Pathogenic variants in actionable MODY genes are associated with type 2 diabetes.

Genome-wide association studies have identified 240 independent loci associated with type 2 diabetes (T2D) risk, but this knowledge has not advanced precision medicine. In contrast, the genetic diagnosis of monogenic forms of diabetes (including maturity-onset diabetes of the young (MODY)) are textbook cases of genomic medicine. Recent studies trying to bridge the gap between monogenic diabetes and T2D have been inconclusive. Here, we show a significant burden of pathogenic variants in genes linked with monogenic diabetes among people with common T2D, particularly in actionable MODY genes, thus implying that there should be a substantial change in care for carriers with T2D. We show that, among 74,629 individuals, this burden is probably driven by the pathogenic variants found in GCK, and to a lesser extent in HNF4A, KCNJ11, HNF1B and ABCC8. The carriers with T2D are leaner, which evidences a functional metabolic effect of these mutations. Pathogenic variants in actionable MODY genes are more frequent than was previously expected in common T2D. These results open avenues for future interventions assessing the clinical interest of these pathogenic mutations in precision medicine.

A Novel Rare Missense Variation of the NOD2 Gene: Evidencesof Implication in Crohn's Disease.

The NOD2 gene, involved in innate immune responses to bacterial peptidoglycan, has been found to be closely associated with Crohn's Disease (CD), with an Odds Ratio ranging from 3⁻36. Families with three or more CD-affected members were related to a high frequency of NOD2 gene variations, such as R702W, G908R, and 1007fs, and were reported in the EPIMAD Registry. However, some rare CD multiplex families were described without identification of common NOD2 linked-to-disease variations. In order to identify new genetic variation(s) closely linked with CD, whole exome sequencing was performed on available subjects, comprising four patients in two generations affected with Crohn's disease without R702W and G908R variation and three unaffected related subjects. A rare and, not yet, reported missense variation of the NOD2 gene, N1010K, was detected and co-segregated across affected patients. In silico evaluation and modelling highlighted evidence for an adverse effect of the N1010K variation with regard to CD. Moreover, cumulative characterization of N1010K and 1007fs as a compound heterozygous state in two, more severe CD family members strongly suggests that N1010K could well be a new risk factor involved in Crohn's disease genetic susceptibility.

Distinct virulence ranges for infection of mice by Bordetella pertussis revealed by engineering of the sensor-kinase BvgS.

The whooping cough agent Bordetella pertussis coordinately regulates the expression of its virulence factors with the two-component system BvgAS. In laboratory conditions, specific chemical modulators are used to trigger phenotypic modulation of B. pertussis from its default virulent Bvg+ phase to avirulent Bvg- or intermediate Bvgi phases, in which no virulence factors or only a subset of them are produced, respectively. Whether phenotypic modulation occurs in the host remains unknown. In this work, recombinant B. pertussis strains harboring BvgS variants were tested in a mouse model of infection and analyzed using transcriptomic approaches. Recombinant BP-BvgΔ65, which is in the Bvgi phase by default and can be up-modulated to the Bvg+ phase in vitro, could colonize the mouse nose but was rapidly cleared from the lungs, while Bvg+-phase strains colonized both organs for up to four weeks. These results indicated that phenotypic modulation, which might have restored the full virulence capability of BP-BvgΔ65, does not occur in mice or is temporally or spatially restricted and has no effect in those conditions. Transcriptomic analyses of this and other recombinant Bvgi and Bvg+-phase strains revealed that two distinct ranges of virulence gene expression allow colonization of the mouse nose and lungs, respectively. We also showed that a recombinant strain expressing moderately lower levels of the virulence genes than its wild type parent was as efficient at colonizing both organs. Altogether, genetic modifications of BvgS generate a range of phenotypic phases, which are useful tools to decipher host-pathogen interactions.

High Prevalence of Rare Monogenic Forms of Obesity in Obese Guadeloupean Afro-Caribbean Children.

