PMC3621145_01
Mapping a balanced chromosomal translocation in ARHGEF9.
A: Patient photographs showing dysmorphic features (long and narrow face, irregular teeth, and micrognathia). B: Ideograms depicting the translocated chromosomes and their normal homologs. C: FISH analysis with chromosome 18 BAC clones with signals proximal and distal to the breakpoint and chromosome X BAC clone RP11-943J20, which spans the X breakpoint. D: Southern blot analysis of the breakpoint within ARHGEF9 intron 6. SacI, XbaI, and RsaI digests revealed patient-specific bands of approximately 6, 11, 3, and 1.3 kb, which are absent in the control DNA. E: Sequence of the junction fragments and the respective genomic regions of the normal homologs. The four nucleotides identical in the breakpoint region and lost during rearrangement from one of the derivative chromosomes are boxed. F: Schematic diagram depicting ARHGEF9 with the location of the chromosome breakpoint (arrow) as determined by FISH, breakpoint cloning, and RT-PCR experiments. The breakpoint-spanning BAC RP11-943J20 is shown below the gene. G:
ARHGEF9 transcript analysis in patient and control cell line RNAs. For the patient cell line, transcripts could be detected from derivative chromosome 18 (e.g., exons 4-6 and 5-6). However, no products could be obtained using primers spanning the breakpoint (exons 5-7) and within exons located proximal to the breakpoint (exons 8-10) on the derivative X chromosome. Specific bands of the expected size are present in all RT-PCRs from the control.
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Dysmorphic features included a long and narrow face, high narrow palate, hypertrophy of the alveolar ridge, irregular teeth, and micrognathia (Fig. 1A). Karyotyping in this individual revealed a de novo balanced chromosomal translocation 46,X,t(X;18) (q11.1;q11.21), depicted in Figure 1B. On chromosome 18, the breakpoint lies within the genomic region of clones RP11-29E21 and RP11-885L22 (chr18: 24,344,568-24,564,620) (Fig. 1C, right panel), which does not contain any known or predicted genes. However, on chromosome X, we noted that BAC clone RP11-943J20, containing ARHGEF9, spanned the breakpoint (Fig. 1C, left panel). Southern blot analysis of patient DNA, which revealed aberrant patient-specific DNA fragments in several digests (Fig. 1D). Subsequent cloning and sequence analysis of the junctional fragment revealed that the breakpoint lies between exons 6 and 7 of ARHGEF9 (Fig. 1E and F). We ruled out a deletion at the breakpoint on derivative chromosome 18 by sequencing the PCR-amplified breakpoint region (Fig. 1F), indicating that the translocation is balanced. RNA derived from an immortalized patient leukocyte cell line was tested for ARHGEF9 transcripts. RT-PCR amplifications with primers located in ARHGEF9 exons 5 and 7, which flank the breakpoint and in exons 8 and 10, which are both located proximal to the breakpoint on the derivative X chromosome, indicated that full-length ARHGEF9 transcripts are absent in the patient, whereas products of the expected size were easily amplifiable from control RNA (Fig. 1G).
Rapid amplification of cDNA ends reveals defective ARHGEF9 transcripts resulting in a loss of the collybistin PH domain and C-terminus. A: Analysis of collybistin transcripts using 3' RACE demonstrates the loss of exons 7-10 from collybistin transcripts, which are replaced by cryptic exons from chromosomes X and 18. B: Splicing of these cryptic exons results in the loss of the collybistin PH domain and C-terminus. C: Using immobilized phosphoinositides and purified GST-collybistin we found that the PH domain is functional, binding PI3P/PtdIns-3-P (phosphatidylinositol-3-phosphate) rather than PIP3/PtdIns-3,4,5-P (phosphatidylinositol 3,4,5-trisphosphate) as predicted by the "membrane activation model" of gephyrin clustering. Controls using centaurin alpha1 verified that these overlay assays were capable of detecting binding to PIP2 and PIP3.
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Supplementary Fig. S2). Using immobilized phosphoinositides and purified GST-collybistin in overlay assays, we determined that the PH domain is required for collybistin function (Fig. 2C) since it binds the phosphoinositide PI3P (PtdIns-3-P/phosphatidylinositol-3-phosphate), a phosphoinositide with an emerging role in membrane trafficking and signal transduction. However, controls using centaurin alpha1, a known PIP2 and PIP3 binding protein, verified that our overlay assays were capable of detecting binding to these phosphoinositides (Fig. 2C).
