New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk.

Levels of circulating glucose are tightly regulated. To identify new loci influencing glycemic traits, we performed meta-analyses of 21 genome-wide association studies informative for fasting glucose, fasting insulin and indices of beta-cell function (HOMA-B) and insulin resistance (HOMA-IR) in up to 46,186 nondiabetic participants. Follow-up of 25 loci in up to 76,558 additional subjects identified 16 loci associated with fasting glucose and HOMA-B and two loci associated with fasting insulin and HOMA-IR. These include nine loci newly associated with fasting glucose (in or near ADCY5, MADD, ADRA2A, CRY2, FADS1, GLIS3, SLC2A2, PROX1 and C2CD4B) and one influencing fasting insulin and HOMA-IR (near IGF1). We also demonstrated association of ADCY5, PROX1, GCK, GCKR and DGKB-TMEM195 with type 2 diabetes. Within these loci, likely biological candidate genes influence signal transduction, cell proliferation, development, glucose-sensing and circadian regulation. Our results demonstrate that genetic studies of glycemic traits can identify type 2 diabetes risk loci, as well as loci containing gene variants that are associated with a modest elevation in glucose levels but are not associated with overt diabetes

Six new loci associated with body mass index highlight a neuronal influence on body weight regulation

Common variants at only two loci, FTO and MC4R, have been reproducibly associated with body mass index (BMI) in humans. To identify additional loci, we conducted meta-analysis of 15 genome-wide association studies for BMI (n > 32,000) and followed up top signals in 14 additional cohorts (n > 59,000). We strongly confirm FTO and MC4R and identify six additional loci (P < 5 × 10−8): TMEM18, KCTD15, GNPDA2, SH2B1, MTCH2 and NEGR1 (where a 45-kb deletion polymorphism is a candidate causal variant). Several of the likely causal genes are highly expressed or known to act in the central nervous system (CNS), emphasizing, as in rare monogenic forms of obesity, the role of the CNS in predisposition to obesity

New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk

Levels of circulating glucose are tightly regulated. To identify new loci influencing glycemic traits, we performed meta-analyses of 21 genome-wide association studies informative for fasting glucose, fasting insulin and indices of beta-cell function (HOMA-B) and insulin resistance (HOMA-IR) in up to 46,186 nondiabetic participants. Follow-up of 25 loci in up to 76,558 additional subjects identified 16 loci associated with fasting glucose and HOMA-B and two loci associated with fasting insulin and HOMA-IR. These include nine loci newly associated with fasting glucose (in or near ADCY5, MADD, ADRA2A, CRY2, FADS1, GLIS3, SLC2A2, PROX1 and C2CD4B) and one influencing fasting insulin and HOMA-IR (near IGF1). We also demonstrated association of ADCY5, PROX1, GCK, GCKR and DGKB-TMEM195 with type 2 diabetes. Within these loci, likely biological candidate genes influence signal transduction, cell proliferation, development, glucose-sensing and circadian regulation. Our results demonstrate that genetic studies of glycemic traits can identify type 2 diabetes risk loci, as well as loci containing gene variants that are associated with a modest elevation in glucose levels but are not associated with overt diabetes

A genome-wide association search for type 2 diabetes genes in African Americans.

African Americans are disproportionately affected by type 2 diabetes (T2DM) yet few studies have examined T2DM using genome-wide association approaches in this ethnicity. The aim of this study was to identify genes associated with T2DM in the African American population. We performed a Genome Wide Association Study (GWAS) using the Affymetrix 6.0 array in 965 African-American cases with T2DM and end-stage renal disease (T2DM-ESRD) and 1029 population-based controls. The most significant SNPs (n = 550 independent loci) were genotyped in a replication cohort and 122 SNPs (n = 98 independent loci) were further tested through genotyping three additional validation cohorts followed by meta-analysis in all five cohorts totaling 3,132 cases and 3,317 controls. Twelve SNPs had evidence of association in the GWAS (P<0.0071), were directionally consistent in the Replication cohort and were associated with T2DM in subjects without nephropathy (P<0.05). Meta-analysis in all cases and controls revealed a single SNP reaching genome-wide significance (P<2.5×10(-8)). SNP rs7560163 (P = 7.0×10(-9), OR (95% CI) = 0.75 (0.67-0.84)) is located intergenically between RND3 and RBM43. Four additional loci (rs7542900, rs4659485, rs2722769 and rs7107217) were associated with T2DM (P<0.05) and reached more nominal levels of significance (P<2.5×10(-5)) in the overall analysis and may represent novel loci that contribute to T2DM. We have identified novel T2DM-susceptibility variants in the African-American population. Notably, T2DM risk was associated with the major allele and implies an interesting genetic architecture in this population. These results suggest that multiple loci underlie T2DM susceptibility in the African-American population and that these loci are distinct from those identified in other ethnic populations

Ca²⁺/Wnt signaling is activated in Fto deficient cells and zebrafish.

