RESUMO
BACKGROUND: Sex-specific predilection in neurological diseases caused by mutations in autosomal genes is a phenomenon whose molecular basis is poorly understood. We studied females of consanguineous Bedouin kindred presenting with severe global developmental delay and epilepsy. METHODS: Linkage analysis, whole exome sequencing, generation of CRISPR/cas9 knock-in mice, mouse behaviour and molecular studies RESULTS: Linkage analysis and whole exome sequencing studies of the affected kindred delineated a ~5 Mbp disease-associated chromosome 2q35 locus, containing a novel homozygous frameshift truncating mutation in ZNF142, in line with recent studies depicting similar ZNF142 putative loss-of-function human phenotypes with female preponderance. We generated knock-in mice with a truncating mutation adjacent to the human mutation in the mouse ortholog. Behaviour studies of homozygous Zfp142R1508* mice showed significant phenotype only in mutant females, with learning and memory deficits, hyperactivity and aberrant loss of fear of open spaces. Bone marrow and spleen of homozygous Zfp142R1508* mice showed depletion of lymphoid and haematopoietic cells, mostly in females. RT-PCR showed lower expression of Zpf142 in brain compartments of female versus male wild-type mice. RNA-seq studies of hippocampus, hypothalamus, cortex and cerebellum of female wild-type versus homozygous Zfp142R1508* mice demonstrated differentially expressed genes. Notably, expression of Taok1 in the cortex and of Mllt6 in the hippocampus was downregulated in homozygous Zfp142R1508* mice. Taok1 mutations have been associated with aberrant neurodevelopment and behaviour. Mllt6 expression is regulated by sex hormones and Mllt6 null-mutant mice present with haematopoietic, immune system and female-specific behaviour phenotypes. CONCLUSION: ZNF142 mutation downregulates Mllt6 and Taok1, causing a neurodevelopmental phenotype in humans and mice with female preponderance.
Assuntos
Mutação , Animais , Feminino , Camundongos , Masculino , Humanos , Linhagem , Proteínas de Ligação a DNA/genética , Fenótipo , Fatores de Transcrição/genética , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/patologia , Sequenciamento do Exoma , Ligação Genética , Epilepsia/genética , Epilepsia/patologiaRESUMO
Microtubule associated protein 11 (MAP11, previously termed C7orf43) encodes a highly conserved protein whose function is unknown. Through genome-wide linkage analysis combined with whole exome sequencing, we demonstrate that human autosomal recessive primary microcephaly is caused by a truncating mutation in MAP11. Moreover, homozygous MAP11-orthologue CRISPR/Cas9 knock-out zebrafish presented with microcephaly and decreased neuronal proliferation, recapitulating the human phenotype. We demonstrate that MAP11 is ubiquitously transcribed with high levels in brain and cerebellum. Immunofluorescence and co-immunoprecipitation studies in SH-SY5Y cells showed that MAP11 associates with mitotic spindles, co-localizing and physically associating with α-tubulin during mitosis. MAP11 expression precedes α-tubulin in gap formation of cell abscission at the midbody and is co-localized with PLK1, a key regulator of cytokinesis, at the edges of microtubule extensions of daughter cells post cytokinesis abscission, implicating a role in mitotic spindle dynamics and in regulation of cell abscission during cytokinesis. Finally, lentiviral-mediated silencing of MAP11 diminished SH-SY5Y cell viability, reducing proliferation rather than affecting apoptosis. Thus, MAP11 encodes a microtubule-associated protein that plays a role in spindle dynamics and cell division, in which mutations cause microcephaly in humans and zebrafish.
Assuntos
Microcefalia/etiologia , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Criança , Pré-Escolar , Citocinese , Modelos Animais de Doenças , Feminino , Células HeLa , Humanos , Masculino , Microcefalia/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/genética , Mitose , Mutação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fuso Acromático/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Quinase 1 Polo-LikeRESUMO
BACKGROUND: Consanguineous kindred presented with an autosomal recessive syndrome of intrauterine growth retardation, marked developmental delay, spastic quadriplegia with profound contractures, pseudobulbar palsy with recurrent aspirations, epilepsy, dysmorphism, neurosensory deafness and optic nerve atrophy with no eye fixation. Affected individuals died by the age of 4. Brain MRI demonstrated microcephaly, semilobar holoprosencephaly and agenesis of corpus callosum. We aimed at elucidating the molecular basis of this disease. METHODS: Genome-wide linkage analysis combined with whole exome sequencing were performed to identify disease-causing variants. Functional consequences were investigated in fruit flies null mutant for the Drosophila SEC31A orthologue. SEC31A knockout SH-SY5Y and HEK293T cell-lines were generated using CRISPR/Cas9 and studied through qRT-PCR, immunoblotting and viability assays. RESULTS: Through genetic studies, we identified a disease-associated homozygous nonsense mutation in SEC31A. We demonstrate that SEC31A is ubiquitously expressed, and that the mutation triggers nonsense-mediated decay of its transcript, comprising a practical null mutation. Similar to the human disease phenotype, knockdown SEC31A flies had defective brains and early lethality. Moreover, in line with SEC31A encoding one of the two coating layers comprising the Coat protein complex II (COP-II) complex, trafficking newly synthesised proteins from the endoplasmic reticulum (ER) to the Golgi, CRISPR/Cas9-mediated SEC31A null mutant cells demonstrated reduced viability through upregulation of ER-stress pathways. CONCLUSION: We demonstrate through human and Drosophila genetic and in vitro molecular studies, that a severe neurological syndrome is caused by a null mutation in SEC31A, reducing cell viability through enhanced ER-stress response, in line with SEC31A's role in the COP-II complex.
