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

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 ¨ller,1 Eamonn R. Maher,1,3 Francis A. Barr,2 and Irene A. Aligianis1,3,16,* Ferenc Mu 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, precisely coordinate vesicular trafficking in the cell. In humans, more than 60 RABs dynamically localize to distinct intracellular membranes, where their recruitment of effector proteins such as sorting adaptors, kinases, phosphatases, motors, and tethering factors regulates membrane identity and vesicle budding, motility, and fusion. RABs function as molecular switches that alternate between two conformational states: the GTP-bound ‘‘active’’ form and the GDP-bound ‘‘inactive’’ form. This allows them to associate and dissociate with target membranes and regulate membrane transport in a spatially and temporarily restricted manner.1,2 The switching of RAB GTPases is governed by four classes of proteins: GTPase-activating proteins (GAPs), guanine nucleotide exchange factors (GEFs), GDP dissociation inhibitors

(GDIs), and GDI displacement factors (GDFs).1 RAB GTPases are reversibly associated with membranes by hydrophobic geranylgeranyl groups that are attached to one or (in most cases) two carboxy-terminal cysteine residues. This prenylation is intrinsic to their role in regulating membrane traffic.1 RAB proteins can bind multiple effectors and have been shown individually and as a group to have diverse roles in human diseases such as immunodeficiency, cancer, and neurodegeneration.1,2 In terms of inherited disorders, loss-of-function mutations in RAB27A (MIM 603868) have been associated with autosomal-recessive Griselli syndrome (MIM 607624); loss-of-function mutations in RAB23 (MIM 604144) have been implicated in autosomal-recessive Carpenter syndrome (MIM 201000), and loss-of-function mutations in RAB39B (MIM 300774) cause X-linked mental retardation associated with autism,

1 Medical 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, ˆ pital d’Enfants Armand-Trousseau, 75571 Paris, France; 11Department University of Chicago, Chicago, IL 60637, USA; 10Service de Ge´ne´tique Me´dicale, Ho of Paediatric Ophthalmology, Birmingham Children’s Hospital, Birmingham B4 6NH, UK 12 Present address: Department of Cell Biology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-087, Japan 13 Present address: Institute of Child Health, London WC1N 1EH, UK 14 Present address: National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA 15 Present address: Institute of Neurology, University College London, London WC1E 6BT, UK 16 Present 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 RAB7 (MIM 602298) are associated with autosomal-dominant Charcot-Marie-Tooth disease type 2B (MIM 600882). Functionally, RAB proteins have been classified into those that regulate the endocytic versus the exocytic (secretory) pathways.2,3 The RAB3 proteins (RAB3A [MIM 179490]; RAB3B [MIM 179510]; RAB3C [MIM 612829], and RAB3D [MIM 604350]) modulate calcium-mediated exocytosis of neurotransmitters and hormones and are regulated by RAB3GAP.4–7 RAB3GAP is a heterodimeric complex consisting of a catalytic subunit (p130), which is encoded by RAB3GAP1 (MIM 602536) on chromosome 2q21.3, and a noncatalytic subunit (p150), encoded by RAB3GAP2 (MIM 609275) on chromosome 1q41.4,5 Mutations in these genes have been implicated in the pathogenesis of two clinically overlapping autosomal-recessive conditions, Warburg Micro syndrome (MIM 600118) and Martsolf syndrome (MIM 212720).8–11 Both disorders are characterized by ocular and neurodevelopmental manifestations; Warburg Micro syndrome is a more severe disorder.8–14 Recent molecular studies have established that these two syndromes represent a phenotypic continuum that appears to be related to the nature and severity of the mutations present in RAB3GAP1 and RAB3GAP2. Severe loss-of-function mutations in RAB3GAP1 have been reported in about 50% (17/35) of Warburg Micro syndrome patients.8,10 Two mutations have been reported in RAB3GAP2; these are a homozygous frameshift mutation in a family affected by Warburg Micro syndrome and a hypomorphic homozygous splicing mutation in a family affected by Martsolf syndrome.9,11 The cognitive deficits observed in these disorders are consistent with the reported role of the RAB3 pathway in cognition. Mutations in RABGDI1 (MIM 300104) (which is a nonspecific regulator of the RAB3 pathway) cause X-linked mental retardation,15 and a missense mutation in RIMS1 (MIM 606629) (a downstream effector of RAB3A) has been implicated in enhanced cognition.16 Furthermore, loss of rab3gap1 in mice resulted in abnormal release of synaptic vesicles and altered short-term synaptic plasticity in the hippocampus; these effects could contribute to the Warburg Micro cognitive phenotype.17 However, these mice were viable and fertile and had no structural eye, brain, or genital abnormalities, suggesting that there is differential species-specific redundancy in these pathways. Further locus heterogeneity has been demonstrated for Warburg Micro and Martsolf syndromes, suggesting that other genes also play a role in the pathogenesis of these disorders.8–11 Here, we report the identification and functional characterization of RAB18 mutations in six families affected by Warburg Micro syndrome. All family members and control subjects in this study provided written informed consent under a research protocol approved by the South Birmingham Local Research Ethics Committee in accordance with the Declaration of Helsinki. Genomic DNA was extracted from venous blood according to standard procedures.

