Loss-Of-Function Mutations in RAB18 Cause Warburg Micro Syndrome

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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 4SD and 6SD 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 by Martsolf syndrome.9,11 The cognitive between 8 and 12 months of age, and upper-limb spasticity deficits observed in these disorders are consistent with developed later, at about 8 years of age. The older children the reported role of the RAB3 pathway in cognition. Muta- are profoundly delayed, are wheelchair bound, and have tions in RABGDI1 (MIM 300104) (which is a nonspecific severe contractures of all four limbs, kyphoscoliosis, and regulator of the RAB3 pathway) cause X-linked mental no voluntary or spontaneous movement. Brain magnetic retardation,15 and a missense mutation in RIMS1 (MIM resonance imaging (MRI) findings are consistent with 606629) (a downstream effector of RAB3A) has been bilateral frontal polymicrogyria and thinning of the corpus implicated in enhanced cognition.16 Furthermore, loss of callosum posteriorly in families 2 and 4 (Figure S1). The rab3gap1 in mice resulted in abnormal release of synaptic brain MRI in family 5 showed bilateral frontal gyration vesicles and altered short-term synaptic plasticity in abnormalities and thinning of the corpus callosum poste- the hippocampus; these effects could contribute to the riorly. Family 6 was reported by Graham et al.14 (as patients Warburg Micro cognitive phenotype.17 However, these 1 and 2) as having typical Warburg Micro syndrome. The mice were viable and fertile and had no structural eye, children from this family are the oldest known children brain, or genital abnormalities, suggesting that there is with Warburg Micro syndrome and are aged 21 and 23. differential species-specific redundancy in these pathways. They have both had severe intractable epilepsy with Further locus heterogeneity has been demonstrated for myoclonic seizures from an early age (2 and 5 years), unlike Warburg Micro and Martsolf syndromes, suggesting that the children in families 1–5 (some of whom have general- other genes also play a role in the pathogenesis of these ized tonic-clonic seizures). Their brain MRIs also show disorders.8–11 bilateral frontal polymicrogyria and thinning, of the Here, we report the identification and functional charac- corpus callosum.14 A nerve conduction study in the boy terization of RAB18 mutations in six families affected by from family 6 was markedly abnormal due to severe loss Warburg Micro syndrome. All family members and control of neurons suggesting an axonal peripheral neuropathy. subjects in this study provided written informed consent Although microgenitalia in boys has been reported as a dis- under a research protocol approved by the South Birming- tinguishing feature in families with RAB3GAP1 mutations, ham Local Research Ethics Committee in accordance with the two boys from family 3 had normal genitalia. The the Declaration of Helsinki. Genomic DNA was extracted children with RAB18 mutations all have facial dysmorphia from venous blood according to standard procedures. similar to that of children with reported RAB3GAP1

500 The American Journal of Human Genetics 88, 499–507, April 8, 2011 I

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1.1 1.2 1.3 1.4 2.1 2.2 2.3 3.1 3.2 3.3 4.1 4.2 4.3 4.4 4.5 5.1 5.2

