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Biallelic variations in SREK1 reduce SNORD115/116 expression, linking severe obesity and Prader-Willi-like traits, offering genetic and molecular insights into a new form of syndromic obesity.Biallelic variations in SREK1 reduce SNORD115/116 expression, linking severe obesity and Prader-Willi-like traits, offering genetic and molecular insights into a new form of syndromic obesity.

Functional genetics has identified drug targets for metabolic disorders. Opioid use impacts metabolic homeostasis, although mechanisms remain elusive. Here, we explore the OPRD1 gene (encoding delta opioid receptor, DOP) to understand its impact on type 2 diabetes. Large-scale sequencing of OPRD1 and in vitro analysis reveal that loss-of-function variants are associated with higher adiposity and lower hyperglycemia risk, whereas gain-of-function variants are associated with lower adiposity and higher type 2 diabetes risk. These findings align with studies of opium addicts. OPRD1 is expressed in human islets and beta cells, with decreased expression under type 2 diabetes conditions. DOP inhibition by an antagonist enhances insulin secretion from human beta cells and islets. RNA-sequencing identifies pathways regulated by DOP antagonism, including nerve growth factor, circadian clock, and nuclear receptor pathways. Our study highlights DOP as a key player between opioids and metabolic homeostasis, suggesting its potential as a therapeutic target for type 2 diabetes. Opioid use impacts metabolic homeostasis, although the mechanisms remain elusive. Here, the authors explore the OPRD1 gene (encoding the delta opioid receptor, DOP) to understand its impact on type 2 diabetes and highlight DOP as a key player between opioids and metabolic homeostasis.

Proteogenomics is becoming a powerful tool in personalized medicine by linking genomics, transcriptomics and mass spectrometry (MS)-based proteomics. Due to increasing evidence of alternative open reading frame-encoded proteins (AltProts), proteogenomics has a high potential to unravel the characteristics, variants, expression levels of the alternative proteome, in addition to already annotated proteins (RefProts). To obtain a broader view of the proteome of ovarian cancer cells compared to ovarian epithelial cells, cell-specific total RNA-sequencing profiles and customized protein databases were generated. In total, 128 RefProts and 30 AltProts were identified exclusively in SKOV-3 and PEO-4 cells. Among them, an AltProt variant of IP_715944, translated from DHX8 , was found mutated (p.Leu44Pro). We show high variation in protein expression levels of RefProts and AltProts in different subcellular compartments. The presence of 117 RefProt and two AltProt variants was described, along with their possible implications in the different physiological/pathological characteristics. To identify the possible involvement of AltProts in cellular processes, cross-linking-MS (XL-MS) was performed in each cell line to identify AltProt-RefProt interactions. This approach revealed an interaction between POLD3 and the AltProt IP_183088, which after molecular docking, was placed between POLD3-POLD2 binding sites, highlighting its possibility of the involvement in DNA replication and repair.

Using multi-omics analyses including RNAseq, RT-PCR, RACE-PCR, and shotgun proteomic with enrichment strategies, we demonstrated that newborn rat astrocytes produce neural immunoglobulin constant and variable heavy chains as well as light chains. However, their edification is different from the ones found in B cells and they resemble aberrant immunoglobulins observed in several cancers. Moreover, the complete enzymatic V(D)J recombination complex has also been identified in astrocytes. In addition, the constant heavy chain is also present in adult rat astrocytes, whereas in primary astrocytes from human fetus we identified constant and variable kappa chains as well as the substitution lambda chains known to be involved in pre-B cells. To gather insights into the function of these neural IgGs, CRISPR-Cas9 of IgG2B constant heavy chain encoding gene ( Igh6) , IgG2B overexpression, proximal labeling of rat astrocytes IgG2B and targets identification through 2D gels were performed. In Igh6 KO astrocytes, overrepresentation of factors involved in hematopoietic cells, neural stem cells, and the regulation of neuritogenesis have been identified. Moreover, overexpression of IgG2B in astrocytes induces the CRTC1-CREB-BDNF signaling pathway known to be involved in gliogenesis, whereas Igh6 KO triggers the BMP/YAP1/TEAD3 pathway activated in astrocytes dedifferentiation into neural progenitors. Proximal labeling experiments revealed that IgG2B is N-glycosylated by the OST complex, addressed to vesicle membranes containing the ATPase complex, and behaves partially like CD98hc through its association with LAT1. These experiments also suggest that proximal IgG2B-LAT1 interaction occurs concomitantly with MACO-1 and C2CD2L, at the heart of a potentially novel cell signaling platform. Finally, we demonstrated that these chains are synthesized individually and associated to recognize specific targets. Indeed, intermediate filaments Eif4a2 and Pdia6 involved in astrocyte fate constitute targets for these neural IgGs. Taken together, we hypothese that neural aberrant IgG chains may act as gatekeepers of astrocytes' fate.

