RESUMO
The genetic diagnosis in inherited optic neuropathies often remains challenging, and the emergence of complex neurological phenotypes that involve optic neuropathy is puzzling. Here we unravel two novel principles of genetic mechanisms in optic neuropathies: deep intronic OPA1 mutations, which explain the disease in several so far unsolved cases; and an intralocus OPA1 modifier, which explains the emergence of syndromic 'optic atrophy plus' phenotypes in several families. First, we unravelled a deep intronic mutation 364 base pairs 3' of exon 4b in OPA1 by in-depth investigation of a family with severe optic atrophy plus syndrome in which conventional OPA1 diagnostics including gene dosage analyses were normal. The mutation creates a new splice acceptor site resulting in aberrant OPA1 transcripts with retained intronic sequence and subsequent translational frameshift as shown by complementary DNA analysis. In patient fibroblasts we demonstrate nonsense mediated messenger RNA decay, reduced levels of OPA1 protein, and impairment of mitochondrial dynamics. Subsequent site-specific screening of >360 subjects with unexplained inherited optic neuropathy revealed three additional families carrying this deep intronic mutation and a base exchange four nucleotides upstream, respectively, thus confirming the clinical significance of this mutational mechanism. Second, in all severely affected patients of the index family, the deep intronic mutation occurred in compound heterozygous state with an exonic OPA1 missense variant (p.I382M; NM_015560.2). The variant alone did not cause a phenotype, even in homozygous state indicating that this long debated OPA1 variant is not pathogenic per se, but acts as a phenotypic modifier if it encounters in trans with an OPA1 mutation. Subsequent screening of whole exomes from >600 index patients identified a second family with severe optic atrophy plus syndrome due to compound heterozygous p.I382M, thus confirming this mechanism. In summary, we provide genetic and functional evidence that deep intronic mutations in OPA1 can cause optic atrophy and explain disease in a substantial share of families with unsolved inherited optic neuropathies. Moreover, we show that an OPA1 modifier variant explains the emergence of optic atrophy plus phenotypes if combined in trans with another OPA1 mutation. Both mutational mechanisms identified in this study-deep intronic mutations and intragenic modifiers-might represent more generalizable mechanisms that could be found also in a wide range of other neurodegenerative and optic neuropathy diseases.
Assuntos
GTP Fosfo-Hidrolases/genética , Genoma Humano/genética , Mutação/genética , Atrofia Óptica Autossômica Dominante/genética , Adolescente , Adulto , Idoso , Éxons/genética , Feminino , Dosagem de Genes/genética , Loci Gênicos/genética , Variação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Atrofia Óptica Autossômica Dominante/classificação , Atrofia Óptica Autossômica Dominante/patologia , Linhagem , Fenótipo , SíndromeRESUMO
Human skin fibroblasts were isolated from a 48-year-old patient carrying compound heterozygous mutations (c.610+364G>A and c.1311A>G) in OPA1, responsible for early onset optic atrophy complicated by ataxia and pyramidal signs (Behr syndrome; OMIM #210000). Fibroblasts were reprogrammed using episomal plasmids carrying hOCT4, hSOX2, hKLF4, hL-MYC and hLIN28. The generated transgene-free line iPS-OPA1-BEHR showed no additional genomic aberrations, maintained the disease-relevant mutations, expressed important pluripotency markers and was capable to differentiate into cells of all three germ layers in vitro. The generated iPS-OPA1-BEHR line might be a useful platform to study the pathomechanism of early onset complicated optic atrophy syndromes.
Assuntos
Ataxia/patologia , Reprogramação Celular , GTP Fosfo-Hidrolases/genética , Perda Auditiva/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Deficiência Intelectual/patologia , Atrofia Óptica/congênito , Espasmo/patologia , Ataxia/genética , Ataxia/metabolismo , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Análise Mutacional de DNA , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Genótipo , Perda Auditiva/genética , Perda Auditiva/metabolismo , Heterozigoto , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Pessoa de Meia-Idade , Atrofia Óptica/genética , Atrofia Óptica/metabolismo , Atrofia Óptica/patologia , Polimorfismo de Nucleotídeo Único , Espasmo/genética , Espasmo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Skin fibroblasts were obtained from a 47-year-old hereditary spastic paraplegia patient carrying a homozygous mutation R486C in CYP7B1 (Cytochrome P450, Family 7, Subfamily B, Polypeptide 1), responsible for causing hereditary spastic paraplegia type 5 (SPG5). Induced pluripotent stem cells (iPSCs) were generated by transfection with episomal plasmids carrying hOCT4, hSOX2, hKLF4, hL-MYC and hLIN28. The generated line iPS-SPG5-R486C was transgene-free, retained the specific mutation with no additional genomic aberrations, expressed pluripotency markers and was able to differentiate into cells of all germ layers in vitro. The generated iPS-SPG5-R486C line may be a useful resource for disease modelling of SPG5.