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Figure 1. Fine-Mapping Data for Families 1–5 Microsatellite studies in the extended families confirmed a founder effect in families 1–4. The affected individuals share a common haplotype between D10S1789 and D10S1228. mutations, as shown in Figure 2. In most families, were not known to be related but originated from the two antenatal diagnosis by ultrasound has been difficult, neighboring villages in Pakistan (Mirpur). Interestingly, but in family 5, the affected fetus was terminated at they shared a core 6056.698 kb haplotype (containing 33 weeks after the identification of cataracts and abnormal 680 SNPs) on chromosome 10p12.1 between rs12263288 brain gyration. (24209.124 kb) and rs3847396 (30265.822 kb) (Figure S3). RAB3GAP1 and RAB3GAP2 mutations were excluded in Microsatellite studies in the extended families confirmed families 1–5 (by linkage and direct sequencing a founder effect in these families; the affected individuals [Figure S2]). We then excluded the involvement of other shared a common homozygous haplotype between genes encoding proteins implicated in the RAB3 pathway D10S1789 (23745.855 kb) and D10S1228 (30167.683 kb) (RAB3A, RIMS1, RIMS2 [MIM 606630], RPH3A [MIM (Figure 1). The genotyping was consistent with linkage; 612159], MADD [MIM 603584], WDR7 [MIM 613473], the parents were heterozygous (as obligate carriers), and DMXL2 [MIM 612186]) by genotyping closely flanking and the unaffected siblings had a haplotype different microsatellite markers (data not shown). We then from that of their affected siblings. A maximum 2-point performed a genome-wide linkage scan in five large consan- LOD score of Z ¼ 8.93 (q ¼ 0 at D10S1749) was calculated guineous families by using the 250K SNP arrays (Affyme- on the assembled haplotypes with GENEHUNTER (version trix) to genotype 11 affected children and four unaffected 2.0b) under a model of autosomal-recessive inheritance siblings. DNA was amplified and hybridized to the Affyme- with full penetrance; the disease-allele frequency was trix 250K SNP chips according to the manufacturer’s estimated at 1 in 1,000, thus confirming linkage to this instructions. To further investigate all significant regions region. of common homozygosity identified on genome-wide The candidate region contained 23 genes (Figure S3). scan, we genotyped polymorphic UniSTS microsatellite Intronic primer pairs were designed for exon-specific markers spaced at 1 Mb intervals with GeneMapper soft- amplification of these 23 candidate genes (Figure S3 ware. In all the affected children from the five families, and Table S2) with Exon Primer. The RAB18 (MIM a 10113.089 kb region of shared homozygosity was identi- 602207) primers are shown in Table S3. After PCR amplifi- fied on chromosome 10p12.1. The region contained 1055 cation, sequencing reactions were performed with BigDye SNPs and was flanked by SNPs rs943124 (20152.733 kb) Terminator v3.1 Cycle Sequencing kit and run on an and rs3847396 (30265.822 kb) (Figure S3). Families 1–4 ABI PRISM 3730 DNA Analyzer (Applied Biosystems). All

The American Journal of Human Genetics 88, 499–507, April 8, 2011 501 Figure 2. Clinical Representation of Patients with Warburg Micro Syndrome at Different Ages All the children have microcephaly, brachycephaly, microphthalmia, microcornea, low anterior hairline, large protruding pinnae, and downturned mouth corners. The older children are wheelchair bound and have kyphoscoliosis, severe spastic quadriplegia with contractures, and diminished muscle bulk. (1a-f) Affected children from family 3 at 8 months (1a); 15 months (1b), and 18 months (1c,1d, and 1e) show truncal hypotonia; 7 years (1f and 1g). (2 a and 2b) 18-month-old from family 1. (3a and 3b) 6-year-old from family 5. (4a–4c) 21-year male from family 6; (4d–4f) 23-year-old female from family 6. Permission was obtained from patients’ parents for publication of these images. sequence variants identified were labeled according to the (Figure 3). This sequence variant segregated with disease most abundant RAB18 isoform, which is also present in the status in the families (the parents were heterozygous, brain (NM_021252.3 or RAB18-001: ENST00000356940). consistent with carrier status, and unaffected siblings had We carried out multiplex ligation-dependent probe ampli- wild-type sequence or were heterozygous). In family 5, fication (MLPA) to detect deletions. Synthetic probes for a homozygous deletion of RAB18 exon 2 was initially de- exons 1, 1a, 1b, 2, 4, 6, and 7 were designed according to tected by PCR (the affected individual did not have an MRC Holland guidelines (Table S4). Probes that met the evident PCR product for exon 2 on agarose gel electropho- recommended guidelines could not be designed for exons resis). This was then confirmed by MLPA (Figure S4) in the 3, 3a, and 5, and dosage analysis was thus not performed two affected children. MLPA results for the parents were for these exons. MLPA analysis was carried out according consistent with their being heterozygous for this deletion, to MRC Holland’s standard guidelines. Products were and they were thus confirmed as carriers (Figure S5). This analyzed via the Applied Biosystems ABI 3130xl genetic deletion is predicted to result in a frameshift. If the trun- analyzer and GeneMarker software (SoftGenetics LLC). cated protein were translated and not subject to nonsense- Two independent checks were performed for all results, mediated mRNA decay, it would result in a 25 amino acid and MLPA was repeated, for confirmation, for any patient protein (amino acids 1–23 would be wild-type) lacking found to have an abnormal result. the key catalytic RAB domains. We then analyzed a further A germline homozygous missense variant in RAB18 58 families affected by Warburg Micro or Martsolf c.71T>A (p.Leu24Gln) was identified in families 1–4 syndromes for mutations in RAB18 by using direct

