RESUMO
The dystrophin gene (Dmd) is recognized for its significance in Duchenne muscular dystrophy (DMD), a lethal and progressive skeletal muscle disease. Some patients with DMD and model mice with muscular dystrophy (mdx) spontaneously develop various types of tumors, among which rhabdomyosarcoma (RMS) is the most prominent. By contrast, spindle cell sarcoma (SCS) has rarely been reported in patients or mdx mice. In this study, we aimed to use metabolomics to better understand the rarity of SCS development in mdx mice. Gas chromatography-mass spectrometry was used to compare the metabolic profiles of spontaneously developed SCS and RMS tumors from mdx mice, and metabolite supplementation assays and silencing experiments were used to assess the effects of metabolic differences in SCS tumor-derived cells. The levels of 75 metabolites exhibited differences between RMS and SCS, 25 of which were significantly altered. Further characterization revealed downregulation of nonessential amino acids, including alanine, in SCS tumors. Alanine supplementation enhanced the growth, epithelial mesenchymal transition, and invasion of SCS cells. Reduction of intracellular alanine via knockdown of the alanine transporter Slc1a5 reduced the growth of SCS cells. Lower metabolite secretion and reduced proliferation of SCS tumors may explain the lower detection rate of SCS in mdx mice. Targeting of alanine depletion pathways may have potential as a novel treatment strategy.NEW & NOTEWORTHY To the best of our knowledge, SCS has rarely been identified in patients with DMD or mdx mice. We observed that RMS and SCS tumors that spontaneously developed from mdx mice with the same Dmd genetic background exhibited differences in metabolic secretion. We proposed that, in addition to dystrophin deficiency, the levels of secreted metabolites may play a role in the determination of tumor-type development in a Dmd-deficient background.
Assuntos
Camundongos Endogâmicos mdx , Rabdomiossarcoma , Sarcoma , Animais , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Rabdomiossarcoma/genética , Camundongos , Sarcoma/metabolismo , Sarcoma/patologia , Sarcoma/genética , Metabolômica/métodos , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Proliferação de Células , Masculino , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/genética , Transição Epitelial-Mesenquimal , Sistema ASC de Transporte de Aminoácidos/metabolismo , Sistema ASC de Transporte de Aminoácidos/genéticaRESUMO
INTRODUCTION/AIMS: Duchenne muscular dystrophy (DMD) presents with skeletal muscle weakness, followed by cardiorespiratory involvement. The need for longitudinal data regarding DMD that could serve as a control for determining treatment efficacy in clinical trials has increased notably. The present study examined the longitudinal data of Japanese DMD patients collectively and assessed individual patients with pathogenic variants eligible for exon-skipping therapy. METHODS: Patients with DMD who visited Kobe University Hospital between March 1991 and March 2019 were enrolled. Data between the patients' first visit until age 20 years were examined. RESULTS: Three hundred thirty-seven patients were included. Serum creatine kinase levels showed extremely high values until the age of 6 years and a rapid decline from ages 7-12 years. Both the median 10-m run/walk velocity and rise-from-floor velocity peaked at the age of 4 years and declined with age. The values for respiratory function declined from the age of 11 years. The median left ventricular ejection fraction was >60% until the age of 12 years and rapidly declined from ages 13-15 years. Examination of the relationship between pathogenic variants eligible for exon-skipping therapy and longitudinal data revealed no characteristic findings. DISCUSSION: We found that creatine kinase levels and motor, respiratory, and cardiac functions each exhibited various changes over time. These findings provide useful information about the longitudinal data of several outcome measures for patients with DMD not receiving corticosteroids. These data may serve as historical controls in comparing the natural history of DMD patients not on regular steroid use in appropriate clinical trials.
Assuntos
Distrofia Muscular de Duchenne , Humanos , Adulto Jovem , Adulto , Criança , Pré-Escolar , Distrofia Muscular de Duchenne/tratamento farmacológico , Volume Sistólico , Função Ventricular Esquerda , Corticosteroides/uso terapêutico , Creatina QuinaseRESUMO
BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder caused by insufficient α-galactosidase A (GLA) activity resulting from variants in the GLA gene, which leads to glycosphingolipid accumulation and life-threatening, multi-organ complications. Approximately 50 variants have been reported that cause splicing abnormalities in GLA. Most were found within canonical splice sites, which are highly conserved GT and AG splice acceptor and donor dinucleotides, whereas one-third were located outside canonical splice sites, making it difficult to interpret their pathogenicity. In this study, we aimed to investigate the genetic pathogenicity of variants located in non-canonical splice sites within the GLA gene. METHODS: 13 variants, including four deep intronic variants, were selected from the Human Gene Variant Database Professional. We performed an in vitro splicing assay to identify splicing abnormalities in the variants. RESULTS: All candidate non-canonical splice site variants in GLA caused aberrant splicing. Additionally, all but one variant was protein-truncating. The four deep intronic variants generated abnormal transcripts, including a cryptic exon, as well as normal transcripts, with the proportion of each differing in a cell-specific manner. CONCLUSIONS: Validation of splicing effects using an in vitro splicing assay is useful for confirming pathogenicity and determining associations with clinical phenotypes.