<p>(A) Wnt signaling phospho antibody microarray.Control (<i>Fto^+/+</i>) and <i>Fto</i> knockout (<i>Fto^−/−</i>) MEFs were treated with Wnt3a and changes in phosphorylation of Wnt signaling proteins analysed by an antibody microarray. P/N: Phospho-Antibody/Non-Phospho-Antibody ratio. For detailed calculations see Methods. (B) Total CamKII, phosphorylated (Thr305) CaMKII, and pan phosphorylated PKC (Thr497) were analysed in control (<i>Fto^+/+</i>) and <i>Fto</i> knockout (<i>Fto^−/−</i>) MEFs treated with Wnt3a conditioned medium (+) for 0, 10, 20 and 40 minutes. Brackets indicate PKC isoforms, asterisks show a non-specific band. (C) Phosphorylated CaMKII (Thr287) is upregulated in the pronephric ducts (PND) of <i>fto</i> morphant embryos compared to uninjected controls, as shown by immunofluorescence at 48 hpf. Scale bar: 50 µm.</p

E15.5 Fto knockout mice embryos display tissue specific cilia defects.

<p>Paraffin sections from wild type and mutant animals showing immunolocalization of acetylated- α-tubulin (green) and γ-tubulin or IFT88 (red) in the choroid plexus (A,B); nasopharynx (C); cochlea (D); kidney (E). Loss of Fto results in shortened cilia in the choroid plexus, nasopharynx and kidney whilst cilia in the cochlea appear unperturbed. Scale bars: in A, B 50 µm; in C,D,E 20 µm.</p

Loss of <i>fto</i> results in a craniofacial zebrafish phenotype.

<p>(A,B) <i>fto</i> knockdown zebrafish display small eyes, shortened dorsal ventral axis (red brackets), reduced pharyngeal width and length (double headed blue arrows), mislocalised melanocytes (blue arrows and red asterisk) and a curved truncated body axis. Scale bar: 500 µm, n =  con 50/50, <i>fto</i>MO 48/50. (C) Hematoxylin and Eosin staining on paraffin sections highlight loss of lamination and reduced size of the eye in <i>fto</i> morphants at 78hpf (n =  con 10/10, <i>fto</i>MO 10/10. Scale bar: 100 µm. pl; photoreceptor layer, opl; outer plexiform layer, bcl; bipolar cell layer, acl; amacrine cell layer, ipl; inner plaxiform layer, gcl; ganglion cell layer, rpe; retinal pigmented epithelium. (D) Dorsal whole mount view of control uninjected and <i>fto</i> morphant embryos at 48 hpf, morphants have reduced optic spacing (blue brackets) indicative of microcephaly. (E) Hematoxlyin and Eosin staining on transverse paraffin sections through the brain at eye level showing microcephaly in morphant embryos at 78 hpf. Scale bar: 100 µm. ot; optic tectum, t; tegmentum, h; hypothalamus. (F) <i>Fto</i> knockdown was confirmed in 48 hpf morphant embryos by western analysis using an anti-human FTO antibody, note the missing band at approximately 65KDa (arrow head). Asterisks indicate non-specific bands. (G) Embryos treated with the <i>fto</i> MO fail to develop the majority of head cartilage at 5 dpf compared to untreated controls, a reduced basal plate remains intact between treatments (asterisks). <i>p53</i> MO was used to counteract off-target morpholino effects, <i>p53</i> MO failed to rescue the <i>fto</i>MO phenotype. Ventral and lateral views displayed in the top and bottom columns, respectively. Scale bar: 200 µm; n =  con 30/30, <i>fto</i>MO 36/41, <i>fto</i>MO;<i>p53</i>MO 28/32. (H) <i>In situ</i> hybridisation for <i>myod</i> and <i>krox20</i> in control and fto morphants at 14 hpf. Arrowheads indicate a reduction in somite size in morphants compared to controls. Scale bar 200 µm; n =  con 52/55, <i>fto</i>MO 45/48. (I) Aberrant migration of NCCs, visualised using <i>sox10</i> probe, was observed in the head (brackets) and trunk (arrows) of <i>fto</i> morphants. Scale bar: 500 µm; n =  con 33/33, <i>fto</i>MO 27/34.</p

Canonical Wnt signaling is downregulated in <i>fto</i> morphants zebrafish.

