Homozygous ARHGEF2 mutation causes intellectual disability and midbrain-hindbrain malformation

Mid-hindbrain malformations can occur during embryogenesis through a disturbance of transient and localized gene expression patterns within these distinct brain structures. Rho guanine nucleotide exchange factor (ARHGEF) family members are key for controlling the spatiotemporal activation of Rho GTPase, to modulate cytoskeleton dynamics, cell division, and cell migration. We identified, by means of whole exome sequencing, a homozygous frameshift mutation in the ARHGEF2 as a cause of intellectual disability, a midbrain-hindbrain malformation, and mild microcephaly in a consanguineous pedigree of Kurdish-Turkish descent. We show that loss of ARHGEF2 perturbs progenitor cell differentiation and that this is associated with a shift of mitotic spindle plane orientation, putatively favoring more symmetric divisions. The ARHGEF2 mutation leads to reduction in the activation of the RhoA/ROCK/MLC pathway crucial for cell migration. We demonstrate that the human brain malformation is recapitulated in Arhgef2 mutant mice and identify an aberrant migration of distinct components of the precerebellar system as a pathomechanism underlying the midbrain-hindbrain phenotype. Our results highlight the crucial function of ARHGEF2 in human brain development and identify a mutation in ARHGEF2 as novel cause of a neurodevelopmental disorder.


Introduction
Brain development depends on spatiotemporally controlled gene expression.[1][2][3] Alterations in the expression pattern of such genes can result in neurodevelopmental disorders by impinging on key processes such as neural progenitor specification, cell division, and differentiation and the migration of newly born neurons from their site of origin to their final destination within the brain.[4][5][6] The latter is crucial for the formation of specific brain structures.[7][8][9] Factors that control localized gene function include Rho GTPase regulators.Here, we present evidence that the loss of function of Rho guanine nucleotide exchange factor 2 (ARHGEF2) causes a human neurodevelopmental disorder characterized by intellectual disability, mild microcephaly, and midbrain-hindbrain malformation.
ARHGEF2 (synonym GEF-H1, murine Lfc) catalyzes the replacement of GDP to GTP bound to Rho-related proteins and thereby controls timing and localization of the activation of Rho GTPases such as RhoA.[10][11][12][13][14] ARHGEF2 connects microtubule and actin cytoskeleton dynamics.[14] In this context ARHGEF2 activity is reduced through microtubule binding and further controlled by upstream regulators.[15][16][17][18][19][20][21][22][23][24][25] ARHGEF2 is key for actin and microtubule reorganization and is required for mitotic spindle formation and orientation.[11] Inhibition of ARHGEF2 results in spindle disorientation and dysfunction, mitotic delay, accumulation of prometaphase cells, and further mitotic aberrations.[11,22] In mouse neocortex, Arhgef2 is expressed in neural precursor and immature neurons and regulates neurogenesis from cortical precursor cells.[24] Arhgef2 down-regulation by shRNA keeps radial precursors cycling, potentially by disrupted spindle plane orientation, and thereby inhibits neurogenesis.In contrast, Arhgef2 overexpression causes an increase of neurons in the cortical plate.[24][25][26] Arhgef2 also plays a role in neural tube closure by regulating morphogenetic movements.[27] Furthermore, Arhgef2 participates in the migration of non-neuronal cells and in Wnt-induced planar cell polarity, via the activation of RhoA.[28,29] Although evidence for a central function of Arhgef2 in cytoskeletal dynamics and critical signal transduction pathways exists and other ARHGEF genes have been linked with neurological disease, [30][31][32] little is known about ARHGEF2 function in humans and no disease phenotype associated with this gene has been reported.

