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1.  Income and Health in Tanzania. An Instrumental Variable Approach 
World Development  2015;66:500-515.
Summary
There is a substantial debate over the direction of the causal relation between income and health. This is important for our understanding of the health production process and for the policy debate over improving healthcare. We instrument income with rainfall measurements by matching satellite information on timing and positioning of 21 rainfall stations to longitudinal data (1991–94) of over 4,000 individuals in 51 villages in Tanzania. A 10% increase in income reduces the number of illnesses by 0.02. We also find that a 10% increase in income implies an increase of about 0.1 vaccinations of children under six.
doi:10.1016/j.worlddev.2014.09.016
PMCID: PMC4307997  PMID: 25648157
Tanzania; rainfall shocks; income; health; spatial interpolation
2.  Common genetic variants highlight the role of insulin resistance and body fat distribution in type 2 diabetes, independently of obesity 
Diabetes  2014;63(12):4378-4387.
We aimed to validate genetic variants as instruments for insulin resistance and secretion, to characterise their association with intermediate phenotypes, and to investigate their role in T2D risk among normal-weight, overweight and obese individuals.We investigated the association of genetic scores with euglycaemic-hyperinsulinaemic clamp- and OGTT-based measures of insulin resistance and secretion, and a range of metabolic measures in up to 18,565 individuals. We also studied their association with T2D risk among normal-weight, overweight and obese individuals in up to 8,124 incident T2D cases. The insulin resistance score was associated with lower insulin sensitivity measured by M/I value (β in SDs-per-allele [95%CI]:−0.03[−0.04,−0.01];p=0.004). This score was associated with lower BMI (−0.01[−0.01,−0.0;p=0.02) and gluteofemoral fat-mass (−0.03[−0.05,−0.02;p=1.4×10−6), and with higher ALT (0.02[0.01,0.03];p=0.002) and gamma-GT (0.02[0.01,0.03];p=0.001). While the secretion score had a stronger association with T2D in leaner individuals (pinteraction=0.001), we saw no difference in the association of the insulin resistance score with T2D among BMI- or waist-strata(pinteraction>0.31). While insulin resistance is often considered secondary to obesity, the association of the insulin resistance score with lower BMI and adiposity and with incident T2D even among individuals of normal weight highlights the role of insulin resistance and ectopic fat distribution in T2D, independently of body size.
doi:10.2337/db14-0319
PMCID: PMC4241116  PMID: 24947364
Genetics; type 2 diabetes; insulin resistance; insulin secretion; adipose expandability
3.  Concomitant angiosarcoma and lymphoproliferative disorder in solid organ transplant recipients 
An increased risk of posttransplant malignancy has been consistently reported following various solid organ transplants. The malignancies most commonly encountered are non-melanoma skin cancers, carcinomas of lung or breast and posttransplant lymphoproliferative disorders. Angiosarcoma, an uncommon vascular mesenchymal neoplasm, is rare in the posttransplant setting. This report describes two patients who developed high-grade angiosarcoma following a solid organ transplant. Notably, in both patients, the diagnosis of angiosarcoma was preceded by diagnosis of a lymphoproliferative disorder with monoclonal immunoglobulin heavy chain rearrangement.
doi:10.1186/2045-3329-4-15
PMCID: PMC4233048  PMID: 25400905
Angiosarcoma; Posttransplant lymphoproliferative disorder; Solid organ transplant
4.  Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance 
The Journal of Clinical Investigation  2014;124(9):4028-4038.
Structural maintenance of chromosomes (SMC) complexes are essential for maintaining chromatin structure and regulating gene expression. Two the three known SMC complexes, cohesin and condensin, are important for sister chromatid cohesion and condensation, respectively; however, the function of the third complex, SMC5–6, which includes the E3 SUMO-ligase NSMCE2 (also widely known as MMS21) is less clear. Here, we characterized 2 patients with primordial dwarfism, extreme insulin resistance, and gonadal failure and identified compound heterozygous frameshift mutations in NSMCE2. Both mutations reduced NSMCE2 expression in patient cells. Primary cells from one patient showed increased micronucleus and nucleoplasmic bridge formation, delayed recovery of DNA synthesis, and reduced formation of foci containing Bloom syndrome helicase (BLM) after hydroxyurea-induced replication fork stalling. These nuclear abnormalities in patient dermal fibroblast were restored by expression of WT NSMCE2, but not a mutant form lacking SUMO-ligase activity. Furthermore, in zebrafish, knockdown of the NSMCE2 ortholog produced dwarfism, which was ameliorated by reexpression of WT, but not SUMO-ligase–deficient NSMCE. Collectively, these findings support a role for NSMCE2 in recovery from DNA damage and raise the possibility that loss of its function produces dwarfism through reduced tolerance of replicative stress.
doi:10.1172/JCI73264
PMCID: PMC4151221  PMID: 25105364
5.  A general protocol for the crystallization of membrane proteins for X-ray structural investigation 
Nature protocols  2009;4(5):619-637.
Protein crystallography is used to generate atomic resolution structures of protein molecules. These structures provide information about biological function, mechanism and interaction of a protein with substrates or effectors including DNA, RNA, cofactors or other small molecules, ions and other proteins. This technique can be applied to membrane proteins resident in the membranes of cells. To accomplish this, membrane proteins first need to be either heterologously expressed or purified from a native source. The protein has to be extracted from the lipid membrane with a mild detergent and purified to a stable, homogeneous population that may then be crystallized. Protein crystals are then used for X-ray diffraction to yield atomic resolution structures of the desired membrane protein target. Below, we present a general protocol for the growth of diffraction quality membrane protein crystals. The process of protein crystallization is highly variable, and obtaining diffraction quality crystals can require weeks to months or even years in some cases.
doi:10.1038/nprot.2009.27
PMCID: PMC4075773  PMID: 19360018
6.  Multivalent Presentation of MPL by Porous Silicon Microparticles Favors T Helper 1 Polarization Enhancing the Anti-Tumor Efficacy of Doxorubicin Nanoliposomes 
PLoS ONE  2014;9(4):e94703.
