The Experts below are selected from a list of 48 Experts worldwide ranked by ideXlab platform
J. R. Miller - One of the best experts on this subject based on the ideXlab platform.
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Use of porcine Interspersed Repeat sequences in PCR-mediated genotyping
Mammalian Genome, 1994Co-Authors: J. R. MillerAbstract:PCR primers derived from porcine short and Long Interspersed Repeat sequences were used to amplify DNA samples isolated from individual members of threegeneration pig reference pedigrees. Subsequent high-resolution gel electrophoresis of both SINE and LINE-PCR products allowed direct visualisation of polymorphisms that segregated in a Mendelian manner. Additional polymorphisms were detected by Southern blotting of the gels described above followed by hybridization with simple sequence DNA. Genotyping by Interspersed Repeat-PCR exploits the natural architecture of the pig genome and allows the typing of polymorphisms by utilizing pre-existing sequence information.
William Bruno - One of the best experts on this subject based on the ideXlab platform.
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duplication of cxc chemokine genes on chromosome 4q13 in a melanoma prone family
Pigment Cell & Melanoma Research, 2012Co-Authors: Xiaohong R. Yang, Paola Ghiorzo, Giorgio Landi, Mai Xu, D. Calista, Kevin K Brown, Celia Badenas, Nicholas K Hayward, Maria Teresa Landi, William BrunoAbstract:Cutaneous malignant melanoma is an etiologically heterogeneous disease with genetic, host, and environmental factors, as well as their interactions contributing to its development (Tucker and Goldstein, 2003). Approximately 10% of melanoma cases occur in a familial setting (Goldstein and Tucker, 2001). To date, two high-risk melanoma susceptibility genes, CDKN2A on chromosome 9p21 and CDK4 on 12q14, have been identified. Germline mutations of these two genes account for only a small proportion of familial melanoma susceptibility, suggesting the existence of other high-risk genes. Recently, copy number variants (CNVs) have been shown to contribute substantially to disease susceptibility in several inherited diseases including cancer (Kuiper et al., 2010). Specifically, a recent genome-wide CNV mapping study reported the identification of a major susceptibility gene for a familial cancer, chordoma (Yang et al., 2009), suggesting that screening for complex genomic rearrangements that co-segregate with disease in families may provide a powerful alternative to traditional gene-mapping approaches. We conducted a genome-wide search for CNVs in 30 high-risk melanoma-prone families without known segregating mutations using a whole-genome human array–comparative genomic hybridization (array-CGH) chip (Nimblegen 385K; average probe spacing, 7 kb). The families were from the United States and ascertained through health care professionals or self referrals. The families included at least two living first degree relatives with a history of invasive melanoma. All family members who were willing to participate in the study provided written informed consent under an NCI IRB-approved protocol. Each underwent a full-body skin examination and completed risk factor questionnaires for sun-related exposures. All diagnoses of melanoma were confirmed by histologic review of pathologic material or by pathology reports. We analyzed blood-derived genomic DNA from 79 individuals including 62 melanoma patients (1–4 patients per family) and 17 spouses. We used the Nexus Copy Number built-in Rank Segmentation algorithm to identify significant CNVs (P=1×10−6; number of probes per segment ≥10; log2 ratio>0.25 for gains and <−0.25 for losses). We focused on CNVs that were not present in the 17 spouses, did not overlap with previously reported CNVs, were located within gene regions, and occurred in all melanoma patients within a family. We identified a duplicated region located on 4q13 in two melanoma patients (II-1 and II-3) in the family shown in Fig. 1. Three individuals (II-1, II-3, and II-4) had a confirmed history of melanoma at the time of initial examination (Fig. 1). Similar to other American melanoma-prone families, all of the melanoma patients had dysplastic nevi (DN), and relatively early onset of melanoma; one patient had multiple primary melanomas. The siblings’ mother (I-2) had confirmed squamous cell carcinomas of the skin and reportedly had lung cancer and melanoma; the maternal grandmother and a maternal cousin also were reported to have melanoma. None of these three melanomas could be confirmed. Fig. 1 The pedigree of the melanoma family with the 4q13 duplication. Age as shown is age at diagnosis for melanoma patients and age at evaluation for unaffected individuals. II-1 and II-3 were analyzed by whole-genome array-CGH; I-1, II-1, II-2, II-3, II-4, ... To validate the