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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
Spinal muscular atrophy (SMA) is a lower motor neuron disease with autosomal recessive inheritance. The first cases of SMA were reported by Werdnig in 1891. Although the phenotypic variation of SMA led to controversy regarding the clinical entity of the disease, the genetic homogeneity of SMA was proved in 1990. Five years later, in 1995, the gene responsible for SMA, SMN1, was identified. Genetic testing of SMN1 has enabled precise epidemiological studies, revealing that SMA occurs in 1 of 10,000 to 20,000 live births and that more than 95% of affected patients are homozygous for SMN1 deletion. In 2016, nusinersen was the first drug approved for treatment of SMA in the United States. Two other drugs were subsequently approved: onasemnogene abeparvovec and risdiplam. Clinical trials with these drugs targeting patients with pre-symptomatic SMA (those who were diagnosed by genetic testing but showed no symptoms) revealed that such patients could achieve the milestones of independent sitting and/or walking. Following the great success of these trials, population-based newborn screening programs for SMA (more precisely, SMN1-deleted SMA) have been increasingly implemented worldwide. Early detection by newborn screening and early treatment with new drugs are expected to soon become the standards in the field of SMA.
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Atrofia Muscular Espinal , Recém-Nascido , Humanos , Atrofia Muscular Espinal/diagnóstico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/terapia , Testes Genéticos , Homozigoto , Triagem Neonatal , Padrões de HerançaRESUMO
The authors wish to make the following correction to this paper [...].
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Spinal muscular atrophy (SMA) is a common devastating neuromuscular disorder, usually involving homozygous deletion of the SMN1 gene. Newly developed drugs can improve the motor functions of infants with SMA when treated in the early stage. To ensure early diagnosis, newborn screening for SMA (SMA-NBS) via PCR-based genetic testing with dried blood spots (DBSs) has been spreading throughout Japan. In Hyogo Prefecture, we performed a pilot study of SMA-NBS to assess newborn infants who underwent routine newborn metabolic screening between February 2021 and August 2022. Hyogo Prefecture has ~40,000 live births per year and the estimated incidence of SMA is 1 in 20,000-25,000 based on genetic testing of symptomatic patients with SMA. Here, we screened 8336 newborns and 12 screen-positive cases were detected by real-time PCR assay. Multiplex ligation-dependent probe amplification assay excluded ten false positives and identified two patients. These false positives might be related to the use of heparinized and/or diluted blood in the DBS sample. Both patients carried two copies of SMN2, one was asymptomatic and the other was symptomatic at the time of diagnosis. SMA-NBS enables us to prevent delayed diagnosis of SMA, even if it does not always allow treatment in the pre-symptomatic stage.
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Atrofia Muscular Espinal , Lactente , Humanos , Recém-Nascido , Homozigoto , Projetos Piloto , Japão/epidemiologia , Deleção de Sequência , Atrofia Muscular Espinal/diagnóstico , Atrofia Muscular Espinal/epidemiologia , Atrofia Muscular Espinal/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Spinal muscular atrophy (SMA) is caused by survival motor neuron 1 SMN1 deletion. The survival motor neuron 2 (SMN2) encodes the same protein as SMN1 does, but it has a splicing defect of exon 7. Some antisense oligonucleotides (ASOs) have been proven to correct this defect. One of these, nusinersen, is effective in SMA-affected infants, but not as much so in advanced-stage patients. Furthermore, the current regimen may exhibit a ceiling effect. To overcome these problems, high-dose ASOs or combined ASOs have been explored. Here, using SMA fibroblasts, we examined the effects of high-concentration ASOs and of combining two ASOs. Three ASOs were examined: one targeting intronic splicing suppressor site N1 (ISS-N1) in intron 7, and two others targeting the 3' splice site and 5' region of exon 8. In our experiments on all ASO types, a low or intermediate concentration (50 or 100 nM) showed better splicing efficiency than a high concentration (200 nM). In addition, a high concentration of each ASO created a cryptic exon in exon 6. When a mixture of two different ASOs (100 nM each) was added to the cells, the cryptic exon was included in the mRNA. In conclusion, ASOs at a high concentration or used in combination may show less splicing correction and cryptic exon creation.
