Relevance of Extending FGFR3 Gene Analysis in Osteochondrodysplasia to Non-Coding Sequences: A Case Report.

Skeletal dysplasia, also called osteochondrodysplasia, is a category of disorders affecting bone development and children's growth. Up to 552 genes, including fibroblast growth factor receptor 3 (FGFR3), have been implicated by pathogenic variations in its genesis. Frequently identified causal mutations in osteochondrodysplasia arise in the coding sequences of the FGFR3 gene: c.1138G>A and c.1138G>C in achondroplasia and c.1620C>A and c.1620C>G in hypochondroplasia. However, in some cases, the diagnostic investigations undertaken thus far have failed to identify the causal anomaly, which strengthens the relevance of the diagnostic strategies being further refined. We observed a Caucasian adult with clinical and radiographic features of achondroplasia, with no common pathogenic variant. Exome sequencing detected an FGFR3(NM_000142.4):c.1075+95C>G heterozygous intronic variation. In vitro studies showed that this variant results in the aberrant exonization of a 90-nucleotide 5' segment of intron 8, resulting in the substitution of the alanine (Ala359) for a glycine (Gly) and the in-frame insertion of 30 amino acids. This change may alter FGFR3's function. Our report provides the first clinical description of an adult carrying this variant, which completes the phenotype description previously provided in children and confirms the recurrence, the autosomal-dominant pathogenicity, and the diagnostic relevance of this FGFR3 intronic variant. We support its inclusion in routinely used diagnostic tests for osteochondrodysplasia. This may increase the detection rate of causal variants and therefore could have a positive impact on patient management. Finally, FGFR3 alteration via non-coding sequence exonization should be considered a recurrent disease mechanism to be taken into account for new drug design and clinical trial strategies.

ß Cell microRNAs Function as Molecular Hubs of Type 1 Diabetes Pathogenesis and as Biomarkers of Diabetes Risk.

MicroRNAs (miRNAs) are small non-coding RNAs that play a crucial role in modulating gene expression and are enriched in cell-derived extracellular vesicles (EVs). We investigated whether miRNAs from human islets and islet-derived EVs could provide insight into ß cell stress pathways activated during type 1 diabetes (T1D) evolution, therefore serving as potential disease biomarkers. We treated human islets from 10 cadaveric donors with IL-1ß and IFN-γ to model T1D ex vivo. MicroRNAs were isolated from islets and islet-derived EVs, and small RNA sequencing was performed. We found 20 and 14 differentially expressed (DE) miRNAs in cytokine- versus control-treated islets and EVs, respectively. Interestingly, the miRNAs found in EVs were mostly different from those found in islets. Only two miRNAs, miR-155-5p and miR-146a-5p, were upregulated in both islets and EVs, suggesting selective sorting of miRNAs into EVs. We used machine learning algorithms to rank DE EV-associated miRNAs, and developed custom label-free Localized Surface Plasmon Resonance-based biosensors to measure top ranked EVs in human plasma. Results from this analysis revealed that miR-155, miR-146, miR-30c, and miR-802 were upregulated and miR-124-3p was downregulated in plasma-derived EVs from children with recent-onset T1D. In addition, miR-146 and miR-30c were upregulated in plasma-derived EVs of autoantibody positive (AAb+) children compared to matched non-diabetic controls, while miR-124 was downregulated in both T1D and AAb+ groups. Furthermore, single-molecule fluorescence in situ hybridization confirmed increased expression of the most highly upregulated islet miRNA, miR-155, in pancreatic sections from organ donors with AAb+ and T1D.

Photoswitchable Machine-Engineered Plasmonic Nanosystem with High Optical Response for Ultrasensitive Detection of microRNAs and Proteins Adaptively.

Modulating optoelectronic properties of inorganic nanostructures tethered with light-responsive molecular switches by their conformational change in the solid state is fundamentally important for advanced nanoscale-device fabrication, specifically in biosensing applications. Herein, we present an entirely new solid-state design approach employing the light-induced reversible conformational change of spiropyran (SP)-merocyanine (MC) covalently attached to gold triangular nanoprisms (Au TNPs) via alkylthiolate self-assembled monolayers to produce a large localized surface plasmon resonance response (∼24 nm). This shift is consistent with the increase in thickness of the local dielectric shell-surrounded TNPs and perhaps short-range dipole-dipole (permanent and induced) interactions between TNPs and the zwitterionic MC form. Water contact angle measurement and Raman spectroscopy characterization unequivocally prove the formation of a stable TNP-MC structural motif. Utilizing this form, we fabricated the first adaptable nanoplasmonic biosensor, which uses an identical structural motif for ultrasensitive, highly specific, and programmable detection of microRNAs and proteins at attomolar concentrations in standard human plasma and urine samples, and at femtomolar concentrations from bladder cancer patient plasma (n = 10) and urine (n = 10), respectively. Most importantly, the TNP-MC structural motif displays a strong binding affinity with receptor molecules (i.e., single-stranded DNA and antibody) producing a highly stable biosensor. Taken together, the TNP-MC structural motif represents a multifunctional super biosensor with the potential to expand clinical diagnostics through simplifying biosensor design and providing highly accurate disease diagnosis.