Results and discussion
We report that patients with a homozygous mutation in ARHGEF2 develop intellectual disability, mild microcephaly, and midbrain-hindbrain malformations.Two affected children of healthy, consanguineous parents of Kurdish-Turkish descent were born at term without complications after an uneventful pregnancy (II.1;II.2,Fig 1A).At birth, mild congenital microcephaly with occipitofrontal head circumferences (OFC) of -1.95 (II.1) and -2.33 (II.2) SDS (standard deviation score) but normal weight and height were apparent (S1 and S2 Tables).In addition, the two boys variously displayed wide intermamillary distance, broad fingers, low posterior hairline, and facial dysmorphism with long philtrum, thin upper lip, high palate, downslanted palpebral eye fissures, long eyelashes, bilateral ptosis, and horizontal pendular nystagmus (S1 Table ).Ophthalmological examination revealed congenital strabismus, astigmatism, amblyopia (II.2), optic disc pallor (II.2), abnormalities of the retinal pigment epithelium (II.2), and abnormal visual-evoked potentials further underlining optic nerve affection.Motor milestones were not severely delayed despite generalized muscular hypotonia observed in patient II.1 and weak tendon reflexes in both children.Both children stumbled frequently and had a disturbance of fine motor movements (S3 Table ).Moderate intellectual disability (IQ <50) and severe developmental speech delay despite normal hearing capacity were diagnosed in both patients (S1 and S3 Tables).Cranial magnetic resonance imaging (MRI) revealed mild microencephaly, elongated midbrain, hypoplasia of the pons, ventral and dorsal longitudinal clefts (grooves) in pons and medulla, and inferior vermis hypoplasia (Fig 1B).The finding of grooves and cerebellar hypoplasia (in absence of the 'dragonfly sign' with hypotrophy of the cerebellar hemispheres rather than of the vermis) argued against the differential diagnosis of pontocerebellar hypoplasia.Results of routine blood tests, extensive metabolic work-up, chromosome analysis, and cardiac function assessment were normal.
To identify the genetic cause of this disease, we performed whole exome sequencing and bioinformatic analysis followed by Sanger sequencing in both affected individuals.We thereby identified a homozygous deletion of a single guanine (G) at the exon12-intron12 boundary (Fig 2A  To evaluate the biological effects of the identified ARHGEF2 mutation on brain development, we utilized both in vitro and in vivo approaches.We used a well-characterized cell culture model of E13 mouse neocortical cells recapitulating the timing of cortical precursor differentiation observed in vivo.[24,33,34] The large majority of these cells are actively dividing radial glial precursors positive for the neural progenitor marker nestin immediately following plating.These cells then differentiate to generate excitatory neurons (DIV1-3) and subsequently astrocytes and oligodendrocytes (DIV5-7).We demonstrated previously, using  this model, that knockdown of Arhgef2 causes a decrease in the proportion of βIII-tubulin positive neurons and a corresponding increase of cycling cortical precursors.[24] We thus expected the human ARHGEF2 frameshift mutation to have a similar impact on neurogenesis, leading to an increase of cycling (and eventually apoptotic) precursors and a decrease of neurons generated during brain development.In a first series of experiments, we transfected murine cortical radial precursors with an Arhgef2 shRNA plasmid and a nuclear EGFP reporter plasmid; the latter allows the visualization of transfected cells.As expected, Arhgef2 knockdown significantly decreased the proportion of double positive cells for EGFP and early neuron marker βIII-tubulin, whereas it increased the proportion of EGFP and proliferation marker Ki67 double positive precursors (Fig 3A, n = 1378-1293 cells, One-way ANOVA).Next, we co-transfected cortical progenitors with the same combination of plasmids and an additional construct encoding for human wildtype ARHGEF2.In this condition, we observed no significant change in the proportion of EGFP and βIII-tubulin double positive cells or EGFP and Ki67 as compared to untransfected cells, demonstrating that wildtype ARHGEF2 rescues the phenotype produced by shRNA (Fig 3A).Last, we co-transfected cortical progenitors with Arhgef2 shRNA, the EGFP reporter, and a construct encoding mutant ARHGEF2 identified in the index patients.Here, the proportion of EGFP and βIII-tubulin double positive cells was reduced, whereas the number of EGFP and Ki67 remained increased (Fig 3A).We conclude that the phenotype produced by Arhgef2 shRNA was rescued by coexpression of wildtype but not of mutant human ARHGEF2, consistent with the interpretation that the patient mutation is a loss-of-function mutation and that this ARHGEF2 mutation impairs neurogenesis.
