Six years' accomplishment of the Initiative on Rare and Undiagnosed Diseases: nationwide project in Japan to discover causes, mechanisms, and cures.

The identification of causative genetic variants for hereditary diseases has revolutionized clinical medicine and an extensive collaborative framework with international cooperation has become a global trend to understand rare disorders. The Initiative on Rare and Undiagnosed Diseases (IRUD) was established in Japan to provide accurate diagnosis, discover causes, and ultimately provide cures for rare and undiagnosed diseases. The fundamental IRUD system consists of three pillars: IRUD diagnostic coordination, analysis centers (IRUD-ACs), and a data center (IRUD-DC). IRUD diagnostic coordination consists of clinical centers (IRUD-CLs) and clinical specialty subgroups (IRUD-CSSs). In addition, the IRUD coordinating center (IRUD-CC) manages the entire IRUD system and temporarily operates the IRUD resource center (IRUD-RC). By the end of March 2021, 6301 pedigrees consisting of 18,136 individuals were registered in the IRUD. The whole-exome sequencing method was completed in 5136 pedigrees, and a final diagnosis was established in 2247 pedigrees (43.8%). The total number of aberrated genes and pathogenic variants was 657 and 1718, among which 1113 (64.8%) were novel. In addition, 39 novel disease entities or phenotypes with 41 aberrated genes were identified. The 6-year endeavor of IRUD has been an overwhelming success, establishing an all-Japan comprehensive diagnostic and research system covering all geographic areas and clinical specialties/subspecialties. IRUD has accurately diagnosed diseases, identified novel aberrated genes or disease entities, discovered many candidate genes, and enriched phenotypic and pathogenic variant databases. Further promotion of the IRUD is essential for determining causes and developing cures for rare and undiagnosed diseases.

Severe cardiac involvement with preserved truncated dystrophin expression in Becker muscular dystrophy by +1G>A DMD splice-site mutation: a case report.

Becker muscular dystrophy (BMD) is caused by specific mutations in the DMD gene that causes progressive muscle weakness and primarily affects skeletal and cardiac muscle. Although cardiac involvement is a significant cause of mortality in BMD, the genetic-phenotype correlation for skeletal and cardiac muscles has not been elucidated. Here, we described a 39-year-old man with BMD, who presented with subtle skeletal muscle weakness in the right leg in his 20s and underwent left ventricular restoration for severe dilated cardiomyopathy at the age of 29. He had difficulty climbing stairs after the age of 35. Neither duplication nor deletion of exons was detected by multiplex ligation-dependent probe amplification. A hemizygous c.264 + 1G>A mutation in intron 4 of the DMD was identified by next-generation sequencing. Furthermore, exon 4 skipping of the DMD was confirmed in both skeletal and cardiac muscles evaluated by reverse transcriptase PCR. Endomyocardial and skeletal muscle biopsies revealed dystrophic pathology characterized by muscle fiber atrophy and hypertrophy with a mild degree of interstitial fibrosis. Interestingly, dystrophin immunohistochemistry demonstrated patchy and faint staining of the skeletal muscle membranes but almost normal staining of the cardiac muscle membranes. Western blot analysis revealed a decreased amount of truncated dystrophin in skeletal muscle but surprisingly almost normal amount in cardiac muscle. This case indicates that BMD patients may have severe cardiac dysfunction despite preserved cardiac truncated dystrophin expression.

Amelioration of intracellular Ca2+ regulation by exon-45 skipping in Duchenne muscular dystrophy-induced pluripotent stem cell-derived cardiomyocytes.

Duchenne muscular dystrophy (DMD) is a devastating muscle disorder caused by frameshift mutations in the DMD gene. DMD involves cardiac muscle, and the presence of ventricular arrhythmias or congestive failure is critical for prognosis. Several novel therapeutic approaches are being evaluated in ongoing clinical trials. Among them, exon-skipping therapy to correct frameshift mutations with antisense oligonucleotides is promising; however, their therapeutic efficacies on cardiac muscle in vivo remain unknown. In this study, we established induced-pluripotent stem cells (iPSCs) from T cells from a DMD patient carrying a DMD-exon 46-55 deletion, differentiated the iPSCs into cardiomyocytes, and treated them with phosphorodiamidate morpholino oligomers. The efficiency of exon-45 skipping increased in a dose-dependent manner and enabled restoration of the DMD gene product, dystrophin. Further, Ca2+-imaging analysis showed a decreased number of arrhythmic cells and improved transient Ca2+ signaling after exon skipping. Thus, exon-45 skipping may be effective for cardiac involvement in DMD patients harboring the DMD-exon 46-55 deletion.