Assuntos
Doença de Fabry , Sítios de Splice de RNA , Humanos , Éxons , Doença de Fabry/genética , Íntrons , Mutação , Sítios de Splice de RNA/genética , Splicing de RNARESUMO
Nephronophthisis is an autosomal-recessive kidney disease that is caused by abnormalities in primary cilia. Nephronophthisis-related ciliopathies (NPHP-RCs) are a common cause of end-stage kidney disease (ESKD) in children and adolescents. NPHP-RCs are often accompanied by extrarenal manifestations, including intellectual disability, retinitis pigmentosa, or polydactyly. Although more than 100 causative genes have been identified, its diagnosis is difficult because the clinical features of each mutation often overlap. From September 2010 to August 2021, we performed genetic analysis, including next-generation sequencing (NGS), in 574 probands with kidney dysfunction and retrospectively studied cases genetically diagnosed with NPHP-RCs. RESULTS: We detected mutations related to NPHP-RCs in 93 patients from 83 families. Members of 60 families were diagnosed using NGS, and the mutations and the corresponding number of families are as follows: NPHP1 (24), NPHP3 (10), OFD1 (7), WDR35 (5), SDCCAG8 (4), BBS10 (3), TMEM67 (3), WDR19 (3), BBS1 (2), BBS2 (2), IFT122 (2), IFT140 (2), IQCB1 (2), MKKS (2), SCLT1 (2), TTC21B (2), ALMS1 (1), ANKS6 (1), BBS4 (1), BBS12 (1), CC2D2A (1), DYNC2H1 (1), IFT172 (1), and MAPKBP1 (1). A total of 39 cases (41.9%) progressed to ESKD at the time of genetic analysis, whereas 58 cases (62.3%) showed extrarenal manifestations, the most common being developmental delay, intellectual disability, and autism spectrum disorder in 44 patients. Comprehensive genetic analysis using NGS is useful for diagnosing patients with NPHP-RCs.
Assuntos
Transtorno do Espectro Autista , Ciliopatias , Deficiência Intelectual , Doenças Renais Císticas , Adolescente , Transtorno do Espectro Autista/genética , Criança , Ciliopatias/diagnóstico , Ciliopatias/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Deficiência Intelectual/genética , Japão , Doenças Renais Císticas/diagnóstico , Doenças Renais Císticas/genética , Mutação , Estudos RetrospectivosRESUMO
Gitelman syndrome (GS) is a rare, autosomal recessive, salt-losing tubulopathy caused by loss of function in the SLC12A3 gene (NM_000339.2), which encodes the natrium chloride cotransporter. The detection of homozygous or compound heterozygous SLC12A3 variants is expected in GS, but 18%-40% of patients with clinical GS carry only one mutant allele. Previous reports identified some pathogenic deep intronic variants in SLC12A3. Here, we report the screening of SLC12A3 deep intronic variants in 13 patients with suspected GS carrying one mutated SLC12A3 allele. Variant screening used the HaloPlex Target Enrichment System Kit capturing whole introns and the promotor region of SLC12A3, followed by SureCall variant analysis. Rare intronic variants (<1% frequency) were identified, and pathogenicity evaluated by the minigene system. Deep intronic variant screening detected seven rare SLC12A3 variants from six patients. Only one variant showed pathogenicity in the minigene system (c.602-16G>A, intron 4) through activation of a cryptic acceptor site. No variants were detected in the promotor region. Deep intronic screening identified only one pathogenic variant in patients with suspected GS carrying monoallelic SLC12A3 variants. Our results suggest that deep intronic variants partially explain the cause of monoallelic variants in patients with GS.