Porous silicon (pSi) microparticles, in diverse sizes and shapes, can be functionalized to present pathogen-associated molecular patterns that activate dendritic cells. Intraperitoneal injection of MPL-adsorbed pSi microparticles, in contrast to free MPL, resulted in the induction of local inflammation, reflected in the recruitment of neutrophils, eosinophils and proinflammatory monocytes, and the depletion of resident macrophages and mast cells at the injection site. Injection of microparticle-bound MPL resulted in enhanced secretion of the T helper 1 associated cytokines IFN-γ and TNF-α by peritoneal exudate and lymph node cells in response to secondary stimuli while decreasing the anti-inflammatory cytokine IL-10. MPL-pSi microparticles independently exhibited anti-tumor effects and enhanced tumor suppression by low dose doxorubicin nanoliposomes. Intravascular injection of the MPL-bound microparticles increased serum IL-1β levels, which was blocked by the IL-1 receptor antagonist Anakinra. The microparticles also potentiated tumor infiltration by dendritic cells, cytotoxic T lymphocytes, and F4/80+ macrophages, however, a specific reduction was observed in CD204+ macrophages.
doi:10.1371/journal.pone.0094703
PMCID: PMC3988134  PMID: 24736547
7.  An in-frame deletion at the polymerase active site of POLD1 causes a multisystem disorder with lipodystrophy 
Nature genetics  2013;45(8):947-950.
DNA polymerase delta, whose catalytic subunit is encoded by POLD1, is responsible for lagging strand DNA synthesis during DNA replication1. It achieves this with high fidelity due to its intrinsic 3′ to 5′ exonuclease activity, which confers proofreading ability. Missense mutations in the exonuclease domain of POLD1 have recently been shown to predispose to colorectal and endometrial cancer2. Here we report a recurring heterozygous single amino acid deletion at the polymerase active site of POLD1 that abolishes DNA polymerase activity but only mildly impairs 3′ to 5′ exonuclease activity. This mutation causes a distinct multisystem disorder that includes subcutaneous lipodystrophy, deafness, mandibular hypoplasia and hypogonadism in males. This suggests that perturbation of function of the ubiquitously expressed POLD1 polymerase has surprisingly tissue-specific effects in man, and argues for an important role for POLD1 function in adipose tissue homeostasis.
doi:10.1038/ng.2670
PMCID: PMC3785143  PMID: 23770608
8.  Perilipin Deficiency and Autosomal Dominant Partial Lipodystrophy 
The New England journal of medicine  2011;364(8):740-748.
Summary
Perilipin is the most abundant adipocyte-specific protein that coats lipid droplets, and it is required for optimal lipid incorporation and release from the droplet. We identified two heterozygous frameshift mutations in the perilipin gene (PLIN1) in three families with partial lipodystrophy, severe dyslipidemia, and insulin-resistant diabetes. Subcutaneous fat from the patients was characterized by smaller-than-normal adipocytes, macrophage infiltration, and fibrosis. In contrast to wild-type perilipin, mutant forms of the protein failed to increase triglyceride accumulation when expressed heterologously in preadipocytes. These findings define a novel dominant form of inherited lipodystrophy and highlight the serious metabolic consequences of a primary defect in the formation of lipid droplets in adipose tissue.
doi:10.1056/NEJMoa1007487
PMCID: PMC3773916  PMID: 21345103
9.  The Bacterial Carbon-Fixing Organelle Is Formed by Shell Envelopment of Preassembled Cargo 
PLoS ONE  2013;8(9):e76127.
Background
Cyanobacteria play a significant role in the global carbon cycle. In Synechococcuselongatus, the carbon-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is concentrated into polyhedral, proteinaceous compartments called carboxysomes.
Methodology/Principal Findings
Using live cell fluorescence microscopy, we show that carboxysomes are first detected as small seeds of RuBisCO that colocalize with existing carboxysomes. These seeds contain little or no shell protein, but increase in RuBisCO content over several hours, during which time they are exposed to the solvent. The maturing seed is then enclosed by shell proteins, a rapid process that seals RuBisCO from the cytosol to establish a distinct, solvent-protected microenvironment that is oxidizing relative to the cytosol. These closure events can be spatially and temporally coincident with the appearance of a nascent daughter RuBisCO seed.
Conclusions/Significance
Carboxysomes assemble in a stepwise fashion, inside-to-outside, revealing that cargo is the principle organizer of this compartment’s biogenesis. Our observations of the spatial relationship of seeds to previously formed carboxysomes lead us to propose a model for carboxysome replication via sequential fission, polymerization, and encapsulation of their internal cargo.
doi:10.1371/journal.pone.0076127
PMCID: PMC3762834  PMID: 24023971
10.  A model for personalized in vivo analysis of human immune responsiveness 
Science translational medicine  2012;4(125):125ra30.
Studies of human immune diseases are generally limited to the analysis of peripheral blood lymphocytes of heterogenous patient populations. Improved models are needed to allow analysis of fundamental immunologic abnormalities predisposing to disease and in which to assess immunotherapies. Immunodeficient mice receiving human fetal thymus grafts and fetal CD34+ cells i.v. produce robust human immune systems, allowing analysis of human T cell development and function. However, to use humanized mice to study human immune-mediated disorders, immune sytsems must be generated from adult hematopoietic cells. Here, we demonstrated robust immune reconstitution in mice with hematopoietic stem cells (HSCs) aspirated from bone marrow of adults with Type 1 diabetes (T1D) and healthy control volunteers. In these humanized mice, cryopreservation of HLA allele-matched fetal thymic tissue prevented allogeneic adult HSC rejection. Newly generated T cells, which included regulatory T cells, were functional, self-tolerant, and had a diverse repertoire. The immune recognition of these mice mimicked that of the adult CD34+ cell donor, but the T cell phenotypes were more predominantly “naïve” than those of the adult donors. HSCs from T1D and control donors generated similar numbers of natural Tregs intrathymically; however, peripheral T cells from T1D subjects showed increased proportions of activated or memory cells compared to controls, suggesting possible HSC-intrinsic differences in T cell homeostasis that might underly immune pathology in T1D. This “Personalized Immune” (PI) mouse provides a new model for individualized analysis of human immune responses that may provide new insights into not only T1D, but other forms of immune function and dysfunction as well.
doi:10.1126/scitranslmed.3003481
PMCID: PMC3697150  PMID: 22422991
11.  Haplotype association analysis of genes within the WNT signalling pathways in diabetic nephropathy 
BMC Nephrology  2013;14:126.
