View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector REPORT Loss-of-Function Mutations in RAB18 Cause Warburg Micro Syndrome Danai Bem,1 Shin-Ichiro Yoshimura,2,12 Ricardo Nunes-Bastos,2 Frances F. Bond,3 Manju A. Kurian,1,13 Fatima Rahman,1 Mark T.W. Handley,4 Yavor Hadzhiev,1 Imran Masood,5 Ania A. Straatman-Iwanowska,1,13 Andrew R. Cullinane,1,14 Alisdair McNeill,1,3,15 Shanaz S. Pasha,1 Gail A. Kirby,1 Katharine Foster,6 Zubair Ahmed,7 Jenny E. Morton,3 Denise Williams,3 John M. Graham,8 William B. Dobyns,9 Lydie Burglen,10 John R. Ainsworth,11 Paul Gissen,1,13 Ferenc Mu¨ller,1 Eamonn R. Maher,1,3 Francis A. Barr,2 and Irene A. Aligianis1,3,16,* Warburg Micro syndrome and Martsolf syndrome are heterogenous autosomal-recessive developmental disorders characterized by brain, eye, and endocrine abnormalities. Previously, identification of mutations in RAB3GAP1 and RAB3GAP2 in both these syndromes implicated dysregulation of the RAB3 cycle (which controls calcium-mediated exocytosis of neurotransmitters and hormones) in disease pathogenesis. RAB3GAP1 and RAB3GAP2 encode the catalytic and noncatalytic subunits of the hetrodimeric enzyme RAB3GAP (RAB3GTPase-activating protein), a key regulator of the RAB3 cycle. We performed autozygosity mapping in five consanguineous families without RAB3GAP1/2 mutations and identified loss-of-function mutations in RAB18. A c.71T > A (p.Leu24Gln) founder mutation was identified in four Pakistani families, and a homozygous exon 2 deletion (predicted to result in a frameshift) was found in the fifth family. A single family whose members were compound heterozygotes for an anti-termination mutation of the stop codon c.619T > C (p.X207QextX20) and an inframe arginine deletion c.277_279 del (p.Arg93 del) were identified after direct gene sequencing and multiplex ligation-dependent probe amplification (MLPA) of a further 58 families. Nucleotide binding assays for RAB18(Leu24Gln) and RAB18(Arg93del) showed that these mutant proteins were functionally null in that they were unable to bind guanine. The clinical features of Warburg Micro syndrome patients with RAB3GAP1 or RAB3GAP2 mutations and RAB18 mutations are indistinguishable, although the role of RAB18 in trafficking is still emerging, and it has not been linked previously to the RAB3 pathway. Knockdown of rab18 in zebrafish suggests that it might have a conserved developmental role. Our findings imply that RAB18 has a critical role in human brain and eye development and neurodegeneration. Rabs, small G proteins belonging to the Ras superfamily, (GDIs), and GDI displacement factors (GDFs).1 RAB precisely coordinate vesicular trafficking in the cell. In GTPases are reversibly associated with membranes by humans, more than 60 RABs dynamically localize to hydrophobic geranylgeranyl groups that are attached to distinct intracellular membranes, where their recruitment one or (in most cases) two carboxy-terminal cysteine resi- of effector proteins such as sorting adaptors, kinases, dues. This prenylation is intrinsic to their role in regulating phosphatases, motors, and tethering factors regulates membrane traffic.1 RAB proteins can bind multiple effectors membrane identity and vesicle budding, motility, and and have been shown individually and as a group to have fusion. RABs function as molecular switches that alternate diverse roles in human diseases such as immunodeficiency, between two conformational states: the GTP-bound cancer, and neurodegeneration.1,2 In terms of inherited ‘‘active’’ form and the GDP-bound ‘‘inactive’’ form. This disorders, loss-of-function mutations in RAB27A (MIM allows them to associate and dissociate with target 603868) have been associated with autosomal-recessive membranes and regulate membrane transport in a spatially Griselli syndrome (MIM 607624); loss-of-function muta- and temporarily restricted manner.1,2 The switching of tions in RAB23 (MIM 604144) have been implicated in auto- RAB GTPases is governed by four classes of proteins: somal-recessive Carpenter syndrome (MIM 201000), and GTPase-activating proteins (GAPs), guanine nucleotide loss-of-function mutations in RAB39B (MIM 300774) cause exchange factors (GEFs), GDP dissociation inhibitors X-linked mental retardation associated with autism, 1Medical and Molecular Genetics, School of Clinical and Experimental Medicine and Centre for Rare Diseases and Personalised Medicine, University of Birmingham, Birmingham B15 2TT, UK; 2Cancer Research Centre, University of Liverpool, Liverpool L3 9TA, UK; 3West Midlands Regional Genetics Service, Birmingham Women’s Hospital, Birmingham B15 2TG, UK; 4Medical and Developmental Genetics, Medical Research Council Human Genetics Unit, Edinburgh EH4 2XU, UK; 