Activity at Hospitals: A Cross-sectional Study in Hyogo Prefecture, Japan.

INTRODUCTION: The coronavirus disease 2019 (COVID-19) pandemic is spreading rapidly all over the world. The Japanese government lifted the state of emergency, announced in April 2020, on May 25, but there are still sporadic clusters. Asymptomatic patients who can transmit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause some of these clusters. It is thus urgent to investigate the seroprevalence of antibodies against SARS-CoV-2 and their neutralizing activity. We conducted a cross-sectional study of >10,000 samples at hospitals in Hyogo Prefecture, Japan. METHODS: Between August 6 and October 1, 2020, we collected samples of residual blood from the patients who visited or were admitted to five hospitals and a foundation in Hyogo. We tested the samples for antibodies against SARS-CoV-2 by electrochemiluminescence immunoassay (ECLIA) and chemiluminescent enzyme immunoassay (CLEIA). Sera that were positive by ECLIA or CLEIA were analyzed by an immunochromatographic (IC) test and neutralizing activity assay. RESULTS: We tested 10,377 samples from patients aged between 0 and 99 years old; 27 cases (0.26%) were positive on the ECLIA, and 51 cases (0.49%) were positive on CLEIA. In the 14 cases that tested positive on both ECLIA and CLEIA, the positive rates on the IC test and for neutralizing activity were high (85% and 92%, respectively). In 50 cases (0.48%) that were positive by either ECLIA or CLEIA, the corresponding rates were low (20% and 6%, respectively). The positive rate of neutralizing antibody was 0.15%. CONCLUSIONS: These results indicate that most Hyogo Prefecture residents still do not have antibodies and should avoid the risk of incurring a SARS-CoV-2 infection. Two or more antibody tests should be required for seroepidemiological studies of the antibody for SARS-CoV-2, and a neutralizing activity assay is also essential.

The First Case of TEMPI Syndrome in Japan.

TEMPI syndrome, a disease entity comprising telangiectasia, erythrocytosis with high erythropoietin, monoclonal gammopathy, perinephric fluid collection, and intrapulmonary shunting, was first described by Sykes et al. in 2011. To our knowledge, only 15 cases have been reported worldwide, none of which were in Japan. We herein report a 47-year-old man who had intractable ascites for 2 and a half years and was referred to our department for a peritoneovenous shunt. In addition to ascites, he had telangiectasia, high erythropoietin, monoclonal gammopathy, and perinephric fluid collection. Thus, this is the first case of TEMPI syndrome in Japan.

Detection of a novel CBFB-MYH11 fusion transcript in acute myeloid leukemia M1 with inv(16)(p13q22).

Acute myeloid leukemia (AML) with an inv(16)(p13q22) or t(16;16)(p13;q22) chromosomal abnormality represents one of the most common subtypes of de novo cases. These chromosomal rearrangements result in multiple CBFB-MYH11 fusion transcripts, with type-A being the most frequent. We here describe a unique case of de novo AML-M1, with inv(16)(p13q22), leading to an unusual CBFB-MYH11 fusion transcript, and der(7)t(7;11)(q31;q21). The fusion transcript involves a CBFB exon 5 with a breakpoint at nucleotide 754, an insertion of a 13-bp sequence of CBFB intron 5 at the fusion point, and the MYH11 exon 27 with a breakpoint at nucleotide 3464. To our knowledge, this CBFB-MYH11 fusion transcript has never been reported previously. The clinical characteristics of the present case are in line with previous reports suggesting that rare CBFB-MYH11 fusion transcripts lead to aberrant characteristics such as an atypical cytomorphology and additional cytogenetic abnormalities.

Relationship of hepatitis B virus infection to the recurrence of hepatocellular carcinoma after direct acting antivirals.

Recently, two conflicting articles about recurrence of hepatocellular carcinoma (HCC) after direct acting antivirals (DAA) against hepatitis C virus (HCV) were published. We investigated the relationship between DAA and HCC recurrence. Eligible patients were (1) history of HCC and treated curatively with interventions, and (2) interferon-free DAA therapy was initiated after eradication of HCC. We analyzed contributing factor for HCC recurrence. Ten out of 23 participants (43%) encountered recurrence of HCC. Age, sex, diabetes mellitus, fibrosis score, chemistry, and alpha-fetoprotein did not differ between patients with recurrence and patients without recurrence. The patients with recurrence had significantly higher values of antibody to hepatitis B core antigen (anti-HBc) than the patients without recurrence, 6.06±3.75 vs. 0.91±2.43 (p=0.0019). The relative risk of HCC recurrence comparing anti-HBc positive to negative was 5.2 (95% confidence interval 1.40 to 19.32). Odds ratio was 22.0 (95% confidence interval 2.5 to 191.1). We conclude that anti-HBc positivity was a strong contributing factor for HCC recurrence after DAA therapy.

