We identified 5 probands of mixed European descent with heterozygous mutations in SH2B1 (Figure A): a frameshift mutation, F344LfsX20, which leads to a truncated protein product, and 3 missense mutations, P90H (2 patients), T175N, and P322S; all were absent from 500 control subjects (P < 0.001). Probands were apparently unrelated over 3 generations, as assessed by medical history. All mutations were inherited from overweight/obese parents, and carriers were hyperphagic and had reduced final height as adults (Table ). Mutation carriers were hyperinsulinemic (fasting plasma insulin >60 pmol/l) and euglycemic; liver function tests and lipid profiles were within the normal range (data not shown). Unexpectedly, we found that mutation carriers were reported to have delayed speech and language development and aggressive behavior by healthcare professionals and by family members (Table ). However, these individuals did not consent to further behavioral testing, so the precise nature and severity of these phenotypes could not be determined. None of the controls were reported to have behavioral abnormalities by healthcare professionals.
Identification of SH2B1 mutations.
Phenotypic characteristics of SH2B1 mutation carriers
We next sought to assess the molecular and cellular basis for the phenotypes associated with these human mutations. SH2B1 is a member of a family of SH2 domain–containing adaptor molecules — SH2B1 (also known as PSM), SH2B2 (also known as APS), and SH2B3 (also known as Lnk) — that bind to activated receptor tyrosine kinases, including insulin receptor and TrkA, the receptor for nerve growth factor (NGF) (13
). SH2B1 can also bind to activated JAK2 (14
), a cytokine receptor–associated tyrosine kinase that is activated after binding of cytokine receptor ligands, such as leptin and growth hormone (GH). SH2B1 has an aminoterminal dimerization domain, nuclear localization and export sequences, a central pleckstrin homology domain, and a carboxyterminal SH2 domain (Figure A). The SH2B1
transcript undergoes alternative splicing at the 3′ end, giving rise to 4 protein products (SH2B1α, SH2B1β, SH2B1γ, and SH2B1δ; ref. 15
) that share their amino termini, nuclear localization sequence, nuclear export sequence, pleckstrin homology domain, and SH2 domain, but differ at their carboxyl termini. We investigated expression of the 4 SH2B1 isoforms in human tissues by RT-PCR (Figure B). As the β isoform is the predominant form in the hypothalamus, we introduced the mutations into SH2B1β and examined their effect on SH2B1β expression, subcellular localization, and ability to enhance NGF-induced neuronal differentiation, cycling through the nucleus, GH-induced macrophage motility, JAK2 activation, leptin signaling, and insulin signaling.
Previous studies implicated SH2B1 in neuronal differentiation induced by NGF or by glial cell line–derived neurotrophic factor (16
). To test the mutations’ effect on the ability of SH2B1β to enhance neuronal differentiation, we transiently expressed GFP-tagged WT or mutant SH2B1β in PC12 cells, treated the cells with NGF to induce neuronal differentiation, and determined the percentage of GFP+
cells that were differentiated (neurite outgrowths >2 times the length of the cell body). The truncation mutation failed to enhance NGF-induced neuronal differentiation, and the P90H, A175N, and P322S mutations significantly impaired the ability of SH2B1β to enhance neuronal differentiation compared with WT (Figure A).
Functional characterization of SH2B1 mutations.
Nuclear shuttling of SH2B1β appears to be required for its stimulatory effect on neuronal differentiation (19
). To test whether the mutations impair the ability of SH2B1β to translocate to the nucleus, we treated 293T cells expressing GFP-tagged forms of SH2B1β with an inhibitor of nuclear export, leptomycin B, for 8 hours. While confocal microscopy revealed that approximately 95% of the cells expressing GFP-tagged WT SH2B1β showed a nuclear/cytoplasmic GFP fluorescence ratio of ≥1, cycling of the mutants into the nucleus was significantly impaired (Figure B and Supplemental Figure 1; supplemental material available online with this article; doi:
SH2B1 has been implicated in GH regulation of the actin cytoskeleton (20
). We therefore examined the ability of WT and mutant forms of SH2B1β to enhance GH-induced motility of cultured RAW264.7 macrophages. In contrast to GFP-tagged WT SH2B1β, which stimulated both basal and GH-induced motility, the point mutants inhibited GH-induced motility (Figure C).
SH2B1β mutants P90H, A175N, and P322S were expressed at the appropriate size and intensities and retained their ability to activate JAK2 (Figure D and data not shown). P90H, A175N, and P322S also exhibited a steady-state subcellular distribution similar to that of WT SH2B1β (Figure ). In contrast, the F344LfsX20 mutant exhibited significantly reduced expression and was unable to activate JAK2 (Figure D and data not shown), presumably due to the fact that it lacks the SH2 domain previously shown to be required for JAK2 activation (21
). SH2B1β F344LfsX20 was also present at lower levels in the plasma membrane relative to the cytoplasm in both 293T and PC12 cells and was found in aggregates in the cytoplasm (Figure and data not shown). We assessed the ability of the SH2B1β mutants to enhance leptin signaling by examining their ability to stimulate leptin-dependent tyrosyl phosphorylation of IRS2. Consistent with the findings on JAK2 activation, the SH2B1β point mutants were as effective as WT SH2B1β in stimulating leptin-dependent tyrosine phosphorylation of IRS2; similar results were seen for insulin-stimulated tyrosine phosphorylation of IRS2 (data not shown). As these latter assays rely on the overexpression of both SH2B1β and IRS2, it is possible that any subtle effects of the SH2B1 mutants were masked.
Subcellular distribution of SH2B1β WT and mutant proteins.
We also postulated that some of these heterozygous mutations may affect the ability of SH2B1 to dimerize. GFP-tagged WT SH2B1β coimmunoprecipitated with Flag-tagged WT SH2B1β, while the mutant 3AD/2FA (in which the phenylalanine zipper that is required for dimerization is mutated) exhibited greatly diminished coimmunoprecipitation (Figure D). Binding of the GFP-tagged P90H and A175N mutants to Flag-tagged WT SH2B1β was similar to that seen with GFP-tagged WT; however, the P322S mutation enhanced dimerization.
We have demonstrated that loss-of-function mutations in SH2B1 were associated with severe early-onset obesity, insulin resistance, and reduced final height. All the mutations were associated with loss of function in assays of GH/NGF-mediated signaling. Intriguingly, apart from the frameshift mutation, the other mutants did not impair leptin signaling. Although this discordance may reflect the differing sensitivities of the assays used, it is plausible that some of the effects of SH2B1 on energy homeostasis may be mediated by leptin-independent pathways.
Unexpectedly, we observed that mutations were associated with a range of behavioral abnormalities, including a tendency for social isolation and high levels of aggression, as reported by healthcare professionals and family members. We recognize that the absence of formal psychiatric testing and disease classification in these individuals and in the controls is a limitation of our study. However, we note that these phenotypes were not seen in association with the other genetic obesity syndromes we have characterized to date (11
), although the socioeconomic status of mutation carriers was comparable. As we observed impaired NGF-induced neuronal differentiation in vitro, a similar response to other ligands, such as centrally expressed neurotrophins, could contribute to these features (22
). The maladaptive behaviors reported in mutation carriers compared with controls were not reported in previous studies in mice. Our studies imply an unexpected role for SH2B1 in human behavior.