The novel Rho-GTPase activating gene MEGAP͞ srGAP3 has a putative role in severe mental retardation Volker Endris*, Birgit Wogatzky*, Uwe Leimer†, Dusan Bartsch†, Malgorzata Zatyka‡, Farida Latif‡, Eamonn R. Maher‡, Gholamali Tariverdian*, Stefan Kirsch*, Dieter Karch§, and Gudrun A. Rappold*¶ *Institut fu¨r Humangenetik, Universita¨t Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany; †Zentralinstitut fu¨r Seelische Gesundheit, J5, 68159 Mannheim, Germany; ‡Section of Medical and Molecular Genetics, Department of Paediatrics and Child Health, University of Birmingham Medical School, Edgbaston, Birmingham B15 2TT, United Kingdom; and §Klinik fu¨r Kinderneurologie und Sozialpaediatrie, Kinderzentrum Maulbronn, 75433 Maulbronn, Germany Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved June 10, 2002 (received for review April 23, 2002) In the last few years, several genes involved in X-specific mental In the present study, we analyzed a patient with a balanced de retardation (MR) have been identified by using genetic analysis. novo translocation t(X;3)(p11.2;p25) with one of its breakpoints Although it is likely that additional genes responsible for idiopathic mapping within the 3pϪ syndrome deleted region (9–12). This MR are also localized on the autosomes, cloning and characteriza- young woman shows hypotonia and severe MR, features char- Ϫ tion of such genes have been elusive so far. Here, we report the acteristic for 3p patients, but not microcephaly, growth failure, Ϫ isolation of a previously uncharacterized gene, MEGAP, which is heart and renal defects, and the 3p typical facial abnormalities disrupted and functionally inactivated by a translocation break- (13, 14). The translocation breakpoint on chromosome X is point in a patient who shares some characteristic clinical features, located outside of any coding region. However, the breakpoint such as hypotonia and severe MR, with the 3p؊ syndrome. By on chromosome 3 interrupts a previously unknown gene, which fluorescence in situ hybridization and loss of heterozygosity anal- we initially termed MEGAP (MEntal disorder-associated GAP ysis, we demonstrated that this gene resides on chromosome 3p25 protein). MEGAP shares structural and functional similarities and is deleted in 3p؊ patients that present MR. MEGAP͞srGAP3 with other members of the RhoGAP protein family and is highly mRNA is predominantly and highly expressed in fetal and adult expressed in fetal and adult brain tissue. The C-terminal part of brain, specifically in the neurons of the hippocampus and cortex, MEGAP corresponds to srGAP3, which recently has been shown structures known to play a pivotal role in higher cognitive function, to play a critical role in the Slit-Robo signal-transduction path- learning, and memory. We describe several MEGAP͞srGAP3 tran- way (15). We suggest that the phenotype observed in our patient script isoforms and show that MEGAP͞srGAP3a and -b represent is caused by a misregulation of a neuronal signal-transduction functional GTPase-activating proteins (GAP) by an in vitro GAP machinery controlling the correct migration of neurons and their axonal connectivity. assay. MEGAP͞srGAP3 has recently been shown to be part of the Slit-Robo pathway regulating neuronal migration and axonal Methods branching, highlighting the important role of MEGAP͞srGAP3 in Case Report. The proband is a 16-year-old female, the second mental development. We propose that haploinsufficiency of ͞ child of healthy nonconsanguineous parents (father, 47 years; MEGAP srGAP3 leads to the abnormal development of neuronal mother, 45 years). Her brother of 26 years is not affected, and structures that are important for normal cognitive function. there are no further affected individuals in this family. Pregnancy was reported to be uneventful. Delivery occurred at 40 weeks of emarkable progress in understanding the genetic traits of gestation by breech presentation. Birth weight was 3,720 g, Ridiopathic mental retardation (MR) has occurred in recent length was 55 cm, and head circumference was 35 cm. Her height years. Several genes could be identified on the human X is 163 cm (25.pc) and head circumference is 55 cm (75.pc). chromosome by using positional cloning strategies, and their Mental and motor developmental delay was evident in the early contribution to the mental handicap was revealed by mutation stages of life. She could stand freely at the age of 4 years and walk detection (1, 2). These analyses showed for most of these genes only at the age of 5.5 years. Clinical findings at the age of 16 years an apparent common molecular mechanism in contributing to included an extremely psychomotoric retardation with an atactic the same intracellular pathways involving the Rho-class of small gait, jerky arm movements, very low mental performance, GTP-coupled proteins. This family of ras-homologous proteins periodical autoaggressive behavior, and absence of speech. She is composed of several members, with RhoA, Rac1, and Cdc42 shows a triangular face with a high nasal bridge, micrognathia, the