Assuntos
Retículo Endoplasmático/metabolismo , Homeostase , Mutação , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Proteínas de Transporte Vesicular/genética , Animais , Consanguinidade , Modelos Animais de Doenças , Drosophila , Eletromiografia , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Doenças do Sistema Nervoso/diagnóstico , Condução Nervosa , Linhagem , Fenótipo , Síndrome , Tomografia Computadorizada por Raios XRESUMO
RSRC1, whose polymorphism is associated with altered brain function in schizophrenia, is a member of the serine and arginine rich-related protein family. Through homozygosity mapping and whole exome sequencing we show that RSRC1 mutation causes an autosomal recessive syndrome of intellectual disability, aberrant behaviour, hypotonia and mild facial dysmorphism with normal brain MRI. Further, we show that RSRC1 is ubiquitously expressed, and that the RSRC1 mutation triggers nonsense-mediated mRNA decay of the RSRC1 transcript in patients' fibroblasts. Short hairpin RNA (shRNA)-mediated lentiviral silencing and overexpression of RSRC1 in SH-SY5Y cells demonstrated that RSRC1 has a role in alternative splicing and transcription regulation. Transcriptome profiling of RSRC1-silenced cells unravelled specific differentially expressed genes previously associated with intellectual disability, hypotonia and schizophrenia, relevant to the disease phenotype. Protein-protein interaction network modelling suggested possible intermediate interactions by which RSRC1 affects gene-specific differential expression. Patient-derived induced pluripotent stem cells, differentiated into neural progenitor cells, showed expression dynamics similar to the RSRC1-silenced SH-SY5Y model. Notably, patient neural progenitor cells had 9.6-fold downregulated expression of IGFBP3, whose brain expression is affected by MECP2, aberrant in Rett syndrome. Interestingly, Igfbp3-null mice have behavioural impairment, abnormal synaptic function and monoaminergic neurotransmission, likely correlating with the disease phenotype.
Assuntos
Processamento Alternativo/genética , Deficiências do Desenvolvimento/genética , Regulação para Baixo/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Deficiência Intelectual/genética , Proteínas Nucleares/genética , Animais , Diferenciação Celular/genética , Linhagem Celular Transformada , Criança , Pré-Escolar , Consanguinidade , Deficiências do Desenvolvimento/complicações , Feminino , Seguimentos , Ontologia Genética , Humanos , Lactente , Deficiência Intelectual/complicações , Masculino , Camundongos , Camundongos Knockout , Células-Tronco Pluripotentes/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Primary microcephaly is a congenital neurodevelopmental disorder of reduced head circumference and brain volume, with fewer neurons in the cortex of the developing brain due to premature transition between symmetrical and asymmetrical cellular division of the neuronal stem cell layer during neurogenesis. We now show through linkage analysis and whole exome sequencing, that a dominant mutation in ALFY, encoding an autophagy scaffold protein, causes human primary microcephaly. We demonstrate the dominant effect of the mutation in drosophila: transgenic flies harboring the human mutant allele display small brain volume, recapitulating the disease phenotype. Moreover, eye-specific expression of human mutant ALFY causes rough eye phenotype. In molecular terms, we demonstrate that normally ALFY attenuates the canonical Wnt signaling pathway via autophagy-dependent removal specifically of aggregates of DVL3 and not of Dvl1 or Dvl2. Thus, autophagic attenuation of Wnt signaling through removal of Dvl3 aggregates by ALFY acts in determining human brain size.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Membrana/genética , Microcefalia/genética , Fosfoproteínas/genética , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados , Proteínas Relacionadas à Autofagia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas Desgrenhadas , Drosophila , Ligação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Microcefalia/patologia , Mutação , Tamanho do Órgão/genética , Via de Sinalização Wnt/genéticaRESUMO
SLC25A1 mutations are associated with combined D,L-2-hydroxyglutaric aciduria (DL- 2HGA; OMIM #615182), characterized by muscular hypotonia, severe neurodevelopmental dysfunction and intractable seizures. SLC25A1 encodes the mitochondrial citrate carrier (CIC), which mediates efflux of the mitochondrial tricarboxylic acid (TCA) cycle intermediates citrate and isocitrate in exchange for cytosolic malate. Only a single family with an SLC25A1 mutation has been described in which mitochondrial respiratory chain dysfunction was documented, specifically in complex IV. Five infants of two consanguineous Bedouin families of the same tribe presented with small head circumference and neonatal-onset encephalopathy with severe muscular weakness, intractable seizures, respiratory distress, and lack of psychomotor development culminating in early death. Ventricular septal defects (VSD) were demonstrated in three patients. Blood and CSF lactate were elevated with normal levels of plasma amino acids and free carnitine and increased 2-OH-glutaric acid urinary exertion. EEG was compatible with white matter disorder. Brain MRI revealed ventriculomegaly, thin corpus callosum with increased lactate peak on spectroscopy. Mitochondrial complex V deficiency was demonstrated in skeletal muscle biopsy of one infant. Homozygosity mapping and sequencing ruled out homozygosity of affected individuals in all known complex V-associated genes. Whole exome sequencing identified a novel homozygous SLC25A1 c.713A>G (p.Asn238Ser) mutation, segregating as expected in the affected kindred and not found in 220 control alleles. Thus, SLC25A1 mutations might be associated with mitochondrial complex V deficiency and should be considered in the differential diagnosis of mitochondrial respiratory chain defects.