Background
Renal interstitial fibrosis and glomerular sclerosis are hallmarks of diabetic nephropathy (DN) and several studies have implicated members of the WNT pathways in these pathological processes. This study comprehensively examined common genetic variation within the WNT pathway for association with DN.
Methods
Genes within the WNT pathways were selected on the basis of nominal significance and consistent direction of effect in the GENIE meta-analysis dataset. Common SNPs and common haplotypes were examined within the selected WNT pathway genes in a white population with type 1 diabetes, discordant for DN (cases: n = 718; controls: n = 749). SNPs were genotyped using Sequenom or Taqman assays. Association analyses were performed using PLINK, to compare allele and haplotype frequencies in cases and controls. Correction for multiple testing was performed by either permutation testing or using false discovery rate.
Results
A logistic regression model including collection centre, duration of diabetes, and average HbA1c as covariates highlighted three SNPs in GSK3B (rs17810235, rs17471, rs334543), two in DAAM1 (rs1253192, rs1252906) and one in NFAT5 (rs17297207) as being significantly (P < 0.05) associated with DN, however these SNPs did not remain significant after correction for multiple testing. Logistic regression of haplotypes, with ESRD as the outcome, and pairwise interaction analyses did not yield any significant results after correction for multiple testing.
Conclusions
These results indicate that both common SNPs and common haplotypes of WNT pathway genes are not strongly associated with DN. However, this does not completely exclude these or the WNT pathways from association with DN, as unidentified rare genetic or copy number variants could still contribute towards the genetic architecture of DN.
doi:10.1186/1471-2369-14-126
PMCID: PMC3701522  PMID: 23777469
Diabetic nephropathy; WNT signalling pathway; Association study; End-stage renal disease
12.  Intrahepatic Lipid Content and Insulin Resistance Are More Strongly Associated with Impaired NEFA Suppression after Oral Glucose Loading Than with Fasting NEFA Levels in Healthy Older Individuals 
Introduction. The mechanisms underlying the association between insulin resistance and intrahepatic lipid (IHL) accumulation are not completely understood. We sought to determine whether this association was explained by differences in fasting non-esterified fatty acid (NEFA) levels and/or NEFA suppression after oral glucose loading. Materials and Methods. We performed a cross-sectional analysis of 70 healthy participants in the Hertfordshire Physical Activity Trial (39 males, age 71.3 ± 2.4 years) who underwent oral glucose tolerance testing with glucose, insulin, and NEFA levels measured over two hours. IHL was quantified with magnetic resonance spectroscopy. Insulin sensitivity was measured with the oral glucose insulin sensitivity (OGIS) model, the leptin: adiponectin ratio (LAR), and the homeostasis model assessment (HOMA). Results. Measures of insulin sensitivity were not associated with fasting NEFA levels, but OGIS was strongly associated with NEFA suppression at 30 minutes and strongly inversely associated with IHL. Moreover, LAR was strongly inversely associated with NEFA suppression and strongly associated with IHL. This latter association (beta = 1.11 [1.01, 1.21], P = 0.026) was explained by reduced NEFA suppression (P = 0.24 after adjustment). Conclusions. Impaired postprandial NEFA suppression, but not fasting NEFA, contributes to the strong and well-established association between whole body insulin resistance and liver fat accumulation.
doi:10.1155/2013/870487
PMCID: PMC3659510  PMID: 23737780
13.  A genome-wide approach accounting for body mass index identifies genetic variants influencing fasting glycemic traits and insulin resistance 
Manning, Alisa K. | Hivert, Marie-France | Scott, Robert A. | Grimsby, Jonna L. | Bouatia-Naji, Nabila | Chen, Han | Rybin, Denis | Liu, Ching-Ti | Bielak, Lawrence F. | Prokopenko, Inga | Amin, Najaf | Barnes, Daniel | Cadby, Gemma | Hottenga, Jouke-Jan | Ingelsson, Erik | Jackson, Anne U. | Johnson, Toby | Kanoni, Stavroula | Ladenvall, Claes | Lagou, Vasiliki | Lahti, Jari | Lecoeur, Cecile | Liu, Yongmei | Martinez-Larrad, Maria Teresa | Montasser, May E. | Navarro, Pau | Perry, John R. B. | Rasmussen-Torvik, Laura J. | Salo, Perttu | Sattar, Naveed | Shungin, Dmitry | Strawbridge, Rona J. | Tanaka, Toshiko | van Duijn, Cornelia M. | An, Ping | de Andrade, Mariza | Andrews, Jeanette