Coexpression of NUP98/TOP1 and TOP1/NUP98 in de novo Acute Myeloid Leukemia with t(11;20)(p15;q12) and t(2;5)(q33;q31).

The t(11;20)(p15;q11∼12) translocation is a very rare but recurrent cytogenetic aberration that occurs in myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). This translocation was shown to form a fusion gene between NUP98 at 11p15 and TOP1 at 20q12. Here, we describe a new case of de novo AML M2 with t(11;20) which was associated with another balanced translocation. An 81-year-old man was admitted to undergo salvage therapy for relapsed AML. G-banding and spectral karyotyping showed 46,XY,t(2;5)(q33;q31),t(11;20)(p15;q12)[20]. Expression of the NUP98/TOP1 fusion transcript was confirmed: NUP98 exon 13 was in-frame fused with TOP1 exon 8. The reciprocal TOP1/NUP98 fusion transcript was also detected: TOP1 exon 7 was fused with NUP98 exon 14. After achieving hematological complete remission, the karyotype converted to 46,XY,t(2;5)(q33;q31)[19]/46,sl,t(11;20)(p15;q12)[1]. FISH analysis demonstrated that the 5q31 breakpoint of t(2;5) was centromeric to EGR1. In all 10 cases described in the literature, the NUP98 exon 13/TOP1 exon 8 fusion transcript was expressed, indicating that it may be responsible for the pathogenesis of MDS/AML with t(11;20). On the other hand, the TOP1/NUP98 transcript was coexpressed in 4 cases of de novo AML, but not in 3 cases of therapy-related MDS. Thus, this reciprocal fusion may be associated with progression to AML.

Long-term evaluation of partial splenic embolization followed by interferon therapy in patients with hepatitis C virus (HCV) cirrhosis and thrombocytopenia.

OBJECTIVE: Patients with hepatitis C virus (HCV) cirrhosis and thrombocytopenia are often excluded from receiving interferon therapy because the treatment results in severe platelet depletion. Surgical splenectomy or partial splenic embolization (PSE) is a promising procedure for increasing the platelet count before interferon therapy. We performed PSE and evaluated the long-term clinical course in HCV cirrhotic patients. METHODS: Patients with HCV cirrhosis and thrombocytopenia were included (n=108) in this study. The straight-coiled PSE procedure (Takatsuka method) was performed. The platelet count, hemodynamic changes, rate of a sustained virological response (SVR) and prevalence of hepatocellular carcinoma (HCC) were evaluated. RESULTS: PSE resulted in a significant increase in the platelet count (before PSE: 7.9±2.3×10(4)/µL, two weeks after PSE: 16.7±6.6×10(4)/µL (p<0.001). Therefore, all participants were started on regular-dose interferon therapy. The SVR rate was 24% for serotype 1 and 62% for serotype 2. In the biochemical responders (BR) with SVR, the overall survival rate was 94.6% over five years and 89.3% over 10 years. In the non-responders (NR), the overall survival rate was 78.7% over five years and 62.2% over 10 years. The overall survival rate of the patients with SVR+BR was significantly higher than that observed in the patients with NR (p=0.0082). There were no differences in the prevalence of HCC between the patients with SVR+BR and NR. CONCLUSION: PSE enabled the induction of regular-dose interferon therapy in patients with HCV cirrhosis and thrombocytopenia. Although the prevalence of HCC did not differ between the SVR+BR and NR patients, there was a significant survival benefit in the patients with SVR+BR.

CCAR1/CoCoA pair-mediated recruitment of the Mediator defines a novel pathway for GATA1 function.