502 The American Journal of Human Genetics 88, 499–507, April 8, 2011 Figure 3. RAB18 Mutations in Individuals c.274_276delAGAc.274_276delAGA with Warburg Micro Syndrome A c.71Tc.71T >A>A c.669T>Cc.669T>C (A) Sequence chromatograms of RAB18 mutations are shown. Affected individuals (top) and healthy controls (bottom). All mutations identiﬁed are shown with their amino acid and nucleotide positions labeled according to NM_021252.3, which is the only coding transcript in human brain and retina. Deletion (B) Sequence alignment of the N-terminal portion of Rab18s from different species. Sequence identity is indicated in blue, and sequence similarity is indicated in red. Switch regions, Leu24, and Arg93 are 1 2 3 4 5 6 7 labeled, secondary structure is indicated in cyan, and residues encoded by exon 2 of Homo sapiens RAB18 are enclosed in an orange box. Sequences were aligned by B the ClustalW algorithm (Thompson et al., 1994). Accession numbers of sequences used in the alignment were Homo sapiens NP_067075; Gallus gallus NP_001006355; Danio rerio rab18b NP_001003449.1; Drosophila melanogaster NP_524744.2, and Caenorhabditis elegans NP_741092.1. (C) Position of Leu24 and Arg93 within the RAB18 crystal structure. Leu24 and Arg93 are shown in yellow, the GTP analog GppNP and the magnesium ion are shown in green, and the switch regions are shown in red. Molecular coordinates for the RAB18 crystal structure were taken from work carried out by Kukimoto-Niino et al. (RCSB PDB code 1X3S).

C being obligate carriers. None of the sequence variants were identified in 400 ethnically matched control chromosomes. The main RAB18 transcript contains seven exons and encodes a 206 amino acid protein that is ubiq- uitously expressed in mammalian tissues and has particularly high expression in epithelia and brain. The first X-ray diffraction crystal of RAB18 released by Kukimoto-Niino et al. in 2005 (Protein Data Bank accession number 1X3S) confirmed the structural homology of RAB18 sequencing and MLPA. Two further RAB18 variants were with other members of the RAB family and showed detected in both affected siblings of family 6. These that it has the key switch 1 and 2 domains (Figure 3). As siblings are compound heterozygotes for an in-frame for other RAB proteins, the subcellular localization and arginine deletion in exon 7 c.277_279del (p.Arg93del) function of RAB18 is determined by its ability to bind and an anti-termination mutation of the stop codon nucleotides. The missense variants p.Leu24Gln and c.619T>C (p.X207GlnextX20), which is predicted to p.Arg93del both occur in highly conserved amino acid resi- extend RAB18 by 20 amino acids and thus abolish dues, and their location on folded RAB18 is shown in C-terminal prenylation and membrane targeting (Figure 3). Figure 3. The Leu24Gln variant occurs within the Their father was shown to be heterozygous for the conserved a1 helical domain, 2 amino acids downstream p.X207GlnextX20 variant, and their mother was heterozy- of the previously published artificially engineered inactive gous for the p.R93 del variant, consistent with their GDP-bound Ser22Asn mutant RAB18.18 To assess the