S. | Aspelund, Thor | Atalay, Mustafa | Aulchenko, Yurii | Balkau, Beverley | Bandinelli, Stefania | Beckmann, Jacques S. | Beilby, John P. | Bellis, Claire | Bergman, Richard N. | Blangero, John | Boban, Mladen | Boehnke, Michael | Boerwinkle, Eric | Bonnycastle, Lori L. | Boomsma, Dorret I. | Borecki, Ingrid B. | Böttcher, Yvonne | Bouchard, Claude | Brunner, Eric | Budimir, Danijela | Campbell, Harry | Carlson, Olga | Chines, Peter S. | Clarke, Robert | Collins, Francis S. | Corbatón-Anchuelo, Arturo | Couper, David | de Faire, Ulf | Dedoussis, George V | Deloukas, Panos | Dimitriou, Maria | Egan, Josephine M | Eiriksdottir, Gudny | Erdos, Michael R. | Eriksson, Johan G. | Eury, Elodie | Ferrucci, Luigi | Ford, Ian | Forouhi, Nita G. | Fox, Caroline S | Franzosi, Maria Grazia | Franks, Paul W | Frayling, Timothy M | Froguel, Philippe | Galan, Pilar | de Geus, Eco | Gigante, Bruna | Glazer, Nicole L. | Goel, Anuj | Groop, Leif | Gudnason, Vilmundur | Hallmans, Göran | Hamsten, Anders | Hansson, Ola | Harris, Tamara B. | Hayward, Caroline | Heath, Simon | Hercberg, Serge | Hicks, Andrew A. | Hingorani, Aroon | Hofman, Albert | Hui, Jennie | Hung, Joseph | Jarvelin, Marjo Riitta | Jhun, Min A. | Johnson, Paul C.D. | Jukema, J Wouter | Jula, Antti | Kao, W.H. | Kaprio, Jaakko | Kardia, Sharon L. R. | Keinanen-Kiukaanniemi, Sirkka | Kivimaki, Mika | Kolcic, Ivana | Kovacs, Peter | Kumari, Meena | Kuusisto, Johanna | Kyvik, Kirsten Ohm | Laakso, Markku | Lakka, Timo | Lannfelt, Lars | Lathrop, G Mark | Launer, Lenore J. | Leander, Karin | Li, Guo | Lind, Lars | Lindstrom, Jaana | Lobbens, Stéphane | Loos, Ruth J. F. | Luan, Jian’an | Lyssenko, Valeriya | Mägi, Reedik | Magnusson, Patrik K. E. | Marmot, Michael | Meneton, Pierre | Mohlke, Karen L. | Mooser, Vincent | Morken, Mario A. | Miljkovic, Iva | Narisu, Narisu | O’Connell, Jeff | Ong, Ken K. | Oostra, Ben A. | Palmer, Lyle J. | Palotie, Aarno | Pankow, James S. | Peden, John F. | Pedersen, Nancy L. | Pehlic, Marina | Peltonen, Leena | Penninx, Brenda | Pericic, Marijana | Perola, Markus | Perusse, Louis | Peyser, Patricia A | Polasek, Ozren | Pramstaller, Peter P. | Province, Michael A. | Räikkönen, Katri | Rauramaa, Rainer | Rehnberg, Emil | Rice, Ken | Rotter, Jerome I. | Rudan, Igor | Ruokonen, Aimo | Saaristo, Timo | Sabater-Lleal, Maria | Salomaa, Veikko | Savage, David B. | Saxena, Richa | Schwarz, Peter | Seedorf, Udo | Sennblad, Bengt | Serrano-Rios, Manuel | Shuldiner, Alan R. | Sijbrands, Eric J.G. | Siscovick, David S. | Smit, Johannes H. | Small, Kerrin S. | Smith, Nicholas L. | Smith, Albert Vernon | Stančáková, Alena | Stirrups, Kathleen | Stumvoll, Michael | Sun, Yan V. | Swift, Amy J. | Tönjes, Anke | Tuomilehto, Jaakko | Trompet, Stella | Uitterlinden, Andre G. | Uusitupa, Matti | Vikström, Max | Vitart, Veronique | Vohl, Marie-Claude | Voight, Benjamin F. | Vollenweider, Peter | Waeber, Gerard | Waterworth, Dawn M | Watkins, Hugh | Wheeler, Eleanor | Widen, Elisabeth | Wild, Sarah H. | Willems, Sara M. | Willemsen, Gonneke | Wilson, James F. | Witteman, Jacqueline C.M. | Wright, Alan F. | Yaghootkar, Hanieh | Zelenika, Diana | Zemunik, Tatijana | Zgaga, Lina | Wareham, Nicholas J. | McCarthy, Mark I. | Barroso, Ines | Watanabe, Richard M. | Florez, Jose C. | Dupuis, Josée | Meigs, James B. | Langenberg, Claudia
Nature genetics  2012;44(6):659-669.
Recent genome-wide association studies have described many loci implicated in type 2 diabetes (T2D) pathophysiology and beta-cell dysfunction, but contributed little to our understanding of the genetic basis of insulin resistance. We hypothesized that genes implicated in insulin resistance pathways may be uncovered by accounting for differences in body mass index (BMI) and potential interaction between BMI and genetic variants. We applied a novel joint meta-analytical approach to test associations with fasting insulin (FI) and glucose (FG) on a genome-wide scale. We present six previously unknown FI loci at P<5×10−8 in combined discovery and follow-up analyses of 52 studies comprising up to 96,496non-diabetic individuals. Risk variants were associated with higher triglyceride and lower HDL cholesterol levels, suggestive of a role for these FI loci in insulin resistance pathways. The localization of these additional loci will aid further characterization of the role of insulin resistance in T2D pathophysiology.
doi:10.1038/ng.2274
PMCID: PMC3613127  PMID: 22581228
14.  Association Analysis of Dyslipidemia-Related Genes in Diabetic Nephropathy 
PLoS ONE  2013;8(3):e58472.
