The c.1617del variant of TMEM260 is identified as the most frequent single gene determinant for Japanese patients with a specific type of congenital heart disease.

Although the molecular mechanisms underlying congenital heart disease (CHD) remain poorly understood, recent advances in genetic analysis have facilitated the exploration of causative genes for CHD. We reported that the pathogenic variant c.1617del of TMEM260, which encodes a transmembrane protein, is highly associated with CHD, specifically persistent truncus arteriosus (PTA), the most severe cardiac outflow tract (OFT) defect. Using whole-exome sequencing, the c.1617del variant was identified in two siblings with PTA in a Japanese family and in three of the 26 DNAs obtained from Japanese individuals with PTA. The c.1617del of TMEM260 has been found only in East Asians, especially Japanese and Korean populations, and the frequency of this variant in PTA is estimated to be next to that of the 22q11.2 deletion, the most well-known genetic cause of PTA. Phenotype of patients with c.1617del appears to be predominantly in the heart, although TMEM260 is responsible for structural heart defects and renal anomalies syndrome (SHDRA). The mouse TMEM260 variant (p.W535Cfs*56), synonymous with the human variant (p.W539Cfs*9), exhibited truncation and downregulation by western blotting, and aggregation by immunocytochemistry. In situ hybridization demonstrated that Tmem260 is expressed ubiquitously during embryogenesis, including in the development of cardiac OFT implicated in PTA. This expression may be regulated by a ~ 0.8 kb genomic region in intron 3 of Tmem260 that includes multiple highly conserved binding sites for essential cardiac transcription factors, thus revealing that the c.1617del variant of TMEM260 is the major single-gene variant responsible for PTA in the Japanese population.

Functional characterization of variants found in Japanese patients with hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant form of vascular dysplasia. Genetic diagnosis is made by identifying loss-of-function variants in genes, such as ENG and ACVRL1. However, the causal mechanisms of various variants of unknown significance remains unclear. In this study, we analyzed 12 Japanese patients from 11 families who were clinically diagnosed with HHT. Sequencing analysis identified 11 distinct variants in ACVRL1 and ENG. Three of the 11 were truncating variants, leading to a definitive diagnosis, whereas the remaining eight were splice-site and missense variants that required functional analyses. In silico splicing analyses demonstrated that three variants, c.526-3C > G and c.598C > G in ACVRL1, and c.690-1G > A in ENG, caused aberrant splicing, as confirmed by a minigene assay. The five remaining missense variants were p.Arg67Gln, p.Ile256Asn, p.Leu285Pro, and p.Pro424Leu in ACVRL and p.Pro165His in ENG. Nanoluciferase-based bioluminescence analyses demonstrated that these ACVRL1 variants impaired cell membrane trafficking, resulting in the loss of bone morphogenetic protein 9 (BMP9) signal transduction. In contrast, the ENG mutation impaired BMP9 signaling despite normal cell membrane expression. The updated functional analysis methods performed in this study will facilitate effective genetic testing and appropriate medical care for patients with HHT.

Difference in Clinical Phenotype, Mutation Position, and Structural Change of RNF213 Rare Variants Between Pediatric and Adult Japanese Patients with Moyamoya Disease.

It is unclear how rare RNF213 variants, other than the p.R4810K founder variant, affect the clinical phenotype or the function of RNF213 in moyamoya disease (MMD). This study included 151 Japanese patients with MMD. After performing targeted resequencing for all coding exons in RNF213, we investigated the clinical phenotype and statistically analyzed the genotype-phenotype correlation. We mapped RNF213 variants on a three-dimensional (3D) model of human RNF213 and analyzed the structural changes due to variants. The RNF213 p.R4810K homozygous variant, p.R4810K heterozygous variant, and wild type were detected in 10 (6.6%), 111 (73.5%), and 30 (19.9%) MMD patients, respectively. In addition, 15 rare variants were detected in 16 (10.6%) patients. In addition to the influence of the p.R4810K homozygous variant, the frequency of cerebral infarction at disease onset was higher in pediatric patients with other rare variants (3/6, 50.0%, P = 0.006) than in those with only the p.R4810K heterozygous variant or with no variants (2/51, 3.9%). Furthermore, on 3D modelling of RNF213, the majority of rare variants found in pediatric patients were located in the E3 module and associated with salt bridge loss, contrary to the results for adult patients. The clinical phenotype of rare RNF213 variants, mapped mutation position, and their predicted structural change differed between pediatric and adult patients with MMD. Rare RNF213 variants, in addition to the founder p.R4810K homozygous variant, can influence MMD clinical phenotypes or structural change which may contribute to the destabilization of RNF213.

Variant spectrum of PIEZO1 and KCNN4 in Japanese patients with dehydrated hereditary stomatocytosis.

