Bedoyan JK et al. (2012), Disruption of RAB40AL function leads to Martin-...

XB-ART-45232

J Med Genet 2012 May 01;495:332-40. doi: 10.1136/jmedgenet-2011-100575.

Show Gene links Show Anatomy links

Disruption of RAB40AL function leads to Martin--Probst syndrome, a rare X-linked multisystem neurodevelopmental human disorder.

Bedoyan JK , Schaibley VM , Peng W , Bai Y , Mondal K , Shetty AC , Durham M , Micucci JA , Dhiraaj A , Skidmore JM , Kaplan JB , Skinner C , Schwartz CE , Antonellis A , Zwick ME , Cavalcoli JD , Li JZ , Martin DM .

Abstract
BACKGROUND AND AIM: Martin--Probst syndrome (MPS) is a rare X-linked disorder characterised by deafness, cognitive impairment, short stature and distinct craniofacial dysmorphisms, among other features. The authors sought to identify the causative mutation for MPS. METHODS AND RESULTS: Massively parallel sequencing in two affected, related male subjects with MPS identified a RAB40AL (also called RLGP) missense mutation (chrX:102,079,078-102,079,079AC→GA p.D59G; hg18). RAB40AL encodes a small Ras-like GTPase protein with one suppressor of cytokine signalling box. The p.D59G variant is located in a highly conserved region of the GTPase domain between β-2 and β-3 strands. Using RT-PCR, the authors show that RAB40AL is expressed in human fetal and adult brain and kidney, and adult lung, heart, liver and skeletal muscle. RAB40AL appears to be a primate innovation, with no orthologues found in mouse, Xenopus or zebrafish. Western analysis and fluorescence microscopy of GFP-tagged RAB40AL constructs from transiently transfected COS7 cells show that the D59G missense change renders RAB40AL unstable and disrupts its cytoplasmic localisation. CONCLUSIONS: This is the first study to show that mutation of RAB40AL is associated with a human disorder. Identification of RAB40AL as the gene mutated in MPS allows for further investigations into the molecular mechanism(s) of RAB40AL and its roles in diverse processes such as cognition, hearing and skeletal development.

PubMed ID: 22581972
PMC ID: PMC3350147
Article link: J Med Genet
Grant support: [+]

Species referenced: Xenopus
Genes referenced: cox4i1 mmut rab40b

Article Images: [+] show captions

	Figure 1. RAB40AL p.D59G variant analysis and segregation. (A) The p.D59G variant identified in the sequenced individuals (III-5 and IV-1) segregates with the phenotype in the family. For individuals with a red dot, p.D59G was identified by Sanger sequencing. Affected individuals are shown in blue. For individuals with a green dot, p.D59G status was not determined. (B) Sequence chromatograms showing the two consecutive nucleotide changes (AC→GA) in a male subject (hemizygous) affected with MPS and an unaffected obligate female carrier (heterozygous). This change results in an Asp to Gly change at codon 59. Sanger sequencing readouts are shown. (C) The p.D59G variant (red) lies within a highly conserved 37 amino acid region of the GTPase domain and is conserved from humans to mosquitoes.
	Figure 2. Phylogenetic relationship of RAB40 family of proteins. Dendogram generated from protein sequences aligned using CLUSTAL W V.2.1 is shown. Synteny analyses of RAB40AL and RAB40A using UCSC Genome Browser did not identify mouse, Xenopus or zebrafish orthologues of human RAB40AL. The X-linked RAB40AL and RAB40A are unique to primates (red rectangle). Un, unknown.
	Figure 3. RAB40AL expression in relevant fetal and adult human tissues. RT-PCR using Clontech multiple tissue cDNA panels from human fetal and adult tissues shows high RAB40AL expression in fetal and adult brain and kidney tissues, with lower levels of expression in fetal lung, heart, liver and skeletal muscle. Single bands corresponding to 812 bp and 437 bp for RAB40AL and β–actin PCR products, respectively, were observed as expected. Bands were also confirmed by Sanger sequencing. A, adult; F, fetal.
	Figure 4. Reduced abundance of green fluorescent protein (GFP)–RAB40AL–p.D59G fusion protein in COS7 cells. Western analysis of whole cell lysates from cells transiently transfected with GFP–RAB40AL vector constructs (V, vector only; WT, GFP–RAB40AL; or MUT, GFP–RAB40AL p.D59G) using anti-GFP (or anti-GAPDH) monoclonal antibody (mAb). Results show reduced abundance of GFP–RAB40AL–p.D59G fusion protein, with essentially unchanged levels of the house-keeping GAPDH protein. Transfection efficiencies were not significantly different among the three vector constructs (see table 2). COS, untransfected COS7 cell lysate.
	Figure 5. p.D59G disrupts normal intracellular protein localisation. Cells transiently transfected with N-terminal green fluorescent protein (GFP)-tagged vector constructs (vector control, A-D; RAB40AL, E-H; and RAB40AL p.D59G, I-L) and stained with anti-mitochondrial COXIV antibody (Mito Ab) and DAPI are shown. GFP–RAB40AL localises to the mitochondria and throughout the cytoplasm (panel H), while this localisation is disrupted with GFP–RAB40AL–p.D59G (panel L). GFP–RAB40AL–p.D59G appears to be accumulated or clustered within the nucleus, nucleolus and/or perinuclear region (panels I and L). Representative transfected cells are shown (arrows).

References [+] :

Abankwa, A novel switch region regulates H-ras membrane orientation and signal output. 2008, Pubmed