Intravenous nicardipine dosing for blood pressure lowering in acute intracerebral hemorrhage: the Stroke Acute Management with Urgent Risk-factor Assessment and Improvement-Intracerebral Hemorrhage study.

BACKGROUND: Intravenous nicardipine is commonly used to reduce elevated blood pressure in acute intracerebral hemorrhage (ICH). We determined factors associated with nicardipine dosing and the association of dose with clinical outcomes in hyperacute ICH. METHODS: Hyperacute (<3 hours from onset) ICH patients with initial systolic blood pressure (SBP) greater than 180 mm Hg were included. All patients initially received 5 mg/hour of intravenous nicardipine. The dose was adjusted to maintain SBP between 120 and 160 mm Hg. Associations of maximum hourly and total doses with early neurologic deterioration (END), hematoma expansion (>33%), and modified Rankin Scale score 4-6 at 3 months were assessed. RESULTS: Two hundred six patients (81 women, 65.8 ± 11.8 years) were studied. Initial SBP was 201.9 ± 15.9 mm Hg. Maximum and total nicardipine doses were 9.1 ± 4.2 mg/hour and 123.7 ± 100.2 mg/day, respectively. Multivariate analyses revealed that men (standardized regression coefficient [ß] = .20, P = .0030 for maximum dose; ß = .25, P = .0002 for total dose), age (ß = -.28, P = .0002; ß = -.25, P = .0005), and initial SBP (ß = .19, P = .0018; ß = .18, P = .0021) were independently associated with both maximum and total doses. Body weight (ß = .20, P = .0084) was independently associated with total dose. After multivariate adjustment, maximum dose (per 1 mg/hour; odds ratio [OR], 1.25; 95% confidence interval [CI], 1.09-1.45) was independently, and total dose (per 10 mg/day; OR, 1.06; 95% CI, .998-1.132) tended to be independently, associated with END. Nicardipine dose was not associated with hematoma expansion or 3-month outcome. CONCLUSIONS: Nicardipine dose is roughly predictable with sex, age, body weight, and initial SBP in acute ICH. The maximum dose was associated with neurologic deterioration.

A symptomatic male carrier of Duchenne muscular dystrophy with Klinefelter's syndrome mimicking Becker muscular dystrophy.

Duchenne and Becker muscular dystrophy (DMD/BMD) are commonly inherited muscle disorders. We report a 31-year-old male who had muscle symptoms with left-right differences and intellectual disability. He was diagnosed with BMD at age 15 primarily based on muscle biopsy findings. A few years later, DMD gene analysis revealed that he was a heterozygous carrier of a normal copy of the gene and a mutated copy with an exon 45-54 deletion, which is expected to result in an out-of-frame mutation. A karyotype analysis was compatible with XXY Klinefelter's syndrome. The analysis of X-chromosome inactivation (XCI) using his skeletal muscle sample revealed a skewed XCI pattern. This is the first reported case of a symptomatic male carrier of DMD caused by skewed XCI in Klinefelter's syndrome with a genetically proven heterozygous mutation of the DMD gene. The skewed XCI pattern could also explain the left-right differences in skeletal muscle symptoms observed in this patient.

Highly sensitive screening of antisense sequences for different types of DMD mutations in patients' urine-derived cells.