Families 1–5 are consanguineous. Families 1–4 are of Pakistani origin, and family 5 is Turkish (pedigrees of families 1–5 are shown in Figure 1). The clinical phenotype for the families is summarized in Table S1 and is typical of Warburg Micro syndrome. The eye phenotype in the oldest affected child from families 1–4 was previously reported.12 All the affected children in these families presented at birth with congenital cataracts, microphthalmia, and microcornea. In addition, they have small atonic pupils that do not react to light or mydriatic agents. Despite early cataract surgery, their vision has remained poor (only light perception) as a result of progressive optic atrophy and severe cortical visual impairment (confirmed by normal electroretinogram [ERG] and absent visually evoked potentials [VEPs]). The progressive neurological deterioration observed in these children is highlighted in Table S1. Although they were born with normal head circumferences (50th–75th percentiles) and develop early milestones, such as smiling, they all have severe truncal hypotonia such that they have limited head control and, at the very most, only learn to sit with support. The children have achieved no developmental milestones beyond those at the 4 month level; they have not learned to crawl, pull up to a standing position, or walk. They developed postnatal acquired microcephaly within the first year of life, and their head circumferences fell to between
4SD and
6SD below the mean. Although some of the younger children have babbled, none have developed any recognizable words or speech. A characteristic pattern of progressive lower-limb spasticity started between 8 and 12 months of age, and upper-limb spasticity developed later, at about 8 years of age. The older children are profoundly delayed, are wheelchair bound, and have severe contractures of all four limbs, kyphoscoliosis, and no voluntary or spontaneous movement. Brain magnetic resonance imaging (MRI) findings are consistent with bilateral frontal polymicrogyria and thinning of the corpus callosum posteriorly in families 2 and 4 (Figure S1). The brain MRI in family 5 showed bilateral frontal gyration abnormalities and thinning of the corpus callosum posteriorly. Family 6 was reported by Graham et al.14 (as patients 1 and 2) as having typical Warburg Micro syndrome. The children from this family are the oldest known children with Warburg Micro syndrome and are aged 21 and 23. They have both had severe intractable epilepsy with myoclonic seizures from an early age (2 and 5 years), unlike the children in families 1–5 (some of whom have generalized tonic-clonic seizures). Their brain MRIs also show bilateral frontal polymicrogyria and thinning, of the corpus callosum.14 A nerve conduction study in the boy from family 6 was markedly abnormal due to severe loss of neurons suggesting an axonal peripheral neuropathy. Although microgenitalia in boys has been reported as a distinguishing feature in families with RAB3GAP1 mutations, the two boys from family 3 had normal genitalia. The children with RAB18 mutations all have facial dysmorphia similar to that of children with reported RAB3GAP1

