FOSL2 truncating variants in the last exon cause a neurodevelopmental disorder with scalp and enamel defects.

PURPOSE: We aimed to investigate the molecular basis of a novel recognizable neurodevelopmental syndrome with scalp and enamel anomalies caused by truncating variants in the last exon of the gene FOSL2, encoding a subunit of the AP-1 complex. METHODS: Exome sequencing was used to identify genetic variants in all cases, recruited through Matchmaker exchange. Gene expression in blood was analyzed using reverse transcription polymerase chain reaction. In vitro coimmunoprecipitation and proteasome inhibition assays in transfected HEK293 cells were performed to explore protein and AP-1 complex stability. RESULTS: We identified 11 individuals from 10 families with mostly de novo truncating FOSL2 variants sharing a strikingly similar phenotype characterized by prenatal growth retardation, localized cutis scalp aplasia with or without skull defects, neurodevelopmental delay with autism spectrum disorder, enamel hypoplasia, and congenital cataracts. Mutant FOSL2 messenger RNAs escaped nonsense-mediated messenger RNA decay. Truncated FOSL2 interacts with c-JUN, thus mutated AP-1 complexes could be formed. CONCLUSION: Truncating variants in the last exon of FOSL2 associate a distinct clinical phenotype by altering the regulatory degradation of the AP-1 complex. These findings reveal a new role for FOSL2 in human pathology.

Drug transporters are implicated in the diffusion of tacrolimus into the T lymphocyte in kidney and liver transplant recipients: Genetic, mRNA, protein expression, and functionality.

Because of a narrow therapeutic index and a wide inter- and intra-patient variability, therapeutic drug monitoring of the immunosuppressant drug tacrolimus (TAC) based on whole-blood concentrations (Cblood) is mandatory in solid organ transplant recipients. Using peripheral blood mononuclear cells concentrations (CPBMC) could improve patient outcomes. The poor correlation between Cblood and CPBMC makes hypothesize that drug transporters are implicated in the intracellular accumulation of TAC. The aim of this work was therefore to clinically study: i) the role of genetic variants and ii) the effect of mRNA and protein expression of 4 drug transporters on the TAC CPBMC/blood ratio. In addition, functional in vitro experiments were performed to mechanistically validate the clinical observations. Genetic variants of ABCB1/P-gp and SLC28A3/CNT3 did not influence TAC CPBMC in liver transplant recipients (LTR). ABCC2/MRP2 at the mRNA level; ABCB1/P-gp, SLC28A3/CNT3 and SLC29A1/ENT1 at the protein level; correlated with the CPBMC/blood in kidney and LTR. In vitro results suing transporter-expressing cells confirmed that TAC is substrate of P-gp but not MRP2, whereas experiments remained inconclusive for CNT3 and ENT1. In conclusion, the genetic-transcription-protein-functional approach presented in this work provides new insights in the understanding of TAC transport at the T lymphocyte plasma membrane.

A Robust and Fast/Multiplex Pharmacogenetics Assay to Simultaneously Analyze 17 Clinically Relevant Genetic Polymorphisms in CYP3A4, CYP3A5, CYP1A2, CYP2C9, CYP2C19, CYP2D6, ABCB1, and VKORC1 Genes.

In the field of pharmacogenetics, the trend is to analyze a panel of several actionable genetic polymorphisms. It may require the use of high-throughput sequencing which demands expensive reagents/instruments and specific skills to interpret results. As an alternative, the aim of this work was to validate an easy, fast, and inexpensive multiplex pharmacogenetics assay to simultaneously genotype a panel of 17 clinically actionable variants involved in drug pharmacokinetics/pharmacodynamics. We designed primers to perform a multiplex PCR assay using a single mix. Primers were labeled by two fluorescent dye markers to discriminate alleles, while the size of the PCR fragments analyzed by electrophoresis allowed identifying amplicon. Polymorphisms of interest were CYP3A4*22, CYP3A5*3, CYP1A2*1F, CYP2C9*2-*3, CYP2C19*2-*3-*17, VKORC1-1639G > A, ABCB1 rs1045642-rs1128503-rs2229109-rs2032582, and CYP2D6*3-*4-*6-*9. The assay was repeatable and a minimum quantity of 10 ng of DNA/ sample was needed to obtain accurate results. The method was applied to a validation cohort of 121 samples and genotyping results were consistent with those obtained with reference methods. The assay was fast and cost-effective with results being available within one working-day. This robust assay can easily be implemented in laboratories as an alternative to cumbersome simplex assays or expensive multiplex approaches. Together it should widespread access to pharmacogenetics in clinical routine practice.

PharmFrag: An Easy and Fast Multiplex Pharmacogenetics Assay to Simultaneously Analyze 9 Genetic Polymorphisms Involved in Response Variability of Anticancer Drugs.

