RESUMO
For neurodevelopmental disorders (NDDs), a molecular diagnosis is key for management, predicting outcome, and counseling. Often, routine DNA-based tests fail to establish a genetic diagnosis in NDDs. Transcriptome analysis (RNA sequencing [RNA-seq]) promises to improve the diagnostic yield but has not been applied to NDDs in routine diagnostics. Here, we explored the diagnostic potential of RNA-seq in 96 individuals including 67 undiagnosed subjects with NDDs. We performed RNA-seq on single individuals' cultured skin fibroblasts, with and without cycloheximide treatment, and used modified OUTRIDER Z scores to detect gene expression outliers and mis-splicing by exonic and intronic outliers. Analysis was performed by a user-friendly web application, and candidate pathogenic transcriptional events were confirmed by secondary assays. We identified intragenic deletions, monoallelic expression, and pseudoexonic insertions but also synonymous and non-synonymous variants with deleterious effects on transcription, increasing the diagnostic yield for NDDs by 13%. We found that cycloheximide treatment and exonic/intronic Z score analysis increased detection and resolution of aberrant splicing. Importantly, in one individual mis-splicing was found in a candidate gene nearly matching the individual's specific phenotype. However, pathogenic splicing occurred in another neuronal-expressed gene and provided a molecular diagnosis, stressing the need to customize RNA-seq. Lastly, our web browser application allowed custom analysis settings that facilitate diagnostic application and ranked pathogenic transcripts as top candidates. Our results demonstrate that RNA-seq is a complementary method in the genomic diagnosis of NDDs and, by providing accessible analysis with improved sensitivity, our transcriptome analysis approach facilitates wider implementation of RNA-seq in routine genome diagnostics.
Assuntos
Perfilação da Expressão Gênica , Transtornos do Neurodesenvolvimento , Humanos , RNA-Seq , Cicloeximida , Análise de Sequência de RNA/métodos , Transtornos do Neurodesenvolvimento/diagnóstico , Transtornos do Neurodesenvolvimento/genéticaRESUMO
Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes.
Assuntos
Proteína BRCA1/genética , Mutação em Linhagem Germinativa , Mutação com Perda de Função , Mutação de Sentido Incorreto , Transtornos do Neurodesenvolvimento/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Adolescente , Proteína BRCA1/imunologia , Criança , Pré-Escolar , Cromatina/química , Cromatina/imunologia , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/imunologia , Família , Feminino , Regulação da Expressão Gênica , Heterozigoto , Histonas/genética , Histonas/imunologia , Fator C1 de Célula Hospedeira/genética , Fator C1 de Célula Hospedeira/imunologia , Humanos , Lactente , Masculino , Transtornos do Neurodesenvolvimento/imunologia , Transtornos do Neurodesenvolvimento/patologia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/imunologia , Ubiquitina/genética , Ubiquitina/imunologia , Ubiquitina Tiolesterase/deficiência , Ubiquitina Tiolesterase/imunologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , UbiquitinaçãoRESUMO
The evolutionarily conserved hedgehog (Hh) pathway is essential for organogenesis and plays critical roles in postnatal tissue maintenance and renewal. A unique feature of the vertebrate Hh pathway is that signal transduction requires the primary cilium (PC) where major pathway components are dynamically enriched. These factors include smoothened (SMO) and patched, which constitute the core reception system for sonic hedgehog (SHH) as well as GLI transcription factors, the key mediators of the pathway. Here, we report bi-allelic loss-of-function variations in SMO in seven individuals from five independent families; these variations cause a wide phenotypic spectrum of developmental anomalies affecting the brain (hypothalamic hamartoma and microcephaly), heart (atrioventricular septal defect), skeleton (postaxial polydactyly, narrow chest, and shortening of long bones), and enteric nervous system (aganglionosis). Cells derived from affected individuals showed normal ciliogenesis but severely altered Hh-signal transduction as a result of either altered PC trafficking or abnormal activation of the pathway downstream of SMO. In addition, Hh-independent GLI2 accumulation at the PC tip in cells from the affected individuals suggests a potential function of SMO in regulating basal ciliary trafficking of GLI2 when the pathway is off. Thus, loss of SMO function results in abnormal PC dynamics of key components of the Hh signaling pathway and leads to a large continuum of malformations in humans.
