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RESEARCH ARTICLE
Homozygous ARHGEF2 mutation causes
intellectual disability and midbrain-hindbrain malformation
Ethiraj Ravindran1,2,3 , Hao Hu4,5 , Scott A. Yuzwa6,7, Luis R. Hernandez-Miranda8, Nadine Kraemer1,2,3, Olaf Ninnemann1, Luciana Musante4, Eugen Boltshauser9, Detlev Schindler10, Angela HuÈ bner11, Hans-Christian Reinecker12, Hans-Hilger Ropers4, Carmen Birchmeier8, Freda D. Miller6,7, Thomas F. Wienker4, Christoph HuÈ bner2, Angela M. Kaindl1,2,3
1Institute of Cell Biology and Neurobiology, Charite University Medicine Berlin, Berlin, Germany, 2Department of Pediatric Neurology, Charite University Medicine Berlin, Berlin, Germany,
3SozialpaÈdiatrisches Zentrum (SPZ), Center for Chronic Sick Children, Charite University, Berlin, Germany, 4Max Planck Institute for Molecular Genetics, Berlin, Germany,5Guangzhou Women and Children's Medical Center, Guangzhou, China,6Department of Molecular Genetics, University of Toronto, Toronto, Canada,7Program in Neurosciences & Mental Health, Hospital for Sick Children, Toronto, Canada,8 Max-DelbruÈck-Center for Molecular Medicine, Berlin, Germany,9Department of Pediatric Neurology, University Children's Hospital of Zurich, Zurich, Switzerland,10Department of Human Genetics, University of WuÈrzburg, WuÈrzburg, Germany,11Pediatrics, University Hospital, Technical University Dresden, Dresden, Germany,12Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America
These authors contributed equally to this work.
Current address: Guangzhou Women and Children's Medical Center, Guangzhou, China angela.kaindl@charite.de
Abstract
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 theARHGEF2as a cause of intellectual disability, a midbrain-hind-brain malformation, and mild microcephaly in a consanguineous pedigree of Kurdish-Turk-ish descent. We show that loss ofARHGEF2perturbs progenitor cell differentiation and that this is associated with a shift of mitotic spindle plane orientation, putatively favoring more symmetric divisions. TheARHGEF2mutation 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 inArhgef2mutant mice and identify an aberrant migration of distinct components of the precerebellar system as a pathomechanism underlying the mid-brain-hindbrain phenotype. Our results highlight the crucial function of ARHGEF2 in human brain development and identify a mutation inARHGEF2as novel cause of a neurodevelop-mental disorder.
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Citation:Ravindran E, Hu H, Yuzwa SA, Hernandez-Miranda LR, Kraemer N, Ninnemann O, et al. (2017) HomozygousARHGEF2mutation causes intellectual disability and midbrain-hindbrain malformation. PLoS Genet 13(4):
e1006746.https://doi.org/10.1371/journal.
pgen.1006746
Editor:Rudolf Korinthenberg, University Medical Center, GERMANY
Received:June 23, 2016 Accepted:April 5, 2017 Published:April 28, 2017
Copyright: 2017 Ravindran et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability Statement:All relevant data are within the paper and its Supporting Information files.
Funding:German Research Foundation (SFB665), the Sonnenfeld Stiftung, the German Academic Exchange Service (DAAD), the Berlin Institute of Health (BIH, CRG1), ChariteÂ, Max-Planck Society, EU FP7 project GENCODYS (241995), National Natural Science Foundation of China (No.
81671067), NIH AI113333, DK068181, and
Author summary
During brain development, localized gene expression is crucial for the formation and function of specific brain regions. Various groups of proteins are known to regulate seg-mentation through controlled gene expression, among them, the Rho GTPase regulator family. In this study, we identified a frameshift mutation in the Rho guanine nucleotide exchange factor 2 gene ( ) in two children presenting with intellectual disability, mild microcephaly, and a midbrain-hindbrain malformation. This phenotype is also observed in mutant mice, highlighting the importance of ARHGEF2 across devel-opment of distinct mammalian species. We show that loss of Arhgef2 affects neurogenesis and also cell migration. In addition, we extended the current knowledge of ARHGEF2 expression and its role in early central nervous system development, with special reference to the formation of the precerebellar system. In addition to extensive literature on ARH-GEF2, we now provide evidence for its significant role in neuronal migration in brain development and link the gene to human neurodevelopmental disease.
