Breakpoint mapping and sequencing results for each apparently balanced translocation case included in this study.

<p>Breakpoint mapping and sequencing results for each apparently balanced translocation case included in this study.</p

Translocation junction sequences identified in each family.

<p>Translocation junction sequences to the base-pair level as identified by mate-pair sequencing and verified by Sanger sequencing in A) family 1 with t(1;7)(p36.1;q22), B) family 2 with t(7;8)(q32;q24.13), C) family 3 with t(4;10)(q35;q11.2), and D) family 4 with t(1;20)(p35.3;q13.3). Translocation junction sequences (middle line) and matching reference sequences (top and bottom lines) are shown with a different colour depending on the chromosome involved (chr1-orange; chr4-purple; chr7-blue; chr8-red; chr10-yellow; chr20-green). Microhomology observed at the translocation breakpoint sites is highlighted in yellow, deleted sequences around the breakpoints are underlined, duplicated sequences are in bold, capital letters, and inserted sequences not aligning to either chromosome are in bold, lower-case letters.</p

The <i>ZNF423</i> gene and quantitative Real-Time PCR (qRT-PCR) results.

<p>A) Schematic illustration of the <i>ZNF423</i> gene (minus strand). The protein-coding exons and introns of the gene are represented with solid, vertical lines and dotted, horizontal lines, respectively (not to scale). Intron 3 is enlarged to demonstrate the approximate <i>ZNF423</i> deletion position (blue line) as predicted by MPS in the affected sibling in family 2, as well as the amplicon (Z-RT1, Z-RT2, and Z-RT3) positions (red lines) from the qRT-PCR analysis. The actual coordinates are given on the left (GRCh37/hg19). B) qRT-PCR results demonstrating a reduced relative <i>ZNF423</i> copy number in the proband and normal results in the non-affected sibling as compared with a control. Parental genomic material was unavailable and thus not included in the analysis.</p

Accurate Breakpoint Mapping in Apparently Balanced Translocation Families with Discordant Phenotypes Using Whole Genome Mate-Pair Sequencing

<div><p>Familial apparently balanced translocations (ABTs) segregating with discordant phenotypes are extremely challenging for interpretation and counseling due to the scarcity of publications and lack of routine techniques for quick investigation. Recently, next generation sequencing has emerged as an efficacious methodology for precise detection of translocation breakpoints. However, studies so far have mainly focused on <i>de novo</i> translocations. The present study focuses specifically on familial cases in order to shed some light to this diagnostic dilemma. Whole-genome mate-pair sequencing (WG-MPS) was applied to map the breakpoints in nine two-way ABT carriers from four families. Translocation breakpoints and patient-specific structural variants were validated by Sanger sequencing and quantitative Real Time PCR, respectively. Identical sequencing patterns and breakpoints were identified in affected and non-affected members carrying the same translocations. <i>PTCD1</i>, <i>ATP5J2-PTCD1</i>, <i>CADPS2</i>, and <i>STPG1</i> were disrupted by the translocations in three families, rendering them initially as possible disease candidate genes. However, subsequent mutation screening and structural variant analysis did not reveal any pathogenic mutations or unique variants in the affected individuals that could explain the phenotypic differences between carriers of the same translocations. In conclusion, we suggest that NGS-based methods, such as WG-MPS, can be successfully used for detailed mapping of translocation breakpoints, which can also be used in routine clinical investigation of ABT cases. Unlike <i>de novo</i> translocations, no associations were determined here between familial two-way ABTs and the phenotype of the affected members, in which the presence of cryptic imbalances and complex chromosomal rearrangements has been excluded. Future whole-exome or whole-genome sequencing will potentially reveal unidentified mutations in the patients underlying the discordant phenotypes within each family. In addition, larger studies are needed to determine the exact percentage for phenotypic risk in families with ABTs.</p></div

Family 1 results with t(1;7) translocation.

<p>A) Family 1 pedigree showing the proband with intellectual disability, psychomotor delay and epilepsy, as well as the non-affected mother and father. The t(1;7)(p36.1;q22) translocation is maternally inherited. B) Ideograms showing the normal and derivative (der) chromosomes (chr) 1 and 7 (not to scale). Genetic material from chr1 and chr7 is shown with a solid, orange and dotted, blue frame line, respectively. The approximate breakpoint positions on 1p36.1 and 7q22 are indicated by arrows. C) UPD7 analysis results from one of the informative microsatellite markers (D71824) in the affected proband and non-affected parents; by comparing the peak sizes between all family members, normal biparental inheritance was concluded. D) Quantitative Real-Time PCR results demonstrating the paternal inheritance of the chr3 duplication predicted from the structural variant analysis in the affected proband.</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

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

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

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

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