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

I 127 129

127 123

123 123

125 123

183 183

183 191

183 187

187 187

194 201

201 196

201 194

196 196

196 196

260 267

260 267

267 257

267 267

244 248

248 248

126 116

116 104

122 104

126 126

126 120

127 119

123 119

193 191

193 193

193 193

199 193

193 199

193 191

193 197

195 197

237 237

237 237

237

237 237

237 237

237 237

237 241

243 247

241 247

247 241

241 247

247 247

247 241

241 247

247 247

247 247

247 247

247 245

243 245

26334192

188 180

180 188

188 180

188 188

180 188

184 188

188 180

188 188

188 180

200 180

D10S611

27647527

142 151

151 142

148

142 151

142 142

142 142

142 142

142 142

147 151

151 151

D10S1228

30167638

233 248

251 233

242 248

242 248

251 231

248 248

242 242

242 248

242 236

242 242

123 127

123 127

183 187

183 187

194 206

196 194

196 206

201 201

267 267

267 267

267 267

267 267

23745855

126 124

124 126

126 124

24612582

193 199

199 193

26ATCDC42 24572502

237 237

D10S572

25637143

D10S1160

D10S466

19559217

D10S595

20636874

123 117

D10S211

21907887

206 194

D10S1662

22936024

D10S1789 D10S1749

117 123

123 123

123 123

II 1.1

1.2

123 123

123 123

1.3

1.4

2.1

2.2

2.3

123 123

123 127

127 127

183 183

183 187

187 187

3.1

3.2

3.3

4.1

4.2

4.3

4.4

4.5

5.1

5.2

127 127

123 127

123 129

127 127

123 125

123 123

183 183

191 183

191 183

183 183

187 187

187 187

201 201

201 201

194 201

194 196

201 201

196 196

196 196

267 267

267 267

267 267

267 257

267 257

267 267

248 248

248 248

126 116

124 116

104 104

126 126

120 126

120 126

126 126

119 119

119 119

193 193

193 193

191 193

193 193

193 193

191 199

191 199

193 193

197 197

197 197

237 237

237 237

237

237

237 237

237 237

241 237

241 237

237 237

247 247

247 247

241 247

241 247

247 247

247 241

247 241

247 247

247 247

247 247

247 247

247 247

245 245

245 245

188 188

180 188

180 188

188 188

188 188

180 188

188 188

188 188

188 188

188 180

188 188

180 180

180 180

142 142

142 142

151 142

151 142

142 142

142 151

148 151

142 142

142 142

142 142

142 142

142 142

151 151

151 151

233 233

233 233

251 233

251 233

242 242

242 248

248 248

231 248

242 242

248 242

248 242

242 242

236 236

236 236

D10S466

19559217

D10S595

20636874

D10S211

21907887

206 206

206 206

194 206

194 206

196 196

196 206

194 206

D10S1662

22936024

267 267

267 267

267 267

267 267

267 267

267 267

D10S1789

23745855

126 126

126 126

124 126

124 126

126 126

D10S1749

24612582

193 193

193 193

199 193

199 193

26ATCDC42 24572502

237 237

237 237

237 237

D10S572

25637143

247 247

247 247

D10S1160

26334192

188 188

D10S611

27647527

D10S1228

30167638

117 123

117 123

123 123

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, antenatal diagnosis by ultrasound has been difficult, but in family 5, the affected fetus was terminated at 33 weeks after the identification of cataracts and abnormal brain gyration. RAB3GAP1 and RAB3GAP2 mutations were excluded in families 1–5 (by linkage and direct sequencing [Figure S2]). We then excluded the involvement of other genes encoding proteins implicated in the RAB3 pathway (RAB3A, RIMS1, RIMS2 [MIM 606630], RPH3A [MIM 612159], MADD [MIM 603584], WDR7 [MIM 613473], and DMXL2 [MIM 612186]) by genotyping closely flanking microsatellite markers (data not shown). We then performed a genome-wide linkage scan in five large consanguineous families by using the 250K SNP arrays (Affymetrix) to genotype 11 affected children and four unaffected siblings. DNA was amplified and hybridized to the Affymetrix 250K SNP chips according to the manufacturer’s instructions. To further investigate all significant regions of common homozygosity identified on genome-wide scan, we genotyped polymorphic UniSTS microsatellite markers spaced at 1 Mb intervals with GeneMapper software. In all the affected children from the five families, a 10113.089 kb region of shared homozygosity was identified on chromosome 10p12.1. The region contained 1055 SNPs and was flanked by SNPs rs943124 (20152.733 kb) and rs3847396 (30265.822 kb) (Figure S3). Families 1–4

were not known to be related but originated from the two neighboring villages in Pakistan (Mirpur). Interestingly, they shared a core 6056.698 kb haplotype (containing 680 SNPs) on chromosome 10p12.1 between rs12263288 (24209.124 kb) and rs3847396 (30265.822 kb) (Figure S3). Microsatellite studies in the extended families confirmed a founder effect in these families; the affected individuals shared a common homozygous haplotype between D10S1789 (23745.855 kb) and D10S1228 (30167.683 kb) (Figure 1). The genotyping was consistent with linkage; the parents were heterozygous (as obligate carriers), and the unaffected siblings had a haplotype different from that of their affected siblings. A maximum 2-point LOD score of Z ¼ 8.93 (q ¼ 0 at D10S1749) was calculated on the assembled haplotypes with GENEHUNTER (version 2.0b) under a model of autosomal-recessive inheritance with full penetrance; the disease-allele frequency was estimated at 1 in 1,000, thus confirming linkage to this region. The candidate region contained 23 genes (Figure S3). Intronic primer pairs were designed for exon-specific amplification of these 23 candidate genes (Figure S3 and Table S2) with Exon Primer. The RAB18 (MIM 602207) primers are shown in Table S3. After PCR amplification, sequencing reactions were performed with BigDye Terminator v3.1 Cycle Sequencing kit and run on an 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 most abundant RAB18 isoform, which is also present in the brain (NM_021252.3 or RAB18-001: ENST00000356940). We carried out multiplex ligation-dependent probe amplification (MLPA) to detect deletions. Synthetic probes for exons 1, 1a, 1b, 2, 4, 6, and 7 were designed according to MRC Holland guidelines (Table S4). Probes that met the recommended guidelines could not be designed for exons 3, 3a, and 5, and dosage analysis was thus not performed for these exons. MLPA analysis was carried out according to MRC Holland’s standard guidelines. Products were analyzed via the Applied Biosystems ABI 3130xl genetic analyzer and GeneMarker software (SoftGenetics LLC). Two independent checks were performed for all results, and MLPA was repeated, for confirmation, for any patient found to have an abnormal result. A germline homozygous missense variant in RAB18 c.71T>A (p.Leu24Gln) was identified in families 1–4