Genetic Analysis of Mu and Kappa Opioid Receptor and COMT Enzyme in Cancer Pain Tunisian Patients Under Opioid Treatment.

BACKGROUND: Pain and its opioid treatments are complex measurable traits. Responses to morphine in terms of pain control is likely to be determined by many factors, including the underlying pain sensitivity of the patient, along with nature and extent of the painful process, concomitant medications, genetic and other clinical and environmental factors. This study investigated genetic polymorphisms implicated in the inter-individual pain response variability to opioid treatment in the Tunisian population. METHODS: This prospective association study investigated seven variations in the OPRM1, OPRK1 and COMT gene, which encode Mu and KAPPA opioid receptors, and Catechol-O-methyltransferase enzyme respectively, in a cohort of 129 Tunisian cancer pain patients under oral morphine treatment. Genotyping was performed by simple probe probes on Light Cyler for rs17174629, rs1799972, rs1799971, rs1051659, rs1051660 and rs4680 and by PCR assay for the indel in the promoter region of OPRK1 (rs35566036). A statistical associations study between dose (continuous), dose escalation (yes/no) and SNP or haplotypes were investigated using linear multiple regressions and logistic regressions respectively adjusted on metastases and pain covariates in the R software. RESULTS: We detected significant association of the rs1051660 adjusted on metastasis and pain (P=0.02), no other association has been detected between the 7 polymorphisms screened and the dose of morphine needed for pain relief. CONCLUSION: This can be explained by the strong genetic heterogeneity in the cosmopolitan areas where our patients were recruited for this study, compared to more homegenous population recruited in other studies.

Decreased ATF4 expression as a mechanism of acquired resistance to long-term amino acid limitation in cancer cells.

The uncontrolled growth of tumor can lead to the formation of area deprived in nutrients. Due to their high genetic instability, tumor cells can adapt and develop resistance to this pro-apoptotic environment. Among the resistance mechanisms, those involved in the resistance to long-term amino acid restriction are not elucidated. A long-term amino acid restriction is particularly deleterious since nine of them cannot be synthetized by the cells. In order to determine how cancer cells face a long-term amino acid deprivation, we developed a cell model selected for its capacity to resist a long-term amino acid limitation. We exerted a selection pressure on mouse embryonic fibroblast to isolate clones able to survive with low amino acid concentration. The study of several clones revealed an alteration of the eiF2α/ATF4 pathway. Compared to the parental cells, the clones exhibited a decreased expression of the transcription factor ATF4 and its target genes. Likewise, the knock-down of ATF4 in parental cells renders them resistant to amino acid deprivation. Moreover, this association between a low level of ATF4 protein and the resistance to amino acid deprivation was also observed in the cancer cell line BxPC-3. This resistance was abolished when ATF4 was overexpressed. Therefore, decreasing ATF4 expression may be one important mechanism for cancer cells to survive under prolonged amino acid deprivation.

GCN2 contributes to mTORC1 inhibition by leucine deprivation through an ATF4 independent mechanism.

It is well known that the GCN2 and mTORC1 signaling pathways are regulated by amino acids and share common functions, in particular the control of translation. The regulation of GCN2 activity by amino acid availability relies on the capacity of GCN2 to sense the increased levels of uncharged tRNAs upon amino acid scarcity. In contrast, despite recent progress in the understanding of the regulation of mTORC1 by amino acids, key aspects of this process remain unsolved. In particular, while leucine is well known to be a potent regulator of mTORC1, the mechanisms by which this amino acid is sensed and control mTORC1 activity are not well defined. Our data establish that GCN2 is involved in the inhibition of mTORC1 upon leucine or arginine deprivation. However, the activation of GCN2 alone is not sufficient to inhibit mTORC1 activity, indicating that leucine and arginine exert regulation via additional mechanisms. While the mechanism by which GCN2 contributes to the initial step of mTORC1 inhibition involves the phosphorylation of eIF2α, we show that it is independent of the downstream transcription factor ATF4. These data point to a novel role for GCN2 and phosphorylation of eIF2α in the control of mTORC1 by certain amino acids.