The MED1 subunit of the Mediator transcriptional coregulator complex coactivates GATA1 and induces erythropoiesis. Here, we show the dual mechanism of GATA1- and MED1-mediated transcription. MED1 expression levels in K562 erythroleukemia cells paralleled the levels of GATA1-targeted gene transcription and erythroid differentiation. An N-terminal fragment of MED1, MED1(1-602), which is incapable of interacting with GATA1, enhanced GATA1-targeted gene transcription and erythroid differentiation, and introduction of MED1(1-602) into Med1(-/-) mouse embryonic fibroblasts (MEFs) partially rescued GATA1-mediated transcription. The C-terminal zinc-finger domain of GATA1 interacts with the MED1(1-602)-interacting coactivator CCAR1, CoCoA and MED1(681-715). CCAR1 and CoCoA synergistically enhanced GATA1-mediated transcription from the γ-globin promoter in MEFs. Recombinant GATA1, CCAR1, CoCoA and MED1(1-602) formed a complex in vitro, and GATA1, CCAR1, CoCoA and MED1 were recruited to the γ-globin promoter in K562 cells during erythroid differentiation. Therefore, in addition to the direct interaction between GATA1 and MED1, CoCoA and CCAR1 appear to relay the GATA1 signal to MED1, and multiple modes of the GATA1-MED1 axis may help to fine-tune GATA1 function during GATA1-mediated homeostasis events.

FGF7 supports hematopoietic stem and progenitor cells and niche-dependent myeloblastoma cells via autocrine action on bone marrow stromal cells in vitro.

FGF1 and FGF2 support hematopoietic stem and progenitor cells (HSPCs) under stress conditions. In this study, we show that fibroblast growth factor (FGF7) may be a novel niche factor for HSPC support and leukemic growth. FGF7 expression was attenuated in mouse embryonic fibroblasts (MEFs) deficient for the MED1 subunit of the Mediator transcriptional coregulator complex. When normal mouse bone marrow (BM) cells were cocultured with Med1(+/+) MEFs or BM stromal cells in the presence of anti-FGF7 antibody, the growth of BM cells and the number of long-time culture-initiating cells (LTC-ICs) decreased significantly. Anti-FGF7 antibody also attenuated the proliferation and cobblestone formation of MB1 stromal cell-dependent myeloblastoma cells. The addition of recombinant FGF7 to the coculture of BM cells and Med1(-/-) MEFs increased BM cells and LTC-ICs. FGF7 and its cognate receptor, FGFR2IIIb, were undetectable in BM cells, but MEFs and BM stromal cells expressed both. FGF7 activated downstream targets of FGFR2IIIb in Med1(+/+) and Med1(-/-) MEFs and BM stromal cells. Taken together, we propose that FGF7 supports HSPCs and leukemia-initiating cells indirectly via FGFR2IIIb expressed on stromal cells.

Mediator subunit MED1 is a T3-dependent and T3-independent coactivator on the thyrotropin ß gene promoter.

The MED1 subunit of the Mediator transcriptional coregulator complex is a nuclear receptor-specific coactivator. A negative feedback mechanism of thyroid-stimulating hormone (TSH, or thyrotropin) expression in the thyrotroph in the presence of triiodothyronine (T3) is employed by liganded thyroid hormone receptor ß (TRß) on the TSHß gene promoter, where conventional histone-modifying coactivators act as corepressors. We now provide evidence that MED1 is a ligand-dependent positive cofactor on this promoter. TSHß gene transcription was attenuated in MED1 mutant mice in which the nuclear receptor-binding ability of MED1 was specifically disrupted. MED1 stimulated GATA2- and Pit1-mediated TSHß gene promoter activity in a ligand-independent manner in cultured cells. MED1 also stimulated transcription from the TSHß gene promoter in a T3-dependent manner. The transcription was further enhanced when the T3-dependent corepressors SRC1, SRC2, and HDAC2 were downregulated. Hence, MED1 is a T3-dependent and -independent coactivator on the TSHß gene promoter.

Significance of the neutrophil myeloperoxidase index in patients with atherosclerotic diseases.