(Figure 3). This sequence variant segregated with disease status in the families (the parents were heterozygous, consistent with carrier status, and unaffected siblings had wild-type sequence or were heterozygous). In family 5, a homozygous deletion of RAB18 exon 2 was initially detected by PCR (the affected individual did not have an evident PCR product for exon 2 on agarose gel electrophoresis). This was then confirmed by MLPA (Figure S4) in the two affected children. MLPA results for the parents were consistent with their being heterozygous for this deletion, and they were thus confirmed as carriers (Figure S5). This deletion is predicted to result in a frameshift. If the truncated protein were translated and not subject to nonsensemediated mRNA decay, it would result in a 25 amino acid protein (amino acids 1–23 would be wild-type) lacking the key catalytic RAB domains. We then analyzed a further 58 families affected by Warburg Micro or Martsolf syndromes for mutations in RAB18 by using direct

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

c.274_276delAGA

c.71T >A

A

c.669T>C

Deletion

1

2

3

4

5

B

6

7

Figure 3. RAB18 Mutations in Individuals with Warburg Micro Syndrome (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. (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 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 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

sequencing and MLPA. Two further RAB18 variants were detected in both affected siblings of family 6. These siblings are compound heterozygotes for an in-frame arginine deletion in exon 7 c.277_279del (p.Arg93del) and an anti-termination mutation of the stop codon c.619T>C (p.X207GlnextX20), which is predicted to extend RAB18 by 20 amino acids and thus abolish C-terminal prenylation and membrane targeting (Figure 3). Their father was shown to be heterozygous for the p.X207GlnextX20 variant, and their mother was heterozygous for the p.R93 del variant, consistent with their

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 ubiquitously 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 with other members of the RAB family and showed that it has the key switch 1 and 2 domains (Figure 3). As for other RAB proteins, the subcellular localization and function of RAB18 is determined by its ability to bind nucleotides. The missense variants p.Leu24Gln and p.Arg93del both occur in highly conserved amino acid residues, and their location on folded RAB18 is shown in Figure 3. The Leu24Gln variant occurs within the conserved a1 helical domain, 2 amino acids downstream of the previously published artificially engineered inactive GDP-bound Ser22Asn mutant RAB18.18 To assess the

The American Journal of Human Genetics 88, 499–507, April 8, 2011 503

A

B

18

70

16

60

GTP binding (pmol)

GDP binding (pmol)

14 12 10 8 6

50 40 30 20

4 10

2 0 56

27

0 56

27 GST

RAB18 Leu24Gln

RAB18 Arg93del

RAB18

GST

RAB18 Leu24Gln

RAB18 Arg93del

RAB18

RAB35

RAB5A

Figure 4. GDP- and GTP-Binding Properties of Wild-Type and Disease-Associated Mutant Forms of RAB18 (A) Wild-type RAB18 binds GDP similarly to RAB5A and RAB35, whereas RAB18(Arg93del), RAB18(Leu24Gln) and the negative control protein GST do bind GDP. (B) RAB18 binds to GTP, but RAB18(Arg93del) and RAB18 (Leu24Gln) are defective for GTP binding. Error bars indicate the standard deviation from the mean (n ¼ 3). The bead-bound pool of the RABs is shown in the stained immunoblots below the graphs. All are of similar quality and bind to the glutathione beads equally.

functional consequences of the missense variants, we therefore performed nucleotide-binding assays. Wild-type RAB18;RAB35 (MIM 604199) and RAB5A (MIM 179512) were cloned into pFAT2 (a His-GST-tagging vector modified from pGAT2 to have a polylinker compatible with pQE32), as described previously.19,20 Several RAB18 transcripts have been reported, but RT-PCR and sequencing of human brain, retina, and lymphoblastoid cell lines identified a single RAB18 transcript that corresponds to NM_021252.3 (Figure S6). Thus, wild-type and Leu24Gln mutant transcripts were amplified from patient and control lymphoblastoid cell lines. The p.Arg93del mutation was introduced by the Quickchange method (Stratagene). His-GST-tagged wild-type and mutant RABs were expressed in E. coli BL21(DE3)-pRIL and bound to glutathione-sepharose (GE Healthcare) as described previously.20,21 Glutathione-sepharose-bound RAB proteins were washed three times at 30 C in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 20 mM EDTA and then eluted in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 20 mM reduced glutathione. For GDP- and GTP-binding assays, either 2.5 ml of [3H]-GDP (Perkin Elmer) and 10 mM of Mg2þ-GDP or 0.1 ml of [35S]-GTPgS (Perkin Elmer) and 10 mM of Mg2þ-GTP were loaded into RAB or GST proteins in 100 ml of reaction mixture (50 mM HEPES-NaOH [pH 6.8], 125 ml EDTA, 1 mg/ml BSA, and 1 mM DTT) for