Assuntos
Atrofia Muscular Espinal , Oligonucleotídeos Antissenso , Fibroblastos/metabolismo , Humanos , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/genética , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico , Sítios de Splice de RNA , Splicing de RNA , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disease characterized by defects of lower motor neurons. Approximately 95% of SMA patients are homozygous for survival motor neuron 1 (SMN1) gene deletion, while ~5% carry an intragenic SMN1 mutation. Here, we investigated the stability and oligomerization ability of mutated SMN1 proteins. Plasmids containing wild- and mutant-type SMN1 cDNA were constructed and transfected into HeLa cells. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated similar abundances of transcripts from the plasmids containing SMN cDNA, but Western blotting showed different expression levels of mutated SMN1 proteins, reflecting the degree of their instability. A mutated SMN1 protein with T274YfsX32 exhibited a much lower expression level than other mutated SMN1 proteins with E134K, Y276H, or Y277C. In immunoprecipitation analysis, the mutated SMN1 protein with T274YfsX32 did not bind to endogenous SMN1 protein in HeLa cells, suggesting that this mutation completely blocks the oligomerization with full-length SMN2 protein in the patient. The patient with T274YfsX32 showed a much more severe phenotype than the other patients with different mutations. In conclusion, the stability and oligomerization ability of mutated SMN1 protein may determine the protein stability and may be associated with the clinical severity of SMA caused by intragenic SMN1 mutation.
Assuntos
Atrofia Muscular Espinal , Proteína 1 de Sobrevivência do Neurônio Motor , DNA Complementar , Células HeLa , Homozigoto , Humanos , Atrofia Muscular Espinal/genética , Mutação , Proteína 1 de Sobrevivência do Neurônio Motor/genéticaRESUMO
Glycogen storage disease type Ia (GSDIa, OMIM #232200) is an autosomal recessive metabolic disease characterized by impaired glucose homeostasis and has a long-term complication of hepatocellular adenoma/carcinoma. GSDIa is caused by deleterious mutations in the glucose-6-phosphatase gene (G6PC). Recent studies have suggested that early treatment by gene replacement therapy may be a good solution to correct the glucose metabolism and prevent serious late complications. Early treatment of the disease needs an early disease detection system. Thus, we aimed to develop a screening system for GSDIa using dried blood spots (DBS) to detect the c.648G>T mutation in G6PC, which is a frequent mutation in the East Asian population. In this study, a total of 51 DBS samples (50 healthy controls and one patient with c.648G>T) were tested by modified competitive oligonucleotide priming PCR (mCOP-PCR). In control DBS samples, the c.648G allele was amplified at lower Cq (quantification cycle) values (<11), while the c.648T allele was amplified at higher Cq values (>14). In the patient DBS sample, the c.648T allele was amplified at a lower Cq value (<11), and the c.648G allele was amplified at a higher Cq value (>14). Based on these findings, we concluded that our mCOP-PCR system clearly differentiated the wild-type and mutant alleles, and may be applicable for screening for GSDIa with the c.648G>T mutation in G6PC.
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Doença de Depósito de Glicogênio Tipo I/diagnóstico , Mutação/genética , Reação em Cadeia da Polimerase/métodos , Estudos de Casos e Controles , Teste em Amostras de Sangue Seco , Doença de Depósito de Glicogênio Tipo I/genética , HumanosRESUMO
Glycogen storage disease type Ia (GSDIa) is an autosomal recessive disorder caused by glucose-6-phosphatase (G6PC) deficiency. GSDIa causes not only life-threatening hypoglycemia in infancy, but also hepatocellular adenoma as a long-term complication. Hepatocellular adenoma may undergo malignant transformation to hepatocellular carcinoma. New treatment approaches are keenly anticipated for the prevention of hepatic tumors. Gene replacement therapy (GRT) is a promising approach, although early treatment in infancy is essential for its safety and efficiency. Thus, GRT requires screening systems for early disease detection. In this study, we developed a screening system for GSDIa using dried blood spots (DBS) on filter paper, which can detect the most common causative mutation in the East-Asian population, c.648G>T in the G6PC gene. Our system consisted of nested PCR analysis with modified competitive oligonucleotide priming (mCOP)-PCR in the second round and melting curve analysis of the amplified products. Here, we tested 54 DBS samples from 50 c.648G (wild type) controls and four c.648T (mutant) patients. This system, using DBS samples, specifically amplified and clearly detected wild-type and mutant alleles from controls and patients, respectively. In conclusion, our system will be applicable to newborn screening for GSDIa in the real world.