Assuntos
Síndrome de Gitelman , Alelos , Síndrome de Gitelman/diagnóstico , Síndrome de Gitelman/genética , Humanos , Íntrons/genética , Mutação , Membro 3 da Família 12 de Carreador de Soluto/genéticaRESUMO
BACKGROUND: Urinary titin N-fragment levels have been used to assess the catabolic state, and we used this biomarker to evaluate the catabolic state of infants. METHODS: We retrospectively measured urinary titin N-fragment levels of urinary samples. The primary outcome was its changes according to postmenstrual age. The secondary outcomes included differences between gestational age, longitudinal change after birth, influence on growth, and relationship with blood tests. RESULTS: This study included 219 patients with 414 measurements. Urinary titin N-fragment exponentially declined with postmenstrual age. These values were 12.5 (7.1-19.6), 8.1 (5.1-13.0), 12.8 (6.0-21.3), 26.4 (16.4-52.0), and 81.9 (63.3-106.4) pmol/mg creatinine in full, late, moderate, very, and extremely preterm infants, respectively (p < 0.01). After birth, urinary levels of titin N-fragment exponentially declined, and the maximum level within a week was associated with the time to return to birth weight in preterm infants (ρ = 0.39, p < 0.01). This was correlated with creatine kinase in full-term infants (ρ = 0.58, p < 0.01) and with blood urea nitrogen in preterm infants (ρ = 0.50, p < 0.01). CONCLUSIONS: The catabolic state was increased during the early course of the postmenstrual age and early preterm infants. IMPACT: Catabolic state in infants, especially in preterm infants, was expected to be increased, but no study has clearly verified this. In this retrospective study of 219 patients with 414 urinary titin measurements, the catabolic state was exponentially elevated during the early postmenstrual age. The use of the urinary titin N-fragment clarified catabolic state was prominently increased in very and extremely preterm infants.
Assuntos
Recém-Nascido Prematuro , Peso ao Nascer , Conectina/urina , Idade Gestacional , Humanos , Lactente , Recém-Nascido , Estudos RetrospectivosRESUMO
BACKGROUND: Autosomal recessive polycystic kidney disease (ARPKD) is caused by mutations in the PKHD1 gene. The clinical spectrum is often more variable than previously considered. We aimed to analyze the clinical features of genetically diagnosed ARPKD in the Japanese population. METHODS: We conducted a genetic analysis of patients with clinically diagnosed or suspected ARPKD in Japan. Moreover, we performed a minigene assay to elucidate the mechanisms that could affect phenotypes. RESULTS: PKHD1 pathogenic variants were identified in 32 patients (0-46 years). Approximately one-third of the patients showed prenatal anomalies, and five patients died within one year after birth. Other manifestations were detected as follows: chronic kidney disease stages 1-2 in 15/26 (57.7%), Caroli disease in 9/32 (28.1%), hepatic fibrosis in 7/32 (21.9%), systemic hypertension in 13/27 (48.1%), and congenital hypothyroidism in 3 patients. There have been reported that truncating mutations in both alleles led to severe phenotypes with perinatal demise. However, one patient without a missense mutation survived the neonatal period. In the minigene assay, c.2713C > T (p.Gln905Ter) and c.6808 + 1G > A expressed a transcript that skipped exon 25 (123 bp) and exon 41 (126 bp), resulting in an in-frame mutation, which might have contributed to the milder phenotype. Missense mutations in cases of neonatal demise did not show splicing abnormalities. CONCLUSION: Clinical manifestations ranged from cases of neonatal demise to those diagnosed in adulthood. The minigene assay results indicate the importance of functional analysis, and call into question the fundamental belief that at least one non-truncating mutation is necessary for perinatal survival.
Assuntos
Rim Policístico Autossômico Recessivo , Adulto , Feminino , Testes Genéticos/métodos , Humanos , Japão , Mutação , Fenótipo , Rim Policístico Autossômico Recessivo/diagnóstico , Rim Policístico Autossômico Recessivo/genética , Gravidez , Receptores de Superfície Celular/genéticaRESUMO
Antisense oligonucleotides (ASOs) are agents that modulate gene function. ASO-mediated out-of-frame exon skipping has been employed to suppress gene function. Myostatin, encoded by the MSTN gene, is a potent negative regulator of skeletal muscle growth. ASOs that induce skipping of out-of-frame exon 2 of the MSTN gene have been studied for their use in increasing muscle mass. However, no ASOs are currently available for clinical use. We hypothesized that ASOs against the splicing enhancer sequence within exon 1 of the MSTN gene would inhibit maturation of pre-mRNA, thereby suppressing gene function. To explore this hypothesis, ASOs against sequences of exon 1 of the MSTN gene were screened for their ability to reduce mature MSTN mRNA levels. One screened ASO, named KMM001, decreased MSTN mRNA levels in a dose-dependent manner and reciprocally increased MSTN pre-mRNA levels. Accordingly, KMM001 decreased myostatin protein levels. KMM001 inhibited SMAD-mediated myostatin signaling in rhabdomyosarcoma cells. Remarkably, it did not decrease GDF11 mRNA levels, indicating myostatin-specific inhibition. As expected, KMM001 enhanced the proliferation of human myoblasts. We conclude that KMM001 is a novel myostatin inhibitor that inhibits pre-mRNA maturation. KMM001 has great promise for clinical applications and should be examined for its ability to treat various muscle-wasting conditions.
