Article MCF2 is linked to a complex perisylvian syndrome and affects cortical lamination MOLINARD-CHENU, Aude, et al. Abstract The combination of congenital bilateral perisylvian syndrome (CBPS) with lower motor neuron dysfunction remains unusual and suggests a potential common genetic insult affecting basic neurodevelopmental processes. Here we identify a putatively pathogenic missense mutation in the MCF2 gene in a boy with CBPS. Using in utero electroporation to genetically manipulate cortical neurons during corticogenesis, we demonstrate that the mouse Mcf2 gene controls the embryonic migration of cortical projection neurons. Strikingly, we find that the CBPS-associated MCF2 mutation impairs cortical laminar positioning, supporting the hypothesis that alterations in the process of embryonic neuronal migration can lead to rare cases of CBPS. Reference MOLINARD-CHENU, Aude, et al. MCF2 is linked to a complex perisylvian syndrome and affects cortical lamination. Annals of Clinical and Translational Neurology, 2020, vol. 7, no. 1, p. 121-125 DOI : 10.1002/acn3.50949 PMID : 31846234 Available at: http://archive-ouverte.unige.ch/unige:154128 Disclaimer: layout of this document may differ from the published version. 1 / 1 BRIEF COMMUNICATION MCF2 is linked to a complex perisylvian syndrome and affects cortical lamination Aude Molinard-Chenu1,2,3 ,Joel€ Fluss4, Sacha Laurent5,Meryle Laurent6, Michel Guipponi5,7 & Alexandre G. Dayer1,2,3 1Department of Psychiatry, University of Geneva Medical School, Geneva, 4 CH-1211, Switzerland 2Department of Basic Neurosciences, University of Geneva Medical School, Geneva, 4 CH-1211, Switzerland 3Institute of Genetics and Genomics in Geneva (IGe3), University of Geneva Medical Center (CMU), Geneva, 4 CH-1211, Switzerland 4Pediatric Neurology Unit, Pediatric Subspecialties Service, University Hospitals of Geneva, Geneva, Switzerland 5Service of Genetic Medicine, University Hospitals of Geneva, Geneva, Switzerland 6Pediatric Radiology Unit, University Hospitals of Geneva, Geneva, Switzerland 7Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland Correspondence Abstract Aude Molinard-Chenu, Department of Psychiatry and Department of Basic The combination of congenital bilateral perisylvian syndrome (CBPS) with Neurosciences, University of Geneva Medical lower motor neuron dysfunction remains unusual and suggests a potential School, Geneva 4 CH-1211, Switzerland. Tel: common genetic insult affecting basic neurodevelopmental processes. Here we +41 22 37 95386; E-mail: aude.molinard- identify a putatively pathogenic missense mutation in the MCF2 gene in a boy [email protected] with CBPS. Using in utero electroporation to genetically manipulate cortical neurons during corticogenesis, we demonstrate that the mouse Mcf2 gene con- Funding information trols the embryonic migration of cortical projection neurons. Strikingly, we find This work was supported by the Institute of that the CBPS-associated MCF2 mutation impairs cortical laminar positioning, Genetics and Genomics of Geneva (to AMC supporting the hypothesis that alterations in the process of embryonic neuronal and AD) and by the NCCR Synapsy (51NF40- migration can lead to rare cases of CBPS. 185897). Received: 12 August 2019; Revised: 20 October 2019; Accepted: 22 October 2019 Annals of Clinical and Translational Neurology 2020; 7(1): 121–125 doi: 10.1002/acn3.50949 Introduction unusual combination of both central and peripheral motor impairment remains unknown, but it most proba- Congenital bilateral perisylvian syndrome (CBPS) is a rare bly involves a common genetic insult affecting basic cellu- malformation of the cerebral cortex, most often associated lar processes regulating the development of MNs and with the following clinical signs: pseudobulbar palsy with cortical projection neurons (PNs). During the embryonic oromotor apraxia, mild bilateral spastic palsy, cognitive period, PNs and MNs are generated in germinal ventricu- impairment, and epilepsy.1 Despite this well-reported lar zones then migrate radially to reach their final clinical phenotype, there is often a diagnostic delay, espe- anatomical position and interestingly, they share common cially when some of the clinical features are missing in molecular regulators.5–7 Here, we identify a missense the early stages of the disorder. CBPS is considered by mutation in the MCF.2 Cell Line Derived Transforming certain authors as a peculiar form of cerebral palsy affect- Sequence (MCF2) gene in a boy with CBPS associated to ing predominantly bulbar muscles, but some patients also lower MN dysfunction. Using functional in vivo studies display congenital contractures as clinical and neurophysi- in mice, we find that this CBPS-associated human muta- ological signs consistent with an involvement of the lower tion has a pathogenic effect on the process of embryonic motor neuron (MN).2–4 The mechanism underlying this PNs migration in vivo. ª 2019 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association. 