Type 1 diabetes (T1D) increases risk of the development of microvascular complications and cardiovascular disease (CVD). Dyslipidemia is a common risk factor in the pathogenesis of both CVD and diabetic nephropathy (DN), with CVD identified as the primary cause of death in patients with DN. In light of this commonality, we assessed single nucleotide polymorphisms (SNPs) in thirty-seven key genetic loci previously associated with dyslipidemia in a T1D cohort using a case-control design. SNPs (n = 53) were genotyped using Sequenom in 1467 individuals with T1D (718 cases with proteinuric nephropathy and 749 controls without nephropathy i.e. normal albumin excretion). Cases and controls were white and recruited from the UK and Ireland. Association analyses were performed using PLINK to compare allele frequencies in cases and controls. In a sensitivity analysis, samples from control individuals with reduced renal function (estimated glomerular filtration rate<60 ml/min/1.73 m2) were excluded. Correction for multiple testing was performed by permutation testing. A total of 1394 samples passed quality control filters. Following regression analysis adjusted by collection center, gender, duration of diabetes, and average HbA1c, two SNPs were significantly associated with DN. rs4420638 in the APOC1 region (odds ratio [OR]  = 1.51; confidence intervals [CI]: 1.19–1.91; P = 0.001) and rs1532624 in CETP (OR = 0.82; CI: 0.69–0.99; P = 0.034); rs4420638 was also significantly associated in a sensitivity analysis (P = 0.016) together with rs7679 (P = 0.027). However, no association was significant following correction for multiple testing. Subgroup analysis of end-stage renal disease status failed to reveal any association. Our results suggest common variants associated with dyslipidemia are not strongly associated with DN in T1D among white individuals. Our findings, cannot entirely exclude these key genes which are central to the process of dyslipidemia, from involvement in DN pathogenesis as our study had limited power to detect variants of small effect size. Analysis in larger independent cohorts is required.
doi:10.1371/journal.pone.0058472
PMCID: PMC3608831  PMID: 23555584
15.  Mosaic Overgrowth with Fibroadipose Hyperplasia is Caused by Somatic Activating Mutations in PIK3CA 
Nature genetics  2012;44(8):928-933.
The phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway is critical for cellular growth and metabolism. Correspondingly, loss of function of PTEN, a negative regulator of PI3K, or activating mutations in AKT1, AKT2, or AKT3 have been found in distinct disorders featuring overgrowth or hypoglycemia. We performed exome sequencing of DNA from unaffected and affected cells of a patient with an unclassified syndrome of congenital, progressive segmental overgrowth of fibrous and adipose tissue and bone and identified the cancer-associated p.His1047Leu mutation in PIK3CA, which encodes the p110α catalytic subunit of PI3K, only in affected cells. Sequencing of PIK3CA in 10 further patients with overlapping syndromes identified either p.His1047Leu or a second cancer-associated mutation, p.His1047Arg, in 9 cases. Affected dermal fibroblasts showed enhanced basal and EGF-stimulated phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation and concomitant activation of downstream signaling. Our findings characterize a distinct overgrowth syndrome, biochemically demonstrate activation of PI3K signaling and thereby identify a rational therapeutic target.
doi:10.1038/ng.2332
PMCID: PMC3461408  PMID: 22729222
16.  Structural basis of aquaporin inhibition by mercury 
Journal of molecular biology  2007;368(3):607-617.
The aquaporin family of channels was defined based on the inhibition of water transport by mercurial compounds. Despite the important role of mercurials, little is known about the structural changes involved upon mercury binding leading to channel inhibition. To elucidate the mechanism we designed a mutant, T183C, of aquaporin Z (AqpZ) patterned after the known mercury-sensitive site of aquaporin 1 (AQP1) and determined the x-ray crystal structures of the unbound and mercury blocked states. Superposition of the two structures shows no conformational rearrangement upon mercury binding. In the blocked structure, there are two mercury sites – one bound to Cys183 and occluding the pore, and a second, also bound to the same cysteine but found buried in an interstitial cavity. To test the mechanism of blockade we designed a different mutant, L170C, to produce a more effective mercury block at the pore site. In a dose-response inhibition study, this mutant was 20 times more sensitive to mercury than wild-type AqpZ and 4 times more sensitive than T183C. The x-ray structure of L170C shows four mercury atoms at, or near, the pore site defined in the T183C structure and no structural change upon mercury binding. Thus, we elucidate a steric inhibition mechanism for this important class of channels by mercury.
doi:10.1016/j.jmb.2007.02.070
PMCID: PMC3535476  PMID: 17376483
aquaporin; integral membrane protein; x-ray crystallography; membrane protein structure
17.  Spatial and Temporal Organization of Chromosome Duplication and Segregation in the Cyanobacterium Synechococcus elongatus PCC 7942 
PLoS ONE  2012;7(10):e47837.
The spatial and temporal control of chromosome duplication and segregation is crucial for proper cell division. While this process is well studied in eukaryotic and some prokaryotic organisms, relatively little is known about it in prokaryotic polyploids such as Synechococcus elongatus PCC 7942, which is known to possess one to eight copies of its single chromosome. Using a fluorescent repressor-operator system, S. elongatus chromosomes and chromosome replication forks were tagged and visualized. We found that chromosomal duplication is asynchronous and that the total number of chromosomes is correlated with cell length. Thus, replication is independent of cell cycle and coupled to cell growth. Replication events occur in a spatially random fashion. However, once assembled, replisomes move in a constrained manner. On the other hand, we found that segregation displays a striking spatial organization in some cells. Chromosomes transiently align along the major axis of the cell and timing of alignment was correlated to cell division. This mechanism likely contributes to the non-random segregation of chromosome copies to daughter cells.