Association of the OPRM1 and COMT genes' polymorphisms with the efficacy of morphine in Tunisian cancer patients: Impact of the high genetic heterogeneity in Tunisia?

BACKGROUND: Genetic causes for inter-individual variability response to opioids are clinical difficulties for treatment efficiency. The aim of the present study was to investigate the possible association of opioid treatment outcome with single nucleotide polymorphisms (SNPs) in the mµ opioid receptor (OPRM1) and catechol-o-methyltransferase (COMT) genes, in Tunisian cancer pain patients. METHODS: We genotyped one hundred and twenty-nine cancer patients treated with different doses of morphine for 3 SNPs in OPRM1 gene (rs17174629, rs1799972 and rs1799971) and one in the COMT gene (rs4680). Associations between dose (continuous), dose escalation (yes/no) and SNP or haplotypes were investigated. RESULTS: Unlike other studies on Caucasian and Chinese populations, no significant association were found between the 4 polymorphisms screened and the dose of morphine needed for pain relief. CONCLUSION: This result can be explained by the genetic heterogeneity and cosmopolitan areas of our Tunisian patients compared to the others homogenous population.

Requirement for lysosomal localization of mTOR for its activation differs between leucine and other amino acids.

The mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and metabolism. It controls many cell functions by integrating nutrient availability and growth factor signals. Amino acids, and in particular leucine, are among the main positive regulators of mTORC1 signaling. The current model for the regulation of mTORC1 by amino acids involves the movement of mTOR to the lysosome mediated by the Rag-GTPases. Here, we have examined the control of mTORC1 signaling and mTOR localization by amino acids and leucine in serum-fed cells, because both serum growth factors (or, e.g., insulin) and amino acids are required for full activation of mTORC1 signaling. We demonstrate that mTORC1 activity does not closely correlate with the lysosomal localization of mTOR. In particular, leucine controls mTORC1 activity without any detectable modification of the lysosomal localization of mTOR, indicating that the signal(s) exerted by leucine is likely distinct from those exerted by other amino acids. In addition, knock-down of the Rag-GTPases attenuated the inhibitory effect of amino acid- or leucine-starvation on the phosphorylation of mTORC1 targets. Furthermore, data from cells where Rag expression has been knocked down revealed that leucine can promote mTORC1 signaling independently of the lysosomal localization of mTOR. Our data complement existing models for the regulation of mTORC1 by amino acids and provide new insights into this important topic.

The amino acid sensor GCN2 biases macronutrient selection during aging.

PURPOSE: Selection of a balanced diet has a determinant impact on human health. Individual food preferences involve socio-cultural as well as physiological factors and evolve during aging. In mammals, physiological mechanisms governing food choices appear to require the sensing of nutrient concentrations in diet. This is particularly the case for dietary amino acids that are sensed by the protein kinase GCN2. It has been reported that GCN2 is involved in the adaptive response to amino acid imbalanced diets at the level of food intake and lipid metabolism. Here, we hypothesized that GCN2 may play a role in macronutrient selection and its age-related changes. METHODS: Two groups of wild-type and GCN2 knock-out mice were subjected to a food self-selection protocol at ages 6, 12, 18 and 24 months. During each test, mice were allowed to create their own diets by selecting between three separate food sources, each containing either protein, fat or carbohydrates. RESULTS: Our results show that the absence of GCN2 had two main age-related effects. First, it exacerbated fat preference at the expense of carbohydrate consumption. Second, it prevented the increase in protein intake. CONCLUSION: These findings indicate that, in omnivores, the GCN2 ancient pathway participates in the control of food preference.

Identification of GCN2 as new redox regulator for oxidative stress prevention in vivo.

Constitution of oxidative defense systems and, correspondingly, oxidative stress prevention are highly dependent on amino acid supply. In vitro, experiments have demonstrated that amino acid availability participates to the homeostasis of reactive oxygen species. However the molecular mechanisms involved in the maintenance of redox homeostasis responsive to circulating amino acid levels remain unclear. As GCN2 is a protein kinase considered to be an important sensor for amino acids availability and a potential regulator of redox homeostasis, we hypothesized that this kinase can modulate redox homeostasis in vivo, in response to an amino acid-imbalanced diet. We investigated the response of GCN2+/+ and GCN2-/- mice to a long-term (24 weeks) leucine-imbalanced diet (EDΔLeu). In order to evaluate the oxidation level in each group of mice, we determined the degree of protein oxidation in the liver. Interestingly, GCN2-/- mice exhibited an increase in protein carbonylation, a marker of oxidative stress, in response to the EDΔLeu diet. These data correlate with a decrease in hepatic GPX1 expression, a major antioxidant enzyme, and a decrease in total GPX activity in the liver. Our results suggest that GCN2 and its downstream signaling pathway have an important role in the protection against oxidative injuries induced by an amino acid-imbalanced diet, and that it can play a critical role in the prevention of oxidative damage.

