Mutation Spectrum in RAB 3 GAP 1 , RAB 3 GAP 2 , and RAB 18 and Genotype-Phenotype Correlations in Warburg Micro Syndrome and Martsolf Syndrome

MUTATION UPDATEOFFICIAL JOURNAL

www.hgvs.org

Mutation Spectrum in RAB3GAP1, RAB3GAP2, and RAB18and Genotype–Phenotype Correlations in Warburg MicroSyndrome and Martsolf Syndrome

Mark T. Handley,1 ‡ Deborah J. Morris-Rosendahl,2,29 ‡† Stephen Brown,1 Fiona Macdonald,3 Carol Hardy,3 Danai Bem,4

Sarah M. Carpanini,1 Guntram Borck,5 Loreto Martorell,6 Claudia Izzi,7 Francesca Faravelli,8 Patrizia Accorsi,9

Lorenzo Pinelli,10 Lina Basel-Vanagaite,11,12 Gabriela Peretz,11 Ghada M.H. Abdel-Salam,13 Maha S. Zaki,13 Anna Jansen,14

David Mowat,15 Ian Glass,16 Helen Stewart,17 Grazia Mancini,18 Damien Lederer,19 Tony Roscioli,20,21 Fabienne Giuliano,22

Astrid S. Plomp,23 Arndt Rolfs,24,25 John M. Graham,26 Eva Seemanova,27 Pilar Poo,28 Angels Garcıa-Cazorla,28

Patrick Edery,29 Ian J. Jackson,1 Eamonn R. Maher,4,30 and Irene A. Aligianis1 ∗

1MRC Human Genetics Unit, Medical Research Council and Institute of Genetics and Molecular Medicine University of Edinburgh, Edinburgh,Scotland, UK; 2Institute of Human Genetics Albert-Ludwigs University Medical Centre Freiburg, Freiburg, Germany; 3West Midlands RegionalGenetics Laboratory Birmingham Women’s Hospital, Birmingham, UK; 4Centre for Rare Diseases and Personalised Medicine, School of Clinicaland Experimental Medicine, College of Medical and Dental Sciences University of Birmingham, Edgbaston, Birmingham, UK; 5Institute of HumanGenetics University of Ulm, Ulm, Germany; 6Molecular Genetics Section Hospital Sant Joan de Deu, Barcelona, Spain; 7Department of Obstetricsand Gynaecology University of Brescia, Spedali Civili, Brescia, Italy; 8Division of Medical Genetics Galliera Hospital, Genova, Italy; 9 Departmentof Child Neurology and Psychiatry, Spedali Civili, Brescia, Italy; 10 Department of Neuroradiology, Spedali Civili, Brescia, Italy; 11SchneiderChildren’s Medical Center of Israel and Raphael Recanati Genetics Institute Rabin Medical Center, Beilinson Campus, Petah Tiqva, Israel;12Sackler School of Medicine Tel Aviv University, Tel Aviv, Israel; 13Department of Clinical Genetics, Human Genetics and Genome ResearchDivision National Research Centre, Cairo, Egypt; 14Pediatric Neurology Unit Department of Pediatrics, UZ, Brussel; 15Department of MedicalGenetics Sydney Children’s Hospital, Sydney, Australia; 16Division of Genetics and Developmental Medicine, Department of Pediatrics Universityof Washington, Seattle, USA; 17Clinical Genetics Churchill Hospital, Oxford, UK; 18Department of Genetics Erasmus University Medical Center,Rotterdam, The Netherlands; 19 Institut de Pathologie et de Genetique, Gosselies, Belgium; 20Department of Human Genetics, Nijmegen Centre forMolecular Life Sciences Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 21School of Women’s and Children’s HealthSydney Children’s Hospital and the University of New South Wales, Sydney, Australia; 22 Centre Hospitalier Universitaire de Nice, Hopital del’Archet 2, Nice, France; 23Department of Clinical Genetics Amsterdam Medical Center, Amsterdam, The Netherlands; 24Albrecht Kossel Institutefor Neuroregeneration University of Rostock, Rostock, Germany; 25Centogene AG Institute for rare diseases, Rostock, Germany; 26Division ofClinical Genetics and Dysmorphology, Medical Genetics Institute Cedars-Sinai Medical Centre, Los Angeles, USA; 27Institute of Biology andMedical Genetics Charles University Prague 2nd Medical School, Prague, Czech Republic; 28Neurology Department Hospital Sant Joan de Deu,Barcelona, Spain; 29Department of Genetics Hospices Civils de Lyon, Bron, France; 30West Midlands Regional Genetics Service BirminghamWomen’s Hospital NHS Trust, Birmingham

Communicated by Graham R. TaylorReceived 6 December 2012; accepted revised manuscript 7 February 2013.Published online 19 February 2013 in Wiley Online Library (www.wiley.com/humanmutation). DOI: 10.1002/humu.22296

ABSTRACT: Warburg Micro syndrome and Martsolf syn-drome (MS) are heterogeneous autosomal-recessive de-

Present address: National Heart and Lung Institute, Imperial College, London, UK

Additional Supporting Information may be found in the online version of this article.†Deborah J. Morris-Rosendahl’s present address is National Heart and Lung Insti-

tute, Imperial College, London, UK.‡These authors have contributed equally to the manuscript.∗Correspondence to: Dr Irene Aligianis, MRC Human Genetics Unit, Institute of

Genetics and Molecular Medicine (IGMM), University of Edinburgh, Western Gen-

eral Hospital, Crewe Road; Edinburgh EH4 2XU; Scotland, United Kingdom E-mail:

[email protected]

Contract grant sponsors: Newlife: Molecular Investigations of Micro and Martsolf

Syndromes (07-08/12); MRC Human Genetics Unit Program leader Track Fellowship

(RA1631 and RA1905).

velopmental disorders characterized by brain, eye, andendocrine abnormalities. Causative biallelic germline mu-tations have been identified in RAB3GAP1, RAB3GAP2,or RAB18, each of which encode proteins involved inmembrane trafficking. This report provides an up to dateoverview of all known disease variants identified in 29previously published families and 52 new families. One-hundred and forty-four Micro and nine Martsolf familieswere investigated, identifying mutations in RAB3GAP1 in41% of cases, mutations in RAB3GAP2 in 7% of cases,and mutations in RAB18 in 5% of cases. These are listedin Leiden Open source Variation Databases, which wascreated by us for all three genes. Genotype–phenotypecorrelations for these genes have now established that theclinical phenotypes in Micro syndrome and MS representa phenotypic continuum related to the nature and severityof the mutations present in the disease genes, with more

C© 2013 WILEY PERIODICALS, INC.

deleterious mutations causing Micro syndrome and mildermutations causing MS. RAB18 has not yet been linked tothe RAB3 pathways, but mutations in all three genes causean indistinguishable phenotype, making it likely that thereis some overlap. There is considerable genetic heterogene-ity for these disorders and further gene identification willhelp delineate these pathways.Hum Mutat 34:686–696, 2013. C© 2013 Wiley Periodicals, Inc.

