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The RAB39B p.G192R mutation causes X-linked dominant Parkinson's disease.

Mata IF, Jang Y, Kim CH, Hanna DS, Dorschner MO, Samii A, Agarwal P, Roberts JW, Klepitskaya O, Shprecher DR, Chung KA, Factor SA, Espay AJ, Revilla FJ, Higgins DS, Litvan I, Leverenz JB, Yearout D, Inca-Martinez M, Martinez E, Thompson TR, Cholerton BA, Hu SC, Edwards KL, Kim KS, Zabetian CP - Mol Neurodegener (2015)

Bottom Line: We identified a missense mutation (c.574G > A; p.G192R) in the RAB39B gene that closely segregated with disease and exhibited X-linked dominant inheritance with reduced penetrance in females.Experiments in PC12 and SK-N-BE(2)C cells demonstrated that p.G192R resulted in mislocalization of the mutant protein, possibly by altering the structure of the hypervariable C-terminal domain which mediates intracellular targeting.Further characterization of normal and aberrant RAB39B function might elucidate important mechanisms underlying neurodegeneration in PD and related disorders.

View Article: PubMed Central - PubMed

Affiliation: Veterans Affairs Puget Sound Health Care System, Seattle, WA, USA. nachofm@uw.edu.

ABSTRACT

Objective: To identify the causal gene in a multi-incident U.S. kindred with Parkinson's disease (PD).

Methods: We characterized a family with a classical PD phenotype in which 7 individuals (5 males and 2 females) were affected with a mean age at onset of 46.1 years (range, 29-57 years). We performed whole exome sequencing on 4 affected and 1 unaffected family members. Sanger-sequencing was then used to verify and genotype all candidate variants in the remainder of the pedigree. Cultured cells transfected with wild-type or mutant constructs were used to characterize proteins of interest.

Results: We identified a missense mutation (c.574G > A; p.G192R) in the RAB39B gene that closely segregated with disease and exhibited X-linked dominant inheritance with reduced penetrance in females. The mutation occurred in a highly conserved amino acid residue and was not observed among 87,725 X chromosomes in the Exome Aggregation Consortium dataset. Sequencing of the RAB39B coding region in 587 familial PD cases yielded two additional mutations (c.428C > G [p.A143G] and c.624_626delGAG [p.R209del]) that were predicted to be deleterious in silico but occurred in families that were not sufficiently informative to assess segregation with disease. Experiments in PC12 and SK-N-BE(2)C cells demonstrated that p.G192R resulted in mislocalization of the mutant protein, possibly by altering the structure of the hypervariable C-terminal domain which mediates intracellular targeting.

Conclusions: Our findings implicate RAB39B, an essential regulator of vesicular-trafficking, in clinically typical PD. Further characterization of normal and aberrant RAB39B function might elucidate important mechanisms underlying neurodegeneration in PD and related disorders.

No MeSH data available.


Related in: MedlinePlus

Effect of p.G192R on RAB39B expression and localization in PC12 cells. a Western blot of rat pheochromocytoma (PC12) cells transfected with a vector that was empty (mock), or contained a wild-type or mutant (p.G192R) construct. There was no difference in protein expression between wild-type and mutant RAB39B. b Immunofluorescent microscopy of nerve growth factor-differentiated PC12 cells transfected with wild-type or mutant (p.G192R) myc-tagged RAB39B. The wild-type protein is visualized throughout the cell bodies and neuritic processes, together with the vesicular marker chromogranin A, whereas the mutant protein is largely confined to cell bodies. Arrowheads indicate terminal regions of neurites. The scale bar corresponds to 10 μm
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Fig2: Effect of p.G192R on RAB39B expression and localization in PC12 cells. a Western blot of rat pheochromocytoma (PC12) cells transfected with a vector that was empty (mock), or contained a wild-type or mutant (p.G192R) construct. There was no difference in protein expression between wild-type and mutant RAB39B. b Immunofluorescent microscopy of nerve growth factor-differentiated PC12 cells transfected with wild-type or mutant (p.G192R) myc-tagged RAB39B. The wild-type protein is visualized throughout the cell bodies and neuritic processes, together with the vesicular marker chromogranin A, whereas the mutant protein is largely confined to cell bodies. Arrowheads indicate terminal regions of neurites. The scale bar corresponds to 10 μm

Mentions: We then investigated the effects of RAB39B p.G192R in vitro. In PC12 and SK-N-BE(2)C cells transfected with mutant and wild-type constructs there was no substantial difference in RAB39B protein expression (Figs. 2a and 3a). In NGF-differentiated PC12 cells wild-type RAB39B protein was visualized throughout the cytoplasm of cell bodies and neuritic processes (Fig. 2b), and co-localized with the vesicular marker chromogranin A. However, mutant (p.G192R) RAB39B was largely restricted to cell bodies with negligible amounts of protein evident in neuritic processes. In these experiments there was robust expression of both mutant and wild type RAB39B protein, but cellular phenotype can sometimes differ based on the level of transgene over-expression [10]. Thus we used an alternate vector and method of transfection to over-express RAB39B at lower levels in retinoic acid-differentiated SK-N-BE(2)C cells. Wild type RAB39B protein was frequently visualized within the cytoplasm and at the plasma membrane (co-localized with EGFR; Fig. 3b). However, while mutant RAB39B protein was also abundant in the cytoplasm, it was less frequently observed at the plasma membrane. To quantify these findings we performed immunoblot analysis of fractionated protein extracts from these cells (Fig. 3c). The proportion of membrane-bound to cytosolic RAB39B protein was significantly lower in cells expressing mutant protein than wild type protein (p < 0.01).Fig. 2


The RAB39B p.G192R mutation causes X-linked dominant Parkinson's disease.

