Browsing by Subject "genetics"

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Open Access
Extreme clonality in lymphoblastoid cell lines with implications for allele specific expression analyses
(2008) Plagnol V.; Uz, E.; Wallace, C.; Stevens H.; Clayton, D.; Ozcelik, T.; Todd J.A.
Lymphoblastoid cell lines (LCL) are being actively and extensively used to examine the expression of specific genes and (genome-wide expression profiles, including allele specific expression assays. However, it has recently been shown that approximately 10% of human genes exhibit random patterns of monoallelic expression within single clones of LCLs. Consequently allelic imbalance studies could be significantly compromised if bulk populations of donor cells are clonal, or near clonal. Here, using X chromosome inactivation as a readout, we confirm and quantify widespread near monoclonality in two independent sets of cell lines. Consequently, we recommend where possible the use of bulk, non cell line, ex vivo cells for allele specific expression assays. © 2008 Plagnol et al.
Open Access
Identification of endogenous reference genes for qRT-PCR analysis in normal matched breast tumor tissues
(Cognizant Communication Corporation, 2009) Gur-Dedeoglu, B.; Konu, O.; Bozkurt, B.; Ergul, G.; Seckin, S.; Yulug, I. G.
Quantitative gene expression measurements from tumor tissue are frequently compared with matched normal and/or adjacent tumor tissue expression for diagnostic marker gene selection as well as assessment of the degree of transcriptional deregulation in cancer. Selection of an appropriate reference gene (RG) or an RG panel, which varies depending on cancer type, molecular subtypes, and the normal tissues used for interindividual calibration, is crucial for the accurate quantification of gene expression. Several RG panels have been suggested in breast cancer for making comparisons among tumor subtypes, cell lines, and benign/malignant tumors. In this study, expression patterns of 15 widely used endogenous RGs (ACTB, TBP, GAPDH, SDHA, HPRT, HMBS, B2M, PPIA, GUSB, YWHAZ2, PGK1, RPLP0, PUM1, MRPL19, and RPL41), and three candidate genes that were selected through analysis of two independent microarray datasets (IL22RA1, TTC22, ZNF224) were determined in 23 primary breast tumors and their matched normal tissues using qRTPCR. Additionally, 18S rRNA, ACTB, and SDHA were tested using randomly primed cDNAs from 13 breast tumor pairs to assess the rRNA/mRNA ratio. The tumors exhibited significantly lower rRNA/mRNA ratio when compared to their normals, on average. The expression of the studied RGs in breast tumors did not exhibit differences in terms of grade, ER, or PR status. The stability of RGs was examined based on two different statistical models, namely GeNorm and NormFinder. Among the 18 tested endogenous reference genes, ACTB and SDHA were identified as the most suitable reference genes for the normalization of qRTPCR data in the analysis of normal matched tumor breast tissue pairs by both programs. In addition, the expression of the gelsolin (GSN) gene, a well-known downregulated target in breast tumors, was analyzed using the two most suitable genes and different RG combinations to validate their effectiveness as a normalization factor (NF). The GSN expression of the tumors used in this study was significantly lower than that of normals showing the effectivity of using ACTB and SDHA as suitable RGs in this set of tumor–normal tissue panel. The combinational use of the best performing two RGs (ACTB and SDHA) as a normalization factor can be recommended to minimize sample variability and to increase the accuracy and resolution of gene expression normalization in tumor–normal paired breast cancer qRT-PCR studies.
Open Access
Mutation in TOR1AIP1 encoding LAP1B in a form of muscular dystrophy: A novel gene related to nuclear envelopathies
(Elsevier Ltd, 2014) Kayman-Kurekci G.; Talim, B.; Korkusuz P.; Sayar, N.; Sarioglu, T.; Oncel I.; Sharafi P.; Gundesli H.; Balci-Hayta, B.; Purali, N.; Serdaroglu-Oflazer P.; Topaloglu H.; Dincer P.