Hereditary stomatocytosis (HSt) is a type of congenital hemolytic anemia caused by abnormally increased cation permeability of erythrocyte membranes. Dehydrated HSt (DHSt) is the most common subtype of HSt and is diagnosed based on clinical and laboratory findings related to erythrocytes. PIEZO1 and KCNN4 have been recognized as causative genes, and many related variants have been reported. We analyzed the genomic background of 23 patients from 20 Japanese families suspected of having DHSt using a target capture sequence and identified pathogenic/likely pathogenic variants of PIEZO1 or KCNN4 in 12 families.

Loss-of-function mutations in SGCE found in Japanese patients with myoclonus-dystonia.

SGCE myoclonus-dystonia is a monogenic form of dystonia with an autosomal dominant mode of inheritance that co-occurs with a myoclonic jerk. In this study, we present 12 Japanese patients from nine families with this disease. Targeted next-generation sequencing covering major causative genes for monogenic dystonias identified nine distinct SGCE mutations from each of the families: three nonsense, two frameshift, two missense, one in-frame 15 bp deletion, and one splice donor site mutations, of which four were previously unreported. One missense mutation (c.662G>T, p.Gly221Val) was located at the 3' end of exon 5 (NM_001099400), which was predicted to cause aberrant splicing according to in silico predictions. Minigene assays performed together with the c.825+1G>C mutation demonstrated complete skipping of exon 5 and 6, respectively, in their transcripts. The other missense (c.1345A>G, p.Met449Val) and 15 bp deletion (c.168_182del, p.Phe58_Leu62del) mutations showed a significant reduction in cell membrane expression via HiBiT bioluminescence assay. Therefore, we concluded that all the detected mutations were disease-causing. Unlike the other detected mutations, p.Met449Val affects only isoform 3 (NP_001092870 encoded by NM_001099400) among the variously known isoforms of SGCE. This isoform is brain-specific and is mostly expressed in the cerebellum, which supports recent studies showing that cerebellar dysfunction is a key element in the pathophysiology of SGCE myoclonus-dystonia.

Dystonic Tremor in Adult-onset DYT-KMT2B.

KMT2B-related dystonia (DYT28, DYT-KMT2B) is an inherited dystonia that generally begins in the lower limbs during childhood and evolves into generalized dystonia. We herein report a case of adult-onset DYT28 with dystonic tremor. A 27-year-old woman initially displayed right upper limb and cervical tremors over the course of 1 year. A neurological examination also revealed cervical and lower limb dystonia. Although the disease generally develops during childhood, we diagnosed the woman with DYT28, as genetic testing revealed a mutation in KMT2B. Adult-onset patients with DYT28 might also show uncommon symptoms as well as DYT-TOR1A (DYT1).

Case Report: Long-Term Suppression of Paroxysmal Kinesigenic Dyskinesia After Bilateral Thalamotomy.

Background: Paroxysmal kinesigenic dyskinesia (PKD) is a movement disorder characterized by transient dyskinetic movements, including dystonia, chorea, or both, triggered by sudden voluntary movements. Carbamazepine and other antiepileptic drugs (AEDs) are widely used in the treatment of PKD, and they provide complete remission in 80-90% of medically treated patients. However, the adverse effects of AEDs include drowsiness and dizziness, which interfere with patients' daily lives. For those with poor compatibility with AEDs, other treatment approaches are warranted. Case Report: A 19-year-old man presented to our institute with right hand and foot dyskinesia. He had a significant family history of PKD; his uncle, grandfather, and grandfather's brother had PKD. The patient first experienced paroxysmal involuntary left hand and toe flexion with left forearm pronation triggered by sudden voluntary movements at the age of 14. Carbamazepine (100 mg/day) was prescribed, which led to a significant reduction in the frequency of attacks. However, carbamazepine induced drowsiness, which significantly interfered with his daily life, especially school life. He underwent right-sided ventro-oral (Vo) thalamotomy at the age of 15, which resulted in complete resolution of PKD attacks immediately after the surgery. Four months after the thalamotomy, he developed right elbow, hand, and toe flexion. He underwent left-sided Vo thalamotomy at the age of 19. Immediately after the surgery, the PKD attacks resolved completely. However, mild dysarthria developed, which spontaneously resolved within three months. Left-sided PKD attacks never developed six years after the right Vo thalamotomy, and right-sided PKD attacks never developed two years after the left Vo thalamotomy without medication. Conclusion: The present case showed long-term suppression of bilateral PKDs after bilateral thalamotomy, which led to drug-free conditions.

A novel non-invasive monitoring assay of 5-azacitidine efficacy using global DNA methylation of peripheral blood in myelodysplastic syndrome.

