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1.  Maternal Diets Trigger Sex-Specific Divergent Trajectories of Gene Expression and Epigenetic Systems in Mouse Placenta 
PLoS ONE  2012;7(11):e47986.
Males and females responses to gestational overnutrition set the stage for subsequent sex-specific differences in adult onset non communicable diseases. Placenta, as a widely recognized programming agent, contibutes to the underlying processes. According to our previous findings, a high-fat diet during gestation triggers sex-specific epigenetic alterations within CpG and throughout the genome, together with the deregulation of clusters of imprinted genes. We further investigated the impact of diet and sex on placental histology, transcriptomic and epigenetic signatures in mice. Both basal gene expression and response to maternal high-fat diet were sexually dimorphic in whole placentas. Numerous genes showed sexually dimorphic expression, but only 11 genes regardless of the diet. In line with the key role of genes belonging to the sex chromosomes, 3 of these genes were Y-specific and 3 were X-specific. Amongst all the genes that were differentially expressed under a high-fat diet, only 16 genes were consistently affected in both males and females. The differences were not only quantitative but remarkably qualitative. The biological functions and networks of genes dysregulated differed markedly between the sexes. Seven genes of the epigenetic machinery were dysregulated, due to effects of diet, sex or both, including the Y- and X-linked histone demethylase paralogues Kdm5c and Kdm5d, which could mark differently male and female epigenomes. The DNA methyltransferase cofactor Dnmt3l gene expression was affected, reminiscent of our previous observation of changes in global DNA methylation. Overall, this striking sexual dimorphism of programming trajectories impose a considerable revision of the current dietary interventions protocols.
PMCID: PMC3489896  PMID: 23144842
2.  Enhancing field GP engagement in hospital-based studies. Rationale, design, main results and participation in the diagest 3-GP motivation study 
BMC Family Practice  2012;13:63.
Diagest 3 was a study aimed at lowering the risk of developing type 2 diabetes within 3 years after childbirth. Women with gestational diabetes were enrolled in the study. After childbirth, the subjects showed little interest in the structured education programme and did not attend workshops. Their general practitioners (GPs) were approached to help motivate the subjects to participate in Diagest 3, but the GPs were reluctant. The present study aimed to understand field GPs’ attitudes towards hospital-based studies, and to develop strategies to enhance their involvement and reduce subject drop-out rates.
We used a three-step process: step one used a phenomenological approach exploring the beliefs, attitudes, motivations and environmental factors contributing to the GPs’ level of interest in the study. Data were collected in face-to-face interviews and coded by hand and with hermeneutic software to develop distinct GP profiles. Step two was a cross-sectional survey by questionnaire to determine the distribution of the profiles in the GP study population and whether completion of an attached case report form (CRF) was associated with a particular GP profile. In step three, we assessed the impact of the motivation study on participation rates in the main study.
Fifteen interviews were conducted to achieve data saturation. Theorisation led to the definition of 4 distinct GP profiles. The response rate to the questionnaire was 73%, but dropped to 52% when a CRF was attached. The link between GP profiles and the rate of CRF completion remains to be verified. The GPs provided data on the CRF that was of comparable quality to those collected in the main trial. Our analysis showed that the motivation study increased overall participation in the main study by 23%, accounting for 16% (24/152) of all final visits for 536 patients who were initially enrolled in the Diagest 3 study.
When a hospital-led study explores issues in primary care, its design must anticipate GP participation early in the trial. Based on our questionnaire response rates, we found that one in two GPs were willing to participate in our hospital-led study, regardless of their initial attitudes.
PMCID: PMC3441219  PMID: 22721372
General practice; Biomedical research; Behavioural research; Research design; Cooperative behaviour; Social identification
3.  Whole-Exome Sequencing and High Throughput Genotyping Identified KCNJ11 as the Thirteenth MODY Gene 
PLoS ONE  2012;7(6):e37423.
Maturity-onset of the young (MODY) is a clinically heterogeneous form of diabetes characterized by an autosomal-dominant mode of inheritance, an onset before the age of 25 years, and a primary defect in the pancreatic beta-cell function. Approximately 30% of MODY families remain genetically unexplained (MODY-X). Here, we aimed to use whole-exome sequencing (WES) in a four-generation MODY-X family to identify a new susceptibility gene for MODY.
WES (Agilent-SureSelect capture/Illumina-GAIIx sequencing) was performed in three affected and one non-affected relatives in the MODY-X family. We then performed a high-throughput multiplex genotyping (Illumina-GoldenGate assay) of the putative causal mutations in the whole family and in 406 controls. A linkage analysis was also carried out.
Principal Findings
By focusing on variants of interest (i.e. gains of stop codon, frameshift, non-synonymous and splice-site variants not reported in dbSNP130) present in the three affected relatives and not present in the control, we found 69 mutations. However, as WES was not uniform between samples, a total of 324 mutations had to be assessed in the whole family and in controls. Only one mutation (p.Glu227Lys in KCNJ11) co-segregated with diabetes in the family (with a LOD-score of 3.68). No KCNJ11 mutation was found in 25 other MODY-X unrelated subjects.
Beyond neonatal diabetes mellitus (NDM), KCNJ11 is also a MODY gene (‘MODY13’), confirming the wide spectrum of diabetes related phenotypes due to mutations in NDM genes (i.e. KCNJ11, ABCC8 and INS). Therefore, the molecular diagnosis of MODY should include KCNJ11 as affected carriers can be ideally treated with oral sulfonylureas.