Exon skipping using short antisense oligonucleotides (AONs) is a promising treatment for Duchenne muscular dystrophy (DMD). Several exon-skipping drugs, including viltolarsen (NS-065/NCNP-01), have been approved worldwide. Immortalized human skeletal muscle cell lines, such as rhabdomyosarcoma cells, are frequently used to screen efficient oligonucleotide sequences. However, rhabdomyosarcoma cells do not recapitulate DMD pathophysiology as they express endogenous dystrophin. To overcome this limitation, we recently established a direct human somatic cell reprogramming technology and successfully developed a cellular skeletal muscle DMD model by using myogenic differentiation 1 (MYOD1)-transduced urine-derived cells (MYOD1-UDCs). Here, we compared in vitro drug screening systems in MYOD1-UDCs and rhabdomyosarcoma cells. We collected UDCs from patients with DMD amenable to exon 51 skipping, and obtained MYOD1-UDCs. We then compared the efficiency of exon 51 skipping induced by various morpholino-based AONs, including eteplirsen in differentiated MYOD1-UDCs (UDC-myotubes) and rhabdomyosarcoma cells. Exon skipping was induced more efficiently in UDC-myotubes than in rhabdomyosarcoma cells even at a low AON concentration (1 µM). Furthermore, exon 51 skipping efficiency was higher in UDC-myotubes with a deletion of exons 49-50 than in those with a deletion of exons 48-50, suggesting that the skipping efficiency may vary depending on the DMD mutation pattern. An essential finding of this study is that the sequence of eteplirsen consistently leads to much lower efficiency than other sequences. These findings underscore the importance of AON sequence optimization by our cellular system, which enables highly sensitive screening of exon skipping drugs that target different types of DMD mutations.

Cricopharyngeal bar on videofluoroscopy: high specificity for inclusion body myositis.

OBJECTIVE: To determine the prevalence and characteristics of the cricopharyngeal bar (CPB), defined as marked protrusion with lacking relaxation and stricture of the upper esophageal sphincter on videofluoroscopy, in patients with inclusion body myositis (IBM). METHODS: We conducted a case-control study of comprehensive series of adult healthy individuals and consecutive patients with neuropsychiatric disorders aged over 45 (52 versus 2486). A standard videofluoroscopy was performed. RESULTS: Overall, 47 individuals with CPB were identified. Of the individuals with CPB, 36% were IBM followed by neurodegenerative disorders, muscular disorders, neuromuscular disorders, and others (32%, 21%, 2.1%, and 8.5%, respectively), indicating the heterogeneity of the etiologies. Against muscular disorders, the sensitivity and specificity of the CPB for IBM were 33% (= 17/52; 95% confidence interval [CI], 20-45%) and 96% (= 264/274; 95% CI, 94-99%), respectively. IBM with CPB showed a higher frequency of obstruction-related dysphagia (88% versus 22%, p < 0.001) and severe CPB (76% versus 23%, p < 0.001) than the control with one. The ratio of the upper esophageal distance at the maximum distension at the level of C6 to that of C4 was lower in IBM with CPB than in the controls with one (0.50 versus 0.77, p < 0.001), which suggests the insufficient opening of the upper esophageal sphincter. CONCLUSION: A CPB could be indicative of IBM. The upper esophagus in IBM with CPB became narrow, like a bottleneck. We provide new perspectives of dysphagia diagnosis by videofluoroscopy, especially for IBM-associated dysphagia, to expand the knowledge on the CPB.

Exon Skipping in Directly Reprogrammed Myotubes Obtained from Human Urine-Derived Cells.

Duchenne muscular dystrophy (DMD), a progressive and fatal muscle disease, is caused by mutations in the DMD gene that result in the absence of dystrophin protein. To date, we have completed an investigator-initiated first-in-human study at the National Center of Neurology and Psychiatry based on the systemic injection of the morpholino oligonucleotides which are prone to exon-53 skipping. For the effective treatment of DMD, in vitro testing with myoblasts derived from DMD patients to screen drugs and assess patient eligibility before undertaking clinical trials is thought to be essential. Very recently, we reported a new MYOD1-converted urine-derived cell (UDC) treated with the histone methyltransferase inhibitor (3-deazaneplanocin A hydrochloride), as a cellular model of DMD. The new autologous UDC might show phenocopy of the disease-specific phenotypes of DMD, leading to the application of precision medicine in a variety of muscle-related diseases. In this article, we describe a detailed protocol for efficient modelling of DMD muscle cells using MYOD1-converted UDCs along with reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry to evaluate the restoration of dystrophin mRNA and protein levels after exon skipping.

Publisher Correction: Modelling Duchenne muscular dystrophy in MYOD1-converted urine-derived cells treated with 3-deazaneplanocin A hydrochloride.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Vogt-Koyanagi-Harada disease associated with brainstem encephalitis.

