RESUMO
Exon-skipping therapy is a promising treatment strategy for Duchenne muscular dystrophy (DMD), which is caused by loss-of-function mutations in the DMD gene encoding dystrophin, leading to progressive cardiomyopathy. In-frame deletion of exons 3-9 (Δ3-9), manifesting a very mild clinical phenotype, is a potential targeted reading frame for exon-skipping by targeting actin-binding domain 1 (ABD1); however, the efficacy of this approach for DMD cardiomyopathy remains uncertain. In this study, we compared three isogenic human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing Δ3-9, frameshifting Δ3-7, or intact DMD. RNA sequencing revealed a resemblance in the expression patterns of mechano-transduction-related genes between Δ3-9 and wild-type samples. Furthermore, we observed similar electrophysiological properties between Δ3-9 and wild-type hiPSC-CMs; Δ3-7 hiPSC-CMs showed electrophysiological alterations with accelerated CaMKII activation. Consistently, Δ3-9 hiPSC-CMs expressed substantial internally truncated dystrophin protein, resulting in maintaining F-actin binding and desmin retention. Antisense oligonucleotides targeting exon 8 efficiently induced skipping exons 8-9 to restore functional dystrophin and electrophysiological parameters in Δ3-7 hiPSC-CMs, bringing the cell characteristics closer to those of Δ3-9 hiPSC-CMs. Collectively, exon-skipping targeting ABD1 to convert the reading frame to Δ3-9 may become a promising therapy for DMD cardiomyopathy.
RESUMO
Induced pluripotent stem cell (iPSC)-based disease model is a useful tool that can represent the pathophysiology of patient organs that are inaccessible due to invasiveness. Here, we present a method to induce differentiation of Duchenne muscular dystrophy (DMD) patient-derived iPSCs into cardiomyocytes and restore dystrophin expression by exon skipping using antisense nucleic acids. This involves a 20-day multi-step culture process for differentiation to cardiomyocytes, followed by exon-skipping experiments. Additionally, RT-PCR, western blotting, and immunocytochemistry are used to confirm the restoration of dystrophin expression.
Assuntos
Células-Tronco Pluripotentes Induzidas , Distrofia Muscular de Duchenne , Humanos , Distrofina/genética , Distrofina/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Éxons/genéticaRESUMO
The treatment of cryptococcal encephalomyelitis is often complicated by an inadequate response to antifungal therapy and the reduction or withdrawal of antifungal agents due to adverse effects. To achieve a favorable prognosis, patients should be treated with appropriate drugs according to the latest guidelines. In addition to systemic antifungal drug administration in refractory cases, intracerebroventricular administration of amphotericin B may be considered.
Assuntos
Meningite Criptocócica , Anfotericina B/efeitos adversos , Antifúngicos/efeitos adversos , Humanos , Meningite Criptocócica/tratamento farmacológicoRESUMO
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.
Assuntos
Distrofia Muscular de Duchenne , Splicing de RNA , Distrofina/genética , Humanos , Distrofia Muscular de Duchenne/genética , Mutação/genética , OligonucleotídeosRESUMO
To investigate the prevalence and genotype-phenotype correlations of phosphatase and tensin homolog induced putative kinase 1 (PINK1) variants in Parkinson's disease (PD) patients, we analyzed 1700 patients (842 familial PD and 858 sporadic PD patients from Japanese origin). We screened the entire exon and exon-intron boundaries of PINK1 using Sanger sequencing and target sequencing by Ion torrent system. We identified 30 patients with heterozygous variants, 3 with homozygous variants, and 3 with digenic variants of PINK1-PRKN. Patients with homozygous variants presented a significantly younger age at onset than those with heterozygous variants. The allele frequency of heterozygous variants in patients with age at onset at 50 years and younger with familial PD and sporadic PD showed no differences. [123I]meta-iodobenzylguanidine (MIBG) myocardial scintigraphy indicated that half of patients harboring PINK1 heterozygous variants showed a decreased heart to mediastinum ratio (12/23). Our findings emphasize the importance of PINK1 variants for the onset of PD in patients with age at onset at 50 years and younger and the broad spectrum of clinical symptoms in patients with PINK1 variants.