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Spinal muscular atrophy (SMA) is a lower motor neuron disease, once considered incurable. The main symptoms are muscle weakness and muscular atrophy. More than 90% of cases of SMA are caused by homozygous deletion of survival motor neuron 1 (SMN1). Emerging treatments, such as splicing modulation of SMN2 and SMN gene replacement therapy, have improved the prognoses and motor functions of patients. However, confirmed diagnosis by SMN1 testing is often delayed, suggesting the presence of diagnosis-delayed or undiagnosed cases. To enable patients to access the right treatments, a screening system for SMA is essential. Even so, the current newborn screening system using dried blood spots is still invasive and cumbersome. Here, we developed a completely non-invasive screening system using dried saliva spots (DSS) as an alternative DNA source to detect SMN1 deletion. In this study, 60 DSS (40 SMA patients and 20 controls) were tested. The combination of modified competitive oligonucleotide priming-polymerase chain reaction and melting peak analysis clearly distinguished DSS samples with and without SMN1. In conclusion, these results suggest that our system with DSS is applicable to SMA patient detection in the real world.
Assuntos
Atrofia Muscular Espinal/diagnóstico , Triagem Neonatal/métodos , Saliva , Estudos de Casos e Controles , Exodesoxirribonucleases/genética , Feminino , Humanos , Recém-Nascido , Masculino , Atrofia Muscular Espinal/genética , Reação em Cadeia da Polimerase/métodosRESUMO
Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by deficiency in dystrophin, a protein product encoded by the DMD gene. Mitochondrial dysfunction is now attracting much attention as a central player in DMD pathology. However, dystrophin has never been explored in human mitochondria. Here, we analyzed dystrophin in cDNAs and mitochondrial fractions of human cells. Mitochondrial fraction was obtained using a magnetic-associated cell sorting (MACS) technology. Dystrophin was analyzed by reverse transcription (RT)-PCR and western blotting using an antibody against the dystrophin C-terminal. In isolated mitochondrial fraction from HEK293 cells, dystrophin was revealed as a band corresponding to Dp71b and Dp71ab subisoforms. Additionally, in mitochondria from HeLa, SH-SY5Y, CCL-136 and HepG2 cells, signals for Dp71b and Dp71ab were revealed as well. Concomitantly, dystrophin mRNAs encoding Dp71b and Dp71ab were disclosed by RT-PCR in these cells. Primary cultured myocytes from three dystrophinopathy patients showed various levels of mitochondrial Dp71 expression. Coherently, levels of mRNA were different in all cells reflecting the protein content, which indicated predominant accumulation of Dp71. Dystrophin was demonstrated to be localized to human mitochondrial fraction, specifically as Dp71 subisoforms. Myocytes derived from dystrophinopathy patients manifested different levels of mitochondrial Dp71, with higher expression revealed in myocytes from Becker muscular dystrophy (BMD) patient-derived myocytes.
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Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder that causes degeneration of anterior horn cells in the human spinal cord and subsequent loss of motor neurons. The severe form of SMA is among the genetic diseases with the highest infant mortality. Although SMA has been considered incurable, newly developed drugs-nusinersen and onasemnogene abeparvovec-improve the life prognoses and motor functions of affected infants. To maximize the efficacy of these drugs, treatments should be started at the pre-symptomatic stage of SMA. Thus, newborn screening for SMA is now strongly recommended. Herein, we provide some data based on our experience of SMA diagnosis by genetic testing in Japan. A total of 515 patients suspected of having SMA or another lower motor neuron disease were tested. Among these patients, 228 were diagnosed as having SMA with survival motor neuron 1 (SMN1) deletion. We analyzed the distribution of clinical subtypes and ages at genetic testing in the SMN1-deleted patients, and estimated the SMA incidence based on data from Osaka and Hyogo prefectures, Japan. Our data showed that confirmed diagnosis by genetic testing was notably delayed, and the estimated incidence was 1 in 30,000-40,000 live births, which seemed notably lower than in other countries. These findings suggest that many diagnosis-delayed or undiagnosed cases may be present in Japan. To prevent this, newborn screening programs for SMA (SMA-NBS) need to be implemented in all Japanese prefectures. In this article, we also introduce our pilot study for SMA-NBS in Osaka Prefecture.