Cellular models of gephyrin clustering demonstrate that hPEM2-18 and hPEM2-X colocalize with gephyrin, but do not direct the formation of submembrane microaggregates. A: Western blots of myc-tagged hPEM2 constructs expressed in HEK293 cells indicate that the aberrant myc-hPEM2-18 and myc-hPEM2-X proteins are stable and expressed at equivalent levels to myc-hPEM2SH3+ and myc-hPEM2SH3- when compared to loading control (alpha-tubulin). B: Schematic diagram showing the domains specified by the different hPEM2 constructs. C-F: Coexpression of myc-tagged hPEM2 proteins with EGFP-gephyrin in HEK293 cells costained with a nuclear marker (ToPro3). As expected, myc-hPEM2SH3+ normally colocalizes with gephyrin in large intracellular aggregates (C), while myc-hPEM2SH3- directs EGFP-gephyrin to submembrane clusters (D). Although they lack the regulatory SH3 domain, myc-hPEM2-18 (E) and myc-hPEM2-X (F) proteins colocalize with gephyrin, but do not direct the formation of submembrane microaggregates.
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Western blot analysis of myc-tagged hPEM constructs expressed in HEK293 cells indicates that the aberrant hPEM2-18 and hPEM2-X proteins are stable and expressed at levels equivalent to hPEM2SH3+ and hPEM2SH3- when normalized to endogenous alpha-tubulin (Fig. 3A and B). HEK293 cells revealed that, as expected, variants containing the regulatory SH3 domain (hPEM2SH3+) colocalize with EGFP-gephyrin in large intracellular aggregates (Fig. 3C), while hPEM2SH3-, lacking the SH3 domain, directs EGFP-gephyrin to submembrane clusters (Fig. 3D). However, although hPEM2-18 (Fig. 3E) and hPEM2-X (Fig. 3F) lack the SH3 domain, these proteins colocalize with EGFP-gephyrin and do not direct the formation of submembrane microaggregates.
Overexpression of truncated collybistin mutants in neurons results in loss of gephyrin and GABAAR clusters. Triple staining of neurons transfected with either myc-hPEM2SH3+ (A,B), myc-hPEM2SH3- (C,D) or mutant constructs myc-hPEM2-X (E,F) and myc-hPEM2-18 (G,H) with antibodies against the GABAA receptor gamma2 subunit (red), gephyrin (green), and myc-tag (blue). Note that myc-hPEM2SH3+ or myc-hPEM2SH3- do not interfere with the colocalization of gephyrin and GABAA receptors in synaptic clusters (A-D). Quantitative analysis shows that hPEM2SH3+ or myc-hPEM2SH3- do not influence gephyrin puncta number (I) although deletion of the regulatory SH3 domain influences puncta size (J). In neurons expressing myc-hPEM2-X (E,F) and myc-hPEM2-18 (G,H), gephyrin and GABAA receptor immunoreactivity is significantly reduced. Note that gephyrin and GABAA receptor clustering in neighboring neurons that do not express the myc-hPEM mutants is unaffected (E-H). Quantitative analysis of gephyrin immunofluorescence (I,J) indicates a statistically-significant loss of gephyrin clusters and a reduction in size of remaining clusters. ***P<0.001, Student's t-test, n = 17-28 cells per construct.
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Overexpression of myc-hPEM2SH3+ or myc-hPEM2SH3- did not interfere with the colocalization of gephyrin or GABAARs in synaptic clusters (Fig. 4A-D). Quantitative analysis revealed that neither hPEM2SH3+ nor myc-hPEM2SH3- influence gephyrin puncta number (Fig. 4I) although deletion of the regulatory SH3 domain caused a modest increase in puncta size (Fig. 4J). By contrast, in neurons expressing myc-hPEM2-X (Fig. 4E, F) and myc-hPEM2-18 (Fig. 4G and H), punctuate gephyrin and GABAAR immunoreactivity was significantly reduced, suggesting that these truncated proteins can act in a dominant-negative manner and interfere with the accumulation of endogenous gephyrin and GABAARs at synaptic sites. Quantitative analyses of the punctuate gephyrin immunofluorescence (Fig. 4I and J) confirmed that the number of gephyrin clusters was dramatically reduced, along with a significant reduction in size of the few remaining clusters.