Regarding several cytotoxic agents, it was evidenced that genetic polymorphisms in genes encoding enzymes involved in their metabolism are associated with higher risk of toxicity. Genotyping these genes before treatment is a valuable strategy to prevent side effects and to predict individual response to drug therapy. This pharmacogenetic approach is recommended for chemotherapies such as thiopurines (azathioprine, 6-mercaptopurine, thioguanine), irinotecan, and fluoropyrimidines (capecitabine and 5-fluorouracil). In this study, we aimed at developing and validating a fast, cost-effective, and easily implementable multiplex genotyping method suitable for analyzing a panel of nine variants involved in the pharmacogenetics of widely prescribed anticancer drugs. We designed a multiplex-specific PCR assay where fragments were labeled by two different fluorescent dye markers (HEX/FAM) identifiable by fragment analysis. These two labels were used to discriminate bi-allelic variants, while the size of the fragment allowed the identification of a particular polymorphism location. Variants of interest were TPMT (rs1800462, rs1142345, rs1800460), NUDT15 (rs116855232), DPYD (rs55886062, rs3918290, rs67376798, rs75017182), and UGT1A1 (rs8175347). The assay was repeatable, and genotypes could be determined when DNA sample amounts ranged from 25 to 100 ng. Primers and dye remained stable in a ready-to-use mixture solution after five freeze-thaw cycles. Accuracy was evidenced by the consistency of 187 genotyping results obtained with our multiplex assay and a reference method. The developed method is fast and cost-effective in simultaneously identifying nine variants involved in the pharmacological response of anticancer drugs. This assay can be easily implemented in laboratories for widespread access to pharmacogenetics in clinical practice.

The expression of EMX2 lead to cell cycle arrest in glioblastoma cell line.

BACKGROUND: Glioblastoma (GB) is a highly invasive primary brain tumor that nearly always systematically recurs at the site of resection despite aggressive radio-chemotherapy. Previously, we reported a gene expression signature related to tumor infiltration. Within this signature, the EMX2 gene encodes a homeodomain transcription factor that we found was down regulated in glioblastoma. As EMX2 is reported to play a role in carcinogenesis, we investigated the impact of EMX2 overexpression in glioma-related cell lines. METHODS: For that purpose, we constructed tetracycline-inducible EMX2 expression lines. Transfected cell phenotypes (proliferation, cell death and cell cycle) were assessed in time-course experiments. RESULTS: Restoration of EMX2 expression in U87 glioblastoma cells significantly inhibited cell proliferation. This inhibition was reversible after EMX2 removal from cells. EMX2-induced proliferative inhibition was very likely due to cell cycle arrest in G1/S transition and was not accompanied by signs of cell death. CONCLUSION: Our results suggest that EMX2 may constitute a putative therapeutic target for GB treatment. Further studies are required to decipher the gene networks and transduction signals involved in EMX2's effect on cell proliferation.

Genome-wide association study identifies TF as a significant modifier gene of iron metabolism in HFE hemochromatosis.

BACKGROUND & AIMS: Hereditary hemochromatosis (HH) is the most common form of genetic iron loading disease. It is mainly related to the homozygous C282Y/C282Y mutation in the HFE gene that is, however, a necessary but not a sufficient condition to develop clinical and even biochemical HH. This suggests that modifier genes are likely involved in the expressivity of the disease. Our aim was to identify such modifier genes. METHODS: We performed a genome-wide association study (GWAS) using DNA collected from 474 unrelated C282Y homozygotes. Associations were examined for both quantitative iron burden indices and clinical outcomes with 534,213 single nucleotide polymorphisms (SNP) genotypes, with replication analyses in an independent sample of 748 C282Y homozygotes from four different European centres. RESULTS: One SNP met genome-wide statistical significance for association with transferrin concentration (rs3811647, GWAS p value of 7×10(-9) and replication p value of 5×10(-13)). This SNP, located within intron 11 of the TF gene, had a pleiotropic effect on serum iron (GWAS p value of 4.9×10(-6) and replication p value of 3.2×10(-6)). Both serum transferrin and iron levels were associated with serum ferritin levels, amount of iron removed and global clinical stage (p<0.01). Serum iron levels were also associated with fibrosis stage (p<0.0001). CONCLUSIONS: This GWAS, the largest one performed so far in unselected HFE-associated HH (HFE-HH) patients, identified the rs3811647 polymorphism in the TF gene as the only SNP significantly associated with iron metabolism through serum transferrin and iron levels. Because these two outcomes were clearly associated with the biochemical and clinical expression of the disease, an indirect link between the rs3811647 polymorphism and the phenotypic presentation of HFE-HH is likely.

In vivo identification of solar radiation-responsive gene network: role of the p38 stress-dependent kinase.

