56 research outputs found
The novel KIF1A missense variant (R169T) strongly reduces microtubule stimulated ATPase activity and is associated with NESCAV syndrome
KIF1A is a microtubule-dependent motor protein responsible for fast anterograde transport of synaptic vesicle precursors in neurons. Pathogenic variants in KIF1A have been associated with a wide spectrum of neurological disorders. Here, we report a patient presenting a severe neurodevelopmental disorder carrying a novel de novo missense variant p.Arg169Thr (R169T) in the KIF1A motor domain. The clinical features present in our patient match with those reported for NESCAV syndrome including severe developmental delay, spastic paraparesis, motor sensory neuropathy, bilateral optic nerve atrophy, progressive cerebellar atrophy, epilepsy, ataxia, and hypotonia. Here, we demonstrate that the microtubule-stimulated ATPase activity of the KIF1A is strongly reduced in the motor domain of the R169T variant. Supporting this, in silico structural modeling suggests that this variant impairs the interaction of the KIF1A motor domain with microtubules. The characterization of the molecular effect of the R169T variant on the KIF1A protein together with the presence of the typical clinical features indicates its causal pathogenic effect
Mutations in NSUN2 cause autosomal-recessive intellectual disability
With a prevalence between 1 and 3%, hereditary forms of intellectual disability (ID) are among the most important problems in health care. Particularly, autosomal-recessive forms of the disorder have a very heterogeneous molecular basis, and genes with an increased number of disease-causing mutations are not common. Here, we report on three different mutations (two nonsense mutations, c.679C>T [p.Gln227( *)] and c.1114C>T [p.Gln372( *)], as well as one splicing mutation, g.6622224A>C [p.Ile179Argfs( *)192]) that cause a loss of the tRNA-methyltransferase-encoding NSUN2 main transcript in homozygotes. We identified the mutations by sequencing exons and exon-intron boundaries within the genomic region where the linkage intervals of three independent consanguineous families of Iranian and Kurdish origin overlapped with the previously described MRT5 locus. In order to gain further evidence concerning the effect of a loss of NSUN2 on memory and learning, we constructed a Drosophila model by deleting the NSUN2 ortholog, CG6133, and investigated the mutants by using molecular and behavioral approaches. When the Drosophila melanogaster NSUN2 ortholog was deleted, severe short-term-memory (STM) deficits were observed; STM could be rescued by re-expression of the wild-type protein in the nervous system. The humans homozygous for NSUN2 mutations showed an overlapping phenotype consisting of moderate to severe ID and facial dysmorphism (which includes a long face, characteristic eyebrows, a long nose, and a small chin), suggesting that mutations in this gene might even induce a syndromic form of ID. Moreover, our observations from the Drosophila model point toward an evolutionarily conserved role of RNA methylation in normal cognitive development
Mutations in NSUN2 Cause Autosomal- Recessive Intellectual Disability
With a prevalence between 1 and 3%, hereditary forms of intellectual disability (ID) are among the most important problems in health care. Particularly, autosomal-recessive forms of the disorder have a very heterogeneous molecular basis, and genes with an increased number of disease-causing mutations are not common. Here, we report on three different mutations (two nonsense mutations, c.679C>T [p.Gln227∗] and c.1114C>T [p.Gln372∗], as well as one splicing mutation, g.6622224A>C [p.Ile179Argfs∗192]) that cause a loss of the tRNA-methyltransferase-encoding NSUN2 main transcript in homozygotes. We identified the mutations by sequencing exons and exon-intron boundaries within the genomic region where the linkage intervals of three independent consanguineous families of Iranian and Kurdish origin overlapped with the previously described MRT5 locus. In order to gain further evidence concerning the effect of a loss of NSUN2 on memory and learning, we constructed a Drosophila model by deleting the NSUN2 ortholog, CG6133, and investigated the mutants by using molecular and behavioral approaches. When the Drosophila melanogaster NSUN2 ortholog was deleted, severe short-term-memory (STM) deficits were observed; STM could be rescued by re-expression of the wild-type protein in the nervous system. The humans homozygous for NSUN2 mutations showed an overlapping phenotype consisting of moderate to severe ID and facial dysmorphism (which includes a long face, characteristic eyebrows, a long nose, and a small chin), suggesting that mutations in this gene might even induce a syndromic form of ID. Moreover, our observations from the Drosophila model point toward an evolutionarily conserved role of RNA methylation in normal cognitive development
BOD1 Is Required for Cognitive Function in Humans and <i>Drosophila</i>