Perinatal undernutrition affects the methylation and expression of the leptin gene in adults: implication for the understanding of metabolic syndrome.

Transient environmental influences, such as perinatal nutritional stress, may induce deleterious metabolic symptoms that last for the entire life of individuals, implying that epigenetic modifications play an important role in this process. We have investigated, in mice, the consequences of maternal undernutrition during gestation and lactation on DNA methylation and expression of the leptin gene, which plays a major regulatory role in coordinating nutritional state with many aspects of mammalian biology. We show that animals born to mothers fed a low-protein-diet (F1-LPD group) have a lower body weight/adiposity and exhibit a higher food intake than animals born to mothers fed a control diet (F1-CD group). These modifications persisted throughout life and were associated with lower levels of leptin mRNA and protein in starved F1-LPD mice, emphasizing that maternal protein-undernutrition affects the balance between food intake and energy expenditure in adults. Moreover, this nutritional stress resulted in the removal of methyls at CpGs located in the promoter of leptin, causing a permanent specific modification in the dynamics of the expression of leptin, which exhibits a stronger induction in the F1-LPD than in F1-CD mice in response to a meal. This study is an example of a molecular rationale linking transient environmental influences to permanent phenotypic consequences.

Cushing's syndrome and fetal features resurgence in adrenal cortex-specific Prkar1a knockout mice.

Carney complex (CNC) is an inherited neoplasia syndrome with endocrine overactivity. Its most frequent endocrine manifestation is primary pigmented nodular adrenocortical disease (PPNAD), a bilateral adrenocortical hyperplasia causing pituitary-independent Cushing's syndrome. Inactivating mutations in PRKAR1A, a gene encoding the type 1 alpha-regulatory subunit (R1alpha) of the cAMP-dependent protein kinase (PKA) have been found in 80% of CNC patients with Cushing's syndrome. To demonstrate the implication of R1alpha loss in the initiation and development of PPNAD, we generated mice lacking Prkar1a specifically in the adrenal cortex (AdKO). AdKO mice develop pituitary-independent Cushing's syndrome with increased PKA activity. This leads to autonomous steroidogenic genes expression and deregulated adreno-cortical cells differentiation, increased proliferation and resistance to apoptosis. Unexpectedly, R1alpha loss results in improper maintenance and centrifugal expansion of cortisol-producing fetal adrenocortical cells with concomitant regression of adult cortex. Our data provide the first in vivo evidence that loss of R1alpha is sufficient to induce autonomous adrenal hyper-activity and bilateral hyperplasia, both observed in human PPNAD. Furthermore, this model demonstrates that deregulated PKA activity favors the emergence of a new cell population potentially arising from the fetal adrenal, giving new insight into the mechanisms leading to PPNAD.

Molecular mechanisms involved in the adaptation to amino acid limitation in mammals.

In mammals, metabolic adaptations are required to cope with episodes of protein deprivation and malnutrition. Consequently, mammals have to adjust physiological functions involved in the adaptation to amino acid availability. Part of this regulation involves the modulation of the expression of numerous genes. In particular, it has been shown that amino acids by themselves can modify the expression of target genes. This review describes the regulation of amino acids homeostasis and the their role as signal molecules. The recent advances in the understanding of the molecular mechanisms involved in the control of mammalian gene expression in response to amino acid limitation will be described.

Constitutive beta-catenin activation induces adrenal hyperplasia and promotes adrenal cancer development.

Adrenocortical carcinoma is a rare but aggressive cancer with unknown aetiology. Constitutive activation of beta-catenin is the most frequent alteration in benign and malignant adrenocortical tumours in patients. Here, we show that constitutive activation of beta-catenin in the adrenal cortex of transgenic mice resulted in progressive steroidogenic and undifferentiated spindle-shaped cells hyperplasia as well as dysplasia of the cortex and medulla. Over a 17 months time course, transgenic adrenals developed malignant characteristics such as uncontrolled neovascularization and loco-regional metastatic invasion. These oncogenic events were accompanied by ectopic differentiation of glomerulosa at the expense of fasciculata cells, which caused primary hyperaldosteronism. Altogether these observations demonstrate that constitutively active beta-catenin is an adrenal oncogene which triggers benign aldosterone-secreting tumour development and promotes malignancy.