Assuntos
Miostatina , Oligonucleotídeos Antissenso , Proteínas Morfogenéticas Ósseas/metabolismo , Elementos Facilitadores Genéticos , Éxons , Fatores de Diferenciação de Crescimento/genética , Humanos , Músculo Esquelético/metabolismo , Miostatina/antagonistas & inibidores , Miostatina/genética , Miostatina/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by out-of-frame or nonsense mutation in the dystrophin gene. It begins with a loss of ambulation between 9 and 14 years of age, followed by various other symptoms including cardiac dysfunction. Exon skipping of patients' DMD pre-mRNA induced by antisense oligonucleotides (AOs) is expected to produce shorter but partly functional dystrophin proteins, such as those possessed by patients with the less severe Becker muscular dystrophy. We are working on developing modified nucleotides, such as 2'-O,4'-C-ethylene-bridged nucleic acids (ENAs), possessing high nuclease resistance and high affinity for complementary RNA strands. Here, we demonstrate the preclinical characteristics (exon-skipping activity in vivo, stability in blood, pharmacokinetics, and tissue distribution) of renadirsen, a novel AO modified with 2'-O-methyl RNA/ENA chimera phosphorothioate designed for dystrophin exon 45 skipping and currently under clinical trials. Notably, systemic delivery of renadirsen sodium promoted dystrophin exon skipping in cardiac muscle, skeletal muscle, and diaphragm, compared with AOs with the same sequence as renadirsen but conventionally modified by PMO and 2'OMePS. These findings suggest the promise of renadirsen sodium as a therapeutic agent that improves not only skeletal muscle symptoms but also other symptoms in DMD patients, such as cardiac dysfunction.
Assuntos
Processamento Alternativo , Distrofina/genética , Oligonucleotídeos Antissenso/genética , Animais , Cromatografia Líquida , Masculino , Camundongos , Camundongos Endogâmicos mdx , Estrutura Molecular , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Oligodesoxirribonucleotídeos/química , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/química , Oligorribonucleotídeos/química , Espectrometria de Massas em Tandem , Distribuição TecidualRESUMO
BACKGROUND: Alport syndrome (AS), which is a rare hereditary disease caused by mutations of genes including COL4A3, COL4A4 and COL4A5, has a wide spectrum of phenotypes. Most disease-causing variants of AS are located in the exons or the conservative splicing sites of these genes, while little is known about the intronic disease-causing variants. METHODS: A Chinese AS family was recruited in this study. All the clinical data of AS patient were collected from medical records. After pedigree analysis, the pathogenic variants were studied by the whole exome sequencing (WES). Minigene assay and in vivo RT-PCR analysis were performed to validate the functions of the variants. RESULTS: Renal biopsy showed a typical histopathology changes of AS. WES revealed compound heterozygous substitution, NM_033380 c.991-14(IVS17) A > G, in the intron 17 of the COL4A5 gene, which were confirmed by Sanger sequencing. Moreover, the variant was co-segregated with the phenotype in this family. Minigene assay in cultured cell lines showed that a splicing error was induced by this intronic variant, which further confirmed by in vivo RT-PCR analysis. CONCLUSION: A novel intronic disease-causing variant in COL4A5 gene was identified by WES, which was the molecular pathogenic basis of AS.
