Placental antiangiogenic prolactin fragments are increased in human and rat maternal diabetes.

INTRODUCTION/OBJECTIVES: The role of the placenta in diabetic mothers on fetal development and programming is unknown. Prolactin (PRL) produced by decidual endometrial cells may have an impact. Although full-length PRL is angiogenic, the processed form by bone morphogenetic protein-1 (BMP-1) and/or cathepsin D (CTSD) is antiangiogenic. The objectives were to investigate the involvement of decidual PRL and its antiangiogenic fragments in placentas from type-1 diabetic women (T1D) and from pregnant diabetic rats with lower offspring weights than controls. METHODS: PRL, BMP-1, and CTSD gene expressions and PRL protein level were assessed in T1D placentas (n=8) at delivery and compared to controls (n=5). Wistar rats received, at day 7 of pregnancy, streptozotocin (STZ) (n=5) or nicotinamide (NCT) plus STZ (n=9) or vehicle (n=9). Placental whole-genome gene expression and PRL western blots were performed at birth. RESULTS: In human placentas, PRL (p<0.05) and BMP-1 (p<0.01) gene expressions were increased with a higher amount of cleaved PRL (p<0.05) in T1D than controls. In rats, diabetes was more pronounced in STZ than in NCT-STZ group with intra-uterine growth restriction. Decidual prolactin-related protein (Dprp) (p<0.01) and Bmp-1 (p<0.001) genes were up-regulated in both diabetic groups, with an increased cleaved PRL amount in the STZ (p<0.05) and NCT-STZ (p<0.05) groups compared to controls. No difference in CTSD gene expression was observed in rats or women. CONCLUSIONS: Alterations in the levels of the PRL family are associated with maternal diabetes in both rats and T1D women suggesting that placental changes in these hormones impact on fetal development.

Transcription factor gene MNX1 is a novel cause of permanent neonatal diabetes in a consanguineous family.

AIM: Permanent neonatal diabetes mellitus (PNDM) is a rare monogenic form of non-autoimmune diabetes. Genetic defects have been identified in∼60% of cases, with mutations in ABCC8, KCNJ11 and INS being the most frequent causes of PNDM. Recognition of genetic subtypes strongly impacts on both patients' care and family counseling. This study aimed to identify the genetic aetiology of PNDM in a diabetic girl born of consanguineous parents. METHODS: DNA samples from both the proband and her non-diabetic parents were analyzed for homozygosity mapping, using Illumina Infinium 660K SNP microarrays, focusing on the runs of homozygosity (ROHs) detected only in the patient. Standard Sanger sequencing of candidate genes (MNX1 and GATA6) present in the ROHs was subsequently performed, as well as expression analyses on human embryonic and adult pancreatic islet samples. RESULTS: A putative causal homozygous mutation in the transcription factor gene MNX1 (c.816C>A/p.Phe272Leu) was identified in the PNDM patient, who was clinically diagnosed as a typical case of PNDM with no developmental pancreatic defects or other clinical features. The probable deleterious mutation was located within the MNX1 homeodomain helix 2 that is highly conserved between species. In human embryonic pancreatic islet samples, it has been shown that MNX1 expression is significantly enriched in pancreatic epithelium compared with mesenchyme, suggesting a role for MNX1 in human pancreatic beta-cell development. CONCLUSION: This study found a new putative cause of PNDM in a consanguineous family. Replication in other cohorts would help to clarify the clinical spectrum of MNX1 mutations in PNDM patients.

Multiple functional polymorphisms in the G6PC2 gene contribute to the association with higher fasting plasma glucose levels.