121 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Perisylvian Syndrome, Cortical Lamination and MCF2 A. Molinard-Chenu et al. 8 Patient, Materials and Methods previously. All constitutive expression of shRNAs and cDNAs was driven by the human U6 promoter, in the PLKO.1 vector for the shRNAs and pUB6/V5-His A Clinical description (pUB6) vector for hMCF2 (kind gift from Danny Manor), The study was approved by the local ethics committee TOM and GFP. The following shRNAs were electropo- and both patient and parental consent were obtained. The rated in equal ratios in control and experimental condi- patient, who is a 19-year-old boy, originates from Leba- tions: Mcf2 shRNA (TRCN0000042653, Thermoscientific), non and was born premature at 31 gestation weeks after a and Scramble shRNA (mature sense: CCTAAGGT- prolonged oligoamnios. Both parents are healthy and not TAAGTCGCCCTCG, Addgene). The G4A missense muta- related. The mother has four healthy female siblings. The tion was induced in hMCF2 plasmid using site-directed eldest sister had three children, including one healthy mutagenesis (InFusion Kit, Takara). boy. One maternal aunt was born with a spina bifida occulta and carries no neurological sequelae. The patient Results is a single child. At birth, he exhibited bilateral clubfeet, hip dislocations, and asymmetric paraparesis leading to a MRI and nerve conduction studies presumed diagnosis of congenital arthrogryposis. During infancy and childhood, a severe developmental speech MRI scans of the brain revealed bilateral asymmetric peri- and language impairment was observed along with exces- sylvian polymicrogyria extending to the parietal cortex on sive drooling and feeding problems. Clinical examination the right side. The sylvian sulci also displayed an abnor- of the patient in our institution in late childhood was mal configuration bilaterally (Fig. 1A–C). MRI scan of characterized by a pseudobulbar palsy, an asymmetric the spinal cord revealed a homogeneous thin spinal cord lower limb distal paresis with no movement below the without the physiological lumbar enlargement (Fig. 1D). knees and severe muscle wasting. Right knee jerk was pre- Additionally, nerve conduction studies demonstrated a sent but left knee jerk was absent, as were ankle jerks pure motor impairment from L4-S2 on both sides typical bilaterally. In addition, the patient did not report neither of a lower MN dysfunction. sphincterial dysfunction nor sensory impairment. Exome sequencing MRI study Exome sequencing of the proband and his healthy parents All MRI data were acquired with a dedicated head coil on revealed a putatively damaging missense mutation inher- a 1.5T machine (Avanto Siemens Erlangen Germany). ited from his mother in the MCF2 gene, located on chro- mosome Xq27, (NM_005369.5): c.4G>A, p.(Ala2Thr) in exon 1. This variant is absent from the gnomAD database Exome sequencing (https://gnomad.broadinstitute.org/) and is predicted as Exome of the patient was captured using the Agilent Sur- pathogenic by all used algorithms except Polyphen2 eSelet QXT Human All Exon V5 kit and sequenced on a which classified it as likely pathogenic. MCF2, also known HiSeq2000 instrument (Illumina). Reads mapping and as DBL, is a member of a large family of guanine nucleo- variant calling were performed using BWA 0.7.13, Picard tide exchange factors (GEFs) that modulates the activity 2.9.0, GATK HaplotypeCaller 3.7 and annotated with of Rho GTPases.9,10 Given that GEFs have been shown to annovar 2017-07-17 and UCSC RefSeq (refGene) down- act as key regulators of cellular migration,11 we aimed to loaded on 2018-08-10. The variants were searched in vari- test whether the CBPS-associated MCF2 mutation affected ous databases including dbSNP151, gnomAD 2.1, ClinVar cortical PNs in vivo. 2018, and HGMD 2016. Pathogenicity prediction scores were obtained for missense variants using SIFT, Poly- In vivo mouse studies Phen2, MutationTaster (MT), CADD. mMcf2 was found to be expressed in control E17.5 PNs in a previously published RNA sequencing dataset8 and Mouse in utero electroporation its subcellular cytoplasmic expression in cortical PNs at Animal experiments were conducted according