KEY WORDS: Micro; Martsolf; Rab; RAB3GAP1;RAB3GAP2; RAB18

BackgroundMicro syndrome (MIM# 600118) and Martsolf syndrome (MS;

MIM# 212720) are phenotypically overlapping autosomal recessivedisorders that were first described in 1993 and 1978, respectively[Martsolf et al., 1978; Warburg et al., 1993]. Multiple case reportsand subsequent gene identification have helped to clearly delineatethe clinical features associated with Micro syndrome [Abdel-Salamet al., 2007; Ainsworth et al., 2001; Aligianis et al., 2005; Bem et al.,2011; Borck et al., 2011; Derbent et al., 2004; Dursun et al., 2012;Graham et al., 2004; Megarbane et al., 1999; Morris-Rosendahlet al., 2010; Rodriguez Criado et al., 1999; Yildirim et al., 2012;Yuksel et al., 2007]. One feature of the disease is significant visualimpairment with eye abnormalities, including congenital bilateralcataracts, microphthalmia, microcornea (<10 mm diameter), andsmall atonic pupils that do not react to light or mydriatic agents.Despite early cataract surgery, patients’ vision remains poor (onlylight perception) as a result of progressive optic atrophy and severecortical visual impairment (confirmed by normal electroretinogramand absent visually evoked potentials). Brain abnormalities associ-ated with Micro syndrome include postnatal microcephaly (–4 to–6 SD), with congenital microcephaly only rarely being observed.Polymicrogyria (PMG) of the frontal and parietal lobes and hypo-genesis of the corpus callosum are typically seen upon magneticresonance imaging (MRI) and may account for the seizures sufferedby a subset of patients. Very severe developmental delay is evident,and although patients may develop early milestones such as smiling,most affected children achieve no developmental milestones beyondthose at the 4-month stage: they do not learn to crawl, pull up toa standing position, walk, or talk. Characteristically, patients showcongenital hypotonia, and from about 8 to 12 months, lower-limbspasticity leading to contractures. This spasticity is progressive andeventually affects the upper limbs, leading to spastic quadriplegialater in life. Nerve conduction studies have shown evidence of aprogressive axonal peripheral neuropathy. The microgenitalia as-sociated with Micro syndrome (micropenis and cryptorchidism),though a useful clue in boys, can be overlooked in affected females(hypoplastic labia minora, clitoral hypoplasia, and small introitus).Clinically, the pattern of genital abnormalities in Micro syndromefits with that seen in hypothalamic hypogonadism. Facially, the pa-tients have soft dysmorphic features of deep set eyes, a wide nasalbridge, and relatively narrow mouth, which, when taken togetherwith the eye phenotype, form a recognizable pattern.

MS shares many of the characteristics of Micro syndrome, al-though it is less severe and is much less frequently reported[Aligianis et al., 2006; Bora et al., 2007; Ehara et al., 2007; Harbordet al., 1989; Hennekam et al., 1988; Sanchez et al., 1985; Strisciuglioet al., 1988]. The eye developmental abnormalities (bilateral con-

genital cataracts and micropthalmia) are present but without opticatrophy and cortical visual impairment, with patients having lessfunctional visual impairment. Microcephaly and intellectual dis-ability are also less pronounced, and the progressive spasticity maybe confined to the lower limbs. Germline mutations in three genesRAB3GAP1, RAB3GAP2, and RAB18 have been identified in thesedisorders [Aligianis et al., 2005, 2006; Bem et al., 2011; Borck et al.,2011].

Rabs, small G proteins belonging to the Ras superfamily, aremaster regulators of vesicular trafficking in the cell (for review, see[Hutagalung and Novick 2011]). In humans, there are more than60 Rab proteins. Both individually and as a group, they have beenshown to have diverse roles in human diseases such as immunodefi-ciency, cancer, and neurodegeneration and skeletal dysplasia [Akaviaet al., 2010; Alshammari et al., 2012; Chia and Tang 2009; Dalfo et al.,2004; del Toro et al., 2009; Dupuis et al., 2012; Jenkins et al., 2007;Menasche et al., 2000; Verhoeven et al., 2003]. Each Rab can recruiteffector proteins such as sorting adaptors, kinases, phosphatases,motors, and tethering factors to regulate membrane identity andprocesses such as vesicle budding, motility, and fusion. Rab pro-teins associate with membranes because they are prenylated, withhydrophobic geranylgeranyl groups attached to one or two carboxy-terminal cysteine residues. However, the specificity and dynamicsof their membrane interactions are conferred by four classes of reg-ulatory proteins: GTPase-activating proteins (GAPs), guanine nu-cleotide exchange factors, GDP dissociation inhibitors (GDIs), andGDI displacement factors. These proteins modulate the “switching”of Rabs between GTP-bound ‘‘active’’ and GDP-bound ‘‘inactive’’conformations, their targeting to and extraction from cellular mem-branes, and their interaction with their effectors [Hutagalung andNovick, 2011].

RAB3GAP is a heterodimeric complex consisting of a catalyticsubunit (p130), which is encoded by RAB3GAP1 (MIM# 602536) onchromosome 2q21.3 and a noncatalytic subunit (p150), which is en-coded by RAB3GAP2 (MIM# 609275) on chromosome 1q41 [Fukuiet al., 1997; Nagano et al., 1998]. GAPs stimulate a Rab protein’sintrinsic GTP-hydrolysis activity so that bound GTP is convertedto GDP, and the protein is rendered susceptible to GDI-mediatedmembrane extraction and inactivation. RAB3GAP shows specificityfor RAB3 proteins (RAB3A, MIM# 179490; RAB3B, MIM# 179510;RAB3C, MIM# 612829; and RAB3D, MIM# 604350), which func-tion in the regulated exocytosis of hormones and neurotransmitters,though RAB3GAP2 is dispensable for this activity in vitro. Knock-out models for Rab3gap1 in mice and rab3-GAP in flies have beendescribed and show similar deficits in synaptic plasticity in hip-pocampal neurons and at the neuromuscular junction [Muller et al.,2011; Sakane et al., 2006]. In mice, loss of Rab3gap1 results in ab-normal release of synaptic vesicles and altered short-term synapticplasticity in the hippocampus, effects that could contribute to theWarburg Micro cognitive phenotype. However, these mice are viableand fertile and have no structural eye, brain, or genital abnormali-ties, suggesting that there is differential species-specific redundancyin these pathways.

RAB18 (MIM# 602207) is located on chromosome 10p12.1. Mul-tiple cellular locations and functions for RAB18 have been re-ported. For example, induction of RAB18 expression in endocrinecells has been linked to repression of secretory granule exocyto-sis [Vazquez-Martinez et al., 2007, 2008]. In adipocyte, fibrob-last, and epithelial cell lines, RAB18 can localize to lipid droplets,and this has been linked to roles in lipogenesis and lipolysis[Martin et al., 2005; Ozeki et al., 2005; Pulido et al., 2011]. Inthe same cell types, RAB18 can associate with the Golgi as wellas the ER (Endoplasmic Reticulum) under some circumstances,

HUMAN MUTATION, Vol. 34, No. 5, 686–696, 2013 687

and a further role in trafficking between these compartmentshas been suggested [Dejgaard et al., 2008; Martin et al., 2005].Despite the variety of cellular roles in which RAB18 might partici-pate, very little is known about the molecular regulators and effectorsof its function, in particular, its relationship to RAB3GAP1 andRAB3GAP2 has not been investigated. In zebrafish, morpholinoknockdown of rab18a or rab18b during the development causedmicrophthalmia, microcephaly, pericardial edema, delayed jaw for-mation, a reduced overall body size, and a general developmentaldelay [Bem et al., 2011]. However, to date, no other animal modelsfor RAB18 deficiency have been described.