The American Journal of Human Genetics 88, 499–507, April 8, 2011 503 A B 20 min at 30 C. The reactions were stopped by the addi- 18 70 tion of 500 ml ice-cold wash buffer (50 mM HEPES-NaOH

16 60 [pH 6.8], 150 mM NaCl, and 1 mM MgCl2). Guanosine -bound RAB proteins were isolated with 20 ml of gluta- 14 50 thione-sepharose. The beads were washed three times 12 with 500 ml of the wash buffer, and scintillation was 40 counted. The RABs used for the nucleotide binding assays 10 were of similar quality and bound to the glutathione 8 30 beads equally (Figure 4). The nucleotide-binding assays

6 showed that although RAB18 bound GDP and GTP GTP binding (pmol) GDP binding (pmol) 20 comparably to other RABs (RAB5A and RAB35), neither 4 mutant protein bound detectable levels of either GDP 10 2 or GTP (Figure 4). The RAB18 (Leu24Gln) and RAB18 (Arg93del) mutations are therefore functionally null. Their 0 0 pathogenicity can be explained by their lack of guanosine 56 56 27 27 nucleotide binding, because, as for other RAB proteins, this RAB5A RAB35 RAB18 RAB18 RAB18 RAB18 RAB18 RAB18 GST GST is a prerequisite for RAB18’s correct subcellular localization and function. Arg93del Arg93delArg93del Leu24Gln Leu24Gln Because loss-of-function mutations in RAB18 cause Warburg Micro syndrome, we investigated the effect of knockdown of rab18 in zebrafish to establish whether Figure 4. GDP- and GTP-Binding Properties of Wild-Type and rab18 has a conserved role in brain and eye development. Disease-Associated Mutant Forms of RAB18 Zebrafish have two rab18 orthologs: rab18a (ZDB-GENE- (A) Wild-type RAB18 binds GDP similarly to RAB5A and RAB35, 030131-3980, with 81% orthology to human RAB18) and whereas RAB18(Arg93del), RAB18(Leu24Gln) and the negative rab18b (ZDB-GENE-040801-185, with 88% orthology to control protein GST do bind GDP. (B) RAB18 binds to GTP, but RAB18(Arg93del) and RAB18 the human RAB18). rab18a and rab18b were cloned into (Leu24Gln) are defective for GTP binding. Error bars indicate the pCS2 from mRNA derived by RT-PCR with total zebrafish standard deviation from the mean (n ¼ 3). The bead-bound pool RNA isolated from 24 dpf embryos. Dygoxigenin-labeled of the RABs is shown in the stained immunoblots below the graphs. antisense probes were synthesized as previously described.9 All are of similar quality and bind to the glutathione beads equally. All the primers used are included in Table S5. In situ hybrid- ization for rab18a and rab18b on whole-mount embryos at functional consequences of the missense variants, we 24 and 48 hr showed ubiquitous expression (Figure S7). therefore performed nucleotide-binding assays. Wild-type Antisense