The pattern of changes in the neutrophil myeloperoxidase (MPO) levels in various atherosclerotic conditions was analyzed by assessing the mean myeloperoxidase index (MPXI), which is calculated during the routine complete blood count (CBC) performed using the flow-cytochemistry blood autoanalyzer ADVIA120/2120 (Siemens), and plasma MPO concentrations. MPXI values of ischemic heart disease (IHD) patients did not differ from those of healthy volunteers. However, MPXI values of IHD patients with arteriosclerosis obliterans (ASO) (-6.1 ± 1.8) were significantly lower than those of IHD patients without ASO (0.8 ± 0.5). In contrast, the MPO values in IHD patients with ASO were significantly elevated. In subjects without IHD, while the MPXI values in mild cases of ASO (Fontaine's stages I/II, 3.4 ± 0.8) were significantly higher than those of healthy volunteers (0.4 ± 0.4), the values of those with severe ASO (stages III/IV, 0.3 ± 0.8) were significantly lower than those of mild cases. However, when ASO patients developed IHD, the MPXI values dramatically decreased (stages I/II, -7.3 ± 1.9; stages III/IV, -5.2 ± 1.6). These results indicate that MPXI is elevated in mild, but not in severe, ASO cases, and that MPXI decreases dramatically when ASO patients develop IHD. MPXI may constitute an informative independent biomarker for diagnosis and follow-up of IHD complicated by ASO.

Mediator subunits MED1 and MED24 cooperatively contribute to pubertal mammary gland development and growth of breast carcinoma cells.

The Mediator subunit MED1 is essential for mammary gland development and lactation, whose contribution through direct interaction with estrogen receptors (ERs) is restricted to involvement in pubertal mammary gland development and luminal cell differentiation. Here, we provide evidence that the MED24-containing submodule of Mediator functionally communicates specifically with MED1 in pubertal mammary gland development. Mammary glands from MED1/MED24 double heterozygous knockout mice showed profound retardation in ductal branching during puberty, while single haploinsufficient glands developed normally. DNA synthesis of both luminal and basal cells were impaired in double mutant mice, and the expression of ER-targeted genes encoding E2F1 and cyclin D1, which promote progression through the G(1)/S phase of the cell cycle, was attenuated. Luciferase reporter assays employing double mutant mouse embryonic fibroblasts showed selective impairment in ER functions. Various breast carcinoma cell lines expressed abundant amounts of MED1, MED24, and MED30, and attenuated expression of MED1 and MED24 in breast carcinoma cells led to attenuated DNA synthesis and growth. These results indicate functional communications between the MED1 subunit and the MED24-containing submodule that mediate estrogen receptor functions and growth of both normal mammary epithelial cells and breast carcinoma cells.

Possible role of early transabdominal ultrasound in patients undergoing cytapheresis for active ulcerative colitis.

BACKGROUND: Cytaphresis (CAP) is an effective modality in the treatment of active ulcerative colitis (UC), but the time lag before a notable clinical response on scheduled therapy frequently causes a significant delay in the modification of treatment. We previously reported that the clinical response after CAP was predicted by early application of transabdominal ultrasound (TAUS), but the predictability of long-term outcome after CAP still remains uncertain. METHODS PATIENTS: Twenty-six patients with active UC who received CAP were followed for 1 year. In addition to CAP they received pharmaceutical regimens, such as corticosteroid, 5-aminosalicylic acid, and immunomodulator, as indicated clinically. The mean UC-DAI score was 9.7 before CAP, and 3.2 at 1 year after CAP. Prognostic factor: Total colonic wall thickness was measured by TAUS at 2 to 3 weeks after the initiation of the treatment, and decrement from baseline was calculated. Early ultrasonographic response (EUR) was defined as a decrement statistically. UC-DAI score of 2 or less at 1 year was defined as sustained clinical remission. Score of 6 or more was defined as clinical relapse. RESULTS: EUR was defined as a decrement in wall thickness by at least 2.5 mm from the baseline. EUR was noted in 11 patients, and the remaining 15 did not attain EUR. OUTCOME MEASURES: In the UC-DAI score measured at 1 year after initiation of treatment 90.9% of patients with EUR, whereas 40.0% with non-EUR (p<0.05) showed sustained clinical remission. Regarding relapse, within 1 year 9.1% of patients with EUR relapsed whereas 46.7% with non-EUR (p<0.05) relapsed. CONCLUSION: Early application of TAUS may predict the long-term clinical outcome after CAP in patients with active UC.

A case of montelukast-induced churg-strauss syndrome associated with liver dysfunction.

