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Case Reports
. 2002 Sep 3;99(18):11754-9.
doi: 10.1073/pnas.162241099. Epub 2002 Aug 23.

The novel Rho-GTPase activating gene MEGAP/ srGAP3 has a putative role in severe mental retardation

Volker Endris  1 , Birgit Wogatzky, Uwe Leimer, Dusan Bartsch, Malgorzata Zatyka, Farida Latif, Eamonn R Maher, Gholamali Tariverdian, Stefan Kirsch, Dieter Karch, Gudrun A Rappold
Affiliations
Case Reports

The novel Rho-GTPase activating gene MEGAP/ srGAP3 has a putative role in severe mental retardation

Volker Endris et al. Proc Natl Acad Sci U S A. .

Abstract

In the last few years, several genes involved in X-specific mental retardation (MR) have been identified by using genetic analysis. Although it is likely that additional genes responsible for idiopathic MR are also localized on the autosomes, cloning and characterization of such genes have been elusive so far. Here, we report the isolation of a previously uncharacterized gene, MEGAP, which is disrupted and functionally inactivated by a translocation breakpoint in a patient who shares some characteristic clinical features, such as hypotonia and severe MR, with the 3p(-) syndrome. By fluorescence in situ hybridization and loss of heterozygosity analysis, we demonstrated that this gene resides on chromosome 3p25 and is deleted in 3p(-) patients that present MR. MEGAP/srGAP3 mRNA is predominantly and highly expressed in fetal and adult brain, specifically in the neurons of the hippocampus and cortex, structures known to play a pivotal role in higher cognitive function, learning, and memory. We describe several MEGAP/srGAP3 transcript isoforms and show that MEGAP/srGAP3a and -b represent functional GTPase-activating proteins (GAP) by an in vitro GAP assay. MEGAP/srGAP3 has recently been shown to be part of the Slit-Robo pathway regulating neuronal migration and axonal branching, highlighting the important role of MEGAP/srGAP3 in mental development. We propose that haploinsufficiency of MEGAP/srGAP3 leads to the abnormal development of neuronal structures that are important for normal cognitive function.

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Figures

Figure 1
Figure 1
Genomic organization of the MEGAP/srGAP3 gene on chromosome 3p25 and localization of the translocation breakpoints in the patient. (a) Physical mapping of MEGAP/srGAP3. The precise order of markers for the chromosomal regions 3p25 and Xp11.2 is shown at Top and Bottom, as well as the orientation of telomere and centromere. BAC, PAC, and cosmid clones used for FISH mapping of the respective breakpoints are shown. Both BAC RP11–203C04 and RP5–1036D20 showed signals on the normal and the derivative chromosome, thus containing the translocation breakpoint. The localization of the 22 exons of MEGAP/srGAP3 is indicated as vertical red lines. The entire gene encompasses approximately 250 kb of genomic sequence, with exon 1 separated from exon 2 by about 100 kb. The breakpoint on the derivative chromosome 3 is located between exons 3 and 4 of MEGAP/srGAP3, as shown in the middle part of the scheme. Exons are indicated here as red boxes. Markers D3S2405 and SHGC-57635 (white boxes) are located within intronic sequences of MEGAP/srGAP3. Marker IB772 (white box) resides on the X chromosome. (b) Schematic drawing of the breaking event. The breakpoint on the X chromosome is located in a remnant human endogenous retroviral element (HERV) next to the flanking LTR sequence. The breakpoint region on chromosome 3 shows several CAA trinucleotide repeats on both sides of the translocation. The position of primers used for amplification of the junction fragments are indicated as arrows.
Figure 2
Figure 2
Peptide sequence of MEGAP/srGAP3a. FES/CIP4 (FCH), GAP, and SH3 domains are highlighted; inverted carets indicate exon junctions.
Figure 3
Figure 3
Expression analysis of MEGAP/srGAP3. (a) Multiple tissue Northern blots from adult and fetal tissues (CLONTECH) containing 1–2 μg polyA+ RNA per lane were hybridized with [α-32P]dCTP-labeled probes containing exons 1 to 4 of MEGAP/srGAP3 (bp 27–569). The filters were hybridized overnight at 65°C, stringently washed at 65°C, and exposed to x-ray films for 24 h at −80°C. A control hybridization with β-actin shows signals at 2.0 kb in all lanes, with additional bands at 1.6–1.8 kb in heart and skeletal muscle. (b and c) Three different isoforms of MEGAP/srGAP3 (I, II, and III). (b) Schematic drawing of exons and motifs of MEGAP/srGAP3. The position of the start (ATG) and stop (TGA) codons are indicated. Introns are depicted as black lines and are not drawn to scale. Light gray boxes, 5′ and 3′-UTR; dark gray boxes, FES/CIP4 (FCH), RhoGAP, and SH3 domains; black bars, localization of probes used for hybridization of the Northern blots; AAAAA, localization of putative polyA tails in the 3′-UTR. (c) Expression of MEGAP/srGAP3 in different brain regions. Hybridization of exons 1–4 results in three signals (I, II, III). The longest band (I) corresponds to the predicted 8.3-kb full-length transcript. Signals at 7 kb (II) and 4.5 kb (III) represent transcripts with differing 3′-UTRs.
Figure 4
Figure 4
Distribution of Megap/srGAP3 mRNA in adult mouse brain. In situ hybridization with digoxigenin-labeled Megap/srGAP3 antisense riboprobe on coronary section showing strong expression in the amygdala, piriform cortex, posterior parietal associative area, the dentate gyrus, and particularly strong expression in the hippocampal formation. Hybridization with Megap/srGAP3 sense DIG-RNA gave no signal. A, amygdala; DG, dentate gyrus; Hi, hippocampus; Pir, piriform cortex; PPtA, posterior parietal associative area.
Figure 5
Figure 5
Alternative splicing of exon 12. (a) Alternative splicing forms in brain and kidney of fetal and adult origin. RT-PCR (33 cycles) was performed with oligo-dT-transcribed cDNAs by using primers derived from exons 9 and 14. PCR products at 482, 412, and 383 bp correspond to isoforms a, b, and c, respectively. Isoforms a and b of MEGAP/srGAP3 differ in the use of an alternative exon 12. The presence of exon 12b leads to a transcript 72-bp shorter than isoform MEGAP/srGAP3a, leading to an ORF of 3,227 bp and coding for a predicted protein of 1,075 amino acids. Isoform MEGAP/srGAP3c lacks exon 12, leading to a frame shift and a premature stop as indicated. (b) Predicted peptide sequences of exons 11–13 of isoforms MEGAP/srGAP3a, -b, and -c. *, stop codon due to the frame shift in exon 13.
Figure 6
Figure 6
Filter binding assay showing GAP activity of MEGAP/srGAP3 toward Rac1 and Cdc42Hs. As described, ARHGAP1 (■) shows strong activity toward RhoA, Rac1, and Cdc42Hs. However, MEGAP/srGAP3 (▴) shows almost no activity toward RhoA, a strong activity toward Rac1, and a lower but still significant activity toward Cdc42Hs. [γ-32P]GTP-labeled p21 (0.3 μM) was incubated at 30°C in the absence or presence of equimolar concentrations of the GAP domains of MEGAP/srGAP3a (amino acid residues 426–939) or ARHGAP1 (amino acid residues 198–438). Aliquots of the reaction were taken every 3 min and immediately passed through a 0.45-μm nitrocellulose membrane. The amount of radioactivity bound to the filter was measured in a scintillation counter. Activity at t = 0 min was set to 100%.
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