Introduction
Brain development depends on spatiotemporally controlled gene expression.[1±3] Alterations in the expression pattern of such genes can result in neurodevelopmental disorders by imping-ing 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±6] The latter is crucial for the formation of specific brain structures.[7±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±14] ARHGEF2 connects microtubule and actin cytoskele-ton dynamics.[14] In this context ARHGEF2 activity is reduced through microtubule binding and further controlled by upstream regulators.[15±25] ARHGEF2 is key for actin and microtu-bule reorganization and is required for mitotic spindle formation and orientation.[11]
Inhibition of ARHGEF2 results in spindle disorientation and dysfunction, mitotic delay, accu-mulation of prometaphase cells, and further mitotic aberrations.[11,22] In mouse neocortex,
is expressed in neural precursor and immature neurons and regulates neurogenesis from cortical precursor cells.[24] down-regulation by shRNA keeps radial precursors cycling, potentially by disrupted spindle plane orientation, and thereby inhibits neurogenesis.
In contrast, overexpression causes an increase of neurons in the cortical plate.[24±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 func-tion of Arhgef2 in cytoskeletal dynamics and critical signal transducfunc-tion pathways exists and other ARHGEF genes have been linked with neurological disease,[30±32] little is known about ARHGEF2 function in humans and no disease phenotype associated with this gene has been reported.
ARHGEF2and developmental disorder
PLOS Genetics |https://doi.org/10.1371/journal.pgen.1006746 April 28, 2017 2 / 24
DK043351 and Major Medical Collaboration and Innovation Program of Guangzhou Science Technology and Innovation Commission (No.201604020020). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests:The authors have declared that no competing interests exist.
Results and discussion
We report that patients with a homozygous mutation in develop intellectual dis-ability, 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 (S1andS2 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 horizon-tal pendular nystagmus (S1 Table). Ophthalmological examination revealed congenihorizon-tal stra-bismus, 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 hear-ing capacity were diagnosed in both patients (S1andS3Tables). 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 assess-ment 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 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 pre-dicted truncation of the protein (p.D488T 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 mutations in six further patients, as detailed in the methods chapter. In lympho-blastoid cell lines (LCLs), generated from the patients, 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.
To evaluate the biological effects of the identified mutation on brain develop-ment, we utilized both and approaches. We used a well-characterized cell cul-ture model of E13 mouse neocortical cells recapitulating the timing of cortical precursor differentiation observed .[24,33,34] The large majority of these cells are actively divid-ing 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
ARHGEF2and developmental disorder
Fig 1. Phenotype of patients with midbrain-hindbrain malformation and intellectual disability.(A) The index patients are children (II.1, II.2) of healthy consanguineous Kurdish parents (first cousins) from Turkey. (B) Cranial MRI revealed microencephaly and hypoplasia of the pons and grooves (II.1, II.2) as well as hypoplasia of the cerebellum (II.2) or the caudal vermis (II.1). Arrow heads indicate hypoplastic pons (II.1, II.2) and cerebellum (II.2). Sagittal and axial T2 images.
https://doi.org/10.1371/journal.pgen.1006746.g001
ARHGEF2and developmental disorder
PLOS Genetics |https://doi.org/10.1371/journal.pgen.1006746 April 28, 2017 4 / 24
Fig 2. HomozygousARHGEF2mutation 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 theARHGEF2(NM_004723.3).
Pictogram representing ARHGEF2 protein domains (DH-DBL homology domain, PH-pleckstrin homology domain, phorbol-ester/Diacyl Glycerol-type zinc finger domain and coiled-coil domain) (B) Electropherogram depicting homozygous deletion of one base pair in theARHGEF2in patient II.1, which is heterozygous in the healthy parents and normal in the healthy control.