To further analyze whether the identified ARHGEF2 loss-of function mutation affects brain development in vivo we in utero electroporated E13.5 mouse cortex with Arhgef2 or Con (control) shRNA constructs along with wildtype or mutant human ARHGEF2 plasmids as well as an EGFP vector.EGFP as a marker for electroporated cells and SatB2 as a marker for newborn neurons in murine cortices were quantified three days after electroporation.We identified a pronounced increase in the proportion of EGFP-positive cells in the ventricular, sub-ventricular, and intermediate zones (VZ/SVZ/IZ) and decreased proportions in the cortical plate (CP) of the Arhgef2 shRNA electroporated mice, as compared to mice that were electroporated with the EGFP reporter only (Fig 3B and 3C).In line with our in vitro data, overexpression of wildtype, but not mutant, ARHGEF2 was able to effectively rescue the cell distribution in the cortex induced by Arhgef2 shRNA.Upon electroporation of Arhgef2 shRNA, we additionally observed a dramatic reduction in the proportion of SatB2/EGFP double positive cells localized to the cortical plate.This is consistent with impaired neurogenesis described above in cultured cortical precursor cells.Overexpression of wildtype ARHGEF2 was able to rescue this impairment of neurogenesis while mutant ARHGEF2 was ineffective (Fig 3B and 3C, n = 3, One-way ANOVA).These data further confirm that the patient mutation in human ARHGEF2 acts as a loss-of-function mutation also in vivo, results in dramatically decreased neurogenesis and is most probably disease-causative (Fig 3D).ARHGEF2 has been associated previously with spindle plane orientation, [11,24] and the latter has been shown to play a crucial role for the fate of neuronal precursors, 'deciding' between self-renewal and differentiation.[35] To evaluate whether human mutant ARHGEF2 alters neurogenesis by impairing mitotic spindle orientation, we performed in utero electroporation with the experimental setup described above and determined spindle plane orientation.We determined the angle between the ventricular surface and a line connecting centrosomes in metaphase/anaphase cells in brain sections.Arhgef2 shRNA led to a significant shift of the spindle orientation towards a more horizontal orientation, characterizing putatively more symmetrically, self-renewing cell divisions.Again, overexpression of human wildtype but not mutant ARHGEF2 was able to rescue significantly this phenotype (Fig 4A, n = 68-70 cells from 3-4 mice, One-way ANOVA).In the developing brain, ARHGEF2 reportedly controls the arrangement for symmetric or asymmetric cell divisions of apical progenitors through plane orientation.[24] Given our results from overexpressing mutant ARHGEF2 in the mouse brain, we concluded that loss of ARHGEF2 activity inhibits neurogenesis by favoring more symmetric divisions of neocortical progenitors (Fig 4B).
To further substantiate the effect of a loss of ARHGEF2 functions in human tissues and other cell types, we analyzed cell cycle apparatus in LCLs from our index patients and healthy controls.We detected an abnormal morphology of the mitotic spindle apparatus, decreased spindle pole distance, and decreased cell size in LCLs derived from the patients compared to controls, significantly (Fig 5A and 5B, n = 100-200 cells, One-way ANOVA).There was, however, no cell cycle defect, increased radiosensitivity, nor abnormal centrosome 'morphology' in the patient LCLs (S2 and S3 Figs).We concluded that regulation of the mitotic spindle apparatus is a primary function of ARHGEF2 in the cell cycle also in human tissues and other cell types.
ARHGEF2 regulates various cellular processes through activation of Rho family GTPases, specifically RhoA and its downstream effectors, such as RhoA/ROCK/MLC pathway members.[12,22,28,36,37] Specifically, RhoA has been implicated in the regulation of neurogenesis and planar cell divisions.[38,39] We thus analyzed the activity of RhoA and its immediate downstream effectors in patient and control LCLs.We detected a significant reduction of active RhoA and, consistently, reduced levels of active phospho-myosin light chain (MLC) in the LCLs of affected individuals with an ARHGEF2 mutation compared to healthy individuals (Fig 5C -5E, n = 3, One-way ANOVA).This indicates that the RhoA/ROCK/MLC pathway is most likely impaired in humans with the ARHGEF2 mutation.