doi:10.1371/journal.pone.0047837
PMCID: PMC3480399  PMID: 23112856
18.  New Susceptibility Loci Associated with Kidney Disease in Type 1 Diabetes 
Sandholm, Niina | Salem, Rany M. | McKnight, Amy Jayne | Brennan, Eoin P. | Forsblom, Carol | Isakova, Tamara | McKay, Gareth J. | Williams, Winfred W. | Sadlier, Denise M. | Mäkinen, Ville-Petteri | Swan, Elizabeth J. | Palmer, Cameron | Boright, Andrew P. | Ahlqvist, Emma | Deshmukh, Harshal A. | Keller, Benjamin J. | Huang, Huateng | Ahola, Aila J. | Fagerholm, Emma | Gordin, Daniel | Harjutsalo, Valma | He, Bing | Heikkilä, Outi | Hietala, Kustaa | Kytö, Janne | Lahermo, Päivi | Lehto, Markku | Lithovius, Raija | Österholm, Anne-May | Parkkonen, Maija | Pitkäniemi, Janne | Rosengård-Bärlund, Milla | Saraheimo, Markku | Sarti, Cinzia | Söderlund, Jenny | Soro-Paavonen, Aino | Syreeni, Anna | Thorn, Lena M. | Tikkanen, Heikki | Tolonen, Nina | Tryggvason, Karl | Tuomilehto, Jaakko | Wadén, Johan | Gill, Geoffrey V. | Prior, Sarah | Guiducci, Candace | Mirel, Daniel B. | Taylor, Andrew | Hosseini, S. Mohsen | Parving, Hans-Henrik | Rossing, Peter | Tarnow, Lise | Ladenvall, Claes | Alhenc-Gelas, François | Lefebvre, Pierre | Rigalleau, Vincent | Roussel, Ronan | Tregouet, David-Alexandre | Maestroni, Anna | Maestroni, Silvia | Falhammar, Henrik | Gu, Tianwei | Möllsten, Anna | Cimponeriu, Danut | Ioana, Mihai | Mota, Maria | Mota, Eugen | Serafinceanu, Cristian | Stavarachi, Monica | Hanson, Robert L. | Nelson, Robert G. | Kretzler, Matthias | Colhoun, Helen M. | Panduru, Nicolae Mircea | Gu, Harvest F. | Brismar, Kerstin | Zerbini, Gianpaolo | Hadjadj, Samy | Marre, Michel | Groop, Leif | Lajer, Maria | Bull, Shelley B. | Waggott, Daryl | Paterson, Andrew D. | Savage, David A. | Bain, Stephen C. | Martin, Finian | Hirschhorn, Joel N. | Godson, Catherine | Florez, Jose C. | Groop, Per-Henrik | Maxwell, Alexander P.
PLoS Genetics  2012;8(9):e1002921.
Diabetic kidney disease, or diabetic nephropathy (DN), is a major complication of diabetes and the leading cause of end-stage renal disease (ESRD) that requires dialysis treatment or kidney transplantation. In addition to the decrease in the quality of life, DN accounts for a large proportion of the excess mortality associated with type 1 diabetes (T1D). Whereas the degree of glycemia plays a pivotal role in DN, a subset of individuals with poorly controlled T1D do not develop DN. Furthermore, strong familial aggregation supports genetic susceptibility to DN. However, the genes and the molecular mechanisms behind the disease remain poorly understood, and current therapeutic strategies rarely result in reversal of DN. In the GEnetics of Nephropathy: an International Effort (GENIE) consortium, we have undertaken a meta-analysis of genome-wide association studies (GWAS) of T1D DN comprising ∼2.4 million single nucleotide polymorphisms (SNPs) imputed in 6,691 individuals. After additional genotyping of 41 top ranked SNPs representing 24 independent signals in 5,873 individuals, combined meta-analysis revealed association of two SNPs with ESRD: rs7583877 in the AFF3 gene (P = 1.2×10−8) and an intergenic SNP on chromosome 15q26 between the genes RGMA and MCTP2, rs12437854 (P = 2.0×10−9). Functional data suggest that AFF3 influences renal tubule fibrosis via the transforming growth factor-beta (TGF-β1) pathway. The strongest association with DN as a primary phenotype was seen for an intronic SNP in the ERBB4 gene (rs7588550, P = 2.1×10−7), a gene with type 2 diabetes DN differential expression and in the same intron as a variant with cis-eQTL expression of ERBB4. All these detected associations represent new signals in the pathogenesis of DN.
Author Summary
The global prevalence of diabetes has reached epidemic proportions, constituting a major health care problem worldwide. Diabetic kidney disease, or diabetic nephropathy (DN)—the major long term microvascular complication of diabetes—is associated with excess mortality among patients with type 1 diabetes. Even though DN has been shown to cluster in families, the underlying genetic and molecular pathways remain poorly defined. We have undertaken the largest genome-wide association study and meta-analysis to date on DN and on its most severe form of kidney disease, end-stage renal disease (ESRD). We identified new loci significantly associated with diabetic ESRD: AFF3 and an intergenic locus on chromosome 15q26 residing between RGMA and MCTP2. Our functional analyses suggest that AFF3 influences renal tubule fibrosis, a pathological hallmark of severe DN. Another locus in ERBB4 was suggestively associated with DN and resides in the same intronic region as a variant affecting the expression of ERBB4. Subsequent pathway analysis of the genes co-expressed with ERBB4 indicated involvement of fibrosis.
doi:10.1371/journal.pgen.1002921
PMCID: PMC3447939  PMID: 23028342
19.  Genetic variation near IRS1 associates with reduced adiposity and an impaired metabolic profile 
Kilpeläinen, Tuomas O | Zillikens, M Carola | Stančáková, Alena | Finucane, Francis M | Ried, Janina S | Langenberg, Claudia | Zhang, Weihua | Beckmann, Jacques S | Luan, Jian’an | Vandenput, Liesbeth | Styrkarsdottir, Unnur | Zhou, Yanhua | Smith, Albert Vernon | Zhao, Jing-Hua | Amin, Najaf | Vedantam, Sailaja | Shin, So Youn | Haritunians, Talin | Fu, Mao | Feitosa, Mary F | Kumari, Meena | Halldorsson, Bjarni V | Tikkanen, Emmi | Mangino, Massimo | Hayward, Caroline | Song, Ci | Arnold, Alice M | Aulchenko, Yurii S | Oostra, Ben A | Campbell, Harry | Cupples, L Adrienne | Davis, Kathryn E | Döring, Angela | Eiriksdottir, Gudny | Estrada, Karol | Fernández-Real, José Manuel | Garcia, Melissa | Gieger, Christian | Glazer, Nicole L | Guiducci, Candace | Hofman, Albert | Humphries, Steve E | Isomaa, Bo | Jacobs, Leonie C | Jula, Antti | Karasik, David | Karlsson, Magnus K | Khaw, Kay-Tee | Kim, Lauren J | Kivimäki, Mika | Klopp, Norman | Kühnel, Brigitte | Kuusisto, Johanna | Liu, Yongmei | Ljunggren, Östen | Lorentzon, Mattias | Luben, Robert N | McKnight, Barbara | Mellström, Dan | Mitchell, Braxton D | Mooser, Vincent | Moreno, José Maria | Männistö, Satu | O’Connell, Jeffery R | Pascoe, Laura | Peltonen, Leena | Peral, Belén | Perola, Markus | Psaty, Bruce M | Salomaa, Veikko | Savage, David B | Semple, Robert K | Skaric-Juric, Tatjana | Sigurdsson, Gunnar | Song, Kijoung S | Spector, Timothy D | Syvänen, Ann-Christine | Talmud, Philippa J | Thorleifsson, Gudmar | Thorsteinsdottir, Unnur | Uitterlinden, André G | van Duijn, Cornelia M | Vidal-Puig, Antonio | Wild, Sarah H | Wright, Alan F | Clegg, Deborah J | Schadt, Eric | Wilson, James F | Rudan, Igor | Ripatti, Samuli | Borecki, Ingrid B | Shuldiner, Alan R | Ingelsson, Erik | Jansson, John-Olov | Kaplan, Robert C | Gudnason, Vilmundur | Harris, Tamara B | Groop, Leif | Kiel, Douglas P | Rivadeneira, Fernando | Walker, Mark | Barroso, Inês | Vollenweider, Peter | Waeber, Gérard | Chambers, John C | Kooner, Jaspal S | Soranzo, Nicole | Hirschhorn, Joel N | Stefansson, Kari | Wichmann, H-Erich | Ohlsson, Claes | O’Rahilly, Stephen | Wareham, Nicholas J | Speliotes, Elizabeth K | Fox, Caroline S | Laakso, Markku | Loos, Ruth J F
Nature Genetics  2011;43(8):753-760.