duplication and to examine the co-segregation of the duplication with melanoma status, we developed 2 quantitative PCR (qPCR) assays targeting two genes in this region, CXCL3 exon 4 and CXCL6 exon 4, respectively, and analyzed all 7 individuals with DNA available in this family. qPCR analyses confirmed the duplication in all three affected siblings, II-1, II-3, and II-4 (Fig. 2a). The unaffected father (I-1), the unaffected sibling (II-2), and an unaffected grandson (IV-1), did not have the duplication. The unaffected offspring (III-1) of one of the affected patients (II-1) also had the duplication. However, she was only 23 years old at ascertainment, had extensive number of nevi including DN, and had used sun protection for most of her life. Fig. 2 The 4q13 duplication identified in the melanoma-prone family. Panel a. Quantitative PCR (qPCR) of CXCL3 in genomic DNA of the melanoma family. Each qPCR assay was performed in duplicate. Results of qPCR assays for each individual are shown as a point ... To further confirm the 4q13 duplication and to better define the breakpoints of the amplicons, we analyzed genomic DNA from fifteen individuals (individuals II-1, II-2, II-3, II-4, and III-1 in the family with the 4q13 duplication, 5 melanoma patients from families without the 4q13 duplication, and 5 unaffected controls) using a Nimblegen custom-made fine-tiling CGH array spanning the 4q13 region (average probe spacing, 15 bp). We confirmed the duplication in all three affected siblings and in individual III-1. As expected, the duplications in these four individuals were identical in size and location. In contrast, no duplication was observed in II-2, or in five other melanoma patients from five families that did not carry the duplication, or in five controls. We subsequently PCR amplified and sequenced the junction fragments from individuals II-1 and III-1 and determined that the duplication was 257kb (74663132 to 74919990 bp, hg19) with a head-to-tail tandem orientation (Fig. 2b, 2c). Bioinformatic analysis revealed that the breakpoints were located at or near repetitive short and Long Interspersed Repeat (SINE and LINE) elements (Fig. 2b). In contrast, no junction fragment was amplified from individual II-2 who did not carry the duplication. The duplicated region was not present in the other individuals evaluated by array-CGH. In addition, the 4q13 duplication was not observed in 318 control chromosomes (159 control subjects) by qPCR, suggesting that the duplication is unlikely a common polymorphism. Furthermore, the duplication was not observed in index patients from additional CDKN2A/CDK4 mutation-negative melanoma-prone families, including 16 American, 182 Italian, 170 Spanish, and 96 Australian families using either qPCR or array-CGH. The duplicated region affected 10 genes, including IL8 (interleukin 8), CXCL6 (granulocyte chemotactic protein-2), PPBPL1 (pro-platelet basic protein-like 1), PF4V1 (platelet factor-4 variant 1), CXCL1 (melanoma growth-stimulating activity α), PF4 (platelet factor-4), PPBP (pro-platelet basic protein), CXCL5 (chemokine (C-X-C motif) ligand 5), CXCL3 (melanoma growth-stimulating activity γ), and PPBPL2 (pro-platelet basic protein-like 2). IL8 and PPBPL2 were partially affected and the remaining eight genes were completely contained in the duplicated region (Fig. 2b). Most of these genes beLong to a family of CXC chemokines, which are characterized by one amino acid between the first and second cysteine residues. Among them, CXCL1, CXCL3, CXCL5, CXCL6, PPBP, and IL8 contain a three amino acid ELR motif (glutamine–leucine–arginine; ELR+) between the N-terminus and the first cysteine, and function as potent promoters of angiogenesis, therefore promoting tumorigenesis and metastasis (Payne and Cornelius, 2002). CXCL1 has been shown to be up-regulated in melanoma cells and play an important role in melanoma pathogenesis (Dhawan and Richmond, 2002), with secretion of CXCL1 in melanoma cell lines 6- to 16-fold higher than in normal melanocytes (Norgauer et al., 1996). Overexpression of CXCL1 in melanocytes has been associated with enhanced growth and tumor formation in nude mice (Balentien et al., 1991). Blocking antibodies to either CXCL1 or its receptor, chemokine (C-X-C motif) receptor 2 (CXCR2), inhibited melanoma cell growth and reduced angiogenic activity (Haghnegahdar et al., 2000). Similarly, IL8 was the first chemokine reported to induce melanoma cell chemotactic and haptotactic migration (Wang et al., 1990). Neutralizing antibodies to the IL8 receptors CXCR1 and CXCR2, as well as to IL8 itself, inhibited melanoma cell proliferation and invasive potential (Varney et al., 2003), suggesting the potential for these chemokines and their receptors in melanoma treatment. In a study identifying gene expression signatures in melanoma progression, Haqq et al. found that