Florin Grigorescu

Identification of the gene altered in Berardinelli-Seip congenital lipodystrophy on chromosome 11q13.

Congenital generalized lipodystrophy, or Berardinelli–Seip syndrome (BSCL), is a rare autosomal recessive disease characterized by a near-absence of adipose tissue from birth or early infancy and severe insulin resistance. Other clinical and biological features include acanthosis nigricans, hyperandrogenism, muscular hypertrophy, hepatomegaly, altered glucose tolerance or diabetes mellitus, and hypertriglyceridemia. A locus (BSCL1) has been mapped to 9q34 with evidence of heterogeneity. Here, we report a genome screen of nine BSCL families from two geographical clusters (in Lebanon and Norway). We identified a new disease locus, designated BSCL2, within the 2.5-Mb interval flanked by markers D11S4076 and D11S480 on chromosome 11q13. Analysis of 20 additional families of various ethnic origins led to the identification of 11 families in which the disease cosegregates with the 11q13 locus; the remaining families provide confirmation of linkage to 9q34. Sequence analysis of genes located in the 11q13 interval disclosed mutations in a gene homologous to the murine guanine nucleotide-binding protein (G protein), γ3-linked gene (Gng3lg) in all BSCL2-linked families. BSCL2 is most highly expressed in brain and testis and encodes a protein (which we have called seipin) of unknown function. Most of the variants are null mutations and probably result in a severe disruption of the protein. These findings are of general importance for understanding the molecular mechanisms underlying regulation of body fat distribution and insulin resistance.

Genotype-phenotype relationships in Berardinelli-Seip congenital lipodystrophy

Generalised lipodystrophy of the Berardinelli-Seip type (BSCL) is a rare autosomal recessive human disorder with severe adverse metabolic consequences. A gene on chromosome 9 (BSCL1) has recently been identified, predominantly in African-American families. More recently, mutations in a previously undescribed gene of unknown function (BSCL2) on chromosome 11, termed seipin, have been found to be responsible for this disorder in a number of European and Middle Eastern families. We have studied the genotype/phenotype relationships in 70 affected subjects from 44 apparently unrelated pedigrees of diverse ethnic origin. In all subjects, hepatic dysfunction, hyperlipidaemia, diabetes mellitus, and hypertrophic cardiomyopathy were significant contributors to morbidity with no clear differences in their prevalence between subjects with BSCL1 or BSCL2 and those with evidence against cosegregation with either chromosome 9 or 11 (designated BSCLX). BSCL2 appears to be a more severe disorder than BSCL1 with a higher incidence of premature death and a lower prevalence of partial and/or delayed onset of lipodystrophy. Notably, subjects with BSCL2 had a significantly higher prevalence of intellectual impairment than those with BSCL1 or BSCLX (p<0.0001, OR 17.0, CI 3.6 to 79.0). The higher prevalence of intellectual impairment and the increased risk of premature death in BSCL2 compared to BSCL1 emphasise the importance of molecular diagnosis of this syndrome and have clear implications for genetic counselling.

Complex haplotypes of IRS2 gene are associated with severe obesity and reveal heterogeneity in the effect of Gly1057Asp mutation

In order to understand the role of the insulin receptor substrate-2 (IRS2) gene (chromosome region: 13q34) in obesity, a complex disorder associated with insulin resistance and glucose intolerance, we determined single nucleotide polymorphims (SNPs) and complex haplotypes in women with morbid obesity and a body mass index (BMI) of 41±0.8xa0kg/m2 (n=99) compared with controls having a BMI of 23.8±0.1xa0kg/m2 (n=92). Sequencing of unphased DNA or digestion of polymerase chain reaction fragments revealed seven SNPs, including a new C/T(−769) replacement at the 5 untranslated region. Considering four or seven SNPs, we reconstructed with the PHASE program nine or 24 haplotypes, respectively, that were well correlated into the cladogram. Logistic regression analysis with nine haplotypes in the whole sample revealed that obesity was associated with haplotype H3, with P<0.025, an odds ratio (OR) of 1.9 and a 95% confidence interval (CI) of 1.1–3.4, or pairs 3/3 (P<0.005, OR=8.7, CI=1.9–40.1) and 3/4 (P<0.023, OR=2.5, CI=1.1–5.6), all containing the the Gly1057Asp allelic variant of IRS2, whereas controls were associated with H5 and H6 (P<0.02, OR=0.2, CI=0.01–0.81). Although obese H5 carriers (also containing Gly1057Asp mutation) were the most insulin resistant, haplotypes of IRS2 were poorly correlated (analysis of variance) with insulin resistance. By contrast, haplotypes H3, H4 and pairs 3/3 were consistently associated with increased 2-h glucose levels during an oral glucose tolerance test in obese individuals (P<0.0005, 0.025 and 0.027, respectively). These data indicate that IRS2 is an influential gene in severe obesity and glucose intolerance in this population, whereas gene-based haplotypes of IRS2 have revealed heterogeneity in the behaviour of the Gly1057Asp mutation in relation to insulin resistance.