Assuntos
Alelos , Deficiências do Desenvolvimento/genética , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Receptor Smoothened/genética , Sequência de Bases , Criança , Pré-Escolar , Cílios/fisiologia , Feminino , Humanos , Lactente , Masculino , Modelos Moleculares , Neoplasias/genética , Proteínas do Tecido Nervoso , Proteínas Nucleares , Linhagem , Proteína Gli2 com Dedos de Zinco , Proteína Gli3 com Dedos de ZincoRESUMO
Aminoacyl-tRNA synthetases (ARSs) are essential enzymes responsible for charging tRNA molecules with cognate amino acids. Consistent with the essential function and ubiquitous expression of ARSs, mutations in 32 of the 37 ARS-encoding loci cause severe, early-onset recessive phenotypes. Previous genetic and functional data suggest a loss-of-function mechanism; however, our understanding of the allelic and locus heterogeneity of ARS-related disease is incomplete. Cysteinyl-tRNA synthetase (CARS) encodes the enzyme that charges tRNACys with cysteine in the cytoplasm. To date, CARS variants have not been implicated in any human disease phenotype. Here, we report on four subjects from three families with complex syndromes that include microcephaly, developmental delay, and brittle hair and nails. Each affected person carries bi-allelic CARS variants: one individual is compound heterozygous for c.1138C>T (p.Gln380∗) and c.1022G>A (p.Arg341His), two related individuals are compound heterozygous for c.1076C>T (p.Ser359Leu) and c.1199T>A (p.Leu400Gln), and one individual is homozygous for c.2061dup (p.Ser688Glnfs∗2). Measurement of protein abundance, yeast complementation assays, and assessments of tRNA charging indicate that each CARS variant causes a loss-of-function effect. Compared to subjects with previously reported ARS-related diseases, individuals with bi-allelic CARS variants are unique in presenting with a brittle-hair-and-nail phenotype, which most likely reflects the high cysteine content in human keratins. In sum, our efforts implicate CARS variants in human inherited disease, expand the locus and clinical heterogeneity of ARS-related clinical phenotypes, and further support impaired tRNA charging as the primary mechanism of recessive ARS-related disease.
Assuntos
Aminoacil-tRNA Sintetases/genética , Doença de Charcot-Marie-Tooth/etiologia , Deficiências do Desenvolvimento/etiologia , Doenças do Cabelo/etiologia , Microcefalia/etiologia , Mutação , Doenças da Unha/etiologia , Adulto , Sequência de Aminoácidos , Doença de Charcot-Marie-Tooth/enzimologia , Doença de Charcot-Marie-Tooth/patologia , Deficiências do Desenvolvimento/enzimologia , Deficiências do Desenvolvimento/patologia , Feminino , Genes Recessivos , Predisposição Genética para Doença , Doenças do Cabelo/enzimologia , Doenças do Cabelo/patologia , Humanos , Masculino , Microcefalia/enzimologia , Microcefalia/patologia , Doenças da Unha/enzimologia , Doenças da Unha/patologia , Linhagem , Fenótipo , Prognóstico , Homologia de Sequência , Adulto JovemRESUMO
Sphingomyelinases generate ceramide from sphingomyelin as a second messenger in intracellular signaling pathways involved in cell proliferation, differentiation, or apoptosis. Children from 12 unrelated families presented with microcephaly, simplified gyral pattern of the cortex, hypomyelination, cerebellar hypoplasia, congenital arthrogryposis, and early fetal/postnatal demise. Genomic analysis revealed bi-allelic loss-of-function variants in SMPD4, coding for the neutral sphingomyelinase-3 (nSMase-3/SMPD4). Overexpression of human Myc-tagged SMPD4 showed localization both to the outer nuclear envelope and the ER and additionally revealed interactions with several nuclear pore complex proteins by proteomics analysis. Fibroblasts from affected individuals showed ER cisternae abnormalities, suspected for increased autophagy, and were more susceptible to apoptosis under stress conditions, while treatment with siSMPD4 caused delayed cell cycle progression. Our data show that SMPD4 links homeostasis of membrane sphingolipids to cell fate by regulating the cross-talk between the ER and the outer nuclear envelope, while its loss reveals a pathogenic mechanism in microcephaly.
