Analysis of genetic variation in Akt2/PKB-beta in severe insulin resistance, lipodystrophy, type 2 diabetes, and related metabolic phenotypes

Abstract

We previously reported a family in which a heterozygous missense mutation in Akt2 led to a dominantly inherited syndrome of insulin-resistant diabetes and partial lipodystrophy. To determine whether genetic variation in AKT2 plays a broader role in human metabolic disease, we sequenced the entire coding region and splice junctions of AKT2 in 94 unrelated patients with severe insulin resistance, 35 of whom had partial lipodystrophy. Two rare missense mutations (R208K and R467W) were identified in single individuals. However, insulin-stimulated kinase activities of these variants were indistinguishable from wild type. In two large case-control studies (total number of participants 2,200), 0 of 11 common single nucleotide polymorphism (SNPs) in AKT2 showed significant association with type 2 diabetes. In a quantitative trait study of 1,721 extensively phenotyped individuals from the U.K., no association was found with any relevant intermediate metabolic trait. In summary, although heterozygous loss-of- function mutations in AKT2 can cause a syndrome of severe insulin resistance and lipodystrophy in humans, such mutations are uncommon causes of these syndromes. Furthermore, genetic variation in and around the AKT2 locus is unlikely to contribute significantly to the risk of type 2 diabetes or related intermediate metabolic traits in U.K. populations.

FIG. 1

Rare mutations in Akt2 in human individuals. A: Location of the identified mutations R208K and R467W in relation to functional domains and known phosphorylation sites. B: In vitro kinase assay of Akt2 mutants. HA-Akt2 and HA-Akt2 mutants were immunoprecipitated from lysates of appropriately transfected CHO-T cells treated with (■) or without (□) 100 nmol/l insulin 10 min before lysis. Kinase assays were performed as described in research design and methods. Kinase activities are adjusted relative to that of unstimulated wild-type control subjects (100%). Data measured are means ± SD of five independent experiments. C: Equal amounts of lysates were immunoblotted with anti-phospho-Thr³⁰⁸ Akt (upper panel [pT309]) and anti-phospho-Ser⁴⁷³ Akt (middle panel [pS474]) antibodies to demonstrate increased phosphorylation in response to insulin. Immunoprecipitates were also immunoblotted with anti-HA antibody (lower panel [HA]) to demonstrate similar levels of immunoprecipitates used in the kinase assays.

FIG. 2

Location and linkage disequilibrium map of AKT2 SNPs genotyped. Thirteen exons of AKT2 are represented by solid bars (numbered 1-13); intronic regions and 5′ and 3′ regions are represented by solid lines. The positions of SNPs 1-11 are indicated. The dbSNP reference numbers are indicated below each SNP. The pairwise linkage disequilibrium coefficient r² (top) and D′ (bottom) for the control subjects in the case-control studies were calculated for genotyped SNPs using Haploview. Haplotype blocks were identified using Haploview.