morpholino (MO) oligonucleotides (GeneTools) RAB18;RAB35 (MIM 604199) and RAB5A (MIM 179512) targeted to interfere with translation were designed against were cloned into pFAT2 (a His-GST-tagging vector modified both the transcription initiation codon ATG and the 50 UTR from pGAT2 to have a polylinker compatible with pQE32), region of the rab18a and rab18b mRNA (Table S5). Morpho- as described previously.19,20 Several RAB18 transcripts linos (400 pl) were injected into the yolk of one-cell-stage have been reported, but RT-PCR and sequencing of embryos at a concentrations of 25 mM (84 pg injected/ human brain, retina, and lymphoblastoid cell lines embryo) and 50 mM (168 pg injected/embryo). As a control identified a single RAB18 transcript that corresponds to for nonspecific effects, a 5 base mismatch of rab18a-MO1 or NM_021252.3 (Figure S6). Thus, wild-type and Leu24Gln rab18b-MO1 morpholinos was used. Embryos were raised at mutant transcripts were amplified from patient and 28C and staged in hours (hpf) and days (dpf) post-fertiliza- control lymphoblastoid cell lines. The p.Arg93del tion. Three independent MO injection experiments were mutation was introduced by the Quickchange method performed for each MO titration. At least 500 injected (Stratagene). His-GST-tagged wild-type and mutant RABs embryos were evaluated in total for each MO. were expressed in E. coli BL21(DE3)-pRIL and bound to The most common abnormalities observed at 3 dpf glutathione-sepharose (GE Healthcare) as described previ- in both the rab18a and rab18b morphants were micro- ously.20,21 Glutathione-sepharose-bound RAB proteins phthalmia, microcephaly, pericardial edema, delayed jaw were washed three times at 30C in 50 mM Tris-HCl formation, a reduced overall body size, and a general devel- (pH 7.4), 150 mM NaCl, and 20 mM EDTA and then eluted opmental delay (Figure S8). Further characterization of in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 20 mM the rab18b (the more conserved ortholog) eye phenotype reduced glutathione. For GDP- and GTP-binding assays, revealed that the rab18b morphants had delayed retinal either 2.5 mlof[3H]-GDP (Perkin Elmer) and 10 mMof development and abnormal retinal lamination, residual þ Mg2 -GDP or 0.1 mlof[35S]-GTPgS (Perkin Elmer) and nucleated lens fiber cells, widely open choroid fissure, þ 10 mMofMg2 -GTP were loaded into RAB or GST proteins and microphthalmia at day 3 (Figure S9). To assess the in 100 ml of reaction mixture (50 mM HEPES-NaOH specificity of the rab18b phenotype, we conducted a rescue [pH 6.8], 125 ml EDTA, 1 mg/ml BSA, and 1 mM DTT) for experiment by synthesizing rab18b mRNA. We performed