Assuntos
Artrogripose/genética , Microcefalia/genética , Transtornos do Neurodesenvolvimento/genética , Esfingomielina Fosfodiesterase/genética , Artrogripose/patologia , Linhagem da Célula , Criança , Retículo Endoplasmático/metabolismo , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Masculino , Microcefalia/patologia , Mitose , Transtornos do Neurodesenvolvimento/patologia , Linhagem , Splicing de RNARESUMO
To date, mutations in 15 actin- or microtubule-associated genes have been associated with the cortical malformation lissencephaly and variable brainstem hypoplasia. During a multicenter review, we recognized a rare lissencephaly variant with a complex brainstem malformation in three unrelated children. We searched our large brain-malformation databases and found another five children with this malformation (as well as one with a less severe variant), analyzed available whole-exome or -genome sequencing data, and tested ciliogenesis in two affected individuals. The brain malformation comprised posterior predominant lissencephaly and midline crossing defects consisting of absent anterior commissure and a striking W-shaped brainstem malformation caused by small or absent pontine crossing fibers. We discovered heterozygous de novo missense variants or an in-frame deletion involving highly conserved zinc-binding residues within the GAR domain of MACF1 in the first eight subjects. We studied cilium formation and found a higher proportion of mutant cells with short cilia than of control cells with short cilia. A ninth child had similar lissencephaly but only subtle brainstem dysplasia associated with a heterozygous de novo missense variant in the spectrin repeat domain of MACF1. Thus, we report variants of the microtubule-binding GAR domain of MACF1 as the cause of a distinctive and most likely pathognomonic brain malformation. A gain-of-function or dominant-negative mechanism appears likely given that many heterozygous mutations leading to protein truncation are included in the ExAC Browser. However, three de novo variants in MACF1 have been observed in large schizophrenia cohorts.
Assuntos
Orientação de Axônios/genética , Movimento Celular/genética , Sequência Conservada/genética , Proteínas dos Microfilamentos/genética , Mutação/genética , Neurônios/patologia , Zinco/metabolismo , Adolescente , Tronco Encefálico/patologia , Criança , Pré-Escolar , Cílios/genética , Feminino , Humanos , Lisencefalia/genética , Masculino , Microtúbulos/genética , Malformações do Sistema Nervoso/genéticaRESUMO
Schizophrenia is highly heritable, yet its underlying pathophysiology remains largely unknown. Among the most well-replicated findings in neurobiological studies of schizophrenia are deficits in myelination and white matter integrity; however, direct etiological genetic and cellular evidence has thus far been lacking. Here, we implement a family-based approach for genetic discovery in schizophrenia combined with functional analysis using induced pluripotent stem cells (iPSCs). We observed familial segregation of two rare missense mutations in Chondroitin Sulfate Proteoglycan 4 (CSPG4) (c.391G > A [p.A131T], MAF 7.79 × 10-5 and c.2702T > G [p.V901G], MAF 2.51 × 10-3). The CSPG4A131T mutation was absent from the Swedish Schizophrenia Exome Sequencing Study (2536 cases, 2543 controls), while the CSPG4V901G mutation was nominally enriched in cases (11 cases vs. 3 controls, P = 0.026, OR 3.77, 95% CI 1.05-13.52). CSPG4/NG2 is a hallmark protein of oligodendrocyte progenitor cells (OPCs). iPSC-derived OPCs from CSPG4A131T mutation carriers exhibited abnormal post-translational processing (P = 0.029), subcellular localization of mutant NG2 (P = 0.007), as well as aberrant cellular morphology (P = 3.0 × 10-8), viability (P = 8.9 × 10-7), and myelination potential (P = 0.038). Moreover, transfection of healthy non-carrier sibling OPCs confirmed a pathogenic effect on cell survival of both the CSPG4A131T (P = 0.006) and CSPG4V901G (P = 3.4 × 10-4) mutations. Finally, in vivo diffusion tensor imaging of CSPG4A131T mutation carriers demonstrated a reduction of brain white matter integrity compared to unaffected sibling and matched general population controls (P = 2.2 × 10-5). Together, our findings provide a convergence of genetic and functional evidence to implicate OPC dysfunction as a candidate pathophysiological mechanism of familial schizophrenia.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas de Membrana/genética , Células Precursoras de Oligodendrócitos/metabolismo , Esquizofrenia/genética , Adulto , Antígenos/genética , Diferenciação Celular/fisiologia , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Imagem de Tensor de Difusão , Família , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Mutação/genética , Células Precursoras de Oligodendrócitos/fisiologia , Oligodendroglia/metabolismo , Linhagem , Proteoglicanas/genética , Esquizofrenia/metabolismo , Substância Branca/metabolismoRESUMO