<p>(A) Dual luciferase assay using the β-catenin responsive TopFlash construct shows loss of reporter assay activity in <i>fto</i>MO embryos analysed at both 24 hpf (con: 1.000 SEM ±0.073, ftoMO: 0.491 SEM ±0.105) and 48 hpf (con:1.000 SEM ±0.103, ftoMO 0.580 SEM ±0.066) stages. (B) <i>Lef1</i> transcripts, a canonical Wnt target gene, were analysed by <i>in situ</i> hybridisation (ISH) at 48 hpf. <i>Fto</i> morphants showed marked loss of <i>lef1</i> expression in the optic-tectum (arrows). Scale bar: 500 µm. n =  con 56/66, <i>fto</i>MO 40/53. (C) Loss of β-Catenin was confirmed in <i>fto</i> morphants by western blotting at 48 hpf. β-Catenin protein levels were quantified relative to the loading control (Actin). (D) ISH analysis of <i>ctnnb1</i> (zebrafish <i>β-catenin 1</i>) at 48 hpf showed upregulation of transcripts specifically in areas of the lateral hindbrain (arrowheads). Scale bar: 500 µm. n =  con 70/70, <i>fto</i>MO 65/68 (E) <i>Fto</i> morphant <i>Tg(7xTCF-Xla.Siam:GFP)^ia4</i> display loss of GFP accumulation in both the Telen- and Diencephalic regions of the brain when compared to uninjected controls at 48 hpf, embryos viewed from a dorsal perspective. Scale bar: 100 µm; n =  con 20/20, <i>fto</i>MO 18/20.</p

β-Catenin dependant canonical Wnt signaling is compromised in Fto deficient cells.

<p>(A) Cytoplasmic, membrane and nuclear fractions of control (<i>Fto^+/+</i>) and Fto knockout (<i>Fto^−/−</i>) MEFs treated with control (−) or Wnt3a -conditioned medium (+) for 4 hours were analysed by Western blot using β-Catenin antibody. Hsp90 and c-Jun were used as loading controls for cytoplasmic and nuclear fractions, respectively. (B) <i>Fto</i> mRNA expression in control and <i>Fto</i> knockout MEFs as determined by RT Real Time PCR. The data shown represent the mean ±SEM, (n = 3) (C) Quantification of cytoplasmic, membrane and nuclear β-catenin by ELISA in control and <i>Fto</i> knockout MEFs treated with control or Wnt3a -conditioned medium for 4 hours. The data shown represent the mean ±SEM, (n = 5). One-way ANOVA with Tukey’s multiple comparison test was performed, ***P<0.05, NS: not significant. (D) Immunofluorescence of control and <i>Fto</i> knockout MEFs treated with control and Wnt3a conditioned medium for 4 hours. Scale bar indicates 20 µm. This image is representative of three separate experiments.</p

The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus

We discovered that the hepatitis C virus (HCV) envelope glycoprotein E2 binds to human hepatoma cell lines independently of the previously proposed HCV receptor CD81. Comparative binding studies using recombinant E2 from the most prevalent 1a and 1b genotypes revealed that E2 recognition by hepatoma cells is independent from the viral isolate, while E2–CD81 interaction is isolate specific. Binding of soluble E2 to human hepatoma cells was impaired by deletion of the hypervariable region 1 (HVR1), but the wild-type phenotype was recovered by introducing a compensatory mutation reported previously to rescue infectivity of an HVR1-deleted HCV infectious clone. We have identified the receptor responsible for E2 binding to human hepatic cells as the human scavenger receptor class B type I (SR-BI). E2–SR-BI interaction is very selective since neither mouse SR-BI nor the closely related human scavenger receptor CD36, were able to bind E2. Finally, E2 recognition by SR-BI was competed out in an isolate-specific manner both on the hepatoma cell line and on the human SR-BI-transfected cell line by an anti-HVR1 monoclonal antibody