ARHGEF2and developmental disorder
this model, that knockdown of causes a decrease in the proportion of III-tubulin positive neurons and a corresponding increase of cycling cortical precursors.[24] We thus expected the human frameshift mutation to have a similar impact on neurogenesis, leading to an increase of cycling (and eventually apoptotic) precursors and a decrease of neu-rons generated during brain development. In a first series of experiments, we transfected murine cortical radial precursors with an shRNA plasmid and a nuclear EGFP reporter plasmid; the latter allows the visualization of transfected cells. As expected,
knockdown significantly decreased the proportion of double positive cells for EGFP and early neuron marker III-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 . In this condition, we observed no significant change in the proportion of EGFP and III-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 progeni-tors with shRNA, the EGFP reporter, and a construct encoding mutant
identified in the index patients. Here, the proportion of EGFP and III-tubulin double positive cells was reduced, whereas the number of EGFP and Ki67 remained increased (Fig 3A). We conclude that the phenotype produced by shRNA was rescued by coexpression of wildtype but not of mutant human , consistent with the interpretation that the patient mutation is a loss-of-function mutation and that this mutation impairs neurogenesis.
To further analyze whether the identified loss-of function mutation affects brain development we electroporated E13.5 mouse cortex with or (con-trol) shRNA constructs along with wildtype or mutant human 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-ventricu-lar, and intermediate zones (VZ/SVZ/IZ) and decreased proportions in the cortical plate (CP) of the shRNA electroporated mice, as compared to mice that were electroporated with the EGFP reporter only (Fig 3B and 3C). In line with our data, overexpression of wildtype, but not mutant, was able to effectively rescue the cell distribution in the cortex induced by shRNA. Upon electroporation of shRNA, we addition-ally observed a dramatic reduction in the proportion of SatB2/EGFP double positive cells local-ized to the cortical plate. This is consistent with impaired neurogenesis described above in cultured cortical precursor cells. Overexpression of wildtype was able to rescue this impairment of neurogenesis while mutant was ineffective (Fig 3B and 3C, n = 3, One-way ANOVA). These data further confirm that the patient mutation in human acts as a loss-of-function mutation also , results in dramatically decreased neurogenesis and is most probably disease-causative (Fig 3D).
(C)This mutation leads to a transfer of the neighboring C in the coding region into the splice donor site, as shown by cDNA sequencing. This is predicted to cause a frameshift mutation (p.D448Tfs498, NP_004714.2) and a null allele. (D) The mutation lies within a highly conserved region of the protein. (E) Quantitative real-time PCR revealed strongly reducedARHGEF2 transcript levels in patients and intermediate levels in heterozygous parents, as would be expected from partial nonsense-mediated mRNA decay (n = 3, One-way ANOVA). (F) ARHGEF2 protein levels were below detection levels on a protein immunoblot of lymphoblastoid cells from the affected subjects compared to a control, with intermediate levels in heterozygous parents (ARHGEF2 120 kDa, actin 43 kDa; n = 3, One-way ANOVA). p 0.05, p 0.01, p 0.001, Error bars
represent SD.
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Fig 3. Loss of ARHGEF2 function inhibits neurogenesis and increases neural precursorsin vitroandin vivo. (A)Cultured mouse cortical precursor cells were transfected with mouseArhgef2shRNA (shArhgef2) or scramble shRNA (shCon), with a nuclear EGFP plasmid and with either an empty vector (EV), wildtype (wt) or mutant (mut) humanARHGEF2. Representative fluorescence micrographs ofshArhgef2knockdown cultured precursors immunostained for EGFP (green), III-tubulin or Ki67 (red), and Hoechst 33258 nuclear staining (blue) 3 days after transfection; right panels show the merged images; arrowheads indicate double-positive cells. TheArhgef2shRNA-induced phenotype of decreased early neuronal marker
III-ARHGEF2and developmental disorder
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
alters neurogenesis by impairing mitotic spindle orientation, we performed electropo-ration 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. 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 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 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, how-ever, no cell cycle defect, increased radiosensitivity, nor abnormal centrosome `morphology' in the patient LCLs (S2andS3Figs). We concluded that regulation of the mitotic spindle appara-tus 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 mem-bers.[12,22,28,36,37] Specifically, RhoA has been implicated in the regulation of neurogen-esis 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 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 mutation.