DNA double-strand breaks (DSBs) are highly toxic DNA lesions that can lead to chromosomal instability, loss of genes and cancer. The MRE11/RAD50/NBN (MRN) complex is keystone involved in signaling processes inducing the repair of DSB by, for example, in activating pathways leading to homologous recombination repair and nonhomologous end joining. Additionally, the MRN complex also plays an important role in the maintenance of telomeres and can act as a stabilizer at replication forks. Mutations in NBN and MRE11 are associated with Nijmegen breakage syndrome (NBS) and ataxia telangiectasia (AT)-like disorder, respectively. So far, only one single patient with biallelic loss of function variants in RAD50 has been reported presenting with features classified as NBS-like disorder. Here, we report a long-term follow-up of an unrelated patient with facial dysmorphisms, microcephaly, skeletal features, and short stature who is homozygous for a novel variant in RAD50. We could show that this variant, c.2524G > A in exon 15 of the RAD50 gene, induces aberrant splicing of RAD50 mRNA mainly leading to premature protein truncation and thereby, most likely, to loss of RAD50 function. Using patient-derived primary fibroblasts, we could show abnormal radioresistant DNA synthesis confirming pathogenicity of the identified variant. Immunoblotting experiments showed strongly reduced protein levels of RAD50 in the patient-derived fibroblasts and provided evidence for a markedly reduced radiation-induced AT-mutated signaling. Comparison with the previously reported case and with patients presenting with NBS confirms that RAD50 mutations lead to a similar, but distinctive phenotype.
Assuntos
Hidrolases Anidrido Ácido/genética , Ataxia Telangiectasia/genética , Distúrbios no Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Transtornos do Crescimento/genética , Microcefalia/genética , Síndrome de Quebra de Nijmegen/genética , Alelos , Ataxia Telangiectasia/complicações , Ataxia Telangiectasia/patologia , Proteínas de Ciclo Celular/genética , Criança , Pré-Escolar , Quebras de DNA de Cadeia Dupla , Distúrbios no Reparo do DNA/complicações , Distúrbios no Reparo do DNA/patologia , Feminino , Transtornos do Crescimento/complicações , Transtornos do Crescimento/patologia , Humanos , Lactente , Recém-Nascido , Proteína Homóloga a MRE11/genética , Microcefalia/complicações , Microcefalia/patologia , Síndrome de Quebra de Nijmegen/complicações , Síndrome de Quebra de Nijmegen/patologia , Proteínas Nucleares/genética , LinhagemRESUMO
Recessive mutations in RTTN, encoding the protein rotatin, were originally identified as cause of polymicrogyria, a cortical malformation. With time, a wide variety of other brain malformations has been ascribed to RTTN mutations, including primary microcephaly. Rotatin is a centrosomal protein possibly involved in centriolar elongation and ciliogenesis. However, the function of rotatin in brain development is largely unknown and the molecular disease mechanism underlying cortical malformations has not yet been elucidated. We performed both clinical and cell biological studies, aimed at clarifying rotatin function and pathogenesis. Review of the 23 published and five unpublished clinical cases and genomic mutations, including the effect of novel deep intronic pathogenic mutations on RTTN transcripts, allowed us to extrapolate the core phenotype, consisting of intellectual disability, short stature, microcephaly, lissencephaly, periventricular heterotopia, polymicrogyria and other malformations. We show that the severity of the phenotype is related to residual function of the protein, not only the level of mRNA expression. Skin fibroblasts from eight affected individuals were studied by high resolution immunomicroscopy and flow cytometry, in parallel with in vitro expression of RTTN in HEK293T cells. We demonstrate that rotatin regulates different phases of the cell cycle and is mislocalized in affected individuals. Mutant cells showed consistent and severe mitotic failure with centrosome amplification and multipolar spindle formation, leading to aneuploidy and apoptosis, which could relate to depletion of neuronal progenitors often observed in microcephaly. We confirmed the role of rotatin in functional and structural maintenance of primary cilia and determined that the protein localized not only to the basal body, but also to the axoneme, proving the functional interconnectivity between ciliogenesis and cell cycle progression. Proteomics analysis of both native and exogenous rotatin uncovered that rotatin interacts with the neuronal (non-muscle) myosin heavy chain subunits, motors of nucleokinesis during neuronal migration, and in human induced pluripotent stem cell-derived bipolar mature neurons rotatin localizes at the centrosome in the leading edge. This illustrates the role of rotatin in neuronal migration. These different functions of rotatin explain why RTTN mutations can lead to heterogeneous cerebral malformations, both related to proliferation and migration defects.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Adulto , Encéfalo/patologia , Proteínas de Transporte/genética , Ciclo Celular/fisiologia , Cílios/metabolismo , Feminino , Estudos de Associação Genética/métodos , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lactente , Recém-Nascido , Masculino , Malformações do Desenvolvimento Cortical/genética , Malformações do Desenvolvimento Cortical/metabolismo , Microcefalia/genética , Mutação , Malformações do Sistema Nervoso/genética , Polimicrogiria/etiologia , Polimicrogiria/patologiaRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1006809.].