Purpose: Monitoring response and resistance to 5-azacitidine (AZA) is essential when treating patients with myelodysplastic syndrome (MDS). To quantify methylated DNA not only in the promoter region but also in the gene body, we established a single-molecule methylation assay (SMMA). Patients and methods: We first investigated the methylation extent (expressed as methylation index [MI]) by SMMA among 28 MDS and 6 post-MDS acute myeloid leukemia patients. We then analyzed the MI in 13 AZA-treated patients. Results: Whole-blood DNA from all 34 patients had low MI values compared with healthy volunteers (P<0.0001). DNA hypomethylation in MDS patients was more evident in neutrophils (P=0.0008) than in peripheral mononuclear cells (P=0.0713). No consistent pattern of genome-wide DNA hypomethylation was found among MDS subtypes or revised International Prognostic Scoring System (IPSS-R) categories; however, we found that the MI was significantly increased for patients at very high risk who were separated by the new cytogenetic scoring system for IPSS-R (P=0.0398). There was no significant difference in MI before AZA, regardless of the response to AZA (P=0.8689); however, sequential measurement of MI in peripheral blood demonstrated that AZA non-responders did not have normalized MI at the time of next course of AZA (P=0.0352). Conclusion: Our results suggest that sequential SMMA of peripheral blood after AZA may represent a non-invasive monitoring marker for AZA efficacy in MDS patients.

Genetic variations of bone marrow mesenchymal stromal cells derived from acute leukemia and myelodysplastic syndrome by targeted deep sequencing.

Bone marrow mesenchymal stromal cells (MSCs), which support proliferation and differentiation of hematopoietic stem cells, may play a crucial role in the pathogenesis of myeloid neoplasms. To determine whether MSCs in myeloid neoplasms harbor distinct somatic mutations that may affect their function, we used a targeted gene sequencing panel containing 50 myeloid neoplasm-associated genes with coverage of ≥500. We compared the genetic alterations between MSCs and bone marrow hematopoietic (BM) cells from patients with acute leukemia (n=5) or myelodysplastic syndrome (MDS, n=5). Non-synonymous somatic mutations, such as DNMT3A-R882H and FLT3-D835Y, were only detected in BM cells with high allelic frequency. We found several non-synonymous genetic variants overlapping BM cells and MSCs, including TP53 and ASXL1, partially owing to the heterogenous cell fraction of MSC samples and lineage fidelity. We also found MSC-specific genetic variants with very low allelic frequency (7% to 8%), such as NF1-G2114D and NF1-G140. Further studies in large cohorts are needed to clarify the molecular properties of MSCs including age-related genetic alterations by targeted deep sequencing.

Ecl1 is a zinc-binding protein involved in the zinc-limitation-dependent extension of chronological life span in fission yeast.

Overexpression of Ecl1-family genes (ecl1 +, ecl2 +, and ecl3 +) results in the extension of the chronological life span in Schizosaccharomyces pombe. However, the mechanism for this extension has not been defined clearly. Ecl1-family proteins consist of approximately 80 amino acids, and four cysteine residues are conserved in their N-terminal domains. This study focused on the Ecl1 protein, mutating its cysteine residues sequentially to confirm their importance. As a result, all mutated Ecl1 proteins nearly lost the function to extend the chronological life span, suggesting that these four cysteine residues are essential for the Ecl1 protein. Utilizing ICP-AES (inductively coupled plasma atomic emission spectroscopy) analysis, we found that wild-type Ecl1 proteins contain zinc, while cysteine-mutated Ecl1 proteins do not. We also analyzed the effect of environmental zinc on the chronological life span. We found that zinc limitation extends the chronological life span, and this extension depends on the Ecl1-family proteins.

Replenishing exosomes from older bone marrow stromal cells with miR-340 inhibits myeloma-related angiogenesis.

The study of bone marrow stromal cells (BMSCs) and the exosomes they secrete is considered promising for cancer therapy. However, little is known about the effect of donor age on BMSCs. In the present study, we investigated the therapeutic potential of BMSC exosomes derived from donors of different ages using an in vivo model of hypoxic bone marrow in multiple myeloma (MM). We found that donor age was strongly related to senescent changes in BMSCs. Exosomes derived from young BMSCs significantly inhibited MM-induced angiogenesis in Matrigel plugs. The exosomal microRNA (miRNA) expression profile was different between young and older BMSCs, despite similarities in the size and quantity of exosomes. Of note was the observation that the antiangiogenic effect of older BMSCs was enhanced by direct transfection of miR-340 that was preferentially expressed in exosomes derived from young BMSCs. We found that miR-340 inhibited angiogenesis via the hepatocyte growth factor/c-MET (HGF/c-MET) signaling pathway in endothelial cells. Our data provide new insights into exosome-based cancer therapy by modification of BMSC-derived exosomes.