Previous studies showed that Arhgef2 is expressed in the neural tube of mice from embryonic day 11 on and maintained at high levels during brain development.[24] Our in situ hybridization studies on embryonic mice showed strong expression of Arhgef2 transcripts in the neuroepithelium of telencephalon, diencephalon, and rhombencephalon of E11 mice (S5 Fig) .At the time of birth, Arhgef2 is maintained in the germinal zones of both the neocortex and the cerebellum, as well as in the pontine gray nuclei (PGN) (S5 Fig) .To further corroborate the pathogenicity of the identified ARHGEF2 mutation in brain development, we analyzed the phenotype of Arhgef2 deficient mice.[40] In adult Arhgef2 mutant mice, we observed a significant reduction in volume of the total brain size (referred to as microencephaly), the   To address the cerebellar phenotype observed in Arhgef2 mutant mice and patients, we measured the size of Purkinje cells and the thickness of the cerebellar molecular layer in Arhgef2 mutant and control mice.Our analysis revealed no significant difference in these parameters between wildtype and mutants (S8 Fig) .This led us to hypothesize that the input to the cerebellum, from precerebellar nuclei, could be affected, and thus contributing to the observed reduction of the cerebellar size.During hindbrain development, precerebellar neurons emerge from the proliferative neuroepithelium of the rhombic lip and migrate tangentially to reach their final destinations in the pons and medulla.These neurons form five distinct nuclei that are located at different positions within the hindbrain: pontine gray nuclei (PGN), reticulotegmental nuclei (RTN), external cuneate nuclei (ECN), lateral reticular nuclei (LRN), and inferior olivary nuclei (IO) [41][42][43][44].The PGN, RTN, ECN, LRN projects mossy fibers to the cerebellum, whereas IO projects climbing fibers, and thereby provide input to the cerebellum.[41][42][43][44] To tackle the hypothesis that deficits in development of precerebellar nuclei result in abnormalities in cerebellar size of Arhgef2 mutant mice, we immunostained sagittal and coronal sections taken from Arhgef2 homozygous and Arhgef2 heterozygous (used as control) mice with antibodies against the transcription factor Mbh2 (which labels PGN, RTN, ECN, LRN) and FoxP2 (that marks IO).This analysis revealed that Arhgef2 mutant mice completely lack, in addition to PGN, the RTN and have a severe reduction in LRN neurons (Figs 7A, 7B, 7D, 8A, 8B and 8D).Interestingly, the rostral part of the ECN was abnormally enlarged in Arhgef2 mutant mice when compared to controls, suggesting that some PGN, RTN and LRN might fail to migrate to their ventral positions and aberrantly locate in the ECN (Figs 7C, 8C and 8D).The formation of the IO was not obviously disturbed in Arhgef2 mutant mice (Fig 7D ).Taken together, our anatomical analysis revealed that specific precerebellar nuclei are either absent or severely reduced in Arhgef2 mutant mice.
These findings draw our attention to two possible interpretations on the role of Arhgef2 in development of precerebellar nuclei: (1) Arhgef2 modulates the generation of precerebellar neurons and/or (2) Arhgef2 regulates the migration of these cells from their progenitor niche towards their final localization.Precerebellar neurons arise from discrete progenitor domains located in the developing dorsal hindbrain, which are defined by the combinatorial expression of the transcription factor Olig3 with other bHLH (basic Helix-Loop-Helix) transcription factors, such as Atoh1 (Protein atonal homolog 1) or Ptf1a (Pancreas transcription factor 1 alpha).[45] Precerebellar neurons that form the PGN, RTN, LRN and ECN are generated from Olig3/Atoh1 positive (known as dA1) progenitors, whereas those that form the IO emerge from Olig3/Ptf1a positive (known as dA4) progenitors, [46,47] reviewed in [45].Arhgef2 is unlikely to modulate generation of precerebellar neurons as its transcript becomes expressed only by E11 in mice, a time point when these cells have already been specified and have started their migration.We thus speculated that Arhgef2 rather modulates precerebellar neuron migration.To test this possibility, we co-stained transverse hindbrain sections, taken from wildtype mice at E11, with an Arhgef2 in situ hybridization probe and antibodies against Olig3 and Mbh2 (BarH-like homeobox 1).This revealed that Arhgef2 is co-expressed largely with Mbh2-positive cells emanating from dA1 progenitors and throughout the anterior and posterior extramural streams precerebellar neurons in the mantle zone of hindbrain (S9 Fig) .Arhgef2 does not follow the expression pattern of Olig3 in dorsal hindbrain (S9 Fig) .It is interesting to note that mutation of Arhgef2 appears to selectively affect the migration of dA1-derived neurons, as other hindbrain centers such as the nucleus of the solitary tract (a Olig3 +/Ascl1+ dA3 derivative) or the IO (dA4 derivative) were not affected (Figs 7E and 8E).