Here we report a stop-mutation in the BOD1 (Biorientation Defective 1) gene, which co-segregates with intellectual disability in a large consanguineous family, where individuals that are homozygous for the mutation have no detectable BOD1 mRNA or protein. The BOD1 protein is required for proper chromosome segregation, regulating phosphorylation of PLK1 substrates by modulating Protein Phosphatase 2A (PP2A) activity during mitosis. We report that fibroblast cell lines derived from homozygous BOD1 mutation carriers show aberrant localisation of the cell cycle kinase PLK1 and its phosphatase PP2A at mitotic kinetochores. However, in contrast to the mitotic arrest observed in BOD1-siRNA treated HeLa cells, patient-derived cells progressed through mitosis with no apparent segregation defects but at an accelerated rate compared to controls. The relatively normal cell cycle progression observed in cultured cells is in line with the absence of gross structural brain abnormalities in the affected individuals. Moreover, we found that in normal adult brain tissues BOD1 expression is maintained at considerable levels, in contrast to PLK1 expression, and provide evidence for synaptic localization of Bod1 in murine neurons. These observations suggest that BOD1 plays a cell cycle-independent role in the nervous system. To address this possibility, we established two Drosophila models, where neuron-specific knockdown of BOD1 caused pronounced learning deficits and significant abnormalities in synapse morphology. Together our results reveal novel postmitotic functions of BOD1 as well as pathogenic mechanisms that strongly support a causative role of BOD1 deficiency in the aetiology of intellectual disability. Moreover, by demonstrating its requirement for cognitive function in humans and Drosophila we provide evidence for a conserved role of BOD1 in the development and maintenance of cognitive features
Identifizierung und funktionelle Charakterisierung eines Gendefekts im Kinetochor Protein BOD1, welches mit autosomal rezessiver mentaler Retardierung und Oligomenorrhö assoziiert ist
In the course of systematic clinical and molecular studies to identify
intellectual disability (ID) causing defects that follow an autosomal
recessive mode of inheritance (ARID), we found a single 4.3 Mbp interval on
chromosome 5q with a LOD score of 4.41 by autozygosity mapping in a family
with four females affected by mild to moderate ID and oligomenorrhea. By
sequencing the coding regions of all 28 genes within this region we discovered
a nonsense mutation in exon 2 of the BOD1 gene. This defect co-segregated with
the disease and was not found in 720 Iranian and German control chromosomes.
All other genes within the interval were not affected by nucleotide changes.
BOD1 is expressed in a wide range of tissues, including brain and ovary. By
RT-PCR, we identified two previously unknown isoforms of BOD1 in control
lymphoblast and fibroblast cells and showed expression of all four transcripts
in a variety of brain tissues. qRT-PCR revealed loss of all BOD1 isoforms in
patient fibroblasts, including splice variants that did not contain exon 2.
For all transcripts except one, this seems to be due to nonsense mediated
decay, as defect of this mRNA could be abrogated by cycloheximide treatment of
the cells. Absence of BOD1 protein in cells of the patients was confirmed by
Western blotting experiments. BOD1 is required for proper chromosome
segregation and correction of synthelic chromosomes during mitosis (Porter et
al. 2007). Fibroblasts from two different patients showed several
abnormalities in cell division (chromosomal bi-orientation, mitotic index,
mitotic timings, increased levels of Plk1 activity, etc.) which are all in
line with the findings in the BOD1 depleted HeLa cells (Porter et al. 2007;
Porter et al. unpublished) and may also provide an explanation for the
oligomenorrhea observed in this family. In patient fibroblasts, confocal
indirect immunoflorescence imaging revealed nuclear structural defects.
Subsequent overexpression experiments using BOD1 cDNA in HaCat cells were
performed to further investigate the cell biology of this protein in non-
dividing cells and provided preliminary evidence for an involvement of BOD1 in
nuclear and chromatin organization. This indicated a putative role in the
regulation of gene expression and whole genome expression profiling in patient
lymphoblast cells showed deregulation of target genes that are critical for
human cognition. Moreover, in patient cells the formation of primary cilia was
found to be defective. Cilia defects have been observed previously in several
other disorders involving ID. Finally, overexpression experiments in murine
primary neuronal cells showed co-localization of GFP-tagged BOD1 with
presynaptic proteins, suggesting a putative function of BOD1 in neuronal
synapses, which could be an explanation for the ID observed in our patients.
In summary, our results provide evidence indicating that BOD1 plays critical
roles in cell cycle progression, the formation of primary cilia, chromatin
organization and the regulation of transcription. Furthermore our observations
suggest that in humans, this protein is required for normal brain function,
which may be related to the role of BOD1 in the regulation of PLK1 during
brain development. However, further in depth functional investigations are
needed to shed more light on the precise role of this protein during brain
development and in fully differentiated neuronal tissues.Im Zuge unserer klinischen und molekularen Studien zur Identifizierung der
genetischen Ursachen autosomal-rezessiver geistiger Behinderung, untersuchten
wir eine iranische Familie mit vier Patientinnen, welche einen milden bis
mittelschweren Grad geistiger Behinderung sowie Oligomenorrhoe aufweisen.