Assuntos
Reprogramação Celular , Família 7 do Citocromo P450/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Paraplegia Espástica Hereditária/patologia , Esteroide Hidroxilases/genética , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Análise Mutacional de DNA , Fibroblastos/citologia , Fibroblastos/metabolismo , Genótipo , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Induced pluripotent stem cells (iPSCs) were generated from dermal fibroblasts from a 60-year-old cerebrotendinous xanthomatosis (CTX) patient, carrying a homozygous mutation c. [1183C>A]; p. R395S in CYP27A1. Episomal plasmids encoding the pluripotency genes OCT4, SOX2, KLF4, L-MYC and LIN28 were introduced via electroporation. The generated line iPS-CTX-R395S has no sign of plasmid integration or chromosomal aberration and retained the mutation site in CYP27A1. Furthermore, iPSCs express pluripotency markers and are able to differentiate in all germ layers in vitro. The generated line may be a useful tool for disease modelling of CTX.
Assuntos
Reprogramação Celular , Colestanotriol 26-Mono-Oxigenase/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Xantomatose Cerebrotendinosa/patologia , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Análise Mutacional de DNA , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Genótipo , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Fator 4 Semelhante a Kruppel , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Xantomatose Cerebrotendinosa/genética , Xantomatose Cerebrotendinosa/metabolismoRESUMO
Skin fibroblasts were obtained from a 47-year-old hereditary spastic paraplegia patient carrying a homozygous mutation Y275X in CYP7B1 (Cytochrome P450, Family 7, Subfamily B, Polypeptide 1), responsible for causing hereditary spastic paraplegia type 5 (SPG5). Induced pluripotent stem cells (iPSCs) were generated by transfection with episomal plasmids carrying hOCT4, hSOX2, hKLF4, hL-MYC and hLIN28. The generated line iPS-SPG5-Y275X was transgene-free, retained the specific mutation with no additional genomic aberrations, expressed pluripotency markers and was able to differentiate into cells of all germ layers in vitro. The generated iPS-SPG5-Y275X line may be a useful resource for disease modelling of SPG5.
Assuntos
Reprogramação Celular , Família 7 do Citocromo P450/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Paraplegia Espástica Hereditária/patologia , Esteroide Hidroxilases/genética , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Análise Mutacional de DNA , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Genótipo , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Human skin fibroblasts were isolated from a 40-year-old hereditary spastic paraplegia patient carrying an intronic splice site mutation (c.1687+2T>A) in SPAST, leading to hereditary spastic paraplegia type 4 (SPG4). Fibroblasts were reprogrammed using episomal plasmids carrying hOCT4, hSOX2, hKLF4, hL-MYC and hLIN28. The generated transgene-free line iPS-SPG4-splice retained the specific mutation with no additional genomic aberrations, expressed pluripotency markers and was able to differentiate into cells of all germ layers in vitro. The generated iPS-SPG4-splice line might be a useful platform to study the pathomechanism of SPG4.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Paraplegia Espástica Hereditária/patologia , Espastina/genética , Adulto , Diferenciação Celular , Linhagem Celular , Reprogramação Celular , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Fibroblastos/citologia , Heterozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Microscopia de Fluorescência , Polimorfismo de Nucleotídeo Único , Sítios de Splice de RNA/genética , Análise de Sequência de DNA , Pele/citologia , Paraplegia Espástica Hereditária/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Inherited optic neuropathies (ION) present an important cause of blindness in the European working-age population. Recently we reported the discovery of four independent families with deep intronic mutations in the main inherited optic neuropathies gene OPA1. These deep intronic mutations cause mis-splicing of the OPA1 pre-messenger-RNA transcripts by creating cryptic acceptor splice sites. As a rescue strategy we sought to prevent mis-splicing of the mutant pre-messenger-RNA by applying 2'O-methyl-antisense oligonucleotides (AONs) with a full-length phosphorothioate backbone that target the cryptic acceptor splice sites and the predicted novel branch point created by the deep intronic mutations, respectively. Transfection of patient-derived primary fibroblasts with these AONs induced correct splicing of the mutant pre-messenger-RNA in a time and concentration dependent mode of action, as detected by pyrosequencing of informative heterozygous variants. The treatment showed strong rescue effects (~55%) using the cryptic acceptor splice sites targeting AON and moderate rescue (~16%) using the branch point targeting AON. The highest efficacy of Splice correction could be observed 4 days after treatment however, significant effects were still seen 14 days post-transfection. Western blot analysis revealed increased amounts of OPA1 protein with maximum amounts at ~3 days post-treatment. In summary, we provide the first mutation-specific in vitro rescue strategy for OPA1 deficiency using synthetic AONs.