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

Initial study on removing cellular residues from hydrostatic high-pressure treated allogeneic tissue using ultrasound

Hydrostatic high-pressure technology (HHD) devitalizes tissue quickly and gently, without negatively affecting the structural properties. HHD-treated tissues must be cleaned from devitalized cells. A partially automated, gentle, reproducible and timesaving rinsing test setup utilizing ultrasound is demonstrated in this study. The test setup is used to clean HHD-treated bone allografts of tissue residues and prevent microbiological contamination. A rinsing procedure is investigated. Residual DNA content determination is utilized to analyze cleaned bone allograft tissue for rinsing procedure evaluation

Functional consequences of the absence of BOD1 in patient-derived fibroblasts.

<p>(A) Flow cytometric analysis of cell cycle profile in WT and <i>BOD1</i>^-/- primary fibroblasts electroporated with control or <i>BOD1</i> siRNA. Error bars represent standard deviation. (B) Immunoblotting of BOD1 and tubulin from cell lysates simultaneously electroporated with samples analysed in (A). (C) Representative DIC timelapse imaging of primary fibroblast cells undergoing mitosis. Nuclear Envelope Breakdown (NEB) and Anaphase Onset (AO) are indicated. (D) Cumulative timing of NEB to AO timing in Primary Fibroblast cell lines. Error bars represent standard deviation. P<0.001 for <i>BOD1</i>^-/- cells to each WT sample. Insufficient data collected for <i>BOD1</i>^-/- cells to determine statistical significance. (E) Immunofluorescence localization of PP2A-B56 in WT and <i>BOD1</i>^-/- Primary fibroblasts. DAPI (blue), centromeres (detected with ACA) (green), anti-PP2A-B56α (red). (F) Mean B56α levels at kinetochores of WT and <i>BOD1</i>^-/- Primary fibroblasts (P<0.001). Error bars represent SEM. (G) Immunofluorescence localization of PLK1 in WT and <i>BOD1</i>^-/- Primary fibroblasts. DAPI (white), anti-PLK1 (green), ACA (blue). (H-J) Mean PLK1 levels at kinetochores and centrosomes of WT and <i>BOD1</i>^-/- Primary fibroblasts, respectively (P<0.001 in each instance). Error bars represent SEM. (K) Immunoblotting of PLK1, BOD1 and tubulin in asynchronous WT and <i>BOD1</i>^-/- primary fibroblasts. (L) Immunoblotting of PLK1, BOD1 and tubulin in asynchronous and Monastrol arrested WT and <i>BOD1</i>^-/- Primary fibroblasts. (M) Immunofluorescence localization of Bod1 in WT Primary Fibroblasts. DAPI (white), ACA (blue), anti-Plk1 (red), anti-Bod1 (green). Scale = 5 μm. Inset shows a single bioriented kinetochore pair. (N) Immunoblotting of PP2A-B56δ, PLK1 and tubulin in WT primary fibroblast electroporated with indicated combinations of CTR, B56-pool or <i>BOD1</i> siRNA. Rescue of WT primary fibroblasts after siRNA depletion of Bod1 with plasmids expressing GFP fused to either siRNA resistant WT Bod1 or Bod1^T95E. (O) Mitotic profile of WT primary fibroblasts and <i>BOD1</i>^-/- fibroblasts 1 hr after release from RO 3306 into the indicated concentrations of BI 2536. Results show average of three independent experiments. A minimum of 100 mitotic cells counted per condition per experiment. Error bars represent SEM.</p

Expression of BOD1 and PLK1 in human tissues.

<p>(A) BOD1-specific quantitative RT-PCR experiments were carried out in triplicates, using RNA from the indicated tissues. All splice variants (indicated by the respective exon combinations) were investigated. Error bars represent the SEM. (B) Expression levels i.e. reads per kilobase of transcript per million reads mapped (RPKM), corresponding to BOD1 (NM_138369.2) and PLK1 (NM_005030.5) obtained by RNA-Sequencing of commercially available RNA-samples from different brain tissues, induced pluripotent stem cells (IPSC) and human embryonic stem cells (hES).</p

Neuronal knockdown of Drosophila Bod1 affects learning and synapse development.