The major transcript of RAB3GAP1 (ENST00000264158) isencoded by 24 exons of the RAB3GAP1 gene, though anothertranscript with a consensus coding sequence (ENST00000442034,CCDS54402.1) includes an additional small exon encoding sevenadditional amino acids. Previously reported pathogenic mutationsin RAB3GAP1 in 21 families include three frameshift mutations, sixnonsense mutations, and three splicing mutations suggesting thatMicro syndrome results from loss-of-function mutations in the gene[Abdel-Salam et al., 2007; Aligianis et al., 2005; Dursun et al., 2012;Morris-Rosendahl et al., 2010; Yildirim et al., 2012; Yuksel et al.,2007]. RAB3GAP2 is a 35-exon gene that encodes a novel 1393-amino-acid protein. Only two pathogenic mutations in this genehave been reported in the literature. The first, a missense mutationthat also affects splicing (c.3154G>T, p.Gly1051Cys) was associatedwith a case of MS [Aligianis et al., 2006]. The second, a small, in-frame deletion (c.499 507delTTCTACACT, p.Phe167 Thr169del)was associated with a case of Micro syndrome [Borck et al., 2011].RAB18 is the most recently discovered disease gene for Micro syn-drome [Bem et al., 2011]. It is a seven-exon gene encoding a206-amino-acid protein. Four pathogenic losses of function mu-tations in six families have been described in this gene. Interestingly,the clinical features of Micro syndrome patients with RAB3GAP1,RAB3GAP2, or RAB18 mutations are indistinguishable. To ex-pand the mutation spectrum and further investigate the pheno-types associated with these genes, we have analyzed a large cohortof families with Micro syndrome and MS for mutations in thesegenes.

OverviewThis report provides an up to date summary of all the known dis-

ease variants in RAB3GAP1, RAB3GAP2, and RAB18, identified in29 previously published families and 52 new families (49 Micro andthree Martsolf). In total, 153 families (144 Micro and nine Mart-solf) with typical phenotypes have been investigated, and mutationsidentified in RAB3GAP1 in 41% (63/153) of families (62 Micro syn-drome and one MS), mutations in RAB3GAP2 in 7% (11/153) offamilies (eight Micro syndrome and three Martoslf syndrome), andmutations in RAB18 in 5% (7/153) of families (seven Micro syn-drome). No mutations in these three genes were identified in 67Micro families and five Martsolf families, highlighting further locusheterogeneity for these disorders.

Details of the mutations discovered in each family and clinicalsummaries for specific mutations are presented in Table 1 and Supp.Tables S1–S5. Mutations are also shown in a schematic in Figure 1,Table 1, and Supp. Table S5. Although Micro syndrome and MS areheterogeneous conditions, patients were clinically indistinguishableon the basis of which gene was mutated (Figs. 2 and 3). Therefore,not surprisingly, screening of an additional 13 families with atypicalphenotypes (Supp. Table S3) did not identify mutations in thesegenes.

DatabasesUsing the Leiden Open source Variation Database package

hosted on the Leiden server, we have created databases forRAB3GAP1 (GenBank ID: D31886); RAB3GAP2 (GenBank ID:AB020646), and RAB18 (GenBank ID: AJ277145) that catalog allknown disease-causing mutations [Fokkema et al., 2011]. Thedatabases can be publicly accessed at www.lovd.nl/RAB3GAP1,www.lovd.nl/RAB3GAP2, and www.lovd.nl/RAB18. Data on clin-ical features and mutations were drawn from published articles, anddetails were provided by referring clinicians.

RAB3GAP1

In line with previous studies [Abdel-Salam et al., 2007; Aligianiset al., 2005; Borck et al., 2011; Morris-Rosendahl et al., 2010; Yildirimet al., 2012; Yuksel et al., 2007], the majority of new RAB3GAP1 mu-tations associated with Micro syndrome were frameshift, splicing, ornonsense mutations that might be expected to dramatically affectthe function of the RAB3GAP1 protein and/or induce nonsense-mediated decay (NMD) of the RAB3GAP1 message (Table 1). Thisis consistent with the assertion that Micro syndrome is caused byloss of functional RAB3GAP1 [Aligianis et al., 2005]. Indeed, infamily K16, we identified a homozygous deletion encompassing thefirst four exons of the gene (c.1 283del, p.Ala2Leufs∗12) that po-tentially abolishes transcription entirely. Interestingly, one of theframeshift mutations identified in this study (c.2865 2868insTTCT,p.Pro955Serfs∗15 in family K12) as well as one identified in a previ-ous study (c.2801delC, p.Pro934Leufs∗87) [Aligianis et al., 2005] arelocated in the last exon of the gene, and family K18 are homozygousfor a deletion of this exon. Since transcripts carrying these muta-tions are unlikely to be subject to NMD, these data indicate that theextreme C-terminal domain of the RAB3GAP1 could be essentialfor protein function or stability.

It could be expected that loss-of-function mutations inRAB3GAP1 are very rare in the general population, and in keep-ing with this suggestion, the majority of pathogenic mutationsdiscovered in this study were in consanguineous families withthe affected being homozygous (34 of 42 families in which aRAB3GAP1 mutation was identified). Compound heterozygousmutations were identified in eight affected patients. Of particu-lar note are the mutations identified in family K38. This fam-ily was described by Seemanova and Lesny (1996). The reportdetailed a possible novel X-linked syndrome with microcephaly,microphthalmia, microcornea, congenital cataract, hypogenital-ism, mental deficiency, growth retardation, and spasticity [See-manova and Lesny 1996]. However, we found the affected childto be a compound heterozygote for the splicing c.899+1G>A muta-tion and a frameshift mutation c.2055 2056insGCTCTCAGATATG-GAGTCT, p.Phe686Alafs.20. The mother, a carrier aunt, and ma-ternal grandmother were heterozygous for the splicing mutation,whereas the father was heterozygous for the frameshift mutation,consistent with the carrier status. In six families of Turkish ori-gin, the c.748+1G>A splicing mutation was identified. Haplotypeanalysis (with microsatellite markers flanking the RAB3GAP1) con-firmed that this is a founder mutation, consistent with the previouspublications [Aligianis et al., 2005; Yuksel et al., 2007].

Of particular interest, two of the novel mutations we have iden-tified in RAB3GAP1 are missense mutations. Pathogenic missensemutations in RAB3GAP1 have not previously been described. Weidentified a homozygous c.52A>C, p.Thr18Pro mutation in five fam-ilies (K1–K5) of different ethnic origins (Turkish, Moroccan, andPakistani). We also identified a homozygous c.71A>T, p.Glu24Valmutation in an Egyptian family (K6). In each case, the sequence

688 HUMAN MUTATION, Vol. 34, No. 5, 686–696, 2013

Table 1. Novel Mutations Identified in RAB3GAP1, RAB3GAP2, and RAB18

Family Ethnic origin Nucleotide change Exon Intron Predicted alteration Class of mutation Hom/Het Phenotype