20 min at 30 C. The reactions were stopped by the addition of 500 ml ice-cold wash buffer (50 mM HEPES-NaOH [pH 6.8], 150 mM NaCl, and 1 mM MgCl2). Guanosine -bound RAB proteins were isolated with 20 ml of glutathione-sepharose. The beads were washed three times with 500 ml of the wash buffer, and scintillation was counted. The RABs used for the nucleotide binding assays were of similar quality and bound to the glutathione beads equally (Figure 4). The nucleotide-binding assays showed that although RAB18 bound GDP and GTP comparably to other RABs (RAB5A and RAB35), neither mutant protein bound detectable levels of either GDP or GTP (Figure 4). The RAB18 (Leu24Gln) and RAB18 (Arg93del) mutations are therefore functionally null. Their pathogenicity can be explained by their lack of guanosine nucleotide binding, because, as for other RAB proteins, this is a prerequisite for RAB18’s correct subcellular localization and function. Because loss-of-function mutations in RAB18 cause Warburg Micro syndrome, we investigated the effect of knockdown of rab18 in zebrafish to establish whether rab18 has a conserved role in brain and eye development. Zebrafish have two rab18 orthologs: rab18a (ZDB-GENE030131-3980, with 81% orthology to human RAB18) and rab18b (ZDB-GENE-040801-185, with 88% orthology to the human RAB18). rab18a and rab18b were cloned into pCS2 from mRNA derived by RT-PCR with total zebrafish RNA isolated from 24 dpf embryos. Dygoxigenin-labeled antisense probes were synthesized as previously described.9 All the primers used are included in Table S5. In situ hybridization for rab18a and rab18b on whole-mount embryos at 24 and 48 hr showed ubiquitous expression (Figure S7). Antisense morpholino (MO) oligonucleotides (GeneTools) targeted to interfere with translation were designed against both the transcription initiation codon ATG and the 50 UTR region of the rab18a and rab18b mRNA (Table S5). Morpholinos (400 pl) were injected into the yolk of one-cell-stage embryos at a concentrations of 25 mM (84 pg injected/ embryo) and 50 mM (168 pg injected/embryo). As a control for nonspecific effects, a 5 base mismatch of rab18a-MO1 or rab18b-MO1 morpholinos was used. Embryos were raised at 28 C and staged in hours (hpf) and days (dpf) post-fertilization. Three independent MO injection experiments were performed for each MO titration. At least 500 injected embryos were evaluated in total for each MO. The most common abnormalities observed at 3 dpf in both the rab18a and rab18b morphants were microphthalmia, microcephaly, pericardial edema, delayed jaw formation, a reduced overall body size, and a general developmental delay (Figure S8). Further characterization of the rab18b (the more conserved ortholog) eye phenotype revealed that the rab18b morphants had delayed retinal development and abnormal retinal lamination, residual nucleated lens fiber cells, widely open choroid fissure, and microphthalmia at day 3 (Figure S9). To assess the specificity of the rab18b phenotype, we conducted a rescue 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 directed against the 50 UTR) at 25 mM (84 pg injected/ embryo) or 5-mis-rab18b-MO together with rhodamine dextran (1 mg/ml) followed by a consecutive injection, still at the one-cell stage, with either rab18b mRNA (100 ng) with CFP (cyan fluorescent protein) mRNA or CFP mRNA alone. Higher concentrations of mRNA were found to be toxic. At 24 hpf, we assayed embryos for rhodamine and CFP fluorescence to select for successfully injected embryos. Partial rescue of the eye defects (Figures S7, S8, and S9) pericardial edema and overall developmental delay (data not shown) was observed at 3 days. The microphthalmia and lens abnormalities would be consistent with the human eye phenotype. However, further experiments controling for off-target effects (which can be due to p53 activation), such as insertion of the human mutations into zebrafish and the creation of zebrafish transgenics, would clarify the specificity of these observed phenotypes. The cellular functions of RAB18 are still emerging, and the regulators and effectors of the RAB18 cycle have not yet been identified. Although initial studies in intestinal and kidney epithelial cells proposed a role for RAB18 in endocytosis,22 later studies in fibroblasts, neuroendocrine cells, and adipocytes failed to localize RAB18 to early or late endosomes. Instead, RAB18 colocalized with specific markers of lipid droplets (LD) in 3T3-L1 adipocytes;18,23,24 secretory granules in neuroendocrine PC12, AtT20, and melanotrope cells;25,26 and the cis-Golgi and endoplasmic reticulum (ER) in NRK, Vero cells, and fibroblasts.27 RAB18 has been shown to act as a negative regulator of the regulated secretory pathway in neuroendocrine cells. Although RAB3A has a similar role, these proteins associate with distinct populations of secretory vesicles in neuroendocrine cells, suggesting that these two GTPases act at different steps of the secretory process.25 Further studies identifying RAB18’s effectors will help to clarify its specific role as an inhibitor of the secretory or exocytic pathway and its relationship to RAB3GAP1, RAB3GAP2, and the RAB3 pathway. Consistent with its role in exocytosis, RAB18 has been implicated in human disease. RAB18 expression is downregulated in growth-hormone-secreting pituitary adenomas of patients with acromegaly26. Interestingly, though, none of the Warburg Micro syndrome patients have growth-hormone or other pituitary deficiencies that would account for their short stature. Several microarray studies have also shown dysregulation of RAB18 in neuroendocrine gastointestinal tumors,28 prostate adenocarcinoma,29 breast cancer,30 and pancreatic adenocarcinoma.31 The exact role of RAB18 in the pathophysiology of these tumors is not known. Although a recent study has suggested a distinct role for 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 RAB18 in anterograde or retrograde Golgi trafficking.21,32 Interestingly, endogenous RAB18 is the only RAB protein