This report demonstrates that intellectual disability, mild microcephaly, and a midbrainhindbrain defect can be caused by a homozygous mutation of the ARHGEF2 in humans.We highlight the importance of ARHGEF2 during brain development and, in particular, in neuronal progenitor cell division and differentiation, as well as in neuronal migration.We show that in both human and murine cells ARHGEF2 deficiency interferes with the normal orientation of mitotic spindles and cell fate choices.As expected, the frameshift mutation of ARHGEF2 deregulates the activity of its downstream effectors, i.e. the RhoA/ROCK/MLC pathway.ARHGEF2 thereby joins other members of the same protein family that have already been associated with neurologic disease such as non-syndromic intellectual disability (ARHGEF6), [30] epileptic encephalopathy (ARHGEF9), [31] and peripheral demyelinating neuropathy (ARHGEF10).[32] We show that Arhgef2 deficiency in mice severely impairs the migration of dA1 progenitors, which results in the incorrect location of precerebellar neurons in the  hindbrain of Arhgef2 mutant mice.In keeping with our data, previous studies have shown that deficits in the RhoA/ROCK pathway affect migration of precerebellar neurons.[48] Thus, increasing evidence support the notion that the ARHGEF2-RhoA/ROCK pathway is essential for the migration and specification of precerebellar neurons.ARHGEF2 has been shown to be involved in Wnt-mediated planar cell polarity pathway through its interaction with the Daam and Dishevelled proteins, which are known to control migratory events.Wnt1 mutant mice have been reported to have a midbrain-hindbrain malformation, [49] and humans with biallelic WNT1 mutations displayed cerebellum and brainstem phenotype.[50] It is interesting to note that the major effects in the hindbrain of Arhgef2 mutant mice occur in the migration of dA1-derived neurons, but not other neuronal cell types that emerge from neighboring progenitor domains.Our data demonstrate thus a marked specificity in the molecular control of neuronal migration in the hindbrain.The combined midbrain-hindbrain malformation phenotype observed in humans was not fully recapitulated in Arhgef2 deficient mice, which lacked an obvious midbrain phenotype.This suggests that during evolutionary divergence of these species, additional molecular mechanisms coordinate the function of ARHGEF2.Identifying additional families with ARHGEF2 mutations will help to consolidate the disease-causative role of ARHGEF2 in humans.

Ethics statement
The human study was approved by the local ethics committees of the Charite ´(approval no.EA1/212/08), and the written consent form was received from the investigated individuals.All animal experiments were carried out in accordance to the guidelines of national ethic principles, and approved by Charite ´(registration no.T0344/12).