Assuntos
Proteínas de Transporte de Ânions/genética , Homozigoto , Mitocôndrias/genética , Proteínas Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Mutação , Adolescente , Adulto , Encéfalo/anormalidades , Encéfalo/diagnóstico por imagem , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética/métodos , Predisposição Genética para Doença , Humanos , Lactente , Recém-Nascido , Masculino , Transportadores de Ânions Orgânicos , Linhagem , Fenótipo , Adulto JovemRESUMO
Whole exome sequencing (WES) has become routine in clinical practice, especially in studies of recessive hereditary diseases in inbred consanguineous families, where homozygosity of a founder mutation is assumed. Multiple members of two consanguineous families of a single Bedouin tribe were diagnosed with apparently autosomal recessive/pseudo-dominant retinitis pigmentosa (RP). Affected individuals exhibited severe visual impairment with nyctalopia, marked constriction of visual fields, markedly reduced and delayed responses on electro-retinography (ERG) and eventual loss of central vision. Combined copy-number variant (CNV) analysis, haplotype reconstruction and WES of the kindred identified two distinct novel mutations in EYS (RP25): a p.(W1817*) nonsense mutation (identified through WES) and a large deletion encompassing 9 of the 43 exons, that was missed by WES and was identified through microarray CNV analysis. Segregation analysis of both mutations demonstrated that all affected individuals were either homozygous for one of the mutations, or compound heterozygous for both. The two mutations are predicted to cause loss of function of the encoded protein and were not present in screening of 200 ethnically-matched controls. Our findings of two distinct mutations in the same gene in a single inbred kindred, identified only through combined WES and microarray CNV analysis, highlight the limitations of either CNV or WES alone, as the heterozygous deletion had normal WES read-depth values. Moreover, they demonstrate pitfalls in homozygosity mapping for disease-causing variant identification in inbred communities.
Assuntos
Consanguinidade , Variações do Número de Cópias de DNA , Sequenciamento do Exoma , Proteínas do Olho/genética , Haplótipos , Mutação , Retinose Pigmentar/diagnóstico , Retinose Pigmentar/genética , Adolescente , Adulto , Idoso , Criança , Feminino , Estudo de Associação Genômica Ampla , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , Grupos Populacionais , Adulto JovemRESUMO
A novel autosomal recessive cerebro-renal syndrome was identified in consanguineous Bedouin kindred: neurological deterioration was evident as of early age, progressing into severe intellectual disability, profound ataxia, camptocormia and oculomotor apraxia. Brain MRI was normal. Four of the six affected individuals also had early-onset nephropathy with features of tubulo-interstitial nephritis, hypertension and tendency for hyperkalemia, though none had rapid deterioration of renal function. Genome wide linkage analysis identified an â¼18 Mb disease-associated locus on chromosome 4 (maximal logarithm of odds score 4.4 at D4S2971; θ = 0). Whole exome sequencing identified a single mutation in SLC30A9 within this locus, segregating as expected within the kindred and not found in a homozygous state in 300 Bedouin controls. We showed that SLC30A9 (solute carrier family 30 member 9; also known as ZnT-9) is ubiquitously expressed with high levels in cerebellum, skeletal muscle, thymus and kidney. Confocal analysis of SH-SY5Y cells overexpressing SLC30A9 fused to enhanced green fluorescent protein demonstrated vesicular cytosolic localization associated with the endoplasmic reticulum, not co-localizing with endosomal or Golgi markers. SLC30A9 encodes a putative zinc transporter (by similarity) previously associated with Wnt signalling. However, using dual-luciferase reporter assay in SH-SY5Y cells we showed that Wnt signalling was not affected by the mutation. Based on protein modelling, the identified mutation is expected to affect SLC30A9's highly conserved cation efflux domain, putatively disrupting its transmembrane helix structure. Cytosolic Zn2+ measurements in HEK293 cells overexpressing wild-type and mutant SLC30A9 showed lower zinc concentration within mutant rather than wild-type SLC30A9 cells. This suggests that SLC30A9 has zinc transport properties affecting intracellular zinc homeostasis, and that the molecular mechanism of the disease is through defective function of this novel activity of SLC30A9 rather than by a defect in its previously described role in transcriptional activation of Wnt signalling.