Assuntos
Estudos de Associação Genética , Variação Genética/genética , Heterozigoto , Homozigoto , Doença de Parkinson/genética , Proteínas Quinases/genética , Fatores Etários , Idade de Início , Feminino , Frequência do Gene , Coração/diagnóstico por imagem , Humanos , Masculino , Mediastino/diagnóstico por imagem , Mediastino/patologia , Imagem de Perfusão do Miocárdio , Miocárdio/patologia , Doença de Parkinson/diagnóstico por imagem , Doença de Parkinson/epidemiologia , Doença de Parkinson/patologiaRESUMO
Hereditary systemic amyloidosis aside from transthyretin-related familial amyloid polyneuropathy is quite uncommon in Japan. We herein report a sporadic case of hereditary apolipoprotein A-I (apoAI) amyloidosis. The patient was a 43-year-old Japanese man who exhibited marked hepatomegaly with spleno-testicular enlargement. While he was initially thought to have primary AL amyloidosis, a proteomics analysis revealed that the amyloid was composed of variant apoAI with an E34K variant. To date, only one patient with apoAI amyloidosis has been reported in Japan. However, our study suggests that more patients may be present in Japan, and the majority may have been diagnosed with other types of amyloidosis due to its clinical similarity.
Assuntos
Neuropatias Amiloides Familiares , Apolipoproteína A-I , Adulto , Hepatomegalia/etiologia , Humanos , Japão , Masculino , Pré-AlbuminaRESUMO
A 61-year-old Japanese man presented with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. NR1 antibodies were detected in his cerebrospinal fluid. Chest computed tomography revealed lung tumor. The patient was diagnosed with paraneoplastic anti-NMDAR encephalitis associated with lung cancer and treated with two cycles of intravenous high-dose methylprednisolone and one cycle of intravenous immunoglobulin. However, he died one year later without improvement. An autopsy confirmed small-cell lung cancer (SCLC). Immunohistochemistry revealed the expression of NR1 subunits in the tumor cells, suggesting that SCLC may trigger NR1 autoimmunity though the expression of NR1 subunits as onconeural antigens, expanding the phenotypic spectrum of paraneoplastic neurological syndrome associated with SCLC.
Assuntos
Encefalite Antirreceptor de N-Metil-D-Aspartato/complicações , Neoplasias Pulmonares/complicações , Carcinoma de Pequenas Células do Pulmão/complicações , Encefalite Antirreceptor de N-Metil-D-Aspartato/diagnóstico , Anticorpos/uso terapêutico , Autoanticorpos , Humanos , Imunoglobulinas/uso terapêutico , Imunoglobulinas Intravenosas/uso terapêutico , Imuno-Histoquímica , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Metilprednisolona/uso terapêutico , Pessoa de Meia-Idade , Receptores de N-Metil-D-Aspartato/imunologia , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológicoRESUMO
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.
Assuntos
Células Cultivadas/química , Éxons/genética , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/genética , Urina/química , Humanos , TransfecçãoRESUMO
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.
Assuntos
Cálcio/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Miócitos Cardíacos/metabolismo , Arritmias Cardíacas/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Núcleo Celular/metabolismo , Distrofina/genética , Éxons , Feminino , Deleção de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Japão , Adulto JovemRESUMO
We herein report the case of a 59-year-old man with anti-leucine-rich glioma-inactivated 1 (LGI1) antibody encephalitis who presented with slowly progressive cognitive impairment mimicking dementia for over 3 years and then developed seizures. Unique brain magnetic resonance imaging (MRI) findings of fluctuating striatal lesions were observed during the disease course. He was treated with intravenous methylprednisolone pulse therapy followed by oral prednisolone, which dramatically improved his neurological function. Taken together, these findings indicate that anti-LGI1 encephalitis may present as slowly progressive cognitive impairment mimicking dementia and that fluctuating MRI striatal lesions may be a characteristic radiological finding of this disorder.