RAB3GAP1K1 Turkish c.52A>C 2 p.Thr18Pro Missense Hom MicroK2 Pakistani c.52A>C 2 p.Thr18Pro Missense Hom MicroK3 Pakistani c.52A>C 2 p.Thr18Pro Missense Hom MicroK4 Turkish c.52A>C 2 p.Thr18Pro Missense Hom MicroK5 Moroccan c.52A>C 2 p.Thr18Pro Missense Hom MicroK6 Egyptian c.71A>T 2 p.Glu24Val Missense Hom MicroK7 Egyptian c.9delC 1 p.Asp4Thrfs∗51 Frameshift Hom MartsolfK8 Turkish c.665delC 8 p.Pro222Hisfs∗29 Frameshift Hom MicroK9 Pakistani c.1417 1423delGGGTTGA 15 p.Gly473Lysfs∗23 Frameshift Hom MicroK10 Turkish c.1335delC 15 p.Tyr445∗ Frameshift Hom MicroK11 Egyptian c.82delT 3 p.Ser28Profs∗7 Frameshift Hom MicroK12 India c.2865 2868insTTCT 24 p.Pro955Serfs∗15 Frameshift Hom MicroK13 Nigerian c.2343 2347delACCTT 20 p.Pro782Cysfs∗22 Frameshift Hom MicroK14 Turkish c.1293 1294delTA 14 p.Asp431Glufs∗1 Frameshift Hom MicroK15 Indian c.129delT 3 p.Leu44Trpfs∗49 Frameshift Hom MicroK16 Saudi Arabian Deletion of exons 1,2,3,4 1,2,3,4 Frameshift Hom MicroK17 Algerian Deletion of exons 4+5 4,5 Frameshift Hom MicroK18 German Deletion of exon 24 24 In-frame deletion Hom MicroK19 Indian c.1326T>G 14 p.Tyr442∗ Nonsense Hom MicroK20 Egyptian c.1039C>T 12 p.Arg347∗ Nonsense Hom MicroK21 Saudi Arabian c.1725G>A 17 p.Trp575∗ Nonsense Hom MicroK22 British Caucasian c.2629C>T 23 p.Gln877∗ Nonsense Hom MicroK23 German c.2491G>T p. Glu831∗ Nonsense Hom MicroK24 Israeli c.641delC 7 p.Ser214∗ Nonsense Hom MicroK25 Pakistani c.151-2A>T 3 Splicing Hom MicroK26 Egyptian c.748+2T>G 8 Splicing Hom MicroK27 Turkish c.748+1G>A 8 Splicing Hom MicroK28 Turkish c.748+1G>A 8 Splicing Hom MicroK29 Turkish c.748+1G>A 8 Splicing Hom MicroK30 Turkish c.748+1G>A 8 Splicing Hom MicroK31 Turkish c.748+1G>A 8 Splicing Hom MicroK32 British Caucasian c.899+1G>A 10 Splicing Hom MicroK33 Egyptian c.899+1G>A 10 Splicing Hom MicroK34 Canadian c.1237-2A>G 13 Splicing Hom MicroK35 American Spanish c.520C>T 7 p.Arg174∗ Nonsense Het Micro

c.657delT 8 p.Leu220∗ Nonsense Het MicroK36 German c.1389T>A 6 p.Cys463∗ Nonsense Het Micro

c.2103G>A 19 p.Trp701∗ Nonsense Het MicroK37 British Caucasian c.1039C>T 12 p.Arg347∗ Nonsense Het Micro

c.691C>T 8 p.Arg231∗ Nonsense Het MicroK38 Czech c.899+1G>A; 10 Splicing Het Micro

c.2055 2056insGCTCTCAGATATGGAGTCT 18 Phe686Alafs∗20 Frameshift Het MicroK39 American Caucasian c.473delA 6 p.Phe158Thrfs∗21 Frameshift Het Micro

c.150+1G>C 3 Splicing Het MicroK40 White European c.929G>A 11 p.Trp310∗ Nonsense Het MicroK41 French c.69G>A 2 p.Trp23∗ Nonsense Het Micro

c.401 407delCACACAG 6 p.Ala134Valfs∗14 Frameshift Het MicroK42 Saudi Arabian/ c.280G>T 4 p.Glu94∗ Nonsense Het Micro

Turkish c.748+1G>A 8 Splicing Het Micro

RAB3GAP2K43 Mexican c.1276C>T 14 p.Arg426Cys Missense Hom MartsolfK44 Gambian c.1276C>T 14 p.Arg426Cys Missense Hom MartsolfK45 Pakistani c.407 408insT 5 p.Cys137Metfs∗16 Frameshift Hom MicroK46 Israeli Muslim Arab c.2178 2181delAAAG 20 p. Ile726fs or p.Lys728∗ Frameshift Hom MicroK47 Tunisian c.1434G>A 14 p.Trp478∗ Nonsense Hom MicroK48 Turkish c.147G>A 2 p.Trp49∗ Nonsense Hom MicroK49 Turkish c.589C>T 7 p.Arg197∗ Nonsense Hom MicroK50 Saudi Arabian c.3637C>T 32 p.Arg1213>∗ Nonsense Hom MicroK51 Dutch c.3085G>T 26 p.Glu1029∗ MicroRAB18K52 Egyptian c.284C>G 5 p.Thr95Arg Missense Hom Micro

Nucleotide numbering reflects cDNA numbering with +1, corresponding to the A of the ATG (adenine; thymine and guanine) translation initiation codon in the referencesequence. Reference sequences for RAB3GAP1, RAB3GAP2, and RAB18 are NM_012233.2, NM_012414.3, and NM_021252.4, respectively. Peptide residue numbering reflectsprotein primary structure with p.Met1, corresponding to the first methionine. Reference sequences for RAB3GAP1, RAB3GAP2, and RAB18 are NP_036365.1, NP_036546.2,and NP_067075.1, respectively. Sequence variants are described according to the standard nomenclature (see http://www.hgvs.org/mutnomen/). Hom: homozygous ; Het:heterozygous.


Figure 1. A schematic of mutations identified in RAB3GAP1, RAB3GAP2, and RAB18. Newly described mutations are indicated above the gene,and previously published mutations are indicated below the gene in each case. Peptide residue numbering reflects protein primary structure withp.Met1, corresponding to the first methionine. Reference sequences for RAB3GAP1, RAB3GAP2, and RAB18 are NP_036365.1, NP_036546.2, andNP_067075.1, respectively. Sequence variants are described according to standard nomenclature (see http://www.hgvs.org/mutnomen/).

variants segregated with disease status in the families and werenot present in 270 control chromosomes. Both Thr18 and Glu24are highly conserved in evolution (Fig. 4A) and the substitutionof a polar for a nonpolar amino acid in each case also points to-ward these mutations being pathogenic. The programs Polyphen(http://genetics.bwh.harvard.edu/ggi/pph2/) and Mutation Taster(www.mutationtaster.org) predicted both these mutations to be“possibly damaging” and “disease causing,” respectively. The clini-cal features of these patients are summarized in Supp. Table S1 andFigures 2 and 3. All the affected children have a typical eye, brain,and genital findings, consistent with a diagnosis of Micro syndrome.

In one of the nine families with a Martsolf phenotype screened,K7, we identified a homozygous RAB3GAP1 mutation, c.9delC,p.Asp4Thrfs∗51. This mutation was identified in both siblings andsegregated with disease status with the parents being confirmed asobligate carriers, and unaffected siblings were either carriers or nor-mal. In the only genetically defined case of MS described in theliterature so far, in which a RAB3GAP2 mutation was identified, ithas been suggested that residual protein expression may be respon-sible for ameliorating the more severe Micro phenotype [Aligianiset al., 2006]. Thus, it seemed likely that the same could be true for theRAB3GAP1 mutation. One possibility was that because the mutation


Figure 2. Clinical photographs of patients with Micro and Martsolf syndromes. Brothers K2.1 (age 15 years) and K2.2 (age 13 years) have Microsyndrome and are homozygous for the RAB3GAP1:p.Thr18Pro mutation. Patient 6.1 (age 13 years) and her brother (age 6 years) are homozygous forthe RAB3GAP1: p.Glu24Val mutation and have Micro syndrome. The patients homozygous for the RAB3GAP1: p.Asp4Thrfs∗51 mutation associatedwith the Martsolf phenotype are K7.1 (age 8 years) and K7.2 (age 4 years). Patient K48 with Micro syndrome is shown at the age of 4 monthsand 3 years and is homozygous for the RAB3GAP2:c.147G>A p.Trp49∗ mutation. The sisters K44.1 (age 17 years) and K44.2 (age 14 years) with theRAB3GAP2:p.Arg426Cys mutation have Martsolf syndrome. Patient K52 has Micro syndrome due to a homozygous RAB18:p.Thr95Arg mutation.

occurred so close to the transcriptional start site, translation mightbe initiated from the next in-frame ATG. However, the full-lengthRAB3GAP1 transcript does not contain another in-frame ATG untilc.301, located in exon 5 of the gene. Another possibility was that analternative RAB3GAP1 transcript might bypass the frameshift muta-tion. We examined this possibility, as similar mechanisms have beenreported to produce phenotypic variation and phenotypic rescuein other inherited disorders [Morisaki et al., 1993]. Searches of En-sembl identified several poorly characterized alternative transcripts,including one, ENST00000539493, with an alternative first codingexon (Supp. Fig. S1). Using primers specific to the coding portionof this transcript, we were able to clone it from human cDNA. Uponsequencing, we confirmed that the cloned transcript contained thealternative first coding exon, but found that the 3′ portion of thecoding sequence was identical to that of the full-length transcript.Thus, the alternative transcript encodes a protein that lacks the first50 N-terminal amino acids of its full-length counterpart, but in-cludes the C-terminal RABGAP domain in its entirety (Fig. 5A). Weobtained cDNA from a patient affected by the Martsolf mutation.RT-PCR analysis specific for the full-length transcript showed thatlevels of product were reduced compared with controls. However, ina PCR to amplify a region common to both the full-length and thealternative transcript, levels of product were increased comparedwith controls (Fig. 5B). This may suggest that the expression of thealternative transcript compensates for loss of the full-length proteinin this patient.