A 64-year-old woman was admitted to hospital due to protracted diarrhea and liver dysfunction. The patient was diagnosed as Churg-Strauss syndrome (CSS) due to asthma, paranasal sinusitis, hypereosinophilia, and polyneuropathy. There was a history of taking montelukast, a leukotriene receptor antagonist (LTRA), which is thought to have some relationship with CSS. The liver biopsy specimen showed eosinophilic infiltration and centrolobular fatty change. In this paper, we review the relationship between LTRA and CSS. Several lines of evidence suggest that leukotriene plays an important role in maintaining neural tissues. We also review the potential relationship between centrolobular fatty change and pivoxil-containing antibiotics, which was prescribed for sinusitis before admission. Carnitine deficiency induced by pivoxil-containing agents may cause impaired fatty acid oxidation in mitochondria.

Two week induction of interferon-beta followed by pegylated interferon alpha-2b and ribavirin for chronic infection with hepatitis C.

OBJECTIVES: To elucidate the efficacy of interferon (IFN)-beta induction therapy followed by pegylated IFN alpha and ribavirin for chronic infection with hepatitis C virus (HCV). METHODS: Patients chronically infected with HCV genotype 1, high titer were enrolled. Twice daily bolus injections of 3 million units IFN-beta were administered for 14 days. Thereafter, weekly injection of pegylated IFN alpha 2b and daily intake of ribavirin were followed. Therapy duration was adjusted according to the response to the therapy. When time to an undetectable HCV-RNA was 1, 2, 4, 8, and 12 weeks, total duration of therapy was 12, 24, 36, 48 and 60 weeks, respectively. Patients who failed to achieve an undetectable HCV-RNA within 12 weeks discontinued therapy on 12 week. RESULTS: Among the 101 patients treated, 56 (55.4%) achieved sustained virological response (SVR). SVR rate for each treatment duration was 10/10 for 12 weeks, 12/14 for 24 weeks, 18/19 for 36 weeks, 15/26 for 48 weeks, 1/4 for 60 weeks and 0/28 for patients who discontinued therapy at 12 weeks. Mean time to an undetectable HCV-RNA was 35.5 +/- 2.7 days. Mean therapy duration was 27.3 +/- 1.4 weeks. Using a cut off value of 21.5 fmol/L of HCV core-antigen in the first week, SVR could be predicted by sensitivity of 0.91 and specificity of 0.78. CONCLUSION: IFN-beta induction therapy resulted in acceptable SVR rates despite short therapy duration. Steep reduction of HCV by IFN-beta enables us to predict SVR in the first week of therapy.

Partial splenic embolization reverses insulin resistance in patients with liver cirrhosis.

BACKGROUND: It is well known that patients with liver cirrhosis often develop insulin resistance and diabetes mellitus. Recently, we encountered a liver cirrhosis patient in whom partial splenic embolization (PSE) improved insulin sensitivity. Therefore, we conducted further investigation about PSE and insulin resistance. METHODS: Thirty-seven consecutive patients with liver cirrhosis underwent PSE. Hemodynamic changes, blood counts, and homeostasis model assessment of insulin resistance (HOMA-IR) were assessed before and 2 weeks after PSE. RESULTS: PSE resulted in decreased splenic venous flow and increased intestinal venous flow to the liver. Platelet counts before and after PSE were 7.7+/-0.5 x 10(4) /microL, 15.0+/-1.4 x 10(4) /microL, respectively (p<0.01). HOMA-IR before and after PSE were 6.5+/-2.1, 3.3+/-0.6, respectively (p<0.05). HCV core antigen before and after PSE were 6,340+/-1,296 fmol/L, 4,112+/-873 fmol/L, respectively (p<0.05). Conclusion PSE significantly reverses insulin resistance in patients with liver cirrhosis. The increase in intestinal venous flow to the liver and reduced HCV viral load were thought to be mechanisms of improvement in insulin sensitivity after PSE.

Novel mutation of plakophilin-2 associated with arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a disease characterized by dilatation and akinesis of the right ventricle, and causes life-threatening ventricular arrhythmia. Mutations of plakophilin-2 (PKP2) have recently been identified as one causative abnormality in ARVC. A case of ARVC with a mutation of PKP2 is reported here. Direct sequencing of the patient's DNA revealed an insertion mutation in exon 8 of PKP2 (1728_1729insGATG). The mutation caused the frameshift and the premature termination of translation (R577DfsX5). This is the first case report of PKP2 mutation found in Japanese ARVC patients.