Context: The population of Guadeloupe Island exhibits a high prevalence of obesity. Objective: We aimed to investigate whether rare genetic mutations in genes involved in monogenic obesity (or diabetes) might be causal in this population of Afro-Caribbean ancestry. Design and Setting: This was a secondary analysis of a study on obesity conducted in schoolchildren from Guadeloupe in 2013 that aimed to assess changes in children's profiles after a lifestyle intervention program. Through next-generation sequencing, we sequenced coding regions of 59 genes involved in monogenic obesity or diabetes in participants from this study. Participants and Interventions: A total of 25 obese schoolchildren from Guadeloupe were screened for rare mutations (nonsynonymous, splice-site, or insertion/deletion) in 59 genes. Main Outcome Measures: Correlation between phenotypes and mutations of interest. Results: We detected five rare heterozygous mutations in five different children with obesity: MC4R p.Ile301Thr and SIM1 p.Val326Thrfs*43 mutations that were pathogenic; SIM1 p.Ser343Pro and SH2B1 p.Pro90His mutations that were likely pathogenic; and NTRK2 p.Leu140Phe that was of uncertain significance. In parallel, we identified seven carriers of mutations in ABCC8 (p.Lys1521Asn and p.Ala625Val) or KCNJ11 (p.Val13Met and p.Val151Met) that were of uncertain significance. Conclusions: We were able to detect pathogenic or likely pathogenic mutations linked to severe obesity in >15% of this population, which is much higher than what we observed in Europeans (∼5%).

Low-dose exposure to bisphenols A, F and S of human primary adipocyte impacts coding and non-coding RNA profiles.

Bisphenol A (BPA) exposure has been suspected to be associated with deleterious effects on health including obesity and metabolically-linked diseases. Although bisphenols F (BPF) and S (BPS) are BPA structural analogs commonly used in many marketed products as a replacement for BPA, only sparse toxicological data are available yet. Our objective was to comprehensively characterize bisphenols gene targets in a human primary adipocyte model, in order to determine whether they may induce cellular dysfunction, using chronic exposure at two concentrations: a "low-dose" similar to the dose usually encountered in human biological fluids and a higher dose. Therefore, BPA, BPF and BPS have been added at 10 nM or 10 µM during the differentiation of human primary adipocytes from subcutaneous fat of three non-diabetic Caucasian female patients. Gene expression (mRNA/lncRNA) arrays and microRNA arrays, have been used to assess coding and non-coding RNA changes. We detected significantly deregulated mRNA/lncRNA and miRNA at low and high doses. Enrichment in "cancer" and "organismal injury and abnormalities" related pathways was found in response to the three products. Some long intergenic non-coding RNAs and small nucleolar RNAs were differentially expressed suggesting that bisphenols may also activate multiple cellular processes and epigenetic modifications. The analysis of upstream regulators of deregulated genes highlighted hormones or hormone-like chemicals suggesting that BPS and BPF can be suspected to interfere, just like BPA, with hormonal regulation and have to be considered as endocrine disruptors. All these results suggest that as BPA, its substitutes BPS and BPF should be used with the same restrictions.

Characterization of a Bvg-regulated fatty acid methyl-transferase in Bordetella pertussis.

The whooping cough agent Bordetella pertussis controls the expression of its large virulence regulon in a coordinated manner through the two-component signal transduction system BvgAS. In addition to the genes coding for bona fide virulence factors, the Bvg regulon comprises genes of unknown function. In this work, we characterized a new Bvg-activated gene called BP2936. Homologs of BP2936 are found in other pathogenic Bordetellae and in several other species, including plant pathogens and environmental bacteria. We showed that the gene product of BP2936 is a membrane-associated methyl-transferase of free fatty acids. We thus propose to name it FmtB, for fatty acid methyl-transferase of Bordetella. The role of this protein was tested in cellular and animal models of infection, but the loss of BP2936 did not appear to affect host-pathogen interactions in those assays. The high level of conservation of BP2936 among B. pertussis isolates nevertheless argues that it probably plays a role in the life cycle of this pathogen.

DNA Damage and the Activation of the p53 Pathway Mediate Alterations in Metabolic and Secretory Functions of Adipocytes.