RESUMO
Integrator is an RNA polymerase II (RNAPII)-associated complex that was recently identified to have a broad role in both RNA processing and transcription regulation. Importantly, its role in human development and disease is so far largely unexplored. Here, we provide evidence that biallelic Integrator Complex Subunit 1 (INTS1) and Subunit 8 (INTS8) gene mutations are associated with rare recessive human neurodevelopmental syndromes. Three unrelated individuals of Dutch ancestry showed the same homozygous truncating INTS1 mutation. Three siblings harboured compound heterozygous INTS8 mutations. Shared features by these six individuals are severe neurodevelopmental delay and a distinctive appearance. The INTS8 family in addition presented with neuronal migration defects (periventricular nodular heterotopia). We show that the first INTS8 mutation, a nine base-pair deletion, leads to a protein that disrupts INT complex stability, while the second missense mutation introduces an alternative splice site leading to an unstable messenger. Cells from patients with INTS8 mutations show increased levels of unprocessed UsnRNA, compatible with the INT function in the 3'-end maturation of UsnRNA, and display significant disruptions in gene expression and RNA processing. Finally, the introduction of the INTS8 deletion mutation in P19 cells using genome editing alters gene expression throughout the course of retinoic acid-induced neural differentiation. Altogether, our results confirm the essential role of Integrator to transcriptome integrity and point to the requirement of the Integrator complex in human brain development.
Assuntos
Deficiências do Desenvolvimento/genética , Deleção de Genes , Mutação de Sentido Incorreto , Subunidades Proteicas/genética , RNA Mensageiro/metabolismo , Adulto , Processamento Alternativo , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Criança , Deficiências do Desenvolvimento/diagnóstico , Feminino , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Heterozigoto , Humanos , Masculino , Mutação , Linhagem , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , Síndrome , Transcriptoma , Proteína Wnt1RESUMO
OBJECTIVE: DNAJC6 mutations were recently described in two families with autosomal recessive juvenile parkinsonism (onset age < 11), prominent atypical signs, poor or absent response to levodopa, and rapid progression (wheelchair-bound within â¼10 years from onset). Here, for the first time, we report DNAJC6 mutations in early-onset Parkinson's disease (PD). METHODS: The DNAJC6 open reading frame was analyzed in 274 patients with early-onset sporadic or familial PD. Selected variants were followed up by cosegregation, homozygosity mapping, linkage analysis, whole-exome sequencing, and protein studies. RESULTS: We identified two families with different novel homozygous DNAJC6 mutations segregating with PD. In each family, the DNAJC6 mutation was flanked by long runs of homozygosity within highest linkage peaks. Exome sequencing did not detect additional pathogenic variants within the linkage regions. In both families, patients showed severely decreased steady-state levels of the auxilin protein in fibroblasts. We also identified a sporadic patient carrying two rare noncoding DNAJC6 variants possibly effecting RNA splicing. All these cases fulfilled the criteria for a clinical diagnosis of early-onset PD, had symptoms onset in the third-to-fifth decade, and slow disease progression. Response to dopaminergic therapies was prominent, but, in some patients, limited by psychiatric side effects. The phenotype overlaps that of other monogenic forms of early-onset PD. INTERPRETATION: Our findings delineate a novel form of hereditary early-onset PD. Screening of DNAJC6 is warranted in all patients with early-onset PD compatible with autosomal recessive inheritance. Our data provide further evidence for the involvement of synaptic vesicles endocytosis and trafficking in PD pathogenesis.