AIMS/HYPOTHESIS: We previously identified the G6PC2 locus as a strong determinant of fasting plasma glucose (FPG) and showed that a common G6PC2 intronic single nucleotide polymorphism (SNP) (rs560887) and two common G6PC2 promoter SNPs (rs573225 and rs13431652) are highly associated with FPG. However, these promoter SNPs have complex effects on G6PC2 fusion gene expression, and our data suggested that only rs13431652 is a potentially causative SNP. Here we examine the effect of rs560887 on G6PC2 pre-mRNA splicing and the contribution of an additional common G6PC2 promoter SNP, rs2232316, to the association signal. METHODS: Minigene analyses were used to characterise the effect of rs560887 on G6PC2 pre-mRNA splicing. Fusion gene and gel retardation analyses characterised the effect of rs2232316 on G6PC2 promoter activity and transcription factor binding. The genetic association of rs2232316 with FPG variation was assessed using regression adjusted for age, sex and BMI in 4,220 Europeans with normal FPG. RESULTS: The rs560887-G allele was shown to enhance G6PC2 pre-mRNA splicing, whereas the rs2232316-A allele enhanced G6PC2 transcription by promoting Foxa2 binding. Genetic analyses provide evidence for association of the rs2232316-A allele with increased FPG (ß = 0.04 mmol/l; p = 4.3 × 10(-3)) as part of the same signal as rs560887, rs573225 and rs13431652. CONCLUSIONS/INTERPRETATION: As with rs13431652, the in situ functional data with rs560887 and rs2232316 are in accord with the putative function of G6PC2 in pancreatic islets, and suggest that all three are potentially causative SNPs that contribute to the association between G6PC2 and FPG.

Contribution of 24 obesity-associated genetic variants to insulin resistance, pancreatic beta-cell function and type 2 diabetes risk in the French population.

CONTEXT: Obesity is the major determinant of type 2 diabetes (T2D), presumably through its effect on insulin resistance. Genome-wide association studies reported many single-nucleotide polymorphisms (SNPs) that increase obesity risk and body mass index (BMI), but their impact on T2D-related traits and risk is unclear. OBJECTIVE: We aimed at analyzing the effect of 24 obesity risk alleles, separately and in combination, on variation of both insulin resistance and ß-cell dysfunction, and on T2D risk. DESIGN: We genotyped 24 obesity-associated SNPs and calculated an obesity genotype score (sum of the obesity risk alleles per individual). We analyzed the contribution of each SNP and this score to the variation of four metabolic indices: homeostasis model assessment of insulin resistance (HOMA-IR), homeostasis model assessment of the pancreatic ß-cell function (HOMA-B), insulin sensitivity index (ISI) and insulinogenic index (II) (in up to 8050 nondiabetic French individuals) and to T2D risk (in 2077 T2D cases and 3085 controls). RESULTS: We found a highly significant effect of the obesity genotype score on increased insulin resistance adjusted for age and gender (ß=0.02; P-value=7.16 × 10(-9) for HOMA-IR). Individually, we identified nominal or significant association between increased insulin resistance and risk alleles in FAIM2, FTO, GNPDA2, MC4R, NPC1, PTER and SH2B1. Most signals, including the obesity genotype score and FTO SNP, were also associated with increased ß-cell function (ß=0.01; P-value=1.05 × 10(-6) and ß=0.04; P-value=3.45 × 10(-4), respectively). In our T2D case-control study, only the obesity genotype score and the well-known FTO locus significantly contributed to T2D risk (OR=1.03; P-value=9.99 × 10(-3) and OR=1.15; P-value=9.46 × 10(-4), respectively). Adjustment for BMI abolished all significant associations. CONCLUSIONS: Genetic predisposition to obesity contributes to increased insulin resistance and to its compensation through increased ß-cell function, and weakly increases the T2D risk. These associations are mediated by BMI.

A new highly penetrant form of obesity due to deletions on chromosome 16p11.2.