that has been specifically localized to lipid droplets.18 Its 30 terminal is monoprenylated (in comparison to most other Rab proteins) and undergoes carboxymethylation— modifications that might increase hydrophobicity and determine the protein’s specificity for the LD.33 LDs are formed in the ER and are the primary site for storage of neutral lipids (including cholesterol) in the cell. They are dynamic organelles that move around the cell and distribute lipids to other compartments by using specific docking machinery.34,35 Overexpression of RAB18 has recently been shown to induce lipid-droplet association with the ER in HepG2 cells by expelling adipocyte differentiation-related protein from the lipid droplet.18 Cholesterol is a major component of lipid droplets, and its several roles in the developing and adult brain could be important in Warburg Micro syndrome pathogenesis. These roles include those affecting glial cell proliferation, neurite outgrowth, microtubule stability, synaptogenesis, myelination, and signaling. It is a precursor for steroid hormones and is an indispensable component of nerve cell membranes, including the synaptic membrane. Cholesterol also has a critical role in synaptic-vesicle biogenesis, exocytosis, and neuronal plasticity.36 Proteomic studies have suggested that RAB18 is associated with synaptic vesicles, and this raises the question as to whether RAB18 might play a role in intracellular lipid trafficking to these vesicles in neurons. Altered cholesterol trafficking in Warburg Micro syndrome could thereby account for the exocytosis phenotype observed with RAB18 and RAB3GAP1. It is also striking that several brain, eye, and genital malformations seen in children with Warburg Micro syndrome also occur in a disorder of cholesterol metabolism, Smith-Lemli-Opitz (MIM 270400) syndrome. Previously, no direct link between RAB3 and RAB18 pathways has been reported, but given that loss-of-function mutations in RAB18 and RAB3GAP1 cause an indistinguishable phenotype, it seems likely that there is some overlap between these pathways. One possibility is that RAB3GAP1 is not a specific regulator of the RAB3 family but that it also regulates RAB18. Further functional studies will help elucidate the connection between these proteins. It is possible that RAB3GAP1 has additional targets or functions that could be key in the pathogenesis of Warburg Micro syndrome. Interestingly, plants have orthologs of the RAB3GAP1 catalytic subunit but lack RAB3 orthologs, which further supports this idea. No knockout animal models for rab18 have been described, but our preliminary zebrafish studies suggest that rab18 might have a conserved developmental role that could account for the structural abnormalities seen in these syndromes. Clearly, in humans RAB18 has a key role in eye and brain development and neurodegeneration.

Supplemental Data Supplemental data include nine figures and five tables and can be found with this article online at http://www.cell.com/AJHG/.

The American Journal of Human Genetics 88, 499–507, April 8, 2011 505

Acknowledgments We thank the families who helped with this research; many colleagues for referring affected families; and the West Midlands Regional Genetics laboratory, with whom we worked closely. We thank the UK Newlife Charity and WellChild for financial support. Work in the group of F.A.B. was supported by a Wellcome Trust programme award (082467/Z/07/Z). I.A.A. is funded through a Programme Leader Development Track Fellowship from the Medical Research Council.

10.

11.

Received: January 21, 2011 Revised: March 8, 2011 Accepted: March 16, 2011 Published online: April 7, 2011

12.

Web Resources

13.

The URLs for data provided herein are as follows: dbSNP, http://www.ncbi.nlm.nih.gov/projects/SNP/ Entrez Gene, http://www.ncbi.nlm.nih.gov/gene/ Ensembl: http://www.ensembl.org Exon Primer: http://ihg2.helmholtz-muenchen.de/ihg/ ExonPrimer.html NCBI: http://www.ncbi.nlm.nih.gov/projects/mapview/map_search Online Mendelian Inheritance in Man (OMIM), http://www.ncbi. nlm.nih.gov/omim/ Protein Data Bank, http://www.rcsb.org/pdb UCSC Genome Bioinformatics: http://genome.ucsc.edu/ UniSTS: http://www.ncbi.nlm.nih.gov/sites/entrez?db¼unists Zebrafish Model Organism Database: http://zfin.org

14.

15.

16.

17.