<p>(A-B') Knockdown of <i>Drosophila</i> Bod1 using the postmitotic, pan-neuronal promoter elav-Gal4 and three inducible RNAi lines affects non-associative learning in the light-off jump habituation paradigm. Jump responses were induced by repeated light-off pulses for 100 trials with a 1s inter-trial interval. Bod1 knockdown flies of genotypes (A) UAS-Bod1^vdrc105166/2xGMR-wIR; elav-Gal4, UAS-Dicer2/+, plotted as red squares, (B) UAS-Bod1^vdrc27445/2xGMR-wIR; elav-Gal4, UAS-Dicer2/+, plotted as blue squares, and (C) 2xGMR-wIR/+; UAS-Bod1^HMS00720/elav-Gal4, UAS-Dicer2, plotted as green squares, failed to habituate, i.e. to efficiently reduce their jump response upon repeated stimulation. The genetic background controls, generated by crossing the driver line to the respective genetic background of the RNAi line, are shown as grey circles (2xGMR-wIR/+; elav-Gal4, UAS-Dicer2/+). (A', B', C’) Quantification of average jump responses revealed that all three mutant genotypes habituated significantly slower (*** p<0,001). Red bar in (A') Bod1^vdrc105166, TTC = 49.75, n = 143 versus controls: TTC = 20.88, n = 134. Blue bar in (B'): Bod1^vdrc27445, TTC = 61.92, n = 93 versus controls TTC = 28.93, n = 87. Green bar in (C)’ Bod1^HMS00720, TTC = 10.03, n = 70 versus controls TTC = 5.51, n = 68. (D) Knockdown of <i>Drosophila</i> Bod1 using the elav-Gal4 promoter and RNAi lines Bod1^vdrc27445 and Bod1^vdrc105166 consistently affects synaptic branching at the <i>Drosophila</i> Neuromuscular Junction (see text). L3 muscle 4 synapses were labelled with anti-dlg1 and quantified using an in house-developed macro. A Bod1^vdrc27445 (UAS-Bod1^vdrc27445/2xGMR-wIR; elav-Gal4, UAS-Dicer2/+) and control (2xGMR-wIR/+; elav-Gal4, UAS-Dicer2/+) synapse is shown. Top panel in red: dlg1 labelling; bottom panels show the macro-annotated, quantified synapse). Asterisks highlight the increased number of synaptic branching points at the mutant synaptic terminal.</p

Presynaptic localisation of BOD1 in murine corticoneuronal cells.

<p>Representative indirect immunofluorescence confocal image (LSM510) of mouse cortical neurons transfected at day 7 after preparation with 0.1μg BOD1-GFP for 7hrs. Arrows indicate co-localization of BOD1-GFP (in green) with the (pre)synaptic marker anti-Bassoon (red). Insets are magnifications of the boxed area. The range indicator (RI) shows that the images are not overexposed.</p

Nonsense Mutation in <i>BOD1</i> co-segregates with Intellectual Disability and leads to loss of BOD1 in patient tissues.

<p>(A) Family pedigree and co-segregation of the mutation within the family. Filled symbols represent affected individuals. Sequence chromatograms from one patient (V:2) and one parent (IV:1) are shown on the upper right. (B) Schematic representation of BOD1 (black) and the exon composition in alternative transcripts. Previously unknown transcripts are shown in green. Arrows indicate the location of primers used for RT-PCR experiments (C) Agarose Gel electrophoresis results of RT-PCR experiment. (D) qRT-PCR was performed on patient and control Fibroblasts. The experiments were performed twice with independent cells, each time in triplicate (Error bars represent the SEM). One representative result is shown. (E) NMD analyses of patient fibroblasts were performed twice with independent cell samples, each time in triplicate. The results are from pooled patient (<i>BOD1</i>^-/-) and control (WT) samples. CHX: cycloheximide, DMSO:Dimethyl sulfoxide. Error bars represent the SEM. (F) Western blot of protein extracts from fibroblast cells using a Bod1 polyclonal antibody. The Bod1 antibody recognizes a 22KDa protein, matching the full-length Bod1 protein. Alpha tubulin was used as a loading control.</p

Distribution of segmental duplications in the context of higher order chromatin organisation of human chromosome 7

Background: Segmental duplications (SDs) are not evenly distributed along chromosomes. The reasons for this biased susceptibility to SD insertion are poorly understood. Accumulation of SDs is associated with increased genomic instability, which can lead to structural variants and genomic disorders such as the Williams-Beuren syndrome. Despite these adverse effects, SDs have become fixed in the human genome. Focusing on chromosome 7, which is particularly rich in interstitial SDs, we have investigated the distribution of SDs in the context of evolution and the three dimensional organisation of the chromosome in order to gain insights into the mutual relationship of SDs and chromatin topology. Results: Intrachromosomal SDs preferentially accumulate in those segments of chromosome 7 that are homologous to marmoset chromosome 2. Although this formerly compact segment has been re-distributed to three different sites during primate evolution, we can show by means of public data on long distance chromatin interactions that these three intervals, and consequently the paralogous SDs mapping to them, have retained their spatial proximity in the nucleus. Focusing on SD clusters implicated in the aetiology of the Williams-Beuren syndrome locus we demonstrate by cross-species comparison that these SDs have inserted at the borders of a topological domain and that they flank regions with distinct DNA conformation. Conclusions: Our study suggests a link of nuclear architecture and the propagation of SDs across chromosome 7, either by promoting regional SD insertion or by contributing to the establishment of higher order chromatin organisation themselves. The latter could compensate for the high risk of structural rearrangements and thus may have contributed to their evolutionary fixation in the human genome