Solar radiation is one of the most common threats to the skin, with exposure eliciting a specific protective cellular response. To decrypt the underlying mechanism, we used whole genome microarrays (Agilent 44K) to study epidermis gene expression in vivo in skin exposed to simulated solar radiation (SSR). We procured epidermis samples from healthy Caucasian patients, with phototypes II or III, and used two different SSR doses (2 and 4 J/cm(2)), the lower of which corresponded to the minimal erythemal dose. Analyses were carried out five hours after irradiation to identify early gene expression events in the photoprotective response. About 1.5% of genes from the human genome showed significant changes in gene expression. The annotations of these affected genes were assessed. They indicated a strengthening of the inflammation process and up-regulation of the JAK-STAT pathway and other pathways. Parallel to the p53 pathway, the p38 stress-responsive pathway was affected, supporting and mediating p53 function. We used an ex vivo assay with a specific inhibitor of p38 (SB203580) to investigate genes the expression of which was associated with active p38 kinase. We identified new direct p38 target genes and further characterized the role of p38. Our findings provide further insight into the physiological response to UV, including cell-cell interactions and cross-talk effects.

Orchestrated transcription of biological processes in the marine picoeukaryote Ostreococcus exposed to light/dark cycles.

BACKGROUND: Picoeukaryotes represent an important, yet poorly characterized component of marine phytoplankton. The recent genome availability for two species of Ostreococcus and Micromonas has led to the emergence of picophytoplankton comparative genomics. Sequencing has revealed many unexpected features about genome structure and led to several hypotheses on Ostreococcus biology and physiology. Despite the accumulation of genomic data, little is known about gene expression in eukaryotic picophytoplankton. RESULTS: We have conducted a genome-wide analysis of gene expression in Ostreococcus tauri cells exposed to light/dark cycles (L/D). A Bayesian Fourier Clustering method was implemented to cluster rhythmic genes according to their expression waveform. In a single L/D condition nearly all expressed genes displayed rhythmic patterns of expression. Clusters of genes were associated with the main biological processes such as transcription in the nucleus and the organelles, photosynthesis, DNA replication and mitosis. CONCLUSIONS: Light/Dark time-dependent transcription of the genes involved in the main steps leading to protein synthesis (transcription basic machinery, ribosome biogenesis, translation and aminoacid synthesis) was observed, to an unprecedented extent in eukaryotes, suggesting a major input of transcriptional regulations in Ostreococcus. We propose that the diurnal co-regulation of genes involved in photoprotection, defence against oxidative stress and DNA repair might be an efficient mechanism, which protects cells against photo-damage thereby, contributing to the ability of O. tauri to grow under a wide range of light intensities.

Iron regulates phosphorylation of Smad1/5/8 and gene expression of Bmp6, Smad7, Id1, and Atoh8 in the mouse liver.

Although hepcidin expression was shown to be induced by the BMP/Smad signaling pathway, it is not yet known how iron regulates this pathway and what its exact molecular targets are. We therefore assessed genome-wide liver transcription profiles of mice of 2 genetic backgrounds fed iron-deficient, -balanced, or -enriched diets. Among 1419 transcripts significantly modulated by the dietary iron content, 4 were regulated similarly to the hepcidin genes Hamp1 and Hamp2. They are coding for Bmp6, Smad7, Id1, and Atoh8 all related to the Bmp/Smad pathway. As shown by Western blot analysis, variations in Bmp6 expression induced by the diet iron content have for functional consequence similar changes in Smad1/5/8 phosphorylation that leads to formation of heteromeric complexes with Smad4 and their translocation to the nucleus. Gene expression variations induced by secondary iron deficiency or iron overload were compared with those consecutive to Smad4 and Hamp1 deficiency. Iron overload developed by Smad4- and Hamp1-deficient mice also increased Bmp6 transcription. However, as shown by analysis of mice with liver-specific disruption of Smad4, activation of Smad7, Id1, and Atoh8 transcription by iron requires Smad4. This study points out molecules that appear to play a critical role in the control of systemic iron balance.

Light-dependent regulation of cell division in Ostreococcus: evidence for a major transcriptional input.

Cell division often occurs at specific times of the day in animal and photosynthetic organisms. Studies in unicellular photosynthetic algae, such as Chlamydomonas or Euglena, have shown that the photoperiodic control of cell division is mediated through the circadian clock. However, the underlying mechanisms remain unknown. We have studied the molecular basis of light-dependent control of cell division in the unicellular green alga Ostreococcus. We found that cell division obeys a circadian oscillator in Ostreococcus. We provide evidence suggesting that the clock may, at least in part, regulate directly cell division independently of the metabolism. Combined microarray and quantitative real-time reverse transcription-polymerase chain reaction analysis of the main core cell cycle gene expression revealed an extensive transcriptional regulation of cell division by the photoperiod in Ostreococcus. Finally, transcription of the main core cell cycle genes, including cyclins and cyclin-dependent kinases, was shown to be under circadian control in Ostreococcus, suggesting that these genes are potential targets of the circadian clock in the control of cell division.