Genetic analyses
DNA samples of the two affected individuals were subjected to whole exome sequencing.Five μg genomic DNA were enriched with the Agilent Human All Exon V3 kit (Agilent, Santa Clara, CA, USA) following the manufacturer's protocol.The libraries were sequenced using Illumina HiSeq 2000 sequencer for single-end 101 bp.Coverage of coding regions was >92.6-93.4% with a minimal depth of 20-fold; the raw data were processed as described previously.[51] In brief, the raw sequences were aligned to human reference genome (hg19), and the alignment results were used to call variants of SNVs, indels, and CNVs.We defined from the exome data the regions of homozygosity by at least four high-quality (genotype quality !20, allele counts ! 10, and allele percentage !0.9) consecutive homozygous SNVs uninterrupted by heterozygous SNVs.The overlapping regions of homozygosity (>1 Mb) between the two patients were listed in S4 Table.Subsequently, the variants were screened by known causal mutation databases (OMIM and HGMD), and polymorphism databases (1000Genome, ESP6500, dbSNP138, and an in-house exome database with 721 individuals of whom >90% are of the Middle East origin) with matching variants not exceeding the prevalence cutoff of 0.5%, and the pathogenicity of variants was further evaluated (analysis pipeline: https:// sourceforge.net/projects/merap/).Sanger sequencing of the ARHGEF2 (NM_004723.3) in our cases was performed to confirm the mutation in the patients, establish the genotype in the other family members, and evaluate the presence of potential mutations in additional six consanguineous pedigrees of Sri Lankan, Italian and Turkish descent with developmental delay and a similar radiological phenotype of pontine and cerebellar hypoplasia.Some of these patients also had facial dysmorphism, ataxia, autism, epilepsy, and further MRI findings including a migration or myelination defect.The healthy status of heterozygous parents argues against a haploinsufficiency mechanism underlying this disease phenotype.Two patients with heterozygous deletions encompassing the ARHGEF2 are listed in the Decipher database: (i) patient 276515 (https://decipher.sanger.ac.uk/patient/276515; 2.76 Mb deletion covering 59 genes; arr 1q22;q23(155,296,964-158,056,876)) with abnormal facial features, intellectual disability, and speech delay and (ii) patient 255240 (https://decipher.sanger.ac.uk/patient/255240; 0.92 Mb deletion spanning 24 genes; arr 1q22(155,192,986-156,108,069)) with facial dysmorphism, brachydactyly, intellectual disability, severe speech delay, muscular hypotonia, but normal cMRI findings.
Apart from the ARHGEF2 candidate variant, there were three further homozygous variants shared by the two patients that we ranked as unlikely disease-causing: (i) EXTL1 (NM_004455, exostosin-like glycosyltransferase 1), g.1:26356156G>A, c.939G>A, p.W313X.However, there were 16 heterozygotes in the ESP6500 database and one healthy individual in our in-house database with a homozygous variant.(ii) HMCN1 (NM_031935, hemicentin 1), g.1:186010193G>A, c.6229G>A, p.D2077N.However, this gene has been reported to be associated with macular degeneration, a phenotype that does not fit the clinical features of our index patients.(iii) IGFN1 (NM_001164586, immunoglobulin-like and fibronectin type III domain containing 1), g.1:201193885G>A, c.10369G>A, p.G3457S.However, there were 7 heterozygotes in the ESP6500 database, and this variant has a prediction score from SIFT (tolerated) and PolyPhen2 (possibly damaging) that makes it unlikely as well as a GERP conservation score of merely 1.101.
Because both patients were male, we checked on the X chromosome for hemizygous variant, but detected no rare deleterious variant shared by the two individuals.

Epstein-Barr virus-transformed LCLs
LCLs were established and cultured according to the protocol published by Neitzel et al. 1986.[52] Quantitative real-time PCR (qPCR) RNA extraction and cDNA synthesis were performed with established methods reported previously.[53] To specifically amplify and detect ARHGEF2 and RPII (RNA polymerase II, reference gene) cDNA, we designed sets of primers using the Primer3 online software (www.primer3.ut.ee).qPCR experiments were run in triplicate using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific, Braunschweig, Germany) according to the manufacturer's protocol with primers specified in S5 Table .Quantification was performed as described previously, [53] and statistical calculations were performed on GraphPad Prism 5 Software (GraphPad Software Inc., La Jolla, CA, USA).

Western blot
Protein extraction and Western blots (run in triplicates) were performed with established methods reported previously; [54] antibodies are listed in S6 Table.