We performed genome-wide homozygosity mapping and mapped a novel myopathic phenotype to chromosomal region 1q25 in a consanguineous family with three affected individuals manifesting proximal and distal weakness and atrophy, rigid spine and contractures of the proximal and distal interphalangeal hand joints. Additionally, cardiomyopathy and respiratory involvement were noted. DNA sequencing of torsinA-interacting protein 1 (TOR1AIP1) gene encoding lamina-associated polypeptide 1B (LAP1B), showed a homozygous c.186delG mutation that causes a frameshift resulting in a premature stop codon (p.E62fsTer25). We observed that expression of LAP1B was absent in the patient skeletal muscle fibres. Ultrastructural examination showed intact sarcomeric organization but alterations of the nuclear envelope including nuclear fragmentation, chromatin bleb formation and naked chromatin. LAP1B is a type-2 integral membrane protein localized in the inner nuclear membrane that binds to both A- and B-type lamins, and is involved in the regulation of torsinA ATPase. Interestingly, luminal domain-like LAP1 (LULL1)-an endoplasmic reticulum-localized partner of torsinA-was overexpressed in the patient's muscle in the absence of LAP1B. Therefore, the findings suggest that LAP1 and LULL1 might have a compensatory effect on each other. This study expands the spectrum of genes associated with nuclear envelopathies and highlights the critical function for LAP1B in striated muscle. © 2014 Elsevier B.V.
Open Access
SIP1 is downregulated in hepatocellular carcinoma by promoter hypermethylation
(2011) Acun, T.; Oztas, E.; Yagci, T.; Yakicier, M.C.
Background: Smad interacting protein-1 is a transcription factor that is implicated in transforming growth factor-β/bone morphogenetic protein signaling and a repressor of E-cadherin and human telomerase reverse transcriptase. It is also involved in epithelial-mesenchymal transition and tumorigenesis. However, genetic and epigenetic alterations of SIP1 have not been fully elucidated in cancers. In this study, we investigated mutations and promoter hypermethylation of the SIP1 gene in human hepatocellular carcinomas.Methods: SIP1 expression was analyzed in HCC cell lines and primary tumors in comparison to normal and non-tumor liver tissues by using semi-quantitative RT-PCR, quantitative real-time RT-PCR and immunohistochemistry. Mutation and deletion screening of the SIP1 gene were performed by direct sequencing in HCC-derived cells. Restoration of SIP1 expression was sought by treating HCC cell lines with the DNA methyl transferase inhibitor, 5-AzaC, and the histone deacetylase inhibitor, TSA. SIP1 promoter methylation was analyzed by the combined bisulfite restriction analysis assay in in silico-predicted putative promoter and CpG island regions.Results: We found that the expression of SIP1 was completely lost or reduced in five of 14 (36%) HCC cell lines and 17 of 23 (74%) primary HCC tumors. Immunohistochemical analysis confirmed that SIP1 mRNA downregulation was associated with decreased expression of the SIP1 protein in HCC tissues (82.8%). No somatic mutation was observed in SIP1 exons in any of the 14 HCC cell lines. Combined treatment with DNA methyl transferase and histone deacetylase inhibitors synergistically restored SIP1 expression in SIP1-negative cell lines. Analysis of three putative gene regulatory regions revealed tumor-specific methylation in more than half of the HCC cases.Conclusions: Epigenetic mechanisms contribute significantly to the downregulation of SIP1 expression in HCC. This finding adds a new level of complexity to the role of SIP1 in hepatocarcinogenesis. © 2011 Acun et al; licensee BioMed Central Ltd.
Open Access
Systematic discovery of Rab GTPases with synaptic functions in Drosophila
(2011) Chan, C.-C.; Scoggin, S.; Wang, D.; Cherry, S.; Dembo, T.; Greenberg, B.; Jin, E.J.; Kuey, C.; Lopez, A.; Mehta, S.Q.; Perkins, T.J.; Brankatschk, M.; Rothenfluh, A.; Buszczak, M.; Hiesinger P.R.
Background: Neurons require highly specialized intracellular membrane trafficking, especially at synapses. Rab GTPases are considered master regulators of membrane trafficking in all cells, and only very few Rabs have known neuron-specific functions. Here, we present the first systematic characterization of neuronal expression, subcellular localization, and function of Rab GTPases in an organism with a brain. Results: We report the surprising discovery that half of all Drosophila Rabs function specifically or predominantly in distinct subsets of neurons in the brain. Furthermore, functional profiling of the GTP/GDP-bound states reveals that these neuronal Rabs are almost exclusively active at synapses and the majority of these synaptic Rabs specifically mark synaptic recycling endosomal compartments. Our profiling strategy is based on Gal4 knockins in large genomic fragments that are additionally designed to generate mutants by ends-out homologous recombination. We generated 36 large genomic targeting vectors and transgenic rab-Gal4 fly strains for 25 rab genes. Proof-of-principle knockout of the synaptic rab27 reveals a sleep phenotype that matches its cell-specific expression. Conclusions: Our findings suggest that up to half of all Drosophila Rabs exert specialized synaptic functions. The tools presented here allow systematic functional studies of these Rabs and provide a method that is applicable to any large gene family in Drosophila. © 2011 Elsevier Ltd. All rights reserved.