504 The American Journal of Human Genetics 88, 499–507, April 8, 2011 the rescue by injecting zygotes with rab18b-MO2 (which is that has been specifically localized to lipid droplets.18 Its directed against the 50 UTR) at 25 mM (84 pg injected/ 30 terminal is monoprenylated (in comparison to most embryo) or 5-mis-rab18b-MO together with rhodamine other Rab proteins) and undergoes carboxymethylation— dextran (1 mg/ml) followed by a consecutive injection, still modifications that might increase hydrophobicity and at the one-cell stage, with either rab18b mRNA (100 ng) determine the protein’s specificity for the LD.33 LDs are with CFP (cyan fluorescent protein) mRNA or CFP mRNA formed in the ER and are the primary site for storage of alone. Higher concentrations of mRNA were found to be neutral lipids (including cholesterol) in the cell. They are toxic. At 24 hpf, we assayed embryos for rhodamine and dynamic organelles that move around the cell and CFP fluorescence to select for successfully injected distribute lipids to other compartments by using specific embryos. Partial rescue of the eye defects (Figures S7, S8, docking machinery.34,35 Overexpression of RAB18 has and S9) pericardial edema and overall developmental recently been shown to induce lipid-droplet association delay (data not shown) was observed at 3 days. The micro- with the ER in HepG2 cells by expelling adipocyte differen- phthalmia and lens abnormalities would be consistent tiation-related protein from the lipid droplet.18 Cholesterol with the human eye phenotype. However, further experi- is a major component of lipid droplets, and its several roles ments controling for off-target effects (which can be due in the developing and adult brain could be important in to p53 activation), such as insertion of the human Warburg Micro syndrome pathogenesis. These roles include mutations into zebrafish and the creation of zebrafish those affecting glial cell proliferation, neurite outgrowth, transgenics, would clarify the specificity of these observed microtubule stability, synaptogenesis, myelination, and phenotypes. signaling. It is a precursor for steroid hormones and is an The cellular functions of RAB18 are still emerging, and indispensable component of nerve cell membranes, in- the regulators and effectors of the RAB18 cycle have not cluding the synaptic membrane. Cholesterol also has a yet been identified. Although initial studies in intestinal critical role in synaptic-vesicle biogenesis, exocytosis, and and kidney epithelial cells proposed a role for RAB18 in neuronal plasticity.36 Proteomic studies have suggested endocytosis,22 later studies in fibroblasts, neuroendocrine that RAB18 is associated with synaptic vesicles, and this cells, and adipocytes failed to localize RAB18 to early or raises the question as to whether RAB18 might play a role late endosomes. Instead, RAB18 colocalized with specific in intracellular lipid trafficking to these vesicles in neurons. markers of lipid droplets (LD) in 3T3-L1 adipocytes;18,23,24 Altered cholesterol trafficking in Warburg Micro syndrome secretory granules in neuroendocrine PC12, AtT20, and could thereby account for the exocytosis phenotype melanotrope cells;25,26 and the cis-Golgi and endoplasmic observed with RAB18 and RAB3GAP1. It is also striking reticulum (ER) in NRK, Vero cells, and fibroblasts.27 RAB18 that several brain, eye, and genital malformations seen in has been shown to act as a negative regulator of the regu- children with Warburg Micro syndrome also occur in a lated secretory pathway in neuroendocrine cells. Although disorder of cholesterol metabolism, Smith-Lemli-Opitz RAB3A has a similar role, these proteins associate with (MIM 270400) syndrome. distinct populations of secretory vesicles in neuroendo- Previously, no direct link between RAB3 and RAB18 crine cells, suggesting that these two GTPases act at pathways has been reported, but given that loss-of-func- different steps of the secretory process.25 Further studies tion mutations in RAB18 and RAB3GAP1 cause an indistin- identifying RAB18’s effectors will help to clarify its specific guishable phenotype, it seems likely that there is some role as an inhibitor of the secretory or exocytic pathway overlap between these pathways. One possibility is that and its relationship to RAB3GAP1, RAB3GAP2, and the RAB3GAP1 is not a specific regulator of the RAB3 family RAB3 pathway. Consistent with its role in exocytosis, but that it also regulates RAB18. Further functional studies RAB18 has been implicated in human disease. RAB18 will help elucidate the connection between these proteins. expression is downregulated in growth-hormone-secreting It is possible that RAB3GAP1 has additional targets or func- pituitary adenomas of patients with acromegaly26. Inter- tions that could be key in the pathogenesis of Warburg estingly, though, none of the Warburg Micro syndrome Micro syndrome. Interestingly, plants have orthologs of patients have growth-hormone or other pituitary defi- the RAB3GAP1 catalytic subunit but lack RAB3 orthologs, ciencies that would account for their short stature. Several which further supports this idea. No knockout animal microarray studies have also shown dysregulation of models for rab18 have been described, but our preliminary RAB18 in neuroendocrine gastointestinal tumors,28 pros- zebrafish studies suggest that rab18 might have a conserved tate adenocarcinoma,29 breast cancer,30 and pancreatic developmental role that could account for the structural adenocarcinoma.31 The exact role of RAB18 in the patho- abnormalities seen in these syndromes. Clearly, in humans physiology of these tumors is not known. RAB18 has a key role in eye and brain development and Although a recent study has suggested a distinct role for neurodegeneration. RAB18 in the retrograde Golgi-ER pathway in the COPI (coat protein)-independent pathway,27 previous systematic screens of all RAB proteins have failed to show a role for Supplemental Data 21,32 RAB18 in anterograde or retrograde Golgi trafficking. Supplemental data include nine figures and five tables and can be Interestingly, endogenous RAB18 is the only RAB protein found with this article online at http://www.cell.com/AJHG/.

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