We report on a 28-year-old man with a 2-year history of Vogt-Koyanagi-Harada disease (VKH), who developed one-and-a-half syndrome, facial hypoesthesia, facial nerve palsy, hearing loss and limb ataxia on the right side. An MRI showed inflammatory lesions in the pons extending into the left middle cerebellar peduncle. Corticosteroid treatment successfully ameliorated his symptoms and lesions. This patient demonstrated the possible association of brainstem encephalitis with VKH.

Application of Urine-Derived Stem Cells to Cellular Modeling in Neuromuscular and Neurodegenerative Diseases.

Neuromuscular and neurodegenerative diseases are mostly modeled using genetically modified animals such as mice. However, animal models do not recapitulate all the phenotypes that are specific to human disease. This is mainly due to the genetic, anatomical and physiological difference in the neuromuscular systems of animals and humans. The emergence of direct and indirect human somatic cell reprogramming technologies may overcome this limitation because they enable the use of disease and patient-specific cellular models as enhanced platforms for drug discovery and autologous cell-based therapy. Induced pluripotent stem cells (iPSCs) and urine-derived stem cells (USCs) are increasingly employed to recapitulate the pathophysiology of various human diseases. Recent cell-based modeling approaches utilize highly complex differentiation systems that faithfully mimic human tissue- and organ-level dysfunctions. In this review, we discuss promising cellular models, such as USC- and iPSC-based approaches, that are currently being used to model human neuromuscular and neurodegenerative diseases.

Modelling Duchenne muscular dystrophy in MYOD1-converted urine-derived cells treated with 3-deazaneplanocin A hydrochloride.

Duchenne muscular dystrophy (DMD) is a severe muscle disorder characterised by mutations in the DMD gene. Recently, we have completed a phase I study in Japan based on systemic administration of the morpholino antisense that is amenable to exon-53 skipping, successfully. However, to achieve the effective treatment of DMD, in vitro assays on patient muscle cells to screen drugs and patient eligibility before clinical trials are indispensable. Here, we report a novel MYOD1-converted, urine-derived cells (UDCs) as a novel DMD muscle cell model. We discovered that 3-deazaneplanocin A hydrochloride, a histone methyltransferase inhibitor, could significantly promote MYOGENIN expression and myotube differentiation. We also demonstrated that our system, based on UDCs from DMD patients, could be used successfully to evaluate exon-skipping drugs targeting DMD exons including 44, 50, 51, and 55. This new autologous UDC-based disease modelling could lead to the application of precision medicine for various muscle diseases.

In Vivo Evaluation of Single-Exon and Multiexon Skipping in mdx52 Mice.

Exon-skipping therapy is an emerging approach that uses synthetic DNA-like molecules called antisense oligonucleotides (ASOs) to splice out frame-disrupting parts of mRNA, restore the reading frame, and produce truncated yet functional proteins. Phosphorodiamidate morpholino oligomer (PMO) is one of the safest among therapeutic ASOs for patients and has recently been approved under the accelerated approval program by the US Food and Drug Administration (FDA) as the first ASO-based drug for Duchenne muscular dystrophy (DMD). Multi-exon skipping utilizing ASOs can theoretically treat 80-90% of patients with DMD. Here, we describe the systemic delivery of a cocktail of ASOs to skip exon 51 and exons 45-55 in the mdx52 mouse, an exon 52 deletion model of DMD produced by gene targeting, and the evaluation of their efficacies in vivo.

Exon Skipping Using Antisense Oligonucleotides for Laminin-Alpha2-Deficient Muscular Dystrophy.

Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is among the more promising approaches available for the treatment of several neuromuscular disorders, including Duchenne muscular dystrophy. The main weakness of this treatment arises from the low efficiency and sporadic nature of delivery of the neutrally charged PMO into muscle fibers, the mechanism of which is unknown.Recently, using wild-type and dystrophic mdx52 mice, we showed that muscle fibers took up PMO more efficiently during myotube formation. Interestingly, through in situ hybridization, we detected PMO mainly in embryonic myosin heavy chain-positive regenerating fibers. Next, we tested the therapeutic potential of PMO in laminin-alpha2 (laminin-α2) chain-null dy 3K/dy 3K mice, a model of merosin-deficient congenital muscular dystrophy 1A (MDC1A) with active muscle regeneration. We confirmed the recovery of the laminin-α2 chain following skipping of the mutated exon 4 in dy 3K/dy 3K mice, which prolonged the life span of the animals slightly. These findings support the theory that PMO entry into fibers is dependent on the developmental stage in myogenesis rather than on dystrophinless muscle membranes, and provide a platform for the future development of PMO-mediated therapies for a variety of muscular disorders, such as MDC1A, that involve active muscle regeneration. Herein, we describe the methods for PMO transfection/injection and evaluation of the efficacy of exon skipping in the laminin-α2-deficient dy 3K/dy 3K mouse model both in vitro and in vivo.