best studied examples. Their functions are diverse but have short philtrum, high and narrow palate, slender hands, muscle been ascribed roles in outgrowth of axons and dendrites in vivo hypotonia, and scoliosis. In the last 3 years, three epileptic and the activation of the MAP kinase cascades and cytoskeleton seizures occurred. The patient presents severe attention deficits; organization (3–5). The correct temporal and spatial activity is therefore, intelligence quotient (IQ) testing was not possible. accomplished by the tight regulation of the active GTP-bound and the inactive GDP state. This situation is achieved by the This paper was submitted directly (Track II) to the PNAS office. mutual function of GEF (guanine exchange factors) and GAP Abbreviations: MR, mental retardation; GAP, GTPase-activating protein; MEGAP, mental (GTPase-activating proteins) proteins. It has become evident disorder-associated GAP protein; FISH, fluorescence in situ hybridization; RT-PCR, reverse that MR seems to be a consequence of the improper regulation transcription–PCR; UTR, untranslated region. of the small GTPases or their downstream targets. For example, Data deposition: The sequences reported in this paper have been deposited in the GenBank ARHGEF6 represents a GEF for Rac1 and Cdc42 and was database (accession nos. for: MEGAPa, AF427144; KIAA0411, AB007871; KIAA1156, AL032982; RP3–339A18, Z97054; RP5–1036D20, AL109851; RP11–203C04, AC066583; RP11– identified by the molecular analysis of an X;21 reciprocal trans- 334L22, AC037193; RP11–19E08, AC034186; RP11–380O24, AC026191; CTC-334F04, location (6). In a similar way, oligophrenin1, a GAP for RhoA, AC055745; srGAP1, XM࿝051143; and srGAP2, XM࿝051949). was found to be interrupted in a female patient carrying a ¶To whom reprint requests should be addressed. E-mail: gudrun࿝[email protected] balanced X;12 translocation associated with MR (7, 8). heidelberg.de. 11754–11759 ͉ PNAS ͉ September 3, 2002 ͉ vol. 99 ͉ no. 18 www.pnas.org͞cgi͞doi͞10.1073͞pnas.162241099 Downloaded by guest on October 1, 2021 Magnetic resonance imagery scan or further imaging diagnostics tial denaturation step of 94°C for 2 min, 33 cycles of 94°C for 30 s, have been rejected by the parents. 60°C for 30 s, 72°C for 30 s, and a final 3-min extension step at Case 150500 is a 3-year-old male diagnosed with psychomotor 72°C. PCR products were analyzed on an agarose gel, re-eluted retardation and epicanthus. Cytogenetically, a terminal deletion from the gel, and cloned into pCR2.1 vector by using the of chromosome 3p was diagnosed, with the breakpoint residing TOPO-TA cloning kit (Invitrogen). Clones were sequenced and in band 3p25. Cases P1–P10 have been described (10, 12). Case compared with the published genomic sequence. P11 is an adult without intellectual impairment, who has a cytogenetically visible deletion of chromosome 3p and a normal GAP Assay. MEGAP͞srGAP3b-GST fusion protein was gener- phenotype. ated by subcloning a SalI fragment of KIAA0411 (residues 1–1468, corresponding to bp 1,377–2,849 of MEGAP͞srGAP3b) Genomic Clones and Deletion Mapping. YAC clones from Xp11.2 containing the GAP and SH3 domain into the pGEX4T3 vector and 3p25 were chosen from the Whitehead radiation hybrid (Amersham Pharmacia). Isoform a was generated by replace- maps WCX.11 and WC3.1 and purchased from the German ment of the PsyI͞Eco81I fragment (bases 1323–1813). After Resource Center (RZPD). PAC and BAC clones were obtained transfection into Escherichia coli BL21, protein expression was from the Resource Center, Oakland, (RPCI 1, 3, 11) or from induced with 1 mM isopropyl ␤-D-thiogalactoside. Recombinant Research Genetics, Huntsville (CIT-B, -C). Cosmids were pur- proteins were purified by using glutathione beads in NETN- chased from the RZPD after screening the LL03NC01 ‘‘AC’’ buffer (20 mM Tris⅐HCl, pH 8.0͞100 mM NaCl͞1 mM EDTA͞ chromosome 3 and LL0XNC01 ‘‘U’’ chromosome X-specific 0.5% Nonidet P-40͞1 mM DTT) and washed with 20 mM human cosmid libraries (Lawrence Livermore National Labo- Tris⅐HCl͞0.1 mM DTT. Vectors containing the full-length cod- ratory, Livermore, CA). Overlapping clones were confirmed by PCR and hybridization analysis. Fluorescence in situ hybridiza- ing sequence for the three small GTPases RhoA, Rac1, and tion (FISH) was performed as described. Loss of heterozygosity Cdc42Hs were obtained from the Guthrie cDNA Resource (LOH) analysis was performed as described by Green et al. (12). Center (Sayre, PA) and subcloned into pQE30 vectors (Qiagen). Proteins were prepared under native conditions as recom- Rapid Amplification of cDNA Ends (RACE) Analysis. Human total mended, but included 5 mM MgCl2 in each of the buffers (16). RNA of adult brain (Invitrogen) was reverse transcribed by using Purified proteins were dialyzed overnight against 10 mM ⅐ ͞ ͞ the gene-specific primer Ex5.1 (5Ј-GAGCAGGTTCATGCT- Tris HCl, pH 7.5 2 mM MgCl2 0.1 mM DTT. Protein concen- ͞ GAGGT-3Ј) and Superscript II reverse transcriptase (GIBCO͞ trations were determined by SDS PAGE and bicinchoninic acid BRL).