On one hand, the above suggestion is consistent with experi-mental findings in a fly knockout model of the RAB3GAP1 ortho-logue rab3-GAP [Muller et al., 2011]. Deficits in a form of synap-tic plasticity in this model are absent in flies that also lack the

rab3 gene. This implies that a neurological deficit results from thefailure of rab3-GAP to properly regulate rab3, an activity presum-ably mediated via the RabGAP domain. This domain is present in theshorter RAB3GAP1 protein encoded by the alternative transcript.Thus, this shorter protein may be able to partly fulfill the role ofthe full-length protein and thereby ameliorate the Micro syndromephenotype when a pathogenic mutation affects the longer isoformonly. On the other hand, the two missense mutations we have de-scribed should not affect the expression of the alternative transcriptbut are nevertheless sufficient to cause typical Micro syndrome. Itis possible that the upregulation of the alternative transcript onlyoccurs when the expression of the full-length isoform is lost entirely.Alternatively, it is possible that the conserved N-terminal domainmutated in these patients possesses a distinct regulatory role, andthat mutations in this domain are capable of suppressing the positiveeffects of the short isoform.

RAB3GAP2

Only one RAB3GAP2 mutation, a small in-frame deletion(c.499 507delTTCTACACT, p.Phe167 Thr169del), has previouslybeen associated with a case of Micro syndrome [Borck et al., 2011].Although suggested to constitute a loss-of-function mutation, it wasformally possible that this deletion only partly disrupted RAB3GAP2function or conversely had a “dominant-negative” effect. How-ever, we have now identified nine new families with RAB3GAP2mutations.

Affected patients in families K45 and K46 carry thehomozygous mutations c.407 408insT, p.Cys137Metfs∗16 andc.2178 2181delAAAG, p.Lys728∗, respectively, that are predicted


Figure 3. Brain MRI scans from a healthy 9-year-old male (A–E) and patients with mutations in the RAB3GAP1 (F–R), RAB3GAP2 (S–W), andRAB18 (X–ZB) genes. The images in (F)–(J) are from a 7-year-old male patient who is homozygous for the previously published p.Lys596GlufsX613mutation in RAB3GAP1 and whose brain MRI is representative of most patients with mutations in RAB3GAP1. Images (K)–(N) represent the milderMartsolf phenotype in patient K7.2 (male) at the age of 4 years and 6 months, who is homozygous for the p.Asp4Thrfs∗51 mutation in RAB3GAP1:predominantly frontal and perisylvian PMG, no increased subdural spaces, and no cerebellar hypoplasia. Patient K1 (male, MRI at 4 years and10 months; O–R) who is homozygous for the p.Thr18Pro mutation in RAB3GAP1 had the most severe phenotype with PMG extending over the entirecortex, an extremely thin CC, and severe cerebellar hypoplasia. Patient K46.1 (S–W; male at 19 months) is homozygous for the p.Ile726fs mutationand shows the relatively milder RAB3GAP2 brain phenotype without substantial brain atrophy or cerebellar hypoplasia. Patient K52 (male, MRI at8 months, X–ZB) is homozygous for the p.Thr95Arg mutation in RAB18 and shows the RAB18 brain phenotype that closely resembles most patientswith RAB3GAP1 mutations.

to result in a frameshift. Five homozygous nonsense muta-tions c.1434G>A, p.Trp478∗ in K47; c.147G>A, p.Trp49∗ in K48;c.589C>T, p.Arg197∗ in K49; c.3637C>T, p.1213R>∗ in K50; andc.3085G>T, p.Glu1029∗ in K51 were identified. All these muta-tions segregate with disease status and were not present in 400control chromosomes. If transcribed, these mutations are pre-dicted to result in truncated transcripts, which are predicted tobe subject to NMD. These data strongly support the suggestionthat loss of functional RAB3GAP2 has an indistinguishable effectto that of loss of functional RAB3GAP1 or RAB18. The clini-cal features in these families (summarized in Supp. Table S2 andFigs. 2 and 3) were very severe, consistent with a diagnosis of Microsyndrome.

One pathogenic mutation in RAB3GAP2, a c.3154G>T,p.Gly1051Cys missense mutation that was also shown to affect

splicing, has previously been associated with MS [Aligianis et al.,2006]. It was suggested that the condition arose because of reducedprotein function as a result of the amino acid change and/or re-duced protein expression as a result of the effects on splicing. Inthis study, we identify two families, K43 and K44, one of Mexicanorigin and the other from Gambia, both carry a distinct missensemutation c.1276C>T, p.Arg426Cys, which is homozygous in the af-fected patients. In both families, the affected patients have a milderphenotype in keeping with a diagnosis of MS (Supp. Table S2 andFigs. 2 and 3). Arginine 426, a basic amino acid, is very highly con-served throughout the evolution (Fig. 4B), and its transition to thenucleophilic amino acid cysteine is predicted to be damaging. Al-though the molecular function and domain structure of RAB3GAP2is yet to be delineated, we conclude that this change disrupts butdoes not abolish protein function.


Figure 4. Missense mutations in RAB3GAP1, RAB3GAP2, and RAB18. A: Both p.Thr18Pro and Glu24Val mutations affect residues within aconserved N-terminal domain of RAB3GAP1. B: A p.Arg426Cys mutation in RAB3GAP2 affects a residue within a conserved WKGYRDA motif. C: Ap.Thr95Arg mutation in RAB18 affects a residue within the α3 helix. Multiple sequence alignments were carried out using ClustalW software.

RAB18

A single novel RAB18 mutation was identified in this study, amissense mutation c.284A>G, p.Thr95Arg, in which a nucleophilicamino acid is changed to a basic amino acid. This mutation ishomozygous in an affected patient from an Egyptian family K52,segregates according to disease status, and is not present in 400control chromosomes. The clinical details indicate that this patientsuffers from classical Micro syndrome (Supp. Table S1 and Figs. 2and 3). The p.Thr95Arg mutation is only two amino acid positionsaway from a pathogenic mutation we previously reported, Arg93del[Bem et al., 2011]. Indeed, according to the crystal structure ofRAB18, both mutations are likely to affect the same alpha helix. Wepreviously showed that the Arg93del mutation abolishes nucleotidebinding and thereby protein function, and conclude it is likely thatthe p.Thr95Arg mutation has the same effect (Fig. 4C).