Assuntos
Colágeno Tipo IV/genética , Íntrons/genética , Nefrite Hereditária/genética , Povo Asiático/genética , Criança , Feminino , Humanos , Mutação , Nefrite Hereditária/diagnóstico por imagem , Nefrite Hereditária/patologia , Linhagem , Reação em Cadeia da Polimerase em Tempo Real , Sequenciamento do ExomaRESUMO
Diamond-Blackfan anemia (DBA) is mainly caused by pathogenic variants in ribosomal proteins and 22 responsible genes have been identified to date. The most common causative gene of DBA is RPS19 [NM_001022.4]. Nearly 180 RPS19 variants have been reported, including three deep intronic variants outside the splicing consensus sequence (c.72-92A > G, c.356 + 18G > C, and c.411 + 6G > C). We also identified one case with a c.412-3C > G intronic variant. Without conducting transcript analysis, the pathogenicity of these variants is unknown. However, it is difficult to assess transcripts because of their fragility. In such cases, in vitro functional splicing assays can be used to assess pathogenicity. Here, we report functional splicing analysis results of four RPS19 deep intronic variants identified in our case and in previously reported cases. One splicing consensus variant (c.411 + 1G > A) was also examined as a positive control. Aberrant splicing with a 2-bp insertion between exons 5 and 6 was identified in the patient samples and minigene assay results also identified exon 6 skipping in our case. The exon 6 skipping transcript was confirmed by further evaluation using quantitative RT-PCR. Additionally, minigene assay analysis of three reported deep intronic variants revealed that none of them showed aberrant splicing and that these variants were not considered to be pathogenic. In conclusion, the minigene assay is a useful method for functional splicing analysis of inherited disease.
Assuntos
Anemia de Diamond-Blackfan , Mutação , Splicing de RNA , Proteínas Ribossômicas , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/metabolismo , Humanos , Recém-Nascido , Masculino , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/genéticaRESUMO
To understand the genetics of steroid-sensitive nephrotic syndrome (SSNS), we conducted a genome-wide association study in 987 childhood SSNS patients and 3,206 healthy controls with Japanese ancestry. Beyond known associations in the HLA-DR/DQ region, common variants in NPHS1-KIRREL2 (rs56117924, P=4.94E-20, odds ratio (OR) =1.90) and TNFSF15 (rs6478109, P=2.54E-8, OR=0.72) regions achieved genome-wide significance and were replicated in Korean, South Asian and African populations. Trans-ethnic meta-analyses including Japanese, Korean, South Asian, African, European, Hispanic and Maghrebian populations confirmed the significant associations of variants in NPHS1-KIRREL2 (Pmeta=6.71E-28, OR=1.88) and TNFSF15 (Pmeta=5.40E-11, OR=1.33) loci. Analysis of the NPHS1 risk alleles with glomerular NPHS1 mRNA expression from the same person revealed allele specific expression with significantly lower expression of the transcript derived from the risk haplotype (Wilcox test p=9.3E-4). Because rare pathogenic variants in NPHS1 cause congenital nephrotic syndrome of the Finnish type (CNSF), the present study provides further evidence that variation along the allele frequency spectrum in the same gene can cause or contribute to both a rare monogenic disease (CNSF) and a more complex, polygenic disease (SSNS).
Assuntos
Síndrome Nefrótica , Alelos , Criança , Estudo de Associação Genômica Ampla , Haplótipos , Humanos , Proteínas de Membrana , Mutação , Síndrome Nefrótica/tratamento farmacológico , Síndrome Nefrótica/genética , Esteroides , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genéticaRESUMO
Duchenne muscular dystrophy (DMD) is caused by a nonsense or frameshift mutation in the DMD gene, while its milder form, Becker muscular dystrophy (BMD) is caused by an in-frame deletion/duplication or a missense mutation. Interestingly, however, some patients with a nonsense mutation exhibit BMD phenotype, which is mostly attributed to the skipping of the exon containing the nonsense mutation, resulting in in-frame deletion. This study aims to find BMD cases with nonsense/frameshift mutations in DMD and to investigate the exon skipping rate of those nonsense/frameshift mutations. We searched for BMD cases with nonsense/frameshift mutations in DMD in the Japanese Registry of Muscular Dystrophy. For each DMD mutation identified, we constructed minigene plasmids containing one exon with/without a mutation and its flanking intronic sequence. We then introduced them into HeLa cells and measured the skipping rate of transcripts of the minigene by RT-qPCR. We found 363 cases with a nonsense/frameshift mutation in DMD gene from a total of 1497 dystrophinopathy cases in the registry. Among them, 14 had BMD phenotype. Exon skipping rates were well correlated with presence or absence of dystrophin, suggesting that 5% exon skipping rate is critical for the presence of dystrophin in the sarcolemma, leading to milder phenotypes. Accurate quantification of the skipping rate is important in understanding the exact functions of the nonsense/frameshift mutations in DMD and for interpreting the phenotypes of the BMD patients.