elevated expression of CXCL1 not only distinguished primary tumors from moles but also was useful in identifying metastases (Haqq et al., 2005). Similarly, data from molecular profiling analyses also found upregulation of CXCL3 and IL8 in melanoma tumors and metastases (Lukk et al., 2010) (Mauerer et al., 2011) (Bertucci et al., 2007). In addition, recent data suggest that ciglitazone, an oral thiazolidinedione anti-diabetic drug, inhibited growth and survival of melanoma cells through repressing the expression and secretion of CXCL1, while the recombinant CXCL1 abrogated the apoptotic effect induced by ciglitazone and inhibition of CXCL1 production mimicked the effects of ciglitazone on growth inhibition (Botton et al., 2011). Furthermore, CXCL1 inhibition by ciglitazone was mediated by the decreased expression of microphthalmia-associated transcription factor (MITF), the master gene of melanocyte differentiation. These results further highlight the importance of CXCL1 in melanoma tumorigenicity and its potential as a therapeutic target. In summary, we identified a germline duplication that may explain the melanoma susceptibility in a melanoma-prone family. The duplicated region contains genes that are known to play important roles in melanoma development and progression. Although the duplication is very rare, other types of variants such as point mutations and small insertions/deletions in these genes may explain susceptibility in additional melanoma families and therefore evaluations of variations within this novel duplication are needed to further determine the role of these genes in melanoma susceptibility.
Mark Meuth - One of the best experts on this subject based on the ideXlab platform.
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DNA sequence analysis of gamma radiation-induced deletions and insertions at the APRT locus of hamster cells.
Molecular carcinogenesis, 2006Co-Authors: Carol Miles, Geoffrey Sargent, Geraldine Phear, Mark MeuthAbstract:Gamma radiation-induced gene rearrangements at the Chinese hamster ovary cell locus coding for the purine salvage enzyme adenine phosphoribosyl transferase (APRT) consist of both simple deletions and more complex alterations that are presumably the result of multiple strand breaks. To characterize these mutations at the DNA sequence level, fragments altered by deletion and insertion mutations were obtained by cloning in lambda phage vectors or by using the polymerase chain reaction. The radiation-induced deletions characterized here eliminate 3-4 kb and have at least one breakpoint in an AT-rich region or near short direct or inverted Repeats. Insertions involve small fragments (102 and 456 bp) of repetitive DNA that appear to be related to B2 (short Interspersed repetitive) and Long Interspersed Repeat families. The novel fragments bear little resemblance to each other or to sequences at the integration sites, and their introduction is accompanied by a small target site deletion.
Xiaohong R. Yang - One of the best experts on this subject based on the ideXlab platform.
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duplication of cxc chemokine genes on chromosome 4q13 in a melanoma prone family
Pigment Cell & Melanoma Research, 2012Co-Authors: Xiaohong R. Yang, Paola Ghiorzo, Giorgio Landi, Mai Xu, D. Calista, Kevin K Brown, Celia Badenas, Nicholas K Hayward, Maria Teresa Landi, William BrunoAbstract:Cutaneous malignant melanoma is an etiologically heterogeneous disease with genetic, host, and environmental factors, as well as their interactions contributing to its development (Tucker and Goldstein, 2003). Approximately 10% of melanoma cases occur in a familial setting (Goldstein and Tucker, 2001). To date, two high-risk melanoma susceptibility genes, CDKN2A on chromosome 9p21 and CDK4 on 12q14, have been identified. Germline mutations of these two genes account for only a small proportion of familial melanoma susceptibility, suggesting the existence of other high-risk genes. Recently, copy number variants (CNVs) have been shown to contribute substantially to disease susceptibility in several inherited diseases including cancer (Kuiper et al., 2010). Specifically, a recent genome-wide CNV mapping study reported the identification of a major susceptibility gene for a familial cancer, chordoma (Yang et al., 2009), suggesting that screening for complex genomic rearrangements that co-segregate with disease in families may provide a powerful alternative to traditional gene-mapping approaches. We conducted a genome-wide search for CNVs in 30 high-risk melanoma-prone families without known segregating mutations using a whole-genome human array–comparative genomic hybridization (array-CGH) chip (Nimblegen 385K; average probe spacing, 7 kb). The families were from the United States and ascertained through health care professionals or self