Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human congenital disorders of Glycosylation type-1 patients (CDG-I)

ABSTRACT: Disruption of N-linked glycosylation has a broad impact on proper glycosylation of nascent glycoproteins in the endoplasmic reticulum, which affect multiple signalling pathways( by changing the stability of membrane proteins or the signalling ability of membrane receptors) and may be responsible of the fibrotic stage associated to CDG type-I. We used microarrays to characterize the global changes in gene expression in three distinct groups of CDG-I patients and we identified a common perturbation in the expression of genes encoding for proteins involved in the stress as well as in the fibrotic responses. Experiment Overall Design: Primary fibroblasts, obtained from forearm skin biopsies of healthy control subjects and three distinct groups of CDG-I patients, were cultured in F12 DMEM supplemented with 4.5 g/l glucose and 10% fetal calf serum prior to RNA extraction and Hybridization on human HG 133 A Affymetrix Microarrays. To avoid bias related to the analysis of a specific CDG-I type or to a specific individual expression pattern, we have considered three patients for the CDG-Ic and -Ie group and two patients for the CDG-Ig group. Moreover the total RNA was extracted from three different independent biological replicates.

ORGANISM(S): Homo sapiens

SUBMITTER: Thierry Hennet

PROVIDER: E-GEOD-8440 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Glycosylation of proteins is an important post-translational modification that comprises two major stages: synthesis and attachment of glycans in the endoplasmic reticulum (ER), and glycan remodeling in the ER and Golgi apparatus (GA). Genetic disorders impairing a step of this process give rise to a group of pathologies named congenital disorders of glycosylation (CDG). The most common CDG type is PMM2-CDG, caused by mutations on PMM2 (phosphomannomutase 2) genes. PMM2-CDG clinical presentation vary among affected individuals. CDG condition is considered a form of chronic ER stress. Inhibition of glycosylation results in the accumulation of misfolded proteins in the ER, which induces a complex protective reaction known as the unfolded protein response (UPR), which includes translational repression, transcriptional activation of ER chaperones, and ER-associated degradation of unfolded proteins. In search for CDG biomarkers, EBV-transformed CDG B-lymphoblastoid cell lines (B-LCL) were used as cellular models due to two main advantages: first, B-LCL are secretory cells that are forced to continuously synthetize proteins such as immunoglobulins, cytokines or other cell to cell communication molecules, so their ER is chronically stressed; second, as immune system cells, they express common genes and share common regulatory mechanisms with nervous system cells such as neurons (affected cell in most CDG patients). In this work we generated a collection of 7 EBV-transformed PMM2-CDG B-LCL by culture of patients' purified blood B lymphocytes with supernatants of the marmoset EBV-leukocyte cell line B95-8, and compared their transcriptome with that of 7 EBV-transformed healthy B-LCL. Our analysis revealed 348 significantly up-regulated and 106 down-regulated protein-coding genes compared to non-CDG cell lines, which included response to stress, transcription factors, glycosylation, motility and cell junction, development and cell (neuron) differentiation and synapse genes. Gene Set Enrichment Analysis (GSEA) identified biological consequences associated to gene expression changes in PMM2-CDG cells related to the unfolded protein response (UPR), RNA metabolism and the endoplasmic reticulum, Golgi apparatus and mitochondria components. Dysregulated important genes were MAN1A1, MGAT2, CHST4, LARGE, ADAM23, SEMA4D, UNC13C, AUTS2, CA2, SMN1, EXOSC2 expressed not only in the immune system but in other tissues including the nervous system that were compatible with CDG pathophysiology. Our results confirm PMM2-CDG EBV-transformed B-LCL as a suitable cell model that expands both our knowledge and tools to study CDG pathology at the cellular level, useful for functional characteristics and potential therapeutic drugs testing.

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Transcription profiling of human congenital disorders of Glycosylation type-1 patients (CDG-I)

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