References 1. Stenmark, H. (2009). Rab GTPases as coordinators of vesicle traffic. Nat. Rev. Mol. Cell Biol. 10, 513–525. 2. Zerial, M., and McBride, H. (2001). Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol. 2, 107–117. 3. Schwartz, S.L., Cao, C., Pylypenko, O., Rak, A., and Wandinger-Ness, A. (2007). Rab GTPases at a glance. J. Cell Sci. 120, 3905–3910. 4. Fukui, K., Sasaki, T., Imazumi, K., Matsuura, Y., Nakanishi, H., and Takai, Y. (1997). Isolation and characterization of a GTPase activating protein specific for the Rab3 subfamily of small G proteins. J. Biol. Chem. 272, 4655–4658. 5. Nagano, F., Sasaki, T., Fukui, K., Asakura, T., Imazumi, K., and Takai, Y. (1998). Molecular cloning and characterization of the noncatalytic subunit of the Rab3 subfamily-specific GTPaseactivating protein. J. Biol. Chem. 273, 24781–24785. 6. Geppert, M., Goda, Y., Stevens, C.F., and Sudhof, T.C. (1997). The small GTP-binding protein Rab3A regulates a late step in synaptic vesicle fusion. Nature 387, 810–814. 7. Geppert, M., Bolshakov, V.Y., Siegelbaum, S.A., Takei, K., De, C.P., Hammer, R.E., and Sudhof, T.C. (1994). The role of Rab3A in neurotransmitter release. Nature 369, 493–497. 8. Aligianis, I.A., Johnson, C.A., Gissen, P., Chen, D., Hampshire, D., Hoffmann, K., Maina, E.N., Morgan, N.V., Tee, L., Morton, J., et al. (2005). Mutations of the catalytic subunit of RAB3GAP cause Warburg Micro syndrome. Nat. Genet. 37, 221–223. 9. Aligianis, I.A., Morgan, N.V., Mione, M., Johnson, C.A., Rosser, E., Hennekam, R.C., Adams, G., Trembath, R.C., Pilz, D.T., Stoodley, N., et al. (2006). Mutation in Rab3 GTPase-activating

18.

19.

20.

21.

22.

23.

24.

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

protein (RAB3GAP) noncatalytic subunit in a kindred with Martsolf syndrome. Am. J. Hum. Genet. 78, 702–707. Morris-Rosendahl, D.J., Segel, R., Born, A.P., Conrad, C., Loeys, B., Brooks, S.S., Muller, L., Zeschnigk, C., Botti, C., Rabinowitz, R., et al. (2010). New RAB3GAP1 mutations in patients with Warburg Micro Syndrome from different ethnic backgrounds and a possible founder effect in the Danish. Eur. J. Hum. Genet. 18, 1100–1106. Borck, G., Wunram, H., Steiert, A., Volk, A.E., Korber, F., Roters, S., Herkenrath, P., Wollnik, B., Morris-Rosendahl, D.J., and Kubisch, C. (2010). A homozygous RAB3GAP2 mutation causes Warburg Micro syndrome. Hum. Genet. 129, 45–50. Ainsworth, J.R., Morton, J.E., Good, P., Woods, C.G., George, N.D., Shield, J.P., Bradbury, J., Henderson, M.J., and Chhina, J. (2001). Micro syndrome in Muslim Pakistan children. Ophthalmology 108, 491–497. Yuksel, A., Yesil, G., Aras, C., and Seven, M. (2007). Warburg Micro syndrome in a Turkish boy. Clin. Dysmorphol. 16, 89–93. Graham, J.M., Jr., Hennekam, R., Dobyns, W.B., Roeder, E., and Busch, D. (2004). MICRO syndrome: An entity distinct from COFS syndrome. Am. J. Med. Genet. A. 128A, 235–245. D’Adamo, P., Menegon, A., Lo, N.C., Grasso, M., Gulisano, M., Tamanini, F., Bienvenu, T., Gedeon, A.K., Oostra, B., Wu, S.K., et al. (1998). Mutations in GDI1 are responsible for X-linked non-specific mental retardation. Nat. Genet. 19, 134–139. Sisodiya, S.M., Thompson, P.J., Need, A., Harris, S.E., Weale, M.E., Wilkie, S.E., Michaelides, M., Free, S.L., Walley, N., Gumbs, C., et al. (2007). Genetic enhancement of cognition in a kindred with cone-rod dystrophy due to RIMS1 mutation. J. Med. Genet. 44, 373–380. Sakane, A., Manabe, S., Ishizaki, H., Tanaka-Okamoto, M., Kiyokage, E., Toida, K., Yoshida, T., Miyoshi, J., Kamiya, H., Takai, Y., and Sasaki, T. (2006). Rab3 GTPase-activating protein regulates synaptic transmission and plasticity through the inactivation of Rab3. Proc. Natl. Acad. Sci. USA 103, 10029– 10034. Ozeki, S., Cheng, J., Tauchi-Sato, K., Hatano, N., Taniguchi, H., and Fujimoto, T. (2005). Rab18 localizes to lipid droplets and induces their close apposition to the endoplasmic reticulumderived membrane. J. Cell Sci. 118, 2601–2611. Haas, A.K., Fuchs, E., Kopajtich, R., and Barr, F.A. (2005). A GTPase-activating protein controls Rab5 function in endocytic trafficking. Nat. Cell Biol. 7, 887–893. Yoshimura, S., Haas, A.K., and Barr, F.A. (2008). Analysis of Rab GTPase and GTPase-activating protein function at primary cilia. Methods Enzymol. 439, 353–364. Fuchs, E., Haas, A.K., Spooner, R.A., Yoshimura, S., Lord, J.M., and Barr, F.A. (2007). Specific Rab GTPase-activating proteins define the Shiga toxin and epidermal growth factor uptake pathways. J. Cell Biol. 177, 1133–1143. Lutcke, A., Parton, R.G., Murphy, C., Olkkonen, V.M., Dupree, P., Valencia, A., Simons, K., and Zerial, M. (1994). Cloning and subcellular localization of novel rab proteins reveals polarized and cell type-specific expression. J. Cell Sci. 107, 3437–3448. Martin, S., Driessen, K., Nixon, S.J., Zerial, M., and Parton, R.G. (2005). Regulated localization of Rab18 to lipid droplets: effects of lipolytic stimulation and inhibition of lipid droplet catabolism. J. Biol. Chem. 280, 42325–42335. Martin, S., and Parton, R.G. (2008). Characterization of Rab18, a lipid droplet-associated small GTPase. Methods Enzymol. 438, 109–129.