Genome-wide association studies have identified 32 loci associated with body mass index (BMI), a measure that does not allow distinguishing lean from fat mass. To identify adiposity loci, we meta-analyzed associations between ~2.5 million SNPs and body fat percentage from 36,626 individuals, and followed up the 14 most significant (P<10−6) independent loci in 39,576 individuals. We confirmed the previously established adiposity locus in FTO (P=3×10−26), and identified two new loci associated with body fat percentage, one near IRS1 (P=4×10−11) and one near SPRY2 (P=3×10−8). Both loci harbour genes with a potential link to adipocyte physiology, of which the locus near IRS1 shows an intriguing association pattern. The body-fat-decreasing allele associates with decreased IRS1 expression and with an impaired metabolic profile, including decreased subcutaneous-to-visceral fat ratio, increased insulin resistance, dyslipidemia, risk of diabetes and coronary artery disease, and decreased adiponectin levels. Our findings provide new insights into adiposity and insulin resistance.
doi:10.1038/ng.866
PMCID: PMC3262230  PMID: 21706003
20.  Mitochondrial Oxidative Phosphorylation Is Impaired in Patients with Congenital Lipodystrophy 
Objective:
Lipid accumulation in skeletal muscle and the liver is strongly implicated in the development of insulin resistance and type 2 diabetes, but the mechanisms underpinning fat accrual in these sites remain incompletely understood. Accumulating evidence of muscle mitochondrial dysfunction in insulin-resistant states has fuelled the notion that primary defects in mitochondrial fat oxidation may be a contributory mechanism. The purpose of our study was to determine whether patients with congenital lipodystrophy, a disorder primarily affecting white adipose tissue, manifest impaired mitochondrial oxidative phosphorylation in skeletal muscle.
Research Design and Methods:
Mitochondrial oxidative phosphorylation was assessed in quadriceps muscle using 31P-magnetic resonance spectroscopy measurements of phosphocreatine recovery kinetics after a standardized exercise bout in nondiabetic patients with congenital lipodystrophy and in age-, gender-, body mass index-, and fitness-matched controls.
Results:
The phosphocreatine recovery rate constant (k) was significantly lower in patients with congenital lipodystrophy than in healthy controls (P < 0.001). This substantial (∼35%) defect in mitochondrial oxidative phosphorylation was not associated with significant changes in basal or sleeping metabolic rates.
Conclusions:
Muscle mitochondrial oxidative phosphorylation is impaired in patients with congenital lipodystrophy, a paradigmatic example of primary adipose tissue dysfunction. This finding suggests that changes in mitochondrial oxidative phosphorylation in skeletal muscle could, at least in some circumstances, be a secondary consequence of adipose tissue failure. These data corroborate accumulating evidence that mitochondrial dysfunction can be a consequence of insulin-resistant states rather than a primary defect. Nevertheless, impaired mitochondrial fat oxidation is likely to accelerate ectopic fat accumulation and worsen insulin resistance.
doi:10.1210/jc.2011-2587
PMCID: PMC3380089  PMID: 22238385
21.  Association Analysis of Canonical Wnt Signalling Genes in Diabetic Nephropathy 
PLoS ONE  2011;6(8):e23904.
Aims/Hypothesis
Several studies have provided compelling evidence implicating the Wnt signalling pathway in the pathogenesis of diabetic nephropathy. Gene expression profiles associated with renal fibrosis have been attenuated through Wnt pathway modulation in model systems implicating Wnt pathway members as potential therapeutic targets for the treatment of diabetic nephropathy. We assessed tag and potentially functional single nucleotide polymorphisms (SNPs; n = 31) in four key Wnt pathway genes (CTNNB1, AXIN2, LRP5 and LRP6) for association with diabetic nephropathy using a case-control design.
Methods
SNPs were genotyped using Sequenom or Taqman technologies in 1351 individuals with type 1 diabetes (651 cases with nephropathy and 700 controls without nephropathy). Cases and controls were white and recruited from the UK and Ireland. Association analyses were performed using PLINK, to compare allele and haplotype frequencies in cases and controls. Adjustment for multiple testing was performed by permutation testing.