Brain MRI in Patients with Mutations inRAB3GAP1, RAB3GAP2, and RAB18

We obtained and evaluated the brain MRIs from 17 new pa-tients with Micro or Martsolf syndromes. Four had mutations inRAB3GAP1, nine had mutations in RAB3GAP2, and four had mu-tations in RAB18. We compared these with the previously pub-

lished MRIs from patients with mutations in RAB3GAP1 [Morris-Rosendahl et al., 2010], RAB3GAP2 [Borck et al., 2011], and RAB18[Bem et al., 2011] and patients who have been found to have mu-tations in RAB3GAP1 and RAB18 subsequent to the publicationof their MRI findings [Graham et al., 2004]. Representative imagesfrom patients with mutations in each of the three genes were selectedand are shown in Figure 3.

We have previously described the brain MRI in patients with Mi-cro syndrome, and mutations in RAB3GAP1 to be characterized byextensive bilateral, predominantly frontal PMG, extending to theSylvian fissure and sometimes toward the temporal lobes; widenedSylvian fissures; hypogenesis of the corpus callosum (CC), particu-larly of the splenium; increased subdural spaces, especially aroundthe temporal poles and cerebellar and cerebellar vermis hypoplasia[Morris-Rosendahl et al., 2010]. These typical features are illustratedagain in Figure 3 F–J in a male patient who is homozygous for a pre-viously described mutation, p.Lys596GlufsX613. Variations on thispattern were seen in two patients: K7.1 and K7.2 with the RAB3GAP1mutation p.Asp4Thrfs∗51 who appeared to have a milder brain phe-notype with PMG restricted to the frontal lobes and right Sylvianfissure, an otherwise well-preserved cortical structure, no appar-ent cerebellar hypoplasia, and no increased subdural spaces. Thisrelatively milder brain phenotype correlates well with the milderMartsolf phenotype of the patient. On the contrary, patient K1 whowas homozygous for the p.Thr18Pro mutation appeared to have a


Figure 5. Human RAB3GAP1 transcripts. A: A schematic to show full-length RAB3GAP1 transcript (NP_036365.1; ENST00000264158) and a shortertranscript lacking the first three coding exons but containing an alternative first exon (as ENST00000539493). B: RT-PCR from cDNA extracted fromcontrol (WT88 and FATO) and affected (EG3) lymphoblastoid cell lines. The first primer pair can amplify from full-length RAB3GAP1 transcript only,whereas the second primer pair can amplify from both transcripts.

more severe phenotype (Fig. 3 O–R) with very severe PMG extend-ing over the entire cortex, severe brain atrophy at 4 years and 10months, and severe cerebellar hypoplasia. Again, this MRI patterncorrelates well with the more severe clinical phenotype observed inthis patient (Supp. Table S1). Our current analysis confirmed thatthe brain MRI phenotype in patients with Micro syndrome andwith mutations in the RAB3GAP2 (Fig. 3 S–W) and RAB18 (Fig. 3X–ZB) genes is remarkably similar to the typical RAB3GAP1 mu-tation phenotype described above. However, the overall impressionof the MRIs viewed in all patients with RAB3GAP2 mutations wasthat brain phenotype was relatively milder than that in patients withmutations in RAB3GAP1 and RAB18, with frontal PMG mostly notextending beyond the perisylvian fissure to the temporal and oc-cipital lobes, no apparent white matter loss, and no cerebellar orcerebellar vermis hypoplasia.

Other Diagnoses and DifferentialGraham et al. (2004) reported a case series of three fam-

ilies with classical Micro syndrome in 2004. Subsequently,we reported that patients 1 and 2 were compound heterozy-gotes for an antitermination mutation of the stop codonc.619T > C (p.∗207Glnext∗20) and an inframe arginine deletionc.277 279 del (p.Arg93 del) in RAB18, whereas patients 5 and6 were homozygous for the 1734G-A (W578∗) mutation inRAB3GAP1 [Aligianis et al., 2005; Bem et al., 2011]. Using a 0.5 MbCytoChip v3.0 BAC (bacterial artificial chromosome) array, we havenow detected that patients 3 and 4 have an unbalanced microdele-

tion/microduplication syndrome arr cgh 1p36.33p36.23(RP5–832C2>RP11–431K24)x1, 21q22.3q22.3(RP11–354C5>RP11–135B17)x3. A contiguous region of 24 BAC clones was found tobe deleted from 1p36.33 to 1p36.23 (RP5–832C2>RP11–431K24),giving a deletion size of between 6.9 and 7.1 Mb. This deletioncontains the critical region for 1p36 deletion syndrome and con-tains a maximum of 74 HGNC (The HUGO Gene NomenclatureCommittee) mapped genes. In addition, a contiguous region of 18BAC clones was found to be duplicated from 21q22.3 to 21qter(RP11–354C5>RP11–135B17), giving a duplication size of between5.6 and 6.0 Mb. This region contains a maximum of 94 HGNCmapped genes distal to the Down syndrome critical region. Thereis significant overlap in the clinical features of monosomy1p36 andMicro syndrome (cataracts, microcephaly, developmental delay,hypotonia, spasticity, and genital abnormalities) [Battaglia et al.,2008]. Parental karyotyping confirmed that the father is a carrierof a balanced translocation. Genotyping of microsatellites coveringthe 1p36 deletion interval in both sisters revealed that they haddistinct maternal haplotypes ruling out the possibility that a newrecessive gene was contributing to the phenotype.

In assessing children with ocular defects and microcephaly,prenatal viral infections and chromosomal abnormalities shouldbe excluded. Several chromosomal rearrangements with overlap-ping clinical features include 1p36 microdeletion and 1q21.1 mi-crodeletion (associated with mild-to-moderate mental retardation,microcephaly, cardiac abnormalities, and cataracts). Infants whohave multiple visceral abnormalities (particularly atrioventricularcanal cardiac defects) should be tested for Smith–Lemli–Opitz syn-drome (MIM# 270400), especially if they have 2–3 syndactly or


polydactly. Warburg Micro can be distinguished clinically fromseveral other disorders such as cerebro-oculo-facio-skeletal (COFS;MIM# 214150) syndrome, cockayne syndrome (CS, CSA; MIM#216400 and CSB; MIM# 133540), cataract, microcephaly, failureto thrive, kyphoscoliosis syndrome (CAMAK and CAMFAK; MIM#212540), MS, lethal Rutledge syndrome (MIM# 268670), lethal Neu-Laxova syndrome (MIM# 256520), and congenital cataracts–facial–dysmorphism–neuropathy (CCFDN) syndrome (MIM# 604168),all of which have microcephaly, mental retardation, childhoodcataract, contractures, and genital hypoplasia as symptoms. COFSsyndrome shares many clinical and cellular similarities with CS inthat cultured cells from patients with both these syndromes showhypersensitivity to ultraviolet radiation because of impaired nu-cleotide excision repair (NER). Various forms of COFS and CS typeA and CS type B have been found to be due to mutations in excision–repair cross-complementing genes (ERCC1, 2, 5, and 6). ImpairedNER has not been shown in patients with Micro syndrome. Clin-ically, Micro syndrome differs from COFS and CS in that patientshave less prenatal and postnatal growth deficiency, cortical dysplasiarather than progressive brain atrophy, preservation of hearing, de-ficient visual perception despite early cataract removal, and a betterprognosis for development and survival.

Biological SignificanceMicro syndrome and MS are heterogeneous disorders caused

by mutations in RAB3GAP1/2 or RAB18 in about 50% of cases,with RAB3GAP1 being the most common gene to have mutations.Clinically, the patients with Micro syndrome are indistinguishable,irrespective of which gene is mutated. No evidence of digenic ortriallelic inheritance is present in this cohort.