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BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by homozygous deletion or intragenic mutation of the SMN1 gene. It is well-known that high copy number of its homologous gene, SMN2, modifies the phenotype of SMN1-deleted patients. However, in the patients with intragenic SMN1 mutation, the relationship between phenotype and SMN2 copy number remains unclear. METHODS: We have analyzed a total of 515 Japanese patients with SMA-like symptoms (delayed developmental milestones, respiratory failures, muscle weakness etc.) from 1996 to 2019. SMN1 and SMN2 copy numbers were determined by quantitative polymerase chain reaction (PCR) method and/or multiplex ligation-dependent probe amplification (MLPA) method. Intragenic SMN1 mutations were identified through DNA and RNA analysis of the fresh blood samples. RESULTS: A total of 241 patients were diagnosed as having SMA. The majority of SMA patients showed complete loss of SMN1 (n = 228, 95%), but some patients retained SMN1 and carried an intragenic mutation in the retaining SMN1 (n = 13, 5%). Ten different mutations were identified in these 13 patients, consisting of missense, nonsense, frameshift and splicing defect-causing mutations. The ten mutations were c.275G > C (p.Trp92Ser), c.819_820insT (p.Thr274Tyrfs*32), c.830A > G (p.Tyr277Cys), c.5C > T (p.Ala2Val), c.826 T > C (p.Tyr276His), c.79C > T (p.Gln27*), c.188C > A (p.Ser63*), c.422 T > C (p.Leu141Pro), c.835-2A > G (exon 7 skipping) and c.835-3C > A (exon 7 skipping). It should be noted here that some patients with milder phenotype carried only a single SMN2 copy (n = 3), while other patients with severe phenotype carried 3 SMN2 copies (n = 4). CONCLUSION: Intragenic mutations in SMN1 may contribute more significantly to clinical severity than SMN2 copy numbers.
Assuntos
Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/fisiopatologia , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Adolescente , Criança , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Gravidade do Paciente , Fenótipo , Proteína 2 de Sobrevivência do Neurônio Motor/genéticaRESUMO
Background and Aim: Spinal muscular atrophy (SMA) is a lower motor neuron disease with autosomal recessive inheritance caused by homozygous SMN1 deletions. Although SMA has been considered as incurable, newly developed drugs improve life prognoses and motor functions of patients. To maximize the efficacy of the drugs, SMA patients should be treated before symptoms become apparent. Thus, newborn screening for SMA is strongly recommended. In this study, we aim to establish a new simple screening system based on DNA melting peak analysis. Materials and Methods: A total of 124 dried blood spot (DBS) on FTA® ELUTE cards (51 SMN1-deleted patients with SMA, 20 carriers, and 53 controls) were punched and subjected to direct amplification of SMN1 and CFTR (reference gene). Melting peak analyses were performed to detect SMN1 deletions from DBS samples. Results: A combination of allele-specific polymerase chain reaction (PCR) and melting peak analyses clearly distinguished the DBS samples with and without SMN1. Compared with the results of fresh blood samples, our new system yielded 100% sensitivity and specificity. The advantages of our system include (1) biosafe collection, transfer, and storage for DBS samples, (2) obviating the need for DNA extraction from DBS preventing contamination, (3) preclusion of fluorescent probes leading to low PCR cost, and (4) fast and high-throughput screening for SMN1 deletions. Conclusion: We demonstrate that our system would be applicable to a real-world newborn screening program for SMA, because our new technology is efficient for use in routine clinical laboratories that do not have highly advanced PCR instruments.