Results
Following logistic regression analysis adjusted by collection centre, duration of T1D, and average HbA1c as covariates, a single SNP in LRP6 (rs1337791) was significantly associated with DN (OR = 0.74; CI: 0.57–0.97; P = 0.028), although this was not maintained following correction for multiple testing. Three additional SNPs (rs2075241 in LRP6; rs3736228 and rs491347 both in LRP5) were marginally associated with diabetic nephropathy, but none of the associations were replicated in an independent dataset. Haplotype and subgroup analysis (according to duration of diabetes, and end-stage renal disease) also failed to reveal an association with diabetic nephropathy.
Conclusions/Interpretation
Our results suggest that analysed common variants in CTNNB1, AXIN2, LRP5 and LRP6 are not strongly associated with diabetic nephropathy in type 1 diabetes among white individuals. Our findings, however, cannot entirely exclude these genes or other members of the Wnt pathway, from involvement in the pathogenesis of diabetic nephropathy as our study had limited power to detect variants with small effect size.
doi:10.1371/journal.pone.0023904
PMCID: PMC3158097  PMID: 21876774
22.  Human Frame Shift Mutations Affecting the Carboxyl Terminus of Perilipin Increase Lipolysis by Failing to Sequester the Adipose Triglyceride Lipase (ATGL) Coactivator AB-hydrolase-containing 5 (ABHD5)* 
The Journal of Biological Chemistry  2011;286(40):34998-35006.
Perilipin (PLIN1) is a constitutive adipocyte lipid droplet coat protein. N-terminal amphipathic helices and central hydrophobic stretches are thought to anchor it on the lipid droplet, where it appears to function as a scaffold protein regulating lipase activity. We recently identified two different C-terminal PLIN1 frame shift mutations (Leu-404fs and Val-398fs) in patients with a novel subtype of partial lipodystrophy, hypertriglyceridemia, severe insulin resistance, and type 2 diabetes (Gandotra, S., Le Dour, C., Bottomley, W., Cervera, P., Giral, P., Reznik, Y., Charpentier, G., Auclair, M., Delépine, M., Barroso, I., Semple, R. K., Lathrop, M., Lascols, O., Capeau, J., O'Rahilly, S., Magré, J., Savage, D. B., and Vigouroux, C. (2011) N. Engl. J. Med. 364, 740–748.) When overexpressed in preadipocytes, both mutants fail to inhibit basal lipolysis. Here we used bimolecular fluorescence complementation assays to show that the mutants fail to bind ABHD5, permitting its constitutive coactivation of ATGL, resulting in increased basal lipolysis. siRNA-mediated knockdown of either ABHD5 or ATGL expression in the stably transfected cells expressing mutant PLIN1 reduced basal lipolysis. These insights from naturally occurring human variants suggest that the C terminus sequesters ABHD5 and thus inhibits basal ATGL activity. The data also suggest that pharmacological inhibition of ATGL could have therapeutic potential in patients with this rare but metabolically serious disorder.
doi:10.1074/jbc.M111.278853
PMCID: PMC3186430  PMID: 21757733
Cell Biology; Lipase; Lipid Droplets; Lipid Metabolism; Lipodystrophy
23.  Mitochondrial dysfunction in patients with primary congenital insulin resistance 
The Journal of Clinical Investigation  2011;121(6):2457-2461.
Mitochondrial dysfunction is associated with insulin resistance and type 2 diabetes. It has thus been suggested that primary and/or genetic abnormalities in mitochondrial function may lead to accumulation of toxic lipid species in muscle and elsewhere, impairing insulin action on glucose metabolism. Alternatively, however, defects in insulin signaling may be primary events that result in mitochondrial dysfunction, or there may be a bidirectional relationship between these phenomena. To investigate this, we examined mitochondrial function in patients with genetic defects in insulin receptor (INSR) signaling. We found that phosphocreatine recovery after exercise, a measure of skeletal muscle mitochondrial function in vivo, was significantly slowed in patients with INSR mutations compared with that in healthy age-, fitness-, and BMI-matched controls. These findings suggest that defective insulin signaling may promote mitochondrial dysfunction. Furthermore, consistent with previous studies of mouse models of mitochondrial dysfunction, basal and sleeping metabolic rates were both significantly increased in genetically insulin-resistant patients, perhaps because mitochondrial dysfunction necessitates increased nutrient oxidation in order to maintain cellular energy levels.
doi:10.1172/JCI46405
PMCID: PMC3104774  PMID: 21555852
25.  Early Diagnosis of Werner's Syndrome Using Exome-Wide Sequencing in a Single, Atypical Patient 
Genetic diagnosis of inherited metabolic disease is conventionally achieved through syndrome recognition and targeted gene sequencing, but many patients receive no specific diagnosis. Next-generation sequencing allied to capture of expressed sequences from genomic DNA now offers a powerful new diagnostic approach. Barriers to routine diagnostic use include cost, and the complexity of interpreting results arising from simultaneous identification of large numbers of variants. We applied exome-wide sequencing to an individual, 16-year-old daughter of consanguineous parents with a novel syndrome of short stature, severe insulin resistance, ptosis, and microcephaly. Pulldown of expressed sequences from genomic DNA followed by massively parallel sequencing was undertaken. Single nucleotide variants were called using SAMtools prior to filtering based on sequence quality and existence in control genomes and exomes. Of 485 genetic variants predicted to alter protein sequence and absent from control data, 24 were homozygous in the patient. One mutation – the p.Arg732X mutation in the WRN gene – has previously been reported in Werner's syndrome (WS). On re-evaluation of the patient several early features of WS were detected including loss of fat from the extremities and frontal hair thinning. Lymphoblastoid cells from the proband exhibited a defective decatenation checkpoint, consistent with loss of WRN activity. We have thus diagnosed WS some 15 years earlier than average, permitting aggressive prophylactic therapy and screening for WS complications, illustrating the potential of exome-wide sequencing to achieve early diagnosis and change management of rare autosomal recessive disease, even in individual patients of consanguineous parentage with apparently novel syndromes.
doi:10.3389/fendo.2011.00008
PMCID: PMC3356119  PMID: 22654791
Werner's syndrome; whole exome sequencing; insulin resistance; diabetes; WRN

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