Obesity has become a major worldwide challenge to public health, owing to an interaction between the Western 'obesogenic' environment and a strong genetic contribution. Recent extensive genome-wide association studies (GWASs) have identified numerous single nucleotide polymorphisms associated with obesity, but these loci together account for only a small fraction of the known heritable component. Thus, the 'common disease, common variant' hypothesis is increasingly coming under challenge. Here we report a highly penetrant form of obesity, initially observed in 31 subjects who were heterozygous for deletions of at least 593 kilobases at 16p11.2 and whose ascertainment included cognitive deficits. Nineteen similar deletions were identified from GWAS data in 16,053 individuals from eight European cohorts. These deletions were absent from healthy non-obese controls and accounted for 0.7% of our morbid obesity cases (body mass index (BMI) >or= 40 kg m(-2) or BMI standard deviation score >or= 4; P = 6.4 x 10(-8), odds ratio 43.0), demonstrating the potential importance in common disease of rare variants with strong effects. This highlights a promising strategy for identifying missing heritability in obesity and other complex traits: cohorts with extreme phenotypes are likely to be enriched for rare variants, thereby improving power for their discovery. Subsequent analysis of the loci so identified may well reveal additional rare variants that further contribute to the missing heritability, as recently reported for SIM1 (ref. 3). The most productive approach may therefore be to combine the 'power of the extreme' in small, well-phenotyped cohorts, with targeted follow-up in case-control and population cohorts.

Hypoadiponectinaemia and high risk of type 2 diabetes are associated with adiponectin-encoding (ACDC) gene promoter variants in morbid obesity: evidence for a role of ACDC in diabesity.

AIMS/HYPOTHESIS: Morbid obesity (BMI>40 kg/m(2)) affecting 0.5-5% of the adult population worldwide is a major risk factor for type 2 diabetes. We aimed to elucidate the genetic bases of diabetes associated with obesity (diabesity), and to analyse the impact of corpulence on the effects of diabetes susceptibility genes. METHODS: We genotyped known single nucleotide polymorphisms (SNPs) in the adiponectin-encoding adipocyte C1q and collagen-domain-containing (ACDC) gene (-11,391G>A, -11,377C>G, +45T>G and +276G>T), the peroxisome proliferator-activated receptor gamma (PPARG) Pro12Ala SNP and ACDC exon 3 variants in 703 French morbidly obese subjects (BMI 47.6+/-7.4 kg/m(2)), 808 non-obese subjects (BMI<30 kg/m(2)) and 493 obese subjects (30< or =BMI<40 kg/m(2)). RESULTS: Two 5'-ACDC SNPs -11,391G>A, -11,377C>G were associated with adiponectin levels (p=0.0003, p=0.008) and defined a "low-level" haplotype associated with decreased adiponectin levels (p=0.0002) and insulin sensitivity (p=0.01) and with a risk of type 2 diabetes that was twice as high (p=0.002). In contrast, the prevalence of the PPARG Pro12Ala was identical in diabetic and normoglycaemic morbidly obese subjects. The PPARG Pro12 allele only displayed a trend of association with type 2 diabetes in the non-obese group. ACDC exon 3 variants were associated with type 2 diabetes in the non-obese group only (odds ratio 7.85, p<0.0001). In contrast, the 5'-ACDC "low-level" haplotype was associated with type 2 diabetes in obese and morbidly obese subjects (odds ratio 1.73 and 1.92) but not in non-obese individuals. CONCLUSIONS/INTERPRETATION: These data clarify the contribution of the 5'-ACDC SNPs to the risk of diabesity. Their interaction with corpulence suggests for the first time a different genetic profile of type 2 diabetes in morbidly obese patients compared with in less obese individuals.

Effect of common polymorphisms in the HNF4alpha promoter on susceptibility to type 2 diabetes in the French Caucasian population.