Assuntos
Códon sem Sentido , Distrofina/genética , Éxons/genética , Mutação da Fase de Leitura , Distrofia Muscular de Duchenne/genética , Adolescente , Adulto , Criança , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
OBJECTIVES: Although skeletal muscle atrophy is common in critically ill patients, biomarkers associated with muscle atrophy have not been identified reliably. Titin is a spring-like protein found in muscles and has become a measurable biomarker for muscle breakdown. We hypothesized that urinary titin is useful for monitoring muscle atrophy in critically ill patients. Therefore, we investigated urinary titin level and its association with muscle atrophy in critically ill patients. DESIGN: Two-center, prospective observational study. SETTING: Mixed medical/surgical ICU in Japan. PATIENTS: Nonsurgical adult patients who were expected to remain in ICU for greater than 5 days. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Urine samples were collected on days 1, 2, 3, 5, and 7 of ICU admission. To assess muscle atrophy, rectus femoris cross-sectional area and diaphragm thickness were measured with ultrasound on days 1, 3, 5, and 7. Secondary outcomes included its relationship with ICU-acquired weakness, ICU Mobility Scale, and ICU mortality. Fifty-six patients and 232 urinary titin measurements were included. Urinary titin (normal range: 1-3 pmol/mg creatinine) was 27.9 (16.8-59.6), 47.6 (23.5-82.4), 46.6 (24.4-97.6), 38.4 (23.6-83.0), and 49.3 (27.4-92.6) pmol/mg creatinine on days 1, 2, 3, 5, and 7, respectively. Cumulative urinary titin level was significantly associated with rectus femoris muscle atrophy on days 3-7 (p ≤ 0.03), although urinary titin level was not associated with change in diaphragm thickness (p = 0.31-0.45). Furthermore, cumulative urinary titin level was associated with occurrence of ICU-acquired weakness (p = 0.01) and ICU mortality (p = 0.02) but not with ICU Mobility Scale (p = 0.18). CONCLUSIONS: In nonsurgical critically ill patients, urinary titin level increased 10-30 times compared with the normal level. The increased urinary titin level was associated with lower limb muscle atrophy, occurrence of ICU-acquired weakness, and ICU mortality.
Assuntos
Conectina/urina , Diafragma/patologia , Unidades de Terapia Intensiva , Atrofia Muscular/patologia , Músculo Quadríceps/patologia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Creatinina/urina , Estado Terminal , Diafragma/diagnóstico por imagem , Feminino , Mortalidade Hospitalar , Humanos , Japão , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Debilidade Muscular/patologia , Atrofia Muscular/diagnóstico por imagem , Desempenho Físico Funcional , Estudos Prospectivos , Músculo Quadríceps/diagnóstico por imagem , UltrassonografiaRESUMO
BACKGROUND: Duchenne muscular dystrophy (DMD) is a fatal progressive muscle-wasting disease caused by mutations in the DMD gene. Dilated cardiomyopathy is the leading cause of death in DMD; therefore, further understanding of this complication is essential to reduce morbidity and mortality. METHODS: A common null variant (R577X) in the ACTN3 gene, which encodes α-actinin-3, has been studied in association with muscle function in healthy individuals; however it has not yet been examined in relationship to the cardiac phenotype in DMD. In this study, we determined the ACTN3 genotype in 163 patients with DMD and examined the correlation between ACTN3 genotypes and echocardiographic findings in 77 of the 163 patients. RESULTS: The genotypes 577RR(RR), 577RX(RX) and 577XX(XX) were identified in 13 (17%), 44 (57%) and 20 (26%) of 77 patients, respectively. We estimated cardiac involvement-free survival rate analyses using Kaplan-Meier curves. Remarkably, the left ventricular dilation (> 55 mm)-free survival rate was significantly lower in patients with the XX null genotype (P < 0.01). The XX null genotype showed a higher risk for LV dilation (hazard ratio 9.04). CONCLUSIONS: This study revealed that the ACTN3 XX null genotype was associated with a lower left ventricular dilation-free survival rate in patients with DMD. These results suggest that the ACTN3 genotype should be determined at the time of diagnosis of DMD to improve patients' cardiac outcomes.