Assuntos
Auxilinas/metabolismo , Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP40/genética , Transtornos Parkinsonianos/genética , Adolescente , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Transtornos Parkinsonianos/metabolismo , Fenótipo , Adulto JovemRESUMO
We describe the clinical presentation and 17 years follow up of a boy, born to consanguineous parents and presenting with intellectual disability (ID), autism, "marfanoid" dysmorphic features, and moderate abnormalities of sulfite metabolism compatible with molybdenum cofactor deficiency, but normal sulfite oxidase activity in cultured skin fibroblasts. Genomic exome analysis revealed a homozygous MOCS3 missense mutation, leading to a p.Ala257Thr substitution in the highly conserved ubiquitin-like-domain of the protein. MOCS3 is the third protein, besides MOCS1 and MOCS2, involved in the biosynthesis of the molybdenum cofactor and has a dual ubiquitin-like function in tRNA thiolation. It is plausible that the phenotype results from deficiency of this dual function, not only from defective synthesis of molybdenum cofactor, which would explain similarities and differences from the MOCS1 and MOCS2-related disorders. This observation should encourage testing of additional ID patients with mild abnormalities of sulfite metabolism for MOCS3 mutations.
Assuntos
Transtorno Autístico/genética , Deficiência Intelectual/genética , Erros Inatos do Metabolismo dos Metais/genética , Nucleotidiltransferases/genética , Sulfurtransferases/genética , Adolescente , Transtorno Autístico/complicações , Transtorno Autístico/fisiopatologia , Expressão Gênica , Homozigoto , Humanos , Deficiência Intelectual/complicações , Deficiência Intelectual/fisiopatologia , Masculino , Erros Inatos do Metabolismo dos Metais/complicações , Erros Inatos do Metabolismo dos Metais/fisiopatologia , Mutação de Sentido Incorreto , FenótipoRESUMO
BACKGROUND: ECHS1 encodes a mitochondrial enzyme involved in the degradation of essential amino acids and fatty acids. Recently, ECHS1 mutations were shown to cause a new severe metabolic disorder presenting as Leigh or Leigh-like syndromes. The objective of this study was to describe a family with 2 siblings affected by different dystonic disorders as a resulting phenotype of ECHS1 mutations. METHODS: Clinical evaluation, MRI imaging, genome-wide linkage, exome sequencing, urine metabolite profiling, and protein expression studies were performed. RESULTS: The first sibling is 17 years old and presents with generalized dystonia and severe bilateral pallidal MRI lesions after 1 episode of infantile subacute metabolic encephalopathy (Leigh-like syndrome). In contrast, the younger sibling (15 years old) only suffers from paroxysmal exercise-induced dystonia and has very mild pallidal MRI abnormalities. Both patients carry compound heterozygous ECHS1 mutations: c.232G>T (predicted protein effect: p.Glu78Ter) and c.518C>T (p.Ala173Val). Linkage analysis, exome sequencing, cosegregation, expression studies, and metabolite profiling support the pathogenicity of these mutations. Expression studies in patients' fibroblasts showed mitochondrial localization and severely reduced levels of ECHS1 protein. Increased urinary S-(2-carboxypropyl)cysteine and N-acetyl-S-(2-carboxypropyl)cysteine levels, proposed metabolic markers of this disorder, were documented in both siblings. Sequencing ECHS1 in 30 unrelated patients with paroxysmal dyskinesias revealed no further mutations. CONCLUSIONS: The phenotype associated with ECHS1 mutations might be milder than reported earlier, compatible with prolonged survival, and also includes isolated paroxysmal exercise-induced dystonia. ECHS1 screening should be considered in patients with otherwise unexplained paroxysmal exercise-induced dystonia, in addition to those with Leigh and Leigh-like syndromes. Diet regimens and detoxifying agents represent potential therapeutic strategies. © 2016 International Parkinson and Movement Disorder Society.
Assuntos
Distúrbios Distônicos/genética , Distúrbios Distônicos/fisiopatologia , Enoil-CoA Hidratase/deficiência , Adolescente , Enoil-CoA Hidratase/genética , Exercício Físico , Humanos , Masculino , LinhagemRESUMO
Polymicrogyria is a malformation of the developing cerebral cortex caused by abnormal organization and characterized by many small gyri and fusion of the outer molecular layer. We have identified autosomal-recessive mutations in RTTN, encoding Rotatin, in individuals with bilateral diffuse polymicrogyria from two separate families. Rotatin determines early embryonic axial rotation, as well as anteroposterior and dorsoventral patterning in the mouse. Human Rotatin has recently been identified as a centrosome-associated protein. The Drosophila melanogaster homolog of Rotatin, Ana3, is needed for structural integrity of centrioles and basal bodies and maintenance of sensory neurons. We show that Rotatin colocalizes with the basal bodies at the primary cilium. Cultured fibroblasts from affected individuals have structural abnormalities of the cilia and exhibit downregulation of BMP4, WNT5A, and WNT2B, which are key regulators of cortical patterning and are expressed at the cortical hem, the cortex-organizing center that gives rise to Cajal-Retzius (CR) neurons. Interestingly, we have shown that in mouse embryos, Rotatin colocalizes with CR neurons at the subpial marginal zone. Knockdown experiments in human fibroblasts and neural stem cells confirm a role for RTTN in cilia structure and function. RTTN mutations therefore link aberrant ciliary function to abnormal development and organization of the cortex in human individuals.