Assuntos
Autoanticorpos/sangue , Encéfalo/patologia , Disfunção Cognitiva/etiologia , Encefalite Límbica/imunologia , Encefalite Límbica/patologia , Proteínas/imunologia , Administração Intravenosa , Anti-Inflamatórios/uso terapêutico , Encéfalo/diagnóstico por imagem , Disfunção Cognitiva/diagnóstico , Disfunção Cognitiva/tratamento farmacológico , Demência/diagnóstico , Diagnóstico Diferencial , Progressão da Doença , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Leucina , Encefalite Límbica/complicações , Encefalite Límbica/tratamento farmacológico , Imageamento por Ressonância Magnética , Masculino , Metilprednisolona/uso terapêutico , Pessoa de Meia-Idade , Prednisolona/uso terapêutico , Convulsões/etiologiaRESUMO
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.
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Mycobacterium abscessus infection tends to occur in patients with an advanced immunocompromised status. We encountered a case of intractable cutaneous M. abscessus infection that developed in a patient with systemic lupus erythematosus (SLE) during maintenance therapy. A 28-year-old woman developed a fever and redness of the skin on her buttocks. General antibacterial therapy was ineffective, and acid-fast bacteria were detected in the biopsy that was conducted to differentiate the dermal symptoms of SLE. The clinical findings eventually improved; however, the symptoms recurred multiple times during treatment. Despite recent advances in SLE treatment, M. abscessus infection remains a considerable complication of SLE.
Assuntos
Antibacterianos/uso terapêutico , Claritromicina/uso terapêutico , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/etiologia , Micobactérias não Tuberculosas/efeitos dos fármacos , Adulto , Feminino , Humanos , Hospedeiro Imunocomprometido/efeitos dos fármacos , Infecções por Mycobacterium não Tuberculosas/microbiologia , Resultado do TratamentoRESUMO
Spinocerebellar ataxia type 31 (SCA31) is known as a late-onset, relatively pure cerebellar form of ataxia, but a longitudinal prospective study on the natural history of SCA31 has not been done yet. In this prospective cohort study, we enrolled 44 patients (mean ± standard deviation 73.6 ± 8.5 years) with genetically confirmed SCA31 from 10 ataxia referral centers in the Nagano area, Japan. Patients were evaluated every year for 4 years using the Scale for the Assessment and Rating of Ataxia (SARA) and the Barthel Index (BI). Of the 176 follow-up visits (91.5%), 161 were completed in this study. Five patients (11.4%) died during the follow-up period, and two patients (4.5%) were lost to follow-up. The annual progression of the SARA score was 0.8 ± 0.1 points/year and that of the BI was -2.3 ± 0.4 points/year (mean ± standard error). Shorter disease duration at baseline was associated with faster progression of the SARA score. Our study indicated the averaged clinical course of SCA31 as follows: the patients develop ataxic symptoms at 58.5 ± 10.3 years, become wheelchair bound at 79.4 ± 1.7 years, and died at 88.5 ± 0.7 years. Our prospective dataset provides important information for clinical trials of forthcoming disease-modifying therapies for cerebellar ataxia. It also represents a useful resource for SCA31 patients and their family members in genetic counseling sessions.
Assuntos
Ataxias Espinocerebelares/fisiopatologia , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Progressão da Doença , Família , Feminino , Seguimentos , Humanos , Japão , Estimativa de Kaplan-Meier , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estudos Prospectivos , Análise de Regressão , Índice de Gravidade de Doença , Ataxias Espinocerebelares/diagnóstico , Ataxias Espinocerebelares/epidemiologia , Ataxias Espinocerebelares/reabilitação , Fatores de Tempo , Cadeiras de RodasRESUMO
Multifocal fibrosclerosis is the term used to represent a combination of similar fibrous lesions occurring at different anatomical sites. We herein report a hypertrophic pachymeningitis patient with a soft tissue mass around the thoracic vertebral bodies. A histopathological analysis of the biopsied tissues from both lesions showed dense fibrosis and a marked infiltration of lymphocytes and plasma cells, which lead to the diagnosis of multifocal fibrosclerosis. This pathological condition closely resembles that of IgG4-related disease and is a very rare combination of manifestations. Our case suggests that hypertrophic pachymeningitis patients need to also undergo a whole body examination.