Importantly, we identify several new mutations in RAB3GAP2as causative in Micro syndrome and a mutation in RAB3GAP1 ascausative in a case of MS. The RAB3GAP2 Micro mutations arenonsense mutations and a frameshift mutation, which are likely toconstitute loss-of-function. We show evidence that the effect of theRAB3GAP1 Martsolf mutation, a frameshift, may be amelioratedbecause of expression of an alternative RAB3GAP1 transcript. Inagreement with previous reports, this strongly suggests that Microsyndrome and MS represent a phenotypic continuum that reflectsthe severity of the causative mutation, with loss-of-function muta-tions causing Micro syndrome and less damaging mutations caus-ing the milder MS. This could explain the more frequent reportsof Micro syndrome as compared with MS because mutations thatreduce the expression or function sufficient to cause MS may berare as compared with mutations that cause loss-of-function andtherefore Micro syndrome. In this context, the eponymous use ofMicro syndrome and MS may be confusing, and we would proposea gene-based phenotypic description.

Future WorkMicro syndrome and MS are genetically heterogeneous disorders,

and further gene identification using exome sequencing will allowdelineation of the pathways these proteins act in. Previously, nodirect link between RAB3 and RAB18 pathways has been reported,but given that loss-of-function mutations in RAB3GAP1 and RAB18cause an indistinguishable phenotype, it seems likely that there issome overlap between these pathways. Functional studies are neces-sary to determine the exact relationship of RAB3GAP1/2 and RAB18and the identification of RAB18’s effectors. One possibility is thatRAB3GAP1 is not a specific regulator of the RAB3 family but that it

also regulates RAB18. It is possible that RAB3GAP1 has additionaltargets or functions (in addition to its catalytic activity) that couldbe the key in the pathogenesis of Warburg Micro syndrome. Interest-ingly, plants have orthologs of the RAB3GAP1 catalytic subunit butlack RAB3 orthologs, which further support this idea. No knockoutanimal models for RAB18 have been described, but our preliminaryzebrafish studies suggest that rab18 might have a conserved devel-opmental role that could account for the structural abnormalitiesseen in these syndromes.

The parents of affected Micro patients appear completely nor-mal, despite lacking one functional allele of the disease gene. It islikely, therefore, that Martsolf mutations significantly attenuate pro-tein expression or function but the remaining activity is sufficientto counteract a proportion of the Micro syndrome pathology. Onthis basis, and given the severity and progressive nature of thesedisorders, it is possible that the subset of Micro syndrome patientscarrying nonsense mutations could benefit from the treatment thatpromotes read through of stop codons. The use of such therapeuticshas shown promise in animal models for other genetic conditions,and though the side-effect profile of available compounds is prob-lematic in extended use, trials of new compounds are underway[Bidou et al., 2012].

Acknowledgments

We thank the families who helped with this research, many colleagues forreferring affected families, the West Midlands Regional Genetics labora-tory with whom we work closely, and the UK Newlife Charity for financialsupport. We are grateful to C. Teller for clinical information and E. Jantz-Schuble, A-S Kaiser, A. Steiert, C. Zeschnig, A. Gallacher, and S. McKay fortechnical assistance.

Disclosure statement: The authors declare no conflict of interest.

References

Abdel-Salam GM, Hassan NA, Kayed HF, Aligianis IA. 2007. Phenotypic variability inMicro syndrome: report of new cases. Genet Couns 18:423–435.

Ainsworth JR, Morton JE, Good P, Woods CG, George ND, Shield JP, Bradbury J,Henderson MJ, Chhina J. 2001. Micro syndrome in Muslim Pakistan children.Ophthalmology 108:491–497.

Akavia UD, Litvin O, Kim J, Sanchez-Garcia F, Kotliar D, Causton HC, Pochanard P,Mozes E, Garraway LA, Pe’er D. 2010. An integrated approach to uncover driversof cancer. Cell 143:1005–1017.

Aligianis IA, Johnson CA, Gissen P, Chen D, Hampshire D, Hoffmann K, Maina EN,Morgan NV, Tee L, Morton J, Ainsworth JR, Horn D, et al. 2005. Mutations ofthe catalytic subunit of RAB3GAP cause Warburg Micro syndrome. Nat Genet37:221–223.

Aligianis IA, Morgan NV, Mione M, Johnson CA, Rosser E, Hennekam RC, AdamsG, Trembath RC, Pilz DT, Stoodley N, Moore AT, Wilson S, Maher ER. 2006.Mutation in Rab3 GTPase-activating protein (RAB3GAP) noncatalytic subunit ina kindred with Martsolf syndrome. Am J Hum Genet 78:702–707.

Alshammari MJ, Al-Otaibi L, Alkuraya FS. 2012. Mutation in RAB33B, which encodesa regulator of retrograde Golgi transport, defines a second Dyggve–Melchior–Clausen locus. J Med Genet 49:455–461.

Battaglia A, Hoyme HE, Dallapiccola B, Zackai E, Hudgins L, McDonald-McGinn D, Bahi-Buisson N, Romano C, Williams CA, Brailey LL, Zuberi SM,Carey JC. 2008. Further delineation of deletion 1p36 syndrome in 60 patients: arecognizable phenotype and common cause of developmental delay and mentalretardation. Pediatrics 121:404–410.

Bem D, Yoshimura S, Nunes-Bastos R, Bond FC, Kurian MA, Rahman F, Handley MT,Hadzhiev Y, Masood I, Straatman-Iwanowska AA, Cullinane AR, McNeill A, et al.2011. Loss-of-function mutations in RAB18 cause Warburg micro syndrome. AmJ Hum Genet 88:499–507.

Bidou L, Allamand V, Rousset JP, Namy O. 2012. Sense from nonsense: therapies forpremature stop codon diseases. Trends Mol Med 18:679–688.

Bora E, Cankaya T, Alpman A, Karaca E, Cogulu O, Tekgul H, Ozkinay F. 2007. A newcase of Martsolf syndrome. Genet Couns 18:71–75.


Borck G, Wunram H, Steiert A, Volk AE, Korber F, Roters S, Herkenrath P, WollnikB, Morris-Rosendahl DJ, Kubisch C. 2011. A homozygous RAB3GAP2 mutationcauses Warburg Micro syndrome. Hum Genet 129:45–50.

Chia WJ, Tang BL. 2009. Emerging roles for Rab family GTPases in human cancer.Biochim Biophys Acta 1795:110–116.

Dalfo E, Gomez-Isla T, Rosa JL, Nieto Bodelon M, Cuadrado Tejedor M, BarrachinaM, Ambrosio S, Ferrer I. 2004. Abnormal alpha–synuclein interactions with Rabproteins in alpha–synuclein A30P transgenic mice. J Neuropathol Exp Neurol63:302–313.

Dejgaard SY, Murshid A, Erman A, Kizilay O, Verbich D, Lodge R, Dejgaard K,Ly-Hartig TB, Pepperkok R, Simpson JC, Presley JF. 2008. Rab18 and Rab43have key roles in ER-Golgi trafficking. J Cell Sci 121:2768–2781.

del Toro D, Alberch J, Lazaro-Dieguez F, Martin-Ibanez R, Xifro X, Egea G, Canals JM.2009. Mutant huntingtin impairs post-Golgi trafficking to lysosomes by delocal-izing optineurin/Rab8 complex from the Golgi apparatus. Mol Biol Cell 20:1478–1492.

Derbent M, Agras PI, Gedik S, Oto S, Alehan F, Saatci U. 2004. Congenital cataract,microphthalmia, hypoplasia of corpus callosum and hypogenitalism: report andreview of Micro syndrome. Am J Med Genet A 128A:232–234.

Dupuis N, Lebon S, Kumar M, Drunat S, Graul-Neumann LM, Gressens P, El GhouzziV. 2012. A novel RAB33B mutation in Smith–McCort dysplasia. Hum Mutat34:283–286.

Dursun F, Guven A, Morris-Rosendahl D. 2012. Warburg Micro syndrome. J PediatrEndocrinol Metab 25:379–382.

