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
View: Text | PDF
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

×

Figure 4

Relative mRNA expression of various ADRA2 isoforms in human adipose tissue, and expression and subcellular localization of ADRA2A wild-type and mutant proteins.

Options: View larger image (or click on image) Download as PowerPoint
Relative mRNA expression of various ADRA2 isoforms in human adipose tiss...
(A–C) Relative mRNA expression of ADRA2A, ADRA2B, and ADRA2C isoforms in the human omental and s.c. abdominal adipose tissue. (A) The expression of ADRA2B and ADRA2C isoforms was compared with that of ADRA2A mRNA in omental adipose tissue, which was arbitrarily assigned a value of 1. The ADRA2A expression was 9.45-fold higher than that of ADRA2C, while ADRA2B was undetectable (Ct > 30). (B) The expression of ADRA2B and ADRA2C isoforms was compared with that of ADRA2A mRNA in s.c. abdominal adipose tissue, which was arbitrarily assigned a value of 1. The ADRA2A expression was 16.67-fold higher than that of ADRA2C, while ADRA2B was undetectable (Ct > 30). (C) The expression of ADRA2A in the s.c. abdominal adipose tissue was about 1.84-fold higher than in the omental adipose tissue. (D and E) Immunoblots for the expression of ADRA2A_V5_WT and ADRA2A_V5_L68F proteins as probed with V5 antibody. The immunoblots were probed (D) in the absence of peptide-N-glycosidase F (PNGase F) treatment (representative blot, n = 2) and (E) in the presence of PNGase F treatment (representative blot, n = 4). PNGase F is an endoglycosidase that specifically removes N-linked glycans from glycoproteins. GAPDH was included for protein loading control. The predicted molecular weight of human ADRA2A is approximately 50 kDa, but because it is a heavily glycosylated protein, it migrates slower than expected, as can be seen in D. (E) Upon PNGase F treatment, the deglycosylated protein reveals the approximate molecular weight of 50 kDa for both for the wild-type and mutant ADRA2A. (F–H) Subcellular localization of human ADRA2A_V5_WT and ADRA2A_V5_L68F proteins expressed in HEK-293 cells. Proteins were fixed in 4% paraformaldehyde and incubated with primary antibody to V5 epitope followed by incubation with AlexaFluor 568–coupled fluorescent secondary antibody (red fluorescence) and counterstaining with DAPI (a nuclear stain; blue fluorescence). Cells were imaged for red and blue fluorescence using scanning confocal microscopy. Shown are single Z-slice images (Z-stacks were deconvolved using IMARIS software) for (F) pcDNA (vector control), (G) ADRA2A_V5_WT, and (H) ADRA2A_V5_L68F proteins. Both the ADRA2A_V5_WT and ADRA2A_V5_L68F localize to plasma membrane as expected (red fluorescence), confirming that there was no subcellular mislocalization of the mutant ADRA2A_V5_L68F protein. Numerous cells were examined visually, but only a few cells were processed.