referrals. The families included at least two living first degree relatives with a history of invasive melanoma. All family members who were willing to participate in the study provided written informed consent under an NCI IRB-approved protocol. Each underwent a full-body skin examination and completed risk factor questionnaires for sun-related exposures. All diagnoses of melanoma were confirmed by histologic review of pathologic material or by pathology reports. We analyzed blood-derived genomic DNA from 79 individuals including 62 melanoma patients (1–4 patients per family) and 17 spouses. We used the Nexus Copy Number built-in Rank Segmentation algorithm to identify significant CNVs (P=1×10−6; number of probes per segment ≥10; log2 ratio>0.25 for gains and <−0.25 for losses). We focused on CNVs that were not present in the 17 spouses, did not overlap with previously reported CNVs, were located within gene regions, and occurred in all melanoma patients within a family. We identified a duplicated region located on 4q13 in two melanoma patients (II-1 and II-3) in the family shown in Fig. 1. Three individuals (II-1, II-3, and II-4) had a confirmed history of melanoma at the time of initial examination (Fig. 1). Similar to other American melanoma-prone families, all of the melanoma patients had dysplastic nevi (DN), and relatively early onset of melanoma; one patient had multiple primary melanomas. The siblings’ mother (I-2) had confirmed squamous cell carcinomas of the skin and reportedly had lung cancer and melanoma; the maternal grandmother and a maternal cousin also were reported to have melanoma. None of these three melanomas could be confirmed. Fig. 1 The pedigree of the melanoma family with the 4q13 duplication. Age as shown is age at diagnosis for melanoma patients and age at evaluation for unaffected individuals. II-1 and II-3 were analyzed by whole-genome array-CGH; I-1, II-1, II-2, II-3, II-4, ... To validate the duplication and to examine the co-segregation of the duplication with melanoma status, we developed 2 quantitative PCR (qPCR) assays targeting two genes in this region, CXCL3 exon 4 and CXCL6 exon 4, respectively, and analyzed all 7 individuals with DNA available in this family. qPCR analyses confirmed the duplication in all three affected siblings, II-1, II-3, and II-4 (Fig. 2a). The unaffected father (I-1), the unaffected sibling (II-2), and an unaffected grandson (IV-1), did not have the duplication. The unaffected offspring (III-1) of one of the affected patients (II-1) also had the duplication. However, she was only 23 years old at ascertainment, had extensive number of nevi including DN, and had used sun protection for most of her life. Fig. 2 The 4q13 duplication identified in the melanoma-prone family. Panel a. Quantitative PCR (qPCR) of CXCL3 in genomic DNA of the melanoma family. Each qPCR assay was performed in duplicate. Results of qPCR assays for each individual are shown as a point ... To further confirm the 4q13 duplication and to better define the breakpoints of the amplicons, we analyzed genomic DNA from fifteen individuals (individuals II-1, II-2, II-3, II-4, and III-1 in the family with the 4q13 duplication, 5 melanoma patients from families without the 4q13 duplication, and 5 unaffected controls) using a Nimblegen custom-made fine-tiling CGH array spanning the 4q13 region (average probe spacing, 15 bp). We confirmed the duplication in all three affected siblings and in individual III-1. As expected, the duplications in these four individuals were identical in size and location. In contrast, no duplication was observed in II-2, or in five other melanoma patients from five families that did not carry the duplication, or in five controls. We subsequently PCR amplified and sequenced the junction fragments from individuals II-1 and III-1 and determined that the duplication was 257kb (74663132 to 74919990 bp, hg19) with a head-to-tail tandem orientation (Fig. 2b, 2c). Bioinformatic analysis revealed that the breakpoints were located at or near repetitive short and Long Interspersed Repeat (SINE and LINE) elements (Fig. 2b). In contrast, no junction fragment was amplified from individual II-2 who did not carry the duplication. The duplicated region was not present in the other individuals evaluated by array-CGH. In addition, the 4q13 duplication was not observed in 318 control chromosomes (159 control subjects) by qPCR, suggesting that the duplication is unlikely a common polymorphism. Furthermore, the duplication was not observed in index patients from additional CDKN2A/CDK4 mutation-negative melanoma-prone families, including 16 American, 182 Italian, 170 Spanish, and 96 Australian families using either qPCR or array-CGH. The duplicated region affected 10 genes, including IL8 (interleukin 8), CXCL6 (granulocyte chemotactic protein-2), PPBPL1 (pro-platelet basic