Project description:Identification of novel, highly penetrant, breast cancer susceptibility genes will require the application of additional strategies beyond that of traditional linkage and candidate gene approaches. Approximately one-third of inherited genetic diseases, including breast cancer susceptibility, are caused by frameshift or nonsense mutations that truncate the protein product [1]. Transcripts harbouring premature termination codons are selectively and rapidly degraded by the nonsense-mediated mRNA decay (NMD) pathway. Blocking the NMD pathway in any given cell will stabilise these mutant transcripts, which can then be detected using gene expression microarrays. This technique, known as gene identification by nonsense-mediated mRNA decay inhibition (GINI), has proved successful in identifying sporadic nonsense mutations involved in many different cancer types. However, the approach has not yet been applied to identify germline mutations involved in breast cancer. We therefore attempted to use GINI on lymphoblastoid cell lines (LCLs) from multiple-case, non-BRCA1/2 breast cancer families in order to identify additional high-risk breast cancer susceptibility genes. We applied GINI to a total of 24 LCLs,established from breast-cancer affected and unaffected women from three multiple-case non-BRCA1/2 breast cancer families. We then used Illumina gene expression microarrays to identify transcripts stabilised by the NMD inhibition. Total RNA obtained from the lymphoblastoid cell lines derived from 24 individuals.
Project description:Identification of novel, highly penetrant, breast cancer susceptibility genes will require the application of additional strategies beyond that of traditional linkage and candidate gene approaches. Approximately one-third of inherited genetic diseases, including breast cancer susceptibility, are caused by frameshift or nonsense mutations that truncate the protein product [1]. Transcripts harbouring premature termination codons are selectively and rapidly degraded by the nonsense-mediated mRNA decay (NMD) pathway. Blocking the NMD pathway in any given cell will stabilise these mutant transcripts, which can then be detected using gene expression microarrays. This technique, known as gene identification by nonsense-mediated mRNA decay inhibition (GINI), has proved successful in identifying sporadic nonsense mutations involved in many different cancer types. However, the approach has not yet been applied to identify germline mutations involved in breast cancer. We therefore attempted to use GINI on lymphoblastoid cell lines (LCLs) from multiple-case, non-BRCA1/2 breast cancer families in order to identify additional high-risk breast cancer susceptibility genes. We applied GINI to a total of 24 LCLs,established from breast-cancer affected and unaffected women from three multiple-case non-BRCA1/2 breast cancer families. We then used Illumina gene expression microarrays to identify transcripts stabilised by the NMD inhibition.
Project description:Recessive retinitis pigmentosa (RP) is often caused by nonsense mutations that lead to low mRNA levels as a result of nonsense-mediated decay. Some RP genes are expressed at detectable levels in leukocytes as well as in the retina. We designed a microarray-based method to find recessive RP genes based on low lymphoblast mRNA expression levels Keywords: Recessive mutations; mRNA expression; nonsense mediated-decay; retinitis pigmentosa; lymphocyte; Affymetrix genechip Human Genome U133Plus2.0.
Project description:Goal of the study is the identification of transcriptome deregulation of smg7 pad4 mutants, which are deficient in nonsense-mediated mRNA decay and are blocked in immune signaling, which should avoid secondary responses from immune signaling
Project description:The cAMP-induced intestinal chloride secretion plays a significant role in the pathogenesis of secretory diarrheas. This study aimed to investigate the anti-secretory effects of α,β-dehydromonacolin K, a derivative of lovastatin from Aspergillus sclerotiorum, on cAMP-induced chloride and fluid secretion in human T84 cells and human colonoids. In T84 cells, α,β-dehydromonacolin K inhibited cAMP-induced chloride secretion with an IC50 of ~ 6.32 μM. Apical chloride current analyses in T84 cells demonstrated that α,β-dehydromonacolin K inhibits CFTR chloride channels, stimulated by cAMP agonists, with IC50 of ~ 1 μM. Interestingly, potency of α,β-dehydromonacolin K on CFTR inhibition was decreased in the presence of AMP-activated protein kinase inhibitor or when genistein, a direct CFTR activator, was as a stimulator. Basolateral potassium current analyses indicated that α,β-dehydromonacolin K did not affect basolateral potassium channel activities. In a three-dimensional (3D) model of human colonoids, α,β-dehydromonacolin K significantly suppressed both cAMP-induced and calcium-induced fluid secretion. Proteomic analysis in human clonoids revealed that α,β-dehydromonacolin K interacted with 33 proteins, including those associated with nonsense-mediated mRNA decay. Notably, inhibitory effects of α,β-dehydromonacolin K on cAMP-induced chloride secretion in T84 cells and cAMP-induced fluid secretion in human colonoids were significantly diminished in the presence of a nonsense-mediated mRNA decay inhibitor SMG 1i, suggesting that α,β-dehydromonacolin K inhibited the cAMP-induced fluid secretion in human intestinal epithelial cells by mechanisms involving the nonsense-mediated mRNA decay pathways. In summary, α,β-dehydromonacolin K represents a promising class of natural compounds that exerts an anti-secretory effect in human intestinal epithelia via a novel mechanism of action.