Previous studies showed that is expressed in the neural tube of mice from embry-onic day 11 on and maintained at high levels during brain development.[24] Our
hybridization studies on embryonic mice showed strong expression of transcripts in the neuroepithelium of telencephalon, diencephalon, and rhombencephalon of E11 mice (S5 Fig). At the time of birth, 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 corrobo-rate the pathogenicity of the identified mutation in brain development, we analyzed the phenotype of deficient mice.[40] In adult mutant mice, we observed a significant reduction in volume of the total brain size (referred to as microencephaly), the tubulin-positive cells and increased proliferation marker Ki67-positive precursors is rescued byARHGEF2(wt) but notARHGEF2(mut) (n = 1378±1293 cells in 4 independent experiments, One-way ANOVA, scale bar 25 m).(B, C)Quantification of EGFP positive cells and SatB2/EGFP double-positive cells (arrowheads) in E13.5 mouse cortices co-electroporated with plasmids specified above and analyzed 3 days later. Representative fluorescence micrographs of sections co-stained for SatB2 (new-born neurons) and EGFP (dotted line indicates boundary between CP and VZ/SVZ/IZ) (n = 3, One-way ANOVA, scale bar 50 m).(D)Pictorial representation: ARHGEF2 favors neurogenesis and its downregulation/dysfunction inhibits neurogenesis by maintaining murine NPCs in cycling phase. p 0.05, p 0.01, p 0.001. Error bars represent S.E.M.
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Fig 4. MutantARHGEF2favors self-renewing (symmetric) precursor proliferation. (A)Quantification of spindle plane orientation of dividing, metaphase and anaphase precursors transfected with EGFP (n = 68±70 cells, Student'st-test, scale bar 20 m).Arhgef2shRNA is associated with a more horizontal orientation of the spindle pole. Overexpression ofARHGEF2(wt), not ARHGEF2(mut), rescues the spindle orientation phenotype in dividing precursors. Representative fluorescence micrographs of sections stained with CDK5RAP2 (centrosomes), DAPI, and EGFP, indicating the plane of division (dotted line) and respective radial diagram.(B)Model indicating the plane of symmetry and cell fate decision in the developing brain. p 0.05. Error bars represent S.
E.M.
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cerebellum and the brainstem, as well as the striking absence of the pontine nuclei(Fig 6, n = 3±4, Student's ). We concluded that the phenotypes observed in the nervous system of deficient mice recapitulate largely those malformations observed in the index patients. Our results showed a clear correspondence between regions displaying high levels of Fig 5. Mitotic spindle defects and reduced RhoA activation in LCLs of index patients withARHGEF2mutation.(A) Abnormal spindle formation with an increase of abnormal misdirected spindles and broad, unfocused microtubules poles in immortalized patient II.1 and II.2 lymphocytes (LCLs, metaphase cells only of patient II.1 and II.2 are shown, centrosome marker CDK5RAP2 (red), spindle marker -tubulin (green), DNA was stained with DAPI (blue)). CDK5RAP2 signals did not differ significantly between patient and control cells; this holds also true for the centrosome marker -tubulin (S3 Fig). (B) Quantification results underlining abnormal spindle morphology, decreased spindle pole distance, and reduced cell size of patient versus control LCLs (n = 100±200, One-way ANOVA, scale bar 5 m): abnormal spindle morphology in patients (II.1 77%, II.2 78%) versus controls (Co-1 16%, Co-2 17%, Co-3 14%), decreased spindle pole distance in patients (II.1 2.70, II.2 2.72 m) versus controls (Co-1 4.46, Co-2 4.05, Co-3 4.61 m), and reduced cell size in patients (II.1 96.14, II.2 94.66 m2) versus controls (Co-1 175.52, Co-2 181.74, Co-3 204.51 m2). We observed no difference in size or proliferation of T-cells betweenArhgef2-/-andArhgef2+/+mice(S4 Fig). (C, D) Reduced levels of active RhoA and active MLC in patient LCLs in protein immunoblots (n = 3, One-way ANOVA). (E) Pictogram of RhoA/ROCK/MLC pathway downstream of ARHGEF2, important for processes such as cytoskeletal dynamics and neurogenesis. p 0.05, p 0.01, p 0.001, p 0.0001. Error bars represent SD.
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