protein-like 1), PF4V1 (platelet factor-4 variant 1), CXCL1 (melanoma growth-stimulating activity α), PF4 (platelet factor-4), PPBP (pro-platelet basic protein), CXCL5 (chemokine (C-X-C motif) ligand 5), CXCL3 (melanoma growth-stimulating activity γ), and PPBPL2 (pro-platelet basic protein-like 2). IL8 and PPBPL2 were partially affected and the remaining eight genes were completely contained in the duplicated region (Fig. 2b). Most of these genes beLong to a family of CXC chemokines, which are characterized by one amino acid between the first and second cysteine residues. Among them, CXCL1, CXCL3, CXCL5, CXCL6, PPBP, and IL8 contain a three amino acid ELR motif (glutamine–leucine–arginine; ELR+) between the N-terminus and the first cysteine, and function as potent promoters of angiogenesis, therefore promoting tumorigenesis and metastasis (Payne and Cornelius, 2002). CXCL1 has been shown to be up-regulated in melanoma cells and play an important role in melanoma pathogenesis (Dhawan and Richmond, 2002), with secretion of CXCL1 in melanoma cell lines 6- to 16-fold higher than in normal melanocytes (Norgauer et al., 1996). Overexpression of CXCL1 in melanocytes has been associated with enhanced growth and tumor formation in nude mice (Balentien et al., 1991). Blocking antibodies to either CXCL1 or its receptor, chemokine (C-X-C motif) receptor 2 (CXCR2), inhibited melanoma cell growth and reduced angiogenic activity (Haghnegahdar et al., 2000). Similarly, IL8 was the first chemokine reported to induce melanoma cell chemotactic and haptotactic migration (Wang et al., 1990). Neutralizing antibodies to the IL8 receptors CXCR1 and CXCR2, as well as to IL8 itself, inhibited melanoma cell proliferation and invasive potential (Varney et al., 2003), suggesting the potential for these chemokines and their receptors in melanoma treatment. In a study identifying gene expression signatures in melanoma progression, Haqq et al. found that elevated expression of CXCL1 not only distinguished primary tumors from moles but also was useful in identifying metastases (Haqq et al., 2005). Similarly, data from molecular profiling analyses also found upregulation of CXCL3 and IL8 in melanoma tumors and metastases (Lukk et al., 2010) (Mauerer et al., 2011) (Bertucci et al., 2007). In addition, recent data suggest that ciglitazone, an oral thiazolidinedione anti-diabetic drug, inhibited growth and survival of melanoma cells through repressing the expression and secretion of CXCL1, while the recombinant CXCL1 abrogated the apoptotic effect induced by ciglitazone and inhibition of CXCL1 production mimicked the effects of ciglitazone on growth inhibition (Botton et al., 2011). Furthermore, CXCL1 inhibition by ciglitazone was mediated by the decreased expression of microphthalmia-associated transcription factor (MITF), the master gene of melanocyte differentiation. These results further highlight the importance of CXCL1 in melanoma tumorigenicity and its potential as a therapeutic target. In summary, we identified a germline duplication that may explain the melanoma susceptibility in a melanoma-prone family. The duplicated region contains genes that are known to play important roles in melanoma development and progression. Although the duplication is very rare, other types of variants such as point mutations and small insertions/deletions in these genes may explain susceptibility in additional melanoma families and therefore evaluations of variations within this novel duplication are needed to further determine the role of these genes in melanoma susceptibility.
Benjamin Haley - One of the best experts on this subject based on the ideXlab platform.
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Repression of Stress-Induced LINE-1 Expression Protects Cancer Cell Subpopulations from Lethal Drug Exposure.
Cancer cell, 2017Co-Authors: Gulfem D. Guler, Charles A Tindell, Robert M. Pitti, Catherine Wilson, Katrina Nichols, Tommy K. Cheung, Hyojin Kim, Matthew Wongchenko, Yibing Yan, Benjamin HaleyAbstract:Maintenance of phenotypic heterogeneity within cell populations is an evolutionarily conserved mechanism that underlies population survival upon stressful exposures. We show that the genomes of a cancer cell subpopulation that survives treatment with otherwise lethal drugs, the drug-tolerant persisters (DTPs), exhibit a repressed chromatin state characterized by increased methylation of histone H3 lysines 9 and 27 (H3K9 and H3K27). We also show that survival of DTPs is, in part, maintained by regulators of H3K9me3-mediated heterochromatin formation and that the observed increase in H3K9me3 in DTPs is most prominent over Long Interspersed Repeat element 1 (LINE-1). Disruption of the repressive chromatin over LINE-1 elements in DTPs results in DTP ablation, which is partially rescued by reducing LINE-1 expression or function.