Downregulation of Plasma miR-215 in Chronic Myeloid Leukemia Patients with Successful Discontinuation of Imatinib.

Approximately 40% of chronic myeloid leukemia (CML) patients who discontinue imatinib (IM) therapy maintain undetectable minimal residual disease (UMRD) for more than one year (stopping IM (STOP-IM)). To determine a possible biomarker for STOP-IM CML, we examined plasma miRNA expression in CML patients who were able to discontinue IM. We first screened candidate miRNAs in unselected STOP-IM patients, who had sustained UMRD after discontinuing IM for more than six months, in comparison with healthy volunteers, by using a TaqMan low-density array for plasma or exosomes. Exosomal miR-215 and plasma miR-215 were downregulated in the STOP-IM group compared to the control, indicating that the biological relevance of the plasma miR-215 level is equivalent to that of the exosomal level. Next, we performed real-time quantitative RT-PCR in 20 STOP-IM patients, 32 patients with UMRD on continued IM therapy (IM group) and 28 healthy volunteers. The plasma miRNA-215 level was significantly downregulated in the STOP-IM group (p < 0.0001); we determined the cut-off level and divided the IM group patients into two groups according to whether the plasma miR-215 was downregulated or not. The IM group patients with a low plasma miR-215 level had a significantly higher total IM intake, compared to the patients with elevated miR-215 levels (p = 0.0229). Functional annotation of miR-215 target genes estimated by the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatic tools involved cell cycle, mitosis, DNA repair and cell cycle checkpoint. Our study suggests a possible role of miR-215 in successful IM discontinuation.

Exosomal miR-135b shed from hypoxic multiple myeloma cells enhances angiogenesis by targeting factor-inhibiting HIF-1.

Exosomes are small endosome-derived vesicles containing a wide range of functional proteins, mRNA, and miRNA. Exosomal miRNA from cancer cells helps modulate the microenvironment. In multiple myeloma (MM), the massive proliferation of malignant plasma cells causes hypoxia. To date, the majority of in vitro hypoxia studies of cancer cells have used acute hypoxic exposure (3-24 hours). Thus, we attempted to clarify the role of MM-derived exosomes in hypoxic bone marrow by using MM cells grown continuously in vitro under chronic hypoxia (hypoxia-resistant MM [HR-MM] cells). The HR-MM cells produced more exosomes than the parental cells under normoxia or acute hypoxia conditions, and miR-135b was significantly upregulated in exosomes from HR-MM cells. Exosomal miR-135b directly suppressed its target factor-inhibiting hypoxia-inducible factor 1 (FIH-1) in endothelial cells. Finally, exosomal miR-135b from HR-MM cells enhanced endothelial tube formation under hypoxia via the HIF-FIH signaling pathway. This in vitro HR myeloma cell model will be useful for investigating MM cell-endothelial cell interactions under hypoxic conditions, which may mimic the in vivo bone marrow microenvironment. Although tumor angiogenesis is regulated by various factors, exosomal miR-135b may be a target for controlling MM angiogenesis.

The fission yeast php2 mutant displays a lengthened chronological lifespan.

The Schizosaccharomyces pombe php2(+) gene encodes a subunit of the CCAAT-binding factor complex. We found that disruption of the php2(+) gene extended the chronological lifespan of the fission yeast. Moreover, the lifespan of the Δphp2 mutant was barely extended under calorie restricted (CR) conditions. Many other phenotypes of the Δphp2 mutant resembled those of wild-type cells grown under CR conditions, suggesting that the Δphp2 mutant might undergo CR. The mutant also showed low respiratory activity concomitant with decreased expression of the cyc1(+) and rip1(+) genes, both of which are involved in mitochondrial electron transport. On the basis of a chromatin immunoprecipitation assay, we determined that Php2 binds to a DNA region upstream of cyc1(+) and rip1(+) in S. pombe. Here we discuss the possible mechanisms by which the chronological lifespan of Δphp2 mutant is extended.

Extension of chronological lifespan by ScEcl1 depends on mitochondria in Saccharomyces cerevisiae.

Ecl1, a product of the YGR146C gene in Saccharomyces cerevisiae, was identified as a factor involved in chronological lifespan. In this study we found evidence that the function of Ecl1 in the extension of chronological lifespan is dependent on mitochondrial function. The respiratory activity of cells increased when Ecl1 was overexpressed or cells were grown under calorie restriction, but there was no additive effect of calorie restriction and Ecl1 overexpression on increases in respiratory activity or on the extension of chronological lifespan. Based on these results, we propose that overexpression of Ecl1 has same effect as caloric restriction and that its function also depends on mitochondria, just like caloric restriction.