Amino acid availability controls TRB3 transcription in liver through the GCN2/eIF2α/ATF4 pathway.

In mammals, plasma amino acid concentrations are markedly affected by dietary or pathological conditions. It has been well established that amino acids are involved in the control of gene expression. Up to now, all the information concerning the molecular mechanisms involved in the regulation of gene transcription by amino acid availability has been obtained in cultured cell lines. The present study aims to investigate the mechanisms involved in transcriptional activation of the TRB3 gene following amino acid limitation in mice liver. The results show that TRB3 is up-regulated in the liver of mice fed a leucine-deficient diet and that this induction is quickly reversible. Using transient transfection and chromatin immunoprecipitation approaches in hepatoma cells, we report the characterization of a functional Amino Acid Response Element (AARE) in the TRB3 promoter and the binding of ATF4, ATF2 and C/EBPß to this AARE sequence. We also provide evidence that only the binding of ATF4 to the AARE plays a crucial role in the amino acid-regulated transcription of TRB3. In mouse liver, we demonstrate that the GCN2/eIF2α/ATF4 pathway is essential for the induction of the TRB3 gene transcription in response to a leucine-deficient diet. Therefore, this work establishes for the first time that the molecular mechanisms involved in the regulation of gene transcription by amino acid availability are functional in mouse liver.

Aldo keto reductase 1B7 and prostaglandin F2alpha are regulators of adrenal endocrine functions.

Prostaglandin F(2alpha) (PGF(2alpha)), represses ovarian steroidogenesis and initiates parturition in mammals but its impact on adrenal gland is unknown. Prostaglandins biosynthesis depends on the sequential action of upstream cyclooxygenases (COX) and terminal synthases but no PGF(2alpha) synthases (PGFS) were functionally identified in mammalian cells. In vitro, the most efficient mammalian PGFS belong to aldo-keto reductase 1B (AKR1B) family. The adrenal gland is a major site of AKR1B expression in both human (AKR1B1) and mouse (AKR1B3, AKR1B7). Thus, we examined the PGF(2alpha) biosynthetic pathway and its functional impact on both cortical and medullary zones. Both compartments produced PGF(2alpha) but expressed different biosynthetic isozymes. In chromaffin cells, PGF(2alpha) secretion appeared constitutive and correlated to continuous expression of COX1 and AKR1B3. In steroidogenic cells, PGF(2alpha) secretion was stimulated by adrenocorticotropic hormone (ACTH) and correlated to ACTH-responsiveness of both COX2 and AKR1B7/B1. The pivotal role of AKR1B7 in ACTH-induced PGF(2alpha) release and functional coupling with COX2 was demonstrated using over- and down-expression in cell lines. PGF(2alpha) receptor was only detected in chromaffin cells, making medulla the primary target of PGF(2alpha) action. By comparing PGF(2alpha)-responsiveness of isolated cells and whole adrenal cultures, we demonstrated that PGF(2alpha) repressed glucocorticoid secretion by an indirect mechanism involving a decrease in catecholamine release which in turn decreased adrenal steroidogenesis. PGF(2alpha) may be regarded as a negative autocrine/paracrine regulator within a novel intra-adrenal feedback loop. The coordinated cell-specific regulation of COX2 and AKR1B7 ensures the generation of this stress-induced corticostatic signal.

A transgenic mouse line with specific Cre recombinase expression in the adrenal cortex.

The Cre-loxP system combined with gene targeting strategies has proven to be very useful for gene inactivation in specific tissues and/or cell types. To achieve adrenal cortex specific recombination in vivo, we used a 0.5-kb fragment of the 5'-flanking region of the akr1b7 gene to drive Cre expression in adrenocortical cells. The resulting 0.5 akr1b7-Cre mice express Cre in all steroidogenic zones of the adrenal cortex but not in the gonads. Although recombination of the ROSA26R reporter locus was not observed in all cortical cells, we provide evidence that Cre is expressed in all the cells of the cortex in adult mice. In addition, Cre activity was found in collecting ducts and maturing glomeruli of the kidney. This line is the first to show specific Cre expression in the adrenal cortex in the absence of Cre expression in the gonads. This transgene thus provides a valuable tool for specific gene recombination in the adrenal cortex and kidney.