PMCID: PMC3372463  PMID: 22701567
4.  Consequences of gestational and pregestational diabetes on placental function and birth weight 
World Journal of Diabetes  2011;2(11):196-203.
Maternal diabetes constitutes an unfavorable environment for embryonic and fetoplacental development. Despite current treatments, pregnant women with pregestational diabetes are at increased risk for congenital malformations, materno-fetal complications, placental abnormalities and intrauterine malprogramming. The complications during pregnancy concern the mother (gravidic hypertension and/or preeclampsia, cesarean section) and the fetus (macrosomia or intrauterine growth restriction, shoulder dystocia, hypoglycemia and respiratory distress). The fetoplacental impairment and intrauterine programming of diseases in the offspring’s later life induced by gestational diabetes are similar to those induced by type 1 and type 2 diabetes mellitus. Despite the existence of several developmental and morphological differences in the placenta from rodents and women, there are similarities in the alterations induced by maternal diabetes in the placenta from diabetic patients and diabetic experimental models. From both human and rodent diabetic experimental models, it has been suggested that the placenta is a compromised target that largely suffers the impact of maternal diabetes. Depending on the maternal metabolic and proinflammatory derangements, macrosomia is explained by an excessive availability of nutrients and an increase in fetal insulin release, a phenotype related to the programming of glucose intolerance. The degree of fetal damage and placental dysfunction and the availability and utilisation of fetal substrates can lead to the induction of macrosomia or intrauterine growth restriction. In maternal diabetes, both the maternal environment and the genetic background are important in the complex and multifactorial processes that induce damage to the embryo, the placenta, the fetus and the offspring. Nevertheless, further research is needed to better understand the mechanisms that govern the early embryo development, the induction of congenital anomalies and fetal overgrowth in maternal diabetes.
PMCID: PMC3215769  PMID: 22087356
Maternal diabetes; Placental function; Birth weight; Macrosomia; Intrauterine growth retardation
5.  Time course analysis of RNA stability in human placenta 
BMC Molecular Biology  2009;10:21.
Evaluation of RNA quality is essential for gene expression analysis, as the presence of degraded samples may influence the interpretation of expression levels. Particularly, qRT-PCR data can be affected by RNA integrity and stability. To explore systematically how RNA quality affects qRT-PCR assay performance, a set of human placenta RNA samples was generated by two protocols handlings of fresh tissue over a progressive time course of 4 days. Protocol A consists of a direct transfer of tissue into RNA-stabilizing solution (RNAlater™) solution. Protocol B uses a dissection of placenta villosities before bio banking. We tested and compared RNA yields, total RNA integrity, mRNA integrity and stability in these two protocols according to the duration of storage.
A long time tissue storage had little effect on the total RNA and mRNA integrity but induced changes in the transcript levels of stress-responsive genes as TNF-alpha or COX2 after 48 h. The loss of the RNA integrity was higher in the placental tissues that underwent a dissection before RNA processing by comparison with those transferred directly into RNA later™ solution. That loss is moderate, with average RIN (RNA Integration Numbers) range values of 4.5–6.05, in comparison with values of 6.44–7.22 in samples directly transferred to RNAlater™ (protocol A). Among the house keeping genes tested, the B2M is the most stable.
This study shows that placental samples can be stored at + 4°C up to 48 h before RNA extraction without altering RNA quality. Rapid tissue handling without dissection and using RNA-stabilizing solution (RNAlater™) is a prerequisite to obtain suitable RNA integrity and stability.
PMCID: PMC2664811  PMID: 19284566
6.  Muscle Oxygen Supply Impairment during Exercise in Poorly Controlled Type 1 Diabetes 
Aerobic fitness, as reflected by maximal oxygen (O2) uptake (V˙O2max), is impaired in poorly controlled patients with type 1 diabetes. The mechanisms underlying this impairment remain to be explored. This study sought to investigate whether type 1 diabetes and high levels of glycated hemoglobin (HbA1c) influence O2 supply including O2 delivery and release to active muscles during maximal exercise.
Two groups of patients with uncomplicated type 1 diabetes (T1D-A, n = 11, with adequate glycemic control, HbA1c <7.0%; T1D-I, n = 12 with inadequate glycemic control, HbA1c >8%) were compared with healthy controls (CON-A, n = 11; CON-I, n = 12, respectively) matched for physical activity and body composition. Subjects performed exhaustive incremental exercise to determine V˙O2max. Throughout the exercise, near-infrared spectroscopy allowed investigation of changes in oxyhemoglobin, deoxyhemoglobin, and total hemoglobin in the vastus lateralis. Venous and arterialized capillary blood was sampled during exercise to assess arterial O2 transport and factors able to shift the oxyhemoglobin dissociation curve.
Arterial O2 content was comparable between groups. However, changes in total hemoglobin (i.e., muscle blood volume) was significantly lower in T1D-I compared with that in CON-I. T1D-I also had impaired changes in deoxyhemoglobin levels and increase during high-intensity exercise despite normal erythrocyte 2,3-diphosphoglycerate levels. Finally, V˙O2max was lower in T1D-I compared with that in CON-I. No differences were observed between T1D-A and CON-A.
Poorly controlled patients displayed lower V˙O2max and blunted muscle deoxyhemoglobin increase. The latter supports the hypotheses of increase in O2 affinity induced by hemoglobin glycation and/or of a disturbed balance between nutritive and nonnutritive muscle blood flow. Furthermore, reduced exercise muscle blood volume in poorly controlled patients may warn clinicians of microvascular dysfunction occurring even before overt microangiopathy.
PMCID: PMC4323553  PMID: 24983346

Results 1-6 (6)