Assuntos
Proteínas de Transporte/genética , Córtex Cerebral/embriologia , Córtex Cerebral/fisiologia , Cílios/fisiologia , Malformações do Desenvolvimento Cortical/genética , Adolescente , Proteínas de Ciclo Celular , Linhagem Celular , Criança , Feminino , Técnicas de Inativação de Genes , Genes Recessivos , Humanos , Imageamento por Ressonância Magnética , Masculino , Malformações do Desenvolvimento Cortical/diagnóstico , MutaçãoRESUMO
We describe a syndrome of primary microcephaly with simplified gyral pattern in combination with severe infantile epileptic encephalopathy and early-onset permanent diabetes in two unrelated consanguineous families with at least three affected children. Linkage analysis revealed a region on chromosome 18 with a significant LOD score of 4.3. In this area, two homozygous nonconserved missense mutations in immediate early response 3 interacting protein 1 (IER3IP1) were found in patients from both families. IER3IP1 is highly expressed in the fetal brain cortex and fetal pancreas and is thought to be involved in endoplasmic reticulum stress response. We reported one of these families previously in a paper on Wolcott-Rallison syndrome (WRS). WRS is characterized by increased apoptotic cell death as part of an uncontrolled unfolded protein response. Increased apoptosis has been shown to be a cause of microcephaly in animal models. An autopsy specimen from one patient showed increased apoptosis in the cerebral cortex and pancreas beta cells, implicating premature cell death as the pathogenetic mechanism. Both patient fibroblasts and control fibroblasts treated with siRNA specific for IER3IP1 showed an increased susceptibility to apoptotic cell death under stress conditions in comparison to controls. This directly implicates IER3IP1 in the regulation of cell survival. Identification of IER3IP1 mutations sheds light on the mechanisms of brain development and on the pathogenesis of infantile epilepsy and early-onset permanent diabetes.
Assuntos
Apoptose , Diabetes Mellitus/patologia , Epilepsia/complicações , Epilepsia/patologia , Microcefalia/complicações , Células-Tronco Neurais/patologia , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Pré-Escolar , Biologia Computacional , Família , Evolução Fatal , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Ligação Genética/efeitos dos fármacos , Humanos , Lactente , Recém-Nascido , Imageamento por Ressonância Magnética , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microcefalia/patologia , Dados de Sequência Molecular , Mutação/genética , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Linhagem , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Mutations in WDR62 are associated with primary microcephaly; however, they have been reported with wide phenotypic variability. We report on six individuals with novel WDR62 mutations who illustrate this variability and describe three in greater detail. Of the three, one lacks neuromotor development and has severe pachygyria on MRI, another has only delayed speech and motor development and moderate polymicrogyria, and the third has an intermediate phenotype. We observed a rare copy number change of unknown significance, a 17q25qter duplication, in the first severely affected individual. The 17q25 duplication included an interesting candidate gene, tubulin cofactor D (TBCD), crucial in microtubule assembly and disassembly. Sequencing of the non-duplicated allele showed a TBCD missense mutation, predicted to cause a deleterious p.Phe1121Val substitution. Sequencing of a cohort of five patients with WDR62 mutations, including one with an identical mutation and different phenotype, plus 12 individuals with diagnosis of microlissencephaly and another individual with mild intellectual disability (ID) and a 17q25 duplication, did not reveal TBCD mutations. However, immunostaining with tubulin antibodies of cells from patients with both WDR62 and TBCD mutation showed abnormal tubulin network when compared to controls and cells with only the WDR62 mutation. Therefore, we propose that genetic factors contribute to modify the severity of the WDR62 phenotype and, although based on suggestive evidence, TBCD could function as one of such factors.