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Atrofia Muscular Espinal/genética , Triagem Neonatal/métodos , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , DNA/genética , Teste em Amostras de Sangue Seco/métodos , Éxons , Feminino , Deleção de Genes , Frequência do Gene , Ensaios de Triagem em Larga Escala/métodos , Humanos , Recém-Nascido , Masculino , Atrofia Muscular Espinal/sangue , Atrofia Muscular Espinal/diagnóstico , Desnaturação de Ácido Nucleico/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
BACKGROUND: Spinal muscular atrophy (SMA) is a neuromuscular disease caused by homozygous deletion of SMN1 exons 7 and 8. However, exon 8 is retained in some cases, where SMN2 exon 7 recombines with SMN1 exon 8, forming a hybrid SMN gene. It remains unknown how the hybrid SMN gene contribute to the SMA phenotype. METHOD: We analyzed 515 patients with clinical suspicion for SMA. SMN1 exons 7 and 8 deletion was detected by PCR followed by enzyme digestion. Hybrid SMN genes were further analyzed by nucleotide sequencing. SMN2 copy number was determined by real-time PCR. RESULTS: SMN1 exon 7 was deleted in 228 out of 515 patients, and SMN1 exon 8 was also deleted in 204 out of the 228 patients. The remaining 24 patients were judged to carry a hybrid SMN gene. In the patients with SMN1 exon 7 deletion, the frequency of the severe phenotype was significantly lower in the patients with hybrid SMN gene than in the patients without hybrid SMN gene. However, as for the distribution of SMN2 exon 7 copy number among the clinical phenotypes, there was no significant difference between both groups of SMA patients with or without hybrid SMN gene. CONCLUSION: Hybrid SMN genes are not rare in Japanese SMA patients, and it appears to be associated with a less severe phenotype. The phenotype of patients with hybrid SMN gene was determined by the copy number of SMN2 exon 7, as similarly for the patients without hybrid SMN gene.
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Atrofia Muscular Espinal/fisiopatologia , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Sequência de Bases , Quimera/genética , Variações do Número de Cópias de DNA/genética , Éxons/genética , Feminino , Deleção de Genes , Dosagem de Genes , Genótipo , Humanos , Japão/epidemiologia , Masculino , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Fenótipo , Reação em Cadeia da Polimerase , Deleção de Sequência , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genéticaRESUMO
Spinal muscular atrophy (SMA) is a common neuromuscular disease with autosomal recessive inheritance. The disease gene, SMN1, is homozygously deleted in 95% of SMA patients. Although SMA has been an incurable disease, treatment in infancy with newly developed drugs has dramatically improved the disease severity. Thus, there is a strong rationale for newborn and carrier screening for SMA, although implementing SMA carrier screening in the general population is controversial. We previously developed a simple, accurate newborn SMA screening system to detect homozygous SMN1 deletions using dried blood spots (DBS) on filter paper. Here, we modified our previous system to detect the heterozygous deletions of SMN1, which indicates SMA carrier status. The system involves a calibrator-normalized relative quantification method using quantitative nested PCR technology. Our system clearly separated the DBS samples with one SMN1 copy (carrier status with a heterozygous deletion of SMN1) from the DBS samples with two SMN1 copies (non-carrier status with no deletion of SMN1). We also analyzed DBS samples from SMA families, confirmed SMA in the affected children, and determined the carrier status of their parents based on the SMN1 copy number. In conclusion, our system will provide essential information for risk assessment and genetic counseling, at least for SMA families.
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BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by degeneration or loss of lower motor neurons. The survival of motor neuron (SMN) 1 gene, which produces the SMN protein, has been identified as a responsible gene for the disease. SMN is ubiquitously expressed in any tissue and may play an important role on the metabolism in the human body. However, no appropriate biomarkers reflecting the alteration in the metabolism in SMA have been identified. METHODS: Low-molecular-weight metabolites were extracted from plasma of 20 human infants (9 SMA type 1 patients and 11 controls) and 9 infant mice (5 SMA-model mice, 4 control mice), and derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide. Finally, the derivatized products were applied to Gas Chromatography/Mass Spectrometry apparatus. To confirm the metabolite abnormality in SMA type 1 patients, we performed SMN-silencing experiment using a hepatocyte-derived cell line (HepG2). RESULTS: We performed a comprehensive metabolomics analysis of plasma from the patients with SMA type 1 and controls, and found that phosphoethanolamine (PEA) was significantly higher in the patients than in the controls. HepG2 experiment also showed that SMN-silencing increased PEA levels. However, comprehensive metabolomics analysis of plasma from SMA-model mice and control mice showed different profile compared to human plasma; there was no increase of PEA even in the SMA-model mice plasma. CONCLUSION: Our data suggested that PEA was one of the possible biomarkers of human SMA reflecting metabolic abnormalities due to the SMN protein deficiency.