Assuntos
Insuficiência Cardíaca , Distrofia Muscular de Duchenne , Actinina/genética , Genótipo , Humanos , Distrofia Muscular de Duchenne/genética , Taxa de SobrevidaRESUMO
BACKGROUND: Approximately 15% of patients with Dent disease have pathogenic variants in the OCRL gene on Xq25-26, a condition that is referred to as Dent disease 2 (Dent-2). Dent-2 patients sometimes show mild extrarenal features of Lowe syndrome, such as mild mental retardation, suggesting that Dent-2 represents a mild form of Lowe syndrome. To date, eight female patients with Lowe syndrome have been reported, but no female Dent-2 patients have been reported. METHODS: In this study, we performed genetic testing of the first female Dent-2 patient to detect the presence of an OCRL variant. Aberrant splicing was demonstrated by in vivo, in vitro, and in silico assays, and skewed X-chromosome inactivation (XCI) in our patient and asymptomatic mothers of three Lowe patients with the heterozygous OCRL variant was evaluated by HUMARA assays using genomic DNA and RNA expression analysis. RESULTS: Our patient had an OCRL heterozygous intronic variant of c.1603-3G > C in intron 15 that led to a 169-bp insertion in exon 16, yielding the truncating mutation r.1602_1603ins (169) (p.Val535Glyfs*6) in exon 16. HUMARA assays of leukocytes obtained from this patient demonstrated incompletely skewed XCI (not extremely skewed). On the other hand, the asymptomatic mothers of 3 Lowe patients demonstrated random XCI. These results may lead to our patient's Dent-2 phenotype. CONCLUSIONS: This is the first report of a female patient clinically and genetically diagnosed with Dent-2 caused by an OCRL heterozygous splicing site variant and skewed XCI. Skewed XCI may be one of the factors associated with phenotypic diversity in female patients with Lowe syndrome and Dent-2.
Assuntos
Cromossomos Humanos X/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Nefrolitíase/genética , Síndrome Oculocerebrorrenal/genética , Monoéster Fosfórico Hidrolases/genética , Criança , Éxons , Feminino , Testes Genéticos , Heterozigoto , Humanos , Íntrons , Masculino , Mutagênese Insercional , Pais , FenótipoRESUMO
BACKGROUND: In recent years, the elucidation of splicing abnormalities as a cause of hereditary diseases has progressed. However, there are no comprehensive reports of suspected splicing variants in the CLCN5 gene in Dent disease cases. We reproduced gene mutations by mutagenesis, inserted the mutated genes into minigene vectors, and investigated the pathogenicity and onset mechanisms of these variants. METHODS: We conducted functional splicing assays using a hybrid minigene for six suspected splicing variants (c.105G>A, c.105+5G>C, c.106-17T>G, c.393+4A>G, c.517-8A>G, c.517-3C>A) in CLCN5. We extracted information on these variants from the Human Gene Mutation Database. We reproduced minigene vectors with the insertion of relevant exons with suspected splicing variants. We then transfected these minigene vectors into cultured cells and extracted and analyzed the mRNA. In addition, we conducted in silico analysis to confirm our minigene assay results. RESULTS: We successfully determined that five of these six variants are pathogenic via the production of splicing abnormalities. One showed only normal transcript production and was thus suspected of not being pathogenic (c.106-17T>G). CONCLUSION: We found that five CLCN5 variants disrupted the original splice site, resulting in aberrant splicing. It is sometimes difficult to obtain mRNA from patient samples because of the fragility of mRNA or its low expression level in peripheral leukocytes. Our in vitro system can be used as an alternative to in vivo assays to determine the pathogenicity of suspected splicing variants.
Assuntos
Canais de Cloreto/genética , DNA Recombinante , Doenças Genéticas Ligadas ao Cromossomo X/genética , Nefrolitíase/genética , RNA Mensageiro/análise , Simulação por Computador , Bases de Dados Genéticas , Células HEK293 , Células HeLa , Humanos , MutaçãoRESUMO
Splicing reporter minigenes are used in cell-based in vitro splicing studies. Exon skippable antisense oligonucleotide (ASO) has been identified using minigene splicing assays, but these assays include a time- and cost-consuming step of reverse transcription PCR amplification. To make in vitro splicing assay easier, a ready-made minigene (FMv2) amenable to quantitative splicing analysis by fluorescence microscopy was constructed. FMv2 was designed to encode two fluorescence proteins namely, mCherry, a transfection marker and split eGFP, a marker of splicing reaction. The split eGFP was intervened by an artificial intron containing a multicloning site sequence. Expectedly, FMv2 transfected HeLa cells produced not only red mCherry but also green eGFP signals. Transfection of FMv2CD44v8, a modified clone of FMv2 carrying an insertion of CD44 exon v8 in the multicloning site, that was applied to screen exon v8 skippable ASO, produced only red signals. Among seven different ASOs tested against exon v8, ASO#14 produced the highest index of green signal positive cells. Hence, ASO#14 was the most efficient exon v8 skippable ASO. Notably, the well containing ASO#14 was clearly identified among the 96 wells containing randomly added ASOs, enabling high throughput screening. A ready-made FMv2 is expected to contribute to identify exon skippable ASOs.