Assuntos
Proteínas de Transporte de Cátions/genética , Proteínas de Ciclo Celular/genética , Neuropatia Hereditária Motora e Sensorial/genética , Homeostase/genética , Deficiência Intelectual/genética , Nefropatias/genética , Proteínas Nucleares/genética , Zinco/metabolismo , Idade de Início , Árabes , Mapeamento Cromossômico , Consanguinidade , Citosol/metabolismo , Citosol/ultraestrutura , Feminino , Estudo de Associação Genômica Ampla , Células HEK293 , Humanos , Lactente , Masculino , Mutação , Linhagem , Síndrome , Fatores de Transcrição , Via de Sinalização Wnt/genéticaRESUMO
PAX7 encodes a transcription factor essential in neural crest formation, myogenesis, and pituitary lineage specification. Pax7 null mice fail to thrive and exhibit muscle weakness, dying within 3 weeks. We describe a human autosomal-recessive syndrome, with failure to thrive, severe global developmental delay, microcephaly, axial hypotonia, pyramidal signs, dystonic postures, seizures, irritability, and self-mutilation. Aside from low blood carnitine levels, biochemical and metabolic screen was normal, with growth hormone deficiency in one patient. Electromyography was normal, with no specific findings in brain MRI/MRS yet nondemonstrable neuropituitary, a finding of unclear significance. Muscle biopsy showed unaffected overall organization of muscle fibers, yet positive fetal alpha myosin staining, suggesting regeneration. Homozygosity mapping with whole-exome sequencing identified a single disease-associated mutation in PAX7, segregating as expected in the kindred with no homozygosity in 200 ethnically matched controls. Transfection experiments showed that the PAX7 splice-site mutation putatively causes nonsense-mediated mRNA decay affecting onlyPAX7 isoform 3. This isoform, expressed specifically in brain, skeletal muscle and testes, is the sole Pax7 variant normally found in mice. The human muscle phenotype is in line with that in conditional Pax7 null mutant mice, where initial aberrant histological findings resolve postnatally through muscle regeneration.
Assuntos
Deficiências do Desenvolvimento/genética , Insuficiência de Crescimento/genética , Hipotonia Muscular/genética , Doenças Neuromusculares/genética , Fator de Transcrição PAX7/genética , Sequência de Aminoácidos , Animais , Aberrações Cromossômicas , Deficiências do Desenvolvimento/patologia , Insuficiência de Crescimento/patologia , Genes Recessivos , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Knockout , Desenvolvimento Muscular/genética , Hipotonia Muscular/patologia , Mutação , Doenças Neuromusculares/patologia , Degradação do RNAm Mediada por Códon sem Sentido , Fenótipo , Alinhamento de Sequência , Transcriptoma , Sequenciamento do ExomaRESUMO
AIM: Pancreatic lipase (triacylglycerol lipase EC 3.1.1.3) is an essential enzyme in hydrolysis of dietary fat. Dietary fat, especially polyunsaturated fatty acids (PUFA), regulate pancreatic lipase (PNLIP); however, the molecular mechanism underlying this regulation is mostly unknown. As PUFA are known to regulate expression of proliferator-activated receptor gamma (PPARγ), and as we identified in-silico putative PPARγ binding sites within the putative PNLIP promoter sequence, we hypothesized that PUFA regulation of PNLIP might be mediated by PPARγ. MATERIALS AND METHODS: We used in silico bioinformatics tools, reporter luciferase assay, PPARγ agonists and antagonists, PPARγ overexpression in exocrine pancreas AR42J and primary cells to study PPARγ regulation of PNLIP. RESULTS: Using in silico bioinformatics tools we mapped PPARγ binding sites (PPRE) to the putative promoter region of PNLIP. Reporter luciferase assay in AR42J rat exocrine pancreas acinar cells transfected with various constructs of the putative PNLIP promoter showed that PNLIP transcription is significantly enhanced by PPARγ dose-dependently, reaching maximal levels with multi PPRE sites. This effect was significantly augmented in the presence of PPARγ agonists and reduced by PPARγ antagonists or mutagenesis abrogating PPRE sites. Over-expression of PPARγ significantly elevated PNLIP transcript and protein levels in AR42J cells and in primary pancreas cells. Moreover, PNLIP expression was up-regulated by PPARγ agonists (pioglitazone and 15dPGJ2) and significantly down-regulated by PPARγ antagonists in non-transfected rat exocrine pancreas AR42J cell line cells. CONCLUSION: PPARγ transcriptionally regulates PNLIP gene expression. This transcript regulation resolves part of the missing link between dietary PUFA direct regulation of PNLIP.