In systemic sclerosis (SSc), B-cells are activated and present in the skin and lung of patients where they can interact with fibroblasts. The precise impact and mechanisms of the interaction of B-cells and fibroblasts at the tissular level are poorly studied. We investigated the impact and mechanisms of B-cell/fibroblast interactions in cocultures between B-cells from patients with SSc and 3-dimensional reconstituted healthy skin model including fibroblasts, keratinocytes and extracellular matrix. The quantification and description of the B-cell infiltration in 3D cocultures were performed using cells imagery strategy and cytometry. The effect of coculture on the transcriptome of B-cells and fibroblasts was studied with bulk and single-cell RNA sequencing approaches. The mechanisms of this interaction were studied by blocking key cytokines like IL-6 and TNF. We showed a significant infiltration of B-cells in the 3D healthy skin model. The amount but not the depth of infiltration was higher with B-cells from SSc patients and with activated B-cells. B-cell infiltrates were mainly composed of naïve and memory cells, whose frequencies differed depending on B-cells origin and activation state: infiltrated B-cells from patients with SSc showed an activated profile and an overexpression of immunoglobulin genes compared to circulating B-cells before infiltration. Our study has shown for the first time that activated B-cells modified the transcriptomic profile of both healthy and SSc fibroblasts, toward a pro-inflammatory (TNF and IL-17 signaling) and interferon profile, with a key role of the TNF pathway. B-cells and 3D skin cocultures allowed the modelization of B-cells infiltration in tissues observed in SSc, uncovering an influence of the underlying disease and the activation state of B-cells. We showed a pro-inflammatory effect on skin fibroblasts and pro-activation effect on infiltrating B-cells during coculture. This reinforces the role of B-cells in SSc and provide potential targets for future therapeutic approach in this disease.

Background We studied a young woman with atypical diabetes associated with mild intellectual disability, lymphedema distichiasis syndrome (LDS) and polymalformative syndrome including distichiasis. We used different genetic tools to identify causative pathogenic mutations and/or copy number variations. Results Although proband’s, diabetes mellitus occurred during childhood, type 1 diabetes was unlikely due to the absence of detectable autoimmunity. DNA microarray analysis first identified a de novo, heterozygous deletion at the chr16q24.2 locus. Previously, thirty-three pathogenic or likely pathogenic deletions encompassing this locus have been reported in patients presenting with intellectual deficiency, obesity and/or lymphedema but not with diabetes. Of note, the deletion encompassed two topological association domains, whose one included FOXC2 that is known to be linked with LDS. Via whole-exome sequencing, we found a heterozygous, likely pathogenic variant in WFS1 (encoding wolframin endoplasmic reticulum [ER] transmembrane glycoprotein) which was inherited from her father who also had diabetes. WFS1 is known to be involved in monogenic diabetes. We also found a likely pathogenic variant in USP9X (encoding ubiquitin specific peptidase 9 X-linked) that is involved in X-linked intellectual disability, which was inherited from her mother who had dyscalculia and dyspraxia. Conclusions Our comprehensive genetic analysis suggested that the peculiar phenotypes of our patient were possibly due to the combination of multiple genetic causes including chr16q24.2 deletion, and two likely pathogenic variants in WFS1 and USP9X .

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.

Human induced pluripotent stem cells (hiPSCs) have the potential to be differentiated into any cell type, making them a relevant tool for therapeutic purposes such as cell-based therapies. In particular, they show great promise for obesity treatment as they represent an unlimited source of brown/beige adipose progenitors (hiPSC-BAPs). However, the low brown/beige adipocyte differentiation potential in 2D cultures represents a strong limitation for clinical use. In adipose tissue, besides its cell cycle regulator functions, the cyclin-dependent kinase inhibitor 2A (CDKN2A) locus modulates the commitment of stem cells to the brown-like type fate, mature adipocyte energy metabolism and the browning of adipose tissue. Here, using a new method of hiPSC-BAPs 3D culture, via the formation of an organoid-like structure, we silenced CDKN2A expression during hiPSC-BAP adipogenic differentiation and observed that knocking down CDKN2A potentiates adipogenesis, oxidative metabolism and the browning process, resulting in brown-like adipocytes by promoting UCP1 expression and beiging markers. Our results suggest that modulating CDKN2A levels could be relevant for hiPSC-BAPs cell-based therapies.

The G-protein-coupled receptor accessory protein MRAP2 is implicated in energy control in rodents, notably via the melanocortin-4 receptor1. Although some MRAP2 mutations have been described in people with obesity1–3, their functional consequences on adiposity remain elusive. Using large-scale sequencing of MRAP2 in 9,418 people, we identified 23 rare heterozygous variants associated with increased obesity risk in both adults and children. Functional assessment of each variant shows that loss-of-function MRAP2 variants are pathogenic for monogenic hyperphagic obesity, hyperglycemia and hypertension. This contrasts with other monogenic forms of obesity characterized by excessive hunger, including melanocortin-4 receptor deficiency, that present with low blood pressure and normal glucose tolerance4. The pleiotropic metabolic effect of loss-of-function mutations in MRAP2 might be due to the failure of different MRAP2-regulated G-protein-coupled receptors in various tissues including pancreatic islets.