Mata IF, Jang Y, Kim CH, Hanna DS, Dorschner MO, Samii A, Agarwal P, Roberts JW, Klepitskaya O, Shprecher DR, Chung KA, Factor SA, Espay AJ, Revilla FJ, Higgins DS, Litvan I, Leverenz JB, Yearout D, Inca-Martinez M, Martinez E, Thompson TR, Cholerton BA, Hu SC, Edwards KL, Kim KS, Zabetian CP - Mol Neurodegener (2015)

Effect of p.G192R on RAB39B expression and localization in PC12 cells. a Western blot of rat pheochromocytoma (PC12) cells transfected with a vector that was empty (mock), or contained a wild-type or mutant (p.G192R) construct. There was no difference in protein expression between wild-type and mutant RAB39B. b Immunofluorescent microscopy of nerve growth factor-differentiated PC12 cells transfected with wild-type or mutant (p.G192R) myc-tagged RAB39B. The wild-type protein is visualized throughout the cell bodies and neuritic processes, together with the vesicular marker chromogranin A, whereas the mutant protein is largely confined to cell bodies. Arrowheads indicate terminal regions of neurites. The scale bar corresponds to 10 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4581468&req=5

Fig2: Effect of p.G192R on RAB39B expression and localization in PC12 cells. a Western blot of rat pheochromocytoma (PC12) cells transfected with a vector that was empty (mock), or contained a wild-type or mutant (p.G192R) construct. There was no difference in protein expression between wild-type and mutant RAB39B. b Immunofluorescent microscopy of nerve growth factor-differentiated PC12 cells transfected with wild-type or mutant (p.G192R) myc-tagged RAB39B. The wild-type protein is visualized throughout the cell bodies and neuritic processes, together with the vesicular marker chromogranin A, whereas the mutant protein is largely confined to cell bodies. Arrowheads indicate terminal regions of neurites. The scale bar corresponds to 10 μm
Mentions: We then investigated the effects of RAB39B p.G192R in vitro. In PC12 and SK-N-BE(2)C cells transfected with mutant and wild-type constructs there was no substantial difference in RAB39B protein expression (Figs. 2a and 3a). In NGF-differentiated PC12 cells wild-type RAB39B protein was visualized throughout the cytoplasm of cell bodies and neuritic processes (Fig. 2b), and co-localized with the vesicular marker chromogranin A. However, mutant (p.G192R) RAB39B was largely restricted to cell bodies with negligible amounts of protein evident in neuritic processes. In these experiments there was robust expression of both mutant and wild type RAB39B protein, but cellular phenotype can sometimes differ based on the level of transgene over-expression [10]. Thus we used an alternate vector and method of transfection to over-express RAB39B at lower levels in retinoic acid-differentiated SK-N-BE(2)C cells. Wild type RAB39B protein was frequently visualized within the cytoplasm and at the plasma membrane (co-localized with EGFR; Fig. 3b). However, while mutant RAB39B protein was also abundant in the cytoplasm, it was less frequently observed at the plasma membrane. To quantify these findings we performed immunoblot analysis of fractionated protein extracts from these cells (Fig. 3c). The proportion of membrane-bound to cytosolic RAB39B protein was significantly lower in cells expressing mutant protein than wild type protein (p < 0.01).Fig. 2

Bottom Line: We identified a missense mutation (c.574G > A; p.G192R) in the RAB39B gene that closely segregated with disease and exhibited X-linked dominant inheritance with reduced penetrance in females.Experiments in PC12 and SK-N-BE(2)C cells demonstrated that p.G192R resulted in mislocalization of the mutant protein, possibly by altering the structure of the hypervariable C-terminal domain which mediates intracellular targeting.Further characterization of normal and aberrant RAB39B function might elucidate important mechanisms underlying neurodegeneration in PD and related disorders.

View Article: PubMed Central - PubMed

Affiliation: Veterans Affairs Puget Sound Health Care System, Seattle, WA, USA. nachofm@uw.edu.

ABSTRACT

Objective: To identify the causal gene in a multi-incident U.S. kindred with Parkinson's disease (PD).

Methods: We characterized a family with a classical PD phenotype in which 7 individuals (5 males and 2 females) were affected with a mean age at onset of 46.1 years (range, 29-57 years). We performed whole exome sequencing on 4 affected and 1 unaffected family members. Sanger-sequencing was then used to verify and genotype all candidate variants in the remainder of the pedigree. Cultured cells transfected with wild-type or mutant constructs were used to characterize proteins of interest.

Results: We identified a missense mutation (c.574G > A; p.G192R) in the RAB39B gene that closely segregated with disease and exhibited X-linked dominant inheritance with reduced penetrance in females. The mutation occurred in a highly conserved amino acid residue and was not observed among 87,725 X chromosomes in the Exome Aggregation Consortium dataset. Sequencing of the RAB39B coding region in 587 familial PD cases yielded two additional mutations (c.428C > G [p.A143G] and c.624_626delGAG [p.R209del]) that were predicted to be deleterious in silico but occurred in families that were not sufficiently informative to assess segregation with disease. Experiments in PC12 and SK-N-BE(2)C cells demonstrated that p.G192R resulted in mislocalization of the mutant protein, possibly by altering the structure of the hypervariable C-terminal domain which mediates intracellular targeting.

Conclusions: Our findings implicate RAB39B, an essential regulator of vesicular-trafficking, in clinically typical PD. Further characterization of normal and aberrant RAB39B function might elucidate important mechanisms underlying neurodegeneration in PD and related disorders.

No MeSH data available.


Related in: MedlinePlus