Insulin and IGF-1 Signaling in Oocyte Maturation

Xenopus laevis oocytes possess insulin and/or insulin-like growth factor-1 (IGF-1) receptors and respond to respective hormones by increasing glucose transport and progressing from the G2 to M phase of the cell cycle (maturation). While molecular transduction mechanisms involving mitogen-activating kinases and cyclin-dependent kinases begin to be elucidated, missing links remain between the initial receptor tyrosine phosphorylation events and downstream signaling. The discovery that phosphotyrosines produced by receptor autophosphorylation or during substrate phosphorylation serve as an anchor for src homology 2 domains of several signaling proteins had a major impact on understanding how cytoplasmic enzymes are recruited at the level of the plasma membrane for subsequent activation.

Association of the FTO gene with obesity and the metabolic syndrome is independent of the IRS-2 gene in the female population of Southern France.

AIMnThe objective of the present study was to investigate the genetic association of the fat-mass-and-obesity-associated (FTO) gene in obese women in the presence of the known influential role of the insulin receptor substrate 2 (IRS-2) gene.nnnMETHODSnThis case-control study was carried out in the Languedoc-Roussillon region of France, and included lean control women (n=128), and women (n=119) of various degrees of obesity (body mass index [BMI] mean+/-S.D.: 39.3+/-7.4kg/m(2)) and a prevalence of 26.9% of the metabolic syndrome (MetS). For the FTO gene, genotyping was performed by sequence-specific oligonucleotide-polymerase chain reaction (SSO-PCR) on the single nucleotide polymorphism (SNP) rs1421085 (C/T) while, for IRS-2, the rs1805097 (G/A) corresponding to variant Gly1057Asp was genotyped by direct sequencing.nnnRESULTSnThe FTO gene (homozygous C/C) was significantly associated to both simple and morbid obesity (P<0.026 and P<0.0034, respectively), with odds-ratios (ORs) of 2.58 (95% CI: 1.1-6.0) and 4.1 (95% CI: 1.6-10.5), respectively, independent of IRS-2. MetS was also associated with FTO (P<0.032, OR: 3.1, 95% CI: 1.1-8.5), but not with IRS-2. Genotypes of FTO were correlated with insulin resistance, and homozygous C/C was positively correlated with an increase in insulin resistance over the value predicted by the increase in BMI.nnnCONCLUSIONnThese data confirm the influential role of the FTO gene in obesity in the French female population and, in addition, revealed the role of FTO in insulin resistance and MetS. These effects appeared to be independent of IRS-2, which is directly involved in insulin action. This study may offer new insights into the genetic determinants of obesity and MetS in women.

Interleukin-10 promoter microsatellite polymorphisms influence the immune response to heparin and the risk of heparin-induced thrombocytopenia

INTRODUCTIONnHeparin-induced thrombocytopenia (HIT) results from an atypical immune response with synthesis of IgG antibodies (Abs) to platelet factor 4/heparin complexes (PF4/H), and probably involves both B and T cells. We investigated whether 3 single nucleotide polymorphisms (SNPs), rs1800896 (-1082G/A), rs1800871 (-819C/T) and rs1800872 (-592C/A) and the polymorphic CA repeat microsatellites IL10R [5325CA(11_15)] and IL10G [8134CA(14_29)] are associated with the synthesis of Abs to PF4/heparin and HIT.nnnMATERIALS AND METHODSnEighty-two patients with definite HIT and two control groups were studied. The first control group (Ab(neg)) consisted of 85 patients without Abs to PF4/heparin after cardiopulmonary bypass (CPB). The second control group (Ab(pos)) consisted of 84 patients who had developed significant levels of PF4-specific antibodies after CPB, but without HIT.nnnRESULTSnAllele frequencies of the 3 SNPs were similar in HIT patients and controls. Fourteen alleles in IL10G (G16 to G29) and 3 alleles in IL10R (R13 to R15) were defined. The short G20 allele of IL10G was more frequent in Ab(neg) patients (8.2%) than in Ab(pos) (2.9%) and HIT patients (3%). It thereby appeared to protect against developing Abs to PF4/heparin (OR 0.29; 95% CI [0.12-0.70], p=0.006). Combined haplotypes cH1/cH8 comprising the short G20 + R13 alleles were less frequent in HIT (OR 0.33; 95% CI [0.11-0.97], p=0.036), and levels of Abs to PF4 in Ab(pos) patients were lower in cH1/cH8 subjects (p=0.019).nnnCONCLUSIONnThese results suggest that IL10 promoter microsatellite polymorphisms might influence the immune response against PF4/heparin and the risk of HIT.