Assuntos
Lipase/genética , Lipase/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Pâncreas Exócrino/metabolismo , Pâncreas/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Biologia Computacional/métodos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Pâncreas/efeitos dos fármacos , Pâncreas Exócrino/efeitos dos fármacos , Pioglitazona , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacologia , Ratos , Alinhamento de Sequência , Tiazolidinedionas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Siblings of non-consanguineous Jewish-Ethiopian ancestry presented with congenital axial hypotonia, weakness of the abducens nerve, psychomotor developmental delay with brain ventriculomegaly, variable thinning of corpus callosum and cardiac septal defects. Homozygosity mapping identified a single disease-associated locus of 3.5 Mb on chromosome 3. Studies of a Bedouin consanguineous kindred affected with a similar recessive phenotype identified a single disease-associated 18 Mb homozygosity locus encompassing the entire 3.5 Mb locus. Whole exome sequencing demonstrated only two homozygous mutations within a shared identical haplotype of 0.6 Mb, common to both Bedouin and Ethiopian affected individuals, suggesting an ancient common founder. Only one of the mutations segregated as expected in both kindreds and was not found in Bedouin and Jewish-Ethiopian controls: c.1404A>G, p.[*468Trpext*6] in CCDC174. We showed that CCDC174 is ubiquitous, restricted to the cell nucleus and co-localized with EIF4A3. In fact, yeast-two-hybrid assay demonstrated interaction of CCDC174 with EIF4A3, a component of exon junction complex. Knockdown of the CCDC174 ortholog in Xenopus laevis embryos resulted in poor neural fold closure at the neurula stage with later embryonic lethality. Knockdown embryos exhibited a sharp reduction in expression of n-tubulin, a marker for differentiating primary neurons, and of hindbrain markers krox20 and hoxb3. The Xenopus phenotype could be rescued by the human normal, yet not the mutant CCDC174 transcripts. Moreover, overexpression of mutant but not normal CCDC174 in neuroblastoma cells caused rapid apoptosis. In line with the hypotonia phenotype, the CCDC174 mutation caused depletion of RYR1 and marked myopathic changes in skeletal muscle of affected individuals.
Assuntos
Éxons , Hipotonia Muscular/genética , Mutação , Proteínas/genética , Transtornos Psicomotores/genética , Cromossomos Humanos Par 3 , RNA Helicases DEAD-box , Fator de Iniciação 4A em Eucariotos , Genes Recessivos , Estudos de Associação Genética , Ligação Genética , Haplótipos , Homozigoto , Humanos , Recém-Nascido , Masculino , Hipotonia Muscular/congênito , Linhagem , Transtornos Psicomotores/congênito , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: A syndrome of profound hypotonia, intellectual disability, intrauterine growth retardation with subsequent failure to thrive, dyskinesia and epilepsy was diagnosed in Bedouin Israeli families. Mild dysmorphism was evident: plagiocephaly, broad forehead with prominent nose, smooth philtrum and congenital esotropia. We set out to decipher the molecular basis of this syndrome. METHODS: Genome-wide linkage analysis and fine mapping were done. Whole exome sequencing data were filtered for candidate variants within locus. Validation and segregation of the mutation was assayed via Sanger sequencing. UNC80 expression pattern was analysed through reverse transcription PCR. RESULTS: Homozygosity mapping followed by fine mapping identified a 7.5â Mb disease-associated locus (logarithm of odds score 3.5) on chromosome 2. Whole exome and Sanger sequencing identified a single homozygous nonsense mutation within this locus, segregating within the families as expected for recessive heredity and not found in a homozygous state in 150 Bedouin controls: c.151C>T, p.(R51*) in UNC80. CONCLUSIONS: The syndrome described is caused by a mutation in UNC80, truncating most of the 3258 amino acids highly conserved encoded protein, that has no known motifs. UNC80 bridges between UNC79 and the cation channel NALCN, enabling NALCN's role in basal Na(+) leak conductance in neurons, essential for neuronal function. The phenotype caused by the UNC80 mutation resembles that previously described for homozygous NALCN mutations.