5Department of Cellular Pathology, Queen Elizabeth Hospital National Health Service Foundation Trust, Birmingham B15 2WB Institute of Neurology, University College London WC1E 6BT, UK; 6Radiology Department, Birmingham Children’s Hospital, Birmingham B4 6NH, UK; 7Molecular Neuroscience Group, School of Clinical and Experimental Medicine, University of Birmingham, Birmingham B15 2TT, UK; 8Clinical Genetics and Dysmorphology, Cedars Sinai Medical Centre, Los Angeles, CA 90048, USA; 9Department of Human Genetics, Neurology and Pediatrics, University of Chicago, Chicago, IL 60637, USA; 10Service de Ge´ne´tique Me´dicale, Hoˆpital d’Enfants Armand-Trousseau, 75571 Paris, France; 11Department of Paediatric Ophthalmology, Birmingham Children’s Hospital, Birmingham B4 6NH, UK 12Present address: Department of Cell Biology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-087, Japan 13Present address: Institute of Child Health, London WC1N 1EH, UK 14Present address: National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA 15Present address: Institute of Neurology, University College London, London WC1E 6BT, UK 16Present address: Medical and Developmental Genetics, Medical Research Council Human Genetics Unit, Edinburgh EH4 2XU, UK *Correspondence: [email protected] DOI 10.1016/j.ajhg.2011.03.012. Ó2011 by The American Society of Human Genetics. All rights reserved. The American Journal of Human Genetics 88, 499–507, April 8, 2011 499 epilepsy, and macrocephaly (MIM 300774). Mutations in Families 1–5 are consanguineous. Families 1–4 are of RAB7 (MIM 602298) are associated with autosomal-domi- Pakistani origin, and family 5 is Turkish (pedigrees of nant Charcot-Marie-Tooth disease type 2B (MIM 600882). families 1–5 are shown in Figure 1). The clinical phenotype Functionally, RAB proteins have been classified into for the families is summarized in Table S1 and is typical of those that regulate the endocytic versus the exocytic Warburg Micro syndrome. The eye phenotype in the oldest (secretory) pathways.2,3 The RAB3 proteins (RAB3A [MIM affected child from families 1–4 was previously reported.12 179490]; RAB3B [MIM 179510]; RAB3C [MIM 612829], All the affected children in these families presented at birth and RAB3D [MIM 604350]) modulate calcium-mediated with congenital cataracts, microphthalmia, and microcor- exocytosis of neurotransmitters and hormones and are nea. In addition, they have small atonic pupils that do not regulated by RAB3GAP.4–7 RAB3GAP is a heterodimeric react to light or mydriatic agents. Despite early cataract complex consisting of a catalytic subunit (p130), which surgery, their vision has remained poor (only light percep- is encoded by RAB3GAP1 (MIM 602536) on chromosome tion) as a result of progressive optic atrophy and severe 2q21.3, and a noncatalytic subunit (p150), encoded by cortical visual impairment (confirmed by normal electrore- RAB3GAP2 (MIM 609275) on chromosome 1q41.4,5 Muta- tinogram [ERG] and absent visually evoked potentials tions in these genes have been implicated in the pathogen- [VEPs]). The progressive neurological deterioration esis of two clinically overlapping autosomal-recessive observed in these children is highlighted in Table S1. conditions, Warburg Micro syndrome (MIM 600118) and Although they were born with normal head circumfer- Martsolf syndrome (MIM 212720).8–11 Both disorders are ences (50th–75th percentiles) and develop early mile- characterized by ocular and neurodevelopmental manifes- stones, such as smiling, they all have severe truncal tations; Warburg Micro syndrome is a more severe hypotonia such that they have limited head control and, disorder.8–14 Recent molecular studies have established at the very most, only learn to sit with support. The that these two syndromes represent a phenotypic children have achieved no developmental milestones continuum that appears to be related to the nature and beyond those at the 4 month level; they have not learned severity of the mutations present in RAB3GAP1 and RAB3- to crawl, pull up to a standing position, or walk. They GAP2. Severe loss-of-function mutations in RAB3GAP1 developed postnatal acquired microcephaly within the have been reported in about 50% (17/35) of Warburg first year of life, and their head circumferences fell to Micro syndrome patients.8,10 Two mutations have been re- between À4SD and À6SD below the mean. Although ported in RAB3GAP2; these are a homozygous frameshift some of the younger children have babbled, none have mutation in a family affected by Warburg Micro syndrome developed any recognizable words or speech. A character- and a hypomorphic homozygous splicing mutation in istic pattern of progressive lower-limb spasticity started a family affected