Activation of the p53 pathway in adipose tissue contributes to insulin resistance associated with obesity. However, the mechanisms of p53 activation and the effect on adipocyte functions are still elusive. Here we found a higher level of DNA oxidation and a reduction in telomere length in adipose tissue of mice fed a high-fat diet and an increase in DNA damage and activation of the p53 pathway in adipocytes. Interestingly, hallmarks of chronic DNA damage are visible at the onset of obesity. Furthermore, injection of lean mice with doxorubicin, a DNA damage-inducing drug, increased the expression of chemokines in adipose tissue and promoted its infiltration by proinflammatory macrophages and neutrophils together with adipocyte insulin resistance. In vitro, DNA damage in adipocytes increased the expression of chemokines and triggered the production of chemotactic factors for macrophages and neutrophils. Insulin signaling and effect on glucose uptake and Glut4 translocation were decreased, and lipolysis was increased. These events were prevented by p53 inhibition, whereas its activation by nutlin-3 reproduced the DNA damage-induced adverse effects. This study reveals that DNA damage in obese adipocytes could trigger p53-dependent signals involved in alteration of adipocyte metabolism and secretory function leading to adipose tissue inflammation, adipocyte dysfunction, and insulin resistance.

What Is the Best NGS Enrichment Method for the Molecular Diagnosis of Monogenic Diabetes and Obesity?

Molecular diagnosis of monogenic diabetes and obesity is of paramount importance for both the patient and society, as it can result in personalized medicine associated with a better life and it eventually saves health care spending. Genetic clinical laboratories are currently switching from Sanger sequencing to next-generation sequencing (NGS) approaches but choosing the optimal protocols is not easy. Here, we compared the sequencing coverage of 43 genes involved in monogenic forms of diabetes and obesity, and variant detection rates, resulting from four enrichment methods based on the sonication of DNA (Agilent SureSelect, RainDance technologies), or using enzymes for DNA fragmentation (Illumina Nextera, Agilent HaloPlex). We analyzed coding exons and untranslated regions of the 43 genes involved in monogenic diabetes and obesity. We found that none of the methods achieves yet full sequencing of the gene targets. Nonetheless, the RainDance, SureSelect and HaloPlex enrichment methods led to the best sequencing coverage of the targets; while the Nextera method resulted in the poorest sequencing coverage. Although the sequencing coverage was high, we unexpectedly found that the HaloPlex method missed 20% of variants detected by the three other methods and Nextera missed 10%. The question of which NGS technique for genetic diagnosis yields the highest diagnosis rate is frequently discussed in the literature and the response is still unclear. Here, we showed that the RainDance enrichment method as well as SureSelect, which are both based on the sonication of DNA, resulted in a good sequencing quality and variant detection, while the use of enzymes to fragment DNA (HaloPlex or Nextera) might not be the best strategy to get an accurate sequencing.

Highly sensitive diagnosis of 43 monogenic forms of diabetes or obesity through one-step PCR-based enrichment in combination with next-generation sequencing.

OBJECTIVE: Accurate etiological diagnosis of monogenic forms of diabetes and obesity is useful as it can lead to marked improvements in patient care and genetic counseling. Currently, molecular diagnosis based on Sanger sequencing is restricted to only a few genes, as this technology is expensive, time-consuming, and labor-intensive. High-throughput next-generation sequencing (NGS) provides an opportunity to develop innovative cost-efficient methods for sensitive diabetes and obesity multigene screening. RESEARCH DESIGN AND METHODS: We assessed a new method based on PCR enrichment in microdroplets (RainDance Technologies) and NGS using the Illumina HiSeq2000 for the molecular diagnosis of 43 forms of monogenic diabetes or obesity. Forty patients carrying a known causal mutation for those subtypes according to diagnostic laboratories were blindly reanalyzed. RESULTS: Except for one variant, we reidentified all causal mutations in each patient associated with an almost-perfect sequencing of the targets (mean of 98.6%). We failed to call one highly complex indel, although we identified a dramatic drop of coverage at this locus. In three patients, we detected other mutations with a putatively deleterious effect in addition to those reported by the genetic diagnostic laboratories. CONCLUSIONS: Our NGS approach provides an efficient means of highly sensitive screening for mutations in genes associated with monogenic forms of diabetes and obesity. As cost and time to deliver results have been key barriers to uncovering a molecular cause in the many undiagnosed cases likely to exist, the present methodology should be considered in patients displaying features of monogenic diabetes or obesity.