Assuntos
Proteínas de Transporte/genética , Cátions/metabolismo , Códon sem Sentido/genética , Discinesias/genética , Deficiência Intelectual/genética , Proteínas de Membrana/genética , Hipotonia Muscular/genética , Canais de Sódio/genética , Epilepsia/genética , Exoma/genética , Feminino , Ligação Genética/genética , Homozigoto , Humanos , Canais Iônicos , Masculino , Neurônios , SíndromeRESUMO
BACKGROUND: Progressive cerebello-cerebral atrophy (PCCA) leading to profound mental retardation, progressive microcephaly, spasticity and early onset epilepsy, was diagnosed in four non-consanguineous apparently unrelated families of Jewish Moroccan ancestry. Common founder mutation(s) were assumed. METHODS: Genome-wide linkage analysis and whole exome sequencing were done, followed by realtime PCR and immunofluorescent microscopy. RESULTS: Genome-wide linkage analysis mapped the disease-associated gene to 0.5 Mb on chromosome 17p13.3. Whole exome sequencing identified only two mutations within this locus, which were common to the affected individuals: compound heterozygous mutations in VPS53, segregating as expected for autosomal recessive heredity within all four families, and common in Moroccan Jews (â¼1:37 carrier rate). The Golgi-associated retrograde protein (GARP) complex is involved in the retrograde pathway recycling endocytic vesicles to Golgi; c.2084A>G and c.1556+5G>A VPS53 founder mutations are predicted to affect the C-terminal domain of VPS53, known to be critical to its role as part of this complex. Immunofluorescent microscopy demonstrated swollen and abnormally numerous CD63 positive vesicular bodies, likely intermediate recycling/late endosomes, in fibroblasts of affected individuals. CONCLUSIONS: Autosomal recessive PCCA type 2 is caused by VPS53 mutations.
Assuntos
Atrofia/genética , Cerebelo/patologia , Mutação , Proteínas de Transporte Vesicular/genética , Adolescente , Adulto , Pré-Escolar , Mapeamento Cromossômico , Feminino , Genes Recessivos , Estudo de Associação Genômica Ampla , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Linhagem , Adulto JovemRESUMO
Chromodomain helicase DNA binding protein 2 (Chd2) is a chromatin remodeller implicated in neurological disease. Here we show that Chaserr, a highly conserved long noncoding RNA transcribed from a region near the transcription start site of Chd2 and on the same strand, acts in concert with the CHD2 protein to maintain proper Chd2 expression levels. Loss of Chaserr in mice leads to early postnatal lethality in homozygous mice, and severe growth retardation in heterozygotes. Mechanistically, loss of Chaserr leads to substantially increased Chd2 mRNA and protein levels, which in turn lead to transcriptional interference by inhibiting promoters found downstream of highly expressed genes. We further show that Chaserr production represses Chd2 expression solely in cis, and that the phenotypic consequences of Chaserr loss are rescued when Chd2 is perturbed as well. Targeting Chaserr is thus a potential strategy for increasing CHD2 levels in haploinsufficient individuals.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Transtornos do Crescimento/genética , RNA Longo não Codificante/genética , RNA Mensageiro/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Genes Letais , Haploinsuficiência , Heterozigoto , Homozigoto , Camundongos , Camundongos Knockout , Regiões Promotoras GenéticasRESUMO
Gout is caused by deposition of monosodium urate crystals in joints when plasma uric acid levels are chronically elevated beyond the saturation threshold, mostly due to renal underexcretion of uric acid. Although molecular pathways of this underexcretion have been elucidated, its etiology remains mostly unknown. We demonstrate that gout can be caused by a mutation in LDHD within the putative catalytic site of the encoded d-lactate dehydrogenase, resulting in augmented blood levels of d-lactate, a stereoisomer of l-lactate, which is normally present in human blood in miniscule amounts. Consequent excessive renal secretion of d-lactate in exchange for uric acid reabsorption culminated in hyperuricemia and gout. We showed that LDHD expression is enriched in tissues with a high metabolic rate and abundant mitochondria and that d-lactate dehydrogenase resides in the mitochondria of cells overexpressing the human LDHD gene. Notably, the p.R370W mutation had no effect on protein localization. In line with the human phenotype, injection of d-lactate into naive mice resulted in hyperuricemia. Thus, hyperuricemia and gout can result from the accumulation of metabolites whose renal excretion is coupled to uric acid reabsorption.
