Using Transcription Modules to Identify Expression Clusters Perturbed in Williams-Beuren Syndrome

The genetic dissection of the phenotypes associated with Williams-Beuren Syndrome (WBS) is advancing thanks to the study of individuals carrying typical or atypical structural rearrangements, as well as in vitro and animal studies. However, little is known about the global dysregulations caused by the WBS deletion. We profiled the transcriptomes of skin fibroblasts from WBS patients and compared them to matched controls. We identified 868 differentially expressed genes that were significantly enriched in extracellular matrix genes, major histocompatibility complex (MHC) genes, as well as genes in which the products localize to the postsynaptic membrane. We then used public expression datasets from human fibroblasts to establish transcription modules, sets of genes coexpressed in this cell type. We identified those sets in which the average gene expression was altered in WBS samples. Dysregulated modules are often interconnected and share multiple common genes, suggesting that intricate regulatory networks connected by a few central genes are disturbed in WBS. This modular approach increases the power to identify pathways dysregulated in WBS patients, thus providing a testable set of additional candidates for genes and their interactions that modulate the WBS phenotypes

Validation d'une méthode de production de cellules myoblastiques en culture

L'accréditation des laboratoires de Biologie Médicale s'assure de la mise en place d'une organisation optimale et des compétences prouvées pour la qualité des opérations effectuées. Dans ce contexte, l'objectif de ce travail a été de proposer une méthode de production des cellules myoblastiques en suivant les exigences de la Norme NF EN ISO 15189 concernant les validations de méthodes. Dans un premier temps, nous avons rappelé des notions essentielles d'histologie, de myogenèse et de culture cellulaire. Ceci afin de mieux appréhender l'intérêt des modèles cellulaires ex vivo pour l'étude de plus de deux cents maladies neuromusculaires identifiées jusqu'à présent. Dans un second temps, après avoir pris connaissance des exigences de la Nonne, nous avons validé une méthode de caractérisation des cellules myoblastiques en culture. Ces données nous ont permis de valider une méthode de production efficiente dans l'environnement du Centre de Biotechnologie CellulaireLYON1-BU Santé (693882101) / SudocSudocFranceF

Cryoconservation (utilisation de méthodes alternatives pour la conservation et la décongélation de cellules humaines en culture)

LYON1-BU Santé (693882101) / SudocSudocFranceF

DNA double-strand breaks induced by mammographic screening procedures in human mammary epithelial cells

International audiencePurpose: To assess in vitro mammographic radiation-induced DNA damage in mammary epithelial cells from 30 patients with low (LR) or high (HR) family risk of breast cancer. Materials and methods: Spontaneous and radiation-induced DNA double-strand breaks (DSB) were quantifi ed by using immunofl uorescence of the phosphorylated H2AX histone ( γ H2AX) in diff erent conditions of mammography irradiation (2, 4, 2 2 mGy). Results: HR patients showed signifi cantly more spontaneous γ H2AX foci than LR patients ( p 0.014). A signifi cant dose-eff ect was observed, with an exacerbation in HR patients ( p 0.01). The dose repetition (2 2 mGy) provided more induced and more unrepaired DSB than 2 mGy and 4 mGy, and was exacerbated in HR ( p 0.006). Conclusions: This study highlights the existence of DSB induced by mammography and revealed by γ H2AX assay with two major radiobiological eff ects occurring: A low-dose eff ect, and a LOw and Repeated Dose (LORD) eff ect. All these eff ects were exacerbated in HR patients. These fi ndings may lead us to reevaluate the number of views performed in screening using a single view (oblique) in women whose mammographic benefi t has not properly been proved such as HR patients

POLG exon 22 skipping induced by different mechanisms in two unrelated cases of Alpers syndrome.

International audienceThe POLG genes were sequenced in two unrelated patients presenting with Alpers syndrome. The novel c.3626_3629dupGATA and the c.3643+2T>C alleles were associated in trans with p.A467T and p.[W748S;E1143G], respectively. POLG transcripts from skin fibroblasts showed complete exon 22 skipping for patient 2, but surprisingly partial exon 22 skipping from the c.3626_3629dupGATA for patient 1. The creation of a putative exonic splicing silencer could be responsible for the splicing anomaly observed in patient 1. Both c.3643+2T>C and c.3626_3629dupGATA create a premature termination codon and a low polymerase γ activity in skin fibroblasts is responsible for the severe phenotype in these patients

Kinetic properties of mutant deoxyguanosine kinase in a case of reversible hepatic mtDNA depletion