AIMS/HYPOTHESIS: The gene encoding HNF-4alpha, an orphan nuclear receptor playing critical roles in embryogenesis and metabolism by regulating gene expression in pancreatic beta cells, liver, and other tissues, is localised to chromosome 20q13, where linkage to type 2 diabetes has been shown in multiple studies. As two reports have independently demonstrated a convincing association with variants adjacent to the HNF-4alpha P2 promoter in Finnish and Ashkenazi Jewish populations, we evaluated their contribution to diabetes risk in the French Caucasian population. METHODS: Genotypes for four haplotype tag SNPs were analysed for association with diabetes in a case-control study of 744 unrelated type 2 diabetic patients and 686 normoglycaemic subjects, and for linkage in 148 diabetic families in whom significant linkage to the HNF4alpha region had been shown. RESULTS: The association seen in the Finnish and Ashkenazi studies for SNPs rs2144908 and rs1884614 located within a haplotype block encompassing the beta cell promoter P2 of HNF-4alpha was not replicated in our study; in French Caucasians the minor allele prevalence was increased in control subjects [odds ratio (OR) 0.80, uncorrected p=0.022 for rs2144908; OR 0.82 uncorrected p=0.058 for rs1884614]. Furthermore, none of the SNPs tested in the French familial sample was associated with diabetes, nor do they appear to contribute to the linkage. CONCLUSIONS/INTERPRETATION: None of the previously associated SNPs confer an increased risk for diabetes in French Caucasians. A large meta-analysis of association studies will determine whether there is a consistent association between particular SNPs upstream of HNF-4alpha and type 2 diabetes in several ethnic groups.

The adiponectin gene SNP+45 is associated with coronary artery disease in Type 2 (non-insulin-dependent) diabetes mellitus.

BACKGROUND: The ACRP30/adiponectin gene on chromosome 3q27, a region linked to the metabolic syndrome, encodes for the abundant adipocyte-specific secreted protein. Consistent rodent and human studies suggested that this adipokine may be a molecular link between metabolic and cardiovascular diseases. AIMS: In order to investigate the role of single nucleotide polymorphisms (SNPs) within the APM1 gene in the susceptibility to coronary artery disease (CAD), we performed a case-control study on Caucasian Type 2 (non-insulin-dependent) diabetic patients, a population at high-risk for CAD. METHODS: Five APM1 SNPs were genotyped in 162 Type 2 diabetic French and Swiss subjects with CAD and in 315 Type 2 diabetic French and Swiss subjects without CAD. RESULTS: In univariate analysis, SNP+45 T>G was associated with CAD (OR 1.9 95% CI 1.2-2.9 P = 0.0036). In multivariate analysis, SNP+45 T>G remained associated with CAD (OR 1.2 95% CI 0.8-1.9 P = 0.017), independently of classical cardiovascular risk factors including components of the metabolic syndrome. SNP haplotype analyses revealed a CAD protective combination of all SNP wild-type alleles (OR 0.5 95% CI 0.3-0.7 P = 0.0006). CONCLUSIONS: Our study, performed in diabetic subjects, revealed an association between individual SNP+45 in the APM1 gene and CAD. Furthermore, the susceptibility for CAD due to SNP+45 was independent of classic cardiovascular risk factors. Further studies will be necessary to confirm the role of SNP+45 in the development of CAD. However, ACRP30/adiponectin may contribute to atherosclerosis susceptibility in high-risk populations such as Type 2 diabetic subjects.

No evidence for linkage or for diabetes-associated mutations in the activin type 2B receptor gene (ACVR2B) in French patients with mature-onset diabetes of the young or type 2 diabetes.

Activins are members of the transforming growth factor-beta superfamily. They have a wide range of biological effects on cell growth and differentiation. For transmembrane signaling, activins bind directly to activin receptor type 2A (ACVR2A) or 2B (ACVR2B). Transgenic and knock-out mice for the ACVR2B gene display various endocrine pancreas-related abnormalities, including islet hypoplasia and glucose intolerance, demonstrating the crucial role of ACVR2B in the regulation of pancreas development. We have thus examined the contribution of this factor to the development of mature-onset diabetes of the young (MODY) and type 2 diabetes. No evidence of linkage at the ACVR2B locus has been detected in MODY families with unknown etiology for diabetes or found in affected sib pairs from families with type 2 diabetes. Mutation screening of the coding sequence in MODY probands and in a family with severe type 2 diabetes, including a case of pancreatic agenesis, showed single nucleotide polymorphisms that did not cosegregate with MODY and were not associated with type 2 diabetes. Our results indicate that ACVR2B does not represent a common cause of either MODY or type 2 diabetes in the French Caucasian population.