Assuntos
Gota/genética , Hiperuricemia/genética , Lactato Desidrogenases/genética , Mutação de Sentido Incorreto , Adulto , Animais , Domínio Catalítico , Criança , DNA/metabolismo , Saúde da Família , Feminino , Células HEK293 , Heterozigoto , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mutação , Linhagem , Estereoisomerismo , Ácido Úrico/metabolismoRESUMO
Studies of ciliopathies have served in elucidating much of our knowledge of structure and function of primary cilia. We report humans with Bardet-Biedl syndrome who display intellectual disability, retinitis pigmentosa, obesity, short stature and brachydactyly, stemming from a homozyogous truncation mutation in SCAPER, a gene previously associated with mitotic progression. Our findings, based on linkage analysis and exome sequencing studies of two remotely related large consanguineous families, are in line with recent reports of SCAPER variants associated with intellectual disability and retinitis pigmentosa. Using immuno-fluorescence and live cell imaging in NIH/3T3 fibroblasts and SH-SY5Y neuroblastoma cell lines over-expressing SCAPER, we demonstrate that both wild type and mutant SCAPER are expressed in primary cilia and co-localize with tubulin, forming bundles of microtubules. While wild type SCAPER was rarely localized along the ciliary axoneme and basal body, the aberrant protein remained sequestered to the cilia, mostly at the ciliary tip. Notably, longer cilia were demonstrated both in human affected fibroblasts compared to controls, as well as in NIH/3T3 cells transfected with mutant versus wildtype SCAPER. As SCAPER expression is known to peak at late G1 and S phase, overlapping the timing of ciliary resorption, our data suggest a possible role of SCAPER in ciliary dynamics and disassembly, also affecting microtubule-related mitotic progression. Thus, we outline a human ciliopathy syndrome and demonstrate that it is caused by a mutation in SCAPER, affecting primary cilia.
Assuntos
Síndrome de Bardet-Biedl , Proteínas de Transporte , Cílios , Deficiência Intelectual , Mutação , Retinose Pigmentar , Animais , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/metabolismo , Síndrome de Bardet-Biedl/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cílios/genética , Cílios/metabolismo , Cílios/patologia , Feminino , Humanos , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Masculino , Camundongos , Células NIH 3T3 , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologiaRESUMO
BACKGROUND: Paroxysmal atrial fibrillation (AF) can be caused by gain-of-function mutations in genes, encoding the cardiac potassium channel subunits KCNJ2, KCNE1, and KCNH2 that mediate the repolarizing potassium currents Ik1, Iks, and Ikr, respectively. METHODS: Linkage analysis, whole-exome sequencing, and Xenopus oocyte electrophysiology studies were used in this study. RESULTS: Through genetic studies, we showed that autosomal dominant early-onset nocturnal paroxysmal AF is caused by p.S447R mutation in KCND2, encoding the pore-forming (α) subunit of the Kv4.2 cardiac potassium channel. Kv4.2, along with Kv4.3, contributes to the cardiac fast transient outward K+ current, Ito. Ito underlies the early phase of repolarization in the cardiac action potential, thereby setting the initial potential of the plateau phase and governing its duration and amplitude. In Xenopus oocytes, the mutation increased the channel's inactivation time constant and affected its regulation: p.S447 resides in a protein kinase C (PKC) phosphorylation site, which normally allows attenuation of Kv4.2 membrane expression. The mutant Kv4.2 exhibited impaired response to PKC; hence, Kv4.2 membrane expression was augmented, enhancing potassium currents. Coexpression of mutant and wild-type channels (recapitulating heterozygosity in affected individuals) showed results similar to the mutant channel alone. Finally, in a hybrid channel composed of Kv4.3 and Kv4.2, simulating the mature endogenous heterotetrameric channel underlying Ito, the p.S447R Kv4.2 mutation exerted a gain-of-function effect on Kv4.3. CONCLUSIONS: The mutation alters Kv4.2's kinetic properties, impairs its inhibitory regulation, and exerts gain-of-function effect on both Kv4.2 homotetramers and Kv4.2-Kv4.3 heterotetramers. These effects presumably increase the repolarizing potassium current Ito, thereby abbreviating action potential duration, creating arrhythmogenic substrate for nocturnal AF. Interestingly, Kv4.2 expression was previously shown to demonstrate circadian variation, with peak expression at daytime in murine hearts (human nighttime), with possible relevance to the nocturnal onset of paroxysmal AF symptoms in our patients. The atrial-specific phenotype suggests that targeting Kv4.2 might be effective in the treatment of nocturnal paroxysmal AF, avoiding adverse ventricular effects.