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Etanolaminas/sangue , Atrofias Musculares Espinais da Infância/sangue , Atrofias Musculares Espinais da Infância/diagnóstico , Animais , Biomarcadores/sangue , Estudos de Casos e Controles , Pré-Escolar , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Lactente , Recém-Nascido , Metaboloma , Metabolômica , CamundongosRESUMO
Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by SMN1 gene deletion/mutation. The drug nusinersen modifies SMN2 mRNA splicing, increasing the production of the full-length SMN protein. Recent studies have demonstrated the beneficial effects of nusinersen in patients with SMA, particularly when treated in early infancy. Because nusinersen treatment can alter disease trajectory, there is a strong rationale for newborn screening. In the current study, we validated the accuracy of a new system for detecting SMN1 deletion (Japanese patent application No. 2017-196967, PCT/JP2018/37732) using dried blood spots (DBS) from 50 patients with genetically confirmed SMA and 50 controls. Our system consists of two steps: (1) targeted pre-amplification of SMN genes by direct polymerase chain reaction (PCR) and (2) detection of SMN1 deletion by real-time modified competitive oligonucleotide priming-PCR (mCOP-PCR) using the pre-amplified products. Compared with PCR analysis results of freshly collected blood samples, our system exhibited a sensitivity of 1.00 (95% confidence interval [CI] 0.96-1.00) and a specificity of 1.00 (95% CI 0.96-1.00). We also conducted a prospective SMA screening study using DBS from 4157 Japanese newborns. All DBS tested negative, and there were no screening failures. Our results indicate that the new system can be reliably used in SMA newborn screening.
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BACKGROUND: Polymerase chain reaction (PCR) analysis using DNA from dried blood spot (DBS) samples on filter paper is a critical technique for spinal muscular atrophy (SMA) newborn screening. However, DNA extraction from DBS is time-consuming, and elimination of PCR inhibitors from DBS is almost impossible. METHODS: Exon 7 of the two homologous SMA-related genes, survival motor neuron (SMN) 1 and SMN2, of five SMA patients and five controls were amplified by PCR with a punched-out circle of the DBS paper. Two types of DNA preparation methods were tested; DNA-extraction (extracted DNA was added in a PCR tube) and non-DNA-extraction (a punched-out DBS circle was placed in a PCR tube). As for the DNA polymerases, two different enzymes were compared; TaKaRa Ex Taq™ and KOD FX Neo™. To test the diagnostic quality of PCR products, RFLP (Restriction fragment length polymorphism) analysis with DraI digestion was performed, differentiating SMN1 and SMN2. RESULTS: In PCR using extracted DNA, sufficient amplification was achieved with TaKaRa Ex Taq™ and KOD FX Neo™, and there was no significant difference in amplification efficiency between them. In direct PCR with a punched-out DBS circle, sufficient amplification was achieved when KOD FX Neo™ polymerase was used, while there was no amplification with TaKaRa Ex Taq™. RFLP analysis of the direct PCR products with KOD FX Neo™ clearly separated SMN1 and SMN2 sequences and proved the presence of both of SMN1 and SMN2 in controls, and only SMN2 in SMA patients, suggesting that the direct PCR products with KOD FX Neo™ were of sufficient diagnostic quality for SMA testing. CONCLUSION: Direct PCR with DNA polymerases like KOD FX NeoTM has potential to be widely used in SMA newborn screening in the near future as it obviates the DNA extraction process from DBS and can precisely amplify the target sequences in spite of the presence of PCR inhibitors.