Mittels Autozygosity Mapping wurde ein 4,3 Mbp Intervall auf Chromosom 5
identifiziert, welches 28 Gene beinhaltet. Die kodierenden Bereiche dieser
Gene wurden sequenziert und als einzige Nukleotidänderung wurde eine Nonsense
Mutation in Exon 2 des BOD1 Gens nachgewiesen. Diese Mutation co-seggregiert
mit der Krankheit und wurde nicht in 720 iranischen und deutschen Kontroll-
Chromosomen gefunden. BOD1 wird in einer Vielzahl von Geweben exprimiert,
einschließlich des Gehirns sowie der Eierstöcke. Durch RT-PCR wurden zwei
bislang unbekannte Isoformen BOD1s entdeckt, die ebenfalls im Gehirn
exprimiert werden. qRT-PCR zeigte den Verlust aller Isoformen in Fibroblasten
der Patientinnen. Für alle Isoformen bis auf eine ist hierfür vermutlich
nonsense mediated decay verantwortlich, da in dieser mRNA der Defekt durch die
Behandlung mit Cycloheximid aufgehoben wurde. Die Abwesenheit von BOD1 in den
Zellen der Patientinnen wurde auch mittels Western Blot nachgewiesen. BOD1
wird für die korrekte Chromosomentrennung und die Korrektur der synthelischen
Chromosomen während der Meiose benötigt (Porter et al. 2007). In
Übereinstimmung mit bereits beschriebenen Anomalien, welche in HeLa Zellen
ohne BOD1 auftreten (Porter et al. 2007; Porter et al. unveröffentlicht),
wiesen Fibroblasten der Patientinnen mehrere Abnormitäten während der
Zellteilung auf (chromosomale Biorientierung, mitotischer Index, zeitliche
Koordinierung der Mitose, erhöhte Plk1 Aktivität, etc.), welche eine Erklärung
für die Oligomenorrhoe der Patientinnen bieten. Die Untersuchung von
Interphase Fibroblasten der Patientinnen mittels konfokaler indirekter
Immunfloureszenz ergab, dass strukturelle Defekte des Zellkerns vorliegen.
Überexpression von BOD1 in sich nicht teilenden HaCat Zellen zeigte eine
Beteiligung von BOD1 an der Zellkern- und Chromatinorganisation, was eine
putative Rolle BOD1s bei der Regulierung der Genexpression nahe legt. Whole
Genome Expression Profiling in Lymphoblasten der Patientinnen zeigte eine
Deregulation von Zielgenen, die entscheidend für die humane Kognition sind.
Darüber hinaus ist die Formation der primären Zilien in den Zellen der
Patientinnen fehlerhaft. Schäden an Zilien wurden in einer Vielzahl von
Krankheiten die mit geistiger Behinderung einhergehen beobachtet. Letztendlich
zeigten Überexpressionsstudien in murinen primären neuronalen Zellen Co-
Lokalisation von GFP getaggten BOD1 mit Page: 168 präsynaptischen Proteinen,
was auf eine putative Funktion von BOD1 in neuronalen Synapsen hinweist, deren
Beeinträchtigung zu der in den Patientinnen beobachteten geistigen Behinderung
führen könnten. Unsere Ergebnisse zeigen die kritische Beteiligung BOD1s am
Zellzyklus, an der Formation primärer Zilien, der Chromatinorganisation sowie
der Regulierung der Transkription. Des Weiteren weisen unsere Beobachtungen
darauf hin, dass BOD1 für die normale Gehirnfunktion des Menschen notwendig
ist, was im Zusammenhang mit der Rolle BOD1 als Regulator von PLK1 während der
Gehirnentwicklung im Zusammenhang stehen kann. Weiterführende funktionelle
Studien sind nötig um die genaue Funktion BOD1s während der Gehirnentwicklung
und besonders im differenzierten neuronalen Gewebe aufzuklären
Epileptic Encephalopathies: New Genes and New Pathways
Epileptic encephalopathies represent a group of devastating epileptic disorders that occur early in life and are often characterized by pharmaco-resistant epilepsy, persistent severe electroencephalographic abnormalities, and cognitive dysfunction or decline. Next generation sequencing technologies have increased the speed of gene discovery tremendously. Whereas ion channel genes were long considered to be the only significant group of genes implicated in the genetic epilepsies, a growing number of non-ion-channel genes are now being identified. As a subgroup of the genetically mediated epilepsies, epileptic encephalopathies are complex and heterogeneous disorders, making diagnosis and treatment decisions difficult. Recent exome sequencing data suggest that mutations causing epileptic encephalopathies are often sporadic, typically resulting from de novo dominant mutations in a single autosomal gene, although inherited autosomal recessive and X-linked forms also exist. In this review we provide a summary of the key features of several early- and mid-childhood onset epileptic encephalopathies including Ohtahara syndrome, Dravet syndrome, Infantile spasms and Lennox Gastaut syndrome. We review the recent next generation sequencing findings that may impact treatment choices. We also describe the use of conventional and newer anti-epileptic and hormonal medications in the various syndromes based on their genetic profile. At a biological level, developments in cellular reprogramming and genome editing represent a new direction in modeling these pediatric epilepsies and could be used in the development of novel and repurposed therapies
Usefulness of exome sequencing in the study of spastic paraparesis and cerebellar atrophy: de novo mutation of the KIF1A gene, a new hope in prognosis
Utilidad del exoma en el estudio de la paraparesia espástica y la atrofia cerebelosa: mutación de novo en el gen KIF1A, una nueva esperanza pronóstica
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