Assuntos
Potenciais de Ação/genética , Fibrilação Atrial , Mutação , Canais de Potássio Shal , Animais , Fibrilação Atrial/genética , Fibrilação Atrial/metabolismo , Fibrilação Atrial/patologia , Fibrilação Atrial/fisiopatologia , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Canais de Potássio Shal/genética , Canais de Potássio Shal/metabolismoRESUMO
Twelve individuals of consanguineous Bedouin kindred presented with autosomal recessive progressive spastic paraplegia evident as of age 0-24 months, with spasticity of lower limbs, hyperreflexia, toe walking and equinus deformity. Kyphoscolisois was evident in older patients. Most had atrophy of the lateral aspects of the tongue and few had intellectual disability. Nerve conduction velocity, electromyography and head and spinal cord magnetic resonance imaging were normal in tested subjects. Muscle biopsy showed occasional central nuclei and fiber size variability with small angular fibers. Genome-wide linkage analysis identified a 6.7Mbp disease-associated locus on chromosome 3q21.3-3q22.2 (LOD score 9.02; D3S1290). Whole-exome sequencing identified a single homozygous variant within this locus, c.51_52ins(28); p.(V18fs56*) in KY, segregating in the family as expected and not found in 190 Bedouin controls. High KY transcript levels were demonstrated in muscular organs with lower expression in the CNS. The phenotype is reminiscent of kyphoscoliosis seen in Ky null mice. Two recent studies done independently and parallel to ours describe somewhat similar phenotypes in one and two patients with KY mutations. KY encodes a tranglutaminase-like peptidase, which interacts with muscle cytoskeletal proteins and is part of a Z-band protein complex, suggesting the disease mechanism may resemble myofibrillar myopathy. However, the mixed myopathic-neurologic features caused by human and mouse Ky mutations are difficult to explain by loss of KY sarcomere stabilizing function alone. KY transcription in CNS tissues may imply that it also has a role in neuromotor function, in line with the irregularity of neuromuscular junction in Ky null mutant mice.
Assuntos
Mutação , Peptídeo Hidrolases/genética , Paraplegia Espástica Hereditária/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Homozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , Linhagem , Peptídeo Hidrolases/metabolismo , Fenótipo , Paraplegia Espástica Hereditária/diagnóstico , Medula Espinal/metabolismoRESUMO
Autosomal recessive Adams-Oliver syndrome was diagnosed in three remotely related Bedouin consanguineous families. Genome-wide linkage analysis ruled out association with known Adams-Oliver syndrome genes, identifying a single-homozygosity â¼1.8-Mb novel locus common to affected individuals (LOD score 3.37). Whole-exome sequencing followed by Sanger sequencing identified only a single mutation within this locus, shared by all affected individuals and found in patients from five additional apparently unrelated Bedouin families: a 1-bp deletion mutation in a predicted alternative splice variant of EOGT, leading to a putative truncated protein. RT-PCR demonstrated that the EOGT-predicted alternative splice variant is ubiquitously expressed. EOGT encodes EGF-domain-specific O-linked N-acetylglucosamine transferase, responsible for extracellular O-GlcNAcylation of epidermal growth factor-like domain-containing proteins, and is essential for epithelial cell-matrix interactions. F-actin staining in diseased fibroblasts showed apparently intact cell cytoskeleton and morphology, suggesting the EOGT mutation acts not through perturbation of cytoskeleton but through other mechanisms yet to be elucidated.
Assuntos
Displasia Ectodérmica/genética , Deformidades Congênitas dos Membros/genética , Mutação , N-Acetilglucosaminiltransferases/genética , Dermatoses do Couro Cabeludo/congênito , Adolescente , Processamento Alternativo , Criança , Pré-Escolar , Displasia Ectodérmica/diagnóstico , Displasia Ectodérmica/enzimologia , Fator de Crescimento Epidérmico/metabolismo , Feminino , Humanos , Lactente , Recém-Nascido , Deformidades Congênitas dos Membros/diagnóstico , Deformidades Congênitas dos Membros/enzimologia , Masculino , N-Acetilglucosaminiltransferases/metabolismo , Linhagem , Dermatoses do Couro Cabeludo/diagnóstico , Dermatoses do Couro Cabeludo/enzimologia , Dermatoses do Couro Cabeludo/genéticaRESUMO
Foveal hypoplasia, always accompanied by nystagmus, is found as part of the clinical spectrum of various eye disorders such as aniridia, albinism and achromatopsia. However, the molecular basis of isolated autosomal recessive foveal hypoplasia is yet unknown. Individuals of apparently unrelated non consanguineous Israeli families of Jewish Indian (Mumbai) ancestry presented with isolated foveal hypoplasia associated with congenital nystagmus and reduced visual acuity. Genome-wide homozygosity mapping followed by fine mapping defined a 830 Kb disease-associated locus (LOD score 3.5). Whole-exome sequencing identified a single missense mutation in the homozygosity region: c.95T>G, p.(Ile32Ser), in a conserved amino acid within the first predicted transmembrane domain of SLC38A8. The mutation fully segregated with the disease-associated phenotype, demonstrating an â¼10% carrier rate in Mumbai Jews. SLC38A8 encodes a putative sodium-dependent amino-acid/proton antiporter, which we showed to be expressed solely in the eye. Thus, a homozygous SLC38A8 mutation likely underlies isolated foveal hypoplasia.