Branched-Chain Amino Acid Database Integrated in MEDIPAD Software as a Tool for Nutritional Investigation of Mediterranean Populations

Branched-chained amino acids (BCAA) are essential dietary components for humans and can act as potential biomarkers for diabetes development. To efficiently estimate dietary intake, we developed a BCAA database for 1331 food items found in the French Centre d’Information sur la Qualité des Aliments (CIQUAL) food table by compiling BCAA content from international tables, published measurements, or by food similarity as well as by calculating 267 items from Greek, Turkish, Romanian, and Moroccan mixed dishes. The database embedded in MEDIPAD software capable of registering 24 h of dietary recalls (24HDR) with clinical and genetic data was evaluated based on archived 24HDR of the Saint Pierre Institute (France) from 2957 subjects, which indicated a BCAA content up to 4.2 g/100 g of food and differences among normal weight and obese subjects across BCAA quartiles. We also evaluated the database of 119 interviews of Romanians, Turkish and Albanians in Greece (27–65 years) during the MEDIGENE program, which indicated mean BCAA intake of 13.84 and 12.91 g/day in males and females, respectively, comparable to other studies. The MEDIPAD is user-friendly, multilingual, and secure software and with the BCAA database is suitable for conducting nutritional assessment in the Mediterranean area with particular facilities for food administration.

PO31 Cartographie génétique dense du locus FTO indique l’association du gène au syndrome des ovaires polykystiques

Introduction Le gene FTO (fat mass and obesity associated gene) a ete associe a l’obesite via des SNP (single nucleotide polymorphisms) localises dans son intron 1, parmi lesquels rs1421085 et rs8050136. Dans une precedente etude en Europe Centrale, nous avons rapporte l’association du SNP rs1421085 au syndrome metabolique (SMet) dans le syndrome des ovaires polykystiques (SOPK). Patients et methodes Afin de realiser la cartographie dense de ce locus, dans le cadre des nouvelles hypotheses lancees par notre programme europeen MEDIGENE (FP7-279171), nous avons defini la structure haplotypique d’une region genomique de 696 pb dans une population de SOPK et de controles (nxa0=xa0288). Le genotypage des SNP rs8050136 (A/C), rs4783819 (C/G), rs8051591 (A/G), rs4783820 (T/A) et rs9935401 (G/A) a ete effectue par sequencage direct alors que la reconstruction des haplotypes a ete realisee par PHASE. Resultats Nous avons reconstruit 4 haplotypes dans la population parmi lesquels H1 (ACGTA) qui est porteur exclusif de l’allele A du SNP rs8050136. L’association genetique (regression logistique) et la correlation genotype-phenotype (ANOVA) ont revele que le genotype A/A de rs8050136, de meme que la paire d’haplotypes H1/H1, sont associes au SMet dans le SOPK (Pxa0 Conclusion Ce travail souligne l’importance de la cartographie haplotypique par rapport aux SNP pris individuellement dans les etudes pangenomiques (GWAS) dans la perspective d’un criblage par les technologies a tres haute densite.

O101 Découverte de l’association du gène FTO avec le SOPK et la résistance à l’insuline dans une population d’Europe Centrale

Introduction Le gene FTO (fat mass and obesity associated) a ete decouvert par criblage systematique du genome comme associe au diabete de type 2 et a l’indice de masse corporelle (IMC). Des associations probantes ont ete demontrees dans l’obesite morbide chez l’enfant. Patients et methodes Afin de comprendre le role du FTO dans la resistance a l’insuline dans le syndrome des ovaires polykystiques (SOPK), nous avons explore les SNP de ce gene dans une population des femmes (nxa0=xa0104) avec SOPK issues d’Europe Centrale par rapport aux controles (nxa0=xa083). Une proportion de 29 % des femmes avec SOPK presente une resistance a l’insuline plus severe par rapport au SOPK maigre (HOMA 6,0xa0±xa01,0 vs 3,1xa0±xa00,3), un Acanthosis Nigricans (syndrome HAIRAN) et obesite (IMC 33,6xa0±xa00,8 vs 23,5xa0±xa00,4). Le SNP-081 (HapMap) localise dans l’intron 1 du gene a ete genotype par SSO-PCR (sequence specific oligonucleotide-PCR) et par sequencage. Ce SNP (C/T) est localise sur un haplotype unique dans la population europeenne. Resultats La frequence allelique du SNP-081 a ete plus importante dans le SOPK (qxa0=xa00,53 vs 0,49) surtout chez les femmes avec obesite. Un effet plus marquant de la prevalence des formes homozygotes (C/C) a ete observe avec un dosage genique respectivement de 18, 28,5 et 35,5 % chez les femmes controles, SOPK maigres et HAIRAN, (P Conclusion Ces resultats montrent pour la premiere fois l’influence du gene FTO dans le SOPK, en relation avec le degre d’obesite ou directement avec la resistance a l’insuline et par ce biais pourrait constituer un marqueur genetique important ayant un role predictif dans la resistance a l’insuline.