25. Vazquez-Martinez, R., Cruz-Garcia, D., Duran-Prado, M., Peinado, J.R., Castano, J.P., and Malagon, M.M. (2007). Rab18 inhibits secretory activity in neuroendocrine cells by interacting with secretory granules. Traffic 8, 867–882. 26. Vazquez-Martinez, R., Martinez-Fuentes, A.J., Pulido, M.R., Jimenez-Reina, L., Quintero, A., Leal-Cerro, A., Soto, A., Webb, S.M., Sucunza, N., Bartumeus, F., et al. (2008). Rab18 is reduced in pituitary tumors causing acromegaly and its overexpression reverts growth hormone hypersecretion. J. Clin. Endocrinol. Metab. 93, 2269–2276. 27. Dejgaard, S.Y., Murshid, A., Erman, A., Kizilay, O., Verbich, D., Lodge, R., Dejgaard, K., Ly-Hartig, T.B., Pepperkok, R., Simpson, J.C., and Presley, J.F. (2008). Rab18 and Rab43 have key roles in ER-Golgi trafficking. J. Cell Sci. 121, 2768–2781. 28. Hofsli, E., Thommesen, L., Yadetie, F., Langaas, M., Kusnierczyk, W., Falkmer, U., Sandvik, A.K., and Laegreid, A. (2005). Identification of novel growth factor-responsive genes in neuroendocrine gastrointestinal tumour cells. Br. J. Cancer 92, 1506–1516. 29. True, L., Coleman, I., Hawley, S., Huang, C.Y., Gifford, D., Coleman, R., Beer, T.M., Gelmann, E., Datta, M., Mostaghel, E., et al. (2006). A molecular correlate to the Gleason grading system for prostate adenocarcinoma. Proc. Natl. Acad. Sci. USA 103, 10991–10996. 30. Yan, L.X., Huang, X.F., Shao, Q., Huang, M.Y., Deng, L., Wu, Q.L., Zeng, Y.X., and Shao, J.Y. (2008). MicroRNA miR-21 overexpression in human breast cancer is associated with

31.

32.

33.

34.

35.

36.

advanced clinical stage, lymph node metastasis and patient poor prognosis. RNA 14, 2348–2360. Kalinina, T., Gungor, C., Thieltges, S., Moller-Krull, M., Penas, E.M., Wicklein, D., Streichert, T., Schumacher, U., Kalinin, V., Simon, R., et al. (2010). Establishment and characterization of a new human pancreatic adenocarcinoma cell line with high metastatic potential to the lung. BMC Cancer 10, 295. Haas, A.K., Yoshimura, S., Stephens, D.J., Preisinger, C., Fuchs, E., and Barr, F.A. (2007). Analysis of GTPase-activating proteins: Rab1 and Rab43 are key Rabs required to maintain a functional Golgi complex in human cells. J. Cell Sci. 120, 2997–3010. Leung, K.F., Baron, R., Ali, B.R., Magee, A.I., and Seabra, M.C. (2007). Rab GTPases containing a CAAX motif are processed post-geranylgeranylation by proteolysis and methylation. J. Biol. Chem. 282, 1487–1497. Liu, P., Bartz, R., Zehmer, J.K., Ying, Y., and Anderson, R.G. (2008). Rab-regulated membrane traffic between adiposomes and multiple endomembrane systems. Methods Enzymol. 439, 327–337. Zehmer, J.K., Huang, Y., Peng, G., Pu, J., Anderson, R.G., and Liu, P. (2009). A role for lipid droplets in inter-membrane lipid traffic. Proteomics 9, 914–921. Liu, J.P., Tang, Y., Zhou, S., Toh, B.H., McLean, C., and Li, H. (2009). Cholesterol involvement in the pathogenesis of neurodegenerative diseases. Mol. Cell. Neurosci. 43, 33–42.

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