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DNA/sangue , Teste em Amostras de Sangue Seco/métodos , Atrofia Muscular Espinal/genética , Triagem Neonatal/métodos , Reação em Cadeia da Polimerase/métodos , Proteína 1 de Sobrevivência do Neurônio Motor/genética , DNA Polimerase Dirigida por DNA , Deleção de Genes , Humanos , Recém-Nascido , Japão , Polimorfismo de Fragmento de Restrição , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Taq Polimerase , Thermococcus/enzimologiaRESUMO
BACKGROUND: Spinal Muscular Atrophy (SMA) is a common autosomal recessive neuromuscular disorder characterized by degeneration or loss of lower motor neurons. More than 95% of SMA patients show homozygous deletion for the survival motor neuron 1 (SMN1) gene. For the screening of SMN1 deletion, it is necessary to differentiate SMN1 from its highly homologous gene, SMN2. We developed a modified competitive oligonucleotide priming-PCR (mCOP-PCR) method using dried blood spot (DBS)-DNA, in which SMN1 and SMN2-specific PCR products are detected with gel-electrophoresis. Next, we added a targeted pre-amplification step prior to the mCOP-PCR step, to avoid unexpected, non-specific amplification. The pre-amplification step enabled us to combine mCOP-PCR and real-time PCR. In this study, we combined real-time mCOP-PCR and PCR-restriction fragment length polymorphism (PCR-RFLP) to develop a new screening system for detection of SMN1 deletion. METHODS: DBS samples of the subjects were stored at room temperature for a period of less than one year. Each subject had already been genotyped by the first PCR-RFLP using fresh blood DNA. SMN1/SMN2 exon 7 was collectively amplified using conventional PCR (targeted pre-amplification), the products of which were then used as a template in the real-time PCR with mCOP-primer sets. To confirm the results, the pre-amplified products were subject to the second PCR-RFLP. RESULTS: The real-time mCOP-PCR separately amplified SMN1 and SMN2 exon7, and clearly demonstrated SMN1 deletion in an SMA patient. The results of the real-time mCOP-PCR using DBS-DNA were completely consistent with those of the first and second PCR-RFLP analysis. CONCLUSION: In our new system for detection of SMN1 deletion, real-time mCOP-PCR rapidly proved the presence or absence of SMN1 and SMN2, and the results were easily tested by PCR-RFLP. This solid genotyping system will be useful for SMA screening.
Assuntos
Atrofia Muscular Espinal/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Estudos de Casos e Controles , Deleção de Genes , Humanos , Atrofia Muscular Espinal/genética , Polimorfismo de Fragmento de Restrição , Proteína 2 de Sobrevivência do Neurônio Motor/genéticaRESUMO
BACKGROUND: Spinal Muscular Atrophy (SMA) is a common autosomal recessive neuromuscular disease characterized by defects of lower motor neurons. More than 95% of SMA patients show homozygous deletion for the survival motor neuron 1 (SMN1) gene. For the screening of SMN1 deletion using dried blood spot (DBS), we developed a new combined system with real-time "modified competitive oligonucleotide priming"-polymerase chain reaction (mCOP-PCR) and PCR restriction fragment length polymorphism (PCR-RFLP). Although our real-time mCOP-PCR method is secured enough to be gene-specific, its amplification efficiency is not as good because the reverse primers carry a nucleotide mismatched with the sequence of the pre-amplified product. The mismatch has consequently been generated in the process of introducing a restriction enzyme site in the pre-amplified products for PCR-RFLP. METHOD: DBS samples of the subjects were stored at room temperature for a period of less than one year. Each subject had already been genotyped by the first PCR-RFLP using fresh blood DNA. SMN1/SMN2 exon 7 was collectively amplified using conventional PCR (targeted pre-amplification). Pre-amplified products were used as template in the real-time mCOP-PCR, and, on the other hand, were digested with DraI enzyme (PCR-RFLP). To improve the amplification efficiency of mCOP-PCR, one nucleotide change was introduced in the original reverse primers (SMN1-COP and SMN2-COP) to eliminate the mismatched nucleotide. RESULTS: The real-time mCOP-PCR with a new primer (SMN1-COP-DRA or SMN2-COP-DRA) more rapidly and specifically amplified SMN1 and SMN2, and clearly demonstrated SMN1 deletion in an SMA patient. With the new primers, the amplification efficiencies of real-time mCOP-PCR were improved and the Cq values of SMN1 (+) and SMN2 (+) samples were significantly lowered. CONCLUSION: In the advanced version of our screening system for homozygous SMN1 deletion using DBS, the real-time mCOP-PCR with newly-designed reverse primers demonstrated the presence or absence of SMN1 and SMN2 within a shorter time, and the results were easily tested by PCR-RFLP. This rapid and accurate screening system will be useful for detection of newborn infants with SMA.