Whole-exome sequencing identifies ADRA2A mutation in atypical familial partial lipodystrophy
Abhimanyu Garg, Shireesha Sankella, Chao Xing, Anil K. Agarwal
Abhimanyu Garg, Shireesha Sankella, Chao Xing, Anil K. Agarwal
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Research Article Endocrinology

Whole-exome sequencing identifies ADRA2A mutation in atypical familial partial lipodystrophy

Abstract

Despite identification of causal genes for various lipodystrophy syndromes, the molecular basis of some peculiar lipodystrophies remains obscure. In an African-American pedigree with a novel autosomal dominant, atypical familial partial lipodystrophy (FPLD), we performed linkage analysis for candidate regions and whole-exome sequencing to identify the disease-causing mutation. Affected adults reported marked loss of fat from the extremities, with excess fat in the face and neck at age 13–15 years, and developed metabolic complications later. A heterozygous g.112837956C>T mutation on chromosome 10 (c.202C>T, p.Leu68Phe) affecting a highly conserved residue in adrenoceptor α 2A (ADRA2A) was found in all affected subjects but not in unaffected relatives. ADRA2A is the main presynaptic inhibitory feedback G protein–coupled receptor regulating norepinephrine release. Activation of ADRA2A inhibits cAMP production and reduces lipolysis in adipocytes. As compared with overexpression of a wild-type ADRA2A construct in human embryonic kidney–293 cells and differentiated 3T3-L1 adipocytes, the mutant ADRA2A produced more cAMP and glycerol, which were resistant to the effects of the α2-adrenergic receptor agonist clonidine and the α2-adrenergic receptor antagonist yohimbine, suggesting loss of function. We conclude that heterozygous p.Leu68Phe ADRA2A mutation causes a rare atypical FPLD, most likely by inducing excessive lipolysis in some adipose tissue depots.

Authors

Abhimanyu Garg, Shireesha Sankella, Chao Xing, Anil K. Agarwal

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Figure 5

Effects of overexpression of wild-type and mutant ADRA2A in HEK-293 and 3T3-L1 preadipocyte cells on cAMP and glycerol production, respectively — response to clonidine and yohimbine.

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Effects of overexpression of wild-type and mutant ADRA2A in HEK-293 and ...
(A and B) HEK-293 cells were transfected with ADRA2A_WT and ADRA2A_L68F containing V5 epitope tag at the amino terminus. The production of cAMP was measured in the presence of various concentrations of clonidine or yohimbine by cAMP-Glo assay and normalized to protein. Dose response curves for (A) clonidine or (B) yohimbine were plotted as a semi-log curves and expressed as nM cAMP produced per μg protein (mean ± SEM, n = 4). For clonidine, ADRA2A_WT has an EC50 value of 27 μM and ADRA2A_L68F has an EC50 value of 48 μM. For yohimbine, ADRA2A_WT has an EC50 value of 79 μM and ADRA2A_L68F has an EC50 value of 143 μM. (C and D) ADRA2A_WT and ADRA2A_L68F containing V5 epitope tag at the amino terminus were expressed in 3T3-L1 cells. Twenty-four hours after transfection, the cells were allowed to differentiate into adipocytes using standard differentiation protocol (see the Methods for details). The cells were differentiated for 8 days and incubated with various concentrations of (C) clonidine or (D) yohimbine dissolved in dimethylsulfoxide for 24 hours. Released glycerol in the media was measured, normalized to protein, and expressed as μM glycerol released/μg protein (mean ± SEM, n = 3 for clonidine and n = 4 for yohimbine). The expression of cAMP or release of glycerol in the cells not treated with constructs or vehicle is also plotted in each panel. cAMP production or glycerol release from mutant ADRA2A compared with wild-type ADRA2A was significant using mixed-effect model, which assessed genotype, dose, and genotype × dose interaction as fixed effects, with each experiment modeled as a random effect. The EC50 values were determined by using GraphPad Prism version 6.04. Since our X coordinates are plotted as logarithms, and since the log of 0 is undefined, we approximated this point with an X coordinate of 0.01 μM, about 2 log units below the lowest “real” X value. For clonidine, ADRA2A_WT has an EC50 value of 3.5 μM and ADRA2A_L68F has an EC50 value of 45 μM. For yohimbine, ADRA2A_WT has an EC50 value of 35 μM and ADRA2A_L68F has an EC50 value of 60 μM. Analyses were performed with SAS 9.4 (SAS Institute). An omnibus P value of less than 0.05 was considered statistically significant.