Ehara H, Utsunomiya Y, Ieshima A, Maegaki Y, Nishimura G, Takeshita K, Ohno K.2007. Martsolf syndrome in Japanese siblings. Am J Med Genet A 143A:973–978.

Fokkema IF, Taschner PE, Schaafsma GC, Celli J, Laros JF, den Dunnen JT. 2011. LOVDv.2.0: the next generation in gene variant databases. Hum Mutat 32:557–563.

Fukui K, Sasaki T, Imazumi K, Matsuura Y, Nakanishi H, Takai Y. 1997. Isolation andcharacterization of a GTPase activating protein specific for the Rab3 subfamily ofsmall G proteins. J Biol Chem 272:4655–4658.

Graham JM Jr, Hennekam R, Dobyns WB, Roeder E, Busch D. 2004. MICRO syndrome:an entity distinct from COFS syndrome. Am J Med Genet A 128A:235–245.

Harbord MG, Baraitser M, Wilson J. 1989. Microcephaly, mental retardation, cataracts,and hypogonadism in sibs: Martsolf’s syndrome. J Med Genet 26:397–400.

Hennekam RC, van de Meeberg AG, van Doorne JM, Dijkstra PF, Bijlsma JB. 1988.Martsolf syndrome in a brother and sister: clinical features and pattern of inheri-tance. Eur J Pediatr 147:539–543.

Hutagalung AH, Novick PJ. 2011. Role of Rab GTPases in membrane traffic and cellphysiology. Physiol Rev 91:119–149.

Jenkins D, Seelow D, Jehee FS, Perlyn CA, Alonso LG, Bueno DF, Donnai D, Josifova D,Mathijssen IM, Morton JE, Orstavik KH, Sweeney E, et al. 2007. RAB23 mutationsin Carpenter syndrome imply an unexpected role for hedgehog signaling in cranial-suture development and obesity. Am J Hum Genet 80:1162–1170.

Martin S, Driessen K, Nixon SJ, Zerial M, Parton RG. 2005. Regulated localizationof Rab18 to lipid droplets: effects of lipolytic stimulation and inhibition of lipiddroplet catabolism. J Biol Chem 280:42325–42335.

Martsolf JT, Hunter AG, Haworth JC. 1978. Severe mental retardation, cataracts, shortstature, and primary hypogonadism in two brothers. Am J Med Genet 1:291–299.

Megarbane A, Choueiri R, Bleik J, Mezzina M, Caillaud C. 1999. Microcephaly, mi-crophthalmia, congenital cataract, optic atrophy, short stature, hypotonia, severepsychomotor retardation, and cerebral malformations: a second family with microsyndrome or a new syndrome? J Med Genet 36:637–640.

Menasche G, Pastural E, Feldmann J, Certain S, Ersoy F, Dupuis S, Wulffraat N,Bianchi D, Fischer A, Le Deist F, de Saint Basile G. 2000. Mutations in RAB27A

cause Griscelli syndrome associated with haemophagocytic syndrome. Nat Genet25:173–176.

Morisaki H, Morisaki T, Newby LK, Holmes EW. 1993. Alternative splicing: a mecha-nism for phenotypic rescue of a common inherited defect. J Clin Invest 91:2275–2280.

Morris-Rosendahl DJ, Segel R, Born AP, Conrad C, Loeys B, Brooks SS, Muller L,Zeschnigk C, Botti C, Rabinowitz R, Uyanik G, Crocq MA, Kraus U, Degen I, FaesF. 2010. New RAB3GAP1 mutations in patients with Warburg Micro Syndromefrom different ethnic backgrounds and a possible founder effect in the Danish.Eur J Hum Genet 18:1100–1106.

Muller M, Pym EC, Tong A, Davis GW. 2011. Rab3-GAP controls the progression ofsynaptic homeostasis at a late stage of vesicle release. Neuron 69:749–762.

Nagano F, Sasaki T, Fukui K, Asakura T, Imazumi K, Takai Y. 1998. Molecular cloningand characterization of the noncatalytic subunit of the Rab3 subfamily-specificGTPase-activating protein. J Biol Chem 273:24781–24785.

Ozeki S, Cheng J, Tauchi-Sato K, Hatano N, Taniguchi H, Fujimoto T. 2005. Rab18localizes to lipid droplets and induces their close apposition to the endoplasmicreticulum-derived membrane. J Cell Sci 118:2601–2611.

Pulido MR, Diaz-Ruiz A, Jimenez-Gomez Y, Garcia-Navarro S, Gracia-Navarro F,Tinahones F, Lopez-Miranda J, Fruhbeck G, Vazquez-Martinez R, Malagon MM.2011. Rab18 dynamics in adipocytes in relation to lipogenesis, lipolysis and obesity.PLoS One 6:e22931.

Rodriguez Criado G, Rufo M, Gomez de Terreros I. 1999. A second family with Microsyndrome. Clin Dysmorphol 8:241–245.

Sakane A, Manabe S, Ishizaki H, Tanaka-Okamoto M, Kiyokage E, Toida K, YoshidaT, Miyoshi J, Kamiya H, Takai Y, Sasaki T. 2006. Rab3 GTPase-activating proteinregulates synaptic transmission and plasticity through the inactivation of Rab3.Proc Natl Acad Sci USA 103:10029–10034.

Sanchez JM, Barreiro C, Freilij H. 1985. Two brothers with Martsolf’s syndrome. J MedGenet 22:308–310.

Seemanova E, Lesny I. 1996. X-linked microcephaly, microphthalmia, microcornea,congenital cataract, hypogenitalism, mental deficiency, growth retardation, spas-ticity: possible new syndrome. Am J Med Genet 66:179–183.

Strisciuglio P, Costabile M, Esposito M, Di Maio S. 1988. Martsolf’s syndrome in anon-Jewish boy. J Med Genet 25:267–269.

Vazquez-Martinez R, Cruz-Garcia D, Duran-Prado M, Peinado JR, Castano JP, MalagonMM. 2007. Rab18 inhibits secretory activity in neuroendocrine cells by interactingwith secretory granules. Traffic 8:867–882.

Vazquez-Martinez R, Martinez-Fuentes AJ, Pulido MR, Jimenez-Reina L, QuinteroA, Leal-Cerro A, Soto A, Webb SM, Sucunza N, Bartumeus F, Benito-Lopez P,Galvez-Moreno MA, Castano JP, Malagon MM. 2008. Rab18 is reduced in pitu-itary tumors causing acromegaly and its overexpression reverts growth hormonehypersecretion. J Clin Endocrinol Metab 93:2269–2276.

Verhoeven K, De Jonghe P, Coen K, Verpoorten N, Auer-Grumbach M, Kwon JM,FitzPatrick D, Schmedding E, De Vriendt E, Jacobs A, Van Gerwen V, Wagner K,Hartung HP, Timmerman V. 2003. Mutations in the small GTP-ase late endosomalprotein RAB7 cause Charcot-Marie-Tooth type 2B neuropathy. Am J Hum Genet72:722–727.

Warburg M, Sjo O, Fledelius HC, Pedersen SA. 1993. Autosomal recessive micro-cephaly, microcornea, congenital cataract, mental retardation, optic atrophy, andhypogenitalism. Micro syndrome. Am J Dis Child 147:1309–1312.

Yildirim MS, Zamani AG, Bozkurt B. 2012. Warburg micro syndrome in two childrenfrom a highly inbred Turkish family. Genet Couns 23:169–174.

Yuksel A, Yesil G, Aras C, Seven M. 2007. Warburg Micro syndrome in a Turkish boy.Clin Dysmorphol 16:89–93.


Documents

Mutation Spectrum in RAB 3 GAP 1 , RAB 3 GAP 2 , and RAB 18 and Genotype-Phenotype Correlations in Warburg Micro Syndrome and Martsolf Syndrome