Exon Skipping Therapy Using Phosphorodiamidate Morpholino Oligomers in the mdx52 Mouse Model of Duchenne Muscular Dystrophy.

Exon skipping therapy using synthetic DNA-like molecules called antisense oligonucleotides (ASOs) is a promising therapeutic candidate for overcoming the dystrophin mutation that causes Duchenne muscular dystrophy (DMD). This treatment involves splicing out the frame-disrupting segment of the dystrophin mRNA, which restores the reading frame and produces a truncated yet functional dystrophin protein. Phosphorodiamidate morpholino oligomer (PMO) is the safest ASO for patients among ASOs and has recently been approved under the accelerated approval pathway by the U.S. Food and Drug Administration (FDA) as the first drug for DMD. Here, we describe the methodology and protocol of PMO transfection and evaluation of the exon skipping efficacy in the mdx52 mouse, an exon 52 deletion model of DMD produced by gene targeting. The mdx52 mouse model offers advantages over the mdx mouse, a spontaneous DMD model with a nonsense mutation in exon 23, in terms of the deletion in a hotspot of deletion mutations in DMD patients, the analysis of caveolae and also Dp140 and Dp260, shorter dystrophin isoforms.

Solid-Phase Synthesis of Difficult Purine-Rich PNAs through Selective Hmb Incorporation: Application to the Total Synthesis of Cell Penetrating Peptide-PNAs.

Antisense oligonucleotide (ASO)-based drug development is gaining significant momentum following the recent FDA approval of Eteplirsen (an ASO based on phosphorodiamidate morpholino) and Spinraza (2'-O-methoxyethyl-phosphorothioate) in late 2016. Their attractiveness is mainly due to the backbone modifications which have improved the in vivo characteristics of oligonucleotide drugs. Another class of ASO, based on peptide nucleic acid (PNA) chemistry, is also gaining popularity as a platform for development of gene-specific therapy for various disorders. However, the chemical synthesis of long PNAs, which are more target-specific, remains an ongoing challenge. Most of the reported methodology for the solid-phase synthesis of PNA suffer from poor coupling efficiency which limits production to short PNA sequences of less than 15 residues. Here, we have studied the effect of backbone modifications with Hmb (2-hydroxy-4-methoxybenzyl) and Dmb (2,4-dimethoxybenzyl) to ameliorate difficult couplings and reduce "on-resin" aggregation. We firstly synthesized a library of PNA dimers incorporating either Hmb or Dmb and identified that Hmb is superior to Dmb in terms of its ease of removal. Subsequently, we used Hmb backbone modification to synthesize a 22-mer purine-rich PNA, targeting dystrophin RNA splicing, which could not be synthesized by standard coupling methodology. Hmb backbone modification allowed this difficult PNA to be synthesized as well as to be continued to include a cell-penetrating peptide on the same solid support. This approach provides a novel and straightforward strategy for facile solid-phase synthesis of difficult purine-rich PNA sequences.

Carotid Ultrasonography Can Identify Stroke Patients Ineligible for Intravenous Thrombolysis Therapy due to Acute Aortic Dissection.

Acute aortic dissection is the most common acute aortic condition requiring urgent surgical therapy. Due to lack of typical symptoms, it is sometimes difficult to identify acute aortic dissection causing ischemic stroke. We report a case of a patient with acute ischemic stroke who was deemed ineligible for intravenous recombinant tissue plasminogen activator treatment based on a finding of acute aortic dissection detected by carotid ultrasonography. After urgent aortic replacement surgery, the patient recovered with no neurological deficit. This case underscores the crucial role of carotid ultrasonography for the investigation of possible underlying acute aortic dissection when considering the use of intravenous recombinant tissue plasminogen activator therapy for hyperacute stroke.