DGUOK [dG (deoxyguanosine) kinase] is one of the two mitochondrial deoxynucleoside salvage pathway enzymes involved in precursor synthesis for mtDNA (mitochondrial DNA) replication. DGUOK is responsible for the initial rate-limiting phosphorylation of the purine deoxynucleosides, using a nucleoside triphosphate as phosphate donor. Mutations in the DGUOK gene are associated with the hepato-specific and hepatocerebral forms of MDS (mtDNA depletion syndrome). We identified two missense mutations (N46S and L266R) in the DGUOK gene of a previously reported child, now 10 years old, who presented with an unusual revertant phenotype of liver MDS. The kinetic properties of normal and mutant DGUOK were studied in mitochondrial preparations from cultured skin fibroblasts, using an optimized methodology. The N46S/L266R DGUOK showed 14 and 10% residual activity as compared with controls with dG and deoxyadenosine as phosphate acceptors respectively. Similar apparent negative co-operativity in the binding of the phosphate acceptors to the wild-type enzyme was found for the mutant. In contrast, abnormal bimodal kinetics were shown with ATP as the phosphate donor, suggesting an impairment of the ATP binding mode at the phosphate donor site. No kinetic behaviours were found for two other patients with splicing defects or premature stop codon. The present study represents the first characterization of the enzymatic kinetic properties of normal and mutant DGUOK in organello and our optimized protocol allowed us to demonstrate a residual activity in skin fibroblast mitochondria from a patient with a revertant phenotype of MDS. The residual DGUOK activity may play a crucial role in the phenotype reversal

Submicroscopic Deletion in Patients with Williams-Beuren Syndrome Influences Expression Levels of the Nonhemizygous Flanking Genes

Genomic imbalance is a common cause of phenotypic abnormalities. We measured the relative expression level of genes that map within the microdeletion that causes Williams-Beuren syndrome and within its flanking regions. We found, unexpectedly, that not only hemizygous genes but also normal-copy neighboring genes show decreased relative levels of expression. Our results suggest that not only the aneuploid genes but also the flanking genes that map several megabases away from a genomic rearrangement should be considered possible contributors to the phenotypic variation in genomic disorders

PRKDC mutations associated with immunodeficiency, granuloma, and autoimmune regulator-dependent autoimmunity.

textabstractBackground PRKDC encodes for DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a kinase that forms part of a complex (DNA-dependent protein kinase [DNA-PK]) crucial for DNA double-strand break repair and V(D)J recombination. In mice DNA-PK also interacts with the transcription factor autoimmune regulator (AIRE) to promote central T-cell tolerance. Objective We sought to understand the causes of an inflammatory disease with granuloma and autoimmunity associated with decreasing T- and B-cell counts over time that had been diagnosed in 2 unrelated patients. Methods Genetic, molecular, and functional analyses were performed to characterize an inflammatory disease evocative of a combined immunodeficiency. Results We identified PRKDC mutations in both patients. These patients exhibited a defect in DNA double-strand break repair and V(D)J recombination. Whole-blood mRNA analysis revealed a strong interferon signature. On activation, memory T cells displayed a skewed cytokine response typical of TH2 and TH1 but not TH17. Moreover, mutated DNA-PKcs did not promote AIRE-dependent transcription of peripheral tissue antigens in vitro. The latter defect correlated in vivo with production of anti-calcium-sensing receptor autoantibodies, which are typically found in AIRE-deficient patients. In addition, 9 months after bone marrow transplantation, patient 1 had Hashimoto thyroiditis, suggesting that organ-specific autoimmunity might be linked to nonhematopoietic cells, such as AIRE-expressing thymic epithelial cells. Conclusion Deficiency of DNA-PKcs, a key AIRE partner, can present as an inflammatory disease with organ-specific autoimmunity, suggesting a role for DNA-PKcs in regulating autoimmune responses and maintaining AIRE-dependent tolerance in human subjects

PRKDC mutations associated with immunodeficiency, granuloma, and autoimmune regulator-dependent autoimmunity

Background PRKDC encodes for DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a kinase that forms part of a complex (DNA-dependent protein kinase [DNA-PK]) crucial for DNA double-strand break repair and V(D)J recombination. In mice DNA-PK also interacts with the transcription factor autoimmune regulator (AIRE) to promote central T-cell tolerance. Objective We sought to understand the causes of an inflammatory disease with granuloma and autoimmunity associated with decreasing T- and B-cell counts over time that had been diagnosed in 2 unrelated patients. Methods Genetic, molecular, and functional analyses were performed to characterize an inflammatory disease evocative of a combined immunodeficiency. Results We identified PRKDC mutations in both patients. These patients exhibited a defect in DNA double-strand break repair and V(D)J recombination. Whole-blood mRNA analysis revealed a strong interferon signature. On activation, memory T cells displayed a skewed cytokine response typical of TH2 and TH1 but not TH17. Moreover, mutated DNA-PKcs did not promote AIRE-dependent transcription of peripheral tissue antigens in vitro. The latter defect correlated in vivo with production of anti-calcium-sensing receptor autoantibodies, which are typically found in AIRE-deficient patients. In addition, 9 months after bone marrow transplantation, patient 1 had Hashimoto thyroiditis, suggesting that organ-specific autoimmunity might be linked to nonhematopoietic cells, such as AIRE-expressing thymic epithelial cells. Conclusion Deficiency of DNA-PKcs, a key AIRE partner, can present as an inflammatory disease with organ-specific autoimmunity, suggesting a role for DNA-PKcs in regulating autoimmune responses and maintaining AIRE-dependent tolerance in human subjects