Assuntos
Predisposição Genética para Doença , Mutação/genética , Proteínas do Tecido Nervoso/genética , Sequência de Bases , Encéfalo/patologia , Proteínas de Ciclo Celular , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Imageamento por Ressonância Magnética , Masculino , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Gravidez , Tubulina (Proteína)/metabolismoRESUMO
Data sharing is essential for a better understanding of genetic disorders. Good phenotype coding plays a key role in this process. Unfortunately, the two most widely used coding systems in medicine, ICD-10 and SNOMED-CT, lack information necessary for the detailed classification and annotation of rare and genetic disorders. This prevents the optimal registration of such patients in databases and thus data-sharing efforts. To improve care and to facilitate research for patients with metabolic disorders, we developed a new coding system for metabolic diseases with a dedicated group of clinical specialists. Next, we compared the resulting codes with those in ICD and SNOMED-CT. No matches were found in 76% of cases in ICD-10 and in 54% in SNOMED-CT. We conclude that there are sizable gaps in the SNOMED-CT and ICD coding systems for metabolic disorders. There may be similar gaps for other classes of rare and genetic disorders. We have demonstrated that expert groups can help in addressing such coding issues. Our coding system has been made available to the ICD and SNOMED-CT organizations as well as to the Orphanet and HPO organizations for further public application and updates will be published online (www.ddrmd.nl and www.cineas.org).
Assuntos
Disseminação de Informação , Classificação Internacional de Doenças , Doenças Metabólicas/classificação , Doenças Metabólicas/genética , Systematized Nomenclature of Medicine , Codificação Clínica , Genótipo , Humanos , Doenças Metabólicas/diagnóstico , FenótipoRESUMO
Spinocerebellar ataxias are phenotypically, neuropathologically, and genetically heterogeneous. The locus of autosomal recessive spinocerebellar ataxia type 7 (SCAR7) was previously linked to chromosome band 11p15. We have identified TPP1 as the causative gene for SCAR7 by exome sequencing. A missense and a splice site variant in TPP1, cosegregating with the disease, were found in a previously described SCAR7 family and also in another patient with a SCAR7 phenotype. TPP1, encoding the tripeptidyl-peptidase 1 enzyme, is known as the causative gene for late infantile neuronal ceroid lipofuscinosis disease 2 (CLN2 disease). CLN2 disease is characterized by epilepsy, loss of vision, ataxia, and a rapidly progressive course, leading to early death. SCAR7 patients showed ataxia and low activity of tripeptidyl-peptidase 1, but no ophthalmologic abnormalities or epilepsy. Also, the slowly progressive evolution of the disease until old age and absence of ultra structural curvilinear profiles is different from the known CLN2 phenotypes. Our findings now expand the phenotypes related to TPP1-variants to SCAR7. In spite of the limited sample size and measurements, a putative genotype-phenotype correlation may be drawn: we hypothesize that loss of function variants abolishing TPP1 enzyme activity lead to CLN2 disease, whereas variants that diminish TPP1 enzyme activity lead to SCAR7.
Assuntos
Aminopeptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Lipofuscinoses Ceroides Neuronais/genética , Serina Proteases/genética , Ataxias Espinocerebelares/genética , Sequência de Aminoácidos , Aminopeptidases/química , Animais , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Exoma , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Dados de Sequência Molecular , Lipofuscinoses Ceroides Neuronais/patologia , Linhagem , RNA/genética , Homologia de Sequência de Aminoácidos , Serina Proteases/química , Tripeptidil-Peptidase 1RESUMO
We present a neurodegenerative disorder starting in early childhood of two brothers consisting of severe progressive polyneuropathy, severe progressive cerebellar atrophy, microcephaly, mild epilepsy, and intellectual disability. The cause of this rare syndrome was found to be a homozygous mutation (c.1250_1266dup, resulting in a frameshift p.Thr424GlyfsX48) in PNKP, identified by applying homozygosity mapping and whole-genome sequencing. Mutations in PNKP have previously been associated with a syndrome of microcephaly, seizures and developmental delay (MIM 613402), but not with a neurodegenerative disorder. PNKP is a dual-function enzyme with a key role in different pathways of DNA damage repair. DNA repair disorders can result in accelerated cell death, leading to underdevelopment and neurodegeneration. In skin fibroblasts from both affected individuals, we show increased susceptibility to apoptosis under stress conditions and reduced PNKP expression. PNKP is known to interact with DNA repair proteins involved in the onset of polyneuropathy and cerebellar degeneration; therefore, our findings explain this novel phenotype.