Cell cycle analysis
Peripheral blood lymphocytes (PBL) were isolated from heparinized blood samples by Ficoll density gradient centrifugation.DNA damage was introduced by exposure of PBLs to 6 MV X-ray photons.Matched cultures were set up from untreated and irradiated cells in RPMI 1640 medium, supplemented with 15% fetal bovine serum (FBS, PAN Biotech, Aidenbach, Germany).Lymphocyte growth activation was achieved by phytohemagglutinin (PHA HA16, Thermo Scientific, Dartford, UK).The cell cycle assay was performed using 5-bromo-2 0 -deoxyuridine (BrdU)-Hoechst 33258 flow cytometry.Cells with replication fork-stalling types of DNA damage become delayed in the G2 phase of the cell cycle.

Phospho-histone H3 analysis
LCLs were washed in PBS, fixed in 2% paraformaldehyde (PFA), and permeabilized with 90% methanol for 30 min on ice.Phospho-histone H3-Ser10 (pH3) was detected with anti-pH3 antibody and corresponding secondary antibody (S6 Table ).DAPI at 2 μg/ml final concentration was used as a counterstain for DNA content and cell cycle distribution.Fluorescence was recorded using the same LSRII flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) as for cell cycle studies.Data analysis was done with WinMDI 2.9 software (MicroSoft, Redmond, WA, USA).

In utero electroporation
In utero electroporation was performed as described previously [55] using a CUY21 EDIT electroporator (TR Tech, Japan) to deliver five 50 ms pulses of 40-50 V with 950 ms intervals per embryo.Per embryonic brain, 4 μg total of plasmid DNA was suspended in the tracer 0.5% trypan blue in a ratio of 37.5% shArhgef2 (Lfc) (or shCon), 37.5% human ARHGEF2 (wt)/human ARHGEF2 (mut)/empty vector, and 25% pEF-EGFP plasmid.Embryos were collected 3 days following electroporation.Following an over-night fixation in 4% PFA, brains were cryopreserved and mounted.Coronal cryosections of 18 μm were incubated with antibodies listed in S6 Table.Images were collected from three brain sections per condition using a Quorom spinning disk confocal microscope system or the Zeiss Axio Imager M2 system.EGFP-positive cells were counted in either the combined ventricular zone/sub-ventricular zone/intermediate zone (VZ/SVZ/IZ) or the cortical plate (CP) and reported as proportion of EGFP-positive cells in each of these regions.SatB2/EGFP-double positive cells in the CP were also counted for each condition.
For mitotic spindle plane analysis, the electroporated brain sections were stained with antibodies directed against Cdk5rap2 and corresponding secondary antibodies (S6 Table ).Nuclei were labeled with DAPI.Images of transfected cells in 10-15 μm z-stacks at an interval of 0.1 μm thickness were collected using a Zeiss Spinning Disc microscopy system CXU-S1 with ZEN 2012 software.The mitotic spindle plane was evaluated through measurement of the angle between the ventricular surface and a line connecting the centrosomes, using the ImageJ software.

RhoA Pull-down assay
Activated RhoA was assessed using the Rho Activation Assay Biochem kit (Cytoskeleton, Denver, USA) according to the manufacturer's protocol.Experiments were run in triplicate.

Analysis of Arhgef2 deficient mice
Arhgef2 deficient mice were genotyped as previously reported.[40] P0 and adult brains were dissected and embedded in paraffin/OCT medium as reported previously.[54] Sagittal/coronal brain sections of 10 μm were collected on histologic slides and stained with Hematoxylin and Eosin (H&E) or Gallyas or 3, 3'-Diaminobenzidine (DAB) staining following standard protocols.Whole brain, brain stem, cerebellum, and pontine nuclei volume were evaluated by measuring respective areas in every tenth section using ImageJ and multiplying by the thickness between sections.Immunohistology was performed on brain sections by blocking in staining buffer (0.2% gelatin, 0.25% Triton X-100, 3% BSA) for 30 min for permeabilization, followed by overnight incubation with primary antibodies and an 2 h incubation with the corresponding secondary antibodies (antibodies listed in S6 Table ).Nuclei were labeled with 4',6-Diamidino-2-phenylindole (DAPI, 1:1000, Sigma-Aldrich).Images were obtained using a Zeiss Spinning Disc microscopy system CXU-S1 with ZEN 2012 software.

