Loss-of-function mutations in RAB18 cause Warburg micro syndrome

Abstract

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.

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.

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.

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).

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 = 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.