Assuntos
Processamento Alternativo , Éxons , Genes Reporter , Receptores de Hialuronatos/genética , Oligonucleotídeos Antissenso/genética , Ordem dos Genes , Vetores Genéticos/genética , Ensaios de Triagem em Larga Escala , Humanos , Proteínas Recombinantes de Fusão/genéticaRESUMO
The DMD gene is one of the largest human genes, being composed of 79 exons, and encodes dystrophin Dp427m which is deficient in Duchenne muscular dystrophy (DMD). In some DMD patient, however, small size dystrophin reacting with antibody to N-terminal but not to C-terminal has been identified. The mechanism to produce N-terminal small size dystrophin remains unknown. Intronic polyadenylation is a mechanism that produces a transcript with a new 3' terminal exon and a C-terminal truncated protein. In this study, intronic alternative polyadenylation was disclosed to occur in the middle of the DMD gene and produce the half-size N-terminal dystrophin Dp427m, Dpm234. The 3'-rapid amplification of cDNA ends revealed 421 bp sequence in the downstream of DMD exon 41 in U-251 glioblastoma cells. The cloned sequence composing of the 5' end sequence of intron 41 was decided as the terminal exon, since it encoded poly (A) signal followed by poly (A) stretch. Subsequently, a fragment from DMD exon M1 to intron 41 was obtained by PCR amplification. This product was named Dpm234 after its molecular weight. However, Dpm234 was not PCR amplified in human skeletal and cardiac muscles. Remarkably, Dpm234 was PCR amplified in iPS-derived cardiomyocytes. Accordingly, Western blotting of cardiomyocyte proteins showed a band of 234 kDa reacting with dystrophin antibody to N-terminal, but not C-terminal. Clinically, DMD patients with mutations in the Dpm234 coding region were found to have a significantly higher likelihood of two ECG abnormal findings. Intronic alternative splicing was first revealed in Dp427m to produce small size dystrophin.
Assuntos
Distrofina/genética , Distrofia Muscular de Duchenne/genética , Mutação , Poliadenilação , Adolescente , Processamento Alternativo , Criança , Pré-Escolar , Distrofina/metabolismo , Eletrocardiografia , Coração/fisiopatologia , Humanos , Íntrons , Masculino , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatologia , Miocárdio/metabolismoRESUMO
Modulation of dystrophin pre-mRNA splicing is an attractive strategy to ameliorate the severe phenotype of Duchenne muscular dystrophy (DMD), although this requires a better understanding of the mechanism of splicing regulation. Aberrant splicing caused by gene mutations provides a good model to study splicing regulatory cis-elements and binding proteins. In this study, we identified skipping of in-frame exon 25 induced by a nonsense mutation (NM_004006.2:c.3340A > T;p.Lys1114*) in the DMD gene. Site-directed mutagenesis study in minigenes suggested that c.3340A > T converts an exonic splicing enhancer sequence (ESE) to a silencer element (ESS). Indeed, RNA pull-down and functional study provided evidence that c.3340A > T abolishes the binding of the splicing enhancer protein Tra2ß and promotes interactions with the repressor proteins hnRNP A1, hnRNP A2, and hnRNP H. By carefully analyzing the sequence motif encompassing the mutation site, we concluded that the skipping of exon 25 was due to disruption of a Tra2ß-dependent ESE and the creation of a new ESS associated with hnRNP A1 and hnRNP A2, which in turn increased the recruitment of hnRNP H to a nearby binding site. Finally, we demonstrated that c.3340A > T impairs the splicing of upstream intron 24 in a splicing minigene assay. In addition, we showed that the correct splicing of exon 25 is finely regulated by multiple splicing regulators that function in opposite directions by binding to closely located ESE and ESS. Our results clarify the detailed molecular mechanism of exon skipping induced by the nonsense mutation c.3340A > T and also provide information on exon 25 splicing.