In situ hybridization of Arhgef2
A 516 bp Arhgef2 PCR product was generated with primers listed in S5 Table and subsequently cloned into a p-AL2-T vector.The RNA probe was generated by in vitro transcription.Chromogenic/fluorescence in situ hybridization was performed on 16 μm thick brain sections from E11/P0 mice post-fixed in 4% PFA for 15 min and permeabilized with Proteinase K for 2.5 min at RT.The reaction was stopped by applying 0.2% glycine in PBS 1x, followed by 20 min post fixation with 20% glutaraldehyde in 4% PFA.The sections were treated with the prehybridization buffer (50% formamide, 5x SSC pH 7.0, 2.5 M EDTA, 0.1% Tween-20, 0.15% CHAPS, 0.1 mg/ml Heparin, 100 μg/ml yeast tRNA, 50 μg/ml salmon sperm DNA, 1x Denhardt's solution) at 65˚C for 2 hours followed by overnight incubation with Arhgef2 RNA probes at 65˚C.Sections were then treated with RNase A for 30 min at 37˚C, subsequently washed with SSC pH 4. Proliferation and cell size assay of WT and Arhgef2-/-mice CD4 + naive T cells were isolated from spleen of C57BL/6 (WT) and Arhgef2-/-mice by using CD4 + CD62L + T Cell Isolation Kit (Miltenyi Biotec, San Diego, CA, USA) and subsequently stained with CellTracer Violet Cell Proliferation Kit (Thermo Fisher Scientific) according to the instructions provided.Cells were then incubated with 2 μg/ml of anti-CD28 antibody (BD, New Jersey, USA) in anti-CD3e antibody (BD, New Jersey, USA) pre-coated wells for 4 days at the cell concentration of 1 × 10 6 cell/ml.4 days later, cells were collected and stained with APC-conjugated anti-CD3e (Biolegend, San Diego, CA, USA) and BV786-conjugated anti-CD4 (BD, New Jersey, USA).Cells were analyzed by FACS Aria II (BD Bioscience, New Jersey, USA) followed by using FlowJo software (Tree Star).All cells were pre-gated on singlet cells (area & width) and on living cells (7AAD − ).
and 2B), causing deletion of a G at the exon12-exon13 transition from normally spliced cDNA and ultimately a frameshift mutation in the affected children (c.1461delG, NM_004723.3,Fig 2C).The mutation lies within a highly conserved region, results in predicted truncation of the protein (p.D488Tfs Ã 11, NP_004714.2,Fig 2D), and segregates with the disease phenotype, i.e., was heterozygous in both parents and not observed in healthy controls.We failed to detect the deletion in various whole exome databases and did not detect biallelic ARHGEF2 mutations in six further patients, as detailed in the methods chapter.In lymphoblastoid cell lines (LCLs), generated from the patients, ARHGEF2 mRNA levels were decreased as can be expected by partial nonsense-mediated decay (Fig 2E, n = 3, One-way ANOVA).Moreover, ARHGEF2 protein levels were virtually absent in patient cells and decreased to intermediate levels in cells from the heterozygous parents (Fig 2F, n = 3, One-way ANOVA).The predicted truncated form of ARHGEF2 protein was below detection levels in patient LCLs (S1 Fig), indicating the total loss of ARHGEF2 in patient cells.

Fig 2 .
Fig 2. Homozygous ARHGEF2 mutation in index patients.(A) By whole exome sequencing, the homozygous mutation c.1461delG (chr1:155,928,110delC, hg19) was identified in exon12-intron 12 boundary of the ARHGEF2 (NM_004723.3).Pictogram representing ARHGEF2 protein domains (DH-DBL homology domain, PH-pleckstrin homology domain, phorbolester/Diacyl Glycerol-type zinc finger domain and coiled-coil domain) (B) Electropherogram depicting homozygous deletion of one base pair in the ARHGEF2 in patient II.1, which is heterozygous in the healthy parents and normal in the healthy control.